ISLAM, ZOHIRUL

称号及び氏名博士（応用生命科学）

学位授与の日付

平成３１年２月２８日

論Expression and functional role of the orphan G protein-coupled文

名receptor Gpr137b in osteoclast differentiation and macrophage
polarization（破骨細胞分化およびマクロファージ極性化におけるリガンド未同定Ｇ蛋白質共役型受容体 Gpr137b の発現と機能）
論文審査委員

主査

乾隆

副査

山地亮一

副査

小森雅之

副査

石橋宰

論文要旨

General Introduction
G-protein-coupled receptors (GPCRs) compose the largest receptor gene family with morethan 800 members in the human genome and play pivotal roles in a variety of physiological andpathological processes. Therefore, this large family of transmembrane receptors has attracted greatinterest as possible therapeutic targets. However, endogenous ligands of more than 100 GPCRs,
which are called “orphan GPCRs”, remain unknown. GPCRs can be broadly classified into twogroups, i.e., odorant/sensory and non-odorant GPCRs. Odorant/sensory GPCRs are confined toparticular cell types and conduct organismal behaviors such as feeding and mating. Conversely,
non-odorant GPCRs comprise about 1% genes in the genome and widely expressed in the body but
usually at low levels. Despite the low abundance, GPCRs are highly enriched or tightly restricted
to a particular cell types, however, have an enormous potential to shed light on the unique traits ofthese cells.
Moreover, non-odorant GPCRs act in response to diverse endogenous ligands and areinvolvedinvariousphysiologicalprocesses

inthehost,including

hematopoiesis,neurotransmission, immune function, etc. Gpr137b is a non-odorant orphan GPCR conserved
among mammals. Gpr137b was first identified in the kidneys, and later shown to be expressed inother tissues and cell types. However, the biological function of Gpr137b is yet to be determined.
Osteoclasts are large multinucleated cells involved in bone resorption and originated fromhematopoietic progenitors. Monocyte in peripheral blood and various types of tissue macrophages
are also developed from the same progenitor cells. Osteoclasts are formed by the fusion ofcommitted monocytic cells and critical for healthy skeletal development, bone remodeling, andcalcium metabolism. In this study, I identified Gpr137b as a gene encoding an orphan GPCRpredominantly expressed in mature osteoclasts by genome-wide transcriptome analysis(RNA-seq).
Chapter 1: Gpr137b-gene knockout by the CRISPR/Cas9 genome editing techniqueSince Gpr137b was demonstrated to be highly expressed in RAW264 cells and markedly
increased during osteoclastic differentiation, Thus, I predict its important role in osteoclastic
differentiation and I generated two Gpr137b-KO RAW264 cells using the CRISPR/Cas9 genome
editing system. PCR to amplify the genomic region near the gRNA-recognizing sequence yielded
products of the same size from both clones, which were demonstrated to have 188-nucleotide
frameshift deletions at a region containing the translation start codon (ATG). I also isolated two
independent cell clones from RAW264 cells transfected with pSpCas9 (BB)-2A-GFP as thecontrols. I verified that no mutation was introduced in a genomic region near the PAM sequence ofGpr137b in these clones, thus designated them Gpr137b-wildtype (WT) clones. Genomic PCR
using primers designed to recognize the deleted sequence yielded a product of the expected sizefrom the Gpr137b-WT clones and no product from the Gpr137b-KO clones. Since a pseudogenethat is 97% homologues to the Gpr137b gene, Gpr137b-ps, exists in mouse, I designed a set ofspecific primers to amplify Gpr137b and Gpr137b-ps. The corresponding region of theGpr137b-ps gene in these clones was shown to be intact. Next, I demonstrated that the deleted
region of the Gpr137b gene was not transcribed in the Gpr137b-KO clones by qRT-PCR using
primers that recognize this region but not the corresponding region of the Gpr137b-ps gene. By
contrast, I confirmed that the corresponding region of the Gpr137b-ps gene is transcribed in theGpr137b-KO clones using primers designed to specifically recognize this region in Gpr137b-ps.
Further, I verified the lack of GPR137B protein expression in Gpr137b-KO clones byimmunostaining using the anti-GPR137B polyclonal antibody generated in house. Fluorescencesignals detected at the peripheral region as well as nuclei of the Gpr137b-WT cells, possiblyrepresenting the localization of GPR137B protein at the cell membrane and nucleus, were absent
in the Gpr137b-KO cells.

Chapter 2: Role of Gpr137b in osteoclast differentiation
At first, I confirmed the predominant Gpr137b expression in mouse and human osteoclast
cells in compare to the representative mouse and human tissues by qRT-PCR. Additionally, I
confirmed that Gpr137b exhibits an osteoclast specific expression pattern among bone cells by
qRT-PCR. Next, to elucidate the role of Gpr137b in osteoclast differentiation, generated
Gpr137b-WT and -KO clones were then treated with soluble receptor activator of nuclear factor
kappa-B ligand (sRANKL) to induce osteoclastic differentiation. Gene expression microarray
analysis revealed that a set of genes are differentially regulated through RANKL-induced
osteoclast differentiation. These genes include representative osteoclast-specific markers such as
Tartrate-resistant acid phosphatase (TRAP), Cathepsin K (CatK), ATPase H+ transporting,lysosomal V0 subunit D2 (Atp6V0d2) and Matrix metalloproteinase 9 (MMP-9), which weredysregulated in the RANKL-treated Gpr137b-KO clones compared to the RANKL-treatedGpr137b-WT clones, suggesting that Gpr137b is possibly involved in RANKL-induced
osteoclastic differentiation. These results were also validated by subsequent quantitative RT-PCR(qRT-PCR) and western blotting. KEGG pathway analysis of the Gpr137b-dependant
RANKL-induced genes revealed the involvement of Gpr137b in osteoclast differentiation. Myfindings strongly suggest that the pharmaceutical targeting of Gpr137b possibly represents a novel
and innovative approach to the prevention and management of bone diseases.
Chapter 3: Role of Gpr137b in macrophage polarization
Macrophages play a crucial role in host defense, disease pathogenesis and maintenance oftissue homeostasis. In general, macrophages can be classified into M1 (classical) and M2(alternative) subtypes based on their phenotypes and functions. M1/M2 macrophage polarization
is well maintained physiologically, and imbalanced polarization causes various pathologicalconditions. M1 macrophages, which are typically induced by lipopolysaccharide (LPS) andinterferon (IFN)-γ, are activated during acute inflammation to produce proinflammatory cytokines,
reactive nitrogens, and oxygen intermediates. By contrast, M2 macrophages are typically activatedby interleukin (IL-4), IL-13, and anti-inflammatory cytokines produced by a variety of innate andadaptive immune cells, thus they play a role in post-inflammatory tissue repair and reconstruction.
Imbalanced M1/M2 macrophage polarization is associated with various disease conditions. M1
macrophages are characterized by an upregulation of Nos2, Cxcl10, Il1a and Gbp5; whereas, adistinctive molecular feature of M2 macrophages is the high-level expression of arginase-1(Arg1); some C-C motif chemokine ligands such as Ccl17, Ccl22, and Ccl4; and Cd206(alternatively designated Mrc1).
Interestingly, KEGG pathway analysis described in Chapter 2 indicated the involvement ofGpr137b in the (IL-4) signaling pathway besides the osteoclast differentiation pathway. Thus, Ihypothesized that Gpr137b could be involved in macrophage polarization as IL-4 is well known toinduce M2 macrophage polarization. Gene expression microarray analysis of Gpr137b-WT and-KO clones treated with IL-4 revealed that Gpr137b-KO attenuated the IL-4-induced expression ofM2 macrophage-related genes such as mannose receptor (Cd-206), C-C motif chemokine ligand(Ccl-22), Arginase1, and heparin-binding EGF-like growth factor (Hb-egf), which wassubsequently verified by qRT-PCR.
To determine whether Gpr137b has any impact on M1 macrophage polarization,
Gpr137b-WT and -KO clones were then treated with LPS, a well-known inducer of M1
macrophage polarization. qRT-PCR analysis revealed that the LPS-induced expression ofrepresentative M1 macrophage markers such as Cxcl10, Il1a, Nos2 and Gbp5 was not affected by
Gpr137b-KO. This result suggests that Gpr137b may not be associated with M1 macrophage
polarization of RAW264 cells.
Conclusion
My findings in this study markedly expanded my views about bone remodeling andmacrophage polarization. Elucidating the function of Gpr137b in osteoclast differentiation will
make it a promising target for the novel anti-resorptive agent. Further, given the fact thatmacrophages affect many physiological or pathological processes, the current study will shed lighton the utilization of Gpr137b as a potential drug target in the clinical application including
helminth infection, inflammation allergy, rheumatoid arthritis and tissue repair.

審査結果の要旨

昨今の高度高齢化社会において，骨粗鬆症や関節リウマチなどの骨量減少・骨破壊をもたらす疾患の罹患者は国内だけで 1 千万人にも上るとされ，その対策は喫緊の課題である。
現在，これらの疾患の治療において，骨吸収を担う破骨細胞を標的とする種々の骨吸収阻害剤が用いられているが，特に治療効果が高いとされるビスフォスフォネート剤服用中の歯科処置による顎関節壊死に代表されるように，重篤な副作用の問題により使用が制限される場合も多い。破骨細胞の分化や機能制御に関わる遺伝子やタンパク質を新たに同定し，これらの機能を分子レベルで解明できれば，作用機序が異なる新規骨吸収阻害剤の開発に繋がり，上記課題の解決への糸口となる。そこで本学位申請者は，破骨細胞への分化能を有するマウスマクロファージ様細胞株 RAW264 を用いて，破骨細胞分化に伴い発現が上昇する遺伝子を探索し，その結果見出された遺伝子の機能についてゲノム編集技術を用いて解析した。
第 1 章では，RAW264 細胞を破骨細胞分化誘導因子である receptor activator of nuclear
factor kappa-B ligand（RANKL）で処理することにより破骨細胞分化を誘導し，それに伴って発現が変動する遺伝子を網羅的遺伝子発現解析（RNA-seq）により探索した。発現量の指標である reads per kilobase of exon per million mapped reads (RPKM)値が 50 以上，かつ破骨細胞分化に伴う発現変動が 10 倍以上という条件を満たす遺伝子を選抜した
結果，発現上昇遺伝子を 56 種類見出し，これらの中で特に Gpr137b に着目した。Gpr137bがコードするタンパク質は，G タンパク質共役型受容体（GPCR）の１つである。GPCRはヒトでは約 800 種類存在し，現在用いられている薬剤の約半数は GPCR を標的としている。また，GPCR のうち約 100 種類は，リガンドが未だに同定されていない，所謂「オーファン GPCR」であり， Gpr137b もこれに属する。オーファン GPCR は，新たな薬剤開発のための標的分子としてリガンド探索を含めた研究が盛んに行われているが，これまでに破骨細胞におけるオーファン GPCR に関する研究報告はほとんど存在しない。そこで，破骨細胞における Gpr137b の機能を解明するために，CRISPR/Cas9 系を用いたゲノム編集により，Gpr137b 遺伝子ノックアウト（ Gpr137b -KO）RAW264 細胞クローンを作製した。なお，マウスでは Gpr137b と 97%の相同性を示す偽遺伝子 Gpr137b-ps が存在するが，得られた Gpr137b -KO 細胞クローンでは， Gpr137b 遺伝子の両アリルのみに 188 塩基対の欠損が認められ， Gpr137b-ps 遺伝子は無傷であることが確認された。
第 2 章では，前章で作製した Gpr137b -KO RAW264 細胞およびコントロール細胞をRANKL 存 在 下 で 培 養 し ， こ れ ら の 破 骨 細 胞 分 化 能 に つ い て 解 析 し た 。 そ の 結 果 ，Gpr137b -KO RAW264 細胞では， Trap ， Ctsk ，および Mmp9 等の破骨細胞マーカー遺伝
子の RANKL による発現誘導が顕著に抑制されていたことから，Gpr137b が破骨細胞分化に重要な役割を担う遺伝子であることが示唆された。さらに，マイクロアレイを用いた網羅的遺伝子発現解析およびパスウェイ解析の結果，Gpr137b が破骨細胞分化に決定的な役割を果たす nuclear factor kappa-Β パスウェイの制御に関わることが示唆された。以上より，マクロファージ様細胞株である RAW264 の破骨細胞分化に伴い発現が上昇する遺伝子として Gpr137b を同定したが，一方で Gpr137b は，分化前の RAW264 細胞やマクロファージ前駆細胞（単球）を含む骨髄細胞においても，他の主要な臓器と比較して顕著に高いレベルで発現していた。
第 3 章では，Gpr137b がマクロファージの機能にも関与している可能性について追求した。マクロファージは炎症性である M1 と抗炎症性である M2 の 2 種類のサブクラスに大別され，様々な薬剤やサイトカインにより各サブクラスへの極性化が制御される。そこで，

Gpr137b -KO RAW264 細胞クローンを用いて解析を行った結果，Gpr137b は interleukin-4が誘導する M2 極性化に関わるが，lipopolysaccharide が誘導する M1 極性化には関与しないことが明らかとなった。
本申請論文は，オーファン GPCR である Gpr137b が破骨細胞に選択的に高発現し，破骨細胞分化に重要な役割を担う遺伝子であることを明らかにした初めての論文である。本
知見は，Gpr137b やその下流のシグナル伝達因子を標的とすることにより，副作用のリスクが低く，且つ既存薬剤と作用機序が異なる新規骨吸収抑制剤の開発の可能性を示すことから，生化学，および創薬科学に大きく貢献するものである。よって，本論文の審査ならびに最終試験の結果と併せて，博士（応用生命科学）の学位を授与することを適当と認める。