ISSN 1340−9662

日本医科大学

先端医学研究所紀要
第１巻 平成27年度

Institute for Advanced Medical Sciences
Nippon Medical School
Year Book

Vol. 1 (2015)

日本医科大学

先端医学研究所紀要
第１巻 平成 27 年度

Institute for Advanced Medical Sciences
Nippon Medical School
Year Book

Vol. 1 (2015)

平成27年５月14日

中庭にて

目

次

第１巻発刊によせて･･･････････････････････････････････････････････････････････ 先端医学研究所・所長 南

史朗･ 

1

１．研究概要 ……………………………………………………………………………………………………

5

２．研究業績 ……………………………………………………………………………………………………

6

３．代表的研究論文……………………………………………………………………………………………

7

Ⅰ．病態解析学部門

Ⅱ．細胞生物学部門
１．研究概要 ……………………………………………………………………………………………………

21

２．研究業績･････････････････････････････････････････････････････････････････････････････････････････････････････ 

22

３．代表的研究論文･ ･･･････････････････････････････････････････････････････････････････････････････････････････ 

25Ⅲ．遺伝子制御学部門
１．研究概要･････････････････････････････････････････････････････････････････････････････････････････････････････ 

39

２．研究業績･････････････････････････････････････････････････････････････････････････････････････････････････････ 

40

３．代表的研究論文････････････････････････････････････････････････････････････････････････････････････････････ 

43

Ⅳ．生体機能制御学部門
１．研究概要･････････････････････････････････････････････････････････････････････････････････････････････････････ 

53

２．研究業績･････････････････････････････････････････････････････････････････････････････････････････････････････ 

55

３．代表的研究論文････････････････････････････････････････････････････････････････････････････････････････････ 

57

Ⅴ．武蔵小杉地区動物実験室･ ･････････････････････････････････････････････････････････････････････････････････････ 

65Ⅵ．平成27（2015）年度老人病研究所公開セミナー･･･････････････････････････････････････････････････････････ 67

Ⅶ．平成27（2015）年度研究補助金、助成金等受け入れ･････････････････････････････････････････････････････ 

70

Ⅷ．教職員，研究者名簿････････････････････････････････････････････････････････････････････････････････････････････ 72

紀要第１巻の発刊によせて

所長

南

史

朗

先端医学研究所紀要第 1 巻をお送り申し上げます。本紀要は、平成 27 年度の本研究所の研究業績を中心にまとめたものです。
平成 27 年 4 月 1 日より、老人病研究所から名称を変更し、「先端医学研究所」となりました。
当研究所は、1954 年に老年医学の研究施設として発足しました。その後、時代とともに内容も変遷し、発生から老化までの加齢科学の研究をはじめ、分子・細胞レベルから個体に至る生命科学の研究へと発展してきました。最近の医学研究の進歩に伴い、病態が細胞・分子レベルで解析されるようになり、疾病の治療も対症療法から根本治療へと変わりつつあります。医学研究のめざましい発展と技術革新を背景に、2015 年 4 月に歴史ある老人病研究所を改組するとともに、名称を先端医学研究所としました。
医学博士課程を担当する 5 つの分野を含む 6 つの部門によって構成されています。本研究所の基本･テーマを「細胞代謝 Cellular metabolism」とし、部門の枠を超え、互いに協力して、医学の広い領域にわたる研究を遂行してゆく体制を作りました。
医学研究の進歩がめざましい今日、研究所の機能強化と社会のニーズに答えることを目的とし、･「自由な研究活動の場」、開かれたラボとして広範な医学研究ができるようにしてゆく所存です。また、病院と一体化した研究所の特徴を生かし、研究の成果がきっと臨床に還元できるように、トランスレー
ショナル研究を推進するように、研究所員ともども努力してゆきたいと思います。
皆様のご指導、ご鞭撻をお願い申し上げます。

−1−Ⅰ．病態解析学部門
Department of Molecular Pathophysiology

病態解析学部門（大学院

分子細胞構造学部門）
病態解析学部門【研究概要】
老人病研究所病理部門では、１）ケロイド発生機序の研究、２）血管形成メカニズムの解析など、形態に主眼をおいた特色のある研究を行っている。
１）ケロイド発生機序の研究

武蔵小杉病院形成外科と無瘢痕創傷を目的に肥厚性瘢痕とケロイド発生の解析を行っている。
･
これまでケロイド発生に関与する遺伝子解析で IL−6 及び c−Abl 遺伝子の発現上昇を見いだし･IL−6 シグナルのケロイド発生への関与を明らかにした。ケロイドでは TGF−ß や PDGF が過剰発現を示すことからこれらのシグナル伝達を同時に抑制する小分子標的治療薬 STI571 のケロイド
線維芽細胞への投与によるプロコラーゲン産生とタイプⅠコラーゲン mRNA の発現の低下を目的に
ケロイド新薬としての可能性の解析を進めている。さらに、TGF−ß と PDGF シグナル伝達の抑制に関与する microRNA 遺伝子を解析しケロイド線維化調節に関わる COLIA2 プロモーターに関する研究にも着手している。
２）血管形成メカニズムの解析

血管の構築メカニズムは未だ不明な点が多い。特に血管の形態は多様性に富んでおり、大動脈から毛細血管まで様々なものが存在する。我々はこれらの形態の多様性がどのように生じるのか、
血管の多様な形態を規定する機構についての研究を行っている。これまでに、アフリカツメガエルのオタマジャクシを用い、発生時にみられる血管のダイナミックな形態学的変化が「いつ」・「どこで」生じるのかを明らかにしてきた。本年度は live cell imaging によってリアルタイムで血管の構築過程を解析できるトランスジェニックカエルを作製した。このトランスジェニックカエルは VEGFR2 のプロモーター・エンハンサーによって血管内皮細胞に GFP や mCherry を特異的に発現させており、血管内皮細胞の挙動を細胞レベルで明らかにすることができる。今後は長期間の観察を可能とする
循環システムの条件検討を行っていく必要がある。血管の形態は疾患と直接関わっていることが多いことから、血管の形態を制御することによって疾患を治療する新たな方法が開発されることが期待される。

−5−

【研究業績】〈英文著書〉〈学会発表〉（国際学会）（一般演題）（国内学会）（一般演題）
１．藤原正和：アフリカツメガエルによる血管の形態に関わる因子の機能解析 . 第２３回日本血管生物医学会学術集会 , 2016. 3.（神戸）２．藤原正和：アフリカツメガエルによる血管形成イメージング解析 . 第４回日本比較病理学研究会 ,2016. 2.（東京）【代表的研究論文】
Shimizu, H., Yamagish S., Chiba H., Ghazizadeh M.: Methionine aminopeptidase 2 as a potential
therapeutic target for human non-small-cell lung cancers. Adv Clin Exp Med. 2016; 25:117-128.

−6−

ORIGINAL PAPERS© Copyright by Wroclaw Medical University
ISSN 1899–5276

Hajime Shimizu1, 3, A–D, F, Shigeki Yamagishi2, B, C, E, Hideki Chiba3, E, F,
Mohammad Ghazizadeh1, A, C–F

Methionine Aminopeptidase 2
as a Potential Therapeutic Target
for Human Non-Small-Cell Lung Cancers
1 Department of Molecular Pathology, Institute of Gerontology, Nippon Medical School, Japan

2 Department of Surgery, Division of Thoracic Surgery, Aizu Chuo Hospital, Fukushima, Japan
3 Department of Basic Pathology, Fukushima Medical University, Fukushima, JapanA – research concept and design; B – collection and/or assembly of data; C – data analysis and interpretation;
D – writing the article; E – critical revision of the article; F – final approval of article

Abstract
Background. Methionine aminopeptidase 2 (MetAP2) is a bi-functional protein that plays a critical role in theregulation of post-translational processing and protein synthesis.
Objectives. We studied whether MetAP2 is activated and expressed in human non-small-cell lung cancer (NSCLC)
tissues and whether inactivation of MetAP2 activity, with its specific inhibitor fumagillin, potentially inhibits proliferation of NSCLC cells.
Material and Methods. The expression and function of MetAP2 were evaluated in NSCLC tissues, primary cell
cultures and cell lines using immunohistochemistry, RT-PCR, Western blot, aminopeptidase activity assay andflow cytometry. MetAP2 expression was also studied in relation to clinicopathological factors.
Results. MetAP2 expression in NSCLS, including adenocarcinoma (ADC) and squamous cell carcinoma (SCC),
showed a moderate to strong positive reaction while normal appearing bronchial epithelium showed weak staining and normal alveolar epithelial cells were widely negative. A high MetAP2 mRNA and protein expression was
found in NSCLC tissues. The aminopeptidase activity in NSCLC was 2-fold higher than that in normal lung tissues.
In a series of 41 ADC patients, MetAP2 expression was significantly correlated with patient’s outcome or survival
time. Inhibition of MetAP2 by fumagillin in SCC cell lines revealed a significant increase in caspase-3 activity ascompared to the control (p = 0.001).
Conclusions. Our results indicate that MetAP2 is involved in NSCLC and is an important regulator of proliferative
and apoptotic targets. Thus inhibition of MetAP2, such as by fumagillin, may be a potential therapeutic modality
for prevention of tumor cell growth, development and progression in NSCLC patients (Adv Clin Exp Med 2016,
25, 1, 117–128).
Key words: apoptosis, methionine aminopeptidase 2 (MetAP2), myristoylation, fumagillin, non-small-cell lungcancer (NSCLC).

Non-small-cell lung cancer (NSCLC) is oneof the most difficult malignancies to treat. Despite
years of research, the prognosis for patients withlung cancer remains poor, with a five-year survival rate of 14%. Nevertheless, lung cancer may be
curable in its early stages and most patients derive some benefit from treatment, such as longer
survival or amelioration of symptoms [1]. DNA
ploidy analysis may be efficient to estimate malignant potential in lung ADCs [2]. Recently, clinicalapplication of molecular targeted therapy such as
epidermal growth factor receptor (EGFR) has improved prognosis. Accordingly, the overall survival of patients with metastatic NSCLC evidencing
EGFR mutations has improved to 27–30.5 months
when treated with EGFR [3]. Therefore, moleculartargeted therapy for the appropriate population,using good predictive markers, is indispensable.
The gene expression profile analysis of human esophageal cell carcinoma cell lines after early−7−

病態解析学部門

Adv Clin Exp Med 2016, 25, 1, 117–128
DOI: 10.17219/acem/60715

118

H. Shimizu et al.

response to ionizing irradiation using cDNA microarray screening disclosed MetAP2 genes thatwere modulated in irradiation [4]. The MetAP2gene may be useful in understanding the molecular basis of radiotherapy and in developing strategies to augment its effect or establish novel, lesshazardous alternative adjuvant therapies. Overexpression of MetAP2 in immortalized bronchial epithelial cell line NL20 accelerated growth andwas reversed using treatment with MetAP2 inhibitors. Thus MetAP2 plays an important role in tumor cell growth and may contribute to tumorigenesis [5, 6].
Protein synthesis starts with an initiator methionine in both prokaryotes and eukaryotes. The
translational process on ribosomes starts withmethionine. In order for the newly synthesized
protein to be transported to its exact intracellular location, the methionine at the NH2-terminal is removed. After the removal of methionine
via MetAP, protein myristoylation takes placeby the enzyme N-myristoyl-transferase (NMT).
NMT is a cytosolic enzyme in eukaryotic cells [7].
The process is essential for further amino terminal modifications (e.g. acetylation by N-α acetyltransferase and myristoylation of glycine by
N-myristoyl-transferase). The structural alterations from these modifications are essential in cellproliferation [8].
In eukaryotes, two isoforms of MetAP havebeen identified as MetAP1 and MetAP2 [9]. Both
MetAP1 and MetAP2 are essential components ofthe cell growth machinery. In yeasts and humans,
two proteins are known to possess MetAP2 activity and are known as MetAP1 and MetAP2. Downregulation of either MetAP1 or MetAP2 protein
expression by small interfering RNA (siRNA) significantly inhibited the proliferation of human endothelial cells [10].
MetAP2 has attracted much more attention
than MetAP1 due to the discovery of MetAP2
as a target molecule of the anti-angiogenic compounds, fumagillin and ovalicin [11]. Identification of MetAP2 as the cellular target of fumagillinclass molecules, and the significant growth inhibition observed in cells sensitive to MetAP2 inhibition suggested the direct involvement of MetAP2
in the regulation of cell proliferation [12, 13].
Targeting the angiogenesis process has become
an important strategy for inhibiting tumor growth.
Fumagillin and its derivatives have been known toexert their inhibitory effects by specifically and covalently binding to MetAP2 [14, 15]. Inhibitors ofangiogenesis can be classified into 2 groups, specific and nonspecific factors. Non-specific inhibitors,
angiostatin, a tissue inhibitor of metalloproteinases-2 (TIMP-2), and endostatin have attracted−8−interest because of their strong antitumor effect.
Recently, much research has focused on TNP-470,
a synthetic analog of fumagillin, and its derivatives(IDR-803, IDR-804, IDR-805, CKD-732) [16].
High expression of MetAP2 has been demonstrated in breast, colorectal and cholangio-carcinoma [17–19]. Suppression of hepatoma growth andangiogenesis by fumagillin has also been reported [20]. As for NSCLC, moderate-to-high MetAP2
staining was identified only in lung carcinoma celllines not lung cancer tissues [5]. Here we demonstrate the first description of increased MetAP expression in NSCLC tissues, primary cell culturesfrom NSCLC and cell lines. Our findings strongly
suggest that the inhibition of MetAP2 by fumagillin or its analogs may be a potential target for tumor cell growth, development and progression in
NSCLC patients.

Material and Methods
Material
We investigated 41 cases of NSCLC histologically classified as ADC [21], ranging in age from45 to 80 years (mean, 67 years). The age, gender,
tumor size, histological grading, nodal metastasis,staging and patient outcome were evaluated by reviewing the medical and pathologic records. Tumor size was evaluated using the greatest perpendicular diameter of each lung lesion. In addition,
we studied surgical tissues from 8 cases of histologically classified primary NSCLC including 4 cases of ADC and 4 cases of SCC in order to evaluate
MetAP2 aminopeptidase enzyme activity, mRNA
expression, immunohistochemical localizationand protein expression. Ten cases of normal lungtissues freshly obtained from resected benign lesions served as control. The other tissue samples
were fixed in neutral-buffered formaldehyde andprocessed for histological and immunohistochemical evaluation. Furthermore, we studied cell cultures from RERF-LC-AI (well differentiated) andLC-1/sq (moderately differentiated) human SCC
cell lines for MetAP2 inhibition assay by a novel
inhibitor of MetAP2, fumagillin.

Methods
Cell Lines and Culture Condition

RERF-LC-AI (well differentiated) and LC-1/sq(moderately differentiated) human SCC cell lines
were purchased from Riken BioResource Center(Tsukuba, Japan). The tumor cells were cultured
in Dulbecco’s modified Eagle’s Medium (DMEM)
containing 10% fetal bovine serum, 100 IU/mL

119Methionine Aminopeptidase in Lung Cancer

Preparation of Tissues

The tissue samples (mean weight, 300 mg)
were homogenized in 10 volumes of 0.01 mol/L
phosphate-buffered saline (PBS) pH 7.4 and sonicated at 40 amplitudes for 2 min to obtain thesoluble fraction. After centrifugation at 4°C, thesupernatants were used to measure MetAP2 aminopeptidase activity and proteins in triplicate. For
caspase-3 activity assay, primary cell cultures from
NSCLC and normal lung tissues were harvested bycentrifugation at 10,000 rpm for 5 min at 4°C andcounted. For each case, 1 × 106 cells re-suspended
in ice cold cell lysis buffer, and sonicated at 40 amplitudes for 30 s.
The cell lysates were centrifuged at 10,000 rpm
for 3 min, and the supernatants were transferred toa microcentrifuge tube for caspase-3 activity assay.

MetAP2 Aminopeptidase
Activity Assay

MetAP2 aminopeptidase activity was determined by hydrolysis of methionine L-Leu-p-nitroanilide as a substrate, described elsewhere [22].
For MetAP2 activity, an assay mixture (500 μL)
containing 50 mM Tris-HCl at pH 7.5 and 0.25 mM
methionine L-Leu-p-nitroanilide as a substratewith an appropriate concentration of the enzyme
was used. The reaction mixtures were incubated at 37°C for 30 min, left for 15 min on ice, andfollowed by spectrophotometric determination at
405 nm. The amount of aminopeptidase activity
that released one micromole of L-Leu-p-nitroanilide per minute under assay conditions was defined as one unit.

Semi-Quantitative RT-PCR Analysis
of MetAP2 mRNA Expression

The total RNA was extracted using an RNeasy
mini kit (Quiagen, Hilden, Germany), according
to the manufacturer’s instructions. Before cDNA
synthesis, the RNA was incubated with DNase
and then precipitation was done using 95% ethanol. The precipitated RNA was then electrophoresed on 2.0% agarose-ethidium bromide (EtBr)
gels to verify the RNA quantity. Subsequently,
RNA concentration was determined by UV spectrophotometry. Total RNA (1.5 μg) was then reversely transcribed utilizing the specific primers.

The following PCR amplification was carried out
in a Thermal Cycler (Takara, Tokyo, Japan). In
each case, the PCR cycles were optimized to confirm amplification within the linear phase. For
each sample, the relative mRNA level was normalized using glyceraldehyde 3-phosphate dehydrogenase (GAPDH). The following primer pairs wereused: MetAP2 sense, 5’ATGGCGGGTGTGGAGGAGGTAGCGGCCT-3’ (nucleotides 135-162)
and anti-sense 5’TTAATAGTCATCTC CTCTGCTGACAACT-3’ (nucleotides 1544–1571). The
expected PCR product for MetAP2 was 1440 bp.
The internal control was GAPDH. For quantification of the PCR bands, we used densitometry with
Quantity One Software (Bio-Rad Labs, Hercules,
CA, USA) relative to GAPDH. The results wereconsidered as the mean ± SE of three experiments.

Immunostainings for MetAP2

Primary cultures of RERF-LC-AI and LC-1/sq human SCC cell lines were stained using
an indirect immunofluorescence method. Cells(2.5 × 104) in chamber slides (Nalge Nunc Int.,
Naperville, Illinois) were cultured overnight. Twochamber slide sets from LSCC were prepared, oneof which received 1 ug/mL fumagillin (BIOMOL
Research Lab, Inc, USA) treatment. After 24 h culture, we fixed the cells in cold acetone, washedin PBS, and treated for 30 min with 10% normal
horse serum to eliminate non-specific reaction.
The cells were treated with 1 : 50 dilution of polyclonal anti-human MetAP2 antibody overnight
followed by PBS washes and reaction with FITC-conjugated goat anti-rabbit IgG at 1 : 100 dilution. Immunoperoxidase reaction for metAP2 wasdone using frozen or deparaffinized sections anda streptavidin-biotin peroxidase complex method. In brief, endogenous peroxidase was blocked
by 10% normal goat serum and the sections werereacted with 1 : 50 polyclonal rabbit anti-human
MetAP2 antibody (Zymed Lab Inc, CA) overnight. After PBS washes, the sections were treatedwith biotinylated goat anti-mouse or anti-rabbit
IgGs, washed and processed using a streptavidinbiotin-peroxidase kit (Histofine, Nichirei, Japan).
The chromogenic reaction was with diaminobenzidine. Counterstaining was done with hematoxylin. For negative control, the primary antibody
was omitted or substituted with non-immune
rabbit serum.

MetAP2 Expression in Lung
Adenocarcinomas

We studied the relationships between MetAp2
expression and pathological and clinical features(tumor grade, tumor stage, tumor size and nodal metastasis). MetAP2 staining was graded by the−9−

病態解析学部門

penicillin, 100 ug/mL streptomycin, and 0.25%
amphotericin B at 37°C in a humidified atmosphere in a 5% CO2 incubator. The cells were treated with trypsin (0.25%), harvested and processed
for secondary cultures in the same culture medium
until they reached 80% confluence.
In this study, tumor cells obtained at the lowpassage cultures (passages at 4–5) were used.

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H. Shimizu et al.

percentage of staining area of tumor cells as follows: 0: 0%, 1: 1~25%, 2: 26~50%, 3: 51~75% and
4: 76~100%. Staining intensity was graded 0: negative, 1: mild, 2: moderate and 3: strong staining.
The staining score (0 to 12) was estimated by multiplying the staining area 0 to 4 (%) by the staining intensity 0 to 3. We set the low expression of
MetAP2 intensity as a score of 0 to < 6 and high expression as a score of 6 to 12.

Western Blot Analysis

We extracted proteins from the primary cultures of three cases each of adenocarcinoma and
SCC of the lung. Each protein sample (20 ug/lane)
was run on SDS gels, transferred onto Immobilon polyvinylidene difluoride membranes (BioRad Lab, Hercules, CA, USA) in a transfer buffer consisting of 0.02% SDS, 25 mM Tris-HCl, pH
8.3, 192 mM glycine, and 20% v/v methanol, thenincubated overnight at 4°C with a blocking buffer (5% nonfat dry milk, 50 mM Tris-HCl, pH 7.5,
0.1% Tween-20, 150 mM NACI). Two sets ofmembranes were made and incubated with 1 : 500
polyclonal rabbit anti-human MetAP2 antibodies(Zymed Lab Inc, CA, USA) overnight at room temperature. Proteins were extracted from the primarycultures of three cases of ADC and SCC of the lungand normal lung tissues. The samples were washedwith 50 mM Tris-HCl, pH 7.5, containing 150 mM
NaCI and 0.1% Tween 20 and then were treatedwith corresponding secondary antibodies conjugated with alkaline phosphatase. The reaction wasdeveloped with the ProtoBlot NBT and BICP System (Promega, Madison, WI, USA).

Caspase-3 Fluorometric Measurement

Caspases-3 production requires that the active
enzymes undergo folding from their large and small
subunit constituents after expressing separately in
Escherichia coli. The methodological details havebeen described previously [23]. The active enzyme
was obtained under optimal conditions for eachenzyme; each subunit from the purified inclusion
bodies were solubilized in 6 M guanidine HCl andsubsequently diluted to a 100 μg/mL final concentration at room temperature.
Measurement of the fluorescence counts inthe wells was done with a 400 nm excitation filter and 505 nm emission filter. The levels of released 7-amino-4-trifluoromethyl coumarin were
measured with a BioLumin 960 spectrofluorometer (Molecular Dynamics Japan, Tokyo, Japan).
The specific activity of caspase-3 present was calculated in each sample. The relative absorbancewas calculated as described according to the manufacturer’s instructions (MBL, Co, LTD, Nagoya,Japan).

−10−

Flow Cytometric Assay

RERF-LC-AI and LC-1/sq human SCC cells
obtained from the low-passage cultures (passages at 4–5) were utilized in this study. To measurepropidium iodide (PI) staining, lung cancer cells(1 × 104) were harvested and stained with FITC-labeled PI (Molecular Probes, Eugene, OR, USA)as specified by the supplier. Briefly, cancer cells(1 × 104) in 1 mL of medium were cultured as indicated for 21h, washed and then stained with PIFITC in a binding buffer and analyzed with CellQuest software (BD Bioscience, San Jose, CA,
USA) with FACSCalibur within 1 h. The data wasexpressed as the mean of three experiments before
and after treatment.

Statistical Analysis
Statistical analyses for comparisons between
the clinicopathological findings were performed
using Fisher’s exact test and Kaplan-Meier survival analyses. The difference between two relatedgroups was examined for statistical significance using the Student’s t-test for paired data. A p < 0.05
was recorded as statistically significant.

Results
MetAP2 Expression in Normal
Lung and NSCLC Tissues
Immunoperoxidase staining for MetAP2 in
a paraffin section of normal alveolus showed negative staining (Fig. 1A). Sections from bronchial epithelium showed a weak degree of staining(Fig. 1B). ADC (well differentiated) and SCC cells
showed moderate to strong intensity (Fig. 1C, D).
The intensity of MetAP2 expression was greater incarcinoma cells as compared to bronchial epithelial tissues. SCC tissues showed stronger staining
than ADC tissues.

Pathological Features of Lung
Adenocarcinoma Patients andExpression of MetAP2
We studied paraffin-embedded lung tissue sections of 41 cases of histologically classified ADCs.
The relationships between pathological and clinical features are shown in Table 1. A comparison
between the low expression of MetAP2 intensity
and high expression of MetAP2 intensity in ADCs
showed that 21 cases had low expression and20 cases had high expression of MetAP2. There was121

Methionine Aminopeptidase in Lung Cancer

Table 1. Pathological features of lung adenocarcinoina patients and expression of MetAP2
Variables

No.

MetAP2 expression
low

high

p-value

13
8

11
9

0.756

15
5

1.000

Age
≤ 70
> 70

24
17

Gendermale
female30
11Tumor size
≤ 30 mm> 30 mm

16
25

9
12

7
13

0.751

Tumor differentiation
w/d & m/d
p/d

28
13

14
7

14
6

1.000

Node metastasis
negative
positive

27
14

16
5

11
9

0.197

Pathological stagestage I
stage II & III

28
13

14
7

10
10

0.350

Patient outcome
alive
dead

10
31

3
18

9
11

0.043

15
6

* w/d & m/d – well differentiated & moderately-differentiated; ** p/d – poorly differentiated.

no significant association between the low and highexpression of MetAP2 and clinical features (tumor
size, tumor differentiation, nodal metastasis andstaging), but patient outcome revealed a significant difference between low and high MetAP2 expressing patients (Table 1; p < 0.04).

−11−

病態解析学部門

Fig. 1.
Immunohistochemical
analysis of human normallung and cancer tissues(A-D, magnification ×100).
MetAP2 in paraffin section from normal alveolus
showed negative staining(Fig. 1A). Bronchial epithelium showed a weak degreeof staining (Fig. 1B, arrow).
ADC (well-differentiated)
and SCC cells showed
moderate to strong intensity (Fig. 1C, D)

122H. Shimizu et al.

MetAP2 Activities in Normal
Lung and NSCLC Tissues

Table 2. Aminopeptidase activities in normal lung and
NSCLC* tissues

Using methionine-p-nitroanilide as a substrate, we measured the spectrophotometric determination of the hydrolysis of methionine p-nitroanilide in their soluble and membrane-boundforms in surgically removed lung carcinoma tissues and normal lung tissues freshly obtained fromresected benign lesions, serving as the control. The
mean aminopeptidase activity was 80.6 ± 13.4 innormal lung tissue vs. 156.9 ± 20.8 in lung carcinoma tissue. Compared to normal lung tissue,
carcinoma tissue had a remarkably higher activity (Table 2). In 4 cases of ADC, each case showeda significantly higher aminopeptidase activity thanan unaffected normal tissue counterpart (Fig. 2A)
and overall, ADC cases had 2 times higher activity than normal tissue counterparts (97.3 ± 16.51
vs. 45.4 ± 8.6; p < 0.03, Fig. 2B). Likewise, 6 cases of SCC tissues had 196.7 ± 20.1 aminopeptidase
activity vs. 104.2 ± 15.2 activity in unaffected adjacent normal lung tissues (Fig. 2C) with an overallsignificant difference (p < 0.04; Fig. 2D).

Case**

Age/Sex

Aminopeptidase activity (unit/L)
normaltumor

1

82 / M

29.311.8

73.3 ± 3.3

277 / F

38.7+1.8

129.3 ± 5,2

3

65 / F69.7± 1.5

122 ± 5.3

471 / M

43.7± 2.0

64.7+3.2

577 / M

102.7± 5.5

115.7± 5.S

664 / M

149.7 ± 2.6

174 ± 3.5

778 / M50± 1.7247 ± 7.8

876 / M

102 ± 3.6

226.7± 9.5

960 / M

141 ± 3.8

182.3 ± 4.6

1074 / M

79.7 ± 2.0

234.3 ± 7.4* NSCLC – non-small cell lung cancer; ** cases 1–4 – adenocarcinoma and 5–10 – squamous cell carcinoma (SCC);
the data presented as mean ± S.E.

Fig. 2. Spectrophotometric determination of hydrolysis of methionine p-nitroanilide in their soluble and membranebound forms in surgically-removed lung carcinoma tissues and unaffected adjacent surrounding tissues. Comparisonof the expression level of MetAP2 between 4 cases of ADC and 4 cases of normal lung tissues separately (A) and incombination (B). Comparison of the expression level of MetAP2 between 6 cases of SCC and 6 cases of unaffectedadjacent normal lung tissues separately (C) and in combination (D)

−12−

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Methionine Aminopeptidase in Lung Cancer

To assess the mRNA expression levels of
MetAP2, we studied surgical operating tissues
from 7 cases of histologically classified primary NSCLC as 4 ADC and 3 SCC patients. Normallung tissues were obtained from surgically resected benign lesions.
The mRNA levels of MetAP2 in the four casesof ADC were significantly different compared tonormal lung tissues (Fig. 3A; p < 0.008). Also, thethree cases of SCC had a significant upregulationof the MetAP2 gene as compared to normal lungtissues (Fig. 3B; p < 0.002).
Proteins were extracted from subconfluent
primary cultures of three representative cases of
ADC and SCC and normal lung tissue counterparts. The levels of MetAP2 in the three cases of

ADC (Fig. 3C) and three cases of SCC (Fig. 3D)
showed a significant difference in the quantitative
measurement of the protein expression compared
to normal lung tissues (p < 0.05; respectively).

MetAp2 Immunofluorescence
Intensity, Caspase-3 Activity andCell Death Effect of Fumagillin
in Cultured SCC Cell Lines
We assessed the effect of fumagillin as an inhibitor of MetAP2 on cell proliferation activity andcell death population using caspase-3 fluorometricand PI (propidium iodide) flow cytometric analysis. We used the primary cultures of RERF-LC-AI(well differentiated) and LC-1/sq (moderately differentiated) human SCC cell lines.
The immunofluorescence intensity of MetAP2
in moderately differentiated SCC cells was decreased after fumagillin (1 µg/mL) treatment for 24 h(Fig. 4A). Also, caspase-3 activity was markedly

Fig. 3. MetAP2 mRNA and protein expression in ADC and SCC. (A) The mRNA levels of MetAP2 by RT-PCR in
4 cases of ADC compared to 4 cases of normal lung tissues showed significant increase in ADC (p < 0.008). (B) The
protein levels of MetAP2 by Western blot in 3 cases of ADC compared to 3 cases of normal lung tissues showed significant increase in ADC (p < 0.05). (C) The mRNA levels of MetAP2 by RT-PCR in 3 cases of SCC compared to 3 casesof normal lung tissues showed significant increase in SCC (p < 0.002). (D) The protein levels of MetAP2 by Western
blot in 3 cases of SCC compared to 3 cases of normal lung tissues showed significant increase in SCC (p < 0.05)−13−

病態解析学部門

Semi-Quantitative RT-PCR
and Western Blot Analysis
of MetAP2 in Normal Lung
and NSCLC Tissues

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H. Shimizu et al.

Fig. 4. Inhibition of MetAP2 by addition of fumagillin in WDSCC and MDSCC cell cultures. (A)Immunofluorescence staining of MetAP2 in untreated and treated (plus fumagillin, 1 µg/mL) MDSCC cell culture
showed decrease in fluorescence intensity in treated cells (magnification ×200). (B) Caspase-3 fluorometric assay after
exposure of MDSCC cells to fumagillin. Both well-differentiated SCC and moderately-differentiated SCC cells showedsignificantly increased caspase-3 activity. (C) Cell death effect using propidium iodide stain and flow cytometry on
MDSCC cells. Both WDSCC and MDSCC cells showed significantly increased cell death by propidium iodide staining

increased in fumagillin-treated well and moderately differentiated SCC cells (Fig. 4B) (p < 0.028
and p < 0.001, respectively) compared to no-treatment control SCC cells. In the moderately differentiated SCC cells, caspase-3 activity was moreincreased than in the no-treatment SCC control
cells (Fig. 4B). In addition, regarding cell death effect, the fumagillin-treated well and moderately
differentiated SCC cells showed significantly increased cell death (Fig. 4C; p < 0.003 and p < 0.001,
respectively).

Discussion
Despite aggressive therapy, patients with advanced stage NSCLC demonstrate a poor survival with significant long-term morbidity in disease
survivors. High-risk disease features are strongly
correlated with tumor vascularity, suggesting thatangiogenesis inhibitors may be a useful addition to
current therapeutic strategies [24].
In the past, NSCLCs were seen together without paying attention to the more specific molecularpathological types. This was thought to be appropriate, because different therapeutic procedures
were not available for the treatment of the subtypes
of NSCLC such as ADC and squamous cell carcinomas. Since the time the first special EGFR mutation was identified, the situation has changed and−14−

nowadays this rapidly evolving field provides new
results [25]. EGFR mutations have been reported
as a predictive factor for favorable prognosis of gefitinib-treated patients with lung ADC. Patient’s
sex and smoking status were not significantly associated with longer overall survival and progression-free survival according to EGFR mutation
status [26]. Patients with advanced NSCLC who
were selected on the basis of EGFR mutations improved progression-free survival with acceptabletoxicity but those with no EGFR mutation did not.
In all living cells, protein synthesis is initiated with either methionine (in the cytosol of eukaryotes) or formylmethionine. MetAP activity is essential for cellular growth and viability.
In yeasts, knockout of either MetAP1 or MetAP2
causes a decrease in growth rates while elimination of both genes is lethal, indicating that the two
MetAPs play essential functions and are together
essential for yeast proliferation [27, 28]. MetAP2
has attracted more attention than MetAP1 by thediscovery of MetAP2 as a target molecule of theanti-angiogenic compounds, fumagillin and ovalicin [13]. A novel MetAP2 inhibitor, fumagillin,
strongly inhibits the growth of human colon cancer HT29 cells, melanoma B16F10 cells and neuroblastoma CHP-134 cells [29–31]. MetAP2 is themolecular target of angiogenesis inhibitors, such as
fumagillin, which can also inhibit cancer cell proliferation, implying that MetAP2 may play a quite

125

Methionine Aminopeptidase in Lung Cancer

compared to normal lung tissues (p = 0.008). Also,
three cases of squamous cell carcinomas had significant upregulation of the MetAP2 gene as compared to normal lung tissues (p = 0.002). In sevencases of NSCLC, MetAP2 mRNA expression levels
were upregulated as compared to normal lung tissues (p = 0.001) (data not shown). Also, the levelsof MetAP2 in three cases of ADC and three cases of squamous cell carcinomas showed significant differences in the quantitative extent of protein expression compared to normal lung tissues(p = 0.05, respectively). MetAP2 proteins were increased in ADC and SCC cases compared to normal lung tissues, according to Western blot analysis. Higher expression of the MetAP2 protein inhuman cancers further supports the contention
that MetAP2 plays a role in cancer development.
We saw moderate-to-strong staining of MetAP2
in all ADC cases examined [37]. S100a4 protein isa calcium-binding agent that regulates tumor metastasis and a variety of cellular processes via interaction with different target proteins, including
MetAP2, a main regulator of the proliferative andapoptotic pathways in mesothelioma cells [33, 34].
Interestingly, the normal bronchial epithelium showed a weak degree of staining; therefore,
the frequent MetAP2 expression in NSCLC seems
to be categorized as an aberrant expression. Thisfinding correlates to the finding that colorectal
normal mucosa far from the cancer shows a mild
degree of MetAP2 staining [11].
The frequent and aberrant MetAP2 expressions in biliary epithelial cells might have acquired MetAP2 through their dysplasia-carcinoma
sequence. Positive MetAP2 expression was observed in a small population of non-dysplastic biliary epithelial cells. Thus, it has been reported that
MetAP2 is a novel biomarker for the early detection of cholangiocarcinoma [19].
In the present study, we asked whether MetAP2is activated and expressed in human NSCLC tissues or inactivation of MetAP2 activity with fumagillin (an angiogenesis inhibitor) may potentiallyinhibit proliferation of lung cancer cell lines.
The expression level of MetAP2 was significantly higher in the 6 cases of SCC than 4 cases of
ADC. Also, semi-quantitative MetAP2 mRNA andprotein levels showed higher expression in SCC
than in ADC. Thus, we chose the SCC cell lines
to assess the effect of fumagillin as an angiogenesis inhibitor on cell proliferation activity and cell
death population using caspase-3 flow cytometric analysis. The immunofluorescence intensity of
MetAp2 in the moderately differentiated SCC cellline was decreased after treatment with fumagillinfor 24 h (Fig. 4A) and caspase-3 activity was markedly increased (Fig. 4B; p = 0.001) as compared to−15−

病態解析学部門

complex role in tumor progression under which
MetAP2 enzyme activity is inactivated through covalent modification [32, 33].
Several studies have demonstrated that themetastasis-associated gene product S100A4 interacts with the angiogenesis-related protein MetAP2.
Other studies also support this conclusion [34].
Recently, small molecule protein p67/MetAP2 wasshown to have a greater affinity toward ERK1/2 kinases, and its N-terminal p26 segment will mask
the phosphorylation sites on ERK1/2 to block theactivation and activity of ERK1/2. This will thenlead to inhibition of the cell cycle activated throughthe growth factor-mediated cell signaling pathway
and thus cell growth and proliferation [35].
Major components of the cell signaling pathways, the ras/mitogen-activated protein kinase(MAPK) systems are altered in lung cancer cellsby oncogenes through overexpression or mutation, leading to dysregulated cell signaling and cellproliferation [36]. It has been reported that NCIH-460 (large cell carcinoma), H1299, A549 (ADC)
and Calu6 (anaplastic carcinoma) cell lines cause
inhibition of tumor cell growth on three distinct
chemical classes of MetAP2 inhibitors as follows:
TNP-470, A800141, and A-357300.
This data prompted us to examine the possible new treatment roles of MetAP2 in lung cancers. There was no significant association of low
and high expression of MetAP2 and clinical features (tumor size, tumor cell differentiation, nodal metastasis and staging) (Table 1). But median survival time of the low and high expressionof MetAP2 disclosed a significant difference(Table 1, p = 0.043). Survival rate was more increased in low MetAP2 expression patients (85.7%)
than high MetAP2 expression patients (55%).
MetAP2 may play quite a complex role in tumor
progression, which inactivates MetAP2 enzyme
activity through covalent modification [32, 33].
Additionally, we measured the spectrophotometric determination of the hydrolysis of methionine p-nitroanilide in their soluble and membrane-bound forms in surgically removed lungcarcinoma tissues and unaffected adjacent normal
surrounding tissues. Total average aminopeptidase
activity was 80.6 ± 13.4 in normal lung tissue versus 156.9 ± 20.8 in lung carcinoma tissue (Table 2).
Compared to normal lung tissue, carcinoma tissue
had significantly higher activities. Histologically,
SCC demonstrated higher MetAP2 activity than
ADC. These results were further confirmed by thesemi-quantitative RT-PCR analysis of MetAP2mRNA expression in normal lung and NSCLC
tissues. The mRNA levels of MetAP2 in four cases of ADC showed significant differences in thequantitative extent of MetAp2 gene expression as126

H. Shimizu et al.

well-differentiated SCC cells (Fig. 4B; p = 0.028).
In addition, the cell death effect was also increasedin fumagillin-treated moderately differentiated SCC
cells (Fig. 4C; p = 0.001) as compared to well-differentiated SCC cells (Fig. 4C; p = 0.003). These
results indicate that fumagillin as an angiogenesis
inhibitor has potentially inhibited lung cancer cellproliferation.
The mechanism by which inhibition of MetAP2
might lead to growth inhibition may be throughboth tumor cell intrinsic and extrinsic mechanisms. A defect in the removal of N-terminal methionine caused by metAP2 inhibition might lead
to aberrant levels of proteins important for cellproliferation and apoptosis [33]. In a mechanismthat remains to be completely elucidated, inhibition of MetAP2 by small molecule inhibitors led
to the transcriptional activation of p53, which inturn activates the expression of p21 that inhibits
cycline E/Cdk2, accounting for the cell cycle blockade by these inhibitors [38]. Fumagillin is likewisea eukaryotic initiation factor 2-associated glycoprotein, p67. Fumagillin increases the stability of
p67 and affinity to ERKs 1 and 2 and causes theinhibition of the phosphorylation of ERKs 1 and 2[38, 39]. MetAP2 inhibitors IDR-803, IDR-804,
IDR-805 and CDK-732, as well as fumagillin analogs, strongly inhibit the growth of cancers ina model of nude mouse xenograft. Inhibition ofangiogenesis is emerging as a promising strategy for the treatment of cancer [41]. Choosing themost appropriate time of day for TNP-470, a synthetic analogue of fumagillin, administration willaid in the treatment of tumors.

The transcription of MetAP2 mRNA is regulated by clock gene proteins of the mCLOCK:mBMAL1 heterodimer in sarcoma180-bearing
mice [42]. Interestingly, MetAP2 was expressedin many cell types, including fibroblasts in idiopathic pulmonary fibrosis. In the bleomycin induced acute lung injury in mice, fumagillin attenuated the deposition of collagen [43]. This finding
was further confirmed when it was found that PPl-2458, a member of the fumagillin class of irreversible MetAP2 inhibitors, potently inhibits the proliferation of human fibroblast-like synoviocytes
derived from rheumatoid arthritis in the late G1phase of the cell cycle [44]. Further, we report thefirst study on MetAP2 expression and function in
NSCLC tissues and cell lines.
A high MetAP2 mRNA and protein expression as well as activity was found in NSCLC tissues
and cell lines and the expression of MetAP2 correlated with patient’s outcome. The higher concentration of MetAP2 in NSCLC tissues than thecorresponding normal bronchial epithelial cells
suggests a greater dependence on this enzyme bymalignant cells for their function and proliferation. Hence, a reduction in the enzyme activity
may be more harmful to cancer cells than normal
epithelial cells. The evaluation of specific inhibitors of MetAP2 in animal models should provide
justification for future selection and evaluation of
MetAP2 inhibitors in clinical trials for NSCLC patients. MetAP2 is an important regulator of proliferative and apoptotic pathways and its inhibition
may provide a potential therapeutic intervention
for lung cancer.

Acknowledgements. The authors would like to thank Dr. Kiyoshi Koizumi, Division of Thoracic Surgery, Departmentof Surgery, Aizu Chuo Hospital, Fukushima, who helped with providing NSCLC patient samples and their medical records data.

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[33] Catalano A, Romano M, Robuffo I, Strizzi L, Procopio A: Methionine aminopeptidase-2 regulates human mesothelioma cell survival: role of Bcl-2 expression and telomerase activity. Am J Pathol 2001, 159, 721–731.
[34] Endo H, Takenaga K, Kanno T, Satoh H, Mori S: Methionine aminopeptidase 2 is a new target for the metastasis-associated protein. S100A4. J Biol Chem 2002, 19, 26396–26402.
[35] Datta B: Roles of P67/MetAP2 as a tumor suppressor. Biochim Biophys Acta 2009, 1796, 281–292.
[36] Dy GK, Adjei AA: Novel targets for lung cancer therapy: part I. J Clin Oncol 2002, 15, 2881–2894.
[37] Tucker LA, Zhang Q, Sheppard GS, Lou P, Jiang F, McKeegan E: Ectopic expression of methionine aminopeptidase-2 causes cell transformation and stimulates proliferation. Oncogene 2008, 27, 3967–3976.
[38] Lu J, Chong CR, Hu X, Liu JO: Fumarranol, a rearranged fumagillin analogue that inhibits angiogenesis in vivo.
J Med Chem 2006, 49, 5645–5648.
[39] Datta B, Majumdar A, Datta R, Balusu R: Treatment of cells with the angiogenic inhibitor fumagillin results inincreased stability of eukaryotic initiation factor 2-associated glycoprotein, p67, and reduced phosphorylation of
extracellular signal-regulated kinases. Biochemistry 2004, 43, 14821–14831.
[40] Datta B: Roles of P67/MetAP2 as a tumor suppressor. Biochim Biophys Acta 2009, 1796, 281–292.
[41] Chun E, Han CK, Yoon JH, Sim TB, Kim YK, Lee KY: Novel inhibitors targeted to methionine aminopeptidase 2(MetAP2) strongly inhibit the growth of cancers in xenografted nude model. Int J Cancer 2005, 10, 124–130.

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Address for correspondence:
Hajime Shimizu
Department of Molecular Pathology
Institute of Gerontology
Nippon Medical School
Nakahara-ku
Kosugi-cho
KawasakiJapan 211-8533
Tel.: +81 44 733 1823
E-mail: hirstone@nms.ac.jp
Conflict of interest: None declared
Received: 1.12.2014
Revised: 24.03.2015
Accepted: 24.11.2015−18−

Ⅱ．細胞生物学部門
Department of Biochemistry and Cell Biology

細胞生物学部門（大学院

細胞生物学部門）
教授

太田

成男

研究室の特色大学院医学研究科細胞生物学分野（先端医学研究所細胞生物学部門）では最新のバイオテクノロジーを駆使して、老化や病気の原因を細胞の中から分子レベルで探り、それを応用できるところまで到達しようとしている。細胞内小器官のミトコンドリアはエネルギー産生だけでなく様々なプラスの機能を持ち、一方、マイナス面としては、活性酸素を発している。エネルギー代謝を促進し、活性酸素の弊害を抑制することで、多くの疾患の予防と治療に貢献し、ひいては老化の抑制に貢献できるものと考えている。
さらに、研究を発展させて、分子状水素を様々な疾患の予防と治療に貢献しようとしている。
研究が本当の意味で社会に役立つためには、しっかりとした基礎が必要である。また、科学は人間の知的欲求を満たしてくれる。そして、科学の成果は私たちの生活の質を向上させる。科学は「知」と「実」を与えてくれる。研究成果を実用化されるよう努めることも私たちの責務である。
今年度の主な研究成果は以下のようである。
（1）分子状水素はフリーラジカル連鎖反応で生じる脂質メディエーターを調節して遺伝子発現を制御する
Iuchi,･ K.,･ Imoto･ A.,･ Kamimura･ N.,･ Ichimiya･ H.,･ Yokota･ T.,･ Ohta･ S.:･ Molecular･ hydrogen･ regulates･
gene･ expression･ by･ modifying･ the･ free･ radical･ chain･ reaction-dependent･ generation･ of･ oxidized･
phospholipid･mediators.･Sci･Rep.･2016･Jan･7;6:18971.･doi:･10.1038/srep18971.
分子状水素が、遺伝子発現を制御するメカニズムを初めて解明した。分子状水素は脂質フリーラジ
カル連鎖反応に干渉し、脂質メディエーターを改変し、その脂質メディエーターがカルシウムシグナ
リングを制御し、遺伝子発現を制御するものである。
（2）水素分子は、脂肪酸代謝を向上させるために、転写活性化補助因子 PGC-1α の遺伝子の発現を刺激する
Kamimura･N,･Ichimiya･H,･Iuchi･K,･Ohta･S.:･Molecular･hydrogen･stimulates･the･gene･expression･of･transcriptional･coactivator･PGC-1α･to･enhance･fatty･acid･metabolism.･npj･Aging･and･Mechanisms･of･
Disease.･2016;2:16008･DOI:10.1038/npjamd.2016.8
分子状水素は、脂質代謝を促進するメカニズムを解明した。分子状水素は、脂質ラジカル連鎖反応を抑制し、過酸化脂質を低下させる。過酸化脂質から生成させる４—ヒドロキシノネナール（4-HNE）を低下させる。4-HNE がシグナル分子として機能して、PGC-1αを増加させ、脂質代謝関連遺伝子発現を亢進させるというものである。
（3）ラットの網膜における一酸化窒素由来ペルオキシナイトライトによって引き起こされる酸化ストレスに対する水素分子の保護効果
Yokota･T,･Kamimura･N,･Igarashi･T,･Takahashi･H,･Ohta･S,･Oharazawa:･Protective･eff･ect･of･molecular･hydrogen･ against･ oxidative･ stress･ caused･ by･ peroxynitrite･ derived･ from･ nitric･ oxide･ in･ rat･ retina.･
Clin･Experiment･Ophthalmol.･2015･Aug;43(6):568-77.

−21−

細胞生物学部門【研究概要】

（4）文脈性恐怖条件付後の急性拘束ストレスによる恐怖記憶の低減：タイミングの重要性
Uwaya A, Lee H, Park J1, Lee H, Muto J, Nakajima S, Ohta S, Mikami T: Acute immobilizationstress following contextual fear conditioning reduces fear memory: timing is essential. Behav Brain
Funct. 2016 Feb 24;12(1):8. 10.1186/s12993-016-0092-1.
学会・社会活動
ミトコンドリア学会機関誌である国際誌「Mitochondrion」の編集委員も引き続き務めている。また、
Nature Partner Journal Aging and Mechanism of Disease が発刊され、Editorial Board となった。
今年度は分子状水素医学シンポジウムから、日本分子状水素医学生物学会を設立し、初代の理事長を務めている。日本ミトコンドリア学会は理事、日本 Cell Death 学会では評議員も務めている。

〈原著論文〉
1. Nicolson, GL.1）, Mattos, GF.2）, Settineri, R.3）, Costa, C.2）, Ellithorpe, R.4）, Rosenblatt, S.5）, Valle, JL.6）,
Jimenez, A.7）, Ohta, S.（1）Dept. Mol. Pathol,, The Inst. Mol. Med., Huntington Beach, USA., 2）Lab.
Ion Channels, Sch. Med., Univ. de la República, Montevideo, Uruguay., 3）Sierra Res., Irvine, USA.,  ･
4）

Tustin Longevity Ctr., Tustin, USA., 5）Saint John's Health Ctr., Santa Monica, USA. 6）ProgressiveMed. Ctr., Orange, USA., 7）Hope Cancer Inst., Playas de Tijuana, Mexico.）: Clinical Effects ofHydrogen Administration:From Animal and Human Diseases to Exercise Medicine. Int J Clin
Med.2016;7:32-76.
2.  Iuchi, K., Imoto, A., Kamimura, N., Ichimiya, H., Yokota, T., Ohta, S.: Molecular hydrogen regulates
gene expression by modifying the free radical chain reaction-dependent generation of oxidized
phospholipid mediators. Sci Rep. 2016; 6:18971.
3.  Kamimura, N., Ichimiya, H., Iuchi, K., Ohta, S.: Molecular hydrogen stimulates the gene expression oftranscriptional coactivator PGC-1α to enhance fatty acid metabolism. npj Aging and Mechanismsof Disease. 2016;2:16008.
4.  Hayashida, K.1）, Sano, M.1）, Kamimura, N., Yokota, T., Suzuki, M.2）, Ohta, S., Fukuda, K.1）, Hori, S.2）.
（1）Dept. Cardiol., Sch. Med., Keio Univ., 2）Dept. Emergency and Critical Care Med., Sch. Med., Keio
Univ.）: Response to Letter Regarding Article, "Hydrogen Inhalation During Normoxic Resuscitation
Improves Neurological Outcome in a Rat Model of Cardiac Arrest Independently of Targeted
Temperature Management". Circulation. 2015;132（11）:e148.
5.  Yokota, T., Kamimura, N., Igarashi, T.1）, Takahashi, H. 1）, Ohta, S., Oharazawa, H. 2）.（ 1）Dept.
Ophthalmol., 2）Dept. Ophthalmol., Musashikosugi Hosp.）: Protective effect of molecular hydrogen
against oxidative stress caused by peroxynitrite derived from nitric oxide in rat retina. Clin
Experiment Ophthalmol. 2015;43（6）:568-77.
6.  Kanamaru, T., Kamimura, N., Yokota, T., Nishimaki, K., Iuchi, K., Lee, H., Takami, S.1）, Akashiba,H.1）, Shitaka, Y.1）, Ueda, M.2）, Katsura, K.2）, Kimura, K.2）, Ohta, S.（1）Pharmacol. Res. Lab., Astellas
Pharma Inc., 2）Dept. Neurological Sci.）:Intravenous transplantation of bone marrow-derived
mononuclear cells prevents memory impairment in transgenic mouse models of Alzheimer's
disease. Brain Res. 2015;1605:49-58.

−22−

7.  Uwaya, A., Lee, H., Park, J.1）, Lee, H.2）, Muto, J.3）, Nakajima, S.4）, Ohta, S., Mikami, T.5）.（1）Dept. Lab.
Med., The Jikei Univ. Sch. Med., 2）Dept. Cell Biol. Neurosci., Juntendo Med. Sch., 3）Grad. Sch. Healthand Sport Sci., Nippon Sport Sci. Univ., 4）Kyoritsu Women's Junior College, 5）Dept. Health and Sport
Sci.）：Acute immobilization stress following contextual fear conditioning reduces fear memory:timing is essential. Behav Brain Funct. 2016;12（1）:8.
8.  Iuchi, K., Yagura, T.1）.（1）Dep. Biosci., Sch. Sci. Technol., Kwansei Gakuin Univ.）: DNA binding
activity of Ku during chemotherapeutic agent-induced early apoptosis. Exp Cell Res. 2016;342（2）:135-44.

1. Ohta, S.: Molecular hydrogen as a novel antioxidant: overview of the advantages of hydrogen for
medical applications. Methods Enzymol. 2015;555:289-317.  
2. 太田成男：分子状水素医学：広範な疾患治療と予防医学の応用へ向かって 臨床麻酔 2015:39(6):832-9.
3. 太田成男：ミトコンドリアとエイジング（老化） アンチエイジング医学の基礎と臨床・第 3 版

メジ

カルルレビュー 編集：日本抗加齢医学会 専門医指導子認定委員会 2015; 80-81.
4. 太田成男：水素医学の創始、展開、今後の可能性：広範な疾患に対する分子状水素の予防ならびに治療の臨床応用へ向かって 生化学 2015;87(1)82-90.
5. 太田成男：医療への水素利用 ケミカルエンジニアリング 化学工業社 2015;60(3)42(206)-8(12).
6. 早坂理恵（太田成男：監修）水素美容のひみつ 産学社 2015. 5:1-112.

〈海外講演〉
1. Ohta, S.: Molecular Hydrogen as a Novel Antioxidant: Overviews of the Advantages of Hydrogen for
Medical Applications. European Pharma Global Congress 2015. Valencia, Spain

2015. 08. 25-27.

2. Kamimura, N., Nishimaki, K., Ichimiya, H., Ohta, S.: Molecular hydrogen improves obesity anddiabetes by regulating hepatic gene expression and stimulating energy metabolism in db/db mice.
The 2015 Gordon Research Conference on Biology of Aging,  Sunday River, USA, 2015. 07. 19-24.
3. Nishimaki, K., Yokota, T., Kamimura, N., Ohsawa, I., Ohta,

S.: Drinking effect of hydrogen water  

prevents progression of Alzheimer's disease. The 2015 Gordon Research Conference on Biology ofAging,  Sunday River, USA, 2015. 07. 19-24.

〈一般学会講演〉1. 上村尚美，Wolf Alexander，一宮治美，井内勝哉，西槙貴代美，横田隆，太田成男：糖尿病モデル
マウスの in vivo 酸化還元状態の測定と分子状水素の効果．日本分子生物学会年会（第 38 回）日本
生化学会大会（第 88 回）合同大会．2015.12.1-4.
2. 井内勝哉，井本明美，西槙貴代美，一宮治美，Lee Hyunjin，横田隆，上村尚美，太田成男 : 分子状
水素はフリーラジカル連鎖反応で生じる脂質メディエーターを調節して遺伝子発現を制御する．日
本分子生物学会年会（第 38 回）日本生化学会大会（第 88 回）合同大会．2015.12.1-4.
3. 横田隆，上村尚美，五十嵐勉，高橋浩，小原澤英彰，太田成男：ペルオキシナイトライト産生酸化
ストレス傷害に対する水素分子（H2）のラット網膜保護効果．日本分子生物学会年会（第 38 回）日
本生化学会大会（第 88 回）合同大会．2015.12.1-4.
4.

上村尚美、Alexander M Wolf、一宮治美、井内勝哉、西槙貴代美、横田隆、太田成男 : 糖尿病モデ
ルマウスにおけるミトコンドリアの in vivo 酸化還元状態の測定と分子状水素の効果
ミトコンドリア学会

福井 ( 福井県国際交流会館 )−23−2015.11.19—20.

第 15 回日本

細胞生物学部門〈総説〉

5. 井内勝哉，井本明美，西槙貴代美，一宮治美，Lee Hyunjin，横田隆，上村尚美，太田成男：TBHP を用いた酸化ストレス誘導性の細胞死に対する分子状水素の保護作用，第 24 回日本 CellDeath 学会学術集会，2015.7.11-12.
6. 中嶋裕也、太田成男、Wolf Alexander:   ブルーライト照射による皮膚内での酸化ストレス誘導
40 回日本香粧品学会

東京（有楽町朝日ホール）2015.6.19（会期 6.18-19）

7. 太田成男：水素医学研究の最新情報

アンチエイジング歯科学会 2015.5.16（会期 5.16-17）

−24−

第

www.nature.com/scientificreports

received: 13 July 2015
Accepted: 02 December 2015
Published: 07 January 2016

Molecular hydrogen regulates
gene expression by modifying
the free radical chain reactiondependent generation of oxidized
phospholipid mediators

細胞生物学部門

OPEN

Katsuya Iuchi1,*, Akemi Imoto1,*,†, Naomi Kamimura1,*, Kiyomi Nishimaki1,*,
Harumi Ichimiya1, Takashi Yokota1 & Shigeo Ohta1,2
We previously showed that H2 acts as a novel antioxidant to protect cells against oxidative stress.
Subsequently, numerous studies have indicated the potential applications of H2 in therapeutic andpreventive medicine. Moreover, H2 regulates various signal transduction pathways and the expressionof many genes. However, the primary targets of H2 in the signal transduction pathways are unknown.
Here, we attempted to determine how H2 regulates gene expression. In a pure chemical system, H2gas (approximately 1%, v/v) suppressed the autoxidation of linoleic acid that proceeds by a free radical
chain reaction, and pure 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphocholine (PAPC), one of the
major phospholipids, was autoxidized in the presence or absence of H2. H2 modified the chemical
production of the autoxidized phospholipid species in the cell-free system. Exposure of cultured cells to
the H2-dependently autoxidized phospholipid species reduced Ca2+ signal transduction and mediatedthe expression of various genes as revealed by comprehensive microarray analysis. In the cultured
cells, H2 suppressed free radical chain reaction-dependent peroxidation and recovered the increased
cellular Ca2+, resulting in the regulation of Ca2+-dependent gene expression. Thus, H2 might regulate
gene expression via the Ca2+ signal transduction pathway by modifying the free radical-dependent
generation of oxidized phospholipid mediators.
Molecular hydrogen (H2) was originally thought to behave as an inert gas in mammalian cells; however, ourprevious studies showed that this is not always the case1, demonstrating that H2 neutralizes the hydroxyl radical(·OH) and peroxynitrite (ONOO–) inside cells and acts as a novel antioxidant to protect the cells against oxidative stress1,2. Inhalation of 1%–4% (v/v) H2 gas is effective for the treatment of ischemia/reperfusion injuries1,3,4.
Recently, inhalation of 1.3% H2 gas from a premixed gas was shown to protect neurons in a cardiac arrest model5.
However, the mechanism of how such a low concentration of H2 exerts the positive effects is not known.
Numerous studies have strongly suggested that H2 has the potential for a variety of therapeutic and preventive
applications6,7. In addition to extensive animal experiments, more than 10 clinical studies examining the efficacyof H2 have been reported6,7, including double-blinded clinical studies in patients with Parkinson’s disease andrheumatism8,9. Based on these studies, the field of hydrogen medicine is rapidly growing.
Subsequently, H2 was shown to exhibit multiple functions, including anti-inflammatory, anti-apoptotic,
anti-allergic, and antioxidant activities, as well as regulation of differentiation and energy metabolism6,7. To exert
multiple functions in addition to anti-oxidative roles, H2 regulates various signal transduction pathways andthe expression of many genes6,7. For examples, H2 protects neural cells and stimulates energy metabolism by
1

Department of Biochemistry and Cell Biology, Graduate School of Medicine, Nippon Medical School, 1–396 Kosugimachi, Nakahara-ku, Kawasaki-city, Kanagawa 211–8533, Japan. 2Department of Neuroregenerative Medicine,
Juntendo University Graduate School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113–8421, Japan. *Theseauthors contributed equally to this work.†Present address: Department of Clinical Chemistry, School of Allied
Health Sciences, Kitasato University, 1-15-1 Kitasato, Minami-ku, Sagamihara-city, Kanagawa 252-0373, Japan.
Correspondence and requests for materials should be addressed to S.O. (email: ohta@nms.ac.jp)

1

Scientific RepoRts | 6:18971 | DOI: 10.1038/srep18971−25−

www.nature.com/scientificreports/***

Octanoic acid

***

2

***

1

0

30
60
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**

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PBS

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***

***

***

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a

**

0.3

PBS
0

Octanoic acid
030
60Time (min)Linolenic acid

120
0.1

0

30

60
90
Time (min)120

Figure 1. Solubility of H2 in fatty acids in the presence of an aqueous solvent. H2-saturated phosphatebuffered saline (PBS) was mixed with the same volume (75 mL) of saturated fatty acid (octanoic acid) (a) or
unsaturated fatty acid (linolenic acid) (b) and maintained for 16 h in a closed aluminum bag as describedin Methods. The same volume (3 mL) of each phase was transferred to each open tube (φ 13 mm), followedby measurement of H2 at the indicated time (n = 4). The experiments were performed at 25 °C. ***P < 0.001
vs. PBS. (a,b) Significance was calculated using an unpaired two-tailed Student’s t-test. (c) Time courses ofretention times of H2 in each phase in the open vessels. **P < 0.01, vs. octanoic acid (n = 4).

stimulating the hormonal expression of ghrelin10 and fibroblast growth factor 21 (FGF21)11, respectively. In contrast, H2 relieves inflammation by decreasing pro-inflammatory cytokines12. However, it is difficult to explain themolecular mechanisms by which H2 exerts these functions by conventional concepts alone. To understand themolecular mechanisms by which H2 exerts these multiple functions, it is essential to identify the primary targetsof H2 that modulate signal transduction and gene expression.
Therefore, in this study, we aimed to elucidate one of the molecular mechanisms by which H2 mediates signaltransduction and gene expression. Our results suggested that low concentrations of H2 modulated Ca2+ signaltransduction and regulated gene expression by modifying the production of oxidized phospholipid species.

Results

H2 accumulated in the lipid phases.

To understand the difference between intracellular conditions andaqueous solutions, we focused on the lipid phases to determine the intracellular localization of H2 accumulation
at room temperature. H2 incorporation was two- or three-fold higher in the liquid fatty acid phases than in the
aqueous phase in the presence of both water and fatty acids, and was retained longer in the fatty acid phases thanin the aqueous phase in open vessels (Fig. 1a,b). In particular, H2 seemed to be retained significantly longer inthe unsaturated fatty acid (linolenic acid) than in the saturated fatty acids (octanoic acid) (Fig. 1c), although thisdifference in retention time might be attributed to the difference in the number of carbons. Since unsaturated
fatty acids are the primary targets for initiating a free radical chain reaction, we assumed that H2 could efficiently
suppress this reaction in biomembranes, even at low concentrations.

Autoxidation of unsaturated fatty acids was suppressed by low concentrations of H 2
gas. Autoxidation of unsaturated fatty acids proceeds by a free radical chain reaction in air13. Thus, we meas-

ured autoxidation of a filmy di-unsaturated fatty acid (linoleic acid: R-CH = CH-CH2-CH = C-R’) at 37 °C for
20 h in the dark in the presence of various concentrations of H2 gas. A conjugated diene, which should be formedby autoxidation, was estimated by the absorption at 234 nm (Fig. 2a). The absorption at 234 nm was increased
depending on the formation of the conjugated diene [R-CH = CH-CH = CH-CH(-OOH)-R’ or R-CH(-OOH)CH = CH-CH = CH-R’] accompanied by peroxidation in a pure chemical system (H2, O2, and N2 were supplied
from gas cylinders) (Fig. 2b). O2 was essential for autoxidation (Fig. 2c). As a result, even only approximately 1%
H2 gas significantly suppressed autoxidation of linoleic acid at 37 °C, even in the absence of any catalysts in thedark in a pure chemical system (Fig. 2c).

Ca2+ signal transduction by H2-dependent chemical oxidation of phospholipids. Phospholipids

are converted into oxidized mediators that modulate various signal transduction pathways by not only enzymatic
reactions, but also by chemical oxidation14,15. Oxidized phospholipids, including 1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphocholine (POVPC) and 1-palmitoyl-2-glutaroyl-sn-glycero-3-phosphocholine (PGPC),
are present in oxidatively modified low-density lipoproteins (oxLDLs) and have been found in atherosclerotic
lesions15. These compounds are important as inducers of different cellular responses, including inflammation,
proliferation, and cell death. Moreover, autoxidation of 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphocholine(PAPC) leads to the chemical production of various bioactive phospholipid species, such as POVPC, PGPC,
1-palmitoyl-2-(5-hydroxy-8-oxooct-6-enoyl)-sn-glycero-3-phosphocholine (HOOA-PC), and 5-hydroxy-8-oxo6-octenedioic acid (HOdiA-PC)14,15.
We assumed that low concentrations of H2 would influence some chemical reactions leading to the productionof putative oxidized lipid mediators for the modulation of signal transduction. Because PAPC is one of the major
phospholipids in mammalian biomembranes, the role of H2 in the chemical production of oxidized phospholipid

2

Scientific RepoRts | 6:18971 | DOI: 10.1038/srep18971−26−

www.nature.com/scientificreports/cno oxidation
H2 0%
H2 1%
H2 80%100
Δ A234 (%)

ΔA234

ΔA234

160μM linolic acid
+5μM HODE+10μM HODE
+20μM HODE

80
60
40

200 220 240 260 280 300
Wavelength (nm)

200 220 240 260 280 300
Wavelength (nm)0H2 (%)
O2 (%)

細胞生物学部門

20

no te
atme
nt

b

a

100 0 0.1 0.3 1
3 10 80
0 20 20 20 20 20 20 20

Figure 2. Suppression of autoxidation of linoleic acid-film by H2 gas. (a) Profile of ultraviolet absorption of
9-hydroxyoctadecadienoic acid; CH3(CH2)4-CH = CH-CH = CH-CH(-OH)-(CH2)7-COOH) (9-HODE) in
cyclohexane, shown as a standard conjugated diene. (b) Linoleic acid-film was autoxidized at 37 °C for 20 h in a
glass tube placed in a closed aluminum bag in the presence of various concentrations of H2 and O2 as describedin Methods. Representative profiles of ultraviolet absorption of the cyclohexane solution of H2-dependent
autoxidized linoleic acid are shown. (c) Linoleic acids autoxidized with various concentrations of H2 were
evaluated by measuring absorption at 234 nm. *P = 0.034 (0.3% H2), #P = 0.069 (1% H2), **P < 0.01 (3% H2,
10% H2), and ***P < 0.001 (80% H2) vs. 0% H2 (n = 15).

mediators was determined by conducting autoxidation of pure PAPC (resulting in OxPAPC) in the absence ofany catalysts in the dark. The peroxidation of PAPC in air was confirmed by an increase in the signal for the
fluorescent dye specific to lipid peroxides, Liperfluo (Fig. 3a). A previous study indicated that OxPAPC activatestranscription factors involved in Ca2+ signaling16. Indeed, when THP-1 cells (a human monocytic cell line derived
from a patient with acute monocytic leukemia) were exposed to OxPAPC, a transient increase in cellular Ca2+
was observed when a Ca2+-sensitive fluorescent dye, Fluo4-AM was used (Fig. 3b). This Ca2+ signaling depended
on OxPAPC in an oxidation time-dependent manner (Fig. 3c).
Next, the H2-dependent production of OxPAPC, which leads to the activation of Ca2+ signaling, was investigated by autoxidizing PAPC for 3 days at 25 °C in air at various concentrations of H2 (designated as H2OxPAPC,
and the notation of H2[x%]OxPAPC was used when autoxidized in the presence of x% H2). H2 suppressed
the generation of total peroxides as revealed by Liperfluro fluorescence intensity (Fig. 3d). Ca2+ signaling wasobserved when PAPC was autoxidized with less than 0.3% H2, whereas more than 1.3% H2 significantly disrupted
this signaling (Fig. 3e).
In order to investigate the molecule(s) influenced by H2, we analyzed H2OxPAPCs by using mass spectrometry on autoxidation day 3. In all, 209 bands were detected, with molecular masses ranging from 126.3754 to
991.6494 Da; this was consistent with the findings of a previous report15 (Supplementary Fig. 1). The differencesin the production of H2OxPAPC and OxPAPC species were presented using a heat map (Supplementary Fig. 1i).
The levels of many bands were increased or decreased with differences in concentrations of H2. For examples
as the relatively increased species, the levels of the Ca2+ signaling inducers POVPC16, HOOA-PC, HOdiA-PC,
and hydroxyeicosatetraenoic acid-3-phosphocholine (HETE-PC)17 were slightly increased in response to H2(Supplementary Fig. 1i).
Because the reduced form of POVPC was reported to function as an antagonist18, it is possible that increased
levels of the reduced form(s) of some OxPAPC species, rather than the decreased levels of putative agonists (suchas POVPC), might have disrupted Ca2+ signaling as a putative antagonist(s). Further studies are warranted to
identify the H2-dependent bioactive mediator(s).

Comprehensive analysis of H2-dependent regulation of gene expression. Next, we investigatedhow H2OxPAPC influences gene expression. PAPC was autoxidized in the absence or presence of various concentrations of H2 for 3 days and then administered to cultured THP-1 cells. In a preliminary experiment, the change
in the expression level of tumor necrosis factor (TNF)-α gene in response to OxPAPC from that to H2OxPAPC
peaked at 4 h. Thus, by using microarray analysis, we comprehensively analyzed the change in gene expression
in response to the H2-dependent mediators at 4 h in three samples under each condition. In all, 86 genes wereselected according to the following criteria as described in the legend of Fig. 4a: a significant increase in OxPAPC(vs. PAPC), and a significant decrease in H2[1.3%]OxPAPC and H2[5%]OxPAPC (vs. OxPAPC) (Supplementary
Table 1). The gene expression profile was presented in a heat map (Fig. 4a). The selected genes were validated bysemi-quantitative real-time polymerase chain reaction (RT-PCR), and marginal changes in the expression levelsof some genes were confirmed (Supplementary Fig. 2).
In addition, the regulatory expression of TNF-α and IL-8 by H2OxPAPC was investigated using THP-1 and adifferent cell type (human aortic endothelial cells: HAEC), respectively (Fig. 4b,c).
According to the Kyoto Encyclopedia of Genes and Genomes (KEGG) Pathway Database (http://www.
genome.jp/kegg/pathway.html), the functions of 7,143 genes were identified and classified (Fig. 4d, upper). We
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a

d
100

120

80

100

60

80

40

60

20

40

1

2

3 4
5
time (day)OxPAPC

ATP

20

6

0

PA

e

20

50

15

Ca2+ response (%)

Fluorescent intensity (AU)

b

10
5
0

0 30

c
Ca2+ response (%)

**

PC
Ox
PA
PC
H2
Ox [0.3%
PA
]
H2 PC
[
Ox 1.3%
PA
]
H2 PC
[
2
Ox .5%
PA
]
PC

0

*

90
150
time(s)

210

40
30
20
10
0

60
50
40
30
20
10
0

0

1 2 3 4 5
incubation (day)

0

*

*

*

0.15 0.3 0.6 1.3
H2 gas (%)

5

80

6

Figure 3. PAPC autoxidized with H2 modulated Ca2+ signaling. (a) Chemically pure PAPC was autoxidized
in air with 100% humidity at 25 °C in a closed aluminum bag for the indicated periods, and time-dependent
production of peroxides in air from chemically pure PAPC was estimated using Liperfluo fluorescence, where
wavelengths of excitation and emission were set at 488 and 535 nm, respectively, as described in Methods. (b)Representative responses in THP-1 by OxPAPC with the fluorescent Ca2+ indicator Fluo4-AM are shown as
described in Methods. The arrow and arrowhead indicate the addition of OxPAPC and ATP, respectively. ATP(a ligand of the Ca2+ channel P2X7) was used as a positive control. (c) PAPC was autoxidized for the indicatedperiods at 25 °C, and subjected to the Ca2+-signaling assay in THP-1 cells. The OxPAPC-induced Ca2+ response
depended on autoxidizing period of OxPAPC. (d) PAPC was autoxidized in air for 3 days in the absence orpresence of the indicated concentrations of H2 (H2OxPAPC), and the peroxide of OxPAPC or H2OxPAPC wasestimated using Liperfluo as described in (a) (n = 3-6). *P = 0.044, **P < 0.01. (e) PAPC was autoxidized in air
for 3 days with the indicated concentrations of H2 (H2OxPAPC) and then subjected to Ca2+ signaling assays as
described in Methods (n = 6). *P = 0.021 (1.3%H2), *P = 0.022 (5% H2), and *P = 0.030 (80% H2) vs. no H2.

classified the 86 selected genes (Fig. 4e, lower). Of these 86 genes, 46.5% belonged to those involved in signaling
pathways (Fig. 4d, upper), whereas 25.8% of the total number of 7,143 genes is involved in signaling pathways(Fig. 4d, lower). Genes encoding factors involved in signal transduction and transcription factors are indicated byblue and black, respectively, on the right in the heat map (Fig. 4a).
Among the genes involved in signaling pathways, the proportion of those belonging to Ca2+ signaling werelower in the selected genes than in those in the entire genome, indicating that H2 regulates fewer componentsof the Ca2+ signaling pathways (Fig. 4e, lower). This was consistent with the finding that H2OxPAPC decreased
Ca2+ signaling. In contrast, the proportion of genes belonging to the mitogen-activate protein kinase (MAPK)

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**

3.0

**

2.0
1.0

PC
PA

Ox

c

PA
PC
H2
Ox [1.3%
PA
P ]
H2 C
Ox [5%]
PA
PC

0.0

2000
IL-8 (pg/ml)

HAEC

1500
1000

**

500
0

d

total genes

selected genes

1 2 4 8 16 32

ef

total genes

Ca 2+
Calcineurin

CaMK

TNF
PLK2
PTGS2(COXII)
IL-8
EDN1
HSPA1B
ATP2B4
CREB
FGF3
NFAT
FLNA
CCL4(MCP-1)
PMAP1
ID3
SOCS1
GADD45G
NFKB2
EGR-1
ATF3
JUNB
KLF2

**

PA
PC
Ox
PA
PC
H2
Ox [1.3%
PA
P ]
H2 C
[
5
Ox %]
PA
PC

PTGS2
HMOX1
ATP1B2
PLA2G4C
SOST
JUN
HSPA1APLK2
LILRB3
KLF2
HSPA1B
TUBB2AFOSBBTG2CAMKK1
CDKN1C
ID3
ASGR1
JUND
SOCS1
JUNB
SGK1
BEST2
ULBP2
ERO1LB
HIST2H2BF
SAT1
FIGF
AQP1
AGPAT4GADD45G
FGF3
RELB
RPS27
RASD1
12-Seq
IL4I1
IHH
CTSK
ATF3
SEMA6CBBC3
RASA4B
DGKA
CYP27A1
NXF1
HIST1H2AL
ATP2B4NFKB2NFATC2
ITGA10
SMG1
CD86
6-Sep
HIST1H4A
NUPR1
TOB1
SNORD3B-1
VAMP1
RASGRP2
FUT1
EDN1
CLK1
ITGAX
CPEB1
PMAIP1
LOC643802
DHDH
PABPC1L
HERC2
PPARG
CPT1B
CLCN2
RPL10
RPA4
SP100
PPP1R12B
PNPLA7FLNA
FBXO32FCAR
MAPK8IP3
SEMA3B

4.0

細胞生物学部門

0.25 0.5

N
FA
C T
R
si EB
gn
al
in
g

PA
PC
xP
AP
C
H
2[
1.
3%
H
]O
2[
5% xP
AP
]O
xP C
AP
C
O

gene

THP-1

5.0
TNF-alpha (pg/ml)baselected genes

signaling pathway
metabolic pathway
diseasesRNA
Proteinneuroninfection
OthersPI3K-Akt signaling pathway
MAPK signaling pathway
Ras signaling pathway
Rap1 signaling pathwaycAMP signaling pathway
Chemokine signaling pathway
Calcium signaling pathwaycGMP-PKG signaling pathway
Oxytocin signaling pathway
Wnt signaling pathway
FoxO signaling pathway
AMPK signaling pathway
TNF signaling pathway
Estrogen signaling pathway
Toll-like receptor signaling pathway
NF-kappa B signaling pathway
B cell receptor signaling pathway
PPAR signaling pathway
VEGF signaling pathway
OthersFigure 4. Changes in gene expression regulated by H2OxPAPC. (a) Three samples of PAPC, OxPAPC, andH2OxPAPC were exposed to THP-1 cells for 4 h , and the gene expression was comprehensively analyzed using
microarray. Eighty-six genes were selected according to the following criteria; genes up-regulated by OxPAPC(more than 2.5-fold, vs. PAPC) and those down-regulated by H2[1.3%]OxPAPC and H2[5%]OxPAPC (lessthan 0.75-fold and 0.5-fold, respectively, vs. OxPAPC) are shown in a heat map (red and green indicate the upregulation vs. PAPC treatment, and the down-regulation vs. OxPAPC treatment, respectively, as shown in thecolor gradient). Possible target genes of NFAT and CREB are marked with red on the right. Genes encoding
factors involved in signal transduction and transcription are indicated by blue and black, respectively, on theright. The release of TNF-α (b) (from THP-1) and IL-8 (c) (from HAEC) was investigated using ELISA as
described in Methods. (d, upper) Ratio of genes belonging to each category for a total of 7,142 genes identified
by the KEGG database. (d, lower) Ratio of genes belonging to each category in the 86 selected genes listed ina. (e, upper) Ratio of genes belonging to each signaling pathway identified by the whole KEGG database. (c,lower) Ratio of genes belonging to each signaling pathway in the selected genes listed in (a). (f) The H2OxPAPCdependent expression of genes transcribed by CREB and NFAT. Transcription factors are indicated in yellow.

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signaling was higher (Fig. 4e, lower), indicating that H2 regulates more components of MAPK signal transduction
pathways (Fig. 4e, lower).
The signal transduction pathways that were regulated by H2 are shown in Supplementary Table 1 according
to the KEGG Pathway Database. These data suggested the possibility that low concentrations of H2 contribute tovarious signal transduction pathways via oxidized phospholipid species.
cAMP response element binding protein (CREB)-target genes were selected according to the CREB Target
Gene Database (http://natural.salk.edu/CREB/), and nuclear factor of activated T cells (NFAT) target genes wereselected by referring to Medline, as shown in Supplementary Table 1. The target genes of CREB and NFAT aremarked by red on the right in the heat map panel as NFAT or CREB (Fig. 4a). A considerable number of theselected genes were targets of CREB or NFAT (Fig. 4 f). These data are consistent with the findings of previous
studies showing the Ca2+-dependent regulation by these transcription factors: CREB is activated via phosphorylation by a calmodulin-dependent kinase (CaMK)19 in a Ca2+-dependent manner, and NFAT is dephosphorylated
by calcineurin (CN) in a Ca2+-dependent manner, translocates to the nucleus, and then functions as a transcription factor with its partner proteins, e.g., activator protein 1 (AP-1), CREB, or nuclear factor-kappa B (NF-κ B)20.
Indeed, exposure of THP-1 to OxPAPC, but not to H2OxPAPC, stimulated the nuclear translocation of NFAT(Supplementary Fig. 3).
Thus, H2-dependent oxidized mediators or putative antagonists could be associated with transcriptional regulation via Ca2+ signaling.

Free radical inducers contributed to the NFAT pathway in cultured cells.

Autoxidation of unsaturated fatty acids, including PAPC, proceeds by a free radical chain reaction13, and·OH is the primary trigger forthis reaction13,21,22. We previously showed that H2 reduces ·OH levels inside cultured cells by using the spin trapping method and a specific fluorescent indicator1. Thus, in this study, we investigated the effects of H2 on the lipid
free radical chain reaction by using cultured cells. To initiate a free radical chain reaction inside the cells, we used
2,2′ -azobis(2-methylpropionamidine)dihydrochloride (AAPH)23, which is not affected by H2 (Supplementary
Fig. 4) and is suitable for the slow generation of free radicals by a spontaneous chemical reaction. The lipid free
radical chain reaction results in the production of lipid peroxides (LPOs)21,24, which can be detected using the fluorescent dye Liperfluo25. Thus, we exposed cultured THP-1 cells to AAPH and estimated LPO production basedon the Liperfluo signal. The Liperfluo signal significantly decreased in the presence of low levels of H2 gas (e.g.,
1.3%; Fig. 5a,b). Thus, even at such low concentrations, H2 has the potential to reduce the generation of LPOs by
suppressing the initiation and/or propagation of free radical chain reactions in cultured cells.
Next, we determined whether the responses induced by chemically produced H2OxPAPC (Figs 3 and 4) could
simulate the effects induced by the free radicals in cultured cells. When THP-1 cells were exposed to AAPH, thecellular Ca2+ levels increased (Fig. 5c) in a time-dependent manner (Fig. 5d), as shown by the analysis of Fluo-3,
and the Ca2+ signaling was suppressed by H2 (Fig. 5c,e). NFAT was also activated, as shown by the translocationof NFAT into the nucleus (Fig. 5f,g), and the nuclear translocation of NFAT were recovered by H2 (Fig. 5f,g).
Moreover, the free radical inducer stimulated the expression of some target genes of NFAT, including TNF-α,early growth response protein 1 (EGR1), and activating transcription factor 3 (ATF3), which have been shown in
Supplementary Table 1, and H2 decreased their expressions (Fig. 5h), suggesting that H2 regulates these genes via
the NFAT pathway.
In contrast, AAPH-mediated activation of CREB was not observed (Supplementary Fig. 5) in this cultured cellline, regardless of the stimulation of cellular Ca2+. In particular, the expression of the CREB-target gene NFKB2(NF-κ B, subunit 2 gene) was not affected by AAPH (Fig. 5i), and the expression of HMOX1 (Heme Oxygenase 1gene), a nuclear factor-E2-related factor 2 (Nrf2)-target, was slightly but not significantly increased by H2 (Fig. 5i).
This result was consistent with those of a previous study26. Thus, the NFAT pathway could mainly contribute to
the H2-dependent transcriptional response induced by free radicals at least in THP-1 cells.
Taken together, these cellular responses, at least partly, are in agreement with those obtained using the in vitro
H2-dependent products of OxPAPC species (Figs 3, 4). Therefore, we proposed a model in which H2 is linked tothe modulation of Ca2+ signal transduction and the NFAT pathway via oxidized phospholipid species, as illustrated in Fig. 6.

Discussion

While the biological effects of H2 have been evaluated in more than 300 animal studies and 10 clinical analyses
in humans6,7, the molecular mechanisms by which H2 at low concentrations exerts its multiple effects on signaltransduction remained unknown. Therefore, in this study, we aimed to examine how H2 regulates signal transduction pathways that mediate gene expression. Our results suggested that low concentrations of H2 modulated
Ca2+ signal transduction and regulated gene expression by modifying the production of oxidized phospholipid
species. Hence, these data provide important insights into one of the molecular mechanisms by which H2 mediates gene expression.
H2 can be ingested via several methods. Drinking of H2-infused water (H2-water) has been shown to be efficacious in the treatment of various diseases in animal models and humans6,7; however, H2 can be infused up to
only 0.8 mM under atmospheric pressure, and drinking saturated H2-water provides a blood concentration up
to only ~10 μ M, with a short dwelling time in the body11,27. Moreover, inhaling 1%–4% (v/v) H2 gas was shown
to be effective, reaching concentrations of 8–32 μ M H2 in the blood1,4,5. However, initiation of cellular signals by
these low concentrations of H2 may be difficult to be explained because H2 should be too inert to react with most
molecules. To activate H2 for reaction with the other molecules, a sufficient level of a putative catalyst must bepresent; however, it is unlikely that such a putative catalyst would be abundant inside cells. Moreover, H2 is verysmall and is unlikely to bind to a putative H2-binding receptor because its intra-molecular fluctuation would be

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Figure 5. H2 suppressed free radical inducer-dependent fatty acid peroxidation and Ca2+ and NFAT
signaling. (a) THP-1 was exposed to a free radical inducer (10 mM AAPH) in the absence or presence of theindicated concentrations of H2 for 4.5 h. Representative flow cytometric profiles are shown to demonstrate lipid
peroxides with Liperfluo signals. (b) The Liperfluo signals were quantified. *P = 0.015, ***P < 0.001 vs. 0% H2(n = 6). (c) THP-1 cells were treated with 10 mM AAPH for 3 h in the presence of the indicated concentrationsof H2. Intracellular Fluo-3 fluorescence intensity was observed using a laser scanning confocal microscope.
Scale bar: 50 μ m. (d) THP-1 cells were treated with 10 mM AAPH for the indicated periods in the absence of H2
and then time dependent increase in Ca2+-signal was monitored by intracellular Fluo-3 fluorescence intensity
as described in (c). (e) Fluo3-positive cells were semi-quantified after the treatment with 10 mM AAPH for 3 h
in the absence or presence of the indicated concentrations of H2. **P < 0.01 vs. no H2 (n = 3). (f) THP-1 was

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treated with 10 mM AAPH for 3 h in the presence of the indicated concentrations of H2. The translocation of
NFAT into the nucleus was examined as described in Methods and shown by immunostaining in yellow. The
nucleus was counter-stained with Hoechst 33342 as shown in blue. Scale bar: 50 μ m. (g) The NFAT-expressing
areas were semi-quantified and shown by the ratio of NFAT in the nucleus with that in cytosol. *P = 0.023 and**P < 0.01 vs. no H2 (n = 10). (h,i) The expressions of the NFAT-target genes (TNF-α, EGR1, and ATF3) (h) andnon-NFAT target gens (NFKB2 and HMOX1) (i) were estimated using RT-PCR coupled with a TaqMan probe(the probes are listed in Supplementary Table 2). The names of the genes are described in Supplementary Table
1. *P = 0.015 (for ATF3) (+ AAPH and + H2 vs. + AAPH and –H2). #P = 0.14 (for HMOX1) (+ AAPH and –H2
vs. + AAPH and + H2), and **P < 0.01 (n = 3)
expected to lead to instability in terms of thermodynamics, as previously discussed28. Thus, it was unknown how
low concentrations of H2 regulate signal transduction and gene expression.
Since increased oxidative stress involving ·OH triggers free radical chain reactions, we assumed that the chemically produced mediators derived from phospholipids could contribute to various pathogenic conditions. In thepresent study, we verified that a small amount of H2 (as low as 1.3%) affected free radical-dependent lipid peroxidation, from which oxidized lipid mediators should be derived22.
Generally, H2 hydrogenates unsaturated fatty acids at higher temperatures with a palladium catalyst. To thebest of our knowledge, no studies have examined autoxidation-dependent hydrogenation at approximately 1%(v/v) H2 gas at 37 °C without any catalysts. Although H2 was thought to be inert in the absence of a catalyst at bodytemperature, we demonstrated that approximately 1% (v/v) H2 suppressed autoxidation of an unsaturated fatty
acid in a chemically pure system in this study; thus, our data provided insights into the biological activities of H2.
There are two possibilities: the effects of oxidized phospholipid species on Ca2+ signaling may be explain bydecreased levels of a putative agonist that induces Ca2+ signaling or by increased levels of a putative antagonist
that disturbs Ca2+ signaling. Although we could not identify these species in this study, it is likely that H2 modified the production of reduced forms of oxidized phospholipid species during free radical chain reactions by thefollowing previous findings: POVPC is a bioactive phospholipid-mediator that is produced by chemical oxidationof PAPC, and the reduced form of POPVC has been shown to function as an antagonist for signal transduction18.
Thus, it is possible that during a lipid free radical chain reaction, H2 contributes to the generation of a reducedform(s) that function(s) as an antagonist(s). Therefore, we proposed a hypothetic model in which H2 is linked tothe modulation of Ca2+ signal transduction and the NFAT pathway via oxidized phospholipid species as illustrated in Fig. 6.
Previous studies have shown that 1%–4% was efficacious in inhaling H2 gas in various animal experiments1,3,4,29–31. Since a mixed gas containing 1.3% H2, 30% O2 and 68.7% N2 is available, the effects of around 1.3%
needed to be investigated in further studies, including clinical ones5. The effective concentrations of H2 gas wereapproximately consistent throughout this study (Figs 2–5).
No receptors involved in Ca2+ signaling were identified in the present study; however, a previous study showedthat some chemically oxidized phospholipid mediators, such as 9-HODE and 11-HETE, could bind a G-protein
coupled receptor (G2A) to induce Ca2+ signaling17. Thus, putative oxidized phospholipid mediators or antagonists might bind to G-protein coupled receptors to modulate signal transduction.
In addition to the anti-oxidative roles of H2, it has shown to function as an immunosuppressant in allografttransplantation32. This immunosuppressant effect can be explained by the suppression of NFAT activation because
an immunosuppressant such as CsA and tacrolimus (FK506) acts through the inactivation of calcineurin. Since
pro-inflammatory cytokines are regulated by NFAT-dependent mechanisms20, the anti-inflammatory effects by
H2 can be explained by the suppression of NFAT. Additionally, the anti-allergic effects of H2 can be explained by
the decrease in Ca2+/NFAT signaling33.
A considerable number of the multiple functions of H2, as shown by previous studies, might be explained by
the link between H2 and NFAT because of the numerous multiple functions of NFAT20,34. For example, decreased
expression of inducible nitric oxide synthase (iNOS) by H235 can be explained by the inactivation of NFAT36. The
suppression of osteoclast differentiation37 and improvement of hypertension38,39 by H2 could involve the NFAT
pathway40,41. Moreover, the decreased expression of gene products through an NFAT-dependent pathway might
be involved in α -synuclein-induced degeneration of midbrain dopaminergic neurons in Parkinson’s disease42.
This NFAT-dependent pathway might explain the beneficial effects of H2 in these patients8. Further studies areneeded to elucidate the mechanisms by which H2 exerts multiple functions in terms of the involvement of the
NFAT pathway.
In summary, in this study, we investigated the link among H2, oxidized phospholipids, and Ca2+ signaling.
Further studies are warranted to identify the H2-dependent bioactive mediator(s). Our data provided important
insights into one of the mechanisms by which H2 regulates signal transduction and gene expression; however,H2 might contribute to other types of signaling pathways as well because H2 regulates many genes belonging
to various signaling pathways. A more detailed understanding of the molecular mechanisms of H2-dependent
signal transduction and gene expression is expected to facilitate the application of H2 in a wide range of medical
applications.

Methods

Measurement of H2.

Gases containing H2 were prepared by mixing H2, O2, N2, and CO2 at various concentrations from each gas cylinder equipped with a flow meter. The H2 concentration in the mixed gas or air wastested in each experiment by using gas chromatography (Breath Gas Analyzer, Model TGA2000; TERAMECS
Co. Ltd., Kyoto, Japan) as described previously1. For the measurement of H2 in the solvent, H2 was transferredto the air phase in a closed aluminum bag, and the concentration of H2 measured by using gas chromatography

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細胞生物学部門

Figure 6. A model of the proposed pathway. When free radical chain oxidation generates oxidized
phospholipid mediators, Ca2+ signaling is induced, followed by the activation of calcineurin and subsequent
induction of the NFAT pathway. On the other hand, H2 modifies the production of oxidized phospholipids bymodulating free radical chain reactions. The putative oxidized phospholipids appear to function as antagonists
and lead to a decline in Ca2+ signaling.
as described previously1. The aluminum used in the bag was covered with a plastic film to avoid any influence ofaluminum.

Autoxidation of linoleic acid-film. Linoleic acid and (± )9-HODE were purchased from Nacalai Tesque(Kyoto, Japan) and CAY (MI, USA), respectively. Linoleic acid was dissolved in cyclohexane to 16 mM, and 2 μ L
was dispensed into each glass tube (φ 10 × 50 mm) that had been filled with argon gas; it was allowed to dry up
to form a linoleic acid-film at the bottom of a glass tube. The glass tubes were placed into a closed aluminum
bag, and the gas in the bag was completely replaced with the indicated mixed gas, where pure H2, O2, and N2
were obtained from separate cylinders. The bag was incubated at 37 °C for 20 h for the autoxidation, and 0.2 mL
cyclohexane was immediately added to the glass tube to obtain 0.16 mM peroxidized linoleic acid. The concentration of conjugated diene was estimated by measuring the absorption at 234 nm while scanning from 200 to
300 nm.
Autoxidation of pure PAPC in air in the absence or presence of H2.

Chemically synthesized pure
PAPC was purchased from Avanti Polar Lipids (Alabaster, AL, USA). PAPC was autoxidized in air as described
43
previously . Briefly, 0.5 mg of PAPC in 50 μ L of chloroform was transferred to a φ 10 × 50 mm glass tube anddried up under a gentle stream of nitrogen. The lipid residue was allowed to autoxidize in air with 100% humidity
at 25 °C in the presence or absence of the indicated concentrations of H2 gas in a closed aluminum bag for theindicated periods, and then suspended in PBS at a concentration of 0.5 mg/mL.

Estimation of OxPAPC with Liperfluo. OxPAPC was assayed in ethanol with Liperfluo as described previously25. Five min after adding OxPAPC to 1 μ M Liperfluo at room temperature, the fluorescence was measured
using a fluorescence spectrophotometer (RF-5300PC; Shimadzu Corporation, Kyoto, Japan), where wavelengthsof excitation and emission were set at 488 and 535 nm, respectively.
Measurement of Ca2+ signaling.

Intracellular Ca2+ in THP-1 cells treated with OxPAPC was measured
using a Calcium Kit-Fluo 4 (CS22; Dojindo, Kumamoto, Japan) according to the manufacturer’s protocol. Briefly,
THP-1 cells were washed with PBS and incubated with 4.5 μ M Fluo 4-AM in recording medium (20 mM HEPES,

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115 mM NaCl, 5.4 mM KCl, 0.8 mM MgCl2, 1.8 mM CaCl2, 13.8 mM glucose) containing 0.064% pluronic F-127
and 1.25 mM probenecid for 30 min at 37 °C. The cells were washed with PBS and resuspended in recording
medium containing 1.25 mM probenecid. The cells were seeded on 35-mm glass-bottomed dishes and then stimulated with 100 μ g/mL OxPAPC or H2OxPAPC, followed by 25 μ M ATP. The changes in Fluo 4-AM fluorescencewere monitored using a laser scanning confocal microscope (FV1200; Olympus Corporation, Tokyo, Japan). The
strength of each fluorescent signal in 400 cells was examined and judged as positive if there was greater than 30%of the ATP signal.
Intracellular Ca2+ of THP-1 cells treated with the free radical inducer AAPH23 was measured by Fluo-3(F-23915; Molecular Probes, Eugene, OR, USA). Briefly, THP-1 cells were pre-incubated with 2 μ M Fluo 3-AM
in HBSS containing 0.02% pluronic F-127 for 30 min at 37 °C, resuspended in RPMI1640 (with 10% FBS) containing 2.5 mM probenecid, seeded in 24-well plates, and then treated with AAPH in the presence or absence of
H2. Changes in Fluo-3 fluorescence signals were observed using a laser scanning confocal microscope (FV1200;
Olympus).

Mass spectrometric analysis and presentation of data using heat maps. OxPAPC (dissolved inchloroform at 2.5 mg/mL) was analyzed using by electrospray ionization-mass spectrometry (ESI-MS) by using
an LTQ ORBITRAP XL mass spectrometer (Thermo Fisher Scientific, San Jose, CA, USA) equipped with a nitrogen sheath gas flow rate of 40 AU at 300 °C. The sample was directly infused. The scanning range was from m/z
250 to 1000 in the positive ion detection mode. The ion spray voltage was set to 4 kV. OxPAPC species were identified according to their m/z values and confirmed using mass spectrometric analysis as described previously14,44,45.
Two independent experiments were performed. The average of the data was used for construction of a heat
map and displayed in mass spectrometric profiles. In the heat map, bands were arranged according to molecularmass from small to large, and the strength of each band obtained from H2OxPAPC was compared with those by
OxPAPC. Red and green bands represented increased and decreased levels as compared with those of OxPAPC,
respectively. The mass spectrometric display indicates the average band from two experiments. Only when bands
were detected by all of 10 experiments (two experiments at 0%, 0.2%, 0.3%, 1.3% and 5% of H2), they wereadopted.
Comprehensive analysis of gene expression. THP-1 cells were exposed for 4 h to PAPC or OxPAPC,
H2[1.3%]OxPAPC, and H2[5%]OxPAPC that had been autoxidized for 3 days with 0%, 1.3%, or 5% H2, respectively. Total RNA was extracted using an RNeasy Mini Kit according to the manufacturer’s protocol (Qiagen,
Valencia, CA, USA) and labeled using a Low-Input QuickAmp Labeling Kit, One-Color (Agilent Technologies,
Santa Clara, CA, USA). Gene expression analysis was performed on samples from three independent experiments
using a microarray (SurePrint G3 Human GE 8 × 60 K v2 Microarray; Agilent Technologies). The raw microarraydata were deposited in the Gene Expression Omnibus (GEO; accession number, GSE62434; http://www.ncbi.nlm.
nih.gov/geo/query/acc.cgi?acc=GSE62434). CREB target genes were selected according to the CREB Target Gene
Database (http://natural.salk.edu/CREB/), while NFAT target genes were selected by reference to Medline, as
listed in Supplementary Table 1. Signal transduction pathways associated with each gene were identified according to the KEGG Pathway Database (http://www.genome.jp/kegg/pathway.html).
Quantitative real-time PCR. To quantify mRNA levels, quantitative real-time PCR was carried out using
TaqMan Probe and Premix Ex Taq (Probe qPCR; TaKaRa Bio Inc., Shiga, Japan) in a TaKaRa PCR Thermal Cycler
Dice TP960 (TaKaRa Bio) according to the manufacturer’s protocols. To normalize mRNA expression levels,
glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an endogenous internal control. Primers andprobes used for RT-PCR are described in Table 2.
ELISA (Enzyme-linked immuno-sorbent assay) HAEC and THP-1 cells were treated with PAPC, OxPAPC
or H2OxPAPC for 22 h. The IL-8 (HAEC) and TNF-α (THP-1) contents in the culture media were determinedusing Human CXCL8/IL-8 Quantikine ELISA Kit (R&D Systems, Minneapolis, MN, USA) and Human TNF-α
Quantikine ELISA Kit (R&D Systems, Minneapolis, MN, USA), respectively, according to the manufacturer’s
protocol.
Detection of lipid peroxidation in cultured cells. THP-1 cells (1 × 105 cells/mL) were stained with 5 μ M

Liperfluo25 for 30 min and then treated with 10 mM of AAPH23 for 4.5 h in the absence or presence of the indicated concentrations of H2 gas in a closed vessel. The cells were analyzed using a Cell Lab Quanta flow cytometer(Beckman Coulter, Miami, FL, USA).

Detection of the translocation of NFAT into the nucleus by immunofluorescence.

THP-1 cells(1 × 105 cells/mL) were treated with OxPAPC (0.1 mg/mL), or H2[2.5%]OxPAPC ( 0.1 mg/mL) for 1.5 h, whichwere used for the Ca2+ signaling assay, and then the translocation of NFAT was determined using immunofluorescence as follows. The cells were fixed for 20 min with 10% neutral buffered formalin (3.8% formaldehyde),
and then permeabilized with 0.2% Triton X-100 in Tris-buffered saline (TBS-T) for 10 min. After the cells were
washed, and blocked with 5% nonfat milk in TBS-T, they were incubated with anti-NFAT1 antibodies (1:100
dilution; 25A10.D6.D2; Abcam, Cambridge, MA, USA) overnight at 4 °C, followed by incubation with Alexa
Fluor 488-conjugated anti-mouse antibodies (1:400 dilution; A-11029; Life Technologies, Carlsbad, CA, USA) for
1 h at 25 °C. The cells were counterstained with Hoechst 33342. Immunofluorescence was observed using a laser
scanning confocal microscope (FV1200; Olympus).

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THP-1 cells (1 × 105 cells/mL) were treated with 10 mM AAPH for 3 h in the absence or presence of indicated concentrations of H2, and the NFAT translocation was investigated using immunofluorescence as describedabove.

Cell culture. THP-1 cells (ATCC) were cultured in RPMI1640 containing 10% FBS. Human aortic endothelial

cells (HAEC) were obtained from Lonza and maintained in endothelial cell growth medium [EBM medium +growth supplements+ FCS (Lonza)]. Cells were cultured at 37 °C in a 5% CO2 humidified atmosphere and wereused for experiments from passage 4 to 8.

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細胞生物学部門

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Acknowledgements

We thank Ms. Mayumi Takeda and Ms. Suga Kato for technical assistance and secretarial work, respectively.
Financial support for this study was provided by Grants-in-Aid for Scientific Research from the Japan Society
for the Promotion of Science (23300257, 24651055, and 26282198 to S.O.; 23500971 and 26350129 to N.K.; and25350907 to K.N.).

Author Contributions

S.O. conceived of and directed the project, analyzed data, and wrote the manuscript. N.K. planned eachexperiment. K.I., A.I., N.K., K.N., T.Y. and H.I. performed experiments on cell culture (by K.I.), Ca2+ signaling(by A.I.), gene expression (by N.K.), and chemical reactions (by K.N.), supported by H.I. and T.Y.

Additional Information

Supplementary information accompanies this paper at http://www.nature.com/srep
Competing financial interests: S.O. holds the right of a patent regarding a medical use of hydrogen gas.
How to cite this article: Iuchi, K. et al. Molecular hydrogen regulates gene expression by modifying the free
radical chain reaction-dependent generation of oxidized phospholipid mediators. Sci. Rep. 6, 18971; doi:
10.1038/srep18971 (2016).
This work is licensed under a Creative Commons Attribution 4.0 International License. The images
or other third party material in this article are included in the article’s Creative Commons license,
unless indicated otherwise in the credit line; if the material is not included under the Creative Commonslicense, users will need to obtain permission from the license holder to reproduce the material. To view a copyof this license, visit http://creativecommons.org/licenses/by/4.0/

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Scientific RepoRts | 6:18971 | DOI: 10.1038/srep18971−36−

Ⅲ．遺伝子制御学部門
Department of Molecular Oncology

遺伝子制御学部門（大学院

遺伝子制御学部門）
教授

田中

信之

【研究概要】
我々は、がん抑制因子 p53 の解析を進めることで、がん化及びがん抑制の分子機構を明らかにすることを目的に研究を続けている。この過程で、p53 の誘導遺伝子の同定を行い、Noxa を始めとする様々な･
p53 の標的遺伝子を発見し解析を進めていると同時に、p53 欠損細胞での様々な細胞応答の変化を･解析する過程で、p53 がグルコース代謝を制御していることを発見した。がん細胞がグルコースの代謝を主なエネルギー供給源として増殖していることはワールブルグ効果と呼ばれ、古くから知られて損細胞では転写因子 NF-κB の転写活性化能が恒常的に高いこと、このことががん化に重要である
ことを発見し、p53 欠損細胞でのグルコース代謝の上昇が NF-κB の活性化を促すというポジティブ
フィードバック機構が存在すること、がん細胞が膨大なエネルギーを産生することにこの機構が重要である事を報告している。
これらの p53 の研究に加えて、我々はがん幹細胞の解析を進めている。がんは少数存在する幹細胞様の細胞から発生・進行することが近年明らかとなりつつある。このがん幹細胞は増殖が遅く、抗がん剤に抵抗性のために、がん再発の原因となっており、がん幹細胞をいかにして排除するかが、がん治療の重要な標的となっている。
がんは少数存在する幹細胞様の細胞から発生・進行することが近年明らかとなりつつある。このがん幹細胞は増殖が遅く、抗がん剤に抵抗性のために、がん再発の原因となっており、がん幹細胞をいかにして攻撃するかが、がん治療の重要な標的となっている。安倍が中心となって hedgehog 経路の転写因子
Gli1 が MEP50/PRMT5 アルギニンメチル基転移酵素複合体（メチロソーム）によって活性化されるという新しい機構を発見し、この機構ががんの微小環境でのがん幹細胞の維持に重要である事を明らかにした。更に、EGFR 陽性非小細胞肺がん症例の解析及び肺がん細胞のヌードマウス移植での解析から、この MEP50/PRMT5/Gli1 の活性化経路が肺がんの再発に重要であり、この経路を抑制することががんの再発の予防に重要であることを明らかにし、論文を投稿すると共に、肺癌治療のための抗癌剤の効果の検査法（特願 : 2016-094888 号）の特許を出願した。同時に上原は、がん微小環境で産生される
I 型 IFN ががん幹細胞の維持に働いていることを見出した。実際、p53 欠損マウスは生後早期に腫瘍が･発生して死亡するが、p53 に加えて I 型 IFN を欠損したマウスは腫瘍の発生が遅れる傾向にあることを見出し、更に I 型 IFN が肺がんや乳がんでのがん幹細胞の維持に重要であることを見出している。更に、清水ががん幹細胞の発生に重要な遺伝子の同定を行い、この遺伝子をゲノム編集により欠損させると、がん化した細胞が腫瘍形成能を失うこと、p53 がこのがん幹細胞の発生を抑制していることを発見している。これらの解析を進めることで、がん幹細胞の発生・維持機構を明らかにすると共に、p53 によるがん化の抑制機構を明らかにしていきたいと考えている。
中嶋は、抗がん剤によるがん細胞のアポトーシス誘導のメカニズムを解析しており、大学院生の松本と共に非小細胞肺がん細胞でのシスプラチンによるアポトーシス誘導の分子機構及びその効果を増強する方法を見出して報告した。更に、抗がん剤で誘導されるオートファジーによるがん細胞の治療
抵抗性獲得の機構を解析する過程で、幾つかの非小細胞肺がん細胞が Beclin 1 や ATG5 依存的に起こる･
マクロオートファジーとは別の、LAMP2A や Hsc70 依存的なシャペロン介在性オートファジーによる−39−

遺伝子制御学部門

いるが、その分子制御機構やがん化そのものに対しての役割は明らかではなかった。我々は、p53 欠

Mcl-1 の分解抑制によって生存していることを、大学院生の鈴木と共に発見し報告しており、がん治療の新たな標的を見出している。同時に岩渕は、EGFR 陽性非小細胞肺がんで、転写因子 HIF-1 がEGFR 特異的分子標的薬ゲフィチニブに対しての耐性の獲得に重要であることを見出すと共に、その分子機構を明らかにしている。
谷村は、炎症組織では p53 の誘導は起こるものの、p53 によるアポトーシスや老化による排除機構が･
働かないことを見出し、その機構の解析を続けていると共に、Toll 様受容体のシグナル伝達分子
MYD88 が転写因子 NF-κB 依存的に HIF-1 を誘導すること、この経路ががん化に重要であることを見出しており、炎症と発がんの新たな分子機構の解明を目指しており、これらの研究を介して、がん化の分子機構の解析と、効果的ながん治療法の開発を進めている。

【研究業績】〈原著〉
1）Suzuki J, Nakajima W, Suzuki H, Asano Y and Tanaka N. Chaperone-mediated autophagy
promotes lung cancer cell survival through selective stabilization of the pro-survival protein,
MCL1. Biochem. Biophys. Res. Commun., http://dx.doi.org/10.1016/j.bbrc.2016.12.037, 2016
2）Matsumoto M, Nakajima W, Seike M, Gemma A and Tanaka N. Cisplatin-induced apoptosis
in non-small-cell lung cancer cells is dependent on Bax- and Bak-induction pathway andsynergistically activated by BH3-mimetic ABT-263 in p53 wild-type and mutant cells. Biochem.
Biophys. Res. Commun., 473:490-496, 20163）Nakajima W, Sharma K, Lee JY, Maxim NT, Hicks MA, Vu TT, Luu A, Yeudall WA, Tanaka N
and Harada H. DNA damaging agent-induced apoptosis is regulated by MCL-1 phosphorylationand degradation mediated by the Noxa/MCL-1/CDK2 complex. Oncotarget, 14:36353-36365, 2016

〈総説〉
1）Abe Y and Tanaka N. Hedgehog signaling pathway in lung cancer: the mechanisms and roles intumor progression and implications for cancer therapy.“Cell and Molecular Mechanics in Healthand Disease”Biomed Res. Int., in press.
2）W. Nakajima, N. Tanaka, BH3 mimetics: their action and efficacy in cancer chemotherapy, Integr.
Cancer Sci. Ther. 3:437e441, 2016

〈学会発表〉
1）田中信之：癌微小環境下での肺癌幹細胞の新たな維持機構の解析とそれを標的とした治療法の検討
第 56 回日本肺癌学会学術集会 シンポジウム、2015、東京
2）阿部芳憲、武内進、田中信之：PRMT5-Gli1 経路の遮断による肺がんの再発および抗がん剤抵抗性腫瘍の抑制効果 第 38 回 日本分子生物学会年会、2015、神戸
3）松本優、中嶋亘、鈴木淳也、田中信之：非小細胞肺癌細胞株におけるシスプラチンによるアポトー
シスについての考察 第 38 回 日本分子生物学会年会、2015、神戸
4）鈴木淳也、中嶋亘、田中信之：肺癌の分子標的薬 crizotinib はオートファジーとネクローシスによる細胞死を誘導する 第 38 回 日本分子生物学会年会、2015、神戸
5）上原郁野、田中信之：I 型インターフェロンによる発がんの促進と癌幹細胞維持への関与

第 38 回

日本分子生物学会年会、2015、神戸
6）谷村篤子、中里茜、田中信之：マウス繊維芽細胞での内在性活性化 K-ras 発現による癌抑制因子
p53 の下流遺伝子転写調節 第 38 回 日本分子生物学会年会、2015、神戸

−40−

〈特許〉
出願
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遺伝子制御学部門−41−

Biochemical and Biophysical Research Communications 473 (2016) 490e496

Contents lists available at ScienceDirect

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

Cisplatin-induced apoptosis in non-small-cell lung cancer cells isdependent on Bax- and Bak-induction pathway and synergistically
activated by BH3-mimetic ABT-263 in p53 wild-type and mutant cells
Masaru Matsumoto a, b, Wataru Nakajima a, Masahiro Seike b, Akihiko Gemma b,
Nobuyuki Tanaka a, *a

Department of Molecular Oncology, Institute for Advanced Medical Sciences, Nippon Medical School, 1-396 Kosugi-cho, Nakahara-ku, Kawasaki 211-8533,
Japan
Department of Pulmonary Medicine and Oncology, Graduate School of Medicine, Nippon Medical School, 1-1-5 Sendagi, Bunkyo-ku, Tokyo 113-8603,
Japanba r t i c l e i n f o

a b s t r a c t

Article history:
Received 4 March 2016
Accepted 14 March 2016
Available online 18 March 2016

Cisplatin is a highly effective anticancer drug for treatment of various tumors including non-small-celllung cancer (NSCLC), and is especially useful in cases nonresponsive to molecular-targeted drugs.
Accumulating evidence has shown that cisplatin activates the p53-dependent apoptotic pathway, but it
also induces apoptosis in p53-mutated cancer cells. Here we demonstrated that DNA-damage inducible
proapoptotic BH3 (Bcl-2 homology region 3)-only Bcl-2 family members, Noxa, Puma, Bim and Bid, are
not involved in cisplatin-induced apoptosis in human NSCLC cell lines. In contrast, the expression ofproapoptotic multidomain Bcl-2-family members, Bak and Bax, was induced by cisplatin in p53dependent and -independent manners, respectively. Moreover, in wild-type p53-expressing cells,
cisplatin mainly used the Bak-dependent apoptotic pathway, but this apoptotic pathway shifted to the
Bax-dependent pathway by loss-of-function of p53. Furthermore, both Bak- and Bax-induced apoptosis
was enhanced by the antiapoptotic Bcl-2 family member, Bcl-XL knockdown, but not by Mcl-1 knockdown. From this result, we tested the effect of ABT-263 (Navitoclax), the speciﬁc inhibitor of Bcl-2 and
Bcl-XL, but not Mcl-1, and found that ABT-263 synergistically enhanced cisplatin-induced apoptosis in
NSCLC cells in the presence or absence of p53. These results indicate a novel regulatory system incisplatin-induced NSCLC cell apoptosis, and a candidate efﬁcient combination chemotherapy method
against lung cancers.
© 2016 Elsevier Inc. All rights reserved.

Keywords:
Cisplatin
Apoptosis
Bax
Bak
ABT-263
NSCLC

1. Introduction
Lung cancer is a leading cause of cancer-related death worldwide in both men and women, and more than 85% of cases areclassiﬁed as non-small-cell lung cancer (NSCLC), including adenocarcinoma, squamous cell carcinoma and large-cell carcinoma [1].
NSCLCs harboring epidermal growth factor receptor (EGFR) mutations or anaplastic lymphoma kinase (ALK) gene rearrangements
have been successfully targeted with tyrosine kinase inhibitors

Abbreviations: NSCLC, non-small-cell lung cancer; CDDP, cisplatin [cisdiamminedichloroplatinum (II)]; shRNA, short hairpin RNA; IP, immunoprecipitation; IB, immunoblotting; WCL, whole cell lysate.
* Corresponding author.
E-mail address: nobuta@nms.ac.jp (N. Tanaka).

(TKIs) [2,3]. However, the rate of EGFR positive mutation is about
20% and ALK rearrangement is only about 5e7% [4], and thus most
NSCLC cases do not respond to these TKIs [1,5]. In such cases,
conventional chemotherapy using platinum-based compounds arerecommended [6,7]. Cisplatin is the ﬁrst discovered and mostcommonly used platinum-based compound [8]. In the cell, cisplatincrosslinks purine bases in DNA, thereby evoking DNA damage responses. The anti-tumor effect of cisplatin is primarily caused by
DNA damage induced apoptosis [9]. The tumor suppressor p53
plays an essential role in DNA damage-induced apoptosis throughinduction of a set of apoptosis inducers [10]. On the other hand,another study showed that cisplatin also induced apoptosis in p53mutated NSCLC cells and other cancer cells [11]. Therefore, themolecular mechanism underlying cisplatin-induced apoptosis in
NSCLC cells has not yet been fully elucidated.

http://dx.doi.org/10.1016/j.bbrc.2016.03.053
0006-291X/© 2016 Elsevier Inc. All rights reserved.

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M. Matsumoto et al. / Biochemical and Biophysical Research Communications 473 (2016) 490e496(Waltham, MA, USA); Mcl-1 (S-19) and Bax (N-20) from Santa Cruz
Biotechnology (CA, USA); alpha-tubulin (DM 1A) from SigmaeAldrich (Tokyo, Japan); and Bak (06e536) from Millipore (Billerica, MA, USA). Anti-Bak (ab-1; Calbiochem) and anti-Bax (6A7;
SigmaeAldrich) antibodies were used for immunoprecipitation.
ABT-263 (Navitoclax) was purchased from AdooQ BioScience (Irvine,
CA, USA). Cisplatin was purchased from Calbiochem (San Diego, CA,USA).

The Bcl-2 proteins are critical regulators of apoptosis. They
possess conserved BH (Bcl-2 homology) domains and are classiﬁed
into “multidomain” and “BH3-only” proteins. The pro-apoptotic
multidomain members Bax and Bak function as apoptosis executers in the mitochondria. Studies on mice lacking both Bax andBak showed that Bax and Bak are essential inducers of
mitochondrion-mediated apoptosis in response to various stimuli,
including DNA damage [12,13]. In contrast, anti-apoptotic multidomain proteins, such as Bcl-2, Bcl-XL and Mcl-1, inhibit Bax/Bakmediated apoptosis. BH3-only proteins, which are critical for
initiating apoptosis, activate Bax and Bak through direct and/or
indirect activation [12,13]. The BH3-only proteins Bim, Bid, Puma
and Noxa directly activate Bax and Bak, and also inactivate antiapoptotic multidomain proteins [14]. Quadruple deﬁciency ofBim, Bid, Puma and Noxa abrogates apoptosis induced by various
stimuli, suggesting the importance of these factors in triggering
Bax/Bak-mediated apoptosis induction [14]. In response to DNA
damage, the expression of Noxa/Puma and Bim is induced by p53
and FOXO3, respectively, resulting in induction of apoptosis[15e18]. Another study also reported the possible role of Bid in the
DNA damage response [19].
Here we examined the mechanism of cisplatin-induced
apoptosis of NSCLC cells with the aim of helping develop new approaches for treatment of NSCLC.

2.2. Immunoprecipitation and immunoblotting
Immunoprecipitation and immunoblotting analyses were performed as previously described [20].
2.3. RNA interference
Short hairpin RNAs (shRNAs) shNoxa, shBim, shBid, shPuma,
shMcl-1 and shBcl-XL were cloned into the pSuper puro vector(Oligoengine; Seattle, WA, USA). The target sequences were as(shNoxa),
50 follows:
50 -GGAAACGGAAGATGGAATA-30
CTACCTCCCTACAGACAGA-30 (shBim), 50 -GGGATGAGTGCATCACAAA-30 (shBid), 50 -GGGTCCTGTACAATCTCAT-30 (shPuma), 50 (shMcl-1),
and
50 -AGGATAGCAAGAGGATTATGGCTAA-30
CAGCTGGAGTCAG -30 (shBcl-XL). Retroviral infection was performed as previously described [21]. The lentiviral sh-p53, sh-Bax
and sh-Bak-expressing constructs were kindly provided by Dr.
Harada (Virginia Commonwealth University, Richmond, VA, USA)[22].

2. Materials and methods
2.1. Cells, antibodies and materials
Human NSCLC cell lines A549, H460, LC2/Ad and PC10 werepurchased from the American Type Culture Collection (Manassas,
VA, USA). Antibodies were purchased from the following manufacturers: Bim (C34C5), Bcl-XL (54H6), cleaved PARP (D64E10) andcleaved caspase-3 (5A1E) from Cell Signaling Technology (Danvers,
MA, USA); Noxa (114C307.1) from Thermo Fisher Scientiﬁcsh-control-+

sh-Noxa

-

+
10kDa

Cleaved
Cas-3

17kDa

Tubulin

50kDa

80
60
40
20

25kDa
50kDa

sh-Bidsh-control

sh-Bidsh-control

H460

relative Puma level (RT-PCR)

Bim
Tubulin
A549

40
20

80
60
40
20
0

l
nt
ro
co
sh
-

80
60
40
20
0

H460
sh-Bimsh-Bimsh-control

A549

sh-control

C

60

0

sh
-c
on
tro
l
sh
-N
ox
a

0

80

sh
-B
id

+

Cell viability (% of control)

-

H460

sh
-c
on
tro
l
sh
-P
um
a
sh
-B
im

sh-Noxa

100

sh
-B
id

+

A549

100

Cell viability (% of control)

-

D

H460

100

sh
-c
on
tro
l
sh
-P
um
a
sh
-B
im

sh-control

A549

N
ox
a

H460

A549
CDDP
Noxa

Cell viability (% of control)

100

Quantitative real-time PCR (qPCR) analysis was performed aspreviously described [21]. The primer and probe sets used werepredesigned primer/probe sets: b-actin, Hs03023880_g1; Bak,

sh
-

B

Cell viability (% of control)

A

2.4. Quantitative real-time polymerase chain reaction (PCR)
analysis

1.2

1

0.8

0.8

0.6

0.6

0.4

0.4

0.2

0.2

0

Bid

20kDa

Tubulin

50kDa

1.2

A549

1

Vehicle

CDDP

0

H460

sh-control
sh-Puma

VehicleCDDP

Fig. 1. Effect of Noxa, PUMA, Bim or Bid knockdown on cisplatin (CDDP)-induced apoptosis of NSCLC cells. (A, B) A549 and H460 cells stably expressing sh-Noxa or sh-control weretreated with cisplatin (20 mM) for 24 h and analyzed by immunoblot analysis using indicated antibodies (A) and by cell viability assay (B). Results are expressed as the mean ± SEM
of at least three independent experiments. P values are relative to the control. *P < 0.05; **P < 0.01. (C, D) A549 and H460 cells stably expressing shRNA targeting Bid (sh-Bid) or shBim were subjected to immunoblot analysis using the indicated antibodies (left panel; C). A549 and H460 cells stably expressing sh-Puma or sh-control were treated with cisplatin(20 mM) for 24 h and the expression of Puma was analyzed by qPCR (right panel; C). Data are mean ± SD (n ¼ 3). Cell viability was measured by trypan blue exclusion analysis (D).
Results are expressed as the mean ± SEM of at least three independent experiments. P values are relative to the control. *P < 0.05; **P < 0.01.

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M. Matsumoto et al. / Biochemical and Biophysical Research Communications 473 (2016) 490e496

Hs00832876_gl;
Bax,
Hs00180269_ml;
and
Puma,
Hs00248075_m1. The mRNA expression levels were normalized tothose of b-actin levels.

were treated with 20 mM cisplatin (IC50 was determined as 12 mM
and 8 mM, respectively) solved in water to prevent solventmediated inactivation [24]. As shown in Fig. 1A, cisplatin induced
the expression of Noxa and also the activation of caspase-3, anapoptosis executer. In contrast, shRNA-mediated silencing of Noxa
expression did not affect the induction of cleaved caspase-3, ahallmark of caspase-3 activation, and induction of apoptosis (Fig. 1A
and B). Silencing of other BH3-only proteins important for triggering apoptosis, Bim, Bid or PUMA, also did not affect cisplatininduced apoptosis (Fig. 1C and D). Because the expression level of
PUMA protein was detected at low levels by immunoblot, we
detected the expression of PUMA by qPCR (Fig. 1C, right).

2.5. Cell death assay
NSCLC cell lines were plated in microtiter plates (12 wells,
Greiner Bio-one, Frickenhausen, Germany) at a concentration of1 � 105 cells per well in 500 ml medium and treated with 20 mM
cisplatin in the presence or absence of 1 mM ABT-263 for 48 h or
72 h. After treatment, cell viability was measured by the trypan
blue dye exclusion method using a VI-Cell image analyzer (Beckman Coulter, Brea, CA, USA). The IC50 value for cisplatin was determined as previously described [20]. The signiﬁcance of differences
between the experimental variables was determined using Student's t-test.

3.2. Cisplatin enhances p53-dependent Bak- and p53-independentBax-expression
To better examine the mechanism underlying apoptosis induction, we next analyzed the expression of proapoptotic multidomain
Bcl-2 family proteins Bax and Bak in response to cisplatin treatment, as a previous study demonstrated that the expression of Baxand Bak is directly regulated by p53 [10,25]. As shown in Fig. 2A andB, the protein and mRNA levels of Bax and Bak were induced bycisplatin. Interestingly, while induction of Bak protein and mRNA bycisplatin was clearly suppressed by silencing of p53 expression, theinduction of Bax was not affected, but caspase-3 activation andcleavage of PARP, other hallmarks of apoptosis, were signiﬁcantly

3. Results
3.1. Noxa, PUMA, Bim and Bid do not contribute to cisplatininduced apoptosis in NSCLC cells
Since a previous study demonstrated that cisplatin induces
Noxa-dependent apoptosis in HeLa cells [23], we ﬁrst analyzed therole of Noxa in cisplatin-induced apoptosis of NSCLC cells. The
NSCLC cell lines A549 and H460, which express wild-type p53,

A

B
A549

8
0

12

24

0

24 (h)

12

Bak

25kDa

Bax

20kDa

Tubulin

50kDa

6
5
4
3
2
1
0

C

bak

0

6

12

6
CDDP
0h
12h
24h

5
4
3
2
1
bak

bax

H460
sh-p53

24

7

0

bax

A549
sh-control
CDDP

H460

8

7

mRNA fold induction

CDDP

H460

mRNA fold induction

A549

0

6

sh-control

12

24

0

6

sh-p53

12

24

0

6

12

24 (h)

p53

53kDa

Bak

25kDa

Bax

20kDa

CleavedCas-3
Cleaved
PARP

17kDa
85kDa

Tubulin

50kDa

A549
sh-cont
sh-p53
**
**

0

12
bak

24

0

12
bax

24 (h)

8
7
6
5
4
3
2
1
0

E

H460
sh-cont
sh-p53

*
*

0

12
bak

24

0

12
bax

24 (h)

cell viability (%)

8
7
6
5
4
3
2
1
0

mRNA fold induction

mRNA fold induction

D

A549

100
80

H460
**

100
80

60

60

40

40

20

20

0

sh-control sh-p53*

0sh-control sh-p53

Fig. 2. Role of p53 in the activation of Bax and Bak in cisplatin-treated NSCLC cells. (A, B) A549 and H460 cells were treated with cisplatin (20 mM) for the indicated times andanalyzed by immunoblot analysis using indicated antibodies (A), and Bax and Bak mRNA levels were determined by qPCR. Data are mean ± SD (n ¼ 3). (C, D, E) A549 and H460 cells
stably expressing sh-p53 or sh-control were treated with cisplatin (20 mM) for the indicated times, and analyzed by immunoblot analysis using the indicated antibodies (C), and Bax
or Bak mRNA were determined by qPCR (D). Data are mean ± SD (n ¼ 3). Cell viability was measured (E). Results are expressed as the mean ± SEM of at least three independent
experiments. Student's t-test was used for statistical analysis. P values are relative to the control. *P < 0.05; **P < 0.01.

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M. Matsumoto et al. / Biochemical and Biophysical Research Communications 473 (2016) 490e496

Although it has been demonstrated that survival of several cancer
cell lines completely depend on Mcl-1 [26,27], apoptosis was notaffected by silencing of Mcl-1 expression (Fig. 4A and B). BH3-onlyproteins associate with antiapoptotic multidomain Bcl-2 family
proteins via their BH3-domain, resulting in induction of apoptosis[12,13]. Recently, BH3 mimetics have been used for cancer
chemotherapy and ABT-263 has proved promising in clinical trials[28,29]. Because ABT-263 is a speciﬁc inhibitor of Bcl-2 and Bcl-XL,
but not Mcl-1, we next analyzed the effect of ABT-263 on cisplatininduced apoptosis. ABT-263 enhanced the cisplatin-induced activation of Bax, Bak, and caspase-3 in A549 cells (Fig. 4C). In contrast,
in A549 cells expressing p53 shRNA, cisplatin-induced activation ofBax and Bak was suppressed; however, ABT-263 clearly enhanced
the activation of Bax and caspase-3 (Fig. 4C). In correlation withthese effects, ABT-263 enhanced apoptosis in A549 cells as well as
A549 cells expressing p53 shRNA, especially at early time points(Fig. 4D). Moreover, cisplatin and ABT-263 synergistically enhanced
the activation of Bax and caspase-3 and the induction of apoptosis
in p53 mutated LC2/Ad and PC10 cells (Fig. 4E and F).

suppressed by silencing p53 (Fig. 2C and D). Induction of apoptosis
was also suppressed by shRNA-mediated silencing of p53 expression (Fig. 2E).
3.3. Induction of apoptosis by cisplatin in NSCLC cells lacking p53
function was suppressed by reduced Bax expression
In A549 cells expressing p53 shRNA, the cisplatin-induced
activation of Bak (detected by antibodies speciﬁc to the activatedform of Bak) was suppressed; in contrast, the activation of Bax was
still observed (Fig. 3A). Therefore, we next analyzed whether
apoptosis induction by cisplatin was also dependent on Bax induction. As shown in Fig. 3B and C, apoptosis induction by cisplatinin A549 cells expressing both p53 shRNA and Bax shRNA wassigniﬁcantly suppressed compared with only p53 shRNAexpressing cells. As expected, in the p53-mutant NSCLC cell lines
LC2/Ad and PC10, cisplatin treatment resulted in the induction ofBax but not Bak (Fig. 3D). The expression and induction of Noxa
were also suppressed in these cells. Furthermore, cisplatin-induced
apoptosis in LC2/Ad cells was suppressed by silencing of Bax but not
Bak expression (Fig. 3E).

4. Discussion
In the apoptosis regulatory pathways mediated by the Bcl-2
family proteins, induction and/or activation of BH3-only proteinsin response to apoptosis-inducing signals result in activation of
pro-apoptotic multidomain Bcl-2 families Bax and Bak throughdirect association and inhibition of anti-apoptotic multidomain Bcl2 families [12,13]. In this triparticle apoptotic switch, BH3-onlyproteins function as an initial triggering factor. The importance of

3.4. Cisplatin-induced apoptosis in NSCLC cells was not affected by
reduced expression of Mcl-1 and enhanced by ABT-263
Antiapoptotic multidomain Bcl-2 family proteins, such as Mcl-1
and Bcl-XL, inhibit apoptosis through direct association with Baxand Bak [12]. As shown in Fig. 4A and B, cisplatin-induced apoptosis
in NSCLC cells was enhanced by silencing of Bcl-XL expression.

A

B

Bak

25kDa

Bax

20kDa

Tubulin

50kDa

x

nt

-Ba

20kDa

-co

-co

*

nt

25kDa

sh-p53x

A549 sh-control

sh

+

sh

-

-Ba

+

Bax
p53
Tubulin**100
Cell viability (% of control)

WCL

-

sh

CDDP
IP : Bak (ab-1)
IB : Bak
IP : Bax (6A7)
IB : BaxC

sh-p53sh

A549 sh-control

*

**

80
60
40
20
0

sh-control sh-p53

sh-Bax sh-p53++

sh-cont sh-Bax

Bak

25kDa

Bax

20kDa

Noxa

10kDa

Tubulin

50kDa

k

sh

(h)

-Ba

nt

48

Bak
TubulinBax
Tubulin

x

24

-Ba

12

sh

0

-co

PC10
48

nt

24

-co

12

sh

0

sh

LC2/AdCDDP

LC2/Ad100
Cell viability (% of control)E

D

LC2/Ad

80
60

**

40
20
0

sh-control sh-Bak

sh-BaxFig. 3. Bax and Bak activation in cisplatin-treated NSCLC cells lacking p53 function. (A) A549 and H460 cells stably expressing sh-p53 or sh-control were treated with cisplatin(20 mM) for 12 h and analyzed by immunoprecipitation (IP) with conformational change-speciﬁc anti-Bak (Ab-1) or conformational change-speciﬁc anti-Bax (6A7) antibodies.
Immunoblot analyses were carried out on precipitated samples with indicated antibodies (upper panel). The asterisk indicates a nonspeciﬁc band. Whole cell lysates (WCLs) were
subjected to immunoblot analysis with the indicated antibodies (lower panel). (B, C) A549 and H460 cells stably expressing sh-p53 or sh-control were further infected withlentivirus encoding sh-Bax or sh-control and analyzed by immunoblot analysis using indicated antibodies (B). Cells were treated with cisplatin (20 mM) for 24 h and cell viability wasmeasured (C). Results are expressed as the mean ± SEM of at least three independent experiments. P values are relative to the control. *P < 0.05; **P < 0.01. (D) LC2/Ad and PC10 cells
were treated with cisplatin (20 mM) for the indicated times, and analyzed by immunoblot analysis using indicated antibodies. (E) LC2/Ad and PC10 cells stably expressing sh-Bak, shBax or sh-control were subjected to immunoblot analysis using the indicated antibodies (left panel). The cells were treated with cisplatin (20 mM) for 24 h, and cell viability wasmeasured (right panel). Results are expressed as the mean ± SEM of at least three independent experiments. P values are relative to the control. **P < 0.01.

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M. Matsumoto et al. / Biochemical and Biophysical Research Communications 473 (2016) 490e496

B

sh
-co
nt
sh
-M
cl1

sh
-co
nt
sh
-M
cl1

A549

30kDa

Tubulin

50kDa

**

0.6
0.4

20kDa

cell viability (%)

25kDa

Cleaved
Cas-3

17kDa
(short exposure)
17kDa(long exposure)

CleavedPARP

85kDa(short exposure)
85kDa(long exposure)

Tubulin

50kDa

E

60
40

0
CDDP

+

*

+

-

-

+

+
+

* 25kDa
20kDa

Bak

25kDa

Bax

20kDa

WCL Cleaved
Cas-3
Cleaved
PARP

17kDa

Tubulin

50kDa-

ABT-263-+++

LC2/Adrelative cell viability--

sh-control
sh-p53

20

PC10

+
+

**

80

F

-

L

25kDa

Bax

+

-1

A549
100

20kDa

-

l-X
Bc

+

Bak

CDDP
ABT-263
IP : Bak (ab-1)
IB : Bak
IP : Bax (6A7)
IB : Bax

sh

l

+
+

25kDa
25k
25kD
5kD
kDa
a

LC2/Ad

cl

-

tro

-

+

on

L

+

D

sh-p53

-

+
+

-c

l-X
Bc

-1

cl

l

-

-

sh

sh

tro

+

-M
sh

0

-M
sh

0.2

on

WCL

sh-control

-

0.4

0

-c

A549
CDDP
ABT-263
IP : Bak (ab-1)
IB : Bak
IP : Bax (6A7)
IB : Bax

*

0.6

0.2

sh

C

0.8

PC10

1

1.0

0.8

0.8

relative cell viability

Bcl-XL1

0.8

relative cell viability

sh
-Bc
l-X

t
sh
-co
n

sh
-co
nt
sh
-Bc
l-X

L

H460
L

A549

relative cell viability

50kDa

1.2

1

0.6
*

0.4

**
0.6
0.4
0.2

0.2

85kDa

0

CDDP
ABT-263--+

Tubulin

1.2

+
-

40kDa

1.4

+
+

0
CDDP
ABT-263

-

-

+

Mcl-1

H460

1.4

+
-

H460

+

A549

-

A

+
+

Fig. 4. Effect of ABT-263 on cisplatin-treated p53 mutant NSCLC cells. (A, B) A549 and H460 cells expressing sh-Bcl-XL, sh-Mcl-1 or sh-control were subjected to immunoblot
analysis using the indicated antibodies (left panel). Cells were treated with cisplatin (20 mM) for 24 h and cell viability was measured (B). Results are expressed as the mean ± SEM of
at least three independent experiments. P values are relative to the control. *P < 0.05; **P < 0.01. (C) A549 cells expressing sh-p53 or sh-control were treated with cisplatin (20 mM)
for 12 h, and the active forms of Bax and Bak were detected as described in Fig. 3A (upper panel). WCLs were subjected to immunoblot analysis with the indicated antibodies (underpanel). (D) LC2/Ad and PC10 cells were treated with cisplatin (20 mM) in the presence or absence of ABT-263 (1 mM) for 12 h and the active forms of Bax and Bak were detected(upper panel). The asterisk indicates a nonspeciﬁc band. WCLs were subjected to immunoblot analysis with the indicated antibodies (lower panel). (E) A549 cells stably expressing
sh-p53 or sh-control and LC2/Ad or PC10 cells were treated with cisplatin (20 mM) in the presence or absence of ABT-263 (1 mM) for 12 h and cell viability was measured. Results are
expressed as the mean ± SEM of at least three independent experiments. P values are relative to the control. *P < 0.05; **P < 0.01.

BH3-only proteins, especially Bim, Bid, Puma and Noxa, was previously demonstrated by the results showing that quadruple deﬁcient cells show almost the same phenotype as Bax and Bak doubledeﬁcient cells [14]. However, our present study indicates that theBH3-only proteins do not regulate cisplatin-induced apoptosis in
NSCLC cells. In our system, only Noxa was induced by cisplatin, butsilencing of Noxa expression and also silencing of Bim, Bid or Puma
expression could not affect cisplatin-induced apoptosis. In contrast,
we determined that cisplatin induces p53-dependent Bak expression and also p53-independent Bax expression. Moreover, in p53

wild-type cells, cisplatin-induced apoptosis is affected by reduced
Bak expression, and in p53 lacking or mutated cells, cisplatininduced apoptosis is affected by reduced Bax expression. Therefore, although we cannot exclude the possibility that another BH3only protein regulates this apoptosis or that the other BH3-onlyproteins compensate for the effect of silencing of single BH3-onlyprotein, our results suggest that Bak- and Bax-induction bycisplatin is important for the induction of apoptosis in NSCLC cells.
MCL-1 is one of the most highly ampliﬁed genes in a variety ofhuman cancers, and its expression is often associated with−47−

M. Matsumoto et al. / Biochemical and Biophysical Research Communications 473 (2016) 490e496

resistance of chemotherapy and relapse of cancer [30]. Moreover,other studies showed that survival of several cancer cell lines
completely depend on Mcl-1 [26,27]. These ﬁndings suggest that
Mcl-1 is an important factor for cancer cell survival and tumor
expansion. However, NSCLC cell lines used in these experiments
could not show a protective effect not only in cell survival but also
in cisplatin-induced apoptosis. Moreover, the expression of Noxa, a
speciﬁc inhibitor of Mcl-1, was efﬁciently induced by cisplatin, but
inhibition of the Noxa expression could not affect cisplatin-induced
apoptosis, suggesting that the Noxa-Mcl-1 pathway could noteffectively regulate cell survival and death in NSCLC. Although Noxa
inhibits Mcl-1 by ubiquitin-mediated degradation [31], otherstudies showed that Noxa, also called APR, is overexpressed inseveral cancer cell lines [20,32]. Therefore, it is possible that Noxa
does not inhibit Mcl-1 through a speciﬁc protein modiﬁcation or
expression of other cofactor(s), and also that Mcl-1 cannot function
under some conditions.
In several cancers, it has been reported that p53 status is an
important determinant of cisplatin sensitivity; however, somestudies suggest that cisplatin-induced cell death is independent of
p53 status [33,34]. Other studies showed that the absence of p53
does not alter cellular sensitivity to cisplatin [11,34], but the precise
mechanism has not been fully elucidated. In the present study, wedemonstrate that cisplatin induces apoptosis by the activation ofboth p53-dependent Bak and p53-independent Bax expression,and inhibition of Bax expression attenuates cisplatin-induced
apoptosis in p53 lacking or mutated cells. These results indicatethat the induction of Bax is involved in p53-independent apoptosis
by cisplatin. A previous study showed that p53 can directly activate
the Bak promoter [25]. The mechanism of p53-independent Bax
induction has not been fully elucidated. However, a previous study
showed that p73, a p53-related transcription factor, activates theBax promoter [35], suggesting its possible role in p53-independent
Bax induction.
BH3 mimetics, such as ABT-263, are expected to contribute as
efﬁcient cancer therapy, but they could not antagonize Mcl-1[28,29]. In small cell lung cancer, it has been shown that ABT-263
enhances the induction of apoptosis by the HDAC inhibitor, vorinostat [36]. In contrast to its importance in cancer, we observedthat Mcl-1 could not affect in cisplatin-induced apoptosis in several
NSCLC cell lines. Moreover, ABT-263 synergistically enhances
cisplatin-induced apoptosis in these cells, even in mutant p53expressing cells. These results suggest the possibility that ABT263 is an efﬁcient therapeutic agent for NSCLC.
Competing ﬁnancial interests
The authors declare no competing ﬁnancial interests.
Acknowledgments
We thank Hisashi Harada, Yoshinori Abe, Ikuno Uehara, andAtsuko Tanimura for helpful critical discussions, and Yumi Asano,
Miho Kawagoe and Yoshimi Takatera for technical support. This
work was supported by Grants-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology of Japan (MEXT)
and by a grant from the MEXT-Supported Program for the Strategic
Research Foundation at Private Universities.
Transparency document
Transparency document related to this article can be foundonline at http://dx.doi.org/10.1016/j.bbrc.2016.03.053.

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Ⅳ．生体機能制御学部門
Department of Bioregulation

生体機能制御学部門（大学院

生体機能制御学部門）
教授

南

史朗

【研究概要】
生体の個体としての機能とその制御機構の解明をめざし、ホルモン・生理活性物質を対象として生理学的研究を行っている。ホルモンの分泌調節機構、ホルモンの作用機序、ホルモンの細胞内シグナル伝達機構を研究し、病態の解明をめざす。
Ｉ．栄養状態と細胞内シグナリング（豊島由香、田口雄亮、時田玲子）
動物は、栄養状態に応答して物質代謝を変動し、恒常性を維持する巧妙な仕組みを持つ。体内の様々なホルモン分泌を変化させると共に、組織ごとに細胞内のシグナル伝達因子を変動させることで物質代謝を調節すると考えられる。私たちは、タンパク質・アミノ酸栄養状態に着目し、それによって変動するホルモン（インスリン、インスリン様成長因子、成長ホルモン、アディポネクチンなど）の分泌調節機構、およびこれらホルモンの細胞内シグナル伝達系を介した代謝調節機構の解明を目指している。
１）インスリンシグナル因子の変化がもたらす脂質代謝調節機構の解明
これまでに、低タンパク質栄養状態のラットの肝ではインスリンシグナリングが増強して中性脂加することを見出してきた。肝臓特異的に 4E-BP1 の発現を抑制したラットを作成して検討した結果、4E-BP1 が低タンパク質栄養状態による脂肪肝の生成に必須な因子であり、肝中性脂肪含量の調節に重要な役割を果たすことを明らかとした。
さらに、IRS-2 の病態生理的意義を明らかにする目的で、ゲノム編集技術の一つである CRISPR/
Cas9 法により IRS-2 ノックアウトラットの作出を試み、成功した。
２）インスリンシグナルの増強による脂質合成酵素の変化
単離肝細胞を用いた検討により、低タンパク質栄養状態のラット肝ではインスリンシグナルの増強によって脂質合成酵素 Acaca 遺伝子の発現量が増加し、脂質合成亢進の一因と考えられた。
Ⅱ．成長ホルモンの新たな生理作用と作用機序（中田朋子、福島

誠、南

史朗）

成長ホルモン（GH）は雄ラットでは 3 時間ごとに分泌されるのに対し雌では不規則に分泌される。この分泌リズムの違いが様々な遺伝子発現の雌雄差を作っていることが知られているが、その機序や意義はよく解明されていない。私たちは、ラット GH 分泌の雌雄差をマーカーとして GH の新たな生理作用を検討してきた。
１）AKR1D1 はステロイド核の 4 位の二重結合を還元する酵素であり、ステロイドホルモンの代謝や胆汁酸の合成に働く。ラット肝の AKR1D1 の mRNA・タンパク量には雌雄差があり、GH 依存的に変動することを明らかにした。また、AKR1D1と異物を認識する核受容体 CAR の mRNA 量に相関があることから、GH の肝におけるステロイド代謝調節機序の解明、および異物認識（解毒作用）機序の解明につながると期待される。

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生体機能制御学部門

肪含量が増加すること、インスリン作用の発揮に重要な IRS-2 および翻訳抑制因子 4E-BP1 の量が増

Ⅲ．メタボリック症候群の脂肪酸合成酵素 FAS の役割（八木孝、矢野宏行、南史朗）

糖尿病や脂肪肝などの代謝異常は、医学的・社会的に重大な問題である。私たちは、栄養状態の変化に対応する肝における糖・脂質代謝異常の機序を解明するために、国際医療センター分子代謝制御研究部（松本道宏博士）と共同研究を行い、脂肪合成酵素 FAS に焦点をあてて研究を進めてきた。
１）肥満・2 型糖尿病における肝 FAS の意義
内性脂肪酸合成（de novo lipogenesis ：DNL）を触媒するリポジェニック酵素群の活性は食後２型
糖尿病肝で亢進している。DNL の鍵となる脂肪酸合成酵素（FAS）は、グルコースからパルミチン酸を合成するリポジェニック酵素であり、その病態生理的意義は不明である。そこで、脂肪肝合併２型
糖尿病モデルである ob/ob マウスの遺伝的背景を導入した肝特異的 FAS 欠損マウス (ob/ob LKO) を作製し、検討した。その結果、肥満・糖尿病肝における FAS の発現亢進は、DNL・糖新生・β 酸化・
肝糖取り込みの制御を介して絶食−摂食サイクルにおける血糖値の変動、すなわち空腹時の血糖低下や摂食後の高血糖を軽減し、血糖の恒常性維持に寄与する可能性を提唱した。
２）非アルコール性脂肪肝炎（NASH）の病態における FAS の役割
炎症・線維化をきたした食餌誘導 NASH の病態における FAS の役割は明らかでない。そこで、ob/ob LKO を、トランス脂肪酸やフルクトースおよびコレステロールを多量に含む NASH 誘導食により飼育し、代謝表現型解析を行った。肝 FAS 欠損によって、通常食飼育では脂肪肝の著明な改善と肝機能障害の悪化を認めた。NASH 誘導食飼育では肝中性脂肪含量の軽度な低下と、肝機能障害の軽減、炎症関連遺伝子の発現低下や線維化の著明な抑制を認めた。FAS は NASH の病態形成の促進因子であり、その寄与は食餌内容によって異なると考えられた。
Ⅳ．性ステロイドの中枢作用（折笠千登世、勝又晴美）
性ステロイドは全身性に作用して、動物の生殖機能のみならず、生理機能を維持する役割を担っている。また、性ステロイドが周生期の脳に作用することによって性差を含む脳の機能的分化をもたらす。統合中枢である脳機能の分化および維持にどのように関与するのか、分子レベルで研究する。
１）ラットの養育行動の神経回路の解析
養育行動は、生存を脅かす様々な要因から仔をまもる行動であり、自然界ではもちろん、人工的に飼育する実験環境においても容易に認められる。本研究の目標は、養育行動関連の神経回路の特定を行い、その回路発現に関わる分子基盤を明らかにすることにある。養育行動の神経回路の組織化学的解析を行い、性的未経験雄の養育行動が単飼または群飼といった環境要因によって異なることを明らかにした（Orikasa et al. Physiol Behav 2015）。この行動を統御する遺伝子がメラニン凝集ホルモンであるか否か cfos 発現を指標にして検討した。

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【研究業績】〈原著〉
1．Orikasa C, Nagaoka K, Katsumata H, Sato M, Kondo Y, Minami S, Sakuma Y. Social isolation
prompts maternal behavior in sexually naïve male ddN mice. Physiol Behav 151: 9-15, 2015

〈総説〉

〈学会発表〉（国際学会）
1．Yuka Toyoshima, Reiko Tokita, Yusuke Taguchi, Fumiaki Yoshizawa, Shin-Ichiro Takahashi,
Hisanori Kato, and Shiro Minami.  Protein malnutrition increases the amount and phosphorylationof 4E-binding protein 1 (4E-BP1) to accumulate lipid in rat liver.  12th Asian Congress of Nutrition,
May 2015, Yokohama
2．Taguchi Y, Toyoshima Y, Tokita R, Kato H, Takahashi S, Minami S.  Insulin-induced acetyl-CoA
carboxylase alpha is responsible for triglyceride accumulation in primary hepatocytes of low
protein diet-fed rats.  12th Asian Congress of Nutrition, May 2015, Yokohama（国内学会）
1．八木

亨、辻村−早川知子、山地大介、矢野宏行、満島勝、長嶋洋治、史朗、春日雅人、松本道宏。肝臓特異的な脂肪酸合成酵素の欠損は ob/ob マウスの脂肪肝と耐糖能を改善するが随時高血糖を惹起する。第 52 回日本臨床分子医学会学術集会、2015 年 4 月、
京都
2．八木
南

孝、酒井真志人、辻村−早川知子、山地大介、矢野宏行、満島勝、内田 亨、長嶋洋治、史朗、春日雅人、松本道宏。肝臓特異的な脂肪酸合成酵素の欠損は ob/ob マウスの脂肪肝と耐糖能を改善するが随時高血糖を惹起する。第 88 回日本内分泌学会学術総会、2015 年 4 月、東京
3．八木
南孝、酒井真志人、辻村−早川知子、山地大介、矢野宏行、満島勝、内田亨、長嶋洋治、史朗、春日雅人、松本道宏。肝臓特異的な脂肪酸合成酵素の欠損は ob/ob マウスの脂肪肝と耐糖能を改善するが随時高血糖を惹起する。第 58 回日本糖尿病学会年次学術集会、2015 年 4 月、
下関
4．村澤恒男、石川真由美、南史朗。2 型糖尿病患者の alogliptin 無効例に対する sitagliptin 切り替

え時の体重、推算 GFR を含めた有効性・安全性の検討。第 58 回日本糖尿病学会年次学術集会、2015 年 4 月、下関
5．石川真由美、豊村順子、田口雄亮、中田朋子、豊島由香、南史朗。成長ホルモンによる膵β細胞の小胞体ストレスの抑制。第 58 回日本糖尿病学会年次学術集会、2015 年 4 月
6．Orikasa C, Nagaoka K, Katsumata H, Sato M, Kondo Y, Sakuma Y, Minami S.
prompts maternal behavior in sexually naïve male ddN mice.
本行動神経内分泌合同学術集会、2015 年 9 月、仙台

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下関
Social isolation

第 42 回日本神経内分泌第 23 回日

生体機能制御学部門南孝、酒井真志人、内田7．福永ヒトミ、望月留美、金子佳世、大槻昌子、八木孝、石川真由美、南史朗、立山尚子、米山剛一。妊娠糖尿病の反復例における患者の意識についての検討。第 31 回糖尿病・妊娠学会、2015 年 10 月、東京
8．大槻昌子、八木孝、南史朗、石川真由美、金城忠志、田島廣之、山崎有人、笹野公伸。サブ

クリニカルクッシング症候群に合併した原発性アルドステロン症の１例。第 24 回内分泌臨床Update、2015 年 11 月、大宮
9．西宏起、山中大介、亀井宏泰、森友美、豊島由香、竹中麻子、潮秀樹、千田和広、伯野史彦、

高橋伸一郎 .   アミノ酸欠乏をシグナルとした肝臓特異的な脂質蓄積の新機構の解明 .   BMB2015（第 38 回日本分子生物学会年会／第 88 回日本生化学大会合同大会）、2015 年 12 月，神戸
10. 中田朋子、平野良隆、勝又晴美、時田玲子、南史朗 .   成長ホルモンはラット肝臓においてAKR1D1 の発現を抑制する。Growth hormone represses expression of AKR1D1 in the rat liver.  
第 38 回日本分子生物学会年会
11. 八木第 88 回日本生化学会大会

孝、酒井真志人、辻村−早川知子、矢野宏行、満島合同大会、2015 年 12 月、神戸
勝、長嶋洋治、南史朗、春日雅人、

松本道宏。脂肪肝合併 2 型糖尿病の病態における肝臓の脂肪酸合成酵素の役割の解明。第 27 回分
子糖尿病シンポジウム、2015 年 12 月、東京
12. 八木孝、酒井真志人、辻村−早川知子、矢野宏行、満島長嶋洋二、南勝、飯田智、長沼孝雄、高峰英、史朗、春日雅人、松本道宏。肝臓特異的な脂肪酸合成酵素の欠損は食餌誘導性非アルコール性脂肪肝炎の進展を抑制する。第 19 回日本病態栄養学会年次学術集会、2016 年 1 月、
横浜

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Physiology & Behavior 151 (2015) 9–15

Contents lists available at ScienceDirect

Physiology & Behavior
journal homepage: www.elsevier.com/locate/phb

Social isolation prompts maternal behavior in sexually naïve maleddN mice
Chitose Orikasa a,⁎, Kentaro Nagaoka b, Harumi Katsumata a, Manami Sato c, Yasuhiko Kondo c,
Shiro Minami a, Yasuo Sakuma daInstitute for Advanced Medical Science, Nippon Medical School, Kanagawa 211-8533, Japan
Department of Veterinary Medicine, Tokyo University of Agriculture and Technology, Tokyo 183-0054, Japan
Department of Animal Sciences, Teikyo University of Science, Tokyo 120-0045, Japand
University of Tokyo Health Science, Tokyo 206-0033, Japanbc

H I G H L I G H T S• Social isolation in sexually naïve male mice promoted the onset of maternal behavior.
• Social isolation during either adolescence or young adulthood was effective.
• Partial isolation (exposure to conspeciﬁc signals) was not effective.

a r t i c l e

i n f oArticle history:
Received 17 March 2015
Received in revised form 2 July 2015
Accepted 3 July 2015
Available online 10 July 2015
Keywords:
Maternal behavior
Social isolation
Chemosensory
ddN male mice
Pubertya b s t r a c tMaternal behavior in mice is considered to be sexually dimorphic; that is, females show maternal care for theiroffspring, whereas this behavior is rarely shown in males. Here, we examined how social isolation affects the interaction of adult male mice with pups. Three weeks of isolation during puberty (5–8 weeks old) induced retrieving and crouching when exposed to pups, while males with 1 week isolation (7–8 weeks old) also showed suchmaternal care, but were less responsive to pups. We also examined the effect of isolation during young adulthood(8–11 weeks old), and found an induction of maternal behavior comparable to that in younger male mice. Thiseffect was blocked by exposure to chemosensory and auditory social signals derived from males in an attachedcompartment separated by doubled opaque barriers. These results demonstrate that social isolation in both puberty and postpuberty facilitates male maternal behavior in sexually naïve mice. The results also indicate that airborne chemicals and/or sounds of male conspeciﬁcs, including ultrasonic vocalization and noise by their movement may be sufﬁcient to interfere with the isolation effect on induction of maternal behavior in male mice.
© 2015 Elsevier Inc. All rights reserved.

1. Introduction
Maternal care is an indispensable aspect of mammalian reproduction, as offspring are fed by lactation. In rodents, parturient females
build a nest as preparation for pups after delivery. After delivery, females immediately retrieve pups to the nest, licking around theiranogenital area and crouching over them. In contrast, virgin females
are less interested in pups and maternal care. However, such females
may become maternal, provided they are exposed to pups for a few
days (so-called priming) [1–3]. In contrast, maternal care is not spontaneous in most male rodents [3]. In laboratory mouse strains, sexually⁎ Corresponding author at: Institute for Advanced Medical Science, Nippon Medical
School, Kosugi, Nakahara, Kanagawa 211-8533, Japan.
E-mail address: orikasa@nms.ac.jp (C. Orikasa).

naïve males rarely show maternal behavior. When these males encounter pups, they sometimes engage in infanticide [4,5]. However, the experience of mating followed by cohabitation with gestating females
has been reported to suppress infanticide and provoke males to show
maternal behavior similar to lactating mothers [6,7]. This indicatesthat functional neural circuits regulating maternal-speciﬁc behaviors
exist in the normal male mouse brain, but might not be activated in sexually naïve mice.
Social isolation exerts a variety of inﬂuences on a wide range ofphysiology and behaviors. In male mice, it induces inter-male aggression [8] and augmented emotionality [9]. In female mice, pubertal isolation disrupts sexual behavior despite the increased expression of
estrogen receptors observed in the anteroventral periventricular nucleus and the ventromedial nucleus of the hypothalamus after pubertal
isolation [10]. Social isolation can be a stressful event in mice and rats
[11,12]. The effect of social stress during puberty can cause long-

http://dx.doi.org/10.1016/j.physbeh.2015.07.007
0031-9384/© 2015 Elsevier Inc. All rights reserved.

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C. Orikasa et al. / Physiology & Behavior 151 (2015) 9–15

lasting alterations in the brain and subsequently in adult behaviors.
Therefore in this experiment, we examined the effect of social isolation
starting at 5 weeks of age, when mice are just beginning to enter puberty.
Interestingly, previous research has demonstrated that social isolation in
adult mice can elevate play behavior [3] and parenting behavior in malemice [13]. Although typically 70–80% of adult male mice will attack a
newborn pup [14], male mice showed decreased infanticide after
isolation [13].
In this study, we examined whether lack of social signals during adolescence increases maternal behavior in sexually naïve male mice aswell as during young adulthood. Pheromone signals may be involvedin these physiological and behavioral responses [15–18]; therefore, we
also examined the effect of modiﬁed social isolation that allowed malemice to smell and hear adjacent male conspeciﬁcs, but not to see ortouch them.
2. Materials and methods
2.1. Animals
Two pairs of inbred strain ddN mice were purchased from the RIKEN
Bio-Resource center (Wako, Japan) and bred in our laboratory. All maleoffspring were separated from female litter mates at weaning (21 daysold) and kept in groups of three to ﬁve male siblings until the start ofisolation. Mice were housed in 19 × 27 × 15 cm polypropylene cageswith wood chip bedding. Throughout all experiments, mice werehoused in our animal facility under controlled illumination (lights on
from 6 a.m. to 8 p.m.), temperature (23 °C) and humidity (50.0 ±10%). Food and water were given ad libitum.

was moved to a separate cage containing a mixture of fresh and soiled
home-cage bedding 10 min before testing. We then followed a standard
pup presentation paradigm [20] to obtain a measure of maternal behavior in virgin males. The observation began by placing three pups aged
4–7 days in the opposite corner farthest from the male or the corner
occupied by the mouse's resting nest.
We recorded the distance between the males and the pups, anddeﬁned “close” as staying in close proximity (b1 cm) of pups and “far”
as staying further away than 1 cm [21]. Maternal behavior was assessedover 10 min by recording retrieval (picking up a pup with its mouth andcarrying it to the nest) and crouching (extending its limbs, assuming anursing-like posture over the pups). Retrieval latencies, and timespent licking and crouching over the pups were recorded. In retrieval,
the number of pups that they picked up was also recorded. For
crouching, we recorded the number of pups they lay on top of.
We categorized males as “Maternal” when they displayed at least
one of the above responses within 10 min, “Attack” if they attacked
the pups (biting a pup, often accompanied by actual wounds on thepup), and “Ignore” when they showed no response to the pups. If amale appeared about to attack a pup, the pups were quickly removed
and observation was terminated. Pups that had been attacked were
not used in subsequent tests and experiments. Behavioral data ofmales that attacked pups were excluded from analysis of maternal
behavior.

2.3. Maternal behavior test

2.3.1. Experiment 1
Each male was assigned to one of three experimental groups duringpuberty. Mice were housed in isolation in two groups; one for 3 weeks
beginning at age 5 weeks (P3I, n = 40), and one for 1 week beginning atage 7 weeks (P1I, n = 11). The third group (PG, n = 15) was continuously group-housed with siblings separated from female litter mates
at weaning (Fig. 1). Each experimental group was made up of littermates. The sexually naïve P3I males at 5 weeks old and P1I males at
7 weeks old were displaced to cages and housed singly for 3 weeks or
1 week, respectively, prior to behavioral tests at 8 weeks of age. PG
males remained in cages with siblings, and just before behavioral testing were displaced to cages alone for behavioral observation. All males
were tested for maternal behavior at 8 weeks after the proceduredescribed above.

Each experimental male was placed in a clean cage with fresh bedding 2 days before testing. All behavioral tests were carried out during
the day, 5–8 h after lights were turned on [19]. On each day of testing,cages containing experimental males were moved from shelves in the
animal room to the observation setting, and acclimated there for10 min. To test group-housed males' maternal behavior, each male

2.3.2. Experiment 2
In Experiment 2, we examined the effect of modiﬁed social housing
on the induction of maternal behavior by previous social isolation during adulthood. Sexually naïve male mice were assigned into threegroups: isolation for 3 weeks (A3I: from 8 to 11 weeks old, n = 30),
group-housing with barriers (A3G′: from 8 to 11 weeks old, n = 10)

2.2. Ethics statement
Experiments and animal housing adhered to the guidelines for theCare and Use of Laboratory Animals of Nippon Medical School, adopting
NIH (National Institutes of Health) guidelines for the care and use of experimental animals. All procedures were approved by the Committee
for Experimental Animal Ethics in Nippon Medical School.

Exprimental Groups

Birth
0

1

2

Weaning
3
4

5

6

7

8

9

10

11 12
(weeks old)

P3I
Experiment 1

P1I
PG
A3I

Experiment 2

AG
A3G’

Group-housing

Isolation

Group-housing with barrier

Behavior tests
Fig. 1. Schematic representation of isolation and behavioral test schedules in each group.

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C. Orikasa et al. / Physiology & Behavior 151 (2015) 9–15

or group-housing without barriers (AG: from 8 to 11 weeks old, n = 13)(see Fig. 1). In restrained social-housing (A3G′), a pair of litter mates
was kept in the same cage (49 × 28.5 × 29 cm), but separated by a barrier made of two interstitial opaque acrylic boards containing six holes(2-cm diameter) at different levels. This barrier prevented physical, visual and involatile chemical interactions, but allowed airborne odors,
audible and ultrasonic sounds (voice and noise) to pass through. The
ﬂoors of both chambers were covered with wood chips, and food andwater were freely available. Each pair was placed in this apparatus for
3 weeks (from 8 to 11 weeks old), which matched the isolation timeof the A3I group. All mice were tested for maternal behaviors when
they were 11 weeks old (Fig. 1).

**

Time spent close to pups (s)500

2.4. Statistics
We used the Chi-squared test with Bonferroni post hoc tests to examine signiﬁcant differences in incidences of the categories, maternal
behaviors, and attacks toward pups. The duration of each behavior
was analyzed by one-way analysis of variance (ANOVA), followed by
Bonferroni post hoc tests to determine the signiﬁcant differencesamong groups. The nonparametric Mann–Whitney U-test was applied
for analysis of latencies, as a maximal latency (observation time) was allotted for mice that did not respond to the pups. All scoring was done
blindly by two observers, and we checked for coherence between theparametric factors using Pearson's correlation (licking duration, r =0.908***; time spent close to pups, r = 0.986*** of pubertal mice, r =0.994*** of young adults; crouching duration, r = 0.993*** of pubertal
mice, r = 0.990 of young adults), and the nonparametric using
Spearman's correlation (latency to pup retrieval, r = 0.996*** of one
pup, r = 0.964*** of three pups, ***p b .001).

*

400
300
200
100
0
P3I

P1I

PG

Fig. 2. Time spent being close to pups. Duration of time spent in close proximity (b1 cm) topups was signiﬁcantly longer in mice after social isolation for 3 weeks (P3I) or for 1 week
(P1I) than in mice that were group-housed (PG). **p b .001, *p b .05, one-way ANOVA with
Bonferroni post hoc analysis.

than that in PG males (Fig. 3B, one pup; all pups: p b .05). No signiﬁcantdifference in retrieval latency was observed between P1I and PG males(one pup: p = .428; all pups: p = .643), or between P3I and P1I males(one pup: p = .215; all pups: p = .356).
Fig. 4 shows data for time spent licking and crouching. While licking
duration did not differ across groups (Fig. 4A: F2,20 = 1.02, p = .38),
crouching duration was signiﬁcantly longer in the P3I group than in
the PG group (Fig. 4B: F2,64 = 7.98, p b .05, Bonferroni post hoc analysis,
PG: p b .05). There was no signiﬁcant difference in crouching duration
between males in the P1I group compared with the other groups(Bonferroni post hoc analysis, P3I: p = .356; PG: p = .380). Crouching
behavior in each group was analyzed in terms of the number of pups
over which they crouched: all pups, some pups, or none (Fig. 4C). Although the number of males that crouched some pups was signiﬁcantly
higher in the P3I and P1I groups than in the PG group (X2 = 9.90, df = 2,
p b .05), the number of males that crouched all pups was signiﬁcantly
higher in the P3I group than in the PG group (X2 = 8.42, df = 2,
p b .05) (Fig. 4C).

3. Results
3.1. Experiment 1
Table 1 shows the number and percentage of males in each response
category (see Materials and methods: Maternal behavior test). A significantly larger number of singly-housed male mice (P3I and P1I groups)
displayed maternal behavior than PG mice did (X2 = 23.32, df = 2, p b.001). Most group-housed males showed indifference toward pups. A
signiﬁcantly higher percentage of mice showed ‘Ignore’ in the PG
group than in the P3I or P1I groups (X2 = 33.97, df = 2, p b .001). No
signiﬁcant difference was found among groups in the number ofattacked pups (X2 = 2.62, df = 2, p = .270).
Isolated males from the P3I and P1I groups spent signiﬁcantly moretime in the vicinity of pups than did PG males (F2,64 = 10.19, p b .001,
Bonferroni post hoc analysis, P3I: p b .001; P1I: p b .05) (Fig. 2).
Retrieval in each group was analyzed in terms of the number of
retrieved pups: all pups, some pups, or no pups (Fig. 3A). Although
the number of males that retrieved some pups was signiﬁcantly higher
in the P1I group than in the PG group (X2 = 10.75, df = 2, p b .05), thenumber of males that retrieved all three pups was signiﬁcantly higher in
the P3I group than in the PG group (X2 = 13.28, df = 2, p b .05).
Additionally, latency of retrieval in P3I males was signiﬁcantly shorter

3.2. Experiment 2
Table 1 includes the number and percentage of males in each response category for Experiment 2. Only A3I showed a signiﬁcantly
higher proportion of males displaying maternal behavior (X2 = 31.15,
df = 2, p b .001). The number of mice in the ‘Ignore’ category was higher
in both the AG and A3G′ groups than in the A3I group (X2 = 22.90, df =2, p b .001), regardless of whether they were separated by a barrier. No
signiﬁcant difference was found among groups in the number ofattacked pups (X2 = .73, df = 2, p = .70).

Table 1
Number and percentage of males in each response category in Experiment 1 and Experiment 2.
Category

Experiment 1

Experiment 2

P3I (40)

Maternal
IgnoredAttacked

P1I (11)

PG (15)A3I (30)

AG (13)A3G′ (10)Number%Number%Number%Number%Number%Number%27
7
6

67.5**
17.5**
15.0

7
3
1

63.6**
27.3**
9.1

0
15
0

0.0
100.0
0

22
5
3

73.3**
16.7**
10.0

0
11
2

0.0
84.6
15.4

0
8
2

0.0
80.0
20.0

Tests were interrupted when attacks occurred in order to avoid infanticide. Thus, males in the “Attack” category were excluded from further analyses. **p b .001, Chi-square test with
Bonferroni post hoc analysis, different from group-housed males.

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C. Orikasa et al. / Physiology & Behavior 151 (2015) 9–15

A
% Mice showing retrieval

100

Retrieve all pups
Retrieve some pups
No retrieval

80
60
40
20
0

P3I

P1I

PG

Median latency to retrieve (s)

B

*

600

*

600

500

500

400

400

300

300

200

200

100

100

0

0
P3I

P1I

PG

P3I

1 pup

P1I

PG

All pups

Fig. 3. Effect of social isolation on retrieving behavior. (A) Incidence of retrieval behavior in each group (carried all pups, some pups, or no pups). (B) Retrieval behavior was assessed byrecording the latency to pup collection. Median times required to collect one pup (left panel), all pups (right panel) are shown for each group, *p b .05, Mann–Whitney U-test.

Three week isolation after maturation also increased several male–
pup interaction parameters. A3I males spent signiﬁcantly more time in
the vicinity of pups than did AG and A3G′ males (F2,45 = 19.68,
p b .001, Bonferroni post hoc analysis, AG; A3G′: p b .001) (Fig. 5A). Approximately 73% of the A3I group retrieved pups, whereas few did so in
the AG and A3G′ groups (Fig. 5B); this difference in incidence of retrieving all pups was signiﬁcant (Fig. 5B: X 2 = 17.57, df = 2, p b .001). There
was no signiﬁcant difference between groups in incidence of retrievingsome pups (X2 = 6.18, df = 2, p = .045). Crouching duration for A3I
males was also signiﬁcantly longer than that for the AG or A3G′ groups(Fig. 6A: F2,45 = 14.86, p b .001, Bonferroni post hoc analysis, AG:p b .001; A3G′: p b .05), as was the number of all pups that theycrouched over (Fig. 6B: X 2 = 16.04, df = 2, p b .001).
4. Discussion
The results of this study show that social isolation induced maternal
behavior in male mice within 10 min, whereas group-housing did not.
There is a large volume of literature demonstrating that social isolation
usually results in various behavioral changes, such as depression-likebehavior [22], enhanced aggressiveness [23], and higher levels of
impulsivity [24]. It therefore seems paradoxical, at least outwardly,that social isolation enhances intermale aggression but reduces attacks
on pups. These behaviors might be regulated by distinct neural circuits
because of different biological signiﬁcation in social behavior. One
report found that social isolation during adolescence enhanced playand contact behaviors [25]. Our current results are also a case of isolation reinforcing sociality. Thus, the inﬂuence of social isolation may
show a wide diversity depending on social context and behaviors
examined.
Time spent with pups was signiﬁcantly longer in mice that had been
isolated for 3 weeks or for 1 week than in those that had been grouphoused. This resulted from the extended time they spent contacting
and crouching over the pups. Three weeks of isolation also shortened
the latency of pup retrieval, but did not affect time spent licking. Thismight be because the cues that trigger licking are different from thosefor other components of maternal behavior. First contact with pups
and pup-licking have been suggested to be inﬂuenced by emotionalarousal or anxiety in mice [26,27]. Thus, neural substrates underlying
licking may be different from those underlying retrieval and crouching
behavior. Although galanin neurons in the preoptic area (POA) havebeen reported to play an important role in male mouse maternal behavior, optogenetic activation of galanin neurons induced pup grooming
but failed to increase crouching behavior in male mice [28].

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C. Orikasa et al. / Physiology & Behavior 151 (2015) 9–15

A

B
120

Time spent crouching (s)Time spent licking (s)

140

100
80
60
40
20
0

*

200
150
100
50
0

P3I

P1I

PG

P3I

P1I

PG

C
% Mice showing crouching100
Crouch all pups
Crouch some pups
No crouching

80
60
40
20
0

P3I

P1I

PG

Fig. 4. Effect of social isolation on pup licking and crouching behavior. (A) Mean duration (s) that pups were licked in each group. Time spent licking pups was not signiﬁcantly different
across groups. (B) Mean duration (s) of crouching behavior (feeding posture) for each group. Males in the P3I group crouched longer than those in the PG group, *p b .05, one-way ANOVA
with Bonferroni post hoc analysis. (C) Incidence of crouching behavior in each group (over all pups, some pups, or no pups).

Although both P3I and P1I males were categorized as ‘Maternal’, thequality of maternal behavior was very different between them. A very
high percentage of P3I males retrieved all pups and crouched over

A

B

**

500

100

*

% Mice showing retrieval

600
Time spent close to pups (s)them, while most P1I males retrieved only one or two pups (Fig. 4).
Thus, it seems that 3 weeks of isolation can induce robust maternal behavior, while 1 week of isolation is effective at slightly increasing

400
300
200
100
0
A3I

AG

A3G’

Retrieve all pups
Retrieve some pups
No retrieval

80
60
40
20
0

A3I

AG

A3G’

Fig. 5. Effects of social isolation after maturation on maternal behavior in male mice. (A) Mean duration (s) of time spent in the vicinity of pups was signiﬁcantly longer in male mice after
social isolation for 3 weeks (A3I) than group-housing males with barriers (A3G′) or without barriers (AG). **p b .001, one-way ANOVA with Bonferroni post hoc analysis. (B) Incidence of
retrieval behavior in each group (carried all pups, some pups, or no pups).

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C. Orikasa et al. / Physiology & Behavior 151 (2015) 9–15

A

B
100

**

200
150

% Mice showing crouching

Time spent crouching (s)

250

*

100
50
0

Crouch all pups
Crouch some pups
No crouching

80
60
40
20
0

A3I

AG

A3G’

A3I

AG

A3G’

Fig. 6. Effect of social isolation after maturation on crouching behavior. (A) Crouching duration (s). A3I males spent more time crouching over pups than did group-housed male mice withor without barriers. **p b .001, *p b .05, one-way ANOVA with Bonferroni post hoc analysis. (B) Incidence of crouching behavior in each group (over all pups, some pups, or no pups).

maternal behavior but not to a sufﬁcient level. We also examined the effect of isolation at different developmental stages, demonstrating thatsocial isolation was not only effective in puberty (5–8 weeks old), butalso post-puberty (8–11 weeks old). Therefore, the effect of social isolation on maternal care does not depend on hormonal conditions speciﬁc
to puberty, but rather depends on neural and/or humoral conditions
produced by the isolation. However, Ghiraldi and Svare [13] reported
that long isolation from weaning to adult age failed to suppress attackof pups. Presumably, isolation for too long during development may disrupt socialization instead of inducing maternal behavior.
In non-monogamous rodents, maternal behavior is generally female
speciﬁc (i.e. sexually dimorphic). In most strains of laboratory mice,males show indifference or attacking in response to pups [14]. Indeed,sexually naïve male laboratory mice rarely show maternal care, andsometimes commit infanticide [6]. Perinatal sex steroids masculinize
the mouse brain, resulting in suppression of maternal behavior andfacilitation of pup attack [29]. Sex steroids during the critical period ofbrain sex differentiation may produce inhibitory substrates for maternal
behavior in male mice. However, a system that activates an inactive
system of maternal behavior in the male mouse brain could be ‘turned
off’ by social isolation, like a sensitization to pups for male rats [30,31].
What is the factor that activates maternal behavior in the male mousebrain through social isolation? In this study, we examined the effect ofrestricted social stimuli (A3G′) on maternal behavior, i.e., cohabiters
were separated by double opaque barriers that prevented direct contact
and visual cues but were permeable to airborne chemical and auditory
signals. In most mammalian species, chemosensory signals derived
from conspeciﬁcs may be the most important social stimulus. A lack ofthese chemosensory signals could hence be responsible for the isolation
effect.
In male mice, copulation and subsequent cohabitation with a pregnant female lead to a transition in behavior from attacking (infanticide)to parenting [6,7]. The current study demonstrates that not only cohabitation but social isolation can cause male mice to be maternal. Currently, we do not have enough data to reveal whether these phenomenashare the same neural substrates. It is noteworthy that the brain regions
activated by pup exposure [6] are also activated during infanticide [7,32]. This might imply that the same or overlapped neural mechanisms
underlie maternal responsiveness that appears after different social experiences. Future investigation into these drastic effects of social experience will be important to fully understand the neural circuitry
controlling maternal behaviors.
Disclosure statement
The authors have nothing to disclose.

Acknowledgments
This work was founded by Grants-in-Aid for scientiﬁc research from
the Ministry of Education, Science, Sports and Culture of Japan (C.O.
20590238).

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parental care in C57BL/6J male mice, Physiol. Behav. 36 (1986) 59–62.
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substances, Nature 183 (1959) 55–56.
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The effects of social experience and gonadal hormones on retrieving behavior of−62−

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−63−

武蔵小杉地区動物実験室

運営委員会委員長

南史朗【運営概要】
武蔵小杉地区動物実験室は、日本医科大学の共同利用研究設備として先端医学研究所と武蔵小杉病院が中心となって管理運営を行っている。
１．動物実験委員会の開催
日時：平成 27 年 4 月 1 日 午後 2 時 30 分より 場所：職員食堂 出席者数：12 名
２．動物実験講習会（教育訓練）の開催
日時：平成 27 年 4 月 30 日 午後 3 時より 場所：C 館 1 階会議室 出席者数：32 名
３．動物実験計画書の申請課題数 36 件
４．感染実験や発がん実験等の注意を要する実験の件数 10 件
５．年間延べ入室者数 4,261 名
６．日平均飼育数

マウス 738 匹、ラット 164 匹

７．年間使用動物数 マウス 3,607 匹、ラット 513 匹
８．SPF 微生物モニタリングの実施
平成 27 年 4 月 20 日、平成 27 年 10 月 5 日
９．定期清掃の実施
SPF 飼育室

平成 27 年 11 月 10−11 日、平成 28 年 3 月 29−30 日

マウス飼育室 平成 28 年 3 月 23−24 日
ラット飼育室 平成 28 年 3 月 22 日
小動物実験室 平成 28 年 3 月 2 日
10．実験動物慰霊祭への参加
日時：平成 27 年 10 月 16 日午後 5 時より 場所：医学部教育棟講堂 出席者数：15 名−65−

平成 27（2015）年度

先端医学研究所公開セミナー（平成 27 年 4 月～平成 28 年 3 月）
4 月 30 日
アフリカツメガエルを用いた血管の形態形成機構の解析
藤原正和（病態解析学部門）

血管は太い大動脈から細い毛細血管まで様々な大きさのものが存在する。また、分岐頻度や微細構造も様々で、血管は形態学的に極めて多様であることが分かっている。しかし、この多様な血管の形態がどの･ように形成されるのか、そのメカニズムについてはほとんど明らかになっていない。そこで、透明な体を持ち、血管の観察が容易に行えるアフリカツメガエルのオタマジャクシを用い、生体内の血管がいつ・どこで多様な形態を獲得するのかを解析し、血管の形態を制御するメカニズムを明らかにすることを目的とした。今回は、これまで用いてきた microangiography による血管の形態変化の解析結果や、現在作製中のトランスジェニックカエル Tg（Xl Flk-1:mCherry）についての経過報告を行いたい。
5 月 28 日
分子状水素は脂質のフリーラジカル連鎖反応に介入してシグナル伝達と遺伝子発現を制御する
井内

勝哉（細胞生物学部門）

新しい概念の抗酸化物質である水素（H2）は、炎症が関与する様々な生活習慣病に対して効果を示す
ことから、H2 の抗炎症作用の分子機構が注目されている。しかし、H2 の抗炎症作用の正確な分子機構は明らかになっておらず、H2 が最初に反応または結合する標的は未知である。そこで本研究では、炎症の引き金になる脂質過酸化に着目し、酸化ストレスによる過酸化脂質の生成や活性に対する H2 の影響を調べた。その結果、酸化ストレスによる脂質過酸化が低濃度の H2 によって調節されることが分かった。さらに、この過酸化脂質の調節を介して、H2 は炎症性シグナルや遺伝子発現を制御することが分かった。
6 月 25 日
肺癌分子標的薬 crizotinib はオートファジーを誘導する
鈴木淳也（遺伝子制御学部門）

オートファジーは飢餓などで誘導されオルガネラを分解しリサイクルすることで生存の為に働いている。
いくつかの抗癌剤がアポトーシスとオートファジーを同時に誘導し、オートファジー阻害によりアポトー･
シスが増強して治療効果が上がることが現在数多く研究されている。一方で、オートファジーが細胞死を引き起こす事が報告されているが、反論も多くまだ実態は分かっていない。我々は肺がん治療薬のCrizotinib が、アポトーシスを全く誘導せずにオートファジーによって強力に細胞死を誘導する事、オート
ファジー阻害剤を併用してもアポトーシスが起こらない事を見出し、その機構を解析している。この解析から、オートファジー細胞死の実態解明が期待できる。

−67−

7 月 30 日
翻訳抑制因子 4E−BP1 は肝臓で中性脂肪量を調節する
豊島

由香（生体機能制御学部門）

我々は、低タンパク質栄養状態の肝臓で、翻訳抑制因子 4E-BP1 の量が増加すると共に、中性脂肪量が増加することを見出してきた。一般的に、4E−BP1 は様々な組織においてタンパク質合成を調節することが知られているが、脂質代謝を調節しているかは不明であった。そこで、低タンパク質栄養状態の肝臓で･起こる 4E−BP1 量の増加と中性脂肪量の増加に因果関係があるか明らかにするために、肝臓特異的に4E−BP1 の発現を抑制させたラットを作出した。今回、このラットモデルを用いた解析で、肝臓において4E−BP1 が中性脂肪量を調節することを明らかにしたので報告する。
9 月 24 日
血管の多様な形態とその形成メカニズムについての研究藤原正和（病態解析学部門）

体の隅々まで張り巡らされた血管は、太さ・分岐頻度・有窓構造など、非常に多様な形態を持つ。しかし、これらの多様な形態がどのように構築されるのか、そのメカニズムについてはほとんど分かっていない。
我々はヒト正常組織では観察することが困難な血管の形成過程を透明な体を持つアフリカツメガエルのオタマジャクシを用いて観察し、生体内の血管がいつ・どこで形態学的な多様性を獲得するのかを経時的に解析する実験系を開発した。今回は、これまでの microangiography の解析結果、作製中のトランスジェ
ニックカエル Tg（Xl Flk-1:mCherry）、その他の計画などについて報告を行いたい。
10 月 29 日
Applying Raman spectroscopy to biomedical studies
Liang-da Chiu 特任助教（東京大学

大学院理学系研究科

化学専攻

分析化学研究室）

   Raman spectroscopy is an optical method that probes the molecular vibrations of a sample bydetecting the energy shift of the scattered light from the incident light. It has a strong potential inbiomedical research because of its non-destructive and label-free characteristics, which enables it toreal-time analyse the chemical composition of a living specimen. In my talk, I will introduce my recent
works on the Raman spectroscopic study of stem cell differentiation and bone formation. In the end, I
will also quickly show how other research hospitals around the world are using Raman spectroscopy inmedical practices, and how Raman spectroscopy can help the further progress in medical research anddiagnosis.
11 月 26 日
感音難聴の根治を目指して～ウイルス感染時に内耳はどう応答するか、その分子メカニズムの解明～
林裕史

講師（帝京大学）

突発性難聴、メニエール病、先天性難聴などウイルス感染の関与が示唆される内耳性難聴の存在は知られているものの、その病態はいまだ明らかではなく、ゆえに決定的な治療法は存在しない。内耳は血液脳関門の存在から、脳、網膜、精巣、毛根、胎盤などと同様、免疫特権部位と呼ばれる強い免疫抑制環境にあるとされてきた臓器であるが、我々はこれまでの研究において有毛細胞周囲に存在する支持細胞群のうち、ヘンゼン細胞・クラウディウス細胞が、ウイルス感染時にウイルスの genome RNA を認識−68−

する細胞内受容体である retinoic acid-inducible gene-I（RIG-I）like receptor（RLR）family のシグナル伝達
経路を介して type I interferon（IFN）を発現することを示し、支持細胞が蝸牛における免疫担当細胞であることを見出している。本セミナーでは最新の知見も含めて内耳のウイルス感染応答について御紹介したい。
1 月 28 日
成長ホルモン（GH）によって減少するタンパクの解析中田

朋子（生体機能制御学部門）

GH の新たな生理機能を追跡する目的で次のような実験を行った。新生児期にグルタミン酸ナトリウム（MSG）を投与することによって下垂体の GH を枯渇させたラットを用いて GH の補充投与の効果を観察した。ラットの肝臓から抽出したタンパクを SDS 電気泳動後 CBB 染色したところ、MSG ラットで増加し、GH 投与で減少するタンパクを検出した。このタンパクは下垂体摘除手術によって増加した。このタンパ
クについて解析したので報告する。
2 月 25 日
細胞はどのように力を感じているか？
−メカノバイオロジーとメカノセラピー−
小川令

大学院教授（形成再建再生医学分野）

メカノバイオロジーは、張力やせん断応力、静水圧や浸透圧といった物理的刺激が、生体にどのような影響を与えるかを解析する研究領域である。臨床医学の領域においては、常に物理的刺激が加わっている筋や骨格を扱う整形外科、心拍動、血圧や血流を扱う血液循環器内科などでは比較的なじみのある研究分野であるが、その他の領域ではまだ一般的ではない。われわれは、皮膚や軟部組織の形成過程また創傷の治癒過程においても細胞は常に物理的刺激を感受しながら遺伝子発現を調節していることを解明してきた。この物理的刺激の生体への作用機序を理解することによって、疾患の原因解明、創傷治癒の促進、再生医療、またがん治療などに応用できる可能性を考え、種々のメカノセラピーを臨床で実践している。今後の医工・医薬連携の必要性について報告する。
3 月 31 日
アフリカツメガエルをモデルとした血管形成のイメージング解析藤原

正和（病態解析学部門）

透明な体を持つアフリカツメガエルのオタマジャクシは発生や変態の過程で生じる様々な血管の形態変化を容易に観察できる優れた実験動物である。今回は Flk-1 プロモーター・エンハンサーによって蛍光･タンパク質の発現が制御されたトランスジェニックカエル Tg（Xl Flk-1:TurboFP-C）を作製したのでそれについて報告をする。作製した F0 個体は TurboFP-C を尾や頭部の血管で限定的に発現した。今後は血管の形態変化を長時間解析するために、全ての血管において TurboFP-C を発現する F2 個体を作製し、血管の形態形成メカニズムを細胞レベルで解析し、触覚、後肢、尾などの特徴的な形態を持つ血管の形態機構を明らかにしていきたい。

−69−平成 27年度（2015）研究補助金、助成金受け入れ細胞生物学部門（1）日本学術振興会科学研究費補助金

基盤研究（B）「健康増進と疾病に寄与する分子状水素の多様な機能を発揮するメカニズムの解明」
太田

成男

上村

尚美

（2）日本学術振興会科学研究費助成事業（学術研究助成基金助成金）基盤研究（C）「水素分子の糖尿病改善効果と遺伝子発現誘導における作用機序の解明」（3）日本学術振興会科学研究費助成事業（学術研究助成基金助成金）基盤研究（C）「水素分子による酸化ストレス防御機構と脳内レドックス動態の解析」
西槙貴代美（4）日本学術振興会科学研究費助成事業（学術研究助成基金助成金）若手研究（B）「ミトコンドリア生体分子の化学修飾に着目した水素の抗炎症作用メカニズムの解明」
井内勝哉（5）日本学術振興会科学研究費助成事業（学術研究助成基金助成金）若手研究（B）「エピジェネティクス制御からみた水素の抗炎症作用のメカニズム」
Lee（6）日本医療研究開発機構

Hyunjin

難治性疾患実用化研究事業（分担）「ミトコンドリア脳筋症 MELAS 脳卒中様発作に対するタウリン療法の開発」
太田成男（7）日本学術振興会科学研究費助成事業（学術研究助成基金助成金）基盤研究（C）（分担）「グルココルチコイドは高強度運動による海馬での神経新生の増加を引き起こす要因か否か」
太田成男（8）日本学術振興会科学研究費助成事業（学術研究助成基金助成金）基盤研究（C）（分担）「脂肪肝炎〜肝発癌の病期に応じた最適な酸化ストレス介入療法の開発」
太田成男

遺伝子制御学部門（1）私立大学等経常費補助金特別補助「戦略的研究基盤支援」「Clinical Rebiopsy Bank Project を基盤とした包括的がん治療開発拠点形」（代表：弦間昭彦）
田中

−70−

信之

生体機能制御学部門（1）日本学術振興会科学研究費助成事業（学術研究助成基金助成金）・若手研究（B）「低タンパク質栄養によるアディポネクチン増加機構の解明とその生理的意義」
鈴木（豊島）由香（2）文科省「オーダーメイド医療の実現プログラム（第３期）」
南

−71−

史朗

先端医学研究所・教職員，研究者等氏名
平成 28 年 3 月 31 日現在
Ⅰ . 病態解析学部門
助

教

藤原

正和

テクニカル・スタッフ

枝川

聖子

大学院研究生村賀香名子

形成外科・講師

土佐眞美子

救命救急センター・助教

原義明

形成外科・助教

桑原大彰

横浜中央看護専門学校・非常勤講師

清水

一

大学院教授

太田

成男

客員教授

鈴木

吉彦

准 教 授

上村

尚美

講

師

Wolf Alexander Martin

助

教 	

井内

Ⅱ . 細胞生物学部門

勝哉

非常勤講師

森

マネジメントサポート・スタッフ 	西槙貴代美

アシスタント・スタッフ 	一宮治美

ポスト ･ ドクター 	

李炫溱

大学院研究生

上家明美

大学院研究生

中島

裕也

秘

加藤

寸賀

書  	

隆

実験補助

武田真由美

皮膚科・教授 	船坂陽子

スポーツ科学・准教授

三上俊夫

形成外科・助教渡邉

真泉

北里大学・助教 	

井本

明美

研 修 生

小田

文乃  （皮膚科）

大学院教授

田中

信之

講

師

中嶋

亘

助

教

阿部

芳憲

助

教

上原

郁野

助

教

谷村

篤子

川内

敬子

Ⅲ . 遺伝子制御学部門

非常勤講師

−72−

テクニカル・スタッフ 	浅野由ミ

ポスト ･ ドクター清水幹容

ポスト ･ ドクター岩渕

千里

大学院生 	

松本

優

大学院生

鈴木

淳也

実験補助  	

河越

美保

実験補助

高寺

俊美

帝京大学・講師 	

林裕史（帝京大学・講師）

研 修 生 	

中里

茜（東京医科歯科大・大学院生）

Ⅳ . 生体機能制御学部門
大学院教授 	

南

史朗

准 教 授

折笠千登世

講

師

豊島

由香

助

教

中田

朋子

マネジメントサポート・スタッフ

勝又晴美テクニカル・スタッフ

時田玲子

ポスト ･ ドクター福島誠

ポスト ･ ドクター

田口

雄亮

大学院生

八木

孝

大学院生

矢野

宏行

大学院研究生

大場るり子

パート事務員

大木佳菜子

内科・講師

石川真由美

東京大学・特任助教

鈴木信周

マネジメントサポート・スタッフ

横田隆テクニカル・スタッフ

梶田

満子

長

田中

信之

放射線取扱主任者

上原

郁野

Ⅴ . 分子生物学部門

Ⅵ . ゲノム医学部門

Ⅶ . アイソトープ実験室室−73−

Ⅷ . 組換え DNA 実験施設
安全主任者

中田

朋子

Ⅹ . 動物実験室
実験動物飼育員

金井祐美子

実験動物飼育員

田口

憲明

事務室長

里見

裕右

主

任

小川

泰子

パート事務員鈴木弓子

パート事務員山田深雪

Ⅸ . 事務室−74−

先端医学研究所紀要

第１巻

平成 28 年 9 月 30 日印刷
平成 28 年10月 1 日発行（非売品）
発 行〒211−8533

日本医科大学
先端医学研究所

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