「機能性磁性ナノビーズ技術を基盤とする難治性疾患におけるタンパク質分解機構の解明と新規治療法の開発」

平成 26 年度～平成 30 年度
私立大学戦略的研究基盤形成支援事業
研究成果報告書

平成 31 年 3 月
学校法人名 東京医科大学
大学

名 東京医科大学

研究組織名 生 化 学 分 野
研究代表者 宮 澤
啓 介
（東京医科大学医学部医学科主任教授）

目

刊行にあたって次････････････････････････････････････････････････････････

1

プロジェクトの概要･･･････････････････････････････････････････････････････ 3・ 学校法人名

・ 大学名 ･･･････････････････････････････････････････････････････････ 3
・ 研究組織名･･･････････････････････････････････････････････････････

・ プロジェクト所在地

3･･･････････････････････････････････････････････ 3

・ 研究プロジェクト名･････････････････････････････････････････････････ 3
・ 研究観点･････････････････････････････････････････････････････････

3

・ 研究代表者･･･････････････････････････････････････････････････････

3

・ プロジェクト参加研究者数・ 該当審査区分･････････････････････････････････････････ 3･････････････････････････････････････････････････････

・ 研究プロジェクトに参加する主な研究者

3･････････････････････････････ 3
4・ 研究の概要･･･････････････････････････････････････････････････････

・ キーワード･･･････････････････････････････････････････････････････ 10

・ 研究発表の状況･･･････････････････････････････････････････････････ 10・ その他の研究成果等･･･････････････････････････････････････････････ 45

・ 「選定時」及び「中間評価時」に付された留意事項及び対応･････････････ 46
・ 施設・装置・設備・研究費の支出状況
・ 施設・装置・設備の整備状況
・ 研究費の支出状況･･･････････････････････････････ 47･･･････････････････････････････････････ 48･････････････････････････････････････････････････ 48

プロジェクトの成果
・

主要論文

・ その他

･･･････････････････････････････････････････････････････ 52･････････････････････････････････････････････････････････ 377

研究成果報告書刊行にあたって
早いものでこの私立大学戦略的研究基盤形成支援事業が採択されて  年間が経過しました。本事業は、タンパク質分解系に軸足を置いた先進的研究を行っている、学内の５つの研
究グループが集結し、立案したものです。事業採択にともない「分子標的探索センター」を新設し、ハイスループット・アフィニテイー精製技術と質量分析を併用することで、細胞内のタンパク質分解系に関わる機能性タンパク質や標的分子の同定を効率的かつ迅速に行い、難治性疾患の病態解明や、これら疾患に対する新規治療法の開発に挑戦してまいりました。

プロジェクトを統括する立場として特に配慮したのは、優れた研究成果を生み出すことは勿論のこと、①個別研究に終始するのではなく、班員間の協力体制のもとで事業全体が有機的に連携していること、②大学教育機関として次世代の人材育成に貢献すること、そして③全学的な研究活動の活性化に寄与することの  点でした。これらを遂行するために、班員の意思統一と情報交換のための進捗報告会を頻回に開催し、年２回開催される学内の医学会総会への各班からの発表を励行してまいりました。また、大学院生はもとより若手医師や医学科学生達の研究活動への参加を可能にするために、「開かれた研究室運営」をお願いしてきました。今回この研究成果報告書の刊行にあたり過去５年間を振り返りますと、個々の研究成果は極めて独創的かつ挑戦的で、安易に時流に流された研究テーマ設定は何一つないことに改めて気づかされます。班員の先生方の高い見識と、本事業に対する深い理解と協力により、真のリサーチマインドを大切に育んでいく学内風土の醸成に、本事業が大きく寄与できたのではないかと考えております。
「優れた基礎研究は必ず応用展開に結び付く」、これは私が敬愛する師の言葉ですが、優れた研究シーズの創出と実臨床応用に向けての研究拠点として、今後も分子標的探索セン
ターが有効に機能し、発展していくことを心より願っております。ご支援ご協力を賜りまし
た文部科学省、学校法人東京医科大学、共同研究者の皆様に心より厚く御礼申し上げます。
東京医科大学 生化学分野
主任教授 宮澤 啓介

-1-

プロジェクトの概要-2-

（様式 2）
法人番号プロジェクト番号131054
Ｓ1411011

平成 26 年度～平成 30 年度「私立大学戦略的研究基盤形成支援事業」
研究成果報告書概要
１ 学校法人名

東京医科大学３ 研究組織名

２ 大学名

東京医科大学

分子標的探索センター

４ プロジェクト所在地

東京都新宿区新宿 6-1-1，東京医科大学 第一校舎内 分子標的探索センター

５ 研究プロジェクト名 機能性磁性ナノビーズ技術を基盤とする難治性疾患におけるタンパク質分解機構の解明と新規治療法の開発
６ 研究観点７ 研究代表者

研究拠点を形成する研究研究代表者名

所属部局名

職名

宮澤 啓介

医学部医学科・生化学分野

主任教授

８ プロジェクト参加研究者数
９ 該当審査区分

48 名

理工・情報

生物・医歯

人文・社会

１０ 研究プロジェクトに参加する主な研究者
研究者名

所属・職名

プロジェクトでの研究課題

プロジェクトでの役割

宮澤 啓介

生化学分野・
主任教授

オートファジーの制御

プロジェクト統括半田 宏

ナノ粒子先端医学応用講座・特任教授

セレブロン(CBRN)ユビキチンリガーゼの基質・制御機構の解明

FG ビーズを用いたアフィニテイー
精製

医学総合研究所・教授
病態生理学分野・主任教授
生化学分野・
准教授
医学総合研究所・教授

ユビキチン E3 リガーゼ・シノビオリンの機能解析と創薬
筋変性疾患のタンパク質処理機構の観点からの病態解明

トランスジェニックマウスの作成
および病態解析
ゼブラフィッシュによるタンパク質の機能解析

オートファジーの制御

質量分析

サイトカインシグナル伝達に関わる ER
シャペロンタンパク質の機能

タンパク質発現調節機構の解析

中島 利博
林 由起子
平本 正樹

善本 隆之
（共同研究機関等）＜研究者の変更状況（研究代表者を含む）＞
旧
ﾌﾟﾛｼﾞｪｸﾄでの研究課題（変更の時期：平成

所属・職名

年

月

研究者氏名

日）

-3-

プロジェクトでの役割䢈䢛䢗䡸䢚䡦䡴䢀䛷䛾◊✲ㄢ㢟㻌ᡤᒓ䞉⫋ྡ㻌

◊✲⪅Ặྡ㻌

䝥䝻䝆䜵䜽䝖䛷䛾ᙺ๭㻌

㻌

㻌

㻌

㻌

䠄ኚ᭦䛾᫬ᮇ䠖ᖹᡂ㻌 㻌 ᖺ㻌 㻌 ᭶㻌 㻌 ᪥䠅㻌㻌㻌᪂㻌
変更前の所属・職名（様式 2）

法人番号プロジェクト番号変更（就任）後の所属・職名

研究者氏名131054
Ｓ1411011

プロジェクトでの役割-3-

１１ 研究の概要（※ 項目全体を１０枚以内で作成）（１）研究プロジェクトの目的・意義及び計画の概要
生命現象は合成と分解との動的平衡により維持され、そのバランス制御の破綻は様々な病態を引き起こす。本プロジェクトは研究代表者・宮澤を中心に参集された本学の班員らにより、「がん」、「筋ジストロフィー」、「リウマチ」などの異なる領域の先駆的な研究を統合し、「タンパク質分解系の異常」という共通基盤を有する難治性疾患について、分子病態の解明と創薬・新規治療法の開発とを行うことを目的としている。
班員の半田らが開発した機能性磁性ナノビーズ(FG ビーズ)を用いたワンステップ・アフィニテ
イー精製をコア技術として導入し、マススペクトル分析装置を含むプロテオーム解析機器を集約した「分子標的探索センター」を開設した。これにより、個々の研究を横断的かつ集約的に行うことで、飛躍的な推進が期待できる。各疾患における病態基盤分子の共通性と特異性とが明らかになることは、病態解明および創薬開発の視点から大きな意義を有する。
班員らは学術論文だけでなく、既に多くの知的財産の確保、創薬開発・産学連携の実績もある
ことより、本学臨床教室ならびに学外との技術協力により、橋渡し研究を推進し、早期に実用化する。これらの先端研究、知財化、創薬開発、実用化の経験・フローは、本学大学院教育のプログラムとしても次世代へと継承する。

（２）研究組織
研究代表者の宮澤がアフィニテイー精製技術を集約させた「分子標的探索センター」を設置。
このセンターを中核としてタンパク質分解機構の観点から病態解明、新規治療法の開発を研究目標とする本学の研究能力の高い 5 つのグループを参集させ、各グループが「得意」とする研究技術・手法の技術協力ならびに情報共有化により、極めて質の高い研究を効率的に遂行できる研究組織を構築した。研究代表者宮澤は班員間の連携・情報共有化を図り、かつ、新規導入
研究機器に関する説明会、技術指導会の開催とともに、毎年一回「中間報告会」「進捗状況報告会」を主催し、各研究プロジェクトの進捗状況・研究成果を班員間ならびに学内外の研究者・学生に公表している。また、平成 30 年度・最終年度の報告会は「分解系から疾患をみる」とのタイトルでシンポジウム形式の大学院特別講義を行った。また，これらの内容は全て分子標的探索センターホームページで公開している。（http://www.tokyo-med.ac.jp/target/）（３）研究施設・設備等【研究施設】 平成 26 年度より「分子標的探索センター」を東京医科大学・新宿キャンパス・
第一校舎一階に開設（施設面積 171ｍ２）（http://www.tokyo-med.ac.jp/target/）。FG ビー
ズとマススペクトル解析装置の併用による標的分子の同定の迅速化を実現し、さらに標的分子の機能解析・パスウエイ解析に必要とされる研究機器を同センター内に設置し、作業の効率化と集約化を図った。
【研究機器】主な研究機器の導入時期・使用状況は以下の通りである。
○共焦点レーザースキャン顕微鏡（ZEISS LSM710 システム）（平成 26 年導入）：タイムラプ
ス撮影を含め、予約制によりほぼ連日、各班員の研究スタッフ 10 名程度が使用。
-4-

（様式 2）
法人番号プロジェクト番号131054
Ｓ1411011○マススペクトル解析装置（AB SCIEX TripleTOF4600 LC/MS/MS システム）（平成 27 年導入）：
専属の研究補助員 1 名を採用し、各班員からの要請に週 3-4 回の頻度で対応している。
○生細胞イメージングシステム（Essen BioScience IncuCyte ZOOM）（平成 28 年度導入）：導入後、ほぼ連日フル稼働の状態で、10 名の研究員が使用。
○網羅的遺伝子発現解析装置（Affimetrix Gene Atlas）（平成 28 年度導入）： 2 名の研究員が各班員の要請に対応。現在，週１回程度の使用頻度。
【本プロジェクトの人員構成】
6 名の班員(宮澤，半田，中島，林，善本，平本)ならびに各班員が主宰する研究室員 21 名、本事業で採用された PD 1 名、研究補助員 6 名、および、大学院生 4 名、臨床研究医 4 名、医学部医学科学生 6 名の計 48 名。

（４）研究成果の概要 ※下記、１３及び１４に対応する成果には下線及び＊を付すこと。
本事業採択により、機能性磁性ナノビーズを用いたハイスループット・アフィニテイー精製技
術をコア技術に据えた「東京医科大学・分子標的探索センター」を新設し学内研究拠点とした。
細胞内タンパク質の「分解系」に軸足をおいた先進的研究を行っている学内の５つの研究室が有機的に連携することで、難治性疾患の分子病態の解明を行い、併せてこれら疾患の新規治療を目的として、新規薬剤および既存薬剤の drug-repositioning の観点から標的分子の同定とパスウエイ解析を推進することでイノベーションの創生を目指した。本事業では単に班員の個別研究に終始することなく、情報交換・技術連携が確実に実施された。これにより多くの上質な研究シーズが誕生し、かつ、その一部は実用化に至った。これと並行して、本事業は学内研究活動の活性化と若手研究者の育成に貢献できたと考える。
宮澤・平本班(研究課題：オートファジーの制御)：
当班員らはオートファジーの人為的制御法(manipulation)の確立という大きな目標を掲げ、その制御に係る新規分子標的の同定に取り組んだ。特にオートファジー阻害剤としてマクロライ
ド抗生剤を、また、オートファジー誘導剤としてチロシンキナーゼ阻害剤(TKI)である EGFR-TKIの gefitinib に着目し、実臨床応用のための proof of concept (POC)確立に向けて、各薬剤の標的分子の同定と薬理効果の検証を行った。その結果、①肺がん細胞株で gefitinib の標的分子である EGFR をノックアウトしても gefitinib 処理によるオートファジーが誘導されることを明らかにした。これより本来の標的分子である EGFR 阻害とは独立した系でオートファジーが誘導されることを報告した(*57)。また、②TKI とオートファジー阻害能を有する薬剤とを併用することで TKI の殺細胞効果が増強することから、TKI により誘導されるオートファジーは「細胞保護的」に機能していることを明らかにした（＊6,34）。加えて③オートファジーの定量的モニタリング法を確立し、種々の TKI の中で sorafenib, dasatinib, gefitinib が特に強いオートファジー誘導能を有することを明らかにした（未発表・投稿準備中）。さらに、④オート
ファジー誘導活性に係る標的分子の同定に成功した（*発表 2018 年・投稿準備中）。興味深い
ことに同分子はがん細胞の上皮間葉転換 (EMT)に深く関与していることも明らかとなった。
（未発表・投稿準備中）
一方、オートファジー阻害法については、アジスロマイシン(AZM)やクロリスロマイシン(CAM)
等のマクロライド抗生剤がオートファジー阻害活性を有することを発見した(*30,58)。「オートファジー阻害剤」としての AZM, CAM の re-positioning を目的として、オートファジー阻害
活性における標的分子の同定にナノビーズ・テクノロジーを用いることで成功した。（*発表
2018 年・投稿準備中）また、これらマクロライド抗生剤は単剤では抗がん作用を発現しないが、①各種 TKI との併用により TKI の効果を増強するアジュバント作用(*34,57)、②プロテアソー
ム阻害剤との併用による小胞体(ER)ストレス負荷を介した殺細胞効果の増強(58)、③アミノ酸枯渇状況下で殺細胞効果を発揮すること(*30)を明らかにした。これにより、がん治療におけるオートファジー阻害剤としてのマクロライド抗生剤の位置づけを明確にした。
-5-

（様式 2）
法人番号プロジェクト番号131054
Ｓ1411011

当班は、細胞内のタンパク質分解処理に係るプロテアソーム、オートファジー、アグリソームの３者間のネットワークにも着目し、これらが相互に不良タンパク質(unfolded protein)処理を補完することで ER ストレス緩和に寄与しているとの独自の仮説を立てた。この仮説のもとに
これら三者を包括的に阻害することで多発性骨髄腫、転移性乳癌を含めた難治性腫瘍疾患において ER ストレス負荷を介した強力な細胞死誘導によるがん治療応用への可能性を示した。さらに、ER ストレスの定量的ライブモニタリング法を確立し、in vitro の細胞系でプロテアソーム
阻害剤、オートファジー阻害活性を有するマクロライド抗生剤、アグリソーム形成阻害能を有する HDAC 阻害剤の 3 者を併用することで ER ストレス負荷が一気に高まり、これと連動して強力な殺細胞効果が得られること明らかにした(*5,31,58)。さらに班員の林・川原との共同研究によりゼブラフィシュへのがん細胞の xenograft により in vivo でのがん細胞の ER ストレス負荷が経時的にモニタリング可能な系を確立し、臨床応用を踏まえた「ER ストレス誘導療法」として薬剤コンビネーションの検討が進行中である。
半田班 (研究課題：セレブロン(CBRN)ユビキチンリガーゼの基質・制御機構の解明) ：当班員は過去にサリドマイドの催奇性のターゲットとしてセレブロン(CBRN)を同定した。本プ
ロジェクトではこの成果を踏まえて CBRN E3 ユビキチンリガーゼ複合体の機能解析を進めた。
セレブロンは DDB1、Cul4、Roc1 と共に E3 ユビキチンリガーゼ複合体を形成し、その基質受容体として働くことと、サリドマイドとその誘導体（免疫調節薬 IMiDs と総称）による多発性骨髄腫への抗がん作用である主作用のターゲットであることを明らかにした。本成果としては、
まず IMiDs とセレブロン複合体の構造を X 線結晶構造解析すると共に、単一効果しか発揮しな
い従来の抗がん剤と比べて、IMiDs は T 細胞を活性化する免疫調節作用とがん細胞の増殖阻害作用という多面的治療効果を発揮する優れた抗がん剤であることを明らかにした (*62,81)。さらに、サリドマイド誘導体の一つ CC-885 が急性骨髄性白血病（AML）の治療薬として有効であることを示した。CC-885 がセレブロンと結合すると、翻訳終結制御因子 GSPT1 が新規基質として CRBN E3 ユビキチン複合体と結合し、Ub 化･分解される。この GSPT1 の分解が CC-885 の AMLへの抗がん効果に必須であることを証明した（*36）。また、セレブロン、CC-885、GSPT1 から成る複合体の構造解析を電子顕微鏡や X 線結晶構造解析により解明し、GSPT1 が CC-885 結合型
セレブロンと結合する際に、分子糊としての役割を果たすことを明らかにした（*62）。サリド
マイドとそれ以降に開発されたサリドマイド類似体は、セレブロンと選択的に結合し、固有の新規基質をリクルートして、その Ub 化･分解を引き起こすことで治療効果を発揮するため、最近、これらを総称して「CRBN modulators(セレブロンモジュレーター)」と呼ぶことになった(*6，
学会，講演会発表)。さらに、サリドマイドは二種類の光学異性体(S 体、R 体)を有し、どちらが主作用や副作用を有するのかについては長年の難題であったが我々により主作用も副作用も両方ともＳ体がほとんど担うことが明らかとなった（*11）。
また、近年、CRBN 研究に基づき、CRBN 依存的に目的のタンパク質(protein of interest:POI)を分解できる薬剤 proteolysis-targeting chimeras(PROTACs)が開発された。これはサリドマ
イドやその誘導体と POI 結合化合物とを連結し、サリドマイドを介して CRBN 近傍にリクルート
される POI を Ub 化･分解することにより、治療効果を発揮すると云うものである。これにより、
今まで undruggable な転写制御因子などでも標的とできるので、これまでにないタイプの新規薬剤開発が期待されている。
また、サリドマイド催奇性に関して、妊婦が服用する時期により、発症する組織が異なり、多様な表現型があることから、催奇性に関わる基質は一つではないことが予想されてきた。近年、サリドマイド結合型 CRBN と選択的に結合し、Ub 化･分解されることにより催奇性が発症する新規基質が同定されつつある。我々も独自に研究を進めてきており、上肢や耳の発達に関わる因子を見出し、その因子とサリドマイドの相互作用を生化学的解析やモデル実験動物を用いて解-6-

（様式 2）
法人番号プロジェクト番号131054
Ｓ1411011

析し、現在、論文投稿中である(*1)。
従って、サリドマイドによる抗がん作用および催奇性発症作用のいずれに関しても、サリドマ
イドが CRBN と特異的に結合し、固有の基質を CRBN 上にリクルートし、その基質を Ub 化･分解することにより、主作用や副作用が発現されることを明らかにすることができ、我々の研究が、生命科学という基礎研究においても、医療技術や創薬という応用面においても、貢献できたことを報告する。
中島班（研究課題：ユビキチンＥ３リガーゼ・シノビオリンの機構解析と創薬開発）：
当班員は、ロコモティブ症候群の代表的疾患である関節リウマチ（以下、リウマチ）の滑膜細胞の過増殖の分子メカニズムを明らかにする過程で、シノビオリン遺伝子を同定した（Genes
Dev 2003）。本研究では同遺伝子がリウマチなどの炎症性疾患のみならず、肥満・脂肪肝、糖尿病などのエネルギー代謝・メタボリック症候群の病態にも深く関与することを世界に先駆けて発見した（*4,64,86）。さらに、その際の分子標的が、過去に班員らがその概念の提唱と実証の先導的役割（Nature 1995, Cell 1996 & 1997, Nat Genet 1998 & 2000 など）を果たしてきた転写統合装置複合体であることを明らかにした（*64）。すでに、世界初となるユビキチン阻害剤の同定（Int. J. Mol. Biol. 2011）に成功していたが、植物（クルミ）成分の中に同様の活性を見出いし、健康食品としての実用化に成功した（*12）。また、膵ラ氏島等特異的シノ
ビオリンノックアウトマウスを用いた研究により、全世界で推定患者数 3 万人、30 歳前後でほとんどの方が亡くなるＩ型糖尿病と視神経萎縮による視力障害・失明を二大症候とする
Wolfram 症候群の糖尿病に関する病態メカニズムを平本班との協力のもとで明らかにし、その革新的治療法の可能性を提示した（*学会発表 2018 年度、論文作成中）。

林班（研究課題：筋変性疾患のタンパク質処理機構の観点からの病態解明）：
小胞体におけるタンパク質品質管理に重要な役割を果たしている SIL1 の機能をノックダウン
したゼブラフィッシュモデルを作製し、ヒトマリネスコ-シェーグレン症候群(MSS)患者で認められる骨格筋の変性、小眼球、小脳プルキンエ細胞の減少といった MSS の臨床症状と ER ストレス、アポトーシス、オートファジーの亢進といった病態を小型魚類で再現することに成功した（*,17）。さらに CRISPR/Cas9 システムを用いてノックアウトフィッシュの作製に成功した（特許出願予定）ことから、今後、治療薬スクリーニングを開始する予定である。②筋特異的ユビ
キチンリガーゼである MuRF 遺伝子のゼブラフィッシュにおけるプロモーター領域を新たに同定し、murf 遺伝子発現を GFP の発現として可視化できるゼブラフィッシュモデルを作製した（＊
学会発表）。このモデルフィッシュの稚魚を用いて低分子化合物のスクリーニングを実施し、MuRF の発現を変化させうる低分子化合物を複数同定した（特許出願）。また，③常染色体優性
遺伝形式をとるミオパチー２家系において、遺伝性末梢神経障害の原因遺伝子として知られる

HSPB8 に２つの新規変異を同定した。患者骨格筋の病理変化からタンパク質品質管理の障害が示唆された。そこで、今回同定したヒト変異 HSPB8 を発現する細胞ならびにゼブラフィッシュ
モデルを作製し、凝集体形成を再現し、病態解明を進めている(＊学会発表 2018 年度、論文作
成中）。
善本班（研究課題：サイトカインシグナル伝達に関わる ER シャペロンタンパク質の機能）：
当班員はサイトカインサブユニット EBI3 の新規発現調節機構を発見した。新しく生合成された
細胞外分泌蛋白質や膜蛋白質は、小胞体でカルネキシンなどの分子シャペロンに結合し、蛋白質の正しい高次構造形成が誘導されると、ゴルジ体を通って細胞膜に運ばれる。これまでは、
この蛋白質の高次構造形成は、定常状態および炎症誘導時という状況を区別して議論されるこ-7-

（様式 2）
法人番号プロジェクト番号131054
Ｓ1411011とがなかったが、善本らは、IL-6/IL-12 ファミリーに属するヘテロダイマーサイトカイン IL-27
や IL-35 の共通サブユニット Epstein-Barr virus induced gene 3 (EBI3)が、炎症誘導時に、樹状細胞のみならず T 細胞でも発現が増強することを見出し、さらにこれに「標的分子」とカ
ルネキシンが結合することでカルネキシンの分子シャペロン活性を介し蛋白質レベルでの安定化を促し、発現増強につながることを発見した。これは炎症誘導時の新しい蛋白質発現増強機構と考えられる (Mizoguchi et al. J Clin Invest. in revision, *発表 2018 年度)。その「標的分子」として、T 細胞では炎症性疾患や自己免疫性疾患発症に重要な IL-23 のレセプターのサブユニットの 1 つ IL-23R（＊学会発表 2018 年度）や、樹状細胞では MHC クラス I（未発表）などを見出した。元々、この EBI3 は活性化した樹状細胞から産生されるサイトカイン IL-27のサブユニットの 1 つとして報告されたが、本来の役割は小胞体で合成されたばかりの蛋白質にカルネキシンと共に結合し、正しい高次構造形成を促進することであり、そこへ、蛋白質合成されたばかりの p28 やｐ35 がリクルートされて EBI3 と分子結合し、そのまま、ゴルジ体を抜けて細胞外へ放出され、ヘテロダイマーサイトカイン IL-27 や IL-35 として機能するようになったと考えられる。

＜優れた成果が上がった点＞
いずれの班員らの研究成果も独創性があり，かつ，新たな学術領域へと展開する可能性のある優れた研究成果である。既に論文発表により評価が確立している成果から順を追って列記する。
１．X 線結晶構造解析によるセレブロンと IMiDs 複合体の構造(Nat. Struct. Mol. Bio., 2014)，
２．IMiDs による多発性骨髄腫の多面的治療効果機構の解明（Br. J. Haematol., 2014），
３．急性骨髄性白血病の新規治療薬としてサリドマイド誘導体 CC-885 を共同開発し、その作用機構を解明した（Nature, 2016），
４．グルタミンによるグルタミン合成酵素発現量のセレブロン依存的制御を解明した。（Mol.
Cell, 2016）。
５．シノビオリン阻害剤を開発し，肥満・脂肪肝などメタボリック症候群にも有効であること
を動物実験で実証した（EMBO J., 2015）。
６．マリネスコ-シェーグレン症候群(MSS)の疾患モデルをゼブラフィッシュで作成することに成功した。 (PLoS One, 2016)。
７．マクロライド抗生剤のオートファジー阻害活性にかかわる標的分子の同定に成功した。
これによりマクロライドの「オートファジー阻害剤」としての実臨床応用への可能性が高まった。（manuscript in preparation）
８．Wolfram 症候群における糖尿病発症の分子病態を解明した。（manuscript in preparation）
９．IL-27、IL-35 のサイトカインサブユニット EBI3 のカルネキシンとの会合を介した安定化が、炎症時のサイトカイン発現増強につながる新規調節機構を発見した。（manuscript
submitted）＜課題となった点＞
研究プロジェクト自体は順調に進行し，優れた研究実績を上げるとともに多くの上質な研究
シーズも誕生した。加えて本学の全学的な研究活動の活性化、若手研究者の育成にも十分に貢献できたと考える。
ただし，マススペクトル解析装置の導入が本事業採択 2 年後の平成 27 年度末であったため、「分
子標的探索センター」の機能が十分に発揮されるまでに若干の時間的遅れを生じた。このために論文発表までに時間を要し、現時点で未だ進行中のプロジェクトも多く認める。

-8-

（様式 2）
法人番号プロジェクト番号131054
Ｓ1411011＜自己評価の実施結果と対応状況＞
事業採択に伴い「分子標的探索センター」を新設し、年度毎の研究業績を HP で公開するとともに、平成 26 年 10 月「第 1 回キックオフシンポジウム」、平成 27 年度「第 1 回進捗報告会」、
平成 28 年度「中間報告会」、平成 29 年度「第 3 回進捗報告会」、平成 30 年度「最終報告会」の計 5 回の公開報告会を実施し、「自己点検」としてきた。併せて東京医科大学医学会総会（6月、11 月の年 2 回開催される学内医学総会）への各班員からの研究成果の発表を促し、班員間での情報共有と相互の進捗状況の把握に努めた。班員達からのポスター発表のいずれかが、ほぼ毎回医学会奨励賞（ポスター発表 50-60 演題中のベスト３）に選出され受賞する快挙を遂げ、本事業の研究内容が高く評価されてきたともいえる。5 年間での計 102 編の原著論文と、現在、論文化を進めている優れた研究内容を総合すれば、本事業は当初の目的を十分に果たすことができたと言えよう。
今後も「分子標的探索センター」の有効利用を図り、研究拠点としての役割を果たしていきたい。

＜外部（第三者）評価の実施結果と対応状況＞
中間報告会を平成 29（2017）年 1 月 30 日、教育研究棟（自主自学館）3 階大会議室に於いて、外部評価委員として河野隆志氏（国立がん研究センター研究所 ゲノム生物学研究分野・分野長）と相澤信氏（日本大学 医学部 機能形態学系生体構造医学分野・教授）を招聘して公開開催し、学内外から多くの参加者があった。外部評価委員からは、新研究技法を核に「タンパク質分解系」を研究テーマとする多分野の研究者が結集したユニークな事業であること、また、極めて質の高い研究成果が得られていることが高く評価された。今後も班員間の連携を高め、臨床系
分野を含めた共同研究の輪を広げていくことで本事業の役割が果たせる、とのコメントであった。
最終報告会は、平成 31 年（2019）年 3 月 22 日、教育研究棟（自主自学館）3 階 A・B 会議室に於いて外部評価委員として中間報告会と同様に河野隆志氏と相澤信氏の両名を招聘して行われた。プロジェクトリーダー宮澤による事業概要と成果の概略の説明に続き、若手ならびに主要メンバー達による研究成果の報告が行われた。また、この最終報告会は「分解系から疾患をみる」とのタイトルで大学院特別講義も兼ねて公開開催された。外部評価委員からは、前回の中間報告会からの 2 年間で研究の進展が大いに認められたことと共に、本事業は若手研究者の育成にも貢献し、今後のさらなる発展も期待されるとの高い評価が得られた。

＜研究期間終了後の展望＞１．半田班らが開発した機能性磁性（FG）ビーズは薬剤ターゲットの探索だけでなく、患者血清中のエクソソームの定量測定を可能にした。また、蛍光磁性（FF）ビーズは、迅速･高感度診断を可能にした。このように開発したビーズは、薬剤開発だけでなく、新たな診断薬や診断装置の開発にも多大な貢献をしている。従って、ビーズ開発から始まった独自の技術は、生命科学の基礎研究は言うに及ばず、新たな薬剤や診断技術の開発に発展し、さらなる貢献が期待されている。
２．宮澤班は、従来の 14 員環マクロライド抗生剤（CAM）や 15 員環マクロライド（AZM）に加えて、北里生命科学研究所の砂塚敏明教授らが合成した 12 員環マクロライド化合物もオートフ
ァジー阻害効果を有することを発見し、抗菌活性を伴わない新規オートファジー阻害剤としての実用化を目指す。
また、チロシンキナーゼ阻害剤の分子標的の一つが、がん細胞の上皮間葉転換（EMT）に深く関与していることを発見した（＊発表 2018 年度）。さらなる解析を進め、「がん転移予防薬」-9-

（様式 2）
法人番号プロジェクト番号131054
Ｓ1411011の開発を進める。
3. 中島班は脂肪、肝臓、膵β細胞特異的シノビオリン・ノックアウトに加え、現在、解析中の筋特異的ノックアウトマウスの表現型について可及的速やかに論文化を目指していく。エネル
ギー代謝に関わる分子・細胞・臓器レベルでの知見を集約することにより個体レベルでの新たなエネルギー調節機構を明らかにした。

＜研究成果の副次的効果＞
半田班はサリドマイド催奇性のターゲットとして発見した CRBN が、脳神経幹細胞の分化･増殖に関わることを見出し、現在、ケミカルを用いて、神経幹細胞の増殖や分化を制御する技術の開発に取り組んでおり、全く新たな生命科学研究に挑戦している(*2)。
宮澤班はマイトファージー(mitophagy)の解析段階で、核内 BRCA1 蛋白質の新規分解機構を偶然に発見した。ミトコンドリア障害が核内タンパク質分解促進につながる新機軸の分子メカニズ
ムとして現在論文化を進めている。（未発表）中島班はシノビオリン抑制活性を有したサプリメントの実用化を果たしたが、さらに、Wolfram症候群に関しては、新規シノビオリンの下流シグナルを明らかとした。その制御に関しては、
すでに臨床上、汎用されている薬物が存在することを突き止め、Drug-repositioning の一環として若年性稀少性致死性疾患である同症候群の治療法を早期に提供する予定である。さらに、これらを通じ国民の健康に寄与したい。

１２ キーワード（当該研究内容をよく表していると思われるものを８項目以内で記載してください。）（１） ユビキチン（２）
オートファジー（３） 小胞体ストレス（４） がん（５）
関節リウマチ（６） 筋変性疾患（７） 多発性骨髄腫
（８）
セレブロン
１３ 研究発表の状況（研究論文等公表状況。印刷中も含む。）
上記、１１(４)に記載した研究成果に対応するものには＊を付すこと。
＜雑誌論文＞【2019】*1. Asatsuma-Okumura T, Ando H, Simone MD Yamamoto J, Sato T, Shimizu N, Yamaguchi Y, Ito T, Guerrini L Handa H.
Identification of critical neosubstrates of CRL4(CRBN) associated with thalidomide teratogenicity. under review*2. Ando H, Sato T Ito T, Yamamoto J., Sakamoto S, Nitta N, Asatsuma-Okumura T, Shimizu, Mizushima R, Aoki I, Imai T,
Yamaguchi Y, Berk AJ, Handa H. Cereblon control of zebrafish brain size by regulation of neural stem cell proliferation.
iScience in press.
3. Ohta K, Ohkuma T, Lorenzo AD, Yokota A, Hino H, Kazama H, Moriya S, Takano N, Hiramoto M, Miyazawa K.
Fingolimod sensitizes EGFR wild-type non-small cell lung cancer cells to lapatinib or sorafenib and induces cell cycle arrest.
Oncol Rep. in press.

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法人番号プロジェクト番号131054
Ｓ1411011*4. Fujita H, Aratani S, Mizoguchi I, Yagishita N, Nakajima T. Enhanced expression of synoviolin in peripheral blood fromobese/overweight donors. EMBO J. 2019 in press【2018】*5. Kazama H, Hiramoto M, Miyahara K, Takano N, Miyazawa K. Designing an effective drug combination for ER stressloading in cancer therapy using a real-time monitoring system. Biochem Biophys Res Commun. 501(1):286-292. 2018.
*6. Saito Y, Moriya S, Kazama H, Hirasawa K, Miyahara K, Kokuba H, Hino H, Kikuchi H, Takano N, Hiramoto M,
Tsukahara K, Miyazawa K. Amino acid starvation culture condition sensitizes EGFR-expressing cancer cell lines togefitinib-mediated cytotoxicity by inducing atypical necroptosis. Int J Oncol. 52(4):1165-1177. 2018.
7. Hiramoto M, Udagawa H, Ishibashi N, Takahashi E, Kaburagi Y, Miyazawa K, Funahashi N, Nammo T, Yasuda K. A type2 diabetes-associated SNP in KCNQ1(rs163184) modulates the binding activity of the locus for Sp3 and Lsd1/Kdm1a,
potentially affecting CDKN1C expression. Int J Mol Med. 41:717-728. 2018.
8. Iriyama N, Hino H, Moriya S, Hiramoto M, Hatta Y, Takei M, Miyazawa K. The cyclin-dependent kinase 4/6 inhibitor,
abemaciclib, exerts dose-dependent cytostatic and cytocidal effects and induces autophagy in multiple myeloma cells. Leuk
Lymphoma. 59(6):1439-1450. 2018.
9. Kabe Y, Suematsu M, Sakamoto S, Hirai M, Koike I, Hishiki T, Matsuda A, Hasegawa Y, Tsujita K, Ono M, Minegishi N,
Hozawa A, Murakami Y, Kubo M, Itonaga M, Handa H. Development of a highly-sensitive device for counting the number of
disease-specific exosomes in human sera. Clin. Chem., 64:1463-1473. 2018.
10. Kimura A, Kitajima M, Nishida K, Serada S, Fujimoto M, Naka T, Fujii-Kuriyama Y, Sakamato S, Ito T, Handa H, Tanaka
T, Yoshimura A. and Suzuki, H. NQO1 inhibits the TLR-dependent production of selective cytokines by promoting IκB-ζ
degradation. J Exp Med. 215:2197-2209. 2018.
*11.

Mori T, Ito T, Liu S, Ando H, Sakamoto S, Yamaguchi Y, Tokunaga E, Shibata N, Handa H, Hakoshima T. Structural

basis of thalidomide enantiomer binding to cereblon. Sci. Rep. 8:1294. 2018.
*12. Ishida Y, Fujita H, Aratani S, Chijiiwa M, Taniguchi N, Yokota M, Ogihara Y, Uoshima N, Nagashima F, Uchino H,
Nakajima T. The NRF2-PGC-1β pathway activates kynurenine aminotransferase 4 via attenuation of an E3 ubiquitin
ligase, Synoviolin, in a cecal ligation/perforation -induced septic mouse model. Mol Med Rep. 18:2467-2475. 2018*13. Maeda T, Fujita Y, Tanabe-Fujimura C, Zou K, Liu J, Liu S, Kikuchi K, Shen X, Nakajima T, Komano H*: An E3
ubiquitin ligase, synoviolin, is involved in the degradation of homocysteine-inducible endoplasmic reticulum protein.
Biol. Pharm. Bull. 41(6):915-919. 2018*14. Fujita H, Aratani S, Yagishita N, Nishioka K, Nakajima T: Identification of the inhibitory activity of E3 ligase Syvn1
in walnut extract. Mol Med Rep.18: 5701-5708. 2018.
15. Matsubara T, Inoue H, Nakajima T, Tanimura K, Sagawa A, Sato Y, Osano K, Nagano S, Ueki Y, Hanyu T, Hashizume K,
Amano N, Tanaka Y, Takeuchi T: Abatacept in combination with methotrexate in Japanese biologic-naïve patients with active-11-

（様式 2）
法人番号プロジェクト番号rheumatoidarthritis: a randomised placebocontrolledphase IV study. RMD Open. 4:4(2)131054
Ｓ1411011

2018.

*16. Uruha A, Hayashi YK, Mori-Yoshimura M, Oya Y, Kanai M, Murata M, Nishino I. A 31-year-old man with slowly
progressive limb muscle weakness and respiratory insufficiency. Brain Pathology 28:123–124. 2018.
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Kashimada A, Hasegawa S, Isagai T, Uchiyama T, Matsuo M, Kawai M, Goto M, Morio T, Hayashi YK, Takagi M.

Targeting the enhanced ER stress response in Marinesco-Sjögren syndrome. J Neurol Sci 385:49-56. 2018.
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HSPB8-related distal hereditary motor neuropathy and myofibrillar myopathy. Eur J Neurol 25:154-163. 201819. Mori-Yoshimura M, Mitsuhashi S, Nakamura H, Komaki H, Goto K, Yonemoto N, Takeuchi F, Hayashi YK, Murata M,
Takahashi Y, Nishino I, Takeda S, Kimura E. Characteristics of Japanese patients with Becker muscular dystrophy andintermediate muscular dystrophy in a Japanese national registry of muscular dystrophy (Remudy): heterogeneity and clinicalvariation. J Neuromuscul Dis 5:193–203. 2018.
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Protective effects against tumors and infection by IL-27 through promotion of expansion and differentiation of hematopoietic
stem cells into myeloid progenitors. Oncoimmunology. 7(5): e1421892. 2018.
22. Chiba Y, Mizoguchi I, Hasegawa H, Ohashi M, Orii N, Nagai T, Sugahara M, Miyamoto Y, Xu M, Owaki T, Yoshimoto T.
Regulation of myelopoiesis by proinflammatory cytokines in infectious diseases. Cell Mol Life Sci. 75: 1363-1376. 2018
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75:120-127. 2017
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enhances the suppressive effect of interferon-γ on the maintenance of hematopoietic stem cells. EMBO J. 36: 2390-2403. 201729. Himoura N, Nagai H, Fujiwara S, Jimbo H, Yoshimoto T, Nishigori C. Interleukin (IL)-18, cooperatively with IL-23,
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Miyazawa K. Macrolide Antibiotics exhibit cytotoxic effect under amino acid-depleted culture condition by blocking
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Ishikawa T, Miyazawa K. Targeting bortezomib-induced aggresome formation using vinorelbine enhances the cytotoxic
effect along with ER stress loading in breast cancer cell lines. Int J Oncol. 49(5):1848-1858. 2016
32. Terayama H, Hirai S, Naito M, Qu N, Katagiri C, Nagahori K, Hayashi S, Sasaki H, Moriya S, Hiramoto M, Miyazawa K,
Hatayama N, Li ZL, Sakabe K, Matsushita M, Itoh M. Specific autoantigens identified by sera obtained from mice that are
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35. Sharma J, Ishizawa S, Yukino R, Takamura T, Hanyu N, Yasuno H, Handa H, Sandhu A. Fast and sensitive medical
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Matyskiela M. E, Lu G, Ito T, Pagarigan B, Lu C-C, Miller K, Fang W, Wang N-Y, Nguyen D, Houston J, Carmel G,

Tran T, Riley M, Nosaka L, Lander G C, Gaidarova S, Xu S, Ruchelman A. L, Handa H, Carmichael J, Daniel T. O, Cathers B.
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Jeong J. S, Reitsma J. M, Park C. S, Hess S, Deshaies R. J. Glutamine triggers acetylation-dependent degradation of
glutamine synthetase via the thalidomide receptor cereblon. Mol Cell 61:809-820. 2016
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Kabe Y, Nakane T, Koike I, Yamamoto T, Sugiura Y, Harada E, Sugase K, Shimamura T, Ohmura M, Muraoka K,

Yamamoto A, Uchida T, Iwata S, Yamaguchi Y, Krayukhina E, Noda M, Handa H, Ishimori K, Uchiyama S, Kobayashi T,
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Med. 37: 1611-8. 2016*43. Maeda T, Tanabe-Fujimura C, Fujita Y, Abe C, Nanakida Y, Kun

Zou, Liu J, Liu S, Nakajima T, Komano H. NAD(P)Hquinone oxidoreductase 1 inhibits the proteasomal degradation of homocysteine-inducible endoplasmic reticulum
protein. Biochem Biophys Res Commun. 473:1276-1280. 201644. Sugie K, Yoshizawa H, Onoue K, Nakanishi Y, Eura N, Ogawa M, Nakano T, Sakaguchi Y, Hayashi YK, Kishimoto T,
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（様式 2）
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Y., Handa, H. SV40 VP1 major capsid protein in its self-assembled form allows VP1 pentamers to coat various types ofartificial beads in vitro regardless of their sizes and shapes. Biotechnol. Rep. 5:105-111. 2015
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Hara N, Uchino H, Eskil E, Nishioka K, Nakajima T. The E3 ligase synoviolin controls body weight and mitochondrial
biogenesis through negative regulation of PGC-1β. EMBO J 34: 425-577. 2015
65. Nakajima F, Aratani S, Fujita H, Yagishita N, Ichinose S, Makita K, Setoguchi Y, Nakajima T. Synoviolin inhibitor LS102
reduces endoplasmic reticulum stress-induced collagen synthesis in an in vitro model of stress-related interstitial pneumonia.
Int J Mol Med 35: 110-116. 2015
66. Yamauchi J, Coler-Reilly A, Sato T, Araya N, Yagishita N, Ando H, Kunitomo Y, Takahashi K, Tanaka Y, Shibagaki Y,
Nishioka K, Nakajima T, Hasegawa Y, Utsunomiya A, Kimura K, Yamano Y. Anti-CCR4 antibody mogamulizumab targets
human T-lymphotropic virus type I-infected CD8+ as well as CD4+ T cells to treat associated myelopathy. J Infect Dis
211:238-48.

2015

67. Nakajima F, Komoda A, Kawate M, Aratani S, Fujita H, Nakatani K, Akiyama M, Makita K, Nakajima T. Effects of Xenon
Irradiation of Stellate Ganglion Region on Fibromyalgia. J Phys Ther Sci 27:209-212. 2015*68. Yuen M, Sandaradura SA, Dowling JJ, Kostyukova AS, Moroz N, Quinlan KG, Lehtokari VL, Ravenscroft G, Todd EJ,
Ceyhan-Birsoy O, Gokhin DS, Maluenda J, Lek M, Nolent F, Pappas CT, Novak SM, D'Amico A, Malfatti E, Thomas BP,
Gabriel SB, Gupta N, Daly MJ, Ilkovski B, Houweling PJ, Davidson AE, Swanson LC, Brownstein CA, Gupta VA, Medne L,
Shannon P, Martin N, Bick DP, Flisberg A, Holmberg E, Van den Bergh P, Lapunzina P, Waddell LB, Sloboda DD, Bertini E,
Chitayat D, Telfer WR, Laquerrière A, Gregorio CC, Ottenheijm CA, Bönnemann CG, Pelin K, Beggs AH, Hayashi YK,
Romero NB, Laing NG, Nishino I, Wallgren-Pettersson C, Melki J, Fowler VM, MacArthur DG, North KN, Clarke NF.
Leiomodin-3 dysfunction results in thin filament disorganization and nemaline myopathy. J Clin Invest 125:456-457.
2015*69. Uruha A, Hayashi YK, Oya Y, Mori-Yoshimura M, Kanai M, Murata M, Kawamura M, Ogata K, Matsumura T, Suzuki S,
Takahashi Y, Kondo T, Kawarabayashi T, Ishii Y, Kokubun N, Yokoi S, Yasuda R, Kira J, Mitsuhashi S, Noguchi S, Nonaka I,
Nishino I. Necklace cytoplasmic bodies in hereditary myopathy with early respiratory failure.

J Neurol, Neurosurg,

Psych 86:483-489. 2015
70. Tanboon J, Viravan S, Hayashi YK, Nishino I, Sangruchi T. 2 month-old male with hypotonia, Brain Pathol 25:651-652.
2015
71. Suzuki S, Nishikawa A, Kuwana M, Nishimura H, Watanabe Y, Nakahara J, Hayashi YK, Suzuki N, Nishino I.
Inflammatory myopathy with anti-signal recognition particle antibodies: case series of 100 patients. Orphanet J Rare Diseases
10:61, 2015
72. Tanboon J1, Hayashi YK, Nishino I, Sangruchi T. Kyphoscoliosis and easy fatigability in a 14-year-old boy. Neuropathol
35:91-93. 2015-16-

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法人番号プロジェクト番号131054
Ｓ1411011

73. Puppo F, Dionnet E, Gaillard MC, Gaildrat P, Castro C, Vovan C, Bertaux K, Bernard R, Attarian S, Goto K, Nishino I,
Hayashi Y, Magdinier F, Krahn M, Helmbacher F, Bartoli M, Lévy N. Identification of variants in the 4q35 gene FAT1 inpatients with a facioscapulohumeral dystrophy-like phenotype. Hum Mut 36:443-453. 201574. Matsuda C, Kiyosue K, Nishino I, Goto Y, Hayashi YK. Dysferlinopathy fibroblasts are defective in plasma membrane
repair. PLoS Currents 7. 201575. Endo Y, Noguchi S, Hara Y, Hayashi YK, Motomura K, Miyatake S, Murakami N, Tanaka S, Yamashita S, Kizu R, Bamba
M, Goto Y, Matsumoto N, Nonaka I, Nishino I. Dominant mutations in ORAI1 cause tubular aggregate myopathy withhypocalcemia via constitutive activation of store-operated Ca2+ channels. Hum Mol Genet 24:637-648. 201576. Endo Y, Dong M, Noguchi S, Ogawa M, Hayashi YK, Kuru S, Sugiyama K, Nagai S, Ozasa S, Nonaka I, Nishino I. Milder
forms of muscular dystrophy associated with POMGNT2 mutations. Neurol Genet 1:e33. 2015
77. Dong M, Noguchi S, Endo Y, Hayashi YK, Yoshida S, Nonaka I, Nishino I. DAG1 mutations associated withasymptomatic hyperCKemia and hypoglycosylation of alpha-dystroglycan. Neurology 84:273-279. 2015
78.Yoshimoto T, Chiba Y, Furusawa J, Xu M, Tsunoda R, Higuchi K, Mizoguchi I. Potential clinical application ofinterleukin-27 as an antitumor agent. Cancer Sci. 106: 1103-1110. 2015
79. Mizoguchi I, Chiba Y, Furusawa J, Xu M, Tsunoda R, Higuchi K, Yoshimoto T. Therapeutic potential of interleukin-27
against cancers in preclinical mouse models. Oncoimmunology 4: e1042200. 2015
80. Toyota H, Yanase N, Yoshimoto T, Harada M, Kato Y, Mizuguchi J. Vaccination with OVA-bound nanoparticles
encapsulating IL-7 inhibits the growth of OVA-expressing E.G7 tumor cells in vivo. Oncol Rep. 33: 292-296. 2015【2014】*81. Chamberlain P. P, Lopez-Girona A, Miller K, Carmel G, Pagarigan B, Chie-Leon B, Rychak E, Corral L. G, Ren Y. J,
Wang M, Riley M, Delker S. L, Ito T, Ando H, Mori T, Hirano Y, Handa H, Hakoshima T, Daniel T. O, Cathers B. E.
Structure of the human Cereblon-DDB1-lenalidomide complex reveals basis for responsiveness to thalidomide analogs.
Nat. Struct. Mol
82.

Biol. 21:803-809. 2014

Pérez-Perarnau A, Preciado S, Palmeri CM, Moncunill-Massaguer C, Iglesias-Serret D, González-Gironès DM, MiguelM, Karasawa S, Sakamoto S, Cosialls AM, Rubio-Patiño C, Saura-Esteller J, Ramón R, Caja L, Fabregat I, Pons G, Handa H,
Albericio F., Gil J, Lavilla R. A trifluorinated thiazoline scaffold leading to pro-apoptotic agents targeting prohibitins. Angew.
Chem Int Ed. 53:10150-10154. 2014
83. Yamamoto J, Hagiwara Y, Chiba K, Isobe T, Narita T, Handa H, Yamaguchi Y. DSIF and NELF interact with Integrator to
specify the correct post-transcriptional fate of snRNA genes. Nat. Commun., 5, 4263, 2014.
84. Sakamoto S, Omagari K, Kita Y, Mochizuki Y, Naito Y, Kawata S, Matsuda S, Itano O, Jinno H, Takeuchi H, Yamaguchi
Y, Kitagawa Y, Handa H. Magnetically promoted rapid immunoreactions using functionalized fluorescent magnetic beads:
Proof of Principle. Clinical Chem. 60:610-620. 2014
85. Terada K, Tanaka T, Hanyu N, Honda T, Handa H. Rapid and sensitive detection of alpha-fetoprotein by a magnetically-17-A

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法人番号プロジェクト番号131054
Ｓ1411011

promoted shake-free immunoassay employing fluorescent magnetic nanobeads. Int. J. Anal. Bio-Sci. 2:101-107. 2014*86. Wu T, Zhao F, Gao B, Tan C, Yagishita N, Nakajima T, Wong PK, Chapman E, Fang D, Zhang DD. Hrd1 suppresses
Nrf2-mediated cellular protection during liver cirrhosis. Genes Dev. 28: 708-22. 2014
87. Araya N, Sato T, Ando H, Tomaru U, Yoshida M, Coler-Reilly A, Yagishita N, Yamauchi J, Hasegawa A, Kannagi M,
Hasegawa Y, Takahashi K, Kunitomo Y, Tanaka Y, Nakajima T, Nishioka K, Utsunomiya A, Jacobson S, Yamano Y. HTLV-1
induces a Th1-like state in CD4+CCR4+ T cells. J Clin Invest. 124: 3431-42. 2014
*88. Nakajima F, Aratani S, Fujita H, Yagishita N, Ichinose S, Makita K, Setoguchi Y, Nakajima T. Synoviolin inhibitor
LS102 reduces endoplasmic reticulum stress-induced coll agen secretion in an in vitro model of stress-related interstitial
pneumonia. Int J Mol Med. 35:110-116. 201489. Yonekawa T, Malicdan MC, Cho A, Hayashi YK, Nonaka I, Mine T, Yamamoto T, Nishino I, Noguchi S. Sialyllactose
ameliorates myopathic phenotypes in symptomatic GNE myopathy model mice. Brain 137:2670-2679. 201490. Suzuki S, Yonekawa T, Kuwana M, Hayashi YK, Okazaki Y, Kawaguchi Y, Suzuki N, Nishino I. Clinical and histologicalfindings associated with autoantibodies detected by RNA immunoprecipitation in inflammatory myopathies. J Neuroimmuno
274:202-208. 2014
91. Mori-Yoshimura M, Oya Y, Yajima H, Yonemoto N, Kobayashi Y, Hayashi YK, Noguchi S, Nishino I, Murata M. GNE
myopathy: a prospective natural history study of disease progression. Neuromuscul Disord 24:380-386. 2014*92. Miyatake S, Koshimizu E, Hayashi YK, Miya K, Shiina M, Nakashima M, Tsurusaki Y, Miyake N, Saitsu H, Ogata K,
Nishino I, Matsumoto N. Deep sequencing detects very-low-grade somatic mosaicism in the unaffected mother of siblings
with nemaline myopathy. Neuromuscul Disord 24:642-647. 2014
93. Kajino S, Ishihara K, Goto K, Ishigaki K, Noguchi S, Nonaka I, Osawa M, Nishino I, Hayashi YK. Congenital fiber typedisproportion myopathy caused by LMNA mutations.

J Neurol Sci 340:94-98. 2014*94. Goto M, Okada M, Komaki H, Sugai K, Sasaki M, Noguchi S, Nonaka I, Nishino I, Hayashi YK. A nationwide survey
on Marinesco-Sjögren syndrome in Japan. Orphanet J Rare Diseases 9:58, 2014*95. Fujii T, Hayashi S, Kawamura N, Higuchi MA, Tsugawa J, Ohyagi Y, Hayashi YK, Nishino I, Kira J. A case of
adult-onset reducing body myopathy presenting a novel clinical feature, asymmetrical involvement of thesternocleidomastoid and trapezius muscles. J Neurol Sci 343:206-210. 2014
96. Cho A, Hayashi YK, Monma K, Oya Y, Noguchi S, Nonaka I, Nishino I. Mutation profile of the GNE gene in Japanesepatients with distal myopathy with rimmed vacuoles (GNE myopathy). J Neurol, Neurosurg, Psych 85:914-917. 2014
97. Anada RP, Wong KT, Malicdan MC, Goh KJ, Hayashi Y, Nishino I, Noguchi S. Absence of beta-amyloid deposition in thecentral nervous system of a transgenic mouse model of distal myopathy with rimmed vacuoles. Amyloid 21:138-139. 2014
98. Mizoguchi I, Yoshimoto T, Katagiri S, Furusawa J, Chiba Y, Mizuguchi J, Tauchi T, Ohyashiki JH, Ohyashiki K.
Immunological control of chronic myeloid leukemia leading to treatment-free remission. J Hematol Transfus. 2: 1024. 2014-18-

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法人番号プロジェクト番号131054
Ｓ1411011

99. Ohyashiki JH, Ohtsuki K, Mizoguchi I, Yoshimoto T, Katagiri S, Umezu T, Ohyashiki K. Down-regulated
microRNA-148b in circulating PBMCs in chronic myeloid leukemia patients with undetectable minimal residual disease: A
possible molecular classifier to stop imatinib safely. Drug Des Devel Ther. 8: 1151-1159. 2014
100. Yoshimoto T, Mizoguchi I, Katagiri S, Tauchi T, Furusawa J, Chiba Y, Mizuguchi J, Ohyashiki JH, Ohyashiki K.
Immunosurveillance markers may predict patients who can discontinue imatinib therapy without relapse. Oncoimmunology. 3:
e28861. 2014
101. Terayama H, Yoshimoto T, Hirai S, Naito M, Qu N, Hatayama N, Hayashi S, Mitobe K, Furusawa J, Mizoguchi I, Kezuka
T, Goto H, Suyama K, Moriyama H, Sakabe K, Itoh M. Contribution of IL-12/IL-35 common subunit p35 to maintaining thetesticular immune privilege. PLoS ONE. 9: e96120. 2014
102. Fujiwara S, Nagai H, Shimoura N, Oniki S, Yoshimoto T, Nishigori C. Intratumoral CD4+ T lymphodepletion sensitizes
poorly immunogenic melanomas to immunotherapy with an OX40 agonist. J Invest Dermatol. 134: 1884-1892. 2014

＜図書＞
【英文著書】
Sandhu A, Handa H. (eds). Magnetic Nanoparticles for Medical Diagnostics: IOP eBooks. Online ISBN 978-0-7503-1584-5.
2018
Ito T, Hatakeyama M, Handa H. Magnetic Nanoparticles for Medical Diagnostics : Chapter3. Development of a new affinity
nanobead technology and target isolation of bioactive compounds. (eds. Sandhu, A. and Handa H.) IOP eBooks 3.1-3.27 2018
Sakamoto S, Hatakeyama M, Handa H. Magnetic Nanoparticles for Medical Diagnostics : Chapter4. Fluorescent magnetic
beads for medical diagnostics. (eds. Sandhu A, Handa H.) IOP eBooks, 4.1-4.12, 2018
Kawano M, Hatakeyama M, Matsui M, Handa H. Magnetic Nanoparticles for Medical Diagnostics : Chapter5. Developmentof surface-modified magnetic nanoparticles for medical applications. (eds. Sandhu A. and Handa H.) IOP eBooks, 5.1-5.29,
2018
Ito T, Sakamoto S, Handa H. Nanoscale Fabrication, Optimization, Scale-up and Biological Aspects of Pharmaceutical
Nanotechnology : Chapter12. Application of one-step affinity purification of drug-binding proteins with practical magnetic
nanoparticles to drug discovery. (ed. Grumezescu A.M.) Elsevier Science Ltd., Amsterdam, 479-510. 2018
【和文著書・総説】
伊藤拓水、半田 宏：医薬品医療機器レギュラトリーサイエンス「サリドマイドの催奇形性のメカニズム解明と将来」50：4-11、(一財)医薬品医療機器レギュラトリーサイエンス財団（2019.1.10）*林由起子．マリネスコ-シェーグレン症候群の原因と症状．新薬と臨床 67:110-113, 2018
伊藤拓水、半田宏：医学のあゆみ「セレブロンモジュレーター」267：10776-1081、医歯薬出版株式会社（2018.12.29）

田中俊行、畠山士、安野寛、羽入尚広、坂本聡、半田 宏：Medical Science Digest(Industry News)「蛍光磁性ビーズを利用した高速高感度免疫測定システムの開発」44：201-204、ニュー・サイエンス社（2018.7.30）
朝妻知子、伊藤拓水、半田 宏：血液内科「セレブロン制御による急性骨髄性白血病治療の可能性」76：117-123、
科学評論社（2018.2.26）
伊藤拓水、半田 宏：臨床血液－第 79 回学術集会 教育講演特集号－「IMiDs とセレブロンをめぐる最近の話題」
58：259-265、日本血液学会（2017.10.30）
伊藤拓水、半田 宏：ファルマシア「抗がん作用を持つ新たなセレブロンモジュレーターの開発」53：328-332、 (公)日本薬学会（2017.4.1）
伊藤拓水、半田宏：ファルマシア「用語解説｢多発性骨髄腫｣」53：343、 (公)日本薬学会（2017.4.1）

畠山士、半田 宏：「コアシェル微粒子の設計･合成技術･応用の展開《普及版》」（監修：川口春馬）（第 7 章 ア
フィニティ磁性微粒子とスクリーニング自動化システム）204-214/246、シーエムシー出版（2016.8.8）-19-

（様式 2）
法人番号プロジェクト番号131054
Ｓ1411011

伊藤拓水、山本淳一、半田 宏：日本臨牀 増刊号 多発性骨髄腫学 最新の診療と基礎研究 （免疫調節薬(immunomodulatory drugs: IMiDs)による抗骨髄腫効果の機序）152-157/591、(株)日本臨牀社（2016.7.20）
伊藤拓水、安藤秀樹、半田 宏：臨床血液（免疫調節役(IMiDs)のターゲットの発見）57： 556-562、日本血液学会（2016.5.30）
伊藤拓水、半田 宏：医学のあゆみ ユビキチン系の破綻と疾患（サリドマイドの標的ユビキチンリガーゼ"セレブロン"とその機能）256：891-895、医歯薬出版(株)（2016.2.20）
畠山士、半田 宏：「磁気便覧」（編集：日本磁気学会）（ナノ磁性カプセル 448-449、蛍光磁性ビーズ 449-450、自動バイオスクリーニング 452-453/904）丸善出版(株)（2016.1.30）
半田 宏：「サイエンスは個性＆プライド
東京医科大学医学会（2015．7.30）

Science is one's originality and pride.」東京医科大学雑誌 73 巻、219-220、

伊藤拓水、安藤秀樹、半田 宏（監修：赤司浩一）：「IMiDs とセレブロン」IMiDs(免疫調節薬)基礎と臨床 2015、26-34／233、 (株)メディカルレビュー社（2015.5.15）
坂本聡、伊藤拓水、半田 宏：「半田ビーズを用いたサリドマイド標的同定」CSJ カレントレビュー第 19 号（生物活性分子の標的同定と機能解明）、86-93、 (株）化学同人、（2015）
伊藤拓水、安藤秀樹、半田 宏：「サリドマイド標的セレブロンの同定と催奇形性（Phacomelia) 」日本臨牀 73 巻
(特集：多発性骨髄腫の病態と最新治療－基礎と臨床の最新情報－)、143-148、(株)日本臨牀社（2015.1.1）
中島利博：リウマチをなおす（監修中谷孝）芸術出版社 （2014）

坂本聡、半田 宏（企画編集：田崎裕人）：「蛍光体と磁性体を収めたナノカプセルの創製と高速疾患診断への応用」マイクロ/ナノカプセルの調整、徐放性制御と応用事例、277-284／510、 (株)技術情報協会（2014.10.31）
川野雅章、半田 宏（企画編集：田崎裕人）：「ウイルス外殻タンパク質から成るナノカプセルの医療応用」マイ
クロ/ナノカプセルの調整、徐放性制御と応用事例、250-256／510、 (株)技術情報協会（2014.10.31）
半田 宏、伊藤拓水、安藤秀樹：「サリドマイド催奇性の原因因子の発見から創薬への展開」Organ Biology 21
巻、134-140、(株)日本医学館（2014）
寺田孝太郎、田中俊行、羽生尚広、本田孝行、半田 宏：「蛍光性ビーズを用いた迅速・高感度免疫測定法における洗浄工程の簡便化・高効率化検討」多摩川技報 31 巻、13-18、多摩川精機(株)（2014．11.21）
加部泰明、末松誠、半田 宏（編集：杉浦悠毅、末松誠）：「低分子薬剤標的因子の精製技術を知りたい」実験医学増刊（驚愕の代謝システム）、142－149／206、羊土社（2014.9.10）＜学会発表＞【2019】*半田宏「サリドマイドの標的セレブロンの発見から新薬開発」日本薬学会第 139 年会 シンポジウム「選択的蛋白
質分解医薬品開発の最前線」幕張メッセ 2019.3.23(3.20-3.23) （千葉）*Hiroshi Handa「Development of functionalized magnetic nanobeads used for biomedical applications」BIT’s
5th Annual World Congress of Smart Materials-2019 (WCSM2019) 2019.3.8(3.6-3.8）Rome（Italy）*半田宏「IMiDs の分子機構に関する最新の知見」International Symposium for Multiple Myeloma 2019

帝国ホテル（東京）2019.2.23*《特別講演》半田宏「サリドマイド催奇形機構の理解」第 3 回サリドマイド胎芽症研究会ァレンスセンター 2019.2.9

（東京）

-20-

ソラシティーカンフ

（様式 2）
法人番号プロジェクト番号131054
Ｓ1411011【2018】*Takano N, Hiramoto M, Miyazawa K. Identification of intracellular target of azithromycin as an autophagy inhibitor.
第 77 回日本癌学会学術集会 2018 年 9 月（大阪）*宮原か奈、風間宏美、宮澤啓介、石川 孝 再発乳癌に対する「小胞体ストレス負荷療法」の確立を目的とした
MDA-MB-231-ERAI-venus システムの応用

第 26 回2018 年 5 月 16 日（京都）日本乳癌学会学術総会*平本正樹、日野浩嗣、徳久真弓、風間宏美、高野直治、宮澤啓介 チロシンキナーゼ阻害薬ゲフィチニブによるオ
第 91 回

ートファジー調節作用に関わる分子標的探索

日本生化学会大会

2018 年 9 月 24 日（京都）日野浩嗣、入山規良、國場 寛子、風間宏美、森谷昇太、高野直治、平本正樹、宮澤啓介
はリソソーム由来の空胞形成を伴った細胞死を誘導する 第 91 回

CDK4/6 阻害薬 abemaciclib

日本生化学会大会 2018 年 9 月 24 日（京都）

Hirotsugu Hino, Hiromi Kazama, Shota Moriya, Naoharu Takano, Masaki Hiramoto, Keisuke Miyazawa
cytocidal effect of CDK4/6 inhibitor abemaciclib

Lysosome-targeted

第 77 回 日本癌学会 2018 年 9 月 27 日（大阪）横田歩香、平本正樹、日野浩嗣、徳久真弓、風間宏美、高野直治、宮澤啓介 HER2 陽性乳癌細胞株における多機能
アダプタータンパク質 p62/SQSTM1 の解析

第 182 回

東京医科大学医学会総会

2018 年 11 月 17 日（東京）武田淳雄、高野直治、森谷昇太、風間宏美、日野浩嗣、山田裕美子、阿部晃久、平本正樹、宮澤啓介、塚原清彰 プ
ロトンポンプ阻害薬とマクロライド系抗生剤との併用による殺細胞効果の増強 第 182 回
会

東京医科大学医学会総2018 年 11 月 17 日（東京）日野浩嗣、國場寛子、風間宏美、森谷昇太、高野直治、平本正樹、宮澤啓介
ソーム機能阻害に起因する空胞形成を伴った細胞死を誘導する 第 182 回

CDK4/6 阻害薬アベマシクリブはリソ
東京医科大学医学会総会

2018 年 11

月 17 日（東京）*平本正樹、日野浩嗣、徳久真弓、風間宏美、高野直治、宮澤啓介 チロシンキナーゼ阻害薬の分子標的によるオー
トファジー調節作用

第 182 回

東京医科大学医学会総会

2018 年 11 月 17 日（東京）*半田 宏「アフィニティ磁性ビーズの開発から創薬および診断技術への展開」 近畿大学分子工学研究所セミナー
近畿大学福岡キャンパス 2018.12.22 （福岡）*《特別講演》伊藤拓水、半田 宏「Cereblon Modulators の最新 Topics」第 1 回 静岡東部 IMiDs 研究会
ヴェルデ沼津プラザ

2018.11.30 （静岡）*《ワークショップ》伊藤拓水、半田 宏「新規セレブロンモジュレーターの分子機構の解析」第 41 回日本分子生物学会年会『ユビキチン研究の新潮流：ユビキチンコードを識る・操る』 パシフィコ横浜 2018.11.30(11.28-11.30)（神奈川）*《key note》Hiroshi Handa「Development of functionalized magnetic nanobeads for their application to drug
discovery and medical diagnosis」Joint Japan-Spain Symposium on Madical Research Ministry of Science,
Innovation and Universities、2018.11.7(11.7-11.8) Madrid (Spain)-21-

（様式 2）
法人番号プロジェクト番号*《特別講演》伊藤拓水、半田 宏「Cereblon Modulators の最新 Topics」Cel Med

131054
Ｓ1411011

WEB 講演会

SUVT スタジオ

2018.11.26 （東京）伊藤拓水、半田 宏「Cereblon Modulators の最新 Topics」Fukuoka IMiDs Seminar

ホテルオークラ福岡 2018.11.21（福岡）*《key note》Hiroshi Handa「Development of functionalized magnetic nanobeads for their application to drug
discovery and medical diagnosis」Joint Japan-Spain Symposium on Madical Research Ministry of Science,
Innovation and Universities、2018.11.7(11.7-11.8)

Madrid （Spain）*《特別講演》半田 宏「サリドマイド催奇性のターゲットの発見から創薬への展開」創薬薬理フォーラム第 26 回シンポジウム日本薬学会

長井記念館 2018.10.12(10.11-10.12）（東京）

田村秀人、石橋真理子、山本淳一、伊藤拓水、半田 宏、高橋秀実、猪口孝一「免疫調節薬による骨髄腫細胞上のPD-L1 発現の増強とその機序」第 80 回日本血液学会学術集会 大阪国際会議場

2018.10.12-10.14（大阪）*佐藤智美、梶原健、半田 宏、永島雅文「ゼブラフィッシュ脳の発生過程における抗うつ剤 SSRI の作用機構」合同
年会 2018

第 40 回日本生物学的精神医学会 第 61 回日本神経化学会大会（ポスター発表）神戸国際会議場

2018.9.6-9.8（神戸）*《特別講演》伊藤拓水、半田 宏「Cereblon Modulators の最新 Topics」Kanto IMiDs Forum 2018 ～ＩＭiDs のさらなる可能性～

ベルサール神田2018.6.23（東京）《パネルディスカッション》パネリスト：内藤幹彦、冨成祐介、半田 宏、大和隆志、モデレーター：久保田文「※
低分子薬の新たなモダリティ低分子薬の新たなモダリティ：標的蛋白質分解誘導薬の最前線」日経 BP 社本館
2018.5.29（東京）*半田 宏「半田ビーズによるサリドマイドターゲットの発見から創薬への展開」低分子薬の新たなモダリティ低分子薬の新たなモダリティ：標的蛋白質分解誘導薬の最前線 日経 BP 社本館

2018.5.29（東京）*《基調講演》半田 宏「サリドマイド催奇性のターゲットの発見から新規抗がん剤開発へ」第 22 回
標的治療学会学術集会

日本都市センターホテル

2018.5.16(5.16-5.18)

日本がん分子（東京）*《招請講演》半田 宏「セレブロンの発見とセレブロンモジュレーターによる治療応用」第 43 回日本骨髄腫学会学術集会

東京ベイ幕張ホール

2018.5.12(5.12-5.13)（千葉）*《特別講演》半田 宏「半田ビーズを用いたサリドマイドターゲットの同定から Cereblon Modulators への展開」
関東・東北骨髄腫カンファレンス in 東京

ベルサール八重洲

2018.4.21 （東京）*《特別講演》半田 宏「半田ビーズを用いた薬剤ターゲットの同定から創薬への応用」第 16 回東海リンパ腫フォー
ラム学術講演会

キャッスルプラザホテル名古屋

2018.4.7（愛知）

武井寛幸(日医大)、中村清吾、日下部守昭、関野正樹、中川貴之、半田 宏、坂本聡、北川雄光、松田祐子、栗田智-22-

（様式 2）
法人番号プロジェクト番号子、垂野香苗「乳腺外科領域における医工連携染色法－」第 118 回日本外科学会

131054
Ｓ1411011－磁気ナノ粒子を用いたセンチネルリンパ節の同定と新たな免疫定期学術集会 第 93 回卒後教育セミナー

東京国際フォーラム

2018.4.7(4.5-4.7) （東京）*伊藤拓水、半田 宏「セレブロンモジュレーターによるユビキチンリガーゼ制御工学」日本薬学会第 138 年会
ンポジウム『化学修飾の制御と創薬』

2018.3.28 (3.25-3.28)

シ

（金沢）

*半田 宏「半田ビーズの開発から医療バイオへの応用」第 51 回神戸ラボ全体研究会議 神戸ポートアイ神戸大 BT
センター

2018.3.23（神戸）*Satoshi Sakamoto, Sachiko Matsuda, Moriaki Kusakabe, Masaki Sekino, Yuko Kitagawa,

Hiroshi Handa「Development ofa rapid disease diagnosis system utilizing antibodycoated fluorescence magnetic beads.（ 機能化された分散性微粒子を利用する生体分子測定技術の開発）」日本化学会第 98 春季年会（ポスター発表）日本大学理工学部
ス

2018.3.20(3.20-3.23)（千葉）*半田 宏「機能性磁性ビーズの開発とその医療バイオ応用」富士フィルム R&D 主催ゼミ
2018.3.13

船橋キャンパ

富士フィルム先進研究所（神奈川）*安藤秀樹、半田 宏「サリドマイドターゲットであるセレブロンの最近の話題（2.セレブロンの神経幹細胞増殖への役割の解析）」私立大学戦略的研究基盤形成事業『機能性磁性ナノビーズ技術を基盤とする難治性疾患におけるタンパク質分解機構の解明と新規治療法の開発』進捗状況報告会

東京医科大学第一看護棟 2018.2.28（東京）*伊藤拓水、半田 宏「サリドマイドターゲットであるセレブロンの最近の話題（1.サリドマイド光学異性体のセレブロン結合における構造基盤）」私立大学戦略的研究基盤形成事業『機能性磁性ナノビーズ技術を基盤とする難治性疾患におけるタンパク質分解機構の解明と新規治療法の開発』進捗状況報告会
2018.2.28

東京医科大学 第一看護棟（東京）*《受賞講演》半田 宏「新しいナノ磁性ビーズによる創薬標的の単離・同定・計測技術の開発」平成 29 年度島津賞
贈呈式

京都ホテルオークラ2018.2.20（京都）*川幡由希香、川原玄理、井上道雄、西野一三、林由起子 「ゼブラフィッシュモデルを用いた HSPB8 ミオパチーにおける凝集体タンパク質の解析」

第 95 回日本生理学会 2018 年 3 月 30 日（高松）林由起子、山下香、華藤恵美、和田英治「エメリン欠損マウスと抗老化」第 95 回日本生理学会

2018 年 3 月 29 日

（高松）

川原玄理、中屋敷真未、前田秀将、花尻（木倉）瑠理、吉田謙一、林由起子「麻薬成分 25D-NBOMe 投与による横紋筋融解症ゼブラフィッシュモデル」 第 95 回日本生理学会 2018 年 3 月 29 日（高松）華藤恵美、山下香、小島瑞代、和田英治、林由起子「核膜病関連心筋症におけるインターロイキン６の役割」第 95回日本生理学会2018 年 3 月 28 日（高松）和田英治、濵野高行、辻田誠、林由起子．「末期腎不全における骨格筋病態と腎移植前後の筋機能評価」第 95 回日-23-（様式 2）
法人番号プロジェクト番号
本生理学会131054
Ｓ1411011

2018 年 3 月 29 日（高松）和田英治、林由起子「Pathomechanisms of muscle degeneration in nuclear envelopathy」第 59 回日本神経学会総会

2018

年 5 月 24 日（札幌）*川幡由希香，川原玄理，林由起子 「Analysis of aggregated proteins in HSPB8 myopathy using zebrafish models」第181 回東京医科大学医学会総会，2018 年 6 月 2 日

東京医科大学病院（東京）

川原玄理、中屋敷真未、前田秀将、吉田謙一、林由起子「麻薬成分 25D-NBOMe を用いた横紋筋融解症ゼブラフィッ
シュモデル」第 181 回

東京医科大学医学会総会 2018 年 6 月（東京）川原玄理、中屋敷真未、前田秀将、花尻（木倉）瑠理、吉田謙一、林由起子「麻薬成分 25D-NBOMe を用いた横紋筋融解症ゼブラフィッシュモデルの解析」 第 4 回 日本筋学会

2018 年 8 月 10 日（岡山）*川幡由希香、川原玄理、井上道雄、西野一三、林由起子「ゼブラフィッシュモデルを用いた HSPB8 ミオパチーにおける凝集体タンパク質の解析」第 4 回 日本筋学会

2018 年 8 月 10 日（岡山）川原玄理、中屋敷真未、前田秀将、花尻（木倉）瑠理、吉田謙一、林由起子「麻薬成分 25D-NBOMe による横紋筋融解症モデルフィッシュの解析」 第 7 回医薬工３大学包括連携推進シンポジウム

2018 年 10 月（東京）

林由起子、和田英治「核膜病における筋障害メカニズムの解析. Pathomechanisms of muscle degeneratin in nuclear
envelopathy」 日本人類遺伝学会第 63 回大会

2018 年 10 月 13 日

パシフィコ横浜（神奈川）*川幡由希香，川原玄理，林由起子「Analysis of aggregated proteins in HSPB8 myopathy using zebrafish models」
第 181 回東京医科大学医学会総会，2018 年 11 月 17 日 東京医科大学病院（東京）

華藤恵美、山下香、小島瑞代、和田英治、林由起子「核膜病関連心筋症におけるインターロイキン６の役割」筋生
理の集い2018 年 12 月 8 日*Nakajima T

東京慈恵会医科大学（東京）What Rheumatoid synovial cells tell us: apoptosis.

RHEUMATOLOGY2018.6.28-29

APLAR’S EXTENSIVE COURSE ON（Tajikistan）

須藤カツ子、大久保ゆかり、持田澄子、天野栄子、荻野令子、林由紀子、小田切優子、中島利博、荒谷聡子、西洋孝、長井美穂、矢野由希子,、真村瑞子、原由紀子、河井健太郎：大学院生における研究環境の現状。第 181 回東京医科大学医学会総会 2018.6.2（東京）*藤田英俊：慢性炎症疾患を司るシノビオリンの分子・細胞内小器官・臓器・個体レベルでのエネルギー調節機構。
第 19 回運動器科学研究会 2018.9.7-8（岐阜）*藤田英俊、石田裕介、荒谷聡子、横田真穂、内野博之、中島利博：脳内炎症に関わるキヌレニンの代謝機構の解明。
日本線維筋痛症学会第 10 回学術集会

2018.9.29-30（東京）大久保ゆかり、林由起子、須藤カツ子、天野栄子、荻野令子、花田尊子、原由紀子、矢野

由希子、長井美穂、中島利博、荒谷聡子、真村瑞子、柏木保代、上野里絵、西洋孝、宮澤 啓介、持田澄子：女性研究者研究活動支援事-24-

（様式 2）
法人番号プロジェクト番号131054
Ｓ1411011

業の取組とその後の成果。第 7 回医薬工 3 大学包括連携推進シンポジウム 2018.10.6（東京）*横田真穂、藤田英俊、荒谷聡子、中島利博：増殖を指標としたリウマチ滑膜細胞の遺伝子発現解析。第 7 回医薬
工 3 大学包括連携推進シンポジウム 2018.10.6（東京）*藤田英俊、荒谷聡子、横田真穂、中島利博：リウマチ滑膜細胞の増殖機構の解明。第 7 回医薬工 3 大学包括連携推進シンポジウム 2018.10.6（東京）*横田真穂、藤田英俊、荒谷聡子、中島利博：リウマチ滑膜細胞における過増殖メカニズムの解明。第 182 回東京
医科大学医学会総会 2018.11.17（東京）*藤田英俊、荒谷聡子、横田真穂、中島利博：リウマチ滑膜細胞における遺伝子発現ネットワークの解明。第 182 回
東京医科大学医学会総会

2018.11.17（東京）*中島利博：タンパク質分解系を基軸とした多臓器間エネルギー調節ネットワークの解析。Annual Meeting 2018 医学総合研究所研究発表会

2018.11.17（東京）*Orii N, Hasegawa H, Xu M, Mizoguchi I, Yoshida H, Yoshimoto T.「Antitumor effects of IL-27 against a mouse chronic
myeloid leukemia model」第 47 回日本免疫学会総会・学術集会 2018.12.10-12. （福岡）
Koda Y, Nakamoto N, Chu P, Yoshimoto T, Kanai T.「Plasmacytoid dendritic cells suppress acute non-viral hepatitis via
induction of interleukin-35 producing regulatory T cells」第 47 回日本免疫学会総会・学術集会 2018.12.10-12. （福岡）
*Mizoguchi I, Ohashi M, Hasegawa H, Chiba Y, Orii N, Kan S, Xu M, Ochiai N, Owaki T, Yoshimoto T.「A novel role for
EBI3 to augment IL-23Rα protein expression through a lectin chaperone calnexin」東京薬科大学生命科学部 設立 25
周年記念シンポジウム 2018.10.20. （東京）
Yoshimoto T, Hasegawa H, Naoko O, Xu M, Mizoguchi I.「Prediction of chemical respiratory sensitizers by OX40L expression
in dendritic cells using a new 3D co-culture system」The 4th International Conference on Toxicity Testing Alternatives &Translational Toxicology and the 2nd Asian Congress on Alternatives. 2018.10.9-12. Guangzhou, Guangdong, China
折井直子、長谷川英哲、溝口出、仲一仁、善本隆之「慢性骨髄性白血病(CML)マウスモデルを用いた CML 発症

における IL-27 の抗腫瘍効果」第 77 回日本癌学会学術総会 2018.9.27-29. （大阪）
善本隆之「サイトカインによる再生・がん・免疫の制御」静岡大学・大学院総合科学技術研究科・特別講演 2018.9.26.
（静岡）
溝口

出、折井直子、長谷川英哲、善本隆之「化学物質の呼吸器感作性 in vitro 評価法の開発」第 6 期 LRI 研究報告会 2018.8.31. （東京）
大脇敏之、溝口

出、杉山大介、善本隆之「ヒト臍帯血由来間葉系幹細胞による免疫調節と IL-27 の関与」第 83 回

日本インターフェロン・サイトカイン学会学術集会 2018.7.26-27. （東京）
善本隆之、大橋美緒、長谷川英哲、折井直子、徐明利、大脇敏之、溝口-25-

出「新しい 3 次元共培養系を用いた呼吸（様式 2）
法人番号プロジェクト番号131054
Ｓ1411011

器感作性代替法の開発」第 45 回日本毒性学会学術年会 2018.7.18-20. （大阪）
Orii N, Mizoguchi I, Chiba Y, Hasegawa H, Xu M, Ochiai N, Mochizuki Y, Owaki T, Yoshimoto T.「Protective role for IL-27
in the development of mouse chronic myeloid leukemia model」The 45th NATITO CONFERENCE ON Immunological andMolecular Bases for Cancer Immunotherapy 2018.6.26-29. （Sapporo）
大脇敏之、菅原京加、落合 央、溝口 出、千葉佑規乃、長谷川英哲、折井直子、宮本泰則、弓場俊輔、善本隆之「ヒト間葉系幹細胞による免疫調節と IL-27 の関係性」第 16 回日本再生医療学会総会 2018.3.7-9. （仙台）
*Mizoguchi I, Ohashi M, Hasegawa H, Chiba Y, Orii N, Kan S, Xu M, Ochiai N, Owaki T, Yoshimoto T.「A novel role for
EBI3 to augment IL-23Rα protein expression through a lectin chaperone calnexin」東京医科大学記念館ポスター発表
懇談会 2018.2.2. （東京）
*善本隆之「炎症時の新しい蛋白質発現の増強機構」平成 26 年度選定戦略的研究基盤形成支援事業「機能性磁性ナ
ノビーズ技術を基盤とする難治性疾患におけるタンパク質分解機構の解明と新規治療法の開発」進捗状況報告会
2018.2.28. （東京）【2017】
阿部晃久，森谷昇太，平本正樹，風間宏美，山田博之

ハルミンはヒトメラノーマ細胞 G361 において抗アポトーシ

スタンパク質であるサバイビンと Mcl-1 のユビキチン-プロテアソーム系による分解を促進しアポトーシスを誘導する 第 90 回日本生化学会大会・2017 年 12 月 6 日・（神戸）*森谷昇太，風間宏美，

平本正樹，

宮澤啓介 Macrolides overcome cell adhesion-mediated bortezomib

resistance and enhance the cytotoxic effect in myeloma cells.

第 76 回日本癌学会学術集会・2017 年 9 月

28 日・（横浜）*齋藤雄，平澤一浩，
宮原か奈，

塚原清彰，

森谷昇太，

風間宏美， 日野浩嗣，

高野直治，

平本正樹，

宮澤啓介，

國場寛子 Amino AcidStarving Culture Condition Enhances GefitinibInduced Cytotoxicity via

Necroptosislike Cell Death in EGFRExpressing Cancer Cells

第 179 回 東京医科大学医学会総会 2017.6.3（東京）阿部晃久，森谷昇太，平本正樹，風間宏美，宮澤啓介 ハルミンはヒトメラノーマ細胞 G361 において抗アポトー
シスタンパク質であるサバイビンと Mcl-1 のユビキチン-プロテアソーム系による分解を促進しアポトーシスを誘導する 第 180 回東京医科大学医学会総会 2017.11.4（東京）*風間宏美，平本正樹， 宮原か奈，高野直治，宮澤啓介 MB231-ERAI-venus システムを用いた難治性乳癌に対する「ER ストレス負荷療法」の確立

第 180 回東京医科大学医学会総会

2017.11.4（東京）日野浩嗣，國場寛子，風間宏美，森谷昇太，高野直治，平本正樹，宮澤啓介

CDK4/6 阻害薬アベマシクリブによる

非アポトーシス/非ネクロプトーシス細胞死を介した抗腫瘍効果 第 180 回東京医科大学医学会総会 2017.11.4（東京）太田行紀，大熊尭，Alberto L.， 平本正樹，

多発性硬化症治療薬 FTY720 の抗腫瘍効果の検討*宮原か奈，風間宏美， 宮澤啓介， 石川孝

日野浩嗣，

風間宏美，

森谷昇太，

第 180 回東京医科大学医学会総会

高野直治，

宮澤啓介

2017.11.4（東京）乳癌細胞株におけるアグリソーム形成を標的としたボルテゾミブと-26-

（様式 2）
法人番号プロジェクト番号131054
Ｓ1411011

ビノレルビンの併用療法の有効性

第 25 回日本乳癌学会学術総会 2017.7.13（福岡）

宮原か奈，風間宏美，

宮澤啓介 転移性乳癌に対するアグリソームを標的とする新規治療薬の可能性

石川孝，

第 26 回日本癌病態治療研究会

2017.6.1（横浜）*半田宏「ケミカルバイオロジーから病態解明・医療開発へ－「サイエンスは個性とプライド」」第 52 回日本ステ
ロイドホルモン学会学術集会

一橋大学（東京）2017.11.18*Hiroshi Handa「Development of functionalized magnetic beads used for medical applications 」NALS(Nanomaterials
Applied to Life Sciences) 2017

Palacio de Congresos Gijon

2017.12.14(12.13-12.15）（Spain）

山本淳一、Fajar Muhamad、瑞慶覧安里、舘野俊平、半田宏、山口雄輝「NELF を中心としたポリ(A)付加部位の選択機構の解析」ConBio2017(生命科学系学会合同年次大会) （ポスター発表）神戸ポートアイランド2017.12.7(12.6-12.9) （兵庫）*半田 宏「Cereblon modulators」Cancer Stem Cell Symposium ホテル日航福岡 2017.11.11 (11.10-11.11) （福岡）*半田 宏「CRBN Modulators」Multiple Myeloma Expert Seminar in Tokyo ベルサール八重洲

2017.10.24（東京）*《特別教育講演》半田 宏「IMiDs とセレブロンをめぐる最近の話題」第 79 回日本血液学会学術集会
ォーラム

東京国際フ

2017.10.22(10.20-10.22) （東京）*半田 宏「Cereblon Modulators 」CHIBA-IBARAKI Hematology joint seminar

成田東部ホテルエアポート 2017.10.7

（千葉）
*半田 宏
「Cereblon Modulators」
第 7 回長崎 IMiDs 研究会

ザ･ホテル長崎 BW プレミアコレクション 2017.10.6 （長崎）*伊藤拓水、朝妻知子、山本淳一、半田 宏「リガンド依存的ユビキチンリガーゼ CRBN の構造基盤」日本ケミカルバ
イオロジー学会第 12 回年会（ポスター発表）北海道大学 2017.6.8 (6.7-6.9)（札幌）*半田 宏「Cereblon Modulators」第 3 回筑後多発性骨髄腫セミナー マリターレ創世久留米 2017.5.26（福岡）*Hiroshi Handa「Development of an affinity magnetic nanobead technology for the identification of drug targets」
Intermag 2017

CCD(The Convention Centre Dublin)2017.4.26(4.22-4.29)（Ireland）*半田 宏「セレブロンの役割と免疫調節薬(IMiDs)の分子機構」第 54 回日本臨床分子医学会学術集会（ランチョン
セミナー２）

東京国際フォーラム

2017.4.15 (4.14-4.15) （東京）

*佐藤智美、梶原健、永島雅文、半田 宏「発生過程のゼブラフィッシュ視蓋におけるセロトニントランスポーターの機能解析」第 122 回日本解剖学会

長崎大学

2017.3.28 (3.28-3.30) （長崎）-27-

（様式 2）
法人番号プロジェクト番号*半田宏「Cereblon Modulators」Kyushu Hematology Forum2017.2.18.

131054
Ｓ1411011

グランドハイアット福岡 2017.2.18（福岡）*半田宏、種市大喜、山本淳一、朝妻知子、佐藤智美、安藤秀樹、伊藤拓水「アフィニティ磁性ビーズによる標的因子の同定から創薬まで」私立大学戦略的研究基盤形成事業「機能性磁性ナノビーズ技術を基盤とする難治性疾患
におけるタンパク質分解機構の解明と新規治療法の開発」中間報告 東京医科大学*伊藤拓水、佐藤智美、安藤秀樹、朝妻知子、山本淳一、種市大喜、半田

2017.1.30（東京）

宏「CRBN の生物学的意義の解析」私立

大学戦略的研究基盤形成事業「機能性磁性ナノビーズ技術を基盤とする難治性疾患におけるタンパク質分解機構の解明と新規治療法の開発」中間報告*半田宏「アフィニティ磁性ビーズの開発から創薬への展開 -サリドマイド催奇性のタ－ゲット同定から抗がん剤

開発-」先進バイオフォーラム 2017半田東京医科大学 2017.1.30（東京）大阪府立大学2017.1.28（大阪）

宏「アフィニティ磁性ビーズの開発から創薬への展開 -サリドマイド催奇性のタ－ゲット同定から抗がん剤開発-」群馬大学大学院理工学府ナノメディシン関連技術講演

群馬大学

2017.1.19（群馬）千葉祐規乃、溝口 出、長谷川英哲、大橋美緒、善本隆之 「IL-27 を用いたがん治療を目指した新しい DC ワクチ
ン療法への応用」*Nakajima T:

第 16 回日本再生医療学会総会

2017.3.7-9（仙台）

Synoviolin as a novel factor for Rhematoid Arthritis, chronic inflammation and metabolic disorders.

18th Myanmar Internal Medicine Conference 2017(in conjunction with 4th AFIM congress and 4th ACP Southeast Asian
Chapter Meeting)2017.10.1（Yangon）*Fujita H, Aratani S, Yokota M and Nakajima T: Development of novel regulatory methods of mitochondrial functions.
The 16th International Symposium on Advanced Technology (ISAT-16)

2017.11.1-2（東京）

小松梨恵、遊道和雄、仁木久照、西岡久寿樹、中島利博、藤井亮爾：SPACI1/SAAL1 が制御する CKD6 発現量の減
少は RA 滑膜細胞の増殖を制御する。第 61 回日本リウマチ学会

総会・学術集会

2017.4.20-22（福岡）*中島利博：ユビキチン化酵素シノビオリンの発見から臨床応用。第 13 回日本臨床プロテオーム研究会

2017.5.13（東京）*荒谷聡子、藤田英俊、中島利博：リウマチ滑膜細胞のタンパク質の転写後修飾による二元的制御機構の解析。第 6
回医薬工 3 大学包括連携推進シンポジウム

2017.6.17（東京）*藤田英俊 、荒谷聡子、横田真穂 、中島利博：関節リウマチにおけるタンパク質分解ネットワークの解明。第 6 回
医薬工 3 大学包括連携推進シンポジウム 2017.6.17（東京）藤田英俊：シノビオリンノックアウトマウスにおけるメタボローム解析。第 18 回 運動器科学研究会

2017.9.1-2（広島）*藤田英俊、荒谷聡子、横田真穂、中島利博：関節リウマチにおけるタンパク質分解ネットワークの解明。第 180 回-28-

（様式 2）
法人番号プロジェクト番号
東京医科大学医学会総会

131054
Ｓ1411011

2017.11.4（東京）*横田真穂、藤田英俊、荒谷聡子、中島利博：リウマチ滑膜細胞の増殖機構の解明。第 180 回東京医科大学医学会
総会

2017.11.4（東京）*Mizoguchi I, Ohashi M, Hasegawa H, Chiba Y, Xu M, Yoshimoto T.「A novel role for Epstein-Barr virus-induced
gene 3 to augment IL-23 receptor α protein expression through a lectin chaperone calnexin」第 46 回日本免疫学会総会・学術集会 2017.12.12-14（仙台）

Ohashi M, Mizoguchi I, Chiba Y, Hasegawa H, Xu M, Yoshimoto T.「Prediction of chemical respiratory and contact sensitizers
by OX40L expression in dendritic cells using a novel 3D co-culture system」第 46 回日本免疫学会総会・学術集会
2017.12.12-14（仙台）

Yoshimoto,T.「Advances in cancer immunotherapy」Institute of Medical University 1st International Symposium on Roles ofAging and Cancer 2017.8. 28. （Tokyo）*溝口出、千葉祐規乃、長谷川英哲、大橋美緒、中村涼乃、折井直子、干詩宇、徐明利、大脇敏之、善本隆之「IL-27/IL-35 共通サブユニット EBI3 による IL-23R の新しい蛋白質発現安定化機構」第 6 回医薬工 3 大学包括連
携推進シンポジウム 2017.6.17 （東京）
Chiba Y, Ohashi M, Hasegawa H, Xu, M, Owaki T, Mizoguchi I, Yoshimoto T.「A novel antitumor activity of IL-27 bypromoting the differentiation of HSCs to antitumorigenic M1-like macrophages in tumor-bearing mice」The 2nd International
Conference on Cytokine Signaling in Cancer. Heraklion Crete 2017.5.30-6.4.（Greece）
千葉祐規乃、溝口 出、長谷川英哲、大橋美緒、善本隆之「IL-27 を用いたがん治療を目指した新しい DC ワクチン
療法への応用」第 16 回日本再生医療学会総会 2017.3.7-9 （仙台）
*善本隆之「炎症性腸疾患発症に関与する IL-23R 蛋白質の新しい発現安定化機構」平成 26 年度選定戦略的研究基盤
形成支援事業「機能性磁性ナノビーズ技術を基盤とする難治性疾患におけるタンパク質分解機構の解明と新規治療法の開発」中間報告会 2017.1.30 （東京）【2016】*宮原か奈 、 森谷昇太、平澤一浩、平本正樹、小松誠一郎、石川孝、宮澤啓介 「Targeting bortezomib-induced

aggresome formation using vinorelbine enhances the cytotoxic effect along with ER-stress loading in breast cancer celllines」

第 177 回 東京医科大学医学会総会東京医科大学病院 2016.6.4(東京)*平澤一浩 、 塚原清彰、森谷昇太、平本正樹、風間宏美、由田淳、阿部晃久、宮澤啓介、宮原か奈、稲津正人「Macrolides enhance bortezomib-induced cytotoxicity and overcome stromal cell-mediated drug resistance inmyeloma cells. 」

第 177 回 東京医科大学医学会総会、 2016 年 6 月 4 日、東京医科大学病院、(東京)

宮澤啓介（教育講演）「*マクロライドとオートファジー」 第 23 回マクロライド新作用研究会. 2016.7.30.

(東京)*平本正樹 、 森谷昇太、武村淳、風間宏美、日野浩嗣、高野直治、阿部晃久、宮澤啓介 「光反応性クロスリンカ
ーと磁性ナノビーズを用いた分子標的探索の試み」 第 89 回

-29-

日本生化学会大会

2016 年 9 月（仙台）（様式 2）
法人番号プロジェクト番号131054
Ｓ1411011*平澤一浩 、 塚原清彰、森谷昇太、平本正樹、宮澤啓介、宮原か奈 「アミノ酸飢餓状態下で、マクロライド系抗菌薬はオートファジー阻害により頭頸部癌細胞株の細胞死を誘導する」 第 75 回日本癌学会学術集会 2016 年 10 月
（横浜）
*宮原か奈 、 森谷昇太、平澤一浩、平本正樹、小松誠一郎、石川孝、阿部晃久、宮澤啓介「ビノレルビンは乳癌細胞株に於いてアグリソーム形成を阻害することによりボルテゾミブ誘導性の細胞毒性を増強する」

第 75

回日本癌学会学術集会 2016 年 10 月（横浜）*森谷昇太 、 風間宏美、

平本正樹、 相澤信、砂塚敏明、半田宏、宮澤啓介 「Macrolides enhancebortezomib-induced cytotoxicity in myeloma cells co-cultured with stroma layer.」 第 78 回

日本血液学会2016 年

10 月（横浜）*森谷昇太 、 風間宏美、

平本正樹、 相澤信、砂塚敏明、半田宏、宮澤啓介 「Macrolides enhancebortezomib-induced cytotoxicity and overcome stromal cell-mediated drug resistance in myeloma cells」
京医科大学医学会総会 2016 年 11 月 5 日

第 178 回 東

東京医科大学病院 （東京）

*平澤一浩 、 塚原清彰、森谷昇太、平本正樹、風間宏美、由田淳、阿部晃久、宮澤啓介、宮原か奈、稲津正人「Macrolide antibiotics exhibit cytotoxic effect under amino acid-depleted culture condition via blocking autophagy
flux in head and neck squamous cell carcinoma cell lines」第 178 回 東京医科大学医学会総会

2016 年 11 月 5 日 東京医科大学病院（東京）*伊藤拓水、半田宏「急性骨髄性白血病に効果を示す新規のセレブロンモジュレーター」第 39 回日本分子生物学会年会

パシフィコ横浜

2016.12.1(11.30-12.2) （横浜）佐藤智美、梶原健、永島雅文、半田宏「発生過程の視蓋におけるセロトニン輸送体の機能解析」 第 39 回日本分子生物学会年会（ポスター発表）パシフィコ横浜 2016.12.1(11.30-12.2)（横浜）*Handa H.「Identification of a target of thalidomide using our affinity nanobead technology and development of novel
drugs based on the target」Japan-Spain Joint Workshop on Nanomedicine Research

2016.12.1(12.1-12.2)

Institute of

Health Carlos III (ISCIII), Madrid (Spain)*半田宏「Cereblon Modulators」ImiDs Forum 2016

がん研究会有明

半田宏、川野雅章「蚕高発現系により医工連携研究

ウイルス外郭タンパク質から成るウイルス様粒子（VLP)の2016.11.7（東京）

工学的技術開発とワクチン製剤としての医療応用」第 89 回日本生化学会大会 バイオインダストリーセミナー 仙台国際センター2016.9.25.(9.25～9.27)（仙台）*伊藤拓水、半田 宏「急性骨髄性白血病に有効な新規セレブロンモジュレーターの開発」
大会

東北大学 2016.9.25(9.25～9.27)

伊藤拓水、半田第 89 回日本生化学会（仙台）宏「急性骨髄性白血病に有効な新規セレブロンモジュレーターの開発」 第 89 回日本生化学会大会（ポスター発表） 仙台国際センター 2016.9.25.(9.25～9.27) （仙台）*半田

宏「セレブロンモジュレーター」Kyoto IMiDs Forum半田宏「医工連携-セレブロン、免疫調節役の標的-」TeFFA 国際産学連携ワークショップ 2016

-30-

京都大学

2016.9.24（京都）-産学連携と医工（様式 2）
法人番号プロジェクト番号2016.9.6

連携-

東京工業大学半田宏「IMiDs 最前線」
第 4 回熊本 IMiDs 研究会

131054
Ｓ1411011（東京）
ANA クラウンプラザホテル 熊本ニュースカイ 2016.8.5 （熊本）*Handa H.「Cereblon; a direct target of immunomodulatory agents」2016 the Japanese Society of Medical Oncology
Annual Meeting (第 14 回日本臨床腫瘍学会学術集会)

Kobe International Exhibition Hall2016.7.30.(7.28-7.30) （神戸）半田宏「磁気ビーズ研究開発の歴史と現状」第 1 回ライフサイエンス分野における時期ビーズ応用技術構築に関する懇談会 2016.7.19
半田バイオフロンティアパートナーズ

宏「ケミカルターゲットの同定から生体制御機構の理解と創薬への応用」新学術領域研究「天然物ケミカル

バイオロジー ～分子標的と活性制御～」研究成果報告シンポジウム 慶應義塾大学

2016.7.1（東京）*Handa H.「Cereblon (CRBN), the common target of multiple pharmacological actions of thalidomide and its
derivatives」UCLA CTSI Seminar Series UCLA David Geffen School of Medicine
半田宏「セレブロン ～IMiDs のターゲット～」第 10 回臨床骨髄腫研究会

2016.3.23（L.A）
メルキュールホテル札幌

2016.2.4（札幌）*半田宏「サリドマイド標的因子セレブロン同定から新たな多発性骨髄腫治療薬の開発」文部科学省私立大学戦略的研究基盤形成支援事業第一回進捗状況報告会
坂本聡、半田ィング

東京医科大学 2016.1.27（東京）

宏「抗体標識蛍光磁気ビーズによる転移がん迅速診断システムの構築」 AMED キックオフミーテ

東京大学2016.1.7 （東京）*Nakajima T.「Synoviolin, as a new stellar factor for understanding chronic inflammation.」 MOSCOW
INTERNATIONAL FORUM OF BONES AND JOINTS DISORDERS/THE INTERNATIONAL SCHOOL CONFERENCE(ISC) 2016.4.18-21（Moscow）
Nazgul Omurzakova, Nakajima T 「Study on Clinical-Functional Peculiarities Assessment of Rheumatic Fheumatic Fever inthe Kyrgyz Republic.」The World Congress of Cardiology and Cardiovascular Health (WCC 2016) 2016.6.4-7（Mexico City）*Aratani S, Nakajima T「 Synoviolin, as a novel factor for understanding obesity and metabolic disorders based on

chronic inflammation.」 International Conference on Obesity and Chronic Diseases (ICOCD-2016) 2016.7.25-27（ Las

Vegas）*Aratani S, Fujita H, Yagishita N, Yokota M, Nishioka K, Nakajima T 「E3 Ubiquitin ligase synoviolin inhibitors has effects
on obesity. 」

International Conference on Obesity and Chronic Diseases (ICOCD-2016) 2016.7.25-27 （Las Vegas）

黒岩義之，平井利明，中村郁朗，中島利博，横田俊平，内山眞幸、林毅、西岡久寿樹 「HPV ワクチン接種後神経
内分泌障害を呈した 104 例：新規症候群のベッドサイド三段階診断群の三段階診断」 第 57 回日本神経学会総会
2016.5.18-21（神戸）平井利明，黒岩義之，内山眞幸、林毅、横田俊平，中村郁朗，中島利博，西岡久寿樹、井口保之 「子宮頚癌ワクチ
ン接種後の中枢神経障害：脳血流検査，脳波，内分泌検査異常について」 第 57 回日本神経学会総会-31-

2016.5.18-21

（様式 2）
法人番号プロジェクト番号131054
Ｓ1411011（神戸）*荒谷聡子、藤田英俊、西岡久寿樹、中島利博 「E3 ユビキチン化酵素シノビリオン阻害剤の臨床応用への可能性」
第 177回東京医科大学医学会総会 2016.6.4（東京）石田祐介、千々岩みゆき、内野博之、荒谷聡子、藤田英俊、中島利博 「肺血症性脳症に対するシノビオリンとの関連」

第 177 回東京医科大学医学会総会

2016.6.4（東京）*荒谷聡子、藤田英俊、八木下尚子、中島若巳、西岡久寿樹、中島利博 「シノビオリン阻害剤の実用化に向けて」
第 5 回医薬工 3 大学包括連携推進シンポジウム 2016.6.18（東京）*藤田英俊、荒谷聡子、須藤カツ子、稲津正人、原直美、内野博之、西岡久寿樹、中島利博 「Synoviolin is a key regulator
in chronic inflammation.」*中島利博
会

第 5 回医薬工 3 大学包括連携推進シンポジウム 2016.6.18（東京）「新規自己炎症症候群モデルマウスの樹立とシノビオリン阻害剤の効果」 第 17 回 運動器科学研究2016.9.2-3（大阪）*藤田英俊「リウマチ発症因子シノビオリンによるエネルギー代謝調節の解明」 第 17 回 運動器科学研究会

2016.9.2-3

（大阪）

*荒谷聡子

「シノビオリン阻害剤の実用化に向けて」 第 17 回 運動器科学研究会

2016.9.2-3（大阪）

河野麻衣、薦田昭宏、橋本聡子、中谷 孝、中島利博 「社会復帰に向け生活リズムの獲得に着目した一症例」日本
線維筋痛症学会第 8 回学術集会

2016.9.17-18（東京）

多尾聡美、姫田 敬、薦田昭宏、中谷 孝、中島 利博 「線維筋痛症患者における当院での作業療法の取り組み」 日
本線維筋痛症学会

第 8 回学術集会

2016.9.17-18（東京）

手島議起、渡邉彩花、薦田昭宏、橋本聡子、黒田祐子、中谷 孝、中島利博 「線維筋痛症患者に対するスーパーラ
イザー照射後の即時効果について」

日本線維筋痛症学会 第 8 回学術集会

2016.9.17-18（東京）

薦田昭宏、橋本聡子、窪内郁恵、黒田祐子、渡邉彩花、手島議起、中島若巳、中谷 孝、中島利博 「線維筋痛症に対する運動学習における運動イメージの活用」

第 8 回学術集会

日本線維筋痛症学会

2016.9.17-18（東京）

黒田祐子、渡邉彩花、薦田昭宏、橋本聡子、手島議起、中谷 孝、中島利博 「生理的コスト指数と主観的運動強度の関係性」

日本線維筋痛症学会

第 8 回学術集会

2016.9.17-18（東京）

斎藤麻衣、矢野由紀、薦田昭宏、中谷 孝、中島利博 「線維筋痛症にともなうドライマウスに対するアプローチ」
日本線維筋痛症学会第 8 回学術集会

2016.9.17-18（東京）

山口結花、藤島みどり、梅原由加里、永森ひさみ、長野愛子、中平律子、北川中行、中谷 孝、中島利博 「線維筋痛症・難病センターにおける外来患者の症例報告」

日本線維筋痛症学会

第 8 回学術集会

2016.9.17-18 （東京）梅原由加里、山口結花、長野愛子、永森ひさみ、北川中行、中谷 孝、中島利博
導」

日本線維筋痛症学会 第 8 回学術集会

2016.9.17-18

-32-

（東京）

「入院中 FM 患者の日常生活指（様式 2）
法人番号プロジェクト番号131054
Ｓ1411011

西森美佐子、山口結花、梅原由加里、澁谷美雪、荒谷聡子、中島若巳、北川中行、中谷 孝、中島利博 「線維筋通第 8 回学術集会症における当院の関節超音波検査の取り組みと役割」日本線維筋痛症学会

2016.9.17-18 （東京）荒谷聡子、藤田英俊、黒岩義之、臼井千恵、西岡久寿樹、中島利博 「ヒトパピローマウイルスワクチン関連神経
免疫異常症候群モデルマウスの解析」 日本線維筋痛症学会 第 8 回学術集会*藤田英俊、荒谷聡子、西岡久寿樹、中島利博
術集会

2016.9.17-18

2016.9.17-18（東京）「シノビオリン阻害剤の探索」 日本線維筋痛症学会

第 8 回学（東京）*藤田英俊、荒谷聡子、西岡久寿樹、中島利博 「マウスモデルを用いたシノビオリン阻害活性を有する天然物の効
第 8 回学術集会

果の検証」日本線維筋痛症学会

2016.9.17-18（東京）*藤田英俊、荒谷聡子、西岡久寿樹、中島利博 「ヒトにおけるシノビオリン阻害剤の効果の検証」 日本線維筋痛症学会

第 8 回学術集会

2016.9.17-18（東京）黒岩義之、横田俊平、中島利博、平井利明、大西孝宏、中村郁朗、西岡久寿樹 「HPV ワクチン接種後に起こる神経内分泌攪乱の発症様式：HANS 137 例にみられた 112 症例の解析から」会

2016.9.17-18

日本線維筋痛症学会 第 8 回学術集

（東京）

大西孝宏、黒岩義之、横田俊平、中島利博、西岡久寿樹 「HANS 諸症状の時系列的な病態解析について」 日本
線維筋痛症学会

第 8 回学術集会

2016.9.17-18 （東京）

西森美佐子、山口結花、梅原由加里、中平律子、荒谷聡子、中島若巳、北川中行、中谷 孝、中島利博 「線維筋痛症における超音波検査の役割」

日本線維筋痛症学会

第 8 回学術集会

2016.9.17-18（東京）

窪内郁恵、薦田昭宏、橋本聡子、渡邉彩花、島津 央、川口 佑、杉田裕介、菅原貴和、渡邊真也、中谷 孝、中島利博 「線維筋痛症例に対する反復性経頭蓋磁気刺激療法と理学療法の関連性について」 日本線維筋痛症学会回学術集会

2016.9.17-18

第 8

（東京）

*中島利博 「シノビオリンが拓く慢性炎症・関節リウマチの病態メカニズムと新規治療法の開発」 第 31 回日本
整形外科学会

基礎学術集会 2016.10.13-14（福岡）

荒谷聡子、藤田英俊、八木下尚子、山野嘉久、西岡久寿樹、中島利博 「関節リウマチにおける転写後修飾による多重化シグナル調節機構の解析」

第 3 回 JCR

ベーシックリサーチカンファレンス 2016.10.14-15（東京）

藤田英俊、荒谷聡子、八木下尚子、山野嘉久、須藤カツ子、内野博之、西岡久寿樹、中島利博 「リウマチ発症因
子シノビオリンによる新規エネルギー代謝機構の解明」 第 3 回 JCR ベーシックリサーチカンファレンス
2016.10.14-15（東京）

中島利博 「臨床医のリサーチマインドを育成・持続させる大学取り組みさせる大学取り組み」 第 178 回東京医科大学 医学会総会 100 周年記念特別 シンポジウム 2016.11.5（東京）*林由起子 「核膜病モデルマウスの筋病態解析」 第 89 回日本薬理学会年会
展開：疾患治療・創薬に向けてパシフィコ横浜 2016.3.9-11

シンポジウム 23

骨格筋研究の新（横浜）*Hayashi HY, Wada E, Kawahara G 「A new mouse model for myopathy associated with nuclear envelopathy.」
-33-

第 93

（様式 2）
法人番号プロジェクト番号回日本生理学会大会 札幌コンベンションホール 2016.3.22-24

131054
Ｓ1411011（札幌）*Kawahara G, Hayashi HY 「Novel zebrafish models of neuromuscular diseases.」 第 93 回日本生理学会大会
2016.3.22-24

ンベンションホール

札幌コ

（札幌）

Wada E, Hamano T, Matsui I, Yoshida M, Hayashi YK, Matsuda R 「Renal involvement in the pathogenesis of mineral andbone disorder in dystrophin-deficient mdx mice.」2016.3.22-24

第 93 回日本生理学会大会

札幌コンベンションホール

（札幌）*Kawahara G, Hayashi YK「Novel zebrafish models of neuromuscular diseases.」 13th International Congress of Human
Genetics 2016.4.3-7（Kyoto）

Hayashi YK, Nishino I 「Characterization of Japanese patients with FHL1 myopathy.」 13th International Congress of Human
Genetics 2016.4.3-7 （Kyoto）
林由起子、西野一三 「FHL1 ミオパチーの臨床病理学的解析」 第 113 回日本内科学会年次総会・講演会 東京国際フォーラム2016.4.15-17

林由起子、西野一三
学会総会

（東京）

「Clinical and pathological characterization of patients with FHL1 myopathy.」 第 57 回日本神経2016.5.18-5.21（神戸）

Wada E, Hayashi YK, Matsuda R「Anti-IL-6 receptor antibody attenuates muscular dystrophy via promoting skeletal muscle
regeneration but not cardiovascular muscles in dystrophin/utrophin double-knockout mice. 」
Biology and Disease of Skeletal Muscle Conference 2016.6.29-7.2

2016 New Directions in（Orlando FL USA）

Wada E 「Preventing dystrophic phenotypes of mdx mice by sevelamer, a phosphate binding drug.」 2016 New Directions in
Biology and Disease of Skeletal Muscle Conference 2016.6.29-7.2（Orlando FL USA）

和田英治、谷端淳、岩村憲、武田伸一、林由起子、松田良一「Anti-IL-6R antibody attenuates muscular dystrophy via

promoting skeletal muscle regeneration in dKO mice.」 第 2 回日本筋学会学術集会 2016.8.5-6. NCNP （東京）*川原玄理、林由起子
術集会「マリネスコ-シェーグレン症候群のゼブラフィッシュモデルの解析」第 2 回日本筋学会学2016.8.5-6. NCNP （東京）

*Kawahara G, Hayashi YK「A zebrafish model of Marinesco-Sjogren syndrome.」 ASHG 2016 Annual Meeting2016.10.18-22 （Vancouver Canada）
宇都宮志保，川原玄理，林由起子 「ゼブラフィッシュモデルを用いたデュシェンヌ型筋ジストロフィーの新規治療薬候補の探索」

第 178 回東京医科大学医学会総会*川原玄理、林由起子

東京医科大学 2016.11.5（東京）「ゼブラフィッシュを用いた神経筋疾患研究」 筋生理の集い 2016.12.17. 慈恵医大

東京

善本隆之、古澤純一、千葉祐規乃、大橋美緒、長谷川英哲、徐明利、中江進、小林富美恵、吉田裕樹、溝口出 「IL-27
による造血幹細胞の分化増殖の誘導と感染防御における役割」 第 81 回日本インターフェロン・サイトカイン学会
学術集会 2016.5.13（長崎）
Yoshimoto T, Ohashi M, Hasegawa H, Chiba Y, Xu M, Mizoguchi-34-「Establishment of a novel in vitro evaluation system for（様式 2）
法人番号プロジェクト番号131054
Ｓ1411011

the prediction of respiratory sensitizing potential of chemicals.」 ICCA-LRI and NIHS Workshop 2016.1.15-16 （Awaji
Island）
Yoshimoto T, Furusawa J, Chiba Y, Xu M, HasegawaH, Nakae S, Kobayashi F, Yoshida H, Mizoguchi I, Ohashi M, Hasegawa
H「Promotion of expansion and differentiation of hematopoietic stem cells by IL-27 into myeloid progenitors to control

infection in emergency myelopoiesis.」 Symposium, 16th International Congress of Immunology, 2016.8.21-26 （Melbourne,
Australia）
Chiba Y, Mizoguchi I, Hasegawa H, Yoshimoto T. 「Antitumor effects of IL-27 by promoting differentiation and expansion ofhematopoietic stem cells to M1 macrophage.」 第 75 回日本癌学会学術総会

2016.10.6-8（横浜）Ohashi M, Mizoguchi I, Hasegawa H, Chiba C, Xu M, Yoshimoto T「Development of a novel 3D co-culture system forevaluation of respiratory sensitizing potential.」 第 45 回日本免疫学会総会・学術集会 2016.12.5-7（沖縄）

Hasegawa H, Mizoguchi I, Chiba Ohashi M, C, Xu M, Yoshimoto T「A pivotal role for IL-23p19 produced by activated CD4+
T cells in development of EAE.」 第 45 回日本免疫学会総会・学術集会 2016.12.5-7（沖縄）

Chiba Y, Mizoguchi I, Hasegawa H, Ohashi M, C, Xu M, Yoshimoto T 「IL-27 induces antitumor activity by promoting
differentiation of HSCs to M1-like antitumorigenic macrophages and their mobiliziation into tumor.」 第 45 回日本免疫学会
総会・学術集会 2016.12.5-7（沖縄）

Nagai T, Yoshimoto T. 「Differences between immature and mature dendritic cells derived from human monocytes.」 第 45回日本免疫学会総会・学術集会

2016.12.5-7（沖縄）【2015】
Nakaoku T, Tsuta K, Ichikawa H, Shiraishi K, Sakamoto H, Enari M, Furuta K, Shimada Y, Ogiwara H, Watanabe SI,
Nokihara H, Yasuda K, Hiramoto M, Nammo T, Kim YH, Mishima M, Yokota J, Yoshida T, Kohno T 「Druggable oncogene
fusions in invasive mucinous lung adenocarcinoma.」 AACR Annual Meeting 2015. April 2015 （Philadelphia PA）
宇田川陽秀 、 舟橋伸昭、 平本正樹、 川口美穂、 西村渉、 南茂隆生、 安田和基 「脂肪細胞培養上清によるグルココルチコイド受容体を介した膵 β 細胞機能変化」第 58 回日本糖尿病学会年次学術集会 2015 年 5 月
南茂 隆生 、 宇田川陽秀、 川口美穂、 舟橋伸昭、 上番増（下関）

喬、 平本正樹、 西村渉、 安田和基 「膵島のゲノム網羅的解析による膵島代償機序/糖尿病発症機序関連因子の同定」 第 58 回日本糖尿病学会年次学術集会 2015 年
5月

（下関）

安田 和基 、 南茂
藤嘉朗、 関隆生、 平本正樹、 西村渉、 宇田川陽秀、 上番増喬、 舟橋伸昭、 金井弥栄、 松本健治、 斎

洋介、 笠間和典 「高度肥満患者由来の脂肪組織の多層的オミックス解析」 第 58 回日本糖尿病学会年次学術集会 2015 年 5 月（下関）安田和基 、 南茂隆生、 平本正樹、 西村
藤嘉朗、 関渉、 宇田川陽秀、 上番増喬、 舟橋伸昭、 金井弥栄、 松本健治、 斎

洋介、 笠間和典 「日本人肥満者由来 NASH 肝の多層的オミックス解析パネルの構築」 第 58 回日本糖尿病学会年次学術集会 (肝臓と糖尿病・代謝研究会) 2015 年 5 月（下関）*森谷昇太 、小松誠一郎、山﨑佳穗、河合優佑、國場寛子、車暁芳、稲津正人、後藤明彦、平本正樹、半田 宏、宮-35-

（様式 2）
法人番号プロジェクト番号澤啓介 「Targeting the integrated networks of aggresome formation, proteasome,131054
Ｓ1411011

and autophagy potentiates ER

stress mediated cell death in multiple myeloma cells.」 医学会奨励賞 第 175 回 東京医科大学医学会総会、2015.6.6
東京医科大学病院（東京）*伊藤謙太郎、杉田翔平、山城優太朗、森谷昇太、車暁芳、平本正樹、横山智央、宮澤啓介 「EGFR-independent
autophagy induction with gefitinib and enhancement of its cytotoxic effect by targeting autophagy with clarithromycin
in non-small cell lung cancer cells.」

第 175 回 東京医科大学医学会総会、2015.6.6

東京医科大学病院（東京）

平本正樹 、 宮澤啓介 「KCNQ1 遺伝子イントロンの 2 型糖尿病感受性 SNP 領域におけるアリル特異的 DNA 結合因子の解析」

第 175 回 東京医科大学医学会総会、2015.6.6

宮澤啓介（特別講演）

東京医科大学病院「ビタミン K2 の抗腫瘍効果とその臨床応用への可能性」（東京）
第 25 回日本産婦人科学会・新生児血液学会学術集会サテライトシンポジウム 2015.6.5 （東京）宇田川陽秀 、 舟橋伸昭、 平本正樹、 川口美穂、 西村渉、 南茂隆生、 安田和基「脂肪細胞培養上清による膵 β細胞機能変化」 第 36 回日本肥満学会 2015 年 10 月

（名古屋）

*Moriya S, Che XF, Yokoyama T, Hiramoto M, Miyazawa K 「Targeting autophagy with clarithromycin enhances
gefitinib-induced cell death in non-small cell lung cancer cells.」

第 74 回日本癌学会学術集会 2015 年 10 月 （名古屋）*森谷昇太、 車暁芳、横山 智央、平本正樹、宮澤啓介「クラリスロマイシン併用によるゲフィチニブ誘導性オートファジーを標的とした肺癌細胞株の殺細胞効果の増強」
2015.10.8-10

第 74 回 日本癌学会学術総会

名古屋国際会議場（名古屋）*向井俊太郎、 森谷昇太、平本正樹、風間宏美、國場寛子、横山智央、半田宏、糸井隆夫、宮澤啓介「Macrolides

sensitize EGFR-TKI-induced non-apoptotic cell death via blocking autophagy flux in pancreatic cancer cell lines.」 第
176 回 東京医科大学医学会総会

東京医科大学病院

2015.11.7（東京）*伊藤謙太郎、 杉田翔平、山城優太朗、森谷昇太、車暁芳、平本正樹、横山智央、宮澤啓介 「EGFR-independent
autophagy induction with gefitinib and enhancement of its cytotoxic effect by targeting autophagy with clarithromycin
in non-small cell lung cancer cells.」

医学会奨励賞 第 176 回 東京医科大学医学会総会

東京医科大学病院

2015.11.7（東京）
平本正樹 、 宇田川陽秀、 高橋枝里、 加納圭子、 鏑木

康志、 宮澤啓介、 石橋奈緒子、 舟橋伸昭、 南茂隆生、

安田和基 「KCNQ1 遺伝子イントロンにおける 2 型糖尿病感受性 SNP rs163184 領域のアリル間比較解析」 第 38 回
日本分子生物学会年会 第 88 回日本生化学会大会 合同大会 2015 年 12 月

（神戸）

阿部晃久 、 平本正樹、 山田博之 「ハルミンはオートファジーを介して抗アポトーシスタンパク質の発現を抑制
しアポトーシスを誘導する」第 38 回日本分子生物学会年会 第 88 回日本生化学会大会 合同大会 2015 年 12 月 （神戸）阿部晃久 、 平本正樹、 山田博之 「A549 細胞におけるパルミトイルセラミド蓄積誘導とネクローシス」 第 38 回
日本分子生物学会年会 第 88 回日本生化学会大会 合同大会 2015 年 12 月
*Handa H（神戸）「Cereblon (CRBN), the common target of multiple pharmacological actions of thalidomide and its-36-

（様式 2）
法人番号プロジェクト番号
derivatives」New developments of zebrafish research

2015.11.24

半田 宏「セレブロン ～IMiDs のターゲット～」第 6 回 Bay area hematology congress
ンチネンタルホテル

2015.11.16

131054
Ｓ1411011

ヨコハマグランドインターコ（横浜）

半田 宏「セレブロン ～IMiDs のターゲット～」Okayama IMiDs Conference

ホテルグランヴィア岡山

2015.11.6（岡山）
半田 宏「Discovery of a target of immunomodulatory drugs (IMiDs)〈免疫調節薬（IMiDs)のターゲットの発見）」第 77
回日本血液学会学術集会 シンポジウム 6「多発性骨髄腫の新規治療薬の感受性あるいは耐性を決定する機序、因子」
石川県立音楽堂 2015.10.17(10.16‐10.18)（石川）半田 宏「サリドマイド催奇性のターゲットから創薬へ」CSJ 化学フェスタ

タワーホール船堀 2015.10.15（東京）
上田 実、細矢孝充、上杉志成、新家一男、山本昇、半田 宏

パネルディスカッション「ケミカルバイオロジーとはどのような化学なのか－化学としての新しさ、社会との関わり」CSJ 化学フェスタ
2015.10.15

タワーホール船堀（東京）

半田 宏「セレブロン ～IMiDs のターゲット～」香川血液疾患フォーラム

ロイヤルパークホテル高松 2015.9.4（高松）
半田 宏「IMiDs の多彩な薬理作用 ～標的分子探索研究の進歩～」Fukuoka IMiDs Forum

ヒルトン福岡シ―ホーク

2015.8.18（福岡）半田 宏「IMiDs の多彩な薬理作用 ～標的分子探索研究の進歩～」Tokyo 3M Forum 2015

東京ドームホテル 2015.7.8（東京）
Handa H「Basic aspects concerning the mechanisms of actions of immunomodulatory drugs (IMiDs) in multiple myeloma」The
20th Congress of EHA-JSH Joint Symposium (Multiple Myeloma)

2015.6.12(6.11-6.14)

Messezentrum Vienna(Wien)

半田 宏、坂本聡「分子標的探索と生物学的評価」新学術領域研究「天然物ケミカルバイオロジー～分子標的と活性制御～」第 8 回公開シンポジウム(ポスター)
半田東北大学 2015.6.8 (6.8-6.9) （仙台）

宏「IMiDs の多彩な薬理作用～標的分子探索研究の進歩～」最新医学セミナー 宮崎大学医学部 2015.4.28

（宮崎）
半田

宏「サイエンスと知財」第 1 回知的財産プロデューサー等連絡会議 講演会

共同通信会館 2015.4.7

（東

京）
半田
会

宏「アフィニティビーズを利用した生物活性物質標的タンパク質の単離・同定・評価」日本薬学会第 135 年

神戸学院大学半田2015.3.26(3.25-3.28) （神戸）宏「セレブロン －IMiDs 標的分子研究の最前線－」Myeloma Forum in Yokohama

ーコンチネンタルホテル
半田

ヨコハマグランドインタ

2015.2.28 （横浜）宏「ケミカルで知り、ケミカルで動かす －サリドマイド催奇性のターゲットの発見から創薬への展開－」サ-37-

（様式 2）
法人番号プロジェクト番号
ントリー生命科学財団 セミナー
半田サントリー生命科学財団 2015.2.4

131054
Ｓ1411011（京都）

宏「サリドマイド催奇性のターゲットの発見から創薬への展開 －ケミカルで知り、ケミカルで動かす-」埼
埼玉医科大学 ゲノム医学研究センター 2015.1.23

玉医科大学 講演会

（埼玉）

小松梨恵、佐藤知雄、山野嘉久、遊道和雄、別府諸兄、西岡久寿樹、中島利博、藤井亮爾
炎における CDK6

「コラーゲン誘導関節

第 59 回日本リウマチ学会総会・学術集会 2015 年 4 月 23 日

阻害剤の効果」（愛知）*藤田英俊、荒谷聡子、中島利博、須藤カツ子、中島若巳、稲津正人、西岡久寿樹、原直美、内野博之 「慢性炎症と肥満とをつなぐシノビリオンにおるエネルギー代謝調節機構」 第 175 回東京医科大学医学会総会

2015.6.6（東京）
Omurzakov N、三浦 直樹、川原 幸一、西 順一郎、八木下 尚子、 荒谷聡子、中島利博 「キルギス共和国における溶連菌感染症ならびにその合併症であるリウマチ性心疾患/リウマチ熱の現状―9 年間のフィールド調査について―Study of Rheumatic Fever /Rheumatic Heart Diseases in Kyrgyz Republic」

海外学術調査フェスタ 2015.6.27（東京）*荒谷聡子、藤田英俊、八木下尚子、中島若巳、西岡久寿樹、中島利博 「私たちが開発した世界初の E3 ユビキチ
ン化酵素シノビオリン阻害剤の可能性」 第 5 回

医薬工 3 大学包括連携推進シンポジウム 2015.6.20（東京）*藤田英俊、荒谷聡子、八木下尚子、須藤カツ子 、内野博之、稲津正人、臼井正彦、西岡久寿樹、中島利博
ウマチ発症因子シノビオリンによるエネルギー代謝調節機構」
2015.6.20「リ

第 5 回 医薬工 3 大学包括連携推進シンポジウム（東京）

Nakajima T 「Synoviolin and metabolic disorder」

Shanghai-Tokyo Workshop on Rheumatology 2015

2015.7.31 （上海）*荒谷聡子 「私達が開発した世界初の E3 ユビキチン化酵素シノビオリン阻害剤 LS シリーズの可能性について」
第 16 回 運動器科学研究会

2015.9.12（鹿児島）

藤田英俊 「慢性炎症に機能するシノビオリンの新しい生体機能」 第 16 回 運動器科学研究会

2015.9.12（鹿児島）
黒岩義之、横田俊平、中村郁朗、中島利博、平井利明 、西岡久寿樹 「Anatomical diagnosis からみた HANS 発症のメカニズム」

日本線維筋痛症学会第 7 回学術集会

横田俊平、黒岩義之、中島利博、西岡久寿樹

2015.10.3（東京）「身体多機能障害疾患としての HANS の症候と一元的病巣の推察」

日本線維筋痛症学会第 7 回学術集会 2015.10.3（東京）平井利明、横田俊平、中村郁朗、黒岩義之、中島利博、西岡久寿樹「HANS の治療について」会第 7 回学術集会

日本線維筋痛症学

2015.10.3（東京）藤田英俊、荒谷聡子、八木下尚子、西岡久寿樹、中島利博 「慢性炎症に関わるシノビオリンの新しい生体機能」
日本線維筋痛症学会第 7 回学術集会

2015.10.3（東京）中村郁朗、横田俊平、黒岩義之、中島利博、西岡久寿樹 「HPV ワクチン副反応の国際的共通性」 日本線維筋痛-38-

（様式 2）
法人番号プロジェクト番号症学会第 7 回学術集会

131054
Ｓ1411011

2015.10.3（東京）

薦田昭宏、窪内郁恵、橋本聡子、黒田祐子、中島若巳、澁谷美雪、中谷孝、中島利博 「線維筋痛症の認知的側面に対するリハビリテーション」日本線維筋痛症学会第 7 回学術集会 2015.10.3（東京）窪内郁恵、薦田昭宏、橋本聡子、渡邉彩花、島津央、河口佑、青山勝、渡邊真也、中谷孝、中島利博
症例に対する反復性経頭蓋磁気刺激法の効果と関連因子」 日本線維筋痛症学会第 7 回学術集会「線維筋痛

2015.10.3（東京）臼井千恵、西岡健弥、荒谷聡子、藤田英俊、山野嘉久、中島利博、西岡久寿樹 「線維筋痛症の心的外傷性ストレスに対する脆弱性: 東日本大震災後 19 ヵ月間の追跡研究」

日本線維筋痛症学会第 7 回学術集会

2015.10.3（東京）中島若巳、荒谷聡子、藤田 英俊、中谷孝、中島利博 「大腿骨頸部骨折が発症の引き金となった背部痛・咽喉頭部
異常感に仙骨硬膜外ブロックが有効であった線維筋痛症の 1 症例」 日本線維筋痛症学会第 7 回学術集会
2015.10.3（東京）西森美佐子、尾崎鈴子、藤島みどり、山口結花、梅原由加里、荒谷聡子、中島若巳、中谷孝、中島利博 「線維筋日本線維筋痛症学会第 7 回学術集会

痛症における超音波検査の有用性の検討」

2015.10.3（東京）山口結花、藤島みどり、梅原由加里、長野愛子、永森ひさみ、北川中行、中谷孝、中島利博 「線維筋痛症・難病
センター外来看護師としての取り組み」

日本線維筋痛症学会第 7 回学術集会

2015.10.3（東京）Nakajima T 「Decade of Kyrgyz - Japanese cooperation: the mission against the chronic rheumatic heart diseases - past,present and future.」KYRGYZ NATIONAL CONGRESS OF RESPIRATORY AND ALLERGICDISEASES/EURO-ASIAN INTERNATIONAL PRIMARY CARE RESPIRATORY GROUP (IPCRG) SYMPOSIUM/ III
KOGRESS OF KYRGYZ THORACIC SOCIETY

2015.10.9（キルギス）

Nakajima T 「About prospects and future plans of the Kyrgyz - Japan cooperation in rheumatic feve.」
CEREMONY OF KYRGYZ - JAPAN RESEARCH CENTER

2015.10.9

OPENING（キルギス）

黒岩義之、横田俊平、中村郁朗、中島利博、西岡久寿樹、平井利明 「ヒト・パピローマウィルス・ワクチン接種後の多彩な神経症候に関する病態考察」

第 68 回日本自律神経学会総会

2015.10.30（愛知）

平井利明、井口保之，横田俊平、中村郁朗、中島利博、西岡久寿樹、黒岩義之 「ヒト・パピローマウィルス・ワ
クチン接種後の情動・自律神経障害：症候学的ならびに脳血流画像所見」 第 68 回日本自律神経学会総会
2015.10.30（愛知）*荒谷聡子、藤田英俊、西岡久寿樹、中島利博 「E3 ユビキチン化酵素シノビリオン阻害剤の臨床応用への可能性」
第 176 回東京医科大学医学会総会 2015.11.7（東京）

黒岩義之、横田俊平、中村郁朗、中島利博、平井利明 、西岡久寿樹 「HPV ワクチン副反応発症の病因・病態」
第 30 回日本臨床リウマチ学会 2015.11.22（兵庫）

藤田英俊、荒谷聡子、八木下尚子、須藤カツ子、中島若巳、内野博之、西岡久寿樹、中島利博 「慢性炎症に関わるシノビオリンの新規エネルギー代謝調節機構」 第 38 回日本分子生物学会 2015.12.4
Hayashi YK（兵庫）「Myopathy with prominent contractures.」 14th Asian and Oceanian Myology Center Annual Scientific-39-

（様式 2）
法人番号プロジェクト番号
Meeting, March 1-4, 2015

131054
Ｓ1411011

Shangri-La Hotel, Bangkok, （Thailand）

Hayashi YK, Suzuki S, Kawahara G, Inoue H. 「Characterization of model mice for nuclear envelopathies」第 120 回解剖学会総会全国学術集会・第 92 回日本生理学会大会

神戸国際会議場・展示場 2015.3.21-23（神戸）

Hayashi YK, Suzuki S, Kawahara G, Noguchi S, Nishino I. 「Different muscle involvement in mouse models of nuclear
envelopathy」 第 56 回日本神経学会総会

朱鷺メッセ

林由起子、鈴木茂文、和田英治、川原玄理

2015.5.20-23（新潟）「核膜病モデルマウスの解析」 第 1 回日本筋学会学術集会 2015.8.8（東京）
川原玄理、林由起子.「筋疾患ゼブラフィッシュモデルの新規開発」 第 1 回日本筋学会学術集会、2015.8.8 （東京）
和田英治、濱野高行、松井功、吉田瑞子、林由起子、松田良一 「腎機能低下が mdx マウスの骨ミネラル代謝異常を惹起する」

第 1 回日本筋学会学術集会

和田英治、濱野高行、林由起子、松田

第 246 回生理学東京談話会 2015.9.26

2015.8.8

良一（東京）「腎機能の低下が mdx マウスの骨ミネラル代謝異常を惹起する」（東京）

林由起子、西野一三 「FHL1 ミオパチーの臨床病理学的解析」日本人類遺伝学会第 60 回大会

2015.10.14-17 （東京）Miki K, Nagaoka K, Bohnenkamp H., Yoshimoto T, Kamigaki, T, Maekawa R.

「Induction of antigen specific T cell

responses using PepTivator® OVA-loaded DCs in mouse model.」 第 12 回日本免疫治療学研究会 2015.2.28（東京）

大屋敷一馬、溝口 出、善本隆之、田内哲三、岡部聖一、片桐誠一朗、梅津知宏、大屋敷純子「Stop-imatinib CML
患者末梢血単核球における免疫関連 miRNA の意義」第 7 回血液疾患免疫療法研究会 2015.9.26（東京）

片桐誠一朗、田内哲三、梅津知宏、田所健一、溝口出、善本隆之、大屋敷純子、大屋敷一馬「慢性骨髄性白血病患者における治療中止を成功させるための予測因子」第 77 回日本血液学会学術集会 2015.10.16/18（金沢）

溝口 出、千葉佑規乃、角田廉、徐明利、善本隆之 「化学物質の呼吸器感作性 in vitro 評価法の開発」 第 3 期 LRI
研究報告会

2015.8.28（東京）*Mizoguchi I, Chiba Y, Xu M,

Yoshimoto T. 「Binding of Epstein-Barr virus-induced gene 3 to calnexin enhances its

chaperone activity and augments interleukin-23 receptor expression, leading to development of colitis」 第 44 回日本免疫学会総会・学術集会 2015.11.18-20（札幌）

善本隆之、千葉佑規乃、角田廉、徐明利、溝口 出
ジウム「化学物質の呼吸器感作性 in vitro 評価法の開発」LRI シンポ

第 28 回日本動物実験代替法学会 2015.12.10-12（横浜）【2014】
西村渉 、川口美穂 、宇田川陽秀 、衛藤弘城 、舟橋伸昭、南茂隆生 、平本正樹 、安田和基
験による膵 β 細胞障害の時間経過の解析」 第 51 回日本臨床分子医学会学術集会「細胞系譜追跡実

2014.4.11

宇田川陽秀 、平本正樹 、舟橋伸昭 、川口美穂 、西村渉 、南茂隆生 、安田和基「東京脂肪細胞由来液性因子に-40-

（様式 2）
法人番号プロジェクト番号131054
Ｓ1411011

よる新規インスリン分泌調節因子の発現誘導と膵 β 細胞機能変化」
第 57 回日本糖尿病学会年次学術集会

2014.5.23（大阪）
南茂

隆生 、宇田川陽秀 、川口美穂 、舟橋伸昭 、上番増喬 、平本正樹 、西村 渉 、安田和基「膵島のゲノム

網羅的解析による糖尿病発症機序の考察」 第 57 回日本糖尿病学会年次学術集会 2014.5.23（大阪）

西村 渉 、 川口美穂 、 宇田川陽秀 、 衛藤弘城 、 舟橋伸昭 、 南茂隆生 、平本正樹 、 安田和基 「転写因子 MafA による膵 β 細胞の分化可塑性制御」 第 57 回日本糖尿病学会年次学術集会

2014.5.23（大阪）*森谷昇太 、 車 暁芳 、 平本正樹 、 宮澤啓介 、 小松誠一郎 、 山﨑佳穗 、 河合優佑 、 國場寛子 、 稲津「Targetng aggresome formation under simultaneous inhibition of proteasome and

正人 、 後藤明彦 、 半田 宏

autophagy potentiates ER-stress mediated cell death in myeloma cells」 第 173 回東京医科大学医学会総会 2014.6.7（東京）*森谷昇太 、 小松誠一郎 、 車

暁芳 、 後藤明彦 、 宮澤啓介 「Simultaneous targeting aggresome, proteasome,and autophagy potentiates ER-s tress mediated cell death in myeloma cells.」

第 73 回日本癌学会

2014.9.25.

（横浜）
宇田川陽秀 、 平本正樹 、 舟橋伸昭 、 川口美穂 、 西村 渉 、 南茂隆生 、 安田和基 「前駆・成熟脂肪細胞由来液性因子による膵 β 細胞機能変化」 第 35 回日本肥満学会

2014.10.25（宮崎）*森谷昇太 、 小松誠一郎 、 山﨑佳穗 、 國場寛子 、 廣田綾子 、 車 暁芳 、 稲津正人 、 後藤明彦 、 平本
正樹 、 宮澤啓介 「Targeting aggresome formation enhances ER-stress mediated cell death in myeloma cells. 」 第76 回日本血液学会学術集会 2014.10.30（大阪）*宮澤啓介（特別講演）「造血器腫瘍における細胞内タンパク質分解ネットワークを標的とする新規治療法」老年者
造血器疾患研究会. 2014.11.20（東京）

Hirayama A 、 Sugawara S 、

Abe H 、

Kasama K 、
spectrometry.」

Saito Y 、

Yasuda K 、Maekawa K 、

Nammo T 、

Hiramoto M 、Tomita M 、Seki Y 、

Soga T 「A metabolomic study of obesity using capillary electrophoresis-mass

第 37 回日本分子生物学会年会

2014.11.25

（横浜）

Handa

H, Kawano M「Medical application of functionalized SV40 virus-like particles」ICSS MEETING 2014.12.9.

Hotel

Kowloon、（Hong Kon)

Eaton

*Ito T, Ando H, Yamaguchi Y, Handa H「Cereblon is a substrate receptor of the Cul4 ubiquitin ligase whose substrate
recognition is modulated by IMiDs」第 37 回日本分子生物学会年回 パシフィコ横浜

2014.11.26（横浜）*片山美樹、伊藤拓水、安藤秀樹、山口雄輝、半田 宏「E3 ユビキチンリガーゼの基質認識レセプターCRBN とポ
マリドマイド依存的な基質 Aiolos の生化学的解析」第 37 回日本分子生物学会年回

パシフィコ横浜 2014.11.25

（横浜）
*半田
場

宏「IMiDs の多彩な薬理作用 ～標的分子探索研究の進歩～」第 76 回日本血液学会学術集会 大阪国際会議

2014.11.1 （大阪）

Sakamoto S、Handa H「Screening and Evaluation of Natural Product Targets」The 3rd International Symposium on Chemical-41-

（様式 2）
法人番号プロジェクト番号
Biology of Natural Products:Target ID and Regulation of Bioactivity(poster)
2014.10.28*半田131054
Ｓ1411011

千里ライフサイエンスセンター（大阪）

宏「機能性ナノビーズの医学・医療への応用」文部科学省 私立大学戦略的研究基盤形成支援事業 「機能性磁性ナノビーズ技術を基盤とする難治性疾患のタンパク質分解制御機構の解明と新規治療法の開発」キックオフシ
ンポジウム

東京医科大学 2014.10.8

（東京）

半田

宏「ケミカルで生命の謎を知る！」第 48 回医科学フォーラム 東京医科大学

半田宏「セレブロン～サリドマイド催奇性から創薬への展開～」F-CHOP 2014 京都国際ホテル 2014.9.13 （京都）半田宏「大学のシーズ研究から実用化に向けて －大学における基礎研究の重要性－」メディカル・ヘルスイノベ

ーション講座 第 8 回定例講演会

信州大学医学部 2014.9.9

2014.9.24（東京）（長野）

宏「IMiDs のセレブロンを介した多様な薬理作用」Sinnjuku Myeloma Conference*半田2014.7.24

国際医療研究センター（東京）*Ito T, Ando H, Yamaguchi Y, Handa H「Cereblon is a substrate receptor of the CUL4-DDB1 ubiquitin ligase whose
activity is directly controlled by thalidomide and its analogs」Protein Modification & Homeostasis
Conference Center2014.6.16-20

Suzhou Duxhu Lake

（China）*Handa H「Idenification of a primary target of thalidomide teratogenecity: towards the debelopment of new drugs」RNA
Biology

McGovern Institute,MIT Bldg.

2014.6.14（Boston）

坂本 聡、Vipul Gupta、劉 舒捷、安藤秀樹、舘野峻平、金子裕生

湯上真人、石井亮平、濡木 理、山口雄輝、半田宏「抗炎症薬サリチル酸の生体内作用機構の解明」第 9 回日本ケミカルバイオロジー学会
2014.6.11-13
半田

（大阪）

宏、坂本聡「分子標的探索と生物学的評価」(ポスター)

子標的と活性制御～」第 6 回公開シンポジウム
半田大阪大学会館

名古屋大学

新学術領域研究「天然物ケミカルバイオロジー～分
2014.5.29 （名古屋）

宏「IMiDs の作用機序 －近年の報告から－」Therapeutic strategies for Multiple Myeloma 20142014.4.12

ホテル日航福岡（福岡）

八木下尚子、荒谷聡子、藤田英俊、山野嘉久、西岡久寿樹、中島利博 「滑膜細胞調節因子シノビオリンによる関節リウマチ骨軟骨破壊機構の解明」

第 58 回日本リウマチ学会 2014.4.25（東京）*藤田英俊、荒谷聡子、八木下尚子、西岡久寿樹、中島利博「E3 ユビキチンリガーセであるシノビオリンによる体重制御機構」

第 58 回日本リウマチ学会 2014.4.25 （東京）

*荒谷聡子、山野嘉久、藤田英俊、八木下尚子、伊藤健司、長田賢一、中村郁朗、西岡久寿樹、中島利博「小胞体ス
トレスと線維筋痛症における抗疼痛薬による肥満の関与について
Omurzakova N, Jumagulova A, NAKAJIMA T.

」第 58 回日本リウマチ学会 2014.4.25 （東京）Study about prevalence of group A streptococcus and clinical-functional

assessment of rheumatic fever in Kyrgyz Republic. World Congress of Cardiology WCC-2014
Omurzakova N, , NAKAJIMA T.

2014.5.4-7（Melbourne）「Prevalence of Group A b-hemolytic Streptococcus and Clinical-Functional Peculiarities-42-

（様式 2）
法人番号プロジェクト番号
Assessment of Rheumatic Fever in the Kyrgyz Republic.」

131054
Ｓ1411011

The 11-th International Congress of Rheumatology Global

Arthritis Research Network -2014 (BRIC -GARN meeting - 2014) in Moscow (Russia) at the International University Forum.
2014.6.5-7（Russia）*荒谷聡子、藤田英俊、西岡久寿樹、中島利博 「肥満に対する創薬開発を目指した E3 ユビキチン化酵素シノビオ
リンの解析」

第 173 回東京医科大学医学会総会 2014.6.7（東京）*藤田英俊「リウマチ発症因子シノビオリンによる新規エネルギー代謝調節機構」 第 15 回運動器科学研究会
2014.9.4-5 （東京）
荒谷聡子:「GABA 構造類似薬の副作用による肥満と小胞体ストレスの関与について」回学術集会

2014.913-14

日本線維筋痛症学会 第 6（長野）

藤田英俊、荒谷聡子、中島利博、河西智子、平津恵美、中村香織、佐藤永一、西岡久寿樹
いた基盤的技術の開発」

第 174 回東京医科大学医学会総会「ゲノム編集技術を用

2014.11.1 （東京）

平津恵美、河西智子、中村香織、佐藤永一、西岡久寿樹、藤田英俊、荒谷聡子、中島利博「関節リウマチ患者由来
滑膜細胞のプライマリーカルチャー手法の確立」 第 174 回東京医科大学医学会総会 2014.11.1（東京）

河西智子、平津恵美、中村香織、佐藤永一、西岡久寿樹、藤田英俊、荒谷聡子、中島利博 「 In-Fusion クローニン
グ法を用いた遺伝子クローニング手法の確立」

第 174 回東京医科大学医学会総会

2014.11.1 （東京）

中村香織、平津恵美、河西智子、佐藤永一、西岡久寿樹、藤田英俊、荒谷聡子、中島利博「フローサイトメトリーを用いた細胞の分画」

第 174 回東京医科大学医学会総会 2014.11.1（東京）

藤田英俊、荒谷聡子、中島利博、中島若巳、西岡久寿樹、諸田沙織、内野博之、須藤カツ子:「*リウマチ発症因子
シノビオリンによるエネルギー代謝調節機構」 第 174 回東京医科大学医学会総会

2014.11.1（東京）

Hayashi HK 「Immunohistochemistry for muscle diseases.」 13th Asian and Oceaninan Muscular Center Annual Scientific
Meeting (AOMC) 2014.4.14-17（Philippine）

Hayashi HK 「Facioscapulohumeral muscular dystrophy.」 13th Asian and Oceaninan Muscular Center Annual Scientific
Meeting (AOMC) 2014.4.14-17（Philippine）

林由起子，後藤加奈子，宮武聡子，輿水江里子，松本直通，埜中征哉，西野一三
伝学的多様性」 第 55 回日本神経学会学術大会 2014.5.21-24「ネマリンミオパチーの臨床遺（福岡）

林由起子，埜中征哉，宮武聡子，輿水絵里子，松本直通，西野一三．「次世代シークエンサーを活用したネマリン
ミオパチーの遺伝子変異解析．」 第 56 回日本小児神経学会学術集会 2014.5.29-31 （浜松）Miki K, Nagaoka K, Bohnenkamp H, Yoshimoto T, Kamigaki T Maekawa R 「Induction of both OVA specific CD4+ andCD8+ T cells by using PepTivator® OVA-pulsed DCs in mouse model.」Annual Meeting 2014. 2014.4.5-9

American Association for Cancer Research 105th（San Diego）

Takahara M, Goto S, Miki K, Saiwaki S, Nagaoka K, Matsushita H, Kondo T Bohnenkamp H, Yoshimoto T, Maekawa R,
Kamigaki T 「Induction of antigen specific T cells using PepTivator®-pulsed DCs.」 International Society For Cellular-43-

（様式 2）
法人番号プロジェクト番号131054
Ｓ1411011

Therapy 20th Annual Meeting 2014. 2014.4.23-26（Paris）
Aoyama K, Yoshimoto T. 「In vivo and in vitro models for prediction of respiratory sensitizers.」 ILSI-HESI Workshop on theAssessment of Respiratory Sensitization.

2014.5.28-29（Alexandria, VA, USA）

Terayama H, Naito M, Yoshimoto T, Hirai S, Qu N, Kuramasu M, Hatayama N, Kanazawa T, Suyama K, Sakabe K, Itoh M.
「Contribution of IL-35 to maintaining the testicular immune privilege」 第 33 回日本アンドロロジー学会学術大会
2014.6.12-13（軽井沢）Miki K, Nagaoka K, Yoshimoto T, Kamigaki T Maekawa R 「PepTivator® OVA-pulsed DCs can induce both OVA specificCD8+ and CD4+ T cells in mouse model. 」第 73 回日本癌学会学術総会 2014.9.25-27（横浜）

Chiba Y, Mizoguchi I, Hisada M, Tsuchida A, Mizuguchi J, Yoshimoto T. 「Regulation of antitumor immune responses
through differentiation and mobilization of hematopoietic stem cells by IL-27」 第 73 回日本癌学会学術総会 2014.9.25-27（横浜）
Furusawa J, Chiba Y, Mitobe K, Mizoguchi I, Mizuguchi J, Yoshimoto T. 「IL-27 promotes the differentiation ofhematopoietic stem cells into DC progenitor cells. 」13th International Symposium on Dendritic Cells, DC2014.
2014.9.14-18（ France）
Chiba Y, Furusawa J, Mizoguchi I, Xu M, Mizuguchi J, Yoshimoto T. 「IL-27 exerts potent antitumor activity by promoting
the differentiation of hematopoietic stem cells to M1-like antitumor macrophages and their mobilization.」第 43 回日本免疫学会総会・学術集会 2014.12.10-12 （京都）Furusawa J， Chiba Y， Mizoguchi I， Xu M， Mizuguchi J， Yoshimoto T. 「IL-27 promotes the differentiation ofhematopoietic stem cells into multipotent myeloid progenitor cells.」 第 43 回日本免疫学会総会・学術集会 2014.12.10-12（京都）＜研究成果の公開状況＞（上記以外）
シンポジウム・学会等の実施状況、インターネットでの公開状況等＜既に実施しているもの＞
本事業に関する研究成果ならびに活動報告、技術講習会等は逐次「東京医科大学・分子標的探索センター」HP（http://www.tokyo-med.ac.jp/target/）で公開している。
＜これから実施する予定のもの＞
特記事項なし-44-

（様式 2）
法人番号プロジェクト番号
１４ その他の研究成果等
特許申請 4 件-45-131054
Ｓ1411011

（様式 2）
法人番号プロジェクト番号

１５ 「選定時」及び「中間評価時」に付された留意事項及び対応＜「選定時」に付された留意事項＞
特記事項なし＜「選定時」に付された留意事項への対応＞
特記事項なし＜「中間評価時」に付された留意事項＞
特記事項なし＜「中間評価時」に付された留意事項への対応＞
特記事項なし-46-

131054
Ｓ1411011

（様式2）
法人番号プロジェクト番号131054
S1411011

１６

（千円）
内
年度・区分 支出額 法 人
平 施 設
成
2 装 置
6 設 備
年
度 研 究費
平 施 設
成
2 装 置
7 設 備
年
度 研 究費
平 施 設
成
2 装 置
8 設 備
年
度 研 究費
平 施 設
成
2 装 置
9 設 備
年
度 研 究費

訳

共同研
受託
究機関
寄付金 その他（
研究等
負担

負 担

私 学
助 成

28,56010,93917,621

28,006

14,40813,59836,90012,30024,60023,003

12,20010,80324,1808,060

16,12024,90914,252

10,65731,26022,749

）

0
0

0
0

0
0

0
0
0
54,009

平 施 設
成
3 装 置
0 設 備
年
度 研 究費54,00354,003

0

施 設

0

0

0

0

0

0

0

総 装 置

0

0

0

0

0

0

0

89,64031,29958,3410000183,930

126,123

57,8070000273,570

157,422

116,148

0

0

0

0

額 設 備
研 究費
総

計

0
0
0

-47-

備 考

（様式2）
法人番号131054

１７《施設》 （私学助成を受けていないものも含め、使用している施設をすべて記載してください。）

施 設 の 名 称

整備年度

分子標的探索センター

研究施設面積H27

研 究 室 等 数 使用者数

171 ㎡
49 ㎡
76 ㎡

ナノ粒子先端医学応用講座

医学総合研究所

6
1
1

28
9
10

事業経費

補助金額

865
0
0

0
0
0

（千円）
補助主体

※ 私学助成による補助事業として行った新増築により、整備前と比較して増加した面積
0㎡《装置・設備》 （私学助成を受けていないものは、主なもののみを記載してください。）
整備年度

装置・設備の名称

型

番

台

数

稼働時間数

事業経費

（千円）
補助金額

補助主体

（研究装置）ｈｈｈｈｈ（研究設備）
網羅的遺伝子発現解析装置

生細胞イメージングシステム

マススペクトル解析装置
共焦点レーザースキャン顕微鏡

00-0394
HD/2CLR
TP14266

H28
H28
H27
H26

1式
1式
1式
1式

LSM700 2Ch UGB

30/月
500/月
100/月
300/月

ｈ
ｈ
ｈ
ｈ
ｈ

6,570
17,61036,90028,5604,380 私学助成
11,740 私学助成
24,600 私学助成
17,621 私学助成

（情報処理関係設備）
ｈ
ｈ
ｈ
ｈ
ｈ

１８ 研究費の支出状況
年
度
平成
小 科 目
消 耗 品 費光 熱 水 費
通信運搬費
印刷製本費
旅費交通費
報酬・委託料（雑務費・雑費）
（修理費）
計
人件費支出
（兼務職員）
教育研究経費支出
計
教育研究用機器備品

図

書
計

（千円）
26

支 出 額
教
13,057年度
金

主 な 使 途育研
究

543 印刷費
122 旅費2,797 委託料
2,312 雑務費・雑費
43 修理費18,874アル
バイト

積 算 内 訳
主 な
額
経
費
支
出
13,057 試薬、実験器具

容543 英文論文投稿料
122 学会旅費
2,797 実験補助業務委託
2,312 検査料、ソフト代
43 機器修理
18,874関係
支出

00
設 備 関 係 支 出（１個又は１組の価格が５００万円未満のもの）
9,132 超低温フリーザー、小型デジタル超音波ホモジナイザー
9,132
遠心濃縮機システムほか
9,132
9,132
研
究
ス
タ
ッ
フ
関
係
支
出

リサーチ・アシスタント
ポスト・ドクター
研究支援推進経費

計

内

0

0
-48-

（様式2）
法人番号
年

度

小 科 目

年度

積 算 内 訳主 な 使 途金 額
主 な 内 容
教
育
研
究
経
費
支
出
9,269 実験材料
9,269 試薬、実験器具
0
0
1 運搬費
1 宅急便代
240 印刷費240 論文投稿料、ポスター印刷代
680 旅費680 学会旅費、打ち合わせ旅費
4,464 委託料
4,464 実験補助業務委託、外国送金手数料
68 保守料68 機器保守料
814 雑務費、雑費
814 解析代、ソフト代
15,536
15,536
ア
ル
バ
イ
ト
関
係
支
出
2,835 研究補助2,835 時給 1,300円，年間時間数 1758時間

支 出 額

消 耗 品 費光 熱 水 費
通信運搬費
印刷製本費
旅費交通費
報酬・委託料（ 保守料 ）
（雑務費・雑費）
計
人件費支出（兼務職員）
教育研究経費支出
計

実人数 1人2,835
2,835
設 備 関 係 支 出（１個又は１組の価格が５００万円未満のもの）
4,632 機器備品
4,632 小型魚類飼育システム、ＰｒｏｔｅｉｎＰｉｌｏｔ解析用ＰＣ、
対物ﾚﾝｽﾞ ﾌﾟﾗﾝｱﾎﾟｸﾛﾏｰﾄ63×1.4 Oil Dicほか
4,632
4,632
研
究
ス
タ
ッ
フ
関
係
支
出

教育研究用機器備品

図

27

平成

131054

書計

リサーチ・アシスタント
ポスト・ドクター
研究支援推進経費

計
年

0

度

小 科 目
消 耗 品 費光 熱 水 費
通信運搬費
印刷製本費
旅費交通費
報酬・委託料（ 保守料 ）
（ 修理費 ）
（雑務費・雑費）
計
人件費支出（兼務職員）
教育研究経費支出
計
教育研究用機器備品

図書
計

リサーチ・アシスタント
ポスト・ドクター
研究支援推進経費

計

28

平成

0

年度

積 算 内 訳主 な 使 途金 額
主 な 内 容
教
育
研
究
経
費
支
出
8,179 実験材料
8,179 試薬、実験器具
0
0
2 運搬費
2 宅急便代00770 旅費770 学会旅費、打ち合わせ旅費
4,406 委託料
4,406 実験補助業務委託、外国送金手数料
2,385 保守料
2,385 機器保守料
1,917 修理費
1,917 機器修理費
1,680 雑務費、雑費
1,680 解析代
19,33919,339
ア
ル
バ
イ
ト
関
係
支
出
1,930 研究補助
1,930 時給 1,300円，年間時間数 1197時間

支 出 額

実人数 1人1,930
1,930
設 備 関 係 支 出（１個又は１組の価格が５００万円未満のもの）
3,640 機器備品
3,640 メディカルフリーザー、NanoDrop OneC超微量分光光度計、
Nano drop ONE C（ｷｭﾍﾞｯﾄﾀｲﾌﾟ）
3,640
3,640
研
究
ス
タ
ッ
フ
関
係
支
出
10,166 研究補助

10,166 学内2人

10,166

10,166-49-

（様式2）
法人番号
年

度

小 科 目

年度

積 算 内 訳主 な 使 途金 額
主 な 内 容
教
育
研
究
経
費
支
出
19,691 実験材料
19,691 試薬、実験器具
005 郵便料、運搬費
5 郵便代、宅急便代
5 印刷代
5 ポスター印刷
1,168 旅費
1,168 学会旅費、打ち合わせ旅費
7,675 委託料
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5,326 機器保守料
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プロジェクトの成果-51-

Biochemical and Biophysical Research Communications 501 (2018) 286e292

Contents lists available at ScienceDirect

Biochemical and Biophysical Research Communications
journal homepage: www.elsevier.com/locate/ybbrc

Designing an effective drug combination for ER stress loading incancer therapy using a real-time monitoring system
Hiromi Kazama a, Masaki Hiramoto a, Kana Miyahara b, Naoharu Takano a,
Keisuke Miyazawa a, *ab

Department of Biochemistry, Tokyo Medical Univeristy, Tokyo, Japan
Department of Breast Oncology and Surgery, Tokyo Medical University, Tokyo, Japana r t i c l e i n f o

a b s t r a c t

Article history:
Received 28 April 2018
Accepted 1 May 2018
Available online 7 May 2018

Excess stress caused by accumulation of misfolded proteins inside the endoplasmic reticulum (ER) lumen
can cause cells to undergo apoptosis. Misfolded proteins exported from ER to cytoplasm are ubiquitinated and mostly degraded by the proteasome, but can also be destroyed by autophagy mediated by thedocking proteins p62 and NBR1. When misfolded proteins accumulate beyond the capacity of theseclearance systems, they are transported to the microtubule organization center to form aggresomes,
which are also degraded by autophagy. Together, these phenomena suggest the existence of a coordinated intracellular network for coping with the accumulation of misfolded proteins. Thus, rational inhibition of this network system might enhance killing of cancer cells subjected to pronounced ER stressloading. Based on this rationale, we sought to establish a quantitative assay for monitoring ER stressloading. MDA-MB231 cells stably transfected with the ERAI-Venus vector exhibited a strong XBP1
splicing signal in response to ER stress. Using the IncuCyte cell imaging system, we monitored theﬂuorescence intensity of XBP1-Venus, normalized against cell density, as an ER stress indicator. Thisparameter correlated closely with other reporters of unfolded protein responses. Assessment of the
XBP1-Venus signal during exposure to various drug combinations revealed that simultaneous inhibitionof the proteasome, autophagy, and aggresome formation led to more effective ER stress loading andhigher cytotoxicity than inhibition of only two components. Our data suggest that this monitoring
system is a useful tool for designing effective drug combinations for ER stress loading in cancer therapy.
© 2018 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY license(http://creativecommons.org/licenses/by/4.0/).

Keywords:
ER stress
ERAI-Venus
XBP1
Proteasome
Autophagy
Aggresom
Breast cancer
Cancer

1. Introduction
ER stress is caused by the accumulation of misfolded proteins
inside the ER lumen. To cope with this stress, cells have evolved aseries of cellular responses designated the unfolded protein responses (UPRs): i) restraint of translation, to suppress synthesis andaccumulation of new misfolded proteins; ii) induction of chaperone
proteins, to promote proper refolding; and iii) export of misfolded
proteins out of the ER lumen, followed by their polyubiquitinationand proteasome-mediated degradation (ER-associated degradation, ERAD) [1,2]. However, when the stress of protein misfolding
exceeds the capacities of these pathways, the cell undergoes
apoptosis via induction of the proapoptotic transcription factor* Corresponding author. Department of Biochemistry, Tokyo Medical University,
6-1-1 Shinjuku, Shinjuku-ku, Tokyo 160-8402, Japan.
E-mail address: miyazawa@tokyo-med.ac.jp (K. Miyazawa).

CHOP/GADD153, activation of caspase-12, and other mechanisms[3,4]. In multiple myeloma characterized by accelerating M-protein
production and secretion, the potent therapeutic effect of proteasome inhibitors such as bortezomib (BZ) is attributed to ER
stressemediated induction of apoptosis [5,6]. We and others havereported that macrolide antibiotics such as clarithromycin (CAM)
and azithromycin (AZM) block autophagic ﬂux [7e9]. We also havereported that combined treatment with BZ plus either CAM or AZM,
simultaneously inhibiting two major intracellular degradation
mechanism, enhances induction of apoptosis by ER stress loading
via up-regulation of CHOP in both myeloma cell lines and breast
cancer cell lines [8,9]. Under these conditions, aggresomes consisting of protein aggregates and vimentin are also induced [10,11].
Ubiquitinated misfolded proteins are selectively degraded,mainly by the proteasome. However, under conditions of proteasome inhibition or accumulation of misfolded proteins beyond thecapacity of proteasomal degradation, they can also be degraded byhttps://doi.org/10.1016/j.bbrc.2018.05.001
0006-291X/© 2018 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

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H. Kazama et al. / Biochemical and Biophysical Research Communications 501 (2018) 286e292

the autophagyelysosome pathway. This process is mediated by two
docking proteins, p62/SQSTM-1 and NBR1, which contain an
ubiquitin (Ub)-associated domain and the LC3 (Atg8)-interacting
region in their C-termini. LC3-II (the lipidated form of LC3-I) localizes on the inner and outer membranes of the autophagosome.
Thus, after binding to p62, Ub-proteins are engulfed into the
autophagosome via the p62eLC3-II interaction [12]. When Ubproteins accumulate beyond the capacity of this system, they aretransported along microtubule tracks by the HDAC6-dynein motor
complex to the MTOC, where they form a perinuclear inclusion
body called the aggresome [13e15]. The aggresome is partiallydisassembled and processed for degradation by the
autophagyelysosome system in cooperation with HDAC6 and theproteasomal deubiquitinating enzyme Poh1 [16]. Thus, a coordinated network encompassing the proteasome, autophagy, andaggresome formation enables the cell to cope with and survive ER
stress loading. This is supported by our ﬁnding that simultaneous
inhibition of the proteasome and autophagy, as well as targeting
aggresome formation by HDAC6 inhibition, results in the efﬁcientkilling of breast cancer cells, probably via ER stress [10]. Moregenerally, these ﬁndings suggest that ER stress loading therapy, inwhich this network is rationally blocked, represents a promising
new approach for cancer therapy [11].
To identify effective drug combinations for cancer therapy, aprecise quantitative mirroring system for ER stress loading isnecessary. In this study, we established a real-time ER stress assay
system combining a breast cancer cell line stably expressing the ER
stresseactivated indicator (ERAI) and the IncuCyte™ live cell imaging system. Using this system, we further assessed ER stressloading under simultaneous inhibition of autophagy, the Ubproteasome pathway, and aggresome formation.
2. Material and methods
2.1. Reagents
Tunicamycin (TN), paclitaxel (PTX), and vinorelbine ditartrate(VNR) were purchased from Wako Pure Chemical Industries, Ltd.
(Osaka, Japan); thapsigargin (TPG) from Nacalai Tesque (Kyoto,
Japan); AZM and CAM from Tokyo Chemical Industry Co., Ltd.
(Tokyo, Japan); BZ from Selleck Chemicals (Houston, TX, USA); andsuberoylanilide hydroxamic acid (SAHA, vorinostat) from Cayman
Chemical Company (Ann Arbor, MI, USA). AZM, CAM, and PTX weredissolved in 95% ethanol at a concentration of 5 mM as stock solutions. BZ and SAHA were dissolved in dimethyl sulfoxide (DMSO)
to make stock solutions at concentrations of 1 mM and 10 mM,
respectively. VNR was dissolved in ultra-pure water to prepare
1 mM stock solutions.
2.2. Cell line and culture conditions
The breast cancer cell line MDA-MB-231 was obtained from theAmerican Type Culture Collection (ATCC, Manassas, VA, USA), andcultured in Dulbecco's Modiﬁed Eagle's Medium-high glucose(DMEM) (Sigma-Aldrich, Merck, St. Louis, MO, USA) supplemented
with 10% fetal bovine serum (FBS) (lot number FB-1061/500; Biosera, Kansas City, MO) and penicillinestreptomycin solution (1/100) (Wako Pure Chemical Industries, Ltd., Osaka, Japan) in a humidiﬁed incubator containing 5% CO2, at 37 � C.
2.3. Establishment of the MDA-MB-231-F-XBP1-Venus cell line
Plasmid pCAX-F-XBP1-Venus was a kind gift from Professor
Masayuki Miura (Department of Genetics, Graduate School of
Pharmaceutical Sciences, The University of Tokyo) [17]. The F-

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287

XBP1-Venus fusion gene was subcloned into the pEZ (OriGene
Technologies Inc., Rockville, MD) vector for puromycin selection.
MDA-MB-231 cells were transfected with pEZ-F-XBP1-Venus using
Lipofectamine® 3000 (Invitrogen, Thermo Fisher Scientiﬁc, Van
Allen Way Carlsbad, CA, USA), and further cultured in the presenceof puromycin (Wako) at 2 mg/ml to isolate stable transfectants. Of 14
clones that expressed XBP1-Venus fusion protein in response to
300 nM TPG, clone #5 exhibited the strongest ﬂuorescence signal,
and was used for the following experiments. The established cellline was used for experiments within ten passages after thawing.
2.4. Real-time monitoring of spliced XBP1-Venus signal using
IncuCyte
The ﬂuorescence intensities derived from spliced XBP1-Venus in
MDA-MB-231-F-XBP1-Venus cells were monitored using an IncuCyte™ ZOOM cell imaging system (Essen BioScience, Ann Arbor,MI) over 48-h exposure to various reagents. At the same time,phase-contrast imaging for conﬂuence, which calculates the density of cells in each imaging ﬁeld, was carried out using IncuCyte. A
10 � objective lens was used in all experiments. All experiments
were performed in quadruplicate using 96-well ﬂat bottom plates
at an initial cell density of 1 � 104 cells/well. Data are expressed asmeans ± SD.
2.5. Counting viable and dead cells
The relative viable cell number was assessed using the CellTiterBlue Cell Viability Assay® kit (Promega, Madison, WI, USA).
Assessment of dead cells was performed by staining with IncuCyte
Cytotox Red Reagent® (Essen BioScience), a sensitive cyanine
nucleic acid dye that yields a >100-fold increase in ﬂuorescenceupon binding to DNA once cells lose plasma membrane integrity,
allowing entry of the dye. Red ﬂuorescence intensity was monitored using IncuCyte during exposure to various reagents.
2.6. Immunoblotting
Cells were lysed in RIPA buffer (Nacalai Tesque) supplemented
with protease and phosphatase inhibitor cocktails (Nacalai Tesque).
Cellular proteins were separated by SDS-PAGE and transferred onto
Immobilon-P transfer membranes (Millipore, Merck, Bedford, MA,
USA). The membranes were probed with the following primary
antibodies (Abs): anti-GRP78 Ab (dilution: 1/500 dilution), antiphospho-PKR-like endoplasmic reticulum kinase (PERK) (Thr981)
Ab (1/500), anti-eIF2a (FL-315) Ab (1/500), anti-ATF-6a (F7)monoclonal (m) Ab (1/1000), and anti-b-actin (C4) mAb (1/1000)
from Santa Cruz Biotechnology (Dallas, TX, USA); anti- IRE1a(Ser724) Ab (1/1000) from Novus Biologicals (Littleton, CO, USA);anti-IRE1a (14C10) mAb (1/1000), anti-PERK (C33E10) mAb (1/1000), anti-phospho-eIF2a (Ser51) Ab (1/1000), and anti-CHOP(L63F7) mAb (1/1000) from Cell Signaling Technologies (Danvers,
MA, USA); anti-FLAG (M2) mAb (1/1000) from Sigma-Aldrich; antiXBP-1s (143F) mAb (1/250) from BioLegend (San Diego, CA, USA);and anti-ATF-4 Ab (1/500) from Abcam (Cambridge, UK). After incubation with the appropriate secondary Ab [e.g., peroxidaseconjugated AfﬁniPure goat anti-mouse IgG (H þ L) (1/2500) or
peroxidase-conjugated AfﬁniPure donkey anti-rabbit IgG (H þ L)
(1/2500); Jackson ImmunoResearch Labs, West Grove, PA, USA] for
1e2 h, immunoreactive proteins were detected with Immobilon
Western Chemiluminescent HRP Substrate (Millipore/Merck,
WBKLS0500). Densitometry was performed using a WSE-6300H/C
Luminograph III (ATTO, Tokyo, Japan).

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289

Fig. 2. Comparison of ER stress signals detected by the ERAI-Venus system and immunoblotting with speciﬁc antibodies for UPR-related proteins. (A) MDA-MB231-XBP1-Venus
cells were cultured with TPG (300 nM) or TN (10 mM). XBP1-Venus/C-C was monitored during a 48-h exposure to these reagents. (B) MDA-MB231-XBP1-Venus cells weretreated with TPG (300 nM) or TN (10 mM) for 24 or 48 h. Immunoblotting using the indicated antibodies was performed to detect UPR signals. Immunoblotting with anti-b-actin
mAb was used as an internal control. Results show one of three independent experiments. (C) Protein expression levels in (B) were measured by densitometry and normalizedagainst the corresponding levels of b-actin.

2.7. Statistics
Statistical analysis was performed using the two-tailed nonpaired Student's t-test. The criterion for statistical signiﬁcance wasp < 0.05.
3. Results and discussion
3.1. Establishment of a real-time assay system for ER stress loading
in a cancer cell line
ERAI is an ER stress activated indicator based on the splicing of
XBP1 mRNA by IRE1 [17]. IRE1, an ER transmembrane kinase thatserves as a stress sensor, oligomerizes in response to ER stressloading [18], leading to activation of an RNase domain located on
the cytosolic side of the membrane. The activated RNase induces
the non-conventional splicing of the mRNA encoding the transcription factor XBP1; the spliced mRNA produces XBP1-s, theactive form of the protein, which contains the bZIP domain [18].
XBP1 activates transcription of genes encoding ER chaperones, aswell as proteins involved in ERAD and lipid synthesis; together, thetargets of XBP1 alleviate ER stress [1,2]. In MDA-MB231 cells stably
expressing ERAI (MDA-MB231-XBP1-Venus cells) the IRE1mediated removal of 26 nucleotides under ER stress eliminates astop codon, resulting in production of the XBP1-Venus fusion
protein (Fig. 1A) [17]. Establishment of the MDA-MB231-XBP1-

Venus cell line was conﬁrmed by monitoring nuclear ﬂuorescence
after treatment with the ER stressor thapsigargin (TPG).
To evaluate the real-time ER stress monitoring system, we used
IncuCyte to monitor the ﬂuorescence intensities derived fromspliced XBP1-Venus during a 48 h-exposure to another ER stressor,
tunicamycin (TN). At the same time, phase contrast imaging of cell
conﬂuence, reﬂected by the density of viable cells on the ﬂat bottom of the culture dish, was carried out in each imaging ﬁeld, alsousing IncuCyte. Fluorescence intensities were divided by cell
conﬂuence, indicated as “XBP1-Venus/C-C”. Cell conﬂuence waswell correlated with the viable cell number, as assessed by
CellTiter-Blue cell viability assay after a 48-h exposure to TN(Supplementary Fig. S1). During the 48-h exposure to TN at
0.1e5 mM, XBP1-Venus/C-C increased in a time- and a dosedependent manner, and became saturated at TN concentrations
above 5 mM (Fig. 1B and C). Simultaneously, dead cell counts weremonitored using Cytox Red staining (Fig. 1D). The ﬂuorescence intensity of Cytox Red was inversely correlated with viable cellnumber as assessed by CellTiter-Blue cell viability assay, and was
also correlated with XBP1-Venus/C-C ratios (Fig. 1E, Supplementary
Fig. S1).
Next we compared the ﬂuorescence intensity of XBP1-Venus
with other UPR indicators (Fig. 2). After treatment with TPG(300 nM) or TN (10 mM) for 24 or 48 h, cells were lysed, and proteins
were separated by SDS-PAGE. Immunoblotting was performed with
antibodies speciﬁc for related proteins. GRP78 (BiP), a HSP70

Fig. 1. ERAI-Venus system for the detection of the spliced XBP1-Venus signal after treatment with TN. (A) Structure of pEZ-F-XBP1-Venus, generated as ERAI constructs: Coding
regions of basic and leucine zipper domains are indicated as yellow and orange boxes, respectively. Transcripts from ERAI constructs are not spliced under normal conditions. The
unspliced mRNA is translated into a truncated XBP-1 with a FLAG tag at its N terminus. Under ER stress, the transcripts are spliced, leading to a frame shift. The spliced mRNA istranslated into an XBP-1-Venus fusion protein with a FLAG tag at its N terminus. Thus, after treatment with TPG (300 nM) for 24 h, nuclear ﬂuorescence is detectable in MDAMB231-XBP1-Venus cells stably transfected with pEZF-XBP1-Venus. “Normal condition” indicates cells cultured in DMEM þ10% FBS, whereas “ER stress condition” indicates cells
cultured in DMEM þ10% FBS in the presence of TPG (300 nM). Scale bar, 10 mm (B) MDA-MB231-XBP1-Venus cells were treated with TN (0.1e10 mM) in a 96-well ﬂat bottom culture
plate. The ﬂuorescence intensities derived from spliced XBP1-Venus were monitored using IncuCyte during a 48-h exposure to TN. For normalization, the detected ﬂuorescenceintensities of Venus were divided by cell conﬂuency, and are indicated as “XBP1-Venus/C-C”. (C) Detection of dead cells using IncuCyte CytoTox Red Reagent. Scale bars, 50 mm.

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H. Kazama et al. / Biochemical and Biophysical Research Communications 501 (2018) 286e292

molecular chaperone located in the ER lumen, is rapidly upregulated upon ER stress [1,19]. Immunoblotting with anti-GRP78 Ab
revealed prominent upregulation of this protein in response to
TPG- and TN-treatment, with an expression pattern similar to thatof XBP1-Venus/C-C. In terms of XBP1-Venus/C-C, 300 nM TPG wassuperior to 10 mM TN during a 48 h-exposure. However, immunoblotting revealed a reduction in the level of GRP78 at 48 h relative
to 24 h, especially in cells exposed to TN. GRP78 is short-lived andmetabolized through N-terminal arginylation, which acts as an Ndegron [20]. Although ER-resident proteins are generally considered to be metabolically stable, the difference in the proﬁles of
XBP1-Venus/C-C and GRP78 appears to be due to differences inprotein turnover.
Immunoblotting revealed that the expression levels of IRE1aand XBP-1s, as well as the phosphorylation state of IRE1a,
responded more strongly to TPG than TN. Moreover, the proﬁles ofthese proteins were also consistent with XBP1-Venus/C-C signals.
PERK is another ER transmembrane kinase that oligomerizes andauto-phosphorylates upon ER stress [21]. The activated PERK oligomer phosphorylates eIF2a, leading to suppression of translationand thereby blocking further accumulation of misfolded proteins
inside the ER lumen [1,21]. As shown in Fig. 2B, the mobility shift of
PERK to a higher molecular weight, a reﬂection of phosphorylation
state, was detected after exposure to both TPG and TN, and was
associated with phosphorylation of eIF2a (p-eIF2a). The ER
stresserelated proapoptotic transcription factor CHOP, which acts
downstream of the p-eIF2aeATF4 axis, was also more strongly
induced by TPG than by TN after a 48-h treatment. Together, thesedata reveal that TPG had a stronger effect than TN on ER stressloading, as determined by XBP1-Venus/C-C, which was consistent
with other UPR signals detected by immunoblotting.
There appeared to be some time lag among the UPRs during the48 h-exposure to TPG and TN. Additionally, it would not be surprising if there were some difference in the expression proﬁle between the artiﬁcial fusion protein XBP1-Venus and endogenous ERresident proteins. In immunoblotting, however, XBP1-Venus/C-C
exhibited a good correlation with the IRE1aeXBP-1s axis, especiallyafter the 48 h-exposure to TPG and TN, indicating that our monitoring system could be used to quantitatively assess ER stressloading.

3.2. Targeting the network comprising the proteasome, autophagy,
and aggresomes enhances ER stress loading and causes pronounced
cytotoxicity in MDA-MB231-XBP1-Venus cells
Many lines of evidence suggest that coordination of the network
consisting of the proteasome, the autophagy pathway, and aggresome formation allows cancer cells to cope with and survive ER
stress [11,12,15]. Because cells undergo apoptosis under excessive
ER stress loading, we next attempted to assess XBP1-Venus/C-C
after strategically blocking this network. In addition, we compared
the cytotoxicity under each condition (Supplementary Fig. S2).
We ﬁrst attempted to simultaneously inhibit the
Ubeproteasome system and autophagyelysosome system using
the proteasome inhibitor BZ in conjunction with CAM or AZM; thelatter compounds are macrolide antibiotics that inhibit autophagic

Fig. 3. ER stress loading and cytotoxic effect of inhibition of the proteasome and autophagy, or the proteasome and aggresome formation. (A, B, and C) MDA-MB231-XBP1-Venus
cells were treated with BZ (15 nM) with or without either CAM (50 mM) or AZM (50 mM) for 1e48 h (D, E and F) MDA-MB231-XBP1-Venus cells were treated with BZ (15 nM) with orwithout either VNR (50 nM) or PTX (50 nM) for 1e48 h (A and D) The ﬂuorescence intensities derived from spliced XBP1-Venus were monitored as described in Fig. 1. (B and E)
Assessment of dead cell number was performed by staining with Cytotox-Red reagent, and red ﬂuorescence intensity was monitored using IncuCyte. (C and F) Assessment of viable
cell number was performed using the CellTiter-Blue cell viability assay.*p < 0.001.

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291

Fig. 4. Simultaneous inhibition of proteasome, autophagy, and aggresome results in pronounced ER stress loading and enhances the cytotoxic effect. MDA-MB231-XBP1-Venus cells
were treated with BZ (10 nM) þ/� CAM (50 mM) þ/� VNR (50 mM) (A and B), and with BZ (10 nM) þ/� CAM (50 mM) þ/� SAHA (1.5 mM) (C and D) for 1e48 h (A and C) Fluorescenceintensities derived from spliced XBP1-Venus were monitored using IncuCyte. (B and D) Assessment of dead cell number was performed by staining with Cytotox-Red Reagent, andred ﬂuorescence intensity was monitored using IncuCyte.

ﬂux [6,7]. As shown in Fig. 3A, treatment of MDA-MB231-XBP1Venus cells with BZ alone caused an increase in XBP1-Venus/C-C
along with a cytotoxic effect, whereas AZM and CAM alone caused
almost no stress loading or cytotoxicity (Fig. 3B and C). Intriguingly,
the combination of BZ plus macrolides resulted in a prominent
increase in XBP1-Venus/C-C, especially in cells treated with
BZ þ CAM. Stress loading by BZ þ CAM was stronger than thatinduced by BZ þ AZM, and was well correlated with cytotoxicity, asassessed by Cytox-Red staining and the CellTiter Blue assay (Fig. 3B
and C).
When misfolded proteins accumulate beyond the cells' clearance capacity, their aggregates are retrogradely transported along
microtubules by the dynein/HDAC6 complex to the MTOC, where
they form aggresomes [14,15]. Under proteasome inhibition, we
compared the effect of VNR and PTX: VNR, a vinca alkaloid, inhibits
microtubule polymerization, whereas PTX, a taxane, inhibits
depolymerization of tubulin, leading to microtubule stabilization.
Therefore, both drugs appear to block the dynamic remodeling of
microtubules by disrupting the scaffold for aggregate transport
toward MTOC. We previously reported that the combination of
BZ þ VNR is superior to BZ þ PTX in terms of the killing effectagainst breast cancer cell lines in vitro [22]. In the MDA-MB231XBP1-Venus system, stress loading differed between VNR andPTX (Fig. 3D and E). In the presence of BZ, VNR exerted more ER
stress loading than PTX at the same concentration, consistent withthe cytotoxic effects described in our previous study (Fig. 3F) [22].
For concomitant inhibition of all misfolded protein processing,
we attempted to simultaneously inhibit the Ubeproteasome-57-

pathway with BZ, the autophagyelysosome system with CAM,
and aggresome formation with VNR (Fig. S2). This drug combination caused more intensive ER stress loading than any combinationof two out of the three drugs, and also exerted the strongest
cytotoxic effect (Fig. 4A and B). Because the deacetylase HDAC6
regulates aggresome formation, we used SAHA (vorinostat), an
inhibitor of HDAC6, instead of VNR to inhibit aggresome formation(Fig. 4C and D) [10,11]. The combination of BZ þ SAHA caused more
efﬁcient ER stress loading and cytotoxicity than BZ þ VNR (max.
XBP1-Venus/C-C: 121.3 at 26 h in BZ þ SAHA vs. 56.8 at 29 h in
BZ þ VNR) (Fig. 4A, C). However, addition of CAM (to block autophagy) to BZ þ SAHA resulted in further enhancement of stressloading, as well as a more pronounced cytotoxic effect (max. XBP1Venus/C-C: 132.3 at 19 h) (Fig. 4C and D). Moreover, peak stressoccurred sooner than in BZ þ SAHA (19 h vs. 26 h). Notably, CAM
treatment alone exerted almost no cytotoxicity or stress loading(Fig. 3). The pronounced killing effect by the combination of thesedrugs was reproduced in other breast cancer cell lines (MDAMB468, BT474), as well as in myeloma cell lines (RPMI-8226, IM-9,
KMS-12-PE) as previously reported (data not shown) [9,10].
Together, these data suggest that simultaneously targeting theentire network responsible for misfolded protein processing canexert an intense cytotoxic effect on cancer cells.
There are many obstacles to putting in vitro data into practice forcancer therapy, especially in terms of pharmacokinetics and drugdelivery. For clinical application, enhancement of adverse effects bydrug combination must also be considered. A recent report showedthat zebraﬁsh implanted with primary tumor cells from a cancer

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patient behaved the same as implanted rodents with the tumor
cells, but much more rapidly, allowing tumors to grow 4 days [23].
The use of such models could guide the development of effective
cancer treatments. Because MDA-MB231-XBP1-Venus cells can betransplanted into zebraﬁsh, it would be intriguing to directly
monitor ER stress in vivo in the implanted cells during tumor
progression and drug response. Our assay system represents a
useful tool for designing effective drug combinations for ER stressloading in vivo, as well as in vitro.
Conﬂicts of interest
The authors declare no conﬂict of interest.
Acknowledgments
We thank Professor Masayuki Miura (Department of Genetics,
Graduate School of Pharmaceutical Sciences, The University ofTokyo) for the kind gift of pCAX-F-XBP1. This study was supported
by funds provided through a MEXT-supported program of theStrategic Research Foundation at Private Universities (S1411011,
2014e2018) from the Ministry of Education, Culture, Sports, Science and Technology of Japan, and partially supported by JSPS
KAKENHI Grant Numbers JP26460478, JP16K21388, andJP16K21387, and a Grant-in-Aid from Tokyo Medical University
Cancer Research (to N. T.).
Transparency document
Transparency document related to this article can be foundonline at https://doi.org/10.1016/j.bbrc.2018.05.001.
Appendix A. Supplementary data
Supplementary data related to this article can be found athttps://doi.org/10.1016/j.bbrc.2018.05.001.
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Amino acid starvation culture condition sensitizes
EGFR-expressing cancer cell lines to gefitinib-mediatedcytotoxicity by inducing atypical necroptosis
YU SAITO1, SHOTA MORIYA2, HIROMI KAZAMA2, KAZUHIRO HIRASAWA1, KANA MIYAHARA3,
HIROKO KOKUBA4, HIROTSUGU HINO2, HIROYUKI KIKUCHI5, NAOHARU TAKANO2,
MASAKI HIRAMOTO2, KIYOAKI TSUKAHARA1 and KEISUKE MIYAZAWA2Departments of 1Otolaryngology (Head and Neck Surgery), 2Biochemistry, 3Breast Oncology and Surgery,4
Joint Research for Basic Medical Science, Institute of Medical Science, and 5Preventive Medicine
and Public Health, Tokyo Medical University, Tokyo 160-8402, Japan
Received November 30, 2017; Accepted December 20, 2017
DOI: 10.3892/ijo.2018.4282
Abstract. The maintenance of the intracellular level of aminoacids is crucial for cellular homeostasis. This is carried out
via the regulation of both the influx from the extracellularenvironment and the recycling of intracellular resources. Since
epidermal growth factor receptor (EGFR)-tyrosine kinase
inhibitors, including gefitinib (GEF) have been reported toinduce the apoptosis of several cancer cell lines, in the present
study, we examined whether the cytotoxic effects of GEF arefurther enhanced under amino acid starvation (AAS) culture
conditions. Under AAS culture conditions, the cell killing
effect of GEF was synergistically pronounced in the EGFRexpressing cell lines, namely, CAL 27, Detroit 562, A549 and
PANC-1 cells compared with those treated with either GEF
or AAS alone. The addition of essential amino acids, but not
non-essential amino acids to the cell culture medium resulted
in the cancellation of this pronounced cytotoxicity. The
knockdown of L-type amino acid transporter 1 (LAT-1) bysiRNA also enhanced GEF-induced cytotoxicity. Therefore,
the shortage of the intracellular amino acid pool appears todetermine the sensitivity to GEF. Notably, this enhanced cytotoxicity is not mediated by the induction of apoptosis, but isaccompanied by the pronounced induction of autophagy. The
presence of necrostatin-1, an inhibitor of receptor-interacting
serine/threonine-protein kinase 1 (RIPK-1), but not that of
Z-VAD-fmk, attenuated the cytotoxic effects of GEF under
AAS culture conditions. Electron microscopy demonstrated

Correspondence to: Professor Keisuke Miyazawa, Departmentof Biochemistry, Tokyo Medical University, 6-1-1 Shinjuku,
Shinjuku-ku, Tokyo 160-8402, Japan
E-mail: miyazawa@tokyo-med.ac.jp

Key words: autophagy, necroptosis, apoptosis, gefitinib, epidermal
growth factor receptor, cancer, tyrosine kinase inhibitorthat the CAL 27 cells treated with GEF under AAS culture
conditions exhibited swelling of the cytosol and organelles
with an increased number of autophagosomes and autolysosomes, but without chromatin condensation and nuclear
fragmentation. Autophagic cell death was excluded as theinhibition of autophagy did not attenuate the cytotoxicity.
These results strongly suggest the induction of necroptosis in
response to GEF under AAS culture conditions. However, we
could not detect any phosphorylation of RIPK-1 and mixed
lineage kinase domain like pseudokinase (MLKL), as well as
any necrosome formation. Therefore, the enhanced cytotoxic
effect of GEF under AAS culture conditions is thought to be
mediated by atypical necroptosis.
Introduction
Amino acids are essential for cellular homeostasis, growthand proliferation via their contribution to a diverse range ofcellular processes. Cells use a number of mechanisms to sense
and maintain their homeostatic levels. Intracellular levels ofamino acids are maintained by the balance between theirinflux, utilization and recycling (1). The transport of serum
amino acids into cells is an active process that is facilitatedby plasma membrane-localized amino acid transporters. The
members of the L-type amino acid transporter (LAT) family
are Na+-independent transporters that deliver neutral aminoacids into cells (2). Although the LAT family plays importantroles in the development and function of normal tissues, they
are frequently increased in cancer cells (3). LAT-1 is mostcommonly upregulated in multiple types of cancer and has
also been used as a biomarker for malignant cancer (4,5).
In contrast to the influx of amino acids, the degradation ofproteins is also the important intracellular mechanism forreleasing free amino acids both under steady-state conditions
and during cellular stresses. Macroautophagy (thereafter
designated as autophagy) is a self-digestive system conservedin all eukaryotic cells. Cellular proteins and organelles are
engulfed into a double-membrane vesicle to form an autophagosome. The cargos are transported to the lysosome and are

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degraded by lysosomal hydrolytic enzymes via membranefusion between the autophagosome and the lysosome, which
is designated as the autolysosome (6). As the recycling ofamino acids by this self-digestive mechanism is essential
for supplying the intracellular amino acid pool for cellular
metabolism, autophagy is markedly upregulated when the cells
are exposed to a nutrient-starving condition (7). On the other
hand, angiogenesis is indispensable for supporting progressive
tumor cell growth. Due to the insufficient vascularization,
autophagy is believed to be accelerated as a form of adaptationof tumor cells particularly in the central region of the tumor
mass in a hyponutrient and hypoxic microenvironment (6,8).
Autophagy can be potentiated by treatment with chemical
inducers or chemotherapeutic agents, as well as in responseto amino acid deprivation. We and others have reported thatepidermal growth factor receptor (EGFR)-tyrosine kinase
inhibitors (TKIs), such as gefitinib (GEF) and erlotinib
potently induce autophagy in several cancer cell lines, as wellas in murine embryonic fibroblasts (MEFs) (9-13). EGFR
is overexpressed or activated in its tyrosine kinase activity
by somatic mutation in a broad range of human cancers,
including non-small cell lung cancer (NSCLC) (14,15). It has
been reported that inactivated endosomal, but not cell surface
EGFR interacts with lysosomal-associated transmembraneprotein B4 (LAPTM-4B), resulting in a complex formation forrecruiting Rubicon from Beclin-1 (Atg6) (16). Since Rubicon
is a potent inhibitory protein for autophagy via the molecularinteraction with Beclin-1, the dissociation of Rubicon from
Beclin-1 results in the initiation of autophagy. Thus, thesurvival of cancer cells with a higher EGFR expression canbe supported by the efficient induction of autophagy undervarious metabolic stresses (16).
We have also reported that macrolide antibiotics, suchas azithromycin (AZM) and clarithromycin (CAM) exert an
inhibitory effect on autophagy flux in myeloma and squamouscell carcinoma cell lines (17,18). Of note, AZM and CAM
exert cytotoxic effects under amino acid-depleted culture
conditions in these cell lines along with the upregulation of the
pro-apoptotic transcription factor, CHOP/GADD153, although
they exhibit no cytotoxicity in complete culture medium (18).
Mammalian target of rapamycin (mTOR), is a master regulator that combines amino acid availability to cell growth andautophagy (19-21). Therefore, the shortage of the intracellularamino acid pool appears to determine the sensitivity to various
cellular stresses. In this context, it is important to examine
whether the effect of GEF is further enhanced when cancer
cells are exposed to an amino acid starvation (AAS) culture
condition.
Historically, three types of cell death have been identified on the basis of morphological criteria, which include
type I (apoptosis), type II (autophagic cell death) and type III(necrosis) (22). Although physiological levels of autophagy
are essential for the maintenance of cellular homeostasis
during various stress conditions, excessive or uncontrolledlevels of autophagy can induce autophagic cell death (22,23).
Autophagic cell death was originally defined as a type ofcell death accompanied by the large-scale autophagic vacuolization of the cytoplasm and was described during animal
development, under tissue homeostasis and in diseased tissues,
as well as in cultured cells treated with chemotherapeutic

agents or other toxic compounds (22). On the other hand,
necrosis has been stereotypically considered as an accidental
and passive cell death, as opposed to apoptosis. Necroptosis,
in which necrosis is regulated, was originally described in a
FADD-deficient variant of human Jurkat T cells treated with
TNF-α, which is characterized as receptor-interacting serine/
threonine-protein kinase (RIPK)1-dependent in association
with the morphological features of cell swelling and plasma
membrane integrity, but without chromatin condensation andnuclear fragmentation (24,25). Thereafter, non-apoptotic cell
death with morphologically necrotic features associated withthe cell death inhibition induced by necrostatin 1 (NEC-1), a
specific inhibitor of RIPK1, has been ‘passively’ described as
necroptosis.
In this study, we found that the cell killing effect of GEF
was apparently pronounced along with the upregulation of
autophagy when the cells were cultured under AAS culture
conditions plus GEF. Of note, this pronounced effect was not
mediated either by the induction of apoptosis or autophagic
cell death, but apparently by an atypical type of cell death. The
molecular mechanisms and phenotype regarding this atypicalcell death are precisely discussed.
Materials and methods
Reagents. GEF, which was purchased from Cayman Chemical
Co. (Ann Arbor, MI, USA), was dissolved in dimethyl sulfoxide(DMSO) to yield 10 mM stock solutions. Z-VAD-fmk, which isa pan-caspase inhibitor, was purchased from Peptide Institute(Osaka, Japan) and 3-methyladenine (3-MA), SP600125, doxycycline hyclate (DOX) and puromycin dihydrochloride wereobtained from Sigma-Aldrich (St. Louis, MO, USA). NEC-1,
a specific inhibitor of RIPK1, was purchased from Enzo
Life Sciences (Farmingdale, NY, USA). Cycloheximide wasobtained from Calbiochem (La Jolla, CA, USA). Recombinanthuman TNF- α, staurosporine, amino acid-free Dulbecco's
modified Eagle's medium (DMEM) (048-33575), MEM
essential amino acids solution (X100; 132-15641) and MEM
non-essential amino acids solution (X50; 139-15651) wereobtained from Wako Pure Chemical Industries (Osaka, Japan).
Cell lines and culture conditions. The human oral squamouscell carcinoma cell line, CAL 27, the human pharyngeal
carcinoma cell line, Detroit 562, the human NSCLC cell line,
A549, the human pancreatic cancer cell line, PANC-1, thehuman colorectal adenocarcinoma cell line, HT-29 and thebreast cancer cell line, MDA-MB-231 were obtained fromthe American Type Culture Collection (ATCC, Manassas,
VA, USA). The human NSCLC cell line, PC-9, which carriesdelE746-A750 in exon 19 of the EGFR gene and exhibits
exquisite sensitivity to EGFR TKIs (26), was obtained fromthe RIKEN BioResource Center (Tsukuba, Japan). The murine
embryonic fibroblast (MEF) cell line (DR-wild-type) established by SV-40 immortalization was also obtained from the
ATCC. All cell lines were cultured in DMEM plus 10% fetal
bovine serum (FBS; Biosera, Ringmer, UK) and 1% penicillin/
streptomycin solution (Wako Pure Chemical Industries). The‘AAS’ culture conditions described in this study indicate
amino acid-free DMEM (048-33575; Wako Pure Chemical
Industries) plus 10% FBS and 1% penicillin/streptomycin.

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The m5-7 cell line, an Atg5 tet-off MEF system, was a kind
gift from Professor Noboru Mizushima (University of Tokyo,
Tokyo, Japan). The m5-7 cells were maintained in DMEM
containing 10% FBS. For the knockdown of the Atg5 gene forthe full inhibition of autophagy, the cells were further cultured
in the presence of 10 ng/ml DOX for 4 days (27). All cell lines
were cultured in a humidified incubator containing 5% CO2
and 95% air at 37˚C. All cell lines were used for the experiments within 10 passages after thawing.
For the typical induction of necroptosis, the HT-29 cells
were pre-treated with Z-VAD-fmk (20 µM) for 30 min
followed by an additional treatment with cycloheximide (CHX,
10 mg/ml ) and human TNF-α (20 ng/ml) for 8 h as previously
described in the literature (28).
Assessment of cell growth inhibition. Cell growth inhibition was measured by the CellTiter-Blue cell viability assay(Promega, Madison, WI, USA). The cells were treated with orwithout GEF (5 to 50 µM) for 24 and 48 h in 96-well plates(Thermo Fisher Scientific, San Jose, CA, USA) in pentaplicate.
During the last 4 h, CellTiter-Blue reagent was added to eachwell, and fluorescence was measured at 560 nm excitation and590 nm emission. The percentage of the mean fluorescencemeasured to that in untreated cells and was expressed as %cell growth inhibition.
Morphological assessment. The cells were spread on
glass slides using a Cytospin 4 Centrifuge (Thermo Fisher
Scientific) to make glass slide preparations, then stained with
May-Grünwald-Giemsa, and examined under a digital microscope (BZ-8000; Keyence Co., Osaka, Japan).
Flow cytometry. For the assessment of apoptosis, the cells
were stained with Annexin V and propidium iodide (PI) using
the Annexin V-FITC Apoptosis Detection kit (Nacalai Tesque,
Kyoto, Japan) according to the manufacturer's instructions
and subjected to flow cytometry using the Attune® Acoustic
Focusing Cytometer (Life Technologies, Carlsbad, CA, USA).
Immunoblotting. Immunoblotting was performed as previously described (29). Briefly, the cells were lysed with RIPA
lysis buffer (Nacalai Tesque) supplemented with a proteaseand phosphatase inhibitor cocktail (Nacalai Tesque). Cellular
proteins were quantified by Bradford assay (Thermo Fisher
Scientific). Equal amounts of proteins were loaded onto thegels, separated by sodium dodecyl sulfate-polyacrylamide
gel electrophoresis (SDS-PAGE) and transferred onto an
Immobilon-P membrane (Millipore, Bedford, MA, USA).
The membranes were probed with primary antibodies (Abs),
such as anti-microtubule-associated protein 1 light chain 3(LC3) B antibody (Ab) (NB600-1384; Novus Biologicals,Inc., Littleton, CO, USA, at 1/4,000 dilution), anti-ATG5 Ab(#12994S, at 1/1,000 dilution), anti-caspase-3 Ab, (#9662S,
1/1,000), anti-poly(ADP-ribose) polymerase (PARP) Ab(#9542S, 1/1,000), anti-LAT1 (#9166S, 1/1,000), anti-RIPK1(#4926S, 1/1,000), anti-phospho-RIPK1 (Ser166) (#65746S,
1/1,000), anti-RIPK3 Abs (#13526S, 1/1,000) (Cell Signaling
Technology, Danvers, MA, USA), anti-mixed lineage kinasedomain like pseudokinase (MLKL), anti-phospho-MLKL(Ser358) (ab184718, 1/1,000) Abs (Abcam, Cambridge, MA,

1167USA) and anti-EGFR Ab (sc-03, 1/1,000), anti-phospho-EGFR(Tyr1173) Ab (sc-101668, 1/1,000), anti-p62 (sequestosome-1)
mAb (sc-28359, 1/1,000), anti-GAPDH mAb (sc-32233,
1/1,000) and anti- β -actin mAb (sc-47778, 1/1,000) (Santa
Cruz Biotechnology, Santa Cruz, CA, USA). Immunoreactive
proteins were detected with horseradish peroxidase-conjugatedsecondary Abs (anti mouse Ab: 115-035-003, at 1/5,000 dilution, anti rabbit Ab: 711-035-152, at 1:5,000 dilution; Jackson
ImmunoResearch, West Grove, PA, USA) and an enhanced
chemiluminescence reagent (ECL) (Millipore). Densitometry
was performed using a Molecular Imager, ChemiDoc XRS
system (Bio-Rad Laboratories, Richmond, CA, USA).
Immunoprecipitation. The harvested cells were washed withphysiological-buffered saline (PBS) and lysed with lysis
buffer (10 mM Tris-HCl, 150 mM NaCl, 1% NP-40, pH 8.0)
supplemented with a protease and phosphatase inhibitor
cocktail (Nacalai Tesque). The cell lysates were spun down
at 16,000 x g for 15 min. The soluble fraction was collected,
and the protein concentration was determined by Bradford
assay. Subsequently, 800 µg of the extracted protein solution
in lysis buffer were immunoprecipitated overnight with either
anti-RIPK1 Ab or anti-RIPK3 Ab at 4˚C. Antibodies werecollected with Protein A SureBeads (Bio-Rad Laboratories)
for 1-h rotation at 4˚C, then washed 3 times with the lysis
buffer. The binding proteins on the beads were eluted with 2X
SDS loading buffer by 5-min boiling in a heat block.
Electron microscopy. Following treatment with/without GEF(25 µM) under either complete culture condition or AAS
culture condition for 24 h, CAL 27 cells were fixed with 2.5%
glutaraldehyde in 0.1 M phosphate buffer (pH 7.3) for 1 h. The
samples were further fixed in 1% osmium tetroxide for 1 h,dehydrated in graded ethanol (30-100%), and embedded in
Quetol 812 Epoxy Resin (Nisshin EM Co., Ltd., Tokyo, Japan).
Ultrathin sections were cut using an Ultracut J microtome(Reichert Jung, Vienna, Austria). These sections were stainedwith lead nitrate and uranium acetate (Merck, Darmstadt,
FRG) and subjected to electron microscopic analysis using ascanning electron microscope (JEM-1200EX II; JEOL Ltd.,Tokyo, Japan).
RNA interference. For the gene silencing of LAT1 and RIPK1
in the CAL 27 cells, the LAT1 siRNA and control siRNAsequences are described as follows: LAT1 sense, GGAAC
AUUGUGCUGGCAUUdTdT and antisense, AAUGCCAGCACAAUGUUCCdTdT; control sense, GUUAAAGGUUUGAC
UCGCGdTdT and antisense, CGCGAGUCAAACCUUUAA
CdTdT. RIPK1 siRNA (HSS112847) and control siRNA(12935-300) were purchased from Life Technologies (Grand
Island, NY, USA). siRNAs were diluted to a final concentration of 10 nM in Opti-MEM I (Life Technologies). Transfection
was performed with the cells at 40% confluency using
Lipofectamine RNAiMAX transfection reagent (LifeTechnologies) according to the manufacturer's instructions.
The knockdown efficiency was assessed by immunoblotting.
Stable transfection of mCherry-EGFP-LC3B plasmid into
A549 and CAL 27 cells. In the present study, a pBABEpuro-mCherry-EGFP-LC3B plasmid vector (no. 22418) was

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purchased from Addgene (Cambridge, MA, USA). The A549
and CAL 27 cells were transfected with this plasmid DNA
using Lipofectamine 2000 (Life Technologies) according to
the manufacturer's instructions and as previously described(30). Briefly, 4 µg of pBABE-puro-mCherry-EGFP-LC3B
plasmid solution and 10 µl of Lipofectamine 2000 were incubated in 500 µl of serum-free Opti-MEM (Life Technologies)
for 20 min and mixed into 2x106 cells cultured in 1.5 ml ofantibiotic-free DMEM with 10% FBS in a 60-mm dish. At
48 h after transfection, the cells were seeded into a 150-mm
dish with puromycin dihydrochloride (at 2 µg/ml for A549
cells and at 0.5 µg/ml for CAL 27 cells). The individual
puromycin-resistant clones were isolated using a cloning ring.
After cloning, A549/pBABE-puro-mCherry-EGFP-LC3B andCAL27/pBABE-puro-mCherry-EGFP-LC3B were used forthe following experiments.
Time-lapse imaging. On the first day, the A549/pBABEpuro-mCherry-EGFP-LC3B cells (clone #3) and CAL 27/
pBABE-puro-mCherry-EGFP-LC3B cells (clone #7) wereseeded into the CELLview™ Cell Culture dish, 35 mm glassbottom (627860; Greiner Bio-One, Frickenhausen, Germany),
and cultured for 24 h to make them adhere to the glass bottom.
On the second day, the cells were washed twice with PBS andincubated in either the AAS culture medium containing 10%
FBS or the complete culture medium containing 10% FBS
with/without GEF (25 µM). Thereafter, observation began at
5-min intervals using a confocal laser scanning microscope(LSM 700; Carl Zeiss, Oberkochen, Germany) for the detection of EGFP at 488 nm laser wavelength and mCherry at
555 nm laser wavelength. In this time-series scanning, a 63X
oil immersion lens was used, and bright field images wereobtained simultaneously. ZEN 2012 Black Edition software(Carl Zeiss) was used for the analysis.
Statistical analysis. All quantitative data are expressed as
the means ± standard deviation (SD). Statistical analysis was
performed using a two-tailed non-paired Student's t-test. To
assess the synergism of the combined GEF and AAS treatment, multivariate linear regression analysis using both GEF
and AAS as independent variables was first performed todetermine whether an additive effect was observed between
GEF and AAS. Subsequently, interaction terms were added in
the model to clarify the existence of a synergistic effect, aspreviously described (31). The criterion for statistical significance was taken as P<0.05.
Results
Amino acid starvation enhances GEF-induced cytotoxicity
in EGFR-expressing cell lines. It is now well known that the
depletion of amino acids in the cell culture medium promotes
the induction of autophagy for the adaptation to the shortageof the intracellular amino acid pool (18-21). It has also beenreported that EGFR-TKIs, such as GEF induce cytoprotective autophagy in various EGFR-expressing cell lines (9-13).
Therefore, in this study, we first examined whether the cytotoxicity of GEF is pronounced under AAS culture conditions
using the head and neck cancer cell lines, CAL 27 and Detroit
562, the NSCLC cell lines, A549 and PC-9, and the pancreaticcancer cell line, PANC-1, all of which express EGFR at a higher
level, as well as immortalized MEFs (Fig. 1A). All the celllines were cultured in the presence of GEF at various concentrations in either amino acid-depleted DMEM containing 10%
FBS or complete culture medium for up to 48 h (Fig. 1B).
Normal DMEM contains a sufficient amount of 20 amino acids
including 75 mg/l L-glutamic acid and 30 mg/l L-methionine,
whereas AAS (amino acid-free DMEM plus 10% FBS) only
contains amino acids less than 1/10 of the normal DMEM
with 10% FBS. Although AAS itself exerted some cell growth
inhibitory effects, the apparent enhanced cytotoxic effect of
GEF was observed even after 24 h of exposure in the CAL 27,
A549 and PANC-1 cells compared to treatment with either
AAS or GEF alone. After 48 h of exposure to GEF under
AAS culture conditions, the effect became more apparent inall cell lines except for the PC-9 cells. As previously reported,
the PC-9 cells have EGFR mutation with a higher sensitivity
to GEF, resulting in a sufficiently strong cell killing effect by
GEF alone, which may produce no further enhancement evenby combining GEF and AAS (26,32). The PANC-1 cells wereresistant to GEF, but exhibited a significant cytotoxicity under
AAS culture conditions. After 48 h of treatment, multivariate
linear regression analysis revealed that these pronounced
effects were synergistic in the CAL 27, Detroit 562, A549 and
PANC-1 cells.
The supplementation of essential amino acids, but not
non-essential amino acids, into the AAS culture medium
almost cancelled the pronounced cytotoxicity in CAL 27 cells(Fig. 2A). This strongly suggests that the enhanced cytotoxicity by the combined treatment with GEF plus AAS is due tothe shortage of the intracellular amino acid pool. To confirm
this hypothesis, we subsequently knock down LAT-1 in the
CAL 27 cells. It has been reported that among the aminoacid transporters, LAT-1 is specifically expressed at a higher
level in cancer cells, by which the essential amino acids areimported into cancer cells for supporting cellular metabolism(3,33). As shown in Fig. 2B, the high LAT-1 expression wasconsiderably suppressed by transfection with LAT-1 siRNA in
the CAL 27 cells. LAT-1 knockdown resulted in a significant
decrease in the viable cell number in the presence of GEF
compared with the cells transfected with control siRNA andtreated with GEF. Notably, LAT-1 knockdown itself exerted
no effect in the absence of GEF, suggesting some metaboliccompensation or adaptation for cell survival when the cells
were cultured in the complete culture medium.
Sensitization of GEF under AAS conditions is not mediated by
the induction of apoptosis. We subsequently examined whether
apoptosis is involved in the enhanced GEF-induced cell deathunder AAS conditions. First, immunoblotting was performed
to confirm the cleavage of caspase-3 and PARP as indicators of apoptosis. As shown in Fig. 3A, GEF treatment undercomplete culture conditions led to some cleavage of caspase-3
and PARP. However, GEF treatment under AAS culture conditions did not lead to any apparent increase in the cleavage of
caspase-3 as compared with GEF treatment in complete culture
medium, although the cytotoxic effect was pronounced. As
shown by flow cytometry, GEF treatment under AAS culture
conditions increased the number of Annexin V+/PI+ doublestained cells compared with either GEF treatment under-62-

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Figure 1. Cell growth inhibition following treatment with gefitinib (GEF) under amino acid starvation (AAS) culture conditions in EGFR-expressing cancer celllines. (A) Cellular proteins in the CAL 27, Detroit 562, A549, PC-9, PANC-1 and MEF cells in the exponential growth phase in the complete culture condition
containing 10% FBS were lysed as described in Materials and methods. The cellular proteins extracted from the 1x105 cells were loaded on each lane and separated by 11.25% SDS-PAGE and immunoblotted with anti-EGFR antibody (Ab) and anti-phospho-EGFR (Tyr1173) Ab. Immunoblotting with anti-GAPHD mAb
was performed as an internal control. The cell lysate of HL-60 leukemia cells was used as a negative control, as previously described (11). Numbers indicate theratios of EGFR to GAPDH and the ratios of the phospho-EGFR to EGFR in each lane. N.D., not detectable. (B) Cells were cultured under the complete culture
condition and AAS culture condition in the presence of GEF (0-25 µM) for 24 and 48 h. Viable cell number was assessed by CellTiter-Blue cell viability assay as
described in Materials and methods (*P<0.05 and **P<0.01, complete culture condition vs. AAS by a two-tailed non-paired Student's t-test). By multivariate linear
regression analysis using both GEF and AAS as independent variables, the synergistic effect was observed in the 24-h treatment of CAL-27 cells (P<0.001)
and PANC-1 cells (P=0.008), as well as in the 48-h treatment of CAL-27 cells (P<0.001), Detroit 562 cells (P=0.023), A549 cells (P<0.001) and PANC-1 cells(P<0.001). In the PC-9 cells, the additive cytotoxic effect was observed in the 48-h exposure (P<0.001), but not in the 24-h exposure (P= 0.335).

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with staurosporine exhibited the typical apoptotic karyorrhexis and blebbing of the plasma membrane. However, weobserved the translucent cytoplasm and rupture of the plasma
membrane of CAL 27 cells (Fig. 3C). These results indicatethat non-apoptotic cell death appeared to be involved in the
pronounced cytotoxicity induced by AAS culture conditionsand GEF treatment.

Figure 2. Shortage of intracellular amino acid pool is involved in the pronounced cytotoxicity of gefitinib (GEF) under amino acid starvation (AAS)
culture conditions in CAL 27 cells. (A) CAL 27 cells were cultured under
AAS conditions supplemented with/without essential and non-essential
amino acids in the presence of GEF (0-20 µM) for 48 h. Viable cell number
was assessed by CellTiter-Blue cell viability assay (*P<0.05, vs. AAS). EAA,
essential amino acids; NEAA, non-essential amino acids. (B) Following pretreatment with LAT-1 siRNA or control siRNA for 24 h, the CAL 27 cells
were further cultured with/without GEF (25 µM) for 48 h in DMEM containing 10% FBS, and viable cell numbers were assessed. Immunoblottingwith anti-LAT-1 anibody (Ab) was performed after 24 h of treatment withsiRNAs. Immunoblotting with anti-GAPDH monoclonal antibody (mAb)
was performed as an internal loading control; *P<0.05, vs. control siRNA.

complete culture medium conditions or culture of the cells in
AAS medium without GEF. Unlike staurosporine treatment
for the typical induction of apoptosis, the number of Annexin
V+/PI- cells indicating the cells undergoing an early phase of
apoptosis was not increased (Fig. 3B). It was recently reported
that phosphatidylserine labeled with Annexin V is exposed tothe cell surface prior to loss of cell integrity during necroptosis
induction (34). Therefore, Annexin V positive staining does
not necessarily indicate cells undergoing apoptosis. As regards
the morphological findings, we could not observe any chromatin condensation and nuclear fragmentation under AAS
culture conditions plus GEF treatment, whereas cells treated

Induction of autophagy in response to GEF and AAS conditions. We then examined whether autophagy is induced in
response to GEF under either complete culture conditions or
AAS culture conditions by immunoblotting with anti-LC3B
Ab and anti-p62 Ab. As shown in Fig. 4A, the expressionof p62, a substrate of autophagy, was decreased in response
to 24 h of treatment with GEF in complete culture medium
compared with that in the cells cultured without GEF incomplete culture medium as a control (lane 1 vs. 2). p62
expression (lane 3) decreased in response to AAS without
GEF and further decreased in response to 24 h of exposure to
GEF plus AAS (lane 4). These results indicate that autophagy
was accelerated by GEF under AAS culture conditions(Fig. 4A). In the CAL 27 cells, the expression ratio of LC3B-II
to β-actin, a hallmark of autophagosome formation (35), wasincreased even under the complete culture condition (lane 1).
This suggests the upregulation of autophagy under the normal
culture condition in this cell line, which was consistent withthe electron microscopic findings with an increased numberof autophagosomes even in the control culture condition (datanot shown). This was possibly due to the fact that the CAL 27
cells expressed a higher level of EGFR (Fig. 1A), implying
a sufficient number of endosomal EGFR for autophagy
induction (16). However, as regards the expression ratio of
LC3B-II to β -actin, the CAL 27 cells under AAS culture
conditions exhibited a decrease in this ratio compared with thecontrol cells (lane 1 vs. 3). In addition, both the LC3B-I and
LC3B-II bands became faint under AAS culture conditions,although AAS should induce more autophagy. We previously
observed the same phenomenon when the cells had markedly
upregulated autophagy along with leading LC3B-II degradation (18). As the expression level of LC3B-II is determinedby the balance of synthesis and breakdown, the degradationof LC3B-II appeared to occur more rapidly than the induction of LC3B-II under AAS culture conditions. To confirm
the accelerated induction of autophagy under AAS culture
conditions plus GEF treatment, we carefully monitoredautophagosome formation during a shorter exposure to GEF
in either complete culture condition or AAS culture conditions
using the A549 and CAL 27 cells, which stably express thetandem fluorescent-tagged LC3B (mCherry-EGFP-LC3B) byconfocal microscopic time-lapse imaging (30). The number offluorescent puncta indicating autophagosomes increased when
the cells were cultured in the AAS medium in the absence of
GEF. Both cell lines treated with GEF in the complete culture
medium exhibited a greater number of mCherry-EGFP-puncta
than the cells cultured in the AAS medium (Fig. 4B). The turnover of puncta appeared to be rapid, and most dots disappeared
within 20 min after formation (data not shown). When the cells
began to be cultured in the presence of GEF under AAS culture
conditions, we observed a marked enhancement of autophagosome formation even within 30 min (Fig. 4B). Therefore, the-64-INTERNATIONAL JOURNAL OF ONCOLOGY 52: 1165-1177, 2018

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Figure 3. Non-apoptotic cell death induction following treatment with gefitinib (GEF) under amino acid starvation (AAS) conditions in CAL 27 cells.
(A) Immunoblotting with caspase-3 antibody (Ab) and PARP Ab from the cell lysates of CAL 27 cells cultured under the complete culture condition or AAS
with/without GEF (25 µM) for 24 h. Immunoblotting with anti-β-actin mAb was used as an internal control. Numbers indicate the ratios of the cleaved PARP toβ-actin and the cleaved caspase-3 to β-actin in each lane. N.D., not detectable. (B) Flow cytometry with Annexin V/PI double staining after 24 h of treatment of
CAL 27 cells with GEF (25 µM) under the complete culture condition or AAS containing 10% FBS. The vertical axis indicates the log fluorescence intensity ofpropidium iodide (PI) and the horizontal axis indicates the log fluorescence intensity of Annexin V. The numbers indicate the percentage of cells in each area.
(C) May-Grünwald-Giemsa staining was performed following treatment with/without GEF (25 µM) under the complete culture condition or AAS for 24 h. As
a positive control for apoptosis induction, CAL 27 cells were treated with staurosporine at 1 µM for 4 h. Scale bar, 10 µm.

induction of autophagy is accelerated in response to GEF plus
AAS.
Induction of atypical necroptosis after treatment with GEF
under AAS culture conditions. To examine whether theenhanced cytotoxicity is due to induction of ‘autophagic cell
death’ (22,23), we used the Atg5 tet-off MEF cell line, named
m5-7 (27). This cell line can be conditionally transformed into
knockout the Atg5 gene, as a useful system for investigating
the effects of autophagy in our study. Additionally, as MEF
cells express EGFR, we intended to investigate whether our
findings in cancer cell lines can be extended to immortalized
fibroblasts.
Pre-treatment of the m5-7 cells with Dox, which leads to
Atg5 knockout, results in the inhibition of autophagy (27). As
shown in Fig. 5, the pronounced cytotoxicity by GEF (50 µM)
plus AAS was observed regardless of the deletion of the
autophagic process in the m5-7 cells.

To investigate the molecular mechanisms responsible forthis pronounced cell killing effect, we further examined thetype of cell death involved in the GEF plus AAS treatmentusing CAL 27 cells. The cells were cultured with GEF either
in the control medium or the AAS culture medium in thepresence or absence of various inhibitors: 3-Methyl adenine(3-MA) for blocking autophagosome formation, SP600125 forblocking autophagic cell death (36), Z-VAD-fmk for inhibiting
pan-caspases for blocking apoptosis, and NEC-1, a RIPK-1
inhibitor for blocking necroptosis (24). As shown in Fig. 6,
treatment with 3-MA and SP600125 enhanced the cytotoxic
effects of GEF and AAS possibly due to the cytotoxicity of theinhibitors. Taking the results of these inhibitors and m5-7 cells
shown in Fig. 5 together, autophagic cell death appeared to be
excluded. Z-VAD-fmk attenuated the GEF-induced cytotoxic
effect, but did not exert any effects on the pronounced cytotoxicity of treatment with GEF plus AAS. Notably, in the presenceof NEC-1, the pronounced cytotoxicity was almost completely

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Figure 5. Effects of autophagy inhibition on gefitinib (GEF)-induced cytotoxicity using the Atg5 tet-off MEF cell line (m5-7). Following pre-treatment
with/without doxycycline (Dox, 10 ng/ml) for 4 days, the m5-7 cells wereseeded in a 96-well culture plate in pentaplicate for 24 h and washed twicewith PBS. The cell culture medium was replaced with complete culture
medium or amino acid starvation (AAS) culture medium in the presenceor absence of GEF (50 µM, at approximately IC50) for 24 h. Assessment of
the viable cell number and immunoblotting with anti-Atg5 antibody were
performed as described above. Immunoblotting with GAPDH monoclonal
antibody was used as an internal control. Significance is indicated as follows:
a vs. a’ shows no statistical significance, and b vs. b’ also shows no statisticalsignificance.

Figure 4. Induction of autophagy following treatment with gefitinib (GEF)
under amino acid starvation (AAS) conditions or complete culture condition. (A) CAL 27 cells were treated with GEF at 25 µM for 24 h. Cellular
proteins were separated by 15% SDS-PAGE, and immunoblotting was performed using anti-LC3B antibody (Ab) and anti-p62 monoclonal antibody(mAb). Immunoblotting with anti-β-actin mAb was performed as an internal
control. As a positive control for p62 and LC3B immunoblotting, the celllysate derived from MDA-MB-231 cells treated with AZM (50 µM) was
usedas described in our literature (29). Numbers indicate the ratios of the
p62 to β-actin and the LCB-II to β-actin in each lane. (B) Assessment of theinduction of autophagy in A549/pBABE-puro-mCherry-EGFP-LC3B stabletransfectants. Following 24 h of culture under the complete culture condition to make the cells adherent, the cells were washed twice with PBS andincubated in either the AAS culture medium containing 10% FBS or control
complete DMEM with/without GEF (25 µM) for time-lapse imaging at 5-mininterval by confocal laser scanning microscopy. Images were merged with
EGFP, mCherry and phase contrast. Scale bar, 10 µm. The image on the left
bottom panel is an enlarged image of the cells treated with AAS plus GEF at
the 1-h time-point. N, nucleus. The numbers of intracellular autophagosome
were assessed by counting the fluorescent puncta (EGFP and/or mCherryalone) in 20 cells in each culture condition, and plotted at the indicated timepoint. Data are representative of three independent experiments and values
are expressed as the means ± SEM (*P<0.05, AAS plus GEF vs. GEF or
AAS alone). CAL 27/pBABE-puro-mCherry-EGFP-LC3B stable transfectants behaved similarly to A549 transfectants in autophagosome formation.
However, the precise assessment of autophagosome number was difficult on
live imaging as the autophagosomes grow in a lump without forming a finesingle cell layer (data not shown).

cancelled. This cytotoxicity cancellation was also observedin the A549 cells treated with GEF and AAS. Therefore, theinduction of necroptosis in response to culture under AAS
conditions plus GEF treatment was strongly suggested.
The CAL 27 cells adhere to the culture flask bottom in
the normal culture condition, and they become non-adherent
while undergoing cell death. As regards electron microscopy,
the non-adherent CAL 27 cells treated with GEF plus AAS
exhibited a translucent cytoplasm, cytoplasmic vacuoles,
mitochondrial swelling, and plasma membrane rupture withan increased number of autophagosomes (Fig. 7). As similarly shown in Fig. 3C, no nuclear fragmentation, chromatincondensation, or apoptotic bodies were observed. Therefore,
all these morphological findings also support the induction of
necroptosis.
It is now well known that necroptosis is a RIPK-1/3dependent form of cell death (38-40). In TNF- α-induced
necroptosis, RIPK-1 is activated in response to TNF- α
stimuli and forms a cytosolic complex, known as the necrosome, which is formed by RIPK-1 in complex with RIPK3
and MLKL (39,40). MLKL is recruited to the necrosome
and phosphorylated by RIPK3. This allows MLKL to form
the MLKL oligomer for insertion into the plasma membrane
to induce necroptosis (41,42). Therefore, in this study, we
knocked down RIPK-1 in CAL 27 cells using siRNA. The
knockdown of RIPK-1 led to a significant attenuation of
GEF plus AAS-induced cytotoxicity compared with the cells
transfected with control siRNA (Fig. 8A). No differenceswere observed between the control siRNA- and RIPK-1
siRNA-treated cells under the GEF-containing culture conditions and AAS conditions. However, we could not detect the-66-

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Figure 6. Effects of various inhibitors on the enhanced cytotoxicity by combining gefitinib (GEF) and amino acid starvation (AAS) in CAL 27 and A549 cells.
The CAL 27 and A549 cells were treated with 3-MA, SP600125, Z-VAD-fmk and necrostatin-1 (NEC-1) at the indicated concentrations in the presence orabsence of GEF (25 µM) under the complete culture condition or AAS for 24 h (*P<0.05 and **P<0.01, vs. AAS without NEC-1).

phosphorylation of RIPK-1 and MLKL by immunoblotting
using specific Abs, which are known to be involved in thesignaling pathways of TNFα-induced necroptosis (40,41)(Fig. 8B). In the immunoprecipitation assay using anti-RIPK-3
Ab, HT-29 cells pre-treated with Z-VAD-fmk followed by anadditional treatment with cycloheximide and human TNF-α
for the induction of necroptosis showed the co-precipitatedphosphorylated (p)-MLKL, as well as p-RIPK-1; however, we
could not detect the co-precipitated p-MLKL and p-RIPK-1 in
the CAL 27 cells treated with GEF plus AAS (Fig. 8C). Thissuggested that culture under AAS conditions plus GEF treatment did not induce necrosome formation in the CAL 27 cells.
Taking all the data together, although the morphologicalfindings and the effect of NEC-1 fit well with the inductionof necroptosis, it would be more appropriate to describe thephenomenon as follows: The enhanced killing effect by the
combined treatment with GEF plus AAS is mediated by‘atypical necroptosis’ or alternatively, ‘necroptosis-like cell
death’ in the CAL 27 cells.
Discussion
To the best of our knowledge, this is the first report showingthat AAS sensitizes the killing effect of GEF in EGFRexpressing cancer cell lines (Fig. 1B). As a solid tumor appears
to proliferate by adapting to nutrient insufficiency alongwith angiogenesis and autophagy, this has important clinicalimplications, such as the application of combination therapyof EGFR-TKI and LAT-1 inhibitors or anti-angiogenesis
therapy for cancer patients. Indeed, it was previously reported
that the phase II clinical trial of the anti-vascular endothelial
growth factor monoclonal antibody bevacizumab in combination with erlotinib for advanced EGFR mutation-positive
NSCLC resulted in an encouraging antitumor activity, and

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Figure 7. Electron microscopy of CAL 27 cells following treatment with gefitinib (GEF) under amino acid starvation (AAS) conditions. The CAL 27 cells
were treated with GEF (25 µM) under AAS culture conditions for 24 h, andelectron microscopy was performed in adherent and non-adherent CAL 27
cells. Scale bar represents the length indicated at each magnification. The
white square box area in the upper panels was enlarged in the lower panels.
N, nucleus; mt, mitochondria; arrowhead, autophagosome/autolysosome;open (white) arrow, swollen mitochondria; closed (black) arrow, membranerupture.

this could be a new first-line regimen for EGFR mutationpositive NSCLC (43,44). In addition, we previously reported
that macrolide antibiotics, which have an inhibitory effect on
autophagy, exerted a CHOP/GADD153-mediated cell killing
effect under amino acid starvation conditions in head and necksquamous cell carcinoma cell lines (18). As shown in Fig. 2A,
the addition of amino acids to the culture medium resulted in
the almost complete cancellation of cytotoxicity, implying thatthe cell killing effect appeared to be mediated by the shortageof intracellular amino acids owing to the blockage of theirrecycling process. Therefore, ‘tumor-starving therapy’ may be
a novel concept for cancer therapy.
However, as shown in Fig. 1B, the response to AAS culture
in terms of cell growth inhibition differed significantly in eachcell line (e.g., the pancreatic cancer cell line, PANC-1, exhibited potent cytotoxicity in response to 48 h of AAS culturecompared with other cancer cell lines). In addition, there was
the significant difference in cell death under AAS conditionsin the MEF cell line used in Fig. 1B and in m5-7, an Atg5
tet-off MEF cell line used in Fig. 5. The m5-7 cell line was
generated by Hosokawa et al (27), and has been cloned forthe complete inhibition of the autophagy machinery. During
this cloning process, the m5-7 cell line appeared to haveacquired a different phenotype including its response to AAS
treatment compared with those in the immortalized MEF cellline. Therefore, the demand for intercellular amino acid poolappears to be varied among the cell lines, which is possibly
due to the difference of cellular metabolism.
We deduced that the enhanced cell killing effect by
GEF plus AAS was mediated by the induction of apoptosis.
However, we could not detect any signs of enhanced apoptosis in the CAL 27 cells during the pronounced cytotoxicity(Figs. 3 and 7). Treatment with GEF alone induced caspase-3
and PARP cleavage to a certain extent, but much less thanthat induced by staurosporine (Fig. 3A). As the CAL 27 cells
treated with staurosporine exhibited typical apoptotic features,such as PARP/caspase-3 cleavage, an increased number of theAnnexin+/PI- cell population as shown by flow cytometry, andmorphological changes showing nuclear fragmentation andchromatin condensation (Fig. 3), the canonical machinery for
apoptosis execution should be conserved in this cell line. The
question that remains to be answered is which type of cell
death phenotype was observed in this study and what cellularsignals determine this phenotype.
According to the results shown in Figs. 3 and 6, autophagic
cell death can be excluded in this case. The enhanced cytotoxicity shown in this study fits well with the necroptosis
definition, that is, the morphological features of cell swellingand plasma membrane integrity, without chromatin condensation and nuclear fragmentation, and cell death inhibition
by NEC-1 (Figs. 3, 6 and 7). The knockdown of RIPK-1 bysiRNA, which significantly attenuated the enhanced cytotoxicity, also supports the induction of necroptosis (Fig. 8A).
However, recent reports have revealed the molecular mechanism of necroptosis (40,41) as follows: Various stresses thatinduce necroptosis appear to be mediated by the interaction ofactivated RIPK3 and MLKL. MLKL is a substrate for RIPK3
kinase activity, and MLKL phosphorylation at Thr357 andSer358 by RIPK3 results in tetramer formation via the fourhelical bundle domain (4HBD) in the N-terminal region. Thisoligomerization subsequently leads to MLKL migration to theplasma membrane to bind phosphatidyl inositol lipids (42).
MLKL tetramer accumulation in the membrane site finallyforms an octamer and acts as the cation channel for ion influx,
which increases the intracellular osmotic pressure leading tocellular destruction (45). As shown in Fig. 4B, autophagy wasconsiderably upregulated in response to GEF and AAS. It has
recently been reported that, in association with the autophagicmachinery for TRAIL-induced necroptosis, p62/SQSTM1
recruits RIPK1 and mediates the formation of a multimolecular complex designated as necrosome consisting of RIPK1,
RIPK3 and MLKL for the upregulation of RIPK3 kinase
activity (46). Therefore, the rapid upregulation of autophagosomes in response to GEF plus AAS appears to function as a
scaffold of necrosome formation, switching toward necroptosis
rather than the induction of apoptosis. However, there were nosigns of the phosphorylation of RIPK1 and MLKL, or necrosome formation (Fig. 8B and C). Although other unclarified
machineries for necroptosis may still exist, these data indicate
a non-standard execution of necroptosis. As the enhanced
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Figure 8. Assessment of the induction of necroptosis in CAL 27 cells following treatment with gefitinib (GEF) under amino acid starvation (AAS) conditions. (A) Following pre-treatment with RIPK1 siRNA or control siRNA for 48 h, the CAL 27 cells were further cultured with/without GEF (25 µM) undereither AAS conditions with 10% FBS or DMEM containing 10% FBS, and viable cell number was assessed. Immunoblotting with anti-RIPK1 antibody
was performed after 24 h of treatment with siRNAs. Immunoblotting with anti-GAPDH monoclonal antibody was performed as an internal loading control(*P<0.05, control siRNA vs. RIPK1 siRNA). (B) Following treatment of the CAL 27 cells with GEF (25 µM) under AAS culture conditions for the indicatedtime periods, the cells were lysed and the cellular proteins were separated by SDS-PAGE, and then immunoblotted with anti-RIPK1 antibody (Ab), anti-RIPK3
Ab, anti-MLKL Ab, and phosphor (p)-specific Abs for RIPK1 MLKL Abs. As for the positive control for the typical necroptosis induction, HT-29 cells werepre-treated with Z-VAD-fmk (20 µM) for 30 min followed by an additional treatment with cycloheximide (CHX, 10 µg/ml ) and human TNF-α (20 ng/ml)
for 8 h as previously reported (28). This treatment is indicated as ZCT. (C) The CAL 27 cells treated with GEF (25 µM) under AAS conditions for 24 h wereused for co-immunoprecipitation assay. The CAL 27 cells treated under the complete culture condition without GEF were used as control. Cell lysates were
immunoprecipitated with anti-RIPK3 Ab or the isotype matched IgG. Immunoprecipitates were separated by SDS-PAGE and immunoblotted with either
anti-p-MLKL, anti-MLKL, anti-p-RIPK1, anti-RIPK1, anti-RIPK3, or anti-p62 Ab. HT-29 cells treated with ZCT as described above were used as positive
control for necrosome formation.

the presence of NEC-1, as well as the knockdown of RIPK1,
this cell death phenotype appears to be dependent on RIPK1,
at least in part (Figs. 6 and 8A). The RIPK3 expression level
was much lower in the CAL 27 cells than in the HT-29 cells(Fig. 8B). Thus, the lower RIPK3 expression level in CAL 27
cells may affect the execution of necroptosis in our case.
In contrast to necroptosis acceleration, autophagy has beenreported to rather suppress necroptosis in RCC4 cells, a humanrenal cell carcinoma cell line (47). The inhibition of mTOR
with CCL-779, a specific mTOR inhibitor, has been shownto stimulate autophagy and lead to the elimination of RIPKs
via autophagy-mediated degradation (47). The simultaneous
inhibition of mTOR with CCL-779 and autophagy with chloroquine resulted in necroptosis induction. Autophagy promotes
the apoptosis induced by Fas ligand/CD95 due to its ability
to degrade Fap-1, a negative regulator of CD95 signaling (48).
On the other hand, autophagy protects the apoptosis inducedby TNF-α by controlling the levels of PUMA, a pro-apoptotic
BCL-2 family protein (49). As shown in Fig. 8B, RIPK1 expression was downregulated during the 24-h exposure to AAS plus
GEF in the CAL 27 cells. Therefore, during the upregulated
autophagic degradation, the delicate balance of the molecularcomponents for cell death execution may also determine thecell fate and cell death phenotype.
It has recently been reported that when the MLKL activation is limited or reversed, the ESCRT-III machinery controls
the duration of plasma membrane integrity, which mayexert an efficient in vivo immune response (34). In contrast

to apoptosis, necrosis and necroptosis induce a series of
immunological responses resulting in inflammation in vivo.
Therefore, a more fine-tuned system consisting of many regulatory molecules might exist. Modulation of the necroptotic
process by degradation of regulators and effectors may havesome unknown biological roles including tumor immunity.
Although the precise molecular mechanism for executing
the pronounced cell killing effect in our case remains to beclarified, the varying amounts of degradation of molecularcomponents for cell death execution might interfere with thecell death phonotype, but ‘not in a stereotypical manner’.
Acknowledgements
We would like to thank Professor Noboru Mizushima(University of Tokyo, Tokyo, Japan) for kindly providing
us with the tet-off Atg5 m5-7 cell line. We would also liketo thank Dr Edward Barroga (http://orcid.org/0000-00028920-2607), Associate Professor and Senior Medical Editorof Tokyo Medical University for editing the manuscript. This
study was supported by funds provided through a MEXTsupported program of the Strategic Research Foundation atPrivate Universities (S1411011, 2014-2018) from the Ministry of
Education, Culture, Sports, Science and Technology of Japan,
and partially supported by JSPS KAKENHI Grant Numbers
JP26460478, JP16K19187, JP16K21388, and JP16K21387,
and a Grant-in-Aid from Tokyo Medical University Cancer
Research to N.T.

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Competing interests
The authors declare that they have no competing interests.
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OPEN

Structural basis of thalidomide
enantiomer binding to cereblon
Tomoyuki Mori 1, Takumi Ito2,3, Shujie Liu4, Hideki Ando2, Satoshi Sakamoto4, Yuki
Yamaguchi4, Etsuko Tokunaga5, Norio Shibata 5, Hiroshi Handa2 & Toshio Hakoshima1

Received: 28 March 2017
Accepted: 22 December 2017
Published: xx xx xxxx

Thalidomide possesses two optical isomers which have been reported to exhibit different
pharmacological and toxicological activities. However, the precise mechanism by which the two
isomers exert their different activities remains poorly understood. Here, we present structural andbiochemical studies of (S)- and (R)-enantiomers bound to the primary target of thalidomide, cereblon(CRBN). Our biochemical studies employed deuterium-substituted thalidomides to suppress opticalisomer conversion, and established that the (S)-enantiomer exhibited ~10-fold stronger binding to
CRBN and inhibition of self-ubiquitylation compared to the (R)-enantiomer. The crystal structures of
the thalidomide-binding domain of CRBN bound to each enantiomer show that both enantiomers bindthe tri-Trp pocket, although the bound form of the (S)-enantiomer exhibited a more relaxed glutarimide
ring conformation. The (S)-enantiomer induced greater teratogenic effects on fins of zebrafish
compared to the (R)-enantiomer. This study has established a mechanism by which thalidomide exerts
its effects in a stereospecific manner at the atomic level.
More than 50 years have passed since thalidomide was first prescribed as a sedative and antiemetic to provide
effective relief from morning sickness during early pregnancy. The teratogenic effects associated with its use weresoon discovered along with severe birth defects such as phocomelia and amelia1–3. Pharmacological studies aimed
at delineating the cause of thalidomide-induced teratogenicity led to the discovery of a number of unexpectedpharmacological activities including anti-inflammatory, inhibition of tumor necrosis factor (TNF)-α production
and anti-angiogenic effects4–6. Other studies demonstrated thalidomide-induced oxidative stress, which resultsin DNA damage or perturbation of the signaling pathways involving NF-κB or Bmp/Dkk1/Wnt7–10. Thalidomide
and its derivatives are now widely used as potent immunomodulatory drugs (IMiDs) in the treatment of several
diseases including multiple myeloma (MM) and leprosy (Hansen’s disease)11–14. Furthermore, thalidomide has
recently been investigated in the treatment of vascular diseases15,16.
Thalidomide is the small synthetic compound α-phthalimido-glutarimide (IUPAC systematic name, 3-(RS)2-(2,6-dioxo-3-piperidyl)isoindole-1,3-dione), which possesses one chiral centre (the C3-carbon atom of theglutarimide ring), and comprises a racemic mixture of two optical isomers, (R)- (also (+)-) and (S)- (also (−)-)
enantiomers, currently in clinical use. The (R)- and (S)-enantiomers were once thought to be responsible for thesedative and teratogenic effects, respectively. This idea was challenged by the findings that the (R)-enantiomer
is also teratogenic in a rabbit model11,12 and that interconversion of the enantiomers could occur under physiological conditions17–19. However, a number of reports, including some describing the use of configuration-stable
thalidomide analogues, have shown that the (S)-enantiomer is more teratogenic and effective at inhibiting TNF-α
production and angiogenesis compared to the (R)-enantiomer20–24.
Thalidomide directly binds cereblon (CRBN), which was originally reported as a cerebral protein associated withmild mental retardation25,26. CRBN is a highly conserved protein that forms a CRL4-type E3 ubiquitin ligase complex,
CRL4CRBN, with Cul4A and damaged DNA binding protein 1 (DDB1), and plays a key role in limb outgrowth andexpression of fibroblast growth factor Fgf8 in zebrafish and chicks25. Thalidomide is suggested to initiate its teratogenic
effects by binding to CRBN and modulating the associated ubiquitin ligase activity25. Moreover, a human MM cell linewith deletion of the CRBN gene was shown to be resistant to thalidomide derivatives, indicating that CRBN is involved
1
Structural Biology Laboratory, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, Nara, 630-0192,
Japan. 2Department of Nanoparticle Translational Research, Tokyo Medical University, Tokyo, 160-8402, Japan.
3
PRESTO, JST, 4-1-8, Honcho, Kawaguchi, Saitama, 332-0012, Japan. 4School of Life Science and Technology,
Tokyo Institute of Technology, Yokohama, 226-8501, Japan. 5Department of Nanopharmaceutical Sciences, Nagoya
Institute of Technology, Gokiso, Showa-ku, Nagoya, 466-8555, Japan. Tomoyuki Mori and Takumi Ito contributed
equally to this work. Correspondence and requests for materials should be addressed to H.H. (email: hhanda@
tokyo-med.ac.jp) or T.H. (email: hakosima@bs.naist.jp)

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Figure 1. Binding assays of deuterium-substituted (S)- and (R)-thalidomides with human CRBN TBD. (a)Chemical structures of deuterated (S)- and (R)-thalidomides, (S)-D-Thal and (R)-D-Thal, respectively. Atom
numbering is shown in the (S)-D-Thal chemical structure. The hydrogen atom at the chiral centre C3 atom of
the glutarimide moiety is substituted with a deuterium atom. (b) Competitive elution assay using thalidomideimmobilized beads coupled with racemic thalidomide. Beads were mixed with extracts from 293 T cells
expressing FLAG-HA-CRBN and washed three times with 0.5% NP-40 lysis buffer and bound proteins were
eluted with wash buffer containing 1 mM deuterated (S)- or (R)-thalidomide ((S)-D-Thal or (R)-D-Thal), (S)or (R)-thalidomide ((S)-Thal or (R)-Thal) or DMSO for the indicated time. The eluate was then analyzed by
SDS-PAGE and immunoblotting (IB). (c) Inhibitory effects of thalidomide enantiomers on auto-ubiquitylationof FH-CRBN were detected in the presence of MG132. Cells were treated with DMSO or the indicatedconcentrations of (S)-D-Thal or (S)-D-Thal for 4 hours prior to harvesting. Full-length blots in (b) and (c) arepresented in Supplementary Fig. 8.

in both the teratogenic and beneficial effects of thalidomide27. Recent crystallographic studies of human, mouse andchick CRBN have succeeded in elucidating the manner by which thalidomide and its derivatives bind the tri-Trp pocket
formed by three conserved surface tryptophan residues in the thalidomide-binding domain (TBD) of CRBN28,29. In
these studies, however, all experiments were performed using racemic compounds, hence enantiomer-specific differences in CRBN binding were not addressed. The precise manner by which these enantiomers exert their different
pharmacological and toxicological effects remained unanswered. Here, we report on a series of crystallographic andbiochemical studies investigating the interaction between each thalidomide enantiomer and CRBN, and provide a
structural basis for the differences in CRBN binding and stereospecific effects of thalidomide on teratogenicity.

Results

Differences in CRBN binding affinity between (S)- and (R)- thalidomides assayed using deuterium-substituted enantiomers. Since interconversion of thalidomide enantiomers could occur under physio-

logical conditions, special precautions are required to delineate any enantiomer-specific differences in the biologicalactivity of thalidomide. To this end, we synthesized each thalidomide enantiomer with deuterium substitution of thehydrogen atom bonded to the chiral carbon atom (C3, Fig. 1a), hereafter deuterated (S)- and (R)-thalidomides arereferred to as (S)-D-Thal and (R)-D-Thal in Figs 1 and 2. Deuterated thalidomides have been found to be at least fivetimes more stable than thalidomide with respect to racemization30. We initially compared the CRBN-binding affinityof the enantiomers in a competitive binding assay with each deuterated thalidomide enantiomer chemically coupled
to ferrite beads (Fig. 1b). CRBN bound to the beads was eluted by the addition of deuterated (S)- or (R)- thalidomide
in a concentration-dependent manner. We found that the binding affinity of CRBN to the (S)-enantiomer was ~10fold stronger than that to the (R)-enantiomer. This is consistent with a previous demonstration that non-deuterated(S)-thalidomide binds CRBN more tightly than the (R)-enantiomer by competition for thalidomide-immobilized
beads28. The aforementioned conclusion is further supported by results of a cell-based assay, in which deuterated(S)-thalidomide inhibited the auto-ubiquitylation activity of the CRBN-containing ubiquitin ligase complex morestrongly than deuterated (R)-thalidomide (Fig. 1c), and is consistent with previous results utilizing non-deuterated thalidomide28. Considering their half-lives of racemization, which occurs in a matter of hours with non-deuterated thalidomide under physiological conditions11,12,18,19,21,24,25, only a very small fraction of each enantiomer isthought to be racemized during the assay. To confirm this hypothesis, we analyzed the enantiomeric purities of all
the thalidomides used before and after the experiments. The purities were monitored by HPLC (DAICEL Chiralpak
IA, 4.6 × 250 mm, MeOH = 100%, flow rate 1.0 ml/min, λ = 254 nm). (Supplementary Table 1 and Supplementary
Fig. 1). No significant racemization was detected for the non-deuterated thalidomides and no racemization was
detected for the deuterated thalidomides. These results are consistent with a previous report indicating that the racemization of thalidomide is slow under lower or neutral pH conditions, while the racemization is accelerated underhigher pH values30. The half-life of racemization of (S)-thalidomide was 31.8 h at 6.18 pH and 29.9 h at pH 7.78 (atScienTific REPORTS | (2018) 8:1294 | DOI:10.1038/s41598-018-19202-7

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Figure 2. Effects of deuterium-substituted (S)- and (R)-thalidomides on IKZF3 degradation and CRBNIKZF3 binding. (a) Effects of deuterated (S)- and (R)-thalidomide enantiomers, (S)-D-Thal and (R)-D-Thal,on IKZF3 protein levels in MM1S cells after 12-h treatment. IB, immunoblotting. (b) Co-immunoprecipitationof FLAG-HA-CRBN in the presence of each deuterated thalidomide enantiomer, (S)-D-Thal or (S)-D-Thal. IP,
immunoprecipitation. Full-length blots in (a) and (b) are presented in Supplementary Fig. 9.
37 °C), while that of deuterated (S)-thalidomide was 156.3 h at pH 6.18 and 59.5 h at pH 7.7830. The pH values ofthe medium solutions used in the current experiments were 5.76 for the Zebrafish E3 medium (5 mM NaCl, 0.17,
mM KCl, 0.33 mM MgSO4, and 0.33 mM CaCl2), 6.54 for the DMEM (Nacalai) Auto-Ub medium and 7.05 for the
RPMI1640 MM1S medium, suggesting that both thalidomide and deuterated thalidomide are rather stable toward
racemization in each medium used in our assays.
We also determined the dissociation constants K D for the binding of non-deuterated (S)- and(R)-thalidomide to human and mouse CRBN TBDs by isothermal titration calorimetry (ITC). The results
show that (S)-thalidomide binds TBD 6- or 9-fold more tightly than (R)-thalidomide in terms of the KD values(Supplementary Fig. 2). These results collectively suggest that differences in the pharmacological activity of theenantiomers are determined, at least in part, by their differential binding affinity to CRBN.

Deuterated (S)- and (R)- thalidomides showed differences in IKZF degradation. Recent studies haveshown that lenalidomide and pomalidomide directly bind CRBN and promote the recruitment of pseudo-substrates
Ikaros (IKZF1) and Aiolos (IKZF3) to the E3 complex containing CRBN, thus leading to ubiquitylation and degradation31,32. We set out to determine whether enantiomers exert differential effects on the down-stream substrate
IKZF1/3. We examined IKZF3 degradation in MM1S cells with deuterated (S)- and (R)-thalidomides (Fig. 2a). The
breakdown of IKZF3 was observed in the presence of deuterated (S)-thalidomide ((S)-D-Thal in Fig. 2a). However,no discernible breakdown of IKZF3 was observed with deuterated (R)-thalidomide. Furthermore, we performed a
co-immunoprecipitation assay for CRBN binding to IKZF3 in the presence of deuterated (S)- or (R)-thalidomide(Fig. 2b). We observed strong CRBN binding to IKZF3 in the presence of deuterated (S)-thalidomide. On the other
hand, the effect by deuterated (R)-thalidomide on the binding was very weak. These results support the notion thatthe binding affinity of CRBN to the (S)-enantiomer was stronger than that to the (R)-enantiomer and, that the difference in binding affinity affects degradation of the down-stream substrates.
Common binding model of (S)- and (R)-thalidomides to the tri-Trp pocket.

In an effort to clarify
the differences in CRBN binding at the structural level, we determined the structure of mouse CRBN TBD boundto (S)- or (R)-thalidomide and refined the structures at 1.8 Å and 2.0 Å resolution, respectively (Tables 1 and 2).
Both structures gave clear electron densities for the bound thalidomide molecules (Fig. 3a). CRBN TBD consistsof eight β-strands (β1-β8) with a single zinc ion, which is coordinated by four conserved cysteine residues fromtwo CXXC motifs located in the β1-β2 and β5-β6 loops (Fig. 3b and Supplementary Fig. 3). The central β-sheet(β4-β5-β6-β7-β3) contains the tri-Trp pocket formed by Trp383 (β4-β5 loop), Trp389 (β5-strand) and Trp403(β6-strand). (S)-thalidomide binds this pocket so that the glutarimide ring is docked into the tri-Trp pocket, andthe phthalimido group is located outside of the pocket (Fig. 3b). Inside the pocket, the glutarimide ring, which is
sandwiched between Trp383 and Trp389, makes nonpolar contacts with Trp403 forming the floor of the pocket
and forms two hydrogen bonds with the protein: the glutarimide 6-carbonyl group forms a hydrogen bond to themain-chain amide of Trp383 and the glutarimide 1-imino group (NH) forms a hydrogen bond to the main-chain
carbonyl group of His381 (Fig. 3c). These characteristics of the binding mode are identical to those observed for(R)-thalidomide (Fig. 3d) and do not differ from those detailed in previous reports28,29. No significant structuraldifferences were found between the overall structures of TBDs bound to (S)- and (R)-thalidomide, as reflected in
the extremely small root-mean-square (rms) deviation (0.13 Å) (Fig. 4a). Additionally, these structures are similar
to the free forms28 (0.45–0.46 Å) and other reported structures28,29 (Fig. 4b,c), although the β2-β3 loop displays
conformational flexibility with relatively high temperature factors, as observed in the free forms28 (Fig. 4d). In
addition to the similarity in the overall structures, the thalidomide-binding sites also exhibit high similarity without any significant structural deviation between the (S)- and (R)-thalidomide-bound forms (Fig. 4e).
The observed high similarity in the structures of the thalidomide-binding sites, the binding modes, and overall
TBD structures of our complexes imply that other factors are responsible for the differences in the binding affinityof (S)- and (R)-thalidomides.

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www.nature.com/scientificreports/(S)-thalidomide(R)-thalidomide

Space group

R3 (H)

R3 (H)(RS)-thalidomide (racemic)
R3 (H)

Unit cell a = b = (Å)

201.90202.24201.38c = (Å)123.61123.82

123.36γ = (°)

120

120

120

Wavelength (Å)

1.282680.90001.0000Resolution rangea (Å)

50–1.850–2.050–2.0(Outer shell)(1.86–1.8)(2.05–2.0)(2.07–2.0)

Completeness (%)

100 (100)

99.9 (99.9)

100 (100)

Oscillation range (°)

180

180

180

Measured

974,617752,877

723,800

Unique173,436

130,317

125,695

Multiplicity

5.6

5.8

5.8

Mosaicity (°)

0.38

0.41

0.33

I/σ(I)

17.8 (3.1)

20.6 (3.9)

20.6 (4.6)

Rmerge(%)

8.1 (50.0)

7.8 (49.3)

7.8 (41.0)

Reflectionsb

Table 1. Crystallographic statistics of the CRBN TBD bound to thalidomides. aValues in parentheses are for thehighest-resolution shell. bData were collected at SPring-8 beamline BL44XU with a MX225HE detector (each 1°
oscillation with 1 s exposure time for the (S)-thalidomide-bound form, 0.5° oscillation with 1.2 s exposure time
for the (R)-thalidomide-bound form and each 1° oscillation with 1 s exposure time for the (RS)-thalidomidebound form) at 100 K.

(S)-thalidomide (R)-thalidomide (RS)-thalidomide (racemic)
Rwork/Rfreea (%)

18.8/21.6

18.7/21.1

18.9/21.0

Number of atoms

13,621

13,077

13,548CRBN molecules

16

16

16

residues

1,505

1,500

1,505

Thalidomide

16

16

16

Zinc ions

16

16

16

Sulphate ions

30

30

30

Water molecules

1,313

809

1,240

CRBN molecules

27.8

32.4

27.6

Thalidomide

26.9

37.3

28.1

Zinc ions

27.8

27.7

24.8

Sulphate ions

43.9

46.6

42.8

Water molecules

40.9

42.2

40.9

bonds (Å)

0.005

0.005

0.005

angles (°)

1.011

1.029

1.066

Averaged B-factors (Å2)

R.m.s.d. from ideal values

Ramachandran plots (%)
Favored

98.8

98.8

98.7

Allowed

1.2

1.2

1.3

Outliers000Table 2. Structural refinement statistics of the CRBN TBD bound to thalidomides. aRfree was calculate on arandom 5% reflections of the data.

Conformational differences between bound (S)- and (R)-thalidomides. The electron density mapsof our complex structures are sufficiently clear to define the conformation of the bound thalidomide molecules(Fig. 3a). We then examined the thalidomide conformation in the CRBN-bound state. Our crystals contain 16 crystallographically independent CRBN TBD-thalidomide complexes in the asymmetric unit (see the Experimental
section). We found that the 16 (S)-thalidomide molecules bound to CRBN TBD display essentially the same conformation with a small averaged rms deviation of 0.056 Å (Supplementary Fig. 4a). Similarly, the 16 (R)-thalidomide
molecules display essentially the same conformation, which is distinct from that of (S)-thalidomide as described
below, and exhibit a small averaged rms deviation of 0.074 Å (Supplementary Fig. 4b). These results suggest that(S)- and (R)-thalidomide molecules bound to CRBN have a defined conformation but no variable conformations.
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Figure 3. (S)- and (R)-thalidomides bound to CRBN TBD. (a) Stereo views of (S)-thalidomide (yellow)
and (R)-thalidomide (green) molecules bound to mouse CRBN TBD in the crystal with composite omit
maps (2mFo-DFc) shown in 1σ contour (blue). (b) Stereo view of mouse CRBN TBD (cyan) bound to (S)thalidomide (yellow). Side chains of residues at the tri-Trp pocket are shown as stick models. (c) Close-up viewof a (S)-thalidomide molecule (yellow) bound to the tri-Trp pocket (cyan). Residues forming the tri-Trp pocket
are shown as stick models (cyan for C, blue for N, and red for O). The (S)-thalidomide molecule is shown as a
stick model (yellow for C, blue for N, and red for O). Two hydrogen bonds formed between the protein and theglutarimide moiety of thalidomide are represented as red dotted lines. (d) As in c but for (R)-thalidomide (green
for C, blue for N, and red for O) bound to the tri-Trp pocket (magenta).

In both enantiomeric structures, the phthalimide ring is tilted against the glutarimide ring by 75° so that two
protruding carbonyl groups of the phthalimide ring are docked into grooves at the pocket entrance: the phthalimido 1-carbonyl group into the groove between Trp383 and Trp403, and the 3-carbonyl group into the groove

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Figure 4. Overall structural comparison of CRBN TBDs in the free and thalidomide-bound forms. (a)Comparison of the (S)- (cyan) and (R)- (magenta) thalidomide-bound forms of CRBN TBD structures. The
structural overlay shows no significant structural differences with a small root-mean-square (rms) deviation(0.13 Å). (b) Comparison of the (S)-thalidomide-bound (cyan) form with the free form28 (grey, PDB code
3WX2) of CRBN TBD structures. The rms deviation is 0.45 Å for Cα carbon atoms except for the mobile
β2-β3 loop (residues 344–359). (c) Comparison of the (R)-thalidomide-bound (magenta) and free (grey)
forms of CRBN TBD structures. The structural overlay shows a large structural deviation in the mobile β2-β3loop (residues 344–359), although the remainder of the domain cores are similar with a relatively small rms
deviation (0.46 Å). (d) Two crystallographically independent molecules A (grey) and B (green) of CRBN TBD
in the free form28 superimposed on each other. Nine residues (351–359) of the flexible β2-β3 loop of molecule
B were invisible in the current map. The rms deviation is 1.82 Å for all Cα carbon atoms and 0.34 Å for the core
domain without the mobile β2-β3 loop (residues 341–361). (e) Structural comparison of the tri-Trp pockets
accommodating (S)-thalidomide (yellow) and (R)-thalidomide (green). Middle, superposition of (S)- and (R)thalidomide-bound tri-Trp pockets. Left, A close-up view of the tri-Trp pocket of mouse CRBN TBD (cyan)bound to (S)-thalidomide (yellow). Right, a close-up view of the tri-Trp pocket of mouse CRBN TBD (magenta)
bound to (R)-thalidomide (green). Side chains of key residues forming the tri-Trp pocket are shown as stickmodels.

between Trp389 and His381 (Fig. 5a,b). Thus, the phthalimide ring is locked at the pocket entrance and therotational conformation of the phthalimide ring around the C-N bond between the phthalimide and glutarimide
rings is the same in both enantiomers. Overlay of the bound (S)- and (R)-thalidomide molecules shows goodoverlap of the molecules with a relatively small shift in orientation of the phthalimide ring (Fig. 5c). This shift
is caused by the different nature of the glutarimide ring conformation. The glutarimide ring of (S)-thalidomide
displays a C4-endo puckered conformation, a relaxed and stable five-membered ring conformation, in which
the C4-carbon atom is displaced from the plane formed by the rest of the ring atoms. This puckered form closely
resembles that observed in the isolated free form of thalidomide33,34 (Fig. 5d). Compared to the free form, the
CRBN-bound form has a small shift (0.5 Å) of the phthalimide ring toward the endo direction (middle in Fig. 5d)so as to fit the grooves of the entrance of the tri-Trp pocket (Fig. 5a).

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Figure 5. Comparison of the bound thalidomide conformations. (a) Phthalimido carbonyl groups of (S)thalidomide docked into grooves formed at the entrance of the tri-Trp pocket. (S)-thalidomide (yellow stickmodel) bound to the Tri-Trp hole of CRBN TBD. The phthalimido 1-carbonyl group (labelled with red 1) isdocked into the groove formed by the side chains of Trp383 and Trp403. The phthalimido 3-carbonyl group(labelled with red 3) is docked into the groove formed by Trp389 and His381 side chains. The van der Waals
surfaces of the two carbonyl groups are shown with dots and labelled with red atom numbers. The grooves
for docking with the phthalimido carbonyl groups are indicated with orange arrows. (b) As in a, but for (R)thalidomide (green stick model) bound to the tri-Trp pocket of the CRBN TBD. (c) Comparison of CRBNbound thalidomide conformations. (S)-thalidomide superimposed onto (R)-thalidomide with overlapping
imide groups, which are linked to CRBN by direct hydrogen bonds and polar interactions. The puckered C4
atoms are separated from each other by 1.0 Å and the puckered C3 atoms by 0.5 Å. The phthalimido groups
are shifted from each other by a maximum of 1.0 Å. (d) Comparison of the glutarimide ring conformations ofthalidomide molecules in the CRBN-bound and free states in crystals. CRBN-bound (S)-thalidomide (yellow)
is superimposed onto the free form (grey) of (S)-thalidomide in the racemic thalidomide crystal (34) withimide groups overlapped. The glutarimide ring of CRBN-bound (S)-thalidomide displays the typical C4-endo
puckered conformation. This conformation is similar to the glutarimide ring of free (S)-thalidomide, whichdisplays a slightly twisted C4-endo puckered conformation. The phthalimido group of the CRBN-bound form
exhibits a displacement (0.5 Å for the carbonyl groups) from that of the free form with overall rms deviation of
the phthalimido groups of 0.9 Å. (e) As in (d) but showing (R)-thalidomides. The glutarimide ring of CRBNbound (R)-thalidomide (green) displays a twisted (C3-endo-C4-exo-C5-endo) conformation. This highly
twisted conformation is in sharp contrast with free (R)-thalidomide (grey) in the racemic thalidomide crystal(34). In contrast to (S)-thalidomide, the phthalimido group of CRBN-bound (R)-thalidomide exhibits marked
displacement (1.7 Å for the 1-carbonyl group) relative to the free form with large overall rms deviation (1.4 Å) of
the phthalimido groups.

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Figure 6. Comparison of (R)-thalidomides in the free form and in the CRBN-bound form found in thecomplex crystal. Superposition of the free form of (R)-thalidomide (grey) on the CRBN-bound form of(R)-thalidomide (green) found in the crystal. The free form of (R)-thalidomide has its glutarimide ring in
a C4-exo conformation, while (R)-thalidomide (green) bound to the tri-Trp pocket of CRBN displays atwisted conformation (see text). The C4-exo conformation of the free form causes serious steric clash between
the phthalimido-1-carbonyl group and the ring carbon atom of Trp383 (2.6 Å indicated by a dotted line).
Additionally, short contacts (~3.6 Å) were found between the C4-carbon atom of the glutarimide moiety andring carbons of Trp383. The twisted conformation of the bound (R)-thalidomide avoids this clash by shifting thephthalimide ring toward the endo direction so that the clashes are relaxed (4.0 Å and 3.8 Å, respectively) in thetwisted form.

In sharp contrast to the relaxed form of the bound (S)-thalidomide, CRBN-bound (R)-thalidomide has theglutarimide ring in a twisted conformation, which is distinct from the stereochemically relaxed C4-exo puckered
conformation of (R)-thalidomide (Fig. 5e), suggesting that bound (R)-thalidomide is structurally constrainedin a metastable conformational state. The twisted conformation is forced primarily by the contacts between thephthalimide ring and the entrance of the tri-Trp pocket and accompanies a large displacement (1.7 Å) of thephthalimide ring toward the endo direction (middle in Fig. 5e). This displacement needs to avoid steric clash of
the phthalimido-1-carbonyl group against the groove formed by Trp403 and Trp383, and enables the protruding
carbonyl groups to dock into the grooves (Fig. 5b). To visualize the steric hindrance, we superimposed the freeform of (R)-thalidomide onto the CRBN-bound form of (R)-thalidomide found in the crystal (Fig. 6). We foundthat the C4-exo conformation of the free form causes serious steric clash (2.6 Å) of the phthalimido-1-carbonylgroup against the ring carbon atom of Trp383 (indicated by a dotted line in Fig. 6). The twisted conformationof the bound (R)-thalidomide avoids this clash by shifting the phthalimide ring toward the endo direction(1.7 Å, green arrow). Moreover, close inspection of the glutarimide ring puckering by modelling revealed that(R)-thalidomide in the C4-exo puckered glutarimide ring conformation causes steric clash of the C4-carbon
atom against Trp383. To avoid this clash, the glutarimide ring is also forced to adopt a twisted conformation.
All of these relatively small but significant differences in the binding mode between (S)- and (R)-thalidomide
molecules suggest that the (S)-thalidomide bound state of the thalidomide-CRBN complex is more stable thanthe (R)-thalidomide bound state. As with the previous structures28,29, our structure of thalidomide-bound CRBN
TBD prepared with racemic thalidomide revealed that the bound thalidomide molecules are all (S)-enantiomers(Supplementary Fig. 5). This is also consistent with the notion that CRBN preferentially binds the (S)-enantiomer.

Dependence of thalidomide-induced teratogenicity on chirality. In an effort to examine the effectsof thalidomide enantiomers on zebrafish development, we transferred dechorionated embryos to media containing different concentrations of thalidomide at 2 hours post fertilization (hpf) and allowed them to develop for 3
days. It was apparent that with thalidomide-treated embryos, the development of pectoral fins and otic vesicles
was disturbed (Fig. 7a). Treatment with 200 μM (S)-thalidomide induced severe defects on fins, whereas treatment with 200 μM (R)-thalidomide resulted in no discernible severe defects (Fig. 7b). At higher concentrations(400 μM) of drug, (S)-thalidomide induced defects in more than 80% of fish with a higher population of defective
embryos, whereas (R)-thalidomide induced defects in only 50% of fish with a lower population of defects. Thus,(S)-thalidomide exerts greater teratogenic effects on fin development of zebrafish, which is consistent with theresults of our binding and ubiquitylation-inhibition assays using deuterium-substituted thalidomide enantiomers(Fig. 1), and also the results of the complex structures between TBD and each thalidomide enantiomer.

Discussion

Based on our complex structures, we are now able to assess the CRBN-binding affinity of thalidomide derivatives.
The glutarimide ring in a relaxed six-membered ring conformation is important for CRBN binding with theimide group, serving as both a hydrogen donor and acceptor. Based on the chemical nature of the glutarimide
ring, five carbon atoms (C1-3, C5 and C6) of the ring favor an in-plane arrangement because of the sp2 configuration of two carbonyl carbon atoms (C2 and C6) with the amido (CONHCO) π electron resonance. Thus,

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Figure 7. (S)-thalidomide treatment causes more sever developmental defects in zebrafish than (R)thalidomide. (a) Dorsal views of pectoral fins of 72-hpf embryos. Fins are indicated by arrowheads. The
teratogenic effects resulting from treatment with thalidomide are classified into three categories. Fins thatstretched out from the body wall and were more than 85% in length compared to control fins were definedas “no effect.” Fins that stretched out but were shortened to 60–85% of control length were defined as “mild”
phenotypes. Fins that showed disc-like morphology and were shortened to less than 60% of control length were
defined as “severe” phenotypes. (b) Incidence of pectoral fin malformations. Treatments with 200 μM or 400 μM(S)-thalidomide (S200 and S400, respectively) induce more severe effects than treatments with 200 μM or 400
μM (R)-thalidomide (R200 and R400, respectively) in 72-hpf embryos.
ring puckering usually occurs at the C4 carbon atom. In the isolated state, the C4-endo conformation of the(S)-thalidomide glutarimide ring minimizes the conformational energy by allowing the N-C bond at the chiral C3
atom to be oriented in a stable equatorial conformation, whereas (R)-thalidomide favors the C4-exo conformerof the glutarimide ring, allowing the N-C bond to adopt an equatorial conformation. In the C4-endo conformerof the (S)-thalidomide glutarimide ring, the chiral centre tilts the phthalimido group slightly toward the endo
direction (bottom in Fig. 5d), which is suitable for binding to the tri-Trp pocket of CRBN TBD. Therefore, small
adjustment of the orientation of the phthalimido group is sufficient for CRBN binding (middle in Fig. 5d). In theC4-exo conformer of the (R)-thalidomide glutarimide ring, however, the chiral centre tilts the phthalimido groupslightly toward the exo direction (bottom in Fig. 5e), which is in the opposite direction required for binding to thetri-Trp pocket. In this case, small adjustment of the orientation of the phthalimido group is insufficient for CRBN
binding, and therefore the glutarimide ring pucker of (R)-thalidomide needs to be changed to the twist conformation, C4-exo-C5-endo, to shift the phthalimido group up toward the endo direction (middle in Fig. 5e) for CRBN
binding with loss of conformational energy. It should be noted that this enantiomer-specific discrimination by
the tri-Trp pocket is mediated by hydrogen bonding interactions between the amide group of the glutarimide ringand the inside of the pocket, in addition to contacts between carbonyl groups of the phthalimido group and theentrance of the pocket.
Lenalidomide and pomalidomide have attracted much attention from pharmaceutical scientists and physicians as hopeful IMiDs in the treatment of multiple myeloma and other cancers11,14. The homeobox transcription
factor MEIS2, has been implicated in various aspects of human development, has been suggested to be an endogenous substrate of CRL4CRBN, and IMiDs block MEIS2 from binding to CRL4CRBN 28. Contrary to this inhibition ofsubstrate binding, these drugs directly bind CRBN and promote CRL4CRBN binding to IKZF1 and IKZF3, leading
to ubiquitylation and degradation31,32. Furthermore, in the treatment of myelodysplastic syndrome (MDS) withdeletion of chromosome 5q (del(5q)), lenalidomide induces ubiquitylation of casein kinase 1A1 (CK1α) and its
degradation35. One attractive hypothesis concerning IMiDs drawn from these IMiD-dependent degradations
is that IMiDs act as modulators of CRL4CRBN substrate recognition by playing a role as a molecular glue36 or
an interfacial drug37 that specifically links CRBN and substrate proteins through direct interactions with bothproteins. The binding mode of IMiDs to CRBN suggests that the phthalimido group should make contact withthe substrate proteins. Since the orientation of the phthalimido group of CRBN-bound IMiDs differs somewhat
between (S) and (R)-enantiomers, we speculate that CRBN bound to the (S) and (R) enantiomers may exhibit
different binding affinity to the substrates. The recent structure of the complex between lenalidomide-bound
CRBN and CK1α has shown that CK1α binding repositions the phthalimido ring of lenalidomide 2.5 Å toward
CRBN residue Glu37738. Interestingly, this shift accompanies a movement of the phthalimido group toward theendo direction as seen in (S)-thalidomide (Fig. 5d), suggesting that the (S)-enantiomer is favored for CK1α binding. However, details of the differences between (S)- and (R)-enantiomers in CK1α binding should be clarified
by further studies.
As with previously reported structures28,29, binding to CRBN is primarily mediated by the glutarimide
ring. Correspondingly, glutarimide alone is able to bind CRBN and inhibit ubiquitylation to a similar extent as(R)-thalidomide (Fig. 8a,b and Supplementary Fig. 6), whereas phthalimide and glutaric anhydride, which lack
the ring amide of glutarimide, exhibit no binding (Fig. 8c,d). Both carbonyl groups of glutarimide are important,and the absence of one carbonyl group results in loss of binding (δ-valerolactam in Fig. 8d). Bulky modificationof the glutarimide C4-carbon atom also results in loss of CRBN binding due to steric clashes (cycloheximide
in Fig. 8d). Upon ingestion, thalidomide undergoes enzymatic and nonenzymatic modification and breakdown, yielding a number of metabolites with a peak plasma concentration at 3–6 h2,39,40. The primary metabolic

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Figure 8. CRBN binding and E3 inhibition by thalidomide derivatives. (a) Competitive elution assay with
racemic thalidomide-immobilized beads. Glutarimide binds FLAG-CRBN with an affinity similar to that of(R)-thalidomide, but weaker than that of (S)-thalidomide. Phthalimide exhibits no binding. FLAG-CRBN wasprocessed as in Fig. 1b. (b) Effect of glutarimide binding on CRBN auto-ubiquitylation by CRBN-containing
E3 ubiquitin ligase CRL4. The method used was that described in Fig. 1c. (c) Competition binding assay using
thalidomide-immobilized beads. The eluate with 1 mM thalidomide and glutarimide yielded bands, but not withother compounds. The intact glutarimide ring in a relaxed six-membered ring conformation is important for
CRBN binding with the imide group serving as both a hydrogen donor and acceptor. Correspondingly, glutarimide
still binds CRBN, although phthalimide and glutaric anhydride, which lack the ring amide of glutarimide,
exhibited no binding. Both carbonyl groups of glutarimide are important, and the absence of one carbonyl groupresults in loss of binding (δ-valerolactam). Bulky modification of the glutarimide C4-carbon atom also results inloss of CRBN binding due to steric clash (cycloheximide). (d) Chemical structures of compounds utilized in thebinding assay. Full-length blots in (a), (b) and (c) are presented in Supplementary Fig. 10.
derivatives generated by cytochrome P450 isozymes are derived from 4- or 5-hydroxylation of the phthalimido
moiety or 5-hydroxylation of the glutarimide moiety, which subsequently undergo spontaneous hydrolysis12,41(Supplementary Fig. 7). Based on our structural data, metabolites with phthalimido modifications may still bind
CRBN, whereas 4- or 5-hydroxylation of the glutarimide moiety is likely to result in loss of binding due to stericclashes. Hydrolysis yields three primary products (I-III in Supplementary Fig. 7) by ring opening followed byfurther degradation into eight minor products42–47. We suggest classifying the hydrolysis processes into threepathways A, B and C. Compounds in pathway A retain the intact glutarimide ring and likely still bind CRBN,
while compounds in pathways B and C with opened glutarimide rings do not bind CRBN.
In conclusion, our study provides a structural framework for further investigations on the mechanisms of thepharmaceutical and teratogenic actions of this drug and for the development of more effective IMiDs.

Methods

Protein expression and purification. Mouse CRBN TBD (residues 322–430) was cloned into pGEX6P-3(GE Healthcare) and transformed into Escherichia coli strain BL21(DE3) Star (Invitrogen). The E. coli cells were
cultured at 25 °C in TB-5052 medium48 containing 50 μM zinc acetate. The supernatant of the cell lysate was applied
to a Glutathione Sepharose 4B resin (GE Healthcare) and resin-bound GST-fusion protein was cleaved from theresin using 4 units/ml HRV3C protease (Merck). Eluted protein fractions were further purified by cation exchange(HiTrap SP HP, GE Healthcare) and gel-filtration (Superdex 75 pg, GE Healthcare) chromatography. Purified CRBN
protein sample was dialyzed against a buffer solution containing 0.1 mM thalidomide (SIGMA), 5 mM sodium acetate (pH 6.0) and 10 mM 2-mercaptoethanol (changed three times). Tris-(2-carboxyethyl)-phosphine (TCEP) was
added to the protein sample solution up to 10 mM. Purified proteins were analyzed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and matrix-assisted laser desorption/ionization time-of-flight mass
spectrometry (MALDI-TOF MS; Bruker Daltonics). MALDI-TOF MS of mouse and human TBD domains gave
peaks at 12321.2 Da (calculated 12321.1 Da) and 12,456.4 Da (12,477.1 Da), respectively.

Crystallization. Crystallization conditions were searched for using the sitting-drop vapour diffusion methodand Hydra II-Plus-One crystallization robot (Matrix Technology) with a commercial crystallization solution kit.
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The best crystals of the (S)-, (R)-, and racemic thalidomide-bound form of mouse TBD were obtained from a
1:1 mixture solution comprising 30 mg/ml of the protein solution (in 5 mM sodium acetate (pH 6.0), 10 mM
2-mercaptoethanol, 10 mM TCEP and 0.1 mM thalidomide) and reservoir solution (100 mM sodium acetate (pH5.0) and 400–600 mM ammonium sulphate). Crystals appeared within 10 days at 4 °C. The 50–100 μm chunky
crystals were transferred into reservoir solution containing 30% glycerol and then flash-cooled in liquid nitrogen.

Data collection. Diffraction tests of the crystals were performed using a Rigaku R-AXIS VII detector equipped with a Rigaku FR-E X-ray generator. For structure determination, diffraction data of native and
SeMet-labelled crystals were collected at 100 K with the MX225HE detector on BL41XU and BL44XU beamlines
at the SPring-8 synchrotron facility (Table 1). Diffraction data were processed using the HKL2000 program49.
Crystals of the three thalidomide-bound forms belong to space group R3(H), with VM values in the range 2.54–
266 Å3/Da, suggesting a solvent content of 51–53% assuming 16 proteins in the asymmetric unit.
Structure determination and refinement. The crystal structures of the thalidomide-bound forms were
determined by molecular replacement using the model structure of mouse CRBN TBD (PDB code: 3WX2)28 asa search model. The built model was refined through alternating cycles using the Coot50 and CNS programs51 or
REFMAC52. For model building and refinements, composite omit maps were calculated with CNS programs51 andligand parameters were generated using PRODRG253. The secondary structures of models were calculated using
DSSP54. The refinement statistics are summarized in Table 2. These crystals contain 16 molecules in the asymmetric unit. Of these, 15 molecules form 5 trimers similar to those of the free form but with non-crystallographic
three-fold axes, while one molecule forms a trimer with symmetry-related molecules related by a crystallographic
three-fold axis. No significant structural deviations were found among these molecules with small averaged rms
deviations (0.38–0.40 Å). No outliers were flagged in the Ramachandran plots using MolProbity55.
Structural comparison.

Proteins and ligands were superposed using the program LSQKAB56. Coordinate
files of the free form of mouse CRBN TBD28 (PDB code: 3WX2) and the free form of thalidomide33 (CCDC
identifier: 1270378) were obtained from the Protein Data Bank (PDB) and the Cambridge Structural Database(CSD), respectively.

Antibodies.

Anti-mouse monoclonal CRBN antibody was generated in house (epitope 1–18). Aside from
this, primary antibodies against FLAG (M2. Sigma), HA (3F10, Roche), IKZF3 (ab139408, Abcam), and GAPDH(3H12, MBL) were used.

Thalidomide and related compounds.

Racemic thalidomide was purchased from Tocris Biosciences.
(S)- and (R)-thalidomides and their deuterated (D-thalidomide) enantiomers were synthesized as previously described30,57. Enantiomeric purities were monitored by HPLC (DAICEL Chiralpak IA, 4.6 × 250 mm,
MeOH = 100%, flow rate 1.0 ml/min, λ = 254 nm) and are summarized in Supplementary Table 1. Thalidomide
and D-thalidomide enantiomers were dissolved in dimethylsulphoxide (DMSO) at room temperature to generate
a 100 mM stock solution. Similarly, glutarimide, phthalimide, glutaric anhydride, δ-valerolactam and cycloheximide (Sigma Aldrich) were each dissolved in DMSO.

In vivo binding assay using thalidomide-immobilized beads. Binding assays were performed
essentially as previously described25. Human CRBN proteins were overexpressed in 293 T cells by transfecting
FLAG and haemagglutinin (HA) epitope–tagged (FH)- human CRBN constructs using Lipofectamine 2000
(Invitrogen). 293 T cells were maintained in DMEM (Nacalai Tesque) supplemented with 10% FBS. Cell extracts
were incubated with thalidomide-immobilized beads, and bound material was eluted with buffer containing
thalidomide, thalidomide-related compounds, or SDS. Lysates (input) and bead-affinity-purified (AP) materials
were immunoblotted (IB). Recombinant FLAG-CRBN proteins were expressed in Sf9 cells using the Bac-to-Bac
system (Invitrogen) and immunopurified using anti-FLAG antibody. Subsequent binding assays were performed
as described above.
In vitro ubiquitylation assay. Auto-ubiquitylation of FH-CRBN was detected in the presence of the protease inhibitor MG132 as previously described25. Cells stably expressing FH-CRBN were treated for 3 hours prior
to harvesting with the proteasome inhibitor MG132 (10 μM) or left untreated. Lysates were incubated with M2
anti-FLAG agarose beads. FH-CRBN was eluted with SDS and then subjected to SDS-PAGE and immunoblotting
using anti-HA antibody (3F10, Roche). When indicated, (S)- or (R)-thalidomide was added to cells 1 hour prior
to MG132 addition.
Thalidomide treatment of zebrafish.

Fish were kept at 28.5 °C on a 14-h light/10-h dark cycle, andembryos were obtained by natural matings of adult fish as previously described25. Zebrafish embryos were dechorionated prior to thalidomide treatment as follows: At 2 hpf, embryos were incubated in E3 medium containing
2 mg/ml Protease type XIV (Sigma) for 3 min at room temperature and then washed five times with E3 medium.
Following dechorionation, embryos were immediately transferred to E3 medium containing thalidomide andfurther incubated for 24 to 73 h at 28.5 °C. The E3 medium was replaced with medium containing freshly prepared thalidomide every 12 h. Zebrafish (Danio rerio) were maintained in accordance with the Animal Research
Guidelines at Tokyo Institute of Technology and Tokyo Medical University. The experimental protocol was
reviewed and approved by the Animal Research Committee and the method was carried out in accordance withthe committee’s approved guidelines.

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IKZF3 degradation assay. MM1S cells were treated with DMSO or deuterated-thalidomide enantiomers
for 12 h.
Whole-cell extracts were prepared using RIPA buffer (50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 0.5% DOC,
0.1% SDS and 1% NP-40) and subjected to immunoblot analysis. MM1S cells were maintained in RPMI1640(Invitrogen) supplemented with 10% FBS.
Co-immunoprecipitation assay. 293 T cells stably expressing FLAG-HA-CRBN were transfected with
HA-Aiolos expression vectors. After 48 hours, cells were collected and lysed using 0.5% NP-40 lysis buffer containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl and 0.5% NP-40. Extracts were incubated with M2 FLAG
magnetic beads (Sigma) in the presence of (S)-D-thalidomide or (R)-D-thalidomide and incubated for 2 h.
Following extensive washing three times with 0.5% NP-40 lysis buffer, bound proteins were eluted with free
3x FLAG-peptides (Sigma) and then subjected to immunoblot analysis.
Binding assay using calorimetry.

Binding studies utilizing isothermal titration calorimetry (ITC) wereconducted using a calorimeter (MicroCal iTC200, GE Healthcare) at 20 °C. Purified proteins were dialyzed overnight in buffer containing 10 mM sodium phosphate (pH 6.8), 150 mM NaCl and 0.5 mM tris(2-carboxyethyl)
phosphine (TCEP). Given the poor solubility of the drugs, reverse titration was employed whereby CRBN TBD
solution (1.2 mM) was injected (1.5 μl each, 5 min pause) into each drug solution (150 μM). Details of each titration are given in the figure legends of Supplementary Figs 2 and 5. Data fitting was performed using ORIGINTM
software supplied with the instrument.

Accession code.

Protein Data Bank: The atomic coordinates and structure factors for the reported crystal
structures are deposited under accession codes 5YJ0 (the (S)-thalidomide-bound form of CRBN TBD), 5YJ1 (the(R)-thalidomide-bound form) and 5YIZ (the (S)-thalidomide-bound form prepared with racemic thalidomide).

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Acknowledgements

We thank R. Kurata and Y. Tawara for technical support in performing the MALDI TOF mass spectroscopy
analysis. The synchrotron radiation experiments were performed at the BL32XU, BL41XU and BL44XU ofSPring-8 with the approval of the Japan Synchrotron Radiation Research Institute (JASRI); Proposal No.
2012A(1058, 6738), 2012B(1205, 6738), 2013A6844, 2013B(1288, 6844), 2014A(1283, 6944), 2014B(6944, 1533),
2015A(1086, 6549), 2015B(2086, 6549), 2016A(2510, 2519, 6648), 2016B(2510, 2519, 6648), 2017A(2502, 6759)
and 2017B6759. We also thank Dr I. Ooki for help with the data collection at SPring-8. This work was supported
by a Grant-in-Aid for Scientific Research on Innovative Area “Structural Cell Biology” from the Ministry of
Education, Culture, Sports, Science and Technology (MEXT) of Japan and in part by a research grant from the
Uehara Memorial Foundation, Japan (to T.H.), and also supported by Grant-in-Aid for Scientific Research on
Innovative Areas “Chemical Biology of Natural Products” from the Ministry of Education, Culture, Sports,
Science and Technology (MEXT) (23102002 to H.H.), by PRESTO, JST (JPMJPR1531 to T.I.) and by a Grantin-Aid for Scientific Research (S) (17H06112 to H.H.) and (B) (17H04213 to T.I.) and Young Scientists (B)(26750374 to T.I.) from MEXT.

Author Contributions

T.H. and H.H. conceived the project. H.H. provided all cDNAs. T.H. designed the project and was responsible
for construct design for protein preparation. T.M. executed protein biochemistry, C.D. measurements, A.U.C.
experiments, crystallization and data collection, and solved and refined the structures, directed by T.H. T.I. and
S.S. synthesized thalidomide-immobilized beads. T.I. performed binding studies and ubiquitylation assays. T.I.,
S.L., and H.A. performed zebrafish experiments. N.S. and E.T. synthesized and analyzed deuterated thalidomides.
T.M., T.I., Y.Y., H.H. and T.H. interpreted the data and T.H., T.I., Y.Y. and H.H. wrote the manuscript.

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Additional Information

Supplementary information accompanies this paper at https://doi.org/10.1038/s41598-018-19202-7.
Competing Interests: The authors declare that they have no competing interests.
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The NRF2‑PGC‑1β pathway activateskynurenine aminotransferase 4 via attenuationof an E3 ubiquitin ligase, synoviolin, in a cecal
ligation/perforation‑induced septic mouse model
YUSUKE ISHIDA1*, HIDETOSHI FUJITA2,3*, SATOKO ARATANI2,3, MIYUKI CHIJIIWA1,NOBORU TANIGUCHI4, MAHO YOKOTA2, YUKIHIKO OGIHARA1, NAOMI UOSHIMA1,FUMIAKI NAGASHIMA1, HIROYUKI UCHINO1 and TOSHIHIRO NAKAJIMA2,3,5-10
1Department of Anesthesiology, Tokyo Medical University Hospital, Tokyo 160‑0023; 2Institute of Medical Science,3
Department of Future Medical Science, Tokyo Medical University, Tokyo 160‑8402; 4Department of Medicine of
Sensory and Motor Organs, Division of Orthopedic Surgery, Faculty of Medicine, University of Miyazaki, Kiyotake,Miyazaki 889‑1692; 5Integrated Gene Editing Section (iGES); 6Medical Research Center; 7Industry‑University Cooperation(BioMimetics Sympathies Inc.), Tokyo Medical University Hospital, Tokyo 160‑0023; 8Institute of Medical Science,
St. Marianna University School of Medicine, Kawasaki, Kanagawa 216‑8511; 9Department of Biomedical Engineering,Osaka Institute of Technology, Osaka 535‑8585; 10Bayside Misato Medical Center, Kochi 781‑0112, Japan
Received June 15, 2017; Accepted March 15, 2018
DOI: 10.3892/mmr.2018.9175
Abstract. Sepsis-associated encephalopathy (SAE) is asystemic inflammatory response syndrome of which the precise
associated mechanisms remain unclear. Synoviolin (Syvn1) isan E3 ubiquitin ligase involved in conditions associated withchronic inflammation, including rheumatoid arthritis, obesity,
fibrosis and liver cirrhosis. However, the role of Syvn1 in acuteinflammation is not clear. The aim of the present study was toinvestigate the role of Syvn1 in a septic mouse model inducedby cecal ligation/perforation (CLP). Metabolome analysis
revealed that kynurenine (KYN), a key factor for the development of neuroinflammation, was increased in CLP‑induced
septic mice. Notably, KYN was not detected in CLP‑induced

Correspondence to: Professor Toshihiro Nakajima, Instituteof Medical Science, Tokyo Medical University, 6‑1‑1 Shinjuku,
Shinjuku, Tokyo 160‑8402, JapanE‑mail: marlin@tokyo‑med.ac.jp*

Contributed equally

Abbreviations: Syvn1, synoviolin; SAE, sepsis‑associatedencephalopathy; CLP, cecal ligation/perforation; KYN, kynurenine;
KYNA, kynurenic acid; KATs, kynurenine aminotransferases;
PGC-1β, Peroxisome proliferator‑activated receptor coactivator 1β;
QA, quinolinic acid; 3‑HAA, 3‑hydroxykynurenine; SPF, specificpathogen‑free
Key words: synoviolin, NRF2, PGC‑1β, kynurenine, septic mousemodel, kynurenine aminotransferase 4, E3 ligase

septic Syvn1‑deficient mice. KYN is converted to kynurenic
acid (KYNA) by kynurenine aminotransferases (KATs),
which has a neuroprotective effect. The expression of KAT4
was significantly increased in Syvn1‑deficient mice comparedto that in wild‑type mice. Promoter analysis demonstrated
that Syvn1 knockdown induced the KAT4 promoter activity,
as assessed by luciferase reporter activity, whereas Syvn1
overexpression repressed this activity in a dose‑dependent
manner. Furthermore, the KAT4 promoter was significantly
activated by the transcriptional factors, NF‑E2‑related factor 2
and peroxisome proliferator‑activated receptor coactivator 1β,which are targets of Syvn1‑induced degradation. In conclusion,
the results of the current study demonstrates that the repression of Syvn1 expression induces the conversion of neurotoxic
KYN to neuroprotective KYNA in a CLP‑induced mousemodel of sepsis, and that Syvn1 is a potential novel target for
the treatment of SAE.
Introduction
Sepsis is a form of systemic inflammation caused by aninfection, and multiple organ failure is known to occur when
this condition intensifies. Clinical studies demonstrated thatthe central nervous system might be one of the first organs
affected by sepsis. Sepsis‑associated encephalopathy (SAE),
characterized by diffuse cerebral dysfunction, is secondary tosepsis and is related to increased morbidity and mortality (1,2).
Clinical symptoms of SAE are delirium, fluctuating changes
in mental status, lack of attention, and disorganized thinking.
Encephalopathy of variable severity occurs in 9‑71% of septic
patients, and those with central nervous disorders have theworst prognosis in terms of cognitive and motor function.

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ISHIDA et al: ROLE OF Syvn1 IN SEPTIC MOUSE MODEL

In addition, it has been reported that the mortality rate of
septic patients with SAE is approximately twice that of septic
patients without SAE; moreover, the mortality rate increases
to 63% when patients with SAE present with a Glasgow Coma
Scale (GCS) value of 3‑8 (1,2). Although several mechanisms
including inflammation or the disturbance of neurotransmission disturbance, have been proposed, the precise mechanisms
responsible for sepsis‑induced cognitive impairment have notbeen fully elucidated.
The kynurenine (KYN) pathway is well known to be amajor mechanism of tryptophan catabolism, and it is activatedduring neuroinflammation during several neurodegenerative
diseases (3,4) such as SAE (5). Tryptophan is converted
to KYN, and KYN is converted into three intermediates,quinolinic acid (QA), 3‑hydroxykynurenine (3‑HAA), andkynurenic acid (KYNA) via two different pathways (Fig. 1).
3‑HAA and QA are neurotoxic, whereas KYNA is neuroprotective. The relative balance between the two branchesof this pathway might play an important role in the development of neuroinflammation. The conversion to KYNA iscatalyzed by kynurenine aminotransferases (KATs), whichhave been detected in the brain and peripheral tissues suchas the skeletal muscle (6,7). Recent studies showed that theperoxisome proliferator‑activated receptor (PPAR)‑PPAR
coactivator‑1 (PGC‑1) pathway induces skeletal muscle
KAT expression during exercise (7,8). The analysis of
PGC-1α1 skeletal muscle‑specific transgenic mice showedthat increased expression of skeletal muscle KATs induced
KYN metabolism. Synthesis of KYNA was enhanced andthe accumulation of KYN was reduced, thereby protecting
against stress‑induced depression (7).
Among several hundreds of E3 ubiquitin ligases, synoviolin (Syvn1), identified from the cDNA of rheumatoid synovial
cells, is the only known regulator of PGC‑1β ubiquitination (9). We recently demonstrated that Syvn1 interacts with
PGC-1β and induces its degradation (9). Global eliminationof Syvn1 in post‑neonatal mice is associated with weight loss
and reduced white adipose tissue through enhanced energy
expenditure, which is mediated by the function of PGC‑1β (9).
PGC-1β and PGC-1α share extensive sequence identity toeach other (10,11), and the primary structure of PGC‑1β has
several unique features of primary structure such as LXXLL
in its middle portion and the absence of a proline‑rich region
at the C‑terminus. Knockout studies have also suggested
functional differences between PGC‑1α and PGC-1β, suchas lethality (12). We previously demonstrated that Syvn1 is
involved in the development of rheumatoid arthritis, fibrosis,
limb girdle muscular dystrophy, and liver cirrhosis (13-17).
We also described another unique function of Syvn1; specifically, Syvn1 entraps and degrades tumor suppressor p53 andNRF2 (17,18). Nrf2 is a transcriptional factor that activates
antioxidant and cytoprotective genes that share a common
a cis‑acting enhancer sequence, termed the antioxidant
response element (ARE), under conditions of oxidative stress.
Several studies indicate that Nrf2 is neuroprotective against
neurotoxicity (19-21). These results suggest that loss of Syvn1
expression might have neuroprotective effects.
In the present study, we therefore investigated whether
Syvn1‑deficient mice were resistant to cecal ligation/perforation (CLP) treatment, which is a mouse model of sepsis, andrevealed that Syvn1 ablation mediates the activation of tryptophan metabolism via KAT4 gene expression.
Materials and methods
Mice. All procedures involving animals were performedin accordance with institutional and national guidelines
for animal experimentation, and were approved by theInstitutional Animal Care and Use Committee of Tokyo
Medical University (#S‑24021). Mice were kept in specificpathogen free (SPF) under standard conditions (20‑26˚Ctemperature; 40‑65% humidity) with a 12‑h light/12‑h darkcycle. F‑1 Foods (5.1% fat, 21.3% protein) were purchasedfrom Funabashi farm (Chiba, Japan). All mice used in the
study were of the C57BL/6J background. Heterozygous Syvn1
mice and tamoxifen (Tam)‑inducible Syvn1 knockout mice
were previously described (9,13).
Sepsis model. Male C57/BL6 mice at 7‑9 weeks of age wereanesthetized with 4‑5% sevoflurane and a middle abdominal
incision was made along the ventral surface of the abdomen
to expose the cecum. Before perforation, feces were gently
relocated towards the distal cecum. The cecum was ligatedat 5 mm from the distal end and then punctured once with a
21‑gauge needle, allowing the exposure of feces (cecal ligationand puncture; CLP). A small droplet of feces was then gently
squeezed through both sides of the puncture. The cecum wasreturned to the peritoneal cavity with careful attention to ensurethat feces did not contaminate the margins of the abdominaland skin wound. The muscle and skin incision were closedwith 3‑0 black silk. This model represents a high‑grade sepsis
with 100% mortality in 72 h (22). Mice received a subcutaneous injection of pre‑warmed saline solution (37˚C; 5 ml per
100 g body weight). Mice receiving sham surgery underwent
the same procedure except that the cecum was neither ligated
nor punctured (sham group; S). At each experimental time
point after performing the procedure, mice were euthanizedby cervical dislocation, and brain and skeletal muscle tissue
samples were obtained.
Measurement of metabolites. Brain tissues were obtained
at 18 h after the CLP procedure (n=2 for each group).
Approximately 20 mg of frozen brain tissues were immersed
into 1,500 µl of 50% acetonitrile/Milli‑Q water containing
internal standards (H3304‑1002; Human Metabolome
Technologies, Inc., Tsuruoka, Japan) at 0˚C in order to inactivate enzymes. The tissue was homogenized three times at
1,500 rpm for 120s using a tissue homogenizer (BMS‑M10N21;
BMS Co., Ltd., Tokyo, Japan) and then the homogenate wascentrifuged at 2,300 x g and 4˚C for 5 min. Subsequently,
800 µl of the upper aqueous layer was centrifugally filteredthrough a Millipore 5‑kDa cut‑off filter at 9,100 x g and 4˚C
for 120 min to remove proteins. The filtrate was centrifugallyconcentrated and resuspended in 50 µl of Milli‑Q water for
CE‑MS analysis. Metabolome measurements were performed
by a facility service at Human Metabolome Technology Inc.,
Tsuruoka, Japan.
Reverse transcription‑quantitative polymerase chain reaction(RT‑qPCR). Total RNA from the skeletal muscle and brain-87-

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2469

Eagle's medium as previously described (9). Transient transfection was performed with Lipofectamine 2000 according
to the manufacturer's protocol (Invitrogen; Thermo Fisher
Scientific, Inc.). Cells were lysed with cell lysis buffer(Promega Corporation, Madison, WI, USA) 24 h after
transfection, and luciferase activity was measured. To ensure
equal amounts of DNA, empty plasmids were added to eachtransfection.

Figure 1. A schematic diagram of the kynurenine pathway.

of the mice was purified by using ISOGEN (Nippon Gene,
Tokyo, Japan) according to the manufacturer's instructions
and reverse transcribed by using ReverTra Ace with random
primers (Toyobo, Osaka, Japan). qPCR was performed byusing LightCycler 480 Probes Master (Roche Diagnostics,
Mannheim, Germany) and the Step One Plus Detection System(Applied Biosystems, Life Technologies, Tokyo, Japan). The
Relative standard curve method (23) was used in this study andexpression levels were determined relative to that of 18s rRNA.
Primers and probes used in this study are shown in Table I.
Plasmids, siRNA and antibodies. PGV-B2 (PicaGene Basic
vector 2; Toyo Ink, Tokyo, Japan) vector and pcDNA3(Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA,
USA) vector were purchased. pcDNA3 hemagglutinin
antigen (HA) was constructed by inserting the HA sequence
into pcDNA3 (Invitrogen; Thermo Fisher Scientific, Inc.).
The coding sequence of human full‑length NRF2 was
PCR‑amplified (primers, 5'‑CAG TGT GCT GGA ATTATGATG GAC TTG GAG CTG CC‑3' and 5'‑GATATC TGC AGA
ATTGTTTTTCTTAACATCTGGCTTCTTAC‑3') from HeLa
cDNA and full‑length NRF2 was inserted into the pcDNA3
HA plasmid (Invitrogen; Thermo Fisher Scientific, Inc.) for
transient transfection assays. The promoter of the KAT4 gene
was PCR‑amplified (primers, 5'‑AAAGCTAGCAAGCTTCATACTGTA AGC‑3' and 5'‑AAACTCGAGAGAGCCGAGATCTGGGGAAG‑3') from the genome of C57BL/6J mice and thefragment (‑2553 to +30) was subcloned into PGV‑B2 (PicaGene
Basic vector 2; Toyo Ink). Plasmid sequences were confirmedby sequencing. SYVN1 plasmids and siRNA against Syvn1
were previously described (9,13). The following antibodies wereused: Anti‑tubulin (Sigma‑Aldrich; Merck KGaA, Darmstadt,
Germany), and anti‑HA (3F10; Roche Diagnostics, Indianapolis,
IN, USA), anti‑KAT1 and KAT4 (Abcam, Cambridge, UK),anti‑KAT2 and KAT3 (Santa Cruz Biotechnology, Inc., Dallas,
Texas, USA). The anti‑Syvn1 rabbit polyclonal antibody thatwas used was previously reported (18).
Cell culture and transient transfection. A total of 293 cells
and C2C12 cells were cultured in Dulbecco's modified

Luciferase assay. The assay was performed as previously described (24-26). Briefly, 293 cells were transiently
transfected with 50 ng KAT4‑luc reporter plasmid, 0.1 ng
pRL‑CMV, and 10, 50, 100 ng pcDNA3 HA‑NRF2. For
Syvn1 knockdown, 50 ng KAT4‑luc reporter plasmid, 0.1 ng
pRL‑CMV, and 10, 20 nM Syvn1 siRNA were transfected.
For SYVN1 overexpression, 50 ng KAT4‑luc reporter plasmid,
0.1 ng pRL‑CMV, and 10, 50, or 100 ng SYVN1 expression
vector were transfected. 293 cells were transiently transfectedwith 50 ng KAT4‑luc reporter plasmid, 0.1 ng pRL‑CMV, and50 ng pcDNA3 HA‑NRF2 and/or 25 ng pcDNA3 HA PGC‑1β.
293 cells were transiently transfected with 50 ng KAT4‑luc
reporter plasmid, 0.1 ng pRL‑CMV, and 50 ng pcDNA3
HA‑NRF2 and/or 25 ng pcDNA3 HA PGC‑1α. After 24 h,cells were lysed with cell lysis buffer, which was followedby measurement of luciferase activity. Each experiment was
performed at least three times.
RNA interference assay. siRNAs for Syvn1 were previously described (9). Transfection with siRNAs (20 µM) was
performed by using Lipofectamine 2000 (Invitrogen; Thermo
Fisher Scientific, Inc.) according to the manufacturer's
protocol. Total RNA from C2C12 cells was purified 3 d after
transfection using ISOGEN (Nippon Gene) according to the
manufacturer's instructions, and reverse transcribed using
ReverTra Ace with random primers (Toyobo).
Statistical analysis. All data are expressed as themeans ± standard deviation. Differences between two groups
were examined by using the Student's t‑test and P<0.05 wasconsidered to indicate a statistically significant difference.
One‑way analysis of variance with Tukey‑Kramer post
hoc analysis was used to determine correlations in datasets
containing multiple groups in luciferase assays. All results
were derived from at least three independent experiments.
Results
Metabolome analysis of Syvn1‑deficient septic mice. To
investigate the role of Syvn1 in sepsis, we performed themetabolome analysis using brain tissue of WT and heterozygous
Syvn1‑deficient mice because the homozygous mice die in utero.
We first examined the expression of Syvn1 in the brain. Western
blotting showed that the expression of Syvn1 was decreased in
the heterozygous mice (Fig. 2). As shown in Table II, KYN was
not detected in sham mice [WT (sham) and heterozygous Syvn1
mice (sham)] and was increased in CLP‑induced septic mice at
18 h [WT (CLP)]. Interestingly, the quantity of KYN decreasedin CLP‑induced septic Syvn1‑deficient mice [heterozygous
Syvn1 mice (CLP)]. This result suggests that decreased expression of Syvn1 might induce KYN metabolism.

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Table I. Primer sequences and length of specific polymerase chain reaction products.
Gene

Directiona

Primer sequence

Probeb

Syvn1

F
R
F
R
F
R
F
R
F
R

5'‑CTGGGTATCCTGGACTTCCTC‑3'
5'‑AAGCACCATGGTCATCAGAA‑3'
5'‑CAGAGCAGCGCTATTGTTTG‑3'
5'‑GCAGACAGTCTAGGCCAGAAA‑3'5'‑TTACACGTGTGCGACTCCTT‑3'5'‑GCTTGATATCGATCCAAAACG‑3'
5'‑ATGGCTGCTGCCTTTCAC‑3'5'‑GATCTGGAGGTCCCATTTCA‑3'
5'‑GCAATTATTCCCCATGAACG‑3'5'‑GGGACTTAATCAACGCAAGC‑3'

89

KAT1
KAT3
KAT4
18sRNA

81
27
17
48

Direction of primer sequences; bprobe number of Universal ProbeLibrary probes (Roche). F, forward; R, reverse; Syvn1, synoviolin; KAT,
kynurenine aminotransferase.
a

KAT1 and KAT3 was similar between WT mice and KO mice.
KAT2 was not detected in the skeletal muscle tissue. Western
blotting using the skeletal muscle extracts revealed that theexpression of Syvn1 was very low and that the expression of
KAT4 was clearly increased in the skeletal muscle tissue ofKO mice (Fig. 3D). These results suggest that KAT4 could be
an important gene and Syvn1 might regulate the expressionof KAT4.

Figure 2. Measurement of kynurenine in Syvn1‑deficient mice. Western blot
analysis of brain tissue of WT and heterozygous Syvn1‑deficient mice using
an anti‑Syvn1 and anti‑tubulin antibodies. Syvn1, synoviolin; WT, wild‑type.

Expression of KAT genes in Syvn1‑KO mice. The conversion
of KYN to KYNA is catalyzed by KATs, which have been
detected in the brain and skeletal muscle. Therefore, we examined the expression level of the KAT1‑4 genes in the brain andthe skeletal muscle tissue of tamoxifen (Tam)‑inducible Syvn1
knockout (KO) mice (the post‑neonatal knockout mice) (9).
Real‑time PCR assay showed that the expression of Syvn1 wasvery low in the brain tissue of KO mice and that the expressionof KAT3 was significantly increased in KO mice (Fig. 3A).
To examine the protein level, we performed western blotting
using brain extracts from KO mice and WT mice. As shownin Fig. 3B, the expression of Syvn1 was very low. However, theexpression level of KAT1‑4 was similar between WT mice andKO mice. We next investigated the mRNA levels by real‑time
PCR assay. As shown in Fig. 3C, the expression of Syvn1
was very low in the skeletal muscle tissue of KO mice. The
expression of KAT4 was significantly increased in KO micecompared to that in WT mice (P<0.05), and the expression of

Syvn1 represses KAT4 expression. To examine the role ofSyvn1 in KAT4 expression, we performed luciferase assay
using the KAT4 promoter (‑2553/+30) reporter constructs.
We used 293 cells, which have high transfection efficiency,
because we have used 293 cells as model cells for a long timeto analyze transcriptional regulation (9,25,27). As shown in
Fig. 4A, treatment with a siRNA against Syvn1 (siSyvn1)
induced the luciferase reporter activity, 1.2‑fold (10 µM) and
1.4‑fold (20 µM), compared to that with control siRNA (siControl). Overexpression of Syvn1 repressed the reporter activity
in a dose‑dependent manner (Fig. 4B). To further examine
whether Syvn1 regulates the KAT4 expression, we performed
knock‑down assays in C2C12 cells. C2C12 cells were treatedwith control siRNA (siControl) or siRNA for Syvn1 (siSyvn1)
for 3 d and total RNA was purified. Then, we performed
RT‑PCR and real‑time PCR assays to measure the expressionof KAT1‑4. With siSyvn1 treatment, the expression of Syvn1
decreased to 40% that of control levels. The expression of
KAT4 was significantly increased in cells treated with siSyvn1
compared to that in cells treated with control siRNA (siControl). In addition, the expression of KAT3 and KAT4 was notdifferent between siSyvn1 and siControl conditions (Fig. 4C).
NRF2 and PGC‑1β activate the KAT4 promoter. We previously demonstrated that Syvn1 regulates the transcriptional
factor, NRF2, and the transcriptional coactivator, PGC‑1β,which activates NRF2‑ and PPAR‑mediated transcription (12).
Therefore, we analyzed the transcriptional factor bindingsites of the above factors in the KAT4 promoter (‑2553/+30)
using TF BIND (http://tfbind.hgc.jp/). As shown in Fig. 5A,
one PPAR responsive element (PRE) and 17 NRF2 binding-89-

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Table II. Metabolome analysis.

Treatment

Case--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Heterozygous
Heterozygous
WT (sham)Syvn1 (sham)WT (CLP)
Syvn1 (CLP)----------------------------------------------------------------------------------------------------------------------------------------------1
2
1
2
1
2
1
2

Kynurenine (nmol/g)

ND

ND

ND

ND

1.1x10-04

3.1x10-04

ND

ND

ND, not detected; WT, wild‑type; CLP, cecal ligation/perforation.

Figure 3. Expression of KAT genes in Syvn1‑knockout (KO) mice. mRNA and protein expression of KATs in (A and B) brain and (C and D) skeletal muscle
extracts. Total RNA from (A) brain and (C) skeletal muscle was isolated from Syvn1 WT (n=8) and Syvn1‑KO mice (n=6) and subjected to qPCR. Individual
measurements were standardized using 18S RNA, and then the average for Syvn1 WT mice was set to 100. Data were analyzed by performing a Student's t‑testand expressed as mean ± SD. The experiment was performed in triplicate. (B) Brain extracts and (D) skeletal muscle extracts were prepared from WT miceand Syvn1‑KO mice. Tissue extracts were separated by SDS‑PAGE, and processed for western blotting with anti‑KAT1, 2, 3, 4, Syvn1 and tubulin antibodies.
KAT, kynurenine aminotransferase; Syvn1, synoviolin; WT, wild‑type.

sites were detected in the KAT4 promoter. To examine the roleof NRF2 and PGC‑1β, we performed luciferase assays using
the KAT4 promoter (‑2553/+30). NRF2 significantly activatedthe luciferase reporter activity in a dose‑dependent manner(Fig. 5B), whereas PPAR‑α did not induce the reporter activity(data not shown). As shown in Fig. 5C, NRF2 and PGC‑1βsynergistically activated the reporter activity. PGC‑1α also
induced reporter activity, and NRF2 and PGC‑1α additively
activated the reporter activity (Fig. 5D). In addition, NRF2
rescued the repressive effect induced by overexpression of
Syvn1 in a dose‑dependent manner (Fig. 5E). These results

suggest that the NRF2‑PGC‑1β pathway induces the expression of KAT4.
Discussion
In the present study, we investigated the role of Syvn1 in amouse model of sepsis. We showed that the level of KYN waselevated in the brain tissue of septic WT mice. However, KYN
was not detected in septic Syvn1‑deficient mice. In addition,
expression of KAT4, which encodes one of the enzymes thatconverts KYN into KYNA, was elevated in the skeletal muscle

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ISHIDA et al: ROLE OF Syvn1 IN SEPTIC MOUSE MODEL

Figure 4. Syvn1 represses KAT4 expression based on KAT4 promoter‑driven luciferase reporter assays. (A) Effect of Syvn1 knockdown by siRNA. 293 cells
were transiently transfected with a reporter plasmid containing the KAT4 promoter, pRL‑CMV, control siRNA (siControl) or siRNA for Syvn1 (siSyvn1). Data
were analyzed by a Student's t‑test and expressed as mean ± SD. Western blotting was performed with anti‑Syvn1 and anti‑tubulin antibodies. (B) Effect ofSyvn1 overexpression. 293 cells were transiently transfected with a reporter plasmid containing the KAT4 promoter, pRL‑CMV, and the HA‑tagged Syvn1(Syvn1‑HA)‑expression vector (10, 50, and 100 ng). Analysis of variance with Tukey‑Kramer post hoc analysis and expressed as mean ± SD. Western blotting
was performed with anti‑Syvn1 and anti‑tubulin antibodies. (C) Effect of Syvn1 on KAT4 expression. C2C12 cells were transiently transfected with siControl
or siSyvn1. After 3 days, total RNA were purified and qPCR was performed. Individual measurements were standardized using 18S RNA, and the average forsiControl was set to 100. Data were analyzed by performing a Student's t‑test and expressed as mean ± SD. The experiment was performed three times. KAT,
kynurenine aminotransferase; Syvn1, synoviolin; HA, hemagglutinin antigen.

tissue of Syvn1‑KO mice. Moreover, Syvn1 knockdown
induced KAT4 promoter‑driven reporter activity, whereas
overexpression of Syvn1 repressed this effect. The KAT4
promoter was also activated by the NRF2‑PGC‑1β pathway.
NRF2 and PGC‑1β are target proteins of Syvn1‑induced
degradation (9,17). Taken together, these results suggest thatSyvn1 deficiency might induce KYN metabolism via the
NRF2‑PGC‑1β‑KAT4 pathway.
KATs are critical enzymes that catalyze the conversion of KYN to KYNA. Recent studies indicates thatexercise induces the expression of KATs expression in theskeletal muscle (7,8,28). Analysis of skeletal muscle‑specific
PGC-1α‑transgenic mice revealed that an increased expression of KATs in skeletal muscle shifts the KYN metabolism
towards enhanced synthesis of KYNA, resulting in a decrease
in KYN levels, and thereby protecting the tissue fromstress‑induced damage. This novel function of PGC‑1α in theregulation of the KYN metabolism suggests communication
between the skeletal muscle and brain. In the present study,

we showed that KAT4 expression was significantly increasedin the skeletal muscle tissue of Syvn1‑deficient mice and that
the NRF2‑PGC‑1β pathway activates the KAT4 promoter.
We previously demonstrated that Syvn1 interacts with NRF2
and PGC-1β, and induces the degradation of these factors
through ubiquitination. Therefore, in Syvn1‑deficient mice,
accumulated NRF2 and PGC‑1β could activate KAT4 expression in the skeletal muscle to enhance the synthesis of KYNA,
resulting in decreased KYN in the brain tissue.
Syvn1 is involved in both acute and chronic inflammation. We previously demonstrated that Syvn1 overexpression
in transgenic mice led to advanced arthropathy through the
suppression of apoptosis in synoviocytes and that heterozygous
Syvn1‑mice were resistant to arthritis and fibrosis (13,15). In
addition, we recently showed that Syvn1 is involved in thedevelopment of obesity, limb girdle muscular dystrophy, andliver cirrhosis. These studies indicate that Syvn1 is a key factor
for diseases associated with chronic inflammation. In thepresent study, we examined the role of Syvn1 in a CLP‑induced-91-

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2473

Figure 5. The NRF2/PGC‑1β pathway activates the KAT4 promoter. (A) Schematic representation of the KAT4 promoter (‑2553/+30). PRE: PPAR responsive
element, black box: NRF2 binding site (‑2123/‑2114, ‑2029/‑2020, ‑1981/‑1972, ‑1957/‑1948, ‑1742/‑1733, ‑1732/‑1723, ‑1459/‑1450, ‑1386/‑1377, ‑1378/‑1369,‑1308/‑1299, ‑1106/‑1097, ‑759/‑750, ‑359/‑350, ‑250/‑241, ‑105/‑96, ‑46/‑37, and +2/+11). (B) Effect of NRF2 overexpression on KAT4 promoter‑driven luciferase reporter activity. 293 cells were transiently transfected with a reporter plasmid containing the KAT4 promoter, pRL‑CMV, and the HA‑NRF2‑expression
vector (10, 50, and 100 ng). Western blotting was performed with an anti‑HA and anti‑tubulin antibodies. (C) Effect of NRF2 and PGC‑1β overexpression
on the KAT4 promoter‑driven luciferase reporter activity. 293 cells were transiently transfected with a reporter plasmid containing the KAT4 promoter,pRL‑CMV, 50 ng of the HA‑NRF2‑expression vector, and 25 ng of the HA‑PGC‑1β ‑expression vector. Western blotting was performed with an anti‑HA
and anti‑tubulin antibodies. (D) Effect of NRF2 and PGC‑1α overexpression on KAT4 promoter‑driven luciferase reporter activity. A total of 293 cells
were transiently transfected with a reporter plasmid containing the KAT4 promoter, pRL‑CMV, 50 ng of the HA‑NRF2‑expression vector, and 25 ng of the
HA‑PGC‑1α‑expression vector. Western blotting was performed with an anti‑HA and anti‑tubulin antibodies. (E) NRF2 overexpression abrogates the effect ofSyvn1 overexpression on KAT4 promoter‑driven luciferase reporter activity. 293 cells were transiently transfected with a reporter plasmid containing the KAT4
promoter, pRL‑CMV, 100 ng of the Syvn1‑HA‑expression vector, and the HA‑NRF2‑expression vector (10, 50, and 100 ng). Western blotting was performed
with an anti‑HA and anti‑tubulin antibodies. (B‑D) Data were analyzed by performing a Tukey‑Kramer post hoc analysis and expressed as mean ± SD(*P<0.05, **P<0.01). The experiment was performed three times. KAT, kynurenine aminotransferase; Syvn1, synoviolin; HA, hemagglutinin antigen; PGC,
proliferator‑activated receptor (PPAR)‑PPAR coactivator.

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septic mouse model, which is an example of acute inflammation. Proinflammatory cytokines such as tumor necrosis factor(TNF)‑α and interleukin (IL)‑1β are induced in the mousesepsis model and septic patients (5,29,30). Previous studies
demonstrated that Syvn1 is a key target for inflammatory cytokines such as TNF‑α, IL-1, and IL-17 (5,31,32), and that theexpression of Syvn1 is transcriptionally regulated by Ets transcription factors, GABPα, GABPβ, and ILF‑3 (33,34), which
are the downstream of inflammatory cytokines signaling.
These results prompted us to speculate that Syvn1 expression
might be induced by inflammatory cytokines during sepsis
and induce the degradation of NRF2 and PGC‑1β in the skeletal muscle tissue. Further studies are warranted to unveil thedetailed role of Syvn1 in sepsis.
KYN is a key immune mediator. During inflammation,
increases in cytokines such as interferon (IFN)‑ γ, TNF‑ α,
and IL-1β activate indoleamine 2,3‑dioxygenase (IDO), andtryptophan is metabolized into the toxic metabolite KYN via
IDO. Several studies indicate that the development of mood
symptoms with inflammation is associated with reduced
circulating tryptophan levels and concomitant increases inserum levels of KYN (35,36). In addition, recent studies showthat KYN and proinflammatory cytokines such as TNF‑α andIL-1β are induced in mouse sepsis models and human septic
patients (5,29,30). Therefore, the KYN pathway is one of targets
for the treatment of SAE. The IDO inhibitor, 1‑methyl‑D, wasshown to attenuate neuroinflammation through the repressionof proinflammatory cytokines and KYN, and protect against
sepsis‑induced cognitive impairment in a mouse model (5).
In the present study, we showed that the induction of KYN
by CLP is not observed in Syvn1‑deficient mice. We previously developed a Syvn1 inhibitor, LS‑102, and demonstrated
that the Syvn1 inhibitor attenuated arthritis, fibrosis, obesity,
limb girdle muscular dystrophy, and liver cirrhosis (13-17).
Although further studies are needed to investigate the effectof this Syvn1 inhibitor on SAE, Syvn1 might represent a novel
target for the treatment of SAE.
Taken together, in the present study, we provide evidence
that Syvn1 regulates the NRF2‑PGC‑1β ‑KAT4 pathway ina mouse model of sepsis. Further analysis of Syvn1 will be
helpful to understand its physiological and clinical significance.

part by funds provided through a MEXT‑Supported programof the Strategic Research Foundation at Private Universities(grant no. S1411011, 2014‑2018) from the Ministry of Education,
Culture, Sports, Science and Technology of Japan. This
study was also supported in part by the Japan Society for
the Promotion of Science KAKENHI (grant nos. 23659176,
26670479, 26461478 and 16H05157) and Industry‑university
cooperation (BioMimetics Sympathies Inc.).
Availability of data and materials
All data generated or analyzed during this study are includedin this published article.
Authors' contributions
YI, HF, HU and TN conceived the project and designed theexperiments. YI, MC, NT, YO, NU, FN and HU performed thesepsis model and analyzed the metabolome data. YI, HF, SA,
MY and TN performed experiments and analyzed data. YI,
HF and TN wrote the manuscript. YI, HF, SA, MC, NT, MY,
YO, NU, FN, HU and TN discussed the results and commented
on the manuscript.
Ethics approval and consent to participate
All procedures involving animals were performed in accordance with institutional and national guidelines for animal
experimentation, and were approved by the Institutional
Animal Care and Use Committee of Tokyo Medical University(approval no. S‑28044).
Consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
References

Acknowledgements
The authors thank Mr. Shyouichiro Shibata (Institute ofMedical Science, Tokyo Medical University) for technicalassistance. We also thank all members of Professor Toshihiro
Nakajima's laboratory (Institute of Medical Science, Tokyo
Medical University).
Funding
This study was funded in part by grants from the Naito Foundation,
Natural Science Scholarship Daiichi‑Sankyo Foundation ofLife Science, Mitsubishi, Tanabe Pharma Corporation, Bureauof Social Welfare and Public Health, Academic contribution of
Pfizer, Eisai, Santen Pharmaceutical, Abbvie, Astellas, Takeda
Science Foundation, AstraZeneca (R&D Grant 2013), and ONO
Medical Research Foundation. This study was supported in-93-

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Identification of the inhibitory activity of
walnut extract on the E3 ligase Syvn1
HIDETOSHI FUJITA1,2, SATOKO ARATANI1,2, NAOKO YAGISHITA3,KUSUKI NISHIOKA1 and TOSHIHIRO NAKAJIMA1‑71Institute of Medical Science; 2Department of Future Medical Science, Tokyo Medical University, Tokyo 160‑8402;
3
Institute of Medical Science, St. Marianna University School of Medicine, Kawasaki, Kanagawa 216‑8511;
4
Integrated Gene Editing Section (iGES); 5Medical Research Center, Tokyo Medical University Hospital,
Tokyo 160‑0023; 6Department of Biomedical Engineering, Osaka Institute of Technology,
Osaka 535‑8585; 7Bayside Misato Medical Center, Kochi 781‑0112, Japan
Received March 7, 2018; Accepted September 13, 2018
DOI: 10.3892/mmr.2018.9576

Abstract. Synoviolin (Syvn1), an E3 ubiquitin ligase in endo‑
plasmic reticulum‑associated protein degradation, is involvedin rheumatoid arthritis, fibrosis, liver cirrhosis and obesity.
We previously demonstrated that Syvn1 negatively regulates
the function of peroxisome proliferator‑activated receptor
gamma coactivator‑1β (PGC‑1β). In addition, treatment witha Syvn1 inhibitor suppressed weight gain in a mouse modelof obesity by activating PGC‑1β via Syvn1 inhibition. It has
been suggested that the Syvn1 inhibitors may have therapeutic
benefits in obese patients. The present study tested the inhibi‑
tory activity of walnut extract, a natural product, on Syvn1
activity. Walnut extract inhibited the effect of Syvn1 on thecell proliferation of rheumatoid synovial cells and repressed
the interaction between PGC‑1β and Syvn1 in an in vitro
binding assay. Polyubiquitination of PGC‑1β by Syvn1 was
suppressed by walnut extract in a concentration‑dependent
manner, but walnut extract did not have an inhibitory effect on
the autoubiquitination of Syvn1. Treatment with walnut extract
in mouse embryonic fibroblasts increased the number of mito‑
chondria, suggesting that exposure to the extract recovered
PGC‑1β function. These results demonstrated that constituentsof walnut extract may serve as lead compounds in drug devel‑
opment efforts aiming to produce drugs to treat patients withobesity and obesity‑associated metabolic diseases.

Correspondence to: Professor Toshihiro Nakajima, Instituteof Medical Science, Tokyo Medical University, 6‑1‑1 Shinjuku,
Shinjuku‑ku, Tokyo 160‑8402, JapanE‑mail: marlin@tokyo‑med.ac.jp
Abbreviations: Syvn1, synoviolin; PGC‑1β, peroxisome
proliferator‑activated receptor coactivator 1β; RSCs, rheumatoid
synovial cells; SPF, specific pathogen‑free
Key words: synoviolin, walnut extract, PGC‑1β, ubiquitination, E3

ligaseIntroduction
Obesity is a global health problem associated with various
metabolic disorders, including diabetes, hypertension, cardio‑
vascular disease, and depression (1). There are two major typesof medication used to treat obese patients (2). Some anti‑obesity
drugs, such as appetite suppressants, reduce food intake byregulating the function of the central nervous system, whereas
other drugs block absorption of lipids from food in the intes‑
tine. In addition, some candidate anti‑obesity drugs directly
modulate energy metabolism without affecting the central
nervous system, such as peroxisome proliferator‑activatedreceptor (PPAR) agonists. However, the undesirable side
effects of currently available agonists significantly limit theiruse.
Natural products have been used for millennia to treat
diseases and mitigate the adverse effects of toxic substances (3).
Recently, berberine and curcumin, which have antioxidant
and anti‑inflammatory properties, have been reported to haveanti‑obesity effects (4‑6). Berberine and curcumin ameliorate
obesity by increasing energy expenditure. Berberine activates
AMP‑activated protein kinase (AMPK) (4), a key energy
sensor that leads to reduced energy storage and increased
energy production. In addition, berberine regulates expressionof uncoupling protein 1 (UCP1), which is found in the mito‑
chondria and generates heat in brown adipose tissue (BAT)
and white adipose tissue (WAT) in an AMPK‑ and PPAR
gamma coactivator‑1 alpha (PGC‑1α)‑dependent manner (6).
These studies suggest that therapeutic chemicals are involvedin natural products with antioxidant and anti‑inflammatory
properties.
Synoviolin (Syvn1), a mam malian homolog of
Hrd1p/Der3p, is involved in the development of obesity, rheu‑
matoid arthritis, fibrosis, limb girdle muscular dystrophy, andliver cirrhosis (7‑11). Syvn1 was identified in rheumatoid syno‑vial cells (RSCs) as an endoplasmic reticulum (ER)‑resident
E3 ubiquitin ligase (7) that plays an important role in RSC
proliferation (12). LS‑102, a Syvn1 inhibitor, repressed prolif‑
eration of RSCs in a Syvn1‑dependent manner (13). We recently
demonstrated that global elimination of Syvn1 in post‑neonatal

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FUJITA et al: ROLE OF WALNUT EXTRACT IN Syvn1 ACTIVITY

mice was associated with weight loss and reduced white
adipose tissue (14). Adipose tissue from Syvn1 knockout mice
showed significant up‑regulation of PGC‑1β‑target genes, aswell as a significant increase in the number of mitochondria,
mitochondrial respiration, and basal energy expenditure.
Syvn1 interacts with PGC‑1β and negatively regulates its
function. Therefore, we propose that knockout or inhibitionof Syvn1 leads to stabilization of PGC‑1β, enhancing energy
expenditure. These results suggest that Syvn1 is a therapeutictarget for anti‑obesity drugs. However, natural products that
inhibit Syvn1 activity have not been found.
To identify Syvn1 inhibitors with antioxidant andanti‑inflammatory properties in this study, we performed ascreening of natural products based on their inhibitory effects
on RSC proliferation. We found that walnut extract inhibited
Syvn1 activity, indicating that walnut extract could be used totreat patients with obesity.
Materials and methods
Ethical considerations. All human experimental protocols in
the present study (no. 2728, 2729, 3758, 3759) were approvedby the Ethics Review Committee of Tokyo Medical University(Tokyo, Japan). RA patients received stable doses of metho‑
trexate (6‑10 mg/week) before joint replacement surgery.
Written informed consent was obtained from all patientsprior to the collection of joint tissue samples. All procedures
involving animals were performed in accordance with insti‑
tutional and national guidelines for animal experimentation,
and were approved by the Institutional Animal Care and
Use Committee of Tokyo Medical University (no. S‑28038,
S‑28040).
Mice. Mice were kept in SPF under conditions (20‑26˚Ctemperature; 40‑65% humidity) on a 12 h light/12 h dark cycle.
F‑1 Foods (5.1% fat, 21.3% protein) were purchased from
Funabashi farm (Chiba, Japan). Mice had free access to waterbottles. Tamoxifen (Tam)‑inducible Syvn1 knockout mice
(CAG‑Cre‑ER; Syvn1flox/flox) was generated previously (14). To
isolate MEFs, embryos were isolated at E13.5, and the head andinternal (including reproductive) organs were removed. The
remaining tissue was physically dissociated and incubated intrypsin at 37˚C for 15 min. Cells were resuspended in DMEM
and plate the cells in 10 cm tissue culture dishes. On the nextday, medium was changed and cells were expanded for two
passages before freezing.
Plasmids, antibodies, and walnut extract. The Syvn1(NM_032431) and PGC‑1β (NM_133249) plasmids aredescribed in the literature (7,14,15). The following antibodies
were used: Anti‑HA (3F10) (Roche Molecular Biochemicals,Indianapolis, IN, USA). Polyclonal antiserum against GST
was generated by immunizing rats with purified GST.
Anti‑PGC‑1β antibody has been described previously (14).
Walnut extracts were prepared from the branches of walnut
trees by the standard ethanol extraction method (16). Briefly,
the air‑dried walnut branches were milled into fine powder inthe blender and the fibrous powder of walnut branches was
extracted twice, on each occasion with 60% ethyl alcohol atroom temperature for 24 h. The combined ethanol extract was

filtered, and the filtrate was concentrated to dryness under
reduced pressure in a rotary evaporator. The ethanol extract
was freeze‑dried. Without any further purification, the plant
crude ethanol extract was used in our study. Aliquot portionsof the ethanol extract was dissolved in DMSO for use of ourexperiments.
Cell culture and assessment of cell proliferation. Rheumatoid
synovial cells were obtained by standard methods (17).
Briefly, the tissue was minced into small pieces and digested
with collagenase (Sigma‑Aldrich; Merck KGaA, Darmstadt,
Germany). The single‑cell suspension was incubated over‑night, and then floating cells were removed, and adherent cells
were cultured in dishes. RSCs and MEFs, which were derived
from Tam‑inducible Syvn1 knockout mice (CAG‑Cre‑ER;
Syvn1flox/flox), were cultured in Dulbecco's modified Eagle's
medium (DMEM) as previously described (14). RSC prolifera‑tion was measured with DMSO or walnut extract treatment (1,3.3, 10, 33.3 µg/ml) for 3 days using the Cell Counting Kit‑8(Dojindo, Tokyo, Japan). MEFs were treated with DMSO or
Tamoxifen (2.5 µM) for 2 days, and were then treated withDMSO or walnut extract (50 µg/ml) for 3 days. Electron
microscopic analysis was then performed.
GST pull‑down assay. The GST pull‑down assay was performed
as previously described (14,18). Briefly, GST‑Syvn1ΔTM and
MBP‑PGC‑1β (1‑367) were expressed and purified using
glutathione sepharose beads and amylose beads, respec‑tively (GE Healthcare Life Sciences, Little Chalfont, UK).
GST‑Syvn1ΔTM was incubated with MBP‑PGC‑1β bound toresin in 1 ml buffer A (20 mM Tris‑HCl, pH 8.0; 100 mM
NaCl; 1 mM ethylenediaminetetraacetic acid (EDTA);
1 mM dithiothreitol (DTT); 0.1% Nonidet P‑40 (NP‑40); 5%
glycerol; 1 mM Na3VO4; 5 mM NaF; 1 µg/ml aprotinin; and1 µg/ml leupeptin) for 4 h at 4˚C. After washing the beads withbuffer A, bound proteins were fractionated by sodium dodecyl
sulfate‑polyacrylamide gel electrophoresis (SDS‑PAGE)
followed by western blotting.
In vitro ubiquitination assay. In vitro ubiquitination assays
were performed as previously described (14). Briefly,
GST‑PGC‑1β (1‑367) was incubated with 0.75 µg HA‑Ub,
125 ng E1 (Biomol International, Plymouth Meeting, PA,
USA), 150 ng UbcH5c, and 150 ng MBP‑Syvn1ΔTM in reac‑tion buffer (50 mM Tris‑HCl, pH 7.5; 5 mM MgCl2; 0.6 mM
DTT; and 2 mM ATP) at 37˚C for 2 h. Glutathione sepharose
was added to the solution, after which the mixture was washedwith GST wash buffer (50 mM Tris‑HCl, pH 7.5; 0.5 M NaCl;
1% Triton X‑100; 1 mM EDTA; 1 mM DTT; and proteaseinhibitors). Ubiquitinated PGC‑1β was analyzed by western
blotting using anti‑PGC‑1β antibodies.
Ubiquitination assay. In vivo ubiquitination assays were
performed as previously described (14). Briefly, 293T cells weretransfected with HA‑PGC‑1β, FLAG Ub, or Syvn1 expression
plasmids. Cells were treated with DMSO or walnut extract(50 µg/ml) for 3 days. Cells were lysed in lysis buffer (50 mM
HEPES, pH 7.9; 150 mM KCl; 1 mM phenylmethanesulfonyl
fluoride, 1% Triton X‑100; 10% glycerol; and protease inhibitors).
Lysates were mixed with 1 µg anti‑HA antibody conjugated to-101-

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Table I. Primers and probes for reverse transcription‑quantitative polymerase chain reaction.
Gene

Type

Primer (5'‑3')

Probe no.

SYVN1

Forward
ReverseForward
ReverseForward
ReverseForward
ReverseForward
Reverseccagtacctcaccgtgctg
tctgagctagggatgctggt
gcaattattccccatgaacg
gggacttaatcaacgcaagc
tcttgctggaaatgatcaaca
gggctctgtcacacagtaagc
tgagagaggtcctatcaaaacca
cctttatcccagtcaccagaa
ctaaggccaaccgtgaaaag
accagaggcatacagggaca

16

18sRNA
MCAD
Atp5b
ACTB

48
88
15
64

SYVN1, synoviolin; MCAD, medium chain acyl‑coenzyme A dehydrogenase; Atp5b, mitochondrial ATP synthase β subunit; ACTB, β‑actin.

Figure 1. Effect of walnut extract on RSC growth. (A) Effect of Syvn1 knockdown by siRNA. RSCs derived from two patients with RA were transiently
transfected with control siRNA (siControl) or siRNA for Syvn1 (siSyvn1). Following 2 days, total RNA were purified and reverse transcription‑quantitative
polymerase chain reaction was performed. Individual measurements were standardized using 18S RNA, and the average for siControl was set to 100. (B) RSCs
were transiently transfected with siControl or siSyvn1. Following 2 days, RSCs were treated with walnut extract (1, 3.3, and 10 µg/ml) for 3 days. Data arepresented as the mean ± standard deviation (n=3). *P<0.05 and **P<0.01, as indicated. n.s., not significant; DMSO, dimethyl sulfoxide; RSC, rheumatoid
synovial cell; RA, rheumatoid arthritis; si‑/siRNA, small interfering RNA; Syvn1, Synoviolin.

protein G‑sepharose beads. After a 4‑h incubation at 4˚C, beads
were washed three times with lysis buffer. Bound proteins werefractionated by SDS‑PAGE and analyzed by immunoblotting.

MitoTracker staining. For analysis of mitochondria using
MitoTracker Red (Molecular Probes, Eugene, OR, USA), MEFs
were treated with DMSO or walnut extract (50 µg/ml) for 3 days.

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FUJITA et al: ROLE OF WALNUT EXTRACT IN Syvn1 ACTIVITY

primers (Toyobo, Osaka, Japan). RT‑qPCR was performedby using LightCycler 480 Probes Master (Roche Diagnostics,
Mannheim, Germany) and the Step One Plus Detection System(Applied Biosystems; Life Technologies Japan, Tokyo, Japan).
The thermocycling conditions were as follows: Initial denatur‑
ation at 95˚C for 10 min, followed by 45 cycles of denaturation
at 95˚C for 10 sec, annealing at 60˚C for 20 sec and extension
at 72˚C for 1 sec. Expression levels were determined relative tothat of 18sRNA (RSCs) or ACTB (MEFs). Primers and probes
used in the present study are shown in Table I. Relative expres‑sion was determined using the 2‑∆∆Cq method (19).
RNA interference assay. siRNAs for Syvn1 were previously
described (14). Transfection with siRNAs (20 µM) was
performed by using Lipofectamine 2000 (Invitrogen; Thermo
Fisher Scientific, Inc., Waltham, MA, USA) according to the
manufacturer's protocol. Total RNA from RSCs was purified
2 days after transfection using ISOGEN (Nippon Gene, Tokyo,
Japan) according to the manufacturer's instructions, andreverse transcribed using ReverTra Ace with random primers(Toyobo).
Statistical analysis. All data are expressed as themean ± standard deviation and were analyzed using Excel
Statistics 2012 version 1.00 (Social Survey ResearchInformation Co., Ltd., Tokyo, Japan). Differences between two
groups were examined by Student's t‑test. One‑way analysis
of variance with Tukey‑Kramer post hoc analysis was used todetermine correlations in datasets containing multiple groups.
P<0.05 was considered to indicate a statistically significant
difference.
Results
Figure 2. Effect of walnut extract on the interactions between SYVN1and PGC‑1β. (A) An in vitro binding assay was performed with GST or
GST‑SYVN1ΔTM and MBP or MBP‑PGC1β (amino acids 1‑367). (B) An
in vitro binding assay was performed using MBP‑PGC‑1β (amino acids
1‑367) and GST‑SYVN1ΔTM in the presence of DMSO or walnut extract (0.5
or 5 µg/ml). WB was performed using anti‑GST antibodies or anti‑PGC1βantibodies. Syvn1, Synoviolin; PGC‑1β, PGC‑1β, peroxisome prolifer‑ator‑activated receptor coactivator 1β; GST, glutathione S‑transferase; TM,
transmembrane domain; MBP, maltose binding protein; DMSO, dimethyl
sulfoxide; WB, western blotting.

Mitochondria were stained with the MitoTracker Red probe for
30 min at 37˚C according to the manufacturer's protocol. Nuclei
were stained with 4',6‑diamidino‑2‑phenylindole (DAPI). The
intensity of staining by mitotracker was measured (n=8).
Reverse transcription‑quantitative polymerase chain reaction(RT‑qPCR). Tam‑inducible Syvn1 knockout MEFs weretreated with DMSO (WT MEFs) or Tam (Syvn1 knockout
MEFs) for 2 days, and then WT MEFs and Syvn1 knockout
MEFs were treated with DMSO or walnut extract (50 µg/ml)
for 3 days. Total RNA from MEFs treated with DMSO or
walnut extract was purified by using ISOGEN (Nippon Gene,
Tokyo, Japan) according to the manufacturer's instructions
and reverse transcribed by using ReverTra Ace with random

Screening of natural products for Syvn1 inhibitors. Syvn1 is a
crucial factor involved in RSC proliferation (7,13,20). To iden‑
tify Syvn1 inhibitors in natural products, we tested the effects
of natural products on RSC proliferation with or without
Syvn1. At first, we performed knockdown experiments with
control siRNA (siControl) or siRNA for Syvn1 (siSyvn1).
RT‑qPCR showed that siSyvn1 induced 60% repression of
Syvn1 expression (Fig. 1A). Walnut extract inhibited prolifera‑tion in a concentration‑dependent manner in two RSC lines
treated with siControl (Fig. 1B). Whereas, the inhibitory effect
was attenuated in siSyvn1‑treated cells (Fig. 1B). In the caseof patient 1, walnut extract did not significantly have any effect(n.s.). In the case of patient 2, walnut extract had still inhibited
cell growth, however, the strength of the effect was reduced ascompared to control siRNA‑treated cells.
Regulation of Syvn1‑PGC‑1β interaction by walnut extract.
Syvn1 negatively regulates PGC‑1β activity via direct inter‑action with PGC‑1β in vitro and in vivo (14). To determine
whether walnut extract inhibits the interaction of Syvn1
with PGC‑1β, we performed in vitro binding assays using
glutathione S‑transferase‑tagged Syvn1 lacking the trans‑
membrane domain (GST‑Syvn1ΔTM) and maltose binding
protein‑tagged PGC‑1β (amino acids 1‑367) (MBP‑PGC‑1β(1‑367) ). As previously reported (14), MBP‑PGC‑1β (1‑367)
directly bound to GST‑Syvn1ΔTM (Fig. 2A). MBP‑PGC‑1β-103-

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Figure 3. Effect of walnut extract on the polyubiquitination of PGC‑1β. (A) In vitro ubiquitination assays were performed with MBP‑SYVN1ΔTM, GST‑PGC‑1β(1‑367), E1 and E2 enzymes, and HA‑Ub in the presence of DMSO or walnut extract (0.5, 5 or 50 µg/ml). WB was performed using anti‑PGC‑1β antibodies andanti‑SYVN1 antibodies. (B) In vivo ubiquitination assays were performed. 293T cells were transfected with HA PGC‑1β, FLAG Ub and/or SYVN1 expression
plasmids and cells were treated with DMSO and walnut extract (50 µg/ml) for 3 days. Whole cell extracts were immunoprecipitated with anti‑HA antibody.
WB was performed using anti‑FLAG and anti‑HA antibodies. Quantification of the data is presented. *P<0.05, as indicated. (C) Effect of walnut extract on
SYVN1 autoubiquitination. In vitro ubiquitination assays were performed with GST‑SYVN1ΔTM, E1 and E2 enzymes, and HA‑Ub in the presence of DMSO
or walnut extract (0.5, 5 or 50 µg/ml). WB was performed using anti‑SYVN1 antibodies. Quantification of the data is presented. (D) In vivo ubiquitination
assays were performed. 293T cells were transfected with FLAG Ub, and SYVN1/HA expression plasmids and cells were treated with DMSO and walnut
extract (50 µg/ml) for 3 days. Whole cell extracts were immunoprecipitated with anti‑HA antibody. WB was performed using anti‑FLAG and anti‑HA anti‑
bodies. Quantification of the data is presented. The positions of molecular weight standards (in kDa) are indicated to the left of each image. Data are expressedas the mean ± standard deviation (n=3). HA, hemagglutinin; Ub, ubiquitin; DMSO, dimethyl sulfoxide; WB, western blotting; Syvn1, Synoviolin; PGC‑1β,
PGC‑1β, peroxisome proliferator‑activated receptor coactivator 1β; ATP, mitochondrial adenosine triphosphate synthase; GST, glutathione S‑transferase; TM,
transmembrane domain; MBP, maltose binding protein; n.s., not significant.

(1‑367) did not bind to GST, and GST‑Syvn1ΔTM did not bindto MBP (Fig. 2A). Walnut extract inhibited the interactionof Syvn1 and PGC‑1β in a concentration‑dependent manner(Fig. 2B).
Inhibition of PGC‑1β ubiquitination by walnut extract.
Syvn1 ubiquitinates PGC‑1β in vitro and in vivo, negatively

regulating PGC‑1β abundance (14). To investigate ubiquitina‑
tion of PGC‑1β by Syvn1 in the presence of walnut extract,
we performed an in vitro assay of ubiquitination with
MBP‑Syvn1ΔTM and GST‑PGC‑1β (1‑367) in the presenceof ATP, hemagglutinin‑tagged ubiquitin (HA‑Ub), E1, and E2(UbcH5c) (14). As previously reported (14), Syvn1 induced
polyubiquitination of PGC‑1β in vitro, and polyubiquitination

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FUJITA et al: ROLE OF WALNUT EXTRACT IN Syvn1 ACTIVITY

Figure 4. Effect of walnut extract on the number of mitochondria. (A) MEFs were treated with DMSO or walnut extract (50 µg/ml) for 3 days. Cells were
immunostained with MitoTracker Red (red), and DAPI (nuclei, blue); scale bars, 20 µm. The intensity of staining was measured by MitoTracker. Data wereexpressed as the mean ± standard deviation (n=8). (B) Representative electron micrographs of walnut extract‑treated cells (50 µg/m) are presented. Increased
mitochondrial volume can be observed in the large cytoplasmic areas in MEFs (mitochondria are indicated by red asterisks). Magnification, x10,000. (C) The
number of mitochondria in the area (1,000x1,000 pixels) was measured. Data were expressed as the mean ± standard deviation (n=13). (D) Total RNA was
isolated from wild‑type MEFs and Syvn1 knockout MEFs treated with DMSO or walnut extract, and reverse transcription‑quantitative polymerase chain
reaction was performed. Individual measurements were standardized using β‑actin, and then the average DMSO value was set to 1. Data were expressed as
the mean ± standard deviation (n=6). **P<0.01, as indicated. MEFs, mouse embryonic fibroblasts; DMSO, dimethyl sulfoxide; Syvn1, Synoviolin; KO, Syvn1
knockout mice; MCAD, medium chain acyl‑coenzyme A dehydrogenase; ATP5b, mitochondrial adenosine triphosphate synthase β subunit.

of PGC‑1β was not observed in the absence of ATP or
Syvn1. Walnut extract inhibited PGC‑1β polyubiquitination(Fig. 3A). To examine the effect of walnut extract in vivo, we
performed in vivo ubiquitination assay. FLAG‑tagged Ub and
HA‑PGC‑1β were coexpressed with Syvn1 in HEK 293T cells
and cells were treated with DMSO or walnut extract (50 µg/ml)
for 3 days. The ubiquitination of PGC‑1β was observed in
Syvn1‑expressing cells (DMSO‑treated cells). The treatment
with walnut extract decreased the ubiquitination of PGC‑1β in
Syvn1‑expressing cells (Fig. 3B). Walnut extract did not inhibit
autoubiquitination of Syvn1 (Fig. 3C). The effect of walnut
extract was also examined in vivo. FLAG‑tagged Ub (FLAG

Ub) and Syvn1/HA were coexpressed in HEK 293T cells andcells were treated with DMSO or walnut extract (50 µg/ml)
for 3 days. Autoubiquitination of Syvn1 was not inhibited inwalnut extract‑treated cells (Fig. 3D).
Effects of walnut extract on PGC‑1β function. PGC‑1β is acoactivator of several transcription factors, including PPARα,and is implicated in various biological processes, including
mitochondrial biogenesis (21,22). LS‑102 exposure increases
the number of mitochondria in cultured cells (14). To investi‑
gate the effect of walnut extract on regulation of mitochondria
by PGC‑1β, we performed mitochondrial staining using-105-

MOLECULAR MEDICINE REPORTS

MitoTracker with mouse embryonic fibroblasts (MEFs) (14).
MitoTracker staining showed increased mitochondria in MEFs
treated with walnut extract compared with MEFs treated withDMSO (Fig. 4A). We used electron microscopy and counted
mitochondria. The cells treated with walnut extract had signif‑
icantly more mitochondria than the cells treated with dimethyl
sulfoxide (DMSO) did (Fig. 4B and C). In addition, the numberof mitochondria in Syvn1 knockout MEFs treated with DMSO
(KO+DMSO) increased compared to that in wildtype MEFs
treated with DMSO. However, walnut extract produced no
additional effect on the number of mitochondria in the Syvn1
KO MEFs (KO+Walnut extract). Furthermore, the expres‑sion of PGC‑1β target genes, medium chain acyl‑coenzyme
A dehydrogenase (MCAD) and mitochondrial ATP synthase
β subunit (ATP5b), was also induced in MEFs treated with
walnut extract and in Syvn1 KO MEFs treated with DMSO
(Fig. 4D). However, walnut extract produced no additional
effect on the induction of MCAD and ATP5b in the Syvn1 KO
MEFs (Fig. 4D).
Discussion
The development of Syvn1 inhibitors is an active field of study
because they have the potential to treat patients with several
diseases, including rheumatoid arthritis, fibrosis, liver cirrhosis,
and obesity (7‑10,13). In a previous study, we demonstrated that
the Syvn1 inhibitor, LS102, suppressed weight gain in a mousemodel of obesity via inhibition of PGC‑1β polyubiquitination
by Syvn1 (14). In this study, we showed that walnut extract, anatural product, inhibits Syvn1 activity. Walnut extract inhib‑
ited the interaction between Syvn1 and PGC‑1β and repressed
polyubiquitination of PGC‑1β by Syvn1. Taken together, theseresults suggest that walnut extract has anti‑obesity activity.
Selectivity and specificity are important characteristics fortargeted drugs. We identified LS‑102 as an inhibitor of autou‑
biquitination of Syvn1 via a high‑throughput screening (13) anddemonstrated its inhibitory effect on the E3 ligase activity of
Syvn1. LS‑102 suppressed polyubiquitination of target proteinsof Syvn1, including nuclear factor erythroid 2‑related factor 2(NRF2), V247M α‑sarcoglycan mutant, and PGC‑1β (10,11,13).
Interestingly, Syvn1 interacts with NRF2 and V247M
α‑sarcoglycan mutant through proline‑rich domains at the
C‑terminus, whereas Syvn1 binds to PGC‑1β via the Syvn1
unique (SyU) domain (10,11,13,14). Walnut extract did not havean inhibitory effect on autoubiquitination of Syvn1. However,
walnut extract decreased polyubiquitination of PGC‑1β byinhibiting the interaction of Syvn1 and PGC‑1β. Therefore,
walnut extract may specifically target the SyU domain of
Syvn1. These results indicate that walnut extract might
improve obesity by selectively inhibiting the interaction of
Syvn1 and PGC‑1β.
The mitochondrion is an important organelle involved incellular energy control that has been reported to be involvedin the process of obesity and chronic inflammation (23,24).
PGC‑1β plays an important role in mitochondrial biogen‑esis and energy metabolism, including β ‑oxidation of fatty
acids (25). Overexpression of PGC‑1β results in increased
numbers of mitochondria and increased mitochondrial respira‑tory function (26). PGC‑1β transgenic mice show high energy
expenditure and resistance to obesity (27). In addition, PGC‑1β7attenuated inflammation. PGC‑1β diminishes the increasein proinflammatory mediators, such as interleukin‑6 (IL‑6)
and macrophage inflammatory protein 1‑alpha (MIP1α), byrepressing the activity of nuclear factor‑κ B (NF‑κ B) (28). Thesestudies indicate that PGC‑1β has anti‑obesity and anti‑inflam‑
matory properties. In this study, we found that walnut extract
inhibited the negative regulation of PGC‑1β activity by Syvn1,
suggesting that walnut extract activates PGC‑1β. Our results
suggest that walnut extract may attenuate not only obesity, butalso diseases involving chronic inflammation. Further analysis
with disease state model will be need to determine whether
walnut extract will be helpful in several disease with chronic
inflammation. Future studies will be aimed at identifying thebioactive constituents in walnut extract that are responsible for
its inhibitory effect on Syvn1.
Acknowledgements
The authors thank Mr. S. Shibata (Tokyo Medical University,
Tokyo, Japan) for their technical assistance. The authors would
also like to thank all of the members of Dr. Nakajima's labora‑
tory and Dr. Khin Thuzar Wynn (Yangon Speciality Hospital,
Yangon, Myanmar).
Funding
The present study was funded in part by grants from the Naito
Foundation, Natural Science Scholarship Daiichi‑Sankyo
Foundation of Life Science, Mitsubishi, Tanabe Pharma
Corporation, Bureau of Social Welfare and Public Health,Academic Contribution of Pfizer, Eisai, Santen Pharmaceutical,
Abbvie, Takeda Science Foundation, AstraZeneca (R&D
Grant 2013) and ONO Medical Research Foundation. The
present study was also supported partly by funds provided
through a MEXT‑Supported Program of the Strategic Research
Foundation at Private Universities (grant no. S1411011;
2014‑2018) from the Ministry of Education, Culture, Sports,
Science and Technology of Japan, as well as the Japan Society
for the Promotion of Science KAKENHI (grant nos. 23659176,
26670479, 26461478 and 16H05157) and Industry‑University
Cooperation (BioMimetics Sympathies Inc.).
Availability of data and materials
All data analyzed in this study are included in this article.
Authors' contributions
HF, SA, KN, NY and TN conceived the project and designed
the experiments. HF, SA and TN performed the experiments
and analyzed the data. HF and TN wrote the manuscript. All
authors discussed the results and commented on the manuscript.
Ethics approval and consent to participate
All human experimental protocols in the present study(nos. 2728 and 2729, 3758, 3759) were approved by the Ethics
Review Committee of Tokyo Medical University (Tokyo,
Japan). Written informed consent was obtained from all of thepatients prior to the collection of joint tissue samples.

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FUJITA et al: ROLE OF WALNUT EXTRACT IN Syvn1 ACTIVITY

Patient consent for publication
Consent for publication was obtained from all of the patients.
Competing interests
The authors declare that they have no competing interests.
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CASE OF THE MONTH JULY 2017 — CASE 2

doi:10.1111/bpa.12575

A 31-YEAR-OLD MAN WITH SLOWLY PROGRESSIVE
LIMB MUSCLE WEAKNESS AND RESPIRATORYINSUFFICIENCY
Akinori Uruha, MD, PhD1,2; Yukiko K. Hayashi, MD, PhD1,2,3; Madoka Mori-Yoshimura, MD, PhD4;
Yasushi Oya, MD4; Masahiro Kanai, MD4; Miho Murata, MD, PhD4;
Ichizo Nishino, MD, PhD1,2
1 Department of Genome Medicine Development, Medical Genome Center, National Center of Neurology and Psychiatry (NCNP), Tokyo, Japan.
2 Department of Neuromuscular Research, National Institute of Neuroscience, NCNP, Tokyo, Japan.
3 Department of Pathophysiology, Tokyo Medical University, Tokyo, Japan.
4 Department of Neurology, National Center Hospital, NCNP, Tokyo, Japan.

CLINICAL HISTORY
A 31-year-old man, with no family history of neuromusculardiseases, received muscle biopsy for slowly progressive muscle
weakness. The ﬁrst symptom he had was difﬁculty dorsiﬂexing
his feet at age 20 years, which was followed by gradual development of gait disturbance. At age 27 years, he started using ahandrail to climb up stairs. At age 28 years, he developed dysphagia for liquid, in addition to difﬁculty raising arms, which
led him to have aspiration pneumonia later in the same year. At
age approximately 30 years, he developed dyspnea on exertion.
Arterial blood gas analysis revealed hypoxemia (65 mmHg, atroom air) and hypercapnia (82 mmHg), with a vital capacity
decreased to 780 mL, leading to the diagnosis of chronic type 2
respiratory failure, and non-invasive ventilation was started. At
age 31 years, he was unable to walk without aid and required awheelchair for long distances. Physical examination revealed
moderate muscle weakness and atrophy in an asymmetric limbgirdle distribution, together with marked muscle atrophy in thetibialis anterior muscles and weakness in ankle dorsiﬂexion
with Medical Research Council grade 1. Mild neck muscle
weakness was also noted. Serum creatine kinase level was 375
IU/L (normal: <287 IU/L). Electromyography showed myopathic changes. Skeletal muscle CT demonstrated remarkablefat tissue replacement in the semitendinosus muscles (Figure
1A, arrows) at the thigh level.

PATHOLOGY
Skeletal muscle pathology from the biceps brachii demonstrated myopathic changes with moderate ﬁber size variation,
mildly disorganized myoﬁbrillar networks, some ﬁbers withcytoplasmic bodies, which were often located in line in subsarcolemmal region of a muscle ﬁber (Figure 1B, modiﬁed
Gomori trichrome stain), and a few ﬁbers with rimmed
vacuoles. What is your diagnosis? What is the next test to
conﬁrm it?

Figure 1.

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Correspondence

DIAGNOSIS
Hereditary myopathy with early respiratory failure (HMERF).
The diagnosis was conﬁrmed by gene analysis showing a heterozygous titin gene (TTN) mutation g.284701T > C (p.Cys31712Arg)in the FN3 119 domain in the A-band region (ENST00000589042).

DISCUSSION
HMERF is a hereditary myopathy caused by mutations in the Aband region of TTN (1, 2). Since the causative gene was reported
in 2012, an increasing number of patients with HMERF have been
identiﬁed worldwide (5).
HMERF is clinically characterized by adult-onset muscle weakness with respiratory insufﬁciency from an early, typically ambulant stage of the disease. A common initial symptom is gait
disturbance because of distal (especially the anterior compartment)
and/or proximal lower limb muscle weakness. Weakness of ankle
dorsiﬂexion is prominent, leading to easy tripping and foot drop.
There is no or minimal, if any, weakness in the upper limbs at disease onset. Respiratory insufﬁciency subsequently appears after amedian of six years post-onset (range: 0 2 16 years) (5), althoughrespiratory symptoms can be the initial manifestation in 24%236%
of patients (1, 5). Most patients are ambulant when they need nocturnal ventilation (1). To date, clinically evident primary cardiomyopathy has not been reported in patients with HMERF, althoughright heart failure secondary to severe respiratory failure is occasionally observed. Nevertheless, a recent study found cardiac conduction abnormalities in 32% (7/22) of patients with thep.Cys31712Arg mutation of TTN and imaging evidence of otherwise unexplained asymptomatic cardiomyopathy in 18% (4/22) (3).
The autosomal dominant trait is common, although a few homozygous patients with severe symptoms have been reported. Somepatients have no apparent positive family history (5).
Skeletal muscle imaging demonstrates that the semitendinosus
muscles and the anterior compartment of the lower legs (preferentially, the peroneus longus muscles) are selectively involved in theearly stage (1, 2, 5). Indeed, the selective involvement of the semitendinosus muscles is often observed (93%2100%) (2, 5). The
pattern is a radiological clue for diagnosis of HMERF, although it
can be observed in other muscle diseases, including desminopathy,
aB-crystallinopathy, and anoctaminopathy (4).
The pathological hallmark of HMERF is necklace cytoplasmic
bodies; at cross section, a number of cytoplasmic bodies are often
circumferentially aligned in the subsarcolemmal region in muscle
ﬁbers, mimicking a necklace (5). The ﬁnding of necklace cytoplasmic bodies is highly speciﬁc for HMERF (sensitivity: 82%, speciﬁcity: 99%) (5). Rimmed vacuoles are also sometimes observed
but less speciﬁc.

Since TTN encodes the largest known protein consisting of
35,991 amino-acid polypeptides in 363 exons among those recognized so far, sequence analysis of all the exons requires considerable labor. Even if next generation sequencing is available for the
genetic analysis, it would still be difﬁcult to determine which variant is truly pathogenic because TTN is a highly polymorphic gene.
Despite this, all HMERF patients reported so far carry a mutationin exon 343 encoding the FN3 119 domain in the A-band region(ENST00000589042). Therefore, exon 343 should be consideredas the primary target for genetic diagnosis of HMERF.
When a patient shows the pathological ﬁnding of necklace cytoplasmic bodies, sequencing of exon 343 of TTN should be considered for deﬁnite diagnosis of HMERF, even though the patientdoes not exhibit respiratory problems at that time. If the patient has
a mutation in exon 343 of TTN, the patient will eventually develop
a severe respiratory insufﬁciency. Early diagnosis of the disease
would enable earlier recognition of asymptomatic respiratory insufﬁciency and appropriate intervention without delay (eg, nocturnal
ventilation).

ACKNOWLEDGMENTS
We thank Ms. Kaoru Tatezawa and Ms. Kanako Goto for theirtechnical assistance. This study was supported partly by JSPS
KAKENHI Grant Number of 24659437; partly by Research on
Rare and Intractable Diseases, Comprehensive Research on Disability Health and Welfare and Applying Health Technology from
the Ministry of Health, Labour and Welfare of Japan; and partly by
Intramural Research Grant 23-5 and 26-8 for Neurological and Psychiatric Disorders of NCNP.
REFERENCES
1. Ohlsson M, Hedberg C, Brådvik B, Lindberg C, Tajsharghi H,
Danielsson O et al (2012) Hereditary myopathy with early respiratory
failure associated with a mutation in A-band titin. Brain 135:1682–1694.
2. Pfeffer G, Elliott HR, Grifﬁn H, Barresi R, Miller J, Marsh J et al (2012)
Titin mutation segregates with hereditary myopathy with early
respiratory failure. Brain 135:1695–1713.
3. Steele HE, Harris E, Barresi R, Marsh J, Beattie A, Bourke JP et al(2016) Cardiac involvement in hereditary myopathy with early
respiratory failure: A cohort study. Neurology 87:1031–1035.
4. Tasca G, Evil€a A, Pane M, Monforte M, Graziano A, Hackman P et al(2014) Isolated semitendinosus involvement in the initial stages of limbgirdle muscular dystrophy 2L. Neuromuscul Disord 24:1118–1119.
5. Uruha A, Hayashi YK, Oya Y, Mori-Yoshimura M, Kanai M,
Murata M et al (2015) Necklace cytoplasmic bodies in hereditary
myopathy with early respiratory failure. J Neurol Neurosurg
Psychiatry 86:483–489.

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Journal of the Neurological Sciences 385 (2018) 49–56

Contents lists available at ScienceDirect

Journal of the Neurological Sciences
journal homepage: www.elsevier.com/locate/jns

Targeting the enhanced ER stress response in Marinesco-Sjögren syndrome
Ayako Kashimadaa, Setsuko Hasegawaa, Takeo Isagaib, Tsuyoshi Uchiyamac, Muneaki Matsuod,⁎
Motoharu Kawaie, Masahide Gotof, Tomohiro Morioa, Yukiko K. Hayashig, Masatoshi Takagia,aDepartment of Pediatrics, Developmental Biology, Tokyo Medical and Dental University, Tokyo, Japan
Department of Pediatrics, Omuta City Hospital, Fukuoka, Japanc
Department of Neurology, Seirei Hamamatsu General Hospital, Shizuoka, Japand
Department of Pediatrics, Saga University, Saga, Japane
Department of Neurology, Graduate School of Medicine, Yamaguchi University, Yamaguchi, Japanf
Department of Pediatrics, Jichi Medical University, Tochigi, Japang
Department of Pathophysiology, Tokyo Medical University, Tokyo, Japanb

A R T I C L E I N F O

A B S T R A C T

Keywords:
Marinesco-Sjögren syndrome
ER stress
Lymphoblastoid cell line
Tunicamycin
Apoptosis
Tauroursodeoxycholic acid

Background and objective: Marinesco-Sjögren syndrome (MSS) is an autosomal recessive infantile-onset disordercharacterized by cataracts, cerebellar ataxia, and progressive myopathy caused by mutation of SIL1. In mice, adefect in SIL1 causes endoplasmic reticulum (ER) chaperone dysfunction, leading to unfolded protein accumulation and increased ER stress. However, ER stress and the unfolded protein response (UPR) have not been
investigated in MSS patient-derived cells.
Methods: Lymphoblastoid cell lines (LCLs) were established from four MSS patients. Spontaneous and tunicamycin-induced ER stress and the UPR were investigated in MSS-LCLs. Expression of UPR markers was analyzed
by western blotting. ER stress-induced apoptosis was analyzed by ﬂow cytometry. The cytoprotective eﬀects of
ER stress modulators were also examined.
Results: MSS-LCLs exhibited increased spontaneous ER stress and were highly susceptible to ER stress-induced
apoptosis. The inositol-requiring protein 1α (IRE1α)-X-box-binding protein 1 (XBP1) pathway was mainly upregulated in MSS-LCLs. Tauroursodeoxycholic acid (TUDCA) attenuated ER stress-induced apoptosis.
Conclusion: MSS patient-derived cells exhibit increased ER stress, an activated UPR, and susceptibility to ER
stress-induced death. TUDCA reduces ER stress-induced death of MSS patient-derived cells. The potential of
TUDCA as a therapeutic agent for MSS could be explored further in preclinical studies.

1. Introduction
Marinesco-Sjögren syndrome (MSS; OMIM 248800) is a rare autosomal recessive infantile-onset multisystem disorder characterized bybilateral cataracts, cerebellar ataxia, intellectual disability, and progressive muscle weakness due to myopathy [1]. Intellectual disability ishighly variable in MSS, and there are also few patients with normal
cognition. Other clinical features include short stature, hypergonadotropic hypogonadism [2], and strabismus [3]. Homozygous or compound heterozygous mutations of the SIL1 gene on chromosome 5q31
are reported to cause MSS [1].
SIL1 is a co-chaperone of the HSP70 molecular chaperone BIP (alsoreferred to as GRP78 or HSPA5) [4]. BIP is located in the lumen of theendoplasmic reticulum (ER) and binds to newly synthesized proteins tomaintain proper protein folding and translocation in the ER. ADPbound BIP binds tightly to its substrates, whereas ATP induces a conformational change that opens the substrate-binding pocket. SIL1
modulates BIP activation via nucleotide exchange during the ATP/ADP
cycle of BIP. SIL1 protein releases ADP from BIP so that it can bind to
ATP and re-start the protein-folding process [5,6]. Therefore, SIL1 deﬁciency causes BIP dysfunction, leading to accumulation of misfolded
proteins in the ER and increased ER stress. The unfolded protein response (UPR) is a cellular adaptive response to ER stress and restoresprotein-folding homeostasis.
In this study, we aimed to evaluate ER stress and the UPR at thecellular level in MSS using patient-derived lymphoblastoid cell lines

Abbreviations: 4-PBA, sodium phenylbutyrate; ATF6α, activating transcription factor 6α; CASP3, caspase-3; DEXA, dexamethasone; EBV, Epstein-Barr virus; eIF2α, eukaryotic translation initiation factor 2α; ER, endoplasmic reticulum; IRE1α, inositol-requiring protein 1α; LCL, lymphoblastoid cell line; MSS, Marinesco-Sjögren syndrome; PDI, protein disulphideisomerase; PERK, PRKR-like ER kinase; TUDCA, tauroursodeoxycholic acid; UPR, unfolded protein response; VPA, sodium valproate; XBP1, X-box-binding protein 1⁎
Corresponding author at: Department of Pediatrics, Developmental Biology, Graduate Medical School, Tokyo Medical and Dental University, Yushima 1-5-45, Bunkyo-ku, Tokyo 1138519, Japan.
E-mail address: m.takagi.ped@tmd.ac.jp (M. Takagi).
https://doi.org/10.1016/j.jns.2017.12.010
Received 27 February 2017; Received in revised form 11 November 2017; Accepted 8 December 2017
Available online 09 December 20170022-510X/ © 2017 Elsevier B.V. All rights reserved.

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A. Kashimada et al.

(LCLs) and to explore a therapeutic approach. MSS patient-derived cells
exhibited spontaneous ER stress and an activated UPR and were highly
susceptible to ER stress-induced apoptosis. Tauroursodeoxycholic acid
(TUDCA) alleviated excessive ER stress-induced apoptosis in these cells.
The potential use of TUDCA as a therapeutic agent for MSS should be
investigated further.

Cont 1.

Pt 1.

Pt 2.

Pt 3.

Pt 4.

SIL1
β-actin

2. Materials and methods

Fig. 1. Expression of SIL1 is defective in MSS-LCLs.
Western blot analysis of SIL1 expression. β-actin was used as a loading control. Cont: An
EBV-transformed LCL derived from a healthy volunteer. Pt: An EBV-transformed LCL
derived from a patient with MSS.

2.1. Patient samples
All patients included in this study were clinically and geneticallydiagnosed with MSS. Blood samples and medical reports were obtainedwith written informed consent of the patients or their legal guardians.
Research protocols were approved by the ethical committee of Tokyo
Medical and Dental University (approval nos. 103 and 196).
Four Japanese patients were enrolled in this study. These patients
carried the homozygous c.936dupG (p.Leu313fs) mutation in exon 9 of
the SIL1 gene, which is highly common in Japanese MSS patients [7].
Their ages at the time point for cell line establishment ranged from
14 months to 49 years (mean = 16.8 ± 18.9 years). All patients had a
low average IQ or moderate intellectual disability and severe muscle
weakness. The patients' phenotypes are described in Table 1.

A

Cont 1. Cont 2.

Pt 1.

Pt 2.

Pt 3.

Pt 4.

Pt 2.

Pt 3.

Pt 4.

BIP
β-actin2.2. Cell lines and cell culture

B

40

Epstein-Barr virus (EBV)-immortalized lymphoblastoid cell lines(EBV-LCLs) from control subjects (control LCLs) and patients with MSS(MSS-LCLs) were established according to standard protocols. LCLs
were maintained in RPMI 1640 supplemented with 15% fetal bovine
serum and 1% penicillin/streptomycin.

**

**

**

*

Cell death (%)

30

2.3. Western blotting
Cells were lysed in ice-cold RIPA buﬀer containing 50 mM Tris-HCl(pH 8.0), 150 mM NaCl, 1.0% NP-40, 0.5% DOC, 0.1% SDS, 50 mM
NaF, 25 mM β-glycerophosphate, 1 mM PMSF, and a protease inhibitor
cocktail (Roche, Basel, Switzerland). Lysates were resolved on SDSpolyacrylamide gels. The gels were transferred to nitrocellulose

20

10

0

Table 1
Clinical features of patients.

Sex
Present age
Age at cell line
establishment
Ocular involvements
Cataract
Cerebellar signs
Cerebellar atrophy
Nystagmus
Dysarthria
AtaxiaPsychomotor delay and
Muscle symptom
Intellectual disability(intelligencequotient)
Head control
Standing
Walk
Activity daily living
Serum CK (IU/L)
Myopathy
Other
Hypogonadism

Cont 2.

Cont 1. Cont 2.
Patient 1

Patient 2

Patient 3

Patient 4

M
10 y
7 y5 m

F
52 y
49 y

F
24 y
21 y

M
9y
1 y2 m

+

+

+

+

+
+
5y
23 m

+
+
N/A
5y

+
+
2y
1y

+
+
3y
3y

+ (35–50)− (81)+ (40)− (85)

6m
3y−
Wheel chair
276
+

6m
5y
−
Wheel chair
27
+

8m
−
−
Wheel chair
N/A
+

2y6 m
4y
−
Wheel chair
261
+

N/A

+

+

N/A

Pt 1.

Fig. 2. Expression of BIP and the dead cell fraction in non-stressed culture conditions.
(A) Western blot images of BIP expression. β-actin was used as a loading control. (B)Percentage of dead cells (n = 5). Data are presented as mean ± standard deviation (SD)from multiple independent experiments. *p < 0.05, **p < 0.005.

membranes (EMD Millipore, Billerica, MA, USA) and blocked with 5%
non-fat milk prepared in TBST. The membranes were incubated withthe following primary antibodies: anti-SIL1 (OriGene Technologies,
Rockville, MD, USA); anti-spliced X-box-binding protein 1 (XBP1s),
anti-BIP, anti-caspase-3 (CASP3), anti-PARP, anti-phospho-ASK1(Thr645), anti-ASK1, anti-phospho-JNK (Thr183/Tyr185) (G9), andanti-JNK (Cell Signaling Technology, Danvers, MA, USA); and anti-βactin (Sigma-Aldrich, St. Louis, MO, USA). Primary antibodies weredetected by binding of a horseradish peroxidase-conjugated anti-rabbit
or anti-mouse secondary antibody with an ECL kit (GE Healthcare,
Little Chalfont, UK).
2.4. Apoptosis assay, cell survival assay, and measurement of mitochondrial
membrane potential
LCLs were plated at a density of 1 × 106 cells/mL and treated with
2–20 μg/mL tunicamycin (Sigma-Aldrich) for 24 h. LCLs (2.5 × 105
cells) were washed with PBS and resuspended in 100 μL of annexin

M: male, F: female, y: year, m: month, N/A: not applicable, CK: creatin kinase.

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B

A
120

†††

Fig. 3. MSS-LCLs are highly susceptible to ER stress-induced apoptosis.
(A) Survival of SH-SY5Y cells after tunicamycin treatment.
Cells were treated with DMSO or tunicamycin (2 μg/mL)
for 12 or 24 h (n = 5), and then their survival was assayed. (B) Percentage of dead cells among EBV-LCLs after
tunicamycin treatment. Cells were treated with tunicamycin (2 μg/mL) for 24 h (n = 5). (C) Percentage ofapoptotic cells among EBV-LCLs after treatment with various concentrations of tunicamycin (2–20 μg/mL). Cells
were treated with tunicamycin for 24 h (n = 3). Data arepresented as mean ± SD from multiple independent experiments. *p < 0.05, **p < 0.005, †p < 0.005,††
p < 0.0005, and †††p < 0.00005. TM: tunicamycin.

†††

30

12h

80

24h
60
40

Cell death (%)

Cell survival (%)

100
†

††

0 2

0 2

20

10

20
0
0

1000 2000 3000 4000 5000 TM (ng/ml)

C
8

**

**

0
0 2

0 2

Cont 1. Cont 2. Pt 3.

Pt 4.

TM (µg/ml)***

Apoptotic cells (%)

Pt 4.
Cont 1.

6

4

2

0
0

5

10

15

20

TM (µg/ml)

binding buﬀer (10 mM HEPES/NaOH, pH 7.4, 140 mM NaCl, and
2.5 mM CaCl2). Annexin V-FITC (MBL, Nagoya, Japan) and 5 μg/mL 7aminoactinomycin D (Sigma-Aldrich) were added. The tubes were incubated at room temperature in the dark for 15 min. Cells were diluted
with 400 μL of annexin binding buﬀer, and ﬂow cytometric analysis
was performed using LSR Fortessa (Becton Dickinson, Franklin Lakes,
NJ, USA). Annexin V-positive and 7-aminoactinomycin D-negative cells
were deﬁned as apoptotic.
Cell survival was assessed using a Cell Counting Kit (Dojindo,
Kumamoto, Japan). Cells were cultured in 96-well plates. After incubation with tunicamycin, 10 μL of WST-8 reagent was added to
100 μL of medium, and samples were incubated for 4 h. Absorbance at
450 nm was measured with a microplate reader (Model 680, Bio-Rad
Laboratories, Hercules, CA, USA).
JC-1 staining was performed to measure changes in the mitochondrial membrane potential. When the mitochondrial membrane potential
is maintained, JC-1 enters mitochondria and forms complexes known as
J-aggregates, which emit red ﬂuorescence. However, when the mitochondrial membrane potential decreases, JC-1 is monomeric andemits green ﬂuorescence, while red ﬂuorescence decreases. Changes inthe ﬂuorescence of JC-1 were monitored by ﬂow cytometry. Cells weretreated with 4 μg/mL JC-1 for 15–30 min at 37 °C in 5% CO2, harvested, washed with PBS, and analyzed by ﬂow cytometry.

apoptotic cells was determined by ﬂow cytometry using annexin V
staining.
2.6. Statistical analysis
Data were analyzed by nonparametric methods. All statistical analyses were performed using the Mann-Whitney U test. P values < 0.05
were considered statistically signiﬁcant.
3. Results
3.1. MSS-derived cells exhibit increased spontaneous ER stressWestern blot analysis demonstrated loss of SIL1 expression in thefour LCLs (Fig. 1). SIL1 functions in combination with BIP to ensure
proper protein folding in the ER; therefore, a defect in SIL1 leads toaccumulation of misfolded proteins and activates the UPR. BIP is an ER
stress marker and is induced when unfolded proteins accumulate. To
determine whether the UPR was continually upregulated in nonstressed culture conditions, BIP expression was examined by western
blotting. As expected, baseline BIP expression in non-stressed culture
conditions was increased in all four MSS-LCLs. However, the expression
level of BIP varied between the cells (Fig. 2A). The patient 2-derived
LCL showed striking upregulation of BIP compared with the control.
The patient 4-derived LCL showed the lowest upregulation of BIP. ER
stress leads to cellular dysfunction and cell death. Therefore, the deadcell fraction in non-stressed culture conditions was investigated. The
percentage of dead cells was signiﬁcantly increased in all MSS-LCLs(Fig. 2B). The patient 2-derived LCL, in which BIP expression was high,exhibited a high level of spontaneous cell death. By contrast, the patient
4-derived LCL, in which BIP expression was lower, exhibited the least

2.5. Apoptosis assay of cells treated with various drugs prior to tunicamycin
LCLs were pretreated with 1 mM sodium valproate (VPA; Wako,
Osaka, Japan) for 24 h, 10 μM dexamethasone (DEXA; Sigma-Aldrich)for 4 h, 500 μM sodium phenylbutyrate (4-PBA; Sigma-Aldrich) for 4 h,or 500 μM TUDCA (Tokyo Chemical Industry, Tokyo, Japan) for 4 h,and then 2 μg/mL tunicamycin was added for 24 h. The percentage of
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A

B
2.5

**

TM (0µg/ml)

2
NTCont 1.Cont 2. Pt 3. Pt 4. Cont 1.Cont 2. Pt 3. Pt 4.

TM

BIP

BIP/ -actin

Short
expo.
Long
expo.

BIP

TM (5µg/ml)

1.5

BIP

Short
expo.

BIP

Long
expo.

1
XBP1s
0.5

0
NT

TM (2µg/ml)

C

*

*
*

60

−

+

BIP

+ TM (5µg/ml)
Short
expo.

16

40

8

20

XBP1s

**

12

*

30

Long
expo.

BIP

Ratio of XBP1s expression
to control

−

50

Pt 4.

Ratio of BIP expression
to control

Cont 1.

*
**

20

4

10
0
0

24

Cont 1.

D

0

24 hrs0024

24 hrs0

Cont 1.

Pt 4.

Pt 4.

E
*
0

1

3

6

Pt.4
18

24 0

1

3

2

6 18 24 hrs

Cont 1.
−

BIP
PDI

XBP1s

+

Pt 4.
−

+

Ratio of PDI expression
to control

Cont.1

**

1

0
0

24
Cont 1.

024 hrsPt 4.

Fig. 4. ER stress activates the IRE1α-XBP1 signaling pathway in MSS-LCLs.
(A) Western blot analysis of BIP and XBP1s expression in SH-SY5Y cells. ER stress was induced by treatment with 5 μg/mL tunicamycin for 24 h. (B) Western blot analysis of BIP and
XBP1s expression. ER stress was induced by treatment with 5 μg/mL tunicamycin for 24 h. (C) Representative western blot images and the ratio of quantitative BIP and XBP1s protein
expression. β-actin was used as a loading control. ER stress was induced by treatment with 5 μg/mL tunicamycin for 24 h. Expression levels in control LCLs not treated with tunicamycin
were set to 1. Data are presented as mean ± SD from multiple independent experiments (BIP: n = 4, XBP1s: n = 6). *p < 0.05 and **p < 0.005. (D) Time-dependent expression of BIP
and XBP1s. ER stress was induced by treatment with 5 μg/mL tunicamycin for 0–24 h. (E) Representative western blot images and the ratio of quantitative PDI protein expression. β-actin
was used as a loading control. The experimental conditions are the same as in (A).

cell death. These diﬀerences in cell responses to ER stress were notassociated with the clinical aspects of the patients (Table 1).

level of spontaneous apoptosis was elevated, and the patient 4-derived
LCL, in which the level of spontaneous apoptosis was lowest. After
treatment with tunicamycin (2 μg/mL) for 24 h, the dead cell fraction
was signiﬁcantly increased in both control LCLs and MSS-LCLs. Even
the patient 4-derived LCL, in which BIP expression was lowest, wasmore susceptible to ER stress-induced cell death (Fig. 3B). Tunicamycin
induced apoptosis in a dose-dependent manner (Fig. 3C). Although theexpression level of BIP diﬀered between the MSS-LCLs in non-stressed
conditions, ER stress-induced apoptosis was obvious in all these celllines. These results indicate that MSS-LCLs are susceptible to ER stress-

3.2. MSS-LCLs are highly susceptible to ER stress-induced apoptosis
Tunicamycin inhibits N-linked glycosylation, which induces misfolded protein accumulation in the ER [8]. Treatment with tunicamycin
eventually reduced the survival of SH-SY5Y neuroblastoma-derived
cells (Fig. 3A). To further elucidate the UPR in MSS, we evaluated ER
stress-mediated apoptosis in the patient 3-derived LCL, in which the
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A

expression of activating transcription factor 6α (ATF6α) varied between LCLs (Supplemental data 1). These data suggest that the IRE1αXBP1 pathway is predominantly upregulated in MSS-LCLs.
Protein disulphide isomerase (PDI) is a disulphide bond-modulating
ER chaperone that can also facilitate ER-associated degradation of
misfolded proteins [9]. Mitochondrion-associated PDI can induce
apoptosis via permeabilization of the outer mitochondrial membrane
when it accumulates at high levels in response to the presence of misfolded proteins [10]. PDI expression was upregulated in control LCLs
and MSS-LCLs following tunicamycin treatment. Although PDI expression was higher in the latter cells than in the former cells, this diﬀerence
was not signiﬁcant (Fig. 4E).

**

Apoptotic cells (%)

***
10

5

0
DMSO DMSO VPA

3.4. TUDCA can protect MSS-LCLs from ER stress-induced apoptosis

DEXA 4-PBA TUDCA
TM

Targeting ER stress and the UPR is a candidate therapeutic approachfor MSS. The eﬀects of VPA [11,12], DEXA [13], 4-PBA [14], and
TUDCA [15–17], which interfere with ER stress and the UPR, on the
UPR and cell death were investigated. Although VPA, DEXA, and 4-PBA
had minimal eﬀects for exacerbated cell death after tunicamycin
treatment, TUDCA reduced cell death (Fig. 5A). ER stress-induced
apoptosis following tunicamycin treatment was also investigated in
MSS-LCLs derived from patients 3 and 4. TUDCA also signiﬁcantly reduced ER stress-induced apoptosis in these cells (Fig. 5B). To elucidate
the cellular mechanism that underlies the inhibition of apoptosis, expression of UPR-related molecules and apoptosis markers was investigated by western blotting. Unexpectedly, the expression levels of
BIP and XBP1s did not diﬀer (Fig. 6A). IRE1α can also interact with
TRAF2 to recruit and activate ASK1 and JNK [18,19]. The ASK1-JNK
pathway triggers apoptosis. Therefore, activation of the IRE1α-TRAF2ASK1-JNK pathway was also investigated. As expected, ASK1 was activated in control LCLs and MSS-LCLs following tunicamycin treatment;
however, JNK was not obviously activated. Activation of ASK1 waslower in MSS-LCLs than in control LCLs. TUDCA treatment did not
downregulate the IRE1α-TRAF2-ASK1-JNK pathway (Fig. 6B).
Next, the downstream apoptosis pathway was investigated.
Mitochondrial integrity was assessed by ﬂow cytometry using JC-1
staining. After tunicamycin treatment, mitochondria were depolarizedin control LCLs and MSS-LCLs, and this was attenuated by TUDCA
treatment. During apoptosis, caspases are activated after mitochondrial
depolarization. Caspase cleavage was investigated following tunicamycin treatment with or without TUDCA (Fig. 7A). CASP3 and PARP
cleavage was signiﬁcantly reduced after TUDCA treatment (Fig. 7B, C).
These data suggest that TUDCA can protect MSS-LCLs from ER stressinduced apoptosis by inhibiting caspase activation to halt the apoptotic
pathway.

*

B
Ratio of apoptotic cells
to control

6

**
4

2

0
TM
TUDCA
Cont 1.

Cont 2.

Pt 3.

Pt 4.

Fig. 5. TUDCA protects MSS-LCLs from ER stress-induced apoptosis.
(A) Percentage of dead cells in the patient 4-derived LCL. Cells were pretreated with
DMSO, VPA (1 mM, 24 h), DEXA (10 μM, 4 h), 4-PBA (500 μM, 4 h), and TUDCA(500 μM, 4 h), and then DMSO or 2 μg/mL tunicamycin was added to pretreated cells.
The percentage of dead cells was determined at 24 h after tunicamycin treatment (n = 4).
(B) The percentage of apoptotic cells in control 1-, control 2-, patient 3-, and patient 4derived LCLs. Cells were pretreated with or without TUDCA and then stimulated withtunicamycin. The assay conditions were the same as in (A). The apoptotic rate was calculated by dividing the percentage of apoptotic cells in the tunicamycin-treated group bythat in the non-treated group (n = 4). Data are presented as mean ± SD from multiple
independent experiments. *p < 0.05, **p < 0.005, and ***p < 0.005. NT: no treatment, TM: tunicamycin.

induced apoptosis.
3.3. ER stress activates the inositol-requiring protein 1α (IRE1α)-X-boxbinding protein 1 (XBP1) pathway in MSS-LCLs

4. Discussion
Upon ER stress, cells activate the UPR to maintain homeostasis,
which degrades misfolded proteins, reduces protein translation, andaccelerates production of molecular chaperones involved in protein
folding. The UPR was investigated after tunicamycin treatment in SHSY5Y cells, control LCLs, and MSS-LCLs. Tunicamycin treatment induced BIP and XBP1s expression in SH-SY5Y cells (Fig. 4A). BIP expression was increased in the patient 3-derived LCL before tunicamycin
treatment. By contrast, expression of XBP1s was similar in control LCLs
and MSS-LCLs before tunicamycin treatment. However, BIP and XBP1s
expression was strikingly higher in MSS-LCLs than in control LCLs after
tunicamycin treatment (Fig. 4B, C). The kinetics of BIP and XBP1s expression after tunicamycin treatment were investigated. Expression of
BIP and XBP1s gradually increased from 3 h after tunicamycin treatment and this was maintained for 24 h in both control LCLs and MSSLCLs (Fig. 4D). Although expression of BIP and XBP1s was much higher
in MSS-LCLs than in control LCLs, the kinetics did not diﬀer between
these two cell types. In terms of other UPR-related molecules, phosphorylation of eukaryotic translation initiation factor 2α (eIF2α) andWe demonstrated that LCLs derived from MSS patients exhibit
spontaneous ER stress and are highly susceptible to ER stress-induced
apoptosis. However, a previous study reported that expression of
HYOU1 (also known as GRP150 and GRP170), BIP, and calreticulin(CALR) is not altered in MSS-LCLs. In the current study, the expression
level of BIP diﬀered between the MSS-LCLs, and the patient 4-derived
LCL exhibited minimal upregulation of BIP. The UPR and cell deathvaried between the patients even though they all carried the same SIL1
mutation. One potential explanation may be that the expression orfunction of molecules associated with ER stress, including HYOU1,
diﬀered between the patients due to gene polymorphisms or epigenetic
modiﬁcations. This is a limitation of experiments using cell lines derived from genetically heterogeneous patients. Although SIL1 was not
expressed in any of the cell lines used in the current or previous study,
the mutation patterns diﬀered between the two studies. This could also
explain the contradictory ﬁndings. Alternatively, diﬀerences in cellpassage may be responsible. Replicative senescence increases BIP
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ACont 1.

Fig. 6. TUDCA does not aﬀect the ER stress activation
pathway.
(A) Western blot analysis of BIP and XBP1s expression incontrol 1- and patient 4-derived LCLs of the various
treatment groups. The assay conditions were the same as inFig. 5A. β-actin was used as a loading control. (B) Western
blot analysis of phospho-ASK1 and phospho-JNK in control
1- and patient 4-derived LCLs of the various treatmentgroups. The assay conditions were the same as in Fig. 5A.
β-actin was used as a loading control. Data are presented asmean ± SD from multiple independent experiments.
(n = 3)

Pt 4.

TM
TUDCA
BIP
XBP1s
β-actin

B

Cont 1.

Cont 1.

Pt 4.

Pt 4.

TM
TUDCA

TM
TUDCA
p-ASK1

p-JNK

54kDa
46kDa

JNK

54kDa
46kDa

ASK1
β-actinβ-actin

4

*

*

*

*

3

P-JNK/JNK

p-ASK1/ASK1

*

2

1

0.5
1

0
TM
TUDCA

0
TM
TUDCA
Cont 1.

Pt 4.

Pt 4.

Cont 1.

expression [20]. All the cell lines used in the current study were between passage #10 and #20. However, the passage frequency diﬀers
according to how cell lines are established and maintained. The level ofsenescence may have been higher in some of our cell lines than in thoseused in the previous study.
Until now, the etiology of MSS has been mainly investigated bypatient-based pathological analysis. Skeletal muscle biopsies from individuals with MSS exhibit various stages of nuclear degeneration,
scattered apoptotic cells [21], and autophagic vacuoles, indicating achronic myopathic process [22]. In brain autopsies, severe loss of
Purkinje cells in the cerebellum, mild loss of granule cells, and disorganization of the cytoarchitecture in the cerebral cortex are observed[23,24]. Sil1-deﬁcient woozy mutant mice exhibit a similar pathological phenotype as humans, as well as activation of the UPR in thesarcoplasmic reticulum of skeletal muscle [25]. The present study links
the previously observed pathological phenotype and cellular biology.
SIL1 is expressed in various organs of mammals; however, thesymptoms of MSS are limited to speciﬁc organs, mainly neuromuscularorgans. HYOU1, a nucleotide-exchange factor, has functional redundancy with SIL1 [26,27]. HYOU1 may compensate for the alteredfunction of SIL1 in non-aﬀected organs in MSS. Another hypothesis isthat the level of cell turnover determines which organs are aﬀected.
Terminally diﬀerentiated organs such as neurons and muscles aremainly aﬀected in MSS. MSS patients do not exhibit symptoms in thehematologic system, digestive system, or skin, in which cell turnover is
rapid. Supply of cells in these organs is dependent on stem cell proliferation, and cells have a relatively short lifespan. Because damaged
cells are rapidly removed, only a low level of ER stress may arise inthese tissues.

In MSS-LCLs, increased ER stress appeared to activate the UPR
predominantly via the IRE1α-XBP1 pathway and induced apoptosis.
The UPR signaling pathway comprises three branches: PRKR-like ER
kinase (PERK)–eIF2α, IRE1α–XBP1, and ATF6α [28]. IRE1α is atransmembrane protein in the ER. Upon ER stress, IRE1α oligomerizes
and displays unconventional RNA splicing activity, removing an intron
from the XBP1 mRNA, which is translated into the functional transcription factor XBP1s [28][29]. The IRE1α-XBP1 pathway enhances
protein folding/degradation to alleviate ER stress and maintain cell
survival. Under chronic ER stress, hyperactivated IRE1α promotes the
mitochondrial apoptotic pathway via a process called regulated IRE1dependent decay (RIDD) [30]. This process induces selective microRNA decay, which results in caspase activation [31]. Therefore, increased IRE1α-XBP1 signaling in MSS patient-derived cells may play animportant role in ER stress-induced degeneration of tissues. The BIPlevel was obviously increased in unstressed non-treated MSS-derived
cells, whereas activation of the IRE1/XBP1 pathway was not detected.
This led us to hypothesize that ER stress can be better detected byimmunoblotting of BIP than of XBP1 or that the increase in the BIP levelis not suﬃcient to activate the IRE1/XBP1 pathway.
The roles of ATF6 and eIF2α were obscure in the present study. Lee
et al. reported that EBV LMP1 (an integral membrane protein) activates
the PERK, IRE1, and ATF6 pathways [32]. Garrido et al. revealed that
EBNA3C (an EBV-encoded nuclear protein) activates phosphorylationof eIF2α at serine 51 by an interaction with Gadd34 [33]. The present
study analyzed EBV-transformed LCLs. Therefore, the EBV-derived
protein LMP1 or EBNA3C may interfere with evaluation of the UPR.
Hayashi et al. analyzed expression of the ER stress-related genes XBP1,
GRP78 (HSPA5), GRP94 (HSP90B1), CHOP (DDIT3), and CALR using
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Cells with depolarized mitochondria(%)

A

**

***

*

35

Fig. 7. TUDCA inhibits caspase activation.
(A) Western blot analysis of BIP and XBP1s expression incontrol 1- and patient 4-derived LCLs of the various
treatment groups. The assay conditions were the same as
in Fig. 5A. β-actin was used as a loading control. (control
n = 6, patient n = 4) (B) Cells with a depolarized mitochondrial membrane potential were analyzed by ﬂow
cytometry. The assay conditions were the same as inFig. 5A. (C) Representative western blot images of PARP
and cleaved PARP. (D) CASP3 and cleaved CASP3 incontrol 1- and patient 4-derived LCLs. β-actin was used asa loading control. The lower graphs indicate the ratio of
quantitative cleaved protein expression. The rate was calculated by dividing the protein expression level by that intunicamycin-treated control LCLs (n = 3). Data are presented as mean ± SD from multiple independent experiments. *p < 0.05.

30
25
20
15
10
5

0
TM
TUDCA
Pt 4.

Cont 1.

B

Cont 1.

Pt 4.

CCont 1.

Pt 4.

TM
TUDCA

TM
TUDCA
PARP

CASP3

Cleaved
PARP

CleavedCASP3β-actinβ -actin**3
Ratio of cleaved CASP 3 expression
to control

Ratio of cleaved PARP expression
to control

2.5

2

1.5

1

0.5

2

1

0
TM
TUDCA

0
TM
TUDCA
Cont 1.

Pt 4.

Cont 1.

EBV-transformed lymphoblastoid cells derived from patients with bipolar disorder. They reported that induction of XBP1s as well as total
XBP1 by thapsigargin is signiﬁcantly attenuated in these patients. Induction of GRP94 by thapsigargin is also decreased. However, levels ofother molecules, including CHOP, are not altered [34]. This explains
why activation of the UPR was only demonstrated by upregulation of
BIP and XBP1 in the current study.
In our evaluation, TUDCA is a good candidate to modulate ER stressin MSS. TUDCA signiﬁcantly reduced ER stress-induced apoptosis in
MSS-LCLs. TUDCA is an endogenous hydrophobic bile acid used to treat
biliary cirrhosis [35]. TUDCA reduces ER stress by inhibiting activationof the ER stress-associated proteins BIP, PERK, XBP1, and eIF2α[16,36,37]. In addition, TUDCA exerts anti-apoptotic eﬀects by inhibiting BAX translocation, release of cytochrome c, and caspase activation [38,39]. TUDCA principally acts by inhibiting activation of the
ER stress-associated proteins GRP78, PERK, eIF2α, ATF4, IRE1α, JNK,
p38, and CHOP. In particular, TUDCA inhibits the dissociation of
GRP78 and PERK, thereby reducing ER stress-mediated cell death [37].
However, we did not observe changes in the level of BIP or XBP1 or theactivation of ASK1 upon TUDCA treatment. Diﬀerences in the cell typeused, the duration of tunicamycin treatment, or the concentration oftunicamycin may explain the discrepancies between our ﬁndings andthose of previous studies. In the current study, TUDCA ameliorated
mitochondrial damage and inhibited caspase activation and subsequentPt 4.

apoptosis induction. TUDCA promotes phosphorylation of pro-apoptotic BAD and its translocation from mitochondria to the cytosol,
thereby inhibiting apoptosis [40]. We speculate that this explains why
TUDCA treatment did not alter the level of BIP or XBP1s in the current
study. ER stress is involved in degenerative and inﬂammatory processes
in various neuronal and skeletal muscle diseases [41,42]. TUDCA has aneuroprotective eﬀect in some neurodegenerative diseases, such as
Huntington's disease [43], Alzheimer's disease [44], and Parkinson's
disease [45]. However, there is no report regarding a treatment strategy
for MSS or prevention of its progression. Considering safety and drug
bioavailability, TUDCA might be a promising drug to treat the myopathy and neurological symptoms of MSS. One obvious limitation of ourdata is that it was obtained in LCLs, which are not aﬀected clinically in
MSS patients and woozy mice and also have been demonstrated to shownormal antibody responses [46] Additional work is needed to conﬁrm apotential use of TUDCA in cell populations aﬀected by the disease andin appropriate animal models.

5. Conclusion
MSS patient-derived cells display spontaneous ER stress and aresusceptible to ER stress-induced death. TUDCA treatment reduces ER
stress-induced apoptosis in MSS-LCLs. The potential of TUDCA as atherapeutic agent for MSS could be explored further in preclinical
55

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studies.
Supplementary data to this article can be found online at https://doi.org/10.1016/j.jns.2017.12.010.

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Conﬂicts of interest
The authors declare that they have no conﬂicts of interest.
Acknowledgements
We would like to thank the patients and their families for participating in this study.
Funding
This work was supported by a Grant-in-Aid from the Japan Agency
for Medical Research and Development (AMED; 15652552), andResearch on Measures for Intractable Diseases grant from the Ministryof Health (H22-128), Labour, and Welfare.
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ORIGINAL ARTICLE

Altered TDP-43-dependent splicing in HSPB8-related distal hereditary
motor neuropathy and myofibrillar myopathy
b, C. Casalic, I. Nishinod, Y. K. Hayashie, S. Magrif, F. Taronif, C. Stuanig,, M. Laura
A. Cortesea,bfhP. Saveri , M. Moggio , M. Ripoloneh, A. Prellei, C. Pisciottaf, A. Sagnellif, A. Pichiecchioa, M. M. Reillyb,
E. Burattig and D. Pareysonfa

EUROPEAN JOURNAL OF NEUROLOGY

C. Mondino National Neurological Institute Foundation, IRCCS, Pavia, Italy; bMRC Centre for Neuromuscular Diseases, UCL Instituteof Neurology, London, UK; cDepartment of Medical and Surgical Sciences and Biotechnologies, Sapienza University of Rome, Latina,
Italy; dDepartment of Neuromuscular Research, National Center of Neurology and Psychiatry, National Institute of Neuroscience, Tokyo;eDepartment of Pathophysiology, Tokyo Medical University, Tokyo, Japan; fUnit of Rare Neurodegenerative and Neurometabolic
Diseases, IRCCS Foundation, C. Besta Neurological Institute, Milan; gInternational Centre for Genetic Engineering and Biotechnology(ICGEB), Trieste; hNeuromuscular and Rare Diseases Unit, Department of Neuroscience, Fondazione IRCCS Ca’ Granda, Ospedale
Maggiore Policlinico, Milan; and iDepartment of Neurology, Ospedale Maggiore, Crema, Italy

Keywords:

distal hereditary motor
neuropathy, HSPB8,
myoﬁbrillar myopathy,
RNA metabolism, TDP43
Received 8 June 2017
Accepted 7 September 2017
European Journal of
Neurology 2018, 25: 154–163
doi:10.1111/ene.13478

Background and purpose: Mutations in the small heat-shock protein 22 gene
(HSPB8) have been associated with Charcot-Marie-Tooth disease type 2L, distal hereditary motor neuropathy (dHMN) type IIa and, more recently, distal
myopathy/myoﬁbrillar myopathy (MFM) with protein aggregates and TDP-43
inclusions. The aim was to report a novel family with HSPB8K141E-related
dHMN/MFM and to investigate, in a patient muscle biopsy, whether the presence of protein aggregates was paralleled by altered TDP-43 function.
Methods: We reviewed clinical and genetic data. We assessed TDP-43 expression by qPCR and alternative splicing of four previously validated direct
TDP-43 target exons in four genes by reverse transcriptase–polymerase chain
reaction.
Results: The triplets and their mother presented in the second to third decade of life with progressive weakness aﬀecting distal and proximal lower limb
and truncal muscles. Nerve conduction study showed a motor axonal neuropathy. The clinical features, moderately raised creatin kinase levels, selective pattern of muscle involvement on magnetic resonance imaging andpathological changes on muscle biopsy, including the presence of protein
aggregates, supported the diagnosis of a contemporary primary muscle
involvement. In aﬀected muscle tissue we observed a consistent alteration of
TDP-43-dependent splicing in three out of four TDP-43-target transcripts(POLDIP3, FNIP1 and BRD8), as well as a signiﬁcant decrease of TDP-43
mRNA levels.
Conclusions: Our study conﬁrmed the role of mutated HSPB8 as a cause of a
combined neuromuscular disorder encompassing dHMN and MFM with protein aggregates. We identiﬁed impaired RNA metabolism, secondary to TDP43 loss of function, as a possible pathological mechanism of HSPB8K141E toxicity, leading to muscle and nerve degeneration.

Introduction
Correspondence: D. Pareyson, Unit of Neurological Rare Diseasesof Adulthood, Department of Clinical Neurosciences – IRCCS
Foundation, ‘C. Besta’ Neurological Institute, via Celoria 11, 20133
Milan, Italy (tel.: +39 02 2394 3001; fax: +39 02 2394 2293; e-mail:davide.pareyson@istituto-besta.it).

154

Mutations in the small heat-shock protein 22 gene
(HSPB8) are associated with Charcot-Marie-Tooth
disease type 2L and distal hereditary motor neuropathy (dHMN) type IIa [1,2].

© 2017 EAN-119-

TDP-43 IN HSPB8-RELATED DHMN AND MFMMore recently, heterozygous HSPB8 mutations(p.K141E and p.P173SfsX43) have been reported in
two unrelated families with distal myopathy andmotor neuropathy [3]. In these cases, muscle biopsy
showed changes consistent with myoﬁbrillar myopathy(MFM) along with protein aggregates and transactive
response DNA binding protein 43 (TDP-43) inclusions. More recently, a p.Gln170GlyfsX45 mutation
causing HSPB8 haploinsuﬃciency was identiﬁed in
ﬁve cases from three unrelated families with dominant
adult-onset axial and distal myopathy, but no evidence of neuropathy [4].
Here, we report a third family with autosomal dominant dHMN and MFM/protein aggregate myopathy.
Using muscle biopsy tissue from a patient carrying
the K141E mutation in HSPB8 we have also investigated whether the presence of protein aggregates wasparalleled by altered TDP-43 function, a ﬁnding of
possible pathogenic relevance to myoﬁber and nerve
degeneration.

Methods
Patients

Patients were assessed at the inherited neuropathy
clinic of the IRCCS Foundation (‘C. Besta’ Neurological Institute, Milan, Italy) (patients I-1, II-1 and II-3)
and the National Hospital for Neurology and Neurosurgery (London, UK) (patient II-2). Detailed history,
clinical examination, neurophysiology and magnetic
resonance imaging of lower limb muscles were performed. When available, muscle biopsy was reviewed.
Muscle strength was scored using the Medical
Research Council (MRC) grading scale.
Standard protocol approvals, registrations and patientconsents

Institutional review boards approved the study and all
examined family members gave written informed
consent.
Muscle pathology

Patient II-1 underwent quadriceps femoris muscle
biopsy. A muscle biopsy from quadriceps femorisfrom a healthy subject was used as a control. Tissue
specimens were frozen in isopentane-cooled liquid
nitrogen and processed according to standard techniques, as previously described [5]. For histology,transverse sections (8 lm) were stained with Gomori
trichrome, hematoxylin and eosin, and Nicotinamide155

adenine dinucleotide (NADH) tetrazolium reductase.
Immunoﬂuorescence was performed with routine
protocols using the following antibodies: mouse antimyotilin (Novocastra, Leica Biosystems, Wetzlar,
Germania), rabbit anti-Alpha-B-crystallin (Millipore,
Billerica, MA, USA), rabbit anti-desmin and mouseanti-myosin (Abcam, Cambridge, MA, USA).
Genetic analysis

Genomic DNA was extracted from peripheral lymphocytes using standard techniques. All exons andtheir franking intronic regions of HSPB8, BAG3,
DES, CRYAB, MYOT, ZASP, FLNC, VCP,DNAJB6 and FHL1 were sequenced directly using an
ABI PRISM 3130 automated sequencer (PE Applied
Biosystems, Foster City, CA, USA). Primer sequences
are available on request.
Sequence variants were assessed by using publicallyavailable databases including the 1000 Genomes Project database (http://www.1000genomes.org/), NHLBI
Exome Sequencing Project 5400 database (http://evs.
gs.washington.edu/EVS/), dbSNP135 (http://www.ncb
i.nlm.nih.gov/SNP/) and Human Gene Mutation
Database (http://www.hgmd.cf.ac.uk/).
RNA purification and reverse transcriptase–polymerase chain reaction analysis and quantitative
real-time polymerase chain reaction analysis

Tissue (1 µg) from muscle biopsy of patient II-1 andthree normal controls (two males and one female, aged
55, 58 and 57 years, respectively) was homogenized in
1 mL of Trizol at 4°C. RNA was isolated using an
RNeasy mini kit (Qiagen, Hilden, Germany). Total
RNA from sample II-1 was puriﬁed using Trizol
reagent (Invitrogen, Carlsbad, CA, USA) according to
the manufacturer’s instructions. The complementary
DNA synthesis was performed using 1 lg of each RNA
sample, Moloney murine leukemia virus reverse transcriptase (Invitrogen) and random hexameric primers.
Polymerase chain reaction (PCR) analysis was performed using the following primers: DNA polymerasedelta-interacting protein 3 (POLDIP3) (NM_001278657) (Fw: 50 -GCTTAATGCCAGACCGGGAG
TTG-30 ; Rev: 50 -TCATCTTCATCCAGGTCATATAAA
TT-30 ); microtubule-associated protein (MAP) kinase-activating death domain (NM_130470) (Fw: 50 -GACCTGA
ATTGGGTGGCGAGTTCCCT-30 ; Rev: 50 -CATTGGT
GTCTTGTACTTGTGGCTC-30 ); folliculin-interacting
protein 1 (FNIP1) (NM_001008738) (Fw: 50 -GCTACA
AGATAGTCTTGAATTCATC-30 ; Rev: 50 -CAGACCGTGCTATGCCACTGTCTCT-30 ); bromodomain-© 2017 EAN

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A. CORTESE ET AL.

containing protein 8 (BRD8) (NM_139199) (Fw:
50 CAATTCTTGGCCACGCAGTTGATTA-30 ; Rev: 50 and
CTCAGAGAGAAAGTGGAGGAGGTTC-30 )TRAF2 and NCK-interacting kinase (TNIK)(NM_015028) (Fw: 50 -CAAAGGCGAGAGAAGGAG
CTG-30 ; Rev: 50 -CTGATGCTGAAGGGAAACTAA
G-30 ).
In all experiments, exon inclusion/exclusion wasquantiﬁed using Image J according to the methodology described previously [6].
The quantiﬁcation of transactive response (TAR)
DNA-binding protein (TARDBP) and HSPB8 gene
expression levels was performed by real-time PCR
using SYBR green technology. Speciﬁc primers for
TARDBP and HSPB8 genes were designed using Beacon designer software (Bio-Rad, Hemel Hempstead,UK) and are as follows: TDP-43 (Fw: 50 -ATCTGGTGTATGTTGTCAACTATCC-30 ; Rev: 50 -GAAandCTTCTCCAAAGGTACTAAAATACTC-30 )HSPB8 (Fw: 50 -GCACAGCTTCAAGCCAGAG-30 ;Rev: 50 -CAAATGTTGAGTAAGGAGGGACC-30 ).
The housekeeping genes GAPDH and RPL13a
were used to normalize the results of TDP-43 and
HSPB8, respectively. All ampliﬁcations were performed on a CFX96 real-time PCR detection system(Bio-Rad).The detailed qPCR conditions comprised
95°C for 3 min, followed by 45 cycles of 95°C for
15 s and 60°C for 30 s. The relative expression levels
were calculated according to the 2DCt method, byusing the equation DCt = Ct(target) � Ct(housekeeping)for Ct normalization. The box plots of the real-time
PCR were created by using the DCt (TDP-43–
RPL13a or HSPB8–glyceraldehyde-3-phosphate dehydrogenase) values of the patient and controls (n = 3).
The results represent the average and SD of
two independent experiments, with two technicalreplicates.
SH-SY5Y cell line small interfering RNA

Human neuroblastoma SH-SY5Y cells were cultured
in Dulbecco’s Modiﬁed Eagle Medium (DMEM)–Glutamax-I (Gibco-BRL, Life Technologies Inc., Frederick, MD, USA) supplemented with 10% fetal bovine
serum (Gibco-BRL, Life Technologies Inc.) andantibiotic–antimycotic-stabilized suspension (SigmaAldrich, St Louis, MO, USA) at 37°C in an incubatorwith a humidiﬁed atmosphere of 5% CO2. The smallinterfering RNA (siRNA) sense sequences used forsilencing the diﬀerent target proteins were as follows:
luciferase (control) (50 -UAAGGCUAUGAAGAGA
UAC-30 ) and TDP-43 (50 -AAGCAAAGCCAAGAUGAGCCU-30 ). The siRNA-mediated TDP-43 silencing
in these cells was described in detail previously [7].

Case report
Family pedigree

The pedigree was consistent with an autosomal dominant neuromuscular disorder, with trizygotic triplets
and their mother aﬀected (Fig. 1a).
Case 1 (II-1)

The index case, a 31-year-old female, reported normal
birth and developmental milestones. She underwent
surgery for pes planus at the age of 6 years. At theage of 20 years, she started complaining of calf muscle
cramps and progressive walking diﬃculties. She
reported repeated ankle sprains and frequent falls in
her late 20s, causing repeat fractures of ﬁbular andperoneal bones. She was referred to our service at theage of 31 years.
Neurological examination showed waddling andhigh-stepping gait. She was unable to stand fromkneeling or sitting and she needed help to sit from thelying position. She had proximal and distal atrophy inthe lower limbs (Fig. 1b). Strength was reduced in
proximal muscle groups (hip ﬂexion 4 bilaterally, knee
extension 3 on the right and 2 on the left) and distal
muscles (foot dorsiﬂexion 4 bilaterally, foot plantar
ﬂexion 4 on the right and normal on the left, toe
extension 3 on the right and 2 on the left). Neck ﬂexors were also weak to grade 3, but neck extensors
were normal. Deep tendon reﬂexes (DTRs) were present in the upper limbs and absent in the lower limbs.
Examination of cranial nerves and upper limbs was
normal. Sensation was normal throughout.
Creatin kinase (CK) was slightly raised (274 IU/L;
normal value = 24–150 IU/L). Nerve conduction studies (NCSs) showed markedly reduced compound
motor action potential amplitudes in the lower limbs(peroneal nerve, 0.2 mV; tibial nerve, 2 mV) and borderline values in the upper limbs (ulnar nerve, 6 mV;
median nerve, 7 mV). Motor and sensory conduction
velocities and sensory action potentials were normal.
Electromyography (EMG) showed mixed myopathic
and neurogenic changes in proximal and distal muscle
groups.
Muscle magnetic resonance imaging showed a selective pattern of muscle fatty degeneration with prominent involvement of quadriceps, adductor magnus,
medial gastrocnemius and peroneus muscle groups(Fig. 2).
A quadriceps muscle biopsy showed the presence ofmarked ﬁber size variability, split ﬁbers, internal
nuclei and grouping of hypotrophic ﬁbers belonging
to both ﬁber types, suggesting a primary myopathy.

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TDP-43 IN HSPB8-RELATED DHMN AND MFM

157

Figure 1 (a) Pedigree and allelic state for c.421A>G, p.K141E in HSPB8 and c.743A>G, p.H248R in BAG3 in patients I-1, II-1,II-2
and II-3. (b) Proximal and distal lower limb atrophy with relatively preserved muscle bulk at upper limbs. (c) Electropherograms
showing the c.421A>G change (red highlighted) of HSPB8 gene in aﬀected family members. [Colour ﬁgure can be viewed at wileyonlinelibrary.com].

Rimmed vacuoles, subsarcolemmal masses and corelike structures on oxidative stains were also observed.
On immunoﬂuorescence analysis, several ﬁbers
contained protein aggregates that were positive for
Alpha-B-crystallin and myotilin (Fig. 3a–f). As a control, core-like structures and myotilin-positive aggregates were absent from healthy control muscle biopsy(Fig. 3g and h). Stains for desmin and myosin had
normal distribution. On electron microscopy, Z-line
streaming was observed in rare sarcomeres (data notshown).
Case 2 (II-2)

This patient had a normal birth and developmental
milestones. Onset of symptoms occurred during adolescence, when he noticed that he could not sprint
during basketball matches. In his 20s, he noticed thathe could not stand from squatting and had diﬃcultyin jumping and climbing stairs. He stopped running at
24 years of age. Since then, he has experienced progressive diﬃculty in standing up and frequent falls.
He denied symptoms in the upper limbs.
Examination at the age of 31 years showed diﬃculty in standing from the chair. He walked with awaddling gait. He could stand on his toes but had difﬁculty in standing on his heels. Cranial nerves werenormal. Tone and strength were normal in the upper
limbs. There was no scapular winging. In the lower
limbs, he had severe proximal and distal wasting thatwas more pronounced proximally. He had proximal
weakness with hip ﬂexion grade 4 on the right and 2
on the left, knee ﬂexion grade 4 bilaterally, and knee
extension grade 4 on the right and 2 on the left. He
had very mild distal weakness with ankle dorsiﬂexion
grade 4+ on the right but normal on the left and toe
extension grade 4 bilaterally. Ankle plantar ﬂexion
was preserved. He had truncal weakness. Reﬂexes© 2017 EAN

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(a)

(b)

(c)

(d)

Figure 2 Magnetic resonance axial T1-weighted (a and c) and T2-STIR (b and d) sequences at the level of the thigh (a and b) and calf
(c and d) in case II-1. In the thigh (a and b), a nearly complete fatty substitution of the quadriceps muscle is evident as hyperintensity
bilaterally (a, arrows); the adductor magnus is also involved, although to a lesser extent (a, *). No edema-like alterations are documented in the T2-STIR sequences (b). In the calf (c and d), there is selective fatty substitution at the level of the peroneal (c, *) andboth gemini muscles, predominantly the medial ones (c, arrows). A slight hyperintensity is also evident in the T2-STIR sequence (d) at
the level of the right soleus (arrow).

were brisk in upper limbs and normal in lower limbs.
Sensory examination was normal.
The CK level was elevated at 656 IU/L (n.v. = 38–
204 IU/L). NCSs showed absent tibial compound
motor action potential bilaterally and reduced peroneal compound motor action potential amplitudewith normal conduction velocities. Sensory responses
were normal. EMG showed myopathic changes inproximal lower limb muscles and bilateral medial gastrocnemius in a patchy distribution. Chronic neurogenic changes with large motor units were recorded
bilaterally at tibialis anterior muscles.
Muscle magnetic resonance imaging showed a selective pattern of fatty degeneration with the adductor
magnus and adductor longus muscles being severely
degenerated and the quadriceps femoris showing variable involvement. Adductor brevis, sartorius, gracilis
and hamstring muscles were less involved. In the calves,
tibialis posterior, both heads of gastrocnemius and peroneus longus muscles showed marked fatty degeneration and volume loss with sparing of other muscles.
Case 3 (II-3)

Onset of symptoms, encompassing diﬃculties with rising from a sitting position and impossibility of running, occurred in her late 20s. At the age of 31 years,

neurological examination showed pes cavus with hammertoes and moderate wasting of the thighs, but only
minimal atrophy of the legs. There was mild weakness, grade 4, of neck ﬂexion, hip ﬂexion and abduction, knee extension and ﬂexion, foot dorsiﬂexion andtoe extension. DTRs were normal throughout, except
for ankle DTRs, which were brisk. She had a steppage
gait but walking was only slightly aﬀected. She wasable to rise on her toes but could not walk on herheels. She needed to push herself with her arms inorder to get up from a chair and also found it diﬃcult
to sit from the lying position. The remainder of theexamination was unremarkable. CK was raised. EMG
and NCSs were only performed at the age of 15 years
and were reportedly normal.
Case 4 (I-1)

The mother of the triplets reported diﬃculties withclimbing stairs since the age of 30 years. Thereafter,
walking progressively worsened and she needed a stickby the age of 52 years and two crutches 3 years later.
By the age of 55 years she needed a wheelchair for
longer distances.
Neurological examination at the age of 58 years
showed diﬀuse atrophy of lower limbs, hyperextended
knees, pes cavus and hammertoes. Independent© 2017 EAN-123-

TDP-43 IN HSPB8-RELATED DHMN AND MFM

Figure 3 Muscle pathology. (a)Gomori’s trichrome 1009. Marked ﬁbersize variability including small groups of
hypotrophic ﬁbers, several splittings andinternal nuclei, and a few ﬁbers withrimmed vacuoles. (b) ATPase pH 4.3
1009. Grouping of hypotrophic ﬁbers
belonging to both ﬁber types. (c)Gomori’s trichrome 4009 [detail of (a)].
Some ﬁbers with hyperchromic cytoplasmic areas. (d) NADH 4009. A few
ﬁbers with irregular distribution of enzymatic activity, including core-like
aspects. (e) Anti-myotilin 4009.
Positivity in a few cytoplasmic areas. (f)Anti-Alpha-B-crystallin 4009. Cytoplasmic positivity in several ﬁbers. (g)NADH 2009. Normal staining. (h)Anti-myotilin 4009. Normal pattern. a–f
are from patient II-1. g and h are froma normal control and are shown forcomparison. [Colour ﬁgure can beviewed at wileyonlinelibrary.com].

(a)

(b)

(c)

(d)

(e)

(f)

(g)

(h)

walking was not possible. There was reduced powerof truncal, upper and lower limb muscles (neck ﬂexion, abductor pollicis brevis, ﬁrst dorsal interosseousand extensor digitorum, all grade 4, hip ﬂexion 4 on
the right, hip extension 2 bilaterally, knee extension 4on the right and 3 on the left, and knee ﬂexion 2bilaterally). No active movements of feet and toes
were possible. DTRs were present in upper limbs andabsent in lower limbs. There was no sensory loss. CK
was moderately increased. NCSs were in keeping withan axonal motor neuropathy with mixed neurogenic/
myopathic changes on EMG.

159

Genetic analysis

A heterozygous c.421A>G (p.Lys141Glu) change inexon 2 of HSPB8 was identiﬁed in all four aﬀectedfamily members (Fig. 1a and c). The unaﬀected father
was not available for testing. This is the same change
as previously reported in dHMN2A/Charcot-MarieTooth disease type 2L and distal myopathy and motor
neuropathy [3,8]. In addition, in cases I-1 and II-2 wefound a heterozygous c.743A>G (p.His248Arg)
change (rs369947845) in exon 3 of BAG3 (Fig. 1a),which is also present, although rare, in public control© 2017 EAN

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A. CORTESE ET AL.

databases (0.000028% of healthy controls in gnomAD
browser). No mutation was found in DES, CRYAB,
MYOT, ZASP, FLNC, VCP, DNAJB6 or FHL1.
Altered TDP-43-dependent splicing in HSPB8K141E
muscle tissue

The TDP-43-positive aggregates were reported to bepresent in numerous ﬁbers from patients with HSPB8related dHMN/MFM and are also a common ﬁnding
in other protein aggregate myopathy [3,9,10]. Therefore, we investigated, in the muscle biopsy of the
HSPB8K141E case (II-1), whether the presence of protein aggregates was paralleled by changes of TDP-43

expression. As shown in Fig. 4, TDP-43 mRNA wassigniﬁcantly lower in the HSPB8K141E muscle biopsy
than in muscle from normal controls (Fig. 4a). Conversely, no changes of expression of HSPB1 weredetected by qPCR (Fig. 4b).
It was then of interest to determine whether thisapparent loss of TDP-43 expression was also accompanied by a loss of function of TDP-43 splicing regulation. The ability of TDP-43 to regulate RNA
splicing is particularly important in neurodegenerative
diseases as a large proportion of genes aﬀected by its
aggregation have been shown to be important for neuronal survival [11–13]. This was evaluated by assessment of alternative exon splicing of four validated

Figure 4 TDP-43 molecular signature in HSPB8K141E-aﬀected muscle tissue. Box plots of TAR DNA-binding protein (TARDBP) (a) and
HSPB8 (b) mRNA levels measured using qPCR of the controls and the HSPB8 proband as DCt values. The box whisker plots visualize
the minimum (end of the bottom whisker), ﬁrst quartile (bottom border of the box), median (line through the box), third quartile (top border of the box) and maximum (end of the top whisker) of the distribution. The RPL13a housekeeping gene was used for normalization of
TDP-43 expression for the upper graph and glyceraldehyde-3-phosphate dehydrogenase for the lower graph. (c) Alternative splicing of
selected genes in controls versus the HSPB8 patient. For each gene, as comparison with a TDP-43 loss-of-function situation, we used SHSY5Y cell lines treated with either control small interfering RNA (siRNA) (lane ) or TDP-43 siRNA (lane +). Standard reverse transcriptase–polymerase chain reaction ampliﬁcations were then performed for the POLDIP3, BRD8, FNIP1, MADD/IG20 and TNIK transcripts. The labeling on the right shows the aﬀected exons in each case. Band image quantiﬁcation was performed using the ImageJ
program. BRD8, bromodomain-containing protein 8; FNIP1, folliculin-interacting protein 1; HSPB8, small heat-shock protein 22; NS,
not signiﬁcant; TNIK, TRAF2 and NCK-interacting kinase. [Colour ﬁgure can be viewed at wileyonlinelibrary.com].

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TDP-43 IN HSPB8-RELATED DHMN AND MFM

TDP-43-dependent target genes, as described in previous works by some of us [7]. The genes examined
were as follows: POLDIP3 (NM_001278657), BRD8
(NM_139199), FNIP1 (NM_001008738), microtubuleassociated protein (MAP) kinase-activating death
domain (NM_130470) and TNIK (NM_015028).
For all of these genes, we evaluated the ratio ofexon skipping:inclusion or the appearance of pseudoexon activation in the case of TNIK. As an additional control, we included in this analysis a neuronal
control cell line (SH-SY5Y) where endogenous TDP43 has been silenced using siRNA. In this way, wewere also able to compare the muscle splicing proﬁleswith neuronal cell line splicing proﬁles.
As shown in Fig. 4c, this analysis showed that,when we compared a representative normal muscle
splicing proﬁle with HSPB8K141E muscle tissue (lanes
3 and 4), there was a decrease of exon 3 inclusion in
the POLDIP3 gene, an increase in exon 20 inclusionin the BRD8 gene and an increase in exon 7 skipping
for the FNIP1 gene (red highlighted). Importantly,
these three results were consistent with the observed
splicing changes detected in the control SH-SY5Y celllines both before and after siRNA silencing on TDP43, thus conﬁrming a change in splicing proﬁle consistent with a loss of TDP-43 function.
However, diﬀerently from cell line results, no changes
were noted in the splicing proﬁle of microtubule-associated protein (MAP) kinase-activating death domainexon 31 splicing in HSPB8K141E muscle compared with
control muscle. In particular, in the HSPB8K141E muscle sample there was no appearance of the pseudoexon
inclusion band that is visible in SH-SY5Y cells silenced
for TDP-43. Finally, no diﬀerence could be observed in
the case of the TNIK gene. However, diﬀerently fromthe neuronal cell line, where depletion of TDP-43 leads
to an increased inclusion of exon 16, in muscle cell thisexon is 100% included and therefore any loss of function of TDP-43 could not have made any detectablechange for this particular gene.
In conclusion, in HSPB8K141E muscle tissue thesplicing of three out of four assessed transcripts (POLDIP3, BRD8 and FNIP1) showed changes of exon
alternative splicing consistent with TDP-43 loss offunction.

161

have been reported in multiple families with autosomal dominant dHMN and Charcot-Marie-Tooth disease type 2 [1,2] and, more recently, in a few families
with MFM. The exact pathogenesis is still unclear
and diﬀerent mechanisms have been hypothesized,
including reduced chaperoning function and impaired
protein turnover, disruption of cytoskeleton integrity
and altered interaction with the survival motor neuron
complex.
The HSPB8-related neuropathy typically has an
onset in the second to third decade of slowly progressive
distal atrophy and weakness, with no or mild sensory
involvement. Our cases, as well as those reported by
Ghaoui et al. [3] carrying the same K141E mutation in
HSPB8, had proximal as well as distal weakness of thelower limb muscles. Moreover, our patients consistently
showed neck ﬂexor and truncal muscle involvementwith early diﬃculties in sitting from the lying position,
which is extremely unusual in conditions aﬀecting
peripheral nerves and prompted us to suspect a concurrent muscle involvement. None of them showed camptocormia. Reassuringly, respiratory and cardiac muscle
do not seem to be aﬀected in HSPB8-related dHMN/
MFM, as opposed to other causes of MFM.
Additionally, two of the cases that we are reporting
also carried a rare His248Arg variant in BAG3. Due
Table 1 Genes and conditions associated with contemporary or
alternative muscle and nerve involvement
Genes and conditions responsible for contemporary nerve andmuscle involvement
HSPB8 [1–4]
BAG3 [15]GBE1 [22] (adult polyglucosan body disease)
Mitochondrial disorders [23]HSPB1 [8,24,25]
Genes and conditions associated with myopathy. Possible
neurogenic changes on EDx study
MYOT [26]ZASP [27]CRYAB (HSPB5) [28]
PABPN1 [29]sIBM [30]
Genes associated with neuropathy/neuronopathy. Possible muscle
involvement or signiﬁcantly raised CK
MATR3 [31], AR (Kennedy’s disease) [32], XK (McLeod
syndrome) [33]Other causes of alternative muscle or nerve involvement (variable
expressivity depending on diﬀerent mutations)
LMNA [34–36], DNM2 [37,38], VCP [17–19], HNRNPA1 [39]Discussion
HSPB8 is a member of a family of 10 small heatshock proteins (HSPs) that are molecularly deﬁned by
the presence of a highly conserved Alpha-B-crystallin
domain and are functionally involved in chaperonemediated autophagy [14]. Mutations of the 141 lysine
residue in the Alpha-B-crystallin domain of HSPB8

AR, androgen receptor; BAG3, BCL2-associated athanogene 3; CK,
creatin kinase; CRYAB, Alpha-B-crystallin; DNM2, dynamin 2;
EDx, electrodiagnostic; HNRNPA1, heterogeneous nuclear ribonucleoprotein A1; HSPB1, small heat-shock protein 27; HSPB8, small
heat-shock protein 22; LMNA, lamin A/C; MYOT, myotilin;
PABPN1, poly(A) binding protein nuclear 1; sIBM, sporadic inclusion body myositis; VCP, valosin-containing protein; XK, X-linked
Kx blood group; ZASP, ZO-2-associated speckle protein.

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to lack of segregation, this latter variant is unlikely tobe the primary cause of the neuromuscular conditionof the patients, who all show an almost identical clinical phenotype. However, a role of the BAG3 as a
modiﬁer cannot be ruled out.
The list of genes and conditions responsible for
combined muscle and nerve involvement is expanding(Table 1). Mutations in genes encoding other heatshock proteins, including HSPB1 and HSPB5/CRYAB, as well as in genes involved in protein
degradation and chaperone-assisted selective autophagy (e.g. VCP, MATR3, BAG3) all show heterogeneous clinical expressivity ranging from myopathic toneuropathic, neuronopathic and mixed phenotypes[15–19]. It is of interest that TDP-43-positive protein
aggregates on muscle biopsy are a common pathological ﬁnding of many of these conditions.
TDP-43 is an RNA-binding protein involved in different stages of RNA processing, including splicing
regulation, and it constitutes the major component ofpathological inclusions present in the brain of patientsaﬀected by amyotrophic lateral sclerosis and frontotemporal lobar degeneration [20]. Numerous studies
have demonstrated that changing the expression levelof TDP-43 is suﬃcient to alter the expression andsplicing of hundreds RNAs in vitro [11,12]. Moreover,changes of RNA processing have been identiﬁed in
induced pluripotent stem cells cell lines derived frompatients with amyotrophic lateral sclerosis carrying
TARDBP mutations and muscle ﬁbers from patientswith sporadic inclusion body myositis [9,21].
Here we show a consistent alteration of TDP-43dependent splicing in three out of four previously validated direct TDP-43 target transcripts. This ﬁnding
points to a loss of function of TDP-43 in HSPB8related dHMN/MFM and suggests that impaired
RNA metabolism could contribute to HSPB8 pathology. Therefore, other transcripts relevant to nerve andmuscle survival may be abnormally spliced as a resultof TDP-43 loss of function.
Unfortunately, due to unavailability of muscle tissue, we were unable to conﬁrm the presence of TDP43 aggregates in the patient muscle biopsy. However,TDP-43 inclusions were present in the myoﬁbers ofpreviously reported patients with HSPB8-related
dHMN/MFM and have been consistently identiﬁed innumerous myopathies with protein aggregates, as previously discussed.
In conclusion, our study conﬁrms the role of mutated
HSPB8 as a cause of a combined neuromuscular disorder encompassing dHMN and MFM/protein aggregate
myopathy. It will be important for clinicians to consider the possibility of both neuropathy and myopathy
being present in any patient with a HSPB8 mutation, as

well as in other conditions traditionally considered toaﬀect only either nerve or muscle, in order to better
counsel patients. This also has clear implications forclinical scientists interpreting data from whole exome
sequencing studies, warranting careful consideration of
all variants in genes associated with neuromuscularconditions and in terms of tissue distribution of future
treatments for these conditions. Finally, we identiﬁed
an altered splicing of several TDP-43 target genes inmuscle tissue from a patient carrying the HSPB8K141E
mutation, suggesting that TDP-43 loss of function andimpaired RNA metabolism could play a role in muscle
and nerve degeneration in HSPB8-related disease.

Acknowledgements
The authors wish to thank The Biobank of Skeletal
Muscle, Peripheral Nerve, DNA and Cell Cultures,
member of the Telethon Network of Genetic Biobanks(Project GTB12001), funded by Telethon Italy and theEurobiobank Network. This work was supported
partly by Research on Rare and Intractable Diseases,
Health and Labor Science Research Grants (I.N. and
Y.K.H.) and partly by an Intramural Research Grant
for Neurological and Psychiatric Disorders of National
Center of Neurology and Psychiatry (NCNP) (23-4, 268) (I.N. and Y.K.H.). This research was also supported(M.L., M.M.R.) by the National Institute for HealthResearch University College London Hospitals Biomedical Research Centre and by the National Institute for
Health Research Biomedical Research Centre at Great
Ormond Street Hospital for Children NHS Foundation
Trust and University College London.

Disclosure of conflicts of interest
The authors declare no ﬁnancial or other conﬂicts ofinterest.

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RESEARCH ARTICLE

Macrolide Antibiotics Exhibit Cytotoxic Effectunder Amino Acid-Depleted Culture Condition
by Blocking Autophagy Flux in Head and Neck
Squamous Cell Carcinoma Cell Lines
Kazuhiro Hirasawa1, Shota Moriya2, Kana Miyahara3, Hiromi Kazama2, Ayako Hirota2,
Jun Takemura2, Akihisa Abe2, Masato Inazu4, Masaki Hiramoto2, Kiyoaki Tsukahara1,
Keisuke Miyazawa2*

a11111

1 Department of Otolaryngology (Head and Neck Surgery), Tokyo Medical University, Tokyo, Japan,
2 Department of Biochemistry, Tokyo Medical University, Tokyo, Japan, 3 Department of Breast Surgery,Tokyo Medical University, Tokyo, Japan, 4 Institute of Medical Science, Tokyo Medical University, Tokyo,Japan* miyazawa@tokyo-med.ac.jp

OPEN ACCESS
Citation: Hirasawa K, Moriya S, Miyahara K,
Kazama H, Hirota A, Takemura J, et al. (2016)
Macrolide Antibiotics Exhibit Cytotoxic Effect underAmino Acid-Depleted Culture Condition by Blocking
Autophagy Flux in Head and Neck Squamous Cell
Carcinoma Cell Lines. PLoS ONE 11(12):e0164529. doi:10.1371/journal.pone.0164529
Editor: Dong-Yan Jin, University of Hong Kong,
HONG KONG
Received: April 25, 2016
Accepted: September 27, 2016
Published: December 15, 2016
Copyright: © 2016 Hirasawa et al. This is an open
access article distributed under the terms of theCreative Commons Attribution License, which
permits unrestricted use, distribution, andreproduction in any medium, provided the original
author and source are credited.
Data Availability Statement: All relevant data arewithin the paper and its Supporting Information
files.
Funding: This study was supported by funds
provided through a MEXT-supported program of
the Strategic Research Foundation at Private
Universities from the Ministry of Education,
Culture, Sports, Science and Technology ofJapan, Grants-in-Aid for Scientific Research (C)from The Ministry of Education, Culture,
Sports, Science and Technology (no. 26460478,

Abstract
Autophagy, a self-digestive system for cytoplasmic components, is required to maintain theamino acid pool for cellular homeostasis. We previously reported that the macrolide antibiotics azithromycin (AZM) and clarithromycin (CAM) have an inhibitory effect on autophagy
flux, and they potently enhance the cytocidal effect of various anticancer reagents in vitro.
This suggests that macrolide antibiotics can be used as an adjuvant for cancer chemotherapy. Since cancer cells require a larger metabolic demand than normal cells because of
their exuberant growth, upregulated autophagy in tumor cells has now become the target forcancer therapy. In the present study, we examined whether macrolides exhibit cytotoxic
effect under an amino acid-starving condition in head and neck squamous cancer cell lines
such as CAL 27 and Detroit 562 as models of solid tumors with an upregulated autophagy inthe central region owing to hypovascularity. AZM and CAM induced cell death under theamino acid-depleted (AAD) culture condition in these cell lines along with CHOP upregulation, although they showed no cytotoxicity under the complete culture medium. CHOP
knockdown by siRNA in the CAL 27 cells significantly suppressed macrolide-induced cell
death under the AAD culture condition. CHOP-/- murine embryonic fibroblast (MEF) celllines also attenuated AZM-induced cell death compared with CHOP+/+ MEF cell lines. Usinga tet-off atg5 MEF cell line, knockout of atg5, an essential gene for autophagy, also induced
cell death and CHOP in the AAD culture medium but not in the complete culture medium.
This suggest that macrolide-induced cell death via CHOP induction is dependent on autophagy inhibition. The cytotoxicity of macrolide with CHOP induction was completely cancelledby the addition of amino acids in the culture medium, indicating that the cytotoxicity is due tothe insufficient amino acid pool. These data suggest the possibility of using macrolides for“tumor-starving therapy”.

PLOS ONE | DOI:10.1371/journal.pone.0164529 December 15, 2016

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Tumor-Starving Therapy Using Macrolide Antibiotics in HNSCC

http://www.mext.go.jp/), and a Grant-in-Aid forYoung Scientist (B) from the Ministry of
Education, Culture, Sports, Science andTechnology of Japan (no. 25860398, http://www.
mext.go.jp/) and a Research Grant from Tokyo
Medical University to SM.
Competing Interests: The authors have declaredthat no competing interests exist.

Introduction
Head and neck squamous cell carcinomas (HNSCCs) occur frequently with more than
500,000 new cases diagnosed worldwide each year [1]. Recurrent and/or metastatic HNSCC
patients receive platinum-based chemotherapy as a first-line treatment [2]. As 80%-100% of
HNSCCs have an increased expression level of epidermal growth factor receptor (EGFR),
cetuximab, a humanized IgG monoclonal antibody against EGFR, has become increasingly
used in combination therapy [3]. However, only few patients show long-term responses, indicating that more effective therapeutic strategies are required for improved therapeutic outcome [4].
Cancer cells grow faster than normal cells in vivo, and they are believed to exhibit a higher
metabolic demand [5]. In particular, HNSCC cells with increased activation of EGFR-related
signals require a larger metabolic demand. To meet the amino acid demand, cancer cells
increase the expression level of amino acid transporters such as LAT1, as well as the activationof autophagy for degradation of their cytoplasmic proteins and organelles to recycle the intracellular amino acids [6, 7]. Autophagy is a self-eating process wherein cells capsulate their own
cytoplasmic proteins and organelles in double-membrane vesicles to form autophagosomes.
They are transported to the lysosome following membrane fusion to each other (autolysosomes). The contents of autolysosomes are finally degraded by lysosomal hydrolases [8]. The
resulting breakdown products are used as a source of energy and new protein synthesis [9].
Autophagy is indispensable for cell survival and is enhanced under a nutrient-starved condition, particularly in solid tumors because of their excessive metabolic requirements and hypovascular condition [10, 11].
We previously reported that clinically well-used macrolide antibiotics, such as azithromycin(AZM) and clarithromycin (CAM), block autophagy flux [12, 13]. In multiple myeloma andbreast cancer cells, we found that apoptosis induction was enhanced by the proteasome
inhibitor bortezomib and AZM or CAM combination treatment together with endoplasmic
reticulum (ER)-stress loading [12, 14]. In terms of drug-repositioning, AZM and CAM areconsidered strong candidates as clinical autophagy inhibitors. There is, however, still a need tofurther clarify the specific molecular mechanism responsible for macrolide-induced autophagy
inhibition [15].
In the current study, we first show that AZM and CAM exert a potent cytotoxic effect on
HNSCC cell lines only under amino acid-depleted (AAD) culture conditions. We further
discuss the possibility of “tumor-starving therapy” using macrolide antibiotics in HNSCC
patients.

Materials and Methods
Reagents
AZM was purchased from Tokyo Chemical Industry (Tokyo, Japan), and was dissolved indimethyl sulfoxide (DMSO) to prepare 50 mM stock solutions. CAM was purchased from
Wako Pure Chemical Industries (Osaka, Japan) and was dissolved in ethanol to prepare 5 mM
stock solutions. E-64d and pepstatin A, which are inhibitors of lysosomal proteases, and
Z-VAD-fmk, which is a pan-caspase inhibitor, were all purchased from Peptide Institute(Osaka, Japan). Necrostatin-1, a specific inhibitor of RIPK1, was purchased from Enzo Life
Sciences (Farmingdale, NY, USA). Thapsigargin was purchased from Nacalai Tesque (Kyoto,
Japan). Doxycycline hydrochloride, Chloroquine diphosphate, DL-α-Tocopherol and Astaxanthin were from Sigma-Aldrich (St. Louis, MO, USA). Amino acid-free Dulbecco’s Modified
Eagle’s Medium (DMEM) (048–33575), MEM Essential Amino Acids Solution (100x) (132–

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15641) and MEM Non-essential Amino Acids Solution (50x) (139–15651) were purchasedfrom Wako Pure Chemical Industries.

Cell lines and culture conditions
The human oral squamous cell carcinoma cell line CAL 27 and the human pharyngeal carcinoma cell line Detroit 562 obtained from the American Type Culture Collection (ATCC,
Manassas, VA, USA) were cultured in DMEM plus 10% fetal bovine serum (FBS) (Biowest,
Nuaillé, France), and 1% penicillin/streptomycin solution (Wako Pure Chemical Industries).
A CHOP-/- murine embryonic fibroblast (MEF) cell line (CHOP-KO-DR) established from a13.5-day-old CHOP-/- mouse embryo by SV-40 immortalization and a CHOP+/+ MEF cell line
(DR-wild-type) established by SV-40 immortalization as a control cell line for CHOP-KO-DR
were also obtained from the ATCC, and were cultured in DMEM plus 10% FBS and 1% penicillin and streptomycin. The “AAD culture medium” described in this manuscript indicatesamino acid-free DMEM plus 10% FBS and 1% penicillin and streptomycin. Normal DMEM
contains a sufficient amount of 20 amino acids including 75 mg/L L-glutamic acid and 30 mg/
L L-methionine, whereas amino acid-free DMEM contains no amino acids; AAD culture
medium (amino acid-free DMEM plus 10% FBS) only contains amino acids less than 1/10 ofcomplete DMEM with 10% FBS. The m5-7 cell line, an atg5 tet-off MEF system, was a kind
gift from Dr. Noboru Mizushima (The University of Tokyo, Tokyo, Japan). Details of the culture conditions for passage and the condition for knock-out of the atg5 gene for complete
autophagy inhibition were previously described [16]. All cell lines were cultured in a humidified incubator containing 5% CO2 and 95% air at 37˚C. All cell lines were used for the experiments within 5 passages after thawing.

Assessment of cell growth inhibition and apoptosis induction
Cell growth inhibition was measured by the Cell Titer-Blue cell viability assay (Promega,
Madison, WI, USA). Cells were treated with or without drugs for 24, 48, and 72 hrs in 96-wellplates. In the last 4 hrs, the Cell Titer-Blue reagent was added to each well, and fluorescence
was measured at 560 nm excitation and 590 nm emission. The percentage of the mean fluorescence measured to that in untreated cells was expressed as % cell growth inhibition.
For assessment of apoptosis, cells were stained with Annexin V and propidium iodide (PI)using APOPCYTOTM Annexin V-Azami-Green Apoptosis Detection kit (MBL, code 4690,
Nagoya, Japan) according to the manufacturer’s instructions and subjected to flow cytometry
using Attune1 Acoustic Focusing Cytometer (Life Technologies, CA, USA).

Immunoblotting
Immunoblotting was performed as previously described in detail [17]. Briefly, cells were lysedwith RIPA lysis buffer (Nacalai Tesque) containing 1 mM PMSF, 0.15 U/ml aprotinin, 10 mM
EDTA, 10 ng/ml sodium fluoride and 2 mM sodium orthovanadate. Cellular proteins were
quantified using a DC Protein Assay (Bio-Rad, Richmond, CA). Equal amounts of proteins
were loaded onto the gels, separated by SDS-PAGE and transferred to an Immobilon-P membrane (Millipore, Bedford, MA, USA). The membranes were probed with primary antibodies(Abs) such as anti-microtubule- associated protein 1 light chain 3 (LC3) B antibody (Ab)(NB600-1384; Novus Biologicals, Inc., Littleton, CO), and anti-phosoho-eIF2α (Ser51) Ab(#9721S), anti-CHOP (GADD153) monoclonal (m) Ab (#2895S), anti-p70S6K Ab (#9202S),
anti-phospho-p70S6K (Thr389) Ab (#9205S), anti-PARP Ab (#9542) (Cell Signaling Technology, Danvers, MA, USA), and anti-p62 (sequestosome-1) mAb (sc-28359), anti-GAPDH mAb(sc-32233), and anti-β-actin mAb (sc-47778) (Santa Cruz Biotechnology, Santa Cruz, CA,

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Tumor-Starving Therapy Using Macrolide Antibiotics in HNSCCUSA) and anti-XBP-1s Ab (S647501) (BioLegend, San Diego, CA). Immunoreactive proteins
were detected with horseradish peroxidase-conjugated second Abs (Jackson, West Grove, PA)
and an enhanced chemiluminescence reagent (ECL) (Millipore). Densitometry was performed
using a Molecular Imager, ChemiDoc XRS system (Bio-Rad).

Detection of intracellular reactive oxygen species
Reactive oxygen species (ROS) was quantified using ROS-Glo™ H2O2 Assay (Promega) according to the manufacture’s instruction. Cells were treated on a white 96-well plate with themedium plus the test compound (total: 80 μL). To each well, 20 μL of H2O2 substrate solution
was added, and cells were incubated at 37˚C/5% CO2 for 6 hrs. After incubation, 100 μL of thedetection solution was added. After 20 min incubation at room temperature, the relative luminescence was measured.

Transfection of CHOP siRNAs
For the gene silence of CHOP in CAL 27 cells, CHOP siRNA (VHS40605) and a control
siRNA (12935–300), whose sequences are described below, were purchased from Thermo
Fisher Scientific., and diluted to a final concentration of 33 nM in Opti-MEM I (Thermo
Fisher Scientific). Transfection was performed with the cells at 50% confluency using Lipofectamine RNAi MAX transfection reagent (Thermo Fisher Scientific) according to the manufacturer’s instruction.
CHOP sense: UUUCCUGCUUGAGCCGUUCAUUCUC, CHOP antisense: GAGAAUGAACGGCUCAAGCAGGAAA, Control sense and antisense: not shown

Assessment of morphology in cultured cells
Cells were spread on slide glasses using Cytospin 4 Centrifuge (Thermo Fisher Scientific, Inc.,
Rockford, IL, USA) to make slide glass preparations. Preparations were then stained with
May-Grünwald-Giemsa, and examined using a degital microscope BZ-8000 (Keyence Co.,
Osaka, Japan).

Statistical analysis
All quantitative data were expressed as mean ± standard deviation (SD). Statistical analysis was
performed using two-tailed nonpaired Student’s t-test. The criterion for statistical significancewas taken as p < 0.05.

Results
AZM and CAM block autophagy flux and induce cell death under theamino acid-depleted culture condition
We initially confirmed that AZM and CAM inhibit autophagy in HNSCC cell lines as previously described in myeloma and breast cancer cell lines by flux analysis [12, 14, 18]. The
conversion from the soluble cytosolic LC3B-I with a molecular weight of 16 kDa to the membrane-bound LC3B-II with a molecular weight of 18 kDa via the conjugation of phosphatidyl
ethanolamine represents the formation of autophagosomes. Therefore, LC3B-II expression is agood marker for autophagosomes [18]. We cultured CAL 27 cells with macrolides in the presence or absence of lysosomal inhibitors (E-64d and pepstatin A) for 24 hrs in the complete culture medium, and performed immunoblotting with anti-LC3B Ab. As shown in Fig 1A, AZM
or CAM treatment upregulated the LC3B-II expression. However, in the presence of the lysosomal inhibitors, the LC3B-II expression did not further increase compared with CAL 27 cells

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Fig 1. Macrolides induce cell death in amino acid-depleted culture by blocking autophagy flux. (A) Immunoblotting with LC3B and p62 Abs fromthe cell lysates of CAL 27 cells cultured in the complete culture medium with/without AZM, CAM (50 μM) in the presence or absence of lysosomal inhibitors(LIs) E-64d (10 μg/mL) and pepstatin A (10 μg/mL) for 24 hrs. Immunoblotting with anti-GAPDH mAb was used as an internal control. Cell lysate derived
from PANC-1 cells treated with AZM for 24 hrs was used as a positive control [13]. (B) (upper panel) Immunoblotting of LC3 from the cell lysates of CAL 27
cells cultured in the complete culture medium or AAD culture medium containing 10% FBS with or without AZM (50 μM) and CAM (50 μM) for up to 60 min.
Immunoblotting with anti-GAPDH mAb was used as an internal control. Cell lysate derived from PANC-1 cells treated with AZM (50 μM) for 24 hrs wasused as a positive control [13]. (lower panel) The ratios of LC3B-II/GAPDH were plotted. (C) Cell growth inhibition of CAL 27, Detroit 562, and MEF cells
treated with AZM (50 μM) and CAM (50 μM) in the complete culture medium (upper panel) and AAD culture medium (lower panel) for 24, 48, and 72 hrs.
Viable cell numbers are expressed as percentage to viable cells cultured in the complete culture medium at each indicated culture period. Data arepresented as means ± SEM. *p < 0.05. (D) CAL 27, Detroit 562, and MEF cells were cultured under the AAD culture condition in the presence of AZM or
CAM at various concentrations for 48 hrs. Data are presented as means ± SEM. *p < 0.05; AZM vs CAM. (E) Effects of supplementation of amino acids to
the AAD culture medium on CAL 27 cells under the AAD culture condition with or without macrolides. ‘Amino acids’ indicate 1% MEM non-essential aminoacids plus 2% MEM essential amino acids at the final concentration (Wako). The numbers of viable cells were determined and the results were expressedas the percentage against the numbers of viable cells cultured under the AAD culture condition without macrolides at various time points, namely, 24-hr,
48-hr, and 72-hr cultures. *p < 0.05 (vs without macrolides). ‘n.s.’ indicates ‘not significant’.
doi:10.1371/journal.pone.0164529.g001

treated with macrolide or lysosomal inhibitors alone. This indicates that both AZM and CAM
block autophagy flux in CAL 27 cells. Thereafter, we examined autophagy induction under the
AAD culture condition in the presence or absence of macrolides by tracing LC3B-II expression(Fig 1B). CAL 27 cells were washed twice with PBS and cultured in AAD Dulbecco’s Modified
Eagle’s Medium (DMEM) containing 10% FBS. After amino acid depletion, the expression
ratios of LC3B-II to GAPDH augmented within 15 min and further increased at 30 min, butattenuated at 45 min. In the presence of AZM or CAM, the ratios of LC3B-II/GAPDH further
increased at 15 min compared with the CAL 27 cells cultured in the AAD culture medium.
This appears to be due to the accumulation of autophagosomes by autophagy induction in
response to amino acid starvation as well as the inhibition of autophagosome degradation bymacrolides simultaneously in the autophagy flux. Worth noting was that the LC3B-II upregulation by CAM was transient, whereas the LC3B-II upregulation by AZM persisted and further
increased during 60 min exposure. This may be because AZM is more potent than CAM inblocking autophagy flux as we previously described in pancreatic cell lines; however, othermolecular mechanisms may be involved [13].
We next examined the cell growth inhibition of CAL 27 and Detroit 562 cells under the
AAD culture condition. The AAD culture medium showed some cell growth inhibition compared with the complete culture medium (Fig 1C). Although the addition of AZM and CAM

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at 50 showed no cytotoxicity in the complete culture medium, these macrolide antibiotics
induced marked reduction in the viable cell number in the AAD culture medium in both celllines as well as in MEF cells in a time- and dose-dependent manner (Fig 1C and 1D). In addition, treatment of CAL 27 cells with chloroquine, a well-known autophagy inhibitor [18],markedly decreased the viable cell number in the AAD culture medium (S1 Fig). The cytotoxicity of AZM was more potent than that of CAM in HNSCC cell lines, and there appeared to
be a good correlation with the efficacy for blocking autophagy shown in Fig 1B. Therefore,
blocking the efficiency of autophagy appeared to be involved in the mechanism of cytotoxicity
under the AAD culture condition. Notably, the addition of amino acids in the AAD medium
completely cancelled the cytotoxic effect of macrolides in CAL 27 cells (Fig 1E). When we
added the essential and non-essential amino acids respectively in the AAD medium, supplementation of the essential amino acids has more efficiently but not completely cancelled the
AZM-induced cell death than supplementation of the non-essential amino acids (S2 Fig). This
indicates that the cytotoxic effect of macrolides can be induced only under the shortage ofintracellular amino acids.
To examine whether apoptosis is involved in the cytotoxicity of macrolides, CAL 27 cells
were treated with AZM/CAM in the AAD culture medium in the presence or absence of apan-caspase inhibitor, Z-VAD-fmk, or an RIP1 kinase inhibitor, necrostatin-1 [13]. As shownin Fig 2A, Z-VAD-fmk, but not necrostatin-1, attenuated the macrolide-induced cytotoxicity
in a dose-dependent manner. In addition, immunoblotting with anti-PARP Ab showed thecleavage of PARP in AZM/CAM-treated CAL 27 cells under the AAD culture condition (Fig
2B). Furthermore, flow cytometry showed an increase in the number of Annexin V/PI-positive
cells (Fig 2C). All these data indicate apoptosis induction in response to AZM/CAM treatment
under the AAD culture condition.
We next confirmed the dependency on autophagy inhibition for macrolide-induced cell
death under the AAD culture condition using the m5-7 cell line, which is the atg5 tet-off MEF
system [16]. Pretreatment of m5-7 cells with doxycycline (Dox) for knockout of the atg5 gene
resulted in complete autophagy inhibition as exhibited by the absence of LC3B-II induction in
any of the conditions tested (Fig 3A). Under the AAD culture condition, Dox-pretreated m5-7

Fig 2. Apoptosis induction after treatment with macrolides under AAD culture condition. (A) CAL 27 cells in the AAD culture medium containing
10% FBS with or without macrolides were treated with various concentrations of either Z-VAD-fmk (upper panel) or necrostatin-1 (lower panel). Data arepresented as means ± SEM. *p < 0.05 (vs without Z-VAD-fmk/necrostatin-1). (B) Immunoblotting with PARP Ab from the cell lysates of CAL 27 cells
cultured in the complete culture medium or AAD culture medium with or without macrolides for 12 hrs. Immunoblotting with anti-GAPDH mAb was used as
an internal control. Cell lysate derived from HL-60 cells treated with vitamin K2 for 48 hrs was used as a positive control for apoptosis induction [17]. (C)Flow cytometry with annexin V/PI double staining after 24-hr treatment of CAL 27 cells with AZM/CAM (50 μM) in the complete culture medium or AAD
culture medium containing 10% FBS. Numbers indicate the percentage of the cells in each area.
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Fig 3. Effects of autophagy inhibition on macrolide-induced cytotoxicity in tet-off atg5 MEF cell line. (A) m5-7 cells were pre-treated with/without
doxycycline (Dox, 10 ng/mL) for 7 days for complete autophagy inhibition. Subsequently, the cells were further treated with/without macrolides for 24 hrs
under the complete culture or AAD culture conditions. The cellular proteins were separated by 15% SDS-PAGE, and immunoblotted with LC3B Ab.
Immunoblotting with anti-GAPDH mAb was used as an internal control. The positive control was the cell lysate derived from PANC-1 cells treated withAZM (50 μM) for 24 hrs and used in a previous report [13]. (B) After Dox treatment, m5-7 cells were cultured in the complete culture medium or AAD
culture medium containing 10% FBS for 24 hrs. Viable cell numbers were assessed and expressed as percentage to the viable cells cultured in thecomplete culture medium at each indicated culture period. Data are presented as means ± SEM. *p < 0.05 (Dox (-) vs Dox (+)). (C) Cell growth inhibition of
m5-7 cells cultured under the AAD culture condition with or without macrolides for 24 hrs. Data are presented as means ± SEM. *p < 0.05 (vs control).
‘n.s.’ indicates ‘not significant’.
doi:10.1371/journal.pone.0164529.g003

cells apparently showed a more reduced cell viability than Dox-untreated control cells (Fig
3B). Under complete autophagy inhibition in Dox (+) m5-7 cells, AZM treatment did not
induce further enhancement of cell growth inhibition (Fig 3C). This indicates that the cytotoxicity of macrolides is directly linked with autophagy inhibition.

Involvement of CHOP induction for macrolide-induced cell death under
AAD culture condition
It was reported that eIF2α is phosphorylated via GCN2 activation in response to nutrient deprivation, following the induction of a pro-apoptotic transcriptional factor CHOP/GADD153[19, 20]. Therefore, we speculated that CHOP might be involved in our system. As shown in
Fig 4A-1, CHOP was induced in a time-dependent manner under the AAD culture condition,and its expression became more pronounced in the presence of AZM/CAM (Fig 4A-1). Alongwith CHOP induction, eIF2α was phosphorylated to some extent, but attenuated at more than
8 hrs longer exposure to AZM under the AAD culture condition. In contrast to the AAD culture condition, we observed no CHOP induction by AZM/CAM in the cells cultured in thecomplete culture medium (Fig 4A-2).
Although the treatment of CAL 27 cells with thapsigargin, a typical ER-stress inducer via
disturbance of the calcium homeostasis, resulted in the induction of XBP-1, macrolide treatment exhibited no XBP-1s induction under the AAD culture condition (Fig 4A-3). Thus,
CHOP induction appears to be independent from the canonical UPR pathways [21]. Furthermore, in atg5 tet-off m5-7 cells, knockout of the atg5 gene resulted in pronounced CHOP
induction under the AAD culture condition (Fig 4B). This indicates that autophagy inhibition
enhances CHOP induction under the AAD culture condition.
To investigate whether CHOP is involved in cell death induction, we used a CHOP knockout MEF cell line. Under the AAD culture condition, CHOP -/- MEF cells maintained theirconsiderably higher viability than wild-type MEF cells in the presence of AZM/CAM in the
AAD culture medium (Fig 4C). Furthermore, we introduced CHOP siRNA into the CAL 27
cells. Knockdown of CHOP significantly attenuated the cytotoxic effects of AZM and CAM(Fig 4D). Therefore, macrolide-induced CHOP appears to be involved in cell death induction.
To examine whether the insufficiency of the amino acid pool is directly linked to the CHOP
induction in our system, we focused on p70S6K, which is a substrate of mammalian target of

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Fig 4. Macrolides induce cell death of CAL 27 cells under AAD culture condition via CHOP induction. (A) (1) CAL 27 cells were cultured in the AAD
culture medium containing 10% FBS with/without macrolides for various time lengths. Cellular proteins were separated by 11.25% SDS-PAGE andimmunoblotted with CHOP and phospho-eIF2α Abs. The positive control was the CAL 27 cell lysates cultured in the complete culture medium withthapsigargin (300 nM) for 6 hrs. Immunoblotting with anti-β-actin Ab was used as an internal control. (2) Immunoblotting was performed with CHOP mAb
from the cell lysates of CAL 27 cells cultured in the complete culture medium with macrolides for the indicated time periods. The same cell lysate of CAL 27
cells cultured in the AAD culture medium with AZM (50 μM) for 10 hrs was also loaded on the gel. Immunoblotting with anti-GAPDH mAb was used as aninternal control. (3) Immunoblotting was performed with XBP-1s Ab from the cell lysates of CAL 27 cells cultured in the AAD culture medium containing
10% FBS with macrolides for 8 hrs. The positive control was the CAL 27 cell lysates cultured in the complete culture medium with thapsigargin (300 nM)
for 6 hrs. Immunoblotting with anti-β-actin mAb was used as an internal control. (B) m5-7 cells with/without pre-treatment with Dox were further cultured
with macrolides in the AAD culture medium containing 10% FBS for 24 hrs. Immunoblotting was performed using anti-CHOP mAb. The positive control
was the CAL 27 cell lysates cultured in the complete culture medium with thapsigargin (300 nM) for 6 hrs. Immunoblotting with anti-GAPDH mAb was used

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as an internal control. (C) Wild-type MEF cells and CHOP -/- MEF cells were cultured in the AAD culture medium containing 10% FBS with/without
macrolides. Data are presented as means ± SEM. *p < 0.05. (D) Effects of CHOP knockdown by siRNA on CAL 27 cells: (upper panel) The expression
levels of CHOP were analyzed by immunoblotting using lysates of CAL 27 cells to which either control siRNA or CHOP siRNA was introduced and cultured
under the AAD culture condition with or without macrolides for 8 hrs. The positive control was the CAL 27 cell lysates cultured in the complete culture
medium with thapsigargin (300 nM) for 6 hrs. After 8 hrs of treatment with control siRNA and CHOP siRNA, the CAL 27 cells were further cultured underthe AAD culture condition in the presence/absence of macrolides for 48 hrs, and viable cell numbers were assessed. Data are presented asmeans ± SEM. *p < 0.05.
doi:10.1371/journal.pone.0164529.g004

rapamycin (mTOR). mTOR senses and responds to amino acid availability to modulate protein
synthesis via the phosphorylation of threonine 389 in p70S6K [22]. To evaluate intracellularamino acid sufficiency, we assessed the phosphorylation of p70S6K using the specific phosphop70S6K (Thr389) Ab, as previously reported [23] (Fig 5A). Under the AAD culture condition,
phosphorylation of p70S6K was attenuated. When AZM or CAM was added in the AAD culture medium, the suppression of the p70S6K phosphorylation was further pronounced. These
results suggest that the amino acid pool was satisfied by autophagy in the absence of macrolides,
whereas the amino acid pool became exhausted in the presence of macrolides. Furthermore, theaddition of amino acids in the AAD culture medium completely abolished CHOP induction(Fig 5B). As thapsigargin treatment induced CHOP regardless of the addition of amino acids(Fig 5C), the inhibition of CHOP induction shown in Fig 5B is not due to the direct transcriptional effect of amino acids, but is a result of insufficiency of the amino acid pool.

Discussion
In the present study, we showed that AZM and CAM blocked autophagy flux and induced cell
death in HNSCC cells via CHOP induction when the cells were cultured in the AAD culture

Fig 5. CHOP induction by macrolides under AAD culture condition in accordance with intracellular amino acid starvation in CAL 27 cells. (A)After treatment of CAL 27 cells with/without macrolides in the AAD culture medium containing 10% FBS for various lengths of time, the cells were lysedand cellular proteins were separated by 11.25% SDS-PAGE and immunoblotting with anti-phosphor-p70S6K (Thr389) and anti-p70S6K Abs. The positive
control was the cell lysate derived from Detroit 562 cells having constitutively activated PI3K mutation. Immunoblotting with anti-GAPDH mAb was used as
an internal control. (B) Addition of amino acids into the culture medium canceled CHOP induction: After 4 hrs of depletion of amino acids in the culture
medium in the presence of AZM/CAM (50 μM), amino acids were added at final concentrations of 1% MEM non-essential amino acids and 2% MEM
essential amino acids, and subsequently cultured for the indicated time periods. Thereafter, cellular proteins were separated by 11.25% SDS-PAGE andimmunoblotting with anti-CHOP mAb. The positive control was the CAL 27 cell lysates cultured in the complete culture medium with thapsigargin (300 nM)
for 6 hrs. Immunoblotting with anti-GAPDH mAb was used as an internal control. (C) CAL 27 cells were cultured with thapsigargin (300 nM) for 6 hrs with/without the addition of amino acids into the cell culture medium as described above, and immunoblotted with anti-CHOP mAb. Immunoblotting with β-actin
mAb was used as an internal control.
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Fig 6. Proposed scheme for cell death induction by macrolide antibiotics under amino acid-depleted culture condition. Under the amino aciddepleted culture condition, autophagy is induced as an adaptive response. AZM/CAM block autophagy flux leading to the shortage of the intracellularamino acid pool and failure of cellular amino acid homeostasis. This results in the induction of an ER-stress-related pro-apoptotic transcription factor,
CHOP.
doi:10.1371/journal.pone.0164529.g006

medium. Thus, we have drawn up a scheme of our observations as shown in Fig 6. In the complete culture medium, AZM and CAM showed no cytotoxicity in HNSCC cells because theamino acid pool was sufficient and CHOP was not upregulated (Figs 1C and 4A-2). Under the
AAD culture condition, autophagy was induced to compensate the amino acid pool by recycling through a self-digestive mechanism. In this case, a pro-apoptotic transcription factor
CHOP is induced at a lower level, which resulted in some reduction of the viable cell number(Figs 1C and 4A-1). However, in the presence of macrolide antibiotics under the AAD culture
condition, the shortage of the amino acid pool cannot be augmented by autophagy because of
the blocking effect on autophagy by the macrolides. This results in pronounced CHOP induction leading to enhanced cell death (Figs 1C and 4A-1).
CHOP is a well-known pro-apoptotic transcription factor induced in response to ER stressand amino acid deprivation [24, 25]. Under the AAD culture condition, the addition of AZM
and CAM prevented the phosphorylation of threonine 389 in p70S6K (Fig 5A), which indicates the failure of amino acid supply by blocking autophagy. The addition of amino acids in
the AAD culture medium completely abolished CHOP induction (Fig 5B). Therefore, CHOP
was induced as a result of amino acid pool insufficiency. Since CHOP-/- MEF attenuated thecytocidal effects of macrolides under the AAD culture condition compared with wild-type
MEF, the transcriptional activation of CHOP is at least partly involved in the macrolideinduced cell death (Fig 4C). However, since CHOP-/- MEF still exhibited some cytocidal effects
in response to macrolides (Fig 4C), other mechanisms may be involved in the cytocidal effect

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of AZM/CAM under the AAD culture condition. It has been reported that nutrient starvation
stimulates ROS production including H2O2 [26], and that the ROS accumulation induces
autophagy [27]. It has also been reported that autophagy is induced to reduce oxidative stress
via the clearance of damaged mitochondria (designated as ‘mitophagy’) and oxidized proteins[28, 29]. Therefore, we speculated that ROS might be accumulated via autophagy inhibition by
AZM/CAM under the AAD culture condition, which may directly induce cell death. However,
the addition of antioxidant reagents such as α-tocopherol and astaxanthin in the AAD culture
medium did not prevent the cytotoxic effect of AZM/CAM on CAL 27 cells (S3A Fig). Furthermore, assessment of oxidative stress by ROS-Glo™ H2O2 assay revealed a higher oxidative
stress loading in the cells cultured in the AAD culture condition regardless of the presence orabsence of macrolides (S3B Fig). Thus, ROS accumulation does not appear to explain theenhanced cell death by macrolides.
In our system, CHOP induction appears to be due to the failure of amino acid homeostasis,
as previously reported [23]. However, other mechanisms for CHOP induction have beenreported such as UPR and ROS accumulation [21, 30]. If the AZM/CAM treatment induced
accumulation of unfolded proteins by blocking the autophagy flux, these proteins should haveinduced more CHOP under the complete culture condition because of the larger source for denovo protein synthesis (i.e., amino acids) under the complete culture condition (Fig 4A-2).
Furthermore, the spliced form of XBP1, which is known to be effectively induced by UPR [31],was not induced by macrolides under the AAD culture condition (Fig 4A-3). Additionally, as
described above, although the AAD cell culture condition induced ROS production, macrolides did not further enhance ROS accumulation (S3B Fig). Thus, amino acid insufficiency
appears to be the main axis for CHOP induction in our system.
We previously reported that AZM was more effective than CAM for blocking autophagy
flux at the same concentration in the pancreatic cancer cell line PANC-1 [13]. In the CAL 27
and Detroit 562 cells, AZM exhibited a stronger cytotoxicity under the AAD culture condition
than CAM (Fig 1B), which might be correlated with their blocking efficiency of autophagy.
However, unlike AZM, CAM reduced the viable cell number in atg5 knocked-out m5-7 cells
which have no ability for autophagosome formation (Fig 3C). Thus, other molecular mechanisms might underlie the cytotoxic effect of CAM. In previous in vivo studies, CAM wasreported to inhibit angiogenesis and TGF-iogenesisc e [32, 33]. To the best of our knowledge,
there appears to be no report regarding the cytotoxic effect of CAM alone on cultured cells.
Our present results indicate that the cytotoxic effect of macrolides under the amino acidstarving condition appears to be due to at least partially apoptosis induction: cleavage of PARP
and increased number of annexin V-positive cells. However, in microscopy, we could notobserve the apparent apoptotic features in the CAL 27 cells treated with AZM under the AAD
culture condition, such as formation of apoptotic bodies, chromatin condensation, andnuclear fragmentation, although the cells showed many intercellular vacuoles (S4 Fig). Thismight be the typical features of type II programmed cell death called ‘autophagic cell death’[34]. However, in tet-off-atg5 m5-7 cells, blocking autophagy did not rescue the macrolideinduced cell death (S5 Fig). This indicates that autophagic cell death is not involved in our system. Recently, other types of cell death with vacuolization called paraptosis [35, 36], oncosis[37], and methuosis [38] have been reported, although these types of cell death should show aslight response or no response to caspase inhibitors, unlike our result shown in Fig 2A [38].
These lines of evidence suggest that the CHOP axis in response to amino acid deprivation
plays an important role for the macrolide-induced cytotoxic effect, although there might bemore than one molecular mechanism as well as type of cell death.
AZM and CAM block autophagy flux as shown in Fig 1A. In the clinical setting, it has beenreported that the long-term use of AZM in patients with cystic fibrosis increased nontuberculous

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mycobacteria infection owing to autophagy inhibition by AZM [39]. However, the molecularmechanism of blocking autophagy flux by AZM and CAM is not well understood. It is reported
that macrolide antibiotics interact with valosin-containing proteins (VCPs) without mediating
their anti-inflammatory activities [40], and that VCPs are essential for the maturation of autophagosomes [41, 42]. However, transfection of various VCP variants including the AAA-ATPasedeficient form into HEK293T cells did not show any effect on AZM-induced autophagy (datanot shown). Therefore, VCPs do not appear to be involved in the macrolide-induced autophagy
inhibition. The selective inhibition of a proton-pumping V-ATPase by bafilomycin A1 (BAF), a
macrolide and a well-known in vitro autophagy inhibitor, was previously initially evaluated [43].
BAF induces vesicular proton gradient disruption and pH increase in acidic vesicles (e.g., lysosomes). The vesicular acidification disruption by BAF possibly prevents the fusion of autophagosomes with lysosomes, causing autophagy inhibition. The reported increase in macrophagelysosomal pH by AZM may result in inhibition of lysosomal hydrolases with an optimal low pH
for their enzymatic activities [39]. According to these reports, AZM and CAM possibly disrupt
vesicular acidification and inhibit the fusion of autophagosomes with lysosomes, resulting in theblockage of autophagy flux similarly to BAF. We are currently investigating the molecular target(s) of AZM/CAM for blocking autophagy.
To date, several drugs targeting tumor angiogenesis, such as the VEGF inhibitor bevacizumab, have been used in the clinical setting [44]. These drugs are believed to starve tumor cellsby ischemia via the induction of hypovascularization, leading to the upregulation of autophagyin the central region of solid tumors [45, 46]. Thus, blocking autophagy with macrolides may
effectively enhance the cytotoxic effect of VEGF inhibitors. In fact, it has been reported thatblocking autophagy with chloroquine enhanced the cytotoxicity of bevacizumab in hepatocarcinoma, although the report suggested that the synergistic anticancer effect was due to ROS
generation [47]. Recently, amino acid transporters have been extensively investigated in cancer
cells. It has been reported that cancer cells highly express an L-type amino acid transporter 1(LAT1), whereas normal cells highly express LAT2 [48, 49]. At present, an LAT1-specificinhibitor is undergoing phase I clinical trial. Therefore, macrolides could be potentially usedby the combination of these drugs in cancer patients.

Conclusions
We demonstrated that AZM and CAM block autophagy flux and cause cell death under the
AAD culture condition via CHOP induction owing to the failure of internal amino acidhomeostasis. This study indicates the possibility of using macrolides as a novel ‘tumor-starving
therapy’ in cancer treatment.

Supporting Information
S1 Fig. Cell growth inhibition of CAL 27 cells under normal or AAD culture condition in
the presence/absence of chloroquine. CAL 27 cells were cultured with/without 50 μM chloroquine either in the complete culture medium or AAD culture medium for the indicated periodof time. Then, viable cell number was assessed and expressed as percentage to the viable cells
cultured in the complete culture medium at each indicated culture period. Data are presented
as means ± SEM.  p < 0.05.
(TIF)
S2 Fig. Effects of supplementation of essential and non-essential amino acids in AZMinduced cytotoxicity under the AAD culture condition on CAL 27 cells. CAL 27 cells were
cultured in the AAD culture medium supplemented with essential and non-essential amino

PLOS ONE | DOI:10.1371/journal.pone.0164529 December 15, 201612 / 16-140-

Tumor-Starving Therapy Using Macrolide Antibiotics in HNSCC

acids with/without AZM (50 μM). ‘Essential’ and ‘non-essential’ indicate 2% MEM essential
amino acids and 1% MEM non-essential amino acids at the final concentration (Wako),respectively. The number of viable cells was determined and compared with that of viable cells
cultured under the AAD culture condition without AZM.  p < 0.05. ‘n.s.’ indicates ‘not significant’.
(TIF)
S3 Fig. Effects of ROS in macrolide-induced cytotoxicity on CAL 27 cells. (A) CAL 27 cells
were cultured with macrolides under the AAD culture condition with 10% FBS with/without
the two types of ROS scavengers, namely, α-tocopherol (50 μM) and astaxanthin acid (25 μM)
for 48 hrs. (B) CAL 27 cells were cultured with macrolides under the complete or AAD culture
condition for 6 hrs. ROS production was assessed using ROS-Glo™ H2O2 Assay (Promega) as
described in Materials and Methods. ‘n.s.’ indicates ‘not significant’.
(TIF)
S4 Fig. Morphological changes after macrolide treatment in CAL 27 cells. May-GrünwaldGiemsa staining was performed after treatment with or without macrolides under the normal
or AAD culture condition for 24 hrs.
(TIF)
S5 Fig. Effects of autophagy inhibition on macrolide-induced cytotoxicity under AAD culture condition. m5-7 cells with/without pretreatment with Dox (10 ng/mL) were cultured
under the normal culture or AAD culture condition with AZM/CAM (50 μM) for 24 hrs. Viable cell number is expressed as the percentage of viable m5-7 cells with/without Dox under thenormal culture condition. Data are presented as means ± SEM. ‘n.s.’ indicates ‘not significant’.
(TIF)

Acknowledgments
We thank Professor Noboru Mizushima (The University of Tokyo, Tokyo, Japan) for the kind
gift of the tet-off atg5 m5-7 cell line.
We also thank Dr. Edward Barroga, Associate Professor and Senior Medical Editor fromthe Department of International Medical Communications of Tokyo Medical University forediting the manuscript.

Author Contributions
Conceptualization: K. Miyazawa.
Data curation: AH K. Miyazawa.
Formal analysis: KH.
Funding acquisition: K. Miyazawa.
Investigation: KH SM K. Miyahara JT AA MH HK.
Methodology: KH SM KM.
Project administration: K. Miyazawa.
Resources: MI.
Supervision: K. Miyazawa.
Validation: SM AH AA MH K. Miyahara.

PLOS ONE | DOI:10.1371/journal.pone.0164529 December 15, 201613 / 16-141-

Tumor-Starving Therapy Using Macrolide Antibiotics in HNSCC

Visualization: AH KM.
Writing – original draft: KH.
Writing – review & editing: K. Miyazawa KT.

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Targeting bortezomib-induced aggresome formation using
vinorelbine enhances the cytotoxic effect along with
ER stress loading in breast cancer cell lines
KANA MIYAHARA1, HIROMI KAZAMA2, HIROKO KOKUBA3, SEIICHIRO KOMATSU1, AYAKO HIROTA2,
JUN TAKEMURA2, KAZUHIRO HIRASAWA4, SHOTA MORIYA2, AKIHISA ABE2,
MASAKI HIRAMOTO2, TAKASHI ISHIKAWA1 and KEISUKE MIYAZAWA2Departments of 1Breast Surgery and 2Biochemistry, 3Laboratory of Electron Microscopy, and4
Department of Otolaryngology (Head and Neck Surgery), Tokyo Medical University, Tokyo, Japan
Received June 17, 2016; Accepted August 8, 2016
DOI: 10.3892/ijo.2016.3673
Abstract. The ubiquitin-proteasome and autophagy-lysosome
pathways are two major self-digestive systems for cellular
proteins. Ubiquitinated misfolded proteins are degradedmostly by proteasome. However, when ubiquitinated proteins
accumulate beyond the capacity of proteasome clearance, they
are transported to the microtubule-organizing center (MTOC)
along the microtubules to form aggresomes, and subsequently
some of them are degraded by the autophagy-lysosome
system. We previously reported that macrolide antibiotics
such as azithromycin and clarithromycin block autophagy
flux, and that concomitant treatment with the proteasome
inhibitor bortezomib (BZ) and macrolide enhances endoplasmic reticulum (ER) stress-mediated apoptosis in breast
cancer cells. As ubiquitinated proteins are concentrated at
the aggresome upon proteasome failure, we focused on the
microtubule as the scaffold of this transport pathway for
aggresome formation. Treatment of metastatic breast cancer
cell lines (e.g., MDA-MB-231 cells) with BZ resulted in induction of aggresomes, which immunocytochemistry detected
as a distinctive eyeball-shaped vimentin-positive inclusion
body that formed in a perinuclear lesion, and that electron
microscopy detected as a sphere of fibrous structure with somedense amorphous deposit. Vinorelbine (VNR), which inhibits
microtubule polymerization, more effectively suppressed
BZ-induced aggresome formation than paclitaxel (PTX),
which stabilizes microtubules. Combined treatment using BZand VNR, but not PTX, enhanced the cytotoxic effect andapoptosis induction along with pronounced ER stress loading

Correspondence to: Dr Keisuke Miyazawa, Department ofBiochemistry, Tokyo Medical University, 6-1-1 Shinjuku, Shinjuku-ku,
Tokyo 160-8402, Japan
E-mail: miyazawa@tokyo-med.ac.jp

Key words: breast cancer, vinorelbine, bortezomib, aggresome, ER

stresssuch as upregulation of GRP78 and CHOP/GADD153. The
addition of azithromycin to block autophagy flux in the BZ
plus VNR-containing cell culture further enhanced the cytotoxicity. These data suggest that suppression of BZ-induced
aggresome formation using an inhibitory drug such as VNR
for microtubule polymerization is a novel strategy for metastatic breast cancer therapy.
Introduction
Breast cancer is the most common cancer, accounting for 25%
of all cancer cases in females worldwide (1). Developmentof novel drugs, including molecular targeting for breast
cancer, has improved some therapeutic outcomes. However,recurrence or metastasis of breast cancer continues to occur,
resulting in 521,900 deaths annually worldwide (1). Therefore,a novel therapeutic approach to recurrent/metastatic breast
cancer is an important issue. Chemotherapeutic, hormone,and molecular targeting drugs are currently used for metastatic breast cancer therapy. Anthracyclines and taxanes are
considered the key chemotherapeutic drugs (2). Several recent
studies focused on autophagy and paclitaxel (PTX), a wellused taxane for metastatic breast cancer therapy (3-5). Some
reports indicate that PTX inhibits autophagy in breast cancer
cell lines (3), whereas others indicate that autophagy promotes
PTX resistance in breast cancer (4,5).
The autophagy-lysosome system and the ubiquitin-proteasome system (UPS) are two major self-digestive mechanisms
for cellular proteins. UPS is the selective degradation pathway
for most cytoplasmic proteins, especially misfolded and shortlived proteins (6). In this process, misfolded proteins taggedwith ubiquitin are selectively degraded by the 26S proteasome.
In contrast, macroautophagy (hereafter, autophagy) is a highly
conserved self-digestive system in eukaryotic cells for degradation of long-lived proteins, as well as clearance of damaged
or old organelles, including endoplasmic reticulum (ER) (7).
These cellular proteins and organelles are engulfed in the
double-membrane vesicle known as the autophagosome andare delivered to the lysosome for degradation (7,8). In contrast
with the UPS, autophagy is a bulk non-selective degradation-145-

MIYAHARA et al: COMBINED TREATMENT WITH VINORELBINE AND BORTEZOMIB IN BREAST CANCER

system. However, recent reports found crosstalk between UPS
and autophagy via docking proteins such as p62/SQSTM-1, or
NBR1 (neighbor of BRCA1 gene product), in which they haveboth the ubiquitin-associated domain and the LC3 (Atg8)interacting lesion in their C-terminus. LC3-II (lipidated formof LC3-I) studs the inner and outer autophagosome membrane.
Thus, after binding to p62, ubiquitinated proteins are engulfed
into autophagosome through the p62-LC3-II interaction (9,10).
The original function of autophagy is cellular quality control
of cytoplasmic components and protection from nutrient starvation by amino acid recycling. However, it is now believedthat cancer cells apply this system to escape from various
stresses under the hypovascular condition (11-14).
We previously reported that combined treatment using theproteasome inhibitor bortezomib (BZ) and macrolide antibiotics, which block autophagy flux, resulted in pronounced
apoptosis induction, along with ER stress loading in breast
cancer and multiple myeloma cells (15-17). ER stress is causedby the accumulation of misfolded or unfolded proteins inside
the ER lumen. A series of cellular responses is evoked as
unfolded protein responses (UPRs) such as: i) attenuation of
the translation for repression of unfolded protein accumulation, ii) induction of chaperon proteins for proper refolding,and iii) exporting of unfolded proteins to outside the ER
lumen, following their polyubiquitination and degradationby proteasome [ER-associated degradation (ERAD)] (18-21).
However, when the stress exceeds this adaptive capacity, thecell undergoes apoptosis via induction of the proapoptotic
transcription factor CHOP/GADD153 and other mechanisms(19,22-25). ER stress itself also induces autophagy directly
through upregulation of GRP78, which appears to compensate
ERAD-mediated degradation (26).
In addition to these cellular digestive systems, the cytoplasmic misfolded proteins aggregate and are transported
to the microtubule-organizing center (MTOC) along the
microtubules by the retrograde microtubule motor dynein and
HDAC6 complex to form aggresomes (27-30). An aggresome
is a cytoplasmic inclusion body containing misfolded and ubiquitinated protein aggregates originally described by Johnston
and colleagues in 1998 (27). Aggresome formation is a protective response of a cell, which involves sequestering toxic
protein species beyond the capacity of UPS clearance (28-33).
Some parts of the aggresome are degraded by autophagy in an
HDAC6-dependent manner (30,32). Therefore, there appears
to be an integrated intracellular network for misfolded protein
clearance among UPS, autophagy-lysosome system, andaggresome formation. Indeed, our previous reports confirmedthat simultaneous inhibition of proteasome by BZ, autophagy
flux by macrolide antibiotics, and aggresome formation using
suberoylanilide hydroxamic acid (SAHA), which has the effectof HDAC6 inhibition, potently induce apoptosis accompaniedwith ER stress loading in breast cancer and multiple myeloma
cells (34,35).
In this study, we focused on the microtubule that functions as the scaffold of the intracellular trafficking pathway
for protein aggregate transport to MTOC to form aggresomes.
Taxanes including PTX and docetaxel, and vinca alkaloids
such as vinorelbine (VNR) are widely used in metastatic
breast cancer therapy (2). Taxanes have the effect of microtubule stabilization, whereas VNR has the inhibitory effect of1849

microtubule polymerization. Therefore, we hypothesized thatthese treatments might have the inhibitory effect of aggresome
formation along with ER stress loading upon proteasome inhibition. Here, we demonstrate that combined treatment using
BZ and VNR, but not PTX, pronounces apoptosis induction
in metastatic breast cancer cell lines, along with ER stressloading and inhibition of BZ-induced aggresome formation.
Materials and methods
Reagents. BZ was purchased from Selleck Chemicals(Houston, TX, USA). Azithromycin dihydrate (AZM) was
purchased from Tokyo Chemical Industry (Tokyo, Japan).
PTX, VNR, and colchicine were obtained from Wako Pure
Chemical Industries (Osaka, Japan). BZ was dissolved indimethyl sulfoxide (DMSO) to make stock solution at aconcentration of 1 mM. AZM and PTX were dissolved in 95%
ethanol at a concentration of 5 mM as stock solutions. VNR
was dissolved in distilled water to prepare a stock solution of
1 mM. Colchicine was dissolved in 95% ethanol at a concentration of 10 mM as stock solution.
Cell lines and culture conditions. MDA-MB-231 cells werea kind gift from Dr Keiichi Iwaya (Department of Basic
Pathology, National Defense Medical College, Saitama,
Japan), and MDA-MB-468 cells were obtained from theAmerican Type Culture Collection (ATCC) (Manassas, VA,
USA). A CHOP-/- MEF cell line (CHOP-KO-DR) establishedfrom a 13.5-day-old CHOP-/- mouse embryo by SV-40
immortalization and a CHOP+/+ MEF cell line (DR-wild-type)established by SV-40 immortalization as a control cell line for
CHOP-KO-DR were obtained from ATCC. MDA-MB-231,
MDA-MB-468, CHOP-KO-DR, and DR-wild-type cells
were cultured in Dulbecco's modified Eagle's medium(Sigma-Aldrich, St. Louis, MO, USA) supplemented with
10% fetal bovine serum (FBS; Biowest, Kiltimagh, Ireland),penicillin (100 U/ml), and streptomycin (100 µg/ml). All celllines were cultured in a humidified incubator containing
5% CO2 and 95% air at 37˚C.
Assessment of the viable number of cells. The number of
viable cells was assessed using CellTiter-Blue®, a cell viability
assay kit (Promega Co., Madison, WI, USA), with fluorescencemeasurements at 560 nm for excitation and 590 nm for emission.
Flow cytometry. For assessment of apoptosis, cells were
stained with Annexin V and propidium iodide (PI) using
APOPCYTOTM Annexin V-Azami-Green Apoptosis Detection
kit (MBL, code 4690, Nagoya, Japan) according to the manufacturer's instructions and subjected to flow cytometry using
Attune® Acoustic Focusing Cytometer (Life Technologies,
CA, USA).
Immunoblotting. Immunoblotting was performed as previously described (15). Cells were lysed with RIPA lysis buffer(Nacalai Tesque, Kyoto, Japan) supplemented with a proteaseand phosphatase inhibitor cocktail (Nacalai Tesque). Cellular
proteins were quantified using a DC Protein assay kit of
Bio-Rad (Richmond, CA, USA). Equal amounts of proteins

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were loaded onto the gels, separated by SDS-PAGE, andtransferred onto Immobilon-P membranes (Millipore Corp.,
Bedford, MA, USA). These membranes were probed with
first antibodies (Abs) such as anti-ubiquitin (P4D1) mAb
and anti-GAPDH (6C5) mAb purchased from Santa Cruz
Biotechnology, Inc. (Santa Cruz, CA, USA). Immunoreactive
proteins were detected using horseradish peroxidaseconjugated second Abs and an enhanced chemiluminescencereagent (ECL) (Millipore). Densitometry was performed usinga Molecular Imager ChemiDoc XRS System (Bio-Rad).
Gene expression analysis. Real-time polymerase chain reaction (PCR) for expression analysis of ER stress-related genes
was performed as previously described in detail (15).
Electron microscopy. Cells were fixed with 2.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.3) for 1 h. The samples
were further fixed in 1% osmium tetroxide for 1 h, dehydrated
in graded ethanol (30-100%), and embedded in Quetol 812
epoxy resin (Nisshin EM Co., Ltd., Tokyo, Japan). Ultrathin
sections were cut using an Ultracut J microtome (Reichert
Jung, Vienna, Austria). These sections were stained withlead nitrate and uranium acetate and subjected to electron
microscopic analysis using a scanning electron microscope
JEM-1200EX II (Jeol, Tokyo, Japan).
Immunofluorescence staining and confocal microscopy.
MDA-MB-231 cells were seeded on 13-mm glass coverslips
in a 24-well culture plate in the presence or absence of BZ,
PTX, and VNR and cultured for 24 h. Coverslips were washedtwice with PBS and fixed for 15 min on ice in methanol.
After washing twice with PBS, cells were permeabilized with
0.1% Triton X-100 in TBST for 5 min at room temperature. The
coverslips were then washed with TBST and further incubatedwith TBST containing 10% normal goat serum (NGS; Thermo
Fisher Scientific, Waltham, MA, USA) for 30 min at room
temperature for blocking nonspecific binding. Cells were incubated with a primary antibody such as mouse anti-vimentin(V9) mAb, mouse anti-ubiquitin (P4D1) mAb, and anti-p62/
SQSTM1 (D-3) mAb (all from Santa Cruz), and diluted in
TBST, 1.5% NGS, 0.1% bovine serum albumin (BSA) at 4˚Covernight. The coverslips were washed three times for 5 minwith TBST at room temperature and subsequently incubatedwith Alexa Fluor® 488 conjugate-goat anti-mouse IgG (H+L)
secondary antibody (Thermo Fisher Scientific), diluted in
TBST containing 1.5% NGS and 0.1% BSA, for 45 min at
37˚C. The coverslips were washed with TBST and mounted
in ProLong ® Diamond Antifade Montant (Thermo Fisher
Scientific). Nuclei were stained with DAPI (Sigma-Aldrich,
D-9542), and the cells were imaged using a confocal laser
scanning fluorescence microscope, LSM 700 (Carl Zeiss,
Germany).
Assessment of aggresome by fractioning of detergent-solubleand -insoluble proteins, followed by immunoblotting with
anti-ubiquitin mAb. Cells were lysed with Triton X-100 lysis
buffer (10 mM Tris-HCl, 150 mM NaCl, 2% Triton X-100,
pH 7.8) supplemented with a protease inhibitor cocktail(Nacalai Tesqucollectede). The lysates were centrifuged at
12,000 g for 30 min at 4˚C. The supernatant was then collected

as a detergent-soluble fraction. The pellets (which containinsoluble proteins) were then resuspended in sodium dodecyl
sulfate (SDS) lysis buffer (10 mM Tris-HCl, 150 mM NaCl,
2% SDS, pH 7.8) and sonicated for 30 sec using a tip sonicator
VP-5S (Taitec, Saitama, Japan) to prepare a detergent-insoluble
fraction. All resuspended pellets and supernatants were boiled
for 5 min in the presence of an equal volume of SDS-PAGE
sample buffer (125 mM Tris-HCl, 4% SDS, 20% glycerol,
0.002% BPB, pH 6.8), and the containing proteins wereseparated by 11.25% SDS-PAGE and immunoblotted with
anti-ubiquitin mAb.
Morphology assessment. After trypsinization, cell suspensions
were sedimented and fixed on the slide glass using a Cytospin 4(Thermo). Preparations were stained with May-GrünwaldGiemsa and examined using a digital microscope BZ-8100(Keyence Co., Osaka, Japan).
Statistical analysis. All the quantitative data were expressed
as mean ± standard deviation (SD). Statistical analysis was
performed with two-tailed nonpaired Student's t-test. The
criterion for statistical significance was taken as p<0.05.
Results
Combined treatment using BZ plus VNR, but not PTX
enhances apoptosis induction in breast cancer cell lines.
It was reported that PTX inhibited autophagy flux in breast
cancer cell lines (3). We previously reported that simultaneous treatment with the proteasome inhibitor BZ and AZM,
which has an inhibitory effect on autophagy flux, resulted
in enhanced cytotoxicity along with ER stress loading in
multiple myeloma cells (17). Therefore, we hypothesized thatcombined treatment using BZ and PTX instead of AZM mightalso enhance the cytotoxic effect in metastatic breast cancer
cell lines. MDA-MB-231 and MDA-MB-468 cells were firsttreated with BZ alone at various concentrations for 24-72 h.
Both cell lines exhibited cell growth inhibition in a dose- andtime-dependent manner. Fifty percent cell growth inhibitory
concentrations (IC50s) at 48-h exposure to BZ were 36.3 nM
in MDA-MB-231 cells and 10.8 nM in MDA-MB-468 cells(Fig. 1A). The cells were then treated with PTX as well as VNR
in the presence or absence of 25 nM BZ in MDA-MB-231 cells
and 7.5 nM in MDA-MB-468 cells. As illustrated in Fig. 1B,
assessment of viable cells indicated significant enhancementof cell growth inhibition by combining 10 nM PTX and 25 nM
BZ. Unexpectedly, pronounced cytotoxicity did not increase
further and reached a plateau at higher concentrations of
PTX up to 100 nM. In contrast with the PTX combination,
VNR plus BZ resulted in more prominent enhancement ofcell growth inhibition in the dose-dependent manner of VNR.
This phenomenon was more pronounced with the addition of
AZM in the cell culture medium. When the cells were treatedwith BZ and AZM in the presence of PTX or VNR, only the
VNR combination exhibited considerable enhancement ofcell growth inhibition in both cell lines (Fig. 1C). Flow cytometric analysis with Annexin V/PI double staining indicateda significant increase of Annexin V-positive/PI-negative cells(cells undergoing early-stage apoptosis) and Annexin V/PI
double-positive cells (undergoing late-stage apoptosis) only

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Figure 1. Cell growth inhibition and apoptosis induction of MDA-MB-231 and MDA-MB-468 cells after combined treatment using PTX or VNR and BZ in
the presence or absence of AZM. (A) MDA-MB-231 and MDA-MB-468 cells were treated with BZ at various concentrations for 24, 48 and 72 h. The viable
cell number was assessed using CellTiter Blue assay as described in Materials and methods. (B) MDA-MB-231 and MDA-MB-468 cells were treated with
PTX or VNR at various concentrations with/without BZ (25 nM for MDA-MB-231 cells and 7.5 nM for MDA-MB-468 cells) for 48 and 72 h. *P<0.05; PTX
vs. PTX+BZ, VNR vs. VNR+BZ. (C) MDA-MB-231 and MDA-MB-468 cells were treated with PTX or VNR in the presence of BZ (25 and 5 nM) and AZM(50 µM) for 48 and 72 h. *P<0.05; PTX vs. PTX+BZ+AZM, VNR vs. VNR+BZ+AZM.

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Figure 1. Continued. (D) MDA-MB-231 cells were treated with BZ (25 nM), PTX (50 nM), or VNR (50 nM) alone or a combination of BZ and PTX, or BZ
and VNR for 24 h. Flow cytometric analysis with Annexin V/PI double staining was performed. Numbers indicate the percentage of the cells in each area tothe cells in whole gating.

after combined treatment using BZ plus VNR, but not using
BZ plus PTX (Fig. 1D). These data indicate that VNR is superior to PTX in combination with BZ for apoptosis induction inbreast cancer cell lines.
Inhibition of BZ-induced aggresome formation using VNR
and PTX in MDA-MB-231 cells. Aggresome formation occurs
when production of aggregation-prone misfolded protein
exceeds the capacity of proteasome digestion. In aggresomes,
protein aggregates are actively degraded by the autophagylysosome pathway (29,31,35). Hence, we next examined whether
aggresome is induced in MDA-MB-231 cells in response toproteasome inhibition by BZ. Immunocytochemistry using
anti-vimentin mAb exhibited a single eyeball-shaped densebody in the perinuclear lesion after 24-h treatment with BZ(Fig. 2A), which appears to indicate the marker of aggresome
formation at MTOC, as previously reported (27,32). Although
the intracellular structure of vimentin filaments changed greatly
after exposure to BZ compared with untreated cells, we could
detect only 20% of the spherical body-positive MDA-MB-231
cells during 24-h exposure to BZ. Extended BZ exposure timeor increased BZ concentration resulted in increasing dead cells
and losing optimal conditions for detecting a single sphericalaggresome. Immunoblottings with anti-ubiquitin and anti-p62
mAbs exhibited the assembly of ubiquitinated proteins as wellas p62 in the perinuclear lesion in response to BZ, but they
were not as dense as the vimentin profile (Fig. 2A). Electron
microscopy indicated that, in agreement with the sphericalbody stained with anti-vimentin Ab, a different density
area was observed in the perinuclear lesion surrounded by
mitochondria (Fig. 2B). At higher magnification, substantial
condensation of fibrous structure with some dense amorphousdeposits became evident in the spherical body area. These data
indicate aggresome formation in response to BZ treatment in
MDA-MB-231 cells.
PTX inhibits microtubule depolymerization, whereas VNR
inhibits microtubule polymerization (2,3). As shown in Fig. 3,morphologic features at metaphase were different after PTXand VNR-treatment. Since protein aggregates are transported
along the microtubules to MTOC for aggresome formation,
we next assessed the inhibitory effect of PTX and VNR on
the blocking efficacy of aggresome formation. As indicatedin Fig. 4, both PTX and VNR inhibited aggresome formation,
along with the scattering of ubiquitinated protein and p62 in
the cytoplasm. Although the fibrous structure appeared tobe somewhat different between PTX and VNR treatments,
quantitative assessment of the efficacy of aggresome inhibition was difficult using immunocytochemistry. Therefore, we
performed immunoblotting using anti-ubiquitin (P4D1) mAb
to detect the polyubiquitinated proteins in detergent-soluble
and detergent-insoluble fractions (Fig. 5). After treatment of
MDA-MB-231 cells with BZ, polyubiquitinated proteins in
the detergent-insoluble fraction, which indicate aggresome,increased (lane 12) with some increase in the detergent-soluble
fraction, compared with untreated control cells. Treatment of
the cells using BZ plus AZM further enhanced aggresome inthe detergent-insoluble fraction (lane 16), as well as the detergent-soluble ubiquitinated-proteins (lane 6). This condition
appears to indicate overflowing of the ubiquitinated proteins
by simultaneous inhibition of two major protein degradation
systems. It is noteworthy that ubiquitinated proteins in thesoluble fraction increased more with BZ plus VNR treatment
than with BZ plus PTX (lanes 7 vs. 8). The fact that treatment with VNR alone did not increase ubiquitinated proteins-149-

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Figure 2. Aggresome formation after treatment using BZ in MDA-MB-231 cells. (A) After 24-h treatment using BZ at 25 nM, immunocytochemistry was
performed using anti-vimentin mAb, anti-ubiquitin mAb, and anti-p62 mAb. DAPI staining indicates the position of the nucleus (blue). Dotted white line
represents cell outline. Scale bar 10 µm. (B) Electron microscopy: MDA-MB-231 cells were treated with BZ (25 nM) for 24 h.

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Figure 3. Comparison of morphological changes in MDA-MB-231 cells with PTX and VNR treatment. MDA-MB-231 cells were stained with May-GrünwaldGiemsa. Cells were cultured with PTX (50 nM) or VNR (50 nM) for 48 h. Scale bar 20 µm.

Figure 4. Inhibitory effect of VNR and PTX on BZ-induced aggresome formation in MDA-MB-231 cells. MDA-MB-231 cells were treated with BZ (25 nM)
with/without either PTX (50 nM) or VNR (50 nM) for 24 h. Immunocytochemistry was performed using anti-vimentin mAb, and anti-Ub mAb. DAPI staining
indicates the position of the nucleus (blue). Scale bar, 10 µm.

in the soluble fraction (lane 5) confirms that VNR inhibits
BZ-induced aggresome formation more efficiently than PTX.
This phenomenon becomes more apparent in the presence of
AZM for blocking autophagy flux (lanes 9 vs. 10).

Enhancement of ER stress loading after combined treatment using BZ and VNR. Our previous report indicated thatblocking BZ-induced aggresome by HDAC6 inhibition using
SAHA resulted in ER stress loading, which led to CHOP--151-

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Figure 5. Assessment of aggresome inhibition by immunoblotting using anti-ubiquitin mAb. (A) MDA-MB-231 cells were treated with BZ (25 nM), AZM(50 µM), PTX (50 nM), or VNR (50 nM) alone or two or three combinations as indicated for 48 h. The method of preparing samples of cellular proteins forsoluble and insoluble fractions is described in detail in Materials and methods. Each fractionated protein was loaded and separated by 11.25% SDS-PAGE, andimmunoblotted with anti-ubiquitin-mAb. In detergent-soluble fractions, immunoblotting with anti-GAPDH mAb was performed as an internal control. (B) After
immunoblotting, the transferred membrane was re-stained with Coomassie Brilliant Blue R-250 for the assessment of protein loading. (C) Densitometry of thepolyubiquitinated proteins in the detergent-soluble and detergent-insoluble fractions: densitometry of the square areas denoted by the dotted line in the upper
panel was performed, and the density ratios to the controls (lanes 1 and 11) were plotted. In the soluble fractions, each lane density was standardized by thedensity ratios to GAPDH as an internal control.

mediated apoptosis induction in myeloma and breast cancer
cells (34,35). Therefore, we assessed ER stress-related gene
expression after combined treatment using BZ and PTX or
VNR in MDA-MB-231 cells. During 48-h exposure to thesereagents, real-time PCR exhibited pronounced expression of
GRP78 and CHOP with combined treatment using BZ plus
VNR, but not using BZ plus PTX, compared with those treatedusing each reagent alone (Fig. 6). Thus, the gene expression

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Figure 6. ER stress loading after combined treatment using BZ and PTX
or VNR in MDA-MB-231 cells. MDA-MB-231 cells were treated with BZ(25 nM), PTX (50 nM), or VNR (50 nM) alone or a combination of BZ and
PTX, or BZ and VNR for 24 h and 48 h. Gene expression of CHOP and
GRP78, which are related to ER stress loading, were assessed using real-time
PCR.

profiles fit well with the result of the cell growth inhibition
presented in Fig. 1B.
Since the proapoptotic transcription factor CHOP plays acentral role in ER stress-related apoptosis induction (23,25),
we next examined whether CHOP is involved in pronounced
cytotoxicity by combined treatment with VNR and BZ+AZM
using immortalized MEF cells derived from CHOP knockout
mouse. VNR enhanced BZ+AZM-induced cytotoxicity inimmortalized wild-type MEF in a dose-dependent manner,
whereas CHOP-/- MEF exhibited less sensitivity to BZ+AZM
treatment and attenuated enhanced cytotoxicity by VNR(Fig. 7). Notably, as well as breast cancer cell lines, PTX exhibited no enhanced cytotoxicity in the presence of BZ+AZM
in MEF cell line (Figs. 1C and 7). These data suggest that
pronounced apoptosis induction by BZ and VNR is at least
partially mediated through CHOP induction.
Discussion
Herein, we demonstrated that VNR, which has inhibitory
effect on microtubule polymerization, blocked BZ-induced
aggresome formation (Fig. 5), and concomitant treatment
of VNR and BZ with/without AZM markedly enhanced
the cytotoxic effect and apoptosis induction, along with

Figure 7. Involvement of CHOP in enhanced cytotoxicity with a combinationof BZ and VNR+AZM. Cell growth inhibition of wild-type MEF cell line
and CHOP-/- cell line after treatment using VNR or PTX with/without BZ(25 nM) + AZM (50 µM) for 24 h. The viable cell number was assessed using
CellTiter Blue assay as described in Materials and methods. *P<0.05; PTX vs.
PTX+BZ+AZM, VNR vs. VNR+BZ+AZM.

pronounced ER stress loading, in metastatic breast cancer celllines (Figs. 1 and 6). Since PTX inhibits autophagy in breast
cancer cells (3), we initially expected that simultaneous inhibition of the two major digestive systems, proteasome by BZ
and autophagy by PTX, would enhance ER stress-mediatedapoptosis induction, as was the case with BZ plus AZM in ourprevious report on myeloma cells (34). However, contrary to
our initial expectation, no prominent pronounced cytotoxicity
was observed with the BZ and PTX combination (Fig. 1). For
now, we cannot clearly explain the difference in the effectsof the drugs regarding the cytotoxicity of BZ. It may be dueto the pharmacological difference in disrupting microtubules(i.e., inhibition of microtubule polymerization by VNR versus
microtubule stabilization by PTX) (Fig. 3). Indeed, colchicine,which has an inhibitory effect on microtubule polymerization,
also exhibited enhanced cytotoxicity of BZ in MDA-MB-231
cells, like VNR (data not shown). In addition, VNR appears
to be more effective for suppressing BZ-induced aggresome
formation than PTX. This results in an increase of cytosolic
detergent-soluble ubiquitinated proteins (Fig. 5) and enhances-153-

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ER stress loading (Fig. 6), following ER stress-mediated apoptosis via CHOP activation (Figs. 1D and 7).
In addition to aggresome inhibition, recent studies
indicated that subcellular lysosomal positioning was also
affected by microtubule formation. Depolymerization of
the microtubules by nocodazole resulted in the scattering oflysosomes and reduced the nutrient-dependent activation ofmTORC1 (36). Proteasome failure promoted the positioningof lysosomes around the aggresome via local blocking of the
microtubule-dependent transport (37). Microtubules support
the assembly of pre-autophagosomal structures and mediate
trafficking of autophagosomes toward lysosomes (38). Thus,
perturbation of microtubules directly affects the encounter
between autophagosome and lysosome to form autolysosome,leading to suppression of protein degradation by the autophagylysosome system. Microtubules also regulate mTORC1 andclass III PI3K complexes, two major complexes involved in
the initiation of autophagic response (39). All this evidence
suggests that disrupting microtubules interrupts the digestive function of autophagy as well as aggresome formation.
However, treatment using VNR or PTX alone did not increase
polyubiquitinated protein in the detergent-soluble fraction,
whereas AZM induced some detergent-insoluble aggresome,
but far less than BZ. Thus, proteasome plays a critical role inubiquitinated protein clearance.
Since intracellular unfolded protein aggregates are cytotoxic, aggresome is assumed to play a role in protecting cells byencapsulating these protein aggregates by vimentin filaments
when unfolded proteins accumulate beyond the capacity of
UPS (31,32). After 24-h exposure to BZ for proteasome inhibition, MDA-MB-231 cells exhibited an eyeball-shaped vimentin
positive-inclusion body in the perinuclear lesion (Fig. 2A). Thismorphological feature seems to fit with the original description
regarding the aggresome by Johnson et al (27): a pericentriolar
membrane-free, cytoplasmic inclusion containing misfolded,
ubiquitinated protein ensheathed in a cage of vimentin, a typeIII intermediate filament protein. However, analysis using
electron microscopy indicated that this spherical body wascomposed mostly of fibrous structure, and electron-dense
deposits in perinuclear lesions were much smaller than the
typical aggresomes observed in various neurodegenerative
disorders such as Parkinson's disease, Alzheimer's disease,and Huntington's disease (27,32,39) (Fig. 2B). This may be
due to the rapid turnover of tumor cells and their upregulated
metabolic state. It was reported that BZ induced aggresome
formation in pancreatic cancer cells but not in immortalized
normal human pancreatic epithelial cells (40), also suggesting ahigher level of protein synthesis and dependency on the proteasome degradation system for survival in cancer cells than innormal cells. In our system, MDA-MB-231 cells might undergo
apoptosis in response to BZ before presenting the characteristicfeatures of aggresome. However, upon proteasome inhibition by
BZ, how dynamic remodeling of vimentin filaments occurs toform a perinuclear sphere body, even with insufficient amounts
of protein aggregate deposits, remains unclear. Other thancellular protein accumulation followed by transport along the
microtubules toward the centriole, some molecular switch may
occur to initiate the aggresome formation. Vimentin filamentsinteract with signaling proteins such as phospholipase A2,
14-3-3 proteins, and bind to phosphorylated ERK and RhoK

1857

(41,42). A recent report indicated that vimentin C328 is essential for the binding site with zinc to lead to optimal vimentin
performance in network expansion, aggresome formation, andlysosomal distribution (43). This may indicate the existence of
signal recognition site(s) of vimentin to initiate conformationalchanges. Thus, clarification of crosstalk between proteasomeand vimentin is an attractive challenge. More precise timecourse study is required.
In this study, we intentionally used clinically available
drugs. VNR and PTX are key drugs for metastatic breast
cancer therapy, whereas BZ is widely used to treat multiple
myeloma. Their safety has been established, and information
regarding pharmacokinetics and adverse effects has been
accumulated. Thus, inhibition of BZ-induced aggresome
formation using VNR could be a potent practicable combination, based on a novel concept for metastatic breast cancer
patients. Furthermore, MDA-MB-231 and MDA-MB-468
cell lines used in this study have the characteristics of triplenegative breast cancer (TNBC), a subtype of tumor estrogen
receptor (ER)-negative, progesterone receptor (PgR)-negative,
and human epidermal receptor 2 (HER2)-negative, BZ plus
VNR might be effective combination for TNBC therapy. Ourdata also suggest that targeting the intracellular proteostatic
regulatory network among proteasome, autophagy-lysosome,and aggresome has potential for cancer therapy.
Acknowledgements
This study was supported by funds provided through a MEXTsupported program of the Strategic Research Foundation atPrivate Universities (S1411011, 2014-2018) from the Ministry of
Education, Culture, Sports, Science and Technology of Japan;
Grants-in-Aid for Scientific Research (C) from The Ministryof Education, Culture, Sports, Science and Technology(no. 26460478); and a Grant-in-Aid from Tokyo Medical
University Cancer Research to K.M and a Grant-in-Aid forYoung Scientist (B) from the Ministry of Education, Culture,
Sports, Science and Technology of Japan (no. 26670589) to S.K.
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Macrolides sensitize EGFR-TKI-induced non-apoptotic cell
death via blocking autophagy flux in pancreatic cancer cell lines
SHuNTARo MuKAI1, SHoTA MoRIyA2, MASAKI HIRAMoTo2, HIRoMI KAZAMA2,
HIRoKo KoKuBA3, XIAo-FANG CHE2, ToMoHISA yoKoyAMA4, SAToSHI SAKAMoTo6,
AKIHIRo SuGAwARA7, ToSHIAKI SuNAZuKA7, SAToSHI ŌMuRA7,
HIRoSHI HANdA5, TAKAo IToI1 and KEISuKE MIyAZAwA2
departments of 1Gastroenterology and Hepatology and 2Biochemistry, 3Laboratory of Electron Microscopy, departmentsof 4Clinical Oncology and 5Nanoparticle Translational Research, Tokyo Medical university, Tokyo; 6department of
Biological Information, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, yokohama;
7
Kitasato Institute for Life Sciences and Graduate School of Infection Control Sciences, Kitasato university, Tokyo, Japan
Received September 3, 2015; Accepted october 15, 2015
doI: 10.3892/ijo.2015.3237
Abstract. Pancreatic cancer is one of the most difficulttypes of cancer to treat because of its high mortality rate
due to chemotherapy resistance. We previously reported thatcombined treatment with gefitinib (GEF) and clarithromycin(CAM) results in enhanced cytotoxicity of GEF along withendoplasmic reticulum (ER) stress loading in non-small celllung cancer cell lines. An epidermal growth factor receptor
tyrosine kinase inhibitor (EGFR-TKI) such as GEF induces
autophagy in a pro-survival role, whereas CAM inhibits
autophagy flux in various cell lines. Pronounced GEF-induced
cytotoxicity therefore appears to depend on the efficacy of
autophagy inhibition. In the present study, we compared
the effect on autophagy inhibition among such macrolides as CAM, azithromycin (AZM), and EM900, a novel
12-membered non-antibiotic macrolide. We then assessed theenhanced GEF-induced cytotoxic effect on pancreatic cancer
cell lines BxPC-3 and PANC-1. Autophagy flux analysis indicated that AZM is the most effective autophagy inhibitor of
the three macrolides. CAM exhibits an inhibitory effect but
less than AZM and EM900. Notably, the enhancing effectof GEF-induced cytotoxicity by combining macrolides correlated well with their efficient autophagy inhibition. However,this pronounced cytotoxicity was not due to upregulationof apoptosis induction, but was at least partially mediatedthrough necroptosis. Our data suggest the possibility of using
macrolides as ‘chemosensitizers’ for EGFR-TKI therapy inCorrespondence to: Professor Keisuke Miyazawa, departmentof Biochemistry, Tokyo Medical university, 6-1-1 Shinjuku,
Shinjuku-ku, Tokyo 160-8402, Japan
E-mail: miyazawa@tokyo-med.ac.jp

Key words: pancreatic cancer, autophagy, EGFR, macrolides,
gefitinib, ER stress, necroptosis

pancreatic cancer patients to enhance non-apoptotic tumor
cell death induction.
Introduction
Pancreatic cancer is one of the most difficult types of cancer andis a major cause of cancer-related deaths worldwide, because
only 20% of patients have resectable-stage cancer at the timeof diagnosis. In addition, pancreatic cancer is largely resistantto systemic chemotherapy (1,2). In patients with metastatic
pancreatic cancer, the 5-year survival rate is only 2%. Recently,
novel molecular agents targeting specific biologic pathways
that are activated and involved in the pathogenesis of cancer
have been developed to treat various types of cancer. Since
the epidermal growth factor receptor (EGFR) is overexpressedin many pancreatic cancers, EGFR-targeting therapy appears
promising (3). In a recent randomized phase III clinical trial,
erlotinib, an EGFR tyrosine kinase inhibitor (EGFR-TKI) in
combination with gemcitabine demonstrated improvementin survival compared to gemcitabine monotherapy (medianoverall survival, 6.24 vs. 5.91 months; P= 0.038) (4). Therefore,
erlotinib in combination with gemcitabine has been approved
as a major chemotherapy drug for advanced pancreatic cancer,
based on high-level evidence (5,6). However, the medianoverall survival rate for these patients indicates extremely poor
prognosis compared to that of other types of cancers. Thus,
more effective therapeutic strategies are required to improvetherapeutic outcome.
Macroautophagy (hereafter, autophagy) is evolutionallywell-conserved in eukaryotes as the major degradation mechanism whereby cellular proteins and organelle are sequesteredinto a double-membrane vesicle (autophagosome) and aredelivered to lysosomes. They are then membrane-fused (autolysosome) and cargos are degraded for metabolic recycling
by lysosomal hydrolytic enzymes, including protease (7,8). It
has become apparent that many aggressive cancer cells haveupregulated autophagy and are dependent on autophagy forsurvival (9). EGFR tyrosine kinase (EGFR-TK) is overex-

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pressed or somatic mutationally activated in a broad rangeof human cancers, including NSCLC and pancreatic cancers(10). Activated EGFR suppresses autophagy via activationof mammalian targets of rapamycin (mToRs), as well as
tyrosine phosphorylation of Beclin-1 (11-13). In response toligand binding, EGFR becomes a dimer and is activated via
tyrosine phosphorylation. Phosphorylated EGFR subsequently
activates intracellular pro-survival and anti-apoptotic signals.
One of the downstream activated EGFR signals involves thePI3K/AKT/mToR pathway, made up of well-established negative regulators for autophagy (14). In response to nutrient andgrowth factor availability, mTOR promotes cell growth while
suppressing autophagy. In addition, the activated EGFR binds
the autophagy initiating protein Beclin-1, leading to multisite
tyrosine phosphorylation and enhanced binding to autophagy
inhibitors, including Rubicon (15). Thus, exposure to EGFRTKIs such as gefitinib (GEF) and erlotinib (ERL) induces
autophagy via cancellation of the PI3K/AKT/mToR pathway,
as well as blocking phosphorylation of Beclin-1, which
subsequently leads to dissociation of Rubicon from Beclin-1
to initiating autophagy (13,15). A recent report confirmed thatinactivated endosomal, but not cell surface EGFR interacts with
LAPTM4B, resulting in a complex formation for recruiting
Rubicon from Beclin-1 (16). As described above, dissociationof Rubicon from Beclin-1 is essential for autophagy initiation. Cancer cells with higher EGFR expression could thus
be supported and survive by efficient autophagy induction
under various metabolic stresses, such as low nutrient andhypoxic conditions due to insufficient vascularization (15,16).
Therefore, if autophagy induction in response to EGFR-TKI
promotes tumor cell survival, combined treatment with an
autophagy inhibitor may promote tumor cell death.
We and others have reported that macrolide antibiotics
such as clarithromycin (CAM) and azithromycin (AZM)
inhibit autophagy flux (17-19). we also reported that combined
treatment with the proteasome inhibitor bortezomib and CAM
for simultaneous blocking of the ubiquitin-proteasome systemand the autophagy-lysosome system resulted in pronounced
apoptosis induction of myeloma cells along with accumulation of intracellular ubiquitinated proteins and aggresome,which results in pronounced endoplasmic reticulum (ER)stress loading (20). under accumulation of unfolded proteins
inside the ER lumen, a series of adaptive responses occur: i)
attenuation of translation for repression of unfolded protein
accumulation; ii) induction of chaperon proteins for proper
refolding; and iii) exporting of unfolded proteins to outside
ER, following their polyubiquitination and degradation byproteasome [ER-associated degradation (ERAd)]. However,when ER stress loading exceeds these adaptive capacities,
apoptosis is induced via transcriptional activation of a proapoptotic transcription factor such as C/EBP homologous
protein (CHoP)/GAdd153 and the other mechanisms (21,22).
Many lines of evidence now confirm that apoptosis induction
via ER stress loading in cancer cells is a novel potential therapeutic strategy (23).
We recently reported that combined treatment using
GEF and CAM resulted in pronounced cytotoxicity as well
as ER stress loading in NSCLC (24). we also reported thatmacrolide antibiotics, including CAM and AZM, enhanced
bortezomib-induced cytotoxicity in multiple myeloma

cells (19). As EGFR-TKI induced cytoprotective autophagyin NSCLC, we hypothesized that the efficiency of macrolides
in blocking autophagy might directly affect the enhanced
cytotoxicity in refractory pancreatic cancer cells. In thepresent study, we used a novel 12-membered EM900, whichshows anti-inflammatory and/or immunomodulatory effects,
without possessing antibacterial activity (25), as well as
CAM and AZM to compare their effects of: i) enhancing
GEF-induced cytotoxicity; ii) blocking autophagy flux; andiii) ER stress loading in pancreatic cancer cell lines. our dataclearly exhibited a positive correlation between blocking
efficiency in autophagy inhibition and pronounced EGFRTKI-induced cytotoxicity. This result suggests the possibilityof using a macrolide in combination with EGFR-TKI therapy
for pancreatic cancer.
Materials and methods
Reagents. GEF and erlotinib hydrochloride (ERL) werepurchased from Biomolecules (Hamburg, Germany), andgemcitabine hydrochloride was purchased from Tokyo
Chemical Industry Co., Ltd. (Tokyo, Japan). They were dissolved
in dimethyl sulfoxide (dMSo) at a concentration of 10 mM asa stock solution. CAM and AZM were purchased from Tokyo
Chemical Industry. Non-antibiotic macrolide EM900 [(8R,9S)8,9- dihydro-6,9- epoxy-8,9- anhydropseudo- erythromycin
A] was synthesized as previously reported (25). CAM, AZM
and EM900 were dissolved in ethanol to prepare stock solutions of 5 mM. E-64d and pepstatin A, which are inhibitors oflysosomal proteases, and pan-caspase inhibitor Z-VAd-FMK
were all purchased from Peptide Institute (osaka, Japan).
Necrostatin-1, a specific inhibitor of RIPK1, was purchasedfrom Enzo Life Sciences (Farmingdale, Ny, uSA).
Cell lines and culture conditions. For the present study,
leukemia cell line K562 and pancreatic cancer cell lines BxPC-3,
Capan-1 and PANC-1 were obtained from the American Type
Culture Collection (ATCC; Manassas, VA, uSA). BxPC-3
cells and K562 cells were maintained in RPMI-1640 medium(Sigma-Aldrich, St. Louis, Mo, uSA) supplemented with 10%
fetal bovine serum (FBS; Biowest, Kiltimagh, Ireland), 2 mM
L-glutamine, penicillin (100 u/ml), and streptomycin (100 µg/ml) (wako Pure Chemicals Industries, osaka, Japan). PANC-1
cells were maintained in dulbecco's modified Eagle's medium(Sigma) supplemented with 10% FBS, penicillin (100 u/ml)
and streptomycin (100 µg/ml). Capan-1 cells were maintainedin Iscove's modified dulbecco's medium (Life Technologies,
Carlsbad, CA, uSA) supplemented with 20% FBS, penicillin(100 u/ml) and streptomycin (100 µg/ml). All cell lines were
cultured in a humidified incubator containing 5% Co2 and
95% air at 37˚C.
Assessment of the viable number of cells. The number of viable
cells was assessed using CellTiter Blue, a cell viability assay
kit (Promega, Madison, wI, uSA), with fluorescence measurements at 570 nm for excitation and 590 nm for fluorescenceemission.
Immunoblotting. Immunoblotting was performed as previously described (19). Cells were lysed with RIPA lysis-157-

INTERNATIONAL JOURNAL OF ONCOLOGY 48: 45-54, 2016buffer (Nacalai Tesque, Kyoto, Japan) supplemented witha protease and phosphatase inhibitor cocktail (Nacalai
Tesque). Cellular proteins were quantified using a dC
protein assay kit of Bio-Rad Laboratories (Richmond, CA,
uSA). Equal amounts of proteins were loaded onto the gels,separated by SdS-PAGE, and transferred onto Immobilon-P
membranes (Millipore Corp., Bedford, MA, uSA). These
membranes were probed with first antibodies (Abs) such as
anti-LC3B antibody (Ab) (Novus Biologicals LLC, Littleton,
Co, uSA), anti-p62 (d-3) monoclonal (m) Ab (sequestsome-1), anti- β actin (C4) mAb, anti-GAPdH (6C5) mAb(Santa Cruz Biotechnology, Santa Cruz, CA, uSA), anticleaved-caspase-3 Ab, and anti-PARP Ab (Cell Signaling
Technology, danvers, MA, uSA). Immunoreactive proteins
were detected using horseradish peroxidase-conjugatedsecond Abs and an enhanced chemiluminescence reagent(ECL) (Millipore). densitometry was performed usinga Molecular Imager Chemidoc XRS System (Bio-Rad
Laboratories).
Gene expression analysis. Real-time polymerase chain reaction (PCR) for gene expression analysis was performed aspreviously described in detail (19).
Electron microscopy. Cells were fixed with 2.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.3) for 1 h. The samples
were further fixed in 1% osmium tetroxide for 1 h, dehydrated
in graded ethanol (30-100%), and embedded in Quetol 812
epoxy resin (Nisshin EM Co., Ltd., Tokyo, Japan). ultrathin
sections were cut using an ultracut J microtome (Reichert
Jung, Vienna, Austria). The sections were stained withlead nitrate and uranium acetate, and subjected to electron
microscopic analysis using a scanning electron microscope
JEM-1200EX II (JEoL, Tokyo, Japan).
Stable transfection of GFP-LC3 plasmid in K562 cells. For
the present study, a pEGFP-LC3 plasmid vector (no. 2490)
was purchased from Addgene (Cambridge, MA, uSA). K562
cells were transfected with plasmid dNA using Lipofectamine
2000 (Life Technologies) according to the manufacturer's
instructions. Briefly, 4 µg of pEGFP-LC3 plasmids solution
and 10 µl of Lipofectamine 2000 were incubated in 500 µl of
serum-free opti-MEM (Life Technologies) for 20 min andmixed into 2x106 cells cultured in 1.5 ml of antibiotic-free
RPMI-1640 with 10% FBS in a 60-mm dish. Forty-eight
hours after transfection, the cells were seeded into a 96-wellplate and selected for limited dilution cloning in the presence of 1 µg/ml of G418 for 1 month. K562/GFP-LC3 clone#2 was used for the following experiments.
After treatment with macrolide, K562/GFP-LC3 cells
were spread and fixed on slide glasses using a Cytospin 4
Centrifuge (Thermo Fisher Scientific, Inc., Rockford, IL,
uSA) to make slide glass preparations. Analysis by confocal
microscopy for the assessment of autophagosomes was
performed using a confocal laser scanning fluorescencemicroscope LSM 700 (Carl Zeiss, Jena, Germany).
Statistical analysis. The data are expressed as mean ± Sd.
Statistical analysis was performed using the Mann-whitneyu test (two-tailed).

47

Figure 1. Cell growth inhibition after treatment with GEF and ERL in pancreatic cancer cell lines. BxPC-3, Capan-1 and PANC-1 cells were treated with
GEF and ERL at various concentrations for 48 h. The number of viable cells
was assessed using CellTiter Blue as described in Materials and methods.

Results
Cell growth inhibition in pancreatic cancer cell lines after
treatment with EGFR-TKI. Pancreatic cancer cell lines
PANC-1 and Capan-1, both of which have activating EGFR
mutations, and BxPC-3 expressing wild-type EGFR, were
cultured in the presence of the EGFR-TKIs GEF and erlotinib hydrochloride (ERL) at various concentrations (Fig. 1).
Although both GEF and ERL inhibited cell growth in thesecell lines in a dose-dependent manner, GEF was more effective than erlotinib in the cytotoxic effect in our culture system.
Therefore, the following in vitro experiments were performed
using mainly GEF. A concentration of 50% cell growth inhibition (IC50) after 48-h treatment with GEF was 20.4 µM for
PANC-1, 39.5 µM for Capan-1 and 19.5 µM for BxPC-3.
Autophagy induction in response to GEF in pancreatic cancer
cell lines. Conversion from the soluble cytosolic LC3B-I to themembrane-bound LC3B-II via conjugation of phosphatidyl
ethanolamine represents the formation of autophagosomes.
Therefore, increased expression of LC3B-II is a good marker
for autophagosome evaluation (27). Treatment with GEF
induced the increased expression of LC3B-II in a dose- anda time-dependent manner in PANC-1 cells and BxPC-3 cells(Fig. 2A). Not shown is that ERL-treatment also increased theexpression of LC3B-II as well as GEF. In addition, combined
treatment with GEF and lysosomal inhibitors such as E-64d
and pepstatin A, which are used for blocking autophagy flux,
further enhanced LC3B-II expression compared to treatment
with GEF alone or with only lysosomal inhibitors (Fig. 2B).
Furthermore, electron microscopy indicated a marked increaseof autophagosomes and autolysosomes in PANC-1 cells after
treatment with GEF (Fig. 2C). These data indicate that GEF
induces autophagy in pancreatic cell lines as well as othercancer cell lines, including NSCLC cells previously reported
by us and others (25,26).
Enhanced cytotoxicity of EGFR-TKI by combined treatment
with macrolides in pancreatic cancer cell lines. Using NSCLC

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Figure 2. Autophagy induction after treatment with GEF. (A) PANC-1 cells and BxPC-3 cells were treated with GEF (10 and 25 µM) for 16 and 24 h.
Cellular proteins were separated by 15% SdS-PAGE and immunoblotted with anti-LC3B Ab. Immunoblotting with anti-GAPdH mAb was performed as aninternal control. Numbers indicating the expression ratios of LC3B-II/GAPdH were determined using densitometry. (B) PANC-1 cells and BxPC-3 cells were
cultured with or without GEF (25 µM) in the presence or absence of lysosomal inhibitors (LI), E-64d (10 µg/ml), and pepstatin A (10 µg/ml) for 16 and 24 h.
Immunoblottings were performed as (A). Numbers indicate the expression ratios of LC3B-II/GAPdH. (C) PANC-1 cells were treated with GEF (25 µM) for
48 h, and electron microscopy was performed. Scale bars, 2 µm. N, nucleus; mit, mitochondria; AP, autophagosome; AL, autolysosome.

cell lines, we have reported that GEF-induced autophagy
functions as cytoprotective, and simultaneous treatment with
GEF and CAM results in enhanced cytotoxicity (25). In thepresent study, we selected three macrolides (CAM, AZM
and EM900) and examined their efficacy for GEF-induced
cytotoxicity as well as blocking autophagy flux in pancreaticcancer cell lines. First, treatment with up to 50 µM CAM,
AZM or EM900 resulted in little cytotoxicity in BxPC-3 cells.
More than 75 µM EM900 exhibited some cytotoxic effect(Fig. 3A). Next, BxPC-3 and PANC-1 cells were treated with
GEF at various concentrations in the presence of 50 µM CAM,
AZM or EM900. Significant enhancement of GEF-induced
cytotoxicity was observed (Fig. 3B). during 48-h exposure,
AZM was more potent than CAM or EM900 for enhancing
GEF-induced cytotoxicity. At 25 and 50 µM GEF, EM900 was
superior to CAM. The concentration of GEF was therefore
fixed at 25 µM, and PANC-1 cells were treated with macrolides at various concentrations. It was noteworthy that all threemacrolides at >5 µM resulted in enhanced reduction of theviable cell number as comparing with treating the cells with
GEF alone (Fig. 3C). Additionally, AZM was superior to CAM
and EM900 in enhancing the killing effect of 25 µM GEF.
Comparing the blocking effect of autophagy flux among
CAM, AZM and EM900 in PANC-1 cells. Based on the above
data, we further assessed the blocking effect of macrolides on
autophagy flux in pancreatic cancer cell lines. After treatment
with 50 µM macrolides for 24 and 48 h, PANC-1 cells exhibited
increased expression of LC3B-II (Fig. 4A). However, unlike
GEF treatment (Fig. 2B), combined treatment with lysosomal
inhibitors and a macrolide did not increase LC3B-II expression
further, compared with treatment with either a macrolide alone
or only lysosome inhibitors. These results indicate that all
tested macrolides effectively block autophagy flux (Fig. 4B).
For comparison of efficiency on blocking autophagy, PANC-1
cells were treated with a macrolide at various concentrations-159-

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49

Figure 4. Flux analysis of autophagosome formation in macrolides.
(A) PANC-1 cells were treated with CAM, AZM and EM900 at 50 µM for
24 and 48 h. Cellular proteins were separated by 15% SdS-PAGE and immunoblotted with anti-LC3B Ab. Immunoblotting with anti-GAPdH mAb was
performed as an internal control for protein loading. Numbers indicate theexpression ratios of LC3B-II/GAPdH. (B) PANC-1 cells were treated with
AZM and EM900 at 50 µM in the presence or absence of lysosomal inhibitors (LIs) for 16 h, as depicted in Fig. 2B. Immunoblotting was performed asin (A). Numbers indicate the expression ratios of LC3B-II/GAPdH.

Figure 3. Enhancement of cell growth inhibition after combined treatment
with GEF and macrolides in PANC-1 and BxPC-3 cells. (A) BxPC-3 cells
were treated with CAM, AZM and EM900 at various concentrations for 24
and 48 h. The number of viable cells was then assessed using CellTiter Blue.
(B) BxPC-3 and PANC-1 cells were treated with GEF at various concentrations in the presence or absence of macrolides (50 µM) for 24 and 48 h.
*
P<0.05 control vs. macrolide. (C) PANC-1 cells were treated with macrolides at various concentrations in the presence or absence of GEF (25 µM)
for 24 and 48 h. The number of viable cells was assessed using CellTiter Blue.
*P<0.05; CAM vs. AZM/EM900.

for 24 h. Immunoblotting with anti-LC3B Ab clearly indicated accumulation of autophagosome in a dose-dependent
manner for all three macrolides (Fig. 5A). P62, a substrate of
autophagy (28), also accumulated after macrolide treatment
in a dose-dependent manner. To standardize for comparison
among the three macrolides, the expression ratios of LC3B-II
to GAPdH were divided by that of untreated controls andplotted (Fig. 5B). AZM is the most potent in blocking efficiency in autophagy; EM900 is the second most potent andsuperior to CAM. It was noteworthy that the inhibitory
efficacy of autophagy in macrolides correlated well with
their enhancing effect for GEF-induced cytotoxicity (Fig. 3B
and C). To confirm their blocking efficiency, K562 cells witha stably transfected GFP-LC3 gene (K562/GFP-LC3) were
cultured in the presence of these macrolides at 50 µM for
24 h. As indicated in Fig. 5C and d, the number of punctaof GFP-LC3 pre-cell, which indicates autophagosome accumulation in response to the macrolide, was consistent withthe result presented in Fig. 5A and B. Increase in the numberof GFP-LC3 puncta was in the order of AZM, EM900 and
CAM. These data together strongly suggested that enhanced
GEF-induced cytotoxicity by combining macrolides is due totheir effect in blocking autophagy flux.
Non-apoptotic cell death induction after treatment with GEF
with/without macrolides. we next assessed ER stress loading
after treatment with GEF and macrolides, because apoptosis isinduced when ER stress is overloaded beyond cellular adaptive
capacity (21,22). Real-time PCR indicated that GEF treatment
upregulated ER stress-related genes such as GRP78 and

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Figure 5. Comparison of macrolides in the efficiency of blocking autophagy flux. (A) PANC-1 cells were treated with AZM, EM900 and CAM at various
concentrations for 24 h. Cellular proteins were separated by 15% SdS-PAGE for LC3B and 11.25% for p62 and immunoblotted with anti-LC3B and anti-p62
mAbs. Immunoblotting with anti-GAPdH mAb was performed as an internal control. Numbers indicate the ratios of LC3B-II/GAPdH and p62/GAPdH as
determined using densitometry. (B) Expression ratios of LC3B-II/GAPdH presented above were plotted. (C) Confocal microscopy: K562-GFP-LC3 cells
were treated with 50 µM AZM, EM900 and CAM for 24 h. upper panels, puncta of green fluorescence indicates accumulating autophagosome. Lower panels,
differential interference contrast microscopy. Scale bars, 10 µm. (d) Numbers of puncta of GFP-LC3 per cell in Fig. 5C. CAM vs. AZM, P<0.001; AZM vs.
EM900, P= 0.02; EM900 vs. CAM, P= 0.03.

Figure 6. Induction of GRP78 and CHoP in PANC-1 cells after treatment
with GEF and macrolides. PANC-1 cells were treated with/without GEF in
the presence or absence of the three macrolides for 24 and 48 h. Expressionsof ER stress-related genes GRP78 and CHoP were assessed using real-time
PCR.

CHoP, whereas, treatment with macrolides had little effect(Fig. 6). Combined treatment with GEF plus CAM resulted
in pronounced expressions of GRP78 and CHoP, which isa pro-apoptotic transcription factor that upregulates various
pro-apoptotic genes such as Bax and Bim (21). unexpectedly,
concomitant AZM or EM900 plus GEF did not significantly
enhance ER stress loading compared with treatment of thecells with GEF alone. A discrepancy between the efficiency of
autophagy inhibition and ER stress loading led us to examine
whether the enhanced cytotoxicity is completely mediatedthough apoptosis.
As indicated in Fig. 7A, treating PANC-1 cells with GEF
in the presence or absence of EM900 produced no apoptotic
features but did produce necrosis. However, cells treated withgemcitabine (gem), which is the most widely used medicine fortreating patients with metastatic pancreatic cancer, produced
nuclear chromatin condensation in some cells (arrows).

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51

Figure 7. Morphological changes in PANC-1 cells after treatment with GEF and macrolides. (A) May-Giemsa staining: May-Giemsa staining was performed
after treatment with either GEF (25 µM) or gemcitabine (100 µM) in the presence or absence of EM900 (50 µM) for 48 h. Scale bars, 10 µm. Arrows indicate
nuclear chromatin condensations. (B) Electron microscopy: PANC-1 cells were processed for electron microscopy after treatment with GEF (25 µM), AZM(50 µM), GEF+AZM, and gemcitabine (100 µM) for 48 h. Lower panels are at higher magnifications of the squared areas in each upper panel. Scale bars, 2 µm.
(C) PANC-1 cells were treated with GEF (25 µM), AZM (50 µM), EM900 (50 µM), GEF+AZM, and gemcitabine (10 and 100 µM) for 48 h. Cellular proteins
were separated by 15% SdS-PAGE for caspase-3 and 7.5% for PARP and immunoblotted with either anti-cleaved caspase-3 Ab or anti-PARP Ab. Cell lysate
derived from HL-60 cells treated with vitamin K2 was used as a positive control for apoptosis induction (42). (d) PANC-1 cells were treated with various
concentrations of either necrostatin-1 or Z-VAd-FMK in the presence or absence of GEF (50 µM), GEF (50 µM) plus EM900 (50 µM), or gem (300 µM) for
48 h. The number of viable cells was assessed using CellTiter Blue.

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MuKAI et al: MACRoLIdES SENSITIZE EGFR-TKI IN PANCREATIC CANCER CELLSElectron microscopy also indicated that, although gem-treated
PANC-1 cells exhibited a sharp nuclear notch as well as nuclear
heterochromatin formation GEF treatment with/without AZM
did not produce prominent nuclear change, but did produce
accumulation of a large number of autophagosomes and autolysosomes, and increased swollen mitochondria with destructionof cristae structure inside them (Fig. 7B). Furthermore, immunoblotting with anti-cleaved capase-3 Ab and anti-PARP Ab
indicated no cleavage of caspase-3 and PARP after treatment
with GEF plus AZM or EM900 (Fig. 7C). All these datasuggest that enhanced cytotoxicity by combining GEF plus
macrolide as well as GEF-induced cytotoxicity itself does not
involve apoptosis in pancreatic cancer cell lines. This result
was also supported by the data presented in Fig. 7d; in thepresence of necrostatin-1, a potent and selective inhibitor of
necroptosis via blocking RIP-1 kinase (29), GEF- and GEF plus
EM900-induced cell death was significantly attenuated, while
Z-VAd-FMK, a pan-caspase inhibitor for apoptosis inhibition,
had little effect. In contrast, gemcitabine-induced cytotoxicity
was significantly suppressed in the presence of Z-VAd-FMK,
but not in the presence of necrostatin-1. Thus, a combinationof GEF plus macrolide induces mainly non-apoptosis cell
death such as necroptosis, whereas GEM induces apoptosis,
in PANC-1 cells.
Discussion
In the present study, we demonstrated that macrolides such as
AZM, CAM and EM900 enhance GEF-induced cytotoxicity
in pancreatic cancer cell lines. In addition, the efficiency ofmacrolides in blocking autophagy flux correlated well with
their enhancement of GEF-induced cytotoxicity (Figs. 3
and 4). Among the three macrolides, AZM was the mostpotent for enhancing the GEF cytotoxicity as well as for inhibiting autophagy. Many previous reports suggest that autophagy
induction by GEF is a metabolic stress response and functions
as cytoprotective, which might be crucial in resistance to
EGFR-TKIs (11-13). Therefore, it is reasonable that blocking
efficiency directly influences the cytotoxic effect of GEF.
However, the precise mechanism for blocking autophagy bymacrolides must still be clarified.
Based on the chemical structure of macrolides, acommon target appears to be involved in the process of
autophagy. Autophagy inhibition by AZM was originallyreported by Renna et al in 2011 (18). Long-term oral administration of AZM against chronic sinopulmonary infectionin adult patients with cystic fibrosis is associated with thedevelopment of infection with non-tuberculous mycobacteria. They found that, in primary human macrophages,
AZM concentrations achieved during therapeutic dosing
blocked autophagosome clearance by preventing lysosomal
acidification, thereby impairing autophagic and phagosomal
degradation. As a consequence, AZM treatment inhibited
intracellular killing of mycobacteria within macrophages,
which resulted in chronic non-tuberculous mycobacteria
infection (18). Electron microscopy indicated accumulationof a large number of autophagosomes and autolysosomes in
PANC-1 cells after treatment with AZM (Fig. 7B). This result
indicated that the membrane fusion between autophagosome
and lysosome (autolysosome formation) is not impaired.

Lysosomes generate and maintain their pH gradients byvacuolar (V)-type ATPase activity for pumping protons into
the lysosome lumen (31). Therefore, preventing lysosomal
acidification by AZM as indicated by a previous reportsuggests that V-type ATPase is a candidate target moleculefor AZM. Indeed, bafilomycin A1 and concanamycin A are
macrolide compounds that have a selective inhibitory effecton V-type ATPase, and they are frequently used in experiments in vitro for blocking autophagy (32). However, both
macrolides have reportedly prevented maturation of autophagic vacuoles by inhibiting fusion between autophagosomes
and lysosomes (32). Valosin-containing protein (VCP)/p97
appears to be another potential target (33). A recent reportdemonstrated that AZM and CAM interact with VCP, basedon AZM- and CAM-immobilized affinity chromatography (33). Pathophysiologically, germline mutations in VCP
genes account for a spectrum of pathological phenotypes
that include inclusion body myopathy with Paget's diseaseof the bone and frontotemporal dementia, hereditary spastic
paraplegia (IBMPFd), and 1-2% of familial amyotrophic
lateral sclerosis (34,35). In addition, VCP/p97 plays a criticalrole in a broad range of cellular activities, including ERAd,
the ubiquitin-proteasome system, prevention of polyglutamine aggregation and autophagosome maturation in
autophagy (35,36). VCP mutation (p.Glu185Lys) segregatingin the autosomal dominant Charcot-Marie-Tooth diseasetype 2 family was also identified (37). Notably, functional
studies confirmed that the Glu185Lys variant impaired
autophagic function, leading to accumulation of immature
autophagosomes (37). Identification of the target molecule(s)of macrolide for autophagy inhibition as well as assessmentof the affinity to the macrolides used in this study helps toclarify the phenomena analyzed in this study.
despite the positive relationship between blocking
efficiency and enhancement of the cytotoxicity of GEF, discrepancies in ER stress loading were observed (Fig. 5 vs. 6). CAM
and GEF was the most potent combination for upregulation of
pro-apoptotic transcription factor CHoP, whereas concomitant AZM or EM900 plus GEF did not significantly enhance
ER stress loading, compared with GEF alone (Fig. 6). otherthan ER stress-mediated apoptosis, alternative pathway(s) suchas non-apoptotic cell death might contribute to the pronounced
cytotoxic effect. we previously reported that a murine embryonic fibroblast (MEF) cell line derived from CHoP knockout
mice still exhibited enhanced cytotoxicity with combined
treatment of GEF and CAM, although they were much lesssensitive to GEF plus CAM than the CHoP+/+ MEF cellline (24). The same phenomenon was observed in PC-9 cell
knocked down CHoP by siRNA versus control PC-9 cells (24).
Although cytotoxicity was enhanced, a series of experiments
performed for assessing apoptosis did not yield any apparent
features of apoptosis (Fig. 7A-C). However, in the presence of
necrostatin-1, a specific inhibitor for necroptosis via blocking
RIP1 kinase (29), GEF- and GEF plus macrolide-induced cell
death was partially but significantly suppressed (Fig. 7d). This
result suggests that necroptosis might contribute to enhanced
cytotoxicity. unlike NSCLC cell lines, PANC-1 and BxPC-3
cells did not exhibit prominent apoptotic features even after
treatment with GEF alone (24). In addition, treatment withgemcitabine resulted in only slight apoptotic change, such as-163-

INTERNATIONAL JOURNAL OF ONCOLOGY 48: 45-54, 2016

weak cleavages of caspase-3 and PARP, as well as partial chromatin condensation without nuclear fragmentation (Fig. 7A-C).
Also, their features were much weaker than those in other celllines (e.g., NSCLC, breast cancer and leukemia cell lines) (datanot shown). Therefore, pancreatic cancer cell lines used in thepresent study may be rather resistant to apoptosis induction.
This result might represent the difficult clinical features ofpancreatic cancer patients with chemoresistance.
It is necessary to determine the most effective way tointerconnect autophagy inhibition and non-apoptotic cell
death. Currently, no study has demonstrated necroptosis
induction by EGFR-TKIs. However, it has been confirmed that
non-apoptotic cell death, including necroptosis, alternatively
becomes prominent when cells are resistant to apoptosis
induction (38-40). In PANC-1 cells, a higher concentrationof gemcitabine (100 µM) was required for apoptosis induction, indicating difficulty in inducing apoptosis in them,
unlike other cancer cell lines (Fig. 7d). It was reported that
autophagy suppresses RIP kinase-dependent necrosis (necroptosis) in RCC4 cells, a human renal cell carcinoma cell line(41). Inhibition of mToR with the specific mToR inhibitor
CCL-779 stimulated autophagy, leading to elimination of RIP
kinases via autophagy-mediated degradation. However, withthe simultaneous inhibition of autophagy using chloroquineand mToR with CCL-779, necroptosis was induced (41).
This result is somewhat similar to our observation, since
EGFR-TKI inhibits the axis of the PI3K/AKT/mToR pathway.
In their system, autophagy of mitochondria (mitophagy) is
required for cell survival, since mTOR inhibition turns off
the Nrf2 antioxidant defense (41). Thus, simultaneous mToR
and autophagy inhibition leads to an imbalance between ROS
production by mitochondria and the defense by Nrf2, whichcauses necroptosis. Pancreatic cell lines appear to have higher
basal autophagy with increased mitochondria than othercancer cell lines (Figs. 2A and 7B). For clinical use of a macrolide as a potent ‘sensitizer’ in EGFR-TKI therapy, it will also
be important to establish the underlying molecular mechanismof enhanced non-apoptotic cell death, as well as to identify thetarget(s) of macrolides.
Acknowledgements
The present study was supported by funds provided throughan MEXT-supported program of the Strategic Research
Foundation at Private universities (S1411011, 2014-2018)from the Ministry of Education, Culture, Sports, Scienceand Technology of Japan to M.H. and K.M., Grants-in-Aid
for Scientific Research (C) from The Ministry of Education,
Culture, Sports, Science and Technology to T.I. (no. 24591018)
and to K.M. (no. 26460478), and a Grant-in-Aid from Tokyo
Medical university Cancer Research to K.M.
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ARTicLE

doi:10.1038/nature18611

A novel cereblon modulator recruits
GSPT1 to the CRL4CRBN ubiquitin ligase

Mary E. Matyskiela1*, Gang Lu1*, Takumi ito2*, Barbra Pagarigan1, chin-chun Lu1, Karen Miller1, Wei Fang1, Nai-yu Wang1,
Derek Nguyen1, Jack Houston1, Gilles carmel1, Tam Tran1, Mariko Riley1, Lyn’Al Nosaka3, Gabriel c. Lander3,
Svetlana Gaidarova1, Shuichan Xu1, Alexander L. Ruchelman1, Hiroshi Handa2, James carmichael1, Thomas O. Daniel1,
Brian E. cathers1, Antonia Lopez-Girona1 & Philip P. chamberlain1

Immunomodulatory drugs bind to cereblon (CRBN) to confer differentiated substrate specificity on the CRL4CRBN E3
ubiquitin ligase. Here we report the identification of a new cereblon modulator, CC-885, with potent anti-tumour activity.
The anti-tumour activity of CC-885 is mediated through the cereblon-dependent ubiquitination and degradation of thetranslation termination factor GSPT1. Patient-derived acute myeloid leukaemia tumour cells exhibit high sensitivity
to CC-885, indicating the clinical potential of this mechanism. Crystallographic studies of the CRBN–DDB1–CC-885–
GSPT1 complex reveal that GSPT1 binds to cereblon through a surface turn containing a glycine residue at a key position,interacting with both CC-885 and a ‘hotspot’ on the cereblon surface. Although GSPT1 possesses no obvious structural,sequence or functional homology to previously known cereblon substrates, mutational analysis and modelling indicatethat the cereblon substrate Ikaros uses a similar structural feature to bind cereblon, suggesting a common motif for
substrate recruitment. These findings define a structural degron underlying cereblon ‘neosubstrate’ selectivity, andidentify an anti-tumour target rendered druggable by cereblon modulation.
The immunomodulatory drugs lenalidomide and pomalidomide
promote recruitment and ubiquitination of substrate proteins to the
CRL4CRBN (CUL4–DDB1–RBX1–CRBN) E3 ubiquitin ligase, leading to their subsequent degradation by the 26S proteasome1,15,16. The
therapeutic benefits of immunomodulatory drug treatment in multiple
myeloma and 5q-deletion-associated myelodysplastic syndrome are
driven by a combination of both anti-proliferative cellular and immunomodulatory effects2–5. The CRL4CRBN E3 ubiquitin ligase complex
consists of a core scaffolding component CUL4, which binds to the
RBX1 subunit, the docking site for E2 recruitment, and DDB1, an adaptor protein. DDB1 provides a binding site for CRL4 substrate receptor
proteins such as cereblon, termed DCAFs (DDB1 and CUL4 associatedfactors), which recruit substrates to the ubiquitin ligase complex anddetermine substrate specificity6–9.
Structural studies have shown that immunomodulatory drugs
bind in a shallow hydrophobic pocket on the surface of cereblon10,11.
Cereblon binding is mediated by a glutarimide ring, a feature common
to the clinical molecules thalidomide, lenalidomide, pomalidomide,
and CC-122. These compounds feature additional chemical groups thatare not contained by the cereblon binding pocket, and have been postulated to interact with cognate substrates resulting in ‘neomorphic’ E3ligase activity. Several proteins have been reported to be ubiquitinatedby the CRL4CRBN E3 ligase upon treatment with immunomodulatory
drugs. Ikaros and Aiolos are zinc finger transcription factors importantin haematological differentiation12,13, which are degraded by additionof several immunomodulatory drugs5,14–16. Recently, lenalidomide,
but not pomalidomide or CC-122, was demonstrated to induce thedegradation of the protein kinase casein kinase 1α (CK1α), thereby
exploiting CK1α haploinsufficiency associated with 5q-deletionassociated myelodysplastic syndrome5. Importantly, these cereblon substrates share no obvious sequence or structural homology to explain
their susceptibility to immunomodulatory drugs.

We investigated whether cereblon could be repurposed to induce
the degradation of previously unidentified targets resulting in antitumour activity. We identified CC-885 as a potent anti-cancer agent
eliciting broad spectrum growth inhibition against cancer cell lines andpatient-derived acute myeloid leukaemia (AML) cells, indicating theclinical potential for this mechanism. We identified GSPT1 (eRF3a) as
a novel CC-885-dependent cereblon substrate mediating the anti-proliferative effects of CC-885. GSPT1 is a translation termination factor
that binds eRF1 to mediate stop codon recognition and nascent protein
release from the ribosome17–19. GSPT1 is not targeted for degradation
by any of the current generation of clinically approved immunomodulatory drugs, and possesses no obvious homology to Ikaros, Aiolos orCK1α. To investigate the structural determinants for substrate recruitment, we solved the structure of cereblon in complex with DDB1,
CC-885, and GSPT1. We found that the main contacts to cereblon–
CC-885 by GSPT1 are mediated by the peptide backbone of a surface
turn. Degrons, molecular motifs that direct E3 ligase interactions,have been defined for other ligases20–22, and include examples whererecognition is dependent upon small molecules binding to the ligase
complex23,24. Mutational evidence indicates that Ikaros is recruited via
a motif similar to that of GSPT1, demonstrating that there is a common
structural degron for ligand-directed recruitment to the CRL4CRBN
E3 ligase.

CC-885 is a cereblon-dependent anti-tumour agent

On the basis of the hypothesis that additional proteins are vulnerable tocereblon-mediated degradation, we sought to identify cereblon ligandswith unique activities from a library of analogues. CC-885 was identified as a compound exhibiting a strong anti-proliferative phenotype intumour cell lines (Extended Data Fig. 1a). The broad activity profile of
CC-885 is highly differentiated from that of thalidomide, lenalidomide
and pomalidomide (Extended Data Fig. 1a, d). To assess the potential

1

Celgene Corporation, 10300 Campus Point Drive, Suite 100, San Diego, California 92121, USA. 2Department of Nanoparticle Translational Research, Tokyo Medical University, Shinjuku-ku, Tokyo
160-8402, Japan. 3The Scripps Research Institute, San Diego, California 92121, USA.
*These authors contributed equally to this work.
2 5 2 | N AT U R E | V O L 5 3 5 | 1 4 J U Ly 2 0 1 6© 2016 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.

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Article reSeArcH
CC-885 promotes cereblon-dependent GSPT1 destruction

clinical utility of CC-885, the molecule was tested in an ex vivo setting to evaluate potency against patient-derived AML tumour cells.
CC-885 exhibited sub-nanomolar potency against 4 out of 5 patient
samples (Extended Data Fig. 1b), with reduced activity against thenormal lymphoid cells obtained from the same donors.
We next established the cellular target of CC-885. Like lenalidomide
and its analogues, CC-885 contains a glutarimide ring that can directlyinteract with cereblon (Fig. 1a). The anti-proliferative effects of CC-885
are cereblon-dependent, as ablation of cereblon gene expression using
clustered regularly interspaced short palindromic repeats (CRISPR)
conferred resistance to CC-885 in 293FT human embryonic kidneycells, as well as in the AML cell lines NB-4, MOLM-13 and OCI-AML2(Fig. 1 b, Extended Data Fig. 1c). To identify potential substrates of
CRL4CRBN regulated by CC-885, we generated 293T HEK cells stably
expressing Flag- and haemagglutinin (HA)-double-tagged cereblon(FH–CRBN). Using large-scale anti-Flag affinity purification followedby mass spectrometry analysis, GSPT1 (eRF3a) was identified as aprotein that associated with FH–CRBN specifically in the presence of
CC-885 (Fig. 1c, Supplementary Information Table 1). The presenceof GSPT1 in the anti-Flag immunoprecipitates of FH–CRBN complex
bound to CC-885 was confirmed by immunoblot analysis (Fig. 1c,bottom). Reciprocal binding assays performed with HA-tagged GSPT1
immobilized on anti-HA resin further supported that CC-885 could
enhance the binding affinity of endogenous cereblon for GSPT1(Extended data Fig. 2). Lenalidomide did not promote the interactions
between cereblon and GSPT1. Conversely, CC-885 enhanced the binding of Ikaros (IKZF1) to cereblon. Consistent with the binding data,
CC-885, but not lenalidomide or pomalidomide, triggered the depletion of GSPT1, whereas all three agents decreased the level of Ikaros in
NB-4 leukaemia cells (Fig. 1d).

GSPT1 depletion drives CC-885 effects

To address whether the cereblon-dependent degradation of GSPT1
was responsible for the cytotoxic effects of CC-885, a GSPT1 mutant
that retains its normal function, but loses CC-885-dependent cereblon
binding, was used to distinguish the role of GSPT1 from that of othersubstrates. The yeast homologue, SUP35, shares a high degree of

Lenalidomide

IP: Flag (CRBN)

c

Mo
ck
DM
SO
CC
-88
5

b
RLU normalized to parental
DMSO control (%)

a

We next determined whether the changes in GSPT1 protein levels
upon CC-885 treatment resulted from CRL4CRBN-dependent ubiquitination and degradation. In 293FT HEK cells, the CC-885-induced
reduction of GSPT1 protein levels can be prevented by depletion of
cereblon via CRISPR or by co-treatment with either a neddylation
inhibitor (MLN4924) or a proteasome inhibitor (MG132) (Extended
Data Fig. 3, and data not shown). The mRNA level of GSPT1 was
not decreased but rather increased in 293FT HEK cells treated with
CC-885, consistent with a non-transcriptional mechanism (Extended
Data Fig. 3). Together, these results indicate that CC-885 treatment
causes cereblon-dependent ubiquitination and subsequent proteosomal
degradation of GSPT1.
CC-885 promoted both the in vivo and in vitro ubiquitination of
GSPT1 by cereblon (Fig. 1 e, f and Extended Data Fig. 4) and decreased
the protein half-life of GSPT1 in 293FT HEK parental but not in
CRBN−/− cells (Extended Data Fig. 4). Reintroduction of wild-typecereblon via lentiviral infection in 293FT CRBN−/− cells restored the
CC-885-dependent degradation of GSPT1, whereas CRBN(W386A)
and CRBN(E377V) mutants displayed diminished activity, with E377V
exhibiting a stronger effect consistent with the critical role of E377
in anchoring CC-885 in the cereblon–GSPT1 complex (Extended
Data Fig. 4).

FH–CRBN –100
NB-4
NB-4 CRBN–/–MOLM-13
MOLM-13 CRBN–/–
OCI-AML2
OCI-AML2 CRBN–/–

50

+

0
0.0001

0.001

0.01

200
DDB1

116
97.4

GSPT1

66

Flag–HA–
CRBN

45

0.11

CC-885 (μM)
Pomalidomide

+

Mass

31

CC-885
IB: GSPT1de+
8×His–Ub+
DMSO
CC-885 (10 μM) –MG-132 (10 μM) +

IKZF1

+
+
–
+

+
–
+
+

– –
– +
– +
– +
– +
– +
+ +
– +

+ + +
+ + +
+ + +
+ + +
+ + +
– – +
+ + +
– + –

+
+
+
+
+
+
+
+

Mass
191
GSPT1

97

GSPT1

64

C

29

3F

T

Tubulin

/–

GSPT1

+
–
+
+

– – –
+ – –
+ – –
+ – –
– + –
– – +
– – –
– – –

2
RB 93F
N– T

4h

Ni-NTA denaturing
pull down

1 μM

0.1 μM

DMSO
0.001 μM
0.01 μM

CC-8851 μM

0.1 μM

0.01 μM

0.001 μM

DMSO

Pom.

1 μM

0.1 μM

DMSO
0.001 μM
0.01 μM

Len.

f Ube1 ATP(E1)
UbcH5a (E2)
Ubiquitin
CUL4A–RBX1
CRBN–DDB1
GSPT1
CC-885Figure 1 | GSPT1 is a substrate of the cereblon E3 ligase complex boundwith CC-885. a, Structures of lenalidomide, pomalidomide and CC885 with the glutarimide ring shown in red, isoindolinone in blue, themethylene urea in green. b, The effect of CC-885 on cell proliferation inparental and CRBN−/− AML cell lines as indicated. Result is representativeof three biological replicates. Data are shown as mean ± s.d., n = 3
technical replicates. c, Silver stained gel of the immunoprecipitationof Flag-tagged cereblon to identify GSPT1 binding in the presence of
CC-885. d, Immunoblot analysis of whole-cell extracts of NB-4 cells
incubated with DMSO, or with lenalidomide, pomalidomide or CC-885
at the concentrations indicated for 4 h. e, The in vivo ubiquitination of
GSPT1 is cereblon dependent. 293FT CRBN−/− cells were transfected withplasmids expressing 8× His–Ub. After 48 h, cells were treated with CC-885
and MG-132 as indicated for 8 h. Whole–cell extract and ubiquitinatedprotein products enriched with nickel sepharose were subjected toimmunoblot analysis. The anti-His ubiquitin immunoblot showing equal
enrichment of ubiquitinated proteins is shown in Extended Data Fig. 3a.
Arrowhead indicates nonspecific band. f, In vitro ubiquitination of GSPT1by the CUL4A–RBX1–DDB1–cereblon complex. Recombinant protein
products as indicated were incubated with or without ATP (10 mM) andCC-885 (100 μM) in the ubiquitination assay buffer at 30 °C for 2 h, andthen analysed by immunoblotting. Results in d–f are representative of
three independent experiments. For gel source data, see Supplementary
Information Fig. 1.
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a

Degradation by
CC-885

GSPT1
SUP35

1–685
1–561

SUP35–GSPT1–SUP35

b

+

1–616

GSPT1–SUP35–GSPT1

H
A–
G
SP
H T1
(G A–G Δ(157 S – 1
5N PT 38
H /E5 1Δ )(G A–G 76 (1–57 S R) 13
8)5N PT
H ) 1Δ(1–(E A–G
1
57 S
38
6R PT
)/
1Δ
)
(1
–1
38
)/

reSeArcH Article

1–612

613– 594644 637–
DMSO +– CC-885 (1 μM) –645–
562–6455685
593 685
5

+

– +
+ –

–
+

+
–

–
+

+
–

–
+

+
–

–
+

(8 h)
HA
GSPT1

575
H. Sapiens GSPT1
S. Cerevisiae SUP35

CRBN

RLU normalized toparental DMSO control (%)

GSPT1Δ(1–138)
GSPT1Δ(1–138)/(G575N)

150

d

GSPT1

100

eRF1

50

CRBN
Tubulin

0
0.001

0.01

0.1

1

OCI-AML2

CC-885(μM)

e

OCI-AML2
Relative fold change
normalized to day 4

293T Parental

c

Pa
re
s h nta
C l
sh NT
G L
sh SP
G T1
sh SPT -1
G 1
sh SP -2
G T1
SP -3
T1
-4

Actin
293T

8

ParentalshCNTLshGSPT1-1
shGSPT1-2
shGSPT1-3
shGSPT1-4

6
4
2
0

4

6
8
Days after KD

10

Figure 2 | GSPT1 degradation is necessary and sufficient for the CC885-associated cytotoxicity. a, Schematic of the human GSPT1 andSaccharomyces cerevisiae SUP35 chimaeras. The CC-885-dependentdegradation motif of human GSPT1, as well as the corresponding region
present in SUP35, are highlighted. Change of protein stability of the
GSPT1–SUP35 fusion proteins in response to CC-885 treatment is shownin Extended Data Fig. 5d. b, Immunoblot analysis of 293T HEK cells
stably producing HA-tagged GSPT1 wild-type and variants. Dotted line
marks where irrelevant lanes were removed. Detailed domain mapping
experiments are shown in Extended Data Fig. 5. c, Effect of CC-885 on
proliferation of 293T parental cells or stably expressing GSPT1 variants.

Result is representative of three biological replicates. Data are presented
as mean ± s.d., n = 3 technical replicates. d, e, Cell growth curve of OCIAML2 parental cells or cells transduced with lentiviral vectors expressing
a control shRNA (shCNTL) or GSPT1 specific shRNA (shGSPT1-1 to 4).
Immunoblot analysis of whole-cell extracts (d) was carried out at day 4
after transduction with lentiviral vectors. Cell growth (e) was quantified
with CellTiter-Glo (CTG) at day 4, 6, and 8 after transduction. Results are
representative of three biological replicates. Data are mean ± s.d. n = 3
technical replicates. For gel source data, see Supplementary Information
Fig. 1.

sequence homology with human GSPT1, but did not show any changes
in protein level in response to CC-885 when stably expressed in 293T
HEK cells (Extended Data Fig. 5). Through a series of domain-swap
experiments, we identified that conversion of GSPT1 glycine 575 into
the corresponding asparagine of SUP35 prevented both the CC-885induced degradation of GSPT1 and the in vitro binding to cereblon(Fig. 2a, Extended Data Fig. 5). We then tested CC-885 in 293T HEK
cells stably expressing the CC-885-sensitive or -resistant GSPT1
variants. Overexpression of a resistant variant GSPT1Δ(1–138)/(G575N) completely abrogated the CC-885-induced anti-proliferation(Fig. 2b, c), whereas overexpression of a CC-885-sensitive variant GSPT1Δ(1–138) only conferred partial protection. Similar
results were obtained in AML cell lines OCI-AML2 and MOLM-13(Extended Data Fig. 6a, b). To determine whether GSPT1 depletion is sufficient to block cell proliferation, we used lentiviralshort hairpin RNA (shRNA) vectors to silence the expression ofendogenous GSPT1 in 293T HEK, OCI-AML2, and MOLM-13
cells and found a marked reduction of GPST1 protein level correlated with decreased cell fitness as compared to parental cells orcells expressing a control shRNA (Fig. 2d, e and Extended Data
Fig. 6c–e). Together, these experiments indicate that CC-885 cytotoxicity largely, if not completely, depends upon loss of GSPT1. This isconsistent with a previously published report showing that mutationof SUP35 prevented the G1 to S phase transition during cell cycle
progression in Saccharomyces cerevisiae25. Similarly, depletion of
GSPT1 led to cell cycle arrest at G1 in the human colorectal cancer cellline HCT116 (ref. 26).

direct interactions with both CC-885 and the proximal cereblon surface(Fig. 3a). GSPT1 domain 2 does not directly contact either CC-885
or cereblon. Cereblon is bound to DDB1 in a manner consistent withthe previously determined structures of cereblon–DDB1 (refs 10, 11).
CC-885 is bound in the tri-Trp pocket of cereblon, where the glutarimide ring makes 3 hydrogen bonds with cereblon, two to the backbone of residues W380 and H378, and one to the side chain of H378
(Fig. 3b). The isoindolinone ring of CC-885 (Fig. 1a) is presented on thesurface of cereblon in a manner similar to lenalidomide, and interacts
with both cereblon and GSPT1, including a hydrogen bond from theisoindolinone carbonyl oxygen to the side chain of N351 of cereblon.
The chemical structure of CC-885 is extended compared to lenalidomide or pomalidomide (Fig. 1a), allowing further interactions withboth cereblon and GSPT1. The urea moiety of CC-885 is positioned
between cereblon residues E377 and H353, with hydrogen bonds toboth side chains. The terminal methyl-chloro-phenyl ring is positioned
proximal to the β-sheet core of GSPT1 domain 3.
CC-885 is critical for the formation of the complex as demonstrated
by surface plasmon resonance experiments showing that GSPT1 binds
to cereblon-DDB1 with a dissociation constant (KD) of ~350 nM in
the presence of CC-885 (Extended Data Fig. 7). No significant interaction was observed in the absence of CC-885. Similarly, negative stain
electron microscopy confirmed that CC-885 was critical for cereblon–
GSPT1 complex formation, as no cereblon–GSPT1 particles wereobserved in the absence of CC-885 (Extended Data Fig. 8).

Structure of cereblon with DDB1, CC-885 and GSPT1

To explore the molecular requirements for ligand-directed substrate
recruitment to cereblon, we determined the crystal structure of fulllength human DDB1 bound to human cereblon (amino acids 40–442),
CC-885, and domains 2 and 3 of human GSPT1 (amino acids 437–633)
to 3.6 Å resolution (Fig. 3a, full data collection and refinement statistics
given in Extended Data Table 1). In the crystal structure, GSPT1
domain 3 docks against cereblon at the site of CC-885 binding with

GSPT1 binds a ‘hotspot’ on the cereblon–CC-885 surface

It was previously hypothesized that immunomodulatory drug binding
forms an interaction hotspot on the cereblon surface by placement ofa planar hydrophobic group (the ligand phthalimide or isoindolinone
ring, Fig. 1a) amongst unsatisfied hydrogen bonds from both ligand
and the protein surface10. In this way, ligands with low molecularweight would be able to achieve marked alterations in binding potency
by promoting contributions towards substrate binding from thecereblon surface. The structure of cereblon–DDB1–CC-885–GSPT1
confirms that GSPT1 interacts directly with CC-885 and also forms

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Article reSeArcHab

may be due to subtle differences in residue side chain bulk or substrate
conformation at the interaction site. Lenalidomide and pomalidomide
do not cause GSPT1 binding and degradation (Fig. 1b and Extended
Data Fig. 2). These molecules lack the urea and chloro-methyl-phenyl
moieties of CC-885 (Fig. 1a) that probably contribute to the affinity of
the complex through interactions with domain 3 of GSPT1.

F150

H353

GSPT1

Y355

E576

Cereblon
G575

S574 H397
H357

E377

N351

W400

V388

GSPT1 features required for recruitment to cereblon

H378

DDB1

c

H103
F150

E377

P352

N351
H357
T376 W386 W400H397
V388
I37I
K406
R373
K407

d

F102
H353 Y355

W386

W380

e

H103
F150

F102

CC-885

H353 Y355
P352
E377
N351
H357
W386 W400
T376
V388 H397
Q325
I37I
K406
R373

GSPT1

Q325

K407

Figure 3 | Crystal structure of the cereblon–DDB1–GSPT1–CC-885
complex. a, Surface representation with DDB1 shown in purple, cereblon
in green and GSPT1 in blue. Position of CC-885 is indicated by an arrow.
b, Details of the interface between cereblon and GSPT1 with CC-885
shown as sticks with carbons in yellow. Hydrogen bonds between CC-885
and cereblon are shown as yellow dotted lines. c–e, The effect of cereblon
surface mutagenesis on substrate binding (see also Extended Data Fig. 9).
Red indicates a strong reduction in substrate binding following mutation
to alanine, orange indicates some reduction, and green indicates no effect.
Blue surface areas were not tested. c, The effect of the mutations on GSPT1
recruitment; d, the mutational results for Ikaros binding; e, the relative
positions of CC-885 (yellow sticks) and the GSPT1 cereblon-interacting
motif (orange).

direct protein–protein interactions, with cereblon residues N351, H357,
and W400 all contributing direct hydrogen bonds to GSPT1. Further
van der Waals interactions are provided from the side chains of cereblon residues Y355, H397, and V388.
Co-immunoprecipitation of GSPT1 with cereblon surface mutants
reveals that residues including N351, H357, E377, V388 and H397 are
critical to recruiting GSPT1 to cereblon in a manner dependent upon
CC-885 (Fig. 3c, Extended Data Fig. 9). Each of the three side chainsthat hydrogen bond to GSPT1 (N351, H357 and W400) is shown to be
critical for GSPT1 recruitment. When mutation sensitivity is mapped
onto the surface of cereblon by colour, a patch of sensitivity is revealed
that corresponds to the interaction site visible in the crystal structure(Fig. 3c, e).
To evaluate whether this patch of the cereblon surface is similarly
purposed for the recruitment of other substrates, the same panel of
cereblon mutants was tested against Ikaros (Fig. 3d, Extended Data
Fig. 9). As shown in Fig. 3c, d, the critical interaction site is similar
between GSPT1 and Ikaros. Interestingly, all three residues that
contribute hydrogen bonds to GSPT1 are also required for Ikaros
recruitment, suggesting a common mechanism of substrate recruitment. Residues E377 and F150, however, are uniquely required for
GSPT1 recruitment. F150 is proximal to CC-885 and contacts domain
3 of GSPT1 in a manner that is dependent on the GSPT1 tertiary structure; it is therefore probably different from other substrates that do notshare a common fold. E377 makes direct interactions with CC-885,
and mutation may therefore affect ligand binding and conformation.
An E377V polymorphism is found in mouse and rats and underlies
resistance to CC-885 treatment in rodents (Extended Data Fig. 9 e andSupplementary Discussion). A further difference in cereblon–substrateinteractions is observed at V388: GSPT1 binding is lost in the V388A
mutant, but some binding is retained in the V388I mutant, which is also
a rodent polymorphism. In contrast, Ikaros binding is abolished by the
V388I mutation, consistent with previous work5; however, the V388A
mutation has no significant effect on Ikaros binding. These differences

The GSPT1 motif that mediates interactions with cereblon–CC-885
is a solvent-exposed region composed of residues 569–578, includinga small anti-parallel β-sheet forming a β-hairpin with an α-turn fromresidues 571–575 (Fig. 4a). Despite the degree of solvent exposure, theturn is well-ordered in both the previous GSPT1 crystal structure17 andin the complex with cereblon. The conformational stability of the turnis probably due to intramolecular bonding, with an ASX-motif and an
ST-turn mediated by D571 and S574, respectively. These intramolecularmotifs might be expected to rigidify and stabilize the conformation of
the turn before cereblon binding (Fig. 4a). The key hydrogen bonds
formed with cereblon are all mediated by backbone carbonyl oxygen
atoms, which are displayed in an array at the end of the turn. Carbonyl
oxygen atoms from GSPT1 K572, K573, and S574 accept hydrogenbonds from cereblon residues N351, H357, and W400, respectively(Fig. 4a). The GSPT1 turn lies on top of the isoindolinone ring of CC-885
making hydrophobic and van der Waals interactions. A further key
interaction occurs between G575 and the isoindolinone moiety. G575
is of particular importance in determining substrate recruitment, as it
not only contributes to binding interactions, but also the close sterics
at this position indicate that no other residue would be tolerated at thisposition. Therefore, the degron is not the linear peptide sequence, butrather the geometric arrangement of three backbone hydrogen bondacceptors at the apex of a turn (positions i, i+1, and i+2), with a glycine
residue at a key position (i+3).
The structural interpretation is supported by site-directed mutagenesis of the GSPT1 turn, which indicates that G575A loses CC-885dependent cereblon binding (Fig. 4c). It should be noted that the
backbone torsion angles of G575 occupy a part of the Ramachandranplot only accessible to glycine residues and mutagenesis may alter bothside chain decoration and backbone position. V570 exhibits van der
Waals interactions with the methylene–urea linker, and accordingly, theV570A mutant displays reduced cereblon binding (Fig. 4a, c). Consistent
with their expected role in stabilizing the turn conformation, both
D571A and S574A mutants lose cereblon binding (Fig. 4c). However,the side chains of residues K572, K573 and E576 do not affect GSPT1
binding to cereblon, consistent with the crystal structure that indicatesthese residues have minimal side chain interactions with cereblon. The
fact that key bonds are formed from the peptide backbone of GSPT1,
rather than from side chain residues, presents the intriguing possibility that there may be considerable sequence tolerance in substrate
recruitment, as long as the backbone remains in the appropriate
conformation.

Ikaros binds via a similar structural motif

GSPT1 contains no obvious structural or sequence homology to thepreviously identified cereblon substrates such as the protein kinase
CK1α or the zinc finger transcription factor Ikaros. However, thesimilar pattern of sensitivity to mutations on the surface of cereblon(Fig. 3c, d) suggests that a similar anchoring motif may be present in
Ikaros. On the basis of the observation that the GSPT1 interactions
with cereblon were mediated by the protein backbone, we constructeda homology model of Ikaros and examined this for a similar surface turn. The homology model of Ikaros indicated that a zinc finger
domain is indeed capable of presenting a turn that can make the three
key hydrogen bonds with a glycine residue at the critical positionon the solvent-accessible surface of the domain (Fig. 4b). Consistent
with this prediction, a minimal domain of Ikaros containing only
this single predicted zinc finger domain (amino acids 140–168),
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reSeArcH Article
b

a
V570

Q146

D571

E576

H353

G575
H353

K572

V388

G575
G151
N148

S574
N351

Y355

H397

Y355
Q149

W400

K573

N351
W400

H357

H357

WT

V570A D571A K572A K573A S574A G575A E576A– + – +

– + – +

– +– + – + – +100 μM CC-885

C

RB
N N–
o D
G D
SP B
T1 1

C

WT

RB
N N–
o D
Ik D
ar B
os 1

d

c

– +

Q146A N148A Q149A G151A Q146H

–

+ – +

– +

– + –

+ 100 μM pomalidomide

DDB1

DDB1

CRBN

CRBN
MBP–Ikaros

GST–GSPT1e

GSPT1 570
Ikaros 146

9'..6*(
4&14&*$

576
152

Figure 4 | Substrate proteins bind cereblon via a surface turn. a, Theanchoring motif of GSPT1 (blue) bound to cereblon (green) and CC-885(yellow). b, A model of Ikaros (magenta) superimposed onto the backbone
of GSPT1 (blue). c, Coomassie-stained gels of cereblon–DDB1 pull-down
by GST–GSPT1 (amino acids 526–633), with alanine mutations of the
GSPT1 anchoring motif. d, Cereblon–DDB1pull-down with MBP–Ikaros(amino acids 140–168) with alanine mutations of the proposed Ikarosanchoring motif. This experiment is representative of three independent
replicates (c, d). e, Sequence alignment of the GSPT1-anchoring motif
with the putative region from Ikaros, with residues shown to be criticalfor binding in red, and those where an alanine mutation was tolerated ingreen. The critical glycine residue is shown in bold. Ikaros C147 and C150
were not mutated as they coordinate a structural zinc ion (not shown). Forgel source data, see Supplementary Information Fig. 1.

is sufficient to pull down cereblon–DDB1 in the presence of pomalidomide (Fig. 4d).
Crucially, there is no sequence homology between Ikaros and GSPT1in the anchoring motif regions as modelled, except for the glycine
residue at position i+3 (Fig. 4e). The implications of this model were
tested by co-immunoprecipitation of cereblon with mutations targeting
each residue of the putative Ikaros anchoring motif. Mutation of the
key glycine residue (G151) to alanine disrupts cereblon interactions(Fig. 4d). It was previously demonstrated that the Q146H mutation
causes a reduction of cereblon binding15,16. The putative anchoring
motif modelled in Ikaros positions the critical residue, Q146, in aposition oriented towards both bound ligand and the cereblon surface. Mutation of the putative anchoring motif in Ikaros confirms that
Q146H causes a reduction in cereblon binding (Fig. 4d, Extended Data
Fig. 2a). In contrast, the Q146A mutation is similar to wild type, suggesting that steric or electronic hindrance of a histidine residue at thisposition, rather than loss of key interactions from the glutamine, is
responsible for the loss of cereblon binding in the Q146H mutants.
N148A and Q149A mutations in the proposed anchoring motif ofIkaros do not affect cereblon binding, in a manner analogous to theequivalent positions K572 and K573 in GSPT1 (Fig. 4 c, d). C147 andC150 are responsible for coordination of a structural zinc ion, and were
therefore not examined by mutagenesis out of concern for disruptionof the zinc finger domain structure. Structural studies on Ikaros family
members in complex with cereblon will be necessary to identify the fullatomic details of the interactions.

provides expanded clinical potential for cereblon modulators, withpotent effects seen in both AML cell lines and in patient-derived
tumour cells. Furthermore, by targeting what may have been considered
an otherwise undruggable protein, the utility of directing cereblon
for targeting diverse protein substrates posits a wide range of new
therapeutic opportunities.
In contrast to linker-based approaches exploiting cereblon27–29,
CC-885 creates an interaction hotspot on the cereblon surface fordirect protein–protein interactions with the substrate, underlying thegain-of-function ‘chemo-neomorphic’ activity. Although this may constrain the potential substrate range, the contribution from the cereblon
surface supports the function of small drug-like molecules without thepharmacokinetic challenges that may accompany ligase modulators
incorporating large flexible linkers.
Our structural work shows that cereblon recruits GSPT1 throughthe CC-885-induced recognition of a surface turn. A key to the stericcompatibility is the presence of a glycine at the precise position within
the turn. Significantly, except the glycine residue, the interactions arecomposed of hydrogen bonds from the backbone of GSPT1, indicatingthat the degron in this instance will not be revealed by primary
sequence, but instead by the geometry and sterics. Our mutational andmodelling work on Ikaros raises the intriguing possibility of a common structural degron occurring amongst diverse substrate proteins.
Furthermore, after submission of this manuscript, a crystal structure of
cereblon in complex with lenalidomide and CK1α was published thatexhibits a binding mode consistent with our degron30. It may therefore
be possible to rationally identify candidate neosubstrates by searchingfor the cereblon response element across the proteome. Such candidate
substrates may then form targets for exploitation by the next generationof cereblon modulators.

Discussion

In this work, we have extended the clinical scope of cereblonmodulating activities with the identification of a novel ligand, CC-885,
which is potently anti-proliferative in cancer cell lines. CC-885 directs
cereblon to target GSPT1, a substrate that is unrelated in fold andfunction to previously described substrates. Targeting of GSPT1Online Content Methods, along with any additional Extended Data display items andSource Data, are available in the online version of the paper; references unique tothese sections appear only in the online paper.

2 5 6 | N AT U R E | V O L 5 3 5 | 1 4 J U Ly 2 0 1 6© 2016 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.

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Article reSeArcH
received 27 November 2015; accepted 31 May 2016.
Published online 22 June 2016.
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127–130 (2016).
Supplementary Information is available in the online version of the paper.
Acknowledgements Thanks to G. Reyes, C. Havens, P. Jackson and
H. Hadjivassiliou for discussions relating to this manuscript, and P. Jackson,
J. Hansen, M. Correa, B. Fahr, M. Abbasian, E. Ambing, E. Rychak, D. Mendy andK. Hughes for technical assistance. We thank K. Motamedchaboki (Proteomics
Core, Sanford Burnham Prebys Medical Discovery Institute) for mass
spectrometry-based proteomic analysis. Thanks to J. Ballesteros and
P. Hernandez at Vivia for patient sample testing. Parts of this work wereconducted at the Advanced Light Source. The Berkeley Center for Structural
Biology is supported in part by the National Institutes of Health, National
Institute of General Medical Sciences, and the Howard Hughes Medical Institute.
The Advanced Light Source is supported by the Director, Office of Science,
Office of Basic Energy Sciences, of the US Department of Energy under contract
No. DE-AC02-05CH11231. This work was also supported by Grant-in-Aid for
Scientific Research on Innovative Areas “Chemical Biology of Natural Products”
from the Ministry of Education, Culture, Sports, Science and Technology(MEXT) (23102002 to H.H.), by JST, PRESTO (T.I.) and by Grant-in-Aid for Young
Scientists (B) from MEXT (26750374 to T.I.).
Author Contributions M.E.M., P.P.C., B.P, W.F., J.H., T.I., G.C., and M.R. performed
biochemical and crystallographic structural studies. G.L., C.-C.L., K.M., N.-Y.W.,
D.N., T.T., S.G., and S.X. performed molecular and cellular biology experiments.
G.C.L, L.N., M.E.M, and P.P.C. performed electron microscopy studies. A.L.R.
performed chemical synthesis. M.E.M., G.L., A.L.-G., J.C., T.I., H.H., T.O.D., B.C., and
P.P.C. planned the work, and all authors contributed to the manuscript.
Author Information Coordinates for the structure of cereblon–DDB1–
CC-885–GSPT1 have been deposited in the Protein Data Bank with theaccession code 5HXB. Reprints and permissions information is available atwww.nature.com/reprints. The authors declare competing financial interests:
details are available in the online version of the paper. Readers are welcome
to comment on the online version of the paper. Correspondence and requests
for materials should be addressed to P.P.C. (pchamberlain@celgene.com) or
G.L. (glu@celgene.com).

1 4 J U Ly 2 0 1 6 | V O L 5 3 5 | N AT U R E | 2 5 7© 2016 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.

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MethOdS

Data reporting. No statistical methods were used to predetermine sample size.
The experiments were not randomized and the investigators were not blinded toallocation during experiments and outcome assessment.
Protein expression and purification. ZZ-domain-6×His-thrombin-tagged
human cereblon (amino acids 40–442) and full-length human DDB1 were coexpressed in SF9 insect cells in ESF921 medium (Expression Systems), in the presenceof 50 μM zinc acetate. Cells were resuspended in buffer containing 50 mM
Tris-HCl (pH 7.5), 500 mM NaCl, 10 mM imidazole, 10% glycerol, 2 mM TCEP,
1× Protease Inhibitor Cocktail (San Diego Bioscience), and 40,000 U Benzonase(Novagen), and sonicated for 30 s. Lysate was clarified by high speed spin at
108,800g for 30 min, and clarified lysate was incubated with Ni-NTA affinity resin(Qiagen) for 1 h. Complex was eluted with buffer containing 500 mM imidazole,and the ZZ-domain-6×His tag removed by thrombin cleavage (Enzyme Research)overnight, combined with dialysis in 10 mM imidazole buffer. Cleaved eluate wasincubated with Ni-NTA affinity resin (Qiagen), and the flow-through diluted to
200 mM NaCl for further purification over an ANX HiTrap ion exchange
column (GE Healthcare). The ANX column was washed with ten column
volumes of 50 mM Tris-HCl (pH 7.5), 200 mM NaCl, 3 mM TCEP, followed by ten
column volumes of 50 mM Bis-Tris (pH 6.0), 200 mM NaCl, 3 mM TCEP, and the
cereblon–DDB1 peak eluted at 210 mM NaCl. This peak was collected and further
purified by size-exclusion chromatography using a Sephacryl S-400 16/60 column(GE Healthcare) in buffer containing 10 mM HEPES (pH 7.0), 240 mM NaCl, and3 mM TCEP. The cereblon–DDB1 complex was concentrated to 30 mg ml−1 forcrystallization trials.
GSPT1 domains 2 and 3 (amino acids 437–633) were expressed as an MBPfusion in Escherichia coli BL21 (DE3) Star cells (Life Technologies) using 2XYT
media (Teknova). Cells were induced at optical density OD600 0.6 for 18 h at 16 °C.
Cells were pelleted, resuspended in buffer containing 200 mM NaCl, 50 mM Tris
(pH 7.5), 1 mM TCEP, 10% glycerol, 0.01 mg ml−1 lysozyme (Sigma), 10,000 U
benzonase (Novagen), and 1× protease inhibitor cocktail (San Diego Bioscience).
Resuspended cells were frozen, thawed for purification, and sonicated for 30 s
before high speed spin at 108,800g for 30 min. Clarified lysate was incubated withamylose resin (NEB) at 4 °C for 1 h before beads were washed with 500 ml wash
buffer containing 200 mM NaCl, 50 mM Tris (pH 7.5), 1 mM TCEP, 10% glycerol.
Protein was eluted with buffer containing 10 mM maltose, and the MBP tag wasremoved by overnight cleavage with thrombin (Enzyme Research). Cleaved GSPT1
was diluted into buffer containing 90 mM NaCl and further separated over a
Heparin HiTrap column (GE healthcare). The GSPT1 peak eluting at 100 mM
NaCl was collected, concentrated, and further separated by size exclusion chromatography using a Superdex 75 16/600 column (GE Healthcare) in buffer containing 10 mM HEPES pH 7, 240 mM NaCl, and 3 mM TECP. The peak containing
GSPT1 domains 2 and 3 eluted at 75 ml and was concentrated to 16 mg ml−1 forcrystallization trials.
MBP–Ikaros 140–168 and mutants were expressed in E.coli BL21 (DE3) Star
cells (Life Technologies) using 2XYT media (Teknova). Cells were induced at
OD600 0.6 for 18 h at 16 °C. Cells were pelleted, resuspended in buffer containing
200 mM NaCl, 50 mM Tris (pH 7.5), 3 mM TCEP, 10% glycerol, 150 μM zincacetate, 0.01 mg ml−1 lysozyme (Sigma), 40,000 U benzonase (Novagen), and1× protease inhibitor cocktail (San Diego Bioscience). Resuspended cells werefrozen, thawed for purification, and sonicated for 30 s before high-speed spin at
108,800g for 30 min. Clarified lysate was incubated with amylose resin (NEB) at
4 °C for 1 h before beads were washed. Protein was eluted with buffer containing
200 mM NaCl, 50 mM Tris (pH 7.5), 3 mM TCEP, 10% glycerol, 150 μM zincacetate, and 10 mM maltose. Eluate was concentrated and further purified by size
exclusion chromatography over a Superdex 200 16/600 column (GE Healthcare) in
buffer containing 200 mM NaCl, 50 mM Tris (pH 7.5), 3 mM TCEP, 10% glycerol,and 150 μM zinc acetate.
GST–GSPT1 domain 3 (amino acids 526–633) and mutants were expressedin E.coli BL21 (DE3) Star cells (Life Technologies) using 2XYT media(Teknova). Cells were induced at OD600 0.6 for 18 h at 16 °C. Cells werepelleted, resuspended in buffer containing 200 mM NaCl, 50 mM Tris (pH 7.5),
3 mM TCEP, 10% glycerol, 0.01 mg ml−1 lysozyme (Sigma), 20,000 U benzonase(Novagen), and 1× protease inhibitor cocktail (San Diego Bioscience).
Resuspended cells were frozen, thawed for purification, and sonicated for 30 s
before high-speed spin at 108,800g for 30 min. Clarified lysate was incubatedwith Glutathione Sepharose 4 Fast Flow (GE Healthcare) at 4 °C for 2 h
before beads were washed. Protein was eluted with buffer containing 200 mM
NaCl, 50 mM Tris (pH 7.5), 3 mM TCEP, 10% glycerol, and 20 mM reduced
glutathione. Eluate was concentrated and further purified by size-exclusion
chromatography over a Superdex 200 16/600 column (GE Healthcare) in
buffer containing 200 mM NaCl, 50 mM Tris (pH 7.5), 3 mM TCEP, and 10%
glycerol.

6×His–eRF1 (human, full length) was expressed in E. coli BL21 (DE3) Star cells(Life Technologies) using 2XYT media (Teknova). Cells were induced at OD600 0.6
for 18 h at 16 °C. Cells were pelleted, resuspended in buffer containing 150 mM
NaCl, 50 mM Tris (pH 7.5), 3 mM TCEP, 10% glycerol, 0.01 mg ml−1 lysozyme(Sigma), 20,000 U benzonase (Novagen), and 1× protease inhibitor cocktail(San Diego Bioscience). Resuspended cells were frozen, thawed for purification,and sonicated for 30 s before high speed spin at 108,800g for 30 min. Clarified lysate
was incubated with Ni-NTA affinity resin (Qiagen) at 4 °C for 1 h before beads
were washed. Protein was eluted with buffer containing 150 mM NaCl, 50 mM
Tris (pH 7.5), 3 mM TCEP, 10% glycerol, and 500 mM imidazole. Eluate wasconcentrated and further purified by size-exclusion chromatography over a
Superdex 200 16/600 column (GE Healthcare) in buffer containing 150 mM NaCl,
50 mM Tris (pH 7.5), 3 mM TCEP, and 10% glycerol.
6×His–DDB1 (human, full length) was expressed in SF9 insect cells in ESF921
medium (Expression Systems). Cells were resuspended in buffer containing 50 mM
Tris-HCl (pH 7.5), 500 mM NaCl, 10 mM imidazole, 10% glycerol, 2 mM TCEP,
1× Protease Inhibitor Cocktail (San Diego Bioscience), and 40,000 U Benzonase(Novagen), and sonicated for 30 s. Lysate was clarified by high-speed spin at
108,800g for 30 min, and clarified lysate was incubated with Ni-NTA affinity resin(Qiagen) for 1 h. Complex was eluted with buffer containing 500 mM imidazole.
Eluate was collected and further purified by size-exclusion chromatography over a
Superdex 200 16/600 column (GE Healthcare) in buffer containing 150 mM NaCl,
50 mM Tris (pH 7.5), 3 mM TCEP, and 10% glycerol.
The V protein of simian virus 5 (SV5-V) (full length) was expressed in E. coli
BL21 (DE3) Star cells (Life Technologies) using 2XYT media (Teknova) as a GSTfusion protein. Cells were induced at OD600 0.6 for 18 h at 16 °C. Cells were pelleted,
resuspended in buffer containing 150 mM NaCl, 50 mM Tris (pH 7.5), 3 mM TCEP,
10% glycerol, 0.01 mg ml−1 lysozyme (Sigma), 20,000 U benzonase (Novagen), and1× protease inhibitor cocktail (San Diego Bioscience). Resuspended cells werefrozen, thawed for purification, and sonicated for 30 s before high speed spin at
108,800g for 30 min. Clarified lysate was incubated with Glutathione Sepharose
4 Fast Flow (GE Healthcare) at 4 °C for 2 h before beads were washed and protein
eluted with buffer containing 150 mM NaCl, 50 mM Tris (pH 7.5), 3 mM TCEP,
10% glycerol, and 20 mM reduced glutathione. One half of the eluate was incubatedwith thrombin to remove the GST tag, while the other half retained the tag. Tagged
and un-tagged proteins were further purified by size-exclusion chromatography
over a Superdex 200 16/600 column (GE Healthcare) in buffer containing 150 mM
NaCl, 50 mM Tris (pH 7.5), 3 mM TCEP, and 10% glycerol.
SV5-V–DDB1 complex for the in vitro ubiquitination assay was generated byincubating stoichiometric amounts of purified, un-tagged SV5-V and purified
6×His–DDB1 overnight at 4 °C before purification of the complex by separation
over a Superdex 200 16/600 column (GE Healthcare) in buffer containing 150 mM
NaCl, 50 mM Tris (pH 7.5), 3 mM TCEP, and 10% glycerol.
Negative stain electron microscopy analysis of GSPT1 binding to cereblon–
DDB1–CC-885. To prepare grids for negative stain analysis of isolated cereblon–
DDB1 or cereblon–DDB1 bound to CC-885 and GST–GSPT1 domains 2 and3 (amino acids 437–633), pre-incubated protein complex samples were rapidly
diluted from 30 μM to 0.3 μM in a buffer containing 50 mM Tris (pH 7.5), 150 mM
NaCl, and 1 mM TCEP. A 4 μl aliquot was applied to freshly plasma-cleaned 400
mesh Cu-Rh maxtaform grids (Electron Microscopy Sciences) that had been coatedwith a thin layer of carbon. After incubating for 1 min, excess protein was wicked
off with Whatman filter paper and the grid was immediately inverted and placedon a 50 μl droplet of 2% (w/v) uranyl formate solution. After 30 s, excess stain was
wicked off from the grid by touching the edge with filter paper. This staining step
was repeated three times for thorough embedding of the sample, and the grids were
air dried on the edge of a fume hood for constant air flow after the last blotting step.
Samples were imaged on a Tecnai Spirit operating at 120 keV. Data were collectedusing the Leginon automated image acquisition software (PMID 15890530), andanalysed using the Appion processing package (PMID 9263523).
Crystallization and structure determination. Crystallization of the complex
was achieved by sitting-drop vapour diffusion. Cereblon–DDB1 and GSPT1 weremixed together to equimolar stoichiometry at a final concentration of 150 μM.
The solution of cereblon–DDB1–GSPT1 in the presence of 500 μM CC-885 wasmixed 1:1 with, and subsequently equilibrated against, a mother liquor solutionof 200 mM sodium citrate, Tris (pH 8.4–8.6), 17–20% PEG 3350 and incubated
at 9 °C. Crystals were cryoprotected in the reservoir solution supplemented with
20% ethylene glycol and cooled under liquid nitrogen. Data was collected froma single crystal at the Advanced Light Source, beamline 5.0.2. The structure ofhuman cereblon–DDB1–GSPT1–CC-885 was solved by molecular replacement
using Phaser31, with human cereblon–DDB1 (PDB code 4TZ4) and GSPT1 (PDB
code 3E1Y) as search models. Subsequent manual model building using Coot andrefinement were performed using Refmac5 with non-crystallographic symmetry
and external structure restraints32,33. Crystallographic statistics are summarized© 2016 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.

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in Extended Data Table 1, sample electron density is shown in Extended Data
Fig. 7d. Ikaros was modelled on the basis of sequence homology to coordinateswith the Protein Data Bank accession number 2I13.
Surface plasmon resonance binding assay. Binding kinetics were measured usinga Biacore T200. Using the GST-capture kit (GE Healthcare, BR-1002-23), ~2000
relative units (RU) of anti-GST antibody was immobilized on a CM5 chip (GEHealthcare). GST–GSPT1 domains 2 and 3 (0.07 μg ml−1) or GST (0.15 μg ml−1,
reference channel) was then flowed over the chip at 10 μl min−1 for 2 min, resulting
in a capture level of approximately 10 RU. A threefold dilution series of cereblon–
DDB1 (3 μM to 3 nM) was then flowed over both channels at a rate of 30 μl min−1
for 300 s contact time, followed by 900 s dissociation time. After each cycle ofbinding and dissociation, the surface was regenerated with 10 mM glycine (pH 2.1)
for 120 s at 30 μl min−1, followed by 3 M MgCl2 for 60 s at 30 μl min−1. All reagents(GST, GST–GSPT1 domain 2 and 3, cereblon–DDB1, and running buffer) were in
10 mM HEPES (pH 7.4), 150 mM NaCl, 0.05% v/v Surfactant P20 containing 0.02%
DMSO and saturating levels of the relevant compound, glutarimide or CC-885(CC-885 at 10 μM, glutarimide at 100 μM). The apparent on- and off-rate constants
were globally fit with a 1:1 kinetic binding model to the sensograms (black lines)
using the Bioacore T200 kinetic analysis software package.
Pull-down binding assays of cereblon and substrates using purified recombinant components. Pull-down of cereblon–DDB1 with either GSPT1 or Ikaros was
performed using the same protocol. Tagged substrate, either GST–GSPT1–domain
3 (amino acids 526–633) or MBP–Ikaros (amino acids 140–168), was expressed inbacteria and purified using affinity and size-exclusion chromatography. Substrate
was then bound to magnetic glutathione (Pierce) or amylose (NEB) beads for 1 h.
Beads were washed three times in pull-down buffer (50 mM Tris pH 7.5, 150 mM
NaCl, 10% glycerol, 0.01% NP-40, and 1 mM TCEP, with the addition 150 μM zincacetate for Ikaros) to remove excess unbound substrate, before the addition of
DMSO or 100 μM compound and 50 μM cereblon–DDB1, purified from insect cells
using affinity, ion exchange, and size-exclusion chromatography. After 1 h incubation with occasional gentle mixing at room temperature, unbound cereblon–DDB1
was removed and beads were washed three times in pull-down buffer, for a total of
30 s. Substrate and bound cereblon–DDB1 was eluted using either 20 mM reduced
glutathione or 10 mM maltose, separated by SDS–PAGE, and Coomassie stained.
Tagged GST–GSPT1 domains 2 and 3 (amino acids 437–633) was expressed inbacteria and purified using affinity and size-exclusion chromatography. GST or
GST–GSPT1 domains 2 and 3 was then bound to magnetic glutathione (Pierce)beads for 1 h. Beads were washed 3 times in pull-down buffer (50 mM Tris pH 7.5,
150 mM NaCl, 10% glycerol, 0.01% NP-40, and 1 mM TCEP) to remove excess
unbound substrate, before the addition of DMSO or 100 μM compound and50 μM cereblon–DDB1, purified from insect cells using affinity, ion exchange,and size-exclusion chromatography. After 1 h incubation with occasional gentle
mixing at room temperature, unbound cereblon–DDB1 was removed and beads
were washed three times in pull-down buffer, for a total of 30 s. Substrate andbound cereblon–DDB1 was eluted using either 20 mM reduced glutathione, separated by SDS–PAGE, and Coomassie stained.
Pull-down of eRF1 or cereblon–DDB1 with GSPT1 domains 2 and 3 andCC-885. Tagged GST–GSPT1 domains 2 and 3 (amino acids 437–633) was
expressed in bacteria and purified using affinity and size exclusion chromatography. GST or GST–GSPT1 domains 2 and 3 was then bound to magnetic glutathione(Pierce) beads for 1 h. Beads were washed three times in pull-down buffer (50 mM
Tris (pH 7.5), 150 mM NaCl, 10% glycerol, 0.01% NP-40, and 1 mM TCEP) toremove excess unbound substrate, before the addition of DMSO or 100 μM compound and 50 μM purified cereblon–DDB1 or 30 μM purified eRF1, ±CC-885.
After 1 h incubation with occasional gentle mixing at room temperature, unboundcereblon–DDB1 or eRF1 was removed and beads were washed three times in pulldown buffer, for a total of 30 s. Substrate and bound cereblon–DDB1 or eRF1
was eluted using either 20 mM reduced glutathione, separated by SDS–PAGE, andCoomassie stained.
Pull-down binding assays of SV5-V, DDB1, and CUL4–RBX1. To confirm
that purified SV5-V binds to purified DDB1, and that this complex then binds
to CUL4–RBX1, we performed two consecutive GST pull-down assays. GST–
SV5-5 or GST alone was purified from bacteria using affinity and size exclusion
chromatography. Purified GST–SV5-V or GST was then bound to magnetic
glutathione beads (Pierce) for 1 h. Beads were washed three times in pull-down
buffer (50 mM Tris (pH 7.5), 150 mM NaCl, 10% glycerol, 0.01% NP-40, and 1 mM
TCEP) to remove excess GST–protein, before the addition of purified DDB1. After
1 h of incubation with occasional mixing at room temperature, unbound DDB1
was removed and beads were washed 3 times in pull-down buffer. Substrate andbound DDB1 were then eluted using either 20 mM reduced glutathione, separated
by SDS–PAGE, and Coomassie stained. For subsequent binding of CUL4–RBX1,
the same pull-down described above was used to generate GST–SV5-V–DDB1 on
beads. However, after the beads were washed to remove excess DDB1, CUL4–RBX1

was added and incubated for 1 h at room temperature before beads were washedthree times in pull-down buffer and bound protein eluted with 20 mM reduced
glutathione, separated by SDS–PAGE, and Coomassie stained.
In vivo degradation of cereblon substrates. 293FT (Invitrogen) CRBN−/− cells
transiently transfected with plasmids expressing V5-tagged IKZF1, Flag-tagged
Ikaros (IKZF3), Myc-tagged GSPT1, GFP and human or mouse cereblon variants.
36 h after transfection, cells were treated with DMSO, 10 μM lenalidomide, or 1 μM
CC-885 for an additional 12 h. Cells were then washed with ice-cold 1× PBS twice,lysed in buffer A (50 mM Tris-HCl, 150 mM NaCl, 1% Triton-X 100, complete
protease inhibitor tablet (Roche), phosphatase inhibitor tablet (Roche)). Whole-cell
extracts were collected and subjected to immunoblot analysis.
Mammalian lysate co-immunoprecipitation. 293FT (Invitrogen) CRBN−/−cells were transiently transfected with plasmids expressing Flag-tagged cereblon
variants and HA-tagged GSPT1(or HA-tagged IKZF1). After 2 days, cells werelysed in Buffer B (50 mM Tris (pH 7.4), 150 mM NaCl, 0.5% NP-40, 10% glycerol,
1× Complete Ultra protease inhibitor (Roche), and 1× PhosphoSTOP (Roche)).
Whole-cell extracts were collected after centrifugation at 16,000g for 10 min.
Whole-cell extracts containing cereblon and GSPT1 (or IKZF1) were then incubated with anti-HA affinity resin (Roche) in the presence of DMSO or 10 μM
CC-885 (or 10 μM lenalidomide). For testing the interaction between cereblon
variants and DDB1, whole-cell extracts expressing Flag–CRBN were incubatedwith anti-Flag affinity resin (Sigma) . After overnight incubation at 4 °C, anti-Flag
and anti-HA resins were washed with Buffer B six times. Anti-Flag and anti-HA
immunoprecipitates were then subjected to immunoblot analysis.
Cell culture and materials. Human embryonic kidney cell lines 293FT(Invitrogen), 293T (ATCC) and Lenti-X 293 (Clontech) were maintained in
Dulbecco’s Modified Eagle’s medium (DMEM; Invitrogen) supplemented with
10% fetal bovine serum (FBS; Invitrogen), 1× sodium pyruvate (Invitrogen),
1× non-essential amino acids (Invitrogen), 100 U ml−1 penicillin (Invitrogen), and100 μg ml−1 streptomycin (Invitrogen). Acute myeloid leukaemia cell lines KG-1,
Kasumi-1, U937, MOLM-13, HL-60, and MV-4-11 were purchased from American
Tissue Culture Collection (ATCC). NB-4, HNT-34, OCI-AML2, and OCI-AML3cell lines were purchased from Deutsche Sammlung von Mikroorganismen und
Zellkulturen GmbH. KG-1, Kasumi-1, U937, MOLM-13, NB-4, and HNT-34 celllines were maintained in Roswell Park Memorial Institute (RPMI) 1640 tissue
culture medium (Invitrogen) supplemented with 10% FBS, 1× sodium pyruvate,
1× non-essential amino acids, 100 U ml−1 penicillin, and 100 μg ml−1 streptomycin. HL-60 and MV-4-11 cell lines were maintained in Iscove’s Modified Dulbecco’s
medium (IMDM; (Invitrogen) supplemented with 10% FBS, 1× sodium pyruvate,
1× non-essential amino acids, 100 U ml−1 penicillin, and 100 μg ml−1 streptomycin. OCI-AML2 and OCI-AML3 cell lines were maintained in minimal essential
medium (MEM; Invitrogen) supplemented with 10% FBS, 1× sodium pyruvate,
1× non-essential amino acid, 100 U ml−1 penicillin, and 100 μg ml−1 streptomycin. All cell lines were cultured at 37 °C with 5% CO2 in the relevant media mentioned above. All cell lines were confirmed to be mycoplasma-negative using the
MycoAlert Mycoplasma Detection Kit (Lonza).
Cell proliferation assay. Human cancer cell lines cultured in the growth medium
recommended by the vendor were seeded into black 384-well plates containing
DMSO or test compounds. The seeding density for each cell line was optimized
to allow the cell growth in the linear range during a 3-day culture period. To
test the compound effect on cell proliferation in AML cell lines, 5,000 to 10,000
cells per well in 200 μl complete culture media were seeded into black 96-wellplates containing DMSO or test compounds. After 48 or 72 h, cell proliferation
was assessed using the CellTiter-Glo (CTG) Luminescent Cell Viability Assay(Promega) according to manufacturer’s instructions. To test the effect of GSPT1
depletion on cell proliferation in AML cell lines, cells were infected with lentiviralshRNA vectors for 4 days, and cell proliferation was quantified using CTG every
other day thereafter. Relative cell proliferation was normalized against day 4 cell
growth values. To determine the effect of GSPT1 depletion on cell proliferationin 293T HEK cells, cells were infected with lentiviral shRNA vectors for 7 days,
and then imaged using the EVOS FL Cell Imaging System (Thermo Fisher). The
effect of CC-885 on AML patient-derived cells was performed by automated flow
cytometry according to the methods described in ref. 34. Briefly, patient-derived
bone marrow samples from adult patients over 18 years of age who were diagnosed
with AML, were obtained from Hospital La Fe, Hospital General Universitario
Gregorio Marañon and Hospital Lucus Augusti hospitals, all in Spain, following
IRB approved protocols and signed informed consent. Bone marrow samples wereextracted under sterile conditions and were received in the laboratory within 24 hof extraction where they were immediately processed. A small portion of the sample was separated for validation and selection of the monoclonal antibodies (mAb)
to optimize the identification of leukaemic cells. The majority of the sample wasdiluted with culture media and plated into 96-well plates previously prepared withthe test compounds. These plates were incubated for 24, 48 and 96 h and then

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processed via the ExviTech platform. For this purpose, red cells were lysed andleukocytes were stained using the mAb previously selected and with Anexin-V to
exclude apoptotic cells.
Antibodies. Rabbit anti-human CRBN65 mAb (Celgene, San Diego, CA)3, rabbit
anti-DYKDDDDK (Flag) (9A3) mAb (#8146, Cell Signaling), mouse anti-Myc-Tag(9B11) mAb (#2276, Cell Signaling), mouse anti-GFP (B-2) mAb (#sc-9996, Santa
Cruz), rabbit anti-human GSPT1 polyclonal antibody (pAb) (ab49878, Abcam),rabbit anti-human GSPT1 pAb (ab126090, Abcam), rabbit anti-GSPT1 pAb(10763-1-AP, Proteintech), rabbit anti-Ikaros (D10E5) mAb (#9034, Cell signaling), mouse anti-HA.11 (16B12) mAb (#901501, Biolegend), rabbit anti-DDB1
pAb (#5428, Cell Signaling), mouse anti-ubiquitin P4D1 mAb (#SC-8017, Santa
Cru), mouse anti-SV5-V (Santa Cruz, sc-58052), mouse anti-HIS mAb antibody(34660, Qiagen), mouse anti- actin mAb (#A5316, Sigma) and mouse anti-tubulinmAb (#T5201, Sigma) were used as primary antibodies. Goat anti-mouse 800
antibody (#926-32210, LICOR Biosciences), goat anti-rabbit 680 antibody (#92668071, LI-COR Biosciences), goat anti-mouse 680 antibody (#926-68070, LI-COR
Biosciences) and goat anti-rabbit 800 antibody (#926-32211, LI-COR Biosciences)were used as secondary antibodies.
Cellular immunoprecipitation. Cells were lysed in Buffer B (50 mM Tris (pH 7.4),
150 mM NaCl, 0.5% NP-40, one tablet of Complete ULTRA protease inhibitor
cocktail (Roche), and one tablet of PhosSTOP phosphatase inhibitor cocktail(Roche)). Whole-cell lysates were clarified by centrifugation at top speed for
10 min. To identify cereblon-associated proteins, CC-885 (2 μM) or DMSO wereadded into whole cell extracts of 293T HEK cells stably expressing Flag–HA–CRBN
for 2 h. FH–CRBN complexes were then captured with anti-Flag beads (Sigma),
eluted by Flag peptide, and separated on SDS–PAGE gel, followed by mass spectrometry or immunoblot analysis. To test the binding of cereblon to its substrates,
293FT HEK cells stably expressing HA-tagged GSPT1, IKZF1, IKZF1(Q146H)
were lysed in Buffer B. HA-tagged recombinant proteins were then immunoprecipitated with anti-HA beads (Roche). After three washes with Buffer B, theanti-HA beads were mixed with cell lysates of 293FT HEK cells stably expressing
shGSPT1-1 in the presence of DMSO, 10 μM lenalidomide or 10 μM CC-885 for
12 h. To test the binding of HA-tagged GSPT1 variants with cereblon and eRF1,
whole-cell lysates of 293T HEK cells stably expressing GSPT1 variants were treatedwith DMSO or 20 μM CC-885 for 2 h, followed by anti-HA immunoprecipitation
for an additional 4 h. Beads were then washed with Buffer B six times, and boundproteins were eluted by boiling in LDS loading buffer and detected by immunoblot
analysis.
Plasmids. Plasmids pDONR221 and pDONR223-IKZF3 were purchased from
Invitrogen and assembled to generate pDONR223. Coding sequences of IKZF1,
SUP35, GSPT1, cereblon and Flag–HA–cereblon were in vitro synthesized by
Invitrogen and then cloned into pDONR223. Open reading frames encoding
IKZF1(Q146H) and chimaeric fusion proteins of SUP35 and GSPT1 were generated via overlapping PCR, and then cloned into pDONR223. GSPT1 mutations
and truncation deletions were introduced into pDONR223-GSPT1 via overlapping
PCR. Cereblon mutants E377V and W386A in pDORN223 were generated via
overlapping PCR.
DNA fragments of gateway-3×HA–mPGK (mouse phosphoglycerate kinase
promoter)-GFP-P2A-Pur-WPRE, 3×HA-gateway-mPGK-Pur-WPRE and gateway-3×HA-mPGK- Pur-WPRE were in vitro synthesized by Invitrogen, andassembled into plenti6.2/v5-DEST (Invitrogen) to generate plenti-CMV-gateway-3×HA-mPGK-GFP-P2A-Pur, plenti-CMV-3xHA-gateway-mPGK-Pur andplenti-CMV-gateway-3×HA-mPGK-Pur, respectively. The Ubc promoter fromplenti-6/Ubc/V5-DEST (Invitrogen) was used to replace the CMV promoter
in plenti-CMV-gateway-3×HA-mPGK-GFP-P2A-Pur, plenti-CMV-3×HAgateway-mPGK-Pur and plenti-CMV-gateway-3×HA-mPGK-Pur to generate
plenti-Ubc-gateway-3×HA-mPGK-GFP-P2A-Pur, plenti-Ubc-3×HA-gateway3×HA-mPGK-Pur, and plenti-Ubc-gateway-3×HA-mPGK-Pur. The EF1α
promoter from pEF-DEST51 (Invitrogen) was cloned into plenti-CMV-gateway-3×HA-mPGK-GFP-P2A-Pur, plenti-CMV-3×HA-gateway-mPGK-Pur
and plenti-CMV-gateway-3×HA-mPGK-Pur to generate plenti-EF1α-gateway3×HA-mPGK-GFP-P2A-Pur, plenti-EF1α-3×HA-gateway-3×HA-mPGK-Pur,and plenti-EF1α-gateway-3×HA-mPGK-Pur.
Next, pDONR233-GSPT1 was shuttled into plenti-CMV-gateway-3×HAmPGK-GFP-P2A-Pur and plenti-EF1α-gateway-3×HA-mPGK-GFP-P2A-Pur
via gateway LR (att L and att R) recombination. Similarly, pDONR223-GSPT1
wild-type and variants, pDONR223-SUP35, as well as pDONR223-GSPT1/SUP35
chimaeric fusion variants were shuttled into plenti-EF1α-3×-HA-mPGK-Pur.
pDONR223-Flag–HA–CRBN and pDONR223-CRBN wild-type and mutants werecloned into plenti-CMV-gateway-3×HA-mPGK-GFP-P2A-Pur and plenti-EF1αgateway-3×HA-mPGK-P2A-Pur. pDONR223-IKZF1, pDONR223-IKZF1-Q146H
and pDONR223-GSPT1 was cloned into plenti-Ubc-gateway-3×HA-mPGKPur or plenti-Ubc-3×HA-gateway-mPGK-Pur. A stuffer sequence was synthe-

sized in vitro by Invitrogen, and cloned into Mission TRC2 pLKO.5-pur empty
vector control plasmid (Sigma) to generate pLKO.5pur-Stuffer. Coding sequenceof 8×His–Ub was in vitro synthesized by Invitrogen and cloned into pcDNA3 togenerated pcDNA3-8×His–Ub.
In-gel digestion and sample preparation for LC-MS/MS Analysis. Gel bands
were excised and de-stained. Proteins were reduced and alkylated by addition of
50 mM DTT and 50 mM iodoacetamide before digestion by Trypsin at a final concentration of 25 ng μl−1 in 50 mM ammonium bicarbonate for 1 h on ice and additionally 16 h more at 37 °C using a shaking incubator to assure complete digestion.
Digested tryptic peptides were extracted from gels by addition of 200 μl of waterand sonication for 10 min, washed in 5% formic acid in water and extracted in 50%
acetonitrile in 5% formic acid in water, once in 70% acetonitrile and last in 100%
acetonitrile. All extracted peptides were pooled together and were vacuum-dried
and re-dissolved in 20 μl of 0.1% TFA. Tryptic peptides were then concentrated
and desalted using a Millipore C18 Zip Tip (Millipore). The eluted peptides
were then vacuum-dried and re-dissolved in 100 μl of LC-MS/MS loading buffer(2% acetonitrile in 0.1% formic acid in water).
One-dimensional LC-MS/MS analysis. Tryptic digested samples were subjected
to an on-line analysis of peptides by high-resolution, high-accuracy LC-MS/MS,
consisting of a Bruker-Michrom paradigm HPLC, a Zorbax C18 peptide trap column(Agilent technologies), a 15-cm Michrom Magic C18 column, a low-flow
ADVANCED Michrom MS source, and a LTQ-Orbitrap XL (Thermo Fisher
Scientific). The LC-MS/MS raw data were submitted to Sorcerer Enterprisev.3.5 release (Sage-N Research Inc.) with the SEQUEST algorithm to search thetarget-decoy ipi.Human.v3.73 database. The search results were viewed, sorted,
filtered, and statically analysed by using comprehensive proteomics data analysis
software, Peptide/Protein prophet v.4.02 (ISB). The differential spectral countanalysis was done by QTools (Sanford Burnham).
Lentiviral shRNA. Pairs of synthetic complementary oligonucleotides
targeting GSPT1 or a non-mammalian control (shCNTL) were annealed andcloned into pLKO.5pur-Stuffer. Targeting sequences are listed below: shCNTL,
5′-CAACAAGATGAAGAGCACCAA-3′; shGSPT1-1, 5′-GATTACCGTTTATT
CCATA-3′; shGSPT1-2, 5′-CCGATGATGTAGAGACTGATA-3′; shGSPT1-3,
5′-CCTGCACAATACTGTGAGGAA-3′; shGSPT1-4, 5′-GCCGAACTTCAATAGATCAGT-3′.
Protein half-life analysis. 293FT HEK parental and CRBN−/− cells were pretreatedwith DMSO or CC-885 (10 μM) for 30 min, followed by the addition of 100 μg ml−1
cycloheximide (EMD) into the culture medium. At various time points, cells werecollected and subjected to immunoblot analysis.
In vivo ubiqutination assay. The ubiquitination assays were carried out asdescribed previously34. Briefly, 293FT HEK parental or CRBN−/− cells cultured
in 10-cm plates were transfected with 12 μg of pcDNA3-8×His-Ub. After 48 h,cells were treated with 10 μM CC-885 and 10 μM MG132. Eight hours later, cells
were washed twice with ice cold PBS and resuspended in 1 ml PBS. 20 μl of the cellsuspension was boiled in LDS loading buffer (Thermo Fisher), and the remaining
cells were collected via centrifugation and lysed in Buffer C (6 M guanidine–HCl,
0.1 M Na2HPO4/NaH2PO4, 20 mM imidazole (pH 8.0)). Next, whole-cell extracts
were sonicated for 12 pulses, and mixed with 30 μl of Ni-NTA agarose beads atroom temperature for 4 h. Ni-NTA beads were then washed twice with Buffer C,
twice with Buffer D (1 volume of Buffer C to 3 volumes of Buffer E), and twice withBuffer E (25 mM Tris HCl, 20 mM imidazole (pH 6.8)). Bound proteins were elutedby boiling in 2× LDS loading buffer and subjected to immunoblot analysis withtwo mouse anti-GSPT1 monoclonal antibodies 17G9 and 8A2 generated in house(Celgene), a mouse anti-His tag monoclonal antibody (Qiagen #34660, Qiagen)and a rabbit anti-human CRBN monoclonal antibody (CRBN65, Celgene). To
detect the ubiqitination of GSPT1, boiled eluates were further diluted with
20 volumes of Buffer B (50 mM Tris (pH 7.4), 150 mM NaCl, 0.5% NP-40, one tabletof Complete ULTRA protease inhibitor cocktail (Roche)), followed by immunoprecipitation with a mouse anti-GSPT1 monoclonal antibody (17G9) immobilized onprotein G beads. Beads were washed with Buffer B six times, and bound proteins
were eluted by boiling in 1× SDS loading buffer and subjected to immunoblot
analysis for GSPT1 with a rabbit anti-GSPT1 antibody (Abcam # 49878) and two
rabbit anti-ubiquitin antibodies (Cell Signaling #3933 and Bethyl #A300-317A).
In vitro ubiquitination assays. Purified E1, E2, ubiquitin, CUL4A–RBX1,
cereblon–DDB1, and GSPT1 proteins were used to reconstitute the ubiquitinationof GSPT1 in vitro. Human GSPT1 domains 2 and 3 (amino acids 437–633) was
expressed in BL21 star DE3 E. coli and purified by maltose affinity resin (NEB),
heparin HiTrap column affinity (GE Healthcare), and Superdex 75 16/60 sizeexclusion chromatography (GE Healthcare), as described above. Human cereblon–
DDB1 (cereblon amino acids 40–442 and full-length DDB1) and 6×his–DDB1
alone were co-expressed in SF9 insect cells in the presence of 50 μM zinc acetate,
and purified by nickel affinity resin (Qiagen), HiTrap ANX column ion exchange(GE Healthcare), and Sephacryl 400 16/60 size-exclusion chromatography© 2016 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.

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Article reSeArcH(GE healthcare), as described above. SV5-V and DDB1 were purified separately,
incubated to form a complex, and the complex purified with size-exclusion chromatography as described above. Human full-length CUL4A and RBX1 wereco-expressed in SF9 insect cells and purified by nickel affinity resin and Superdex
200 16/60 size-exclusion chromatography. Purified recombinant human Ube1
E1 (E-305), UbcH5a E2 (E2-616), and ubiquitin (U-100H) were purchasedfrom R&D systems. Components were mixed to final concentrations of 1 μM
Ube1, 25 μM UbcH5a, 200 μM Ub, 1 μM CUL4–RBX1, 25 μM GSPT1, and 1 μM
cereblon–DDB1, 6×His–DDB1, or SV5-V–DDB1,with or without 100 μM CC-885
in ubiquitination assay buffer (20 mM HEPES (pH 7.5), 150 mM NaCl, 10 mM
MgCl2). After preincubation for 10 min at room temperature, ubiquitination
reactions were started by addition of ATP to a final concentration of 10 mM.
Reactions were incubated at 30 °C for 2 h before separation by SDS–PAGE followedby coomassie staining or immunoblot analysis for GSPT1, ubiquitin, cereblon
or SV5-V.
qRT–PCR Analysis. Following incubation with test compounds, cells werecollected via centrifugation at 872g for 2 min. Cell pellets were then washed oncein ice-cold PBS and snap-frozen in liquid nitrogen. Total RNA was extractedusing RNeasy Mini Kit (Qiagen) according to manufacturer’s instruction,and reverse-transcribed into first-strand cDNA using AffinityScript QPCR
cDNA Synthesis Kit (Agilent) with random primer. Real-time PCR was performed in triplicate using ViiA 7 Real-Time PCR System with TaqMan Gene
Expression Assay probes (Invitrogen) for GSPT1 (# 4331182) and GAPDH(#4326317E).
Lentiviral production and infection. Lentiviral plasmid was cotransfectedwith the 2nd Generation packaging system (ABM) into Lenti-X 293 cells using
Lipofectamine 2000 (Thermo Fisher). After 12 h of incubation, media was changed

to fresh DMEM media supplemented with 20% FBS. At 36 h after transfection,
viral supernatant was collected and fresh media was replenished. At 56 h, viralsupernatant was collected again, combined with the first viral supernatant, cleared
via centrifugation at 872g for 5 min, and then filtered through a 0.45 micron
cellulose acetate or nylon filter unit. All lentiviral supernatants, except those of
lentiviral shRNA vectors, were concentrated using a Lenti-X concentrator according
to the manufacturer’s instructions. AML cell lines were spin-inoculated withlentivirus at 872g for 90 min. After 12 h, viral supernatant was removed and complete culture media was added to the cells. 48 h later, cells were incubated with
1–2 μg ml−1 puromycin or 10–20 μg ml−1 blasticidin for an additional 3 days toselect cells stably integrated with lentiviral vectors.
Data analysis. All cell proliferation assays were independently performed threetimes with three technical replicates for each sample. All qRT–PCR assays werecarried out two times with three technical replicates for each sample. Whenpossible, results were confirmed in multiple cell lines. For quantification of cellproliferation and qRT–PCR, data were analysed using GraphPad Prism and Excel,
respectively. P < 0.05, unpaired two-sided t-test, is considered as significant.
31. McCoy, A. J. et al. Phaser crystallographic software. J. Appl. Crystallogr. 40,
658–674 (2007).
32. Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of
Coot. Acta Crystallogr. D 66, 486–501 (2010).
33. Murshudov, G. N. et al. REFMAC5 for the refinement of macromolecular crystal
structures. Acta Crystallogr. D 67, 355–367 (2011).
34. Bennett, T. A. et al. Pharmacological profiles of acute myeloid leukemia
treatments in patient samples by automated flow cytometry: a bridge toindividualized medicine. Clin. Lymphoma Myeloma Leuk. 14, 305–318(2014).

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Extended Data Figure 1 | See next page for caption.

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Extended Data Figure 1 | The anti-tumour effects of CC-885 are CRBN
dependent. a, The growth inhibitory IC50 value of CC-885 in humancancer cell lines. The cell proliferation of 132 human cancer cell lines
treated with varying concentrations of CC-885 for 3 days was assessed by
Cell Titre Glo assay. Cancer types as indicated at the bottom are labelledwith different colours. The IC50 value of growth inhibition was determinedusing ActivityBase (IDBS). Data are the mean of two or more biologicalreplicates. The n number for each cell line is shown on the right of thepanel. b, The effect of CC-885 on AML samples taken from patients. The
top panel shows the effects on leukaemic cells; the bottom panel showsthe effects on normal lymphoid cells. The sample from patient B did notcontain sufficient normal lymphoid cells for analysis. c, The effect of
CC-885 on cell proliferation in parental and CRBN−/− 293FT HEK cells.
Result is representative of three biological replicates. Data are shownas mean ± s.d., n = 3 technical replicates. d, The effect of lenalidomide,
pomalidomide and CC-885 on cell proliferation in AML cell lines, THLE-2
and human PBMC. Cells were treated with varying concentrations oflenalidomide, pomalidomide or CC-885. At day 3, cell proliferation wasassessed using CTG assay. Numbers shown are the growth inhibitory
IC50 values of lenalidomide, pomalidomide and CC-885, from biologicalreplicates with n = 5, 3, and 20, respectively.

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Extended Data Figure 2 | CC-885, but not lenalidomide, promotes
the interaction between GSPT1 and CRBN in vitro. a, b, Immunoblot
analysis of anti-HA immunoprecipitates (a) and whole-cell lysates (WCL)(b). HA-tagged GSPT1 or IKZF1 produced in 293FT CRBN−/− HEK cells
were used to capture CRBN from 293FT HEK cells expressing GSPT1specific shRNA, shGPST1-1. DMSO, 10 μM lenalidomide or 10 μM
CC-885 were included in the binding assay. The IKZF1 Q146H mutation
results in a reduction of cereblon binding mediated by either lenalidomide
or CC-885. Some residual binding is observed with CC-885, consistent
with Fig. 1d, which shows that CC-885 is more potent than lenalidomide

against IKZF1. c, Coomassie stain of CRBN–DDB1 pull-down with
GSPT1 using purified components. Purified GST or GST–GSPT1
domains 2 and 3 (amino acids 437–633) bound to magnetic glutathione
beads was incubated with purified CRBN–DDB1 in the presences of
CC-885, lenalidomide (len), pomolidamide (pom), or DMSO (vehicle)
for 1 h at room temperature before three rapid washes. CC-885, but notlenalidomide, pomalidomide or DMSO, mediated the binding of GST–
GSPT1 to CRBN–DDB1. This experiment was performed three times. Forgel source data, see Supplementary Information Fig. 1.

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Extended Data Figure 3 | Transcriptional and post-transcriptional
regulation of GSPT1 by CC-885. a, Ubiquitinated protein products,
as shown in Fig. 1e, were blotted for GSPT1 with mouse anti-GSPT1
monoclonal antibody 8A2, CRBN, and His-tagged Ubiquitin. Arrowheads
mark nonspecific bands. b, 293FT parental and CRBN−/− cells transiently
expressing His-tagged ubiquitin was treated with CC-885 and MG132
as indicated for 8 h. Ubiquitinated protein products were pulled
down with Ni-NTA agarose under denaturing conditions, followedby elution via boiling in SDS loading buffer. Eluates were diluted withimmunoprecipiation buffer and GSPT1 was immunoprecipitated witha mouse anti-GSPT1 monoclonal antibody 17G9. Immunoprecipitates
were then subjected to immunoblot analysis for GSPT1 and ubiquitinwith two different rabbit anti-ubiquitin antibodies. c, Immunoblot
analysis of 293FT parental and CRBN−/− cells treated with CC-885 for
4 h. Cells were pretreated with DMSO, CC-885, MLN4924 or MG132 as
indicated. d, 293FT parental or CRBN−/− cells treated with DMSO or
CC-885 for 24 h were subjected to real-time qPCR (top) and immunoblot
analysis (bottom). Result is representative of two biological replicates.
Data are mean ± s.d., n = 3 technical replicates. For gel source data, see
Supplementary Information Fig. 1.

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Extended Data Figure 4 | See next page for caption.

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Extended Data Figure 4 | The CC-885 induced GSPT1 ubiquitinationand degradation relies on CRBN. a, Controls for the in vitro
ubiquitination assay shown in Fig. 1f. Recombinant protein products
as indicated were incubated with or without ATP (10 mM) and CC-885(100 μM) in the ubiquitination assay buffer at 30 °C for 2 h, and thenseparated by SDS–PAGE followed by Coomassie staining or immunoblot
analysis with anti-GSPT1, anti-cereblon, and anti-ubiquitin antibodies.
The anti-GSPT1 blot shown is the same one shown in Fig. 1f. The
additional blots and Coomassie staining analysis was performed on
samples from the same in vitro ubiquitination reactions. Anti-GSPT1
blotting and Coomassie staining was performed three times, anti-cereblon
and anti-ubiquitin blotting was performed twice. b–d, To demonstrate
the CRBN-dependence of GSPT1 ubiquitination by CC-885-CRL4,
we reconstituted DDB1 with an alternative DCAF, SV5-V, and showedthat SV5-V is not capable of recruiting GSPT1 for polyubiquitination.
b, DDB1 binds to GST–SV5-V, but not GST alone. Coomassie stain withlanes 1, 2, and 3 showing individual proteins, and lanes 4 and 5 showing
pull-down of DDB1 incubated with GST or GST–SV5-V bound to
glutathione magnetic beads and washed three times. This experiment
was performed twice. c, GST–SV5-V–DDB1 protein complex generated
by pull-down in b was used to show binding of CUL4–RBX1 to the
GST–SV5-V–DDB1 protein complex but not GST alone. Coomassie
stain with lanes 1, 2, and 3 showing individual proteins or protein
complexes used in the pull-down, and lanes 4 and 5 showing pull-downof CUL4–RBX1 incubated with GST or GST–SV5-V–DDB1 bound to
glutathione magnetic beads and washed three times. This experiment was
performed once. d, In vitro ubiquitination of GSPT1 by CRBN–DDB1
but not SV5-V–DDB1. SV5-V–DDB1 complex was formed by incubationof individually purified proteins and purification over size-exclusion
chromatography. Recombinant protein products as indicated wereincubated with either CRBN–DDB1, SV5-V–DDB1, DDB1 alone, orthe absence of any DDB1 complex, with and without ATP (10 mM) or
CC-885 (100 μM) in ubiquitination assay buffer at 30 °C for 2 h, andthen separated by SDS–PAGE followed by immunoblot analysis with
anti-GSPT1 and anti-SV5-V antibodies. Asterisk indicates backgroundbands present from the SV5-V protein purification. The anti-SV5-V
western blot indicates that SV5-V is auto-ubiquitinated, as expected fora functional DCAF that is bound to the DDB1–CUL4–RBX1 complex.
e, Immunoblot analysis of 293FT parental and CRBN−/− cells treatedwith 100 μg ml−1 cyclohexamide (CHX) for the indicated periods. Where
indicated, cells were pretreated with DMSO or 10 μM CC-885 for 30 min.
This experiment was performed once. f, Immunoblot analysis of 293FT
parental cells, CRBN−/− cells, and CRBN−/− cells stably expressing CRBN
wild-type or variants as indicated. CRBNiso2 (CRBN isoform 2) showedsimilar E3 ligase activity towards GSPT1 as compared to CRBN isoform 1(data not shown). CRBN isoform 2 lacks an alanine residue at position
23 of CRBN isoform 1, and as such the numbering is shifted compared
to isoform 1. Note that the CRBN(W385A) mutant showed diminishedactivity in cells treated with 0.1 μM CC-885, and similar activity in cells
treated with 1 μM CC-885, compared to wild-type CRBN. CRBN(E376V)
mutant had no activity at both concentrations. Immunoblot analysisof 293FT parental and CRBN−/− cells treated with 100 μg ml−1
cyclohexamide for the indicated periods. Where indicated, cells werepretreated with DMSO or 10 μM CC-885 for 30 min. For gel source data,
see Supplementary Information Fig. 1.

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Extended Data Figure 5 | Identification of the region of GSPT1
indispensable for CC-885-dependent destruction. a, Schematic of
human GSPT1 and Saccharomyces cerevisiae SUP35 chimaeras. Truncation
analysis of GSPT1 revealed that domains 2 and 3 of GSPT1 contain the
CC-885-dependent CRBN-binding motif (data not shown). In this region,
GSPT1 and SUP35 share 53% sequence identity. Chimaeric fusion sites
were selected from several stretches of identical regions to ensure proper
folding of the resultant fusion product. Changes in protein level of thesefusion proteins in 293T HEK cells in response to CC-885 treatment,
as shown in b–d, is summarized on the right. +, protein degraded; −, nochange of protein level. b–d, Immunoblot analysis of 293T HEK parental

cells or cells stably expressing HA-tagged GSPT1, SUP35 or GSPT1–
SUP35 chimaeric proteins. Note that SUP35 expression level is relatively
low compared to GSPT1, possibly owing to its intrinsic instability when
expressed in human cell lines. e, Immunoblot analysis of 293T HEK
cells stably producing HA-tagged GSPT1 wild-type and variants. Nonconserved amino acids in GSPT1 as shown in Fig. 2a were replaced withcorresponding residues in SUP35. f, g, Immunoblot analysis of anti-HA
immunoprecipitates (f) and whole-cell extracts (g) of 293T HEK cells
expressing GSPT1 wild-type and mutants. DMSO or CC-885 was added
into the whole-cell extract after lysis for 6 h. For gel source data, see
Supplementary Information Fig. 1.

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Extended Data Figure 6 | Loss of GSPT1 is the cause of growth
inhibition induced by CC-885. a, Immunoblot analysis of MOLM13 and OCI-AML2 parental cells or cells stably expressing HA-tagged
GSPT1(G575N). Cells were treated with CC-885. b, Cells shown in awere incubated with CC-885 at the indicated concentration for 3 days.
Cell proliferation was determined by CellTiter-Glo (CTG). Result
is representative of three biological replicates. Data are presented asmean ± s.d., n = 3 technical replicates. c, 293T HEK cells were infectedwith an increased amount of empty lentiviral vector (EV) or vectors
expressing control shRNA or any of the four GSPT1-specific shRNAs.
Seven days after infection, cells were imaged using phase-contrast

microscope (bottom) and collected for immunoblot analysis (top). Scalebars measure 0.5 mm. Images shown are representative of three captured
images. d, e, Cell growth curve of MOLM-13 parental cells or cells
transduced with lentiviral vectors expressing a control shRNA (shCNTL)
or GSPT1 specific shRNA (shGSPT1-1 to 4). Immunoblot analyses ofwhole-cell extracts (d) were carried out at day 4 after transduction withlentiviral vectors. Cell growth (e) was quantified with CTG at day 4, 6, and
8 after transduction. Result is representative of three biological replicates.
Data are mean ± s.d., n = 3 technical replicates. For gel source data, see
Supplementary Information Fig. 1.

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Extended Data Figure 7 | See next page for caption.

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Extended Data Figure 7 | Kinetic parameters for cereblon–DDB1–CC-885
binding to GSPT1, and sample electron density from the crystal
structure of the complex. a, Reference-corrected surface plasmon
resonance binding curves for various concentrations of cereblon–DDB1(threefold dilutions from 1 μM, coloured traces) flowed over a surfaceof covalently immobilized anti-GST antibody bound to GST–GSPT1
domains 2 and 3 at 10 °C in the presence of saturating levels of CC-885
or control compound glutarimide. Kinetic parameters shown were
determined by fitting with a 1:1 kinetic binding model (black lines) using
the Bioacore T200 kinetic analysis software package. Binding in thepresence of gluatrimide could not be quantified. We show a representative
set of curves from three independent experiments. For a 1:1 binding
model where A + B ⇆ AB, the net rate of complex formation is givenby the equation d[AB]/dt = ka[A][B] – kd[AB] and the rate of complex
disassociation is given by kd[AB], where ka is the association rate constant(M−1s−1) and kd is the dissociation rate constant (s−1). KD, the equilibrium
dissociation constant, is defined by KD = kd/ka. Rmax is a measurement of
the analyte binding capacity, or maximum response. b, Analysis of thesteady-state response versus the concentration of analyte in the presenceof CC-885, and the determined affinity constant. c, GST–GSPT1 interacts
with endogenous binding partner eRF1. We show that the purified
GST–GSPT1 domains 2 and 3 (amino acids 437–633) protein used in thisSPR binding assay and the electron miscroscopy experiments (Extended
Data Fig. 8) is competent to bind purified eRF1 as reported in ref. 17.
Coomassie stain, with lanes 1, 2 and 3 showing individual proteins. Lanes
4 and 5 show pull-down of GST; lanes 6 and 7 show pull-down of
GST–GSPT1 domains 2 and 3, all were bound to magnetic glutathione
beads incubated with eRF1 ± CC-885 for 1 h and washed three times.
Lanes 8 and 9 show pull-down of GST-GSPT1 bound to magnetic
glutathione beads incubated with CRBN–DDB1 ± CC-885 for 1 h andwashed three times. This experiment was performed twice. For gel
source data, see Supplementary Information Fig. 1. d, Sample electron
density from the cereblon–DDB1–CC-885–GSPT1 crystal structurewith cereblon residues shown in purple, GSPT1 residues shown in grey,
and CC-885 shown in green. Refined 2Fo − Fc density is shown as a blue
mesh contoured at 1.4σ. Fo − Fc difference density, shown as a green mesh
contoured at 4σ, was generated by a single round of Refmac5 refinement
calculated in the absence of GSPT1 residues 570–577.

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Extended Data Figure 8 | See next page for caption.

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Extended Data Figure 8 | Imaging of GSPT1 binding to cereblon–
DDB1–CC-885 by negative stain electron microscopy. a, Negative stainclass averages of N-terminal-tagged GST–GSPT1 domain 2 and 3 (aminoacids 437–633) bound to purified cereblon–DDB1 in the presence of
CC-885. As the GST-tag was fused to the N terminus of domain 2 of GSPT1,
domain 3 can be identified as mediating the interaction with cereblon.
Whereas the GST tag appears flexible in position, the two domains of
GSPT1 are consistent in their orientation with the cereblon–DDB1
complex. GST-dimerization mediates the binding of a second substratein the majority of the classes. Each class average is composed of between
40 and 70 individual particles. This experiment was performed threetimes. b, Negative stain class averages of cereblon–DDB1 and GST–GSPT1
domains 2 and 3 (amino acids 437–633) in the presence of DMSO insteadof CC-885. No classes containing bound substrate were observed in theabsence of CC-885. Each class average is composed of between 40 and 70
individual particles. This experiment was performed once. c, A negative
stain class average of GST–GSPT1 domains 2 and 3 bound to cereblon–
DDB1–CC-885, with DDB1 shaded purple, cereblon shaded green,
GSPT1 domains 2 and 3 shaded blue, and the second dimerized GST
tag and second GSPT1 left uncoloured. d, For comparison, the crystal
structure of GSPT1 domains 2 and 3 bound to cereblon–DDB1–CC-885
with DDB1 in purple, cereblon in green, and GSPT1 in blue. The electron
micrographs revealed a consistent configuration of GSPT1 with cereblon
in all complex class averages and confirmed that domain 3 mediates
cereblon interactions on the basis of the orientation of the GST-tag.

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Extended Data Figure 9 | Effects of cereblon surface mutations on
substrate binding. Cereblon and substrate proteins were co-expressedin 293FT CRBN−/− cells, co-immunoprecipitated in the presence orabsence of CC-885 and lenalidomide, and analysed by western blot.
a, Co-immunoprecipitation of GSPT1 with wild-type and mutant cereblon.
veh, vehicle, DMSO; 885, 10 μM CC-885. b, Co-immunoprecipitationof Ikaros with wild-type and mutant cereblon. veh, vehicle, DMSO;
LEN, 10 μM lenalidomide. c, Co-immunoprecipitation of DDB1 withwild-type and mutant cereblon. Results are representative of threebiological replicates. d, e, Western blots showing the effect of lenalidomide
or CC-885 on Ikaros and GSPT1 degradation; d, effect with humancereblon, e, effect with mouse cereblon. For convenience, the humanamino acid numbering is used to discuss the corresponding residues inmouse. GFP and actin are shown as transfection and loading controls,
respectively. This is a representative experiment of three biologicalreplicates. For gel source data, see Supplementary Information Fig. 1.

© 2016 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.

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extended data table 1 | data collection and refinement statistics from the cereblon–ddB1–CC-885–GSPt1 crystal structure© 2016 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.

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Published as: Mol Cell. 2016 March 17; 61(6): 809–820.

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Glutamine triggers acetylation-dependent degradation of
glutamine synthetase via the thalidomide receptor cereblon
Thang Van Nguyen1, J. Eugene Lee1,2, Michael J. Sweredoski3, Seung-Joo Yang4, SeungJe Jeon4, Joseph S. Harrison5, Jung-Hyuk Yim2, Sang Ghil Lee2,6, Hiroshi Handa7, Brian
Kuhlman5, Ji-Seon Jeong2, Justin M. Reitsma1, Chul-Seung Park4, Sonja Hess3, andRaymond J. Deshaies1,8,*
1Division of Biology and Biological Engineering, Box 114-96, California Institute of Technology,

Pasadena, CA 91125, United States of America
2Center for Bioanalysis, Division of Metrology for Quality of Life, Korea Research Institute ofStandards and Science, Daejeon 305-340, Korea

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3Proteome Exploration Laboratory, Division of Biology and Biological Engineering, Beckman

Institute, California Institute of Technology, Pasadena, CA 91125, United States of America
4School of Life Sciences, National Leading Research Laboratory, and Integrative Aging ResearchCenter, Gwangju Institute Science and Technology (GIST), Gwangju, 500-712, Korea
5Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill, North

Carolina 27599-7260, United States of America
6Department of Biochemistry, Yonsei University, Seoul 120-749, Korea
7Department of Nanoparticle Translational Research, Tokyo Medical University, 6-1-1, Shinjuku,

Shinjuku-ku, Tokyo, 160-8402, Japan
8Howard Hughes Medical Institute

SUMMARY
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Cereblon (CRBN), a substrate receptor for the cullin–RING ubiquitin ligase 4 (CRL4) complex, isa direct protein target for thalidomide teratogenicity and antitumor activity of immunomodulatory
drugs (IMiDs). Here we report that glutamine synthetase (GS) is an endogenous substrate of
CRL4CRBN. Upon exposing cells to high glutamine concentration, GS is acetylated at lysines 11
and 14, yielding a degron that is necessary and sufficient for binding and ubiquitylation by*

Correspondence: ; Email: deshaies@caltech.edu (R.J.D.)
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Author Contributions
T.V.N. and R.J.D.: study conception and design, data analysis, drafting of manuscript with editorial assistance from other authors.
T.V.N.: all cellular and molecular experiments. J. E.L.: mass spectrometry experiments in Figures 1A and S5, table S1. M.S. and S.H.:
mass spectrometry data analysis. M.S.: protein sequence analysis. J.M.R. and S.G.L.: analysis of the mass spectrometry data in Figure
S5. J.H. and B.K.: computational predictions of effects of mutations in the GS-binding site of CRBN and analysis of human GS
structure in Figure S7B, C. J.H.Y. and J.S.J.: quantification of glutamine and glutamate in serum and analysis of data in Figure 2G andtable S3. S.J.Y., S.J. J. and C.S.P.: mouse studies in Figure 2D–F. H.H.: provided essential reagents.

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CRL4CRBN and degradation by the proteasome. Binding of acetylated degron peptides to CRBN
depends on an intact thalidomide-binding pocket but is not competitive with IMiDs. Thesefindings reveal a feedback loop involving CRL4CRBN that adjusts GS protein levels in response to
glutamine and uncover a new function for lysine acetylation.

Graphical abstract

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Introduction

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Cereblon (CRBN) is a putative substrate receptor for a cullin–RING ubiquitin ligase 4(CRL4) complex (Angers et al., 2006). Human CRBN was discovered as a gene that whenmutated results in mild mental retardation (Higgins et al., 2004). Subsequent analysis ofCrbn−/− knockout mice revealed that they are less prone to developing insulin resistance,
fatty liver and visceral fat accumulation when fed a high-fat diet (Lee et al., 2013). To date,
only four putative targets of CRBN have been identified: large conductance Ca2+-activated
K+ channels (Jo et al., 2005), voltage-gated chloride channel (Hohberger and Enz, 2009), thedevelopmental regulator MEIS2 (Fischer et al., 2014), and adenosine monophosphateactivated protein kinase (AMPK) (Lee et al., 2011b; Lee et al., 2014; Lee et al., 2013).
AMPK activity is elevated in Crbn−/− mice, which could contribute to the resistance of theseanimals to developing metabolic syndrome on a high-fat diet. However, the mechanism bywhich CRBN recognizes AMPK or its other natural substrates remains unknown.
CRBN has achieved notoriety as the target that accounts for the teratogenic effects ofthalidomide (Ito et al., 2010), which caused over 10,000 birth defects in humans before it
was withdrawn from the market in 1962 (Lenz et al., 1962; Mcbride, 1961). Subsequently,
CRBN was implicated as the target that accounts for the therapeutic activity of thalidomide
and the related ‘immunomodulatory’ (IMiD) compounds lenalidomide and pomalidomide in

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multiple myeloma (Lopez-Girona et al., 2012; Zhu et al., 2011). CRBN must be present for
myeloma cells to respond to IMiDs, suggesting that formation of an IMiD–CRBN complex
underlies the therapeutic response. Recent studies have revealed that binding of IMiDs to
CRBN promotes recruitment of neosubstrates, including Ikaros (IKZF1), Aiolos (IKZF3)(Kronke et al., 2014; Lu et al., 2014) and casein kinase 1A1 (CK1α) (Kronke et al., 2015) toCRL4CRBN, leading to their increased ubiquitylation and proteasome-dependentdegradation. In contrast, IMiDs block endogenous CRBN substrate MEIS2 from binding to
CRL4CRBN, resulting in inhibition of its ubiquitylation and degradation (Fischer et al.,2014). Because of the paucity of known CRL4CRBN ubiquitylation substrates, it remains
unclear whether the therapeutic action of IMiDs might be modulated by general stabilizationof CRBN’s natural substrates.

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Glutamine synthetase (GS) plays a central role in metabolism, as glutamine is the key
metabolite connecting carbon and nitrogen metabolism through the citric acid cycle. GS has
different functions in different tissues. For example in liver it detoxifies ammonia, in brain it
protects neurons against excitotoxicity by converting glutamate into glutamine, and in
kidney it contributes to pH regulation (Taylor and Curthoys, 2004). Moreover, glutamine hasa critical role in regulating mTOR signaling, translation, and autophagy to coordinate cell
growth and proliferation (Nicklin et al., 2009). Mutations and deregulation of GS have been
linked to human diseases, including congenital glutamine deficiency, Alzheimer’s disease,and cancers (Bott et al., 2015; Christa et al., 1994; Gunnersen and Haley, 1992; Haberle etal., 2005; Kung et al., 2011; Tardito et al., 2015). In keeping with its central role in carbon
and nitrogen metabolism, prokaryotic GS is allosterically regulated by multiple end productsof glutamine metabolism working in concert with cycles of reversible adenylylation/
deadenylylation (Krajewski et al., 2008; Stadtman, 2001). Eukaryotic GS, by contrast, is notadenylylated and its regulation is poorly understood. It has been shown that GS is subject tofeedback control by glutamine, which promotes its post-translational modification anddegradation (Arad et al., 1976; Crook and Tomkins, 1978). However, the molecular basis forthis regulation remains elusive.

Results
GS is an endogenous substrate of CRL4CRBN

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To search for candidate CRBN substrates, including those that might be modulated by
IMiDs, we used stable isotope labeling of amino acids in cell culture (SILAC)-based
quantitative mass spectrometry (Lee et al., 2011a) (Figure S1A). Comparison of heavy:lightratios of peptides indicated that CRBN, subunits of CRL4 (CUL4, DDB1, RBX1), andsubunits of the CRL regulator CSN were recovered in equal amounts from DMSO or
thalidomide-treated 293T cells stably expressing CRBN tagged with a Flag epitope at its Nterminus (FlagCRBN; all tagged proteins are indicated by a superscripted tag either before orafter the name to indicate tagging at the N- or C-terminus) (Figure 1A). A number ofputative substrates behaved like MEIS2 (Fischer et al., 2014) in that they were recovered inlesser amounts from cells treated with thalidomide (Table S1). Notably, glutaminesynthetase (GS) behaved like Ikaros, Aiolos, and CK1α in that it was recovered in greater
amounts (Figure 1A). Essentially identical results were obtained in a label-swap experiment

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HHMI Author Manuscript(data not shown). To validate the mass spectrometry data, we immunoprecipitated FlagCRBN
and immunoblotted for CRL4CRBN subunits and GS. Recovery of GS but not CRL subunits
was stimulated by thalidomide (Figure S1B) and its analog lenalidomide (Len)(Figure 1B).
A similar result was obtained when we evaluated the interaction of endogenous GS withendogenous CRBN (Figure 1C). An important distinction between GS and CRBN’s
neosubstrates including IKZF1, IKZF3 and CK1α (Kronke et al., 2015; Kronke et al., 2014;
Lu et al., 2014), is that we observed significant GS association with CRBN in the absence of
IMiDs (Figures 1B, 1C, and S1B). Thus, we did not pursue further the relationship between
GS and IMiDs.

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The apparent constitutive association of GS with CRBN suggested that GS might be anatural substrate for CRL4CRBN, albeit one that behaves markedly differently from MEIS2.
To pursue this further, we sought to test whether ubiquitylation of GS was dependent on
CRL4CRBN. In co-transfection assays, we observed incorporation of HAubiquitin into FlagGS(Figure 1D). Significantly, ubiquitin-modified FlagGS accumulated in cells in which theproteasome was inhibited with MG132, but was almost entirely absent upon depletion ofendogenous CRBN (depletion was confirmed by immunoblot; Figure S1D). In
addition, FlagCRBN promoted the in vitro ubiquitylation of co-precipitated endogenous GS
when supplemented with E1, E2, ubiquitin, and ATP (Figure 1E, lane 6). GS
polyubiquitylation was markedly enhanced by the addition of recombinant CUL4A-RBX1
purified from insect cells (Figure 1E, lane 3), whereas it was inhibited by addition of
methylated ubiquitin. Collectively, these results argue that GS is an endogenous
ubiquitylation substrate of CRL4CRBN.
CRL4CRBN directly controls the glutamine-induced degradation of GS
Glutamine regulates GS by altering the rate of degradation of the enzyme (Arad et al., 1976;
Crook and Tomkins, 1978). Consistent with these reports, we observed that glutamine
downregulated GS protein levels upon addition to glutamine-starved Hep3B cells (Figure2A) as well as to multiple lung, breast, and glioblastoma cancer cell lines (Figure S1E). Thiseffect was intermediate at the normal serum glutamine concentration (0.5 mM) and was
saturated at 2 mM glutamine (Figure S1F), as reported previously (Crook and Tomkins,1978). The glutamine-induced downregulation of GS in Hep3B cells was blocked by the

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addition of the proteasome inhibitor bortezomib or the NEDD8-activating enzyme inhibitor
MLN4924 (Figure 2B), which inactivates Cullin-RING E3 ubiquitin ligase activity (Soucy etal., 2009). MLN4924 also inhibited glutamine-induced GS degradation in myeloma, breast,and lung cancer cell lines (Figure S2A–C). Most importantly, the glutamine-induced
downregulation of GS in Hep3B cells was blunted upon disruption of CRBN loci by
CRISPR/Cas9 (Figure 2C) or depletion of CRBN by shRNA knockdown (Figure S2D).
Similar results were observed upon shRNA knockdown of CRBN in myeloma and lungcancer cells (Figure S2E & F). Consistent with a role for CRBN in GS degradation, thesteady-state level of GS was elevated in CRBN-depleted cells (Figure S2G). For the Hep3B,
myeloma, and lung cancer cell lines we confirmed that CRBN-dependent effects on GS
downregulation were not due to changes in its mRNA level (Figure S2I–K). Two generaltrends in the data from different cell types are worth noting. First, glutamine does not induce
complete degradation of GS; depending upon the cell line, the reduction ranged from 50–

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80%. Second, there remains a modest glutamine-stimulated loss of GS in cell lines depletedof CRBN by either shRNA or CRISPR/Cas9. It remains unclear if this is due to residual
CRBN or the operation of an unknown secondary pathway. Nevertheless, it is clear that
CRBN promotes rapid, glutamine-dependent downregulation of GS by a post-transcriptional
mechanism that requires Nedd8 conjugation and proteasome activity.
We next sought additional evidence for a physiological role of CRBN in regulating theabundance of GS in vivo. In mice, GS is highly expressed in the brain, liver, kidney andskeletal muscle (BioGPS.org; table S2). We therefore used immunoblotting to examine GSabundance in brain, liver, kidney, skeletal muscle and lung tissues of wild-type andCrbn−/−mice that were starved for 24 hours and re-fed for 4 hours to mimic glutamine
starvation/re-feeding in cultured cells. As shown in Figures 2D–F, GS was elevated in thekidney, skeletal muscle and lung of Crbn−/− mice, but not in the brain and liver (data notshown). Skeletal muscle and lung GS plays a significant role in regulating the concentrationof plasma glutamine (Hensley et al., 2013). Consistent with the accumulation of GS in thesetissues, Crbn−/− mice exhibited an increased glutamine to glutamate ratio in serum (Figure
2G and table S3). Taken together, our data suggest that endogenous GS protein levels arenegatively regulated by glutamine through a feedback loop involving CRL4CRBN.

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The N-terminal extension of GS is required for its CRBN-dependent ubiquitylation

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To identify the sequence in GS recognized by CRBN, we generated a series of deletionmutants and found that the N-terminal 24–amino acids were required for its constitutive
interaction with CRBN (Figures 3A and 3B) and degradation (Figure 3C) in glutaminesupplemented medium. Intriguingly, this N-terminal segment is absent from bacterial GS
enzymes but is conserved in GS throughout the chordate lineage (Figure S3). By analogy toother CRL enzyme–substrate interactions, we speculated that GS degradation might becontrolled by a glutamine-dependent post-translational modification of its N-terminal
segment. To test this idea, we mutated all Ser, Thr, and Tyr residues in the N-terminal 24
amino acids of GS to Ala, either individually or in combination. Notably, all mutantsretained some binding to CRBN (Figure S4A), suggesting that phosphorylation does notplay a major role. We next investigated whether a KxxK motif (lysines 11 and 14) that is
highly conserved in chordates (Figure S3) was required for GS binding to CRBN. We
analyzed double arginine substitutions (RR; keeps the positive charges) and double alanine
substitutions (AA; neutralizes the positive charges) at K11 and K14 and found that thebinding to CRBN, ubiquitylation, and degradation of GS were enhanced by the AA
mutations but diminished or unaffected by the RR mutations (Figures 3D–F). To further
investigate the N-terminal degron, we generated chimeric fusion proteins consisting of GS
amino acids 1–25 fused to Myc-tagged GFP (Figure 4A). The N-terminal region of GS was
sufficient to confer binding to and ubiquitylation by CRBN, as well as degradation (Figures
4B–D). However, all of these activities of the N-terminal degron were blocked by the RR
substitutions. Together, these findings suggest that modification of lysine(s) in the Nterminal extension of GS was critical for its binding to and ubiquitylation by CRBN.

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Glutamine-dependent acetylation of lysines 11 and 14 by CBP/p300 regulates GS
degradation

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Lysine residues can be targeted by multiple modifications, such as ubiquitylation,
SUMOylation, methylation, and acetylation. Our observation that K->R substitutions at K11
and K14 in GS disrupted its binding to CRBN, ubiquitylation and degradation, whereas
neutralizing K->A substitutions had the opposite effect, implicated a potential role foracetylation in neutralizing positive charges that impede interaction with CRBN. Acetylation
was of particular interest, because proteomic studies uncovered modification of K14 on
human, mouse and rat GS (Chen et al., 2012; Lundby et al., 2012; Svinkina et al., 2015;
Weinert et al., 2013), and more generally revealed that reversible lysine acetylation plays animportant role in regulating metabolic enzymes (Wang et al., 2010; Zhao et al., 2010).
Moreover, a KxxK motif is known to be a potential target of the closely-related
acetyltransferases CBP and p300 (Thompson et al., 2001). Consistent with GS being a
potential CBP/p300 substrate, GSMyc co-immunoprecipitated with both p300HA and CBPHA(Figure S4C). Moreover, recombinant p300-HAT domain acetylated recombinant GS6xHis invitro (Figure S4D). Furthermore, in 293FT CRBN knockout cells (CRBN-KO 293FT) (Lu etal., 2014), GSFlag acetylation was significantly enhanced upon coexpression with p300HA or

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treatment with histone deacetylase inhibitors (HDACi) (Figure 5A). To map the acetylationsites of GS, which has 19 lysines, GSFlag was immuno-purified from CRBN-KO 293FT cells
and analyzed by mass spectrometry analysis. Two lysine residues, K11 and K14, were foundto be independently acetylated based on the conclusive mass spectra (Figure S5A and S5B,
and Table S4). However, we were unable to detect a tryptic peptide in which both residues
were acetylated (see legend of Figure S5 for a detailed discussion). We also obtained strong
evidence for acetylation of K25, K189 and K291, and modest evidence for K241 and K268
(Table S4). Given that our mutagenesis studies pointed to a critical role for lysine 11 and/or
lysine 14 in mediating GS ubiquitylation and degradation via CRL4CRBN, we evaluatedpoint mutants to determine whether these residues contributed to the signal observed in ananti-acetyllysine immunoblot of immunoprecipitated GS. Wild-type GSMyc, but not thecorresponding AA or RR mutants, yielded a robust signal (Figure 5B and Figure S4E). We
conclude that GS can be acetylated on K11 and K14 in cells.

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Covalent modifications that target CRL substrates for ubiquitylation are often tightly
regulated. We therefore sought to address if K11 and/or K14 were acetylated in a glutamineand 300/CBP-dependent manner. To address these questions, CRBN-KO 293FT cells
expressing wild type or RR-GSFlag and starved for glutamine were re-fed or mock treated,lysates of these cells were immunoprecipitated with anti-Flag, and the precipitates were
blotted with acetyllysine antibody. Aetylation of GSFlag was markedly stimulated byglutamine but this effect was largely blocked by the RR mutations and the CBP/p300
inhibitor C646 (Bowers et al., 2010) (Figure 5C). A similar result was obtained for
endogenous GS (Figure S4F), but the stimulatory effect of glutamine was less prominent.
If glutamine-stimulated acetylation of K11 and K14 on GS triggers its ubiquitylation anddegradation, we reasoned that histone deacetylase inhibitors (HDACi) might enhanceglutamine-induced downregulation of endogenous GS. Indeed, this was observed to be thecase (Figure 5D; mRNA analysis in Figure S6A). The destabilizing effect of glutamine and

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HDACi was blunted by simultaneous treatment with C646 or a distinct p300/CBP inhibitor,
garcinol (Figures 5E and S6B). HDACi-induced degradation of endogenous GS was
mediated by CRBN because it was significantly attenuated by genetic (Figure 5F) or
chemical (Figure S6C & D) reduction of CRL4CRBN activity.
The hypothesis that emerged from these studies is that high glutamine triggers acetylation of
GS lysines 11 and/or 14, which then mediates binding to CRBN. To test this, we took
advantage of our observation that acetylation at K11 and/or K14 made a dominant
contribution to binding of the anti-acetyllysine antibody to GS. Lysates of 293T cells thatstably expressed FlagCRBN were immunoprecipitated with anti-Flag and anti-GS, and theamount of each immunoprecipitate analyzed by blotting was adjusted such that the amountof GS was similar. As shown in Figure 5G, the GS bound to CRBN was enriched foracetylation compared to the unbound GS. We conclude that CRBN exhibits a strong
preference for binding acetylated GS.
The N-terminal extension of GS comprises an acetyllysine degron that binds the Cterminal domain of CRBN

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We next mapped the GS-binding region on CRBN by deletion analysis and found that the
carboxy-terminal domain (CTD) was necessary and sufficient for binding endogenous GS(Figures 6A and B). The CTD harbors the IMiD binding pocket formed by Trp380, Trp386
and Trp400, with a phenylalanine residue at the base (Phe402) (Chamberlain et al., 2014;
Fischer et al., 2014). These residues form a small hydrophobic pocket (tri-Trp pocket),which shows 100% conservation in CRBN orthologs across animal and plant kingdoms(Chamberlain et al., 2014; Fischer et al., 2014) (Figure S7A). We therefore speculated thatthe tri-Trp pocket might be important for GS binding. FlagCRBN-W386E and FlagCRBNW400E, and the double point mutant FlagCRBN-Y384A/W386A (FlagCRBN-YW/AA,
which is defective in IMiD binding (Ito et al., 2010; Lopez-Girona et al., 2012)) failed tobind endogenous GS (Figure 6C).

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To define in greater detail the molecular basis for GS–CRBN interaction, we generated
biotinylated synthetic peptides (amino acids 5–22 of GS) that were unmodified, monoacetylated on either K11 or K14, or di-acetylated (Figure 6D). The peptide acetylated at both
K11 and K14, but not non-acetylated or mono-acetylated peptides, efficiently pulled-down
recombinant FlagCRBN (Figure 6E). We further examined the effects of each acetylation site
mutation of GSMyc on CRBN binding and ubiquitylation in vivo. Consistent with the peptide
binding analysis, single substitutions at either K11 or K14 with alanine (A) resulted insignificantly less binding and ubiquitylation, compared with double AA mutations at both
K11 and K14 (Figure S6E, F). These results indicate that acetylation of GS on both lysine
residues 11 and 14 contributes to CRBN binding, ubiquitylation and degradation.
To gain further insight into the mechanism by which CRBN binds the di-acetylated degronof GS, we made asparagine 351 to arginine (N351R) and histidine 357 to tyrosine (H357Y)(Figure S7A) point mutants based on examination of the crystal structure. We reasoned thatthe substitution of N351R might block degron binding by occluding the IMiD pocket
whereas H357Y might enhance degron binding by decreasing the cationic charge
surrounding the IMiD pocket. Remarkably, production and testing of the
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indicated FlagCRBN mutants confirmed these predictions (Figures 6F – G), suggesting that
the GS-peptide binding site overlaps or is adjacent to the IMiD pocket. Enhanced binding of
the H357Y mutant to the mono-acetylated K11 peptide but not the mono-acetylated K14
peptide (Figure S6G) further implicates electrostatics as contributing to recognition of theacetylated GS peptides.
Recent studies have reported that IMiD binding to CRBN promotes recruitment of
neosubstrates (Kronke et al., 2014; Lu et al., 2014)(Kronke et al., 2015), whereas IMiDs
block endogenous CRBN substrate MEIS2 from binding to CRL4CRBN (Fischer et al.,2014). We therefore examined whether IMiD binding to CRBN influences the interaction
between the acetyl-degron and CRBN in vitro. Consistent with our initial findings in 293T
and MM.1S cells (Figures 1B, 1C, and S1B), pomalidomide did not block FlagCRBN
binding to di-acetylated peptide, but promoted the interaction between FlagCRBN andpeptide mono-acetylated at K11 or K14 (Figure 6H).

Discussion
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Almost sixty years ago, it was reported that mammalian GS is inactivated by extracellularglutamine (Demars, 1958; Paul and Fottrell, 1963). Subsequent work done prior to thediscovery of the ubiquitin system suggested that glutamine stimulates the modification anddegradation of GS enzyme through an unknown mechanism (Arad et al., 1976; Crook andTomkins, 1978). Based on the findings reported here, we propose a model for regulation of

glutamine-induced degradation of GS by CRL4CRBN (Figure 7). After exposure of cells tohigh glutamine, p300/CPB proteins acetylate GS at lysines 11 and 14 to create a degron thatbinds CRBN. Acetylated GS bound to CRBN is ubiquitylated and subsequently is degradedby the proteasome. Although we provide strong evidence to support these conclusions, wenote that it has been difficult to identify by mass spectrometry acetylation of lysines 11 and14 on GS bound to CRBN. This is probably due to low sequence coverage obtained for
endogenous GS bound to CRBN. However, we have used antibodies against acetyl-lysine toshow that CRBN enriches for acetylated forms of GS.
In mammals, GS is a homodecamer composed of two pentameric rings (Krajewski et al.,2008). The pentamer interface contains a network of electrostatic interactions involving both

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K11 and K14. In particular the ε-amino group of Lysine 11 is normally engaged in a salt
bridge with aspartate 174 in the neighboring subunit (Figure S7B, C). We propose that highglutamine concentrations cause a conformational change in GS that disrupts this ionicinterface and exposes the N-terminal extension such that lysines 11 and 14 can be bound by
p300/CBP and acetylated. We currently do not understand how cells sense extracellularglutamine levels to induce K11 and K14 acetylation. Glutamine could potentially be sensed
by mTOR (Nicklin et al., 2009) or perhaps directly by GS. Another question of interest iswhether high glutamine results in processive or distributive acetylation of subunits in the
decamer. Presumably, acetylation of a single subunit would suffice to target the decamer to
CRBN. We do not know if all subunits are ubiquitinated and degraded in concert or the
decamer is disassembled to enable degradation of only acetylated subunits. We note that a
prior study reported that GS degradation in Schwann cells is mediated by ubiquitin ligase
ZNRF1 (Saitoh and Araki, 2010), but regulation by glutamine levels was not investigated.
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Page 9ZNFR1 is expressed primarily in the nervous system (Araki et al., 2001), suggesting that it isunlikely to play a ‘pan-organismal’ role. ZNFR1 may contribute to residual GS degradationthat we observed in CRBN-depleted cells.

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Reversible lysine acetylation influences diverse biological processes. Recent proteomic
analyses suggest that it regulates many metabolic enzymes (Choudhary et al., 2009; Wang etal., 2010; Zhao et al., 2010). It has been thought that lysine acetylation controls protein–protein interactions that regulate protein activity via acetyl-lysine-binding domains(Choudhary et al., 2014; Mujtaba et al., 2007). In some cases, lysine acetylation has beenshown to compete with other post-translational modifications (e.g. ubiquitylation or
SUMOylation), thereby controlling protein stability or transcriptional activity (Gronroos etal., 2002; Ito et al., 2002; Van Nguyen et al., 2012). Recent studies suggest that lysine
acetylation can regulate the steady-state levels of metabolic enzymes by promoting theirdegradation through the ubiquitin-proteasome system or chaperone-mediated autophagy(Jiang et al., 2011; Lv et al., 2011). Collectively, these prior findings together with thosereported here may have important implications for interpreting the clinical action ofdeacetylase inhibitors that alter the acetylome, since they may activate degradation ofmultiple proteins via CRBN or other pathways.

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CRBN and its ‘tri-Trp’ pocket are evolutionarily conserved in plants and animals. Thisaromatic pocket binds thalidomide, lenalidomide, and pomalidomide, and is reminiscent of
pockets found in proteins containing bromodomains (Dhalluin et al., 1999), plant
homeodomain (PHD) fingers (Wysocka et al., 2006), chromodomains, Tudor domains, andmalignant brain tumor (MBT) repeats, all of which have been implicated in binding toacetylated or methylated lysine (Taverna et al., 2007). Consistent with an important role forthe tri-Trp pocket in recognition of natural substrates, mutations of residues that form thepocket eliminated binding of GS and its acetylated degron peptide, and also disrupt bindingof the endogenous substrate MEIS2 (Fischer et al., 2014). However, IMiDs did not compete
out binding of the acetylated GS degron peptide to CRBN. In fact, IMiDs actually enhanced
binding of monoacetylated GS peptides. Moreover, mutation of the critical lysines 11 and 14
of GS to alanine enhanced binding and ubiquitylation of GS by CRBN. Together, theseresults suggest that acetylation neutralizes the positive charges on K11 and K14, which
otherwise interfere with binding to CRBN. Our data suggest that the acetylated peptide
binds adjacent to the IMiD pocket by a novel mechanism, the description of which awaits acrystal structure of the GS degron peptide bound to CRBN.
A role for CRBN in regulation of AMPK and fat accumulation has been demonstrated in a
Crbn−/− mouse model (Lee et al., 2013). Our work establishes an unexpected molecular link
between CRBN, acetylation, and metabolic control. We suggest that acetylation-dependentubiquitylation by CRL4CRBN may be a general feature of metabolic regulation.

Experimental Procedures
In vitro ubiquitylation assay
The assays were performed as described (Duan et al., 2012; Kleiger et al., 2009). Briefly,
293T cells stably expressing FlagCRBN or empty vector were treated with Bortezomib (1Mol Cell. Author manuscript; available in PMC 2017 March 17.

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HHMI Author ManuscriptµM) for 6 h. Then, the cells were harvested and lysed in IP buffer and immunoprecipitatedwith Flag M2 agarose beads for 2–4 h at 4°C. After washing five times with IP lysis buffer
and two times with ubiquitylation buffer (50 mM Tris-HCl [pH 8.0], 10 mM MgCl2, 0.2 mM
CaCl2, 1 mM DTT, and 100 nM MG132), the beads were incubated at 300C for 1 h in 30 µlof ubiquitylation buffer containing E1 (0.5 µM), UbcH5a (0.5 µM), UbcH3 (1.67 µM),
recombinant RBX1-CUL4A (250 nM), ubiquitin (60 µM), and ATP (4 mM). Where
indicated, methylated ubiquitin (Me-Ub) was also added. Reactions were stopped by adding
SDS sample buffer, separated by SDS-PAGE, transferred to a PVDF membrane, andsubjected to immunoblot analysis.
Peptide pull-down assay

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The assay was performed as described (Wysocka et al., 2005). FlagCRBN protein was
immunoprecipitated from 293T cells stably expressing FlagCRBN using Flag antibodyconjugated agarose beads, and then eluted with Flag peptides. Biotinylated GS peptides weresynthesized (Biomatik) and 5 µg was incubated with 20 µl of Dynabeads M-280 streptavidin(Life Technologies) in PBS for 1–2 h at room temperature. After washing three times with
PBS-T (PBS containing 0.1% Tween-20), the beads were mixed with purified FlagCRBN inbinding buffer (10 mM Tris [pH 7.6], 150 mM NaCl, 0.5% NP-40) containing a proteaseinhibitor cocktail, 1 mM DTT and 0.1% BSA for 2–4 h at 4°C. After binding, the beads
were washed extensively in binding buffer containing 300 mM NaCl (stringent washing).
The bound proteins were eluted in 2x SDS loading buffer, and analyzed by immunoblot.

Supplementary Material
Refer to Web version on PubMed Central for supplementary material.

Acknowledgments

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We thank W. Kaelin (Dana Farber Cancer Institute, Boston) for CRBN-KO 293FT cells. We also thank the Deshaies
lab for helpful discussions, particularly E. Blythe, R. Mosadeghi, W. den Besten, A. Moradian (Proteome
Exploration Laboratory/PEL) for their assistance and R. Verma for insightful advice. J.E.L. was supported by agrant from National Research Council of Science and Technology (DRC-14-2-KRISS). The PEL is funded by the
Gordon and Betty Moore Foundation (Grant GBMF775) and the Beckman Institute. T.V.N is supported by the
Vietnam Education Foundation, a Brian D. Novis Research Award from the International Myeloma Foundation, andthe Leukemia & Lymphoma Society. R.J.D. is an Investigator of the Howard Hughes Medical Institute and thiswork was supported in part by HHMI.

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Page 14Highlights

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GS is an endogenous substrate of CRL4CRBN.

➢

CRL4CRBN directly mediates the glutamine-induced degradationof GS.

➢

Glutamine-stimulated acetylation of lysines 11 and 14 regulates
GS degradation.

➢

The thalidomide-binding domain of CRBN binds to anacetyllysine degron of GS.

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Figure 1. GS is an endogenous substrate of CRL4CRBN

HHMI Author Manuscript(A) Identification of GS as a CRBN-interacting protein. 293T cells stably
expressing FlagCRBN and grown in either ‘heavy’ or ‘light’ SILAC medium were treatedwith DMSO (light) or 50 µM thalidomide (heavy) for 4 h prior to lysis andimmunoprecipitation (IP) with anti-Flag followed by mass spectrometry. The heavy:lightratios for GS and subunits of CRL4 and CSN are shown. The asterisk indicates a ratio thatdiffers significantly from 1 (p-value 1×10−19). The data are an average of two experiments.
Error bars indicate ± SD.
(B) GS binds CRBN. 293T cells stably expressing empty vector or wild-type FlagCRBN
were treated with or without lenalidomide (10 µM) for 3 h. Protein extracts were
immunoprecipitated with Flag antibody followed by Western blot analysis with the indicatedantibodies. The ratio of GS bound to CRBN normalized to input GS is shown.
(C) Endogenous CRBN and GS interact. MM.1S cells were supplemented with DMSO or
1 µM lenalidomide 2 h prior to lysis and IP with mouse IgG control or CRBN antibodies. IP
and input samples were fractionated by SDS-PAGE and immunoblotted with the indicatedantibodies. Quantification was as described in (B).
(D–E) CRBN promotes GS ubiquitylation in cells (D) and in vitro (E). (D) 293T cells
were transiently transfected with plasmids expressing GSFlag and HAubiquitin (HAUb). After
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30h, cells were treated with 10 µM MG132 for 4 h, followed by cell lysis and Flag IP underdenaturing conditions. The input and bound fractions were evaluated by immunoblottingwith HA and Flag antibodies. Ubiquitin conjugates in the input are shown in Figure S1C. (E)293T cells stably expressing FlagCRBN were treated with proteasome inhibitor (1 µM
bortezomib) for 6 h. After IP with Flag antibody, in vitro ubiquitylation of endogenous, coprecipitated GS was carried out for 1 h at 30°C in the presence or absence of E1+E2
and HAUb. Where indicated, methylated ubiquitin (Me-Ub) or recombinant (r) CUL4ARBX1 was added. Reactions were analyzed by SDS-PAGE and immunoblotting with GS
antibody. (Ub)n indicates polyubiquitylation. S.E., L.E.: short and long exposures.

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Figure 2. CRBN is required for glutamine-induced degradation of GS(A) Glutamine regulates GS protein abundance. Hep3B cells were maintained in DMEM
10% FCS without glutamine for 48 h. The cells were then treated with glutamine (4 mM) for
the indicated times. Equal amounts of protein extracts were analyzed by SDS-PAGE andimmunoblotting with the indicated antibodies. GAPDH served as a loading control.
(B) Glutamine-induced GS degradation is blocked by the proteasome inhibitor
bortezomib or the Nedd8-activating enzyme inhibitor MLN4924. Hep3B cells werestarved of glutamine for 36 h, and then pretreated with or without bortezomib (200 nM) or
MLN4924 (2 µM) for 30 min, followed by 4 mM glutamine treatment for 7 h. Cell lysates
were analyzed by SDS-PAGE and immunoblotting with antibodies against GS, CRBN, and
GAPDH. The relative ratio of GS:GAPDH, normalized to lane 1, is shown.
(C) Glutamine-induced GS degradation is promoted by CRBN. Wild-type (WT) and
CRISPR/Cas9-derived CRBN-knockout (KO) Hep3B cells were starved of glutamine for 36
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HHMI Author Manuscripth, followed by addition of 4 mM glutamine for 0, 12 and 16 h. The relative ratio of
GS:GAPDH protein level, normalized to that at 0-time, is shown. Note that this experiment
was done with a pool of KO cells (i.e. non-clonal) and there appears to be a small amount ofresidual CRBN in the population.
(D–F) GS protein levels are elevated in the kidneys, skeletal muscles and lungs of
Crbn−/− mice. Left panels: Tissue extracts prepared from total kidneys and skeletal musclesof wild-type (WT) and Crbn−/− (KO) mice were analyzed by SDS-PAGE and Western
blotting, using GS, CRBN and GAPDH antibodies. n = 3–4 mice per group. Right panels:
densitometric quantification of relative band intensities. Error bars represent the SEM.
(G) Crbn−/− mice exhibit an increased glutamine/glutamate ratio in serum. Glutamineand glutamate levels in serum of wild-type (WT) and homozygous mutant Crbn−/− mice(KO) were quantified by mass spectrometry. Glutamine/glutamate ratio was calculated andrepresented as mean ± SD; n = 6 mice per group (P = 0.02615 by t-test).

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Figure 3. The N-terminal extension of GS and its KxxK motif promote binding to CRBN,
ubiquitylation, and degradation(A–B) The N-terminal extension of GS is required to bind CRBN. (A) Schematic
diagram of full-length (FL) human GS protein structure and deletion constructs used in (B).
The GS degron (amino acids 1–24) recognized by CRBN is highlighted. (B) 293T cells
stably expressing FlagCRBN were transfected with the indicated plasmids. After 36 h, cells
were treated with 10 µM MG132 for 4 h. Cellular extracts were immunoprecipitated with
Flag antibody, fractionated by SDS-PAGE and immunoblotted with Myc and Flag
antibodies. *, indicates a non-specific band. A band ∼25 kDa represents IgG light chains(IgG-LC).
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HHMI Author Manuscript(C) The N-terminal extension of GS is required for degradation. 293T cells stably
expressing Flag-HA-tagged GS (FHGS) or GS with deletion of the N-terminal 24 aminoacids (d1FHGS) were cultured in complete DMEM with 2 mM glutamine, and treated withcycloheximide (CHX; 100 µg/ml) for 0, 2, 4, and 6 h. Cell lysates were analyzed by SDSPAGE and immunoblotting with Flag and GAPDH antibodies. The relative ratio of
GS:GAPDH, normalized to that of zero-time, is shown.
(D) The N-terminal KxxK motif modulates binding of GS to CRBN. 293T cells stably
expressing FlagCRBN were transfected with empty vector (EV) or plasmids encoding theindicated GS mutants. After 36 h, cells were treated with 10 µM MG132 for 4 h. Cellextracts were immunoprecipitated with Flag antibody and the precipitated and input
fractions were analyzed by SDS-PAGE and immunoblotting with DDB1, Myc, and Flag
antibodies. WT: wild type. RR: K11R, K14R. AA: K11A, K14A.
(E) The N-terminal KxxK motif modulates ubiquitylation of GS. 293T cells weretransfected with plasmids encoding HAUb and the indicated Myc-tagged GS mutants. After
24 h, the cells were treated with 10 µM MG132 for 4 h, followed by cell lysis, denaturationof the lysate proteins, and IP with anti-Myc. The input lysates and bound fractions were
evaluated by SDS-PAGE and immunoblotting with HA and Myc antibodies.
(F) The N-terminal KxxK motif modulates degradation of GS. 293T cells weretransfected with plasmids encoding wild type GSMyc or the RR and AA mutants. After 24–
30 h, the cells growing in medium containing 2 mM glutamine were treated with 100 µg/ml
cycloheximide (CHX). At the indicated times following addition of CHX, cells were
harvested, and their content of GS and GAPDH was evaluated by immunoblotting. GSRR:
K11R, K14R GS. GSAA: K11A, K14A GS.

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Figure 4. The N-terminal extension of GS comprises a sufficient, KxxK-dependent ubiquitylationand degradation signal(A) Schematic of GS Degron-GFP fusion proteins. Wild type (GS-NWT) or mutant (GSNRR) versions of the N-terminal extension (amino acids 1–25) of GS were fused to Myctagged GFP. RR refers to the double mutant in which K11 and K14 were changed to R.
(B) The N-terminal extension of GS is sufficient to bind CRBN in a manner that
depends on an intact KxxK motif. CRBN-KO 293FT cells stably expressing MycGFP, GSNWT_MycGFP, or GS-NRR_MycGFP fusion proteins were transfected with empty plasmid

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HHMI Author Manuscript(lanes 1–3) or plasmid expressing FlagCRBN (lanes 4–6). After 36 h, cell extracts were
immunoprecipitated with Flag antibody, fractionated by SDS-PAGE and immunoblottedwith the indicated antibodies.
(C) The N-terminal extension of GS is sufficient to confer CRBN- and KxxK-dependentubiquitylation. CRBN-KO 293FT cells stably expressing MycGFP, GS-NWT_MycGFP, and
GS-NRR_MycGFP fusion proteins were transfected with plasmid expressing HAUb (lanes 1–6) and empty plasmid (lanes 1–3) or plasmid expressing FlagCRBN (lanes 4–6). After 48 h,cells were treated with bortezomib (1 µM) for 4 h prior to lysis and IP with HA antibody.
Immunoprecipitates and input samples were fractionated by SDS-PAGE and immunoblottedwith the indicated antibodies. The anti-HA blots are in Figure S4B.
(D) The N-terminal region of wild type GS is sufficient to confer degradation. 293T
cells, stably expressing MycGFP, GS-NWT_MycGFP, and GS-NRR_MycGFP fusion proteins,grown in 2 mM glutamine were treated with 100 µg/ml cycloheximide (CHX) for theindicated times. Extracts were evaluated by SDS-PAGE and immunoblotting with Myc and
GAPDH antibodies. The relative ratio of test protein:GAPDH, normalized to that of 0-time,is shown.

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Figure 5. p300-mediated acetylation promotes the degradation of GS

HHMI Author Manuscript(A) p300 promotes GS acetylation in cells. CRBN-KO 293FT cells were transfected with
GSFlag and HA-tagged p300 (p300HA) plasmids. After 36 h, the cells were treated with orwithout HDAC inhibitors (1 µM TSA and 10 mM NAM) for 12 h. Cell lysates were
immunoprecipitated with anti-Flag and precipitated and input fractions were analyzed by
SDS-PAGE and immunoblotting with the indicated antibodies. Ac-Lys refers to antibody
that recognizes acetylated lysine.
(B) Lysines 11 and/or 14 are acetylation sites. Lysates from CRBN-KO 293FT cells
transfected with plasmids expressing Myc-tagged wild type or RR (K11R/K14R) mutant GS
were immunoprecipitated with anti-Myc, eluted with Myc peptide, and then analyzed by
SDS-PAGE and immunoblotting with the indicated antibodies. S.E., L.E.: short and longexposures.
(C) Glutamine induces p300-mediated acetylation of GS. CRBN-KO 293FT cells weretransiently transfected with plasmids expressing wild type or RR mutant GSFlag. After 24 h,cells were starved of glutamine for 24 h, then pre-treated with or without 10 µM p300/CBP
inhibitor C646 in fetal bovine serum-free DMEM medium for 2 h, followed by treatment
with 4 mM glutamine for 2 h. The cell lysates were immunoprecipitated with anti-Flag, andthen analyzed by SDS-PAGE and immunoblotting (IB) with the indicated antibodies. The
relative ratio of acetylated GSFlag to total GSFlag protein (Ac-Lys/Flag ratio), normalized tothat of untreated cells, is shown.
(D) HDAC inhibitors enhance glutamine-induced GS degradation. Hep3B cells werestarved of glutamine for 24 h, and then supplemented (or not) with 4 mM glutamine for 12 hin the presence or absence of HDAC inhibitors SAHA (1 µM) and NAM (10 mM). Equal
amounts of cell extracts were analyzed by SDS-PAGE and immunoblotting with antibodies

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HHMI Author Manuscript

against GS, CRBN, and GAPDH. The relative ratio of GS:GAPDH, normalized to that ofuntreated cells, is shown. S.E., L.E.: short and long exposures.
(E) Inhibition of the acetyltransferases p300 and CBP by C646 counteracts HDAC
inhibitor-induced GS degradation. 293T cells were starved of glutamine for 24 h and thenpretreated (or not) with 10 µM C646 in fetal bovine serum-free medium for 1 h. Afterwards,
cells were treated with 4 mM glutamine in the presence or absence of HDAC inhibitors (1µM TSA and 10 mM NAM) for 4 h. Equal amounts of cell extracts were analyzed by SDSPAGE and immunoblotting with the indicated antibodies. The relative ratio of GS:GAPDH,
normalized to that of untreated cells, is shown.
(F) HDAC inhibitor-induced GS degradation requires CRBN. Hep3B cells stably
expressing control shRNA or different CRBN shRNAs were starved of glutamine for 48 h.
Starved cells were mock-treated or supplemented with 4 mM glutamine and 1 µM TSA plus
10 mM NAM, as indicated, for 7 h. Cell lysates were analyzed by SDS-PAGE andimmunoblotting with the indicated antibodies. The relative ratio of GS:GAPDH, normalized
to that of untreated cells, is shown. S.E., L.E.: short and long exposures.
(G) CRBN interacts with acetylated endogenous GS. Cell extracts were prepared from293T cells stably expressing FlagCRBN or empty vector. Immunoprecipitation (1st IP) was
performed with anti-Flag antibody. One twenty-fifth of the unbound fractions was
precipitated with anti-GS antibody (2nd IP; it was previously determined that using 25-foldless material in the 2nd IP would yield an equivalent amount of GS as the 1st IP). The
precipitated fractions from 1st IP and 2nd IP were analyzed by SDS-PAGE andimmunoblotting (IB) with indicated antibodies.

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Figure 6. The N-terminal extension of GS comprises an acetylation-dependent degron for
CRL4CRBN

HHMI Author Manuscript(A–B) GS binds the C-terminal domain of CRBN. (A) Schematic diagram of the structureof full-length (FL) human CRBN and the deletion constructs used in panel B. CRBN
consists of the amino-terminal domain (NTD), the helical bundle domain (HBD) involved in
DDB1 binding and the carboxy-terminal domain (CTD). (B) Cell extracts from CRBN-KO
293FT cells stably expressing full length FlagCRBN or deletion mutants were
immunoprecipitated with Flag antibody and analyzed by SDS-PAGE and immunoblottingwith GS and Flag antibodies. *, indicates uncleaved FlagCRBN-T2A–GFP forms, whichwere visible for all constructs on the uncropped film.
(C) Integrity of the ‘tri-Trp’ cavity in the CTD of CRBN is required for binding GS.
Cellular extracts prepared from CRBN-KO 293FT cells stably expressing wild type
(WT) FlagCRBN or the indicated mutants were subjected to IP with Flag antibody followed
by SDS-PAGE and immunoblotting the precipitated and input fractions with the indicatedantibodies. YW/AA corresponds to Y384A/W386A mutant. S.E., L.E.: short and longexposures.
(D) Design of GS N-terminal peptides. Where indicated, the K11 and K14 residues areacetylated.
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HHMI Author Manuscript(E) CRBN binds specifically to a GS N-terminal peptide acetylated on K11 and K14.
Pull-down assays were performed using purified recombinant human FlagCRBN andimmobilized non-acetylated or acetylated K11, K14, or K11K14 GS peptides (panel D) as
indicated, and analyzed by SDS-PAGE and immunoblotting with anti-Flag.
(F–G) CRBN-N351R mutant does not bind to endogenous GS and thalidomide. (F)Cellular extracts prepared from CRBN-KO 293FT cells stably expressing wild type
(WT) FlagCRBN or the indicated mutants were subjected to IP with Flag antibody followed
by SDS-PAGE and immunoblotting the bound and input fractions with the indicatedantibodies. (G) Thalidomide (Thal)-binding CRBN proteins were purified from CRBN-KO
293FT cells stably expressing empty vector or FlagCRBN (wild type or mutant) by using
thalidomide-immobilized (+) or control (–) beads, and analyzed by SDS-PAGE andimmunoblotting with Flag antibody. S.E., L.E.: short and long exposures.
(H) IMiDs do not compete out binding of GS to CRBN. Pull-down assays were
performed in the presence or absence of pomalidomide (pom) as indicated, using FlagCRBN
purified from CRBN-KO 293FT cells stably expressing FlagCRBN, and non-acetylated oracetylated biotin-GS peptides immobilized on streptavidin resin. The input and boundfractions were analyzed by immunoblotting with Flag and DDB1 antibodies. S.E., L.E.:
short and long exposures.

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Figure 7. Proposed model for regulation of glutamine-induced degradation of GS by CRL4CRBN

After exposure of cells to high glutamine, the N-terminal peptide of GS becomes exposed
and p300/CPB acetylates it at lysines 11 and 14 to create a degron that binds CRBN,
resulting in ubiquitylation and degradation of GS. For the sake of simplicity, other aminoacids in the N-terminal extension of GS are omitted.

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ARTICLE
Received 2 May 2015 | Accepted 15 Feb 2016 | Published 18 Mar 2016

DOI: 10.1038/ncomms11030

OPEN

Haem-dependent dimerization of
PGRMC1/Sigma-2 receptor facilitates
cancer proliferation and chemoresistanceYasuaki Kabe1,*, Takanori Nakane2,3,*, Ikko Koike1, Tatsuya Yamamoto4, Yuki Sugiura1, Erisa Harada4,
Kenji Sugase4, Tatsuro Shimamura2, Mitsuyo Ohmura1, Kazumi Muraoka1, Ayumi Yamamoto5, Takeshi Uchida5,
So Iwata2,6, Yuki Yamaguchi7, Elena Krayukhina8, Masanori Noda8, Hiroshi Handa9, Koichiro Ishimori5,
Susumu Uchiyama8,10, Takuya Kobayashi2,11,12 & Makoto Suematsu13

Progesterone-receptor membrane component 1 (PGRMC1/Sigma-2 receptor) is ahaem-containing protein that interacts with epidermal growth factor receptor (EGFR) andcytochromes P450 to regulate cancer proliferation and chemoresistance; its structural basis
remains unknown. Here crystallographic analyses of the PGRMC1 cytosolic domain at 1.95 Å
resolution reveal that it forms a stable dimer through stacking interactions of two
protruding haem molecules. The haem iron is ﬁve-coordinated by Tyr113, and the open
surface of the haem mediates dimerization. Carbon monoxide (CO) interferes with PGRMC1
dimerization by binding to the sixth coordination site of the haem. Haem-mediated PGRMC1
dimerization is required for interactions with EGFR and cytochromes P450, cancer
proliferation and chemoresistance against anti-cancer drugs; these events are attenuated byeither CO or haem deprivation in cancer cells. This study demonstrates protein dimerization
via haem–haem stacking, which has not been seen in eukaryotes, and provides insights into
its functional signiﬁcance in cancer.

1 Department of Biochemistry, Keio University School of Medicine, and Japan Science and Technology Agency (JST), Core Research for Evolutional Scienceand Technology (CREST), Tokyo 160-8582, Japan. 2 Department of Medical Chemistry and Cell Biology, Graduate School of Medicine, Kyoto University,
Kyoto 606-8501, Japan. 3 Department of Statistical Genetics, Graduate School of Medicine, Kyoto University, Kyoto 606-8501, Japan. 4 Bioorganic ResearchInstitute, Suntory Foundation for Life Sciences, Kyoto 619-0284, Japan. 5 Department of Chemistry, Faculty of Science, Hokkaido University, Sapporo
060-0810, Japan. 6 JST, Research Acceleration Program, Membrane Protein Crystallography Project, Kyoto 606-8501, Japan. 7 Department of Biological
Information, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Yokohama 226-8501, Japan. 8 Department of Biotechnology,
Graduate School of Engineering, Osaka University, Osaka 565-0871, Japan. 9 Department of Nanoparticle Translational Research, Tokyo Medical University,
Tokyo 160-8402, Japan. 10 Okazaki Institute for Integrative Bioscience, National Institutes of Natural Sciences, Okazaki 444-8787, Japan. 11 JST, CREST,
Kyoto 606-8501, Japan. 12 Platform for Drug Discovery, Informatics, and Structural Life Science, JST, Kyoto 606-8501, Japan. 13 Department of Biochemistry,
Keio University School of Medicine, JST, Exploratory Research for Advanced Technology (ERATO), Suematsu Gas Biology Project, Tokyo 160-8582, Japan.
* These authors contributed equally to this work. Correspondence and requests for materials should be addressed to Y.K. (email: ykabe@z3.keio.jp) or to T.K.
(email: t-coba@mfour.med.kyoto-u.ac.jp) or to M.S. (email: gasbiology@z6.keio.jp).
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Much attention has been paid to the roles of haem-iron incancer development. Increased dietary intake of haem is
a risk factor for several types of cancer1–3. Previous
studies showed that deprivation of iron or haem suppresses
tumourigenesis4,5. On the other hand, carbon monoxide (CO),the gaseous mediator generated by oxidative degradation of haem
via haem oxygenase (HO), inhibits tumour growth6. Thus, atenuous balance between free haem and CO plays key roles incancer development and chemoresistance7, although theunderlying mechanisms are not fully understood.
To gain insight into the underlying mechanisms, we took
chemical biological approaches using afﬁnity nanobeads8
carrying haem and identiﬁed progesterone-receptor membranecomponent 1 (PGRMC1) as a haem-binding protein from mouse
liver extracts (Supplementary Fig. 1). PGRMC1 is a member ofthe membrane-associated progesterone receptor (MAPR) family9
with a cytochrome b5-like haem-binding region, and is known tobe highly expressed in various types of cancers10–17. PGRMC1 isanchored to the cell membrane through the N-terminaltransmembrane helix and interacts with epidermal growth
factor receptor (EGFR)18 and cytochromes P450 (ref 19). WhilePGRMC1 is implicated in cell proliferation and cholesterol
biosynthesis20,21, the structural basis on which PGRMC1 exerts
its function remains largely unknown.
Here we show that PGRMC1 exhibits a unique haemdependent dimerization. The dimer binds to EGFR andcytochromes P450 to enhance tumour cell proliferation andchemoresistance. The dimer is dissociated to monomers byphysiological levels of CO, suggesting that PGRMC1 serves as a
CO-sensitive molecular switch regulating cancer cell proliferation.

Results
X-ray crystal structure of PGRMC1. We solved the crystal
structure of the haem-bound PGRMC1 cytosolic domain(a.a.72–195) at 1.95 Å resolution (Supplementary Fig. 2).
In the presence of haem, PGRMC1 forms a dimeric structure
largely through hydrophobic interactions between the haem
moieties of two monomers (Fig. 1a, Table 1 and Supplementary
Fig. 3; a stereo-structural image is shown in Supplementary
Fig 4). While the overall fold of PGRMC1 is similar to that of
canonical cytochrome b5, their haem irons are coordinateddifferently. In cytochrome b5, the haem iron is six-coordinatedby two axial histidine residues. These histidines are missingin PGRMC1, and the haem iron is ﬁve-coordinated byTyr113 (Y113) alone (Fig. 1b and Supplementary Fig. 3).
A homologous helix that holds haem in cytochrome b5 is longer,
shifts away from haem, and does not form a coordinate bond in
PGRMC1 (Fig. 1c). Consequently, the ﬁve-coordinated haem of
PGRMC1 has an open surface that allows its dimerization
through hydrophobic haem–haem stacking. Contrary to our
ﬁnding, Kaluka et al.22 recently reported that Tyr164 of
PGRMC1 is the axial ligand of haem because mutation of thisresidue impairs haem binding. Our structural data revealed
that Tyr164 and a few other residues such as Tyr107 and Lys163
are in fact hydrogen-bonded to haem propionates. This isconsistent with observations by Min et al.23 that Tyr 107 andTyr113 of PGRMC1 are involved in binding with haem.
These amino acid residues are conserved among MAPR
family members (Supplementary Fig. 5a), suggesting that these
proteins share the ability to exhibit haem-dependent
dimerization.

a
90°

C

N

Phe128
Leu130’
Cys129Cys129’cb

Tyr107

Tyr113
2.2 Å

163

Tyr164

Lys163PGRMC1147

Cyt b5Figure 1 | X-ray crystal structure of PGRMC1. (a) Structure of the PGRMC1 dimer formed through stacked haems. Two PGRMC1 subunits (blue andgreen ribbons) dimerize via stacking of the haem molecules. (b) Haem coordination of PGRMC1 with Tyr113. Comparison of PGRMC1 (blue) andcytochrome b5 (yellow, ID: 3NER). (c) PGRMC1 has a longer helix (a.a.147–163), which is shifted away from the haem (arrow).
2

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PGRMC1 exhibits haem-dependent dimerization in solution.
In the PGRMC1 crystal, two different types of crystal contacts(chain A–A00 and A–B) were observed in addition to the haemmediated dimer (chain A–A0 ) (Supplementary Figs 3 and 6a). To
conﬁrm that haem-assisted dimerization of PGRMC1 occurs insolution, we analysed the structure of apo- and haem-bound
PGMRC1 by two-dimensional nuclear magnetic resonance(NMR) using heteronuclear single-quantum coherence andtransverse relaxation-optimized spectroscopy (Supplementary
Figs 6b and 7). NMR signals from some amino acid residues of
PGRMC1 disappeared due to the paramagnetic relaxation effectof haem (Supplementary Figs 6b); these residues were located in
the haem-binding region. When chemical shifts of
apo- and haem-bound forms of PGMRC1 were compared, someamino acid residues close to those which disappeared because of
the paramagnetic relaxation effect of haem exhibit notablechemical shifts (Supplementary Fig. 6a,b; dark yellow).
However, at the interfaces of the other possible dimeric structures(Supplementary Fig. 6a, chain A–A00 ; cyan and chain A–B; violet),no signiﬁcant difference was observed. Furthermore, free energyof dissociation predicted by PISA24 suggested that the haemmediated dimer is stable in solution while the other potential
interactions are not. We also attempted to predict
the secondary structure of PGRMC1 through NMR data by
calculating with TALOS þ program25 (Supplementary Fig. 8); theprediction suggested that the overall secondary structure iscomparable between apo- and haem-bound forms of PGRMC1 insolution.
We analysed the haem-dependent dimerization of the
PGRMC1 cytosolic domain (a.a.44–195) in solution (Fig. 2 andTable 2). Mass spectrometry (MS) analyses under non-denaturingcondition demonstrated that the apo-monomer PGRMC1

Table 1 | Data collection and reﬁnement statistics.

Data collection
Space group
Cell dimensions
a, b, c (Å)
a, b, g ()Wavelength (Å)
Resolution (Å)
Rmeas
I/sI
Completeness (%)
Multiplicity
CC1/2
Reﬁnement
Resolution (Å)
Number of reﬂections
Rwork/Rfree
Number of atoms
Protein
Ligand/ion
Water
B-factors
Protein
Ligand/ion
Water
R.M.S deviations
Bond lengths (Å)
Bond angles ()NativePhasing

I4122

I4122

167.23, 167.23, 63.46
90, 90, 90
1.000
20.0–1.95(2.00–1.95)
0.067 (1.168)
22.83 (2.39)
97.8 (99.0)
11.2 (13.8)
100 (81.8)

168.11, 168.11, 63.65
90, 90, 90
1.738
20.0–2.50(2.56–2.50)
0.010 (0.850)
22.43 (4.54)
99.1 (97.6)
14.9 (15.2)
99.9 (93.5)

19.72–1.95
32,298 (2,384)
0.1834/0.2123
1,776
86
109
54.6
42.9
46.6
0.008
1.164

*Highest resolution shell is shown in parenthesis.

resulted in dimerization by binding with haem (Fig. 2a).
It should be noted that a disulﬁde bond between two Cys129
residues is observed in the crystal of PGRMC1 (Fig. 1a), while
Cys129 is not conserved among the MAPR family proteins(Supplementary Fig. 5a). This observation led us to examine
whether or not the disulﬁde bond contributes to PGRMC1
dimerization. MS analyses under non-denaturing conditions
clearly showed that the Cys129Ser (C129S) mutant is dimerized
in the presence of haem, indicating that the haem-mediateddimerization of PGRMC1 occurs independently of thedisulﬁde bond formation via Cys129 (Fig. 2a). Supporting this,MS analyses under denaturing conditions showed that
haem-mediated PGRMC1 dimer is completely dissociated intomonomer, indicating that dimerization of this kind is not
mediated by any covalent bond such as disulﬁde bond(Supplementary Fig. 9).
We also analysed the haem-dependent dimerization of
PGRMC1 by diffusion-ordered NMR spectroscopy (DOSY)
analyses (Table 2, Supplementary Fig. 10). The results suggested
that the hydrodynamic radius of haem-bound PGRMC1 is larger
than that of apo-PGRMC1. To further evaluate changes inmolecular weights in dimerization of PGRMC1, sedimentation
velocity analytical ultracentrifugation (SV-AUC) analysis was
carried out. Whereas the wild-type (wt) apo-PGRMC1 appeared
at a 1.9 S peak as monomer, the haem-binding PGRMC1 wasconverted into dimer at a 3.1 S peak (Fig. 2b). Similarly, theC129S mutant of PGRMC1 converted from monomer to dimerby binding to haem (Fig. 2b). SV-AUC analyses also allowed us to
examine the stability of haem/PGRMC1 dimer. To this end, weused different concentrations (3.5–147 mmol l � 1) of haem-bound
PGRMC1 protein (a.a. 72–195), which were identical to that usedin the crystallographic analysis. The sedimentation coefﬁcients
calculated on the basis of the crystal structure were 1.71 S formonomer and 2.56 S for dimer (Supplementary Fig. 11, upper
panel). The results showed that the PGRMC1 dimer is notdissociated into monomer at all concentrations examined(Supplementary Fig. 11, lower panel), suggesting that the Kdvalue of haem-mediated dimer of PGRMC1 is under
3.5 mmol l � 1. A value of this kind implies that the PGRMC1
dimer is more stable than other dimers of extracellular domain of
membrane proteins such as Toll like receptor 9 (dimerization
Kd of 20 mmol l � 1) (ref. 26) and plexin A2 receptor (dimerization
Kd higher than 300 mmol l � 1) (ref. 27). The current analytical
data conﬁrmed that apo-PGRMC1 monomer converts into dimerby binding to haem in solution (Table 2).
We also showed by haem titration experiments that haembinding to PGRMC1 was of low afﬁnity with a Kd value of
50 nmol l � 1; this is comparable with that of iron regulatory
protein 2, which is known to be regulated by intracellular levels of
haem28 (Fig. 2c and Supplementary Table 1). These results raised
the possibility that the function of PGRMC1 is regulated byintracellular haem concentrations.
CO inhibits haem-dependent dimerization of PGRMC1.
Crystallographic analyses revealed that Tyr113 of PGRMC1 is anaxial ligand for haem and contributes to haem-dependent
dimerization (Fig. 1a). Analysis of UV-visible spectra revealed
that the heme of PGRMC1 is reducible from ferric to ferrous
state, thus allowing CO binding (Fig. 3a). Furthermore, the UVvisible spectrum of the wild type PGRMC1 was thesame as that of the C129S mutant of PGRMC1, and the R/Z ratio
determined by the intensities between the Soret band (394 nm)
peak and the 274-nm peak showed that these proteins were fully
loaded with haem (Supplementary Fig. 12). Analysis of the ferric
form of PGRMC1 using resonance Raman spectroscopy

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b

Experimental mass:17,844.15±0.52

2,231.465

100

8+

10

Theoretical mass:17,843.65

wt apo7+%

1,983.646
1,623.182

2,550.103
2,978.026

0

1,000

c(s) (AU/S)

8

2,000

100

3,000

4,000

5,000

m/z
6,000

Monomer

42

12+3,077.784

63,357.472 Experimental mass:36,920.19±0.98
Theoretical mass:36,918.06

0

0

1

2

%

11+

6

1.0
2,638.079
2,308.455

1,000

100

2,000
2,229.485

10+3,692.960

3,000

4,000

8+

5,000

m/z
6,000

Experimental mass:17,827.91±0.37
Theoretical mass:17,827.59

%

2,000

3,075.046

4,000

5,000

m/z
6,000

12+

Theoretical mass:36,885.60

%

11+

1,000

1

2

2.5

Experimental mass:36,887.07±0.69

3,354.487

0

Dimer

0.4

0

3,000

100

2,306.441

0.6

3,075.143c(s) (AU/S)

1,000

wt haem

0.0

2,547.832

0

3,000

6

7

8

C129S apo

2.0
1.5

Monomer

1.0

4,000

5,000

m/z
6,000

0.0

0

1

2

c
0.3

400

3
4 5
S20,W (S)0.5

2,635.791

2,000

8

0.2

7+

1,621.7227

0.8c(s) (AU/S)

0

1.2

0.3

3 4 5
S20,W (S)67

8

C129S haem

1.0

0.2
0.1

Kd = 50 nmol l–1

0.2c(s) (AU/S)

ΔAbs402

ΔAbs

3 4 5
S20,W (S)

0.1

0.8
0.6

Dimer

0.4
0.2

0.0

0.0
300

400 500 600 700
Wavelength (nm)

800

0.0
0.0

1.0
2.0
Haem (equivalent)

3.0

0

1

2

3 4 5
S20,W (S)67

8

Figure 2 | PGRCM1 is dimerized by binding with haem. (a) Mass spectrometric analyses of the wild-type (wt) PGRMC1 or the C129S mutant in thepresence or absence of haem under non-denaturing condition. Both proteins had identical lengths (a.a.44–195). Data of experimental mass show
mean±s.d. (b) SV-AUC analyses of the wt-PGRMC1 and the C129S mutant (a.a.44–195) in the presence or absence of haem. SV-AUC experiments were
performed with 1.5 mg ml � 1 of PGRMC1 proteins. The major peak with sedimentation coefﬁcient S20,w of 1.9B2.0 S (monomer) or 3.1 S (dimer) was
detected. (c) Difference absorption spectra of PGRMC1 (a.a.44–195) titrated with haem (left panel). The titration curve of haem to PGRMC1 (right panel).
The absorbance difference at 400 nm is plotted against the haem concentration.

(Supplementary Fig. 13) showed that the relative intensity ofoxidation and spin state marker bands (n4 and n3) is close to 1.0,
which is consistent with it being a haem protein with aproximal Tyr coordination29. A speciﬁc Raman shift peaking at
vFe–CO ¼ 500 cm � 1 demonstrated that the CO-bound haem of
PGRMC1 is six-coordinated (Supplementary Fig. 13).
Since PGRMC1 dimerization involves the open surface ofhaem on the opposite side of the axial Tyr113, no space for CO
binding is available in the dimeric structure (Fig. 3b). This
prompted us to ask if CO binding to haem causes dissociation of
the PGRMC1 dimer. Analysis by gel ﬁltration chromatography
revealed that the relative molecular sizes of the wild-type and theC129S mutant of PGRMC1 are increased by adding haem to apoPGRMC1 regardless of the oxidation state of the iron (Fig. 3c),which is in agreement with the results in Table 1. CO application4

to ferrous PGRMC1 abolished the haem-dependent increase in its
molecular size. Under this reducing condition in the presenceof dithionite, analyses of UV-visible spectra indicated thatCO-binding with haem-PGRMC1 is stable, showing only 20%
reduction of the absorbance at 412 nm within 2 h (Supplementary
Fig. 14). Furthermore, the Tyr113Phe (Y113F) mutant of
PGRMC1 was not responsive to haem. These results suggest that
CO favours the six-coordinate form of haem and interfereswith the haem-mediated dimerization of PGRMC1. To examine
the inhibitory effects of CO on haem-mediated PGRMC1
dimerization, SV-AUC analysis was carried out. The peakcorresponding to the haem/PGRMC1 dimer was detected under
reducing conditions in the presence of dithionite (Supplementary
Fig. 15, middle panel). Under these circumstances, CO application induced dissociation of the haem-mediated dimers of

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Table 2 | PGRMC1 proteins exhibit haem-dependent dimerization in solution.
Apo form

Haem-bound form
Mass (Da)a PGRMC1 wt (a.a.44–195)
ESI-MS
Theoretical
DOSY
SV-AUC—
Hydrodynamic radius 10 � 9 (m)2.04–2.15
S20,w (S)
1.9

b PGRMC1 C129S (a.a.44–195)
ESI-MS
Theoretical
SV-AUC—
S20,w (S)2.0

17,844.14
17,843.65
MW (kDa)
20
MW (kDa)
17.6

Mass (Da)—
Hydrodynamic radius 10 � 9 (m)2.94–3.02
S20,w (S)
3.1

17,827.91
17,827.59
MW (kDa)
18.1—
S20,w (S)3.1

36,920.19
36,918.06
MW (kDa)42
MW (kDa)
35.5

36,887.07
36,885.6
MW (kDa)
35.8

Differences in molecular weights of the wild-type (wt; a) and the C129S mutant (b) PGRMC1 proteins in the absence (apo form) or the presence of haem (haem-bound form). The protein sizes of the wt
and C129S PGRMC1 cytosolic domains (a.a.44–195) in the presence or absence of haem were estimated by ESI-MS, DOSY and SV-AUC.

PGRMC1 to generate a peak of monomers (Supplementary
Fig. 15, lower panel). These observations raised the transition
model for structural regulation of PGRMC1 in response to haem(Fig. 3d). As mentioned above, apo-PGRMC1 exists as monomer.
By binding with haem (binding Kd ¼ 50 nmol l � 1), PGRMC1
forms a stable dimer (dimerization Kdoo3.5 mmol l � 1) throughstacking of the two open surfaces of the ﬁve-coordinated haem
molecules in each monomer. CO induces the dissociation of thehaem-mediated dimer of PGRMC1 by interfering with thehaem-stacking interface via formation of the six-coordinatedCO-haem-PGRMC1 complex. Such a dynamic structural
regulation led us to further examine the regulation of PGRMC1
functions in cancer cells.
PGRMC1 dimerization is required for binding to EGFR.
Because PGRMC1 is known to interact with EGFR and toaccelerate tumour progression18, we examined the effect of haemdependent dimerization of PGRMC1 on its interaction with
EGFR by using puriﬁed proteins. As shown in Fig. 4a, thecytosolic domain of wild-type PGRMC1, but not the Y113F
mutant, interacted with puriﬁed EGFR in a haem-dependent
manner. This interaction was disrupted by the ruthenium-based
CO-releasing molecule, CORM3, but not by RuCl3 as a control
reagent (Fig. 4b). We further analysed the intracellular interaction
between PGRMC1 and EGFR. FLAG-tagged PGRMC1
ectopically expressed in human colon cancer HCT116 cells was
immunoprecipitated with anti-FLAG antibody, and coimmunoprecipitated EGFR and endogenous PGRMC1 binding
to FLAG-PGRMC1 were detected by Western blotting (Fig. 4c).
The C129S mutant of PGRMC1 also interacted with endogenous
PGRMC1 and EGFR (Supplementary Fig. 16). Whereas FLAGtagged wild-type PGRMC1 interacted with endogenous PGRMC1
and EGFR, the Y113F mutant did not. We also examined theeffect of succinylacetone (SA), an inhibitor of haem biosynthesis(Fig. 4d). As expected, SA signiﬁcantly reduced PGRMC1
dimerization and its interaction with EGFR (Fig. 4e), indicatingthat haem-mediated dimerization of PGMRC1 is critical for its
binding to EGFR.
PGRMC1 dimer facilitates EGFR-mediated cancer growth.
Next, we investigated the functional signiﬁcance of PGRMC1
dimerization in EGFR signaling. EGF-induced phosphorylationsof EGFR and its downstream targets AKT and ERK weredecreased by PGRMC1 knockdown (PGRMC1-KD) (Fig. 4f).
Similarly, EGFR signaling was suppressed by treatment of
HCT116 cells with SA (Fig. 4g) or CORM3 (Fig. 4h). These
results suggested that haem-mediated dimerization of PGRMC1
is critical for EGFR signaling.
To further investigate the role of the dimerized form of
PGRMC1 in cancer proliferation, we performed PGRMC1
knockdown-rescue experiments using FLAG-tagged wild-type
and Y113F PGRMC1 expression vectors, in which silent
mutations were introduced into the nucleotide sequence targetedby shRNA (Fig. 5a). While proliferation of HCT116 cells was notaffected by knocking down PGRMC1, PGRMC1-KD cells weremore sensitive to the EGFR inhibitor erlotinib than control
HCT116 cells, and the knockdown effect was reversed byco-expression of shRNA-resistant wild-type PGRMC1 but notof the Y113F mutant (Fig. 5b). Chemosensitivity enhancement by
two different shRNAs to PGRMC1 was seen also in HCT116 cells
and human hepatoma HuH7 cells (Supplementary Fig. 17).
Furthermore, PGRMC1-KD inhibited spheroid formation of
HCT116 cells in culture, and this inhibition was reversed byco-expression of wild-type PGRMC1 but not of the Y113F
mutant (Fig. 5c and Supplementary Fig. 18). Thus, PGRMC1
dimerization is important for cancer cell proliferation andchemoresistance.
We examined the role of PGRMC1 in metastatic progression
by xenograft transplantation assays using super-immunodeﬁcient
NOD/scid/gnull (NOG) mice7,30,31. Ten days after intra-splenic
implantation of HCT116 cells that were genetically tagged with aﬂuorescent protein Venus, the group implanted with PGRMC1KD cells showed a signiﬁcant decrease of liver metastasis incomparison with the control group (Fig. 5d).
Interaction of PGRMC1 dimer with cytochromes P450. Since
PGRMC1 has been shown to interact with cytochromes P450(ref 19), we investigated whether the haem-mediateddimerization of PGRMC1 is necessary for their interactions.
Recombinant CYP1A2 and CYP3A4 including a microsomal
formulation containing cytochrome b5 and cytochrome P450
reductase, drug-metabolizing cytochromes P450, interacted withwild-type PGRMC1, but not with the Y113F mutant, in a haemdependent manner (Fig. 6a,b). Moreover, the interaction of
PGRMC1 with CYP1A2 was blocked by CORM3 under reducing
conditions (Fig. 6c), indicating that PGRMC1 dimerization is

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AbsorbanceAbsorbance

0.12

0.5

0.08

State of haem iron

0.04

Peaks (nm)PGRMC1 wt

0.00
500

550

600

Wavelength (nm)Ferric394, 516, 534

Ferrous415, 568

Ferrous+CO

414, 535, 567

0.0
400

600

500
Wavelength (nm)

c

b

669
Fraction no.
wt

1

2

200

150

66

29 (kDa)3 4 5 6 7 8 9 10 11 12 13 14 15

Apo

21

Ferric haem

21

Apo + dithionite

21

Ferrous haem

21

Ferrous + CO
Y113F

21

21

Apo

21

Ferric haem
C129S

21

Apo

21
(kDa)

Ferric haemd
Haem b
CO

Kd<<3.5 μmol l–1

Apo monomer

Haem monomer

Haem dimer

CO-haem monomer

Figure 3 | Carbon monoxide inhibits haem-dependent PGRMC1 dimerization. (a) UV-visible absorption spectra of PGRMC1 (a.a.44–195).
Measurements were performed in the presence of the oxidized form of haem (ferric), the reduced form of haem (ferrous) and the reduced formof haem plus CO gas (ferrous þ CO). (b) Close-up view of haem stacking. Spheres are drawn with van der Waals radii. (c) Gel-ﬁltration chromatography
analyses of PGRMC1 (a.a.44–195) wild-type (wt) and the Y113F or C129S mutant in the presence or absence of haem, dithionite and/or CO.
(d) Transition model for structural regulation of PGRMC1 in response to haem and CO.

necessary for its interaction with cytochromes P450. Doxorubicin
is an anti-cancer reagent that is metabolized into inactive doxorubicinol by CYP2D6 and CYP3A4 (Fig. 6d)32,33. PGRMC1-KD
signiﬁcantly suppressed the conversion of doxorubicin todoxorubicinol (Fig. 6d) and increased sensitivity to doxorubicin(Fig. 6e). Enhanced doxorubicin sensitivity was modestly but
signiﬁcantly induced by PGRMC1-KD. This effect was reversedby co-expression of the wild-type PGRMC1 but not of the Y113F
mutant, suggesting that PGRMC1 enhances doxorubicin
resistance of cancer cells by facilitating its degradation via
cytochromes P450. To gain further insight into the interaction
between PGRMC1 and cytochromes P450, surface plasmon
resonance analyses were conducted using recombinant CYP51
and PGRMC1. This was based on a previous study showing thatPGRMC1 binds to CYP51 and enhances cholesterol biosynthesis
by CYP51 (refs 19,34). CYP51 interacted with PGRMC1 in aconcentration-dependent manner in the presence of haem, but
6

not in its absence (Supplementary Fig. 19), suggesting therequirement for the haem-dependent dimerization of PGRMC1.
The Kd value of PGRMC1 binding to CYP51 was in a micromolar
range and comparable with those of other haem proteins, such as
cytochrome P450 reductase35 and neuroglobin/Gai1 (ref. 36),
suggesting that haem-dependent PGRMC1 interaction with
CYP51 is biologically relevant.
Discussion
In this study, we showed that PGRMC1 dimerizes by stacking
interactions of haem molecules from each monomer. Recently,
Lucas et al.37 reported that translationally-controlled tumour
protein was dimerized by binding with haem, but its structural
basis remains unclear. This is the report showing crystallographic
evidence that indicates roles of the direct haem–haem stacking in
haem-mediated dimerization in eukaryotes, although a few

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a
Input

b

PGRMC1Y113F

PGRMC1
wt

–
EGFR

c

RuCI3CORM3
+
+
150
25
(kDa)

FLAG-PGRMC1
IP

Input
–

FLAG-PGRMC1 expression

+

EGFR

150
25
(kDa)

FLAG-PGRMC1 IP–

Haem/PGRMC1

Apo Haem Apo HaemIP

wt Y113F–

wt Y113F

EGFR

150

FLAG-PGRMC1
25
(kDa)

Endogenous
PGRMC1de

Relative % of
intracellular haem amount

140
120

FLAG-PGRMC1
expression –

100

SA –

80

pEGFR
EGFR
pAKT

ERK
PGRMC1

–

+

+

–

+

–

–

+
150

FLAG-PGRMC140–Endogenous
PGRMC1**

20
Control

+

–

25
(kDa)

SA

g
SA –
EGF –

+

+
+

–
+

h

+
+

EGF –

RuCI3CORM3
+

+

+

150

150

150

150
75

150
75

150
75

75

75

75

50

50

50

50

50

50

25
(kDa)

25
(kDa)

25
(kDa)

AKT
pERK

+

EGFR

Control PGRMC1-KD
EGF

+

60

0

f

IP

Input

Figure 4 | Haem-dependent dimerization of PGRMC1 is necessary for tumour proliferation mediated by EGFR signalling. (a) FLAG-PGRMC1 wild-type(wt) and Y113F mutant proteins (a.a.44–195), in either apo- or haem-bound form, were incubated with puriﬁed EGFR and co-immunoprecipitated with
anti-FLAG antibody-conjugated beads. Input and bound proteins were detected by Western blotting. (b) In vitro binding assay was performed as in (a)using haem-bound FLAG-PGRMC1 wt (a.a.44–195) and puriﬁed EGFR with or without treatment of RuCl3 and CORM3. (c) FLAG-PGRMC1 wt or Y113F(full length) was over-expressed in HCT116 cells and immunoprecipitated with anti-FLAG antibody-conjugated beads. Co-immunoprecipitated proteins(FLAG-PGRMC1, endogenous PGRMC1 and EGFR) were detected with Western blotting by using anti-PGRMC1 or anti-EGFR antibody. (d) HCT116 cells
were treated with or without 250 mmol l  1 of succinylacetone (SA) for 48 h. The intracellular haem was extracted and quantiﬁed by reverse-phase HPLC.
The data represent mean±s.d. of four separate experiments. **Po0.01 using unpaired Student’s t-test. (e) Co-immunoprecipitation assay was performed
as in (c) with or without SA treatment in HCT116 cells. (f) HCT116 cells expressing control shRNA or those knocking down PGRMC1 (PGRMC1-KD) weretreated with EGF or left untreated, and components of the EGFR signaling pathway were detected by Western blotting. (g,h) HCT116 cells were treated withor without EGF, SA, RuCl3 and CORM3 as indicated, and components of the EGFR signaling pathway were detected by Western blotting.

examples are known in bacteria38. Sequence alignments show that
haem-binding residues (Tyr113, Tyr107, Lys163 and Tyr164) in
PGRMC1 are conserved among MAPR proteins (Supplementary
Fig. 5). In the current study, the Y113 residue plays a crucial role
for the haem-dependent dimerization of PGRMC1 and resultant

regulation of cancer proliferation and chemoresistance (Figs 5c
and 6e). Since the Y113 residue is involved in the putative
consensus motif of phosphorylation by tyrosine kinases
such as Abl and Lck39, we investigated whether phosphorylated
Y113 is present in HCT116 cells by ESI-MS analysis. It was,

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ab

Nucleotide mutation
without amino acid changelle

Phe Leu Leu Tyr

Cont PGRMC1-KD

PGRMC1 shRNA
resistant vector––

wt Y113F
37kDa
FLAG-PGRMC1
Endogenous
PGRMC137kDa

Anti-PGRMC1
Anti-FLAGc

Control

Control120

PGRMC1-KD

100
80

KD→Y113F

60

*

*

40
20

d

PGRMC1-KD

KD→wt

*

*

0
Erlotinib (μmol l–1) 0

50kDa

Anti-actin

140

Relative % of cell viability

PGRMC1 shRNAtargeting sequence

HCT116
control10

20

HCT116PGRMC1-KD

Venus

KD→Y113F

KD→wt

25

*

20

*

20

Occupancy % ofmetastatic cancer

Spheroid volume(×104 μm3)

HE

15
10
5
0

Control

KD

KD
wt

KD
Y113F

15
10

*

5
0

Control

PGRMC1-KD

PGRMC1knock-inFigure 5 | Haem-dependent dimerization of PGRMC1 accelerates tumour growth through the EGFR signaling pathway. (a) Nucleotide sequences of
PGRMC1 targeted by shRNA and of the shRNA-resistant full length PGRMC1 expression vector. Stable PGRMC1-knockdown (PGRMC1-KD) HCT116 cells
were transiently transfected with the shRNA-resistant expression vector of wild-type PGRMC1 (wt) or the Y113F mutant (Y113F). (b) Erlotinib was added to
HCT116 (control) cells, PGRMC1-KD cells or PGRMC1-KD cells expressing shRNA-resistant PGRMC1 wt or Y113F, and cell viability was examined by MTT
assay. The data represent mean±s.d. of four separate experiments. *Po0.01 using ANOVA with Fischer’s LSD test. (c) Spheroid formation in control and
PGRMC1-KD HCT116 cells. Pictures indicate representative micrographs of spheroids formed under each condition. Spheroid size were measured andcompared among groups. The graph represents mean±s.e. of each spheroid size. *Po0.01 using ANOVA with Fischer’s LSD test. Scale bar: 0.1 mm.
(d) Tumour-bearing livers of NOG mice at 10 days after intrasplenic injection of HCT116 (control) or PGRMC1-KD cells. Percentages of cross-sectional
areas showing metastatic tumours were calculated. Data represent mean±s.d. of 10 separate experiments. *Po0.05 using unpaired Student’s t-test.
Scale bar: 5 mm.

however, undetectable under current experimental conditions(Supplementary Fig. 20). Recently, Peluso et al.40 reported thatPGRMC1 binds to PGRMC2, suggesting that MAPR family
members may also undergo haem-mediated heterodimerization.
We showed that the haem-mediated dimer of PGRMC1
enables interaction with different subclasses of cytochromes
P450 (CYP) (Fig. 6). While the effects of PGRMC1 on cholesterolsynthesis mediated by CYP51 have been well documented inyeast19,41 and human cells34, it has not been clear whether
drug-metabolizing CYP activities are regulated by PGRMC1.
Szczesna-Skorupa and Kemper34 reported that PGRMC1
exhibited an inhibitory effect on CYP3A4 drug metabolizing
8

activity by competitively binding with cytochrome P450 reductase(CPR) in HEK293 or HepG2 cells. On the other hand,
Oda et al.42 reported that PGRMC1 had no effect to CYP2E1
and CYP3A4 activities in HepG2 cell. Several other groups
showed that PGRMC1 enhanced chemoresistance in several
cancer cells such as uterine sarcoma43, breast cancer17,
endometrial tumour13 and ovarian cancer44,45; however, noevidence of PGRMC1-dependent regulation of CYP activity
was provided. Our results showed that PGRMC1 contributes
to enhancement of the doxorubicin metabolism, which ismediated by CYP2D6 or CYP3A4 in human colon cancer
HCT116 cells (Fig. 6d). While the effects of structural diversity of

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ab

PGRMC1 PGRMC1
wt
Y113F

–

–

Apo Haem Apo Haem

Input

Input
75

CYP1A2

c

–
Input

RuCl3

25
(kDa)

FLAG-PGRMC1 IP

d
O

CH2OH

CH2OH

OH

H3C

OH

O

OCH3

CYP2D6, CYP3A4

O

140

OH

OH

O

H3C

O

OH
O

NH2

NH2

OH

OH

Doxorubicinol (metabolite)

Doxorubicin

Relative % of
doxorubicinol/doxorubicin ratio

O

OH

OCH3 O

120
100
80
60

Relative % of cell viability

120

* *

100

**

40
20
0

e

25
(kDa)

FLAG-PGRMC1 IP

CORM3
–1
3
10 (μmol l )75

CYP1A2OH

75

PGRMC1 wt–

O

Apo Haem Apo HaemCYP3A425(kDa)

FLAG-PGRMC1 IP

PGRMC1 PGRMC1
wt
Y113F

Control

PGRMC1-KD

Control
PGRMC1-KD

** **

KD→wt

80

KD→Y113F

**

60

**
** **

40
20
0

0

0.1

0.2

0.5

1

Doxorubicin (μmol l–1)

Figure 6 | Haem-dependent PGRMC1 dimerization enhances tumour chemoresistance through interaction with cytochromes P450.
(a,b) FLAG-PGRMC1 wild-type (wt) and Y113F mutant proteins (a.a.44–195), in either apo or haem-bound form, were incubated with CYP1A2(a) or CYP3A4 (b) and immunoprecipitated with anti-FLAG antibody-conjugated beads. Input and bound proteins were detected by Western blotting.
(c) Binding assay was performed as in (a) using haem-bound FLAG-PGRMC1 wt and CYP1A2 with or without RuCl3 and CORM3. (d) Schematic illustrationof doxorubicin metabolism is shown on the left. Doxorubicin was incubated with HCT116 cells expressing control shRNA or shPGRMC1 (PGRMC1-KD),and the doxorubicinol/doxorubicin ratios in cell pellets were determined using LC-MS. Data represent mean±s.d. of four separate experiments.
**Po0.01 versus control using unpaired Student’s t-test. (e) Indicated amounts of doxorubicin were added to HCT116 (control) cells, PGRMC1-KD cells,
or PGRMC1-KD cells expressing shRNA-resistant full-length PGRMC1 wt or Y113F, and cell viability was examined by MTT assay. Data represent
mean±s.d. of four separate experiments. *Po0.01 using ANOVA with Fischer’s LSD test.

CYP family proteins and interactions with different xenobiotic
substrates should further be examined, the current results suggestthat the interaction of drug-metabolizing CYPs with thehaem-mediated dimer of PGRMC1 plays a crucial role inregulating their activities.
We showed that haem-mediated dimerization of PGRMC1
enhances proliferation and chemoresistance of cancer cells
through binding to and regulating EGFR and cytochromes
P450 (illustrated in Fig. 7). Since the haem-binding afﬁnity of
PGRMC1 is lower than those of constitutive haem-binding
proteins such as myoglobin, PGMRC1 is probably interconverted
between apo-monomer and haem-bound dimer forms in

response to changes in the intracellular haem concentration.
Considering microenvironments in and around malignant
tumours, the haem concentration in cancer cells is likely to beelevated through multiple mechanisms, such as (i) an increased
intake of haem, (ii) mutation of enzymes in TCA cycle (forexample, fumarate hydratase) that increases the level of succinyl
CoA, a substrate for haem biosynthesis and (iii) metastasis tohaem-rich organs such as liver, brain and bone marrow46–48.
Moreover, exposure of cancer cells to stimuli such as hypoxia,
radiation and chemotherapy causes cell damages and leads toprotein degradation, resulting in increased levels of TCA cycle
intermediates and in an enhanced haem biosynthesis49,50. On the

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N

NATURE COMMUNICATIONS | DOI: 10.1038/ncomms11030

Apo monomer
PGRMC1(inactive)

N

N

N

Fe
CH3

N

was ampliﬁed with PCR (with primers containing the factor Xa site) and ligatedinto the Bam HI and Sal I sites of pGEX6P-1. The full-length PGRMC1 cDNA
fragment containing resistant sequences for shRNA was generated by using theprimers (Supplementary Methods), and ligated into the Eco RI and Bam HI sitesof the C-terminus of the 3xFLAG-tagged expression vector p3xFLAG CMV14(Sigma).

N

CH3

CH=CH2
CH3

Haem-stacked dimer
PGRMC1(active)

N

Haem A’

Tyr113

CH3CH2COOH CH2COOH

Fe

Haem bHaem A7.5 Å

Fe’

Tyr113’

Transverse plane of haem stacking

COInteraction
Cytochromes P450
EGFR
(CYP1A2, CYP3A4, CYP51)↓↓⇑ Cancer proliferation
anti-apoptosis⇑ Xenobiotic metabolism
cholesterol synthesis

Figure 7 | Schematic diagram for the regulation of PGRMC1 functions.
Apo-PGRMC1 exists as an inactive monomer. On binding to haem, PGRMC1
forms a dimer through stacking interactions between the haem moieties,
which enables PGRMC1 to interact with EGFR and cytochromes P450,
leading to an enhanced proliferation and chemoresistance of cancer cells.
CO interferes with the stacking interactions of the haems and thereby
inhibits PGRMC1 functions.

other hand, excessive haem induces HO-1, the enzyme thatoxidatively degrades haem and generates CO. Thus, HO-1
induction in cancer cells may inhibit the haem-mediateddimerization of PGRMC1 through the production of CO andthereby suppress tumour progression. This idea is consistent withthe observation that HO-1 induction or CO inhibits tumour
growth6,51.
Besides the regulatory roles of PGRMC1/Sigma-2 receptor inproliferation and chemoresistance in cancer cells (ref. 52), recent
reports show that PGRMC1 is able to bind to amyloid betaoligomer53 to enhance its neurotoxicity53,54. Furthermore, Sigma2 ligand-binding is decreased in transgenic amyloid beta
deposition model APP/PS1 female mice55. These results suggest
a possible involvement of PGRMC1 in Alzheimer’s disease. The
roles of haem-dependent dimerization of PGRMC1 in thefunctional regulation of its target proteins deserve further
studies to ﬁnd evidence that therapeutic interventions tointerfere with the function of the dimer may control varieddisease conditions.
Methods

Materials. Recombinant EGF, CYP1A2 and CYP3A4 proteins were purchasedfrom Sigma. Erlotinib was purchased from Cayman. Doxorubicin was purchasedfrom Wako. Anti-FLAG (M2) antibody, FLAG peptide and anti-FLAG antibodyconjugated agarose were purchased from Sigma. Haemin and protoporphyrin-IX(PP-IX) were purchased from Porphyrin Science.
Plasmid constructions. Human PGRMC1 cDNA was cloned from the cDNA
library of HuH7 cells. The PGMRC1 (a.a.44–195 for in vitro studies and a.a.72–195
for crystallographic analyses and SV-AUC) cDNA fragment was ampliﬁed
with PCR, digested with Bam HI and Sal I and then ligated into pGEX6P-1(GE Healthcare). For NMR analysis, the PGRMC1 (a.a.44–195) cDNA fragment
10

Preparations of recombinant proteins. pGEX-PGRMC1 wt, Y113F or C129S
mutant expression vectors were transformed into BL21 (DE3), and the bacteria
were incubated in LB with ampicillin at 37 C until OD600 reached at 0.8. Protein
expression was induced by 1 mmol l  1 isopropyl-b-thiogalactopyranoside for 4 h
at 37 C. Cell pellets were resuspended in the buffer containing 20 mmol l  1
Tris-HCl (pH 7.5), 100 mmol l  1 NaCl and 0.1% Tween 20, sonicated twice for5 min at 4 C and centrifuged at 20,000  g for 30 min. The supernatant was
incubated with glutathione Sepharose 4B (GE Healthcare) for 1 h at 4 C. The resin
was then washed ﬁve times with the same buffer, and the GST tag was cleared byaddition of Precision Protease (GE Healthcare) and further incubation for 16 h at
4 C. The apo-PGRMC1 was prepared by eliminating the bacterial holo-PGRMC1
with size-exclusion chromatography (Superdex 200; GE Healthcare). Haem-bound
PGRMC1 were prepared by treatment with 100 mmol l  1 haemin and puriﬁed bysize-exclusion chromatography. The PGRMC1 protein treated with Precision
Protease to cleave the GST-tag contained additional amino acid residues(GPLGSEF) derived from the restriction site and the protease site for Precision
Protease at the N-terminal region of PGRMC1.
Isotope-labelled PGRMC1 proteins for NMR analyses were prepared by
growing cells (BL21 (DE3)) in minimal M9 media in H2O or 99.9% 2H2O,
including ampicillin, metals, vitamins, 15N-ammonium chloride and 13C or 12C
glucose as sources of nitrogen and carbon, respectively. These procedures were
followed by addition of 1 mmol l  1 isopropyl-b-thiogalactopyranoside for 40 h at
20 C. Protein puriﬁcation was performed as mentioned above. The GST tag wascleaved with Factor Xa (GE Healthcare). The proteins were treated with Factor
Xa to cleave the GST tag at the direct site of N-terminal region of PGRCM1(a.a.44–195).
X-ray crystallography. PGRMC1 (a.a.72–195) crystals were grown at 20 C using
hanging-drop vapour diffusion by mixing equal volumes of protein solution
and reservoir solution containing 100 mmol l  1 sodium cacodylate (pH 6.5)
and 1.26–1.45 mol l  1 ammonium sulphate. Brown crystals reached maximum
size in three weeks. The crystals were soaked in reservoir solution containing 30%
trehalose and then ﬂash-frozen in liquid nitrogen. The X-ray diffraction data for
PGRMC1 crystals were collected at SPring-8 BL41XU and processed with XDS56.
The initial phase was obtained by single-wavelength anomalous dispersion, using adataset collected at 1.73 Å with PHENIX AutoSol57. Manual modeling andreﬁnement were performed with COOT58 and phenix.reﬁne59. The depositedmodel was reﬁned to a resolution of 1.95 Å. In this model, 95.4% of the residues
were in favoured regions of Ramachandran plot, and all the others were in allowed
regions. Data collection and reﬁnement statistics are shown in Table 1. Molecularﬁgures were created by PyMOL (Schrodinger, LLC. The PyMOL Molecular
Graphics System, Version 1.5.0.3).
Mass spectrometry analyses. The puriﬁed PGRMC1 (a.a.44–195) proteins, thewild-type (apo and haem) and the C129S mutant (apo and haem), which included
additional amino acid residues (GPLGSEF), were buffer-exchanged into
100 mmol l  1 ammonium acetate, pH 7.5, by passing the proteins through a
Bio-Spin 6 column (Bio-Rad). The buffer-exchanged PGRMC1 wild-type (apo andhaem) and PGMRC1 C129S mutants were immediately analysed by nanoﬂow
electrospray ionization MS using gold-coated glass capillaries made in house. In thecase of ESI-MS analyses under denaturing conditions, buffer-exchanged proteins
were denatured before ESI-MS analyses by adding aliquots of formic acid at ﬁnal
concentration of 30%). Spectra were recorded on a SYNAPT G2 HDMS mass
spectrometer (Waters, Manchester, UK) in positive ionization mode at 1.20 kV
with a 120 V sampling cone voltage. The spectra were calibrated using 1 mg ml  1
caesium iodide and analysed with Mass Lynx software (Waters).
SV-AUC analyses. SV-AUC experiments were performed in a ProteomeLab XL-I
analytical ultracentrifuge (Beckman Coulter) equipped with 4-hole An60Ti rotors
at 20 C using Beckman Coulter 12-mm double-sector aluminium centerpieces andsapphire windows. Recombinant PGRMC1 proteins were diluted with the buffer(20 mmol l  1 Tris-HCl (pH 7.5) and 100 mmol l  1 NaCl) at the indicatedconcentration. Scanning was performed as quickly as possible at 262,080 g at 6.5 cm(60,000 rpm), between 6.0 and 7.2 cm from the axis of rotation with a radial
increment of 30 mm using an absorbance optical system. The sedimentation
coefﬁcient distributions were obtained using the c(s) method of SEDFIT60. The
partial speciﬁc volume, buffer density and viscosity were calculated using the
program SEDNTERP 1.09 and were 0.7216 cm3 g  1, 1.00293 g ml  1, and 1.017 cP,
respectively. The sedimentation coefﬁcient of PGRMC1 (a.a.72–195) was calculatedwith the UltraScan Solution Modeler (US-SOMO) suite61 using the crystal
structure determined in this study. To analyse the effect of CO, protein samples
were prepared in a deaerated solution and treated with dithionite at 5 mmol l  1
and/or CO gas.

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DOSY analysis. Diffusion-ordered 2D NMR spectroscopy (DOSY) was used toinvestigate the oligomerization state of PGRMC1 (a.a.44–195) induced by haembinding. Apo- or haem-bound PGRMC1 and reference proteins (including hen
egg lysozyme, ovalbumin and bovine serum albumin (BSA)) dissolved in
50 mmol l � 1 phosphate buffer (pH 7.0) containing 5% D2O were measured at
25 C. The protein concentrations were 0.15–0.2 mmol l � 1. DOSY spectra were
measured using the stimulated echo sequence with a longitudinal-eddy-current
delay62,63, and diffusion coefﬁcients were calculated from signals in the aliphatic
regions using the software TOPSPIN (Bruker). The signal intensities ﬁt the
Stejskal-Tanner equation:
2 2 2

I=I ð0Þ ¼ e � g g d ðD � 3ÞD
dwhere I represents the signal intensity when gradient pulses of length d areapplied at strength g, varying from 2 to 95% of the full gradient strength(55 G cm � 1). The diffusion coefﬁcient D25 is estimated by curve ﬁtting with theterm I(0), which corresponds to the signal intensity at a gradient strength of 0. The
term g represents the gyromagnetic ratio, and D represents the delay between two
sets of gradients responsible for the stimulated echo. In this study, d was set to 8 or
10 ms, and D was set to 40 ms. The hydrodynamic radii of the proteins were
estimated on the basis of the Stokes–Einstein equation as follows:
D¼

kB T
6pZr

where T is the absolute temperature, r is the hydrodynamic radius of the sphericalmolecule, Z is the viscosity of the solvent, and kB is the Boltzmann constant. The
molecular weights (MWs) of apo- and haem-bound PGRMC1 proteins were
estimated from a relationship between r and MWs. MWs of apo and haem-bound
PGRMC1 proteins were obtained from the linear-ﬁtting of measured r values forthe reference proteins with known MWs according to the following equation:
MW ¼ 1.2864 (r3) þ 8.0411.
UV-visible absorption spectrometry and haem titration analysis. UV-visible
absorption spectra of the protein were recorded with a V-660 (Jasco) spectrophotometer at room temperature. Haem binding was tracked by difference
spectroscopy in the Soret region of the UV-visible spectrum. Successive aliquotsof 0.5 mmol l � 1 haemin in N,N-dimethylformamide were added to both thesample cuvette, which contained 10 mmol l � 1 apo-PGRMC1 (a.a.44–195), and thereference cuvette. Spectra were recorded 3 min after the addition of each haem
aliquot. The absorbance difference at 400 nm was plotted as a function of haem
concentration, and the dissociation constant (Kd) was calculated using a quadraticbinding equation.
Gel ﬁltration chromatography. Recombinant PGRMC1 (a.a.44–195) (10 mg) wt,
Y113F or C129S mutant, treated with 5 mmol l � 1 sodium dithionite and/or CO gas
or left untreated, was separated on a Superdex 200 column equilibrated in buffer
containing 20 mmol l � 1 Tris-HCl (pH 7.5) and 100 mmol l � 1 NaCl using a
SMART system (GE Healthcare). To prepare the reducing conditions for ferrous
haem proteins, the running buffer was deaerated by boiling and saturating itwith argon gas according to modiﬁed versions of previously reported methods64,65.
Namely, immediately after adding dithionite to give a ﬁnal concentration of
5 mmol l � 1, the buffer was equilibrated into the column. The SMART system wassealed with gas-tight taping to maintain anaerobic conditions. Separations ofproteins were completed within 1 h. Protein samples were also prepared in the
deaerated solution and treated with dithionite at 5 mmol l � 1 and/or CO gas, rightbefore being injected into the column. Fractions were then subjected to SDS-PAGE
under ambient conditions and visualized by silver staining. The size of proteins wasestimated using molecular mass markers (thyroglobin, 669 kDa; catalase, 232 kDa;
aldolase, 150 kDa; bovine albumin, 66 kDa and b-amylase, 20 kDa). Results
showing that the molecular size of PGRMC1 became smaller in CO-treatedconditions (Fig. 3) were collected B60 min after the start of experiments. The
stability of CO-binding to PGRMC1 was examined with UV-visible absorption
spectra to chase temporal alterations for 2 h, as shown in Supplementary Fig. 14.
In vitro binding assays. For in vitro binding assays, EGFR protein was obtained
from ENZO (BML-SE116) as full length protein isolated from human A431 cells.
Human CYP1A2, CYP3A4 proteins puriﬁed as a microsomal formulation
containing cytochrome b5 and cytochrome P450 reductase were obtained from
Sigma (C1561 and C4982, respectively). Proteins for human CYP1A2, CYP3A4 or
EGFR (1 mg) were incubated with 10 mg of FLAG-PGRMC1 (a.a.44–195) treatedwith or without 50 mmol l � 1 haemin in 500 ml of binding buffer containing
20 mmol l � 1 HEPES-NaOH (pH 7.9), 100 mmol l � 1 NaCl, 0.2 mmol l � 1 EDTA,
10% glycerol and 0.1% NP40 for 60 min at room temperature. 5 mmol l � 1 sodium
dithionite was added to produce the reducing conditions speciﬁed in the
aforementioned methods, and the effects of CORM3 or RuCl3 at 10 mmol l � 1 were
examined. Then, 10 ml of equilibrated anti-FLAG (M2) agarose was added to the
mixture, which was then incubated for 60 min at room temperature. Boundproteins were washed three times with 200 ml of binding buffer and eluted with
10 ml of 2 mg ml � 1 FLAG peptide. The eluates were subjected to SDS-PAGE
and visualized by Western blotting using antibodies against CYP1A2, CYP3A4(Santa Cruz: sc-30085 and sc-53850, respectively), FLAG and EGFR (Cell signaling:#2232S).
Cell culture analyses. The human colon cancer cell line HCT116 and humanhepatoma cell line HuH7 were maintained in DMEM medium containing 10%
FCS. To generate a stable PGRMC1 knockdown cell line, lentivirus vectors
encoding a control or PGRMC1 targeting shRNA sequence were transfected into
293FT cells. The lentivirus was prepared according to the manufacturer’s
instructions (Invitrogen). HCT116 and HuH7 cells were infected with thelentivirus, and a stable cell line was selected by maintaining the cells in medium
containing 10 mg ml � 1 blasticidin (Invitrogen) for 1 week.
For co-immunoprecipitation assay, the expression vector of FLAG-PGRMC1 oran empty vector into HCT116 by using a transfection reagent Lipofectamine 2000
(Invitrogen). Cells were incubated with or without 250 m mol l � 1 succinylacetone(SA) for 48 h, and the cells were then lysed with NP40 lysis buffer (20 mmol l � 1
Tris-HCl (pH 7.5), 150 mmol l � 1 NaCl, 1% NP40). The lysates were incubatedwith 10 ml of equilibrated anti-FLAG (M2) agarose for 60 min at room temperature.
Bound proteins were washed three times, and were subjected to SDS-PAGE andvisualized by Western blotting using antibodies against PGRMC1 (NOVUS:
NBP1–83220) and EGFR.
For analysis of EGFR signaling, cells were incubated overnight with serumdeprived medium, and then 100 ng ml � 1 EGF was added for 5 min. Cells werelysed with RIPA buffer, and the lysates were subjected to SDS-PAGE and visualizedby Western blotting using antibodies against PGRMC1, EGFR, phospho-Y1068
EGFR (Cell signaling: #2234S), AKT (Cell signaling: #9272S), phospho-S473AKT(Cell signaling: #4060S), ERK (Cell signaling: #4695S) and phospho-T185 Y187
ERK (Invitrogen: 44680 G).
To analyse proliferation of HCT116 cells, Lipofectamine 2000 (Invitrogen) wasused to transfect the shRNA-resistant expression vector of FLAG-PGRMC1 or an
empty vector into HCT116 control or PGRMC1-knockdown cells. After 24 h, thecells were seeded and incubated for 12 h on a 96-well plate, after which erlotinib or
doxorubicin was added for 24 h. Cell viability was determined by using an MTT
assay kit (Millipore) according to the manufacturer’s instructions.
For analysis of spheroid formation of HCT116 cells, the shRNA-resistant
expression vector of FLAG-PGRMC1 or an empty vector was transfected as
described above. After 24 h, cells were seeded at 1 � 104 cells per well onto a
96-well spheroid culture plate (NanoCulture plate with a microsquare pattern,
SCIVAX Corp.) and incubated for three days. The size of individual spheroid wasdetermined by measuring their optical areas using Image-J and by calculating theapparent radius (r) to estimate their apparent volume (v) according to thefollowing formula: v ¼ 4/3 � pr3.
Measurements of intracellular haem concentrations. To measure protohaem(haem b) concentrations, LC-UV and LC-MS analyses were performed forquantiﬁcation and molecular identiﬁcation, respectively. Brieﬂy, HCT116 cells(1 � 107 cells) were treated with vehicle or 250 mmol l � 1 SA for 48 h. Aftercentrifugation, haem b was extracted from cell pellets twice by adding acetone
containing 30% formate, followed by a 5 min sonication and centrifugation. The
supernatant was collected, and the solvent was evaporated. The dried residues werere-dissolved in acetonitrile containing 0.2% formate and subjected to a LCMS-8030
system equipped with photodiode array (PDA) detector (SPD-20A) (Shimadzu
Corporation, Kyoto, Japan). Haem b was detected by monitoring the absorption at
400 nm. Its identity was conﬁrmed by simultaneous mass spectrometric analysis
at m/z 616.
Analyses of doxorubicin metabolism. HCT116 cells (5 � 106 cells/10 cm dish)were cultured for 2 days, after which the cells were cultured in the presence of
0.3 mmol l � 1 doxorubicin overnight. The cells were lysed with methanol containing
internal standard compounds, and then water-soluble fraction was separated byliquid-liquid extraction (chloroform: methanol: water ¼ 1: 2: 1). The amountsof doxorubicin and its metabolites were quantiﬁed using LC-MS/MS. Brieﬂy,a triple-quadrupole mass spectrometer equipped with an electrospray ionization(ESI) ion source (LCMS-8030; Shimadzu Corporation) was used in the positive-ESI
and multiple reaction monitoring modes. The samples were resolved on an
ACQUITY UPLC BEH C18 column (100 � 2.1 mm i.d., 1.7 mm particle) using
water and acetonitrile as mobile phases A and B, respectively, at a ﬂow rate of
0.15 ml min � 1 and a column temperature of 40 C. Ion transitions from m/z 544 tom/z 130 and from m/z 546 to m/z 399 for doxorubicin and doxorubicinol,
respectively, were monitored for their quantiﬁcation.
Xenograft implantation of HCT116 cells. All the protocols for animal
experiments in this study were approved by the Experimental Animal Committeeof Keio University School of Medicine (the approved number; 08037-(7)). A modelof liver metastases of human colon cancer was prepared according to our previous
methods with minor modiﬁcations7,30. Brieﬂy, HCT116 cells transfected with thecDNA of Venus (1 � 106 cells/mice), a highly sensitive ﬂuorescent protein on tissue
slice sections,66 were transplanted into the spleens of 10-week-old male NOG mice.
Ten days after transplantation, the animals were anesthetized with sevoﬂurane(Maruishi Pharmaceutical), and their livers were removed, embedded in super-

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cryoembedding medium and frozen quickly in liquid nitrogen. The frozen tissues
were sliced with a cryostat (Leica CM1900) at a thickness of 5 mm, andhaematoxylin-eosin staining was performed. Fluorescent microscopy (Keyence:
BIOREVO, BZ-9000) was used to observe Venus ﬂuorescence. Percentages ofcross-sectional areas showing metastatic tumours were calculated by Image-J
as previously described7.

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Acknowledgements
X-ray crystallographic data was collected at the BL-41XU of SPring-8 with the approvalof the Japan Synchrotron Radiation Research Institute (JASRI) (proposal no. 2011B1229,
2012A1184 and 2012B1253). This work was supported by the Japan Science andTechnology Agency (JST), Exploratory Research for Advanced Technology (ERATO)
Suematsu Gas Biology Project (to M.S.). The present afﬁliation of M.S. is President,
Japan Agency for Medical Research and Development (AMED). Mass spectrometry
infrastructure in this work was partly supported by AMED-CREST (to Y.K.), andPlatform for Drug Discovery, Informatics, and Structural Life Science from the ministryof Education, Culture, Sports, Science and Technology, Japan (T.K.).

Author contributions
Y.K., T.K. and M.S. conceived the project; Y.K., T.N. and M.S. designed experiments andwrote the manuscript; Y.K., T.N. and T.S. performed X-ray crystallography. I.K., T.Y. and
Y.S. performed cell biology experiments. E.H. and K.S. contributed to NMR experiments.
E.K., M.N. and S.U. performed ESI-MS and SV-AUC analyses. M.O. and K.M. performed
xenograft experiments in vivo. A.Y., T.U. and K.I. contributed to Raman spectrometricanalyses. Y.Y., H.H. and S.I. contributed to consultations based on their expertise.

Additional information
Accession code: Structural information on PGRMC1 is available from the Protein Data
Bank under accession code 4X8Y.
Supplementary Information accompanies this paper at http://www.nature.com/
naturecommunications
Competing ﬁnancial interests: The authors declare no competing ﬁnancial interests.
Reprints and permission information is available online at http://npg.nature.com/
reprintsandpermissions/
How to cite this article: Kabe, Y. et al. Haem-dependent dimerization of
PGRMC1/sigma-2 receptor facilitates cancer proliferation and chemoresistance. Nat.
Commun. 7:11030 doi: 10.1038/ncomms11030 (2016).
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INTERNATIONAL JOURNAL OF MOLECULAR MEDICINE 37: 1611-1618, 2016

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HIDETOSHI FUJITA1,2, SATOKO ARATANI1,2, RYOUJI FUJII3, YOSHIHISA YAMANO3, NAOKO YAGISHITA3,NATSUMI ARAYA3, TOSHIHIKO IZUMI3, KAZUKO AZAKAMI3, DAISUKE HASEGAWA3,
KUSUKI NISHIOKA1 and TOSHIHIRO NAKAJIMA1-7
1Institute of Medical Science and 2Department of Future Medical Science, Tokyo Medical University, Tokyo 160-8402;
3
Institute of Medical Science, St. Marianna University School of Medicine, Kawasaki, Kanagawa 216-8511;
4
Integrated Gene Editing Section (iGES) and 5Medical Research Center, Tokyo Medical University Hospital,
Tokyo 160-0023; 6Department of Biomedical Engineering, Osaka Institute of Technology,
Osaka 535-8585; 7Bayside Misato Medical Center, Kochi, Kochi 781-0112, Japan
Received November 16, 2015; Accepted April 6, 2016
DOI: 10.3892/ijmm.2016.2566

Abstract. The nuclear factor-κ B (NF-κ B) transcription factor
family members control various biological processes, such as
apoptosis and proliferation. The endoplasmic reticulum (ER)has emerged as a major site of cellular homeostasis regulation.
The accumulation of misfolded protein in the ER causes stressand ER stress-induced NF-κ B activation to protect cells from
apoptosis. In this study, we found a putative ER stress-response
element (ERSE) on the promoter of mitochondrial ubiquitin
ligase activator of NF-κ B (MULAN), and that MULAN
expression was upregulated by ER stress. MULAN specifically
activated NF-κ B dependent gene expression in an E3 ligase
activity-dependent manner. The ectopic expression of MULAN
induced the nuclear translocation of endogenous p65 and thedegradation of Iκ B. Binding assay revealed that MULAN
was associated with transforming growth factor β-activatedkinase (TAK1). The knockdown of MULAN using siRNAinhibited the activation of NF-κB in the cells subjected to ER
stress. The findings of our study indicate that MULAN is an
E3 ligase that regulates NF-κB activation to protect cells from
ER stress-induced apoptosis.

Correspondence to: Professor Toshihiro Nakajima, Instituteof Medical Science, Tokyo Medical University, 6-1-1 Shinjuku,
Shinjuku-ku, Tokyo 160-8402, Japan
E-mail: marlin@tokyo-med.ac.jp

Abbreviations: TAK1, transforming growth factor β-activatedkinase; MULAN, mitochondrial ubiquitin ligase activator of nuclear
factor-κ B; ERSE, endoplasmic reticulum stress-response element;
TRAF, tumor necrosis factor receptor-associated factor; Iκ Bα,inhibitor of κ Bα; IKK, Iκ B kinase

Key words: endoplasmic reticulum stress, nuclear factor-κB

transcription, mitochondrial ubiquitin ligase activator of nuclear
factor-κ B, E3 ubiquitin ligase, transforming growth factor β-activatedkinaseIntroduction
Nuclear factor-κ B (NF-κ B) is a dimeric transcription factor
that controls the expression of genes involved in multiple
processes, including immunity, inflammation, apoptosis andcell cycle progression (1). Briefly, in resting cells, NF-κB is kept
inactive in the cytoplasm by its association with the inhibitorof κBα (IκBα). Stimulating cells with an agonist, such as tumor
necrosis factor α (TNFα) or interleukin (IL)-1β) activates theIκB kinase (IKK) complex, which is composed of two catalytic
subunits, IKKα and IKKβ, and a regulatory subunit, NEMO. Thissignal-induced phosphorylation targets IκBα for Lys48-linked
polyubiquitination and subsequent degradation by the ubiquitinproteasome system (2,3). In this way, NF-κ B is released fromIκBα, and translocates into the nucleus to trigger the expressionof its target genes. Recent studies have revealed several enzymes
involved in the ubiquitination and deubiquitination of signaling
proteins that mediate IKK activation through a degradationindependent mechanism (4). TNFR-associated factor (TRAF)
proteins, ubiquitin E3 ligases (5) have a pivotal role in signaling
pathways that are involved in the activation of NF-κB by manycell-surface receptors, including the TNFR superfamily, the IL-1
receptor (IL-1R) and Toll-like receptors (TLRs) (6). Following
ligand binding to IL-1R or TLRs, TRAF6 is recruited to thereceptor complexes and forms oligomers. TRAF6 oligomerization activates its ligase activity, leading to the Lys63-linked
polyubiquitination of target proteins, including TRAF6 andNEMO. It is also known that receptor-interacting protein (RIP)
is ubiquitinated by TRAF2 through the Lys63 linkage immediately following TNFα stimulation (7). Both the Lys63-linked
polyubiquitylation of TRAF6 and RIP recruits transforming
growth factor β (TGFβ)-activated kinase (TAK1) and two
adaptor proteins, TAK1-binding protein (TAB)1 and TAB2.
TAB2 contains a highly conserved novel zinc-finger domainthat binds preferentially to polyubiquitin chains that are linked
by Lys63 (8). TAB2-associated TAK1 phosphorylates IKKβ at
two serine residues in the activation loop, thereby activating the
IKK complex (9,10).

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Ubiquitination has been shown to play important roles in
the regulation of NF-κB signaling pathways, as well as in endoplasmic reticulum (ER) stress. There are two branches of ER
quality control that address the situation. One is the unfolded
protein response (UPR) pathway (11). The activation of UPR
results in attenuation of protein synthesis, and upregulation ofgenes encoding chaperones that facilitate the protein folding
process in the ER. Thus, UPR reduces the accumulation andaggregation of malfolded proteins, giving the cell the possibility of correcting the environment inside the ER (11,12). A
second major response is known as the ER-associated degradation (ERAD) pathway to reduce the misfolded proteins in theER, which involves retro-translocation, polyubiquitination anddegradation in the cytosol through the 26S proteasome (13,14).
Synoviolin, a representative ERAD-associated E3 ubiquitin
ligase, is regulated by ER stress through the ER stress-response
element (ERSE) which is a regulatory element located in manyof these ER stress-response genes (15). Synoviolin, a mammalian homolog of Hrd1p/Der3p, was identified from the cDNAof rheumatoid synovial cells and is involved in the developmentof obesity, rheumatoid arthritis, fibrosis, limb girdle muscular
dystrophy and liver cirrhosis (16-20).
In the present study, we searched for E3 ligases which areassociated with ER stress and regulate NF-κ B signaling. We
identified an E3 ligase, mitochondrial ubiquitin ligase activator of
NF-κB (MULAN). Previous studies have reported that MULAN
is one of the NF-κB-activating genes by large scale screening (21),and regulates mitochondrial trafficking and morphology (22). In
addition, mitochondrial hyperfusion promotes NF-κB activation
in a TAK1- and IKK-dependent manner via MULAN (23). In
this study, we found that the expression of MULAN is upregulated by ER stress. MULAN associates with TAK1 and activates
NF-κB signaling in an E3 ligase activity-dependent manner. The
knockdown of MULAN by siRNA resulted in the downregulation of NF-κB signaling in cells subjected to ER stress. Our
results suggest that MULAN is an E3 ligase which regulates
NF-κB signaling under conditions of ER stress.
Materials and methods
Plasmids. The coding sequences for full-length MULAN
genes were PCR-amplified (primers, 5'-acagaattcATGGAGAGCGGAGGGC-3' and 5'-tgtgtcgacGCTGTTGTACAGGGGT
ATCA-3') from reversed-transcribed HeLa cell RNA. PCR
products were digested with EcoRI and SalI, and ligated into
pGEX-5X-1 (Amersham Pharmacia Biotechnology, Piscataway,
NJ, USA) for in vitro ubiquitin (Ub) assays. For expression in
mammalian cells, the fragments of MULAN and mutants
were inserted into pcDNA3 hemagglutinin antigen (HA) or
pcDNA3 FLAG, which were constructed by inserting the
HA sequence or FLAG sequence into pcDNA3 (Invitrogen,
Carlsbad, CA, USA), respectively. Fragments of deletion mutants,
termed MULAN-A, MULAN-B, MULAN-N and MULAN-C
were obtained by PCR amplification. Fragments of additionaldeletion mutants, termed MULAN delta transmembrane (dTM)
and MULAN 3C were generated by PCR-based methods. The
sequences of all plasmids generated by PCR were confirmed bysequence analysis. The IKKβ, IKKβ K44A, TRAF6, TRAF6
dominant negative (DN; delta RING), TRAF2, TRAF2 DN(delta RING) and RIP1 plasmids used in this study were obtainedby a PCR-based method. The ERSE-Luc reporter plasmid was
also obtained by a PCR-based method and possesses 4 copies
of synoviolin ERSE. The TAK1, TAK1 K63W and TAB1
plasmids were a kindly gif from Dr Kunihiro Matsumoto. The
pcDNA3 HA-Ub expression plasmid, NF-κ B-Luc reporter
plasmids, Som-Luc and CMV- β -gal plasmid have been
described in previous studies (16,24-26).
Cell culture and antibodies. HeLa cells and 293 cells [provided
by RIKEN BRC through the National Bio-Resource Projectof MEXT (Ibaraki, Japan)] were maintained in Dulbecco's
modified Eagle's medium supplemented with 10% fetal bovine
serum plus penicillin and streptomycin at 37˚C in 5% CO2.
The following antibodies were used: anti-FLAG (M2; F3165)
and anti-β-actin (A5441) from Sigma Chemical Co. (St. Louis,
MO, USA), anti-HA [12CA5 (1583816) and 3F10 (1867423);
Boehringer Mannheim GmbH, Mannheim, Germany], anti-IκB(sc-203) and anti-p65 (sc-372) were purchased from Santa Cruz
Biotechnology, Inc., Santa Cruz, CA, USA.
Induction of ER stress. To induce ER stress, the cells were
stimulated with tunicamycin at concentrations of 2 µg/ml for
0, 3, 6 and 9 h and total RNA was collected following stimulation. ER stress was also induced by thapsigargin in luciferase
assay (Fig. 5). The cells were stimulated with thapsigargin atconcentrations of 2 µM for 4 h.
TNFα and IL-1β treatment. TNFα is known to activate NF-κB
signaling by promoting the degradation of IκBα and the nuclear
translocation of p65. We treated the cells with TNFα at 10 nM
for 4 h as a positive control (Fig. 4) and an inducer of NF-κ B
signaling (Fig. 5). IL-1β was also used at 10 ng/ml for 4 h.
Transfection and immunofluorescence. For immunofluorescence experiments, trypsinized cells were seeded on a coverglass and incubated for 24 h prior to transfection. Transfection
was performed using Lipofectamine 2000 (Invitrogen) with
0.5 µg of MULAN expression plasmid, according to the manufacturer's instructions. The cells were incubated for a further
12 h after transfection, washed 3 times with phosphate-bufferedsaline (PBS), and fixed with 3.7% formaldehyde in PBS for
30 min, followed by permeabilization with 0.1% Triton X-100
for 30 min. The cells then were incubated with the primary
antibody followed by staining with Alexa Fluor 488 antimouse second antibody or Alexa Fluor 594 anti-rabbit second
antibody (Molecular Probes, Eugene, OR, USA). Samples were
scanned with a Zeiss LSM 510 confocal laser scanning microscopy (Carl Zeiss Microscopy, Jena, Germany). Co-localization
scatter diagrams were generated using LSM 510 software (27).
Transient transfection assay. Transient transfection assays were
performed using the 293 cells, as previously described (24).
The cells were lysed with cell lysis buffer (Toyo Ink, Tokyo,
Japan) 24 h following transfection and luciferase activities were
measured. The recorded activity was normalized to the β-gal
activity from CMV-β-gal. All experiments were performedin triplicate. The 293 cells were transfected with 100 ng of
NF-κ B-Luc, 50 ng of CMV-β -gal control plasmid, and 0,
50 and 100 ng of MULAN or mutants, and/or DN-mutants
plasmid, or truncated forms of MULAN expression vector. To-231-

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Figure 1. Induction of mitochondrial ubiquitin ligase activator of nuclear factor-κ B (MULAN) expression by endoplasmic reticulum (ER) stress. (A) Nucleotide
sequence of the 5'-region of the MULAN gene. The numbers on the right indicate the nucleotide position with respect to the transcription start site (+1). Putative
ER stress-response elements (ERSEs) are boxed. (B) HeLa cells were treated with 2 µg/ml tunicamycin for the indicated periods of time. Following stimulation,
total RNA was collected, and RT-PCR and real-time PCR were performed. The signals were calculated by normalization to human GAPDH. Data were analyzed
using the Student's t-test and represent the means ± SD (n=3 experiments). *P<0.05.

ensure an equal amount of DNA, empty plasmids were addedin each transfection.
Immunoprecipitation assay. The HeLa cells were transfectedwith HA TAK1, HA TAB1, HA RIP1, HA IKKβ, and/or
MULAN/FLAG expression vector. After 12 h of transfection, thecells were lysed in 1 ml of lysis buffer (20 mM HEPES, pH 7.5,
100 mM NaCl, 1 mM EDTA, 1 mM DTT, 0.1% NP-40, 5% glycerol and protease inhibitors). The lysates were mixed with 1 µgof anti-FLAG antibody (M2) conjugated to protein G-sepharose
beads (GE Healthcare Bio-Sciences, Uppsala, Sweden). Following
4 h of incubation at 4˚C, the beads were washed 3 times with lysis
buffer. Bound proteins were fractionated by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) andexamined by western blot analysis.
Western blot analysis. The protein extracts were resolved by
10% SDS-PAGE, transferred onto a PVDF membrane, andincubated with primary antibodies [anti-FLAG M2 (F3165),
anti- β -actin (A5441), anti-HA 3F10 (1867423), anti-Iκ B(sc-203) and anti-p65 (sc-372)] followed by horseradish peroxidase-conjugated secondary antibodies. The antigen-antibody
complexes were visualized using an ECL detection system(Promega, Madison, WI, USA).
RNA interference assay, RT-PCR and real-time PCR. siRNAs(Stealth RNAi) for MULAN and GFP were purchased from
Invitrogen. The sequence of the MULAN siRNA-1 was
5'-UUUCCACAAACUGGCUGUUAAGCGU-3', and that ofcontrol siRNAwas 5'-AAGAAGUCGUGCUGCUUCAUGUGGU-3'. Transfection with siRNAs was carried out using
Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. Isogen was used for total RNA isolation

and RT-PCR was performed with ReverTra Ace (Toyobo,
Tokyo, Japan) according to the manufacturer's instructions.
Total RNA (1 µg) was used for cDNA preparation. Real-time
PCR was carried out using the Universal ProbeLibrary system(Roche Diagnostic, Mannheim, Germany). Signals from eachsample were normalized to values obtained for the humanglyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene,which was assayed simultaneously with the experimental
samples. Analyses were performed with a sequence detector(model 7500; Applied Biosystems, Foster City, CA, USA).
Statistical analysis. The non-paired Student's t-test was used
to analyze mean differences. A P-value <0.05 was considered
to indicate a statistically significant difference.
Results
MULAN is regulated by ER stress. Recent studies have revealed
that ER stress induces the activation of NF-κ B (36,37). It is
well known that ubiquitination is critical in both the response
to ER stress and NF-κ B activation. Therefore, we wished todetermine whether E3 ligases, which are related to ER stress,
regulate NF-κB signaling. We found the putative ERSE locatedat positions -118 to -100 on the promoter region of MULAN,
which is one of the NF-κB-activating genes (Fig. 1A). First, weanalyzed the mRNA expression of MULAN in cells subjected
to ER stress by real-time PCR. MULAN mRNA expression
was increased by the induction of ER stress by stimulation withtunicamycin (Fig. 1B). These results suggest that the expressionof MULAN is regulated by ER stress.
MULAN activates NF- κ B gene expression in an E3 ligase
activity-dependent manner. To determine whether MULAN

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Figure 2. Mitochondrial ubiquitin ligase activator of nuclear factor-κ B (MULAN) activates nuclear factor-κ B (NF-κ B)-dependent transcription via E3 ligase
activity and the transmembrane domain (TM). (A) Reporter assays of 293 cells using NF-κ B-Luc (left panel), Som-Luc (middle panel), ER stress-response
element (ERSE)-Luc (right panel). The luciferase activity of cells transfected with pcDNA3 HA vector alone was designated 1. Data were analyzed with theStudent's t-test and represent the mean ± SD (n=3). *P<0.05. (B and D) HEK-293 cells were transfected with NF-κB-Luc reporter, and expression vectors of wildtype MULAN or MULAN mutants. The luciferase activity of cells transfected with pcDNA3 HA vector alone was designated as 1. Data were analyzed using theStudent's t-test and represent the means ± SD (n=3 experiments). *P<0.05. (C) Schematic representation of deletion mutants of MULAN. RING, RING finger domain.

specifically activates NF-κ B-Luc, the luciferase reporter
plasmids were co-transfected with MULAN/HA-expressing
plasmid into the 293 cells. Twenty-four hours after transfection,
the luciferase activities were measured. MULAN enhanced thereporter activity of NF-κ B-Luc, but not that of Som-Luc and
ERSE-Luc (Fig. 2A). To further examine the mechanism of
induction of NF-κ B-dependent gene expression by MULAN,
we utilized several mutants of MULAN and performed reporter
assays. MULAN increased NF-κB-dependent gene expression
by 21.9-fold in a dose-dependent manner (Fig. 2B). By contrast,
dTM, which lacks the second transmembrane domain, and
MULAN 3C, which includes a cysteine to serine substitution in the RING finger domain and lacks E3 ubiquitin ligase
activity, did not stimulate the reporter activity. To determine
which domains contributes to the activation of NF-κ B-Luc,several truncations of MULAN were expressed as HA fusion
proteins (Fig. 2C) and examined by reporter assay. MULAN
induced the reporter activity 16.4-fold. Among these 4 mutants,
only MULAN-C markedly activated transcription 33.4fold, whereas MULAN-A, MULAN-B and MULAN-N didnot (Fig. 2D). These results suggest that both the transmembrane domain (TM) and RING finger domain are important for
the MULAN-dependent activation of NF-κB.
The mechanism of MULAN-dependent NF-κB activation. To
further investigate the target of MULAN, we utilized dominantnegative (DN) mutants of major factors in NF-κB signals, such as
IKKβ, TAK1, TRAF6 and TRAF2. First, we examined the inhibitory effects of these DN mutants in TNFα-treated cells, and we

found that they suppressed the reporter activity of NF-κB-Luc ina dose-dependent manner. As shown in Fig. 3A, the introductionof DN-IKKβ plasmids markedly inhibited the MULAN-induced
activation of NF-κB in a dose-dependent manner. In addition,transfection with DN-TAK1 also yielded the same result (Fig. 3B).
Conversely, DN-TRAF2 and DN-TRAF6 failed to suppress the
MULAN-dependent activation of NF-κB (Fig. 3C and D). These
results suggest that MULAN plays a role in NF-κB activation
upstream of TAK1 and downstream of TRAF2 and TRAF6.
These results prompted us to search the interactants of MULAN.
To determine whether MULAN interacts with TAK1 TAB1,
RIP1 and IKKβ, we transiently transfected HeLa cells withHA TAK1, HA TAB1, HA RIP1, HA IKKβ expression plasmids
and/or MULAN/FLAG expression plasmids. We performed an
immunoprecipitation assay following western blot analysis. As
shown in Fig. 3E (upper panel), HA TAK1 was detected in the
immunoprecipitate with anti-FLAG antibody, but HA TAB1,HA RIP1 and HA IKKβ were not. These results suggest that
TAK1 may be one of the targets of MULAN through which it
induces the activation of NF-κB.
MULAN activates endogenous NF-κB signals. It is well known
that the NF-κ B signaling pathway can be activated throughvarious processes; however, the release of cytoplasmic NF-κB
proteins from IκB proteins and nuclear translocation are pivotal
for signal transduction (28). To determine whether MULAN
induces endogenous NF-κB activation, we observed the translocation of endogenous p65 into the nucleus. MULAN/FLAG or
MULAN 3C/FLAG were transiently transfected into the HeLa-233-

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Figure 3. Mitochondrial ubiquitin ligase activator of nuclear factor-κ B (MULAN) associates with TGFβ-activated kinase (TAK1). (A-D) 293 cells were transfected with (A) NF-κ B-Luc reporter, MULAN expression plasmids and/or Iκ B kinase (IKK) K44A expression plasmids, (B) TAK1 K63W expression plasmids,(C) TNFR-associated factor 6 (TRAF6) DN expression plasmids, or (D) TRAF2 DN expression plasmids. The luciferase activity of cells transfected with empty
vector alone was designated as 1. Data were analyzed using the Student's t-test and represent the means ± SD (n=3 experiments). *P<0.05. (E) Interaction of
MULAN with TAK1 in vivo. HeLa cells were transfected with HA TAK1, HA TAK1-binding proteins 1 (TAB1), HA RIP1, HA IKKβ, and/or MULAN/FLAG
expression plasmids. After 12 h, the cells were harvested and lysates were immunoprecipitated with anti-FLAG antibody and then immunoblotted with anti-HA
antibody. The asterisks indicates the immunoprecipitated IgG heavy chain.

cells. The nuclear localization of endogenous p65 was only
observed in the cells that expressed MULAN/FLAG (Fig. 4A).
To further confirm the nuclear localization of p65, we usednuclear extraction and western blot analysis with anti-p65

specific antibody. In the cells transfected with the pcDNA3 HA
vector or MULAN 3C/HA expression vector, a small amountof p65 was found in the nuclear extracts. On the other hand,stimulation with TNFα and the expression of MULAN led

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Figure 4. Mitochondrial ubiquitin ligase activator of nuclear factor-κ B (MULAN) induces endogenous nuclear factor-κ B (NF-κ B) activation. (A) Intracellular
localization of MULAN and endogenous p65. HeLa cells transfected with the plasmids of MULAN/FLAG or MULAN 3C/FLAG, which includes a cysteine toserine substitution in the RING finger domain, were fixed at 12 h post-transfection and stained with antibody specific to p65. Bar, 20 µm. (B and C) HeLa cells
were transfected with MULAN/HA or MULAN 3C/HA. After 12 h of transfection, the cells were harvested. (B) Nuclear extracts were immunoblotted with
anti-p65 antibody. (C) Whole cell lysates were immunoblotted with anti-Iκ B, anti-β-actin and anti-HA antibody.

Figure 5. Downregulation of mitochondrial ubiquitin ligase activator of nuclear factor-κ B (MULAN) leads to the reduced induction of NF-κ B activation in
endoplasmic reticulum (ER) stress-stimulated cells. (A) HeLa cells were transiently transfected with MULAN siRNA or control siRNA. Real-time PCR assays
were performed. The expression of GAPDH was used as an internal control. (B) HeLa cells were transiently transfected with MULAN siRNA or control
siRNA. After 72 h, NF-κ B-Luc reporter plasmids were transfected, and cells were further incubated 24 h. The cells were then stimulated with tumor necrosis
factor α (TNFα) (10 nM), or thapsigargin (2 µM) for 4 h. The luciferase activity of the cells transfected with empty vector alone was designated as 1. Data wereanalyzed using the Student's t-test and represent the means ± SD (n=3 experiments). *P<0.05.

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to an increase in p65 levels in the nucleus (Fig. 4B). We thenexamined the degradation of endogenous IκBα. We transfected
MULAN/HA or MULAN 3C/HA into the HeLa cells, andcell lysates were prepared 12 h following transfection. In
the cells transfected with pcDNA3 HA vector or MULAN
3C/HA, hardly any Iκ Bα degradation was observed, whereas
noticeable degradation was detected in the cells expressing
wild-type MULAN (Fig. 4C). These results indicate that
MULAN activates NF-κ B-dependent gene expression in an
enzymatic activity-dependent manner.
Knockdown of MUL A N induces a decrease in ER
stress-dependent NF-κB activity. To examine the upper signalof MULAN-dependent NF-κ B activation, we performed
reporter assay with siRNA against MULAN. To confirm
the effects of siRNAs, MULAN siRNA and control siRNA
were transiently transfected into the HeLa cells. After 48 h,
total RNA was isolated and the mRNA levels were measured
by real-time PCR. MULAN siRNA induced the downregulation of MULAN (Fig. 5A). We then performed a reporter
assay with NF-κ B-Luc. The cells stimulated with TNFα andtransfected with control siRNA exhibited an enhanced reporter
activity by 7.3-fold and the MULAN siRNA-transfected cells
exhibited a low reporter activity (Fig. 5B). We also observed
no difference in reporter activity when the cells were stimulated with IL-1β (data not shown). When the cells were treatedwith thapsigargin, a decrease in reporter activity of approximately 50% was observed in the MULAN siRNA-transfected
cells compared with the cells transfected with control siRNA.
These results indicate that the activation of NF-κB by MULAN
plays an important in cells subjected to ER stress.
Discussion
MULAN is regulated by ER stress. We found putative ERSE on
the promoter region of MULAN gene (Fig. 1). ERSE is foundin many of these ER stress-response genes and is regulated by
the ER responsive transcriptional factors, activating transcription factor 6 (ATF6) and X-box binding protein 1 (XBP1).
ERSE, with a consensus sequence of CCAAT-N9-CCACG, is a
cis-acting element that is necessary and sufficient for transcriptional induction of ER chaperone genes (29-31). The putative
ERSE of MULAN, CCAAT-N9-ACACC, differs slighlty fromthe consensus sequence of ERSE. Many ER stress-responsive
genes also have a different ERSE from a consensus sequence;for example, Bip/Grp78, CCAAT-N9-CCAAC and PDI,CCAGT-N9-ACAGC. In addition, the expression of MULAN
was induced by ER stress (Fig. 1). However, we cannot not
rule out possibility of the contribution of other elements on
the MULAN promoter. These results suggest that the putative
ERSE of MULAN may function as an ERSE and it is certainthat MULAN plays a role in the response to ER stress.
The mechanism of MULAN-induced NF- κ B activation.
Ubiquitination is important in NF-κ B activation. It has been
demonstrated that TRAF2, which is recruited to the TNFR
complex, ubiquitinates RIP, and polyubiquitinated RIP associateswith TNFR as well as with TAB2 (8,32,33). These studies suggestthat the polyubiquitination of RIP recruits the TAK1-TAB2
complex, which subsequently activates the IKK complex. It has

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been shown that TRAF6 recruited to the receptor complexes
polyubiquitinates these complexes, and polyubiquitinated
TRAF6 associates with the TAB2/TAB1/TAK1 complex (8). In
this study, we revealed that MULAN activated NF-κB dependenttranscription via E3 ubiquitin ligase activity. The activation of
NF-κB via MULAN was inhibited by IKK DN and TAK1 DN,
but not by TRAF2 and TRAF6. In addition, our binding assay
revealed that MULAN associated with TAK1. TAK1 has beenreported to be induced by polyubiquitination. TAK1 has Lys63linked ubiquitination sites and the polyubiquitination of TAK1
induces IK-dependent NF-κ B activation (34,35). Therefore, it
is possible that MULAN may induce the polyubiqutination of
TAK1 to activate NF-κB signaling.
MULAN-induced NF-κB activation in cells subjected to ER
stress. The ER stress-induced activation of NF-κ B has been
demonstrated to be involved in both the protection of cells from
ER stress-induced apoptosis (36) and in the induction of apoptosis (37). TRAF2 mediates the activation of both the c-Jun
N-terminal kinase (JNK)/stress-activated protein kinase (SAPK)
and the NF-κB pathways following ER stress (38,39). Therefore,
TRAF2 simultaneously mediates the activation of the NF-κ B
survival pathway and the pro-apoptotic JNK pathway, and thefate of the cell would be determined by the interplay between
these opposing signals. TAK1 also activates both the NF-κ B
survival pathway and the pro-apoptotic JNK pathway. The activation of TAK1 is induced by K63-linked polyubiquitination
reactions by TRAF2 or TRAF6 at the K158, K34 and K209
residues (35). Recently, Lys562 of TAK1 was identified as anovel Lys63-linked ubiquitination site. The accumulation ofpolyubiquitination at Lys562 induces IKK-dependent NF-κ B
activation, but not JNK/p38 pathway activation (34). In thisstudy, MULAN activated NF-κ B dependent gene expression,and the expression of MULAN was regulated by ER stress.
In addition, the MULAN-dependent NF-κ B activation was
TAK1-dependent and MULAN interacted with TAK1. We
thus hypothesized that MULAN may induce NF-κB activation
via TAK1 for cell survival following ER stress; MULAN may
activate NF-κB-dependent gene expression by the induction of
de novo MULAN by ER stress.
In conclusion, in the present study we provide evidence that
MULAN is an E3 ligase that mediates NF-κB-dependent gene
expression in cells subjected to ER stress. Further analysis of
MULAN will be helpful in order to broaden our understandingof the physiological significance of MULAN and of the mechanisms of NF-κB activation by MULAN.
Acknowledgements
We would like to thank Dr Kunihiro Matsumoto for kindly
providing the TAK1, TAK1 K63W and TAB1 expression
vectors. We would also like to thank Ms. Yukari Nakagawa
and Mrs. Sanae Shinkawa for their technical assistance andthe members of the Nakajima Laboratory for their helpfulcomments on our manuscript. This study was funded inpart by grants from the Naito Foundation, Inoue Foundationfor Science, Natural Science Scholarship Daiichi-Sankyo
Foundation of Life Science, Mitsubishi Tanabe Pharma
Corporation, Bureau of Social Welfare and Public Health,

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Academic contribution of Pfizer, Eisai, Asahi Kasei Pharma,
Abbvie, Santen, Takeda Science Foundation and AstraZeneca(R&D grant 2013). This study was supported in part by funds
provided through an MEXT-Supported program of the Strategic
Research Foundation at Private Universities (nos. S1411011
and 2014-2018) from the Ministry of Education, Culture,
Sports, Science and Technology of Japan. This study was also
supported in part by the Japan Society for the Promotion of
Science KAKENHI and Industry-university cooperation(BioMimetics Sympathies Inc., Tokyo, Japan).
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Contents lists available at ScienceDirect

Biochemical and Biophysical Research Communications
journal homepage: www.elsevier.com/locate/ybbrc

NAD(P)H quinone oxidoreductase 1 inhibits the proteasomal
degradation of homocysteine-induced endoplasmic reticulum protein
Tomoji Maeda a, *, Chiaki Tanabe-Fujimura a, Yu Fujita a, Chihiro Abe a,
Yoshino Nanakida a, Kun Zou a, Junjun Liu a, Shuyu Liu a, Toshihiro Nakajima b,
Hiroto Komano a, **ab

Department of Neuroscience, School of Pharmacy, Iwate Medical University, 2-1-1 Nishitokuta, Yahaba-Cho, Shiwagun, Iwate, 028-3603, Japan
Institute of Medical Science, Tokyo Medical University, 6-1-1 Shinjyuku, Shinjyuku, Tokyo, Tokyo, 160-8402, Japana r t i c l e i n f o

a b s t r a c t

Article history:
Received 31 March 2016
Accepted 12 April 2016
Available online 13 April 2016

Homocysteine-induced endoplasmic reticulum (ER) protein (Herp) is an ER stress-inducible key regulatory component of ER-associated degradation (ERAD) that has been implicated in insulin hypersecretion in diabetic mouse models. Herp expression is tightly regulated. Additionally, Herp is a highlylabile protein and interacts with various proteins, which are characteristic features of ubiquitinatedprotein. Previously, we reported that ubiquitination is not required for Herp degradation. In addition, we
found that the lysine residues of Herp (which are ubiquitinated by E3 ubiquitin ligase) are not sufﬁcient
for regulation of Herp degradation. In this study, we found that NAD(P)H quinone oxidoreductase 1(NQO1)-mediated targeting of Herp to the proteasome was involved in Herp degradation. In addition, we
found that Herp protein levels were markedly elevated in synoviolin-null cells. The E3 ubiquitin ligase
synoviolin is a central component of ERAD and is involved in the degradation of nuclear factor E2-related
factor-2 (Nrf2), which regulates cellular reactive oxygen species. Additionally, NQO1 is a target of Nrf2.
Thus, our ﬁndings indicated that NQO1 could stabilize Herp protein expression via indirect regulation ofsynoviolin.
© 2016 Elsevier Inc. All rights reserved.

Keywords:
Homocysteine-induced endoplasmic
reticulum protein
NAD(P)H quinone oxidoreductase 1
Degradation
Proteasome

1. Introduction
Homocysteine-induced endoplasmic reticulum (ER) protein(Herp) is strongly upregulated by the unfolded protein response(UPR). Herp has been shown to be involved in the ER-associateddegradation (ERAD) of nonglycosylated Bip substrates [1e4] andN-glycan-independent substrates [5] and to exhibit anti-apoptotic
effects in response to ER stress [3].
Herp is a short-lived protein with a half-life of approximately
2 h. The half-life of Herp is regulated by the ubiquitin proteasome* Corresponding author. Current address: Department of Pharmacology, Nihon
Pharmaceutical University, 10281 Komuro, Ina-machi, Kitaadachi-gun, Saitama,362-0806, Japan.
** Corresponding author. Department of Neuroscience, School of Pharmacy, Iwate
Medical University, 2-1-1 Nishitokuta, Yahaba-Cho, Shiwagun, Iwate, 028-3603
Japan.
E-mail addresses: t-maeda@nichiyaku.ac.jp (T. Maeda), hkomano@iwate-med.
ac.jp (H. Komano).

pathway via major E3 proteins, such as gp78 [6] or RNF5 [7]. On
the other hand, we have previously demonstrated that theubiquitin-like domain (ULD) of Herp plays a role in the regulationof intracellular Herp levels during ER stress [8], suggesting thepresence of ubiquitin-independent proteasome degradation
systems. Proteasome-mediated degradation occurs via two
mechanisms. In the conventional ubiquitin-dependent mechanism, polyubiquitinated proteins are targeted to the 26 S proteasome for degradation. Alternatively, a few proteins can bedegraded by the 20 S proteasome in a ubiquitin-independent
pathway [9,10]. The ubiquitination-independent 20 S proteasome pathway is associated with NAD(P)H quinone oxidoreductase 1 (NQO1), which is known to bind to and stabilize proteinssuch as p53 in order to prevent 20 S proteasomal degradation[11e14]. NQO1 is thought to interact with the 20 S proteasomeand may consequently block access of target proteins to thedegradation core.
Therefore, in this study, we sought to determine whether NQO1mediated ubiquitin-independent proteasomal degradation was
involved in Herp degradation. Our data demonstrated that NQO1

http://dx.doi.org/10.1016/j.bbrc.2016.04.057
0006-291X/© 2016 Elsevier Inc. All rights reserved.

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was involved in mediating the stability of Herp, providing important insights into the regulation of this essential protein.
2. Materials and methods

1277

treatment, cells were harvested and lysed in radio-immune precipitation assay (RIPA) buffer (150 mM NaCl, 10 mM TriseHCl [pH7.5], 1% Nonidet P-40, 0.1% sodium dodecyl sulfate [SDS], and 0.2%
sodium deoxycholate) containing a complete protease-inhibitor
mixture (Roche Applied Science).

2.1. Antibodies, reagents, and cell lines
2.4. Treatment of cells with dicoumarol
Afﬁnity-puriﬁed rabbit anti-Herp antibodies were prepared aspreviously described [1]. Mouse anti-b-actin, anti-FLAG, and antiNQO1 antibodies were purchased from Sigma (St. Louis, MO,
USA). Anti-a-tubulin antibodies were purchased from Santa Cruz
Biotechnology, Inc. (Santa Cruz, CA, USA). Additional commercial
reagents used in this study are as follows: cycloheximide (Wako
Pure Chemical Industries Ltd., Osaka, Japan), MG132, and dicoumarol (Sigma). HEK293 cells, synoviolin-null cells, and wild-type(wt) cells [15] were cultured in Dulbecco's modiﬁed Eagle's medium (DMEM; Wako) containing 10% fetal calf serum (Thermo
Scientiﬁc Molecular Biology, Rockford, IL, USA).
2.2. Plasmids and retroviral-mediated infection
pMX-Herp, pMX-DUb-Herp, pcDNA3.1-Herp-F (C-terminally
FLAG-tagged Herp), and pcDNA3.1-DUb-Herp-F (C-terminally
FLAG-tagged DUb-Herp) constructs were generated as previously
described [8]. pMX-Herp9KR was constructed by two-step polymerase chain reaction (PCR) mutagenesis through mutation of allnine lysine residues to arginine; these constructs were generated
by TaKaRa Bio Inc (Shiga, Japan; Fig. 1). The mouse sequence
encoding NQO1 was excised from the mouse NQO1 pCMV-SPORT6
vector (Thermo Scientiﬁc Molecular Biology) by double digestion
with EcoRI and XhoI and inserted into the pMX vector. Retroviralmediated infection was carried out according to publishedmethods [16].
2.3. Cycloheximide treatments
Cycloheximide-chase experiments were performed as previously described [8]. Cells were plated on 60 mm tissue culture
dishes (5 � 105 cells/dish) and cultured for 24 h. After 24 h,
cycloheximide (20 mg/mL) was added to the cultured cells. At
various time points (0, 2, 3, 4, and 6 h) after cycloheximide
(A)
Herp

1

Herp9KR

Cells were seeded in 60 mm tissue culture dishes (5 � 105 cells/dish), incubated overnight at 37 � C, treated with 200 or 400 mg/mL
dicoumarol at 37 � C for 4 h, and then harvested.
2.5. RNA interference
Synoviolin wt cells overexpressing Herp were seeded in 24-wellplates at a density of 6 � 104 cells per well and transfected with
NQO1 siRNA or negative control RNA using HiPerfect Transfection
Reagent (Qiagen Inc., Valencia, CA, USA) and DMEM. The target
sequence used in this study was 50 -AAGATTCGTTGTCTATAATTA-30(Qiagen). All Stars Negative Control siRNA Alexa Fluor 488 Labeled(RNAi Human/Mouse Starter Kit; Qiagen) was used as a negative
control. After 3 days of incubation, cells were collected, and Herp
levels were measured by western blotting.
2.6. Immunoprecipitation and immunoblotting
To detect the interaction between Herp and NQO1, HEK293 cells
transfected with pcDNA3.1 (mock), pcDNA3.1-Herp-F, or pcDNA3.1DUb-Herp-F were collected and solubilized on ice in RIPA buffer
containing a protease-inhibitor mixture. Lysates were immunoprecipitated overnight with primary antibodies or normal IgG(Santa Cruz Biotechnology, Inc.). Antibody-bound complexes wereisolated by incubation with protein G-Sepharose beads for 4 h andthen washed in RIPA buffer. The protein complexes were eluted in
2 � SDS sample buffer and analyzed by western blotting [17].
2.7. Densitometric analysis and statistical analysis
Band intensities from western blots were densitometricallyquantiﬁed using a luminescent image analyzer LAS-3000 (Fuji
Photo Film Co. Ltd.). Statistical analysis was performed with

ULD
89 93 100KKKKKK K K

375
K

RRRRR R R

R

R

(B)

Herp9KR

Herp
0

2

3

4

6

0

2

3

4

6
Herp
GFP

% remaining

100
80

391

Herp
Herp9KR6040
200Time (h)Fig. 1. Effects of lysine residues on Herp degradation. Herp and Herp9KR were transduced and expressed in synoviolin wt cells, together with green ﬂuorescent protein (GFP).
Cells were treated with 20 mg/mL cycloheximide and then harvested at the indicated times. Herp and Herp9KR were detected in RIPA-solubilized lysates (20 mg) by immunoblotting
using an anti-Herp antibody. The GFP in the lysates was used as an internal loading control. Herp levels were quantiﬁed from three independent experiments, as shown in the left
panel (right graph). The intensity of bands corresponding to Herp in panels B was densitometrically quantiﬁed using a luminescent image analyzer (LAS-3000). The Herp levels at
each time point were calculated as a percentage of the intensity at time 0.

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Student's t-tests.
3. Results
3.1. Effect of lysine residues on Herp degradation
To determine whether Herp degradation proceeded through theubiquitin-independent proteasomal pathway, we investigated thestability of Herp and Herp9KR, in which all nine lysine residues
were mutated to arginine (Fig. 1A) to prevent ubiquitination by E3
ubiquitin ligase. We then used immunoblotting to compare theintracellular degradation of Herp and Herp9KR following cycloheximide treatment (Fig. 1B). Our results indicated that Herp and
Herp9KR were degraded in up to 2 h (Fig. 1B). These results suggested the existence of another degradation pathway in addition to
the lysine-mediated ubiquitination-proteasome degradation
pathway.
3.2. Herp interacted with NQO1
Proteasome-mediated ubiquitin-independent degradation ismodulated via the 20 S proteasome pathway [18]. Therefore, weinvestigated whether the ubiquitin-independent proteasome
pathway may regulate Herp degradation via stabilization by NQO1,
which has been shown to protect several proteins, including p53,
p73 [18], elF4GI [13], and c-Fos [12], from degradation by the proteasome. In the molecular mechanism responsible for stabilization
by NQO1, NQO1 and the protected protein have been shown to exist
within the same complex. Therefore, we hypothesized that a HerpNQO1 complex would be present if Herp was protected by NQO1. To
test this, we attempted to immunoprecipitate this putative complex from HEK293 cells overexpressing Herp-FLAG, DUb-HerpFLAG, or vector only together with NQO1 using anti-FLAG antibodies, followed by detection of NQO1 levels by western blotting.
NQO1 was co-immunoprecipitated with Herp and DUb-Herp(Fig. 2), indicating that Herp and DUb-Herp could interact with
NQO1. This result also indicated that the Herp ULD did not containan NQO1-binding site.
3.3. Effects of NQO1 on the stability of Herp
Recent studies have shown that the transcription factor nuclear
factor E2-related factor-2 (Nrf2) can be degraded by synoviolin, aubiquitin E3 ligase. Nrf2 regulates cellular reactive oxygen species(ROS) levels, and NQO1 and glutamate-cysteine ligase modiﬁer
subunit (GCLM) are target genes of Nrf2 [19]. In fact, the absence insynoviolin in synoviolin-null cells induces the expression of Nrf2
and NQO1. Therefore, we investigated the expression level of Herpin synoviolin-null cells. As a result, endogenous Herp levels werehigher in synoviolin-null cells than in wt cells (Fig. 3A). Next, weinvestigated the effects of NQO1 overexpression and NQO siRNA on
Herp degradation. The level of endogenous Herp was higher in cells
overexpressing NQO1 than in control cells (Fig. 3B). In addition,
Herp levels were decreased by NQO1 siRNA in comparison withthat in the control (Fig. 3C). Downregulation of NQO1 was
conﬁrmed by western blotting (Fig. 3C). These results wereconsistent with the results of NQO1 overexpression. Taken
together, these data indicated that the NQO1-mediated proteasome
pathway regulated Herp degradation.
3.4. Effects of dicoumarol on Herp degradation
NQO1-dependent protection from proteasomal degradation canbe relieved by pharmacological inhibition of the enzyme. Upon
treatment of cells with dicoumarol, the proteins p53 and elF4GI aretargeted for destruction by the proteasome [18,13]. However, themolecular mechanisms underlying the effects of dicoumarol ontargeting proteins for degradation are not completely understood.
Following treatment with dicoumarol, we observed induction of
Herp degradation, whereas MG132, a proteasome inhibitor, blocked
Herp degradation (Fig. 4A). As a control, dicoumarol induced p53
degradation (Fig. S1). These results suggested that the ubiquitinindependent degradation of Herp was regulated by the proteasome pathway. In addition, to further investigate the effects of
dicoumarol on Herp degradation, we next analyzed the degradation of Herp over time after treatment with dicoumarol. Our data
demonstrated that Herp expression was reduced at 4 and 24 h after
treatment to 44.9% ± 4.2% (n ¼ 6, p < 0.001) and 35.1% ± 3.0% (n ¼ 6,
p < 0.001), respectively, but was enhanced at 8 and 12 h after
dicoumarol treatment to 116.8% ± 8.8% (n ¼ 6, p ¼ 0.11),
101.3% ± 9.7% (n ¼ 6, p ¼ 0.91), respectively, compared with that at
0 h (Fig. 4B). In addition, the cell numbers after treatment with
dicoumarol for 4, 8, 12, and 24 h decreased to 89.7% ± 4.2% (n ¼ 3,
p ¼ 0.07), 95.4% ± 4.9% (n ¼ 3, p ¼ 0.40), 92.1% ± 5.9% (n ¼ 3,
p ¼ 0.25), and 76.0% ± 2.0% (n ¼ 3, p < 0.001), respectively,
compared with that at 0 h.
Next, we examined the role of the Herp ULD in the ubiquitinindependent proteasome pathway. The expression levels of Herp
were reduced by treatment with dicoumarol, while DUb-Herp was
resistant to dicoumarol-induced degradation (Fig. 4C), suggestingthat the ULD made Herp susceptible to degradation by theproteasome.
4. Discussion

Fig. 2. Herp interacted with NQO1. HEK293 cells overexpressing Herp-FLAG, DUbHerp-FLAG, or pcDNA3 (mock) together with NQO1 were immunoprecipitated using
an anti-FLAG antibody (FL) or normal mouse IgG (IgG), followed by western blot
analysis of the immunoprecipitated (IP) fraction using anti-Herp or anti-NQO1
antibodies.

This study showed that the stability of Herp was regulated by
NQO1, which interacted with Herp and partly inhibited its degradation via the ubiquitin-independent proteasome. In addition,
synoviolin is involved in Nrf2 degradation, which regulates cellular
ROS, NQO1, and GCLM [19]. Therefore, the absence of synoviolin insynoviolin-null cells induced the expression of NQO1 and promoted the stability of Herp.
The ubiquitin-independent proteasomal degradation pathway
could be involved in Herp degradation because deletion of the Herp
ULD from Herp was found to enhance Herp stability, although it did
not affect the polyubiquitination level of Herp [8]. Our results
suggested that Herp could be degraded by the proteasome throughan NQO1-dependent pathway. Furthermore, our immunoprecipitation results suggested that NQO1 interacted with Herp and DUb-

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1279

Fig. 3. Effects of NQO1 on Herp stability. (A) Western blot analysis of lysates prepared from wt and synoviolin-null cells with the indicated antibodies. Protein (20 mg) was loadedin each lane, and a-tubulin was used as an internal control (left panel). wt, wt cells; �/�, synoviolin-null cells. Quantiﬁcation of Herp levels from three independent experiments isshown in the left panel (right graph). Values are expressed as the mean ± SEs. *, P < 0.01 (two-tailed Student's t-test). (B) Cell lysates (20 mg) from synoviolin-null cells overexpressing pMX (�) or pMX-NQO1 (NQO1) were detected through immunoblotting using anti-Herp and anti-NQO1 antibodies. b-Actin in the lysates was used as an internal loading
control. Herp levels were quantiﬁed from three independent experiments, as shown in the left panel (right graph). Values are expressed as means ± SEs. *, P < 0.01 (two-tailed
Student's t-test). (C) Synoviolin wt cells were transfected with control siRNA (siCTRL) or NQO1 siRNA (siNQO1), as indicated. After 72 h, cell extracts were resolved by SDS-PAGE, andproteins were detected by western blotting with the indicated antibodies. Herp levels were quantiﬁed from four independent experiments, as shown in the left panel (right graph).
Values are expressed as means ± SEs. *, P < 0.01 (two-tailed Student's t-test).

Fig. 4. Effects of dicoumarol on Herp stability. (A) Synoviolin-null cells were incubated for 4 h without (�) or with 200 or 400 mM dicoumarol (Dic) and without (�) or with (þ)
10 mM MG132. Herp levels were quantiﬁed from four independent experiments, as shown in the left panel (right graph). (B) Synoviolin-null cells were treated with 200 mM
dicoumarol and harvested at the indicated times. Synoviolin-null cells (20 mg) were detected by immunoblotting with an anti-Herp antibody. b-Actin in the lysate was also detected
as an internal control. Herp levels were quantiﬁed from three independent experiments, as shown in the left panel (right graph). Values are expressed as means ± SEs. *, P < 0.01
(two-tailed Student's t-test). (C) Synoviolin-null cells overexpressing Herp or DUb-Herp, were incubated for 4 h without (�) or with 200 or 400 mM dicoumarol (Dic) and without(�) or with (þ) 10 mM MG132.

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Herp. However, the degradation of the ULD-deletion mutant DUbHerp was not promoted by dicoumarol, a pharmacological inhibitorof NQO1. This result suggested that the Herp ULD may function as aproteasome-interacting domain, as has been demonstrated for
Parkin [20], through two possible mechanisms. First, NQO1 maycompete with the 20 S proteasome for C/EBPa binding, which leads
to its stabilization [21]; deletion of the Herp ULD could result inincreased NQO1 binding or decreased 20 S proteasome binding.
Therefore, NQO1 could have bound tightly to DUb-Herp and prevented its degradation by the 20 S proteasome. The other possibility is that the human p53 mutants R175H, R248H, and R273H are
resistant to dicoumarol-induced degradation [22]; therefore, arginine residues in the Herp ULD could be involved in dicoumarolinduced degradation.
p53, a tumor suppressor involved in cell growth and function, ismaintained at low levels through degradation mediated by multiple E3 ligases, including synoviolin or RNF5, and by a ubiquitinindependent pathway including NQO1-mediated proteasomal
degradation. In this regard, the regulation of Herp degradation
appeared to be similar to that of p53. Since Herp is involved inseveral important functions, such as ERAD and Ca2þ homeostasis,
Herp levels may be strictly regulated by degradation throughmultiple pathways in response to various physiological conditions.
On the other hand, in this study, the expression level of Herp wastransiently decreased by treatment with dicoumarol and recoveredfrom this decrease (Fig. 4B), suggesting the presence of opposing
pathways. In fact, Herp-deﬁcient mice have been reported to
exhibit impaired glucose homeostasis, and Herp modulates insulin
secretion in pancreatic beta cells [23,24]. In addition, NQO1knockout mice have been reported to show decreased glucose
and fatty acid metabolism [25]. Therefore, NQO-1-mediated protection of Herp may provide a good molecular explanation for thedecrease in glucose metabolism.
In conclusion, the proteasomal degradation of Herp was regulated through both ubiquitin-dependent and -independent degradation pathways, which were indirectly regulated by synoviolin.
Declaration of interest
Competing ﬁnancial interests
The authors declare no competing ﬁnancial interests.
Acknowledgments
We thank Dr. Koichi Kokame (National Cerebral and Cardiovascular Center) for providing the anti-Herp antibody.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.bbrc.2016.04.057.
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RESEARCH ARTICLE

Characterization of Zebrafish Models ofMarinesco-Sjögren Syndrome
Genri Kawahara, Yukiko K. Hayashi*
Department of Pathophysiology, Tokyo Medical University, Tokyo, Japan* yhayashi@tokyo-med.ac.jp

Abstract
a11111

OPEN ACCESS
Citation: Kawahara G, Hayashi YK (2016)
Characterization of Zebrafish Models of MarinescoSjögren Syndrome. PLoS ONE 11(10): e0165563.
doi:10.1371/journal.pone.0165563

SIL1 is a nucleotide exchange factor for the endoplasmic reticulum chaperone, BiP. Mutations in the SIL1 gene cause Marinesco-Sjögren syndrome (MSS), an autosomal recessive
disease characterized by cerebellar ataxia, mental retardation, congenital cataracts, andmyopathy. To create novel zebrafish models of MSS for therapeutic drug screening, weanalyzed phenotypes in sil1 knock down fish by two different antisense oligo morpholinos.
Both sil1 morphants had abnormal formation of muscle fibers and irregularity of the myosepta. Moreover, they showed smaller-sized eyes and loss of purkinje cells in cerebellar
area compared to controls. Immunoblotting analysis revealed increased protein amounts of
BiP, lipidated LC3, and caspase 3. These data supported that the sil1 morphants can represent mimicking phenotypes of human MSS. The sil1 morphants phenocopy the human
MSS disease pathology and are a good animal model for therapeutic studies.

Editor: Linda M. Hendershot, UNITED STATES
Received: July 14, 2016
Accepted: October 13, 2016
Published: October 28, 2016
Copyright: © 2016 Kawahara, Hayashi. This is an
open access article distributed under the terms of
the Creative Commons Attribution License, which
permits unrestricted use, distribution, andreproduction in any medium, provided the original
author and source are credited.
Data Availability Statement: All relevant data arewithin the paper and its Supporting Information
files.
Funding: This work was supported partly by aGrant-in-Aid for challenging Exploratory Researchfrom Japan Society for the Promotion of Science(JSPS), partly by MEXT-Supported Program for
the Strategic Research Foundation at Private
Universities, partly by Research on rare andintractable diseases, Health and Labor Science
Research Grants, and partly by Intramural
Research Grant for Neurological and Psychiatric
Disorders of NCNP (26-8).

Introduction
Marinesco-Sjögren syndrome (MSS; OMIM 248800) is an autosomal recessive disorder clinically characterized by cerebellar ataxia, mental retardation, congenital cataracts, and progressive muscle weakness [1–4]. Mutations in SIL1 (Gene ID: 64374) were reported to be causativefor MSS [5, 6]. SIL1 is also known as binding immunoglobulin protein (BiP)-associated protein(BAP) [7]. BiP plays a key role in protein quality control as a heat shock protein (HSP) 70
chaperone family member located in the endoplasmic reticulum (ER). SIL1 regulates theATPase cycle of BiP as an adenine nucleotide exchange factor [7, 8]. SIL1-null woozy mutant
mice exhibit progressive ataxia caused by loss of Purkinje cells via ER stress, together withmyopathy [9–12]. These reports suggest that SIL1 plays essential roles in ER function in multiple tissues.
Zebrafish is a useful model to elucidate the pathomechanisms of a lot of human diseases
including muscular dystrophies [13–15]. The analyses of zebrafish models for human muscle
disorders have been facilitated because of rapid development and easy-to-identify muscle
structural abnormalities using birefringence assay [16]. They are easily analyzed by the use ofmorpholinos during early development. Zebrafish models of muscle disease have also beenused to rapid therapeutic drug screening for small molecules [17].

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Competing Interests: The authors have declaredthat no competing interests exist.

To generate a model fish of MSS for studies on the functions of zebrafish sil1 and for therapeutic drug screenings, morpholino antisense oligos that targeted zebrafish sil1 mRNA were
designed and injected into zebrafish eggs. Our data indicates the reduction of sil1 expression
causes the abnormal formation of muscles, small sized eyes and reduction of purkinje cells
associated with increased marker proteins for ER-stress, autophagy, and apoptosis.

Materials and Methods
Fish and fish culture
Zebrafish (the AB line) were cultured at 28.5°C according to standard procedures [18] andstandard criteria [19]. Fertilized eggs were collected and used for injection. For anesthesia,
euthanasia we used tricaine solution. Our IACUC, Tokyo Medical University animal facility,
indicates that they approved this research (the approval number:S28029).

Morpholino oligonucleotide injections
Two different anti-sense morpholino oligo-nucleotides (MO) targeted to disrupt splicing ofsil1 mRNA were designed by Gene Tools LLC. The morpholino sequences were sil1 MO1: 5’CAGCATGGGAATAAACTCACCTGGT -3’ and sil1 MO2: 5’- GGTGACTGTGTAAACAGAACAAATC-3’. Morpholinos (3 and 6 ng) were injected into the yolk of one- to two-cell stageembryos. For the injection control, we also used a control morpholino (CMO; standard control
oligo, Gene Tools): 5’-CCTCTTACCTCAGTTACAATTTAT-3’.
To confirm with the effect of morpholino injections, zebrafish total RNA was extracted from4 days post fertilization (dpf) embryos using RNeasy micro kit (Qiagen) and was converted to
cDNA (Superscript III, Life science). To detect misspliced products, PCR was performed with
ExTaq DNA Polymerase (Takara Bio) at 95°C for 30 s, 55°C for 30 s, and 72°C for 30 s for 35
cycles) with the following primer sets: sil1 for exon 2 and 3, forward 5’-GGCAAACAAGTGGAGAGTCAG-3’, reverse 5’- ATGGCTGTCCATGTTCATCA-3’ and beta-actin, forward: 5’ATCAGCAT GGCTTCTGCTCT-3’, reverse: 5’-CACCCTGGCTTACATTTTCAA-3’.

Detection of muscle phenotype of sil1 morphants by birefringence
Birefringence assay was performed to detect abnormal skeletal muscle structure by placing
anesthesized fish on a polarizing filter and subsequently covering them with a second polarizing filter [17]. The filters were placed on an underlit dissection scope and the top-polarizing filter twisted until only the light refracting through the striated muscle was visible. Since thedegree of birefringence is affected by the horizontal orientation of the fish, the fish were oscillated back and forth to account for differences in positioning.

Measurement of size of eyes
The diameter of the eye of 4 dpf embryos was measured under a dissection scope (Olympus,
SZX10) with DP controller (Olympus) software and ImageJ (http://imagej.en.softonic.com/).

Zebrafish SIL1 antibody
A rabbit polyclonal antibody against zebrafish sil1 was produced by synthesized peptides(C+EDLEVFRPTDKWQTLRPGQ; 63–81) as an antigen (Sigma Aldrich). The synthesizedpeptides were also used to absorb the antibodies to confirm the specificity of this antibody.

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Immunohistochemistry
Whole fish embryos were fixed in 4% paraformaldehyde overnight at 4°C and stored in 100%
methanol at -20°C. After rehydration with a 50% methanol in PBS and blocking with 2% casein
in PBS containing 0.05% Tween 20 (PBS-T) to reduce non-specific binding, embryos wereincubated separately with either anti-beta dystroglycan (1:100, Novocastra), anti-parvalbumin(1:1000, Chemicon), or anti-myosin heavy chain (F59, 1:25, Santa Cruz Biotechnology) antibodies at 4°C overnight. After washing several times, the samples were incubated with secondary antibodies (1:500, anti-mouse AlexaFluor 488, 1:500, anti-rabbit AlexaFluor 568,
Invitrogen) and DAPI (DAKO) solution for 30 min. The stained embryos were observed underconfocal microscopies (Leica LCM710).

Western blotting analysis
Proteins were extracted from 20 embryos of 4 dpf in RIPA buffer (Sigma) containing proteaseinhibitors and phosphatase inhibitors (Roche). A total of 30 micrograms of proteins were separated by electrophoresis on 5–20% gradient Tris—glycine gels (Wako Chemicals) and transferred onto a PVDF membrane (Bio-Rad). After blocking the membrane in PBS-T containing
2% casein, blotted proteins were incubated with each primary antibody of anti-zebrafish sil1
(1:200), anti-tubulin (1:500, Cell Signaling Bio), anti-BiP (1:250, abcam), anti-LC3 (1:250, Cell
Signaling Bio), anti-activated caspase 3 (1:250, abcam), and anti-beta actin (1:500, Sigma) at
4°C overnight. After washing, the membranes were incubated with horseradish peroxidase(HRP) secondary antibody (anti-rabbit or mouse IgG, 1:15,000, abcam). Proteins were detectedusing a western blotting detection kit (Bio-Rad). Relative band intensities were quantified bydensitometry using ImageJ, and fold differences determined between each proteins/beta-actin
ratio.

Cloning of zebrafish sil1 cDNA and in vitro transcription of RNAs
Primers used to amplify the full-length zebrafish sil1 cDNA coding sequence (XM009291221)
were forward; 5’-TCTTTTTGCAGGATCATGTTGACAAGT CGTGTAATG-3’, reverse; 5’CGAATCGATGGGATCTTACACCTGTCGCATT TTTAC-3’. PCR was performed with PrimeSTAR HS DNA Polymerase (Takara) at 98°C for 15 s, 55°C for 15 s, 72°C for 1 min for 35
cycles according to the manufacturer’s protocol. The PCR product of the zebrafish sil1 cDNA
was cloned into pCS2+ vector using In-Fusion1 HD Cloning Kit (Takara). All PCR products
and cloned fragments were sequenced by using sequencing primers: pCS2+ forward primer:
5’-CGGAGCAAGCTTGATTTAG-3’, pCS2+ reverse primer: 5’-CCCCCTGAACCTGAAACAT
AA-3’, zebrafish sil1 forward primer2: 5’-CTCTGGACATGCTTGTGGAA-3’ and zebrafish
sil1 forward primer3: 5’-GCAGGTGGGACTGGACATTA-3’. Sequencing results were analyzed
using NCBI database and program on BLAST. Zebrafish sil1 mRNA was synthesized from
Asp718-digested pCS2+ plasmids using the sp6 mMessage mMachine kit (Ambion) and 50 pg
of mRNA was co-injected into 1-cell-stage embryos with zebrafish sil1 MO1 and 2. Following
injection, embryos were cultured in aquatic system at 28.5°C.

Results
Zebrafish sil1 morphant morphology
Two different anti-sense morpholino oligonucleotides (MO) 1 and 2 were designed to disrupt
splicing pattern of sil1 mRNA during development (Fig 1A). The injected embryos were examined at 4 dpf. RT-PCR and sequence analysis confirmed that the MO1 and MO2 injection

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Fig 1. RT-PCR and protein expression of sil1 morpholino-injected embryos. A: Morpholino oligo-nucleotides
target for zebrafish sil1. Two different anti-sense morpholino oligo-nucleotides targeted to disrupt splicing of sil1
mRNA are designed by Gene Tools LLC. B: A single RT-PCR product of 355 bp is seen in 4 dpf embryos injected 3
ng of control morpholino (CMO), whereas 4 dpf embryos injected 3 ng of sil1 morpholino 1 (MO1) had two different
sized products of 355 and 446 bp. Two RT-PCR products of 355 bp and 407 bp were detected in 4 dpf embryos
injected morphant 2 (MO2). C: Western blotting analysis shows reduced amounts of sil1 protein extracted from 20
each of MO1- or MO2-injected morphants compared to 4 dpf wild-type embryos (WT) and CMO. D: Injections of
MO1 or MO2 are effective to reduce the expression of sil1 protein to 66.5% (MO1, light gray) and 46.9% (MO2,gray) compared to CMO-injected fish (black), respectively.
doi:10.1371/journal.pone.0165563.g001
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Fig 2. Altered skeletal muscle in sil1-morpholino injected fish. A: Left panels are pictures under brightfield, and left panels are birefringence assay (BR). Abnormal structure of muscle is obvious in sil1 morpholino
injected fish (MO1 and MO2) compared to control (CMO) under birefringence assay. B: Histogram of thepercentage of normal, affected, and dead fish by morpholino-injection. Injection of 3 ng of MO1 or MO2
resulted in approximately 39.0% and 21.8% of injected embryos exhibiting reduced birefringence,
respectively. C: Restoration of sil1 morphant with co-injection of fish sil1 mRNA (50 pg). Histogram of thepercentage of dead, affected fish of morphants and recovered fish. Co-injection of zebrafish sil1 mRNA witheach MO (3 ng) rescued the phenotypes. White bar shows normal %, gray shows affected % and blackshows dead fish %. Non: non injected fish, MO1: MO1 injected fish, MO2: MO2 injected fish.
doi:10.1371/journal.pone.0165563.g002
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resulted in an in-frame insertion of a whole intron 2 (91 bps) and insertion of partial intron 2(52 bps), respectively (Fig 1B).
To confirm the knock down effect to the expression of fish sil1 by morpholino injections,endogenous zebrafish sil1 protein was analyzed with antibody against zebrafish sil1. In wild
type and CMO injected fish, the sil1 antibody recognized a 52 kDa protein on western blots, aconsistent size predicted by the fish sil1 sequence (Fig 1C). The expression of sil1 protein of
MO1 or MO2 injected fishes was reduced to 66.5% and 46.9%, respectively compared to thewild type (Fig 1D). This result indicated that injections of MO1 or MO2 were effective todecrease the expression of sil1 protein.

Birefringence assay
Following injection of the two different morpholinos, some embryos showed abnormal shapevisible upon light microscopy (Fig 2A). To better visualize the structure and organization ofmuscle fibers of the morphants, MO1, MO2 or CMO injected embryos and wild type embryos
were analyzed by birefringence assays at 4 dpf (Fig 2A). The sil1 morphant embryos were
found to have markedly reduced normal patterns of birefringence compared to wild type andcontrol morphants (Fig 2A). Injection of 3 ng of MO1 or MO2 resulted in approximately
39.0±1.8% and 21.8±3.1% of injected embryos exhibiting reduced birefringence, 36.5±4.7%
and 64.5±1.6% were normal looking, and 24.5±4.7% and 13.7±1.6% of dead, respectively (Fig
2B). These percentages are an average of the results from three different experiments. The

Fig 3. Immunohistochemistry of skeletal muscle tissue of morpholino-injected fish. Immunostainingof CMO injected fish (A, B) and sil1 morphant (MO2) injected fish (C, D) with antibodies against betadystroglycan (beta-DG) (A, C) and myosin heavy chain (MHC) (B, D). Beta-dystroglycan expression at the
myosepta of MO2 injected 4 dpf embryos is misshapen and has a less clear v-shaped structure. Staining
with anti-MHC indicated that formation of myofibers is disturbed in MO2. Arrowheads indicate thedisturbance of myosepta in MO2-injected fish. Bar: 100 μm. Arrows indicate the abnormal structure of
myofibers.
doi:10.1371/journal.pone.0165563.g003

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effects of morpholinos were dose-dependent and the ratio of abnormal embryos were increased
when 6 ng of morpholinos were injected (S1 Fig). Importantly, co-injection of zebrafish sil1
mRNA with each MOs rescued the phenotypes (Fig 2C).

Immunohistochemistry of sil1 morphants with muscle structure’s
components
To examine the expression of muscle proteins, antibodies against beta-dystroglycan, laminin(data not shown), and myosin heavy chain (MHC, slow fibers) were used for

Fig 4. Smaller sized eyes in sil1-morpholino injected fish (4 dpf). The diameter of eyes in MO1 or MO2 injected 4 dpf
embryos are smaller than those of CMO injected embryos. Co-injection of zebrafish sil1 mRNA with each MOs rescues theeye size. Eye size is measured under a dissection scope with DP controller software and ImageJ. (A) wild type, (B) CMO
injected fish, and (C) MO2 injected fish, showing white lines. Measurement of the diameter of eyes (D: MO1, E: MO2). (F):Restoration of eye diameter of sil1 morphant with co-injection of fish sil1 mRNA. Single asterisk indicates Student’s t-test
p = 1.25E-07 (MO1; n = 19, CMO; n = 14), double asterisks indicate p = 7.36E-08 (MO2; n = 16, CMO; n = 28) and triple
asterisks indicate p = 0.00441 (MO2; n = 19, MO2+sil1 mRNA 50 pg; n = 14, CMO; n = 14).
doi:10.1371/journal.pone.0165563.g004

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immunohistochemistry. Beta-dystroglycan expression at the myosepta of MO1 or MO2
injected 4 dpf embryos was misshapen and had a less clear v-shaped structure as observed inwild type and CMO injected embryos (Fig 3A and 3C). Staining with anti-MHC indicated thatformation of myofibers was disturbed in MO1 and 2 (in Fig 3B and 3D). This result is consistent with that observed by birefringence assay. Co-injection of zebrafish sil1 mRNA with each
MO reduced the number of fishes showing marked myofibril disruptions by anti-MHC stain.

Fig 5. Staining of purkinje cells with anti-purvalbumin in cerebellar area. The number of purkinje cells
detected with anti-parvalbumin shows reduced number of positive cells in MO1 or MO2 injected embryos. (A,B and C): CMO injected fish, (D, E and F): MO1 injected fish, (G, H and I): MO2 injected fish. (A, D and G:
Staining Purkinje cell. (B, E and H): DAPI, nuclei. (C, F and I): Merged images. (J): Increased number of
Purkinje cells in sil1 morphants with co-injection of fish sil1 mRNA (50 pg) from 23.1% (MO2, n = 13) to
64.3% (MO2+sil1 mRNA, n = 14).
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Abnormal size of eyes and reduction of purkinje cells in cerebellar areaof sil1 morphants
To examine influence of sil1 morpholinos to neural tissues, the size of eyes and number of purkinje cells in cerebellar area were analyzed. The diameter of eyes in MO1 or MO2 injected 4

Fig 6. Protein expression analysis of sil1-morpholino injected fish. Western blot analysis of control
morpholino (CMO) and sil1-morpholino2 injected fish (MO). The protein amounts of BiP, lipidated form of LC3(LC3-II), and activated caspase 3 are significantly increased in sil1 morphant embryos compared to those of CMOinjected embryos. A: Immunoblot with anti-BiP, LC3-II, activated-Caspase 3, and beta-actin. B: Relative
expression level is analyzed via immunoblot (*p = 0.0226, ** p = 0.000102 and *** p = 0.000705, versus CMO,
Student’s t-test, n = 3).
doi:10.1371/journal.pone.0165563.g006

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dpf embryos was smaller than those of CMO injected embryos (Fig 4C, 4D and 4E). Co-injection of zebrafish sil1 mRNA with each MOs rescued the eye size (Fig 4F).
The number of purkinje cells detected with anti-parvalbumin, a purkinje cell marker [20],showed reduced number of positive cells in MO1 or MO2 injected embryos compared to thoseof controls (Fig 5D and 5G). Co-injection of zebrafish sil1 mRNA with each MO increased thenumber of positive cells in MO1 or MO2 injected embryos (MO2: 76.9%, n = 13, MO2+sil1
mRNA: 35.7%, n = 14) (Fig 5J).

Increased expression of marker proteins of ER-stress, autophagy, andapoptosis
To examine expression levels of marker proteins of ER-stress, autophagy and apoptosis, western blotting was performed. The protein amounts of BiP, lipidated form of LC3 (LC3-II), andactivated caspase 3 were significantly increased in sil1 morphant embryos compared to those of
CMO-injected embryos (Fig 6A and 6B).

Discussion
Here, we showed a zebrafish is a good model of MSS, an autosomal recessive multisystem disorder characterized by cerebellar ataxia, mental retardation, cataract, and myopathy [1–6]. The
sil1 morphants showed altered skeletal muscle structures, judged by unshapen myosepta anddisturbed muscle fibers. In addition, the sil1 morphants have smaller sized eyes and loss of purkinje cells.
Our MSS zebrafish models display an increase in the levels of markers associated with ER
stress, autophagy and apoptosis, which are similar to those observed in humans and a mousemodel of MSS [9–11]. SIL1-deficiency is expected to reduce BiP activity, an ER chaperone protein, which leads to accumulation of abnormal proteins and subsequent ER stress, increased
autophagy and apoptosis. In the reduced expression of sil1, morphants might have clear phenotypes in muscle, abnormal eye size and loss of purkinje cells via activated unfolded protein
response, ER stress, autophagy and apoptosis. Interestingly, sil1 morphants showed smallersized eyes instead of cataracts, which suggests that sil1 may have important roles in development of eyes in zebrafish. Recently SIL1 was reported to play a role in regulation of motor neuron subtype-selective ER stress in amyotrophic lateral sclerosis (ALS) [21]. Further analysis of
the MSS model fish might reveal molecular crosstalk of ER stress, autophagy, and apoptosis invariable tissues and cells.
Further analysis of sil1 function in zebrafish should be undertaken CRISPR-Cas9 system foranalysis of sil1 functions and therapeutic intervention studies for MSS.

Supporting Information
S1 Fig. Histogram of the percentage of dead, affected fish of MO1 (A), MO2 (B) or control
MO injected fish (3 ng and 6 ng). White bar shows normal %, gray shows affected % andblack shows dead fish %. The effects of morpholinos are dose-dependent and the ratio ofabnormal embryos are increased when 6 ng of morpholinos were injected.
(TIF)

Acknowledgments
We would like to acknowledge Dr. Kataaki Okubo and Dr. Mathew S. Alexander for their great
help and suggestions to perform the experiments and to draft of this manuscript. We also
thank Chris Lawrence for providing wild type fish.

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Author Contributions
Conceptualization: GK YKH.
Data curation: YKH.
Formal analysis: GK.
Funding acquisition: GK YKH.
Investigation: GK.
Methodology: GK YKH.
Project administration: YKH.
Resources: GK.
Software: GK.
Supervision: YKH.
Validation: YKH.
Visualization: GK.
Writing – original draft: GK.
Writing – review & editing: YKH.

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Biochemical and Biophysical Research Communications 461 (2015) 28e34

Contents lists available at ScienceDirect

Biochemical and Biophysical Research Communications
journal homepage: www.elsevier.com/locate/ybbrc

EGFR-independent autophagy induction with geﬁtinib andenhancement of its cytotoxic effect by targeting autophagy withclarithromycin in non-small cell lung cancer cells
Shohei Sugita a, 1, Kentaro Ito a, 1, Yutaro Yamashiro a, Shota Moriya a, Xiao-Fang Che a,
Tomohisa Yokoyama b, Masaki Hiramoto a, Keisuke Miyazawa a, *ab

Department of Biochemistry, Tokyo Medical University, Tokyo, Japan
Department of Clinical Oncology, Tokyo Medical University, Tokyo, Japana r t i c l e i n f o

a b s t r a c t

Article history:
Received 13 March 2015
Available online 7 April 2015

Geﬁtinib (GEF), an inhibitor for EGFR tyrosine kinase, potently induces autophagy in non-small cell lungcancer (NSCLC) cell lines such as PC-9 cells expressing constitutively activated EGFR kinase by EGFR genemutation as well as A549 and H226 cells with wild-type EGFR. Unexpectedly, GEF-induced autophagy
was also observed in non-NSCLC cells such as murine embryonic ﬁbroblasts (MEF) and leukemia celllines K562 and HL-60 without EGFR expression. Knockout of EGFR gene in A549 cells by CRISPR/Cas9
system still exhibited autophagy induction after treatment with GEF, indicating that the autophagy induction by GEF is not mediated through inhibiting EGFR kinase activity. Combined treatment with GEF
and clarithromycin (CAM), a macrolide antibiotic having the effect of inhibiting autophagy ﬂux, enhances
the cytotoxic effect in NSCLC cell lines, although treatment with CAM alone exhibits no cytotoxicity. GEF
treatment induced up-regulation of endoplasmic reticulum (ER)-stress related genes such as CHOP/GADD153 and GRP78. Knockdown of CHOP in PC-9 cells and Chop-knockout MEF both exhibited lesssensitivity to GEF than controls. Addition of CAM in culture medium resulted in further pronounced GEFinduced ER stress loading, while CAM alone exhibited no effect. These data suggest that GEF-induced
autophagy functions as cytoprotective and indicates the potential therapeutic possibility of using CAM
for GEF therapy. Furthermore, it is suggested that the intracellular signaling for autophagy initiation in
response to GEF can be completely dissociated from EGFR, but unknown target molecule(s) of GEF for
autophagy induction might exist.
© 2015 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND
license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Keywords:
Autophagy
Geﬁtinib
Non-small cell lung cancer
EGFR
Macrolide antibiotics
ER-stress

1. Introduction
Lung cancer has become the most common cause of death fromcancer, accounting for approximately 23% of all cancer-related
deaths in the world. In particular, non-small cell lung cancer(NSCLC) accounts for about nine out of ten cases of all lung cancers[1]. Small molecules for tyrosine kinase inhibitor (TKI) of epidermal
growth factor receptor (EGFR) such as geﬁtinib (GEF) and erlotinib
have become an effective therapy of choice for NSCLC patients
having EGFR-mutation [2,3]. Despite the initial response to EGFR-* Corresponding author. Department of Biochemistry, Tokyo Medical University,
6-1-1 Shinjuku, Shinjuku-ku, Tokyo 160-8402, Japan. Fax: þ81 3 3351 6466.
E-mail address: miyazawa@tokyo-med.ac.jp (K. Miyazawa).
1
These authors contributed equally to this work and should be regarded as coﬁrst authors.

TKIs, most patients develop resistance, often due to the emergence of a secondary mutation such as T790M, and eventuallyrelapse. The median survival of relapsed patients is reported to be
ﬁve to eight months at present [4]. Thus, a novel therapeuticstrategy for both aiming the cure at initial treatment, when the
NSCLC still has sensitivity to TKIs, and overcoming the resistance toa secondary mutation appears to be urgent for improving therapeutic outcomes of NSCLC.
Autophagy, a highly conserved and regulated cellular process
employed by living cells to degrade proteins and organelles, hasbeen reported to be induced after treatment with EGFR-TKIs in
NSCLC and breast cancer cell lines [5,6]. Recent papers have suggested that the induction of autophagy is indispensable for thecytotoxic effect of EGFR-TKI in primary and resistant cells withmutant EGFR-in NSCLC cells [7,8], while others have reported that
autophagy functions as cytoprotective as a response to metabolic

http://dx.doi.org/10.1016/j.bbrc.2015.03.162
0006-291X/© 2015 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

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stress [5,6,9e11]. Thus, it remains controversial whether autophagy
is associated with sensitivity or resistance to EGFR-TKIs. The mostrecent report demonstrated that the accumulating inactivated
EGFR at endosome is critical for autophagy initiation via formationof a complex with the autophagy inhibitor Rubicon in tumor cells[12]. Association of inactivated EGFR with Rubicon in turn disassociates Beclin 1 from Rubicon for autophagy initiation, which issuggested to control tumor cell metabolism and promote tumor cell
survival [12].
We and others have reported that macrolide antibiotics
including clarithromycin (CAM) and azithromycin, both of which
are clinically well used antibiotics, have the effect of blocking
autophagy ﬂux [13e15]. We have also reported that bortezomib, aproteasome inhibitor used for the treatment of patients withmultiple myeloma, induces autophagy in myeloma cell lines in vitro[16]. Simultaneous inhibition of intracellular proteolytic processes
such as an ubiquitin-proteasome system with bortezomib and an
autophagy-lysosome system with CAM resulted in enhancement of
the apoptosis induction in myeloma cells along with accumulationof intracellular polyubiquitinated proteins and aggresome formation [15,17]. Under the concomitant inhibition of two major intracellular protein degradation systems, ER-tress loading was
pronounced, probably as a result of suppression of ERAD (ERassociated degradation) of ubiquitinated unfolded proteins. This
leads to ER-stress mediated apoptosis induction in myeloma andbreast cancer cells via up-regulation of CHOP (GADD153), a proapoptotic-transcription factor [16e20]. In this context, ER-stressloading appears to play a considerable role in apoptosis induction
in some cancer cells [21e23].
It is of interest that TKIs for BCR-ABL including imatinib anddasatinib have been reported to induce autophagy in chronic
myeloid leukemia (CML) cells along with ER-stress [24,25]. CAMenhanced dasatinib-induced cell death in CML cells in vitro by
inhibiting late stage autophagy has also been reported [26]. In
several patients with resistant CML, oral administration of CAM(500 mg, b.i.d.) was suggested to potentiate TKI-treatment,
although the molecular mechanism remains to be clariﬁed [27].
Based on these lines of evidence, we hypothesized that CAM
could enhance the cytotoxic effect of GEF along with ER-stressloading in NSCLC. In the present study, we examined the combined effect of GEF plus CAM as well as the biological roles of GEFinduced autophagy in NSCLC cells by using CAM as an autophagy
inhibitor.
2. Material and methods
2.1. ReagentsGeﬁtinib (GEF) purchased from Cayman Chemical (Ann Arbor,MI) was dissolved in dimethyl sulfoxide to make stock solutions atconcentrations of 10 mM. Clarithromycin (CAM) purchased from
Tokyo Chemical Industry (Tokyo, Japan) was dissolved in ethanol toprepare stock solutions of 5 mg/ml. Lysosomal inhibitors E-64d andpepstatin A were obtained from Peptide Institute (Osaka, Japan).
Doxycycline hydrochloride was purchased from Wako Pure
Chemicals Industries (Tokyo).
2.2. Cell lines and culture conditions
For this study, NSCLC cell lines such as PC-9 were obtained fromthe RIKEN Bio Resource Center (Tsukuba, Japan), and A549 andH226 were from the American Type Culture Collection (ATCC)(Manassas, VA). Leukemia cell lines HL-60 and K562, along with a
Chop�/� murine embryonic ﬁbroblast (MEF) cell line (CHOP-KODR) established from a 13.5-day-old Chop�/� mouse embryo by SV-

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29

40 immortalization and a Chopþ/þ MEF cell line (DR-wild-type)established by SV-40 immortalization as a control cell line for
CHOP-KO-DR, were also obtained from the ATCC. These authorizedcell lines were expanded and frozen in aliquots within one monthafter they were obtained from the cell banks. Each aliquot was
thawed and the cells were used for the experiments within twomonths after thawing. The NSCLC and leukemia cell lines were
cultured in RPMI-1640 medium (Sigma) supplemented with 10%
fetal bovine serum (FBS) (Biowest, Nuaille, France), 2 mM L-glutamine, penicillin (100 U/ml), and streptomycin (100 mg/ml) (Wako).
CHOP-KO-DR and DR-wild-type cells were maintained in Dulbecco's modiﬁed Eagle's medium (Sigma) supplemented with 10% FBS,
2 mM L-glutamine, penicillin (100 U/ml), and streptomycin (100 mg/
ml). The m5-7 cell line, an Atg5 Tet-off MEF system, was a kind giftfrom Dr. Noboru Mizushima (Tokyo University, Graduate School
and Faculty of Medicine, Tokyo, Japan). The culture conditions forknockout of the Atg5 gene for inhibiting autophagy were previously
described in detail [28]. All cell lines were cultured in a humidiﬁed
incubator containing 5% CO2 and 95% air at 37 � C.
2.3. Assessment of viable number of cells
The number of viable cells was assessed using CellTiter Blue, acell viability assay kit (Promega, Madison, WI), according to the
manufacturer's instructions as previously described in detail [15].
2.4. Immunoblotting
Immunoblotting was performed as previously described [15]. In
brief, cells were lysed with RIPA lysis buffer (Nacalai Tesque, Kyoto,
Japan) supplemented with a protease and phosphatase inhibitor
cocktail (Nacalai Tesque). Cellular proteins were separated by SDSPAGE and transferred onto Immobilon-P membrane (Millipore,
Billerica, MA). The membranes were probed with primary antibodies (Abs): anti-LC3B Ab (Novus Biologicals, Littleton, CO), antip62 (D-3) monoclonal (m) Ab, anti-GAPDH (6C5) mAb, anti-EGFR(1005) Ab (Santa Cruz Biotechnology, Santa Cruz, CA), and anticleaved caspase-3 Ab, anti-PARP Ab (Cell Signaling Technologies,
Danvers, MA). Speciﬁc Abs against tyrosine phosphorylated proteins, such as anti-phosphotyrosine (4G10) mAb, were purchasedfrom Millipore, and anti-phospho-EGFR (Tyr1173) Ab was obtained
from Santa Cruz Biotechnology. Immunoreactive proteins weredetected with horseradish peroxidase-conjugated secondary Abs(Cell Signaling Technologies) and an enhanced chemiluminescencereagent (Millipore). Densitometry was performed using a Molecular Imager, ChemiDoc XRS System (Bio-Rad).
2.5. Gene expression analysis
A real-time polymerase chain reaction (PCR) for gene expression
analysis was performed as we previously described in detail [15].
2.6. Knockout of ERGR gene by CRISPR/Cas9 emediated genome
editing and gene silence of CHOP by siRNA
Target sequences for CRISPR interference [29] were designed at
CRISPR direct (http://crispr.dbcls.jp/), provided by the Database
Center for Life Science (Chiba, Japan). The target sequences forhuman EGFR are GGAGCAGCGATGCGACCCTC (Exon 1) or
TGCAAATAAAACCGGACTGA (Exon 3). Two complementary oligonucleotides with BpiI restriction sites for guide RNAs (gRNAs) weresynthesized at Euroﬁns Genomics (Tokyo, Japan), and cloned into
pX459 CRISPR/Cas9-Puro vector (Addgene, Cambridge, MA)
deposited by the Feng Zhang Lab.

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S. Sugita et al. / Biochemical and Biophysical Research Communications 461 (2015) 28e34

Fig. 1. Cell-growth inhibition and changes of intracellular protein tyrosine phosphorylation pattern after treatment with GEF. (A) All cell lines were treated with GEF at various
concentrations for 48 h. The viable cell number was assessed by CellTiter Blue as described in Materials and methods. (B) Cells were treated with/without 5 mM of GEF for 12 h. Cells
were lysed, and cellular proteins were separated by 7.5% SDS-PAGE and immunoblotted with anti-phosphotyrosine mAb, anti-EGFR Ab, and anti-phospho-EGFR (Tyr1173) Ab.
Immunoblotting with anti-GAPDH mAb was performed as an internal control.

Fig. 2. Autophagy induction after treatment with GEF. (A) Cells were treated with GEF at 5 and 25 mM for 24 he72 h. Cellular proteins were separated by 15% SDS-PAGE for LC3B and11.25% SDS-PAGE for p62. Immunoblottings were performed using anti-LC3B Ab and anti-p62 mAb. Immunoblottings with anti-GAPDH mAb were performed as an internal control.
Because of the high cytotoxicity of GEF in some cell lines, it was difﬁcult to load the same amount of protein after 72 h exposure to GEF. Anti-p62 (D-3) mAb used in these experiments reacts with human p62 but not with murine origin. (B) A549 cells were cultured with/without GEF (25 mM) in the presence or absence of lysosomal inhibitors (LIs), E-64d(10 mg/ml) and pepstatin A (10 mg/ml) for 24 h. Cellular proteins were separated by 15% SDS-PAGE and immunoblotted with anti-LC3B Ab. (C) Two clones of A549 with EGFR geneknocked out by the CRISPR/Cas9 method were treated with GEF at 5 and 25 mM for 72 h. Immunoblottings were performed using anti-EGFR Ab and LC3B Ab.

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A549 cells were transfected with pX459-gRNA using Lipofectamine 2000 (Life Technologies, Carlsbad, CA) according to the
manufacturer's instructions. Beginning the day after transfection,these cells were treated with 2 mg/ml of puromycin for two days.
Surviving cells were reseeded at 0.5 cells per well of a 96-well plate.
Expressions of EGFR in the expanded colonies were detected byimmunoblotting using anti-EGFR (1005) Ab to select the EGFRdepleted colonies. The genome sequences of the edited locus inselected colonies were conﬁrmed by sequence analyses performed
at Euroﬁns Genomics.
Gene silence of CHOP in PC-9 cells by siRNA was performed aspreviously described in detail [20].
2.7. Statistical analysis
All data are expressed as mean ± SD. The statistical analysis was
performed using ManneWhitney's U test (two-tailed).
3. Results and discussion
3.1. Cell growth inhibition in NSCLC and other cell lines after
treatment with GEF
As seen in Fig. 1A, GEF inhibited cell growth in NSCLC cell lines
tested in a dose-dependent manner: PC-9 cells with EGFR mutation
exhibited the most potent cell-growth inhibition in response to GEF
as compared with A549 and H226 cells, which are the cell lines
expressed with wild-type EGFR [30]. IC50 (50% inhibitory concentration) after 48 h-exposure to GEF was 10.4 mM in PC-9 but was

31

33.4 mM in A459 and 26.0 mM in H226 cells. This was well correlatedwith the clinical response, demonstrating that sensitizing EGFR
mutations are the most predictive factor for clinical beneﬁt with
EGFR TKIs [2,3]. The higher sensitivity of PC-9 cells to GEF can be
explained by the fact that they at least in part depend on the cell
growth and/or survival signals produced by ligand-independent
activation of mutant EGFR kinase. This is supported by our data
in Fig. 1B showing that, in contrast to A549 and H226 cells,
constitutive activated EGFR was observed in PC-9 cells, and that thetyrosine phosphorylation of EGFR disappeared in response to GEF.
However, GEF exhibits considerable cell-growth inhibition in
MEF and non-EGFR expressing cell lines such as K562 (a CML cellline with activated BCR-ABL kinase) and HL-60 cells (an AML cellline) (Fig. 1A). (IC50s for these cell lines were 13.1 mM in MEF,
16.4 mM in K562, and 16.2 mM in HL-60.)
3.2. GEF induces autophagy in an EGFR-independent manner
Treating the cells with GEF resulted in an increased expression
ratio of LC3B-II to LC3B-I, which is a hallmark of autophagosome
formation by the conversion of cytosolic LC3B-I into lipidated LC3BII associated with autophagosome membrane in NSCLC cell lines(Fig. 2A) [31]. P62, a substrate of autophagy, decreased in response
to GEF treatment. In addition, in the presence of lysosomal inhibitors (LIs) for blocking autophagy ﬂux, accumulation of GEFinduced LC3B-II isoform was further enhanced as compared totreatment with either GEF or LIs (Fig. 2A, B). All of these data
indicate that GEF induces autophagy in NSCLC cell lines as previously reported by others [5,8,9]. Strikingly, autophagy induction in

Fig. 3. CAM enhances the cytotoxic effect of GEF. (A) Autophagy ﬂux analysis in A549 cells: Cells were treated with CAM at 50 mg/ml in the presence or absence of LIs for 48 h as
described in Fig. 2B. Cellular proteins were separated by 15% SDS-PAGE and immunoblotted with anti-LC3B Ab. Immunoblotting with anti-GAPDH mAb was performed as aninternal control for protein loading. (B) Cells were cultured with GEF at various concentrations for 48 h in the presence or absence of CAM (50 mg/ml). The viable cell number wasassessed as described in Materials and methods. (*p < 0.05：CAM (�) vs. CAM (þ)) (C) For complete inhibition of GEF-induced autophagy, a Atg5 Tet-off MEF cell line, m5-7, wasused [28]. After pre-treatment with/without doxycycline (Dox: 10 ng/ml) for 4 days, m5-7 cells were further cultured in the presence or absence of GEF (25 mM) for 48 h. Assessmentof the viable cell number and immunoblotting with anti-LC3B Ab were performed as described above. (*p < 0.05： Dox (�) vs. Dox (þ)).

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S. Sugita et al. / Biochemical and Biophysical Research Communications 461 (2015) 28e34

response to GEF was still observed in non-lung cancer cell lines
such as MEF, K562, and HL-60 cells. Since K562 and HL-60 are
leukemia cell lines derived from a mesoderm, they do not express
EGFR (Fig. 1B). Therefore, autophagy initiation by GEF appeared not
to be mediated through the inhibition of EGFR kinase activity. To
conﬁrm this, we knocked out EGFR with a CRISPR/Cas9 system in
A549 cells and examined whether autophagy still could be induced
by GEF treatment. As seen in Fig. 2C, A549-EGFR-KO clones (#1 and#2) exhibited autophagy induction in response to GEF as well as
parental and control clones. Thus, the underlying molecularmechanism for autophagy initiation is dissociated from EGFR kinase inactivation and even from EGFR expression.
3.3. CAM enhances GEF-induced cytotoxicity in NSCLC
In contrast to GEF treatment, autophagy ﬂux analysis revealed
that CAM treatment did not increase the LC3B-II isoform any
further as compared to either treatment with LIs or CAM plus LIs in
A549 cells (Fig. 3A), indicating that CAM inhibits autophagy ﬂux in
A549 cells as previously reported in myeloma and breast cancer
cells [15,20]. Next, we added CAM into the culture medium forblocking GEF-induced autophagy. As seen in Fig. 3B, combined
treatment with GEF and CAM at 50 mg/ml efﬁciently created pronounced cytotoxicity in NSCLC cell lines, whereas treatment with
CAM alone yielded no cytotoxicity. Notably, CAM also enhanced thecytotoxic effect of GEF in K562, HL-60, and MEF cells. To further
conﬁrm that the enhanced cytotoxicity is due to blocking of autophagy, we used a Atg5 Tet-off MEF cell line m5-7 [28]. Pretreatment with doxycycline (Dox) resulted in complete inhibitionof GEF-induced autophagy by knockout of the Atg5 gene, which isessential for autophagy induction. Under this condition, GEFinduced cytotoxicity was signiﬁcantly pronounced as comparedwith that in Atg5þ/þ MEF cells (Fig. 3C).

3.4. ER-stress loading in NSCLC after combined treatment with GEF
and CAMWe next examined whether ER-stress loading is involved in theenhanced cytotoxicity. Expression of ER-stress related genes suchas CHOP and GRP78 was up-regulated in response to GEF treatment
within 24 h. Combined treatment with GEF and CAM resulted inpronounced gene expression, whereas treatment with CAM alone
produced almost no effect as compared with untreated control cells(Fig. 4A). CHOP is an ER-stress related transcription factor that upregulates pro-apoptotic molecules such as Bim and Bax [15,21,22],
so we compared the cytotoxicity between Chop�/�MEF and Chopþ/þ
MEF cell lines. Chop�/�MEF cells were less sensitive to GEF thanthe Chopþ/þ MEF cell line (Fig. 4B). In addition, knockdown of CHOP
in PC-9 cells by siRNA (PC-9 CHOP-KD cells) resulted in attenuationof GEF-induced cytotoxicity (Fig. 4C), indicating that ER-stressmediated CHOP induction is involved in the cytotoxic effect of
GEF. However, enhanced cytotoxicity by combining CAM plus GEF
was still detectable in both the Chop�/� MEF cell line and in PC-9
CHOP-KD cells (Fig. 4B, C). These data suggest that GEF plus CAM
did increase ER-stress loading including CHOP, but other molecule(s) appeared to be involved in the pronounced cytotoxicity by
GEF plus CAM.

Fig. 4. ER-stress loading after treatment with GEF and/or CAM in NSCLC cell lines. (A) Kinetics of CHOP and GRP78 expressions assessed by quantitative real-time PCR during 24 h
and 48 h of exposure to GEF (25 mM), CAM (50 mg/ml), and GEF (25 mM) plus CAM (50 mg/ml) in NCSLC cell lines. The data of the real-time PCR products for each gene
were standardized to GAPDH as an internal control. The expression levels of GRP78 and CHOP were compared with those in untreated control cells. (B) A Chop�/�MEF cell line andChopþ/þMEF cell line were cultured in the presence of GEF at various concentrations with/without CAM (50 mg/ml) for 48 h. The number of viable cells was assessed by Cell TiterBlue
as described in Materials and methods. (*p < 0.05：Chop�/�MEF vs. Chopþ/þMEF, #p < 0.05: GEF alone vs. GEF þ CAM) (C) PC-9 cells were treated with CHOP siRNA and control
siRNA for 48 h. The cells were treated with/without GEF (25 mM; G) and CAM (50 mg/ml; C) for 24 h. Cellular proteins were separated by 11.25% SDS-PAGE and immunoblotted with
anti-CHOP mAb. Immunoblotting with anti-GAPDH mAb was performed as an internal control for protein loading. Alternatively, after siRNA treatment, the cells were further treated
with/without GEF at various concentrations in the presence or absence of CAM (50 mg/ml) for 48 h. The number of viable cells was assessed by Cell TiterBlue. (*p < 0.05：control
siRNA vs. CHOP siRNA, #p < 0.05: GEF alone vs. GEF þ CAM).

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We here demonstrate that GEF induces autophagy in all celllines regardless of EGFR expression (Fig. 2). Since all of these celllines inhibited cell growth in response to GEF to a greater or lesser
degree, autophagy might be induced as a metabolic stress response.
However, comparing these cell lines, autophagy induction was not
well-correlated to cell-growth inhibition but occurred to almost thesame extent at 25 mM of GEF (Fig. 2A). According to our previous
report, imatinib induces autophagy in various cell lines regardlessof BCR-ABL expression, and, surprisingly, imatinib exhibited thecytoprotective effect in non-BCR-ABL expressing cells [32]. In thepresence of imatinib, the variable cell number was signiﬁcantly
increased in HL-60 and MEF cells under serum deprivation [32].
These data suggest the existence of target molecule(s) of TKIs forcytoprotective autophagy initiation that might be completely
dissociated from the primary target.
We here also present that CAM enhances the cytotoxicity of GEF
along with blocking GEF-induced cytoprotective autophagy. Since
CAM is a clinically well used macrolide antibiotic, this drug appears
to be a strong candidate for TKI-therapy for improving therapeutic
outcomes in NSCLC patients. Although CAM enhances the cytotoxicity of GEF along with ER-stress loading (Figs. 3 and 4), we could
not clearly detect pronounced apoptosis induction as assessed byAnnexin V staining nor could we detect cleavages of caspase-3 and
PARP or morphological apoptotic features such as chromatincondensation, nuclear fragmentation, and formation of an
apoptotic body (Supplementary data, Figs. S1 to S3.). In addition,
Chop/MEF and PC-9 CHOP-KD cells, both of which should beresistant against ER-stress mediated apoptosis, still exhibited pronounced cytotoxicity after combined treatment (Fig. 4B, C).
Therefore, non-apoptotic cell death including necroptosis might beinvolved in this phenomenon [33,34]. Further studies for identifying the target molecule(s) for autophagy induction as well as
identifying the true executor(s) for pronounced cell death areworthy of being carried out as the next step.

Conﬂict of interest
The authors declare no conﬂict of interest.

Acknowledgments
We thank Dr. Noboru Mizushima (The University of Tokyo,
Graduate School and Faculty of Medicine, Tokyo, Japan) for the kind
gift of Tet-off Atg5 m5-7 cells. We also thank Ms. Ayako Hirota forher assistance with preparing the manuscript. This study was
supported by funds provided through an MEXT-Supported programof the Strategic Research Foundation at Private Universities(S1411011, 2014e2018) from the Ministry of Education, Culture,
Sports, Science and Technology of Japan to M.H. and K.M., and a
Grant-in-Aid from Tokyo Medical University Cancer Research to
K.M.

Appendix A. Supplementary data
Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.bbrc.2015.03.162.

Transparency document
Transparency document related to this article can be foundonline at http://dx.doi.org/10.1016/j.bbrc.2015.03.162.

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international journal of oncology 46: 474-486, 2015

Targeting the integrated networks of aggresome
formation, proteasome, and autophagy potentiates
ER stress‑mediated cell death in multiple myeloma cells
SHota Moriya1, SeiicHiro KoMatSu2, KaHo yaMaSaKi1, yuSuKe Kawai1,
HiroKo KoKuBa3, ayaKo Hirota1, Xiao-fang cHe1, MaSato inaZu4,
aKiHiKo gotoH5, MaSaKi HiraMoto1 and KeiSuKe MiyaZawa1
Departments of 1Biochemistry and 2Breast oncology, 3laboratory of electron Microscopy, 4institute ofMedical Science, tokyo Medical university; 5Department of Hematology, juntendo university, tokyo, japan
received September 22, 2014; accepted october 30, 2014
Doi: 10.3892/ijo.2014.2773
Abstract. the inhibitory effects of macrolide antibiotics
including clarithromycin (caM) on autophagy flux havebeen reported. although a macrolide antibiotic exhibits
no cytotoxicity, its combination with bortezomib (BZ), a
proteasome inhibitor, for the simultaneous blocking of theubiquitin (ub)-proteasome and autophagy-lysosome pathways
leads to enhanced multiple myeloma (MM) cell apoptosis induction via stress overloading of the endoplasmic reticulum (er).
as misfolded protein cargo is recruited by histone deacetylase 6 (HDac6) to dynein motors for aggresome transport,
serving to sequester misfolded proteins, we further investigated the cellular effects of targeting proteolytic pathways andaggresome formation concomitantly in MM cells. Pronounced
apoptosis was induced by the combination of vorinostat [suberoylanilide hydroxamic acid (SaHa); potently inhibits
HDac6] with caM and BZ compared with each reagent or a2-reagent combination. caM/BZ treatment induced vimentin
positive-aggresome formation along with the accumulation of
autolysosomes in the perinuclear region, whereas they were
inhibited in the presence of SaHa. the SaHa/caM/BZ
combination treatment maximally upregulated genes related
to er stress including c/eBP homologous protein (cHoP).
Similarly to MM cell lines, enhanced cytotoxicity with cHoP
upregulation following SaHa/caM/BZ treatment was shownby a wild‑type murine embryonic fibroblast (MEF) cell line;
however, a CHOP‑deficient MEF cell line almost completely
canceled this pronounced cytotoxicity. Knockdown of HDac6
with sirna exhibited further enhanced caM/BZ-induced

Correspondence to: Dr Keisuke Miyazawa, Department of Biochemistry, tokyo Medical university, 6-1-1 Shinjuku, Shinjuku-ku, tokyo
160-8402, japan
e-mail: miyazawa@tokyo-med.ac.jp
Key words: multiple myeloma, aggresome, proteasome, autophagy,
er stress

cytotoxicity and cHoP induction along with the cancellationof aggresome formation. targeting the integrated networks ofaggresome, proteasome, and autophagy is suggested to induce
efficient ER stress‑mediated apoptosis in MM cells.
Introduction
Multiple myeloma (MM) is a refractory hematopoietic malignancy showing clonal plasma cell accumulation. a major
breakthrough in MM treatment has been the introduction of
the first‑in‑class proteasome inhibitor bortezomib (BZ) (1,2).
Moreover, the treatment of relapsed and refractory MM is now
possible with carfilzomib, a second‑generation proteasome
inhibitor, and immunomodulatory agents. this has offered
new alternatives for vulnerable patients (3,4). However, patientswith relapsed and refractory MM are still urgent issues andrequire treatment combinations (5,6). increasing lines ofevidence indicate that proteasome inhibition induces misfolded
protein accumulation in the endoplasmic reticulum (er) (7,8).
this evokes er stress followed by the unfolded protein
response (uPr) (9,10). uPr mainly functions: i) to decrease
protein entry into the er by suppressing translational rate;and ii) to increase the folding capacity of the er via chaperon
protein translational activations. following incorrect foldingin the er, proteins are retro-translocated for degradation in
the cytoplasm via the ubiquitin (ub)-proteasome pathway[i.e., er-associated degradation (eraD)]. a failure in all theadaptation strategies triggers apoptosis and induces c/eBP
homologous protein (cHoP) (gaDD153), a pro-apoptotic
transcription factor and other pathways (7-10). Since MM ischaracterized by the uncontrolled cell growth of monoclonal
antibody (mab)-producing plasma cells, production of large
quantities of unfolded or misfolded immunoglobulin triggers
er stress. thus, therapeutic manipulation of the uPr pathway
appears to disrupt cellular mechanisms for processing highprotein loads and cellular stress, and further leads to death of
MM cells.
Macroautophagy (hereafter, ‘autophagy’) occurs whencellular proteins and organelles (e.g., er) are enveloped in
an autophagosome and degraded in lysosomes by lysosomal-262-

Moriya et al: aggreSoMe, ProteaSoMe, anD autoPHagy in MyeloMa cellShydrolases (11,12). although autophagy is considered abulk non-selective degradation of long-lived proteins andorganelles, recent reports revealed the selective degradation pathway of ubiquitinated protein via autophagy using
docking proteins (e.g., p62) and related proteins (e.g.,nBr1), having both a microtubule-associated protein 1
light chain 3 (lc3)-interacting region and a ub-associateddomain (13,14). Thus, overflowed ubiquitinated proteins arebound to p62 and subsequently engulfed into an autophagosome via the lc3-interacting region in p62. this indicates that
autophagy also acts as a compensatory degradation system
when the proteasome system is impaired (14). we previously
reported that the inhibition of autophagy using the autophagy
inhibitor bafilomycin A1 (Baf) enhanced BZ-induced apoptosis by burdening er stress in MM cell lines (15). we also
reported that macrolide antibiotics [e.g., clarithromycin (caM)
and azithromycin (aZM)] attenuated or blocked autophagy
flux, possibly mediated through the inhibition of the lysosomal
function, and that er stress loading was enhanced by both the
BZ-induced inhibition of the ub-proteasome system and thecaM- or aZM-induced inhibition of the autophagy-lysosome
system. this is followed by cHoP transcriptional activationand the induction of apoptosis in MM and breast cancer
cells (16,17). therefore, concomitant blocking of proteasome
and autophagy appears to be a promising combination therapy.
Moreover, misfolded/unfolded proteins are sequestrated
into aggregates and transported. they are then discarded
from the cytoplasm by dynein motors through the microtubule network to the aggresome (18). The class Ⅱ histone
deacetylase 6 (HDac6), which is a microtubule-associateddeacetylase and an aggresome component, has the capacity
to bind both polyubiquitinated misfolded proteins anddynein motors (19). By acting as an adaptor between ubiquitinated protein aggregates and dynein, HDac6 enables
aggregated protein loading onto the dynein motor protein
complex (20). thus, in the formation of aggresome at the
microtubule-organizing center (Mtoc), HDac6, dynein,
and polyubiquitinated proteins functionally interact with eachother (19,20). if HDac6 is lacking, cells will not be able toclear cytoplasmic unfolded protein aggregates; cells cannot
appropriately form aggresomes; and cells become hypersensitive to unfolded protein accumulation (19). therefore, HDac6
appears to be another critical factor in the management of
unfolded protein-induced stress at the cellular level. Moreover,some parts of the aggresome are reported to be degraded via
the autophagy-lysosome system (21,22). a recent study also
revealed that p62 regulates the accumulation and autophagic
clearance of protein aggregates by directly binding to HDac6,
and this interaction appears to regulate HDac6 activity (23).
all these lines of evidence suggest the integrated intracellular networks of proteasome, autophagy, and aggresome forunfolded protein processing. er stress loading may be further
enhanced by targeting both intracellular proteolytic pathways
and aggresome formation (24). to prove this hypothesis aimed
for clinical application, we intentionally attempted to use
well-approved drugs such as caM, BZ, and vorinostat [suberoylanilide hydroxamic acid (SaHa)], an orally bioavailable
inhibitor of HDac with a half maximal inhibitory concentration (ic50) of 37 nM and which has passed the assessmentof the food and Drug administration for cutaneous t-cell475

lymphoma treatment (25,26). in the present investigation, weclearly demonstrated that the SaHa/caM/BZ combination
treatment induced marked er stress-mediated MM cell death.
this provides a promising treatment for MM patients in theform of ‘er stress-loading therapy’.
Materials and methods
Reagents. BZ was purchased from Selleck chemicals (Houston,tX, uSa). caM was purchased from tokyo chemicalindustry co., ltd. (tokyo, japan), SaHa was from cayman
chemical co. (ann arbor, Mi, uSa), and tubacin was from
Sigma-aldrich (St. louis, Mo, uSa). BZ, SaHa and tubacinwere dissolved in dimethyl sulfoxide to make stock solutions
at concentrations of 1, 10 and 1 mM, respectively. caM wasdissolved in ethanol to prepare stock solutions of 5 mg/ml.
Cell lines and culture conditions. for this study, the MM
cell lines iM-9 and rPMi-8226, and the lung carcinoma
cell line H226 were obtained from the american typeculture collection (atcc) (Manassas, Va, uSa). thehuman MM cell line KMS-12-Pe was obtained from thejapanese collection of research Bioresources (jcrB) (osaka,japan). a cHoP-/- murine embryonic fibroblast (MEF) cellline (cHoP-Ko-Dr) established from a 13.5-day-old cHoP-/mouse embryo by SV-40 immortalization and a cHoP+/+ Mef
cell line (Dr-wild-type) established by SV-40 immortalization
as a control cell line for cHoP-Ko-Dr were also obtained
from the atcc. these authorized cell lines were expanded
and frozen in aliquots within 1 month after obtaining fromthe cell banks. each aliquot was thawed and the cells wereused for the experiments within 2 months after thawing. iM-9,
H226, rPMi-8226, and KMS-12-Pe cells were cultured inrPMi-1640 medium (Sigma-aldrich) supplemented with 10%
fetal bovine serum (fBS) (Biowest SaS, nuaillé, france),
2 mM l-glutamine, penicillin (100 u/ml), and streptomycin(100 µg/ml) (wako Pure chemicals industries, tokyo, japan).
cHoP-Ko-Dr and Dr-wild-type cells were maintainedin Dulbecco's modified eagle's medium (Sigma-aldrich)
supplemented with 10% fBS, 2 mM l-glutamine, penicillin(100 u/ml), and streptomycin (100 µg/ml). all cell lines were
cultured in a humidified incubator containing 5% CO2 and
95% air at 37̊C.
Assessment of viable number of cells. the number of viable
cells was assessed using celltiter-Blue cell Viability assay
kit (Promega corp., Madison, wi, uSa) according to the manufacturer's instructions as previously described in detail (16).
Immunoblotting. immunoblotting was performed as previously described (15,16). in brief, cells were lysed with riPa
lysis buffer supplemented with a protease and phosphataseinhibitor cocktail (both from nacalai tesque, Kyoto, japan).
Cellular proteins were quantified using a DC Protein Assaykit (Bio-rad, Hercules, ca, uSa). equal amounts of proteins
were loaded onto the gels, separated by sodium dodecyl sulfate
polyacrylamide gel electrophoresis (SDS-Page), and transferred onto immobilon-P membrane (Millipore, Billerica,
Ma, uSa). the membranes were probed with primary
antibodies (abs) such as anti-acetylated-α-tubulin (6-11B-1)

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international journal of oncology 46: 474-486, 2015

figure 1. cell growth inhibition and α-tubulin acetylation after treatment with suberoylanilide hydroxamic acid (SaHa) in myeloma cell lines. (a) the
myeloma cells KMS-12-Pe, rPMi-8226, and iM-9 were cultured in the presence of SaHa at various concentrations for 48 h. cell growth inhibition wasassessed as described in Materials and methods. (B) after treatment with SaHa (at 0.5 µM in KMS-12-Pe, rPMi-8226, and at 1.0 µM in iM-9) for 16-48 h,cellular proteins were lysed, separated by 11.25% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‑PAGE), and immunoblotted using a specificmonoclonal antibody (mab) against the acetylated α-tubulin. immunoblotting with anti-α-tubulin mab was performed as an internal control.

mab, anti- α-tubulin (B-7) mab, anti-gaPDH (6c5) mab,anti-HDac6 (H-300) ab, and anti-ub (P4D1) mab, which were
all purchased from Santa cruz Biotechnology, inc. (Santa cruz,
ca, uSa) and anti-ParP ab (cell Signaling technology, inc.,
Danvers, Ma, uSa). immunoreactive proteins were detectedwith horseradish peroxidase-conjugated secondary abs (cellSignaling technology, inc.) and an enhanced chemiluminescence reagent (Millipore). Densitometry was performed usinga Molecular imager chemiDoc XrS System (Bio-rad).
RNA interference. for the gene silencing of HDac6 inrPMi-8226 and H226 cells, HDac6 sirna and control
sirna were purchased from life technologies (grand island,ny, uSa) and whose sequences are described as follows:
HDac6 sense, ccagcacagucuuauggauggcuau
and antisense, auagccauccauaagacugugcugg.
sirnas were diluted to a final concentration of 33 nM in
opti-MeM Ⅰ (Life Technologies). Transfection was performed
with the cells at 40% confluency using lipofectamine
rnaiMaX transfection reagent (life technologies) according
to the manufacturer's instructions. Knockdown efficiency wasassessed by immunoblotting.
Gene expression analysis. total rna was isolated fromcell pellets using isogen (wako Pure chemicals industries)
and genomic Dna was removed using rQ1 rnase-freeDnase (Promega corp.) at 37̊C for 30 min, followed byextraction with phenol chloroform and ethanol precipitation. reverse-transcription using a PrimeScript rt MasterMix (takara Bio, inc., Shiga, japan) was performed according
to the manufacturer's instructions. real-time polymerase
chain reaction (Pcr) was performed on 3 ng of cDna using
validated SyBr-green gene expression assays for human er
stress-related genes (CHOP, BAX, BIM, DR5, GADD34 and
GRP78) in combination with SyBr Premix ex Taq Ⅱ (Takara
Bio, inc.). the sequences of primers and reaction conditions were previously described (16). Quantitative real-time

Pcr was performed in duplicates in a thermal cycler Dicereal-time System tP800 (takara Bio, inc.). the data wereanalyzed using thermal cycler Dice real-time System
Software version 5.00 (takara Bio, inc.), and the comparative
ct method (2-ΔΔct) was used for the relative quantification ofgene expression. the data of real-time Pcr products werestandardized to GAPDH as an internal control. To confirm thespecific amplification of target genes, each gene product was
further separated by using 1.5% agarose gel after real-time
Pcr to detect a single band at the theoretical product size,
as well as by analysis of the dissociation curve for detecting asingle peak.
Immunocytochemistry and confocal microscopy. cells werespread on slide glasses using cytospin 4 centrifuge (thermo
Fisher Scientific, Inc., Rockford, IL, USA) to make slide glass
preparations. Cells were fixed for 20 min in ice‑cold methanoland permeabilized with 0.1% triton X-100 for 20 min, followedby blocking with 2% bovine serum albumin in tBSt (25 mM
tris, 137 mM nacl, 2.7 mM Kcl, 0.05% tween-20, pH 7.4)
for 1 h. cells were immunostained with primary abs suchas mouse anti-vimentin (V9) mab, mouse anti-ub mab, andlysosomal-associated membrane protein-1 (laMP-1) (H4a3)
mabs (all from Santa cruz Biotechnology, inc.). thesecondary abs used for f luorescence detection were
alexa fluor ® 488 f(ab')2 fragment of goat anti-mouseigg (H+l) ab (life technologies). nuclei were stained with
4',6-diamidino-2-phenylindole (DaPi) (wako Pure chemicals
industries). Slides were mounted with Slowfade gold antifade
reagent (life technologies). analysis by confocal microscopy
was performed using the confocal laser scanning fluorescencemicroscope fV10i-Doc (olympus corp., tokyo, japan).
Assessment of aggresome by fractionation of detergent‑solubleand ‑insoluble proteins. cells were lysed with triton X-100
lysis buffer (10 mM tris-Hcl, 150 mM nacl, 2% triton X-100,
pH 7.8) supplemented with a protease inhibitor cocktail (nacalai-264-

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477

figure 2. cell growth inhibition after treatment with suberoylanilide hydroxamic acid (SaHa), bortezomib (BZ), and clarithromycin (caM) in myeloma celllines. (a) upper panels: myeloma cell lines rPMi-8226, iM-9, and KMS-12-Pe were cultured in the presence of various concentrations of SaHa with/without
either BZ (5 nM) or caM (50 µg/ml) for 48 h. lower panels: myeloma cell lines were cultured in the presence of various concentrations of BZ with/without
either SaHa (at 0.5 µM in KMS-12-Pe, rPMi-8226, and at 1.0 µM in iM-9) or caM (50 µg/ml) for 48 h. Viable cell numbers were assessed as describedin Materials and methods. *P<0.05 SaHa/BZ/caM vs. SaHa/BZ and SaHa/caM and BZ/caM. (B) iM-9 cells were treated with either BZ (5 nM) or
caM (50 µg/ml) in the presence or absence or SaHa (1 µM) for 48 h. cellular proteins were separated by 11.25% sodium dodecyl sulfate polyacrylamide
gel electrophoresis (SDS-Page), and immunoblotted with anti-ParP monoclonal antibody (mab). immunoblotting with gaPDH mab was performed as aninternal control for protein loading.

tesque). the lysates were centrifuged at 12,000 g for 30 min at
4̊C. The supernatant was then collected as the soluble fraction. the pellets (which contain the insoluble protein) werethen resuspended in sodium dodecyl sulfate (SDS) lysis buffer(10 mM tris-Hcl, 150 mM nacl, 2% SDS, pH 7.8) and sonicated for 30 sec with a tip sonicator VP-5S (taitec, Saitama,
japan) to prepare the insoluble fraction. equal volumes of eachpellet and supernatants were boiled for 5 min in SDS-Pagesample buffer (125 mM tris-Hcl, 4% SDS, 20% glycerol,
0.002% BPB, pH 6.8) and analyzed by SDS-Page.
Electron microscopy. Cells were fixed with 2.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4) for 1 h. the samples
were further fixed in 1% osmium tetroxide for 1 h, dehydrated
in graded ethanol (30-100%), and embedded in Quetol 812
epoxy resin (nisshin eM co., ltd., tokyo, japan). ultrathin

sections were cut with an ultracut j microtome (reichert-jung,
Vienna, austria). the sections were stained with lead nitrateand uranium acetate, and subjected to electron microscopic analysis using the scanning electron microscope
jeM-1200 EXⅡ (JEOL, Tokyo, Japan).
Statistical analysis. all data are expressed as mean ± SD.
Statistical analysis was performed using Mann-whitneyu test (two-tailed).
Results
SAHA, BZ, and CAM combination treatment potently enhanced
MM cell apoptosis. treatment with SaHa for 48 h resulted ina dose-dependent inhibition of cellular growth in all MM celllines (fig. 1a). the ic50 was 1.2 µM in KMS-12-Pe, 1.5 µM in

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international journal of oncology 46: 474-486, 2015

figure 3. effects of histone deacetylase 6 (HDac6) inhibition on myeloma cell growth. (a) Suppression of HDac6 expression using HDac6 sirna:rPMi-8226 cells were pre-treated with either HDac6 sirna or control sirna for 48 h. Subsequently, the cells were cultured in the presence or absenceof bortezomib (BZ) (10 nM) with/without clarithromycin (caM) (50 µg/ml) for 48 h. *P<0.05 cont sirna vs. HDac6 sirna. (B) enzymatic inhibitionof HDAC6 with a specific HDAC6 inhibitor, tubacin: RPMI‑8226 cells were treated with tubacin at indicated concentrations in the presence or absence ofeither BZ (5 nM) or caM (50 µg/ml) for 48 h. Viable cell numbers were assessed as described in Materials and methods. *P<0.05 tB/BZ/caM vs. tB/BZ
and tB/caM.

rPMi-8226, and 2.1 µM in iM-9 cells. SaHa-treated MM cells
exhibited apoptotic morphologic features such as fragmentationof the nucleus and formation of an apoptotic body along withcleavage of ParP and caspase-3 (data not shown). the regulationof the stability and function of a microtubule has been associatedwith α-tubulin reversible acetylation, and HDac6 functions as
α‑tubulin deacetylase (27). Immunoblotting using a specific Ab
for the acetylated α-tubulin revealed that, in response to SaHa
(at 0.5 µM for KMS-12-Pe and rPMi-8226, at 1 µM for iM-9),
the acetylation of α-tubulin was detectable within 16 h andpersisted for at least 48 h in all three cell lines tested (fig. 1B).
in our previous studies, autophagy flux was shown to be
blocked by caM, and that the BZ and caM combination treatment resulted in er-stress overloading followed by enhanced
induction of apoptosis in MM and breast cancer cells (16,17).
Since HDac6 has been implicated in aggresome formationthat results in sequestering overabundant intracellular unfolded
proteins (18), we examined whether the SaHa plus BZ and/or
caM combination treatment further increased cytotoxicity via
er stress loading. as shown in fig. 2a, the combined treatment with SaHa/BZ, but not with SaHa/caM, potentiatedcell growth inhibition. notably, although caM treatment alone
at 50 µg/ml did not inhibit the growth of cells, the combined
SaHa/BZ/caM treatment showed a clearly pronounced cytotoxicity compared with the SaHa/BZ or BZ/caM treatment
in all MM cell lines. this enhanced cytotoxicity was mediatedthrough apoptosis induction since the pronounced expression of
the cleaved ParP was observed in response to the suboptimal
concentrations of the SaHa/BZ/caM combination in iM-9
cells (fig. 2B).
to confirm that this enhanced cytotoxicity is mediatedthrough HDac6 inhibition, we next attempted to knock down
HDac6 with sirna. Pre-treatment with HDac6 sirna effectively suppressed the expression level of HDac6 in rPMi-8226
cells. under this condition, the cell growth inhibition by the
BZ/caM treatment was pronounced compared with the cells
pre‑treated with control siRNA. In addition, tubacin, a specificHDac6 inhibitor, reproduced the pronounced cytotoxicity whencombined with BZ and BZ/caM (fig. 3a and B) (28). theseresults suggest that HDac6 inhibition appears to be involvedin the enhancement of cytotoxicity induced by the addition of
SaHa to BZ/caM-containing cell culture medium.
SAHA suppressed the aggresome formation induced by simul‑
taneous inhibition of proteasome and autophagy in MM cells.
To further confirm that the pronounced cytotoxicity by SAHA
shown in fig. 2a was due to the inhibition of HDac6 activity
involved in aggresome formation, we assessed aggresome
formation in the presence or absence of SaHa. in rPMi-8226
cells, immunocytochemistry using anti-vimentin mab showedthat the combined treatment with BZ/caM induced a dense
deposit of vimentin in the perinuclear region, which is awell-known marker for aggresome (29). this vimentin deposit
was clearly suppressed in the presence of SaHa (fig. 4a).
However, since the MM cell lines including rPMi-8226
were very sensitive to the SaHa/BZ/caM treatment, inwhich apoptosis was induced within 18 h of exposure to thesereagents (data not shown), precise analysis of aggresome
formation was practically difficult using MM cells. wetherefore used the lung squamous carcinoma cell line H226
which is less sensitive, but shows pronounced cytotoxicity inresponse to the SaHa/BZ/caM combination similarly to
MM cell lines (fig. 4B). as shown in fig. 4c, the prominent
vimentin deposit in the perinuclear region was observed
after combined treatment with BZ/caM for 24 h. thevimentin deposit with high fluorescent intensity was evidently
suppressed in the presence of SaHa, indicating that SaHa
inhibits BZ/caM-induced aggresome formation (fig. 4c).
Similarly to SaHa, treatment with HDac6 sirna suppressedthe vimentin condensation in the perinuclear region in H226
cells (fig. 4D-f).
in addition, immunocytochemistry using anti-ub ab
exhibited the clustering of ub-positive dots in the perinuclear
region by BZ/caM, whereas they were dispersed in the presence of SaHa in H226 cells (data not shown). as an alternative
assessment of aggresome, we treated KMS-12-Pe with BZ,
caM, or BZ/caM in the presence or absence of SaHa.
the cells were then treated with lysis buffer containing 2%-266-

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479

figure 4. assessment of aggresome formation by immunocytochemistry
using anti-vimentin monoclonal antibody (mab). (a) rPMi-8226 cells
were treated with either bortezomib (BZ) (5 nM) or clarithromycin (caM)(50 µg/ml) in the presence/absence of suberoylanilide hydroxamic
acid (SaHa) (0.5 µM) for 16 h. immunocytochemistry using anti-vimentin
mab was performed. 4',6-diamidino-2-phenylindole (DaPi) stainingshows the position of nucleus (blue). (B) H226 cells were cultured in thepresence of various concentrations of SaHa with/without either BZ (5 nM)
or caM (50 µg/ml) for 48 h. Viable cell numbers were assessed as describedin Materials and methods. *P<0.05 SaHa/BZ/caM vs. SaHa/BZ
and SaHa/caM. (c) H226 cells were treated with either BZ (5 nM) or
caM (50 µg/ml) in the presence/absence of SaHa (0.5 µM) for 24 h.
immunocytochemistry using anti-vimentin mab was performed. (D) H226
cells were pre-treated with histone deacetylase 6 (HDac6) sirna orcontrol sirna for 48 h. then, the expression levels of HDac6 and acetylated α‑tubulin were assessed by immunoblotting. (Ε) After pre‑treatment
with sirna, H226 cells were further exposed to BZ (5 nM) and/or
caM (50 µg/ml) for 24 h. immunocytochemistry was performed using
anti-vimentin mab.

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figure 4. continued. assessment of aggresome formation by immunocytochemistry using anti-vimentin monoclonal antibody (mab). (f) Percentage of‘aggresome-positive’ cells in H226 cells after treatment with either BZ±caM±SaHa or BZ±caM pre-treated with HDac6 sirna or control sirna. one
hundred cells stained with anti-vimentin mab were assessed for their staining pattern. the cells with high dense staining in the perinuclear region (indicatedby * in the left lower panel) were defined and plotted as ‘aggresome‑positive’ cells. Each scale bar indicates 10 µm.

triton X-100, and cellular proteins were subsequently fractionated into the detergent-soluble and the -insoluble fraction.
the detergent-insoluble fraction (cell pellets) was sonicatedfor 30 sec in the presence of 2% SDS and boiled for 5 min.
thereafter, the samples were loaded on the gels, separated
by 11.25% SDS-Page, and immunoblotted with anti-ub ab.
the samples derived from the detergent-soluble fractions
were processed similarly. as shown in fig. 5, the increased
intracellular polyubiquitinated proteins were detectable
after treatment with BZ and BZ/caM, but not with caM or
SaHa in the detergent-soluble fraction (upper panel). in thedetergent-insoluble fraction (lower panel) at higher molecularweights, prominent polyubiquitinated proteins were detected
as aggresome contents in the cells treated with BZ/caM.
figure 5. assessment of aggresome formation by immunoblotting using
anti-ubiquitin (ub) monoclonal antibody (mab). KMS-12-Pe cells were treatedwith either bortezomib (B) (5 nM) or clarithromycin (c) (50 µg/ml) in the presence/absence of suberoylanilide hydroxamic acid (SaHa) (0.5 µM) for 16 h.
then cellular proteins were lysed with lysis buffer containing 2% triton X-100
for 30 min at 0̊C and followed by centrifugation at 14,000 x g for 30 min. The
supernatants were used for ‘soluble fractions’. cell pellets were resuspended inbuffer containing 2% sodium dodecyl sulfate (SDS) and sonicated for 30 sec
and further used as ‘insoluble fraction’. each fractionated protein was loaded
and separated by 11.25% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-Page), and immunoblotted with anti-ub mab.

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figure 6. lysosome positioning after treatment with bortezomib (BZ) and/or clarithromycin (caM) in the presence or absence of suberoylanilide hydroxamic
acid (SaHa) in H226 cells. (a) H226 cells were treated with BZ and/or caM in the presence or absence of SaHa for 24 h. then, immunocytochemistry was
performed using anti-laMP-1 monoclonal antibody (mab) for detecting lysosome positioning. Merged images with phase contrast, 4',6-diamidino-2-phenylindole (DaPi) (blue), and lysosomal-associated membrane protein-1 (laMP-1) (green). each scale bar indicates 10 µm. (B-D) electron microscopy of H226
cells after treatment with BZ/caM. (B) the region surrounded by the dashed line shows the clustering of organelles including lysosomes and mitochondria.
(c) the arrows indicate autophagosomes and autolysosomes. the asterisk (*) appears to represent a precursor form of aggresome. (D) clustering autolysosomes/lysosomes might represent ‘lysophagy’ (30). n, nucleus. each scale bar indicates (B) 2 µm and (c and D) 500 nm.

treatment with BZ alone also induced accumulation of thedetergent-insoluble polyubiquitinated proteins but to a lesser
extent than that induced by treatment with BZ/caM. it was
noteworthy that the increased ubiquitinated proteins in thedetergent-insoluble fraction were suppressed in the presenceof SaHa. this also indicates the inhibition of aggresome inresponse to SaHa.

immunocytochemistry using anti-laMP-1 mab showedlysosome accumulation in the perinuclear region along withaggresome formation after BZ/caM treatment. as is thecase of vimentin-positive aggresome formation, the clusteringof laMP-1-positive dots was suppressed in the presence of
SaHa (fig. 6a). we therefore performed electron microscopy for precise analysis of the cytosolic area strongly stained

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with anti-vimentin ab. However, contrary to what we had
expected, electron microscopy exhibited no typical featuresof the aggresome structure such as massive accumulation ofclosely packed electron-dense particles surrounded by a cageof bundles of intermediated filaments in the region of thecentrosome, which was first described by Johnston et al in1998 (30). instead, there was the clustering of a number oflysosomes, autophagosomes, autolysosomes, and mitochondria with increased filaments in response to the BZ/CAM
treatment (fig. 6B). a few numbers of unstructured particles
with a rather high density, which might be the aggresome
precursor, were detectable in this area (fig. 6c). it was noteworthy that many lysosomal fusions or lysosomal aggregatesin various sizes were observed. these might represent thephenomenon termed ‘lysophagy’ which has recently beenreported by others, that is, the lysosome engulfed into an
autophagosome or autolysosome (fig. 6D) (31). in the presence of SaHa, the clustering of these organelles in theperinuclear region was suppressed to some extent along withthe decreased filamentous structures. However, an increased
number of lysosomes including autolysosomes was stillobserved compared with the cells treated with either caM
or BZ alone (fig. 6a).
Concomitant aggresome, proteasome, and autophagy
targeting enhanced ER stress loading. Since aggresome isreported to form as an adaption process against overabundant
intracellular unfolded or misfolded proteins, we hypothesized that HDac6 inhibition with SaHa further enhances
BZ/caM-induced er stress loading to the cells. as we
expected, the expression levels of er stress-related genes
including CHOP were markedly pronounced in the presenceof SaHa; the expression ratios of cHoP to untreated control
cells increased to 5-fold by BZ/caM treatment, and further
increased to 25-fold by BZ/caM/SaHa treatment in iM-9
cells (fig. 7a). in H226 cells, similarly to SaHa, HDac6
sirna treatment enhanced BZ/caM-induced cHoP
expression compared with the cells treated with control
sirna (fig. 7B). cHoP is an er stress-related pro-apoptotic transcription factor which upregulates pro-apoptotic
genes (e.g., BIM, BAX, DR5) and one of the critical molecules
playing a role in the induction of apoptosis in response to er
stress (9,10). in addition, enhanced cytotoxicity was shownby a wild-type Mef cell line along with cHoP upregulation by the combination of the three reagents, whereas thecHoP-/- Mef cell line derived from a CHOP-deficientmouse almost completely canceled this pronounced cytotoxicity (fig. 7c). therefore, the pronounced cytotoxicity andapoptosis in response to the SaHa/BZ/caM combination
treatment shown in fig. 2a is strongly suggested to be due tothe upregulation of cHoP in response to er stress loading.
Discussion
we showed that er stress-mediated apoptosis in MM cells
was potently enhanced by the simultaneous targeting of
aggresome formation, proteasome, and the autophagy-lysosome system. concomitant SaHa, BZ, and caM treatment
markedly enhanced cHoP induction via er stress loading in
MM cells (fig. 7a), whereas a cHoP-/- Mef cell line nearlycompletely cancelled the pronounced cytotoxicity in responseto the SaHa/BZ/caM combination treatment (fig. 7c).
these results strongly suggest that cHoP upregulation
appears to be involved, at least in part, in the pronounced
cytotoxicity shown in fig. 2a. our recent report also showedthat combined treatment with SaHa/BZ/caM exhibited
potent cytotoxicity along with upregulation of cHoP inbreast cancer cell lines (24). Since this enhanced cytotoxicity by er stress loading is associated with inhibition of
the BZ/caM-induced aggresome formation (figs. 4 and 5),
induction of aggresome itself appears to function as cytoprotective against intracellular overloading of unfolded proteinsas previously demonstrated in various neurodegenerative
disorders (32,33).
in this study, we intentionally attempted to use the clinically approved drugs for blocking aggresome formation andthe proteolytic pathways. although exhibiting a potent inhibitory effect on HDac6, SaHa (vorinostat) is a pan-HDacinhibitor; therefore, the inhibitory effects on other HDacs
cannot be completely excluded for the pronounced er stressloading and apoptosis induction shown in figs. 2a and 7a.
However, tubacin, which is a specific inhibitor of HDAC6,
also exhibited pronounced cytotoxicity (fig. 3B), andknockdown of HDac6 superimposed the effects of SaHa,
such as suppression of vimentin-positive aggresome formation induced by BZ/caM treatment (fig. 4c and e),
and further enhanced the er stress-related gene upregulation along with the enhanced BZ/caM treatment
cytotoxicity (figs. 3a and 7B). therefore, HDac6 appears to
be a pivotal target for efficient ER stress loading under proteasome/autophagy inhibition. from the standpoint of er stressloading, intracellular active protein synthesis appears to betheoretically an important factor for determining sensitivity.
MM cell sensitivity to BZ/SaHa-induced cell death is regulated by Myc (34). intracellular er content, protein synthesis
rates, percentage of aggresome-positive cells, and sensitivity
to BZ/SaHa-induced cell death directly correlated withMyc expression. we previously reported that cycloheximide
suppressed BZ/caM-induced er stress loading and cytotoxicity in MM cells (16). thus, the potent effects shown inthis study should depend in part on de novo protein synthesis.
this may explain some differences among the MM cell lines,
H226 cells, and Mef cell lines in terms of cytotoxicity ander stress loading in response to these reagents shown in thisstudy.
along with vimentin-positive aggresome formation in the perinuclear region, fig. 6 shows a number of
autophagosomes and autolysosomes after BZ/caM treatment. electron microscopy demonstrated the clustering ofautolysosomes in this area. These findings are in agreementwith a recent study demonstrating that proteasome inhibition causes the formation of a zone around the centrosome
where microtubular transport of lysosomes is suppressed,
resulting in lysosome entrapment and accumulation (35).
interestingly, the authors of the previous study reported thatthe microtubule-dependent transport of other organelles,
including autophagosomes, mitochondria and endosomes,
is also blocked in this entrapment zone. therefore, theperinuclear region stained with anti-vimentin ab, whereautolysosomes clustered along with the increased number-270-

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483

figure 7. endoplasmic reticulum (er) stress loading after combined treatment with suberoylanilide hydroxamic acid (SaHa), bortezomib (BZ), and clarithromycin (caM). (a) iM-9 cells were treated with BZ (5 nM) and/or caM (50 µg/ml) in the presence or absence of SaHa (1 µM) for 16 h. gene expressions
related to er stress were assessed by real-time polymerase chain reaction (Pcr). (B) effects of histone deacetylase 6 (HDac6) sirna on er stress-related
gene expressions in H226 cells.

of mitochondria, may in part overlap to ‘the entrapmentzone’ (figs. 4e and 6B) (35). it is also suggested that, upon

proteasome inhibition, the targeting of aggregated proteins toaggresome is coordinated with lysosome positioning around

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international journal of oncology 46: 474-486, 2015

figure 7. continued. endoplasmic reticulum (er) stress loading after combined treatment with suberoylanilide hydroxamic acid (SaHa), bortezomib (BZ),
and clarithromycin (CAM). (C) Cell growth inhibition of wild‑type murine embryonic fibroblast (MEF) cell line and CHOP-/- Mef cell line (16) after treatment with BZ in the presence/absence of SaHa (2.5 µM) and/or caM (50 µg/ml) for 48 h. Viable cell numbers were assessed as described in Materials andmethods. *P<0.05 BZ/caM/SaHa vs. BZ/SaHa and BZ/caM.

figure 8. Proposed scheme for intracellular networks of unfolded protein
processing and endoplasmic reticulum (er) stress loading via systematic
blockage of proteolytic pathways and aggresome formation.

the body to facilitate degradation via autophagy. However,our data showed that lysosomal clustering including
autolysosome further became prominent by the combined
treatment with caM/BZ compared with that by the BZ
treatment alone (fig. 4f). indeed, the ad dition of SaHa
in the caM/BZ culture medium suppressed lysosomal
clustering in the perinuclear region; however, an increased

number of cytosolic autolysosomes were still detectable(data not shown). this was possibly because of the suppression of lysosomal clearance upon autophagy inhibition by
caM. we and others have reported that macrolide antibiotics inhibit autophagy flux, although the precise molecularmechanism still remains to be clarified (16,17,36). themacrolide antibiotic Baf, which is a well-used autophagy
inhibitor for in vitro experiments, was initially evaluated forits selective inhibition of a proton-pumping V-atPase (37).
Baf induces the disruption of vesicular proton gradientsand raises the pH of acidic vesicles at nanomolar concentrations. This disruption of vesicular acidification in response
to Baf appears to prevent autophagosome fusion withlysosomes, resulting in the inhibition of autophagy. it wasalso reported that treatment with aZM increased lysosomal
pH in macrophages, which may lead to the inhibition oflysosomal hydrolases having an optimal low pH for their
enzymatic activities (38). in this context, if caM exerts thesame effect on lysosomes, accumulation of autolysosomesby the suppression of lysosomal clearance itself appears tomake ‘lysophagy’ apparent under proteasome inhibition.
a similar phenomenon regarding the selective sequestration of damaged lysosome by autophagy was previously
reported, in which under the condition of lysosomal damage
induced by silica, monosodium urate, and a lysomotropic
reagent, autophagy loss results in the in vitro inhibition oflysosomal biogenesis and the in vivo deterioration of acutekidney injury (39). Identification of the target molecules ofmacrolide antibiotics involved in autophagy flux inhibition,
as well as the molecular mechanism of lysophagy including
the recognition process of impaired lysosomes appears to beimportant issues that need to be clarified.

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Moriya et al: aggreSoMe, ProteaSoMe, anD autoPHagy in MyeloMa cellStaking our data and accumulating lines of evidence
together, we can draw an integrated network scheme
among aggresome, proteasome, and autophagy as shownin fig. 8 (9,10,13,14). upon inhibition of proteasome andautophagy, overabundant ubiquitinated protein aggregatesare transported to Mtoc to form aggresomes. under thiscondition, further inhibition of aggresome formation mostefficiently induces er stress loading in cells with a highprotein synthesis rate such as MM cells. thus, by combining
clinically available drugs such as vorinostat, BZ, and caM,
this systematic strategy for blocking the processing of
intracellular unfolded proteins appears to be applicable torefractory/relapsed MM patients.
Acknowledgements
the authors are indebted to Dr edward f. Barroga, associate
Professor and Senior Medical editor of the Department ofinternational Medical communications of tokyo Medical
university for the editorial review of the report. this study
was supported by funds of a grant-in-aid for Scientificresearch (c) (no. 26460478) and MeXt-Supported Program
for the Strategic research foundation at Private universities(S1411011, 2014-2018) from the Ministry of education, culture,
Sports, Science and technology of japan, a grant-in-aid fromtokyo Medical university cancer research, and a grant-in-aid
from taisho toyama Pharmaceutical co., ltd. (tokyo, japan)
to K.M.; and a grant-in-aid for young Scientist (B) from
the Ministry of education, culture, Sports, Science andtechnology of japan (no. 25860398) and a research grantfrom tokyo Medical university to S.M.
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NEWS & VIEWS RESEARCH
retinal body and sequenced its DNA, whichcontained a much higher proportion of DNA ofplastid origin than equivalent samples from thewhole cell.
Although ocelloids are exceptionallycomplex, warnowiids are not the only microbial cells with eye-like subcellular structures.
A diversity of eukaryotic microorganisms
perceive light using different kinds of eyespots. One such structure is the eyespot of
the green alga Chlamydomonas reinhardtii(Fig. 1b), a unicellular relative of land plants.
This eyespot is located at the edge of the alga’s
plastid and is made up of lipid globules, rich inorange carotenoid pigments, that are stacked
in compartments inside the plastid envelope.
As such, this globule layer is thought to provide
directionality and contrast by shielding andreflecting light from one side of the organism
on to two light-sensitive proteins called type 1
rhodopsins that localize with this eyespot5–7.
These two proteins have intrinsic light-gatedcation-channel activity (and are therefore
named channelrhodopsins) and have been
demonstrated to act as photoreceptors thattrigger movement in response to light5–7.
Cryptophyte algae such as Guillardia theta
also build eyespot structures that are locatedin plastids8, and movement of these cells in
response to light is mediated by the functionof at least two type 1 rhodopsin proteins9,
similar to Chlamydomonas. The alga Euglena
gracilis also has an orange-red eyespot,
although, in contrast to the previous examples,
this structure is associated with the base of theflagellum3, the cells’ swimming propeller. The
photoreceptor in Euglena has been identified
as a photoactivated adenylyl cyclase10 protein.
In yet another branch of the tree of life are
the eyespot-like structures of the swimming
spores of Blastocladiomycota fungi (Fig. 1c).
These structures are lipid-filled vesicles calledside-body complexes that are located close
to the large mitochondrion of these fungal
cells11. The side-body complex is overlaid with
type 1 rhodopsin proteins. In Blastocladiellaemersonii, the type 1 rhodopsin photosensor
contains a guanylyl cyclase domain, whichallows the protein to control the productionof cyclic GMP (ref. 12), a key chemical messenger in vertebrate vision. Recent work13 on
warnowiid ocelloids has also suggested thatmessenger RNA encoding a type 1 rhodopsin
is associated with the retinal body.
These examples demonstrate the wealth ofsubcellular structures and associated lightreceptor proteins across diverse microbial
groups. Indeed, all of these examples represent distinct evolutionary branches in separate
major groups of eukaryotes3. Even the plastidassociated eyespots are unlikely to be theproduct of direct vertical evolution, because
the Chlamydomonas plastid is derived from a
primary endosymbiosis and assimilation of acyanobacterium, whereas the Guillardia plastid is derived from a secondary endosymbiosis

in which the plastid was acquired ‘secondhand’ by intracellular incorporation of a red
alga14. Using gene sequences recovered fromthe warnowiid retinal body, Gavelis et al. investigated the ancestry of this organelle by building phylogenetic trees for the plastid-derived
genes. Their analysis demonstrated that thismodified plastid is also of secondary endosymbiotic origin from a red alga.
Although derived independently, there arecommon themesin the evolution
The ocelloidof these eye-likedemonstrates apeak in subcellular structures. Manyof them involve
complexity
the reconfiguraachieved throughtion of cellular
repurposing
membrane sysmultiple
tems to produce
components.
an opaque bodyproximal to a sensory surface, a surface that in four of the fiveexamples probably involves type 1 rhodopsins.
Given the evolutionary derivation of these systems, this represents a complex case of convergent evolution, in which photo-responsive
subcellular systems are built up separatelyfrom similar components to achieve similarfunctions. The ocelloid example is striking
because it demonstrates a peak in subcellularcomplexity achieved through repurposing

multiple components. Collectively, thesefindings show that evolution has stumbled on
similar solutions to perceiving light time andtime again. ■Thomas A. Richards is in the Departmentof Biosciences, University of Exeter, Exeter
EX4 4QD, UK. Suely L. Gomes is in the
Departamento de Bioquímica, Instituto deQuímica, Universidade de São Paulo,São Paulo 05508-000, Brazil.
e-mails: t.a.richards@exeter.ac.uk;
sulgomes@iq.usp.br
1. Galen. Galen on the Usefulness of the Parts of the
Body (Cornell Univ. Press, 1968).
2. Kepler, J. Astronomiae Pars Optica Manuscript
presented to Rudolf II, Holy Roman Emperor (1604).
3. Jékely, G. Phil. Trans. R. Soc. B 364, 2795–2808(2009).
4. Gavelis, G. S. et al. Nature 523, 204–207 (2015).
5. Schmidt, M. et al. Plant Cell 18, 1908–1930 (2006).
6. Sineshchekov, O. A., Jung, K.-H. & Spudich, J. L.
Proc. Natl Acad. Sci. USA 99, 8689–8694 (2002).
7. Berthold, P. et al. Plant Cell 20, 1665–1677 (2008).
8. Lucas, I. A. N. Br. Phycol. J. 17, 13–19 (1982).
9. Sineshchekov, O. A. et al. Biophys. J. 89, 4310–4319(2005).
10. Ntefidou, M., Iseki, M., Watanabe, M., Lebert, M. &Häder, D.-P. Plant Phys. 133, 1517–1521 (2003).
11. Lovett, J. S. Bacteriol. Rev. 39, 345–404 (1975).
12. Avelar, G. M. et al. Curr. Biol. 24, 1234–1240 (2014).
13. Hayakawa, S. et al. PLoS ONE 10, e0118415 (2015).
14. Archibald, J. M. Curr. Biol. 19, R81–R88 (2009).
This article was published online on 1 July 2015.

M Y ELO I D D I S E AS E

Another action of athalidomide derivative
Lenalidomide effectively treats a blood disorder caused by the 5q chromosomal
deletion. A study shows that the drug binds to its target, CRBN, to promote thebreakdown of an enzyme encoded by a gene in the 5q region. See Article p.183
TA K U M I I T O & H I R O S H I H A N D AAround 60 years ago, thalidomide wasdeveloped as a sedative and sold in
more than 40 countries. But the drug
was soon banned because of its association
with serious developmental defects, such aslimb deformities, in children whose mothers
had taken it while pregnant. Now, thalidomide
is being re-evaluated and is recognized as aneffective treatment for myeloma, a cancer ofplasma cells of the immune system. Moreover,derivatives of thalidomide have been developed; these compounds, which include lenalidomide and pomalidomide, make up a classof immunomodulatory drug termed IMiDs1.
As well as being effective against myeloma,
lenalidomide can treat 2 a type of myelodysplastic syndrome (MDS) — a disorderof blood stem cells (haematopoietic cells) —that is caused by a deletion of the long arm ofchromosome 5. In this issue, Krönke et al.3
(page 183) provide a model of lenalidomide
action in the context of this mutation.
The protein CRBN was identified as a directtarget of thalidomide through affinity-bead
technology4. CRBN functions as a substraterecognition component of an E3 ubiquitin
ligase enzyme complex that catalyses the
conjugation of ubiquitin molecules to specific substrate proteins, thereby marking theproteins for degradation. CRBN is also boundby lenalidomide and pomalidomide5,6 and is
now regarded as a primary target of IMiDs —this binding is required for both the damaging
and the therapeutic effects of the drugs. Previous research7–9 showed that lenalidomide
and pomalidomide promote the degradation
9 J U LY 2 0 1 5 | V O L 5 2 3 | N AT U R E | 1 6 7© 2015 Macmillan Publishers Limited. All rights reserved-275-

RESEARCH NEWS & VIEWSaThalidomide(and derivatives)
O O

O

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ThalidomidebLenalidomide
O O

O

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N

Glutarimide
Lenalidomide

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NH2
CK1α

Figure 1 | Thalidomide and its derivatives confer the substrate specificity of CRBN. a, The
immunomodulatory drugs thalidomide, lenalidomide, pomalidomide and CC-122 all bind to the protein
CRBN, which is a substrate-recognition subunit of an E3 ubiquitin ligase enzyme complex. Binding of thedrugs to CRBN induces the enzyme complex to attach ubiquitin molecules to the transcription factors
Aiolos and Ikaros, which marks them for degradation7–9. This process explains the efficacy of these drugs
for myelomas — cancers that arise from dysregulated proliferation of plasma cells, for which Aiolos andIkaros are survival factors. b, Krönke et al. show that binding of CRBN by lenalidomide, but not the otherrelated drugs, also induces ubiquitination and degradation of the regulatory enzyme CK1α. This activity
underlies the drug’s effective treatment of myelodysplastic syndrome (MDS), which is caused by the 5q
chromosomal deletion and results in the loss of one copy of the gene encoding CK1α. The glutarimide
moiety common to these drugs is marked.

of the transcription factors Ikaros (IKZF1)
and Aiolos (IKZF3) by modulating the activity of a CRBN–ubiquitin ligase complex, andthat this process underlies the drugs’ efficacy
against myeloma. However, it has been unclear
whether all IMiDs confer the same substratespecificity on CRBN.
The deletion of the long arm of chromosome 5, called del(5q), is seen in some people
with MDS, and leads to hyperproliferationof haematopoietic cells and their ineffective
differentiation. Lenalidomide is known toselectively induce apoptotic cell death in cellswith del(5q). The deletion means that people
affected have only one copy of the genes locatedin that chromosomal region. Krönke et al. suggest that this haploinsufficiency might explain
the efficacy of lenalidomide in this disease.
By studying the effect of lenalidomide treatment on protein ubiquitination and abundanceof myeloid blood cells, the authors identify the
enzyme casein kinase 1α (CK1α) as a target ofubiquitin-mediated degradation in the presence of the drug. Deletion of the CRBN geneusing CRISPR/Cas9 genome-editing technology abolished this degradation, suggesting that
this effect is crucially dependent on CRBN.
The gene that encodes CK1α, CSNK1A1, is on
the long arm of chromosome 5, so it seems thatthe result of this degradation is to compound
the already lower than normal levels of thisenzyme that result from the deletion.
CK1α regulates the activity of multiple
proteins. For example, it negatively regulates
p53, a tumour-suppressor protein. The CK1αinhibitor D4476 has been shown to activate
p53 and induce apoptosis in cells with only
one copy of CSNK1A1 (ref. 10). Krönke andcolleagues demonstrate that CK1α depletion
sensitizes normal human haematopoietic
cells to lenalidomide. They also confirm thatoverexpression of CK1α confers lenalidomide
resistance on cells with the del(5q) mutation.
By contrast, overexpression of Ikaros did not
suppress lenalidomide-mediated therapeutic
effects on del(5q) cells.
Rodents have been shown to be resistant to
IMiDs1,6, and the authors found that lenalidomide did not decrease CK1α levels in normalmouse cells. They demonstrate that a singleamino-acid difference between the humanand mouse forms of CRBN is responsible forthis different response to the drug, and thatmouse cells expressing mouse CRBN with thesubstituted human amino acid were subject
to lenalidomide-dependent CK1α degradation. The authors then generated ‘humanized’mouse haematopoietic cells, which expressed
the modified CRBN protein and had onlya single copy of CSNK1A1. They show thatlenalidomide treatment induced increased
apoptotic death of the cells. Moreover, theincrease in apoptosis in these cells was countered when p53 levels in the cells were reduced,
which fits well with the previous reportof p53 involvement in this pathway10. Thisdevelopment represents substantial progress in the field of IMiDs and thalidomide
research — at last, through genetic modification of mouse CRBN, investigators can

1 6 8 | N AT U R E | V O L 5 2 3 | 9 J U LY 2 0 1 5© 2015 Macmillan Publishers Limited. All rights reserved-276-

use mice to study these drugs.
Krönke et al. then compared the effectof lenalidomide on a human MDS del(5q)cell line with that of other IMiDs, including
CC-122, a compound currently undergoingphase I clinical trials for blood cancers andsolid tumours. All IMiDs that were examineddecreased levels of Aiolos and Ikaros, and theefficacy of CC-122 was stronger than that oflenalidomide. But only lenalidomide degraded
CK1α (Fig. 1). Interestingly, high concentrations of CC-122 suppressed lenalidomideinduced CK1α degradation, which suggeststhat lenalidomide competes with CC-122 forbinding to CRBN in cells and that lenalidomide confers a distinct substrate specificity
on CRBN.
Accumulating evidence3,7–9 indicates thatthe substrate recognition of CRBN is alteredin response to each ligand that binds it.
X-ray crystal structures of CRBN bound to
IMiDs have revealed that a common glutarimide moiety in these compounds (Fig. 1) issufficient for this binding to occur5,6. It is
possible that the remaining structure of eachligand might be important for determining
the enzyme’s substrate specificity. Recently,
uridine, a nucleotide base, was found to bindto the same ligand-binding pocket of CRBN11,
and it is likely that CRBN also has cellular
ligands that alter its substrate specificity.
In plants, the hormone auxin functions as amolecular glue to attach the hormone’s target
TIR1, a substrate-recognition component of an
SCF ubiquitin ligase, to its substrate AUX/IAA
proteins12. It is unclear whether ligands suchas IMiDs also function to glue CRBN to its
substrate or whether they act through anothermechanism. Elucidation of the 3D structureof the CRBN–ligand–substrate complex will
provide a deeper understanding of the substrate-recognition process and contributeto the development of potent and clinicallyeffective drugs. ■Takumi Ito and Hiroshi Handa are in the
Department of Nanoparticle Translational
Research, Tokyo Medical University,
Shinjuku-ku, Tokyo, 160-8402, Japan.
e-mail: hhanda@tokyo-med.ac.jp
1. Ito, T., Ando, H. & Handa, H. Cell. Mol. Life Sci. 68,
1569–1579 (2011).
2. List, A. et al. N. Engl. J. Med. 355, 1456–1465(2006).
3. Krönke, J. et al. Nature 523, 183–188 (2015).
4. Ito, T. et al. Science 327, 1345–1350 (2010).
5. Fischer, E. S. et al. Nature 512, 49–53 (2014).
6. Chamberlain, P. P. et al. Nature Struct. Mol. Biol. 21,
803–809 (2014).
7. Krönke, J. et al. Science 343, 301–305 (2014).
8. Lu, G. et al. Science 343, 305–309 (2013).
9. Gandhi, A. K. et al. Br. J. Haematol. 164, 811–821(2014)
10. Järås, M. et al. J. Exp. Med. 211, 605–612 (2014).
11. Hartmann, M. D. et al. J. Struct. Biol. 188, 225–232(2015).
12. Tan, X. et al. Nature 446, 640–645 (2007).
This article was published online on 1 July 2015.

Article

The E3 ligase synoviolin controls body weight andmitochondrial biogenesis through negative
regulation of PGC-1b
Hidetoshi Fujita1,2,†, Naoko Yagishita3,†, Satoko Aratani1,2,†, Tomoko Saito-Fujita4, Saori Morota5,
Yoshihisa Yamano3, Magnus J Hansson6, Masato Inazu1, Hiroko Kokuba1, Katsuko Sudo7, Eiichi Sato1,8,
Ko-ichi Kawahara9, Fukami Nakajima1, Daisuke Hasegawa3, Itsuro Higuchi10, Tomoo Sato3, Natsumi
Araya3, Chie Usui11, Kenya Nishioka12, Yu Nakatani2, Ikuro Maruyama13, Masahiko Usui1, Naomi Hara5,
Hiroyuki Uchino5, Eskil Elmer6, Kusuki Nishioka1 & Toshihiro Nakajima1,2,3,8,9,14,15,*

Abstract

Subject Categories Metabolism; Post-translational Modifications, Proteolysis& Proteomics

Obesity is a major global public health problem, and understanding its pathogenesis is critical for identifying a cure. In this study, agene knockout strategy was used in post-neonatal mice to delete
synoviolin (Syvn)1/Hrd1/Der3, an ER-resident E3 ubiquitin ligase
with known roles in homeostasis maintenance. Syvn1 deficiency
resulted in weight loss and lower accumulation of white adipose
tissue in otherwise wild-type animals as well as in geneticallyobese (ob/ob and db/db) and adipose tissue-specific knockout miceas compared to control animals. SYVN1 interacted with and ubiquitinated the thermogenic coactivator peroxisome proliferatoractivated receptor coactivator (PGC)-1b, and Syvn1 mutants
showed upregulation of PGC-1b target genes and increase in mitochondrion number, respiration, and basal energy expenditure in
adipose tissue relative to control animals. Moreover, the selective
SYVN1 inhibitor LS-102 abolished the negative regulation of
PGC-1b by SYVN1 and prevented weight gain in mice. Thus, SYVN1
is a novel post-translational regulator of PGC-1b and a potential
therapeutic target in obesity treatment.
Keywords endoplasmic reticulum; mitochondria; obesity; PGC-1b; synoviolin

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1042

DOI 10.15252/embj.201489897 | Received 26 August 2014 | Revised 16 January
2015 | Accepted 19 January 2015 | Published online 19 February 2015
The EMBO Journal (2015) 34: 1042–1055
See also: MS Soustek & P Puigserver (April 2015)

Introduction
Obesity is characterized by the excessive accumulation of adipose
tissue, as well as increased risk of diabetes, hypertension, cardiovascular diseases, and depression, and is an enormous economic andsocial burden (Wickelgren, 1998). White adipose tissue (WAT) functions as an energy reservoir and as an endocrine organ, and various
inflammatory mediators and cytokines are overexpressed in the
WAT of obese individuals, which leads to chronic inflammation(Hotamisligil, 2006). Metabolism in adipocytes has been extensively
studied (Rosen & MacDougald, 2006; Lefterova & Lazar, 2009);
however, there are presently no effective and safe pharmacologicaloptions for obesity prevention and treatment. A better understanding

Institute of Medical Science, Tokyo Medical University, Shinjuku-ku, Tokyo, Japan
Department of Future Medical Science, Tokyo Medical University, Shinjuku-ku, Tokyo, Japan
Institute of Medical Science, St. Marianna University School of Medicine, Kawasaki, Kanagawa, Japan
Department of Obstetrics and Gynecology, University of Tokyo, Bunkyo-ku, Tokyo, Japan
Department of Anesthesiology, Tokyo Medical University, Shinjuku-ku, Tokyo, Japan
Mitochondrial Pathophysiology Unit, Department of Clinical Sciences, Lund University, Lund, Sweden
Animal Research Center, Tokyo Medical University, Shinjuku-ku, Tokyo, Japan
Medical Research Center, Tokyo Medical University Hospital, Shinjuku-ku, Tokyo, Japan
Department of Biomedical Engineering, Osaka Institute of Technology, Asahi-ku, 11Neurology and Geriatrics, Japan
Neurology and Geriatrics, Faculty of Medicine, Graduate School of Medical and Dental Sciences, Kagoshima University, Sakuragaoka, Kagoshima, Japan
Department of Psychiatry, Juntendo University Nerima Hospital, Nerima-ku, Tokyo, Japan
Department of Neurology, Juntendo University School of Medicine, Bunkyo-ku, Tokyo, Japan
Laboratory and Vascular Medicine, Graduate School of Medical and Dental Sciences, Kagoshima University, Sakuragaoka, Kagoshima, Japan
integrated Gene Editing Section (iGES), Tokyo Medical University Hospital, Shinjuku-ku, Tokyo, Japanchi, Japan
Bayside Misato Medical Center, Niida, Ko*Corresponding author. Tel: +81 3 3345 0165; Fax: +81 3 6302 0265; E-mail: marlin@tokyo-med.ac.jp†
These three authors contributed equally to this workª 2015 The Authors

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The EMBO Journal

E3 ligase synoviolin and energy expenditureof the underlying mechanisms is therefore necessary to develop suitable treatments for obesity and associated metabolic diseases.
The mitochondrion is a key organelle in cellular energy control
and has been implicated in obesity (Bournat & Brown, 2010).
Peroxisome proliferator-activated receptors (PPARs) (Viana
Abranches et al, 2011) and their coactivators (PPAR coactivator(PGC)-1a, PGC-1b, and PGC-1-related coactivator) (Puigserver
et al, 1998; Andersson & Scarpulla, 2001; Lin, 2001; Kressler et al,2002) play important roles in mitochondrion biogenesis and energy
metabolism, including H2O2-based respiration and b-oxidation of
fatty acids. Gene knockout studies have suggested functional
differences between PGC-1a and PGC-1b, for instance in terms oflethality (Scarpulla, 2008). Interestingly, PGC-1b overexpression
results in an increase in mitochondrion number and respiratory
function in cultured cells (St-Pierre et al, 2003); furthermore,
PGC-1b transgenic mice show high energy expenditure andresistance to obesity (Kamei et al, 2003). In addition to the mitochondrion, the endoplasmic reticulum (ER) is also thought to play
an important role in obesity (Cnop et al, 2012). For example,
mutations or deficiency in genes that function in the unfolded
protein response produces insulin resistance in a mouse model(Cnop et al, 2012; Wang & Kaufman, 2012). Nonetheless, the roleof the ER-associated protein degradation (ERAD) pathway inobesity remains obscure.
Synoviolin (Syvn1), a mammalian homolog of Hrd1p/Der3p, isan E3 ubiquitin (Ub) ligase that was identified from the cDNA ofrheumatoid synovial cells (Amano et al, 2003) and plays importantroles in the ERAD pathway (Yamasaki et al, 2005). In a series ofstudies, we have demonstrated the importance of SYVN1 expressionin arthritis (Amano et al, 2003) and fibrosis (Hasegawa et al, 2010).
Inflammation plays a critical role in regulating metabolic status, andSyvn1 is a key target for inflammatory cytokines such as tumor
necrosis factor a (TNFa), interleukin (IL)-1, and IL-17 (Gao et al,
2006; Toh et al, 2006, 2010).
In the present study, conditional Syvn1 knockout mice weregenerated to clarify the role of Syvn1 in obesity. The results highlight a novel function for SYVN1 in the control of body weight andmitochondrial biogenesis through negative regulation of PGC-1b.

Results
Generation of Syvn1-deficient mice
To study the post-neonatal function of SYVN1, tamoxifen (Tam)inducible Syvn1 knockout mice were generated that carry homozygous floxed-Syvn1 alleles and a Cre-estrogen receptor (ER) transgene(Hayashi & McMahon, 2002) (Fig 1A). Efficient recombination
was confirmed in Syvn1 conditional knockout (CAG-Cre-ER;
Syvn1flox/flox) and control (Syvn1flox/flox and Syvn1flox/+) micefollowing Tam administration by PCR (genome), real-time PCR(mRNA), and Western blotting (protein) (Fig 1B–D). A significant
reduction in body weight was observed in CAG-Cre-ER;Syvn1flox/flox
mice as early as 1 week after Tam administration until it wasapproximately 80% that of control mice (Fig 1E and F). A slightdecrease in weight was also observed in Syvn1 heterozygous CAGCre-ER;Syvn1flox/+ mice (Supplementary Fig S1). Three experiments
were used to determine whether the weight loss was due to low

food intake, absorption-related malnutrition, or both. First, the daily
food intake was measured; however, differences were almost notfound in terms of food consumption between CAG-Cre-ER;
Syvn1flox/flox and control mice at day 1 and 7, and higher foodconsumption was observed in CAG-Cre-ER;Syvn1flox/flox mice thanin control mice at day 14 (Fig 1G). Second, macroscopic and microscopic examinations showed comparable amounts of food residue
in the gut and no histological abnormalities in the intestine of
CAG-Cre-ER;Syvn1flox/flox mice compared to controls. Third, serum
biochemical tests showed no significant differences in the levels of
several biomarkers of nutrition and liver and kidney function
between the two groups (Supplementary Table S1). However, adetailed anatomical analysis showed a marked reduction of WAT in
CAG-Cre-ER;Syvn1flox/flox mice relative to controls (Fig 1H–J).
Loss of Syvn1 in ob/ob mice and db/db mice causes
body weight loss
Two well-established mouse models of obesity (ob/ob and db/db) inwhich the leptin signal is constitutively inactivated (Tartaglia et al,1995) were used to determine whether Syvn1 deficiency is associatedwith a reduction in body weight at the level of the central nervous
system under conditions of constitutive food intake. The expression
level of SYVN1 was higher in ob/ob and db/db than in ob/+ anddb/+ mice (Fig 2A). Moreover, Tam administration resulted in asignificant loss of body weight in CAG-Cre-ER;Syvn1flox/flox;ob/ob
and CAG-Cre-ER;Syvn1flox/flox;db/db compound mutants (Fig 2B and
C). An anatomical dissection revealed a reduction in fat mass in
CAG-Cre-ER;Syvn1flox/flox;ob/ob and CAG-Cre-ER;Syvn1flox/flox;db/db
mice compared to Syvn1flox/flox;ob/ob and Syvn1flox/flox;db/db mice,
respectively (Fig 2D and E, and Supplementary Fig S2). No differences in food intake were noted across groups (Fig 2F and G). Taken
together, these results indicate that SYVN1 directly controls bodyweight at the level of peripheral energy expenditure, and not at thelevel of the central nervous system.
WAT is the source of body weight loss resulting from
Syvn1 deletion
To directly test the effect of Syvn1 knockout on peripheral energy
expenditure in WAT, adipose-specific Syvn1 knockout mice weregenerated by crossing Syvn1flox/flox mice with the adiponectin(Adipoq)-Cre line (Herman et al, 2012; Kleiner et al, 2012) to obtain
Adipoq-Cre;Syvn1flox/flox mice. Cre-mediated Syvn1 deletion in WAT
was confirmed by PCR (Fig 3A) and Western blotting (Fig 3B). The
body weight of Adipoq-Cre;Syvn1flox/flox mice was approximately
90% that of controls (Fig 3C and D), an effect that was associatedwith a reduction in WAT (Fig 3E and F). These results indicate that
SYVN1 directly targets WAT to control body weight.
SYVN1 directly interacts with PGC-1b
To understand the molecular mechanism, we first used microarray
analysis to establish the expression of genes in WAT. Genes
involved in b oxidation and mitochondria biogenesis (Supplementary Fig S3) were increased in CAG-Cre-ER;Syvn1flox/flox micecompared with Syvn1flox/flox mice. Several studies have indicatedthat thermogenic nuclear receptors PPARa and b and theirª 2015 The Authors

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E3 ligase synoviolin and energy expenditure

B

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Hidetoshi Fujita et al

Figure 1. Body weight and WAT in post-neonatal Syvn1 mutant mice.
A

Schematic depiction of Syvn1 gene targeting strategy. Homologous recombination resulted in exons 2–12 being flanked by loxP sites; deletion was achieved by
Tam-induced Cre recombinase-mediated excision.
B
PCR products amplified from genomic DNA isolated from tails on day 7 after Tam administration.
C
Real-time PCR analysis of adipocyte mRNA from control (Control) and CAG-Cre-ER;Syvn1flox/flox (KO) mice on day 7 after Tam administration.
D
Western blotting of spleen proteins on day 7 after Tam administration.
E, F Changes in body weight. *P < 0.05, **P < 0.01 for Tam-treated CAG-Cre-ER;Syvn1flox/flox mice versus vehicle-treated CAG-Cre-ER;Syvn1flox/flox mice, Tam- or vehicletreated Syvn1flox/flox mice, Tam- or vehicle-treated C57BL/6J mice (analysis of variance with Tukey–Kramer post hoc analysis).
G
Average daily food intake measured after Tam injection.
H–J Fat accumulation in post-neonatal Syvn1 KO mice on 7 day after Tam administration. Subcutaneous adipose (H), epididymal adipose (I), and mesentery adipose (J)tissues are shown (Control, n = 8; KO, n = 3). Mesentery fat is shown by black arrows.
Data information: All data are expressed as mean  SD. Data were analyzed with the Student’s t-test. *P < 0.05, **P < 0.01.
Source data are available online for this figure.

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The EMBO Journal

E3 ligase synoviolin and energy expenditureob/+ ob/ob db/+ db/db

A
SYVN1
Tublin

80

*

0
5
Tam
(125 mg/kg BW )10*
15 Days

Control;ob/ob

KO;ob/obE

130
Body weightchange (%)*

Control; ob/ob (n=12)KO; ob/ob (n=6)

40C

Subcutaneous adipose tissue

100

60

F

D

* P < 0.05

Food intake (mg per g)

Body weightchange (%)

120* P < 0.05

G
Subcutaneous adipose tissue

110
90
Control; db/db (n=12) *70

KO; db/db (n=10)

50
0

5

Tam
(125 mg/kg BW )10

15 Days
Control;db/db

KO;db/db

Food intake (mg per g)

BControl; ob/ob (n=12)KO; ob/ob (n=6)
P = 0.19200** P = 0.43
150
100
50
0
day 2-3

day8-9 day 14-15

Control; db/db (n=12)
KO; db/db (n=10)250
P = 0.19 P = 0.37 P = 0.09
200
150
100
50
0
day 2-3

day8-9 day 14-15

Figure 2. Changes in body weight and WAT in post-neonatal Syvn1 knockout (KO) and genetically obese (ob/ob and db/db) mice.
Compound mutants of Syvn1 and ob/ob or db/db were generated as described in Materials and Methods.
A
SYVN1 expression in the WAT of ob/+, ob/ob, db/+, and db/db mice.
B, C Changes in body weight in Tam-treated mice. CAG-Cre-ER;Syvn1flox/flox;ob/ob (KO;ob/ob) and CAG-Cre-ER;Syvn1flox/flox;db/db (KO;db/db) mice, red line; Syvn1flox/flox;
ob/ob, Syvn1flox/+;ob/ob, and Syvn1+/+;ob/ob (Control;ob/ob) and Syvn1flox/flox;db/db, Syvn1flox/+;db/db, and Syvn1+/+;db/db (Control;db/db) mice, blue line.
D, E Subcutaneous fat accumulation in CAG-Cre-ER;Syvn1flox/flox;ob/ob and CAG-Cre-ER;Syvn1flox/flox;db/db mice after post-neonatal Syvn1 knockout. Histological analysisof adipose tissue from Control;ob/ob and Control;db/db mice (left), and CAG-Cre-ER;Syvn1flox/flox;ob/ob and CAG-Cre-ER;Syvn1flox/flox;db/db mice (right) after Tam
administration. Subcutaneous fat is shown by white arrows.
F, G Average daily food intake measured after Tam injection in CAG-Cre-ER;Syvn1flox/flox;ob/ob (D) and CAG-Cre-ER;Syvn1flox/flox;db/db (G) mice.
Data information: Data were analyzed by the Student’s t-test and are expressed as mean  SD. *P < 0.05, **P < 0.01.
Source data are available online for this figure.

coactivators of the PGC-1 family transcriptionally regulate peripheral energy expenditure via b oxidation and mitochondria biogenesis (Lowell & Spiegelman, 2000; Scarpulla, 2006); therefore, theinteraction of SYVN1 with these factors was tested in vitro. Glutathione-S-transferase (GST)-tagged SYVN1 lacking the transmembrane
domain (GST-SYVN1DTM) bound HA-tagged PGC1b but not PPARa
or c or PGC-1a (Fig 4A). In a GST pull-down assay, PGC-1b boundthe SyU domain (amino acids, aa 236–270) of SYVN1 (Fig 4B),
which is highly conserved from Caenorhabditis elegans to humans,
but not in yeast SYVN1 orthologs (Supplementary Fig S4A). In addition, an R266A/R267A double mutation in the SyU domain
decreased this interaction (Fig 4C), but had no effect on the E3
ligase activity of SYVN1 (Supplementary Fig S4B). The GST pulldown assay mapped the SYVN1-binding domain of PGC-1b to aa
195–367 containing an LXXLL motif of middle portion (Supplementary Fig S4C). To verify the interaction in cellulo, HA-PGC-1b and
FLAG-tagged SYVN1 (SYVN1/FLAG) were co-transfected into HEK
293T cells. HA-PGC-1b co-immunoprecipitated with SYVN1/FLAG

but not the control FLAG vector (Fig 4D). To further investigate theinteraction between SYVN1 and PGC-1b, whole-cell lysates of HEK
293 cells, in which SYVN1 and PGC-1b were expressed, were precipitated with anti-SYVN1 antibody or a control non-immune mouse
immunoglobulin (Ig)G and probed with an antibody against PGC-1b
in an immunoblotting assay. Endogenous PGC-1b was detected in
the precipitate with anti-SYVN1 but not with IgG (Fig 4E). These
results clearly indicate that SYVN1 interacts in vivo with PGC-1b
under normal physiological conditions.
Since SYVN1 is an ER-resident protein and PGC-1b translocates
into the nucleus, their subcellular localization was investigated byimmunofluorescence staining in transiently transfected HEK 293T
cells. HA-PGC-1b was mainly detected in the nucleus (Fig 4F), as
previously reported (Kelly et al, 2009). However, when coexpressed with SYVN1/FLAG, the two proteins predominantly colocalized in the perinuclear region, but were not observed in the
nucleus (Fig 4F). In contrast, the coexpression of HA-PGC-1b with
SYVN1R266A, R267A/FLAG, or SYVN1DSyU/FLAG, which do notª 2015 The Authors

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E3 ligase synoviolin and energy expenditure

B

Control
KO
Control
KO
Control
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KO

WAT Liver Tail Muscle

C
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SYVN1* P < 0.05
Weight (g)ADeleted allele Tublin
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*
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E Subcutaneous adipose tissue F
Epididymal adipose tissue
400
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Weight (mg)

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KO (n=10)18

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Hidetoshi Fujita et al

60

80

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400

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200
100

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0
ControlKO

Figure 3. Effect of adipose tissue-specific Syvn1 deletion on body weight and fat mass.
A
PCR products amplified from genomic DNA isolated from WAT, liver, tail, and muscle of Adipoq-Cre;Syvn1flox/flox (KO) and Syvn1flox/flox mice (Control).
B
Western blotting of WAT proteins 15 weeks after birth.
C, D Changes in body weight after weaning. Adipoq-Cre;Syvn1flox/flox mice (KO), red line; control (Syvn1flox/flox and Syvn1flox/+) mice, blue line. Data were analyzed with
the Student’s t-test and represent the mean  SD. *P < 0.05. (D) Control and KO mice 15 weeks after birth.
E, F Reduction in subcutaneous fat (E) and epididymal adipose tissue (F) in KO mice 15 weeks after birth. Data were analyzed with the Student’s t-test and represent
the mean  SD (Control, n = 10; KO, n = 8). *P < 0.05, **P < 0.01.
Source data are available online for this figure.

interact with PGC-1b (Fig 4B and C), resulted in the nuclear localization of HA-PGC-1b (Fig 4F, Supplementary Fig S4D). These
results indicate that SYVN1 traps PGC-1b in the perinuclear
region.
PGC-1b is a novel substrate of SYVN1
An in vitro assay was carried out to determine whether PGC-1b is a
substrate of SYVN1, which is an E3 Ub ligase (Amano et al, 2003).
Polyubiquitinated PGC-1b was detected in the presence of ATP,
HA-Ub, E1, E2, and SYVN1 (Fig 4G). The ubiquitination of PGC-1b
was also examined in vivo. FLAG-tagged Ub and HA-PGC-1b were
coexpressed with wild-type (WT) or mutant SYVN1 (3S)—whichlacks Ub ligase activity (Amano et al, 2003)—in HEK 293T cells.
Ubiquitinated HA-PGC-1b was observed in WT SYVN1- but not
SYVN1 3S-expressing cells (Fig 4H). Collectively, these results
suggest that PGC-1b is a SYVN1 substrate.
Since ubiquitinated proteins are degraded by the proteasome,experiments were performed to verify whether the level of PGC-1b
protein is regulated by SYVN1. PGC-1b protein level was markedly
elevated in the WAT of CAG-Cre-ER;Syvn1flox/flox (Fig 4I) andAdipoq-Cre;Syvn1flox/flox (Supplementary Fig S4E) mice; however,
the transcript level of Ppargc1b (encoding PGC-1b) was unaltered(Supplementary Fig S4F). PGC-1b protein level was 1.4-fold higher
in Tam-treated skin fibroblasts from CAG-Cre-ER;Syvn1flox/flox micethan in vehicle-treated cells (Fig 4J). A similar observation wasmade by knocking down Syvn1 expression using small interfering1046

RNA (siRNA), which resulted in a near-complete disappearance ofSYVN1 expression in HEK 293 cells: PGC-1b protein expression in
Syvn1 siRNA-treated cells was 2.5-fold higher than in controls(Fig 4K). Finally, to investigate the role of SYVN1 in PGC-1b degradation, the proteasome inhibitor MG-132 was applied to cultured
skin fibroblasts. Similar to Tam treatment, MG-132 upregulated
PGC-1b protein levels in vehicle-treated cells by 1.6-fold, but
produced no additional effects on Tam-treated skin fibroblasts(Fig 4J). To confirm whether the SYVN1–PGC-1b interaction is critical for SYVN1-mediated PGC-1b degradation, the half-life of PGC-1b
was measured in mouse embryonic fibroblasts (MEFs) coexpressing
HA-PGC-1b and WT SYVN1 or SYVN1DSyU (Supplementary Fig
S4G). WT SYVN1 overexpression significantly shortened the half-lifeof HA-PGC-1b, whereas SYVN1DSyU had no effect (Fig 4L). These
results indicate that PGC-1b protein expression is negatively regulated by SYVN1 at the post-transcriptional level and strongly suggestthat SYVN1 is a major E3 ligase for PGC-1b in cells.
Negative regulation of PGC-1b by SYVN1
PGC-1b functions as a coactivator of several transcription factors
including PPARa and is implicated in various biological processes
such as mitochondrial biogenesis, b oxidation, and body weightcontrol (Scarpulla, 2008). To investigate the role of SYVN1 in theregulation of the coactivator and mitochondrial biogenesis functionsof PGC-1b, the activity of PPAR luciferase (PPRE X3-TK-luc) (Kimet al, 1998)—which contains three PPAR binding sites and isª 2015 The Authors

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PGC-1βPPARα 150
75
PPARγ

PR2
617

150

10% Input
GST
WT
R266A
R266A, R267A
C307S
ΔSyU

WB: anti-HADE+

HA PGC-1β
SYVN1/FLAG+++

WB: anti-HA

75

ATP
HA-Ub
E1
UbcH5cSYVN1 ΔTM
GST-PGC-1β(aa 1-367)

FLAG Abs* SYVN1

WB: anti-FLAGG

HA AbsHA PGC-1β HA PGC-1β+SYVN1
HA PGC-1β +SYVN1
R266A, R267A
/FLAG

+
+

PGC-1β150

WB: anti-HA

+
+ +
+ +
+ +
+ +
+ +

*

150

WB: anti-PGC-1β

H+ + + + ++ + + +
+
+ + +
+ +
+ +
+ + +
+
+ + + ++ +HA PGC-1β
SYVN1 3S
SYVN1 WTFLAG Ub

IP : anti-HA

250

+
+ +

+
+
+

150

150

WB: anti-FLAG

DAPI

100
75

10% input

Merge

WB: anti-HA

Tam+

PGC-1βPGC-1βPGC-1β

SYVN1

SYVN1

SYVN1

TublinTublinTublin

-

0.5

1

2

Empty
vector4 (h)Half life
4.8 h

WT

1.8 h

ΔSyU

3.6 h
WB: anti-PGC-1β

Intensity (%)

DMSO
+

L

Syvn1
siRNA

MG-132

K

Control
siRNA

KO

J

WB: anti-HA
WB: anti-SYVN1

WB: anti-PGC-1β

I
Control

236-338
ΔSyU
SyU

PR1

75

PGC-1β(aa 1-367)

F

SyU RING

PGC-1αWB: anti-HA
WB: anti-HA
GST-SYVN1
ΔTM
C

75

TM

GST
ΔTM

SP

SYVN1 1SYVN1 ΔTMSYVN1 236-338
SYVN1 ΔTM ΔSyU
SYVN1 SyU

GST-SYVN1

SYVN1 ABs

10% input
GST
GST SYVN1 ΔTM
10% input
GST
GST SYVN1 ΔTM75

B
5 % in put

PGC-1αPGC-1β

mouse IgGPPARα PPARγ
10% input
GST
GST SYVN1 ΔTM
10% input
GST
GST SYVN1 ΔTMAThe EMBO Journal

E3 ligase synoviolin and energy expenditure

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Hidetoshi Fujita et al

100
80
60
40

Empty vector
WT
ΔSyU

20
0
0

1
TIme (h)2Figure 4.

specifically regulated by PPAR and its coactivators—was measured.
As previously reported (Lin et al, 2003), reporter activity was
induced by treatment with Wy-14643, a PPARa agonist. Syvn1 but
not control siRNA transfection significantly enhanced reporter
activity under Wy-14643 induction (Fig 5A). Furthermore, the cotransfection of PGC-1b and Syvn1 siRNA activated reporter activity.
These results indicate that loss of Syvn1 enhances PPARa-mediatedtranscription in a PGC-1b-dependent manner. Conversely, SYVN1
overexpression inhibited the coactivator function of PGC-1b(Fig 5B), but this effect was decreased for the SYVN1R266A, R267A
mutant (Fig 5C).
The role of SYVN1 in the regulation of mitochondrial biogenesis
was also examined. Syvn1 siRNA-treated cells contained large

numbers of mitochondria compared to those treated with control
siRNA, and the increase was not observed in cells that wereco-transfected with both Syvn1 and Ppargc1b siRNAs (Fig 5D).
Furthermore, transcript levels of medium chain acyl-coenzyme A
dehydrogenase and mitochondrial ATP synthase b subunit, two
known PGC-1b target genes (Rodriguez-Calvo et al, 2006; Shaoet al, 2010), were upregulated in Adipoq-Cre;Syvn1flox/flox (Fig 5E
and F) and CAG-Cre-ER;Syvn1flox/flox compared to control mice
(Supplementary Fig S5A). An electron microscopic analysis revealed
increases in both the number and size of mitochondria in CAGCre-ER;Syvn1flox/flox compared to controls (Fig 5G). To investigate
whether the mitochondria in CAG-Cre-ER;Syvn1flox/flox mice werefunctional, O2 consumption in a single-cell suspension of primaryª 2015 The Authors

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E3 ligase synoviolin and energy expenditure

Hidetoshi Fujita et al

Figure 4. PGC-1b is a substrate of SYVN1.
A

GST and GST-tagged SYVN1 lacking the transmembrane domain (GST-SYVN1DTM) were incubated with HEK 293T whole-cell extracts expressing HA-PPARa, HAPPARc, HA-PGC-1a, or HA-PGC-1b.
B, C Interaction of SYVN1 with PGC-1b. (B) Schematic representation of SYVN1. TM, transmembrane domain; SyU, SYVN1 unique domain. (B, C) An in vitro binding assay
was performed with HA-PGC-1b and GST or GST-tagged SYVN1 deletion mutants.
D, E SYVN1 interacts with PGC-1b in vivo. (D) Whole-cell extracts from HEK 293T cells transfected with HA-PGC-1b and/or SYVN1/FLAG expression plasmids were
immunoprecipitated with anti-FLAG antibody. *Immunoprecipitated IgG heavy chain. (E) Whole-cell extracts from HEK 293 cells were immunoprecipitated with
anti-SYVN1 antibody. Western blotting was performed using anti-PGC-1b antibody. *Non-specific band.
F
HEK 293T cells were transfected with HA-PGC-1b (green) and/or SYVN1/FLAG, SYVN1 R266A, R267A/FLAG (red) expression plasmids. After 24 h, cells were
immunolabeled with anti-HA and anti-FLAG antibodies.
G
In vitro ubiquitination assay was performed with maltose-binding protein (MBP)-tagged SYVN1DTM-His, GST-PGC-1b (aa 1–367), E1 and E2 enzymes, and HA-Ub.
Lower panel shows the loading control.
H
Whole-cell extracts from HEK 293T cells transfected with HA-PGC-1b, Ub/FLAG, and WT or mutant SYVN1 (SYVN1 WT or 3S, respectively) expression plasmids were
immunoprecipitated with anti-HA antibody. Lower panels show the loading controls.
I
Cell extracts from adipose tissues were obtained from control and CAG-Cre-ER;Syvn1flox/flox mice 7 days after Tam administration. Western blotting was performed
using an anti-PGC-1b antibody. Representative data from three sets of control and CAG-Cre-ER;Syvn1flox/flox mice are shown.
J
Skin fibroblasts from CAG-Cre-ER;Syvn1flox/flox mice were cultured, and 48 h after Tam or vehicle treatment and addition of 10 lM MG-132 for 2 h, cell lysates werecollected for Western blotting.
K
HEK 293 cells were transiently transfected with control or Syvn1 siRNA. After 48 h, cells were incubated with 10 lM MG-132 for 2 h. Western blotting was
performed with anti-SYVN1 and anti-PGC-1b antibodies.
L
Measurement of PGC-1b half-life. Mouse embryonic fibroblasts derived from Syvn1 knockout mice were transfected with HA-PGC-1b and SYVN1/FLAG or
SYVN1DSyU/FLAG; 48 h later, cells were treated with 40 lM cycloheximide for the indicated times, and immunoblotting was performed with anti-PGC-1b
antibody. Intensity of bands was measured (right panel). Each experiment was performed at least three times.
Source data are available online for this figure.

mouse adipocytes was measured and was found to be higher in cells
from CAG-Cre-ER;Syvn1flox/flox than from control mice (Supplementary Fig S5B). Moreover, mitochondrial respiration and activity werehigher in CAG-Cre-ER;Syvn1flox/flox than in control mice (Fig 5H).
The high respiration rate was also observed in Syvn1 knockout preadipocyte cells compared to Syvn1 WT cells, and the high respiration was not observed by treatment of Syvn1 knockout cells with
Ppargc1b siRNAs (Supplementary Fig S5C). These results indicatedthat the respiratory phenotype of Syvn1 knockout is PGC1b dependent. Finally, the basal energy expenditure was increased in CAGCre-ER;Syvn1flox/flox relative to control mice (Fig 5I). These results
indicate that Syvn1 deletion enhances mitochondrial activity in vivo
and strongly suggests that PGC-1b is a functional target of SYVN1.
Selective SYVN1 inhibition attenuates weight gain
We previously demonstrated that LS-102 selectively inhibits the E3
Ub ligase activity of SYVN1 (Yagishita et al, 2012) and suppresses
rheumatoid arthritis (Yagishita et al, 2012), liver cirrhosis (Wuet al, 2014), and sarcoglycanopathy (Bianchini et al, 2014) in amouse model. Here, it was used to examine the effects of SYVN1
inhibition on obesity. LS-102 suppressed the regulation of PGC-1b
function by SYVN1, as evidenced by reduced ubiquitination of
PGC-1b (Fig 6A) and activation of PPARa-mediated transcription in
HEK 293 cells (Fig 6B). In addition, LS-102 induced the expressionof PGC-1b in WT MEFs, but not SYVN1 KO MEFs (Supplementary
Fig S6). Next, C57BL/6J mice were treated with the vehicle control
dimethylsulfoxide (DMSO) or LS-102 and their body weight wasmonitored over a 2-month treatment period (Fig 6C). Mice treatedwith LS-102 showed no weight gain as a result of normal foodintake, while food intake itself was unaffected (Fig 6D); analysis
by dissection revealed a reduction in WAT fat mass of the epididymis and fewer lipid droplets in these mice compared to controls(Fig 6E and F). Similar to the WAT of CAG-Cre-ER;Syvn1flox/flox
mice, C57BL/6J mice treated with LS-102 had greater numbers of
mitochondria (Fig 6G). The effect of LS-102 on obesity was

1048

examined in db/db mice. The body weight of LS-102-treatedanimals gradually decreased to approximately 85% of that of
DMSO-treated mice (Fig 6H and I), but there was no effect on foodintake (Fig 6J). LS102 also significantly decreased blood glucose in
db/db mice compared to controls (Fig 6K). These findings indicatethat the inhibition of SYVN1 can suppress weight gain in a mousemodel of obesity.

Discussion
Gene knockout technology is a useful method for assessing genefunction and often yields unexpected results, such as the reductions
in body weight and WAT accumulation observed in post-neonatal
Syvn1 knockout mice in the present study (Fig 1). This weight loss
was also observed in crosses with two genetically obese mouselines, ob/ob and db/db, as well as in adipose tissue-specific Syvn1
knockout mice (Figs 2 and 3). Taken together, these results indicatethat the loss of Syvn1 causes peripheral activation of energy expenditure in WAT, eventually leading to weight loss.
Obesity is a risk factor for other chronic diseases such as cardiovascular disorders and diabetes (Wickelgren, 1998). Recently, therelationship between obesity and chronic inflammation—especiallythe contributions of adipokines such as TNFa and IL-1 that linkobesity to rheumatic diseases (Abella et al, 2014)—has been closely
examined (Johnson et al, 2012). For instance, recent studies haveshown that obesity impairs the efficacy of anti-TNFa therapy inrheumatoid arthritis (RA) patients (Gonzalez-Gay & GonzalezJuanatey, 2012). We have identified SYVN1 as a causative factor for
RA, and many studies have confirmed that SYVN1 is an important
target of cytokines (Yamasaki et al, 2005; Gao et al, 2006; Toh et al,2010). Our study is the first demonstration that SYVN1 is a key forunderstanding the common feature among obesity, chronic inflammatory, and RA.
The thermogenic transcriptional coactivator PGC-1b was identified as a SYVN1-interacting molecule (Fig 4). PGC-1b, PGC-1a, andª 2015 The Authors

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A

Relative Luc activity

E
**

**

Syvn1 siRNA

1 μm
Ppargc1b siRNA+Syvn1 siRNA

number of mitochondria

25

15

1

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5

1 μm

1 μm

1.5

20

0

MCAD
*

Control Ppargc1b

Syvn1

siRNA

siRNA

siRNA

0
Control

Relative expression

Relative Luc activity**
Ppargc1b siRNA

1 μm* P < 0.05** P < 0.01
25*

* P < 0.05**
40 ** P < 0.01
20
*
35
30
15
25
10
20
**
15
5
10
5
0
SYVN1
0
SYVN1 R266A,R267A
SYVN1
PGC-1βPGC-1β SYVN1 C307S
PPARα
DMSO
WY-14643
DMSO

Relative Luc activity

D

C

B

**
45 ** P < 0.01
**
40
35
**
30
25
20
15
10
5
0
Control
+
+
+
+
+
+
+
+
+
+
+
Syvn1siRNA ++ + + +
+ + + +
PPARα
+ +
+ +
PGC-1 βDMSO
WY-14643

Control siRNA

The EMBO Journal

E3 ligase synoviolin and energy expenditure

Relative expression

Hidetoshi Fujita et al

KO

3

Atp5b
*

**

PPARα
WY-14643

F
Control KO

2

MCAD

1

Tublin0
ControlKO

Ppargc1b
siRNA+Syvn1siRNA

I

Adipose tissueMM
500 nM

500 nM
Control

Respiration/mg tissue

600
400

* P < 0.05

*
*

200
0

*

*

*

Energy expenditure(kcal/hr/mouse)G% of paired Control

H

0.4
0.3
0.2
0.1
0

KO

*

ControlKO

Figure 5. Regulation of PGC-1b function by SYVN1.
A–C HEK 293 cells (1 × 105 cells) were transiently co-transfected with a reporter plasmid containing PPAR binding sites (PPRE X3-TK-luc) and expression constructs or
siRNAs (A). For WT and mutant SYVN1 overexpression, 50 or 100 lg SYVN1 expression vector was co-transfected (B, C); 16 h later, cells were treated with vehicle or
10 lM Wy-14643 for 6 h. Each experiment was performed at least three times.
D
Representative electron micrographs of siRNA-treated cells. An increase in mitochondrial volume can be seen as the large cytoplasmic area in 3T3 L1 cells (amitochondrion is shown by the white arrow). Magnification, 5,000×. The number of mitochondria in the area (500 pixel × 500 pixel) was measured (right panel).
Each experiment was performed at least three times.
E
Total RNA was isolated from adipose tissue 15 weeks after birth, and mRNA expression was measured by real-time PCR relative to 18s rRNA level, with the averagefor control mice set to 1 (n = 3).
F
Cell extracts from adipose tissue were obtained from control and Adipoq-Cre;Syvn1flox/flox (KO) mice 15 weeks after birth. Western blotting was performed using
anti-medium chain acyl-coenzyme A dehydrogenase (MCAD) antibody.
G
Representative electron micrographs of adipose tissue of control (left) and CAG-Cre-ER;Syvn1flox/flox (right) mice. An increase in mitochondrial volume can be seen as
an enlargement in cytoplasmic area (M, mitochondria). Magnification, 10,000×.
H
Mitochondrial respiration was measured in WAT 7 days after Tam administration (the value for control mice was set to 100%) (n = 3).
I
Basal energy expenditure of control and CAG-Cre-ER;Syvn1flox/flox mice 7 days after Tam administration.
Data information: Data were analyzed with the Student’s t-test (A–E, I) or Mann–Whitney U-test (H) and represent the mean  SD. *P < 0.05, **P < 0.01.
Source data are available online for this figure.

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100*

DMSO (n=3) **LS102 50mg/kg (n=8)

90
80
0

20

40

Days

20

G

DMSO

Days

60

500 nmDMSO

LS-102 50 mg/kg

J

* **
80

500 nm120

P = 0.051
P = 0.89 P = 0.38

100
80
60
40
20
0

5

7 10
Weeks

60

20

*

15
10
5
0

LS-10250 mg/kg

DMSO (n=3)
LS102 50mg/kg (n=8)
14040Adipose tissue

50 μmLS-102DMSO 50 mg/kg
*

*

50

0
DMSO LS-102
50 mg/kg

110

**

**

50 μm

Food intake (mg per g)

Body weight change (%)

0* P < 0.05

100

*
**

90

150

I

120

95

200

H
130

100

Adipose tissue

Epididymal adipose tissue**50
Day 3 Day 29Day 54* **** * ****
* ***
* **
*
*
*
***
**
** **** ***
*

105

F

100

0

110

LS-102

P =0.98

E* P < 0.05
DMSO (n=5)
LS-102 50mg/kg (n=7)
115

120

K
DMSO (n=3)

Blood glucose (mg/dL)

150

P =0.34
P =0.32

DMSO WY-14643
PPARα

Weight (g)Food intake (mg per g)

200

**

DMSO

D

35
30
25
20
15
10
5
0

LS-102DMSO (n=5)
LS-102 50mg/kg (n=7)

CDMSO
5 μ M LS-102

number of mitochondria

WB: anti-HA

B

Body weight change (%)

DMSO LS-102
1 10 30 (μM)++ + + + +HA-Ub
SYVN1 ΔTM
+ + + + +FLAG-PGC-1β + + + + +Relative Luc Activity

A

Hidetoshi Fujita et al

DMSO

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LS102 50mg/kg (n=8)
800
600

**

**

400

200
0
13

27

Days

Figure 6. Effect of SYVN1 inhibition on diet-induced weight gain or genetic obesity.
A

HEK 293 cells were transfected with FLAG-PGC-1b plasmid and immunoprecipitated with anti-FLAG antibody. An in vitro ubiquitination assay was performed with
GST-SYVN1DTM, immunoprecipitated FLAG-PGC-1b, E1 and E2 enzymes, and HA-Ub in the presence or absence of LS-102. Each experiment was performed at least
three times.
B
HEK 293 cells were transiently transfected with PPRE X3-TK-luc and PPARa expression plasmid; 16 h later, cells were treated with vehicle or 10 lM Wy-14643 for
6 h. **P < 0.01. Each experiment was performed at least three times.
C, D Body weight (C) and average food intake (D) were measured daily for C57BL/6J mice treated once a day with DMSO or 50 mg/kg LS-102. *P < 0.05 for C57BL/6J
LS-102-treated versus C57BL/6J DMSO-treated mice.
E
Epididymal adipose tissue of C57BL/6J mice treated with DMSO (left) or LS-102 (right) on day 57 (n = 4).
F
Hematoxylin and eosin staining of epididymal adipose tissue of C57BL/6J mice treated with DMSO (left) or LS-102 (right) (lipid droplets are shown by black arrows).
Magnification, 400×.
G
Representative electron micrographs of adipose tissue from DMSO- (left) and LS-102-treated (right) mice on day 57. An increase in mitochondrial volume can beseen as enlarged cytoplasmic areas (a mitochondrion is shown by the white arrow). Magnification, 10,000×. The number of mitochondria in the area (2,000
pixel × 2,000 pixel) was measured (right panel).
H–J Obese db/db mice were treated daily with DMSO or LS-102, and body weight (H, I) and average daily food intake (J) were measured (Control, n = 3; LS-102, n = 8).
K
Blood glucose was measured on days 13 and 27 (Control, n = 3; LS-102, n = 8).
Data information: All data were analyzed with the Student’s t-test and are shown as mean  SD. *P < 0.05, **P < 0.01.
Source data are available online for this figure.

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E3 ligase synoviolin and energy expenditure

PRC regulate mitochondrial function in the control of energy expenditure (Lin, 2001; Kamei et al, 2003; Liu & Lin, 2011). Althoughthese coactivators share functional and structural identities theprimary structure of PGC-1b has several unique features—including
an middle LXXLL motif (Lin, 2001)—, and knockout studies havesuggested functional differences among these proteins (Scarpulla,2008). In the present study, the aa 195–367 region of PGC-1b
encompassing the middle LXXLL motif was found to interact withand serve as a target of negative regulation by SYVN1 (Fig 4). This
novel regulatory mechanism was also confirmed in vivo in global
and tissue-specific Syvn1 knockout mice (Figs 4 and 5). The selective regulation of PGC-1b by SYVN1 could be evidenced for theunique role of PGC-1b compared to PGC-1a. Thus, a novel function
for SYVN1 in energy metabolism could be exerted via negative regulation of PGC-1b. Recently, knockout mice of Sel-1 suppressor of lin12-like protein (Sel1L), which is an adaptor protein for SYVN1 andfunctions in the ERAD pathway, were shown to be resistant to dietinduced obesity (Sha et al, 2014). Therefore, these studies point to a
new direction for research on obesity that includes a role for the
ERAD pathway in energy control.
Nuclear receptors are transcription factors involved in various
metabolic processes and are therefore attractive targets for therapeutic interventions. Several studies on PPARs—the main regulators oflipid, glucose, and energy metabolism—have been published in thepast two decades and have led to the development of PPAR-related
drugs. Although many PPAR agonists are clinically efficacious, theiruse is associated with many side effects (Swanson et al, 2013). The
PPAR coactivators PGC-1a and b are also possible drug targets, andadequate modulation or activation of PGC-1 expression has great
potential in the treatment of diseases associated with mitochondrial
dysfunction and dysregulation of oxidative metabolism (Liu & Lin,2011). Although targeting a coactivator in new drug development is
attractive, the pharmacological upregulation of nuclear coactivators
is challenging. In the present study, LS-102—a selective inhibitor of
the E3 ligase activity of SYVN1—induced activation of PGC-1b andprevented weight gain and WAT accumulation in mice (Fig 6). In
addition, SYVN1 was more highly expressed in obese (ob/ob) micethan in their non-obese (ob/+) counterparts (Fig 2). These results
provide novel insight into the mechanistic basis of obesity, since thepharmacological inhibition of SYVN1 enhances energy expenditureby preventing PGC-1b degradation. In addition, LS-102 treatment
improved blood glucose before stimulating weight loss (Fig 6).
Although further studies are needed to clarify the role of SYVN1 inglucose metabolism, SYVN1 is an important drug candidate forobesity and associated metabolic diseases. In conclusion, this study
demonstrated a novel role for SYVN1 in the control of energy
metabolism via negative regulation of PGC-1b.

12-h light/12-h dark cycle. F-1 Foods (5.1% fat, 21.3% protein) werepurchased from Funabashi farm (Chiba, Japan). All mice used in the
study were of the C57BL/6J background. Conditional Syvn1loxP/loxP
mice were generated through homologous recombination in embryonic stem (ES) cells (Fig 1). The mouse Syvn1 locus was cloned froma BAC clone, and the targeting construct was linearized and transfected into ES cells by electroporation. Recombinant ES cell clones
expressing the neomycin gene were selected in medium supplemented with G418 and injected into C57BL/6 mouse-derived blastocysts using standard procedures. The neomycin selection cassette
was flanked by Frt recombination sites and excised in vivo by crossing with the general FLP deleter strain (Jackson Laboratories, West
Grove, PA, USA). Floxed heterozygous Syvn1flox/+ and heterozygous
CAG-Cre-ER mice (Jackson Laboratories) were crossed to generate
double heterozygous CAG-Cre-ER;Syvn1flox/+ mice, which were bredwith homozygous Syvn1flox/flox mice to produce conditional Syvn1
homozygous (CAG-Cre-ER;Syvn1flox/flox) and Syvn1 heterozygous(CAG-Cre-ER;Syvn1flox/+), homozygous Syvn1flox/flox, and heterozygous Syvn1flox/+ mice. Control mice lacking the Cre transgene(Syvn1flox/flox or Syvn1flox/+) received Tam injections to eliminate any
potential effects of Tam. To generate the CAG-Cre-ER;Syvn1flox/flox;
ob/ob and CAG-Cre-ER;Syvn1flox/flox;db/db genotypes, CAG-Cre-ER;
Syvn1flox/flox mice were bred with ob/+ or db/+ mice (Jackson Laboratories), yielding the compound heterozygotes CAG-Cre-ER;
Syvn1flox/+;ob/+ and CAG-Cre-ER;Syvn1flox/+;db/+. These wereinterbred to obtain CAG-Cre-ER;Syvn1flox/flox;ob/ob, Syvn1flox/flox;ob/
ob, and Syvn1flox/+;ob/ob mice or CAG-Cre-ER;Syvn1flox/flox;db/db,
Syvn1flox/flox;db/db, and Syvn1flox/+;db/db mice. The genotypes
Syvn1flox/flox;ob/ob, Syvn1flox/+;ob/ob, Syvn1+/+;ob/ob, Syvn1flox/flox;
db/db, Syvn1flox/+;db/db, and Syvn1+/+;db/db were used ascontrol mice. To generate adipose-specific Syvn1 knockout mice,
Syvn1flox/flox and Adipoq-Cre mice (Jackson Laboratories) werecrossed to generate Adipoq-Cre;Syvn1flox/+ compound heterozygotes,
which were mated with Syvn1flox/flox mice. For blinding and randomization, genotyping and measurement of body weight were separately performed by different person, respectively. In addition,
treatment with LS-102 and measurement of body weight were also
performed by different person, respectively. We randomly allocatedmice and estimated sample size as the number was more than 3 atleast. We excluded mice with sickness or injury from experiment
before starting the experiment.
Tamoxifen, LS-102 administration

Materials and Methods

Tamoxifen stock solution was prepared using 100 mg tamoxifen
suspended in corn oil (both from Sigma, St. Louis, MO, USA).
Starting from 7 to 8 weeks after birth, mice were injected intraperitoneally with 125 mg/kg tamoxifen per day for five consecutive
days. LS-102 solution (50 mg/kg) or DMSO was injected intraperitoneally once a day.

Mice

Plasmids and antibodies

All procedures involving animals were performed in accordance withinstitutional and national guidelines for animal experimentation andwere approved by the Institutional Animal Care and Use Committeeof Tokyo Medical University (#S-24021). Mice were kept in SPF
under conditions (20–26°C temperature; 40–65% humidity) on a

Coding sequences of full-length Ppara, Pparg, Ppargc1a, andPpargc1b were PCR-amplified from mouse 3T3-L1 cDNA. Fragmentsof Ppargc1b deletion mutants were obtained by PCR amplification,and these along with full-length Ppargc1b were inserted into the
pcDNA3 HA plasmid (Invitrogen, Carlsbad, CA, USA) for GSTª 2015 The Authors

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Hidetoshi Fujita et alpull-down and transient transfection assays. Plasmid sequenceswere confirmed by sequencing. PPRE X3-TK-luc was purchasedfrom Addgene (Cambridge, MA, USA). SYVN1 plasmids were previously described (Amano et al, 2003; Yamasaki et al, 2007), andSyvn1 point mutants were obtained by PCR amplification. The
following antibodies were used: anti-FLAG (M2), anti-tubulin (bothfrom Sigma), and anti-HA (12CA5 and 3F10; Roche, Indianapolis,
IN, USA). The anti-SYVN1 rabbit polyclonal antibody that was used
was previously reported (Yamasaki et al, 2007). Polyclonal antiserum against PGC-1b was generated by immunizing rabbits withpurified GST-PGC-1b (1–367 aa).

Western blotting with anti-HA antibody. For inhibition of ubiquitination by LS102, FLAG-PGC-1b was purified from HEK 293T cells
transfected with FLAG-PGC-1b with an anti-FLAG antibody andincubated with 1.5 lg HA-Ub, 250 ng E1, 300 ng UBE2G2, and
150 ng GST-SYVN1 (aa 233–338) in reaction buffer (50 mM Tris–
HCl, pH 7.5; 5 mM MgCl2; 0.6 mM DTT; and 2 mM ATP) at 37°C
for 2 h. The ubiquitinated PGC-1b was re-immunoprecipitated with
anti-FLAG antibody and analyzed by Western blotting with anti-HA
antibody.

GST pull-down assay

HEK 293T cells were transfected with HA-PGC-1b, Ub/FLAG,SYVN1, or SYVN1 3S expression plasmids; 24 h later, 20 lM MG132 was added, and cells were lysed in lysis buffer (50 mM HEPES,
pH 7.9; 150 mM KCl; 1 mM phenylmethanesulfonyl fluoride; 1%
Triton X-100; 10% glycerol; and protease inhibitors). Lysates weremixed with 1 lg anti-HA antibody conjugated to protein G-sepharose beads. After a 4-h incubation at 4°C, beads were washed threetimes with lysis buffer. Bound proteins were fractionated by SDS–
PAGE and analyzed by immunoblotting.

GST fusion proteins were expressed and purified using glutathionesepharose beads (GE Healthcare, Little Chalfont, UK) (Aratani et al,
2001; Fujita et al, 2003). Cell extracts were incubated with each
GST fusion protein bound to resin in 1 ml buffer A [20 mM Tris–
HCl, pH 8.0; 100 mM NaCl; 1 mM ethylenediaminetetraacetic acid
(EDTA); 1 mM dithiothreitol (DTT); 0.1% Nonidet P-40 (NP-40);
5% glycerol; 1 mM Na3VO4; 5 mM NaF; 1 lg/ml aprotinin; and1 lg/ml leupeptin] for 4 h at 4°C. After washing with buffer A,
bound proteins were fractionated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) followed byWestern blotting.
Immunoprecipitation assays
HEK 293T cells were transfected with HA-PGC-1b and SYVN1/FLAG
expression vectors; 24 h later, cells were lysed in 200 ll lysis buffer(20 mM Tris–HCl, pH 8.0; 100 mM NaCl; 1 mM EDTA; 1 mM DTT;
1% NP-40; 5% glycerol; and protease inhibitors). Lysates werediluted in binding buffer (of the same composition as lysis buffer,
except that it contained 0.1% NP-40) and mixed with 2 lg antiFLAG (M2) antibody conjugated to protein G-sepharose beads. Aftera 4-h incubation at 4°C, beads were washed three times with binding buffer. Bound proteins were fractionated by SDS–PAGE andanalyzed by Western blotting. To detect the interaction between
endogenous SYVN1 and PGC-1b, HEK 293 cells were lysed in asolution composed of 100 mM Tris–HCl, 80 mM NaCl, 1 mM
EDTA, 5 mM ethylene glycol tetraacetic acid, 5% glycerol, 2%(w/v) digitonin, 0.1% Brij 35, protease inhibitor cocktail, and20 mM MG132. Immunoprecipitation was carried out with antiSYVN1 antibody or control IgG, followed by Western blot using
anti-PGC-1b antibody.

In vivo ubiquitination assay

Cells cultures and transient transfection assay
HEK 293, HEK 293T, and 3T3-L1 cell lines; MEF; and skin fibroblasts were cultured in Dulbecco’s modified Eagle’s medium aspreviously described (Yamasaki et al, 2007). Transient transfection was performed with Lipofectamine 2000 or LTX according to
the manufacturer’s protocol (Invitrogen). Cells were lysed withcell lysis buffer (Promega, Madison, WI, USA) 24 h after transfection, and luciferase activity was measured. To ensure equalamounts of DNA, empty plasmids were added to each transfection.
Immunocytochemistry
The detailed procedure has been described in detail elsewhere(Fujita et al, 2003, 2005). Briefly, cells were transfected with plasmids and fixed 24 h later with 3.7% formaldehyde, permeabilizedwith 0.2% Triton X-100, and blocked with 2% bovine serum albumin. Cells were incubated with rat anti-HA (1:1,000) or rabbit antiFLAG (1:1,000) primary antibody, followed by staining with Alexa
Fluor 594 anti-mouse or Alexa Fluor 498 anti-rat secondary antibody(1:1,000; Molecular Probes, Eugene, OR, USA).
Measurement of protein half-life

In vitro ubiquitination assays
The in vitro ubiquitination assay was performed as previously
described (Yamasaki et al, 2007). Briefly, GST-PGC-1b (aa 1–367)
was incubated with 0.75 lg HA-Ub, 125 ng E1 (Biomol International, Plymouth Meeting, PA, USA), 150 ng UBcH5C, and 150 ng
maltose-binding protein-tagged SYVN1DTM-His in reaction buffer(50 mM Tris–HCl, pH 7.5; 5 mM MgCl2; 0.6 mM DTT; and 2 mM
ATP) at 37°C for 2 h. Glutathione sepharose was then added, andthe mixture was washed with GST wash buffer (50 mM Tris–HCl,
pH 7.5; 0.5 M NaCl; 1% Triton X-100; 1 mM EDTA; 1 mM DTT;
and protease inhibitors). The ubiquitinated PGC-1b was analyzed by1052

The assay was performed using a previously described method withsome modifications (Yamasaki et al, 2007; Bernasconi et al, 2010).
MEFs derived from Syvn knockout mice (Yamasaki et al, 2007) weretransfected with 1 lg pcDNA3 Syvn/FLAG, empty vector, 1.5 lg
pcDNA3 SyvnDSyU/FLAG, and 0.75 lg pcDNA3 HA-PGC-1b; 48 hlater, cells were treated with 40 lM cycloheximide for various
times, then lysed with buffer (10 mM Tris–HCl, pH 8.0; 150 mM
NaCl; 1 mM EDTA; 1% NP-40; 1 mM DTT; and protease inhibitors),
and analyzed by immunoblotting with antibodies against PGC-1b,
SYVN1, or a-tubulin (loading control). Each experiment was
performed at least three times.

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E3 ligase synoviolin and energy expenditure

Luciferase assay

mitochondrial respiration assay. A P value < 0.05 was considered
statistically significant.

The assay was performed as previously described (Chakravarti et al,
1996; Nakajima et al, 1996). HEK 293 cells were transiently transfected with 12.5 ng PPRE X3-TK-luc reporter plasmid, 25 ng
pcDNA3 HA-PPARa, 25 ng pcDNA3 HA-PGC-1b, 0.1 ng pRL-CMV,and 15 nM Syvn1 siRNA. For SYVN1 overexpression, 12.5 ng PPREX3-TK-luc reporter plasmid, 25 ng pcDNA3 HA-PPARa, 75 ng
pcDNA3 HA-PGC-1b, 0.1 ng pRL-CMV, and 50 or 100 ng SYVN1
expression vector were transfected. After 12 h, cells were treatedwith 10 lM WY-14643 for 6 h and lysed with cell lysis buffer,
followed by measurement of luciferase activity. Each experiment
was performed at least three times.

Supplementary information for this article is available online:
http://emboj.embopress.org

Acknowledgements
We thank Dr Allan M Weissman for scientific advice. We also thank S. Shibata,
H. Nagai, K. Takahashi, Y. Suzuki, T. Kasai, K. Nakamura, E. Hiratsu and M.
Chijiiwa for the technical assistance. We also thank all members of Dr Nakajima’s laboratory, and OYC and Charles River Japan for their assistance inanimal production, and also all members of the Bozo Research Center forpathological analysis. In addition, we thank E. Morita and S. Yoshihara of the
Mitsubishi Chemical Medicine Corporation for basal metabolism analysis of

RNA interference and real-time PCR

mice. We thank Dr Bruce Spiegelman for providing PPRE X3-TK-luc plasmid.
This work was funded in part by grants from the Naito Foundation, Natural

SiRNAs for human SYVN1 have been previously described (Yamasaki et al, 2007). SiRNAs for mouse Syvn1 and Ppargc1b werepurchased from Ambion Inc. (Austin, TX, USA). SiRNA transfection
was performed with Lipofectamine 2000. Total RNA from adipose
tissue was purified 7 days after Tam injection using ISOGEN (Nippon Gene, Tokyo, Japan) according to the manufacturer’s instructions and reverse-transcribed using ReverTra Ace with random
primers (Toyobo, Osaka, Japan). Real-time PCR was performed
using LightCycler 480 Probes Master (Roche Diagnostics, Mannheim, Germany). Expression levels were determined relative to thatof 18s rRNA. Primers and probes used in this study are shown in
Supplementary Table S2.

Science Scholarship Daiichi-Sankyo Foundation of Life Science, Mitsubishi
Tanabe Pharma Corporation, Bureau of Social Welfare and Public Health,Takeda Science Foundation, AstraZeneca R&D Grant 2013. This work was also
supported in part by Health Labour Sciences Research Grant (201229044A)
and Japan Society for the Promotion of Science KAKENHI Grant Numbers
20249052, 23659176, 23659502, 22790947, 24791006, 26670479 and 26461478.

Author contributions
HF, NY, SA, and TN conceived the project and designed the experiments. HF,
NY, SA, KS, HK, TS-F, IH, SM, NH, and TN performed the experiments andanalyzed the data. HF and TN wrote the manuscript. All authors discussed theresults and commented on the manuscript.

Conflict of interest

Mitochondrial respiration

The authors declare that they have no conflict of interest.

WAT was collected from control and CAG-Cre-ER;Syvn1flox/flox or
Adipoq-Cre;Syvn1flox/flox mice. The detailed procedure for measurement of mitochondrial respiration is described elsewhere (Sjovall
et al, 2013). Pre-adipocyte cells were obtained from CAG-Cre-ER;
Syvn1flox/flox mice. White adipose tissues were minced and incubated with collagenase solution for 45 min at 37°C. Then, cellsuspension was filtered twice. Cells were cultured in DMEM/F12.
Cells were treated with DMSO or tamoxifen, and transient transfection of siRNA was performed 5 days before experiment.

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110

Synoviolin inhibitor LS-102 reduces endoplasmic reticulum
stress-induced collagen secretion in an in vitro
model of stress-related interstitial pneumonia
FUKAMI NAKAJIMA1,2, SATOKO ARATANI2,3, HIDETOSHI FUJITA2,3, NAOKO YAGISHITA4,SHIZUKO ICHINOSE5, KOSHI MAKITA1, YASUHIRO SETOGUCHI6 and TOSHIHIRO NAKAJIMA2,3,7
1

Department of Anesthesiology, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University,
Bunkyo-ku, Tokyo 113-8519; 2Department of Locomotor Science, Institute of Medical Science, and3
Future Medical Science, Institute of Medical Science, Tokyo Medical University, Shinjuku-ku, Tokyo 160-8402;
4
Institute of Medical Science, St. Marianna University School of Medicine, Kawasaki, Kanagawa 216-8512;
5
Research Center for Medical and Dental Science, Tokyo Medical and Dental University, Bunkyo-ku,
Tokyo 113-8510; 6Department of Respiratory Medicine, and 7Integrated Genome Editing Section (iGES),Tokyo Medical University, Shinjuku-ku, Tokyo 160-8402, Japan
Received July 4, 2014; Accepted October 10, 2014
DOI: 10.3892/ijmm.2014.1984

Abstract. The deletion mutation of exon 4 in surfactant
protein C (SP-C), a lung surfactant protein, has been identified inparent-child cases of familial interstitial pneumonia. It has beenshown that this mutation induces endoplasmic reticulum (ER)stress. Synoviolin is an E3 ubiquitin ligase that is localized to the
ER and is an important factor in the degradation of ER-related
proteins. It has been demonstrated that synoviolin is involved inliver fibrosis. In the present study, we investigated the involvement of synoviolin in the pathogenesis of interstitial pneumonia
caused by the exon 4 deletion in the SP-C gene. We transfectedwild-type and exon 4-deleted SP-C genes into A549 human lungadenocarcinoma cells and measured the secretion of collagen,which is a representative extracellular matrix protein involved
in fibrosis. Secreted collagen levels were increased in the culture
medium in SP-C mutants compared to the wild-type cells.
Furthermore, the transcription of mRNAs coding for factors
associated with fibrosis was increased. Subsequently, to assess
the involvement of synoviolin, we constructed plasmids witha luciferase gene under the control of the synoviolin promoter.
The A549 cells were transfected with the construct along withthe exon 4-deleted SP-C plasmid for use in the luciferase assay.
We found a 1.6-fold increase in luciferase activaty in the cells
carrying exon 4 deleted SP-C, as well as an increase in intrinsic
synoviolin expression at the mRNA and protein levels. Collagen

Correspondence to: Professor Toshihiro Nakajima, Department
of Locomotor Science, Tokyo Medical University, 6-1-1 Shinjuku,
Shinjuku-ku, Tokyo 160-8402, Japan
E-mail: marlin@tokyo-med.ac.jp
Key words: endoplasmic reticulum stress, fibrosis, E3 ubiquitin ligase

synoviolin

secretion was decreased by the addition of LS-102, a synoviolin
inhibitor, to the A549 culture medium following transfection withwild-type and exon 4-deleted SP-C. These results demonstratethat synoviolin is involved in the onset of interstitial pneumonia
induced by exon 4-deleted SP-C, which suggests that synoviolin
inhibitors may be used in the treatment of the disease.
Introduction
Idiopathic pulmonary fibrosis (IPF) is part of a large group of
diffuse parenchymal lung diseases, that are characterized byprogressive parenchymal fibrosis, without any known cause andpoor prognosis. Nogee et al conducted a genetic analysis of amother (onset age, 1 year) with desquamative interstitial pneumonia (DIP) and a child (onset age, 6 weeks) with non-specificinterstitial pneumonia (NSIP), and identified a heterozygous
mutation of the surfactant protein C (SP-C) gene (deletionof exon 4, SP-CΔexon4) for the first time (1). SP-C is a component of the pulmonary surfactant, which is a heterogeneouscomplex of lipids and proteins that lowers lung-surface tension
and contributes to the host defense system. The SP-C geneis located on the short arm of chromosome 8, and comprises
6 exons and 5 introns (2). The precursor of SP-C, proSP-C,
consists of 197 amino acids and is transformed into mature
SP-C (consisting of 24-58 amino acids) through intracellularcleavage and modification (3,4). ProSP-CΔexon4 has been foundto induce endoplasmic reticulum (ER) stress (5,6).
Fibrosis is defined by the accumulation of excess extracellular matrix (ECM) components, such as collagen, in and aroundinflamed or damaged tissue (7,8). Fibrosis can develop in the
majority of tissue types, and advanced fibrosis causes fibrotic
diseases, such as pulmonary fibrosis, liver cirrhosis and renal
fibrosis, resulting in organ dysfunction and eventually death.
According to recent studies, synoviolin/HRD1 is involved in liver
fibrosis (9) and collagen synthesis in the kidneys (10). Synoviolin,-291-

NAKAJIMA et al: LS-102 REDUCES ENDOPLASMIC RETICULUM STRESS-INDUCED COLLAGEN SECRETION

which we have previously cloned (11), is an ER-resident E3 ubiquitin ligase with a RING finger domain that activates the
ER-associated degradation (ERAD) pathway (11). Synoviolin is
ubiquitously expressed in all organs of the body, particularly inthe liver, pancreas and skeletal muscle (12). Synoviolin is upregulated in response to ER stress and the overexpression of humaninositol-requiring kinase 1 (IRE1) and activating transcription
factor 6 (ATF6) (12,13). We have previously demonstrated
that heterozygous synoviolin knockout mice are resistant to
CCl4-induced liver fibrosis. In addition, we have demonstrated
that collagen secretion is reduced by synoviolin deficiency inmouse embryonic fibroblasts, suggesting the involvement ofsynoviolin in collagen secretion (9). Li et al (10) demonstrated
that synoviolin expression was increased in a mouse model ofrenal interstitial fibrosis caused by unilateral ureteral obstruction(UUO). In addition, they demonstrated that collagen secretionis reduced by synoviolin deletion in NRK-49F cells (normal
rat kidney interstitial fibroblasts) (10). These data suggest thatsynoviolin expression is increased by ER stress and that synoviolin promotes collagen synthesis. Thus, we hypothesized thatsynoviolin is involved in lung fibrosis, and in particular, in ER
stress-related lung fibrosis. In order to confirm our hypothesis, inthe present study, we investigated the involvement of synoviolin
and synoviolin inhibitor, LS-102, in collagen secretion in an
in vitro model of IPF by transfecting A549 cells (a human lungadenocarcinoma epithelial cell line) with SP-C lacking exon 4.
To investigate our hypothesis that synoviolin is involvedin lung fibrosis and that synoviolin inhibitors are candidate
antifibrotic agents, we investigated the association between
synoviolin and collagen secretion in an in vitro model, in which
A549 cells were transfected with SP-CΔexon4, which has beenreported to induce ER stress (5,6). In the present study, we did
not use any chemical or physical method to induce fibrosis;
instead, we focused on an SP-C mutation, SP-CΔexon4, which
has been identified in parent-child cases of familial interstitial
pneumonia (1).
Materials and methods
Cell culture. The A549 human lung adenocarcinoma cell line
was purchased from the American Type Culture Collection(ATCC, Manassas, VA, USA). The A549 cells were cultured
in minimum essential medium (Sigma-Aldrich, St. Louis, MO,
USA) supplemented with 1% penicillin, 1% streptomycin and
10% (heat-inactivated) fetal bovine serum (FBS).
Transfection of cells with plasmid constructs and siRNA. The
wild-type proSP-C expression vector was constructed using
pcDNA3-FLAG (Invitrogen, Carlsbad, CA, USA) by theinsertion of wild-type proSP-C cDNA that was isolated froma human lung cDNA library (Takara Bio Co., Tokyo, Japan).
SP-CΔexon4 (deletion of exon 4) was constructed according
to previously published techniques (14). All constructs wereconfirmed by DNA sequencing. The A549 cells were grownfor 24 h and transfected with wild-type SP-C or mutant SP-C
constructs using Lipofectamine 2000 (Invitrogen) as per the
manufacturer's instructions.
The sequences of the synoviolin siRNA and scrambled
synoviolin siRNA were identical to those described in aprevious study (15), and were chemically synthesized at111

Hokkaido System Science (Hokkaido, Japan). The A549 cells
were seeded at 40-50% confluency in 6-well plates and cultured
for 24 h. Subsequently, 50 nmol of annealed RNA duplex wastransfected using Lipofectamine 2000 according to the manufacturer's instructions.
Antibodies. The antibodies used in this study were as
follows: anti-synoviolin/HRD1 monoclonal antibody as
previously described (11), anti-FLAG antibody (anti-FLAG
M2 monoclonal antibody, F3165; Sigma-Aldrich), antihemagglutinin-A (HA) 3F10 monoclonal antibody (1867423;
Roche Applied Science) and anti-β-actin monoclonal antibody(A5441; Sigma-Aldrich).
Preparation of cellular protein. Cell cultures were harvested
and proteins were extracted using a cell extraction buffer
containing 0.05 M Tris-HCl (pH 8.0), 0.15 M NaCl, 5.0 mM
ethylenediaminetetraacetic acid (EDTA), 1% NP-40 andprotease inhibitors (1 µg/ml leupeptin, 1 µg/ml dithiothreitol (DTT), 1 µg/ml pepstatin and 1.5 µg/ml aprotinin) at 4˚C.
Reporter assay. The A549 cells were plated on 24-wellplates (5x10 4 cells/well). The synoviolin promoter(SyG-2055/+845 PGV-B2) was prepared as described in aprevious study (16). For transient transfection into the A549
cells, 20 ng/well synoviolin promoter and 50 ng/well test
plasmids (SP-C1-197 or SP-CΔexon4) were co-transfected using
Lipofectamine 2000 (Invitrogen) according to the manufacturer's recommendations. After 24 h, the cultures were aspirated
and the cells were added to 100 µl of passive lysis buffer(Promega, Madison, WI, USA). The cell debris was pelleted and5 µl of supernatant were collected and immediately analyzed for
luciferase activity using the Promega Luciferase assay system(Promega) and a microplate luminometer (Centro XS3 LB 960;
Berthold Technologies, Wildbad, Germany). The 5 µl supernatant samples were used to measure the protein concentrationin each well using a multi-spectrophotometer (Viento XS;
Dainippon Sumitomo Pharma, Tokyo, Japan). The assay was
performed in triplicate and the mean values were calculated foreach sample. The luciferase activity results were normalized tothe protein concentration in each sample.
Western blot analysis. The A549 cells were seeded at 40-50%
confluency in 100-mm plates and harvested 18 h following
transfection. Tunicamycin-treated (10 µg/ml) A549 cells
were prepared as a model of chemical induction of ER stress.
Cellular protein extracts were resolved by 10% SDS-PAGE,
transferred onto a nitrocellulose membrane and immunoblotted with anti-synoviolin/HRD1 monoclonal antibodies
followed by horseradish peroxidase-conjugated secondary
antibodies. The antigen-antibody complexes were visualizedusing an ECL detection system (Promega). β-actin expression
was used as an internal control. Immunoblots were scannedand band densities were quantified using ImageJ software.
Real-time polymerase chain reaction (PCR). The A549 cells
were grown in 100-mm culture plates. Forty-eight hours
following transfection, total RNA from the A549 cells was
isolated using Isogen II (Nippon Gene, Tokyo, Japan) andcDNA was synthesized using RiverTra Ace (Toyobo, Tokyo,

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Figure 1. Effect of surfactant protein C (SP-C) overexpression on collagen secretion in A549 cells. The mutant SP-C gene increased the mRNA expression levelsof (A) transforming growth factor-β (TGF-β), (B) connective tissue growth factor (CTGF) and (C) prolyl 4-hydroxylase subunit alpha 1 (P4HA1) by approximately
2.3-, 1.5- and 2.9-fold, respectively, compared to the wild-type. (D) A549 cells transfected with mutant SP-C secreted an approximately 1.3-fold greater amountof collagen into the medium than the cells transfected with wild-type SP-C. Data are the means ± standard error of the mean (SEM) of 3 experiments. **P<0.01.
WT, wild-type.

Japan) according to the manufacturer's instructions. Real-time
PCR relative quantification analysis was performed to measure
the mRNA expression levels of transforming growth factor-β(TGF- β), connective tissue growth factor (CTGF), prolyl
4-hydroxylase subunit alpha 1 (P4HA1), X-box binding
protein 1 (XBP1) and synoviolin using the FastStart Universal
Probe Master mix (ROX; Roche Applied Science, Indianapolis,
IN, USA), primers from the Universal Probe Library Assay
Design Center and probes from the universal probe library set(both from Roche Applied Science, Tokyo, Japan) with theStep One Plus Detection System and software (Applied
Biosystems, Life Technologies Japan, Tokyo, Japan). The
primers and probes used are as follows: TGF- β forward,
GCAGCACGTGGAGCTGTA and reverse, CAGCCGGTTGCTGAGGTA (probe: Universal probe library probe #72); CTGF
forward, AGCTGACCTGGAAGAGAACATT and reverse,
GCTCGGTATGTCTTCATGCTG (probe: Universal probelibrary probe #71); P4HA1 forward, TCGTCAAAGACCTAG
CAAAACC and reverse, CCGTCTCCAAGTCTCCTGTTA(probe: Universal probe library probe #30); XBP1 forward,
GGAGTTAAGACAGCGCTTGG and reverse, CACTGG
CCTCACTTCATTCC (probe: Universal probe libraryprobe #37); synoviolin forward, TTCGTCAGCCACGCCTAT
and reverse, GAGCACCATCGTCATCAGG (probe: Universal
probe library probe #89). The protocol included 1 cycle at
95˚C for 10 min, followed by 40 cycles at 95˚C for 1 sec and
60˚C for 20 sec, according to the manufacturer's
recommendations. The mRNA level was normalized relative
to the amount of the transcript coding for 18S rRNA.
Measurement of soluble secreted collagen. The A549 cells wereseeded at 40-50% confluency in 6-well plates. Twelve hours
following transfection, the medium was replaced with serumfree Opti-MEM (Gibco, Life Technologies Japan, Tokyo, Japan)
to eliminate the effects of serum on collagen measurement. At
30 h following transfection, the total soluble collagen in theculture supernatants was measured using the Sircol collagenassay method (Biocolor, County Antrim, UK) according to the
manufacturer's instructions. The assay was performed in triplicate and the mean values of each sample were calculated.

Immunoprecipitation. The cells were plated on 100-mm
tissue culture dishes and MG132 was added at a concentration of 20 µM 24 h following transfection. The cells wereco-transfected with hemagglutinin (HA)-tagged ubiquitin(Ub) and an SP-C wild-type, SP-C mutant, or empty vector.
Co-transfected with the synoviolin gene were also prepared.
Twenty-seven hours following transfection, the cells were
harvested and lysed with 200 µl of lysis buffer [50 mM
HEPES-KOH (pH 7.9), 150 mM KCl, 1% Triton X-100,
1 mM phenylmethylsulfonyl fluoride (PMSF), 10% glycerol,
2 µg/ml aprotinin, 2 µg/ml leupeptin, 2 µg/ml pepstatin A
and 20 µM MG132 (a proteasome inhibitor)]. Following
centrifugation at 5,000 rpm for 10 min, 150 µl of the clear
supernatant was incubated with 5 µl of normal mouse IgG(Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), 30 µlof protein G-Sepharose (GE Healthcare, Tokyo, Japan) and350 µl of lysis buffer at 4˚C with rotation for 30 min. The
supernatant was incubated with 2 µl of anti-FLAG M2 monoclonal antibody at 4˚C with rotation for 2 h, after which 40 µlof protein G were added, followed by incubation at 4˚C withrotation for 1 h. The immunoprecipitates were solubilized in
SDS sample buffer [0.078 M Tris-HCl (pH 6.8), 2% SDS, 10%
glycerol, 5% 2-mercaptoethanol and 0.003% bromophenol
blue] and then subjected to 4-15% gradient SDS-PAGE(Bio-Rad, Hercules, CA, USA).
Synoviolin inhibitor, LS-102. Synoviolin inhibitor, LS-102, wasidentified by screening approximately 4 million compounds
for potential synoviolin enzyme inhibitors as described in aprevious study (5). Dimethyl sulfoxide (DMSO) is used assolvent for LS-102; thus, we used DMSO as a control. Twelve
hours following transfection, Opti-MEM (no serum added)
was replaced, and LS-102 (5 µM) or DMSO (5 µM) was added
to the medium to evaluate the effect of LS-102.
Statistical analysis. All data are expressed as the means ± standard deviation (SD). Differences between groups were
examined using the Student's t-test and considered statisticallysignificant at P<0.05. All results were derived from at least
3 independent experiments.

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Figure 2. Effect of surfactant protein C (SP-C)Δexon4 overexpression on synoviolin expression in A549 cells. (A) Luciferase assay revealed that the A549 cells
expressing mutant SP-C showed a 1.6-fold higher luciferase activity than the wild-type SP-C-expressing cells. The mRNA expression levels of (B) X-box binding
protein 1 (XBP1) and (C) synoviolin were increased in the mutant SP-C-expressing A549 cells compared with the wild-type SP-C-expressing cells. (D) Transfectionof A549 cells with mutant SP-C increased the expression of synoviolin protein compared with the cells transfected with wild-type SP-C. Data are the means ± standard error of the mean (SEM) of 3 experiments. WT, wild-type; TM, tunicamycin [positive control of endoplasmic reticulum (ER) stress]. *P<0.05 and **P<0.01.

Results
Overexpression of SP-CΔexon4 promotes collagen secretion in
A549 cells. Studies have found an association between mutations in the SP-C gene and familial pulmonary fibrosis (1,17,18).
Therefore, in this study we focused on the SP-CΔexon4 mutation.
To assess the association between the SP-CΔexon4 mutation andfibrosis in the current experimental system, we measured themRNA expression levels of transforming growth factor- β(TGF- β), connective tissue growth factor (CTGF) andprolyl 4-hydroxylase subunit alpha 1 (P4HA1), which are responsible for enhancing the production of collagen protein, a typical
ECM component involved in fibrosis (19-21). Transfection of thecells with the mutant SP-C gene increased the mRNA expression
levels of TGF-β, CTGF and P4HA1 by approximately 2.3-, 1.5and 2.9-fold, respectively, compared to the wild-type (Fig. 1A-C).
Fibrosis is attributed to the excess deposition of ECM components in and around inflamed or damaged tissue (8). Collagen is amajor component of the ECM; therefore, we measured collagensecretion following transfection of the cells with mutant SP-C.

To confirm that mutant SP-C promoted collagen secretion frommutant SP-C-expressing A549 cells, collagen secretion wasmeasured following transfection with a wild-type or mutant SP-C
gene. The A549 cells transfected with the mutant SP-C genesecreted approximately 1.3-fold more collagen into the medium
than the cells transfected with wild-type SP-C (Fig. 1D).
SP-CΔexon4 increases the expression of synoviolin. To examine
the hypothesis that synoviolin plays a role in fibrosis induced
by SP-CΔexon4, we evaluated the level of synoviolin expression
in SP-CΔexon4 -expressing A549 cells. First, we constructed aplasmid carrying a luciferase gene with expression regulated
by a synoviolin promoter, which was co-transfected together
with the wild-type SP-C or mutant SP-C gene expression
plasmid into the A549 cells. Twenty-four hours following
transfection, a luciferase assay was conducted. The A549 cells
expressing mutant SP-C showed 1.6-fold greater luciferase
activity than the wild-type SP-C-expressing cells (Fig. 2A).
These data suggest that the transcriptional activity of synoviolin was enhanced in the SP-CΔexon4 gene-transfected cells.

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Figure 3. Effect of synoviolin siRNA on synoviolin secretion in A549 cells transfected with surfactant protein C (SP-C)Δexon4. (A) Collagen secretion was
decreased by synoviolin siRNA in A549 cells. (B) A549 cells were co-transfected with SP-CΔexon4 and synoviolin siRNA or scrambled siRNA. The amount ofsoluble secreted collagen in the supernatant of synoviolin siRNA-transfected SP-CΔexon4-expressing A549 cells was less than that produced by scrambled siRNAtransfected SP-CΔexon4-expressing A549 cells. Data are the means ± standard error of the mean (SEM) of 3 experiments. **P<0.01.

Subsequently, to confirm that ER stress is induced in thisexperimental system and that the mRNA expression of synoviolin
is activated in the mutant SP-C-transfected cells, we examinedthe expression levels of X-box binding protein 1 (XBP1) andsynoviolin in the A549 cells by real-time PCR. The mRNA
expression levels of XBP1 and synoviolin were increased in
the mutant SP-C-expressing A549 cells in comparison to thewild-type SP-C-expressing cells (Fig. 2B and C).
Finally, to assess the protein expression of synoviolin,
we performed western blot analysis with an anti-synoviolin
antibody. Transfection of the A549 cells with mutant SP-C
increased the protein expression of synoviolin compared withthe cells transfected with wild-type SP-C (Fig. 2D). Thus, theoverexpression of SP-CΔexon4 in the A549 cells increased thetranscriptional activity of synoviolin and the expression ofsynoviolin at the mRNA and protein levels.
Synoviolin knockdown decreases collagen secretion in
A549 cells transfected with SP-CΔexon4. To explore the role ofsynoviolin in collagen secretion in the A549 cells, we evaluated collagen production in A549 cells following transfection
with siRNA targeting synoviolin. We found that the level ofsoluble secreted collagen in the supernatant of A549 cells
after the knockdown of synoviolin was approximately 20%
less than that produced by the scrambled siRNA-transfected
A549 cells (Fig. 3A). Furthermore, we analyzed the effects
of synoviolin siRNA on collagen secretion in SP-C Δexon4 expressing A549 cells. The A549 cells were co-transfected with
SP-CΔexon4 and either synoviolin siRNA or scrambled siRNA.
The amount of soluble secreted collagen in the supernatantof the synoviolin siRNA-transfected SP-CΔexon4 -expressing
A549 cells was approximately 30% less than that producedby scrambled siRNA-transfected SP-CΔexon4-expressing A549
cells (Fig. 3B).

Synoviolin ubiquitinates proSP-CΔ exon4 as a substrate. To
demonstrate the ubiquitination of proSP-CΔexon4, the A549
cells were co-transfected with hemagglutinin (HA)-tagged
ubiquitin (Ub) and an SP-C wild-type, SP-C mutant, or empty
vector. In addition, to determine whether synoviolin ubiquitinates proSP-CΔexon4 as a substrate, cells co-transfected with thesynoviolin gene were also prepared. Following immunoprecipitation with an anti-FLAG antibody, western blot analysis was
performed using an anti-HA antibody. The cells transfectedwith SP-CΔexon4 showed a ladder pattern, suggesting ubiquitination. The synoviolin-containing cells showed a more intense
band (Fig. 4). These results suggested that the proSP-CΔexon4
protein was ubiquitinated in this experimental system and thatsynoviolin may participate in this ubiquitination.
Collagen secretion is suppressed by the synoviolin inhibitor,
LS-102. To investigate the effects of synoviolin inhibition oncollagen secretion in A549 cells, we performed experiments
using the synoviolin inhibitor, LS-102, as described in a previous
study of ours (22), in which we identified LS-102 by screening
approximately 4 million compounds for potential synoviolin
enzyme inhibitors. As a result, LS-101 and LS-102 were identified as compounds that inhibited the autoubiquitination activity
of synoviolin. These compounds were administered in a mousemodel of collagen-induced arthritis, in which they demonstrated
protective effects against arthritis. LS-101 had more potent
inhibitory effects on the synoviolin enzyme than LS-102.
However, LS-102 was used in this study as it is more selective
for synoviolin than LS-101. Twelve hours following transfection, Opti-MEM (no serum added) was replaced, and LS-102
or DMSO (5 µM; as a control) was added to the medium. The
tunicamycin (10 µg/ml)-treated A549 cells were also prepared
as a positive control for ER stress induction. LS-102 significantly
decreased collagen secretion into the medium (Fig. 5).

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Figure 4. Ubiquitination of precursor of surfactant protein C (proSP-C)Δexon4. A549 cells were co-transfected with hemagglutinin (HA)-tagged ubiquitin (Ub) and
SP-C wild-type, SP-C mutant, or an empty vector. In addition, samples were also prepared that were co-transfected with the synoviolin gene. After immunoprecipitation with an anti-FLAG antibody, western blot analysis was performed using an anti-HA antibody. The cells transfected with SP-CΔexon4 showed a ladder pattern,suggesting ubiquitination. The synoviolin-containing cells showed a more intense band. Data are the means ± standard error of the mean (SEM) of 3 experiments.
Syno, synoviolin.

Figure 5. Effect of the synoviolin inhibitor, LS-102, on collagen secretion in A549 cells. LS-102 significantly decreased collagen secretion into the medium. Data
are the means ± standard error of the mean (SEM) of 3 experiments. WT, wild-type; TM, Tunicamycin [positive control of endoplasmic reticulum (ER) stress].

Discussion
In the present study, we demonstrated that the expressionof synoviolin and collagen secretion were enhanced by theoverexpression of SP-CΔexon4 in A549 cells and that collagensecretion was suppressed by the use of the synoviolin inhibitor,
LS-102.

The pathogenic mechanisms of fibrosis have recently
attracted the attention of researches, and studies haveindicated that diverse factors, pathways and systems are
involved in the progression of fibrosis. However, the detailed
mechanisms involved are not yet fully understood. Wynn
et al (8), Tanjore et al (23,24) and Zhong et al (25) reported
that various pathways are involved in fibrosis, including the

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ER-stress pathway, and that ER stress leads to fibrosis. In thepresent study, we did not use any chemical or physical method
to induce fibrosis, but instead we focused on a mutation in
SP-C that was identified in mother-child cases of familial
interstitial pneumonia, and which produced ER stress in A549
cells. Considering our results in the context of the dual roleof synoviolin as an E3 ubiquitin ligase and collagen synthesis
promoter, this study suggests the existence of a novel pathway:
ER stress increases synoviolin synthesis, which promotes
collagen synthesis, contributing to fibrosis. This result confirm
the association between ER stress and fibrosis that has beenpreviously reported (23,25). Further studies are required to
fully determine the association between synoviolin and fibrosis.
Excessive fibrosis affects various organs and tissues,
including the endocardium, the heart, the liver, the kidneys,
the skin, the bone marrow, the retroperitoneum and the lungs.
Advanced fibrosis is a serious disorder that causes organ
dysfunction and death; accordingly, antifibrotic drugs are indevelopment. Various factors [TGF-β, CTGF, tumor necrosis
factor (TNF), endothelin, chemokines, chemokine receptors,
and matrix metalloproteinases (MMPs)] and effector cells are
involved in fibrosis (19,20,26-28). As a result, there are several
potential therapeutic vectors which may be used in the treatment of fibrosis, and thus a wide range of candidate therapeutic
agents is available (8).
In the present study, we demonstrate that synoviolin
expression and collagen secretion are enhanced by transfecting A549 cells with SP-CΔexon4 and that collagen secretionis suppressed by transfection with synoviolin siRNA or the
synoviolin inhibitor, LS-102. Based on these findings and therecent studies indicating that synoviolin is involved in liver
fibrosis (9) and renal fibrosis (10), our data suggest that thesynoviolin inhibitor, LS-102 represents a candidate antifibrotic
agent for the treatment of interstitial pneumonia; however,further studies, including in vivo studies, are required in order
to confirm this therapeutic potential.
Acknowledgements
We thank the staff at the Future Medical Science at the Instituteof Medical Science and the Integrated Gene Editing Sectionof Tokyo Medical University for their assistance. This study
was funded in part by grants from the Naito Foundation,
Natural Science Scholarship Daiichi-Sankyo Foundationof Life Science, Mitsubishi Tanabe Pharma Corporation,
Bureau of Social Welfare and Public Health, The Ministryof Health Labour and Welfare, The Ministry of Education,
Culture, Sports, Science and Technology, Japan Society for
the Promotion of Science, Health Labour Sciences Research
Grant, Takeda Science Foundation, AstraZeneca R&D Grant
2013, Grant-in-Aid for Exploratory Research, Grant-in-Aid forYoung Scientists (B).
References
1. Nogee LM, Dunbar AE III, Wert SE, Askin F, Hamvas A and
Whitsett JA: A mutation in the surfactant protein C gene associated with familial interstitial lung disease. N Engl J Med 344:573-579, 2001.
2. Beers MF and Mulugeta S: Surfactant protein C biosynthesis
and its emerging role in conformational lung disease. Annu Rev
Physiol 67: 663-696, 2005.

3. Mulugeta S and Beers MF: Surfactant protein C: its uniqueproperties and emerging immunomodulatory role in the lung.
Microbes Infect 8: 2317-2323, 2006.
4. Nogee LM: Genetics of the hydrophobic surfactant proteins.
Biochim Biophys Acta 1408: 323-333, 1998.
5. Mulugeta S, Nguyen V, Russo SJ, Muniswamy M and Beers MF:
A surfactant protein C precursor protein BRICHOS domain
mutation causes endoplasmic reticulum stress, proteasome
dysfunction, and caspase 3 activation. Am J Respir Cell Mol
Biol 32: 521-530, 2005.
6. Maguire JA, Mulugeta S and Beers MF: Endoplasmic reticulum
stress induced by surfactant protein C BRICHOS mutants
promotes proinflammatory signaling by epithelial cells. Am J
Respir Cell Mol Biol 44: 404-414, 2011.
7. Wynn TA: Cellular and molecular mechanisms of fibrosis. J
Pathol 214: 199-210, 2008.
8. Wynn TA and Ramalingam TR: Mechanisms of fibrosis: therapeutic translation for fibrotic disease. Nat Med 18: 1028-1040, 2012.
9. Hasegawa D, Fujii R, Yagishita N, et al: E3 ubiquitin ligase
synoviolin is involved in liver fibrogenesis. PLoS One 5: e13590,
2010.
10. Li LY Shen, Ding Y, Liu Y, Su D and Liang X: Hrd1 participatesin the regulation of collagen I synthesis in renal fibrosis. Mol Cell
Biochem 386: 35-44, 2014.
11. Amano T, Yamasaki S, Yagishita N, et al: Synoviolin/Hrd1, an
E3 ubiquitin ligase, as a novel pathogenic factor for arthropathy.
Genes Dev 17: 2436-2449, 2003.
12. Kaneko M, Ishiguro M, Niinuma Y, Uesugi M and Nomura Y:
Human HRD1 protects against ER stress-induced apoptosis
through ER-associated degradation. FEBS Lett 532: 147-152, 2002.
13. Kaneko M and Nomura Y: ER signaling in unfolded protein
response. Life Sci 74: 199-205, 2003.
14. Wang WJ, Mulugeta S, Russo SJ, et al: Deletion of exon 4 fromhuman surfactant protein C results in aggresome formation andgeneration of a dominant negative. J Cell Sci 116: 683-692, 2003.
15. Yamasaki S, Yagishita N, Sasaki T, et al: Cytoplasmic destructionof p53 by the endoplasmic reticulum-resident ubiquitin ligase‘Synoviolin’. EMBO J 26: 113-122, 2007.
16. Tsuchimochi K, Yagishita N, Yamasaki S, et al: Identificationof a crucial site for synoviolin expression. Mol Cell Biol 25:
7344-7356, 2005.
17. Nogee LM, Dunbar AE III, Wert S, Askin F, Hamvas A and
Whitsett JA: Mutations in the surfactant protein C gene associated with interstitial lung disease. Chest 121: S20S-S21, 2002.
18. Thomas AQ, Lane K, Phillips J III, et al: Heterozygosity fora surfactant protein C gene mutation associated with usualinterstitial pneumonitis and cellular nonspecific interstitial
pneumonitis in one kindred. Am J Respir Crit Care Med 165:
1322-138, 2002.
19. Border WA and Noble NA: Transforming growth factor beta intissue fibrosis. N Engl J Med 331: 1286-1292, 1994.
20. Bonniaud P, Margetts PJ, Kolb M, Haberberger T, Kelly M,
Robertson J and Gauldie J: Adenoviral gene transfer of connective
tissue growth factor in the lung induces transient fibrosis. Am J
Respir Crit Care Med 168: 770-778, 2003.
21. Myllyharju J: Prolyl 4-hydroxylases, the key enzymes of collagenbiosynthesis. Matrix Biol 22: 15-24, 2003.
22. Yagishita N, Aratani S, Leach C, et al: RING-finger type E3
ubiquitin ligase inhibitors as novel candidates for the treatmentof rheumatoid arthritis. Int J Mol Med 30: 1281-1286, 2012.
23. Tanjore H, Cheng DS, Degryse AL, Zoz DF, Abdolrasulnia R,
Lawson WE and Blackwell TS: Alveolar epithelial cells undergo
epithelial-to-mesenchymal transition in response to endoplasmic
reticulum stress. J Biol Chem 286: 30972-30980, 2011.
24. Tanjore H, Lawson WE and Blackwell TS: Endoplasmic
reticulum stress as a pro-fibrotic stimulus. Biochim Biophys
Acta 1832: 940-947, 2013.
25. Zhong Q, Zhou B, Ann DK, et al: Role of endoplasmic reticulum
stress in epithelial-mesenchymal transition of alveolar epithelial
cells: effects of misfolded surfactant protein. Am J Respir Cell
Mol Biol 45: 498-509, 2011.
26. Hogaboam CM, Steinhauser ML, Chensue SW and Kunkel SL:
Novel roles for chemokines and fibroblasts in interstitial fibrosis.
Kidney Int 54: 2152-2159, 1998.
27. Arai M, Ikawa Y, Chujo S, et al: Chemokine receptors CCR2 and
CX3CR1 regulate skin fibrosis in the mouse model of cytokineinduced systemic sclerosis. J Dermatol Sci 69: 250-258, 2013.
28. Fernandez IE and Eickelberg O: New cellular and molecularmechanisms of lung injury and fibrosis in idiopathic pulmonary
fibrosis. Lancet 380: 680-688, 2012.

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Article Amendments

The Journal of Clinical Investigation

Corrigendum
Leiomodin-3 dysfunction results in thin filament disorganization and nemaline myopathy
Michaela Yuen, Sarah A. Sandaradura, James J. Dowling, Alla S. Kostyukova, Natalia Moroz, Kate G. Quinlan, Vilma-Lotta Lehtokari,
Gianina Ravenscroft, Emily J. Todd, Ozge Ceyhan-Birsoy, David S. Gokhin, Jérome Maluenda, Monkol Lek, Flora Nolent,
Christopher T. Pappas, Stefanie M. Novak, Adele D’Amico, Edoardo Malfatti, Brett P. Thomas, Stacey B. Gabriel, Namrata Gupta,
Mark J. Daly, Biljana Ilkovski, Peter J. Houweling, Ann E. Davidson, Lindsay C. Swanson, Catherine A. Brownstein, Vandana A. Gupta,
Livija Medne, Patrick Shannon, Nicole Martin, David P. Bick, Anders Flisberg, Eva Holmberg, Peter Van den Bergh, Pablo Lapunzina,
Leigh B. Waddell, Darcée D. Sloboda, Enrico Bertini, David Chitayat, William R. Telfer, Annie Laquerrière, Carol C. Gregorio,
Coen A.C. Ottenheijm, Carsten G. Bönnemann, Katarina Pelin, Alan H. Beggs, Yukiko K. Hayashi, Norma B. Romero, Nigel G. Laing,
Ichizo Nishino, Carina Wallgren-Pettersson, Judith Melki, Velia M. Fowler, Daniel G. MacArthur, Kathryn N. North, and Nigel F. Clarke
Original citation: J Clin Invest. 2014;124(11):4693–4708. doi:10.1172/JCI75199.
Citation for this corrigendum: J Clin Invest. 2015;125(1):456–457. doi:10.1172/JCI80057.
In the original version of the article, the mutation c.601_602delGA in LMOD3 was incorrectly described; the correct predicted proteinchange is p.D201Qfs*9. In addition, the LMOD3 mutation p.S47Qfs*13 was incorrectly indicated; the correct mutation is S47Qfs*13. Theseerrors affected portions of Table 1, Table 2,
Figure 1, and Supplemental Table 2. The
corrected versions of Table 1, Table 2, andTable 1. LMOD3 variants and protein expression
Figure 1 appear below, and Supplemental
Table 2 has been updated online.
A
B
Patient ID
LMOD3 genotype
Protein expression
1a
1b
2
3
4
5

hom c.138dupC, p.S47Qfs*13
hom c.138dupC, p.S47Qfs*13
hom c.138dupC, p.S47Qfs*13
hom c.154delA, p.M52*
hom c.231G>A, p.W77*
het c.300_304delGACTC, p.T101Rfs*4
het c.601_602delGA, p.D201Qfs*9
hom c.349C>T, p.Q117*
het c.349C>T, p.Q117*
het c.1218delA, p.K406Nfs*11
het c.723_733del, p.D242Efs*4
het c.976G>C, p.G326R
hom c.860delT, p.F287Sfs*3
het c.976G>C, p.G326R
het c.1372C>T, p.Q458*
hom c.1069G>T, p.E357*
hom c.1069G>T, p.E357*
hom c.1099_1100delAA, p.N367Qfs*11
hom c.1099_1100delAA, pN367Qfs*11
hom c.1099_1100delAA, p.N367Qfs*11
hom c.1099_1100delAA, p.N367Qfs*11
hom c.1099_1100delAA, p.N367Qfs*11
het c.1100_1102delACA, p.N367delhet c.1201C>T, p.R401*
het c.1100_1102delACA, p.N367delhet c.1201C>T, p.R401*

6
7
8
9
10
11a
11b
12aC
12b
12c
13a
13b
14a
14b

p.S47Qfs*13 (6.6 kDa), no expression
p.S47Qfs*13 (6.6 kDa), N/D
p.S47Qfs*13 (6.6 kDa), N/D
3a and 3b - p.M52* (5.7 kDa), N/D
p.W77*(8.7 kDa), no expression
p.T101Rfs*4 (12 kDa), no expression
p.D201Qfs*9(24 kDa), no expression
p.Q117*(13.6 kDa), inconclusive
p.Q117* (13.6 kDa), inconclusive
p.K406Nfs*11 (48.2 kDa), inconclusive
p.G326R
p.D242Efs*4 (28.3 kDa), N/D
p.F287Sfs*3 (33 kDa), no expression
p.Q458* (52.9 kDa), N/D
p.G326R
p.E357* (41.1 kDa), N/D
p.E357* (41.1 kDa), N/D
p.N367Qfs*11 (43 kDa), no expression
p.N367Qfs*11 (43 kDa), N/D
p.N367Qfs*11 (43 kDa), no expression
p.N367Qfs*11 (43 kDa), N/D
p.N367Qfs*11 (43 kDa), N/D
p.R401* (48 kDa), expressed
p.N367del (64.8 kDa), expressed
p.N367del, N/D
p.R401*, N/D

Numbering of the nucleotide and protein changes is relative to NM_198271.3 (gene) and NP_938012(protein).BFull-length LMOD3 protein is 560–amino acids long and has a predicted molecular weightof 64.9 kDa. Molecular weight predictions were performed using ExPASy (http://web.expasy.org/
compute_pi/). The presence or absence of protein expression was assessed by Western blot analysis.
C
Corresponds to BOS-1120. Hom, homozygous; het, heterozygous; N/D, not determined (no muscle
was available for analysis); inconclusive, biopsy contained only small amounts of muscle tissue, andthe presence or absence of mutant LMOD3 expression could not be determined conclusively.
A

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The authors regret the errors.

Article Amendments

The Journal of Clinical Investigation

Table 2. Clinical features of LMOD3-NM
Patient ID

Genotype

Ethnicity

Sex

Clinical
subtypeA

1a

p.S47Qfs*13

Algerian

F

SC

1b
2

p.S47Qfs*13
p.S47Qfs*13

Algerian
Belgian

F
M

SC
SC

3a
3b
4
5

p.M52*
p.M52*
p.W77*
p.T101Rfs*4 / p.D201Qfs*9

Portuguese
Portuguese
JapaneseJapanese

F
F
M
F

SC
SC
SC
SC

6

p.Q117*

Japanese

FSC

7

p.Q117* / p.K406Nfs*11

Japanese

F

SC

8
9

p.D242Efs*4 / p.G326R
p.F287Sfs*3

South American
Italian

F
F

SC
SC

10

p.G326R / p.Q458*

Ecuadorian

M

SC

11a
11b
12aC

p.E357*
p.E357*
p.N367Qfs*11

SwedishSwedishAfghani

M
M
M

SC
SC
SC

12b
12c
13a
13b
14a

p.N367Qfs*11
p.N367Qfs*11
p.N367Qfs*11
p.N367Qfs*11
p.N367del / p.R401*

AfghaniAfghaniPakistani
Pakistani
Australian

M

SC

F
F
F

SC
SC
TC

14b

p.N367del / p.R401*

AustralianF

TC

Clinical features
Deceased (neonatal period)B. Polyhydramnios, preterm delivery (30/40), arthrogryposis,
fractures (bilateral femoral).
Deceased (neonatal period). Preterm delivery (36/40).
Deceased (10 months). Decreased fetal movements, breech presentation, arthrogryposis,
ophthalmoplegia.
Deceased (neonatal period). Polyhydramnios, decreased fetal movements, contractures.
Alive at 1 month, lost to follow-up. Polyhydramnios, contractures.
Alive at 4 months, lost to follow-up.
Alive at 2 months, lost to follow-up. Polyhydramnios, decreased fetal movements, subdural
hematoma.
Alive at 10 months, lost to follow-up. Polyhydramnios, decreased fetal movements, fetal edema,preterm delivery (32/40), microcephaly, contractures.
Alive at 1 year 7 months, lost to follow-up. Polyhydramnios, decreased fetal movements,
ophthalmoplegia.
Deceased (neonatal period).
Deceased (4 months). Polyhydramnios, decreased fetal movements, preterm delivery (34/40),
ophthalmoplegia, contractures.
Deceased (6 weeks). Polyhydramnios, decreased fetal movements, preterm delivery (35/40),
breech presentation, ophthalmoplegia, arthrogryposis, fractures (bilateral humeral).
Deceased (5 months). Polyhydramnios, arthrogryposis.
Deceased (neonatal period).
Deceased (2 months). Absent fetal movements, preterm delivery (31/40), breech presentation,
arthrogryposis.
Deceased (neonatal period). Preterm delivery (33/40), arthrogryposis.
Affected fetus.
Deceased (3 months). Polyhydramnios, breech presentation, ophthalmoplegia, kyphosis.
Deceased (neonatal period). Polyhydramnios, decreased fetal movements.
Alive (10 years). Polyhydramnios, decreased fetal movements, bulbar weakness,
ophthalmoplegia, percutaneous endoscopic gastrostomy, nocturnal noninvasive ventilation.
Walks independently. Normal cardiac assessment and echocardiogram.
Alive (4 years). Polyhydramnios, bulbar weakness, percutaneous endoscopic gastrostomy,
nocturnal noninvasive ventilation. Walks with truncal support. Normal cardiac assessment andechocardiogram.

Classification of clinical subtype of NM relates to the European Neuromuscular Centre classification of NM (1). BNeonatal period indicates less than
28 days of age. CCorresponds to BOS-1120. TC, typical congenital NM; SC, severe congenital NM; F, female; M, male; 30/40, gestational age of 30 weeks.
A

Figure 1

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RESEARCH PAPER

Necklace cytoplasmic bodies in hereditary myopathy
with early respiratory failure
Akinori Uruha,1,2,3 Yukiko K Hayashi,1,2,4 Yasushi Oya,5 Madoka Mori-Yoshimura,5Masahiro Kanai,5 Miho Murata,5 Mayumi Kawamura,6 Katsuhisa Ogata,7Tsuyoshi Matsumura,8 Shigeaki Suzuki,9 Yukako Takahashi,10,11 Takayuki Kondo,11Takeshi Kawarabayashi,12 Yuko Ishii,13 Norito Kokubun,13 Satoshi Yokoi,14
Rei Yasuda,15 Jun-ichi Kira,16 Satomi Mitsuhashi,1,2 Satoru Noguchi,1,2
Ikuya Nonaka,2,7 Ichizo Nishino1,2▸ Additional material ispublished online only. To viewplease visit the journal online(http://dx.doi.org/10.1136/
jnnp-2014-309009).
For numbered afﬁliations see
end of article.
Correspondence to
Dr Ichizo Nishino, Departmentof Neuromuscular Research,
National Institute of
Neuroscience, NCNP, 4-11 Ogawa-Higashi, Kodaira,Tokyo 187-8502, Japan;
nishino@ncnp.go.jp
Received 15 July 2014
Revised 2 September 2014
Accepted 7 September 2014
Published Online First
24 September 2014

ABSTRACT
Background In hereditary myopathy with early
respiratory failure (HMERF), cytoplasmic bodies (CBs) areoften localised in subsarcolemmal regions, withnecklace-like alignment (necklace CBs), in muscle ﬁbres
although their sensitivity and speciﬁcity are unknown.
Objective To elucidate the diagnostic value of thenecklace CBs in the pathological diagnosis of HMERF
among myoﬁbrillar myopathies (MFMs).
Methods We sequenced the exon 343 of TTN gene(based on ENST00000589042), which encodes theﬁbronectin-3 (FN3) 119 domain of the A-band and is amutational hot spot for HMERF, in genomic DNA from
187 patients from 175 unrelated families who were
pathologically diagnosed as MFM. We assessed thesensitivity and speciﬁcity of the necklace CBs for HMERF
by re-evaluating the muscle pathology of our patientswith MFM.
Results TTN mutations were identiﬁed in 17 patientsfrom 14 families, whose phenotypes were consistent
with HMERF. Among them, 14 patients had necklace
CBs. In contrast, none of other patients with MFM had
necklace CBs except for one patient with reducing body
myopathy. The sensitivity and speciﬁcity were 82% and99%, respectively. Positive predictive value was 93% in
the MFM cohort.
Conclusions The necklace CB is a useful diagnostic
marker for HMERF. When muscle pathology shows
necklace CBs, sequencing the FN3 119 domain of
A-band in TTN should be considered.

INTRODUCTION▸ http://dx.doi.org/10.1136/
jnnp-2014-309564

To cite: Uruha A,
Hayashi YK, Oya Y, et al. J
Neurol Neurosurg Psychiatry
2015;86:483–489.

Hereditary myopathy with early respiratory failure(HMERF; OMIM 603689) is an adult-onset progressive myopathy characterised by early presentation of respiratory insufﬁciency usually during
ambulant stage.1–3 Pathologically, HMERF sharesfeatures of myoﬁbrillar myopathy (MFM) besides
the key ﬁnding of cytoplasmic bodies (CBs).2 3 Itscausative gene TTN, which encodes a gigantic
protein, titin,2 3 is known to be causative also fortibial muscular dystrophy, limb girdle muscular dystrophy type 2J, early-onset myopathy with fatal cardiomyopathy and dilated or hypertrophic
cardiomyopathy.4–10 Interestingly, all patients with
HMERF so far identiﬁed carry a mutation in exon

343 (based on ENST00000589042) encoding theﬁbronectin-3 (FN3) 119 domain in the A-band
region of titin.2 3 11–17
CBs are abnormal protein aggregates visualised
usually as red-colored objects on modiﬁed Gomori
trichrome stain and can be observed in a wide rangeof myopathic conditions. Nevertheless, they are often
conspicuous in MFM and considered as one of therepresentative pathological ﬁndings in MFM.18 In
muscle specimens of HMERF, CBs are often locatedin the subsarcolemmal region,12 13 14 19 with anecklace-like alignment, which here we call ‘necklace
CBs’. However, the utility of necklace CBs in thepathological diagnosis of HMERF is unknown. We
therefore tested the sensitivity and speciﬁcity of necklace CBs in the diagnosis of HMERF.

METHODS
Patients

National Center of Neurology and Psychiatry(NCNP) functions as a referral centre for muscle
pathology and muscle biopsy samples are sent fromall over Japan. From 1991 to 2013, 187 patientsfrom unrelated 175 Japanese families have beenpathologically diagnosed as MFM at NCNP. In thiscohort, mutations were found in known
MFM-related genes: DES: 8 families (4.6%), VCP: 8
families (4.6%), FLNC: 6 families (3.4%), DNAJB6:
6 families (3.4%), ZASP: 5 families (2.9%), FHL1: 5
families (2.9%), MYOT: 4 families (2.3%) and BAG3:
1 family (0.6%). No mutation was identiﬁed in
CRYAB. Clinical information at the time of muscle
biopsy was available in all patients.

Genetic analysis
Genomic DNA was isolated from peripheral lymphocytes or frozen muscle as previously described.20
Exon 343 of TTN was directly sequenced using ABI
PRISM 3130 automated sequencer (PE Applied
Biosystems). Sequence variants were assessed using
publically available databases including 1000
Genomes
Project
database(http://www.
1000genomes.org/), NHLBI Exome Sequencing
Project 5400 database (http://evs.gs.washington.edu/
EVS/), dbSNP135 (http://www.ncbi.nlm.nih.gov/
SNP/) and Human Genetic Variation Browser (http://
www.genome.med.kyoto-u.ac.jp/SnpDB/);andUruha A, et al. J Neurol Neurosurg Psychiatry 2015;86:483–489. doi:10.1136/jnnp-2014-309009

-300-

483

J Neurol Neurosurg Psychiatry: first published as 10.1136/jnnp-2014-309009 on 24 September 2014. Downloaded from http://jnnp.bmj.com/ on 9 April 2019 by guest. Protected by copyright.

Neuromuscular

484

Uruha A, et al. J Neurol Neurosurg Psychiatry 2015;86:483–489. doi:10.1136/jnnp-2014-309009

-301-

43/M

42/F

38/M
44/F

40/M

46/M

I

J

K
L

M

N

FatherSon

FatherSon
Older brother
Youngerbrother

p.R31783_V31785delp.G31791V

p.G31791D
p.G31791R

p.C31712Y

p.C31712R

p.C31712R

p.C31712R
p.C31712R

p.C31712R

p.C31712R

p.C31712R

p.C31712R

p.C31712R

Fatiguability;
respiratory failure (31)Difficulty in lifting
thigh (20)

Fatiguability (26)Mother and younger
sister: lower leg muscle
weakness

Son: distal myopathy;
sudden death

Foot drop; difficulty inopening a bottle (41)

Fatiguability; weightloss (39)
Difficulty lifting thighs(36)
Gait disturbance (28)
Fatiguability; loss ofappetite (40)Gait disturbance (24)Difficulty in standing
on right toe (56)Foot drop (47)Grandfather of G-2: died Tripping (57)of respiratory failure
Tripping (20)(45)

Mother: sudden death(38)
Older sister: proximal
muscle weakness;
respiratory failure (35)Father: Foot drop;
sudden death (42)Oldest brother: gait
disturbance (32); sudden
death (40)

Tripping (46)
Tripping (20)Foot drop (31)Foot drop (27)

Yes (41)

Yes (27)

24/yesNo

No

No

Yes (30)No

41/yesYes (31)

Yes*

Yes*

Yes*
Yes (40)

Yes*

Yes (39)

Yes (68)

Yes (47)
Yes (50)
Yes (58)
Yes*
Yes (20 s) Yes (29)

Yes (NA)

28/Yes41/yes

38/Yes42/yes56/yes47/yes57/yes20/yes31/yesYes (20 s) Yes (29)

20/no (32)

Yes (26)

Yes (48)
Yes*
Yes (44)
Yes (34)

Respiratory
disturbance(years)Yes (31)

Yes (NA)
Yes (22)
Yes (31)
Yes (27)

Foot
drop(years)29/yes46/yes22/yes31/yes27/yesAge at gait
Initial manifestations disturbance/(years)ambulant––

Yes (38)
Yes (44)–

Yes (42)

Yes (68)

Yes (52)–
Yes (29)

Yes (37)

Yes (29)

Yes (31)

Yes (49)
No
Yes (45)
Yes (37)

Artificial
ventilation(years)–2° AVB (type 1)

–
STC

–

–

RHF, Af––
RHF

RHF, PH

STC

–

RHF
No
No
No

Cardiacinvolve-ment*Asymptomatic but found by laboratory tests.
2° AVB (type 1), Mobitz type 1 second degree atrioventricular block; Af, atrial fibrillation; F, female; M, male; NA, not available; PH, pulmonary hypertension; RHF, right-sided heart failure; STC, sinus tachycardia.

68/F

40/M

E

H

38/M

D

52/M
58/M
29/M

34/F

C

F
G-1
G-2

49/M
26/M
45/M
37/M

Family history (years)

A-1
A-2
B-1
B-2

RelationshipAge(years)/sex

Patient
NumberMutation(protein level)

Clinical features of the patients with TTN variants

Table 1––

Yes (36)––

Yes (42)–

Yes (47)––

Yes (28)
Artificial
nutrition (35)
–

No

No
No
No
No

Dysphagia(years)Myalgia

Head drop

Head drop;
forward bent
postureMyalgia
muscle cramp

Difficulty inopening
mouth

No
No
No
No

Other
featuresJ Neurol Neurosurg Psychiatry: first published as 10.1136/jnnp-2014-309009 on 24 September 2014. Downloaded from http://jnnp.bmj.com/ on 9 April 2019 by guest. Protected by copyright.

Neuromuscularsoftwares to predict functional effects of mutations such as
PolyPhen-2 (http://genetics.bwh.harvard.edu/pph2/) and Mutation
Taster (http://www.mutationtaster.org/). The description of mutationsofTTN
conforms
toEnsemblesequence
ENST00000589042.
In this study a diagnosis of HMERF was made based on thepresence of a mutation in exon 343 of TTN, although the possibility that mutations in other parts of the gene cause HMERF
cannot be totally excluded.

Re-evaluation of clinical data
The clinical information in our cohort together with previous
reports2 3 11–14 showed that respiratory insufﬁciency before
being wheelchair users and selective involvement of semitendinosus muscles on muscle imaging were frequently observed,
raising a possibility that these ﬁndings might be clues for the
diagnosis of HMERF. We therefore reviewed clinical data of ourpatients with MFM in order to calculate sensitivities, speciﬁcities and PPVs of them at the time of muscle biopsy.

EthicsRe-evaluation of muscle pathology

All of the clinical information and materials used in this study
were obtained for diagnostic purpose and permitted for scientiﬁc use with written informed consent. All experiments in thisstudy were approved by the Ethical Committee of National
Center of Neurology and Psychiatry.

Muscle pathology was re-evaluated focusing on necklace CBs on
modiﬁed Gomori trichrome. In the present study, we tentatively
deﬁned the presence of necklace CBs as at least two muscle
ﬁbres containing CBs exclusively localised in the subsarcolemmal area, covering more than 50% of circumference of eachmuscle ﬁbre in three non-serial sections (each section was atleast 250 μm apart and included at least 300 muscle ﬁbres). CBs
were evaluated in all MFM cases, and subsequently the sensitivity and speciﬁcity of necklace CBs for the diagnosis of HMERF
were calculated. The positive predictive value (PPV) and 95%
CIs were calculated with GraphPad Prism V.5.0 (Graph Pad
Software, California, USA).

RESULTS
Mutation analysis

Six different heterozygous mutations in exon 343 of TTN wereidentiﬁed in 17 patients from 14 families (table 1). Among them,
two mutations, g.284701T>C (c.95134T>C; p.C31712R) andg.284939G>A (c.95372G>A; p.G31791D) were previously
reported.2 3 11 12 14 The former mutation was reported to be themost common in other populations.11 12 This was also the case in
our cohort and the mutation was shared by nine families, while allothers were found in single families. The latter mutation was previously reported in a European-American family.14 Among fournovel mutations that we identiﬁed, three were missense:
g.284702G>A (c.95135G>A; p.C31712Y), g.284938G>C(c.95371G>C; p.G31791R) and g.284939G>T (c.95372G>T;
p.G31791V); and one was non-frameshift deletion:g.284913_284921delGAGGGCAGT (c.95346_95354del; p.
R31783_V31785del). None of the variants was listed in theElectron microscopic observation
Biopsied muscle specimens were ﬁxed in 2.5% glutaraldehyde
and post-ﬁxed with 2% osmium tetroxide. Semithin sections
stained with toluidine blue were examined by light microscopy.
Ultrastructural analysis was carried out on longitudinal andtransverse ultrathin sections of muscles after staining withuranyl acetate and lead citrate, using Tecnai spirit transmission
electron microscope (FEI, Hillsboro, Oregon, USA).
Table 2 Laboratory findings at the time of muscle biopsy

Selective involvement of muscles on
imaging test

Muscle pathology

CK IU/L(normal value)

Respiratory function(%VC, sitting position)

Semitendinosus
muscleAnterior
compartmentof lower legs

CB

Necklacesof CBs

RV

Abnormal (value: NA.)

NA

NA

+

+

+

A-2 (26 years)
B-1 (39 years)
B-2 (32 years)
C (31 years)
D (31 years)
E (40 years)

Normal (value:NA)
65 (20–190)
425 (51–197)
659 (−200)
488 (45–170)
375 (51–197)
234 (62–287)+++
NA (+, 34 years)
+
+

+
+
+
NA (+, 34 years)
−*
+

+
+
+
+
+
+

+
+
+
+
+
+

+
−
+
−
−
−

F (50 years)
G-1 (58 years)
G-2 (29 years)
H (68 years)
I (43 years)
J (42 years)
K (34 years)
L (44 years)
M (40 years)
N (46 years)

61 (NA)146 (50–170)
142 (50–170)
140 (50–170)
179 (62–287)
364 (45–163)
645 (51–197)
139 (43–165)
190 (62–287)
799 (62–287)

77% (68%, lying)
84% → 67% (45 years)
82% → 63% (37 years)
32%
VC: 0.97 L
ABG: PaCO2 86 mm Hg,
PaO2 56 mm Hg (RA)
41%
59%
31%
ABG: PaCO2 60 mm Hg
40%64%
67% (55%, lying)
36%
61%
67%

+
+
+
+
+
+
+
+
−*
+

+
+
+
+
+
+
+
+
+
+

+
+
+
+
+
+
+
+
+
+

−
−
+
+
−
+
+
+
+
+

−
−
+
−
−
+
+
−
+
+

Patient
A-1 (49 years)

In the column of Patient, the same alphabet indicates that they belong to the same family.
*Diffuse muscle involvement.
ABG, arterial blood gas; CB, cytoplasmic body; NA, not available; RV, rimmed vacuole; VC, vital capacity.

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NeuromuscularTable 3 Cross tabulation of necklace cytoplasmic bodies andhereditary myopathy with early respiratory failure
Patients with myofibrillar
myopathies
Necklace CBs

HMERF

Non-HMERF

Row total+% within column% within row–% within column% within row
Column total

14
82.4%
93.3%
3
17.6%
1.7%
17

1
0.6%
6.7%
169
99.4%
98.3%
170

15

172

187

CB, cytoplasmic body; HMERF, hereditary myopathy with early respiratory failure.

Figure 1 Necklace cytoplasmic bodies. Cytoplasmic bodies arelocated in line in subsarcolemmal region, often covering the total
circumference of a muscle ﬁbre. Modiﬁed Gomori trichrome stain.
Bar: 50 μm.

31 years (range 20–57 years). Four patients developed dysphagia, and one of them required tube feeding.

Sensitivity and speciﬁcity of the necklace of CBs

genomic variation databases. The mutated amino acids werehighly conserved among species (UCSC Genome Browser).
PolyPhen-2 predicted p.C31712Y, p.G31791R and p.G31791V
mutations as probably damaging with scores 0.996, 1.000 and
1.000, respectively. Likewise, Mutation Taster predicted the p.
C31712Y, p.G31791R, p.G31791V and p.V31782_A31784delmutations to be disease-causing with a probability of 1.000, 1.000,
1.000 and 0.998, respectively. No segregation analysis was possible
in any family.
Clinical information of 17 participants with TTN mutations
are summarised in tables 1 and 2 and online supplementarytable. All patients show clinical signs consistent with HMERF
previously reported.1–3 11–14 The median age of onset wasAmong 17 genetically-conﬁrmed patients with HMERF, necklace CBs were found in 14 patients, comprising 0.1–0.8% of themuscle ﬁbres (ﬁgure 1). In contrast, none of the 170 patients
who had MFM other than HMERF had necklace CBs except
for only one patient who had reducing body myopathy, which
had been conﬁrmed by the presence of reducing bodies inmuscle ﬁbres on menadione-linked α-glycerophosphate
dehydrogenase (MAG) stain without substrate and a mutation in
the second LIM domain of FHL1 (g.60438G>A; c.377G>A;
p.C126Y, based on ENST00000543669; online supplementaryﬁgure S1). Based on these results, the sensitivity and speciﬁcityof the necklace CBs in HMERF were calculated as 82% (14/ 17,
95% CI 57% to 96%) and 99% (169/170, 95% CI 97% to100%), respectively (table 3). Since the prevalence of HMERF
in the MFM cohort was 9.1% (17/ 187), the PPV was calculated
as 93% (95% CI 68% to 100%) based on Bayes’ theorem.

Figure 2 Electron microscope images. (A and B) Sarcomeric disarrangement limited to one sarcomere is observed. (C) Multiple electron-dense
inclusions are present in association with Z lines. (D) A ﬁbre containing necklace cytoplasmic bodies (CBs) shows marked myoﬁbrillar disorganisation(asterisk), especially around the CBs. (E) CBs showing a necklace alignment with lower electron density as compared to that of Z line. ▵: a remnantof Z line. (F) Thin ﬁlamentous structure around the CBs. Lattice-like structure is not seen in the CBs. Bar: 0.5 μm (A, B and F), 1 μm (E), 2 μm (C),and 5 μm (D).
486

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NeuromuscularMuscle specimens of the three patients with HMERF had
CBs, which are usually located in the subsarcolemmal regions,but did not show the deﬁnite necklace-like alignment pattern(online supplementary ﬁgure S2). Those three patients shared
the same mutation, g.284701T>C ( p.C31712R), albeit othernine patients harbouring the same mutation had deﬁnite necklace CBs.
Fibres with rimmed vacuoles were occasionally seen, but thesensitivity, speciﬁcity and PPV for HMERF were lower thanthose of the necklace CBs: 47% (8/ 17, 95% CI 23% to 72%),
61% (104/ 170, 95% CI 53% to 69%) and 11% (95% CI 20%
to 48%), respectively.

Ultrastructural features
EM samples were available from four patients with HMERF.
Sarcomeric disarrangement limited to one sarcomere wasobserved in all patients (ﬁgure 2A, B). In some areas, multiple
electron-dense inclusions associated with Z line were surrounded by disorganised myoﬁbrils (ﬁgure 2C). Fibres withnecklace CBs were included only in one sample. These ﬁbres
showed marked myoﬁbrillar disorganisation (ﬁgure 2D, asterisk), especially in the vicinity of the CBs (ﬁgure 2D, E). The
CBs had a lower electron density as compared with that of theZ line and contained dense and mildly ﬁlamentous components,
without lattice-like structure (ﬁgure 2E, F). Small number of
CBs were partly surrounded by thin ﬁlaments (ﬁgure 2F).

Sensitivity and speciﬁcity of respiratory dysfunction andmuscle imaging data

Using the data at the time of muscle biopsy about the respiratory
function of 102 participants in the MFM cohort, the sensitivity,
speciﬁcity and PPV of the respiratory insufﬁciency before being
wheelchair users (below 80% of vital capacity or over
45 mm Hg of PaCO2) were calculated as 88% (14/16, 95% CI
62% to 99%), 94% (81/86, 95% CI 87% to 98%) and 74%(95% CI 49% to 91%), respectively (table 2 and 4). In ﬁve
non-HMERF participants presenting respiratory insufﬁciency
during the ambulant stage, one had a mutation in VCP, but nocausative genes were identiﬁed in four participants as far as wehave screened.
As for the muscle imaging at the time of muscle biopsy, thesensitivity of selective muscle involvement of semitendinosus
muscles as shown in ﬁgure 3 was 93% (14/ 15; table 4). The
speciﬁcity could not be calculated due to the limited number ofimages available in non-HMERF participants.

DISCUSSION
We have demonstrated that necklace CBs had high speciﬁcity fora diagnosis of HMERF (99%), with sensitivity 82% and PPV
93% in our MFM cohort. In this study, all 15 patients withnecklace CBs had HMERF except for only one who had reducing body myopathy due to a mutation in FHL1, without mutation in exon 343 of TTN. Although we judged this case as
having necklace CBs retrospectively, CBs in this case are smaller
in size and scattered in the subsarcolemmal region in muscle
ﬁbres, giving an appearance different from typical necklace CBs
in HMERF (online supplementary ﬁgure S1), suggesting thatthe speciﬁcity of necklace CBs could be substantially 100%. In
addition, muscle pathology of this patient showed typical reducing bodies in scattered ﬁbres on MAG stain without substrate,
and thus differential diagnosis between reducing body myopathyand HMERF was not a problem in practise.
Interestingly, even in three patients with HMERF without
typical necklace CBs, CBs are aligned in the subsarcolemmal
regions in muscle ﬁbres, albeit they do not encompass morethan half of the myoﬁbre circumference (online supplementaryﬁgure S2), suggesting that this unique subsarcolemmal alignment
pattern is likely to be related to the dysfunction of titin due toexon 343 mutations although the detailed mechanism is still
unknown.
On electron microscopy (EM), CBs in the patients differ
slightly from typical CBs seen in other various diseases. While
typical CBs consists of a core with electron density similar to Z
line,21 the CBs in HMERF show lower density. Furthermore,thin ﬁlaments, which compose a halo of typical CBs, are rarely
seen around the CBs. Interestingly, the sarcomeric disarrangement appears to progress in order of ﬁgure 2A, C, suggesting a
possibility that this sarcomeric disarrangement may ultimately
produce the CBs.
Several groups reported that CBs in HMREF are reactive toantibodies to myoﬁbrillar proteins including myotilin,
αB-crystallin, actin (phalloidin), ﬁlamin C, dystrophin, andγ-sarcoglycan, but not for titin on immunohistochemical analysis.11 12 14 19 Although similar ﬁndings were obtained in ourobservation (data not shown), the pathophysiological signiﬁcance of CBs in HMERF has still not been elucidated.
We found four novel variants in exon 343 of TTN including
three heterozygous missense mutations [g.284702G>A ( p.
C31712Y), g.284938G>C ( p.G31791R) and g.284939G>T(p.G31791V)], and a heterozygous deletion variant[g.284913_284921del ( p.R31783_V31785del)]. These variants
were not described in any of the publically available databases.
Mutated amino acids are highly conserved among species. Also

Table 4 Sensitivity, specificity, and positive predictive value oflaboratory findings for HMERF

Sensitivity
Specificity
PPV

Respiratory
disturbance in theambulant stage

Selective affected
semitendinosus muscles
on muscle imaging

Necklace CBs
on muscle
pathology

88% (14/16)
94% (81/86)
74%

93% (14/15)NANA

82% (14/17)
99% (169/170)
93%

At the time of muscle biopsy.
Respiratory disturbance: <80% of %VC or >45 mm Hg of PaCO2. PPVs were
calculated by the HMERF prevalence of 9.1% in the MFM cohort.
CB, cytoplasmic body; HMERF, hereditary myopathy with early respiratory failure;
MFM, myofibrillar myopathy; NA, not available, PPV, positive predictive value;
VC, vital capacity.

Figure 3 Muscle image. Representative image showing preferential
involvement of semitendinosus muscles. CT images of skeletal muscles
in proximal legs of the patient K. ▵: semitendinosus muscles.

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NeuromuscularTable 5 Mutations in the FN3 119 domain of A-band in TTN in HMERF
DNA change

Amino acid change

OriginFamilyg.284693C>G
g.284701T>C

p.P31709R (p.P30068R)
p.C31712R (p.C30071R)

g.284702G>A
g.284752T>C
g.284754G>C
g.284753G>T
g.284762C>T
g.284913_284921delg.284925C>G
g.284939G>A
g.284938G>C
g.284939G>T

p.C31712Y (p.C30071Y)
p.W31729R (p.W30088R)
p.W31729C (p.W30088C)
p.W31729L (p.W30088L)
p.P31732L (p.P30091 L)
p.R31783_V31785del (p.R30142_V30144del)p.N31786K (p.N30145 K)
p.G31791D (p.G30150D)
p.G31791R (p.G30150R)
p.G31791V (p.G30150V)

FrenchSwedish, British, Finnish, Italian, Spanish, Argentinian(European ancestry), East Indian, JapaneseJapanese
BritishGermanJapanese
Italian, French, British, Portuguese, Swedish
Japanese
BritishAmerican (European ancestry), JapaneseJapaneseJapanese

Reference
3

A, B, C, D, E, F, G, H, I

2, 3, 11, 12, 14

J

*
12
12
13
11, 12, 15, 16, 17

N

*
11

K
L
M

14

*
*

*Possible novel mutation. Titin reference: ENST00000589042 and ENST00000591111, a former transcript, inside the brackets.
HMERF, hereditary myopathy with early respiratory failure.

the variants were predicted to be pathogenic by the plural prediction software programs. Furthermore, in single-base substitutions, other types of substitutions of the same amino acids ( p.
C31712R and p.G31791D) have already been reported in otherfamilies with HMERF (table 5).2 3 11 12 14 Thus, although segregation analysis was not possible, the variants are highly likely
to be pathogenic.
Previous reports suggested that HMERF might not be
extremely rare in Caucasian populations.11 12 In UK, in patientswith HMERF with p.C31712R ( p.C30071R, based on
ENST00000591111), mutation in exon 343 of TTN was identiﬁed in 5.5% of the MFM cohort.11 Patients have also been
identiﬁed in Asian populations including Japanese and Indian,suggesting that patients with HMERF are likely to be distributed
worldwide.13 14 22 Here, we conﬁrmed the presence of patientswith HMERF in Japan. Furthermore, among all the 175 MFM
families in our Japanese cohort, 14 families (8%) had HMERF
with mutations in the exon 343 of TTN, which renders TTN
the most frequent causative gene for MFM in our cohort
although there still remains a possibility that there may be an
undisclosed major causative gene as causative mutations havenot been identiﬁed in more than 60% of the MFM families.
Clinical features of participants with HMERF described inthis study coincide with those in previous reports for mostparts: affected individuals usually present with predominantdistal leg muscle weakness followed by chronic respiratory
failure.1–3 11–14 Interestingly, dysphagia was seen in 4 of the 17,
which was rarely described in the literature.13 Dysphagia seems
to be mostly mild, but was severe in one patient, who required
tube feeding.
Skeletal muscle imaging has been reported to show preferential involvement of semitendinosus, obturator, sartorius, gracilis,
iliopsoas muscles and anterior compartment of lower legs, suggesting such imaging ﬁndings are useful for the diagnosis of
HMERF.3 11 12 19 Particularly, selective involvement of semitendinosus muscles is commonly observed. Our study showed asensitivity of 93%, which is compatible with 95–100% reported
by previous studies.3 12 Unfortunately, skeletal muscle imaging
was not available in many of our patients with MFM other than
HMERF, and thus it was impossible to calculate the speciﬁcity
and PPV of the selective involvement of semitendinosus muscles.
However, it may not be so speciﬁc since such ﬁnding wasobserved also in other MFMs caused by mutations in DES,CRYAB and MYOT.23 24
488

In conclusion, the necklace CB is a useful pathological marker
in the diagnosis of HMERF. When muscle pathology shows
necklace CBs, sequencing the FN3 119 domain of the A-bandin TTN should be considered.
Author afﬁliations
1
Department of Clinical Development, Translational Medical Center, National Centerof Neurology and Psychiatry (NCNP), Tokyo, Japan2
Department of Neuromuscular Research, National Institute of Neuroscience, NCNP,
Tokyo, Japan3
Department of Education, Interdisciplinary Graduate School of Medicine andEngineering, University of Yamanashi, Yamanashi, Japan
4
Department of Neurophysiology, Tokyo Medical University, Tokyo, Japan
5
Department of Neurology, National Center Hospital, NCNP, Tokyo, Japan
6
Department of Neurology, Japanese Red Cross Society, Wakayama Medical Center,
Wakayama, Japan
7
Institute of Clinical Research/Department of Neurology, National Hospital
Organization Higashisaitama Hospital, Saitama, Japan
8
Department of Neurology, National Hospital Organization Toneyama National
Hospital, Osaka, Japan
9
Department of Neurology, Keio University School of Medicine, Tokyo, Japan10
Department of Neurology, Osaka Red Cross Hospital, Osaka, Japan
11
Department of Neurology, Graduate School of Medicine, Kyoto University, Kyoto,Japan
12
Department of Neurology, Institute of Brain Science, Hirosaki University Graduate
School of Medicine, Aomori, Japan
13
Department of Neurology, Dokkyo Medical University, Tochigi, Japan
14
Department of Neurology, Nagoya University Graduate School of Medicine,
Nagoya, Japan
15
Department of Neurology, National Hospital Organization Maizuru Medical Center,
Kyoto, Japan
16
Department of Neurology, Neurological Institute, Graduate School of Medical
Sciences, Kyushu University, Fukuoka, Japan
Acknowledgements The authors thank Ms Kaoru Tatezawa, Ms Yoriko Kojima,
Ms Kazu Iwasawa, Mr Takao Uchikai, and Ms Kanako Goto in NCNP for theirtechnical assistance.
Contributors AU was involved in the conceptualisation and design of the study,
data analysis and interpretation, literature review and drafting the manuscript. YKH
was involved in the conceptualisation and design of the study, data analysis andinterpretation, and manuscript revision for intellectual content. YO, MM-Y, MK, MM,
MK, KO, TM, SS, YT, TK, TK, YI, NK, SY, RY, and JK were involved in the collectionof clinical data. SM and SN were involved in the data interpretation (molecular data)
and manuscript revision for intellectual content. IkN was involved in the supervisionof pathological analysis and interpretation and manuscript revision for intellectual
content. IcN was involved in the supervision of all aspects, including study design,
data analysis and interpretation, and manuscript preparation.
Funding This study was supported partly by JSPS KAKENHI Grant Number of24659437; partly by Research on Rare and Intractable Diseases, Comprehensive
Research on Disability Health and Welfare and Applying Health Technology from the
Ministry of Health, Labour and Welfare of Japan; and partly by Intramural Research
Grant 23-5 and 26-8 for Neurological and Psychiatric Disorders of NCNP.

Uruha A, et al. J Neurol Neurosurg Psychiatry 2015;86:483–489. doi:10.1136/jnnp-2014-309009-305-

J Neurol Neurosurg Psychiatry: first published as 10.1136/jnnp-2014-309009 on 24 September 2014. Downloaded from http://jnnp.bmj.com/ on 9 April 2019 by guest. Protected by copyright.

NeuromuscularCompeting interests None.
Ethics approval Ethical Committee of National Center of Neurology andPsychiatry.
Provenance and peer review Not commissioned; externally peer reviewed.

12
13

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failure (HMERF) may not be rare. J Neurol Neurosurg Psychiatry 2014;85:248.
Fisher D, Kley RA, Stratch K, et al. Distinct muscle imaging patterns in myoﬁbrillar
myopathies. Neurology 2008;71:758–65.
Wattjes MP, Kley RA, Fischer D. Neuromuscular imaging in inherited muscle
diseases. Eur Radiol 2010;20:2447–60.

Uruha A, et al. J Neurol Neurosurg Psychiatry 2015;86:483–489. doi:10.1136/jnnp-2014-309009

-306-

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J Neurol Neurosurg Psychiatry: first published as 10.1136/jnnp-2014-309009 on 24 September 2014. Downloaded from http://jnnp.bmj.com/ on 9 April 2019 by guest. Protected by copyright.

Neuromusculararticlesnpg© 2014 Nature America, Inc. All rights reserved.

Structure of the human Cereblon–DDB1–lenalidomide
complex reveals basis for responsiveness to thalidomide
analogs
Philip P Chamberlain1, Antonia Lopez-Girona1, Karen Miller1, Gilles Carmel1, Barbra Pagarigan1,
Barbara Chie-Leon1, Emily Rychak1, Laura G Corral1, Yan J Ren1, Maria Wang1, Mariko Riley1, Silvia L Delker1,
Takumi Ito2, Hideki Ando2, Tomoyuki Mori3, Yoshinori Hirano3, Hiroshi Handa2, Toshio Hakoshima3,
Thomas O Daniel1 & Brian E Cathers1
The Cul4–Rbx1–DDB1–Cereblon E3 ubiquitin ligase complex is the target of thalidomide, lenalidomide and pomalidomide,
therapeutically important drugs for multiple myeloma and other B-cell malignancies. These drugs directly bind Cereblon(CRBN) and promote the recruitment of substrates Ikaros (IKZF1) and Aiolos (IKZF3) to the E3 complex, thus leading tosubstrate ubiquitination and degradation. Here we present the crystal structure of human CRBN bound to DDB1 and the drug
lenalidomide. A hydrophobic pocket in the thalidomide-binding domain (TBD) of CRBN accommodates the glutarimide moietyof lenalidomide, whereas the isoindolinone ring is exposed to solvent. We also solved the structures of the mouse TBD in the apo
state and with thalidomide or pomalidomide. Site-directed mutagenesis in lentiviral-expression myeloma models showed that key
drug-binding residues are critical for antiproliferative effects.
The ubiquitin-proteasome pathway is the principal mechanism bywhich proteins become marked for degradation contributing to intracellular protein homeostasis. Dysregulation of this key protein homeostatic mechanism is implicated in the etiology of diseases including
cancer, neurological conditions and metabolic diseases1. Drugs thatmodulate the ubiquitin-proteasome pathway therefore have thepotential to affect a wide range of biological processes and diseases.
Thalidomide and related IMiD immunomodulatory agents, such aslenalidomide and pomalidomide, have shown anticancer effects indifferent indications by the direct inhibition of cancer cell proliferation as well as modulation of the immune system and the tumor
microenvironment2–5. This drug class is characterized by a common
structural feature, the glutarimide ring (Fig. 1a–c). The remainder ofthe molecule is also quite similar among the three drugs, with only thepresence or absence of an amino group or a carbonyl group roundingout the structural differences (Fig. 1a–c).
CRBN, first identified as a genetic cause of inherited autosomal
recessive mental retardation6, is a component of a ubiquitin E3 cullin
4–RING ligase (CRL4) complex and is the direct target of thalidomide and other IMiD drugs7,8. CRL4 ubiquitin ligases are formed
by a cullin protein (Cul4), which acts as an assembly factor that provides a scaffold for assembly of a RING box–domain protein (RBX1)
and the adaptor protein damaged DNA–binding protein 1 (DDB1)9.
RBX1 is the docking site for the activated E2 protein, and DDB1
recruits substrate-specificity receptors or DCAFs (DDB1–cullin

4–associated factors) to form the substrate-presenting side of
the CRL4 complex. Roughly 60 DCAFs have been identified9–12.
These DCAFs are typically characterized by the presence of a
WD-repeat domain11. Biochemical and genetic evidence has shownthat CRBN is a DCAF for CRL4 (refs. 7,9) and that binding of IMiD
compounds to CRBN affects the ubiquitin E3 ligase activity of CRBNbound CRL4 (CRL4CRBN), thus mediating the antiproliferative
effects on multiple myeloma (MM) cells and the immunomodulatory effects on T cells7,8,13. Recently, several groups have identified the transcription factors Ikaros and Aiolos as substrates of the
CRL4CRBN–IMiD drug complex, thus explaining many of thetherapeutic effects of IMiD compounds on immune and tumor
cells14–17. These reports describe that binding of IMiD compounds
to CRBN promotes recruitment of Ikaros and Aiolos to CRL4CRBN,
resulting in the increased ubiquitination and proteasome-dependentdegradation of these transcription factors in both MM and T cells. In
myeloma cells, targeted knockdown of Ikaros and Aiolos mimics thedecrease of the myeloma survival factor IRF4 as well as the decreasein cell viability observed with IMiD drug treatment. In T cells, Ikaros
and Aiolos are known repressors of interleukin-2 (IL-2) transcription,and knockdown of either protein produces an increase in IL-2 similar
to that after IMiD drug treatment14,15,17. These findings demonstrated
that IMiD drug binding confers new functionality to the CRL4 CRBN
complex, thus raising the possibility that a neomorphic structure
is involved.

1Celgene Corporation, San Diego, California, USA. 2Tokyo Medical University, Tokyo, Japan. 3Nara Institute of Science and Technology, Nara, Japan.

Correspondence should be addressed to P.C. (pchamberlain@celgene.com).
Received 27 June; accepted 16 July; published online 10 August 2014; doi:10.1038/nsmb.2874

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articlesadthat the IMiD compound–binding site is critical for the druginduced antiproliferative and Aiolos-mediated degradation effects.
We further solved drug complexes of pomalidomide and thalidomide
with the mouse TBD and demonstrated that the binding pocket anddrug binding mode are conserved. Interestingly, we identified tworodent polymorphisms, proximal to the lenalidomide-binding site,that may be responsible for the lack of IL-2–inducing effects observedin mouse splenocytes treated with pomalidomide.

TBD

N

CRBN
LLD
C

BPC

b

BPA

c

DDB1

npg

© 2014 Nature America, Inc. All rights reserved.

BPB

Figure 1 The structures of thalidomide analogs and human CRBN
in complex with human DDB1 and lenalidomide. (a) The chemical
structure of thalidomide, with the glutarimide ring shown in red
and the phthalimide moiety in black, along with an Fo − Fc electron
density difference map calculated in the absence of the molecule andcontoured at 3σ. (b) As in a, showing pomalidomide. (c) As in a, showing
lenalidomide with the isoindolinone moiety in black. (d) Structure of
CRBN–DDB1. The CRBN LLD is shown in yellow, the CRBN TBD in blue
and the region deleted in a human polymorphism associated with mild
mental retardation in red. DDB1 is shown in green with a gray surface,with β-propeller domains labeled BPA, BPB and BPC. Lenalidomide isshown as yellow sticks and the zinc ion as a gray sphere. N and C terminiof Cereblon are labeled.

To better define the molecular mechanisms underlying distinct
actions of lenalidomide compared with other IMiD drugs and todelineate species-specific pharmacologic actions, we solved the crystal
structure of lenalidomide bound to human CRBN in complex with its
native partner DDB1. The structure reveals that CRBN is composedof a Lon protease–like scaffold with a helical insertion that mediates
DDB1 binding. The TBD occurs at the C terminus and has structural
homology to methionine sulfoxide reductase and to RIG-I–like doublestranded RNA (dsRNA)-binding domains. Lenalidomide binding occurs at a small hydrophobic pocket composed of three tryptophan residues, thus providing a structural rationale for the loss ofthalidomide binding to CRBNYW/AA mutants first described by Ito
et al.7. Interestingly, a portion of the drug is
exposed on the surface of the CRBN–
alenalidomide complex, thus indicating that thispart of the IMiD molecule would be available
to form a component of a neomorphic interface in substrate recruitment. Site-specificmutagenesis studies in MM cell lines indicate
Figure 2 Structural comparisons of CRBN–
DDB1. (a) Structural superposition of CRBN
with B. subtilis Lon protease (red, PDB 3M65).
The CRBN LLD is shown in yellow and the TBD
in blue. The DDB1-binding motif is inserted
into the LLD. The TBD exhibits no similarity to
Lon protease and occurs at the C terminus.
(b) Structural superposition of CRBN–DDB1(yellow and green, respectively) with DDB–DDB1(DDB2 shown in blue; DDB1 omitted). CRBN
helices interacting with DDB1 are labeled A–E.

804

RESULTS
Structure of CRBN in complex with DDB1 and lenalidomide
The structure of CRBN reveals that CRBN is a DCAF that does notexhibit WD-repeat architecture typical of the class. The CRBN structure (Fig. 1d) revealed two distinct domains: an N-terminal Lon-likedomain (LLD) and a C-terminal TBD. The structure also revealed
that the DDB1- and lenalidomide-binding sites occur on opposite
sides of the CRBN surface. A human polymorphism linked to mental
retardation, resulting in premature truncation at amino acid 419, hasbeen found in CRBN. The deleted region occurs within the TBD, witha short helical region and a β-strand removed (Fig. 1d). The deleted
region occurs at the interface between the LLD and the TBD, andtruncation of these residues might be expected to deleteriously affectthe domain structure and stability of CRBN.
The Lon-like domain (LLD) spans residues 76–318 and contains
the DDB1-binding motif. Structural alignment with Bacillus subtilis
Lon N-terminal domain18 yields an r.m.s. deviation of 2.4 Å over
165 aligned residues (Fig. 2a). However, the DDB1-binding motif
and the TBD do not exhibit any homology with the examined Lon
domains (Fig. 2a). When compared to B. subtilis Lon, the DDB1binding motif appears to have been inserted into the LLD, and it
is composed of a series of helices between CRBN residues 188
and 248. CRBN binds between the β-propeller A (BPA) and theβ-propeller C (BPC) domains of DDB1 in a similar location to other
DCAFs (for example, DDB2 or DCAF9), as well as to the viral DDB1binding proteins Hbx and SV5V19. However, although CRBN binds to
DDB1 in the same cleft, the nature of the interaction with DDB1 exhibits differences compared to that of DDB2, SV5V or Hbx. Previously
studied DCAFs position a helix-turn-helix motif in the DDB1 binding
site, to predominantly form interactions with the DDB1 BPC domain(helices A and B, Fig. 2b)19. CRBN interacts with DDB1 via a seriesof helices; however, these do not superpose with the DCAFs that havebeen structurally characterized (Fig. 2b). In CRBN, residues 221–248
form helices that interact with the DDB1 BPC domain (helices C

b

A

E

D

C

B

DDB1 BPA
DDB1binding
motif

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articles
Figure 3 Structure of the TBD of human andmouse CRBN. (a) The human TBD is shownin blue, lenalidomide in yellow sticks and the
LLD in yellow. Backbone atoms are represented
only for residues 376–380 to show bonding.
Three hydrogen bonds between the TBD andlenalidomide are shown as dashed lines tothe protein backbone at His378 and Trp380
and also to the side chain of His378. The
zinc-binding site is shown as a gray sphereand is ~18 Å from lenalidomide. The β-sheet
region, which varies in the other structures, ismarked by a β. (b) Mouse TBD in complex withthalidomide. (c) Mouse TBD in complex withpomalidomide.

a

b

β
H353
P352
H378

N351

H380

V379

W382
W402

H357

E377

W386

I390

W388

W380 W400

I373

V388
C394

I371

c

H380

C391
Zn
Y384

C326

V379

W382
W402

npg

© 2014 Nature America, Inc. All rights reserved.

W388

and D, Fig. 2b). CRBN residues 191–197 alsointeract with the DDB1 BPC in a proximal
region to DDB2. However, as well as interacting with the BPC domain, CRBN also positions a helix composed ofresidues 198 to 209 to interact with the BPA domain of DDB1; to ourknowledge, this is the first example of such an interaction (helix E,
Fig. 2b). After the DDB1-binding motif, structural homology with
the LLD is restored between residues 249 and 318, which form afour-helix bundle leading to the TBD at residues 319–428.
The TBD is located on the face of CRBN that is oriented awayfrom the DDB1-binding site. The TBD is composed of a six-stranded
antiparallel β-sheet core, with a structural zinc ion coordinated byfour cysteine residues (Cys323, Cys326, Cys391 and Cys394). The
Table 1 Data collection and refinement statistics
hCRBN–
DDB1–
lenalidomide

mCRBN TBD–
thalidomide

mCRBN TBD–
pomalidomide

P212121

I23

I23

Data collection
Space group
Cell dimensions
a, b, c (Å)
Resolution (Å)

71.8, 129.1,

143.18,143.34,198.7

143.18, 143.18

143.34, 143.34

50–3.050–1.88

50–2.0(3.12–3.01)a(1.95–1.88)(2.11–2.00)
9.0 (24.5)

Rmerge (%)

18.1 (72.3)

8.9 (59.9)

I / σI

10.1 (2.6)

38.4 (6.6)

10.1 (5.4)

Completeness (%)

98.5 (85.8)

99.9 (100.0)

92.3 (92.4)

Redundancy

6.1 (5.8)

44.2 (45.1)

2.9 (2.9)

Resolution (Å)

50.0–3.01

50.0–1.88

30.0–2.0

No. reflections

35,39637,58428,7500.201 / 0.271

0.173 / 0.220

0.195 / 0.241

Refinement

Rwork / Rfree
No. atoms

11,043

3,093

2,857

Ligand/ion

Protein

20

120

124

Water

18

368

181

Protein

30.7

25.8

18.5

Ligand/ion

37.7

27.7

36.6

Water

25.5

40.8

35.7

Bond lengths (Å)

0.008

0.013

0.016

Bond angles (°)

1.211

1.644

1.695

B factors

r.m.s. deviations

aValues in parentheses are for highest-resolution shell. H, human; m, mouse.

nature structural & molecular biology

VOLUME 21

I390
I373

zinc-binding site is located ~18 Å from the lenalidomide-bindingsite (Fig. 3a). The IMiD-compound pocket is itself formed by threetryptophan residues, Trp380, Trp386 and Trp400, with a phenylalanine residue at the base (Phe402) (Fig. 3a). These residues form asmall hydrophobic pocket (tri-Trp pocket) in which the glutarimide
portion of lenalidomide is accommodated. A combination of mutations at both Tyr384 and Trp386 has been reported to cause a loss of
the IMiD-compound effects (Fig. 3a)7,14,15,17. Trp386 is part of thelenalidomide-binding site (Fig. 3), and mutation of that residue would
be expected to have consequences for drug binding. Tyr384, however, does not make any direct interactions
with lenalidomide (Fig. 3a), and it might be
expected to have a more general effect on
the stability of the domain. Within the triApo-mCRBN
Trp pocket, there are two hydrogen bonds
TBD
between the glutarimide ring and the protein backbone at residues His378 and Trp380(Fig. 3a) and a further hydrogen bond withI213
the side chain of His378. The isoindolinone
ring of lenalidomide is not enclosed within
117.73,the hydrophobic pocket and instead presents
117.73, 117.73
on the surface of the protein. In this position,
50–2.0one face of the isoindolinone ring interacts(2.07–2.00)
with a β-turn encompassing residues 351–
9.2 (43.1)
353 (β in Fig. 3a), with contacts to Pro352,
35.6 (9.6)
His353 and Asn351. The carbonyl oxygen of
100.0 (100.0)
the isoindolinone ring is oriented toward the22.5 (22.4)
side chain of His357; however, the distance isslightly too long for a hydrogen bond to form
(4.1 Å). Similarly, the aniline amino group of
3.15–2.0
lenalidomide is oriented toward Glu377 but
17,462
at a distance too great for an interaction to
0.191 / 0.225
be made (5.5 Å).
It has been proposed that the IMiD
1,612
drug interaction with CRBN may serve22
to alter the specificity of the Cul4 ubiqui139
tin ligase complex by recruiting new partners or altering the substrate affinity of its
37.6
client substrates14,15,17. It is therefore intrigu41.3
ing that the isoindolinone ring is exposed on
46.0
the surface of the CRBN–lenalidomide complex, thus indicating that this part of the IMiD
0.006
molecule would be available to form part of a
1.011
neomorphic interface in substrate recruitment.

NUMBER 9-309-

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articlesa HomoMacacab Macaca fascicularis

WT

IB: CRBN

CRBNW400A

DF15R hCRBN

WT

DF15R hCRBNW386A
DF15R hCRBNW400A

50

DF15R
CRBNW386A

Le
n
Po
m

DF15R
CRBNWT

Le
n
Po
m
V

V

DF15R RFP control

DF15R

Po
m

DF15R

100
CRBN

DF15

Le
n

V

c

DF15

V

3 V 30 3bIn

Glycine max(Fig. 3c). The same is true for chain A in the thalidomide structure(Fig. 3b); however, in chains B and D the β-strands have adopted analternative conformation in which these residues bind the phthalimide ring of thalidomide from an adjacent molecule in the asymmetricunit (Supplementary Fig. 2). This reciprocal arrangement forms a
dimer in the crystal lattice, with Tyr355 stacking against the accessible surface of the IMiD compound. There is no evidence of dimerization in the presence or absence of an IMiD compound (data notshown). The change in conformation provides a substantial change
in the surface of CRBN in the IMiD-compound-site vicinity, with
the intriguing potential to influence binding-partner recruitment.
However, the extent to which different conformations are influenced
by protein truncation or the lattice environment is unknown. We havealso solved the structure of the apoprotein and have confirmed that
the TBD and IMiD compound–binding site retain a similar structurein the absence of ligand (Supplementary Fig. 3). Furthermore, wehave solved structures of both human and mouse TBDs, in which weobserved the same conformations (data not shown), thus suggestingthat the structural changes observed are not likely to be a consequenceof species differences.
The alternative substitutions on the isoindolinone or phthalimide
ring provide a further mechanism to selectively recruit binding
partners to the CRL4CRBN E3 ligase complex. We previously demonstrated that the binding affinities of lenalidomide and pomalidomide
for CRBN are similar, whereas the affinity of thalidomide for CRBN
is slightly weaker8. Furthermore, glutarimide alone interacted with
CRBN. The narrow range of binding affinities, taken with the structural studies, is consistent with the glutarimide moiety providing the

n
Po
m

V 30

Pom
(µM)

Drosophila melanogaster

Le

Thal
(µM)

Mus musculus

V

a

414
414
416
417
547
519

Mus
RattusDrosophila
Glycine max

In this instance, the unused hydrophobic and polar bonding potentialof lenalidomide and the adjacent protein surface could play a key partin altering or enhancing binding-partner recruitment. Furthermore,
the structural and spatial differences of the functional groups on
thalidomide, pomalidomide and lenalidomide (Fig. 1a–c) could
provide unique interactions thereby recruiting different sets ofsubstrates to the CRL4CRBN–IMiD drug complex.
The TBD can be expressed and purified in isolation, and we haveused this system to obtain crystal structures of the mouse CRBN TBD
in complex with thalidomide and pomalidomide. We initially solved
the TBD structure by taking advantage of the predicted zinc-bindingsite in a single-wavelength anomalous dispersion experiment at a
normal synchrotron wavelength of 1.0 Å. We noted that the electron
density favored the S enantiomer of thalidomide, which was crystallized as a racemic mixture. This is consistent with the demonstrationthat S-thalidomide binds CRBN more tightly than the R enantiomer
by competition for thalidomide-immobilized beads (Supplementary
Fig. 1) and is also consistent with previous results with conformationally stable R- and S-methyl-pomalidomide analogs8. The core featuresof the IMiD-compound site that interact with the glutarimide ring are
well conserved between the lenalidomide, thalidomide and pomalidomide structures, with the three tryptophan residues providing the
key interactions to the glutarimide ring (Fig. 3a,b). The two hydrogenbonds to His378 and Trp380 are also conserved; however, the thirdhydrogen bond to the side chain of His378 is not made. There is a
striking difference between the lenalidomide structure and the others:
the two β-strands composed of residues 346–363 are absent in boththe thalidomide and the pomalidomide structures (Fig. 3a–c). There
are four protein-drug pairs in the asymmetric unit for both the thalidomide and pomalidomide structures. In all proteins in the asymmetricunit in the pomalidomide structure, the β-strand region is disordered% viable cells

npg© 2014 Nature America, Inc. All rights reserved.

Figure 4 Sequence-alignment differences mapped onto the surface of
the TBD of CRBN. (a) Alignment with tryptophan residues forming thetri-Trp pocket shown in bold. Variable residues proximal to the tri-Trp
pocket are shown in red. (b) Sequence alignment from a mapped onto
the surface of the human CRBN TBD. Conserved residues relative to thehuman protein are shown in red, conservative changes in orange andnonconservative changes in white. The IMiD compound–binding pocket
is indicated by a blue arrow.

CRBN
Aiolos
c-Myc
IRF4
ppRb

CRBNW386

β-tubulin

00.01 0.1 1
10 100
Pomalidomide (µM)

Figure 5 Conserved tryptophan residues (Trp386 and Trp400) confer binding of CRBN to IMiD compounds and are required for drug function incells. (a) Immunoblot (IB) analysis of extracts prepared from DF15R cells reexpressing full-length CRBN wild type or W386A or W400A mutants andpreincubated with vehicle DMSO (V), thalidomide (Thal) or pomalidomide (Pom) at the indicated concentrations and bound to thalidomide-analog
affinity beads, washed and eluted with SDS buffer. Input extract before bead purification (In) is also shown. Immunoblots are representative of three
independent experiments. (b) Cell proliferation assay of DF15 cells (sensitive), DF15R cells (resistant; CRBN null) and DF15R cells transduced with a
red fluorescent protein (RFP) control construct, human (h) CRBN WT, CRBNW386A or CRBNW400A, treated with a dose response of pomalidomide for 5 d.
Values are mean and s.d. from three independent experiments performed on different days. Data for each cell line are normalized to treatment withvehicle (DMSO). (c) Immunoblot analysis of CRBN, Aiolos, c-Myc, IRF4, phospho-Rb (ppRb) and β-tubulin loading control in DF15, DF15R, DF15R
CRBNWT and DF15R CRBNW386A treated with vehicle DMSO (V), 10 µM lenalidomide (Len) or 1 µM pomalidomide (Pom) for 48 h.

806

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Figure 6 Mouse CRBN does not rescue pomalidomide effects in DF15R
cells. (a) Cell proliferation assay of DF15 (sensitive), DF15R (resistant;
CRBNnull), DF15R-hCRBNWT (human CRBN) and DF15R-mCRBNWT(mouse CRBN) cells treated with a dose response of pomalidomide for
5 d. Values are mean and s.d. from three independent experiments
performed on different days. Data for each cell line were normalized totreatment with vehicle (DMSO). (b) CRBN immunoblot (IB) of extracts
from DF15, DF15R and DF15R cells reexpressing full-length mouse CRBN
wild type (mCRBNWT). β-tubulin is a loading control. (c) Aiolos andIkaros immunoblots of extracts from DF15 and DF15R cells reexpressing
mCRBN treated with pomalidomide at the indicated concentrations for
48 h. β-tubulin is a loading control.

DF15 DF15R DF15R
WT

mCRBN

DF15R hCRBN
DF15R mCRBNWT

IB: CRBN

100% viable cells

IB: β-tubulin

c
50

DF15R
Pom (µM)

DF15
0
10

mCRBNWT
010

IB: Aiolos

Mutational analysis of the lenalidomide-binding site
To better understand the role of the residues involved in the
CRBN–IMiD compound interactions in a cellular context, we performed mutagenesis of residues Trp386 or Trp400 to alanine andused lentiviral expression vectors to reexpress full-length wild-type(CRBNWT), W386A (CRBNW386A) or W400A (CRBNW400A) mutant
versions of CRBN in a CRBN-deficient myeloma cell line, DF15R 8.
Transduction of the lentiviral constructs into DF15R cells and selection
with blasticidin generated cell lines that stably expressed wild-type
VOLUME 21b

DF15R
DF15R RFP control
WT

majority of CRBN binding. The CRBN binding affinities, however, are
not proportional to the cellular potency of these drugs, thus suggesting that additional interactions with the phthalimide or isoindolinone
part of the molecules are differentiating cellular effects. Lenalidomide
and pomalidomide differ from thalidomide in possessing an amino
substitution on the isoindolinone or phthalimide ring (Fig. 1a–c).
In the lenalidomide structure, this amino moiety is oriented toward
Glu377, whereas in the pomalidomide structure, the electron
density supports differing orientations in the different chains within
the asymmetric unit (Figs. 1b and 3c and data not shown). As new
substrates are discovered for the CRL4CRBN E3 ligase, it will be interesting to see how structural differences among IMiD drug analogs
will influence the potency and selectivity of substrate recognitionand degradation.
A search for structural homologs to the TBD has revealed that
despite very low sequence identity (<14%), the same fold is found inother proteins, including methionine sulfoxide reductase, an enzyme
involved in the repair of oxidized proteins20, dsRNA–binding proteins RIG-I, LGP2 and melanoma differentiation-associated gene 5(MDA5) (Supplementary Fig. 4), which are involved in sensing viralRNAs as part of the innate immune response21–23, and mammaliansuppressor of Sec4 (MSS4), a regulator of stress response and apoptosis24. Despite sharing the same fold, the tri-Trp-pocket residues
are not conserved in any of these structurally similar proteins, thus
indicating that these cannot bind IMiD drugs in the same manner as
CRBN. However, CRBN orthologs can be identified across animal andplant kingdoms, and these proteins exhibit 100% sequence conservation in the tri-Trp pocket (Fig. 4). This level of sequence conservation
indicates a strong functional constraint and suggests that there may
be an endogenous ligand with which IMiD drugs compete. The
glutarimide-binding pocket formed by these three tryptophan residues is reminiscent of the aromatic pockets used for binding methylated lysine and/or arginine residues found in proteins containing
chromodomains, Tudor domains, plant homeodomain (PHD) fingers,
malignant brain tumor (MBT) repeats, bromodomains for binding to acetylated lysines, or tryptophan boxes as in betaine-binding
proteins such as ProX31 and BetP32 (Supplementary Fig. 5)25–27.
However, our isothermal titration calorimetry analysis showed nosignificant binding affinity to modified lysines, arginines or betaines(data not shown).

nature structural & molecular biology

DF15

a

IB: Ikaros
IB: β-tubulin

00.01 0.1
1
10Pomalidomide (µM)

100or mutant forms of CRBN at levels similar to the matched IMiD
compound–sensitive DF15 cell line (Supplementary Fig. 5a,b).
Immunoprecipitation and western blotting analysis confirmed
that, similarly to recombinant CRBNWT, mutants CRBNW386A andCRBNW400A interacted with DDB1, thus indicating that these proteins
were properly folded and were competent to form CRL4CRBN complexes in cells (Supplementary Fig. 5c)7. We then used cell extracts
from these lines to assay the ability of CRBN to bind IMiD compounds.
We used the thalidomide analog–coupled affinity bead assay7,8(Supplementary Fig. 5d) to demonstrate binding of recombinant
CRBN to IMiD compounds in cell extracts. Similarly to the endogenous
protein in DF15 cell extracts, the recombinant CRBNWT expressed
in DF15R was able to bind thalidomide-analog beads (Fig. 5a),and preincubation of cell extracts with free thalidomide or pomalidomide competed for binding to the beads. In contrast, the tryptophan mutants CRBNW386A and CRBNW400A were unable to bindthalidomide-analog beads (Fig. 5a), in agreement with the structural
information that both tryptophan residues in CRBN are needed forthe interaction with IMiD compounds.
The antiproliferative effect of IMiD compounds on myeloma cells
is mediated through CRBN protein. Thus, engineered cells or cells
made resistant to IMiD drugs that lack detectable levels of CRBN, forexample, DF15R, become insensitive to the antiproliferative effectsof the IMiD compounds (Fig. 5b)8,13. Importantly, the reexpressionof a single recombinant protein, CRBNWT, is sufficient to restore thesensitivity to IMiD compounds in the resistant line DF15R (Fig. 5b)
as well as in other cell lines (data not shown). In these cells, reexpression of CRBNWT also restores IMiD compound–induced degradationof Aiolos and, as expected, restores the inhibition of c-Myc, IRF4
and phopho-Rb expression (Fig. 5c). All of these downstream effects
are consistent with the regained antiproliferative effect of pomalidomide14,15,17,28. In contrast, expression of the mutants defectivein IMiD-compound binding, CRBNW386A or CRBNW400A, did notresensitize DF15R cells to the antiproliferative effects of IMiD drugs(Fig. 5b,c). These cellular data are consistent with the structural data
indicating that both Trp386 and Trp400 residues mediate CRBN binding to IMiD compounds and that CRBN mediates the antiproliferative effects characteristic of IMiD compounds in myeloma cells. It is
known that rodents do not respond to IMiD agents in the same waythat humans do; rodents appear to be resistant to the IL-2 induction(Supplementary Fig. 6), to antiproliferative effects and also to IMiD
compound–induced teratogenicity29. A key step in understanding
IMiD biology and ultimately engineering thalidomide analogs without teratogenic effects may indeed come from pinpointing the key

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articlesnpg© 2014 Nature America, Inc. All rights reserved.

features that underlie the biological differences seen between rodents
and humans. Mouse CRBN is unable to rescue the antiproliferative
effects of pomalidomide in DF15R cells (Fig. 6a). Furthermore, themouse CRBN-expressing cell line does not exhibit the pomalidomidedependent degradation of Ikaros or Aiolos (Fig. 6c). Within the TBD
there are four amino acid differences between mouse and humanproteins. Two of the variable residues are proximal to the lenalidomidebinding site (Fig. 4). One of these residues features a substantialchange in properties from an acidic residue (Glu377) in humans to ahydrophobic residue (valine) in rodents. The other is a conservativechange from valine in humans (Val388) to isoleucine in rodents. We
speculate that these polymorphic differences could account for thelack of rodent responsiveness to IMiD agents and suggest that a morethorough exploration of the functional consequences of these polymorphic differences is warranted. Furthermore, detailed structural
studies of CRL4CRBN in complex with substrates such as Ikaros andAiolos will be needed to fully understand the structural requirements
for substrate recruitment and subsequent ubiquitination.
DISCUSSION
Ito et al. first reported CRBN as the primary molecular target of thalidomide and through mutagenesis demonstrated that the C-terminal
domain encompassed the thalidomide-binding domain7. Herein, we
have presented the crystal structure of human CRBN in complex with
DDB1 and lenalidomide, thus providing the first structural description, to our knowledge, of IMiD drug binding. The IMiD compound–binding site is a shallow hydrophobic pocket on the surface of CRBN.
Three tryptophan residues form the binding site for the glutarimide
ring of lenalidomide, with three hydrogen bonds between CRBN andthe glutarimide ring. The glutarimide ring is a defining feature of
the IMiD class of molecules, and all IMiD drug–CRBN structures
solved to date have exhibited highly similar intermolecular interactions. We have demonstrated that IMiD-compound binding andcellular function is dependent upon these key tryptophan interactions by comparing mutant CRBN to wild-type CRBN in a CRBNdeficient myeloma cell line, DF15R. We also observed that expressionof mouse CRBN behaves in an identical manner to that of mutated
CRBN in our human myeloma cell line, in that mouse CRBN isunable to rescue the pomalidomide-induced antiproliferative or
Aiolos- or Ikaros-associated degradation effects (Fig. 6). Sequence
differences identified in mouse CRBN that are in proximity to thelenalidomide-binding site may have a role in the phenotypic differences observed, thus implying that a single amino acid change could
mediate resistance to IMiD drugs. However, few CRBN mutations
have been found in patients with MM30,31, and these have not yet beencharacterized biochemically.
IMiD drug binding to CRBN has been demonstrated to increase
the recruitment of substrates (for example, Ikaros and Aiolos) to theubiquitin ligase complex and to enhance their degradation14,15,17.
Interestingly, the lenalidomide isoindolinone group is presented
on the surface of the CRBN protein. This binding mode generates
a surface with a number of available hydrogen bonds from both theprotein and lenalidomide in the proximity of an exposed hydrophobicgroup. The unsatisfied bonding potential around the bound lenalidomide could therefore form the basis of an artificially introduced
interaction hotspot, thereby conferring a pharmacologically induced
neomorphic function to this E3 ligase. However, in the case of CRBN,
we have shown that the tri-Trp pocket is highly conserved across the
known orthologs, and this indicates that IMiD drugs may be binding in place of an endogenous ligand that has yet to be identified.
There are several examples of endogenous ligands that have been
808

VOLUME 21

demonstrated to regulate substrate recruitment to ubiquitin ligase
complexes in plant systems. For example, auxin and jasmonate aresmall-molecule regulators of ligases in plants32,33. Similarly, a numberof natural products exhibit pharmacological activity by scaffolding
macromolecular interactions34.
The ability for a small molecule to scaffold a specific macromolecular interaction to an E3 ubiquitin ligase has particularly exciting
implications for drug discovery. We have observed several categories
of variation proximal to the IMiD drug–binding site: variations in
the presentation and substitution pattern of the solvent-exposed ligand
groups, protein conformational differences and species sequence
differences. To fully rationalize the role of these in IMiD biology,
we anticipate several further advancements, such as identificationof the endogenous ligand(s), identification of substrates uniquely
targeted by different IMiD drug molecules, characterization of theeffects of species polymorphisms and determination of the binding
modes of substrate proteins such as Aiolos after recruitment to the
CRL4CRBN complex upon IMiD drug treatment. By describing the
crystal structure of lenalidomide bound to CRBN, a substrate adaptor
for an E3 ubiquitin ligase, we hope to further enable progress toward
the rational design of additional ubiquitin ligase modulators.
METHODS
Methods and any associated references are available in the online
version of the paper.
Accession codes. Coordinates and structure factors have been
deposited in the Protein Data Bank under accession codes 4TZ4(CRBN–DDB1–lenalidomide), 4TZU (CRBN–pomalidomide), 4TZC
(CRBN–thalidomide) and 3WX2 (apo-CRBN).
Note: Any Supplementary Information and Source Data files are available in the online
version of the paper.
ACKnOWLEDGMEnTS
Thanks to G. Reyes, R. Chopra, P. Schafer, P. Jackson, A. Mahmoudi, G. Lu andW. Fang for discussions regarding this manuscript. Thanks to G. Ranieri andR. Walter for data-collection services. Thanks to M. Abbasian, J. Evans and
W. Kehayias for technical assistance. We would like to thank BL41XU and BL44XU
beamlines at the SPring-8 synchrotron facility for the provision of synchrotron
data-collection facilities (proposal nos. 2012A6738, 2012B1205 and 2012B6738).
Use of the Advanced Photon Source was supported by the US Department ofEnergy, Office of Science, Office of Basic Energy Sciences, under contract no.
W-31-109-Eng-38. Portions of this research were conducted at the Advanced
Light Source, a national user facility operated by the Lawrence Berkeley National
Laboratory, on behalf of the US Department of Energy, Office of Basic Energy
Sciences. The Berkeley Center for Structural Biology is supported in part by the
Department of Energy, Office of Biological and Environmental Research, andby the US National Institutes of Health, National Institute of General Medical
Sciences. Research described in this paper was performed at beamline 08ID-1
at the Canadian Light Source, which is supported by the Natural Sciences andEngineering Research Council of Canada, the National Research Council Canada,
the Canadian Institutes of Health Research, the Province of Saskatchewan,
Western Economic Diversification Canada and the University of Saskatchewan.
Research was funded by a Grant-in-Aid for Scientific Research on Innovative Areas
from the Japanese Ministry of Education, Culture, Sports, Science and Technology,
Grant-in-Aid for Scientific Research (A) from the Japan Society for the Promotionof Science (H.H. and T.H.).
AUTHOR COnTRIBUTIOnS
P.P.C., G.C., B.P., B.C.-L., S.L.D., M.R., T.M., Y.H. and T.H. performed protein
chemistry and structural studies. A.L.-G., K.M., E.R., L.G.C., Y.J.R., M.W., T.I. and
H.A. performed biochemical and cellular experiments. P.P.C., A.L.-G., H.H., T.H.,
T.O.D. and B.E.C. planned the work, and all authors contributed to the manuscript.
COMPETING FINANCIAL INTERESTS
The authors declare competing financial interests: details are available in the online
version of the paper.

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ONLINE METHODS

Structure determination. CRBN TBD was purified as follows. Residues 321–429
of mouse CRBN fused to GST were expressed in E. coli BL21 (DE3) Star cells(Life Technologies) in TB medium supplemented with 50 µM zinc acetate. Cells
were lysed by sonication and the soluble fraction purified with GST-affinity,
ion-exchange and size-exclusion chromatography. Protein was concentrated to
28 mg/ml in 50 mM acetate buffer, pH 6.0, 1 mM DTT and 10 mM TCEP. Crystals
were obtained by sitting-drop vapor diffusion by mixing the protein with buffer
1:1 and equilibrating against a mother liquor containing 100 mM sodium acetate,
pH 5, 600–800 mM ammonium sulfate. Crystals were cryoprotected by additionof 20% glycerol and cooled under liquid nitrogen.
The structure of the mouse TBD was solved by single-wavelength anomalous
dispersion with intrinsically bound zinc ions. Briefly, 480° of data were collectedfrom a single crystal at 1.0-Å wavelength at the Advanced Photon Source SER-CAT
beamline 22ID. Data were integrated and scaled with HKL2000 (ref. 35). Phasing
and automated model building were performed with Crank in CCP4i, with thefollowing subprograms: AFRO/CRUNCH2/BP3/SOLOMON/Buccaneer36–39.
Subsequent manual model building and refinement were performed with Cootand Refmac5, respectively40,41. Subsequent structures were solved by molecular replacement with Phaser42. Figures were prepared with PyMOL (DeLano
Scientific) and Maestro (Schrödinger).
Human CRBN with a thrombin-cleavable N-terminal ZZ-histidine tag (residues 40–442) and full-length human DDB1 with a C-terminal strep tag were
coexpressed in SF9 insect cells in ESF921 medium (Expression Systems) supplemented with 50 µM zinc acetate. Cells were subsequently centrifuged andresuspended in eight volumes lysis buffer (50 mM Tris, pH 7.5, 500 mM NaCl,
10% glycerol, 10 mM imidazole and 1 mM TCEP) plus 1× protease-inhibitor
cocktail (S.D. Bioscience) and 5,000 units TurboNuclease (Accelagen) for 1 h
at 4 °C. The lysate was centrifuged at 105,000g for 1 h, and the supernatant wasloaded to 5 mL Ni-NTA preequilibrated in lysis buffer. CRBN–DDB1 was eluted
with a stepwise method with elution buffer (50 mM Tris, pH 7.5, 500 mM NaCl,
10% glycerol, 500 mM imidazole and 1 mM TCEP). The ZZ-histidine tag wasremoved by cleavage overnight with thrombin (Enzyme Research). Peak fractions were pooled, diluted to a final concentration of 200 mM NaCl and immediately applied to a HiTrap ANX 5-mL column (GE Life Sciences). The buffer
was changed on column to Bis-Tris, pH 6.0, and protein was eluted with a linear
gradient to 1 M NaCl. CRBN–DDB1 was further purified by S-400 gel filtration(GE Life Sciences). CRBN–DDB1 at 30.2 mg/ml, in a buffer consisting of 10 mM
MES, pH 6.0, 200 mM NaCl and 5 mM TCEP, was crystallized by sitting-drop
vapor diffusion. CRBN–DDB1 plus 1 mM lenalidomide was mixed 1:1 with,and subsequently equilibrated against, a reservoir solution containing 18% (w/v)
PEG 10,000 (Hampton Research, Aliso Viejo, California, USA) and 100 mM
HEPES, pH 7.5 (Hampton Research, Aliso Viejo, California, USA). Cocrystals
appeared in 7 d and reached dimensions of 0.10 mm × 0.025 mm × 0.025 mm
by 21 d. Before data collection, crystals were cryoprotected in the reservoir solution supplemented with 20% ethylene glycol and cooled under liquid nitrogen.
Data were collected from a single crystal at the Canadian Light Source beamline
CMCF-08ID. The structure of human CRBN–DDB1 was solved by molecularreplacement with Phaser42, with DDB1 (PDB 3EOC) and the mouse CRBN TBD
as search models. Subsequent manual model building and refinement were performed with Coot and Refmac5, respectively40,41. Crystallographic statistics areshown in Table 1, and difference density calculated following a round of refinement with the drug molecule omitted is shown in Figure 1a–c.
Cell lines. The DF15 and DF15R cell lines, previously shown to lack detectable
CRBN protein, were grown and cultured as previously described8.
Wild-type and mutant CRBN plasmids and CRBN pulldown assays. Lentiviral
expression vectors were produced in house. Lentiviral particles were generated
with HEK293T cells. and infection was done with spinoculation methodology.
Selection was performed 1 week after initial infection. A diagram of the plasmids used is shown in Supplementary Figure 1b. DF15 and DF15R cells weretransduced, and a pool of blasticidin-resistant cells were selected and screened bywestern blotting analysis with rabbit monoclonal anti-CRBN antibody (CRBN65;
1:10,000)8,43. We confirmed the presence of the corresponding CRBN mutant
versions by DNA genomic sequencing in all cell lines.

Immunoblot and immunohistochemistry. Western blotting was performed withmouse monoclonal anti-DDB1 (2B12D1; 1:2,000) from Invitrogen, rabbit polyclonals anti-Ikaros (D10E5; 1:1,000), anti-Aiolos (12720; 1:1,000), anti–c-Myc(9402; 1:1,000), anti-p21 (12D1; 1:1,000), anti-IRF4 (D43H70; 1:1,000) andanti-ppRb (Ser608) (2181; 1:1,000) from Cell Signaling Technology and mousemonoclonal anti–β-tubulin (AA2; 1:40,000) and mouse monoclonal anti–β-actin(AC74; 1:10,000) from Sigma. Validation for the antibodies used is available
on the respective manufacturers’ websites. CRBN65 antibody (1:10,000) wasused for CRBN protein detection and has been validated in previous work8,43.
CRBN immunohistochemistry was performed on the Bond-Max automated slide
stainer (Leica Microsystems, Buffalo Grove, Illinois, USA) with the Bond Polymer
Refine Detection system. Formalin-fixed, paraffin-embedded (FFPE) cell pellets
were sectioned at 4 µm thick and deparaffinized on the Bond-Max instrument. Antigen retrieval was performed with Epitope Retrieval 2 (ER2, pH 9.0)
for 20 min at 100 °C. The slides were blocked for endogenous peroxidase activity with Peroxide Block for 5 min at room temperature. Sections were thenincubated with the rabbit monoclonal antibody CRBN65 at a 1:4,000 dilution
for 15 min at room temperature. Post-primary and horseradish peroxidase(HRP)-labeled polymers were incubated at the instrument’s default conditions.
Antigen–antibody complex was then visualized with hydrogen peroxide substrate and diaminobenzidine tetrahydrochloride (DAB) chromogen. Slides werecounterstained with hematoxylin.
Thalidomide-analog bead assay to measure compound binding toendogenous CRBN. Coupling of thalidomide analog to FG magnetic nanoparticle beads (Tamagawa Seiko Co., Tokyo) was carried out as described by Ito et al.7,
and cell-extract binding assays to these beads were performed with minor
modifications. DF15-, DF15R- or DF15R-derived cell-line extracts were preparedin NP-40 lysis buffer (0.5% NP-40, 50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.5 mM
DTT, 0.25 mM PMSF and 1× protease-inhibitor mix (Roche, Indianapolis) at
approximately 2 × 108 cells per ml (20 mg protein/ml). Cell debris and nucleic
acids were cleared by centrifugation (14,000 r.p.m., 30 min, 4 °C). In competition experiments, 0.5-ml aliquots (3–5 mg protein) of the resulting extracts werepreincubated (15 min, room temperature) with 5 µl DMSO (control) or 5 µlcompound at varying concentrations in DMSO. Thalidomide analog–coupledbeads (0.3–0.5 mg) were added to protein extracts and samples rotated (2 h, 4 °C).
Beads were washed three times with 0.5 ml NP-40 buffer, and then bound proteins
were eluted with SDS-PAGE sample buffer. Samples were subjected to SDS-PAGE
and immunoblot analysis performed (as described above) with anti-CRBN(1:10,000 dilution) for all studies; other antisera dilutions were DDB1 (1:2,000
dilution) or β-actin (1:10,000 dilution). In thalidomide-affinity bead competition
assays, a LI-COR Odyssey system was used to quantify CRBN band density, andrelative amounts of CRBN were determined by averaging at least three DMSO
controls and expressing CRBN in each competition sample as percentage inhibition of CRBN protein relative to the averaged controls set at 100% binding.
Original images of gels, autoradiographs and blots used in this study can be foundin Supplementary Data Set 1.

35. Otwinowski, Z. & Minor, W. Processing of X-ray diffraction data collected inoscillation mode. Methods Enzymol. 276, 307–326 (1997).
36. Abrahams, J.P. & Leslie, A.G. Methods used in the structure determination of bovine
mitochondrial F1 ATPase. Acta Crystallogr. D Biol. Crystallogr. 52, 30–42 (1996).
37. de Graaff, R.A., Hilge, M., van der Plas, J.L. & Abrahams, J.P. Matrix methods
for solving protein substructures of chlorine and sulfur from anomalous data.
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improves single-wavelength anomalous diffraction phasing. Acta Crystallogr. D Biol.
Crystallogr. 60, 22–27 (2004).
39. Pannu, N.S. et al. Recent advances in the CRANK software suite for experimental
phasing. Acta Crystallogr. D Biol. Crystallogr. 67, 331–337 (2011).
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43. Gandhi, A.K. et al. Measuring cereblon as a biomarker of response or resistance tolenalidomide and pomalidomide requires use of standardized reagents andunderstanding of gene complexity. Br. J. Haematol. 164, 233–244 (2014).

doi:10.1038/nsmb.2874

nature structural & molecular biology-314-

ARTICLE
Received 28 Mar 2014 | Accepted 1 Jun 2014 | Published 27 Jun 2014

DOI: 10.1038/ncomms5263

DSIF and NELF interact with Integrator to
specify the correct post-transcriptional fateof snRNA genes
Junichi Yamamoto1, Yuri Hagiwara1, Kunitoshi Chiba1, Tomoyasu Isobe1, Takashi Narita2, Hiroshi Handa3& Yuki Yamaguchi1,4The elongation factors DSIF and NELF are responsible for promoter-proximal RNA polymerase
II (Pol II) pausing. NELF is also involved in 30 processing of replication-dependent histone
genes, which produce non-polyadenylated mRNAs. Here we show that DSIF and NELF
contribute to the synthesis of small nuclear RNAs (snRNAs) through their association with
Integrator, the large multisubunit complex responsible for 30 processing of pre-snRNAs. In
HeLa cells, Pol II, Integrator, DSIF and NELF accumulate at the 30 end of the U1 snRNA gene.
Knockdown of NELF results in misprocessing of U1, U2, U4 and U5 snRNAs, while DSIF
is required for proper transcription of these genes. Knocking down NELF also disrupts
transcription termination and induces the production of polyadenylated U1 transcripts causedby an enhanced recruitment of cleavage stimulation factor. Our results indicate that NELF
plays a key role in determining the post-transcriptional fate of Pol II-transcribed genes.

1 Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Yokohama 226-8501, Japan. 2 Graduate School of Frontier Biosciences,
Osaka University, Suita 565-0871, Japan. 3 Department of Nanoparticle Translational Research, Tokyo Medical University, Tokyo 160-8402, Japan.
4 PRESTO, Japan Science and Technology Agency, Kawaguchi 332-0012, Japan. Correspondence and requests for materials should be addressed to Y.Y.
(email: yyamaguc@bio.titech.ac.jp).

NATURE COMMUNICATIONS | 5:4263 | DOI: 10.1038/ncomms5263 | www.nature.com/naturecommunications& 2014 Macmillan Publishers Limited. All rights reserved.

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NATURE COMMUNICATIONS | DOI: 10.1038/ncomms5263

150

Flag-Spt5
Rpb2

100

Ints1
Ints2
Ints3
Ints6
Ints4
Ints5
Ints7

200

Spt5

Ints10
Ints9

75

NELF-A
NELF-B
NELF-C/D

Ints11
Ints1250
NELF-E

116

97.4
CBP80

NELF-AMediator

Rpb2

Ints5

Ints5

Ints6

Ints6

Ints7

Ints7

Ints8

Ints8

Ints9

Ints9

Ints10

Ints11

Ints11

Med17

Rec. Ints
GST-NELF-Ad

Flag-Ints1

Ints4

Ints4

(kDa)

Affinity-purified integrator

Rpb1

Ints3
Rpb1

c

10% Input
GST
GST-DSIF Spt5

Ints1
Rpb1

Integrator

250

EluateControl
Flag-NELF-E
Flag-Spt5(kDa)

2% Input
Control
Flag-NELF-E
Flag-Spt5b

Flag-NELF-E
Control

Flag-Spt5

ControlaHowever, recent studies have revealed that NELF alsofunctions at the 30 ends of certain genes to facilitate 30 processing
and/or transcription termination. For example, 30 processing of
histone genes requires binding of stem–loop-binding protein andU7 snRNP to evolutionarily conserved stem–loop and histone
downstream elements, respectively, and is catalysed by cleavage
and polyadenylation speciﬁcity factor (CPSF)1; NELF interacts
with the nuclear cap-binding complex and stem–loop-binding
protein to facilitate histone-type 30 processing20. Inhibition ofthese factors disrupts the canonical processing pathway andinduces the production of aberrant polyadenylated histonemRNAs through the use of cryptic downstream poly(A) signals(PASs). Moreover, NELF is also involved in transcription
termination of U2 snRNA21.
Genes encoding snRNAs have TATA-less promoters that
contain evolutionarily conserved distal and proximal sequence
elements; these elements bind to Oct-1 and SNAPc/PTF,
respectively, and direct transcription along with a set of generaltranscription factors and Pol II22. Primary snRNA transcripts arethen cleaved, most likely co-transcriptionally, at a conserved 30
box by Integrator, a 1.5-MDa complex that contains 12 subunits(Ints1–12), including two CPSF-like subunits23. Studies using
kinase inhibitors and CTD mutants revealed that
phosphorylation of the CTD at Ser2 and Ser7 is critical for thisprocessing reaction24–27. Consistent with these ﬁndings,
Integrator is able to bind tightly to the Pol II CTD
phosphorylated at both Ser2 and Ser7 in vitro28.
The present study was motivated by the identiﬁcation ofIntegrator as a complex that interacts with both DSIF and
NELF. Here we show that, along with Pol II and Integrator,
DSIF and NELF accumulate at the 30 end of the U1 snRNA geneand contribute to its synthesis in different ways. This study

20% Input
GST
GST-DSIF Spt5
GST-DSIF Spt4
GST-NELF-A
GST-NELF-B
GST-NELF-C/D
GST-NELF-E

I

n higher eukaryotes, transcripts produced by RNA polymerase
II (Pol II) undergo one of three types of 30 processing. Whilemost protein-coding genes produce polyadenylated mRNAs,
replication-dependent histone genes produce mRNAs lacking
poly(A) tails and require a distinct set of cis elements and transacting factors for 30 processing1,2. Pol II is also responsible fortranscribing small, non-coding, non-polyadenylated small nuclear
RNAs (snRNAs), which possess 30 ends that are generated via aseries of processing reactions starting with site-speciﬁc cleavage at
an evolutionarily conserved 30 box3. During the production ofpolyadenylated mRNAs, transcription and 30 processing aretightly coupled; cleavage and polyadenylation factors arerecruited during transcription, and transcript cleavage triggers
transcription termination2,4,5. However, it is unclear whether thistight coupling holds true for the other types of 30 processing.
Moreover, little is known about the mechanisms by which theappropriate processing pathway is selected for a speciﬁc gene.
Promoter-proximal pausing is a widespread checkpoint that
occurs during the transcription cycle6–8. The elongation factors
DSIF (comprising subunits Spt5 and Spt4) and NELF (comprising
subunits NELF-A, -B, -C/D and -E) induce transcriptionalpausing by interacting with early-elongation complexes. Positivetranscription elongation factor b (P-TEFb) then phosphorylates
the C-terminal domain (CTD) of Pol II at Ser2, thus reactivating
the paused complex9–13. Furthermore, P-TEFb phosphorylates
the C-terminal repetitive sequence (CTR) of DSIF, which isanalogous to the Pol II CTD, and this modiﬁcation converts DSIF
from a transcriptional repressor to an activator14,15. A number ofchromatin immunoprecipitation (ChIP) studies of Drosophila
and mammals have supported the idea that NELF associates onlywith the 50 end of protein-coding genes, while DSIF travels withPol II throughout the elongation process16–19.

Flag-Ints2*

Flag-Ints3
Flag-Ints4
Flag-Ints5
Flag-Ints6
Flag-Ints7
Flag-Ints8
Flag-Ints9
Flag-Ints10

Cdk8
Coomassie

CycC

Flag-Ints11
Flag-Ints12

Actin

66

Figure 1 | DSIF and NELF interact with Integrator. (a) SDS–PAGE analyses of DSIF- and NELF-associated proteins afﬁnity-puriﬁed from nuclear
extracts of HeLa cells stably expressing Flag-Spt5 (left) or Flag-NELF-E (right). The extracts were also subjected to an LC-MS/MS analysis. See also
Supplementary Table 1. (b) Immunoblot analyses of the samples described above using antibodies against speciﬁc subunits of Integrator and Mediator.
Actin was detected as a loading control. (c,d) Equal amounts of GST or GST-tagged subunits of DSIF and NELF were immobilized to glutathione Sepharose
beads and then incubated with Integrator afﬁnity-puriﬁed from HeLa cells expressing Flag-Ints10 (c) or recombinant Integrator subunits expressedindividually in insect cells (d). Rpb1 and the Integrator subunits were detected using immunoblotting. The asterisk denotes a nonspeciﬁc signal.
See also Supplementary Fig. 1. Uncropped blots are shown in Supplementary Fig. 6.
2

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provides new insights into the mechanism by which the posttranscriptional fate of Pol II-transcribed genes is determined.
Results
DSIF and NELF interact with Integrator. To understand thephysiological roles of DSIF and NELF in more detail, proteinsassociated with Flag-tagged Spt5 (the large subunit of DSIF)
and Flag-tagged NELF-E were afﬁnity-puriﬁed from HeLa cell
nuclear extracts and analysed using mass spectrometry (Fig. 1aand Supplementary Table 1). As expected, DSIF and NELF
co-precipitated with each other and with Pol II. In addition, thecap-binding complex was co-puriﬁed with NELF, as reported
previously20. Notably, 11 of the 12 subunits of Integrator, as well
as C12orf11, which might be an additional subunit ofIntegrator29,30, were also co-puriﬁed with DSIF or NELF
(Fig. 1a and Supplementary Table 1). To exclude the possibility
that the Integrator complex was co-puriﬁed due to interaction of
DSIF and NELF with Pol II, the afﬁnity-puriﬁed samples wereanalysed by immunoblotting with a panel of antibodies against
subunits of Integrator and Mediator, the latter of which is anotherlarge protein complex that associates with Pol II. This experimentshowed that Integrator subunits, but not Mediator subunits, coprecipitated with DSIF and NELF (Fig. 1b). Moreover, whenaIntegrator was afﬁnity-puriﬁed from HeLa cells expressing
Flag-tagged Ints10, Spt5, NELF-E and the Pol II subunit Rpb1
were co-puriﬁed (Supplementary Fig. 1a), suggesting that
Integrator interacts speciﬁcally with DSIF and NELF.
To study these interactions under equilibrium conditions,
HeLa cell nuclear extracts were subjected to sucrose gradientsedimentation analyses. The levels of DSIF, NELF, Integrator and
Pol II components peaked in fraction number 11 (41 MDa;
Supplementary Fig. 1b), suggesting the presence of a higher-ordercomplex containing all of these factors. A large proportionof Ints3 was separated from the other Integrator subunits(Supplementary Fig. 1b). This additional peak may represent
the recently identiﬁed SSOS complex that contains Ints3 and thehuman single-stranded DNA-binding protein homologues hSSB1
and hSSB2 (refs 31–34).
DSIF and NELF interact with Integrator via Spt5 and NELF-A.
To determine whether DSIF and NELF interact directly with
Integrator, pull-down assays were performed using afﬁnitypuriﬁed Integrator and GST-tagged subunits of DSIF and NELF.
GST-Spt5 and GST-NELF-A pulled down Integrator, although
only Ints6 was co-precipitated efﬁciently by GST-Spt5 (Fig. 1c).
Ints6 also interacted with GST-Spt5 in binding studies using

3′ box

+1

+250

RNU1

Ser7P

Spt5

4

NELF-E
Ints9

2

Actin
Uncl.

7SK

U4 total

U4 uncl.

4
3
2
0

U1 total

U1 uncl.

7SK

U1
6
5

Fold change

Total

5

Control
NELF-E KD
Spt5 KD
Ints9 KD

1

Actin
0

d

6
Fold change

Ser5P

6

7
Ints9 KD

Ser2P

c

Flavo–
Flavo+

Spt5 KD+
Fold change

Flavo –
Rpb1

8

Controlb

NELF-E KD

Cleaved RNA
Uncleaved RNA
Total
AmpliconUncleaved

4

Control
NELF-E KD
Spt5 KD
Ints9 KD3
2
1
0

U2 total

U2 uncl.

U5 total

U5 uncl.

7SK

Figure 2 | Inhibiting P-TEFb, Integrator or NELF induces misprocessing of snRNAs. (a) Schematic illustration of the human RNU1 gene showing thepositions of the primers used to amplify total and uncleaved U1 snRNA using qRT–PCR. (b) The left panel shows an immunoblot analysis of HeLa
cells treated with DMSO (Flavo � ) or 0.5 mM Flavopiridol (Flavo þ ) for 4 h. Antibodies speciﬁc for Rpb1 or Pol II phosphorylated at Ser2, Ser5 or Ser7
were used to conﬁrm the inhibitory effect of Flavopiridol on P-TEFb. Actin was detected as a loading control. The right panel shows qRT–PCR analysesof total and uncleaved U1 snRNA in the same cells. (c) The left panel shows an immunoblot analysis of HeLa cells transfected with a control plasmid
or a plasmid expressing a speciﬁc shRNAs to knockdown (KD) Spt5, NELF or Ints9. Cells were harvested for analysis 84 h after transfection. The rightpanel shows qRT–PCR analyses of total and uncleaved U1 snRNA in the same cells. (d) Quantitative RT–PCR analyses of the RNA samples shown inc using primers targeting the RNU2, RNU4 and RNU5 genes. (b–d) Expression of 7SK RNA was measured as an internal control in qRT–PCR experiments.
The qRT–PCR data were normalized to those of the corresponding control cells and are represented as the mean±s.d. of n ¼ 3 replicates. See also
Supplementary Fig. 2. Uncropped blots are shown in Supplementary Fig. 7.
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+1

b

c

0.4
d

1.6

Rpb4
% Input

0.4bcControl
2.0

d

e

a

b

c

d

a

b

c

d

Spt5

c

d

0

e

a

b

c

d

e

b

c

d

e

b

c

d

e

b

c

d

e

0.4 Ints9

NELF-A

0.2
0.1

0.3
0.2
0.1

a

b

c

d

0

e

0.6

1.6 NELF-A

1.0

2.0

Rpb4

1.6

0.8
0.4

1.2
0.8
0.4

a

b

c

d

0

e

1.2

Spt5

1.2
0.8
0.4

b

b

0.2

a

a

b

c

d

0.4
0.2
0

e

Ints9

a

Spt5 KD

1.2

a

a

0.3

0

e

1.5

0

e

% Input% Input

0.4

Spt5

0.5

Control

0

0

e

0.4

2.0

0.5

1.6

d

0.8

2.5

Rpb4

1.0

e

c

0.2

Ints9

0.4

Ints9 KD

1.5

0

b

1.2

0

% Input

2.5% Inputaa

0.4

NELF-E KD

0.8

d

0

e

1.2

0

0.4

0.6

NELF-A% Input

Control
1.6% Inputc

0.8% Inputcb1.2% Input% Input% Input

0.8

0.6

Spt5

% Input

1.6

1.2

a

e

Flavo+

1.6 Rpb4

0+1,250d% Input

Flavo–+1,000

RNU1

a

b

+750

% Input

PSE

+500

+250

c

d

e

0

a

b

c

d

0.8
0.4
0

e

0.6

NELF-A% Input–100% Input

a

a

b

c

d

e

Ints9

0.4
0.2
0

a

Figure 3 | Interplay of P-TEFb, NELF, Integrator and DSIF on the RNU1 gene. (a) Schematic illustration of the RNU1 locus showing the positions of
the ﬁve amplicons used for ChIP analysis. The numbers are relative to the TSS. PSE, proximal sequence element. (b–e) ChIP analyses of the indicatedproteins binding the RNU1 locus. HeLa cells were treated with DMSO (Flavo � ) or 0.5 mM Flavopiridol (Flavo þ ) for 4 h before harvesting (b).
Alternatively, NELF-E (c), Ints9 (d) or Spt5 (e) was KD (as in Fig. 2c) and soluble chromatin was prepared at 72 h post transfection. The data arerepresented as the mean±s.d. of n ¼ 3 independent experiments. The dashed lines indicate background signals at an intergenic region of chromosome 2.
See also Supplementary Fig. 3.

insect cells expressing the 12 Integrator subunits individually(Fig. 1d), indicating a direct interaction between these proteins. In
the same experiments, GST-NELF-A pulled down Ints4 most
efﬁciently, whereas Ints6, Ints9, Ints11 and Ints12 were pulled
down to a lesser extent (Fig. 1d). These data suggest that both
DSIF and NELF interact directly with Integrator; the former
probably occurs via binding of Spt5 to Ints6 and the latter likely
occurs via binding of NELF-A to Ints4.
The ﬁndings described above prompted us to investigate therole of the CTR, the CTD-like domain of Spt5, in Integrator
binding. GST pull-down assays were performed using GST-Pol II
CTD and GST-Spt5 CTR, with or without pre-phosphorylationby P-TEFb. Only the phosphorylated CTD was able to pull-down
Integrator from HeLa nuclear extracts efﬁciently (Supplementary
4Fig. 1c), suggesting that the Spt5 CTR is not involved in
Integrator binding.
Inhibition of NELF induces misprocessing of snRNAs. Next, weinvestigated the potential role of DSIF and NELF in 30 processingof snRNAs in HeLa cells. Cleavage efﬁciency at the correct site
was estimated using quantitative (q) RT–PCR using two sets ofprimers speciﬁc to total and cleaved snRNAs encoded by human
RNU1, RNU2, RNU4 and RNU5 (Fig. 2a; RNU1 is shown as an
example). Consistent with the results of previous studies23–26,
inhibition of P-TEFb using Flavopiridol or short-hairpin RNA(shRNA)-mediated knockdown of Ints9, one of the CPSF-like
subunits of Integrator, induced an accumulation of the uncleaved

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forms of U1, U2, U4 and U5, with only a minor effect on the total
levels of these snRNAs, most of which are long-lived mature
species (Fig. 2b–d). Knockdown of NELF using a NELF-E-speciﬁcshRNA also caused accumulation of the uncleaved forms of U1,
U2, U4 and U5 in HeLa cells (Fig. 2c,d). To explore the possibility
that the observed accumulation of the uncleaved forms of thesesnRNAs was because of an increase in their stability, the half-lifeof uncleaved U1 was determined by treating cells with thetranscription inhibitor actinomycin D. The half-life of uncleaved
U1 was not affected signiﬁcantly by knockdown of NELF
(Supplementary Fig. 2a,b), suggesting that NELF is involved in
the cleavage of U1 pre-snRNA. By contrast, knockdown of Spt5
reduced the levels of the uncleaved forms of the U1, U2, U4 andU5 snRNAs (Fig. 2c,d), which may be attributable to the fact that
DSIF contributes to transcription of the corresponding genes.
To deﬁne the 50 and 30 ends of the read-through products inmore detail and to provide a more quantitative perspective,
absolute quantiﬁcation of U1 transcripts in HeLa cells was
performed using various primer sets and in vitro synthesized
RNA as a standard. In control cells, the read-through products
were expressed at 42±3 molecules per cell for þ 6/ þ 196 RNA,24±4 molecules per cell for þ 6/ þ 418 RNA, 7.3±2.1 molecules
per cell for þ 6/ þ 658 RNA and 2.1±0.6 molecules per cellfor þ 6/ þ 911 RNA (Supplementary Fig. 2c). For reference,þ 21/ þ 146 RNA, largely representing mature U1 snRNA, waspresent at 6.8±1.1 � 104 molecules per cell. On the other hand,transcripts upstream of the known transcription start site (TSS) of
RNU1 were rare (1.4±0.3 molecules per cell for � 90/ þ 10RNA), indicating that upstream transcription is negligible(Supplementary Fig. 2c). When NELF or Integrator was knocked
down, the production of read-through products longer than
196 nt was induced 2.4- or 4.9-fold, respectively, whereas the
production of transcripts longer than 911 nt was induced 5.8- to
6.7-fold, respectively (Supplementary Fig. 2c). These results
indicate that longer read-through products are induced potently
by knockdown of NELF or Integrator.
Interplay of NELF, Integrator and DSIF on the U1 gene. Next,
we performed ChIP analysis of the RNU1 locus (Fig. 3). Analysis
of TBP showed that the resolution of our ChIP analysis was
B200 bp and that there was little overlap between ChIP signals
from the 50 and 30 ends of the U1 gene (Supplementary Fig. 3). In
control HeLa cells, Pol II (Rpb4), DSIF (Spt5), NELF (NELF-A)
and Integrator (Ints9) displayed a similar pattern of distribution
on the U1 gene; a peak was identiﬁed at the 30 end (b), and a very
weak signal was detected at position þ 1250 relative to the TSS(e). The 50 to 30 signal ratios (a:b) of DSIF and NELF werereproducibly lower than that of Pol II, suggesting that thesefactors are recruited to the U1 gene at a later stage oftranscription.
NELF is generally considered to act only at promoter-proximal
regions. To determine whether recruitment of NELF to the 30
region of the U1 gene is simply because of the small size of thisgene or whether it reﬂects a functional difference between snRNAand protein-coding genes, we compared the NELF with Pol II
signal ratios of the genes encoding U1 and beta-actin as afunction of the distance from the TSS (Supplementary Fig. 3). On
the beta-actin gene, the normalized NELF signal around the TSS
was higher than the corresponding signal at þ 300, which agrees
with recent genome-wide NELF-binding studies showing thatpeak binding of this protein occurs close to the TSS35,36.
Conversely, the normalized NELF signal around the TSS of theU1 gene was lower than the signals at positions þ 180 and þ 370(Supplementary Fig. 3). Thus, there is a subtle but deﬁnite
difference in the distribution of NELF at these two gene loci.

Knockdown of NELF in HeLa cells resulted in a marked
relocalization of Pol II, DSIF and Integrator to the region
downstream of the U1 gene (Fig. 3c). Together with the resultsof the qRT–PCR analysis (Fig. 2c), these ﬁndings suggest thatknockdown of NELF causes profound defects in both 30
processing and transcription termination of the U1 gene. Similarchanges were also induced by knockdown of Ints9 (Fig. 3d) and,to a lesser extent, by Flavopiridol treatment (Fig. 3b). These data
indicate that, as occurs for protein-coding genes, 30 processing of
the U1 gene is coupled to transcription termination. Furthermore,
NELF, Integrator and P-TEFb appear to contribute to theseprocesses. While inhibition of Pol II CTD Ser2 phosphorylation
blocked the recruitment of Ints9 to the 30 region of the U1 gene(Fig. 3b), knockdown of NELF had little effect on the recruitment
of Ints9 to this region (Fig. 3c), and knockdown of Ints9 also had
little effect on the recruitment of NELF to this region (Fig. 3d).
When DSIF was knocked down, occupancy of the U1 gene by
Pol II, NELF-A and Ints9 was reduced substantially (Fig. 3e).
Under these conditions, knockdown of DSIF had little effect on
the distribution of Pol II over the beta-actin gene (data notshown), suggesting that the knockdown did not have general,
growth-related effects. These results are consistent with thenotion that DSIF controls U1 gene transcription. It is unclear
whether DSIF also contributes to the recruitment of NELF andIntegrator, since knockdown of this protein results in a
substantial reduction of Pol II occupancy.
NELF inhibits aberrant polyadenylation of U1 transcripts. The
ﬁndings described above are reminiscent of what happens during
30 processing of histone pre-mRNAs, another class of nonpolyadenylated Pol II transcripts. Knockdown of NELF or acomponent of the histone 30 processing machinery blocks histone
pre-mRNA cleavage at the correct sites, resulting in an accumulation of aberrant polyadenylated histone mRNAs20,37,38.
Therefore, RNA ligase-mediated rapid ampliﬁcation of cDNA
ends (RLM-RACE), which was modiﬁed to ensure that onlytranscripts with six or more adenosines at the 30 ends were
ampliﬁed (Fig. 4a), was used to determine whether aberrant
polyadenylation of U1 transcripts also occurs as a result ofknockdown of NELF. Quantitative TaqMan PCR analyses showedthat knockdown of NELF or Integrator in HeLa cells induced the
production of polyadenylated U1 transcripts but did not affectpolyadenylation of GAPDH transcripts at the correct site
(Fig. 4b).
Next, RLM-RACE products were cloned and sequenced to map
the polyadenylation sites. Clones with fewer than six untemplated
adenosine residues at their 30 end were excluded from theanalyses. A large number of polyadenylation sites were identiﬁedin the NELF knockdown and Integrator knockdown samples(Fig. 4c and Supplementary Fig. 4). There was no detectable PAS(AAUAAA or its variants) around these sites. The absence of a
PAS is not surprising because no PAS-like sequences are present
in the 1-kb region downstream of the 30 box of the U1 gene,
where transcription termination mainly occurs. On the other
hand, only a single polyadenylation site was identiﬁed from thecontrol sample (Fig. 4c and Supplementary Fig. 4).
NELF prevents the recruitment of cleavage stimulation factor.
The ﬁndings described above raised a question as to whether
aberrant polyadenylation is carried out by the canonical mRNA
30 -processing machinery. To address this question, CPSF-73 andthe cleavage stimulation factor CstF-64 were knocked down bytransfection of HeLa cells with recombinant lentiviruses expressing speciﬁc shRNAs (Fig. 5a). Knockdown of CPSF-73 resulted
in a signiﬁcant decrease in the amount of polyadenylated U1NATURE COMMUNICATIONS | 5:4263 | DOI: 10.1038/ncomms5263 | www.nature.com/naturecommunications& 2014 Macmillan Publishers Limited. All rights reserved.

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aTotal RNA
AAAAA
AAAAA
AAAAA
AAAAA

6

AAAAA

**

*

3
2
1

c

Ctrl

+1

1
0.5

+500

Ctrl

+750

NELF-E Ints9
KD
KD+1,000Control, ploy(A)≥10
Control, ploy(A)<10

RNU1

+1

+250

+500

+750

+1,000

+250

+500

+750

+1,000

RNU1+1

Cloning andsequencing (C)1.5

0

NELF-E Ints9
KD
KD
+250

TTT
AAAAA
TTT

Poly(A)+GAPDH transcripts
240

Poly(A)-specific PCR
Quantification (B)
AAAAA

AAAAA

Poly(A)+U1 transcripts

5
Fold change

Reverse transcription

AAAAA
AAAAA
AAAAA
AAAAA

Fold changebAdapter ligationP

RNU1

NELF-E KD, poly(A)≥10
NELF-E KD, poly(A)<10

Ints9 KD, poly(A)≥10
Ints9 KD, poly(A)<10

Figure 4 | Inhibition of NELF or Integrator leads to the accumulation of aberrant polyadenylated U1 transcripts. (a) Schematic representation of the
RLM-RACE procedure. (b) TaqMan PCR analyses of poly(A) þ U1 transcripts and poly(A) þ GAPDH transcripts in control (Ctrl) HeLa cells and HeLa cells
transfected with shRNAs to KD NELF-E or Ints9. The expression levels were normalized to the amount of 7SK RNA and to those of the control cells.
Data are represented as the mean±s.e.m. of n ¼ 5 independent experiments. *Po0.05 and **Po0.01 (two-tailed Student’s t-test). (c) Cryptic
polyadenylation sites of the RNU1 gene. Untemplated poly(A) tails were detected in 3/48, 50/96 and 34/96 of the control, NELF-E knockdown and Ints9
knockdown clones, respectively. The polyadenylation sites of the positive clones were mapped on the RNU1 locus. Clones containing short poly(A) tails(o10 nt) and those containing long poly(A) tails (Z10 nt) are indicated by open and closed symbols, respectively. See also Supplementary Fig. 4.

transcripts, while knockdown of CstF-64 did not (Fig. 5b).
Moreover, double knockdown studies showed that theaccumulation of the polyadenylated U1 transcripts in NELF
knockdown cells was reversed by concurrent knockdown of
CPSF-73 or CstF-64 (Fig. 5b), suggesting that NELF prevents
CPSF- and CstF-mediated cleavage and polyadenylation of U1
transcripts.
To test this proposal, the effect of knockdown of NELF on the
recruitment of CPSF and CstF to the U1 gene was examined(Fig. 5c and Supplementary Fig. 5). Knockdown of NELF
increased the ratio of CstF-64 to Pol II at regions downstreamof the U1 gene signiﬁcantly (b and d), while the ratio of CPSF-73
to Pol II was only affected negligibly. These results suggest that
NELF inhibits aberrant polyadenylation of U1 transcripts by
preventing the recruitment of CstF to the U1 gene locus. Notably,knockdown of NELF also resulted in a signiﬁcant increase in therelative ChIP signal of CstF-64 at the promoter region but not the
30 region of the GAPDH gene (Fig. 5c and Supplementary Fig. 5).
Therefore, NELF may also prevent the recruitment of CstF to thepromoter-proximal region of protein-coding genes.
Discussion
On the basis of the results of this study, we offer a model fortranscription-coupled 30 processing of snRNA genes (Fig. 6).
In this model, after the transcription initiation (step 1), DSIF
6

(Spt4–Spt5) is recruited to Pol II to stimulate transcription
elongation, while P-TEFb phosphorylates the Pol II CTD (step 2).
Integrator is then recruited to the snRNA gene through its
interactions with the phosphorylated-CTD and possibly DSIF.
NELF is also recruited by DSIF and contributes to 30 boxdependent 30 processing by preventing the recruitment of CstF
(step 3). Finally, Integrator performs 30 processing and triggers
transcription termination (step 4).
Processing and termination at the 30 end are tightly coupledduring transcription of protein-coding genes, and, according to
the widely accepted torpedo model, termination is induced by the
50 to 30 exoribonuclease Xrn2, which attacks the newly formed
50 end of cleaved pre-mRNAs4,5. Since inhibition of NELF,
Integrator and P-TEFb all resulted in defective 30 processing andtermination of the U1 gene (Figs 2 and 3), it is likely that theseprocesses occur in a coordinated manner by a mechanism similar
to that of protein-coding genes. This view is consistent with arecent study that suggested the coupling of transcription
termination and 30 processing of U1 snRNA39.
A few pieces of evidence suggest that DSIF is involved in U1
gene transcription. Speciﬁcally, knockdown of DSIF reduced thelevels of both nascent (unstable) and processed (stable) U1
snRNA transcripts (Fig. 2) and reduced the Pol II density acrossthe RNU1 locus (Fig. 3e). Since DSIF functions as a dual regulatorof transcription elongation for protein-coding genes, it may play asimilar role in regulating the U1 gene. It may seem strange that an

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CPSF-73
CstF-64b

Poly(A)+ U1 transcripts

8

***
**

6
Fold change

CstF-64 KD

Controla

CPSF-73 KD

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***

4
2

Actin

c

Control
0.12

CstF-64/Rpb4

KD
N
C EL
st FF- E
64 K
KD D
C
PS
F73
KD
C NE
PS LF
F- -E
73 K
KD D

EL
F

st
F-

N

C

64

-E

C

KD

trl

0

NELF-E KD

*

0.8

*

CstF-64/Rpb4

0.08

a.u.

a.u.

0.6

0.04

0

0.16

*

0.2

U1 PSE(a)

U1 +200(b)0

U1 +500(d)

0.6

CPSF-73/Rpb4

0.12

GAPDH
promoter

GAPDH
3′ pause

CPSF-73/Rpb4

0.4
a.u.

a.u.

0.4

0.08

0.2
0.04
0

U1 PSE(a)

U1 +200(b)

U1 +500
(d)

0

GAPDH
promoter

GAPDH
3′ pause

Figure 5 | NELF inhibits aberrant polyadenylation of U1 transcripts by preventing the recruitment of CstF-64. (a) Immunoblot analyses of HeLa cells
transduced with recombinant lentiviruses expressing no shRNA (Control) or shRNAs designed to KD CstF-64 or CPSF-73. The cells were harvested at
7 days post infection. Actin was detected as a loading control. (b) TaqMan PCR analyses of poly(A) þ U1 transcripts and poly(A) þ GAPDH transcripts
in control (Ctrl) HeLa cells and HeLa cells transfected with shRNAs to KD NELF-E, CstF-64 or CPSF-73. The expression levels were normalized to theamount of 7SK RNA and to those of the control cells. Data are represented as the mean±s.e.m. of n ¼ 4 independent experiments. **Po0.01 and***Po0.001 (two-tailed Student’s t-test). (c) ChIP analyses of control HeLa cells and HeLa cells transfected with a shRNA to KD NELF. The ChIP
assays were performed as described in Fig. 3. After background subtraction, the ChIP signals of CstF-64 and CPSF-73 were normalized to those of Rpb4.
The data are represented as the mean±s.e.m. of n ¼ 5 (CstF-64/Rpb4) or n ¼ 3 (CPSF-73/Rpb4) independent experiments. *Po0.05 (two-tailed
Student’s t-test). See also Supplementary Fig. 5. Uncropped blots are shown in Supplementary Fig. 8.

elongation factor is critical for the synthesis of such a small RNA;
however, recent studies have shown that the little elongation
complex, which comprises ELL and a few other subunits, is also
required for the initiation and elongation phases of snRNA genetranscription40,41. It is not immediately clear why elongation
factors are required for transcription of short snRNA genes. One
possible reason is that elongation factors may maintain an open
chromatin structure of highly transcribed snRNA genes. The
binding pattern of histone H3 over the RNU1 and RNU2 genes is
opposite to that of Pol II (refs 21,39 and our unpublished data),suggesting transcription-dependent eviction of histones. DSIF
may contribute to a high level of expression of snRNA genes bycounteracting the formation of a repressive chromatin structure.
Since Integrator is implicated not only in 30 processing of snRNAs

but also in transcription of their genes per se23,26,42, it is also
possible that DSIF contributes to snRNA transcription byinteracting and cooperating with Integrator.
On the basis of the results of recent genome-wide studies,
NELF is generally considered to bind only to promoter-proximal
regions35. However, our previous study showed that NELF isaccumulated at both the 50 and 30 ends of replication-dependent
histone genes and facilitates histone-type 30 processing20.
Moreover, NELF is accumulated at regions downstream of the
U1 and U2 genes (refs 21,39 and this study). These ﬁndings
led us to hypothesize that, aside from its established role inpromoter-proximal pausing, NELF may also play a distinctrole in the regulation of snRNA and histone genes. Unlike
most Pol II-transcribed genes, snRNA and histone genes produce

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1
SNAPc

Pol IIU1 gene3′ box

PSE
P

P

P

CTD

P-TEFb

2

Spt4
Spt5

SNAPc
PSE

P P

P

3′ boxP

P

P

Phosphorylation
CstF

3

4

NELF
6

Spt4
Spt5

SNAPc
PSE

P P

P

3′ box
P

P

P

NELF
4

Spt4
Spt5

SNAPc

3′ box

PSE

P P

P

P

P

P

Figure 6 | Proposed model of the functions of DSIF, NELF and Integrator during U1 gene transcription and 30 processing. After the transcription
initiation (step 1), DSIF (Spt4–Spt5) is recruited to Pol II, while P-TEFb phosphorylates the Pol II CTD (step 2). Integrator is then recruited through its
interactions with the phosphorylated-CTD and possibly DSIF. NELF is also recruited and contributes to 30 box-dependent 30 processing by preventing therecruitment of CstF (step 3). Finally, Integrator performs 30 processing and triggers transcription termination (step 4).

non-polyadenylated transcripts that follow the ‘minor’ 30
processing pathways, and knockdown of NELF results in the
production of aberrant polyadenylated species (ref. 20 and thisstudy). Therefore, we speculate that polyadenylation is the default
30 processing pathway and that NELF plays a key regulatory rolein the post-transcriptional fate of Pol II-transcribed genes.
As to the mechanism of aberrant polyadenylation, cryptic PASs
are almost always located downstream of replication-dependent
histone genes and are utilized when NELF or the histone-speciﬁc
processing pathway is blocked. Polyadenylated histone mRNAs
are stable but are not fully functional because they are notproperly regulated during the cell cycle37,43. The cryptic PASs ofhistone genes may serve to ensure transcription termination andto prevent read-through into downstream genes. By contrast,
PAS-like sequences were not identiﬁed within the region
comprising a few kilobases downstream of the snRNA genes
examined here. Despite the absence of a consensus PAS,polyadenylation of U1 transcripts apparently occurs at random
when NELF or Integrator is knocked down (Fig. 4c andSupplementary Fig. 4). This aberrant processing is dependent
on CstF and CPSF (Fig. 5), but it remains unclear which enzyme
is responsible for poly(A) addition. Analysis of the half-life of
U1 read-through products suggested that polyadenylated U1
transcripts are not very stable (Supplementary Fig. 2a,b). In
Saccharomyces cerevisiae, the TRAMP complex attaches shortoligo(A) tails to cryptic unstable transcripts and targets them for
degradation by the nuclear exosome44, while mammalian
homologues of TRAMP complex components have not been
8

well characterized. Since U1 transcripts with both long andshort poly(A) tails were identiﬁed by sequencing of RLM-RACE
products (Fig. 4c and Supplementary Fig. 4), aberrant
polyadenylation may involve both canonical nuclear poly(A)polymerase and the TRAMP complex.
The ﬁndings presented here suggest that NELF inhibitsaberrant polyadenylation of U1 transcripts by preventing therecruitment of CstF. Regarding the mechanism by which CstF is
recruited under normal circumstances, a recent study showed that
Ser-65-phosphorylated TFIIB binds to and recruits CstF-64 tosome protein-coding genes45. Another report suggested that
CstF-50 interacts with the Pol II CTD in a phosphorylationindependent manner46. The way in which NELF interferes withthe recruitment of CstF-64 to the U1 gene is an interesting but as
yet unanswered question. Notably, knockdown of a histonespeciﬁc 30 processing factor results in an increased association of
CstF-50 with histone gene loci47. Therefore, the recruitment of
CstF may be a common critical step in the determination of the
post-transcriptional fate.
Here we showed that knockdown of NELF also results in an
increased association of CstF-64 with the 50 end but not the 30 endof the GAPDH gene (Fig. 5c and Supplementary Fig. 5b). Hence,
NELF appears to exclude CstF from the promoter-proximal
region of protein-coding genes while bound to Pol II, implyingthat a similar mechanism operates in both snRNA and proteincoding genes. Although a more detailed study is necessary to
conﬁrm the hypothesis, NELF may function to prevent prematuretermination at cryptic PASs located close to the TSSs of

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protein-coding genes. In fact, recent studies point to a new role ofU1 snRNP in the suppression of premature termination48,49.
Base-pairing of U1 snRNP to pre-mRNAs protects them frompremature polyadenylation and transcription termination at
nearby cryptic PASs within introns. To provide an integrated
view of the post-transcriptional role of NELF, future studies will
be aimed at reinvestigating the role of NELF in controlling
protein-coding genes at a post-transcriptional level.
Methods

Cell culture. HeLa cells stably expressing Flag-NELF-E or Flag-Spt5 weregenerated as described previously14,50. HeLa cells stably expressing Flag-Ints10
were established by transfecting the cells with a pcDNA3-based expression vector
and selecting neomycin-resistant colonies for 2 weeks. Nuclear extracts wereprepared from HeLa cells using Dignam’s method.

Analyses of protein–protein interactions. Anti-Flag afﬁnity puriﬁcation was
performed as described previously20. Brieﬂy, NE buffer (20 mM HEPES-NaOH(pH 7.9), 20% glycerol, 100 mM KCl, 0.2 mM EDTA and 0.1% NP-40) was used forthe binding and washing steps. Bound proteins were eluted with the same buffer
containing 0.1 mg ml � l Flag peptide, excised from Coomassie-stained SDS
polyacrylamide gels and subjected to LC-MS/MS analysis. Sucrose density-gradientcentrifugation was performed as described previously51. For GST pull-down assays,
GST fusion proteins and recombinant P-TEFb were prepared as described
previously14,20,52. Afﬁnity-puriﬁed Integrator was prepared from HeLa cells
expressing Flag-Ints10, as described above. Recombinant Integrator subunits with
an N-terminal Flag tag were expressed in Sf9 cells using the baculovirus expression
system. Integrator was incubated with equal amounts of GST or GST fusion
proteins immobilized to glutathione Sepharose (GE Healthcare) for 2 h at 4 C.
After extensive washing, bound materials were eluted with Laemmli buffer andanalysed by immunoblotting. In Supplementary Fig. 1c, equal amounts of GST,
GST-Pol II CTD or GST-DSIF CTR were immobilized to glutathione Sepharose
and then incubated with or without recombinant P-TEFb and ATP for 10 min at
30 C. After removal of P-TEFb and ATP, the matrices were incubated with HeLa
nuclear extract in the presence of phosphatase inhibitor cocktail (Sigma) for 2 h at
4 C. After extensive washing, bound materials were eluted with Laemmli buffer
and analysed by immunoblotting.
Immunoblotting. Immunoblotting was performed according to the standard
procedures using the following primary antibodies: anti-Flag M2 (Sigma,
5 mg ml � l), anti-Rpb1 8WG16 (Covance, 1:1,000), anti-Spt5 (in-house, rabbit
polyclonal, 1:1,000), anti-NELF-E (in-house, mouse monoclonal, 1 mg ml � 1), antiInts1 (A300–361A, Bethyl, 1 mg ml � 1), anti-Ints3 (A300–427A, Bethyl, 1 mg ml � 1),
anti-Ints4 (A301-296A, Bethyl, 0.2 mg ml � 1), anti-Ints5 (A301-268A, Bethyl,
0.2 mg ml � 1), anti-Ints6 (A300-254A, Bethyl, 0.2 mg ml � 1), anti-Ints7 (A300-271A,
Bethyl, 0.2 mg ml � 1), anti-Ints8 (A300-269A, Bethyl, 1 mg ml � 1), anti-Ints9(A300-412A, Bethyl, 0.5 mg ml � 1), anti-Ints10 (15271-1-AP, ProteinTech,
0.4 mg ml � 1), anti-Ints11 (A301-274A, Bethyl, 0.2 mg ml � 1), anti-Med17(ARP34192, Aviva, 0.5 mg ml � 1), anti-Cdk8 (RB-018, NeoMarkers, 1 mg ml � 1),
anti-Cyclin C (RB-009, NeoMarkers, 2 mg ml � 1), anti-Ser2P H5 (Covance,
1:1,000), anti-Ser5P H14 (Covance, 1:3,000), anti-Ser7P 4E12 (Millipore, 1:3,000),
anti-actin (MAB1501, Millipore, 0.2 mg ml � 1), anti-CstF-64 (sc16473, Santa Cruz,
1 mg ml � 1), and anti-CPSF-73 (A301-091A, Bethyl, 0.2 mg ml � 1).
RNA preparation and qRT–PCR. Total RNA was prepared using Sepasol I Super(Nacalai Tesque) and then treated with RQ1 RNase-free DNase (Promega).
Subsequently, qRT–PCR analyses were performed using the QuantiTect SYBRGreen RT–PCR Kit (Qiagen), an ABI 7300 real-time PCR system (Applied
Biosystems), and the primers listed in Supplementary Table 2.
RLM-RACE. The RLM-RACE procedure was modiﬁed to ensure that onlytranscripts with six or more adenosines at the 30 end were ampliﬁed53. First, RNAligase was used to ligate a 50 phosphorylated RNA/DNA chimeric oligonucleotide
to the 30 ends of transcripts in the total RNA sample, prepared as described above.
Reverse transcription was then performed with a complementary DNA
oligonucleotide and Superscript III reverse transcriptase (Life Technologies). Next,
ﬁrst-round PCR was carried out using a U1-speciﬁc forward primer and the same
reverse primer amended to contain three additional adenosine residues at the 30
end. Nested PCR was then performed using another U1-speciﬁc primer and areverse primer with six adenosine residues. For cleavage site mapping, secondround PCR products of 250–2,000 bp were recovered from agarose gels, digested
with BamHI and HindIII, and then cloned into pBluescript SK þ . In total, 48 or 96
clones were sequenced, most of which harboured RNU1-1, RNU1–2 or RNU1–3.
Sequence analyses indicated that some clones were derived from transcripts with 30
A-rich sequences rather than from polyadenylated transcripts; these irrelevantclones were excluded from further analyses. To quantify polyadenylated species ofpotentially variable lengths, ﬁrst-round PCR products were subjected to TaqMan
real-time PCR analyses.

Gene knockdown using shRNAs. The shRNA sequences were cloned into aversion of pBluescript containing the mouse U6 promoter, as described previously14. The following sequences were used as shRNA targets: Spt5, 50 -AAGAACUGGGCGAGUAUUACA-30 ; NELF-E, 50 -AAGAUGGAGUCAGCAGAUCAG-30 ;Ints9, 50 -AAGAUCGAGAUCAUGCCAGAG-30 ; CstF-64, 50 -AAGACCAAGAGACAGCACUUA-30 ; and CPSF-73, 50 -AAGAUUCUGUAACACUGUCCA-30 .
To knock down Spt5, NELF-E and Ints9, HeLa cells were transfected with a control
plasmid (U6 promoter alone) or the speciﬁc shRNA-containing plasmid using
Lipofectamine 2000 (Life Technologies), and transfectants were harvested at 72 h(for RNA analysis) or 84 h (for ChIP) post transfection. To knockdown CstF-64
and CPSF-73, a cassette containing the mouse U6 promoter and the speciﬁcshRNA sequence was excised from pBluescript and subcloned into pLenti6 (LifeTechnologies), as described previously51. Recombinant lentiviruses were produced
according to the manufacturer’s recommendations and were used to infect HeLa
cells. Infectants were selected in growth medium containing 4 mg ml � 1 blasticidin
and were harvested at 7 days post infection.

ChIP assays. ChIP assays were performed as described previously14. Brieﬂy, HeLa
cells were crosslinked with 1% formaldehyde for 10 min. Nuclei were isolated andresuspended in lysis buffer (10 mM Tris–HCl, pH 8.0, 100 mM NaCl, 1 mM EDTA,
0.5 mM EGTA, 0.1% SDS, protease inhibitor cocktail (Nacalai Tesque)).
Chromatins were sheared by sonication using a Misonix S-4000 sonicator(Misonix). Soluble chromatins were incubated overnight at 4 C with antibodies
described below. Immunocomplexes were captured by protein G Sepharose FF (GEHealthcare) or Dynabeads Protein G (Dynal). Beads were sequentially washed threetimes with RIPA buffer (10 mM Tris–HCl, pH 8.0, 150 mM NaCl, 1 mM EDTA,
0.5 mM EGTA, 0.1% SDS, 1% Triton X-100, 0.1% sodium deoxycholate), threetimes with high-salt ChIP wash buffer (10 mM Tris–HCl, pH 8.0, 500 mM NaCl,
1 mM EDTA, 0.5 mM EGTA, 0.1% SDS, 1% Triton X-100, 0.1% sodium
deoxycholate), twice with LiCl ChIP wash buffer (10 mM Tris–HCl, pH 8.0,
250 mM LiCl, 1 mM EDTA, 1 mM EGTA, 1% NP-40, 1% sodium deoxycholate),
twice with TE (10 mM Tris–HCl, pH 8.0, 1 mM EDTA). Immunoprecipitates were
eluted with elution buffer (100 mM NaHCO3, 1% SDS, 10 mM DTT) and incubatedovernight at 65 C. Genomic DNA fragments in inputs and eluates were puriﬁed
and analysed using qPCR. The antibodies used for the assays were as follows: 2.5 mlof anti-Rpb4 sera (a gift from Koji Hisatake, University of Tsukuba; rabbit
polyclonal), 2.5 ml of anti-Spt5 sera (in-house, rabbit polyclonal), 2.5 ml of antiNELF-A sera (in-house, rabbit polyclonal), 1 mg of anti-Ints9 (A300–412A, Bethyl),
1 mg of anti-CstF-64 (A301-092A, Bethyl) and 1 mg of anti-CPSF-73 (A301-091A,
Bethyl). The primer sequences are listed in Supplementary Table 2.

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Acknowledgements
We thank Shona Murphy for providing plasmids and for helpful discussions, and Koji
Hisatake for providing antibodies. We also thank Naoto Inukai and Yasunori Tsuboi for
their contributions during the ﬁrst stage of this work, and Junko Kato for technicalassistance. This work was supported by a Grant-in-Aid for Scientiﬁc Research on
Innovative Areas (no. 3408) from MEXT and by the PRESTO programme from JST(to Y.Y.). J.Y. was supported by the Yoshida Scholarship Foundation.

Author contributions
J.Y. performed most of the experiments; Y.H. performed some IP and GST pull-down
assays; K.C. and T.I. prepared several DNA constructs; and T.N. performed interactome
analysis. H.H. and Y.Y. supervised research and wrote the manuscript.

Additional information
Supplementary Information accompanies this paper at http://www.nature.com/
naturecommunications
Competing ﬁnancial interests: The authors declare no competing ﬁnancial interests.
Reprints and permission information is available online at http://npg.nature.com/
reprintsandpermissions/
How to cite this article: Yamamoto, J. et al. DSIF and NELF interact with Integrator
to specify the correct post-transcriptional fate of snRNA genes. Nat. Commun. 5:4263
doi: 10.1038/ncomms5263 (2014).

NATURE COMMUNICATIONS | 5:4263 | DOI: 10.1038/ncomms5263 | www.nature.com/naturecommunications& 2014 Macmillan Publishers Limited. All rights reserved.

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Automation and Analytical Techniques

Clinical Chemistry 60:4
610–620 (2014)

Magnetically Promoted Rapid Immunoreactions Using
Functionalized Fluorescent Magnetic Beads:
A Proof of Principle
Satoshi Sakamoto,1† Kenshi Omagari,2† Yoshinori Kita,1 Yusuke Mochizuki,1 Yasuyuki Naito,1
Shintaro Kawata,1 Sachiko Matsuda,2 Osamu Itano,2 Hiromitsu Jinno,2 Hiroya Takeuchi,2 Yuki Yamaguchi,1
Yuko Kitagawa,2 and Hiroshi Handa1*

BACKGROUND: Accurate detection and monitoring of
disease-related biomarkers is important in understanding pathophysiology. We devised a rapid immunoreaction system that uses submicrometer polymercoated fluorescent ferrite (FF) beads containing both
ferrites (magnetic iron oxide) and fluorescent europium complexes.
METHODS: FF beads were prepared by encapsulation ofhydrophobic europium complexes into the polymer
layers of affinity magnetic beads using organic solvent.
A sandwich immunoassay using magnetic collection of
antibody-coated FF beads to a specific place was performed. Brain natriuretic peptide and prostate-specificantigen were selected as target detection antigens todemonstrate the feasibility of this approach. An immunohistochemical staining using magnetic collection of
antibody-coated FF beads onto carcinoma cell samples
was also performed.
RESULTS:

The sandwich immunoassays, taking advantage of the magnetic collection of antibody-coated FF
beads, detected target antigens within 5 min of sample
addition. Without magnetic collection, the sandwich
immunoassay using antibody-coated FF beads required
long times, similar to conventional immunoassays. Using the magnetic collection of antibody-coated FF
beads, immunohistochemical staining enabled discrimination of carcinoma cells within 20 min.

CONCLUSIONS:This proof of principle system demonstrates that immunoreactions involving the magnetic
collection of antibody-coated FF beads allow acceleration of the antigen–antibody reaction. The simple
magnetic collection of antibody-coated FF beads to a

1

Department of Biological Information, Graduate School of Bioscience andBiotechnology, Tokyo Institute of Technology, Yokohama, Japan; 2 Departmentof Surgery, School of Medicine, Keio University, Tokyo, Japan.
†
Satoshi Sakamoto and Kenshi Omagari contributed equally to the work, andboth should be considered as first authors.
* Address correspondence to this author at: Department of Biological Information, Graduate School of Bioscience and Biotechnology, Tokyo Institute ofspecific space enables rapid detection of disease-related
biomarkers and identification of carcinoma cells.
© 2013 American Association for Clinical Chemistry

Biomarkers provide information about disease progression and prognosis (1– 4 ). They are also widelyused in the diagnosis of disease, often at the point ofcare. Immunoassays are commonly used in the measurement of biomarkers in both the research and clinical arenas (5, 6 ). Although ELISA is commonly used
(7, 8 ), it is a time-consuming and relatively complex
approach. Thus, platforms that shorten assay time andreduce the number of steps involved would be beneficial. Immunoreaction using magnetic particles has attracted attention as a means to improve assay efficiency(9, 10 ). Reports of immunoassays with magnetic particles have mainly involved magnetically assisted separation of antibody-coated magnetic particles from reaction medium to facilitate assay processes. Here we
describe a magnetically prompted immunoreaction
system with unique polymer-coated magnetic beads
containing highly analytically sensitive fluorophores.
The immunoassay and the immunohistochemical
staining described here feature simple magnetic collection of the beads in a specific space and require no
signal amplification step for the detection of biomarkers, thus enabling the analytically sensitive and rapid
detection of disease-related biomarkers.
Materials and Methods
Affinity magnetic beads with carboxylic acid were prepared according to previously reported proceduresTechnology, 4259 Nagatsuta-cho, Midori-ku, Yokohama 226-8501, Japan. Fax
81-45-924-5834; e-mail hhanda@bio.titech.ac.jp.
Received July 10, 2013; accepted December 2, 2013.
Previously published online at DOI: 10.1373/clinchem.2013.211433

610

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Rapid Immunoreaction System with Fluorescent Magnetic Beads(11, 12 ). Eu(TTA)3(TOPO)2 [thenoyltrifluoroacetylacetone (TTA)3; tri-n-octylphosphine oxide (TOPO)]
was prepared from europium(III) acetate hydrate(Sigma), TTA, and TOPO (13 ). Transmission electron
microscope (TEM) images were measured by JEM2010F (JEOL) at the Center for Advanced Material
Analysis, Tokyo Institute of Technology. Brain natriuretic peptide (BNP) and anti-BNP antibodies (KYBNP-II and BC203) were kind gifts from Shionogi &Co. Prostate-specific antigen (PSA) and anti-PSA antibodies (5A6 and 1H12) were obtained from HyTest. A
magnetic plate with 96 magnets was obtained from OZ
Biosciences. For the comparison study, the Access Hybritech PSA (Beckman Coulter) was used for measurement of PSA concentrations. Anti– epidermal growth
factor receptor (EGFR) antibodies were obtained fromthe hybridoma cell line 528 (HB-8509), which produces mouse monoclonal antibody against human
EGFR, and purified using the Mab Trap™ kit (GEHealthcare). Anti– epithelial cell adhesion molecule(EpCAM) and anti-CA19-9 antibodies were obtained
from Abcam. Epidermoid carcinoma cells A431 (CRL1555) and small-cell lung cancer cells H69 (HTB-119)
were obtained from ATCC. The A431-GFP stable cellline was obtained by transfection of pEGFP-N1 (Promega) followed by G418 selection. A431 cells and H69
cells were maintained in DMEM and in RPMI 1640
supplemented with 10% fetal bovine serum in a 5% CO2
humidified incubator at 37 °C. A431 or H69 cells (1  106cells) in 100 L PBS were subcutaneously implanted into
nude mice in 2 regions of the upper back. When the tumors reached approximately 5 mm in diameter, they were
removed and one was fixed in 10% neutral buffered formalin for 24 h. Fixed tumors were processed in a tissue
processor and embedded in paraffin. The second tumor
was embedded in optimum cutting temperature (OCT)compound and stored at 80 °C.
Experiments on immunohistochemical staining
were approved by the institutional ethics committee
and were conducted in accordance with the guidelinesof the Declaration of Helsinki. Informed consent was
obtained from each patient. Needle-core biopsy samples were obtained from breast cancer patients (2006 –2009). Samples from esophageal cancer patients undergoing surgery (2001–2006) were used for EpCAM
and cytokeratin 19 staining.

PREPARATION OF FLUORESCENT FERRITE BEADS

A suspension of affinity magnetic beads with carboxylic acid (1.0 mg) was incubated with 10 mmol/L
Eu(TTA)3(TOPO)2 solution in acetone in the darkwith vigorous shaking for 1 h at room temperature.
Distilled water was added and acetone was evaporated under vacuum at 60 °C. The pellet was washed with washingbuffer [50 mmol/L HEPES (pH 7.9), 0.1% NP-40], andthe prepared fluorescent ferrite (FF) beads were dispersed
in distilled water and stored in the dark at 4 °C.
PREPARATION OF ANTI-BNP ANTIBODY–COATED FF BEADS

A suspension of FF beads with carboxylic acid (1.0 mg) in
distilled water was incubated with 20 g/L 1-ethyl-3-(3dimethylaminopropyl)carbodiimide hydrochloride (EDCHCl) in distilled water for 30 min at 4 °C. The activated FF
beads were washed with 2-morpholinoethanesulfonic acidsodium hydroxide (MES-NaOH) (pH 6.0) and were incubated with 1.0 g/L anti-BNP antibody [F(ab)2 fragment of KY-BNP-II] in MES-NaOH (pH 6.0) for 2 h at
4 °C. Then, 1.0 mol/L ethanolamine solution in MESNaOH (pH 6.0) was added, and the suspension was
mixed overnight at 4 °C. Finally, anti-BNP antibody–coated FF beads were washed and stored in HEPES buffer[10 mmol/L HEPES (pH 7.9), 50 mmol/L KCl, 1.0mmol/L EDTA, 0.1% Tween 20] at 4 °C.
PREPARATION OF ANTI-PSA ANTIBODY–COATED FF BEADS

A suspension of FF beads with carboxylic acid in PBS(pH 7.4) was incubated with 200 mmol/L EDCHCl in
PBS and 200 mmol/L N-hydroxysuccinimide in PBS
for 4 h at 4 °C. The activated FF beads were washed withacetate (pH 5.2) and were incubated with 1.0 g/L antiPSA antibody (5A6) in acetate for 1 h at 4 °C. Then, 1.0
mol/L ethanolamine solution in PBS was added and thesuspension was mixed for 1 h at 4 °C. Finally, anti-PSA
antibody– coated FF beads were washed and stored in
HEPES buffer at 4 °C.
PREPARATION OF ANTI-EGFR, ANTI-EpCAM, AND ANTI-CA19-9
ANTIBODY–COATED FF BEADSFor the preparation of anti-EGFR, anti-EpCAM, andanti-CA19-9 antibody– coated FF beads, the procedure
was the same as the procedure for anti-BNP antibody–coated FF beads. Antibody-coated FF beads were stored
in HEPES buffer at 4 °C.
MAGNETICALLY INDUCED SANDWICH IMMUNOASSAY WITH FF
BEADS FOR DETECTION OF BNP

3

Nonstandard abbreviations: TTA, thenoyltrifluoroacetylacetone; TOPO, tri-noctylphosphine oxide; TEM, transmission electron microscopy; BNP, brain natriuretic
peptide; PSA, prostate specific antigen; EGFR, epidermal growth factor receptor;
EpCAM, epithelial cell adhesion molecule; OCT, optimum cutting temperature; FF,fluorescent ferrite; EDCHCl, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride; MES-NaOH, 2-morpholinoethanesulfonic acid-sodium hydroxide; TBS, Trisbuffered saline; DAB, 3,3-diaminobendizine; CLEIA, chemiluminescent EIA; LOD,
limit of detection; LOQ, limit of quantitation; HE, hematoxylin eosin.

Polystyrene 96-well microplates (Matrix Technologies) were coated with 10 mg/L anti-BNP antibody(BC203) in carbonate (pH 9.6) overnight at 4 °C.
After removal of carbonate, blocking solution (10mmol/L HEPES (pH 7.9), 50 mmol/L KCl, 1.0mmol/L EDTA, 0.1% Tween 20, 1% skim milk) was
Clinical Chemistry 60:4 (2014)

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611

added. BNP in reaction buffer (10 mmol/L HEPES(pH 7.9), 50 mmol/L KCl, 1.0 mmol/L EDTA, 0.1%
Tween 20, 1% BSA) was added to each well and incubated for 20 s at 4 °C. Next, 50 mg/L anti-BNP
antibody (KY-BNP-II)-coated FF beads in reaction
buffer (20 L) were added, the plate was gently
shaken for 10 s, and placed on the magnet plate forincubation times of 0.5–3 min. The wells were thenwashed with reaction buffer and time-resolved fluorescence measurements were performed using aplate reader (ArvoSx1420; PerkinElmer).

were incubated with antibody-coated FF beads with amagnet beneath for 10 min, washed with TBS with gentle movement of the magnet, and examined using afluorescence microscope (ECLIPS E1000; Nikon).
OCT-embedded samples were sectioned on a cryostat
microtome, dried for 1 min at 60 °C, fixed with coldacetone for 2 min, hydrated, and treated with blocking
solution.
Results
PRODUCTION OF UNIQUE 140-nm–DIAMETER FF BEADSMAGNETICALLY INDUCED SANDWICH IMMUNOASSAY WITH FF
BEADS FOR DETECTION OF PSA

Polystyrene 96-well microplates were coated with 5.0
mg/L anti-PSA antibody (1H12) in carbonate (pH 9.6)
overnight at 4 °C. After removal of carbonate, blocking
solution [25 mmol/L Tris-HCl (pH 7.9), 150 mmol/L
KCl, 5.0 mmol/L EDTA, 0.1% Tween 20, 1% skimmilk] was added. PSA in blocking solution was added
to each well and incubated for 3 min at 4 °C. Next, 12.5
mg/L anti-PSA antibody (5A6)-coated FF beads inblocking solution (20 L) were added, and the plate
was placed on the magnet plate for incubation timesof 1–30 min. The wells were then washed with blocking solution and time-resolved fluorescence measurements were performed using a plate reader(ArvoSx1420; PerkinElmer).
IMMUNOHISTOCHEMICAL STAINING OF
PARAFFIN-EMBEDDED SAMPLESParaffin sections (4.0 m thick) were mounted onslides, deparaffinized in xylol, and rehydrated in adescending ethanol series for immunostaining. Rehydrated samples were treated with proteinase K(0.4 g/L; Dako) for 30 min, incubated with 0.5%
periodic acid for 10 min at room temperature,
washed with water, and then blocked with 4% Block
Ace (Snow Brand Milk Products) in Tris-bufferedsaline (TBS) for 30 min at 37 °C. After removal of theblocking solution, samples were incubated with primary antibody in blocking solution overnight at
4 °C, washed with TBS, and then incubated with
ENVISON (Dako) for 30 min. After washing, images
were visualized using 3,3-diaminobendizine (DAB),
with hematoxylin counterstaining for 1 min. OCTembedded frozen samples were sectioned on a cryostat
microtome and dried for 1 min at 60 °C, fixed with coldacetone for 2 min, hydrated, and then treated with blocking solution.

We previously developed unique multifunctionalbeads of 200 nm in diameter in which both fluorophores and ferrite nanoparticles were completely encapsulated into the organic polymer (14 ). In light ofthe beads’ features, we expected that the FF beads
would have the potential to be a practical probe in an
immunoassay. Meanwhile, we also created 140-nm–
diameter affinity magnetic beads (12 ). Because the140-nm beads were more magnetically responsive thanthe 200-nm– diameter beads (11, 15 ), we prepared
140-nm– diameter FF beads. We assumed that the useof hydrophilic fluorophores would result in fluorophore leakage from the beads and would complicate
assay outcomes. Thus, the hydrophobic fluorescentcomplex Eu(TTA)3(TOPO)2 (16 –19 ) was selected as
the fluorophore for encapsulation into the polymer
layer of the affinity magnetic beads. Using methanol toencapsulate Eu(TTA)3(TOPO)2 into the beads, we successfully produced 140-nm– diameter FF beads (Fig.
1A). TEM images showed that the shape of the FF beads
was exactly the same as that of the affinity magnetic
beads and the beads suffered no serious damage despite
the use of organic solvents in the process of incorporating fluorophores into the beads (see Fig. 1A in the
Data Supplement that accompanies the online versionof this report at http://www.clinchem.org/content/
vol60/issue4). The FF beads dispersed well in PBS, inwhich they appeared as a dark-brownish suspension (derived from the ferrite nanoparticles) under visible light.
Under ultraviolet exposure, the suspension emitted
bright red fluorescence derived from naked
Eu(TTA)3(TOPO)2. Dispersed FF beads were collectedeasily using a permanent magnet and only the accumulated pellets showed strong fluorescence under ultraviolet irradiation (Fig. 1B). No leaking of fluorophores
from FF beads into PBS was observed during many
repeated collections of the magnetic beads.
MAGNETICALLY PROMPTED RAPID SANDWICH IMMUNOASSAY

RAPID IMMUNOHISTOCHEMICAL STAINING OF TISSUES USING

USING FF BEADSFF BEADS

Our sandwich immunoassay with FF beads is illustrated in Fig. 2. Detection antibody– coated FF beads
and samples were added on a capture antibody–

Samples were treated with 5% BSA blocking solution
for 2 min. After removal of blocking solution, samples
612 Clinical Chemistry 60:4 (2014)-327-

Rapid Immunoreaction System with Fluorescent Magnetic Beads

Fig. 1. FF beads.
(A), Schematic of the structure of FF beads. (B), FF beads
dispersed in PBS (left) or collected by magnet (right) invisible light (above) and under ultraviolet irradiation (below). GMA, glycidyl methacrylate.

coated microplate, a magnet was attached under theplate for 1–2 min to concentrate the FF beads onto
the immobilized capture antibody, unbound FF
beads were washed out (similar to standard immu-

noassays), and the fluorescence of the remaining
FF beads held on the plate through the antigen–antibody reaction was measured directly.
To verify the feasibility of the newly devised system, we examined a sandwich immunoassay with FF
beads using BNP, which is a hormone secreted by theheart and a basic biomarker of heart failure, as the target antigen (20, 21 ). The BNP concentration in the
blood plasma of healthy persons is usually 20 pg/mL,
and concentrations of 100 pg/mL BNP are associatedwith heart disease (22, 23 ). Magnetic collection of antiBNP antibody– coated FF beads in an immunoreaction
allowed dose-dependent detection of BNP within 1
min, after which saturation was observed (Fig. 3A). Although background fluorescence was observed in theabsence of BNP, antigen concentrations equivalent to
2.0 pg/mL could be detected with a high signal-to-noise
ratio. By contrast, the assay without magnetic collection of FF beads could detect only 200 pg/mL BNP (Fig.
3B). Without magnets, the fluorescence intensity derived from the FF beads held on the plate through theantigen–antibody reaction was highly variable. Fig. 3,
C–E, show the detection of BNP at single incubation
times of 1, 2, and 3 min. These graphs indicate that themagnetic force clearly enhanced the detection of BNP.
Notably, a specific antigen–antibody reaction could be
detected rapidly by only 1-min magnetic collection of FF
beadswithouttheuseofanyenzymeandenzymaticreaction.
We next investigated detection of PSA, a widelyused biomarker in patients with prostate cancer (24 ).
Healthy individuals generally have low concentrationsof PSA (0.1 ng/mL), whereas patients with prostateFig. 2. Schematic of a standard immunoassay scheme (top) and magnetically prompted sandwich immunoassay
using antibody-coated FF beads (bottom).

Clinical Chemistry 60:4 (2014)

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613

Fig. 3. Detection of BNP by sandwich immunoassay using FF beads coated with anti-BNP antibody.
Results of BNP detection with magnet (A) and without magnet (B). Red, blue, green, and orange lines represent 200, 20, 2.0, and 0
pg/mL of BNP, respectively. Results of BNP detection at a single incubation time: 1 min (C); 2 min (D); 3 min (E). Blue and red markers
represent BNP detection with magnet and without magnet, respectively. All data are expressed as mean (SD) of 3 measurements.

cancer have concentrations 4.0 ng/mL; hence, clinical examination of prostate cancer requires detectionof PSA in serum ranging from 0.1 to 10 ng/mL (25, 26 ).
PSA could be detected using anti-PSA antibody–coated FF beads either with or without magnetic collection of the beads (Fig. 4); however, there was a large
difference in detection capability. With magnetic collection of FF beads, a wide range of PSA concentrations
could be monitored (Fig. 4A). By contrast, in all cases
in which magnetic collection was not used, the detection signal was less stable than the cases with magnetic
collection (Fig. 4B). Using the magnetic system, we
successfully detected as little as 0.02 ng/mL PSA
within 5 min of sample addition. Fig. 4, C–F, show
the detection of PSA at single incubation times of 1,
3, 10, and 30 min. As is the case in BNP, these graphs
indicate that the magnetic force clearly enhanced thedetection of PSA. On receiving the results, the performance of the sandwich immunoassay with FF
beads was compared with conventional immunoassays. Using PSA samples whose concentrations had
been measured by chemiluminescent EIA (CLEIA),
we measured over 50 PSA samples by the sandwich
immunoassay with FF beads (Fig. 5). Good correlation was observed between PSA concentrations measured

by the sandwich immunoassay with FF beads and PSA
concentrations measured by CLEIA. The correlation between the 2 assays was: y  0.472  0.879x, r 2  0.953.
LIMIT OF DETECTION, LIMIT OF QUANTIFICATION, AND
REPRODUCIBILITY IN THE SANDWICH IMMUNOASSAY
WITH FF BEADSThe limit of detection (LOD) was defined as the lowest measured value for which the measured value
minus 2 SDs was higher than the blank mean value
plus 2 SDs. Based on this criterion, the LOD of the
sandwich immunoassay with FF beads was estimatedto be around 5.0 pg/mL for BNP in plasma andaround 0.005 ng/mL for PSA in serum (see online
Supplemental Figs. 2 and 3). The limit of quantification (LOQ) was defined as the lowest concentration
measurable at an interassay CV 20% in the sandwich
immunoassay with FF beads. Based on this criterion, theLOQ of the sandwich immunoassay with FF beads wasestimated to be 10 pg/mL for BNP in plasma and 0.02
ng/mL for PSA in serum (see online Supplemental Figs. 2
and 3). For reproducibility of the sandwich immunoassay
with FF beads, the mean value, SD, and CV were calculated. For the medium PSA concentration, the meanvalue, SD, and CV were 5.63 pg/mL, 0.489 pg/mL, and

614 Clinical Chemistry 60:4 (2014)-329-

Rapid Immunoreaction System with Fluorescent Magnetic Beads

Fig. 4. Detection of PSA by sandwich immunoassay using FF beads coated with anti-PSA antibody.
Results of PSA detection with magnet (A) and without magnet (B). Red, blue, green, red broken, blue broken, green broken,
and orange lines represent 6.3, 2.0, 0.64, 0.20, 0.064, 0.020, and 0 ng/mL of PSA, respectively. Results of PSA detection at asingle incubation time: 1 min (C), 3 min (D), 10 min (E), 30 min (F). Blue and red markers represent PSA detection with magnet
and without magnet, respectively. All data are expressed as mean (SD) of 3 measurements.

8.7%, respectively. For the high PSA concentration, themean value, SD, and CV were 10.1 pg/mL, 1.35 pg/mL,
and 13.3%, respectively.
MAGNETICALLY PROMPTED RAPID IMMUNOHISTOCHEMISTRYUSING FF BEADSThe scheme of an advanced immunohistochemical
staining using magnetic collection of FF beads is illus-

trated in Fig. 6. FF beads coated with antibodies recognizing specific carcinoma cell-surface antigens areadded onto fixed samples of carcinoma cells, and amagnet is attached beneath them to enhance theantigen–antibody reaction (10 min). After removal ofthe magnet, the samples are washed and observed directly by fluorescence microscopy. In this system,
washing, a key step in immunohistochemical staining
Clinical Chemistry 60:4 (2014)

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615

Fig. 5. Comparison of PSA concentrations measured by the conventional immunoassay (CLEIA) and by the sandwich
immunoassay with FF beads.
All data are expressed as mean (SD) of 3 measurements.

to eliminate undesired signal noise, was simplified by
the use of a magnet.
To confirm the magnetically promoted immunohistochemical staining with FF beads, we selected
xenograft samples of A431 human epidermoid cancer
cells, in which EGFR is highly expressed. EGFR is a key

disease marker to understand various types of malignancy, including breast cancer, because it plays an important role in tumor cell survival and proliferation(27, 28 ). Fig. 7A shows the results of immunohistochemical staining of A431 cells by hematoxylin– eosin(HE) staining, conventional polymer-labeled immuno-

Fig. 6. Schematic of standard staining (top) and magnetically prompted immunohistochemical staining of carcinoma
cells using antibody-coated FF beads (bottom).

616 Clinical Chemistry 60:4 (2014)-331-

Rapid Immunoreaction System with Fluorescent Magnetic Beads

Fig. 7. Immunohistochemical staining of A431 cells (human epidermoid cancer cell, high EGFR expression level) (A);H69 cells (small-cell lung cancer cell, low EGFR expression level) (B); and needle-core biopsy samples of breast
cancer using anti-EGFR antibody-coated FF beads (C).
Left, HE staining; middle, paraffin-embedded staining; right, immunohistochemical staining with magnetic collection of FF
beads.

staining with DAB, and anti-EGFR antibody– coated FF
beads. Samples treated with FF beads showed vivid red
florescence, and the areas of staining corresponded tothose identified by the conventional polymer-labeled
method. By contrast, samples prepared from H69 cells(small-cell lung cancer cells, EGFR expression level isrelatively low) treated with anti-EGFR antibody–coated FF beads produced negligible levels of red fluorescence (Fig. 7B).
Next, immunohistochemical staining of breast
cancer needle-core biopsies was investigated. Similar to the results with A431 cells, clear red fluorescence derived from FF beads was observed in thesame area stained using the polymer-labeled method(Fig. 7C). These results indicate that antibodycoated FF beads can accurately and rapidly recognize

corresponding antigens expressed on cells and tissues through a magnetically enhanced antigenantibody reaction.
Discussion
Conventional immunoassays often exhibit low-levelsignals due to the use of enzyme-modified detection
antibodies. Investigators have been trying to resolve theintrinsic problems of analytical sensitivity; for example, immunoassay systems using antibody-coatednanoparticle doping enzymes instead of enzymebound antibodies provide improvements in analyticalsensitivity (29 ). These types of immunoassays use enzymes in the detection of biomarkers and take several
hours to complete testing.
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617

Magnetic particles are attractive functional materials in the development of immunoreactions, because
magnetic force often improves efficiency and analyticalsensitivity (30 ). Various types of immunoassays withmagnetic particles have been developed and some haveenabled shorter assay times (31–33 ). Mirkin and coworkers reported an analytically sensitive immunoPCR method using magnetic microbeads coated withmonoclonal antibodies to PSA as well as gold nanoparticles functionalized with bar-coded double-stranded
DNA and polyclonal antibodies (34 ). However, theimmuno-PCR method with magnetic microbeads andgold nanoparticles still requires several steps from sample addition to signal acquisition and takes at least
1.5–2 h for detection of PSA. Although there are highly
analytically sensitive immunoassays that simply involve magnetic collection of functionalized magnetic
particles, construction of a rapid immunoassay (10min) or assays without amplification steps has not previously been achieved. Similarly, conventional immunohistochemical staining generally requires overnightincubation of samples with primary antibodies for highanalytical sensitivity. Staining of sentinel lymph node
samples using pigments, including polymer-embedded
staining, has been used for intraoperative diagnosis;
however, pathological diagnosis with pigments is timeconsuming and occasionally leads to misdiagnosis as aresult of human error.
The FF beads that we have developed are uniquematerials in that both hydrophobic fluorophores andferrite nanoparticles are completely encapsulated into
an organic polymer that can be modified chemically(11, 15 ). The lack of fluorophore leakage from FF
beads during magnetic separation indicates that all
fluorophores are retained firmly inside the polymer ofthe beads. The FF beads were quite stable and could be
stored for at least 6 months without any damage andloss of fluorescence intensity. In addition, the fluorescence intensity of FF beads changed depending on theconcentration of the beads (see online Supplemental
Fig. 1B). Thus, the FF beads should be highly advantageous for their application as probes in analytically sensitive bioassays capable of detecting specific biomarkers. The results of the sandwich immunoassay, using FF
beads on which antibodies were coated, clearly demonstrate that magnetic collection of FF beads in the immunoreaction step enabled the detection of disease
markers within 5 min after sample addition (Figs. 3A
and 4A). In addition, the data of the biomarker detection at a single incubation time indicated that several
minutes of magnetic collection of FF beads markedly
improved the detection capability (Fig. 3, C–E, and Fig.
4, C–F). Although a 3–10-min immunoreaction withthe magnet seemed to be most effective for the detection of the biomarkers by the sandwich immunoassay

with FF beads, we recognized that 1-min immunoreaction with the magnet was enough for the detection of
the biomarkers. Additionally, the results of the immunohistochemical staining with antibody-coated FF
beads also demonstrated that a 10-min magnetic collection of FF beads in the incubation step enabled theultrarapid and reliable discrimination of carcinoma
cells (Fig. 7). By contrast, without magnetic collection,
both immunoassays resulted in low detection of
disease markers and required the same time as conventional methods to obtain comparable analyticalsensitivity (Figs. 3B and 4B). Furthermore, we also
found that under the same conditions, including theassay time, disease-related markers in the plasma or
serum could be detected without decreases of florescence counts (see online Supplemental Figs. 2 and 3).
These results clearly indicate that the magnetic force
enhances and accelerates specific interactions between
antigens and antibodies, and the application of magnetic collection of FF beads in a particular space to theimmunoreaction can dramatically improve both thetotal assay time and the number of steps required,regardless of the antibodies used and the presence ofthe plasma or the serum.
The LOQ values of 10 pg/mL for BNP and 0.02
ng/mL are comparable to those for currently used immunoassays. The relatively low LOQ indicates that the
sandwich immunoassay with magnetic collection of FF
beads is a practical and reliable immunoassay systemwith sufficient analytical sensitivity. Often the analytical sensitivity of immunoassays depends on antibody
quality (35 ). We speculate that the difference of detection pattern between BNP and PSA would result fromdifferences in the quality of the antibodies used in the
sandwich immunoassay with FF beads.
The successful rapid and analytically sensitive detection of target molecules (BNP, PSA, and EGFR) by
magnetic collection of FF beads in the immunoreaction steps can be explained by the magnetic concentration of both antigens and antibodies locally on the plateor samples, followed by direct florescent measurement
without the need for signal amplification. The results suggest that the magnetic concentration of biomoleculecoated FF beads to a specific place has the potential toaccelerate the interaction between biomolecules. We
also investigated the versatility of the immunohistochemical staining with FF beads using several carcinoma cells and found that carcinoma cells other thanA431 cells could be discriminated within 20 min (seeonline Supplemental Fig. 4).
To date, several immunoreaction platforms thatuse magnetic force to improve their efficiency havebeen reported (30 –33 ). These mainly involve removalof unbound capture antibodies and, to the best of ourknowledge, there are few bioassays that accelerate spe-

618 Clinical Chemistry 60:4 (2014)-333-

Rapid Immunoreaction System with Fluorescent Magnetic Beads

cific biomolecule interactions and shorten assay
times using magnetic force (36 ). The immunoreaction systems with FF beads described here used magnetic force, not to remove the magnetic beads but toenhance the signal for detection of biomarkers. The
simple operation of collecting FF beads magneticallyat a specific space in the immunoreaction enables
improvements in both analytical sensitivity and total
assay time. Compared to conventional platforms,
the advanced immunoreaction with FF beads enables rapid detection of biomarkers without the useof enzyme reactions.
In summary, we successfully developed a rapid
immunoreaction system using uniquely developed
fluorescent magnetic beads, FF beads. Two distinguishing properties of FF beads, strong magnetic force
and high fluorescence intensity, enable the shortening of
assay time dramatically without loss of sensitivity andeliminate the signal amplification step. Immunoreaction
systems with FF beads would be feasible alternatives toconventional immunoassays and immunohistochemical
staining.

Author Contributions: All authors confirmed they have contributed tothe intellectual content of this paper and have met the following 3 requirements: (a) significant contributions to the conception and design,acquisition of data, or analysis and interpretation of data; (b) draftingor revising the article for intellectual content; and (c) final approval of
the published article.

Authors’ Disclosures or Potential Conflicts of Interest: Upon manuscript submission, all authors completed the author disclosure form.
Disclosures and/or potential conflicts of interest:
Employment or Leadership: None declared.
Consultant or Advisory Role: None declared.
Stock Ownership: None declared.
Honoraria: None declared.
Research Funding: Y. Yamaguchi, Grant-in-Aid for Scientific Research on Innovative Areas “Transcription Cycle” from MEXT; Y.
Kiatagawa, Grant for Research and Development Projects in Cooperation with Academic Institutions from the New Energy and Industrial Technology Development Organization (NEDO); H. Handa,
Grant for Research and Development Projects in Cooperation with
Academic Institutions from the New Energy and Industrial Technology Development Organization (NEDO), Grant-in-Aid for Scientific
Research on Innovative Areas “Chemical Biology of Natural Products” from the Ministry of Education, Culture, Sports, Science andTechnology (MEXT), Grant-in-Aid for Scientific Research (A) fromJapan Society for the Promotion of Science (JSPS), Health Labour
Sciences Research Grants (Research on New Drug Development)from the Ministry of Health, Labour and Welfare (MHLW), andDevelopment of Systems and Technologies for Advanced Measurement and Analysis from the Japan Science and Technology
Agency (JST).
Expert Testimony: None declared.
Patents: None declared.
Role of Sponsor: The funding organizations played no role in thedesign of study, choice of enrolled patients, review and interpretationof data, or preparation or approval of manuscript.
Acknowledgments: We deeply thank Shionogi & Co. for the kind giftof BNP-related materials. We also thank Prof. Haruma Kawaguchi(Kanagawa University) for TEM observations and Naohiro Hanyu
and Dr. Toshiyuki Tanaka (Tamagawa Seiki) for fruitful discussions
on immunohistochemical stainings.

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Hrd1 suppresses Nrf2-mediated cellular
protection during liver cirrhosis
Tongde Wu,1 Fei Zhao,1 Beixue Gao,2 Can Tan,2 Naoko Yagishita,3 Toshihiro Nakajima,3,4
Pak K. Wong,5 Eli Chapman,1 Deyu Fang,2,6 and Donna D. Zhang1,61

Department of Pharmacology and Toxicology, College of Pharmacy, The University of Arizona, Tucson, Arizona 85721, USA:
Department of Pathology, Northwestern University Feinberg School of Medicine, Chicago, Illinois 60611, USA; 3Institute ofMedical Science, St. Marianna University School of Medicine, Kawasaki 216-8511, Japan; 4Institution of Medical Science, Tokyo
Medical University, Tokyo 160-8402, Japan; 5Department of Aerospace and Mechanical Engineering, The University of Arizona,
Tucson, Arizona 85721, USA
2

Increased endoplasmic reticulum (ER) stress and reactive oxygen species (ROS) are the salient features of end-stage
liver diseases. Using liver tissues from liver cirrhosis patients, we observed up-regulation of the XBP1–Hrd1 arm of
the ER stress response pathway and down-regulation of the Nrf2-mediated antioxidant response pathway. We
further confirmed this negative regulation of Nrf2 by Hrd1 using Hrd1 conditional knockout mice. Downregulation of Nrf2 was a surprising result, since the high levels of ROS should have inactivated Keap1, the primaryubiquitin ligase regulating Nrf2 levels. Here, we identified Hrd1 as a novel E3 ubiquitin ligase responsible forcompromised Nrf2 response during liver cirrhosis. In cirrhotic livers, activation of the XBP1–Hrd1 arm of ER
stress transcriptionally up-regulated Hrd1, resulting in enhanced Nrf2 ubiquitylation and degradation andattenuation of the Nrf2 signaling pathway. Our study reveals not only the convergence of ER and oxidative stressresponse pathways but also the pathological importance of this cross-talk in liver cirrhosis. Finally, we showed thetherapeutic importance of targeting Hrd1, rather than Keap1, to prevent Nrf2 loss and suppress liver cirrhosis.
[Keywords: Nrf2; Hrd1; liver cirrhosis]
Supplemental material is available for this article.
Received January 13, 2014; revised version accepted February 24, 2014.

Liver cirrhosis is a pathological state in which the normalliver tissue is replaced with scar tissue. Most commonly,
liver cirrhosis is caused by alcohol consumption, viral
hepatitis, chronic or excessive drug use, or exposure to
hepatotoxic chemicals (Lotersztajn et al. 2005; HernandezGea and Friedman 2011; Wynn and Ramalingam 2012).
Currently, there are no effective treatments available toprevent or suppress the pathogenesis of liver cirrhosis.
Therefore, understanding the molecular mechanisms underlying the pathological progression of liver cirrhosis anddeveloping new therapeutic strategies for prevention orreversal of liver cirrhosis are urgently needed.
Studies suggest the involvement of endoplasmic reticulum (ER) stress in the pathogenesis of liver cirrhosis.
Using experimental disease models, for example, it has
been demonstrated that the ER is enlarged and stressed in
response to intraperitoneal (IP) injection of carbon tetrachloride or by feeding a methionine- and choline-deficient diet (Mu et al. 2010; Zheng et al. 2011b; Wanget al. 2013). To cope with ER stress, mammalian cells
6

Corresponding authors
Email dzhang@pharmacy.arizona.edu
E-mail fangd@northwestern.edu
Article published online ahead of print. Article and publication date areonline at http://www.genesdev.org/cgi/doi/10.1101/gad.238246.114.

708

have developed an adaptive mechanism called the unfolded protein response (UPR), signaled by three sensors
residing on the ER membrane: IRE1, PERK, and ATF6
(Travers et al. 2000; Carvalho et al. 2006; Tsang et al.
2010). The cellular decisions in activating or coordinatingthese pathways are crucial in determining cell fate anddisease outcome in a variety of pathological conditions(Tsang et al. 2010).
Hrd1, an E3 ubiquitin ligase sometimes called synoviolin, is a multipass ER membrane protein encoded bysynovial apoptosis inhibitor 1 (SYVN1 or Hrd1). Recent
studies have identified Hrd1 as a downstream effector of
the IRE1 branch of the UPR. In response to ER stress,
XBP1 mRNA is spliced by IRE1 to produce XBP1s, which
encodes an active transcription factor, resulting in transcriptional up-regulation of Hrd1 through a UPR element
in its promoter (Yoshida et al. 2001; Yamamoto et al.
2008). Hrd1 is essential for embryogenesis, as demonstrated by the embryonic lethality of Hrd1/ mice (Yagishita 2014 Wu et al. This article is distributed exclusively by Cold Spring
Harbor Laboratory Press for the first six months after the full-issue
publication date (see http://genesdev.cshlp.org/site/misc/terms.xhtml).
After six months, it is available under a Creative Commons License(Attribution-NonCommercial 4.0 International), as described at http://
creativecommons.org/licenses/by-nc/4.0/.

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Hrd1 suppresses Nrf2 during liver cirrhosis

et al. 2005). It also plays a crucial role in the pathogenesisof arthropathy and liver cirrhosis/fibrosis (Amano et al.
2003; Hasegawa et al. 2010). Although Hrd1 was initiallycharacterized as an E3 ubiquitin ligase controlling ERassociated degradation (ERAD), recent reports demonstrate that Hrd1 can also control the turnover of nonERAD substrates such as p53, IRE1, and Nrf1, a memberof the cap‘n’collar (CNC) transcription factor family(Yamasaki et al. 2007; Gao et al. 2008; Steffen et al. 2010;
Tsuchiya et al. 2011).
In addition to the UPR, reactive oxygen species (ROS)
have been shown to play a major role in the pathogenesisof liver cirrhosis (Lotersztajn et al. 2005; Wynn and
Ramalingam 2012). Cellular ROS levels are regulated by
the Nrf2 transcription factor that also belongs to the
CNC family (Kensler et al. 2007; Jaramillo and Zhang2013). Under basal conditions, Nrf2 levels are low due totight regulation by Keap1, a substrate adaptor protein for
the Cullin3 (Cul3)-based E3 ubiquitin ligase (Itoh et al.
1999; Kobayashi et al. 2004; Zhang et al. 2004). Activation of Nrf2 has been shown to confer protection against
liver cirrhosis (Kawata et al. 2010; Ramani et al. 2012;
Chen et al. 2013). Conversely, compromised Nrf2 signaling and elevation of ROS were detected in end-stage
alcoholic liver disease (Kurzawski et al. 2012). Although
the functional importance of the UPR and Nrf2 responses
in liver cirrhosis has recently emerged, the interplay
between these two major stress response pathways during
the pathogenesis of liver cirrhosis remains unclear. Using
liver tissues from cirrhosis patients, we detected upregulation of the XBP1–Hrd1 arm of the ER stresspathway and down-regulation of the Nrf2-mediated antioxidant response. Here, we demonstrate that these twostress response pathways converge, and therapeutic modulation through inhibition of the IRE1–XBP1–Hrd1 pathway can prevent loss of the Nrf2-mediated protection,
thus mitigating liver cirrhosis in vivo.
Results
The XBP1–Hrd1 arm of the ER stress pathway is upregulated, and the Nrf2-mediated antioxidant response
pathway is down-regulated in human cirrhotic livers
To investigate the role of the ER and Nrf2-mediated stressresponses in the pathogenesis of liver cirrhosis, wemeasured the protein expression of several key proteins
involved in both pathways in human cirrhotic livertissues. Immunohistochemistry (IHC) and immunoblot
analyses of cirrhotic liver tissues revealed increased
levels of XBP1s and Hrd1 proteins that corresponded tocompromised Nrf2 signaling, as evidenced by the decreased protein levels of Nrf2 and Nrf2-regulated proteins,
NQO1, and GCLM (Fig. 1A,B). Consistently, the mRNAlevel of HRD1 was increased, whereas mRNA levels ofNrf2 target genes, such as NQO1 and GCLM, were decreased in cirrhotic tissues compared with normal tissues(Fig. 1C). In contrast, no change in NRF2 mRNA expression was observed (Fig. 1C), indicating that Hrd1 is alteredat the transcriptional level, while Nrf2 is regulated at the

post-transcriptional level during cirrhosis. These results
demonstrate an up-regulation of the XBP1–Hrd1 arm of the
ER stress response pathway and a down-regulation of theNrf2 antioxidant response pathway in liver tissues fromend-stage alcoholic cirrhosis patients. To further confirm
this, we used a murine CCl4-induced liver cirrhosis model.
The protein levels of XBP1s and Hrd1 were increased incirrhotic liver tissues from both Nrf2+/+ and Nrf2/ mice
(Fig. 1D,E), suggesting activation of the XBP1–Hrd1 pathway during the course of liver cirrhosis. Interestingly, theprotein levels of Nrf2, GCLM, and NQO1 decreased incirrhotic livers, and altered expression of GCLM and NQO1
was Nrf2-dependent, since no changes in their expression
were detected in Nrf2/ mice (Fig. 1D,E). Similar to theresults obtained using human specimens, the mRNAlevel of Hrd1 increased in CCl4-treated groups regardlessof genotype, whereas the mRNA levels of Nrf2 (in Nrf2+/+mice) and Keap1 were not affected by CCl4 treatment(Fig. 1F). In contrast, the mRNA levels of NQO1 andGCLM decreased in Nrf2+/+ but not Nrf2/ mice (Fig. 1F).
This inverse activation status of the XBP1–Hrd1 and Nrf2
pathways observed in human and mouse cirrhotic livers
prompted us to test whether there is cross-talk between
these two pathways using Hrd1 conditional knockout
mice. Hrd1 was deleted through tail vein injection of
adenoviruses containing Cre recombinase. Silencingof Hrd1, which occurred only in Hrd1f/f but not Hrd1+/+
mice, resulted in a robust increase in the protein level of
Nrf2, NQO1, and GCLM as well as an increase in themRNA level of NQO1 and GCLM without changing Nrf2
or Keap1 (Fig. 1G –I).
XBP1s and Nrf2 expression levels are inversely
correlated
Next, using cultured cells, we investigated the inverse
correlation between the activation of the XBP1–Hrd1
pathway and the Nrf2 pathway and the underlying
molecular mechanism of their negative correlation.
Overexpression of XBP1s decreased the protein level ofNrf2 under both basal (untreated) and induced (tertbutylhydroquinone [tBHQ]-treated) conditions (Fig. 2A)
in both HEK293T (Fig. 2A) and MDA-MB-231 (data notshown) cells. Overexpression of XBP1s also reduced theexpression of NQO1, GCLM, and HO-1 but not Keap1(Fig. 2A). Ectopic expression of XBP1s decreased AREluciferase activity in a dose-dependent manner underboth basal and induced conditions (Fig. 2B) and also
decreased the mRNA levels of NQO1 and HO-1 without
affecting NRF2 or KEAP1 (Fig. 2C). Next, we used XBP1+/+
and XBP1/ mouse embryonic fibroblast (MEF) cells to
confirm these observations. Cell status was validated by
the absence of both unspliced XBP1 (XBP1u) and XBP1s in
XBP1/ cells (Fig. 2D). The protein levels of Nrf2, HO-1,
and GCLM were higher in XBP1/ than in XBP1+/+ cells(Fig. 2E), as were the mRNA levels of NQO1, GCLM, andHO-1 but not Nrf2 (Fig. 2F). Increased expression of NQO1and GCLM in XBP1/ cells was found to correlate withenhanced activities of NQO1 (Fig. 2G) and a higher level ofglutathione under both basal and induced conditions in

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Figure 1. The XBP1–Hrd1 arm of the ER stress pathway is up-regulated, and the Nrf2-mediated antioxidant response pathway is downregulated in human cirrhotic liver. (A–C) Normal or cirrhotic liver tissues from patients with end-stage liver cirrhosis. (A)Representative hematoxylin and eosin (H&E) and IHC staining of XBP1s, HRD1, and NRF2 from liver tissues. (B) Expression levelsof the indicated proteins detected using immunoblot analysis. (C) mRNA levels of NRF2, HRD1, NQO1, and GCLM measured byquantitative real-time PCR (qRT-PCR). (D–F) Liver tissues from control or CCl4-treated mice. (D) Representative H&E and IHC stainingof Hrd1, Nrf2, and GCLM from liver tissues. (E) Expression levels of the indicated proteins detected using immunoblot analysis. (F)mRNA levels of Nrf2, Keap1, Hrd1, NQO1, and GCLM measured by qPCR. (G–I) Liver tissues from Hrd1+/+ and Hrd1f/f mice tail veininjected with Cre-containing viruses for 5 d. (G) Representative H&E and IHC staining of Hrd1, Nrf2, and GCLM from liver tissues. (H)Expression levels of the indicated proteins detected using immunoblot analysis. (I) mRNA levels of Nrf2, Keap1, Hrd1, NQO1, andGCLM measured by qPCR. For immunoblot analysis, each lane contains a tissue lysate from an individual person (B) or mouse (E,H).
For qRT-PCR, results are expressed as means 6 SD; n = 4, four people per group (C); n = 4, three mice per group (F); and n = 3, three mice
per group (I).

XBP1/ cells (Fig. 2H). It is notable that more prominent
differences were observed under induced conditions (Fig.
2A–C,E–H). Furthermore, the higher Nrf2 protein level in
XBP1/ cells was due to a longer half-life of the Nrf2
protein in XBP1/ cells (40.5 min vs. 19.8 min in XBP1/
and XBP1+/+ cells, respectively) (Fig. 2I).
Nrf2 is negatively regulated by Hrd1
Next, we explored the relationship between Hrd1, a newly
identified XBP1s target gene, and Nrf2. Similar to the
XBP1 results, overexpression of Hrd1 reduced the protein

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level of Nrf2, GCLM, and HO-1 (Fig. 3A), whereas siRNAmediated silencing of Hrd1 enhanced these proteins (datanot shown). In accordance with these results, expressionof Nrf2, GCLM, and HO-1 was much higher in Hrd1/
compared with Hrd1+/+ cells (Fig. 3B). mRNA expressionof HO-1 was also higher in Hrd1/ cells (Fig. 3C).
Furthermore, Nrf2 protein was more stable with a longer
half-life in Hrd1/ compared with Hrd+/+ cells (39.7 min
vs. 16.3 min) (Fig. 3D). Since Hrd1 is an E3 ubiquitin
ligase, we tested the possibility that Hrd1 ubiquitylates
Nrf2. Overexpression of Hrd1 enhanced Nrf2 ubiquitylation, as shown in a cell-based ubiquitylation analysis (Fig.

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Hrd1 suppresses Nrf2 during liver cirrhosis

Figure 2. XBP1s and Nrf2 expression levels are inversely correlated. (A) Immunoblot analysis of the indicated proteins with celllysates from HEK293T cells transfected with an empty vector or Flag-XBP1s. At 24 h post-transfection, cells were left untreated ortreated with 50 mM tBHQ for 16 h. (B) Luciferase activities were measured in HEK293T cells cotransfected with NQO1-ARE-firefly
luciferase and TK-Renilla luciferase along with the indicated amount of Flag-XBP1s for 36 h. Relative luciferase activities and SDs were
calculated from three independent experiments (n = 3). (C) mRNA levels of Nrf2, Keap1, NQO1, and HO-1 in HEK293T cells
transfected and treated as described in A (n = 3 independent experiments). (D) Splicing analysis of XBP1 mRNA (unspliced [u] andspliced [s]) was performed as described in the Materials and Methods. Total mRNA was extracted from XBP1+/+ and XBP1/ MEF cells
untreated or treated with 5 mg/mL tunicamycin for 16 h. (E–H) Protein levels of the indicated proteins (E), mRNA levels of the indicatedgenes (F), NQO1 activities (G), and intracellular glutathione levels (H) were measured in XBP1+/+ and XBP1/ MEF cells left untreatedor treated with 50 mM tBHQ for 16 h. (I) The Nrf2 protein half-life in XBP1+/+ and XBP1/ MEF cells was measured in the presence of
50 mM cycloheximide at the indicated time points. Cell lysates were subjected to immunoblot analysis, with Nrf2 intensity normalized
to tubulin plotted using a semilogarithmic scale.

3E). In addition, coexpression of cyan fluorescent protein (CFP)-Hrd1 and red fluorescent protein (RFP)-Nrf2
showed that in the perinuclear clusters where Hrd1
localized, the Nrf2 signal was reduced (appeared as holes
in the RFP channel), suggesting that ectopically expressed
Hrd1 down-regulated Nrf2 (Fig. 3F). This inverse colocalization pattern was diminished by MG132 treatment(80% vs. 20% in untreated and MG132-treated cells, respectively) (Fig. 3F). These results suggest that Hrd1 ubiquitylates Nrf2.
To further confirm that XBP1-mediated down-regulation of the Nrf2 signaling pathway is through Hrd1,

several additional experiments were performed. First,
overexpression of XBP1s, but not the unspliced mutant
XBP1-3K/3R (XBP1u), up-regulated Hrd1 and decreased
Nrf2, NQO1, GCLM, and HO-1 under both basal andinduced conditions (Fig. 4A). Second, the XBP1s-mediateddown-regulation of Nrf2 signaling was blunted by transfection of Hrd1-siRNA (Fig. 4B). Third, a decreased Nrf2
expression by XBP1s overexpression was observed only in
Hrd+/+ but not in Hrd/ cells (Fig. 4C). Fourth, upregulation of endogenous XBP1s induced by tunicamycin
also decreased Nrf2, NQO1, and GCLM (Fig. 4D). Finally,
the mRNA levels of Hrd1 and HO-1 were inversely

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Figure 3. Nrf2 is negatively regulated by Hrd1. (A) Immunoblot analysis of the indicated proteins with cell lysates from HEK293T cells
transfected with an empty vector or Flag-Hrd1. At 24 h post-transfection, cells were left untreated or treated with 50 mM tBHQ for 16 h.
(B,C) Protein levels of the indicated proteins (B) and mRNA levels of the indicated genes (C) were measured in Hrd1+/+ and Hrd1/MEF cells left untreated or treated with 50 mM tBHQ for 16 h. (D) The Nrf2 protein half-life in Hrd1+/+ and Hrd1/ MEF cells wasmeasured as described in Figure 2I. (E) Cell-based ubiquitylation analysis was performed in HEK293T cells cotransfected with theindicated expression vectors for 48 h. (F) The cellular localization of Hrd1 and Nrf2 was detected by live-cell imaging, withrepresentative images shown in the left panel. NIH3T3 cells were cotransfected with CFP-Hrd1 and RFP-Nrf2 for 24 h. Inversed
colocalization of CFP-Hrd1 and RFP-Nrf2 is indicated with a white arrow. A cell that contains more than two inversed colocalization
dots was considered as ‘‘positive.’’ At least 50 cells from either the control or the MG132-treated group were counted to get thepercentage of cells showing an inverse correlation in their signal intensities between Hrd1 and Nrf2.

correlated with higher Hrd1 and lower HO-1 in XBP1+/+
cells compared with XBP1/ cells (Fig. 4E).
Hrd1 and Nrf2 interact directly
Immunoprecipitation analyses demonstrated that exogenously and endogenously expressed Hrd1 and Nrf2 coexisted in precipitated complexes (Fig. 5A,B). To map the
Hrd1-interacting domains in Nrf2, several deletion proteins purified from bacteria were used (Fig. 5C). Deletionof either Neh4 or Neh5 diminished the interaction, while
deletion of both (Neh4–5) completely abolished the interaction (Fig. 5D), indicating a direct interaction between Hrd1 and the Neh4–5 domains of Nrf2. This was
further confirmed in a cell-based assay by the fact that Nrf2DNeh4 or Nrf2-DNeh5 immunoprecipitated Flag-Hrd1 lessefficiently compared with wild-type Nrf2 (Nrf2-WT), andNrf2-DNeh4-5 did not immunoprecipitate Flag-Hrd1 at all

712(Fig. 5E). Moreover, overexpression of Hrd1 reduced
Nrf2-WT but was unable to reduce the protein level of
Nrf2-DNeh4-5 (Fig. 5F), which supports the notion that
Neh4–5 domains interact with Hrd1 and that this interaction is important for down-regulation of Nrf2 by
Hrd1.
To map the domain within Hrd1 that interacts with
Nrf2, we performed an in vitro binding assay using Histagged Nrf2 purified from bacteria and Flag-tagged Hrd1
proteins immunoprecipitated from HEK293T cells (Fig.
5G). As expected, His-Nrf2 was immunoprecipitated by
Flag-Hrd1, whereas the interaction of His-Nrf2 with FlagHrd1-N or Flag-Hrd1-N-RING was completely lost when
the C-terminal domain (amino acids 337–617) of Hrd1
was deleted (Fig. 5G). The C-terminal domain of Hrd1
was found to be sufficient to immunoprecipitate Nrf2
(Fig. 5G). These results suggest a direct interaction between

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Hrd1 suppresses Nrf2 during liver cirrhosis

Figure 4. XBP1s down-regulates Nrf2 through Hrd1. (A) The protein levels of the indicated proteins in HEK293T cells transfected with
vector, Flag-XBP1s, or XBP1-3K/3R and treated with tBHQ for 16 h. (B) The protein levels of the indicated proteins in HEK293T cells
cotransfected with Hrd1-siRNA along with an empty vector or Flag-XBP1s. Cells were then treated with tBHQ for 16 h. (C) The protein
levels of the indicated proteins in Hrd1+/+ and Hrd1/ MEF cells transfected with an empty vector or Flag-XBP1s for 24 h. (D) The
protein levels of the indicated proteins in HEK293T cells left untreated or treated with transmembrane (TM), tBHQ, or both for 16 h. (E)The mRNA levels of HO-1 and Hrd1 in XBP1+/+ and XBP1/ MEF cells treated with TM, tBHQ, or both for 16 h. Data are shown asmean 6 SD (n = 3 independent experiments).

the C-terminal domain of Hrd1 and the Neh4–5 domainsof Nrf2, which is consistent with the notion that Nrf2 isa cytosolic protein and interacts with the cytosolic
C-terminal domain of Hrd1.
Hrd1 is a novel E3 ubiquitin ligase of Nrf2
The RING domain of Hrd1 contains a cysteine critical for
its ubiquitin ligase activity. To identify this criticalcysteine residue in the RING domain, six Hrd1 mutant
proteins in which each cysteine was replaced with serine
were constructed (Fig. 6A). Hrd1-C291S was found to be
critical for its function because overexpression of wildtype Hrd1 (Hrd1-WT), not Hrd1-C291S, reduced Nrf2 andHO-1 levels (Fig. 6B). Consistently, only Hrd1-WT, not
Hrd1-C291S, was able to enhance ubiquitylation of Nrf2
both in a cell-based assay (Fig. 6C) and in vitro (Fig. 6D).
Overexpression of Hrd1-WT accelerated degradation of
Nrf2 (from 38.7 min to 17.7 min), while Hrd1-C291S had
no effect on Nrf2 protein decay (38.7 min to 40.2 min)(Fig. 6E). Collectively, these results demonstrate that
Cys291 within the RING domain of Hrd1 is indispensable
for its ubiquitin ligase activity.

To demonstrate that Hrd1 functions independently of
Keap1 or b-TrCP, previously defined E3 ligases that
control Nrf2 protein stability (Kobayashi et al. 2004;
Zhang et al. 2004; Rada et al. 2011, 2012; Chowdhry
et al. 2013), we performed several additional experiments.
Overexpression of XBP1s or Hrd1 reduced Nrf2 levels in
Keap1/ cells (Fig. 6F). In addition, ectopically expressed
Hrd1 decreased not only Nrf2-WT but also Nrf2-ETGE/AAAA and Nrf2-DLG/AAA mutant proteins that werepreviously proven to lose interaction with Keap1, preventing degradation by Keap1-mediated ubiquitylation andsubsequent proteasomal degradation (Fig. 6G; McMahon
et al. 2006; Tong et al. 2006). These results suggest that
Hrd1-mediated Nrf2 degradation is Keap1-independent.
Next, to prove that Hrd1-mediated Nrf2 degradation is
also independent of b-TrCP, we made two additional
constructs, Nrf2-ETGE+S2 and Nrf2-ETGE-DLG +S2, thatwere confirmed to be resistant to both Keap1- and b-TrCPmediated degradation (data not shown). However, Hrd1
was still able to reduce the protein level of these twomutants (Fig. 6H), indicating that the ligase activity of
Hrd1 in ubiquitylating Nrf2 is not associated with Keap1
or b-TrCP. Next, we tested whether the seven lysines

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Figure 5. Hrd1 and Nrf2 interact directly. (A) Immunoprecipitation analysis was performed using cell lysates from HEK293T cells
cotransfected with Flag-Hrd1 and hemagglutinin (HA)-tagged Nrf2 (HA-Nrf2). (B) Immunoprecipitation analysis was performed using
cell lysates from HEK293T cells, immunoprecipitated by normal rabbit IgG or anti-Nrf2 antibody. (C) Schematic illustration of
glutathione S-transferase (GST)-tagged Nrf2-WT and its deletion mutants used for interaction domain mapping. (D) Sepharose affinity
chromatography analysis of the interaction between Nrf2 and Hrd1. [35S]-Hrd1-WT was generated by in vitro transcription andtranslation. GST-tagged Nrf2-WT and its indicated deletion mutants were expressed and purified from Escherichia coli cells. Equal
amounts of Nrf2 proteins were used for each pull-down assay. (E) Immunoprecipitation analysis was performed using cell lysates of
HEK293T cells cotransfected with Flag-Hrd1 and either HA-Nrf2, HA-Nrf2-DNeh4, HA-Nrf2-DNeh5, or HA-Nrf2-DNeh4-5. (F)Immunoblot analysis was performed with cell lysates from HEK293T cells cotransfected with Flag-Hrd1 and either Nrf2-WT or
Nrf2-DNeh4-5 for 24 h. (G) Immunoprecipitation of Nrf2 by Flag-Hrd1 and its mutants. HEK293T cells were transfected with Flag-Hrd1
and the indicated truncation mutants. Hrd1 proteins were first immunoprecipitated using Flag-M2 beads and then incubated with Histagged Nrf2 that was expressed and purified from E. coli cells. Equal amounts of Hrd1 proteins were used for each experiment.

within the Neh2 domain of Nrf2 are also required for
Hrd1-mediated ubiquitylation. The same seven lysines in
Nrf2 that are required for Keap1-dependent ubiquitylation of Nrf2 (Zhang et al. 2004) were also critical for Hrd1mediated Nrf2 ubiquitylation and degradation, sinceNrf2-K7/R7 was not affected by overexpression of Hrd1
(Fig. 6I). In addition, Hrd1-mediated ubiquitylation anddegradation of Nrf2 occurs in the cytosol, since Hrd1

714

reduced only the protein levels of Nrf2-WT and thecytoplasmic-only version of Nrf2 (Nrf2-NLS2) in which
the nuclear localization signal RKRK was mutated to
AAAA, but Hrd1 had no effect on Nrf2-NES1 and Nrf2NES2, which were forced to localize in the nucleus due tomutations made in their nuclear export signals (Fig. 6J;Sun et al. 2007). Taken together, these results clearly
demonstrate that Hrd1, an ER-associated E3 ubiquitin

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Hrd1 suppresses Nrf2 during liver cirrhosis

Figure 6. Hrd1 is a novel E3 ubiquitin ligase of Nrf2. (A) Schematic illustration of the amino acid sequence of the RING domain of
Hrd1. Conserved cysteine residues that are mutated to serines are labeled with arrows. (B) Immunoblot analysis of cell lysates from
HEK293T cells transfected with an empty vector, Flag-Hrd1, or Flag-C291S. Cells were untreated or treated with tBHQ for 16 h. (C)Cell-based ubiquitylation analysis was performed in HEK293T cells cotransfected with Nrf2, Flag-Hrd1, or Flag-C291S mutant and HAubiquitin for 48 h. (D) In vitro ubiquitylation of Nrf2 in the presence of Flag-Hrd1 and Flag-C291S was measured. HA-Nrf2-containing
complexes were immunoprecipitated using HA beads from HEK293T cells transfected with Nrf2 and either Flag-Hrd1-WT or FlagHrd1-C291S. HA bead-bound proteins were incubated with purified E1, E2-UbcH5c, ubiquitin, and ATP. Following denaturation byboiling, Nrf2 was immunoprecipitated with anti-Nrf2 antibodies, and ubiquitylation of Nrf2 was detected by immunoblot analysis
with an anti-ubiquitin antibody. (E) The Nrf2 protein half-life in HEK293T cells transfected with either an empty vector, Flag-Hrd1, or
Flag-C291S was measured as described above. (F) Immunoblot analysis of Nrf2 in Keap1/ MEF cells transfected with an empty vector,
Flag-Hrd1, or Flag-XBP1s for 24 h. (G) Immunoblot analysis of the indicated proteins. HEK293T cells were cotransfected with Flag-Hrd1
and either Nrf2-WT, Nrf2-ETGE/AAAA, or Nrf2-DLG/AAA. (H) Immunoblot analysis of the indicated proteins. HEK293T cells were
cotransfected with Flag-Hrd1 and either Nrf2-WT, Nrf2-ETGE+2S, or Nrf2-ETGE-DLG+2S. (I) Immunoblot analysis of the indicatedproteins. HEK293T cells were cotransfected with Flag-Hrd1 and either Nrf2-WT or Nrf2-K7/R7 for 24 h. (J) Immunoblot analysis of theindicated proteins. HEK293T cells were cotransfected with Flag-Hrd1 and either Nrf2-WT, Nrf2-NLS2, Nrf2-NES1, or Nrf2-NES2.

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ligase, is a novel E3 ubiquitin ligase controlling Nrf2
though an interaction between the C-terminal domain of
Hrd1 and the Neh4–5 domains of Nrf2. This Hrd1mediated ubiquitylation of Nrf2 is independent of both
Keap1 and b-TrCP.
Pharmacological inhibition of Hrd1 as a strategy
to prevent loss of the Nrf2-mediated protective
mechanism
Our results indicate that loss of the Nrf2-mediatedcellular protection through Hrd1-mediated ubiquitylation and subsequent proteasomal degradation of Nrf2
may be crucial in determining liver disease outcome.
This observation argues for a novel therapeutic strategy
for treating liver cirrhosis by targeting Hrd1 to preserve
the Nrf2 protective response rather than using currently
available Nrf2 inducers that only block Keap1-dependentubiquitylation of Nrf2. Therefore, we tested whether
Hrd1 is a potential therapeutic target for preventing/mitigating liver cirrhosis through enhancement of the
Nrf2-regulated protective mechanism. Two compounds,
4U8C (IRE1 inhibitor) (Cross et al. 2012; Qiu et al. 2013)
and LS-102 (Hrd1 inhibitor) (Yagishita et al. 2012), were
tested for their ability to alleviate liver cirrhosis in Nrf2+/+
and Nrf2/ mice. In Nrf2+/+ mice, CCl4 increased XBP1s
and Hrd1 protein levels while decreasing Nrf2, NQO1, andGCLM protein levels (Fig. 7A,B). 4U8C suppressed CCl4mediated up-regulation of XBP1s and Hrd1 and restored
the protein levels of Nrf2, NQO1, and GCLM (Fig. 7A,B).
LS-102 suppressed down-regulation of Nrf2 and its target
genes induced by CCl4 treatment while having no effecton the protein levels of XBP1s and Hrd1, consistent withits mode of action (Fig. 7A,B; Yagishita et al. 2012).
Although the expression pattern of XBP1s and Hrd1 inresponse to both inhibitors was similar in Nrf2+/+ andNrf2/ mice, the expression levels of NQO1 and GCLM
remained the same in the inhibitor-treated Nrf2/groups (Fig. 7A,B). Furthermore, neither CCl4 nor thetwo inhibitors affected Nrf2 or Keap1 mRNA levels (Fig.

Figure 7. Pharmacological inhibition of Hrd1 as a strategy to prevent loss of the Nrf2-mediated protective mechanism. (A–I) IHC
staining (A); the protein levels of the indicated proteins (B); the mRNA levels of the indicated genes (C); serum levels of alanine
aminotransferase (ALT), a key index for liver function (D); H&E staining (E); malonyl dialdehyde (MDA), an indicator for lipid
peroxidation (F); 8-hydroxydeoxyguanosine (8-OH-dG) for oxidative DNA damage (G); apoptotic cell death as indicated by TUNEL
staining (H); and collagen deposition measured using Masson’s trichrome staining (I) were conducted with liver tissues from Nrf2+/+ andNrf2/ mice treated with vehicle control, CCl4, CCl4 plus 4U8C, and CCl4 plus LS-102. Four mice were used in each group. Treatment
regimens are described in the Materials and Methods. (B) For immunoblot analysis, each lane contains a tissue lysate from an individual
mouse. For results expressed as bar graphs, means and SD were from four mice (n = 4).

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Hrd1 suppresses Nrf2 during liver cirrhosis

7C). 4U8C, but not LS-102, suppressed CCl4-induced upregulation of Hrd1 mRNA in both Nrf2+/+ and Nrf2/
mice (Fig. 7C). However, NQO1 mRNA was restored bydrugs only in Nrf2+/+ mice (Fig. 7C). Next, the effect of
4U8C and LS-102 in alleviating liver cirrhosis was tested.
Both 4U8C and LS-102 were able to suppress CCl4induced elevation of alanine aminotransferase (ALT) in
Nrf2+/+ but not in Nrf2/ mice (Fig. 7D). Hematoxylin
and eosin (H&E) staining showed that both LS-102 and
4U8C restored normal liver morphology in Nrf2+/+ but
not in Nrf2/ mice, although LS-102 had the larger effect(Fig. 7E). Moreover, lipid peroxidation and oxidative DNA
damage, measured as malonyl dialdehyde (MDA) and
8-hydroxydeoxyguanosine (8-OH-dG), respectively, werecorrected to a level almost similar to the untreated groupby LS-102 and 4U8C (Fig. 7F,G). In addition, apoptotic cell
death was significantly reduced in Nrf2+/+ mice but only
slightly in Nrf2/ mice (Fig. 7H), and both drugs reduced
collagen deposition in Nrf2+/+ but not in Nrf2/ mice, as
revealed by trichrome staining (Fig. 7I).
Discussion
It has been well documented that boosting the Nrf2mediated cellular protective response offers protection
against chronic diseases induced by accumulation ofoxidatively damaged biomolecules (Kensler et al. 2007;
Jaramillo and Zhang 2013). Therefore, identifying anddeveloping small-molecule Nrf2 activators for disease
prevention and intervention have been areas of intensive
research. Interestingly, the basal Nrf2 levels were foundto be low in aged organisms and certain pathologicalconditions (Sykiotis and Bohmann 2010; Kurzawski et al.
2012). The detailed mechanisms underlying age- anddisease-related down-regulation of Nrf2 are unclear, but
this knowledge is critical for developing a rational strategy to prevent loss of this major cellular protective response.
Keap1 has been regarded as the primary E3 ubiquitin ligase
controlling the protein level of Nrf2 and its downstream
response (Kobayashi et al. 2004; Zhang et al. 2004). Accordingly, all Nrf2 activators known to date function by targeting
Keap1 (Ma and He 2012; Magesh et al. 2012).
In this study, we discovered that the E3 ubiquitin ligasethat compromised the Nrf2 response in cirrhotic liver is
Hrd1 and not Keap1 or b-TrCP. Hrd1 has an essential
developmental role, and aberrant expression of Hrd1
has been associated with a number of chronic diseases(Amano et al. 2003; Yagishita et al. 2005; Hasegawa et al.
2010). For instance, it was shown to be up-regulated inrheumatoid synovial cells, and overexpression of Hrd1
resulted in spontaneous arthropathy, whereas Hrd1+/
mice were more resistant to collagen-induced arthritis(Amano et al. 2003). Recently, Hrd1 was also found to be
up-regulated in CCl4-induced fibrotic livers, and Hrd1+/
mice had fewer activated hepatic stellate cells, lesscollagen accumulation, and less hepatic injury comparedwith Hrd1+/+ mice (Hasegawa et al. 2010). The molecularmechanisms underlying the pathological roles of Hrd1 inthese diseases are presumably derived from its function as
an E3 ubiquitin ligase. Initially, it was thought that Hrd1

only regulated the turnover of proteins in the ERAD
system, since Hrd1 resides in the ER membrane. Morerecently, however, Hrd1 substrates that control specificsignaling pathways have been identified, including p53,
Nrf1, Rer1, and IRE1 (Yamasaki et al. 2007; Gao et al.
2008; Steffen et al. 2010; Tanabe et al. 2012). In this study,
we show that Nrf2 is a bona fide substrate of Hrd1. This
activity is mediated through the direct binding between
the Neh4–5 domains of Nrf2 and the cytosolic C-terminal
domain of Hrd1. The inverse correlation in the expressionof Nrf2 and Hrd1 was observed in both human and mousecirrhotic livers. Furthermore, silencing of Hrd1 in thelivers of conditional knockout mice markedly enhanced
the expression of Nrf2 and its target genes. All of theseobservations support the functional importance of Hrd1mediated control of Nrf2 in disease settings.
In support of our finding, negative regulation of ER
stress on Nrf2 was observed in an in vivo study comparing the gene expression profile in the small intestine andliver in response to tunicamycin treatment. Many phase
II detoxifying genes, including glutathione S-transferase(GST) isoforms and GCLM, were found to be downregulated in an Nrf2-dependent manner (Nair et al.
2007). Combined with the results from our study, it islikely that the observed down-regulation of the Nrf2mediated response in the early study was due to the upregulation of Hrd1 by tunicamycin. Inconsistent with ourdata, a recent study comparing gene expression profiles
from human liver tissues from normal, steatosis, alcohol
cirrhosis, and diabetic cirrhosis showed increased Nrf2
levels and increased downstream genes in alcoholic anddiabetic cirrhotic livers compared with normal nonsteatotic
livers (More et al. 2013). The alcoholic cirrhotic livertissues used in this study are from the same repository as
ours. Therefore, it is unclear why the opposite outcomes
regarding Nrf2 activation were obtained. However, theobserved changes in the expression of Nrf2 and NQO1 inboth studies are marginal, which may be due to the heterogeneity of human samples. Crucially, the experiments
carried out using a mouse model clearly show the negative
regulation between Hrd1 and Nrf2 or Nrf2 target genes(Fig. 1, cf. A–C and D–F or G–I). Furthermore, while our dataexplain pathological conditions when the overly activated
IRE1–XBP1 arm of ER stress suppressed the Nrf2 protective
mechanism, it is unclear what role, if any, the negative
regulation of Nrf2 by Hrd1 may have under physiologicalconditions. We believe that Cul3–Keap1–Rbx1 is the primary E3 ligase that targets Nrf2 for ubiquitylation anddegradation under physiological conditions. Therefore, it isreasonable to speculate from an evolutionary point of viewthat Hrd1-mediated regulation of Nrf2 is a way to get rid ofcells that have activated the IRE1–XBP1 ER stress pathway,
and cellular homeostasis can no longer be achieved.
As illustrated in Supplemental Table 1, Keap1–Cul3–
Rbx1 was the first Nrf2 E3 ubiquitin ligase identified(Kobayashi et al. 2004; Zhang et al. 2004). Structural
studies have demonstrated that a Keap1 dimer binds
Nrf2. Each Kelch domain in Keap1 interacts with eithera DLG or an ETGE motif in the Neh2 domain of Nrf2
(McMahon et al. 2006; Tong et al. 2006). The seven

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lysines between the DLG motif and the ETGE motif of
Nrf2 were required for receiving a polyubiquitin chain(Zhang et al. 2004). Presently, all small chemical Nrf2
activators or endogenous proteins identified disrupt the
DLG –Kelch interaction to suppress Nrf2 ubiquitylation,
resulting in stabilization of Nrf2 and activation of thepathway (Magesh et al. 2012; Jaramillo and Zhang 2013).
Recently, b-TrCP–Skp1–Cul1–Rbx1 was also identified as
an E3 ubiquitin ligase for Nrf2 (Rada et al. 2011, 2012;
Chowdhry et al. 2013). Two motifs, DSGIS and DSAPGS,in the Neh6 domains of Nrf2 were determined to interact
with b-TrCP. Phosphorylation of the serine residues in
DSGIS resulted in recruitment of Nrf2 by b-TrCP into theE3 ligase complex, with subsequent ubiquitylation of
Nrf2 (Rada et al. 2011, 2012; Chowdhry et al. 2013).
However, phosphorylation of DSAPGS is not required(Chowdhry et al. 2013). In the present study, we identified
Hrd1 as another E3 ubiquitin ligase that down-regulates
Nrf2 during the course of liver cirrhosis. Until now,
Keap1, b-TrCP, and Hrd1 are three E3 ubiquitin ligasesof Nrf2 that have been discovered (Supplemental Table 1).
It is also worth mentioning that only b-TrCP and Hrd1, but
not Keap1, are the E3 ubiquitin ligases for Nrf1, anothermember of the CNC family, even though Nrf1 also contains
Keap1-binding DLG and ETGE motifs (Supplemental Table
1; Zhang et al. 2006; Steffen et al. 2010; Tsuchiya et al.
2011). Understanding how Nrf2 is controlled in distinct
pathophysiological conditions and developing Nrf2 modulators to inactivate the appropriate E3 ligases are crucial fortargeted disease prevention and intervention. To illustrate,
we demonstrated the importance of targeting Hrd1, insteadof Keap1, to preserve the Nrf2 defense system, which
suppresses the progression of liver cirrhosis. In cirrhotic
livers, high levels of ROS inactivate Keap1, which should
lead to high Nrf2 levels. However, we observed low Nrf2
levels in cirrhotic liver tissues compared with normal livertissues. Hrd1 turned out to be the primary E3 ubiquitin
ligase controlling the Nrf2 protective response in cirrhotic
livers. We showed that inhibition of Hrd1 directly with LS102 or indirectly through IRE1 inhibition with 4U8C wasable to restore the Nrf2 response, reduce oxidative damage,and alleviate liver injury and cirrhosis.
In summary, we discovered cross-talk between two
major cellular stress response pathways: the IRE1–XBP1–
Hrd1 arm of the ER stress response pathway and thecytoprotective Nrf2-mediated oxidative stress response
pathway. These two pathways converge through Hrd1mediated Nrf2 ubiquitylation. We demonstrated that
Hrd1-mediated suppression of the Nrf2-dependent cytoprotective pathway plays a crucial role in the pathogenesis of liver cirrhosis. Finally, the therapeutic importanceof our findings was demonstrated by showing that pharmacological inhibition of Hrd1 alleviated liver injury andcirrhosis in an Nrf2-dependent manner.
Materials and methods
Liver samples and preparations
Normal human liver tissues and cirrhotic liver tissues from endstage alcoholic cirrhosis patients were obtained through the718

Liver Tissue Cell Distribution System, Minneapolis, Minnesota,
which was funded by National Institutes of Health contract
number HHSN276201200017C.
Generation of Hrd1f/f mice
The Hrd1 gene contains 16 exons; we floxed exons 8–11, which
encode a large region of the Hrd1 protein from its fifth transmembrane domain to the proline-rich sequence (Fig. 4G). To
exclude the potential effects of the neomycin selection cassetteon Hrd1 expression, this cassette was flanked by two flippase
recognition target (FRT) sites, which can be deleted by FLP
recombinase. This targeting vector was transfected into anembryonic stem cell line generated from C57/BL6 mice. The
neomycin selection marker was screened by PCR. Seven clones
were obtained and confirmed by Southern blotting. Blastocyst
injections resulted in several chimeric mice with the capacity for
germline transmission. Breeding of heterozygous mice yielded
Hrd1+/+, Hrd1+/f, and Hrd1f/f mice with the expected Mendelian
ratios. No obvious phenotypic abnormalities were observed inall genotypes.
Animal treatment
For the liver cirrhosis model, male Nrf2+/+ and Nrf2/ mice at
the age of 6 wk were IP-injected with 1 mL/kg 50% (v/v) CCl4 or
olive oil three times a week for 4 wk. For cotreatment groups,
animals received IP injection of 5 mg/kg 4U8C and 1.3 mg/kg LS102 daily. Animals were sacrificed 24 h after the last injection of
CCl4, and liver tissues and serum were collected for analysis.
H&E, IHC analysis, and Masson’s trichrome staining
H&E, IHC, and Masson’s trichrome staining analyses were
performed as described previously (Jiang et al. 2009; Zheng
et al. 2011a). Briefly, paraffin-embedded liver tissues were cut
into 4- to 5-mm sections and stained with H&E or the indicatedantibodies. The trichrome stain (Masson) kit was purchased from
Sigma, and the procedure was carried out according to the
manufacturer’s instructions.

Construction of recombinant DNA molecules
Plasmids expressing Flag-tagged XBP1s and XBP1-3K/3R weregenerous gifts from Dr. Laurie H. Glimcher (Harvard School of
Public Health). Flag-tagged wild-type and truncated forms of
Hrd1 were PCR-amplified using cDNAs reverse-transcribedfrom mRNAs of HEK293T cells and subcloned into the pCMVFlag-5a vector using EcoRI/BamHI restriction sites. The Hrd1C291S mutation was generated by site-directed mutagenesis
using the PCR and DpnI method. Hemagglutinin (HA)-tagged,
His-tagged, and GST fusion proteins of Nrf2-WT and Nrf2
domain deletion mutants have been previously described(Sun et al. 2009). To construct the fluorescent-tagged proteinsfor live-cell imaging, Hrd1 and Nrf2 were cloned into CFP and
RFP vectors, respectively. The following restriction enzyme
cutting sites were used to generate the fluorescently tagged
proteins: XhoI/BamHI (Nrf2-RFP) and Hind III/BamHI (Hrd1CFP). Plasmids expressing wild-type HA-Nrf2 proteins havebeen previously described (Zhang and Hannink 2003). Plasmids for the Nrf2 domain deletion mutants were originallygenerated by PCR and three-way ligation into the pCMV-HA
vector (Clontech) using SalI/KpnI and KpnI/NotI cutting sites.
The primers used for Nrf2 domain deletion mutants are listed
in Table 1.

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Table 1.

Primers used for Nrf2 domain deletion mutants

Wild type
DNeh2 (DAA1–86)

DNeh4 (DAA112–134)

DNeh5 (DAA182–200)

DNeh4–5 (DAA112–200)

DNeh6 (DAA336–386)

DNeh1 (DAA434–561)

DNeh3 (DAA561–604)

59-ACACACGGGTCGACGCTCATCATGATGGACTTGGAGCTGCCGCCG-39
59-GGTCAAATCCGCGGCCGCCTAGTTTTTCTTAACATCTGGCTT-39
59-ACACACGGGTCGACGCTCATCATGATGGACTTGGAGCTGCCGCCG-39
59-GGTGAATTTCTCGGTACCCAGCCAGCCCAG-39
59-GGTCAAATCCGCGGCCGCCTAGTTTTTCTTAACATCTGGCTT-39
59-ACACACGGGTCGACGCTCATCATGATGGACTTGGAGCTGCCGCCG-39
59-CATCAAAGTACAAGGTACCTGATTTGGGAAT-39
59-GTTTGTAGATGACGGTACCGTTTCTTCGGCT-39
59-GGTCAAATCCGCGGCCGCCTAGTTTTTCTTAACATCTGGCTT-39
59-ACACACGGGTCGACGCTCATCATGATGGACTTGGAGCTGCCGCCG-39
59-CAAACTTGCTCAATGGTACCTTGCATACCGTC-39
59-CCTGAGTTACAGGGTACCAATATTGAAAATG-39
59-GGTCAAATCCGCGGCCGCCTAGTTTTTCTTAACATCTGGCTT-39
59-ACACACGGGTCGACGCTCATCATGATGGACTTGGAGCTGCCGCCG-39
59-CATCAAAGTACAAGGTACCTGATTTGGGAAT-39
59-CCTGAGTTACAGGGTACCAATATTGAAAATG-39
59-GGTCAAATCCGCGGCCGCCTAGTTTTTCTTAACATCTGGCTT-39
59-ACACACGGGTCGACGCTCATCATGATGGACTTGGAGCTGCCGCCG-39
59-ATCATTGAATTCGGTACCGCTTTCAGGGTG-39
59-GATAGTGCCGGTACCAGTGTCAAACAG-39
59-GGTCAAATCCGCGGCCGCCTAGTTTTTCTTAACATCTGGCTT-39
59-ACACACGGGTCGACGCTCATCATGATGGACTTGGAGCTGCCGCCG-39
59-GGTTTTCCGATGGGTACCACTTACAGGCAA-39
59-CAACTCAGCACCGGTACCCTCGAAGTTTTC-39
59-GGTCAAATCCGCGGCCGCCTAGTTTTTCTTAACATCTGGCTT-39
59-ACACACGGGTCGACGCTCATCATGATGGACTTGGAGCTGCCGCCG-39
59-CATCACGTAGCATGGCGGCCGCTTAGAGATAAAGGTG-39
59-GGTCAAATCCGCGGCCGCCTAGTTTTTCTTAACATCTGGCTT-39

These plasmids were then used as templates for subcloning
the GST-Nrf2 by PCR and ligation into the pGEX-5X-3 vector
using BamHI and XhoI cutting sites.

instructions. In general, concentrations of 20 pmol and 40 pmol
or siRNA gave the best silencing results and thus were used inthis study.

Cell culture, transfection, and RNAi

Antibodies, immunoprecipitation, and immunoblot analysis

HEK293T and MDA-MB-231 cells were purchased from American Type Culture Collection (ATCC). XBP1+/+ and XBP1/ MEFcells were a generous gift from Dr. Laurie H. Glimcher. Hrd1+/+and Hrd1/ MEF cells were generated by our team (in thelaboratory of T.N.). HEK293T cells were maintained in minimum essential medium Eagle’s medium (MEM) (Cellgro) in thepresence of 10% fetal bovine serum (FBS), 1% L-glutamine (LifeTechnologies), 1.0 mM sodium pyruvate, 0.1 mM nonessential
amino acids (Hyclone), and 0.1% gentamycin (Life Technologies). MDA-MB-231 cells were maintained in MEM (Cellgro) in
the presence of 10% FBS, 1% L-glutamine (Life Technologies),
6 ng/mL insulin (Sigma), and 0.1 mM HEPES (Life Technologies).
All MEF cell lines were maintained in Dulbecco’s modified
Eagle’s medium (DMEM) in the presence of 10%–20% FBS and
0.1% gentamycin. All cells were incubated at 37°C in a humidified incubator containing 5% CO2. All cell culture dishes usedfor HEK293T cells were coated with 0.1 mg/mL poly-D-lysine(Sigma). Transfections of plasmid DNA were performed with
Lipofectamine Plus reagent (Invitrogen) according to the manufacturer’s instructions. siRNA against Hrd1 and scrambled
control siRNA were purchased from Thermo Scientifics. The
four Hrd1-siRNAs (used as a mixture) were 59-CAACAAGGCUGUGUACAUG, 59-UGUCUGGCCUUCACCGUUU-39, 59-GG
AGAUGCCUGAGGAUGGA-39, and 59-CCAAGAGACUGCC
CUGCAA-39 (Thermo Scientific catalog nos. siGENOME
SMARTpool siRNA D-007090-01, D-007090-02, D-007090-03,
and D-007090-04). Transfection of siRNA was performed with
Lipofectamine 2000 (Invitrogen) according to the manufacturer’s

Rabbit anti-Nrf2 (Santa Cruz Biotechnology), anti-Hrd1 (Santa
Cruz Biotechnology), anti-HO-1 (Santa Cruz Biotechnology);
mouse anti-tubulin (Santa Cruz Biotechnology), anti-GAPDH(glyceraldehyde-3-phosphate dehydrogenase; Santa Cruz Biotechnology), anti-NQO1 (Santa Cruz Biotechnology), anti-8oxo-dG (Trevigen), anti-His epitope (Santa Cruz Biotechnology),
anti-HA epitope (Covance), and mouse anti-Flag (Sigma) werepurchased from commercial sources. To detect protein expression in total cell lysates, cells were lysed in sample buffer (50mM Tris-HCl at pH 6.8, 2% sodium dodecyl sulfate [SDS], 10%
glycerol, 100 mM dithiothreitol [DTT], 0.1% bromophenol blue)
24–48 h after transfection.
For immunoprecipitation, cell lysates were collected at 48 h
post-transfection in radio immunoprecipitation assay (RIPA)
buffer containing 10 mM sodium phosphate (pH 8.0), 150 mM
NaCl, 1% Triton X-100, 1% sodium deoxycholate, and 0.1% SDS
in the presence of 1 mM DTT, 1 mM phenylmethylsulfonylfluoride (PMSF), and a protease inhibitor cocktail (PIC) (Sigma).
Cell lysates were precleared with protein A beads and thenincubated with either HA beads (Sigma) for ectopically expressed
proteins or 1 mg of antibodies against specific endogenous proteins with protein A-agarose beads on a rotator overnight at 4°C.
After three washes with RIPA buffer, immunoprecipitated complexes were eluted in sample buffer by boiling, electrophoresed
through SDS–polyacrylamide gels, and subjected to immunoblot
analysis.
For the in vitro pull-down assay, the purified His-tagged Nrf2
protein was incubated with the Flag-tagged wild-type or trun-

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Wu et al.

cated forms of Hrd1 and anti-Flag M2 beads in binding buffer
containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1mM
EDTA, and 1% Triton X-100 in the presence of 1 mM PMSF
and PIC (Sigma) on a rotator for 12 hat 4°C. After five washes
with washing buffer (50 mM Tris-HCl, 150 mM NaCl, 1 mM
PMSF, 0.1 mM PIC), complexes that were pulled down were
eluted in sample buffer by boiling, electrophoresed through SDS–
polyacrylamide gels, and subjected to immunoblot analysis.
Luciferase reporter gene assay
For the dual-luciferase reporter gene assay, HEK293T cells weretransfected with the NQO1 ARE-luciferase plasmid along with
the Renilla luciferase expression plasmid pGL4.74 (hRluc/TK)(Promega) and different amounts of expression vectors for FlagXBP1s. At 24 h post-transfection, cells were treated with a known
inducer of Nrf2, tBHQ (Sigma), for 16 h. The transfected cells
were then lysed with passive lysis buffer (Promega), and both
firefly and Renilla luciferase activities were measured with thedual-luciferase reporter assay system (Promega). Firefly luciferase activity was normalized to Renilla luciferase activity. The
experiment was repeated three times with triplicate samples,
and the data are expressed as mean 6 standard deviation (SD).
Intracellular glutathione level
The intracellular glutathione concentration was measured usinga QuantiChrom glutathione assay kit (BioAssay Systems) according to the manufacturer’s instructions (Chen et al. 2009). All of
the experiments were repeated three times with triplicate
samples. The results are presented as mean 6 SD.

For ubiquitylation of Nrf2 in vitro, HEK293T cells weretransfected with HA-Nrf2 and either Flag-Hrd1 or FlagHrd1(C291S). The transfected cells were lysed in lysis buffer(50 mM Tris-HCl at pH 7.4, 150 mM NaCl, 1 mM EDTA, 1%
Triton X-100) containing 1 mM DTT, 1 mM PMSF, and proteaseinhibitor cocktail. The lysates were precleared with protein A
beads prior to incubation with Flag M2 beads (Sigma) for 12 h.
Flag M2 beads were washed three times with TBS wash buffer (50mM Tris-HCl, 150 mM NaCl at pH 7.4) containing 1 mM DTT,
1 mM PMSF, and protease inhibitor cocktail. The pellets wereincubated with 300 pmol of ubiquitin, 2 pmol of E1, and 10 pmolof E2-UbcH5c in 13 reaction buffer (20 mM Tris-HCl at pH 7.4,
5 mM MgCl2, 2 mM ATP) in a total volume of 40 mL for 90 min at
37°C. Ubiquitin, E1, and E2-UbcH5c were purchased from
Boston Biochem. The Flag M2 was centrifuged at 3000g, andthe pellets were resuspended in 2% SDS, and 150 mM Tris-HCl(pH 8.0) and boiled for 5 min to release bound proteins anddisrupt protein–protein interactions. The supernatant was diluted with buffer lacking SDS prior to immunoprecipitation with
anti-Nrf2 antibodies. Immunoprecipitated proteins were subjected to immunoblot analysis with anti-ubiquitin antibodies.
Protein half-life measurement
To measure the half-life of Nrf2, 50 mM cycloheximide (Sigma)
was added to block protein synthesis. Total cell lysates werecollected at different time points and subjected to immunoblot
analysis with anti-Nrf2 and anti-tubulin antibodies. The intensity of the bands was quantified using the ChemiDoc CRS
gel documentation system and Quantity One software (Bio-Rad).
Relative intensity (Nrf2 vs. tubulin) was plotted using a semilogarithmic scale.

NQO1 activity assay
Cells were washed with PBS twice, harvested in 0.5 mL of
homogenization buffer (20 mM Tris-HCl, 2 mM EDTA at pH7.4), and subjected to three cycles of freezing in a 80°C freezer
and thawing. Cell debris was removed by centrifugation at
12,000g for 5 min at 4°C. The supernatants were transferred to
new microcentrifuge tubes for determination of protein concentration using the BCA protein assay kit (Thermo Scientific)
following the manufacturer’s instructions. NQO1 activity wasdetermined by the continuous spectrophotometric assay toquantitate the dicumarol-inhibitable reduction of its substrate,
DCPIP (Sigma). The rate of DCPIP reduction was monitored over
1.0 min at 600 nm with an extinction coefficient of 2.1
mM1cm1. The NQO1 activity was calculated as the decreasein absorbance per minute per microgram of total protein of thesample in the presence or absence of the NQO1 enzyme inhibitor dicumarol (Sigma).

Protein radiolabeling and in vitro binding assay
Hrd1-WT and its mutants were radiolabeled with [35S]methionine
using the in vitro TNT Quick PCR transcription/translation
system (Promega). GST-tagged Nrf2 and domain deletion proteins were expressed in Escherichia coli Rosetta (DE3) LysS cells
and purified with glutathione sepharose 4B matrix (Amersham
Biosciences). For the in vitro binding assay, radiolabeled proteinsand purified proteins were incubated in binding buffer (4.2 mM
Na2HPO4, 2 mM KHPO4, 140 mM NaCl, 10 mM KCl, 0.2%
bovine serum albumin [BSA], 0.02% Triton X-100, 1 mM DTT)in the presence of sepharose beads for 4–6 h at 4°C. The beads
were then washed six times with binding buffer. The proteins
were eluted by boiling in SDS sample buffer followed by SDSPAGE and autoradiography analysis.
Fluorescently tagged proteins and immunofluorescence

Ubiquitylation of Nrf2
To detect ubiquitylated Nrf2 in cultured cells, HEK293T cells
transfected with expression plasmids for HA-Ub and the indicated proteins for 48 h were treated for 4 h with 10 mM MG132(Sigma) to block protein degradation. Cells were then lysed ina buffer containing 2% SDS, 150 mM NaCl, 10 mM Tris-HCl,and 1 mM DTT. The cell lysates were boiled immediately for 10
min to inactivate cellular ubiquitin hydrolases to preserveubiquitin–protein conjugates. The heated lysates were thencooled and diluted five times with a Tris-buffered salt (TBS)solution without SDS and used for immunoprecipitation with anantibody against Nrf2 (C-20) or normal rabbit IgG. Immunoprecipitated proteins were subjected to immunoblot analysis withan antibody against HA.

720

Cells were grown on 35-mm glass-bottom dishes (In Vivo
Scientific) for live-cell imaging. Cells were transfected with theindicated fluorescent-labeled proteins. At 24 h post-transfection,
the medium was replaced with phenol red-free DMEM supplemented with 10% FBS. All images were taken with the Zeiss
Observer using the Slidebook 4.2.0.11 computer program (Intelligent Imaging Innovations, Inc.).

mRNA extraction and quantitative real-time PCR (qRT-PCR)
Total mRNA was extracted from cells using TRI reagent (Sigma).
Equal amounts of RNA were used for reverse transcription
using a Transcriptor first strand cDNA synthesis kit (Roche).
The following TaqMan probes from the universal probe library

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Hrd1 suppresses Nrf2 during liver cirrhosis(Roche) were used: human Nrf2 (no.70), human NQO1 (no. 87),
human HO-1 (no. 25), human Hrd1 (no. 25), human GAPDH (no.
25), mouse Nrf2 (no. 56), mouse Hrd1 (no. 2), mouse NQO1 (no.
50), mouse HO-1 (no. 25), and mouse b-actin (no. 56). The
following primers were synthesized by Integrated DNA Technologies: hNrf2, forward (59-ACACGGTCCACAGCTCATC-39)
and reverse (59-TGTCAATCAAATCCATGTCCTG-39); hNQO1,
forward (59-ATGTATGACAAAGGACCCTTCC-39) and reverse(59-TCCCTTGCAGAGAGTACATGG-39); hHO-1, forward (59AACTTTCAGAAGGGCCAGGT-39) and reverse (59-CTGGG
CTCTCCTTGTTGC-39); hHrd1, forward (59-CCAGTACCTC
ACCGTGCTG-39) and reverse (59-GCCTCTGAGCTAGGGA
TGC-39); hGAPDH, forward (59-CTGACTTCAACAGCGAC
ACC-39) and reverse (59-TGCTGTAGCCAAATTCGTTGT-39);
mNrf2, forward (59-TTTTCCATTCCCGAATTACAGT-39) andreverse (59-AGGAGATCGATGAGTAAAAATGGT-39); andmNQO1, forward (59-AGGGTTCGGTATTACGATCC-39) and reverse (59-AGTACAATCAGGGCTCTTCTCG-39). qRT-PCR was
performed on the LightCycler 480 system (Roche) as follows: one
cycle of initial denaturation (4 min at 95°C), 45 cycles ofamplification (10 sec at 95°C and 30 sec at 60°C), and a cooling
period. The data presented are relative mRNA levels normalized
to the level of GAPDH, and the value from the untreated cells
was set as 1. PCR assays were performed three times with
duplicate samples, which were used to determine the means 6
standard deviations. Student’s t-test was used to evaluate statistically significant differences.
XBP1 splicing assay
The XBP1 splicing assay was performed as previously described(Calfon et al. 2002). In brief, RNA from XBP1+/+ and XBP1/
MEF cells were reverse-transcribed, followed by PCR using thesense primer (59-AAACAGAGTAGCAGCGCAGACTGC-39)
and the antisense primer (59-TCCTTCTGGGTAGACCTCTGG
GAG-39). PCR products were resolved on 2.5% (w/v) agarose
gels, stained with ethidium bromide, and visualized.
Analysis of lipid peroxidation
Liver samples were analyzed in an indirect assessment for lipid
peroxidation using the thiobarbituric acid-reactive substances(TBARS) assay kit (Cayman Chemical). Liver samples were
homogenized in RIPA buffer (50 mM Tris-HCl at pH 7.6,
containing 150 mM sodium chloride, 1% NP-40, 0.5% sodium
deoxycholate, 0.1% SDS) with protease inhibitors. The assay wasthen carried out according to the manufacturer’s instructions
and analyzed by reading absorbance at 540 nm.
Terminal dUTP nick end-labeling (TUNEL) staining
Apoptotic cell death in liver tissues was detected using TUNEL,
an in situ cell death detection kit (Roche), according to the
manufacturer’s instruction. Images were taken using a fluorescence microscope (Zeiss Observer Z1, Marianas digital microscopy workstation).
Statistical analysis
Data are presented as means 6 SD. Differences were determinedby two-tailed Student’s t-test. A P-value <0.05 was consideredsignificant.

Acknowledgments
This study was funded by ES015010 and CA154377 (to D.D.Z.),
AI079056 (to D.F.), and ES006694 (a center grant).

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Hrd1 suppresses Nrf2-mediated cellular protection during livercirrhosis
Tongde Wu, Fei Zhao, Beixue Gao, et al.
Genes Dev. 2014, 28: originally published online March 17, 2014
Access the most recent version at doi:10.1101/gad.238246.114

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INTERNATIONAL JOURNAL OF MOLECULAR MEDICINE 35: 110-116, 2015

110

Synoviolin inhibitor LS-102 reduces endoplasmic reticulum
stress-induced collagen secretion in an in vitro
model of stress-related interstitial pneumonia
FUKAMI NAKAJIMA1,2, SATOKO ARATANI2,3, HIDETOSHI FUJITA2,3, NAOKO YAGISHITA4,SHIZUKO ICHINOSE5, KOSHI MAKITA1, YASUHIRO SETOGUCHI6 and TOSHIHIRO NAKAJIMA2,3,7
1

Department of Anesthesiology, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University,
Bunkyo-ku, Tokyo 113-8519; 2Department of Locomotor Science, Institute of Medical Science, and3
Future Medical Science, Institute of Medical Science, Tokyo Medical University, Shinjuku-ku, Tokyo 160-8402;
4
Institute of Medical Science, St. Marianna University School of Medicine, Kawasaki, Kanagawa 216-8512;
5
Research Center for Medical and Dental Science, Tokyo Medical and Dental University, Bunkyo-ku,
Tokyo 113-8510; 6Department of Respiratory Medicine, and 7Integrated Genome Editing Section (iGES),Tokyo Medical University, Shinjuku-ku, Tokyo 160-8402, Japan
Received July 4, 2014; Accepted October 10, 2014
DOI: 10.3892/ijmm.2014.1984

Abstract. The deletion mutation of exon 4 in surfactant
protein C (SP-C), a lung surfactant protein, has been identified inparent-child cases of familial interstitial pneumonia. It has beenshown that this mutation induces endoplasmic reticulum (ER)stress. Synoviolin is an E3 ubiquitin ligase that is localized to the
ER and is an important factor in the degradation of ER-related
proteins. It has been demonstrated that synoviolin is involved inliver fibrosis. In the present study, we investigated the involvement of synoviolin in the pathogenesis of interstitial pneumonia
caused by the exon 4 deletion in the SP-C gene. We transfectedwild-type and exon 4-deleted SP-C genes into A549 human lungadenocarcinoma cells and measured the secretion of collagen,which is a representative extracellular matrix protein involved
in fibrosis. Secreted collagen levels were increased in the culture
medium in SP-C mutants compared to the wild-type cells.
Furthermore, the transcription of mRNAs coding for factors
associated with fibrosis was increased. Subsequently, to assess
the involvement of synoviolin, we constructed plasmids witha luciferase gene under the control of the synoviolin promoter.
The A549 cells were transfected with the construct along withthe exon 4-deleted SP-C plasmid for use in the luciferase assay.
We found a 1.6-fold increase in luciferase activaty in the cells
carrying exon 4 deleted SP-C, as well as an increase in intrinsic
synoviolin expression at the mRNA and protein levels. Collagen

Correspondence to: Professor Toshihiro Nakajima, Department
of Locomotor Science, Tokyo Medical University, 6-1-1 Shinjuku,
Shinjuku-ku, Tokyo 160-8402, Japan
E-mail: marlin@tokyo-med.ac.jp
Key words: endoplasmic reticulum stress, fibrosis, E3 ubiquitin ligase

synoviolin

secretion was decreased by the addition of LS-102, a synoviolin
inhibitor, to the A549 culture medium following transfection withwild-type and exon 4-deleted SP-C. These results demonstratethat synoviolin is involved in the onset of interstitial pneumonia
induced by exon 4-deleted SP-C, which suggests that synoviolin
inhibitors may be used in the treatment of the disease.
Introduction
Idiopathic pulmonary fibrosis (IPF) is part of a large group of
diffuse parenchymal lung diseases, that are characterized byprogressive parenchymal fibrosis, without any known cause andpoor prognosis. Nogee et al conducted a genetic analysis of amother (onset age, 1 year) with desquamative interstitial pneumonia (DIP) and a child (onset age, 6 weeks) with non-specificinterstitial pneumonia (NSIP), and identified a heterozygous
mutation of the surfactant protein C (SP-C) gene (deletionof exon 4, SP-CΔexon4) for the first time (1). SP-C is a component of the pulmonary surfactant, which is a heterogeneouscomplex of lipids and proteins that lowers lung-surface tension
and contributes to the host defense system. The SP-C geneis located on the short arm of chromosome 8, and comprises
6 exons and 5 introns (2). The precursor of SP-C, proSP-C,
consists of 197 amino acids and is transformed into mature
SP-C (consisting of 24-58 amino acids) through intracellularcleavage and modification (3,4). ProSP-CΔexon4 has been foundto induce endoplasmic reticulum (ER) stress (5,6).
Fibrosis is defined by the accumulation of excess extracellular matrix (ECM) components, such as collagen, in and aroundinflamed or damaged tissue (7,8). Fibrosis can develop in the
majority of tissue types, and advanced fibrosis causes fibrotic
diseases, such as pulmonary fibrosis, liver cirrhosis and renal
fibrosis, resulting in organ dysfunction and eventually death.
According to recent studies, synoviolin/HRD1 is involved in liver
fibrosis (9) and collagen synthesis in the kidneys (10). Synoviolin,-352-

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which we have previously cloned (11), is an ER-resident E3 ubiquitin ligase with a RING finger domain that activates the
ER-associated degradation (ERAD) pathway (11). Synoviolin is
ubiquitously expressed in all organs of the body, particularly inthe liver, pancreas and skeletal muscle (12). Synoviolin is upregulated in response to ER stress and the overexpression of humaninositol-requiring kinase 1 (IRE1) and activating transcription
factor 6 (ATF6) (12,13). We have previously demonstrated
that heterozygous synoviolin knockout mice are resistant to
CCl4-induced liver fibrosis. In addition, we have demonstrated
that collagen secretion is reduced by synoviolin deficiency inmouse embryonic fibroblasts, suggesting the involvement ofsynoviolin in collagen secretion (9). Li et al (10) demonstrated
that synoviolin expression was increased in a mouse model ofrenal interstitial fibrosis caused by unilateral ureteral obstruction(UUO). In addition, they demonstrated that collagen secretionis reduced by synoviolin deletion in NRK-49F cells (normal
rat kidney interstitial fibroblasts) (10). These data suggest thatsynoviolin expression is increased by ER stress and that synoviolin promotes collagen synthesis. Thus, we hypothesized thatsynoviolin is involved in lung fibrosis, and in particular, in ER
stress-related lung fibrosis. In order to confirm our hypothesis, inthe present study, we investigated the involvement of synoviolin
and synoviolin inhibitor, LS-102, in collagen secretion in an
in vitro model of IPF by transfecting A549 cells (a human lungadenocarcinoma epithelial cell line) with SP-C lacking exon 4.
To investigate our hypothesis that synoviolin is involvedin lung fibrosis and that synoviolin inhibitors are candidate
antifibrotic agents, we investigated the association between
synoviolin and collagen secretion in an in vitro model, in which
A549 cells were transfected with SP-CΔexon4, which has beenreported to induce ER stress (5,6). In the present study, we did
not use any chemical or physical method to induce fibrosis;
instead, we focused on an SP-C mutation, SP-CΔexon4, which
has been identified in parent-child cases of familial interstitial
pneumonia (1).
Materials and methods
Cell culture. The A549 human lung adenocarcinoma cell line
was purchased from the American Type Culture Collection(ATCC, Manassas, VA, USA). The A549 cells were cultured
in minimum essential medium (Sigma-Aldrich, St. Louis, MO,
USA) supplemented with 1% penicillin, 1% streptomycin and
10% (heat-inactivated) fetal bovine serum (FBS).
Transfection of cells with plasmid constructs and siRNA. The
wild-type proSP-C expression vector was constructed using
pcDNA3-FLAG (Invitrogen, Carlsbad, CA, USA) by theinsertion of wild-type proSP-C cDNA that was isolated froma human lung cDNA library (Takara Bio Co., Tokyo, Japan).
SP-CΔexon4 (deletion of exon 4) was constructed according
to previously published techniques (14). All constructs wereconfirmed by DNA sequencing. The A549 cells were grownfor 24 h and transfected with wild-type SP-C or mutant SP-C
constructs using Lipofectamine 2000 (Invitrogen) as per the
manufacturer's instructions.
The sequences of the synoviolin siRNA and scrambled
synoviolin siRNA were identical to those described in aprevious study (15), and were chemically synthesized at111

Hokkaido System Science (Hokkaido, Japan). The A549 cells
were seeded at 40-50% confluency in 6-well plates and cultured
for 24 h. Subsequently, 50 nmol of annealed RNA duplex wastransfected using Lipofectamine 2000 according to the manufacturer's instructions.
Antibodies. The antibodies used in this study were as
follows: anti-synoviolin/HRD1 monoclonal antibody as
previously described (11), anti-FLAG antibody (anti-FLAG
M2 monoclonal antibody, F3165; Sigma-Aldrich), antihemagglutinin-A (HA) 3F10 monoclonal antibody (1867423;
Roche Applied Science) and anti-β-actin monoclonal antibody(A5441; Sigma-Aldrich).
Preparation of cellular protein. Cell cultures were harvested
and proteins were extracted using a cell extraction buffer
containing 0.05 M Tris-HCl (pH 8.0), 0.15 M NaCl, 5.0 mM
ethylenediaminetetraacetic acid (EDTA), 1% NP-40 andprotease inhibitors (1 µg/ml leupeptin, 1 µg/ml dithiothreitol (DTT), 1 µg/ml pepstatin and 1.5 µg/ml aprotinin) at 4˚C.
Reporter assay. The A549 cells were plated on 24-wellplates (5x10 4 cells/well). The synoviolin promoter(SyG-2055/+845 PGV-B2) was prepared as described in aprevious study (16). For transient transfection into the A549
cells, 20 ng/well synoviolin promoter and 50 ng/well test
plasmids (SP-C1-197 or SP-CΔexon4) were co-transfected using
Lipofectamine 2000 (Invitrogen) according to the manufacturer's recommendations. After 24 h, the cultures were aspirated
and the cells were added to 100 µl of passive lysis buffer(Promega, Madison, WI, USA). The cell debris was pelleted and5 µl of supernatant were collected and immediately analyzed for
luciferase activity using the Promega Luciferase assay system(Promega) and a microplate luminometer (Centro XS3 LB 960;
Berthold Technologies, Wildbad, Germany). The 5 µl supernatant samples were used to measure the protein concentrationin each well using a multi-spectrophotometer (Viento XS;
Dainippon Sumitomo Pharma, Tokyo, Japan). The assay was
performed in triplicate and the mean values were calculated foreach sample. The luciferase activity results were normalized tothe protein concentration in each sample.
Western blot analysis. The A549 cells were seeded at 40-50%
confluency in 100-mm plates and harvested 18 h following
transfection. Tunicamycin-treated (10 µg/ml) A549 cells
were prepared as a model of chemical induction of ER stress.
Cellular protein extracts were resolved by 10% SDS-PAGE,
transferred onto a nitrocellulose membrane and immunoblotted with anti-synoviolin/HRD1 monoclonal antibodies
followed by horseradish peroxidase-conjugated secondary
antibodies. The antigen-antibody complexes were visualizedusing an ECL detection system (Promega). β-actin expression
was used as an internal control. Immunoblots were scannedand band densities were quantified using ImageJ software.
Real-time polymerase chain reaction (PCR). The A549 cells
were grown in 100-mm culture plates. Forty-eight hours
following transfection, total RNA from the A549 cells was
isolated using Isogen II (Nippon Gene, Tokyo, Japan) andcDNA was synthesized using RiverTra Ace (Toyobo, Tokyo,

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Figure 1. Effect of surfactant protein C (SP-C) overexpression on collagen secretion in A549 cells. The mutant SP-C gene increased the mRNA expression levelsof (A) transforming growth factor-β (TGF-β), (B) connective tissue growth factor (CTGF) and (C) prolyl 4-hydroxylase subunit alpha 1 (P4HA1) by approximately
2.3-, 1.5- and 2.9-fold, respectively, compared to the wild-type. (D) A549 cells transfected with mutant SP-C secreted an approximately 1.3-fold greater amountof collagen into the medium than the cells transfected with wild-type SP-C. Data are the means ± standard error of the mean (SEM) of 3 experiments. **P<0.01.
WT, wild-type.

Japan) according to the manufacturer's instructions. Real-time
PCR relative quantification analysis was performed to measure
the mRNA expression levels of transforming growth factor-β(TGF- β), connective tissue growth factor (CTGF), prolyl
4-hydroxylase subunit alpha 1 (P4HA1), X-box binding
protein 1 (XBP1) and synoviolin using the FastStart Universal
Probe Master mix (ROX; Roche Applied Science, Indianapolis,
IN, USA), primers from the Universal Probe Library Assay
Design Center and probes from the universal probe library set(both from Roche Applied Science, Tokyo, Japan) with theStep One Plus Detection System and software (Applied
Biosystems, Life Technologies Japan, Tokyo, Japan). The
primers and probes used are as follows: TGF- β forward,
GCAGCACGTGGAGCTGTA and reverse, CAGCCGGTTGCTGAGGTA (probe: Universal probe library probe #72); CTGF
forward, AGCTGACCTGGAAGAGAACATT and reverse,
GCTCGGTATGTCTTCATGCTG (probe: Universal probelibrary probe #71); P4HA1 forward, TCGTCAAAGACCTAG
CAAAACC and reverse, CCGTCTCCAAGTCTCCTGTTA(probe: Universal probe library probe #30); XBP1 forward,
GGAGTTAAGACAGCGCTTGG and reverse, CACTGG
CCTCACTTCATTCC (probe: Universal probe libraryprobe #37); synoviolin forward, TTCGTCAGCCACGCCTAT
and reverse, GAGCACCATCGTCATCAGG (probe: Universal
probe library probe #89). The protocol included 1 cycle at
95˚C for 10 min, followed by 40 cycles at 95˚C for 1 sec and
60˚C for 20 sec, according to the manufacturer's
recommendations. The mRNA level was normalized relative
to the amount of the transcript coding for 18S rRNA.
Measurement of soluble secreted collagen. The A549 cells wereseeded at 40-50% confluency in 6-well plates. Twelve hours
following transfection, the medium was replaced with serumfree Opti-MEM (Gibco, Life Technologies Japan, Tokyo, Japan)
to eliminate the effects of serum on collagen measurement. At
30 h following transfection, the total soluble collagen in theculture supernatants was measured using the Sircol collagenassay method (Biocolor, County Antrim, UK) according to the
manufacturer's instructions. The assay was performed in triplicate and the mean values of each sample were calculated.

Immunoprecipitation. The cells were plated on 100-mm
tissue culture dishes and MG132 was added at a concentration of 20 µM 24 h following transfection. The cells wereco-transfected with hemagglutinin (HA)-tagged ubiquitin(Ub) and an SP-C wild-type, SP-C mutant, or empty vector.
Co-transfected with the synoviolin gene were also prepared.
Twenty-seven hours following transfection, the cells were
harvested and lysed with 200 µl of lysis buffer [50 mM
HEPES-KOH (pH 7.9), 150 mM KCl, 1% Triton X-100,
1 mM phenylmethylsulfonyl fluoride (PMSF), 10% glycerol,
2 µg/ml aprotinin, 2 µg/ml leupeptin, 2 µg/ml pepstatin A
and 20 µM MG132 (a proteasome inhibitor)]. Following
centrifugation at 5,000 rpm for 10 min, 150 µl of the clear
supernatant was incubated with 5 µl of normal mouse IgG(Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), 30 µlof protein G-Sepharose (GE Healthcare, Tokyo, Japan) and350 µl of lysis buffer at 4˚C with rotation for 30 min. The
supernatant was incubated with 2 µl of anti-FLAG M2 monoclonal antibody at 4˚C with rotation for 2 h, after which 40 µlof protein G were added, followed by incubation at 4˚C withrotation for 1 h. The immunoprecipitates were solubilized in
SDS sample buffer [0.078 M Tris-HCl (pH 6.8), 2% SDS, 10%
glycerol, 5% 2-mercaptoethanol and 0.003% bromophenol
blue] and then subjected to 4-15% gradient SDS-PAGE(Bio-Rad, Hercules, CA, USA).
Synoviolin inhibitor, LS-102. Synoviolin inhibitor, LS-102, wasidentified by screening approximately 4 million compounds
for potential synoviolin enzyme inhibitors as described in aprevious study (5). Dimethyl sulfoxide (DMSO) is used assolvent for LS-102; thus, we used DMSO as a control. Twelve
hours following transfection, Opti-MEM (no serum added)
was replaced, and LS-102 (5 µM) or DMSO (5 µM) was added
to the medium to evaluate the effect of LS-102.
Statistical analysis. All data are expressed as the means ± standard deviation (SD). Differences between groups were
examined using the Student's t-test and considered statisticallysignificant at P<0.05. All results were derived from at least
3 independent experiments.

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113

Figure 2. Effect of surfactant protein C (SP-C)Δexon4 overexpression on synoviolin expression in A549 cells. (A) Luciferase assay revealed that the A549 cells
expressing mutant SP-C showed a 1.6-fold higher luciferase activity than the wild-type SP-C-expressing cells. The mRNA expression levels of (B) X-box binding
protein 1 (XBP1) and (C) synoviolin were increased in the mutant SP-C-expressing A549 cells compared with the wild-type SP-C-expressing cells. (D) Transfectionof A549 cells with mutant SP-C increased the expression of synoviolin protein compared with the cells transfected with wild-type SP-C. Data are the means ± standard error of the mean (SEM) of 3 experiments. WT, wild-type; TM, tunicamycin [positive control of endoplasmic reticulum (ER) stress]. *P<0.05 and **P<0.01.

Results
Overexpression of SP-CΔexon4 promotes collagen secretion in
A549 cells. Studies have found an association between mutations in the SP-C gene and familial pulmonary fibrosis (1,17,18).
Therefore, in this study we focused on the SP-CΔexon4 mutation.
To assess the association between the SP-CΔexon4 mutation andfibrosis in the current experimental system, we measured themRNA expression levels of transforming growth factor- β(TGF- β), connective tissue growth factor (CTGF) andprolyl 4-hydroxylase subunit alpha 1 (P4HA1), which are responsible for enhancing the production of collagen protein, a typical
ECM component involved in fibrosis (19-21). Transfection of thecells with the mutant SP-C gene increased the mRNA expression
levels of TGF-β, CTGF and P4HA1 by approximately 2.3-, 1.5and 2.9-fold, respectively, compared to the wild-type (Fig. 1A-C).
Fibrosis is attributed to the excess deposition of ECM components in and around inflamed or damaged tissue (8). Collagen is amajor component of the ECM; therefore, we measured collagensecretion following transfection of the cells with mutant SP-C.

To confirm that mutant SP-C promoted collagen secretion frommutant SP-C-expressing A549 cells, collagen secretion wasmeasured following transfection with a wild-type or mutant SP-C
gene. The A549 cells transfected with the mutant SP-C genesecreted approximately 1.3-fold more collagen into the medium
than the cells transfected with wild-type SP-C (Fig. 1D).
SP-CΔexon4 increases the expression of synoviolin. To examine
the hypothesis that synoviolin plays a role in fibrosis induced
by SP-CΔexon4, we evaluated the level of synoviolin expression
in SP-CΔexon4 -expressing A549 cells. First, we constructed aplasmid carrying a luciferase gene with expression regulated
by a synoviolin promoter, which was co-transfected together
with the wild-type SP-C or mutant SP-C gene expression
plasmid into the A549 cells. Twenty-four hours following
transfection, a luciferase assay was conducted. The A549 cells
expressing mutant SP-C showed 1.6-fold greater luciferase
activity than the wild-type SP-C-expressing cells (Fig. 2A).
These data suggest that the transcriptional activity of synoviolin was enhanced in the SP-CΔexon4 gene-transfected cells.

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Figure 3. Effect of synoviolin siRNA on synoviolin secretion in A549 cells transfected with surfactant protein C (SP-C)Δexon4. (A) Collagen secretion was
decreased by synoviolin siRNA in A549 cells. (B) A549 cells were co-transfected with SP-CΔexon4 and synoviolin siRNA or scrambled siRNA. The amount ofsoluble secreted collagen in the supernatant of synoviolin siRNA-transfected SP-CΔexon4-expressing A549 cells was less than that produced by scrambled siRNAtransfected SP-CΔexon4-expressing A549 cells. Data are the means ± standard error of the mean (SEM) of 3 experiments. **P<0.01.

Subsequently, to confirm that ER stress is induced in thisexperimental system and that the mRNA expression of synoviolin
is activated in the mutant SP-C-transfected cells, we examinedthe expression levels of X-box binding protein 1 (XBP1) andsynoviolin in the A549 cells by real-time PCR. The mRNA
expression levels of XBP1 and synoviolin were increased in
the mutant SP-C-expressing A549 cells in comparison to thewild-type SP-C-expressing cells (Fig. 2B and C).
Finally, to assess the protein expression of synoviolin,
we performed western blot analysis with an anti-synoviolin
antibody. Transfection of the A549 cells with mutant SP-C
increased the protein expression of synoviolin compared withthe cells transfected with wild-type SP-C (Fig. 2D). Thus, theoverexpression of SP-CΔexon4 in the A549 cells increased thetranscriptional activity of synoviolin and the expression ofsynoviolin at the mRNA and protein levels.
Synoviolin knockdown decreases collagen secretion in
A549 cells transfected with SP-CΔexon4. To explore the role ofsynoviolin in collagen secretion in the A549 cells, we evaluated collagen production in A549 cells following transfection
with siRNA targeting synoviolin. We found that the level ofsoluble secreted collagen in the supernatant of A549 cells
after the knockdown of synoviolin was approximately 20%
less than that produced by the scrambled siRNA-transfected
A549 cells (Fig. 3A). Furthermore, we analyzed the effects
of synoviolin siRNA on collagen secretion in SP-C Δexon4 expressing A549 cells. The A549 cells were co-transfected with
SP-CΔexon4 and either synoviolin siRNA or scrambled siRNA.
The amount of soluble secreted collagen in the supernatantof the synoviolin siRNA-transfected SP-CΔexon4 -expressing
A549 cells was approximately 30% less than that producedby scrambled siRNA-transfected SP-CΔexon4-expressing A549
cells (Fig. 3B).

Synoviolin ubiquitinates proSP-CΔ exon4 as a substrate. To
demonstrate the ubiquitination of proSP-CΔexon4, the A549
cells were co-transfected with hemagglutinin (HA)-tagged
ubiquitin (Ub) and an SP-C wild-type, SP-C mutant, or empty
vector. In addition, to determine whether synoviolin ubiquitinates proSP-CΔexon4 as a substrate, cells co-transfected with thesynoviolin gene were also prepared. Following immunoprecipitation with an anti-FLAG antibody, western blot analysis was
performed using an anti-HA antibody. The cells transfectedwith SP-CΔexon4 showed a ladder pattern, suggesting ubiquitination. The synoviolin-containing cells showed a more intense
band (Fig. 4). These results suggested that the proSP-CΔexon4
protein was ubiquitinated in this experimental system and thatsynoviolin may participate in this ubiquitination.
Collagen secretion is suppressed by the synoviolin inhibitor,
LS-102. To investigate the effects of synoviolin inhibition oncollagen secretion in A549 cells, we performed experiments
using the synoviolin inhibitor, LS-102, as described in a previous
study of ours (22), in which we identified LS-102 by screening
approximately 4 million compounds for potential synoviolin
enzyme inhibitors. As a result, LS-101 and LS-102 were identified as compounds that inhibited the autoubiquitination activity
of synoviolin. These compounds were administered in a mousemodel of collagen-induced arthritis, in which they demonstrated
protective effects against arthritis. LS-101 had more potent
inhibitory effects on the synoviolin enzyme than LS-102.
However, LS-102 was used in this study as it is more selective
for synoviolin than LS-101. Twelve hours following transfection, Opti-MEM (no serum added) was replaced, and LS-102
or DMSO (5 µM; as a control) was added to the medium. The
tunicamycin (10 µg/ml)-treated A549 cells were also prepared
as a positive control for ER stress induction. LS-102 significantly
decreased collagen secretion into the medium (Fig. 5).

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115

Figure 4. Ubiquitination of precursor of surfactant protein C (proSP-C)Δexon4. A549 cells were co-transfected with hemagglutinin (HA)-tagged ubiquitin (Ub) and
SP-C wild-type, SP-C mutant, or an empty vector. In addition, samples were also prepared that were co-transfected with the synoviolin gene. After immunoprecipitation with an anti-FLAG antibody, western blot analysis was performed using an anti-HA antibody. The cells transfected with SP-CΔexon4 showed a ladder pattern,suggesting ubiquitination. The synoviolin-containing cells showed a more intense band. Data are the means ± standard error of the mean (SEM) of 3 experiments.
Syno, synoviolin.

Figure 5. Effect of the synoviolin inhibitor, LS-102, on collagen secretion in A549 cells. LS-102 significantly decreased collagen secretion into the medium. Data
are the means ± standard error of the mean (SEM) of 3 experiments. WT, wild-type; TM, Tunicamycin [positive control of endoplasmic reticulum (ER) stress].

Discussion
In the present study, we demonstrated that the expressionof synoviolin and collagen secretion were enhanced by theoverexpression of SP-CΔexon4 in A549 cells and that collagensecretion was suppressed by the use of the synoviolin inhibitor,
LS-102.

The pathogenic mechanisms of fibrosis have recently
attracted the attention of researches, and studies haveindicated that diverse factors, pathways and systems are
involved in the progression of fibrosis. However, the detailed
mechanisms involved are not yet fully understood. Wynn
et al (8), Tanjore et al (23,24) and Zhong et al (25) reported
that various pathways are involved in fibrosis, including the

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ER-stress pathway, and that ER stress leads to fibrosis. In thepresent study, we did not use any chemical or physical method
to induce fibrosis, but instead we focused on a mutation in
SP-C that was identified in mother-child cases of familial
interstitial pneumonia, and which produced ER stress in A549
cells. Considering our results in the context of the dual roleof synoviolin as an E3 ubiquitin ligase and collagen synthesis
promoter, this study suggests the existence of a novel pathway:
ER stress increases synoviolin synthesis, which promotes
collagen synthesis, contributing to fibrosis. This result confirm
the association between ER stress and fibrosis that has beenpreviously reported (23,25). Further studies are required to
fully determine the association between synoviolin and fibrosis.
Excessive fibrosis affects various organs and tissues,
including the endocardium, the heart, the liver, the kidneys,
the skin, the bone marrow, the retroperitoneum and the lungs.
Advanced fibrosis is a serious disorder that causes organ
dysfunction and death; accordingly, antifibrotic drugs are indevelopment. Various factors [TGF-β, CTGF, tumor necrosis
factor (TNF), endothelin, chemokines, chemokine receptors,
and matrix metalloproteinases (MMPs)] and effector cells are
involved in fibrosis (19,20,26-28). As a result, there are several
potential therapeutic vectors which may be used in the treatment of fibrosis, and thus a wide range of candidate therapeutic
agents is available (8).
In the present study, we demonstrate that synoviolin
expression and collagen secretion are enhanced by transfecting A549 cells with SP-CΔexon4 and that collagen secretionis suppressed by transfection with synoviolin siRNA or the
synoviolin inhibitor, LS-102. Based on these findings and therecent studies indicating that synoviolin is involved in liver
fibrosis (9) and renal fibrosis (10), our data suggest that thesynoviolin inhibitor, LS-102 represents a candidate antifibrotic
agent for the treatment of interstitial pneumonia; however,further studies, including in vivo studies, are required in order
to confirm this therapeutic potential.
Acknowledgements
We thank the staff at the Future Medical Science at the Instituteof Medical Science and the Integrated Gene Editing Sectionof Tokyo Medical University for their assistance. This study
was funded in part by grants from the Naito Foundation,
Natural Science Scholarship Daiichi-Sankyo Foundationof Life Science, Mitsubishi Tanabe Pharma Corporation,
Bureau of Social Welfare and Public Health, The Ministryof Health Labour and Welfare, The Ministry of Education,
Culture, Sports, Science and Technology, Japan Society for
the Promotion of Science, Health Labour Sciences Research
Grant, Takeda Science Foundation, AstraZeneca R&D Grant
2013, Grant-in-Aid for Exploratory Research, Grant-in-Aid forYoung Scientists (B).
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Goto et al. Orphanet Journal of Rare Diseases 2014, 9:58
http://www.ojrd.com/content/9/1/58

RESEARCH

Open Access

A nationwide survey on Marinesco-Sjögren
syndrome in Japan
Masahide Goto1,2, Mari Okada3, Hirofumi Komaki1, Kenji Sugai1, Masayuki Sasaki1, Satoru Noguchi3,4, Ikuya Nonaka3,
Ichizo Nishino3,4 and Yukiko K Hayashi3,4,5*Abstract
Background: Marinesco-Sjögren syndrome (MSS) is an autosomal recessive multisystem disorder characterized by
the tetralogy of cerebellar ataxia, congenital cataracts, intellectual disability, and progressive muscle weakness due
to myopathy. MSS is extremely rare, and its clinical, pathological, and genetic features are not yet fully understood.
Methods: We conducted a nationwide, questionnaire-based survey on MSS in Japan and carefully reviewed the
medical records of 36 patients suspected of having this disease. In addition, pathological examinations of muscles,sequence and haplotype analysis in SIL1 were performed.
Results: The patients had been examined between the ages of 2 and 52 years. Delayed psychomotor development
and cataracts from early childhood were observed in all patients, whereas no life-threatening events were observed.
Mutations in SIL1 were identified in 24 of the 27 patients tested, and 43 of the 48 chromosomes possessed the SIL1
c.936dupG (p.Leu313fs) mutation. The haplotype analysis revealed that 31 of the 32 chromosomes (96.9%) with thec.936dupG mutation had the same haplotype.
Conclusions: The results of haplotype analysis suggested the presence of a founder effect. The clinical features ofpatients without SIL1 mutations were indistinguishable from those with SIL1 mutations, suggesting the genetic
heterogeneity of MSS.
Keywords: Marinesco-Sjögren syndrome (MSS), SIL1, Founder effect, Cataracts, Intellectual disability, Ataxia, Rimmed
vacuolar myopathy

Background
Marinesco-Sjögren syndrome (MSS; OMIM 248800) is
an autosomal recessive multisystem disorder clinicallycharacterized by the tetralogy of cerebellar ataxia, congenital cataracts, intellectual disability, and progressive
muscle weakness due to myopathy [1-3]. Additional clinical features, including short stature, hypergonadotropic
hypogonadism [4], and strabismus [5], are also observed.
Mutations in SIL1 (Gene ID: 64374) were reported tobe causative for MSS [6,7]. This gene encodes SIL1, also
known as BiP-associated protein (BAP), which is an endoplasmic reticulum (ER)-resident protein. Bip is an HSP70
chaperone family member located in the ER, and plays a* Correspondence: yhayashi@tokyo-med.ac.jp
3
Department of Neuromuscular Research, National Institute of Neuroscience,
National Center of Neurology and Psychiatry, Tokyo, Japan
4
Department of Clinical Development, Translational Medical Center, National
Center of Neurology and Psychiatry, Tokyo, Japan
Full list of author information is available at the end of the article

key role in protein quality control. SIL1 regulates theATPase cycle of BiP for proper protein folding [8,9].
SIL1-deficient woozy mutant mice exhibit progressive
ataxia caused by the loss of Purkinje cells via ER stress [10].
MSS is an extremely rare disease, and very few cases
have been reported. In this study, we performed a nationwide, questionnaire-based survey on MSS with theaim of characterizing its prevalence, clinical features,
natural history, muscle pathological findings, and mutation status.

Methods
All clinical materials used in this study were obtainedfor diagnostic purposes with written informed consent.
All surveys and experiments performed in this study
were approved by the Ethical Committee of the National
Center of Neurology and Psychiatry.

© 2014 Goto et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative
Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, andreproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain
Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article,
unless otherwise stated.

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The nationwide, questionnaire-based survey

To elucidate the clinical characteristics of MSS, we
conducted a nationwide, questionnaire-based survey inJapan. The first set of questionnaires, which focused on
the experience of treating patients suspected of having
MSS, was sent to a total of 5,452 Japanese specialists forneurology or pediatric neurology. The second set of questionnaires, which focused on the clinical information ofpatients with suspected MSS, was sent to their attending
physicians. These patients’ medical records were carefully
reviewed by 2 of our specialists (M.G., H.K.).
Histochemistry

Biopsied muscle specimens were flash-frozen in isopentane
cooled in liquid nitrogen. Transverse serial frozen sectionsof 10 μm in thickness were subjected to various types ofhistochemical staining, including hematoxylin and eosin(H&E), modified Gomori-trichrome (mGT), and ATPases.
We obtained biopsied skeletal muscles from a total of 17
unrelated patients clinically suspected of having MSS.
Sequence analysis of SIL1

Genomic DNA was extracted from either frozen muscle
or peripheral blood lymphocytes using standard protocols. The PCR primers were designed to amplify all theexons of SIL1 together with their flanking intronic regions.
The primer sequences are available upon request. Direct
sequencing was performed using the BigDye Terminatorv3.1 Cycle Sequencing system and an ABI3100 automatedGenetic Analyzer (Applied Biosystems, Foster City, CA).
The sequence data obtained were analyzed using the
SeqScape (Applied Biosystems) program and comparedwith the genomic sequence of SIL1 in the database(NM_022464). Genetic analysis was performed on DNA
from 27 unrelated patients and the parents of 3 of them.
Two hundred control chromosomes from healthy individuals were examined for each novel mutation in SIL1
by direct sequencing.
Haplotype analysis of SIL1

For the haplotype analysis of the SIL1 genomic region, we
performed direct sequencing of the following 11 common
single nucleotide variants (SNVs) in the Japanese population: rs11748097, rs929775, rs10045761, rs1433008, rs1
1958050, rs7717375, rs7722413, rs3763016, rs6596456,
rs700629, and rs12653845 (http://www.ncbi.nlm.nih.gov/
SNP/). Samples from 21 patients homozygous for thecommon c.936dupG mutation, the parents of 3 patients,
and 92 control Japanese individuals were analyzed.

Results
Patients

A total of 1,875 responses (34.4% response rate) were received to the first set of questionnaires. The second setof questionnaires was sent to a total of 37 attending
physicians (2.0%) who had treated patients suspected ofhaving MSS. The detailed clinical records of a total of 36
patients were carefully reviewed. The shortfall in thenumber of patients was due to an overlap of 1 case.
Sequence analysis of SIL1

Frozen muscle or peripheral blood lymphocytes wereobtained from 27 of the 36 patients suspected of having
MSS, for genetic analysis. Mutations in SIL1 were identified in 24 out of the 27 patients. Twenty-one patientswere homozygous for the previously reported c.936dupG(p.Leu313fs) mutation in exon 9. Patient 4 was homozygous
for the previously reported c.603_607del5 (p.Glu201fs)
mutation in exon 6 [11]. Patient 12 was homozygous forthe previously reported c.331C > T (p.Arg111X) mutationin exon 4 [6,7]. Patient 17 was a compound heterozygote
for the novel c.617_618TC > AA (p.Leu206Glu) mutationin exon 6 and the c.936dupG (p.Leu313fs) mutation inexon 9 [12]. All mutations except for p.Leu206Glu are
predicted to induce premature termination. Mutation of
Leu206, which is highly conserved among species, waspredicted to exert a deleterious impact on protein function
by the Polymorphism Phenotyping v2 (PolyPhen-2; http://
genetics.bwh.harvard.edu/pph2/) and the Sorting Intolerant From Tolerant software (SIFT: http://sift.bii.a-star.edu.
sg/). None of these nucleic acid changes were found in the
200 chromosomes from healthy Japanese controls or in
the Japanese Single Nucleotide Polymorphisms database(http://snp.ims.u-tokyo.ac.jp/index_ja.html).
Haplotype analysis of SIL1

The SIL1 c.936dupG mutation was identified in 43 of
the 48 chromosomes (89.6%) in our cohort. The patients
carrying this mutation were reported from different
areas in Japan. To determine whether this was a resultof a founder effect, we performed a haplotype analysis
using 11 SNVs within or close to SIL1. The results
revealed that 31 of the 32 chromosomes (96.9%) with thec.936dupG mutation had the same haplotype (P1-P24,
Table 1). This haplotype was only found in 18 of the 184
chromosomes (9.8%) from the control group, suggesting afounder effect, although 1 chromosome from Patient 1 hada different haplotype.
Clinical features

Table 2 and Additional file 1: Table S1 show a clinicalsummary of the patients in our series.
The age at which the examination was performed in
the 24 patients (10 men and 14 women) with SIL1 mutations varied from 2 to 52 (mean = 20.1 ± 18.1) years.
Bilateral cataracts requiring prompt surgical intervention
had appeared and rapidly progressed in all 24 patients at
the mean age of 3.5 ± 1.2 years. Strabismus was also-366-

-367-

C/A

C

P26

P27

T

T/G

T/G

T

T

T

T

T

T

T

T

T

T

T

T

T

T

T

T

T

T/G

G: 0.18

rs929775

G

G/A

G/A

G

G

G

G

G

G

G

G

G

G

G

G

G

G

G

G

G

G

A: 0.15rs10045761

N

N

N

#

*

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

*/#

@: c.936dupG, *: c.331C > T, #: c.603_607del, JPT: Japanese frequency.
N: Normal, H: Homozygous.

C

C

P12

C/A

C

P24

P25

C

P4

C

C

P13

P21

C

P10

P20

C

P9

C

C

P7

C

C

P6

P18

C

P5

P16

C

P3

C

C

P2

C

C/A

P1

P15

A: 0.24

JPT

P14

rs11748097

rs No

Table 1 Haplotype analysis

T

T/C

T/C

T

T

T

T

T

T

T

T

T

T

T

T

T

T

T

T

T

T

C: 0.15

rs1433008

A

A/G

A/G

A

A

A

A

A

A

A

A

A

A

A

A

A

A

A

A

A

A/G

A: 0.76rs11958050

A

A/G

A/G

A

A

A

A

A

A

A

A

A

A

A

A

A

A

A

A

A

A

A: 0.82

rs7717375

N

N

N

N

N

H

H

H

H

H

H

H

H

H

H

H

H

H

H

H

H

@

T

T

T

T

T

T

T

T

T

T

T

T

T

T

T

T

T

T

T

T

T/C

C: 0.11

rs7722413

C

C

C

C

C

C

C

C

C

C

C

C

C

C

C

C

C

C

C

C

C/G

G: 0.11

rs3763016

G

G

G

G

A

A

A

A

A

A

A

A

A

A

A

A

A

A

A

A

A

A: 0.13rs6596456

C

C

C

C

A

A

A

A

A

A

A

A

A

A

A

A

A

A

A

A

A

A: 0.14rs700629

T

T/G

T/G

T

T

T

T

T

T

T

T

T

T

T

T

T

T

T

T

T

T

G: 0.11rs12653845

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OthersSkeletal abnormalities

Cerebellar signs

Motor functions

Ocular involvements

5 m-8 m0/3
0/3

7/22 (32%)
5/22 (23%)
28-2000
19/19 (100%)
16/16 (100%)

Flat footShort fingers
Serum CK (IU/L)
Cerebellar atrophy

Rimmed vacuoles in muscles

0/1

3/3 (100%)

144-3010

1/3 (33%)

8/22 (36%)

Spinal deformities

1/3 (33%)

12/18 (67%),

0/1

3/3 (100%)

2/3 (67%)

0/3

2/3 (100%)

3/3 (100%)

2/2 (100%) 15 m, 24 m

15 m, 24 m

12 m-18 m

Short stature

3/8 (38%)

8/24 (33%), 2y-48yDysarthria

Hypogonadism

11/24 (46%), 2y-45y
20/22 (91%), IQ(DQ):24-100

16/24 (67%), 2y-52yAtaxiaNystagmus

21/24 (88%)

Walk with support
5/16, 13y-28yStand with support

Hypotonia

16/20 (80%), 1y-4y
16/20 (80%), 2y-22y

Sit

Loss of ambulation

21/21 (100%), 4 m-18 m
20/20 (100%), 10 m-36 m

Head control

1/3 (33%)
3/3 (100%)

10/18 (56%)
21/22 (95%), 2y-52yStrabismus
Muscle weakness

Negative (n = 3)
3/3 (100%)

Positive (n = 24)
24/24 (100%), 2y-6yCataracts

Psychomotor delay

Development

SIL1 mutations

Table 2 Clinical summary of patients

2/6 (33%)

9/9(100%)

95-6002/8 (25%)

1/7 (14%)

3/7 (43%)

3/8 (38%)

2/3 (67%)

9/9 (100%)

4/9 (44%)

5/8 (63%)

6/8 (75%)

9/9 (100%)

3/3 (100%) 15 m-6y

15 m-6y

12 m-36 m

4 m-7 m9/9 (100%)

5/7 (71%)

9/9 (100%)

Not examined (n = 9)

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observed in 55.6% (10/18) of the patients. Cerebellar
signs included hypotonia (21/24; 88%), ataxia (16/24;67%), nystagmus (11/24; 46%), and dysarthria (8/24;
33%) were seen. Brain MRI demonstrated marked atrophy of the cerebellum, particularly the vermis, in all thepatients examined (19/19). Mild to moderate intellectual
disability, diagnosed by intelligence quotient/developmental quotient between 35 and 70, was seen in 91%(20/22) of the patients. Acquisition of meaningful words
occurred at the age of 2.0 ± 0.8 years, and most of thepatients had required special-needs education. Muscle
weakness was observed in 95% (21/22) of the patients,
with delays in motor milestones. Head control was firstseen in all the patients at a certain time point between 4
and 18 (mean = 7.8 ± 3.7) months, and sitting at a certain
time point between 10 and 36 (mean = 20.0 ± 8.7) months.
Eighty percent (16/20) of the patients could stand withsupport at a certain point between the ages of 1 and4 years, and walk with support at a certain point between
the ages of 2 and 22 (mean = 5.8 ± 2.6) years; however,none of the patients acquired the ability to walk independently. Muscle weakness was slowly progressive
and predominantly in the proximal muscles, with thepatients becoming wheelchair-bound at a certain time
point between the ages of 13 and 28 (mean = 17.4 ± 6.3)
years. Serum creatine kinase levels were normal tomoderately elevated (28–2000, mean = 389 ± 464; normal < 200 IU/L). Short stature (< −2 SD) was seen in
67% (12/18, mean = −3.6 SD) of the patients, and spinal
deformity (8/22; 36%), flat foot (7/22; 32%), and shortfingers (5/22; 23%) were also reported. Hypogonadotropic hypogonadism was seen in 3 of 8 (38%) patients(1 with microtestis, 2 with amenorrhea). No marked
clinical differences were observed among patients withdifferent SIL1 mutations. No patient had cardiac andrespiratory problems.
The 3 patients with no SIL1 mutation (Patients 25, 26,
and 27) and the 9 genetically unexamined patients showedclinical features indistinguishable from the patients with
SIL1 mutations, including cerebellar signs with cerebellar

atrophy on brain images, intellectual disability, congenital
cataracts, and muscle weakness. Elevation of serum CKlevels was also seen in 2 patients (Table 2).
Pathological findings of skeletal muscles

Biopsied skeletal muscles were obtained from 16 patientswith SIL1 mutations and one patient without (Patient 27).
All muscle specimens showed myopathic changes of variation in fiber size and endomysial fibrosis. A few necrotic
and regenerating fibers were seen in some patients with
SIL1 mutations. No neurogenic changes, including fiber
type grouping and grouped atrophy, were observed in anyof the patients. Importantly, scattered rimmed vacuoles(RVs) were seen in all 16 patients with SIL1 mutations, but
not in the patient without (Figure 1).

Discussion
We conducted a nationwide, questionnaire-based survey
to clarify the prevalence, clinical and pathological characteristics, and long-term course of MSS in Japanese patients.
The total number of patients with MSS was only 36.
From a clinical point of view, it is important to carryout careful ophthalmological examination of MSS patients at a young age if visual acuity is to be preserved,
as the cataracts characteristic of MSS usually appear
abruptly and develop rapidly from an early age [13,14].
Indeed, all of the patients in our present series required
early and prompt surgical intervention. Marked cerebellar
atrophy on brain MRI is another characteristic of this disease, however cerebellar ataxia can be difficult to identify,
especially in younger patients with muscle weakness. Skeletal muscle weakness is also a prominent characteristic.
Almost all the MSS patients with SIL1 mutations in thisseries had muscle weakness initially noticed as a delayed
motor milestone, which was detected at an earlier age
than cataracts, as reported previously [3,15,16]. Regarding
muscle biopsy, myopathic changes, including RV formation are a characteristic of patients with SIL1 mutations.
RVs are not disease-specific, and are often seen in adultonset chronic myopathies such as inclusion body myositis,

Figure 1 Modified gomori trichrome stain of the biopsied skeletal muscles. A muscle from a MSS patient with SIL1 mutation shows rimmed
vacuoles (arrow, A), whereas no vacuole is seen in a patient without SIL1 mutation (B). Bar = 20 μm.

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distal myopathy with RVs, oculopharyngeal muscular dystrophy, and myofibrillar myopathies. They are rarely observed, however, in childhood-onset myopathies. The
presence of RVs in muscle biopsy tissue can be helpful informulating an early diagnosis of MSS, allowing the ophthalmologist to perform surgery for cataracts to prevent
total visual loss. The life prognosis of MSS appears to becomparatively good, as respiratory, cardiac, and swallowing functions are well preserved, even in the patients who
are over 50 years of age.
Most of the reported SIL1 gene mutations have been
predicted to induce premature termination and loss offunction of SIL1. Based on a putative model of SIL1-BiP
interaction, the C-terminal 5 amino acids of SIL1 arethought to play a key role in its association with BiP [17].
This concept is further strengthened by the fact that thep.Arg111X, p.Glu201fs, and p.Leu313fs mutations cause
the generation of SIL1 proteins lacking the C-terminal
region. Complete loss of function due to nonsensemediated mRNA decay should also be considered. On
the other hand, Leu206 in exon 6 is well preserved
among species, and the novel nonsynonymous mutation p.Leu206Glu is predicted to exert a deleterious impact on protein function by both SIFT and PolyPhen2.
The c.936dupG (p.Leu313fs) mutation in SIL1, which
was first reported from Japan [12], is highly common in
Japanese MSS patients. Haplotype analysis revealed
that whereas 96.9% of chromosomes from MSS patients
possessing the c.936dupG mutation had the same
haplotype, less than 10% of the chromosomes of thecontrols did so, suggesting a founder effect.
The results of this study also strongly suggest the genetic
heterogeneity of MSS. Three of the 27 patients (11.1%) had
no SIL1 mutation, but demonstrated the cardinal featuresof MSS, including congenital cataracts, ataxia, intellectual
disability, and myopathy. We could not exclude the possibility of the mutation occurred in the promoter orother non-coding region of SIL1 in these 3 patients.
Previous reports also showed that approximately onehalf of the MSS patients were genetically diagnosed as
MSS from mutations in SIL1 [7,16]. The absence of RVs
in the muscle biopsy tissue of one patient with no SIL1
mutation suggests the existence of a different diseasemechanism(s) in such patients. Further analysis isrequired to identify the other causative genes for MSS.

Conclusions
MSS is an extremely rare disease, but a possible foundereffect was present in Japan. The life prognosis of MSS is
comparatively good, and early diagnosis is important for
prevention of a total visual loss. Other causative genes
for MSS can cause indistinguishable clinical features via
different disease mechanisms.

Additional file
Additional file 1: Table S1. Clinical findings of each patient with orwithout SIL1 mutations.
Competing interests
The authors declare that they have no competing of interest.
Authors’ contributions
MG had full access to all the data in the study and wrote the manuscript;
MO performed the mutation analysis; HK participated in analyzing all theclinical data; KS, MS, SN, I Nonaka, and I Nishino were involved in datainterpretation and also supervised manuscript preparation. YKH supervised all
aspects of the study, including study design, data interpretation, andmanuscript preparation. All authors read and approved the final manuscript.
Acknowledgements
The authors would like to thank Kanako Goto and Maki Takami for theirinvaluable technical support and assistance with the genetic analysis. The
authors also thank Jeremy Williams (MA, Professor), Dr. Helena Popiel (MS, PhD,
Lecturer), and Dr. Edward Barroga (DVM, PhD, Associate Professor) of the
Department of International Medical Communications of Tokyo Medical
University for editing and reviewing the manuscript.
Funding
This study was supported partly by grants for Research on Intractable
Diseases, Comprehensive Research on Disability Health and Welfare, andApplying Health Technology, from the Ministry of Health, Labour andWelfare, Japan; partly by Intramural Research Grants 23–4, 23–5, 22–5 for
Neurological and Psychiatric Disorders from the National Center of
Neurology and Psychiatry; and partly by JSPS KAKENHI Grant Numbers
23390236 and 24390227.
Author details
Department of Child Neurology, National Center Hospital, National Center of
Neurology and Psychiatry, Tokyo, Japan. 2Department of Pediatrics,
Hitachiomiya Saiseikai Hospital, Ibaraki, Japan. 3Department of Neuromuscular
Research, National Institute of Neuroscience, National Center of Neurology andPsychiatry, Tokyo, Japan. 4Department of Clinical Development, Translational
Medical Center, National Center of Neurology and Psychiatry, Tokyo, Japan.
5
Department of Neurophysiology, Tokyo Medical University, Tokyo, Japan.
1

Received: 12 December 2013 Accepted: 14 April 2014
Published: 23 April 2014
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Journal of the Neurological Sciences 343 (2014) 206–210

Contents lists available at ScienceDirect

Journal of the Neurological Sciences
journal homepage: www.elsevier.com/locate/jns

Short communication

A case of adult-onset reducing body myopathy presenting a novel clinicalfeature, asymmetrical involvement of the sternocleidomastoid andtrapezius muscles
Takayuki Fujii a, Shintaro Hayashi a, Nobutoshi Kawamura a, Masa-aki Higuchi b, Jun Tsugawa b,
Yasumasa Ohyagi a, Yukiko K. Hayashi c,d, Ichizo Nishino c, Jun-ichi Kira a,⁎a

Department of Neurology, Neurological Institute, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan
Department of Neurology, Fukuoka University, Fukuoka, Japan
Department of Neuromuscular Research, National Institute of Neuroscience, National Center of Neurology and Psychiatry, Tokyo, Japand
Department of Neurophysiology, Tokyo Medical University, Tokyo, Japanbca r t i c l e

i n f oArticle history:
Received 14 December 2013
Received in revised form 22 May 2014
Accepted 24 May 2014
Available online 2 June 2014
Keywords:
Reducing body myopathy
Adult onset
FHL1
Gene
Mutationa b s t r a c t
We herein report a 32-year-old woman with adult-onset reducing body myopathy (RBM) who had a mutation in
the four-and-a-half LIM domain 1 gene (FHL1) and showed a marked asymmetrical involvement ofsternocleidomastoid and trapezius muscles. At 30 years of age she noticed bilateral foot drop, and over the
next two years developed difﬁculty raising her right arm. At 32 years of age she was admitted to our hospitalfor a diagnostic evaluation. Neurological examination showed moderate weakness and atrophy of her rightsternocleidomastoid muscle, right trapezius muscle, and bilateral upper proximal muscles. There were severe
weakness and atrophy of her bilateral tibialis anterior muscles. Her deep tendon reﬂexes were hypoactive inher upper extremities. Her serum creatine kinase level was mildly increased. Muscle biopsy specimens fromthe left tibialis anterior muscle revealed marked variation in ﬁber size, some necrotic or regenerating ﬁbers,
and reducing bodies. Gene analysis of FHL1 demonstrated a mutation: a heterozygous missense mutation ofc.377G N A (p. C126T) in FHL1. Compared with previous adult-onset RBM cases harboring mutations in FHL1,
our case was characterized by asymmetrical atrophy of the sternocleidomastoid and trapezius muscles.
© 2014 Elsevier B.V. All rights reserved.

1. Introduction
Reducing body myopathy (RBM) is an extremely rare muscle disorder. It is characterized by the presence of intracytoplasmic inclusion
bodies that reduce nitroblue tetrazolium in the absence of substrate
α-glycerophosphate, in a menadione-linked α-glycerophosphate dehydrogenase (MAG) reaction [1]. RBM was ﬁrst described as a congenital
disorder in 1972 [1], and in 1999, the ﬁrst adult-onset RBM patient
was reported [2]. In 2008, Schessl et al. [3] identiﬁed the Xchromosomal four-and-a-half LIM domain 1 gene (FHL1) as a causative
gene for RBM. FHL1 encodes the FHL1 protein, composed of four LIM domains preceded by a single N-terminal zinc ﬁnger. FHL1 is highly
expressed in skeletal and cardiac muscles and considered to regulate
muscle mass and strength enhancement [4].
At present, detailed clinical features of adult-onset RBM remain to bedelineated owing to its extreme rarity. Here, we report a case of a 32year-old woman with adult-onset RBM harboring a mutation in the⁎ Corresponding author at: Department of Neurology, Neurological Institute, Graduate
School of Medical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka
812-8582, Japan. Tel.: +81 926425340; fax: +81 926425352.
E-mail address: kira@neuro.med.kyushu-u.ac.jp (J. Kira).

FHL1 gene, and describe its clinical presentation. Together with a literature survey on adult onset RBM cases, this case study could provide newclinical ﬁndings of adult-onset RBM, and might give some insight on aquestion about what deﬁnes the clinical course of RBM.

Patient
Fig. 1. A pedigree of the present case. An affected individual is indicated in black, and unaffected individuals in white. An arrow indicates the index patient.

http://dx.doi.org/10.1016/j.jns.2014.05.056
0022-510X/© 2014 Elsevier B.V. All rights reserved.

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T. Fujii et al. / Journal of the Neurological Sciences 343 (2014) 206–210

207

B

A

C

R
Fig. 2. Clinical picture. (A) The right sternocleidomastoid muscle shows severe atrophy (red arrowhead). (B) The left sternocleidomastoid muscle is intact (black arrowhead). (C) The righttrapezius muscle is mildly atrophic (red arrowhead) compared with the contralateral muscle (black arrowhead).

2. Case report
A 32-year-old woman had been in good health and could participate
regularly in sports activities until the age of 29. She had no family historyof neuromuscular disease (Fig. 1). At 30 years of age, she experienced bilateral foot drop, and over the next two years developed difﬁculty raising
her right arm. At the age of 32 years, she was admitted to our hospital fora diagnostic evaluation. A neurological examination showed moderate

A

R
B

R
Fig. 3. Magnetic resonance imaging of calf muscles (A and B). (A) T1-weighted axial image at calf level (TR, 730.34 ms; TE, 10 ms) shows high-intensity areas and atrophy in the bilateral
tibialis anterior muscles, predominantly on the right (arrows). (B) T1-weighted SPIR axial image at calf level (TR, 627.05 ms; TE, 10 ms) shows no high-intensity areas in the bilateral
tibialis anterior muscles (arrows).

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T. Fujii et al. / Journal of the Neurological Sciences 343 (2014) 206–210

Fig. 4. Muscle biopsy ﬁndings (specimen: left tibialis anterior muscle). (A) Hematoxylin and eosin staining reveals marked variation in ﬁber size, increased central nuclei, and some necrotic ﬁbers. Eosinophilic inclusions are present (arrows). (B) On modiﬁed Gomori trichrome staining, cytoplasmic bodies are scattered (arrow). Some rimmed vacuoles are seen (arrowhead). (C) On ATPase (pH10.5) staining, type 2C ﬁbers are observed (arrows). (D) On menadione-linked α-glycerophosphate dehydrogenase (MAG) staining without substrate, reducing
bodies are seen (arrows). Scale bars: A and D, 100 μm; B, 50 μm; C, 200 μm.

weakness and atrophy of her right sternocleidomastoid muscle, right trapezius muscle, and proximal muscles of bilateral upper limbs (Fig. 2) andsevere weakness and atrophy of her bilateral tibialis anterior muscles(Medical Research Council (MRC) score (right/left): neck ﬂexors 4;
sternocleidomastoid muscle 4/5; trapezius muscle 4/5; deltoid muscle
2/3; biceps brachii muscle 4/4; triceps brachii muscle 4/4; foot extensor;
1/2; toe extensor; 3/3). Her higher brain function was normal, and thefacial and extraocular muscles were intact. Tendon reﬂexes were
hypoactive in both of her upper extremities, and pathological reﬂexes

Z

104

were negative throughout. Cerebellar ataxia, sensory, and autonomic
disturbances were absent. Examination of cardiorespiratory functions
by 24-hour electrocardiography, transthoracic echocardiography and a
spirometer disclosed no abnormality. Her serum creatine kinase level
was high (501 IU/l; normal range 45–163 IU/l). Anti-nuclear and antiJo-1 antibodies in the serum were negative. On needle electromyography, short duration and low amplitude motor unit potentials were observed in the right sternocleidomastoid and trapezius muscles,
bilateral deltoid muscles, and bilateral tibialis anterior muscles.

LIM2

LIM1

LIM4

LIM3

G D Q N V

E

G S

A

Y

T

V

K

G

I
A

G
T

I
V

K
F

V
W 123

Q
K

Y

C

H

C

C

G
101 K
C

Zn

G
K 126
C

F

132

N
129 S
C
T

F F

P

K
G
E
D
F

Zn

150

V
T 153
C

Fig. 5. A schematic drawing of four-and-a-half LIM domain 1 (FHL1) protein consisting of four LIM domains (LIM 1-4) with an additional N-terminal half-LIM domain (Z), and a schematic
illustration of the second LIM domain of FHL1 (LIM 2 domain). An affected residue of the present case (missense mutation of C126T) is indicated in bold black. C, cysteine; K, lysine;G, glycine; F, phenylalanine; A, alanine; I, isoleucine; V, valine; D, aspartic acid; Q, glutamine; N, asparagine; E, glutamic acid; Y, tyrosine; T, threonine; W, tryptophan; H, histidine; S, serine;P, proline.

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T. Fujii et al. / Journal of the Neurological Sciences 343 (2014) 206–210

C

C

Pt

C

0.40

Magnetic resonance imaging (MRI) at calf level showed atrophy andfatty degeneration in the bilateral tibialis anterior muscles, predominantly on the right (Fig. 3). Muscle biopsy specimens from the left
tibialis anterior muscle revealed marked variation in ﬁber size, some necrotic or regenerating ﬁbers, and eosinophilic inclusions on hematoxylin and eosin staining (Fig. 4A). On modiﬁed Gomori trichrome
staining, cytoplasmic bodies were scattered. Some rimmed vacuoles
were also seen (Fig. 4B). On ATPase (pH10.5) staining, type 2C ﬁbers
were present (Fig. 4C). Reducing bodies were found on menadionelinked α-glycerophosphate dehydrogenase (MAG) staining without
substrate (Fig. 4D). After obtaining the patient's informed consent, a peripheral blood sample was submitted for genetic analysis using standardized techniques. The gene analysis of FHL1 demonstrated aheterozygous missense mutation of c. 377G N A (p. C126T) in the
FHL1 gene. The mutation is located within the second LIM domain andaffects a zinc-binding cysteine residue, consistent with the previous
RBM cases (Fig. 5) [5]. Endogenous FHL1 protein expression was analyzed by western blot [6] which revealed, after normalization to actin,
no change in the levels of FHL1 protein (Fig. 6).

0.20

3. Discussion

FHL1

Actin

Amount of FHL1 protein(relative to actin)

1.00
0.80
0.60

There are only ten adult-onset RBM cases diagnosed by muscle biopsy and gene analysis in the previous literature (Table 1) [5–10]. Although there are some adult-onset male patients in other FHL1associated myopathies such as Emery-Dreifuss muscular dystrophy[10] and X-linked myopathy with postural muscle atrophy [11], alladult-onset RBM cases were females. The onset ages were mostly 20s
to 30s. The lower legs were involved most frequently at onset, andthen muscle symptoms gradually spread in an asymmetrical fashion[12]. To the best of our knowledge, our case is the ﬁrst to show asymmetrical involvement of the sternocleidomastoid and trapezius muscles
among previously reported RBM patients.

0.00
C

C

Pt

C

Fig. 6. Amounts of four-and-a-half LIM domain 1 (FHL1) protein in muscle specimens detected by a western blot analysis. Amounts of FHL1 protein are unchanged in patient’s
muscles (Pt) as compared with three normal controls (C) after normalization to actin
amounts. A rabbit polyclonal anti- FHL1 antibodies (AVIVA Systems Biology, San Diego,CA, x1000) and a mouse monoclonal anti-actin antibody (Nichirei, Tokyo, Japan, ×2000)
are used. FHL1/Actin ration; C: 0.69, C: 0.78, Pt: 0.85, C: 1.0.

Table 1
Reported cases of adult-onset reducing body myopathy.
AuthorSex Onset Familyagehistory

Initial symptom

Muscle involvement

Prognosis (disease duration)

FHL1 mutation

Case 1
Schessl et al. [5]

F

30

Son

Difﬁculties climbing up or
down stairs

Wheelchair required for longer
distances (several years)

Case 2
Shalaby et al. [6]

F

32

None

Right foot drop

Case 3
Shalaby et al. [6]

F

29

Son

Left foot drop

Predominantly proximal muscles,
asymmetrical trapezius andshoulder muscles
Tibialis anterior, deltoid, biceps,
and iliopsoas muscles,
predominantly on the right
Bil. lower limbs, left upper limbs

Case 4
Schessl et al. [7]Case 5
Selcen et al. [8]F

50Grand-son

Weakness of the legs

Bil. legs and hands

F

30

None

Right biceps atrophy

Case 6
Schreckenbach et al.
[9]

F

32

Mother,brothers

Weakness of the proximal
upper limbs

Case 7
Malfatti et al. [10]Case 8
Malfatti et al. [10]Case 9Figarella- Branger andMalfatti et al. [2,10]Case 10
Malfatti et al. [10]Present case

F

21

None

F

20

None

F

24

First- degree
cousinWeakness of lt. arm andlimb
Walking difﬁculties,
waddling gaitLt. scapuloperoneal
weakness

Right biceps, gastrocnemius,
extensor pollicis, and bothbrachio-radialis muscles
Supraspinatus muscles, biceps
Wheelchair-bound (13 years)
brachii muscles, small hand muscles,
predominantly on the right, neck
ﬂexor muscle, bil. lower limbs
Asymmetric weakness of four limbs Dysphagia (5 years)
with facial involvement
Severe lower limb girdle weakness Details unknownc. 458G N A
p. C153Y
heterozygousc. 304-312 del.
AAGGGGTGC
p. 102-104 del. KFC
c. 310 T N C
p. C104R
heterozygous
p. C150R
heterozygous
c.448 T N C p. C150R

F

29

Son

F

30

None

Myalgias, lt. hand and rt.
arm weakness
Bil. foot drop

Four limb weakness

Difﬁculty running and atendency to fall (10 years)
Wheelchair-bound (5 years)

Ambulatory for short distances(28 years)
Grade 3-4 on the MRC score(5 years)

Details unknown The clinicalcourse is slowly progressive.

Asymmetric proximo-distal
Details unknown
weakness
Bil. tibialis anterior muscles, rt.
A tendency to fall (4 years)
sternocleidomastoid muscle, rt.
trapezius muscle, upper proximal
muscle, predominantly on the rightc. 449G N C
p. C150S
heterozygousc. 458G N C
p. C153S
c. 448 T N C
p. C150A
c. 377G N A
p. C126T
c. 377G N A
p. C126T
c. 377G N A
p. C126T
heterozygous

Bil., bilateral; Rt., right; Lt., left; FHL1, four-and-a-half LIM domain 1; c., coding DNA (A, adenine; G, guanine; T, thymine; C, cytosine); p., protein (K, lysine; F, phenylalanine; C, cysteine;R, arginine; Y, tyrosine; T, threonine); del., deletion.

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The present case showed a heterozygous missense mutation ofc. 377G N A (p. C126T) in the FHL1 gene, which is the same mutation reported by Malfatti et al. in three RBM cases (one childhoood onset andtwo adult onset) (Table 1) [10]. However, a detailed case description
was not given in this study, which focused on muscle biopsy [10]. Allof the mutations detected in the previously reported RBM patientswere found in the second LIM domain of the FHL1 (LIM 2 domain;
Fig. 5) gene [5]. Mutations at histidine (H) 123 and cysteine (C) 132
seem to be associated with clinically severe courses, showing earlyonset, rapidly progressive muscle weakness resulting in loss of ability
to ambulate, and respiratory failure within several years after onset [5,12]. By contrast, mutations at the cysteine residues more distal to the
LIM 2 domain central core (C101, C104, C150, and C153) might beassociated with milder clinical courses, presenting with adult-onset
and slowly progressive muscle weakness [5,12]. Based on the ﬁndingsof these literature cases, our patient, harboring a mutation at C 126
near the central core, should have shown a severe clinical course. However, her muscle weakness slowly worsened and she remained ambulatory 4 years after onset. Similarly, the Case 9 reported by FigarellaBranger and Malfatti et al. was also slowly progressive [2,10]. Thissuggests that the clinical course of RBM patients is not deﬁned solely
by localization of the mutation and but may involve other factors. One
such candidate could be X-inactivation. Although an X-inactivation
study was not performed in the present case, it could be associatedwith the clinical course of RBM patients in the same way as Duchenne
muscular dystrophy carriers, that is, extreme bias of X-inactivation results in decreased muscular volume and strength, and causes early clinical symptoms [13].
Diagnosis of RBM by clinical presentation might be difﬁcult, because
no clinical proﬁles speciﬁc to RBM have been established [14]. Information of the present report is somewhat limited because of the inclusionof only a single patient. However, a new clinical feature of adult onset
RBM presented by this case, showing asymmetrical atrophy of thesternocleidomastoid and trapezius muscles, might further support a notion that an asymmetrical involvement of muscles is one of the clues tosuspect adult-onset RBM.

Conﬂict of interest statement
We declare that there are no conﬂicts of interest associated with thismanuscript, and there have been no ﬁnancial supports for this work that
could inﬂuence its outcome.
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[2] Figarella-Branger D, Putzu GA, Bouvier-Labit C, Pouget J, Chateau D, Fardeau M, et al.
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1உ30ଐ உ୴ଐ 17Ჴ00᳸19Ჴ30‫ܖٻ‬၏ᨈ ᐯɼᐯ‫ܖ‬᫾ 3F‫˟ٻ‬ᜭܴ
* ƲƳƨưǋƝᐯဌƴƝӋь᪬ƚǇƢǇƨŴኳʕࢸ၏ᨈǫȕǧȆȩǹƴƯটᚃ˟Ǜ͵ƠǇƢƷư଱᩼ƝӋьƘƩƞƍ-377-

૨ᢿᅹ‫ܖ‬Ⴞ ᅶᇌ‫ܖٻ‬৆ဦႎᄂᆮؕႴ࢟঺ૅੲʙಅ

ೞᏡࣱᄬࣱȊȎȓᲧǺ২ᘐǛؕႴƱƢǔᩊ඙ࣱ၌धƷǿȳȑǯឋЎᚐСࣂೞನƷᚐଢƱૼᙹ඙ၲඥƷ᧏ႆ

最終報告会
学長挨拶
 事業総括プロジェクトリーダー

第一部 17:00～

宮澤啓介 （生化学分野 主任教授）

 マクロライドのオートファジー阻害活性における標的分子の探索
高野直治 （生化学分野 講師） チロシンキナーゼ阻害薬の分子標的によるアクチン細胞骨格の再構成
平本正樹 （生化学分野 准教授）
 マリネスコ/シェーグレン症候群のゼブラフィッシュモデルの解析
川原玄理 （病態生理学分野 准教授）
 HSPB8ミオパチーにおけるタンパク質凝集体の解析
川幡由希香 （病態生理学分野 助教）

休憩

5分

第二部 18:10～

 慢性炎症を司るシノビオリンによる分子・細胞内小器官・臓器・個体レベルでのエネルギー調節機構の解明 -基礎から実用化まで中島利博 （医学総合研究所 運動器科学研究部門 教授）

 炎症誘導時の新しい蛋白質発現増強機構
善本隆之 （医学総合研究所 免疫制御研究部門 教授）
 セレブロン(CRBN) E3ユビキチンリガーゼの基質・制御機構の解明
半田宏 （ナノ粒子先端医学応用講座 特任教授）

外部評価委員の先生方の総評

3月22日 金曜日
17：00～19：30
大学病院 自主自学館3階 会議室A・B※終了後カフェテリアにて懇親会がございます-378-