Annual Report (2014)

No.26
The NOVARTIS Foundation (Japan)
for the Promotion of Science

平成 26 年度

財団年報 第 26 号

Contents
Ⅰ . Introduction はじめに ............................................................................................ 7
Akimichi Kaneko, MD, PhD
Chairman of the Board of Trustees

Ⅱ . Reports from the Recipients of Novartis Research Grants
研究報告 .................................................................................................................. 9
1. Inﬂammation caused by secreted lysosomal enzymes. ............................................... 10
Rikinari Hanayama
Osaka University, Immunology Frontier Research Center

2. RAC1 inhibitor is an anti-cancer metastasis drug? ..................................................... 13
Satoru Yokoyama
University of Toyama, Institute of Natural Medicine

3. Analysis of induction mechanism of human TERT gene expression in humanpluripotent stem cells. .............................................................................................. 15
Koichiro Nishino
Laboratory of Veterinary Biochemistry and Molecular Biology, Faculty of Agriculture,
University of Miyazaki

4. Molecular genetical analysis of temperature adaptation that is regulated
by secretary signaling in Caenorhabdites elegans ................................................... 18
Atsushi Kuhara & Akane Ohta
Institute of integrative neurobiology & Department of Biology, Konan University

5. Role of Rab GTPases in the intracellular logistics of insulin granule......................... 21
Kohichi Matsunaga
Institute for Molecular and Cellular Regulation, Gunma University

6. Chemical Biology Based on the Unnatural Steroidal Compounds ............................. 23
Shuji Yamashita
Tohoku University

7. Roles of store-operated Ca2+ channels in skeletal muscle ........................................... 26
Yuji Hara
Tokyo Women's Medical University / Kyoto University

8. Transcriptional quiescence for the establishment of germ cell fate in Drosophila. .... 29
Akira Nakamura
Institute of Molecular Embryology and Genetics, Kumamoto University

9. Regulation of plasma membrane receptor-like kinase functions
by endoplasmic reticulum quality control in plant cells .......................................... 33
Shuh-ichi Nishikawa
Niigata University

10. Analysis of functional molecules which regulate production of type I interferonsin plasmacytoid dendritic cells ................................................................................ 36
Katsuaki Hoshino
Faculty of Medicine, Kagawa University-1-11. Prevention tumor metastasis by targeting tumor endothelial cells .............................. 39
Kyoko Hida, Yasuhiro Hida
Vascular Biology, Frontier Research Unit, Institute for Genetic Medicine,
Hokkaido University

12. Deriving Photoreceptors from Human Somatic Cells ................................................. 40
Yuko Seko
National Rehabilitation Center for Persons with Disabilities

13. Identiﬁcation of cytidine deaminase activity for elucidating the molecular mechanismof plant organelle RNA editing ................................................................................ 43
Mamoru Sugita
Nagoya University, Center for Gene Research

14. Clariﬁcation of novel mechanism of catecholamine biosynthesis by ERK5 .............. 46
Yutaro Obara
Yamagata University, School of Medicine

15. Assessing the biological signiﬁcance and functionof neuron-speciﬁc imprinted genes in brain plasticity............................................. 49
Shin-ichi Horike
Advanced Science Research Center, Kanazawa University

16. Intracellular logistics regulated by LIS1 and lissencephaly ........................................ 52
Masami Yamada
Graduate School of Medicine, Osaka-City University

17. Elucidation of molecular functions of Imp3 in osteosarcoma progression,and development of novel anti-Imp3 targeted therapeutic approach....................... 56
Takatsune Shimizu
Hoshi University

18. Development of metal nanoparticles for organic synthesis
aiming a substitution and reduction of rare metals .................................................. 59
Mitsuhiro Arisawa
Osaka University

19. Epigenetic regulation by nuclear pore complex in cancer progression ....................... 62
Richard Wong
Department of Biology, Institute of Science and Enginnering, Kanazawa University

20. Artiﬁcial Regulation of Protein Degradation by the Proteasome ................................ 65
INOBE, Tomonao
Frontier Research Core for Life Sciences, University of Toyama

21. Regulatory mechanism of mucosal healing by DNA methylation .............................. 68
Hiroyuki Tezuka
Tokyo Medical and Dental University

22. Actin Cytoskeleton Structure Regulation by TRIOBP,
a novel gene responsible to human deafness ........................................................... 70
Shin-ichiro Kitajiri
Department of Otolaryngology, Kyoto University Hospital-2-

23. Study of chromosome instability caused by the impairment ofcentrosome maturation ............................................................................................ 73
Yuko Ozaki
Research Institute for Radiation Biology & Medicine, Hiroshima University

24. Elucidation of the mechanisms by which impaired clearance of apoptotic cells
results in severe inﬂammation ................................................................................. 76
Hiroyasu Nakano
Juntendo University Graduate School of Medicine

25. Molecular mechanisms for the production and maintenance of proliferative cells
in the subventricular zone. ....................................................................................... 78
Hidenori Tabata
Department of Molecular Neurobiology, Institute for Developmental Research,
Aichi Human Service Center

26. Role of the VIPLa-mediated glycoprotein trafﬁcking in locomotion in zebraﬁsh ...... 82
Kazuhide Asakawa
National Institute of Genetics

27. Explorative study on lysine demethylase inhibitors by rational drug design .............. 85
Takayoshi Suzuki
Kyoto Prefectural University of Medicine

28. Comprehensive proteomic analysis of molecules that are targeted
for cancer prognosis or therapy ............................................................................... 88
Kiyotsugu Yoshida
Jikei University School of Medicine

29. Molecular mechanism of Derlin family-mediated novel endoplasmic reticulum
quality control system .............................................................................................. 90
Hideki Nishitoh
Division of Biochemistry and Molecular Biology, Department of Medical Sciences,
University of Miyazaki

30. Development of NO donor drug based on furoxan structure ...................................... 93
Ryosuke Matsubara
Kobe University

31. Development of Novel Genetic Screening Assay
using Next-Generation Sequencer and Clinical Application of
Personalized Medicine in Inherited Arrhythmia Syndromes ................................... 95
Hiroshi Watanabe
Department of Cardiovascular Biology and Medicine

32. Functional Regulation of Nicotinic Acetylcholine Receptors
by a Novel Family of Endogenous Neurotoxin-like Molecules .............................. 98
Hidemi Misawa
Department of Pharmacology, Faculty of Pharmacy, Keio University-3-33. Development of orange-colored bimolecular ﬂuorescencecomplementation (BiFC) assay ............................................................................. 101
Yutaka Kodama
Center for Bioscience Research and Education, Utsunomiya University

34. Regulation of polarized secretion of Wnt by post-translational modiﬁcationin epithelial cells .................................................................................................... 104
Akira Kikuchi
Graduate School of Medicine, Osaka University

35. Synthetic studies on the virulent cell wall lipid of Mycobacterium tuberculosis ...... 107
Seijiro Hosokawa
Waseda University

36. Synthesis of anticancer anthracycline antibiotics having aza-spiro structure ........... 111
Mitsuru Kitamura
Kyushu Institute of Technology

37. Genetic and Epigenetic Landscape of Endothelial Cells Differentiation Reveal
the Transcriptional Factors Network ..................................................................... 114
Yasuharu Kanki
The University of Tokyo

38. Essential roles of class II PI3-kinase C2α in vascular formation and homeostasis. .. 118
Kazuaki Yoshioka
Department of Physiology, Kanazawa University School of Medicine

39. Mechanisms of Eph family receptor-mediated regulation of bone mineralization ... 122
Koichi Matsuo
School of Medicine, Keio University

Ⅲ . Reports from the Recipients of Grants for International Meetings
研究集会報告 ...................................................................................................... 125
1. Joint meeting :The 76th Annual Meeting of Japanese Society of Interferon and Cytokine Research
The 21th International Symposium on Molecular and Cell Biology of Macrophages .... 126
Akihiko Yoshimura
Professor Keio University School of Medicine

2. The 3rd International Moyamoya Meeting ................................................................. 127
Kiyohiro Houkin

3. The 4th International Symposium on Dynamics of Mitochondria (DynaMito2013) ... 128
Koji Okamoto
Associate Professor, Graduate School of Frontier Biosciences, Osaka University
DynaMito2013, Chair-4-4. The 8th International Conference on Cutting-Edge Organic Chemistry in Asia
The 4th New Phase International Conference on Cutting-Edge Organic
Chemistry in Asia ...................................................................................................... 129
Michio Murata
Professor, Department of Chemistry, Osaka University
Atsushi Nishida
Professor, Department of Pharmaceutical Science, Chiba University

5. “New Perspectives of Molecular Imaging on Alzheimer's disease” : A Satellite
Symposium of the 87th Annual Meeting of The Japanese PharmacologicalSociety ................................................................................................................... 131
Kazuhiko Yanai
Professor, Department of Pharmacology, Tohoku University School of Medicine

Ⅳ . The 27th (ﬁscal year 2013) Promotion Report
第 27 期 （2013 年度） 助成事業報告 ............................................................... 132
Ⅴ . The 27th (ﬁscal year 2013) Financial Report
第 27 期 （2013 年度） 財務報告 ....................................................................... 138
Ⅵ . List of the Board Members
役員名簿 .............................................................................................................. 140
Ⅶ . Information from the Secretariat
事務局便り ........................................................................................................... 144

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-6-

Introduction

Akimichi Kaneko, MD, PhD
Chairman of the Board of Trustees
This booklet includes research and meeting reports written by the 2013 grantees. The
Foundation was originally established on September 4, 1987 with basic assets of JPY 1
billion donated by Ciba-Geigy AG, Switzerland for the purpose of contributing to academic
development and thus improving public health and welfare by means of promoting creative
research and international exchange in the field of life science and related chemistry. Since
then, the Foundation has granted nearly JPY 1.8 billion to approximately 1,550 researches andinternational exchange activities. The Foundation supports basic researches in the field of life
science and related chemistry. They may not develop directly into applications, but accumulationof new findings and theories will open a road to application research and business someday.
The tireless effort of researchers including the present grantees is the driving force topromote and to keep the high standard of the Japanese life science. I strongly believe that theaim of the Foundation is to help keeping their activity and we will do our best toward this goal.
We are encouraged by warm acknowledgements to the Foundation written in many publicationsof grantees.
I sincerely appreciate the assistance and warm encouragement extended by the membersof the Board of Trustees, the auditors, the Board of Councilors and the Selection Committee.
The powerful support by the Novartis Pharma KK enabled us to sustain our activity without
interruption.

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はじめに
代表理事 金 子 章 道
ここに 2013 年度にノバルティス科学振興財団研究助成金を受けられた方々の研究報告を収録いたしました。 当財団は 1987 年 9 月 4 日、 スイス、 チバガイギー社からの 10 億円のご寄附をもとに、 「生物・生命科学および関連する化学の領域において、創造的な研究ならびに国際交流への助成を行うことにより、 学術の振興を図り国民の健康と福祉の向上に寄与する」ことを目的に設立されました。 爾来 27 年間に約 1,550
件、 金額にしておよそ 18 億円の助成を行ってまいりました。 当財団がご提供する研究費は、 研究に要する総費用のうち微々たるものかもしれませんが、 研究をスムーズに遂行するための役に立てていただくのがその目的です。 事務局に寄せられた発表
論文の謝辞の中にも受賞者のそうしたお気持ちが垣間見られ、 大変嬉しく思っております。
当財団が助成の対象とする研究は生物 ・ 生命科学および関連する化学の領域における基礎的な研究です。 その成果が即応用につながらなくても、 新発見や新しい理論が応用研究のアイディアとして役立つことになるもしれません。 基礎的な発見や理論を積み上げることが、 いつの日か新分野を開き、 新産業を生み出すもとになるものと信じております。
この年報は受賞者の皆様の素晴らしい研究がまとめられたエッセイ集です。 研究者
お一人おひとりの努力の結晶は我が国の学術水準を発展させていく原動力です。 こ
れらの優れた研究を選考していただいた選考委員の皆様をはじめ、 財団の活動を支えて下さっている関係者の皆様に深く感謝いたします。

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II.
Reports from the Recipients ofNovartis Research Grants-9-

Inflammation caused by secreted lysosomal enzymes.
Rikinari Hanayama
Osaka University, Immunology Frontier Research Center
hanayama@ifrec.osaka-u.ac.jp

Abstract
We identified a novel protein that can be a mediator of lysosomal fusion in macrophages. It is
expressed at the cell plasma membrane and mediates fusion between the membrane and lysosomes
upon calcium stimuli, causing the release of undigested debris and the secretion of lysosomal
enzymes. Our findings would help to elucidate the molecular mechanisms of heterolysis that can bea critical process for the development of chronic inflammation.
Key words : Macrophages, Lysosomal enzymes, Heterolysis, Inflammation

Introduction
During inflammation, macrophages phagocytose many dead cells and/or bacteria into phagosomes
and digest them into a series of peptides by the fusion of phagosomes with lysosomes (1). These
peptides bind to MHC molecules and are transported to the surface of macrophages by the fusionof phago-lysosomes with cell plasma membrane. Using a similar mechanism, undigested debris inphago-lysosomes can be released from macrophages. During these processes, lysosomal enzymes
are also secreted, causing the degradation of the surrounding tissues. This process is called
heterolysis (2), but its molecular mechanisms as well as its relevance to the development of chronic
inflammation have been unclear.

Results
We recently identified a type II transmembrane protein (named TIP1) with multiple protein/lipidinteraction domains in the cytoplasmic region. Notably, the first domain is a C2 domain which canbinds to dynamin, caveolin and phosphatidylserine. It is a protein expressed at the plasma membraneof cells undergoing fusion, and has been implicated in the membrane fusion, vesicular fusion andendocytosis. According to the BioGPS database, TIP1 is highly expressed in placenta and bladder,but weakly in other tissues. Interestingly, at cellular level, TIP1 is highly expressed in thioglycollateelicited peritoneal macrophages, bone-marrow derived macrophages, osteoclasts, microglia and
RAW 264 macrophage cell line, but not in any subset of T cell, B cell, or mast cells. We confirmedthat TIP1 is highly expressed in various macrophages and BMDC, but not in T or B cells, suggestingthat TIP1 is a macrophage specific protein. We next examine the subcellular localization of TIP1
using NIH3T3 cells. We found TIP1 is localized to small puncta that can be stained with LAMP1,
indicating that TIP1 is localized to lysosomes.
To clarify the functions of TIP1 in lysosomes, we established NIH3T3 transformants expressing- 10 -

short-hairpin RNA against TIP1. The LAMP1 staining revealed that the TIP1 knockdown cells
significantly had more lysosomes, compared to the control cells. The FACS staining with lysotracker
which also stain lysosomes confirmed the 2-fold increase of lysosomes in the TIP1 knockdown cells.
These data suggest that lysosomes are accumulated by TIP1 knockdown. To examine the details,
we did electron microscopic analysis of the NIH3T3 cells. The control cells carried many vesicles
without any materials inside them. On the other hand, the TIP1 knockdown cells carried many
vesicles filled with undigested debris and some membranous materials inside them. These datasuggested that accumulation of debris in lysosomes can be due to the impaired lysosomal secretion
by TIP1 knockdown.
We therefore established the following model: TIP1 is expressed both at plasma membrane andlysosomal membrane. Upon calcium stimuli, TIP1 mediates interaction with one another by binding
to phosphatidylserine on each membrane. Thus, TIP1 may regulate fusion for lysosomal secretion,
which causes the release of debris and lysosomal enzymes for heterolysis. This process is very
similar to the synaptotagmin-mediated exocytosis in the nervous system. Synaptotagmin also has
multiple C2 domains that bind to phosphatidylserine in a calcium-dependent manner. Synaptotagmin
is expressed at synaptic vesicles and upon a calcium stimuli, it regulates the fusion for exocytosisof neuro-transmitters. To directly examine whether TIP1 regulates the lysosomal secretion, we
examined the secretion of beta-hexosaminidase, one of the common enzymes in lysosomes. For thisassay, we introduced short hairpin RNA against TIP1 into the bone marrow derived macrophages.
The macrophages were stimulated with A23187, a calcium ionophore which induces calcium influx.
The supernatant was collected and reacted with fluorescent substrate for the quantification of thelysosomal enzyme. The secretion of the beta-hexosaminidase was significantly impaired by TIP1
knockdown, supporting the idea that TIP1 regulates the fusion for lysosomal secretion.
As TIP1 can account for only a part of the mechanisms of lysosomal secretion, we performed
screening for the other exocytosis mediators by introducing shRNA library into K562 leukemia
cell line. The cells carry lysosomes that can be stained with lysotracker. Upon calcium stimuli,
some lysosomes are secreted, causing the decrease of lysotracker staining. However, after calcium
removal, the lysosomes re-develop within 1hr. If the knockdown cells have impaired fusion ortransport of lysosomes, the lysotracker staining after calcium stimuli shall be stronger than WT.
Or if the knockdown cells have impaired development of lysosomes, the lysotracker staining after
calcium removal shall be weaker than WT. We sorted out these cells by FACS Aria and repeated
growth and sorting several times. Thus, we established knockdown clones and identified the othertarget genes.

Discussion & Conclusion
We are currently trying to clarify the functions of TIP1 in the release of debris and enzymes fromlysosomes. The exocytosis by macrophages is also involved in the antigen presentation from phagolysosomes and secretion of exosomes from multi-vesicular body. We will examine whether TIP1

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regulates the membrane fusion events in these processes. As heterolysis is a novel mechanism
to maintain homeostasis by lysosomes, we will try to clarify its physiological functions and its
relevance to the development of immune disorders. For this purpose, we are generating TIP1 KO
mice. Using these mice, we plan to examine the effect of the impaired lysosomal secretion on thedevelopment of inflammation.

References
1. Fairn GD, Grinstein S. How nascent phagosomes mature to become phagolysosomes. Trends
Immunol. 33(8):387-405. (2012)
2. Hibbs JB Jr. Heterocytolysis by macrophages activated by bacillus Calmette-Guerin: lysosome
exocytosis into tumor cells. Science. 184:468-71. (1974)

一般の皆様へ
炎症の場でマクロファージという免疫細胞は、消化酵素を細胞外へと放出します。消化酵素の放出が過剰になると周囲の組織を破壊し、更なる炎症を惹起します。この過程は「他者融解」と呼ばれ、４０年程前から報告されている現象ですが、その制御機構はこれまで不明でありました。私達はこの過程を制御する分子を最近同定しました。消化酵素の放出による他者融解は、マクロファージによる死細胞の貪食除去機構やオートファジーに続く第三の消化酵素による生体の恒常性維持機構である可能性があり、今後その生理的・病理的意義を明らかにしてゆきたいと考えております。

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RAC1 inhibitor is an anti-cancer metastasis drug?
Satoru Yokoyama
University of Toyama, Institute of Natural Medicine
yokoyama@inm.u-toyama.ac.jp

Abstract
Although epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitors (EGFR-TKIs),
including gefitinib, provide a significant clinical benefit in non-small cell lung cancer (NSCLC)
patients, the acquisition of drug-resistance has been known to limit the efficacy of EGFR-TKIs
therapy. In this study, we demonstrated the involvement of EGF-EGFR signaling in NSCLC cell
migration and the requirement of RAC1 in EGFR-mediated progression of NSCLC. This research
opens up a new opportunity to control the cancer progression by targeting RAC1 pathway to
overcome the resistance to EGFR-TKIs in NSCLC patients.
Key words : RAC1; NSCLC; gefitinib; migration

Introduction
Non-small-cell lung cancer (NSCLC) accounts for almost 80% of lung cancer and the
median survival of metastatic NSCLC is around only 1 year, even when treated with platinum
chemotherapy. To identify the new molecular targets in gefitinib-resistant NSCLC cells, we focus on
RAC1 signaling because RAC1 inhibition suppresses cell migration. RAC1 is a member of the Rho
family of small GTPases and its overexpression is correlated with a poor prognosis in NSCLC.
RAC1 is also involved in cancer metastasis; however, it is not well understood whether RAC1 isinvolved in the migration and metastasis of NSCLC cells.

Results
To understand the role of EGFR signaling in other than NSCLC cell growth or survival, we firstly
investigated the effects of EGFR signaling on cell migration by using an in vitro wound healing
assay. NSCLC cell migration could be suppressed after gefitinib treatment at 300 nM for 24 hr; thatin the condition cell growth was not affected.
Among the downstream molecules, RAC1 is identified as the candidate, because RAC1 inhibitorand siRAC1 could suppress the cell migration at the similar levels as gefitinib. In addition,overexpression of constitutive active RAC1 could rescue the gefitinib-suppressing cell migration,suggesting that RAC1 is one of downstream molecules of EGFR.
Considering the clinical significance of resistance to gefitinib therapy in NSCLC, we next
determined whether RAC1 inhibition is clinically applicable to targeting gefitinib-resistant NSCLC.
Interestingly, RAC1 inhibitor, NSC23766, could suppress the cell migration not only in gefitinibsensitive NSCLC cells but also in gefitinib-resistant NSCLC cells. Furthermore, we examined that- 13 -

RAC1 inhibitor could affect the proliferation of gefitinib-resistant NSCLC. Similar as cell migration,
the proliferation in gefitinib-resistant NSCLC cells was inhibited with RAC1 inhibitor, suggestingthat RAC1 is a potential therapeutic target for gefitinib-resistant NSCLC through inhibiting bothcell growth and migration. We also confirmed that RAC1 pathway is independent on MEK-ERK
and PI3K-AKT pathways.
Finally, to investigate the in vivo effects of RAC1 inhibition, we inoculated a human gefitinibresistant NSCLC cells subcutaneously into mice. Consistent with in vitro data, the RAC1 inhibitor,
NSC23766, significantly reduced tumor size and tumor weight without any loss of the body weightof mice. This suggested that NSC23766 could inhibit gefitinib-resistant NSCLC tumor growth
without severe side effects.

Discussion & Conclusion
In this study, we determined the importance of RAC1 for cell migration and growth in NSCLC.
Importantly, the small molecule RAC1 inhibitor NSC23766 could effectively suppress both cell
migration and growth even in gefitinib-resistant NSCLC cells in vitro, and suppress gefitinibresistant NSCLC tumor growth in vivo.
There are some benefits to apply a RAC1 inhibitor in NSCLC patients. Firstly, RAC1 inhibition
could suppress NSCLC cell migration, suggesting that cancer
metastasis might be inhibited with RAC1 inhibitor. Secondly, even in gefitinib-resistant NSCLC
patients, RAC1 inhibitor could still give the therapeutic opportunity. Thirdly, RAC1-mediatedpathway is independent on other EGFR-downstream pathway such as MEK1/2-ERK1/2 and PI3KAKT, suggesting that multiple inhibitors targeting these three pathways could be an attractive
strategy to treat NSCLC patients.

References
Kaneto, N., Yokoyama, S., Hayakawa, Y., Kato, S., Sakurai, H., Saiki, I. (2014) RAC1 inhibition
as a therapeutic target for gefitinib-resistant non-small-cell lung cancer. Cancer Sci.,
In press. doi: 10.1111/cas.12425

一般の皆様へ
日本での死因の第一位を占めているのは悪性新生物であり、その中でも現在特に多いのが肺がんである。また肺がんの一番の問題は転移であり、肺がんの摘出後の生存率を上げるためには、“ 抗がん転移剤 ” の開発が急務である。今回、治療標的として同定した RAC1 の阻害剤は、肺がんの増殖の阻害のみではなく、その転移も抑える可能性がある。また、変異型 EGFR 阻害剤である
gefitinib が効かない肺がんにおいても、RAC1 阻害剤は効果がある可能性が示され、肺がんの新規治療法となりうると考えられる。

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Analysis of induction mechanism of human TERT gene expression
in human pluripotent stem cells.
Koichiro Nishino
Laboratory of Veterinary Biochemistry and Molecular Biology, Faculty of Agriculture,
University of Miyazaki
aknishino@cc.miyazaki-u.ac.jp

Abstract
Telomerase reverse transcriptase (TERT) is a catalytic subunit of telomerase, which extends
telomere at the end of chromosome. Human TERT (hTERT ) expression is induced during
reprogramming from somatic cells to induced pluripotent stem cells (iPSCs), however, mechanisms
of induction of hTERT gene are unclear. To elucidate this issue, we examined epigenetic regulationof hTERT gene in human iPSCs. In this study, differentially DNA methylated region (TERT-DMR)
was identified, which was hyper-methylated in iPSCs. In addition, demethylation at TERT-DMR
down-regulated hTERT expression. These findings suggest that human TERT genes are up-regulated
by DNA methylation.
Key words : Human TERT, Human iPS cells, Epigenetics, DNA methylation

Introduction
Two main characters in pluripotent stem cells such as embryonic stem cells (ESCs) and iPSCs are
infinity proliferation and pluripotency. There are many studies about maintenance of pluripotency,
induction of differentiation and methods of reprogramming into iPSCs, but there are few studies
about the infinity proliferation of stem cells. Eukaryotic cell contains telomere, repeated sequence atthe end of chromosome. The telomere ends become shorter each cell division, therefore, maintaining
or extending telomere length by the enzyme telomerase is a critical event in the immortal cells
such as ESCs, iPSCs and embryonal carcinoma cells (ECCs). Human TERT is a key subunit oftelomerase, which is induced in iPSCs during reprogramming. However, mechanisms of inductionof hTERT gene are unclear. In this study, we focused on epigenetic regulation of hTERT gene inhuman pluripotent stem cells.

Results
In this study, biological analyses were performed by using twenty-two human cell lines
including 6 iPSC lines, 4 ESC lines, 2 ECC lines, and 10 somatic cell lines. Analysis of RT-PCR
indicated that TERT mRNA was detected in immortal cell such as ESCs, iPSCs and ECCs, but
not in somatic cells. Next, global analysis of DNA methylation was performed by using Illumina
HumanMethylation450K, revealing that the differentially DNA methylated region (TERT-DMR) was
identified at 1kb up-stream from the transcription start site (TSS) of hTERT gene. The TERT-DMR
was hyper-methylated in cells expressing hTERT gene compared with that in hTERT non-expressing- 15 -

cells. Rate of DNA methylation was low at proximal region to TSS regardless of TERT expression.
To validate the hyper-methylation in the stem cells in global methylation analysis, bisulfite
sequencing was conducted to analyze the DNA methylation state of 125 CpG cites around hTERT
promoter. Similarly to the result of global methylation analysis, TERT-DMR was highly methylated
in hTERT expressing cells, and was low methylated in not expressing hTERT cells.
To demonstrate whether hTERT expression depends on the methylation status of TERT-DMR,
human iPSCs was treated with a demethylating agent and hTERT expression was examined. TERTDMR was demethylated by the treatment of demethylating agent, 5-aza-deoxycytydine (5-azadC), for 4 days. TERT-DMR was demethylated by the treatment of demethylating agent, 5-azadeoxycytydine (5-aza-dC), for 4 days. TERT expression was undetected by RT-PCR in 5-aza-dC
treated cells though the expression of genes related with pluripotency including SOX2, EPHA1,
SALL4 and NANOG was not affected by the treatment of 5-aza-dC, suggesting that the DNA
methylation in TERT-DMR maintains the hTERT expression. In order to examine whether the
methylation in TERT-DMR affects on hTERT promoter activity, we performed methylated promoter
assay. Interestingly, methylated TERT-DMR induced expression of the reporter gene, whereas,
unmethylated TERT-DMR suppressed the promoter activity in hTERT expressing cells. Bothmethylated and umnethylated TERT-DMR did not induced expression of the reporter gene in hTERT
non-expressing cells. These results suggested that methylation at TERT-DMR play an important rolefor induction of hTERT expression

Discussion & Conclusion
In previous study, transcription factors regulating hTERT expression is widely known, such as
c-Myc, p53 and Rb, but the epigenetic regulation is unknown. There are two reasons why theinvestigation of epigenetic regulation of hTERT is difficult. The first reason is that regulation of
TERT gene in human is different from mouse, therefore, analyses using human cells or tissues are
absolutely imperative for studying hTERT gene. Expression of hTERT is extremely limited in suchcancer cells, germ cells, ESCs, ECCs, hence, cancer cells or tissue have been commonly used for
hTERT experiments. However, those have abnormalities including gene deletion, mutation andchromosome translocation as well as epigenetic mutation. The second reason is that the previous
epigenetic study of hTERT focused on the core promoter region, about -300 to +50 nt.
In this study, we used human iPSCs and ESCs as hTERT expressing cells, which have normal
karyotype. We identified TERT-DMR at 1kb upstream of the TSS. Interestingly, DNA methylation at
the TERT-DMR induced hTERT expression, meaning that hTERT gene is under control of epigenetic
regulation such as DNA methylatyion. Our findings suggest two possibilities; one is that TERTDMR is a binding site of certain suppressors. Certain suppressors bind unmethylated TERT-DMR
and suppress hTERT expression in hTERT non-expressing cells, whereas, methylated TERT-DMR
block binding of the suppressor, then hTERT expression is induced in ESCs/iPSCs. Another is thatcertain promoting factors bind especially at methylated TERT-DMR and induce hTERT expression- 16 -

in hTERT expressing cells. Identification of binding factors that contact TERT-DMR is needed infurther analyses.
In conclusion, we identified differentially methylated region at hTERT upstream region of hTERT
gene. DNA methylarion at TERT-DMR up-regulated hTERT expression. These findings are clues for
elucidating mechanisms of reprograming as well as malignant alteration.

References
1. Chan, S.R.W.L, et al. Telomerase and telomerase. Philos. Trans. R. Soc. Lond. B. Biol. Sci. 359,
109-21 (2004)
2. Li, H, et al. A novel tricomplex of BRCA1, Nmi, and c-Myc inhibits c-Myc-induced humantelomerase reverse transcriptase gene (hTERT) promoter activity in breast cancer. J. Biol. Chem.
277, 20965–73 (2002)
3. Lopatina, et al. Control mechanisms in the regulation of telomerase reverse transcriptase
expression in differentiating human teratocarcinoma cells. Biochem. Biophys. Res. Commun. 306,
650–659 (2003)

一般の皆様へ
体細胞が iPS 細胞へとリプログラミングされると、細胞は無限増殖能と多分化能を獲得します。本
研究では、ヒト iPS 細胞が獲得する無限増殖能に関わる重要な遺伝子、TERT 遺伝子が、DNA メチ
ル化によって発現制御されていることを明らかにしました。ヒトTERT 遺伝子の発現制御機構を明らかにした本研究結果は、iPS 細胞を用いた再生医療、癌化のメカニズムの解明や癌化予測・予防・治療などへの応用へ繫がる成果です。

- 17 -

Molecular genetical analysis of temperature adaptation that is regulated
by secretary signaling in Caenorhabdites elegans
Atsushi Kuhara & Akane Ohta
Institute of integrative neurobiology & Department of Biology, Konan University
atsushi_kuhara@me.com

Abstract
Temperature is critical environmental stimuli and causes biochemical changes. Animals can
respond to the changes in ambient temperature, yet how animals habituate to temperature is poorly
understood. We are using temperature experience-dependent cold tolerance of nematode, C. elegans,as a model for studying temperature sensation and memory. After cultivation at 20℃, wild-type weredestroyed by cold stimuli. In contrast, after cultivation at 15℃, most of animals can survive. We
found that secretary hormonal signaling is important for this cold habituation, and intestine andneurons are essential for regulating cold habituation.
Key words : Temperature response, habituation, C. elegans

Introduction
Temperature is one of the most important environmental stimuli and causes biochemical changes
in the body. Animals including human, therefore, can respond and habituate to the changes inambient temperature. We are investigating about the molecular physiological mechanism underlying
a temperature experience-dependent temperature tolerance in nematode Caenorhabditis. elegans. 20
or 25℃-cultivated wild-type
animals were died by cold stimuli. By contrast, most of 15℃-cultivated
Results1
The nematode C. elegans can store temperature experience in neurons that induces

temperature
habituation-linked
cold tolerance.
For example,
15 degree-cultivatedanimals canwild-type animals can survive at cold
condition.
To examine
molecular
mechanismsof temperaturesurvive at 2 degree, while 25 degree-cultivated animals can not survive at 2 degree (Fig.1). In

thiswestudy,
we foundgeneticalthat cold toleranceof C. elegans is regulated by the insulin-dependent
experience-dependent cold tolerance,
utilized
analysis.

signaling pathway, by the combination analysis of molecular genetics and optical calcium
imaging. We tested many mutant animals defective in secretary signaling pathway. We foundthat mutant animals defective in insulin-like molecules showed partial enhancement of cold
tolerance. To determine the essential cells for secreting insulin in the cold habituation, we
expressed insulin gene in insulin secretary neurons of insulin mutant animals. We found that
Result 1
expressing one of the insulin cDNA in insulin secretary sensoryneurons of insulin mutant
The nematode C. elegans can store
temperatureexperiencein ofneurons
thatandinduces
animalrescued the abnormal
cold tolerance
insulin mutant,
expressingtemperatureinsulin cDNA inother sensoryneurons partially rescued the abnormal cold tolerance of insulin mutant. These
habit uation-lin ked cold tolerance.
For that insulin act cell non-autonomously, and are partially dependent on neuron
results suggest
type.
Figure 1

Results

example, 15 degree-cultivated animals

degree (Fig.1). In this study, we found thatcold tolerance of C. elegans is regulated by
the insulin-dependent signaling pathway,
by the combination analysis of molecular
genetics and optical calcium imaging.

live or dead

13、15、17、18、19、20、21、22、23、25、27℃

cul va ontemperature

2℃

live or dead

Each cul va on temperature → 2℃

100

survival rate(%)

cultivated animals can not survive at 2

Cold shock
temperature

Cul va ontemperature

can survive at 2 degree, while 25 degreeFig. 1

80
60
40
20
0

13

15

17

18

19

20

21

22

23

25

27

cul va on temp. (℃)

In order to determine the downstream molecules of insulin-molecules in cultivation
temperature-dependent cold tolerance, we tested various mutant strains, such as mutant animals
defective in insulin receptor and insulin-dependent pathway (Fig.2). We found that the mutant
animals defective in the insulin receptor or its downstream molecules showed abnormal cold
tolerance. By the genetic epistasis analysis, we revealed that two insulins are both positive- 18 agonists and work redundantly for insulin receptor in cold tolerance. Additional genetic
epistasis analysis indicated that one of the insulin molecules genetically inhibits insulin
receptor through a negative regulation of other insulin.

type.
Cold shock
temperature

Cul va ontemperature

Fig. 1

live or dead

13、15、17、18、19、20、21、22、23、25、27℃

cul va ontemperature

2℃

live or deadWe tested many mutant animals defective in secretary signaling pathway. We found that mutant
Each cul va on temperature → 2℃

survival rate(%)

100
animals defective in insulin-like molecules showed
partial enhancement of cold tolerance. To
8060

determine the essential cells for secreting insulin in40the cold habituation, we expressed insulin gene
20

0
in insulin secretary neurons of insulin mutant animals.
We found that expressing one of the insulin

13

15

17

18

19

20

21

22

23

25

27

cul va on temp.
(℃)
cDNA in insulin secretary sensoryneurons of insulin mutant animal
rescuedthe abnormal cold

tolerance of insulin mutant, andInexpressing
insulinthecDNA
in othersensoryneurons
partially
rescuedorder to determine
downstream
moleculesof insulin-moleculesin cultivation
temperature-dependent cold tolerance, we tested various mutant strains, such as mutant animals

the abnormal cold tolerancedefectiveof insulin
mutant. These results suggest that insulin act cell nonin insulin receptor and insulin-dependent pathway (Fig.2). We found that the mutant
animals
defective onin theinsulin type.
receptor or its downstream molecules showed abnormal coldautonomously, and are partiallydependent
neurontolerance. By the genetic epistasis analysis, we revealed that two insulins are both positive

In order to determine the downstream
moleculesof insulin-molecules
temperatureagonists and work
redundantly
for insulin receptor in in
coldcultivation
tolerance. Additional
genetic
epistasis analysis indicated that one of the insulin molecules genetically inhibits insulin
receptor through a negative regulation of other insulin.

dependent cold tolerance, we tested various mutant strains, such as mutant animals defective ininsulin receptor and insulin-dependent Figure 2
pathway (Fig.2). We found that
Fig. the2mutant animals defective in the insulin
receptor or its downstream molecules
showed abnor mal cold toler a nce.

Insulin–mediated signaling pathway
In su lin -lik em o lecu leINS-1 INS-6（INS-2,3…)
DAF-28
PTEN
DAF-2
DAF-18By the genetic epistasis analysis, we

In su lin
recep to r

AGE-1
PI3 -K

revealed that two insulins are both
positive agonists and work redundantly

A K T/PK B
k in ase

AKT-1,2

PDK-1
DAF-16

DAF-16

FOXO
tra n scrip tio n
f a cto r

for insulin receptor in cold tolerance.

Additional genetic epistasis analysis indicated that one of the insulin molecules genetically inhibits
insulin receptor through a negative regulation of other2／4
insulin.
Results2
Since insulin signaling pathway is involved in dauer lava formation in C. elegans, we tested othersignaling molecules important for dauer formation, such as molecules in TGF-beta and steroid
hormonal signaling pathway. We measured cold habituation phenotype of many mutants defective
in TGF-beta and steroid hormonal signaling pathway (Fig.3). We did not observe strong defects oftemperature-dependent cold tolerance
mutants.
These
insulinsignaling
Results2 in theseSince
insulin signaling
pathwayresults
is involvedsuggest
in dauer lava thatformation
in C. elegans,
we testedother signaling molecules important for dauer formation, such as molecules in TGF-beta andpathway is essential for the cold tolerance, but
TGF-beta
andpathway.
steroidhormoneare notphenotype
essential.
steroidhormonal signaling
We measured
cold habituationof many mutants
defective in TGF-beta and steroid hormonal signaling pathway (Fig.3). We did not observe

Insulin is a secretory molecule, and insulin
receptor
is expressed coldin many
tissuessuchasresults
neuron,strongdefects of temperature-dependent
tolerance inthese mutants.
Thesesuggestthat insulin signaling pathway is essential for the cold tolerance, but TGF-beta and steroid

muscle and intestine. To investigate the tissues
whereinsulin receptor function in the cold tolerance,
hormoneare not essential.
Fig. 3 3
we tested temperature-dependent cold Figure

P4 5 015℃

DAF-12

DAF-5

20℃

2℃

2℃

Dauer
forma onN u clear h o rm o n e
recep to r

Dauerforma on
15℃

2℃

20℃

2℃

100
80
60

-1

-5

da
f

-ty

-7

Survival rate(%)

Survival rate(%)

100
80
60
40
20
0

ild

intestine and neuron. These results

DAF-3

Sn o

W

receptor at least functions in both

Sm ad

DAF-8/14
Sm ad

Unexpectedly, the tissue specific rescue
experiments revealed that insulin

DAF-9

TGF-β DAF-1/4
recep to rdaf

cDNA in neurons, intestine or muscles.

TGF-β

ch o lestero l

DAF-7

pe

animals expressing insulin receptor

Steroid-hormone mediated signal

TGF-β mediated signaldaftolerance of insulin receptor mutant

TGFβ signaling

40
20
0

W

ild

pe
2
-ty
f-1
da

Steroid hormone signaling

Insulin is a secretory molecule, and insulin receptor is expressed in many tissues such asneuron, muscle and intestine. To investigate the tissues where insulin receptor function in thecold tolerance, we tested temperature-dependent cold tolerance of insulin receptor mutant
animals expressing insulin receptor cDNA in neurons, intestine or muscles. Unexpectedly, thetissue specific rescue experiments revealed that insulin receptor at least functions in both
intestineand neuron. These results suggest that intestine and neuron are essential for cold
- 19
tolerance, and insulin is received by intestine and neurons.
To isolate more genes for regulating cold habituation, we introduced genetic screening ofmutants defective in cold habituation. Through 2000 genomes screen by using mutagen, EMS,

suggest that intestine and neuron are essential for cold tolerance, and insulin is received by intestineand neurons.
To isolate more genes for regulating cold habituation, we introduced genetic screening of mutants
defective in cold habituation. Through 2000 genomes screen by using mutagen, EMS, we isolated
10 mutants defective in the cold tolerance. We outcrossed these mutants with wild-type N2 strain
to eliminate background mutations, and several mutant strains remained showing abnormal cold
habituation phenotype. To determine responsible genes for the abnormal cold habituation, we areusing SNP analysis.

Discussion & Conclusion
Temperature habituation are highly complex processes whose mechanisms are extremely intricate.
At the cellular level, the temperature information that is constantly received by the sensory nervous
system and other tissues are processed and stored in the neural circuit and tissues for transient orpermanent storage. These mechanisms are highly complicated since cell network consist of manycells. The results in this study showed that important cell-network for temperature habituation,
where the secretary hormonal signaling acts as a key signaling. Our results allow us to demonstratecell circuit directly regulating temperature habituation. We believe that the molecular andcellular analysis of this system should shed light into understanding fundamental mechanisms oftemperature habituation.

References
1. The genetics of Caenorhabditis elegans. Brenner, S. Genetics, 1974, 77, 71-94.
2. daf-2, an insulin receptor-like gene that regulates longevity and diapause in Caenorhabditis
elegans. Kimura, K. D., Tissenbaum, H. A., Liu, Y., Ruvkun, G., Science, 1997, 277, 942-6
3. Temperature sensing by an olfactory neuron in a circuit controlling behavior of C. elegans.
Kuhara, A., Okumura M., Kimata T., Tanizawa Y., Takano R., Kimura D. K., Inada H.,
Matsumoto K., Mori I., Science, 320, 803-807, 2008
4. Molecular mechanism for trimetric G protein-coupled thermosensation and synaptic regulation in
the temperature response circuit of Caenorhabditis elegans. Ohta, A. & Kuhara, A., Neuroscience
Research, 76, 3, 119–124, 2013

一般の皆様へ
温度は、地球上で常に存在する環境情報であり、生体内の生化学反応に直結した情報である。
そのため、動物は、環境温度に適応することで生存繁栄することができる。本申請者は、動物がどのように温度感知するのか、そしてどのように過去の温度情報を記憶するのかに関して、線虫
C. elegans をモデル系として解析してきた (Kuhara et al., Science, 2008; Kuhara et al., Nature commun.,2011)。しかし、温度環境の変化に適応するための分子機構に関しては、依然としてブラックボックスとなっている。我々は、シンプルな実験動物である線虫を利用し、特に分泌分子を介した温度適応に関わる遺伝子メカニズムの解明に向けて日夜解析を行っている。

- 20 -

Role of Rab GTPases in the intracellular logistics of insulin granule
Kohichi MATSUNAGA
Institute for Molecular and Cellular Regulation, Gunma University
matsunag@gunma-u.ac.jp

Abstract
While Rab27 small GTPase is required for late step pathway of insulin secretion, Rab2a is requiredfor insulin biogenesis and early step of insulin secretion. Here, we report that Rab27a and Rab2ainteract with a novel dual effector in GTP-dependent manner. Rab27a and Rab2a regulate early andlate step of insulin secretion pathways through cooperative bindings with their dual effector.
Key words : insulin, Rab27, beta-cells

Introduction
Regulated secretion is a main pathway for secretory cells to deliver bioactive molecules to thesurface or outside of the cells. The pathway comprises coordinated sequential steps such as secretory
granule biogenesis, maturation, trafficking, and fusion with plasma membrane. Although themolecular machinery for these processes have been characterized, precise molecular mechanisms toconnect each process are poorly understood.

Results
Previous studies have shown that the small GTPase Rab27 is required for late steps of this pathway,
granule trafficking, docking, and fusion (ref1 J Clin Invest. 115(2):388-96(2005)). To fully elucidate
Rab27 function, we comprehensively identified Rab27a interacting proteins in pancreatic beta cellsby a tandem affinity purification system based on MEF-tag immunoprecipitation and LC-MS/MS(ref2 J Biol. Chem. 280, 13187-13194 (2005)). We found that another small GTPase Rab2a interacts
with Rab27a through a novel dual effector of these GTPases. Although Rab27a localized on almost
all granules, Rab2a specifically localized on immature granules in the rat beta-cell line Ins1 832/13.
Furthermore, knockdown of Rab27a and Rab2a decreased glucose-induced insulin secretion andconversion of proinsulin to insulin respectively, whereas that of the dual effector inhibited bothprocesses.

Discussion & Conclusion
These data suggest that the Rab27a-Rab2a complex containing the dual effector is involved in thetransition from granule maturation to secretion in the regulated secretory pathway. Rab27a andRab2a regulate insulin secretion pathways through cooperative bindings with their dual effector.

- 21 -

References
1 Kasai, K. et al. Rab27a mediates the tight docking of insulin granules onto the plasma membrane
during glucose stimulation. J Clin Invest 115, 388-396 (2005).
2 Ichimura, T. et al. 14-3-3 proteins modulate the expression of epithelial Na+ channels byphosphorylation-dependent interaction with Nedd4-2 ubiquitin ligase. J Biol Chem 280, 1318713194 (2005).

一般の皆様へ
糖尿病は人類が抱える最も深刻な疾患の一つです。血糖値を下げるインスリンは膵β細胞内の顆粒に貯蔵され、血中のグルコース濃度を感知して分泌します。糖尿病の主な原因のひとつとして、特に我が国では重要な要因になっている、β細胞の機能低下によるインスリン分泌の減少が挙げられます。しかしながら分泌の詳細なメカニズムは未だ不明な点が多く、分泌能低下の理由もよくわかっていないため、その治療戦略を立てられないのが現状です。本研究はインスリンが合成されてから分泌されるまでのメカニズムの過程の一つを分子のレベルで明らかにしました。

- 22 -

Chemical Biology Based on the Unnatural Steroidal Compounds
Shuji Yamashita, Ph.D.
Tohoku Universitys-yamashita@m.tohoku.ac.jp

Abstract
We established a versatile method to install functionality to the non-activated C-ring of
epiandrosterone. Synthetic route to the C8-methyl steroids was also developed by using tandem
radical cyclization as a key reaction.
Key words : C-H amination, steroid derivatives, tandem radical cyclization

Introduction
Chemical derivatization of steroids is an effective tool for natural product synthesis and canprovide important bioactive agents. However, only a few functionalizations of the non-activated
C-ring of the androstanes have been reported. On the other hand, unnatural steroidal skeletons are
attractive scaffolds for creating novel bioactive compounds. We describe here a new method tofunctionalize the androstane C-ring through a remote C-H amination. Syntheses of the C8-methylsteroids are also reported.

Results
Functionalization of the androstane C-ring was investigated using epiandrosterone (1, Scheme1). Protection of the secondary alcohol of 1, stereoselective reduction of the ketone, and subsequent
treatment with chlorosulfonyl isocyanate and formic acid afforded sulfamate 2 in 80% overall
yield. Rhodium-catalyzed C-H bond amination of 2 according to the Du Bois method successfully
provided the C12,17-oxathiazinane
3 in 86% yield. It is noteworthy
that the C16- and C18-aminatedproducts were not detected. This
exclu sive chemoselect iv it y is
attributed to the long S-O and S-Nbonds and the obtuse N-S-O angleof the sulfamate, as well as thedifference in reactivity between
the primary and secondary C-H
bonds. The str uct ure of 3 was
u na mbig uou sly con f i r med by
X-ray crystallographic analysis of
the corresponding alcohol 4.

- 23 -

We next examined the cleavage of the S-O and/or S-N bonds of oxathiazinane 3 (Table 1). Birch
reduction resulted in no reaction and the starting material was recovered (entry 1). Treatment of 3
with LiAlH4 in THF afforded no products (entry 2), whereas LiAlH4 reduction in toluene followed
by Boc-protection furnished carbamate 5 in 39% yield (entry 3). After careful screening of reagentsand conditions, we found that refluxing with AlH3 in toluene afforded amino alcohol 6 in 90% yield(entry 5).

Selective oxidation of the amino group was achieved by treatment of 6 with 3,5-di-t-butyl-1,2benzoquinone 7 to give ketone 8 in 95% yield (Scheme 2). Stereoselective reduction of ketone 8 was
realized by treatment with sodium metal in the presence of isopropanol to give the C12β-hydroxy
product 9 (77%). The C11,12-dehydro derivative 10 was also prepared under Shapiro’s conditions in
63% overall yield.
Unnatural C8-methyl steroids
13 and 15 were prepared by
n e wl y d e velo p e d t a n d e mradical reaction (Scheme 3).
Using farnesol 11 as a starting
material, C8-methl steroidal
core 12 was sy nthesizedin 7 synthetic steps. After
functional group manipulation,
the key compou nd 13 was
prepared. On the other hand, C19-oxy derivative 15 was also prepared from geraniol 14 by thealmost same procedure described above. The structure of 15 was unambiguously confirmed by
X-ray crystallographic analysis.

- 24 -

Discussion & Conclusion
We established a versatile method to install functionality to the non-activated C-ring of
epiandrosterone. Rhodium-catalyzed remote C-H amination efficiently furnished the oxathiazinane,
which was converted to the C12-amino, oxo, hydoxy, and C11,12-dehydro androstanes. Syntheticroute to the C8-methyl steroids was established by an efficient manner. Natural and unnatural
product syntheses using these strategies will be reported in due course.

References
1 Yamashita, S.; Himuro, M.; Hayashi, Y.; Hirama, M. Tetrahedron Lett. 2013, 54, 1307.
2 Yamashita, S.; Naruko A.; Yamada, T.; Hayashi, Y.; Hirama, M. Chem. Lett. 2013, 42, 220.
(Editor’s choice Article)

一般の皆様へ生体機能を制御する物質としてステロイドは有名であり、性ホルモンや膜脂質など、生命活動の根底に作用することが知られています。天然から得られるステロイドなどを基盤として、様々な医薬品が開発され、現在、人間の社会活動を豊かにするのに貢献しています。本研究では、新しい機能を持った医薬品候補の探索を視野に入れて、天然からは得られないステロイド誘導体を人工合成することに成功しました。安価で効率的に生理活性候補を合成するために、新しい方法を開発しました。
今後、薬効や毒性を評価し、有用な物質を見つけたいと考えています。

- 25 -

Roles of store-operated Ca2+ channels in skeletal muscle
Yuji Hara
Tokyo Women’s Medical University / Kyoto University
hara@sbchem.kyoto-u.ac.jp

Abstract
Calcium ion (Ca2+) plays crucial roles in various tissues including skeletal muscle. We investigate
the function of Store-operated Ca2+ channel (SOCCs), which is activated by depletion of intracellular
Ca 2+ store. To elucidate the downstream pathway of SOCCs, we identified several candidate
pathways of SOCCs by microarray analyses.
Key words : Skeletal muscle, Calcium ion, Store-operated calcium channel (SOCC), myopathy

Introduction
Ca 2+ influx across the plasma membrane evokes various cellular processes including muscle
contraction, cellular excitability, and transcriptional activation (Ref.1). Recent literatures identified
store-operated Ca2+ channels (SOCCs) as a new Ca2+ entry machinery, and its activation is involvedin a variety of events in cells (Ref. 2). With regard to the function of SOCCs in skeletal muscle,
SOCCs are thought to play a crucial role because mutations in its components cause skeletal muscle
diseases (myopathy) (Ref. 3). However, little is known about what the function of SOCCs in skeletal
muscle is, and how its dysfunction leads to myopathy. To address these questions, we investigate thedown-stream pathways of SOCCs in skeletal muscle cells.

Results
1: Identification of SOCC-dependent pathways in skeletal muscle:
SOCCs are composed of two main subunits: a pore forming subunit at the plasma membrane
ORAI1, and a Ca 2+ sensor in the endoplasmic and sarcoplasmic reticulum STIM1 (Figure 1).
Previous literatures have shown that conditional STIM1 knockout leads to myopathy phenotypes
including myofibers with centrally localized nuclei, atrophy, swollen mitochondria in skeletal
muscle.
Moreover, gain-of-f unction
mut at ion s i n OR A I1 a nd
STIM1 genes cause Tubular
aggregate myopathies, which
are characterized by presence of
aggregates on the Sarcoplasmic
reticulum. Thus, these facts
indicate that Ca 2+ inf lu x by

- 26 -

SOCCs is crucial for the function of skeletal muscle. However, it remains to be elucidated themolecular basis for the SOCC-dependent pathways.
To address this question, we carried out microarray analyses. In this experiment, we utilized
C2C12 cells, a mouse myoblast cell line that has a capacity to be differentiated into myotubes,
and that has been established as a model system for skeletal muscle studies. Differentiated C2C12
cells were prepared by incubating in differentiation media supplemented with 2% horse serum. To
activate SOCC-dependent pathways, we used Thapsigargin, an inhibitor of Ca2+-ATPase which acts
as a replenishment pathway of Ca2+ into the Sarcoplasmic reticulum. Thus Thapsigalgin treatments
cause reduction in the content of Ca2+ in sarcoplasmic reticulum, thereby leading to activation of
SOCCs. To identify genes involved in the SOCC-dependent pathways, we prepared three types ofdifferentiated C2C12 myotubes as below:
Group-1: C2C12 cells incubated in differentiation media supplemented with DMSO (this sample isconsidered as a control).
Group-2: C2C12 cells treated with 0.3 micro M Thapsigargin in the presence of 1.8 mM CaCl2.
Group-3: C2C12 cells treated with thapsigargin but cultured in low Ca2+ media plus 10 micro M La3+(a broad Ca2+ channel blocker that also blocks SOCCs).
Those cells were harvested at 4 hours post-incubation. Total RNA samples wereisolated using RNA
isolation kit (Takara), and were subject to microarray analyses. We utilized Mouse Genome 430 2.0
Array (GeneChip, Takara) for microarray analyses, and searched genes that matched the following
criteria:(A) Genes which were up-regulated in response to Thapsigargin treatment (a comparison between
Group-1 and -2).
(B) Genes which were not significantly up-regulated, compared between Group-1 and -3
These experiments were carried out twice. Basedon the criteria above, candidate molecules have been
identified (See the figure). We initially focused ontranscriptional factors which play an essential role inmyogenic differentiation, but we could not detect thedifferences in the expression levels which matched thecriteria above. Instead, there were several candidate
molecules involved in metabolism, signal transduction,and secretory hormonal molecules.
The results obtained by microarray analyses wereconfirmed by semi-quantitative RT-PCR analyses.
We are now trying to elucidate the physiological significance of those proteins in C2C12 cells andmouse skeletal muscle.
2: Molecular basis of the machinery that protects myofibers from contraction-induced
membrane injury:
Repeated contraction of myofibers causes membrane damages. It has been reported that Ca 2+-- 27 -

dependent membrane repair machineries are crucial for maintaining the membrane integrity inskeletal muscle, but it is still obscure about the details of the repair pathways. In addition to theexperiment mentioned above, we are trying to isolate the molecules involved in the membrane repair
pathway by proteome analyses.

Discussion & Conclusion
Accumulating evidence has suggested that SOCCs plays an important role in skeletal muscle.
However little is known about the molecular basis of downstream pathways of SOCC-induced Ca2+
influx. In this study, we identified several candidate molecules, including metabolic regulators,
secretory factors. Further experiments using mouse knockout models would be necessary to clarify
the pathogenesis of Tubular aggregate myopathies.

References
1. Berridge MJ, et al.. Calcium signaling: dynamics, homeostasis and remodeling. Nat Rev Mol Cell
Biol. 2003 Jul;4(7):517-29.
2. Soboloff J, et al.. STIM proteins: dynamic calcium signal transducers. Nat Rev Mol Cell Biol.
2012 Sep;13(9):549-65.
3. Böhm J et al., Constitutive activation of the calcium sensor STIM1 causes tubular-aggregate
myopathy. Am J Hum Genet. 2013 Feb 7;92(2):271-8.

一般の皆様へカルシウムイオンは通常細胞内では非常に低濃度に保たれています。刺激により細胞内カルシウム
イオンが増加すると、多彩な生理応答が引き起こされます。骨格筋では筋収縮への寄与が代表的でありますが、骨格筋自体の恒常性維持のため、様々な経路が活性化されます。その異常に伴い筋ジストロフィーをはじめとする筋疾患がもたらされると考えられています。当研究遂行により、骨格筋疾患の発症機構の解明およびその治療法開発を行っていく所存です。

- 28 -

Transcriptional quiescence for the establishment ofgerm cell fate in Drosophila.
Akira Nakamura
Institute of Molecular Embryology and Genetics, Kumamoto University
akiran@kumamoto-u.ac.jp

Abstract
In many animals, germline development depends on localized determinants in the germ plasm.
Drosophila pgc is the germ plasm factor that transiently represses mRNA transcription in germ(pole) cells and promotes their survival. However, the mechanism linking them remains elusive. We
found that pole cells lacking pgc show ectopic expression of numerous miRNAs that are normally
expressed only in somatic cells. We also found that these miRNAs can target germ plasm RNAs
including nanos, which encodes factors essential for germ cell development. Thus, transcriptionalquiescence in germ cells is essential to protect germ plasm RNAs from miRNA-mediated RNA
silencing.
Key words : germ cell, germ plasm, microRNA, Drosophila, transcriptional control

Introduction
In many animals, germ cell formation depends on maternal factors localized in the germ plasm.
The common function reserved in the germ plasm is an active repression of mRNA transcription,
which is thought to ensure germ cell fate by preventing the somatic transcriptional program(1). Drosophila Pgc is the germ plasm factor that inhibits mRNA transcription in pole cells
by interfering with the transcription machinery (2). Although pgc has been implicated in themaintenance of nanos (3, 4), which is essential for germ cell development (5), the mechanism bywhich Pgc-mediated transcriptional repression regulates nos remains unknown.

Results
We first examined the distributions of germ plasm markers such as nanos mRNA as well as Nanos
and Vasa proteins in embryos from pgc-null mothers (hereafter termed pgc– embryos). In pgc–
embryos, nanos mRNA was normally concentrated in the germ plasm during the cleavage stage(stage 2), but precociously disappeared from pole cells at the cellular blastoderm stage (stage 5). The
distributions of both Nanos and Vasa in pgc– embryos were also normal during the blastoderm stages.
In subsequent development, however, Nos, but not Vas, was precociously disappeared from polecells.
We next tested if the increase in nanos mRNA level in the germ plasm suppressed pgc mutant
phenotypes. To produce Nanos in pole cells independently from endogenous nanos regulation, we
expressed a transgene in which the nanos coding sequence was fused to the pgc 3′ UTR, which- 29 -

contains RNA localization signals to the germ plasm, during oogenesis. In pgc– embryos expressing
the nanos-pgc 3′ UTR, the number of pole cells in the gonad was significantly increased. Therefore,
one of the critical roles of pgc-mediated transcriptional repression is the maintenance of maternal
nanos mRNA in pole cells.
The quantification of maternal nanos mRNA distribution has revealed that the majority of themRNA is present throughout the cytoplasm in cleavage-stage embryo (6). This unlocalized nanos
mRNA, like many other maternal mRNAs, is degraded during the maternal-to-zygotic transition(MZT) by the combined action of maternal and zygotic factors involved in RNA degradation (7). We
assumed that Pgc-mediated transcriptional repression in pole cells might prevent the activation of thezygotic pathway that usually acts in the somatic region. The mir-309 cluster, which contains eightmiRNA genes, is one of early zygotic genes known to be involved in the degradation of hundreds ofmaternal mRNAs in the somatic region (8). While miR-309 primary transcript was never detected inpole cells in wild- type embryos, it was ectopically transcribed in pole cells lacking pgc. However,
the removal of the mir-309 cluster gene did not rescue the pgc– mutant phenotype.
We hypothesized that other early zygotic miRNAs might be misexpressed in pgc– pole cells to
promote maternal nanos mRNA degradation, and found that mir-1, mir-2a-2 cluster, mir-9a and mir10 were misexpressed in pgc– pole cells. Furthermore, luciferase reporter assays in Drosophila S2cells revealed that the reporter containing the nanos 3′ UTR, but not a control 3′ UTR, was repressedby the co-expression of miR-1 or miR-10.
We next asked whether inhibiting the miRNA pathway is important for pole cell survival. Since
the miRNA pathway components, such as AGO1 and Dicer-1, play essential roles in oogenesis (9,10), we were unable to produce eggs lacking AGO1 or Dicer-1. We therefore removed one copy of
AGO1 or Dicer-1 in pgc– mothers, and analyzed the fate of pole cells in the embryos. Reduction of
the activity for either pre-miRNA processing (by removing one copy of Dicer-1) or RNA-induced
silencing complex (by removing one copy of AGO1) resulted in the significant increase in thenumber of pole cells in pgc– embryos. Consequently, the fertility of adult progeny was restored.
Furthermore, the depletion of Dicer-1 or gawky, which encodes a binding partner of AGO1, by RNA
interference, resulted in the increase in the number of pole cells reached in the embryonic gonads in
pgc– embryos.
Finally, we examined the effect of RNAi on the stability of nanos mRNA in pole cells. We foundthat, in Dicer-1 or gawky knockdown embryos, nanos mRNA was stabilized in pole cells even in the
pgc– background. These results indicate that the suppression of miRNA activity is important for thestabilization of nanos mRNA in pole cells, allowing efficient nanos mRNA translation, and thereby
the maintenance of pole cells during embryogenesis.

Discussion & Conclusion
During the MZT, the majority of maternal mRNAs is eliminated and zygotic transcription is
initiated (12, 13). Degradation of maternal mRNA erases the oogenic (germline) program andfacilitates somatic developmental program. In contrast, the MZT in germ cells is delayed relative
to that in the soma (14). This process is essential to maintain the germ plasm that stores all the- 30 -

information necessary for the establishment of germ cell fate. Our results demonstrate that pgc
regulates the timing of MZT in germ cells by preventing zygotic expression of miRNAs that
promote maternal mRNA degradation.
Notably, in zebrafish embryos, germ plasm mRNAs, such as nanos1 and tdrd7, are also
protected from miRNA-mediated degradation. However, inhibition does not occur at the level oftranscriptional control, but rather by maternally provided RNA-binding proteins, which bind thegerm plasm mRNAs to antagonize the miRNA-dependent RNA silencing (15–17). Therefore, theprotection of germ plasm mRNAs from miRNA-mediated degradation is common to many animals,
although the mechanisms appear to be established as a result of convergent evolution.

References
1. Nakamura, A. et al. Repression of early zygotic transcription in the germline. Curr. Opin. Cell
Biol. 22, 709-714 (2010).
2. Hanyu-Nakamura, K. et al., Drosophila Pgc protein inhibits P-TEFb recruitment to chromatin in
primordial germ cells. Nature 451, 730-733 (2008).
3. Nakamura, A. et al., Requirement for a noncoding RNA in Drosophila polar granules for germ
cell establishment. Science 274, 2075-2079 (1996).
4. Deshpande, G. et al., Maintaining sufficient Nanos is a critical function for polar granulecomponent in the specification of primordial germ cells. G3 2, 1397-1403 (2012).
5. Kobayashi S. et al., Essential role of the posterior morphogen nanos for germline development in
Drosophila. Nature 380, 708-711 (1996).
6. Bergsten, S. E. et al., Role for mRNA localization in translational activation but not spatial
restriction of nanos RNA. Development 126, 659-669 (1999).
7. Bashirullah, A. et al., Joint action of two RNA degradation pathways controls the timing ofmaternal transcript elimination at the midblastula transition in Drosophila melanogaster. EMBO
J. 18, 2610-2620 (1999).
8. Bushati, N. et al., Temporal reciprocity of miRNAs and their targets during the maternal-tozygotic transition in Drosophila. Curr. Biol. 18, 501-506 (2008).
9. Hatfield, S. D. et al., Stem cell division is regulated by the microRNA pathway. Nature 435, 974978 (2005).
10. Yang, L. et al., Argonaute 1 regulates the fate of germline stem cells in Drosophila. Development
134, 4265-4272 (2007).
11. Eulalio, A. et al., The GW182 protein family in animal cells: New insights into domains required
for miRNA-mediated gene silencing. RNA 15, 1433-1442 (2009).
12. Giraldez, A. J. microRNAs, the cell’s nepenthe: clearing the past during the maternal-to-zygotic
transition and cellular reprogramming. Curr. Opin. Genet. Dev. 20, 369-375 (2010).
13. Walser, C. B. and Lipstiz H. D. Transcript clearance during the maternal-to-zygotic transition.
Curr. Opin. Genet. Dev. 21, 431-443 (2011).
14. Suddiqui, N. U. et al., Genome-wide analysis of the maternal-to-zygotic transition in Drosophila
primordial germ cells. Genome Biol. 31, R11 (2012).
15. Mishima, Y. et al., Differential regulation of germline mRNAs in soma and germ cells byzebrafish miR-430. Curr. Biol. 16, 2135-2142 (2006).
16. Kedde, M. et al., RNA-binding protein Dnd1 inhibits microRNA access to target mRNA. Cell
131, 1273-1286 (2007).
17. Takeda, Y. et al., DAZL relieves miRNA-mediated repression of germline mRNAs by controllingpoly(A) tail length in zebrafish. PLoS One 4, e7513 (2009).

- 31 -

一般の皆様へ
ショウジョウバエの生殖細胞形成に中心的役割を担う Pgc の生理機能について解析を進めた。Pgc
は生殖細胞における転写を胚発生初期に一過的に抑制する。私たちは，Pgc による転写抑制が胚発
生初期に体細胞で発現する複数の miRNA の発現を抑えることにより，生殖細胞形成・分化に必須の機能を持つ nanos mRNA の miRNA 依存的分解を防いでいることを見出した。生殖細胞におけるmiRNA 経路の抑制は，脊椎動物においても報告されており，生殖細胞の特質を維持する分子機構
として普遍的である可能性が考えられた。

- 32 -

Regulation of plasma membrane receptor-like kinase functions
by endoplasmic reticulum quality control in plant cells
Shuh-ichi Nishikawa
Niigata University
shuh@bio.sc.niigata-u.ac.jp

Abstract
Molecular chaperones of the endoplasmic reticulum (ER) play key roles in the quality control
system of the ER. Recently, leucine rich-repeat receptor-like kinases (LRR-RLK) have been shown
to be the major target of ER quality control in plant cells. Using in vitro synthesized proteins, weanalyzed interactions between ER-resident molecular chaperones and the extracellular domain ofvarious LRR-RLKs of Arabidopsis thaliana. The results suggest relations between phenotypes ofchaperone mutants under heat-stressed conditions and affinities of chaperone-LRR-RLK interactions.
Key words : molecular chaperone, endoplasmic reticulum, quality control, receptor-like kinase,Arabidopsis thaliana

Introduction
Molecular chaperones of the endoplasmic reticulum (ER) play central roles in the quality control
system in the ER, which ensures that misfolded proteins are not transported to the Golgi apparatus.
Recent analyses have shown that leucine-rich repeat receptor-like protein kinases (LRR-RLK) are
the major target of ER quality control in plant cells. While Arabidopsis thaliana contains >200
LRR-RLK genes, mutants of the ER quality control system show defects in processes specific tosome LRR-RLKs. Therefore, specific interactions between ER-resident molecular chaperones and
LRR-RLKs have been suggested.

Results
Previously, we identified five ER-resident J proteins of Arabidopsis thaliana, which regulate
functions of BiP, a molecular chaperone Hsp70 in the ER. We showed that three J proteins,
AtERdj3A, AtERdj3B and AtP58IPK function in ER quality control(1). Analyses of mutants of these
J protein functions revealed that the mutant of AtERdj3B is defective in pollen maturation at hightemperature (under heat stress). We found this was due to defects in the function of an LRR-RLK
required for pollen development. Although at least five LRR-RLKs function in pollen development,
the phenotype of the aterdj3b mutant was specific to that LRR-RLK. Since J proteins interact withclient proteins of BiP, we hypothesized that the specific defect came from specificities in interaction
between J proteins and LRR-RLKs.

1. Synthesis of ER-resident molecular chaperones and the extracellular domain of LRRRLKs by in vitro translation
We used in vitro translation system using wheat germ extract to synthesize proteins used for theprotein-protein interaction assays. Three ER-resident J proteins, AtERdj3A, AtERdj3B and AtP58IPK,- 33 -

were synthesized with the C-terminal biotynilated tag. The extracellular domains of LRR-RLKs
were synthesized with the C-terminal FLAG tag. Western-blotting analyses showed that all proteins
were efficiently synthesized as soluble proteins.

2. Analyses of interaction between chaperones and LRR-RLKs
We used the AlphaScreen assay system (Perkin Elmer) in collaboration with Dr. Yasuomi Tada’sgroup (Kagawa University) to analyze interactions between chaperones and LRR-RKKs. We first
focused on interactions between chaperones and LRR-RLKs involved in pollen development.
The obtained results showed that AtERdj3A and AtERdj3B but not AtP58IPK interacted with the
LRR-RLK’s tested. Interestingly, our results suggest that the LRR-RLK whose functions arecompromised in the mutant of AtERdj3B interacted with AtERdj3B more efficiently than with
AtERdj3A. These results suggest relations between phenotypes of chaperone mutants under heatstressed conditions and affinities of chaperone-LRR-RLK interactions.
We also tried pull-down assays. However, we did not detect chaperone-LRR-RLK interactions by
this method. This was probably due to that affinities between chaperones and LRR-RLKs are too
weak enough to be detected by pull-down assay.

3. Analyses of ER chaperone mutants
From the above results, we expected that simultaneous depletion of AtERdj3A and AtERdj3B
would compromise functions of the other LRR-RLKs in pollen development at high temperatureand constructed the aterdj3a aterdj3b double mutant. Histochemical analyses showed that the double
mutant showed additional defects in pollen development, suggesting that functions of the other
LRR-RLK’s are compromised. We also constructed a double mutant of AtERdj3B and a chaperoneof another chaperone system in the ER. Histochemical analyses of the double mutant also showed
defects in the function of another LRR-RLK in plant development. Analyses of expression of genes
downstream of the LRR-RLK supports that functions of the LRR-RLK are compromised in thechaperone double mutant.

4. Analyses of subcellular localization of LRR-RLKs in chaperone mutants
We constructed transgenic plants expressing C-terminally myc- or GFP-tagged LRR-RLKs
involved in pollen development from their own promoter. Western-blotting analyses showed thatthe constructed plants express the fusion proteins. We are now introducing the fusion genes to themutants of ER-resident molecular chaperones by genetic crossing.

Discussion & Conclusion
Our protein interaction assays show that there are specificities in interactions between ER-resident
J proteins and LRR-RLKs. The obtained results suggest that the specific pollen developmental
defects of the aterdj3b mutant are due to that AtERdj3B alone is able to participate in quality
control of the relevant LRR-RLK. Analyses of the aterdj3b mutants expressing C-terminally GFPfused LRR-RLK involved in pollen development will reveal whether transport of specific LRRRLKs is affected in this mutant. Since the aterdj3a aterdj3b double mutant showed additional pollen- 34 -

developmental defects, detailed analyses of the double mutant would lead to identification of LRRRLK(s) whose maturation relies on both AtERdj3A and AtERdj3B.

References
Yamamoto, M., Maruyama, D., Endo, T., and Nishikawa, S. (2008) Arabidopsis thaliana has a setof J proteins in the endoplasmic reticulum that are conserved from yeast to animals and plants.
Plant Cell Physiol. 49: 1547-1562

一般の皆様へ
小胞体の品質管理機構は、合成された分泌タンパク質や細胞膜タンパク質の高次構造形成を監視し、異常タンパク質を処理する品質管理の場でもあります。小胞体品質管理で中心となるのが小胞体分子シャペロンです。本研究では、植物細胞における小胞体品質管理の主要なターゲットである
細胞膜の受容体キナーゼと、異常タンパク質の選別で機能する小胞体 J タンパク質間の相互作用を解析し、小胞体 J タンパク質変異株が示す特異的な欠損は、受容体キナーゼと J タンパク質間の相互作用の特異性のためであることを示唆する結果を得ました。これは、高次構造の似たタンパク質
でも分子シャペロンによる認識には特異性があることを示す例となります。

- 35 -

Analysis of functional molecules which regulate production oftype I interferons in plasmacytoid dendritic cells
Katsuaki Hoshino
Faculty of Medicine, Kagawa University
hoshino@med.kagawa-u.ac.jp

Abstract
We have compared gene expression profiles in dendritic cell subsets by DNA microarray analysis,
and discovered an Ets family transcription factor, Spi-B, is expressed high level in plasmacytoid
dendritic cells (pDCs). Spi-B can transactivate type I interferon promoter in synergy with interferon
regulatory factor 7 (IRF7). Spi-B-deficient mice and pDCs showed defective production of type
I interferon in response to agonists for Toll-like receptor 7 (TLR7) or TLR9. In conclusion, Spi-B
plays critical roles in production of type I interferons in pDCs
Key words : Plasmacytoid dendritic cell, type I interferon, Toll-like receptor

Introduction
pDCs recognize nucleic acids by endosomal TLR7 or TLR9, and produce a large amount oftype I interferons (IFNs) including IFN-α and IFN-β. We have discovered that the IκB kinase
α contributes to phosphorylation and activation of IFN regulatory factor 7 (IRF7), which is an
essential transcription factor for type I IFN production in pDCs. However, IRF7 is not enough for
type I IFN production by pDCs, because IRF7 is induced in conventional DCs (cDCs) by nucleic
acid stimulation, and the cDCs are unable to produce IFN-α. Therefore, we assume that additional
molecule which is specifically expressed in pDCs is involved in this event. To understand themolecular mechanisms that control this characteristic of pDCs, we searched some genes specifically
expressed in pDCs, and analyzed their functions.

Results
We have compared gene expression profiles of pDCs with those of other DC subsets in mice by
DNA microarray analyses, and identified a group of pDC-specific genes. Among them, we havefocused on a transcription factor, Spi-B, which belongs to an Ets family transcription factor. ThisEts family consists of more than 30 members. All of them have an Ets domain that bind to GGAA
or GGAT consensus sequence.
We examined whether Spi-B could transactivate type I IFN promoters using the luciferase assay.
Spi-B expression alone could not transactivate the Ifna4 promoter. However, when Spi-B was
co-expressed with IRF7, synergistic activation of Ifna4 promoter was observed. Spi-B did notshow such synergistic effect with other IRFs (IRF1, IRF3, IRF4, IRF5 or IRF8). Unlike in thecase of Ifna4 promoter, Spi-B alone could transactivate the Ifnb1 promoter. This activation was- 36 -

synergistically enhanced by co-expression of IRF7, IRF4 or IRF8, but IRF7 showed much stronger
synergism than others. We conclude that Spi-B can transactivate the type I IFN promoters insynergy with IRF7.
We have then analyzed whether Spi-B can associate with IRF family members. We transfected
expression vectors for HA-tagged Spi-B and FLAG-tagged IRFs into HEK293T cells. Spi-B was coimmunoprecipitated with IRF7, but not with other IRF family members.
In order to analyze the in vivo function of Spi-B in pDCs, we have generated Spi-B-deficient mice.
In Spi-B-deficient mice, pDCs were detected in the spleen, inguinal lymph node and bone marrow.
When the mutant mice were injected intravenously with polyU (a TLR7 agonist) or CpG DNA (aTLR9 agonist), elevation of serum IFN-α level was not observed. The mutant mice also manifested
defects in IL-12p40 and IFN-β production, although the defects were not so prominent as in IFN-α
production.
We also analyzed cytokine production from ex vivo pDCs in response to TLR7 or TLR9 agonist.
Bone marrow pDCs were isolated and stimulated with 8-mercaptoguanosine (a TLR7 agonist),
polyU, vesicular stomatitis virus (a TLR7 agonist), R848 (a TLR7 agonist), or CpG DNA. Wild typebone marrow pDCs could produce significant amounts of IFN-α , IFN-β, IL-12p40 and TNF-α.
However, Spi-B-deficient bone marrow pDCs failed to produce these cytokines. TLR7 or TLR9
stimuli induced up-regulation of CD86 expression in both wild type and Spi-B-deficient pDCs,
indicating that TLR signaling-induced CD86 up-regulation is intact in Spi-B-deficient pDCs.
Next, we searched our own gene expression database to find genes encoding pDC-specifictransmembrane proteins, and identified a candidate gene (hereinafter referred to as “pDC-M”).
Then, we searched public database to check expression profile of human homolog of pDC-M. We
have found that the human homolog of mouse pDC-M gene is also expressed in human pDCs.
Then, we analyzed expression level of pDC-M protein on DC subsets by flow cytometry. We haveidentified that pDC-M is specifically expressed on both mouse and human pDCs.

Discussion & Conclusion
In this study, we demonstrated that Spi-B could cooperate with IRF7 to transactivate type I IFN
promoters. The interaction of Spi-B with IRF7 should be the molecular basis for this synergistic
activity. In Spi-B-deficient mice, elevation of serum cytokine including type I IFN and IL-12p40
was impaired in response to TLR7 or TLR9 signaling. Spi-B-deficient ex vivo pDCs also showed
defect in production of these cytokines. Spi-B should be essential for transactivating not only type I
IFN promoters, but also IL12p40 promoter in pDC. We have revealed that Spi-B plays unique roles
in regulating pDC functions. We have also identified a pDC-specific membrane protein, pDC-M.
We are going to analyze the role of pDC-M in pDCs.

- 37 -

References
Sasaki, I., K. Hoshino, T. Sugiyama, C. Yamazaki, T. Yano, A. Iizuka, H. Hemmi, T. Tanaka, M.
Saito, M. Sugiyama, Y. Fukuda, T. Ohta, K. Sato, A. Ainai, T. Suzuki, H. Hasegawa, N. ToyamaSorimachi, H. Kohara, T. Nagasawa, and T. Kaisho. 2012. Spi-B is critical for plasmacytoid
dendritic cell function and development. Blood 120: 4733-4743.

一般の皆様へ
形質細胞様樹状細胞は、Toll 様受容体７（TLR7）と TLR9 を発現しており、その活性化により大量のⅠ型インターフェロン（IFN）を産生する特性がある。この特性は、ウイルス感染時には感染防御反応として機能するが、ある種の自己免疫疾患（全身性エリテマトーデスなど）では病態形成に関与している。今回我々は、転写因子 Spi-B が形質細胞様樹状細胞のⅠ型 IFN 産生に働く重要な分子である事を明らかにした。形質細胞様樹状細胞の特性を解明する事により、自己免疫疾患の診断や治療法の開発が進むことを期待する。

- 38 -

Prevention tumor metastasis by targeting tumor endothelial cells
KYOKO HIDA, YASUHIRO HIDA
Vascular Biology, Frontier Research Unit, Institute for Genetic Medicine, Hokkaido University
khida@igm.hokudai.ac.jp

Abstract
In this study, we analyzed the role of tumor endothelial cell-derived factor in tumor metastasis.
Key words : Angiogenesis, tumor , blood vessels, tumor endothelial cells

Introduction
Tumor endothelial cells (TECs) differed from normal endothelial cells (NECs) in many aspects.

Results
We demonstrate that TECs actively promote tumor metastasis through secretion of TEC-factor by
utilising two types of TECs: HM-TECs and LM-TECs isolated from high and low metastatic tumors,
respectively. Co-implantation of HM-TECs enhanced lung metastases by low metastatic tumors
concomitant with an increase in circulating tumor cells.
HM-TECs also promote tumor cell migration in vitro. All of these phenotypes either in vivo or invitro are abrogated by TEC-factor knockdown in HM-TECs.

Discussion & Conclusion
Because blood vessels provide tumor cells with an escape route from a tumor into the circulation,
we propose that TECs pave the way for tumor metastasis, particularly during the initial stage.

References
1. Joyce, J.A. & Pollard, J.W. Microenvironmental regulation of metastasis. Nat. Rev. Cancer 9,239–252 (2009).
2. Hida, K. et al. Tumor-associated endothelial cells with cytogenetic abnormalities. Cancer Res. 64,
8249–8255 (2004).
3. Ohga, N. et al. Heterogeneity of tumor endothelial cells: comparison between tumor endothelial
cells isolated from high- and low-metastatic tumors. Am. J. Pathol. 180, 1294–1307 (2012).

一般の皆様へ
腫瘍組織ではがん細胞だけではなく間質細胞も異常性を持っていることが知られています．がんの血管も最近では正常の血管とは異なることがわかってきました．本研究ではわれわれはがんの血管内皮細胞が分泌する分子によってがんの転移がおこりやすくなることを見出しました．がん血管の特性の解明が，新たな転移の阻害剤の開発につながる可能性があることが示されました．

- 39 -

Deriving Photoreceptors from Human Somatic Cells
Yuko Seko
National Rehabilitation Center for Persons with Disabilities
seko-yuko@rehab.go.jp

Abstract
Redirecting differentiation of somatic cells by overexpression of transcription factors is apromising approach for regenerative medicine. We found that human dermal fibroblasts aredifferentiated to photoreceptor cells by a transcription factor combination. Transduction of a
combination of the CRX, RAX, NEUROD and OTX2 genes up-regulated expression of several
photoreceptor-specific genes. Global gene expression data by microarray analysis further showedthat photoreceptor-related functional genes were significantly increased in induced-photoreceptor
cells. Functional analysis; i.e. patch clamp recordings, clearly revealed that induced-photoreceptor
cells from fibroblasts responded to light. Exogenous CRX, RAX, OTX2 and NEUROD, are sufficientto generate photoreceptor cells.
Key words : human dermal fibroblast, photoreceptor, transcription factors, redirecting differentiation

Introduction
We recently employed the strategy of “direct reprogramming” to generate retinal photoreceptor
cells from human somatic cells, defining a combination of transcription factors, CRX, RAX andNEUROD, that induce light responsive photoreceptor cells (Seko et al., 2012). In that study, we
induced “iris cells” into photoreceptor cells. Here we demonstrate that the same combination ofgenes used for human iris cells, i.e. CRX, RAX and NEUROD, generate human photoreceptor cells
from human dermal fibroblasts, and that additional OTX2 gene transduction further amplifies theexpression of the retina-specific genes. Our data therefore indicate that human dermal fibroblasts
are a superior cell source for reprogramming into photoreceptor cells.

Results
Human dermal fibroblasts are induced into a rod- or cone-specific phenotype by definedtranscription factors, CRX, NEUROD and RAX.

- 40 -

Additional OTX2 gene transduction increases upregulation levels of photoreceptor-specific genes
in induced photoreceptor from human dermal
fibroblasts.
OTX2 is not an essential factor but an amplifier
for induction of photoreceptor cells from humandermal fibroblasts.
Photoreceptor-related functional genes are clearly
up-regulated in induced photoreceptor cells fromhuman dermal fibroblasts.
Induced photoreceptor cells from fibroblasts arephotoresponsive in vitro.

Ref. 1

Discussion & Conclusion
This is the first report that human dermal fibroblasts can differentiate into photoreceptor cells by a
combination of transcription factors, CRX, RAX, NEUROD and OTX2. Our data suggest that OTX2
plays a role as an amplifier of photoreceptor-related functions. Functional analysis also revealed thatinduced-photoreceptor cells from fibroblasts by CRX, RX, NEUROD and OTX2 are photoresponsivein vitro.
The difference in induced endogenous expression of transcription factors CRX, RX and NEUROD
between CRN-infected fibroblasts and CRN-infected Iris cells as well as the difference in upregulated photoreceptor-specific genes may suggest a difference in reprogramming potential
between the human dermal fibroblasts and the human iris cells. It may be possible to improvedermal fibroblasts as a source by use of other transcription factors or manipulating the histone
methylation signature. However, dermal fibroblasts have an important advantage in that these cells
are obtained safely and easily from patients. Because the direct reprogramming method may besuitable to provide the small numbers of cells required for individualized drug screening and diseasemodeling, dermal fibroblasts may be useful for such purposes despite their limitations.

References
1. Seko Y, Azuma N, Ishii T, Komuta Y, Miyamoto K, Miyagawa Y, Kaneda M, Umezawa A.
Derivation of human differential photoreceptor cells from adult human dermal fibroblasts bydefined combinations of CRX, RAX, OTX2 and NEUROD. Genes Cells. 19(3):198-208. 2014
2 Seko Y, Azuma N, Kaneda M, Nakatani K, Miyagawa Y, Noshiro Y, Kurokawa R, Okano H,
Umezawa A. Derivation of human differential photoreceptor-like cells from the iris by defined
combinations of CRX, RX and NEUROD. PLoS One. 2012;7(4): e35611. Epub 2012 Apr 25.

- 41 -

一般の皆様へ
網膜の再生研究は近年急速に進み、ES 細胞や iPS 細胞から網膜組織が in vitro でつくられている。
一方、多能性幹細胞ではなく“ 体細胞 ” に転写因子遺伝子ミックスを導入すると iPS 細胞を経ることなく必要な細胞を得ることができる “ 直接的分化誘導法 ” と呼ばれる技術も開発され、すでに心臓、膵臓、神経、血小板などがつくられている。直接的分化誘導法は、方法が確実・簡単で早く、実験を小型化できるため、巨大投資なしで巨大疾患コホート研究が可能であり、疾患モデルとしては、多能性幹細胞より優れているとも言われている。本研究は、この直接的分化誘導法で網膜視細胞を作製することを目指した研究である。ヒト皮膚線維芽細胞に転写因子遺伝子ミックスを導入することによって、光応答のある網膜視細胞様の細胞に分化誘導できることがわかった。この分化誘導技術は、
遺伝性網膜変性疾患の診断、病態解明や薬剤のスクリーニング等に有用な疾患モデル作製に応用できる。

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Identification of cytidine deaminase activity for elucidating
the molecular mechanism of plant organelle RNA editing
Mamoru SUGITA
Nagoya University, Center for Gene Researchsugita@gene.nagoya-u.ac.jp

Abstract
In this study, we identified three novel DYW subclass pentatricopeptide repeat (PPR) proteins as
RNA editing factors in the moss Physcomitrella patens. Our present and previous data provide thefirst evidence that all known editing events require DYW subclass PPR proteins in this moss. In
addition, an in vivo functional analysis revealed that the DYW domain of PPR protein was requiredfor RNA editing.
Key words : RNA editing, plant organelles, pentatricopeptide repeat protein, cytidine deaminase

Introduction
RNA editing frequently occurs in many transcripts of plant organelles. More than 400 RNA editing
sites, involving cytidine (C)-to-uridine (U) transitions, have been identified in flowering plants.
However, the molecular mechanism of RNA editing is not fully understood. Recent genetics studies
have shown robust involvement of PPR proteins in RNA editing in plant organelles. In the moss
Physcomitella patens, C-to-U RNA editing occurs at only one site in the chloroplasts and at 11 sites
in the mitochondria (Sugita et al. RNA Biol. 2013). This raises the questions whether PPR proteins
are responsible for all RNA editing events in mosses and possess cytidine deaminase activity for
RNA editing.

Results(1) Two mitochondrial PPR proteins, PpPPR_65 and 98, were identified as RNA editing factors(Ichinose et al. Plant Cell Physiol 2013). Accordingly, the present and previous data provide thefirst evidence that all 11 known editing events in mitochondria require eight DYW subclass PPR
proteins in P. patens (see Table).
RNA editing site
nad3-C230
nad4-C272
ccmFc-C103
ccmFc-C122
ccmFc-C122
cox2-C370
cox3-C733

Editing factor
PpPPR_56

Editing factor
PpPPR_78

PpPPR_65

RNA editing site
rps14-C138
cox1-C755
nad5-C598

PpPPR_71
PpPPR_77

nad5-C730
atp9-C92

PpPPR_91
PpPPR_98

PpPPR_79(2) To confirm PpPPR_98 as an editing site-specific recognition factor, we performed anelectrophoresis mobility shift assay (EMSA) using the recombinant PpPPR_98 and RNA probes.
Prior to this analysis, we checked the extent of atp9-C92 editing because the atp9 gene is- 43 -

interrupted by three introns and the atp9-C92 site lies within the third exon, being only 8 nt long.
In the wild type protonemata, the atp9-C92 site was completely edited in fully spliced mRNA
while this site was unedited in unspliced transcripts. Accordingly, we used two different oligoRNA probes, spliced and unspliced (34 nt) for EMSA experiments. The RNA was incubated withthe recombinant protein. The protein-RNA complex was detected as shifted bands that migrated
more slowly than free RNA probe in the gel. The recombinant PpPPR_98 strongly bound to thespliced atp9 RNA but very weakly bound to the unspliced atp9 RNA. This result indicated that
PpPPR_98 is a bona fide site-specific recognition factor for the spliced atp9 transcript (Ichinose
et al. Plant Cell Physiol 2013).
(3) RNA editing occurs at only one site, rps14-C2, to create a translation initiation codon AUG in
the P. patens chloroplasts. To investigate whether chloroplast-localized PpPPR_45 is involved in
RNA editing, we constructed and characterized PpPPR_45 knockdown mutants. The analysis
revealed that PpPPR_45 was a site-specific recognition factor for rps14-C2 editing. This suggeststhat RNA editing regulates translation of rps14 mRNA.
(4) The DYW domain usually contains a conserved region, which includes invariant residues
that match the active site of cytidine deaminases (C/HxE….PCxxC) from various organisms.
Therefore, a hypothesis was provided in which the DYW domains are responsible for RNA
editing in plant organelles and catalyze RNA editing. The DYW domain itself is expected to
be an RNA editing enzyme. To investigate this possibility, we performed an in vivo functional
analysis of DYW domain using the generated transgenic mosses. This analysis revealed thatthe full DYW domain and its putative cytidine deaminase catalytic site are essential for RNA
editing. In addition, three amino acid residues at the C-terminus of DYW domain were shone to
be indispensable for RNA editing.

Discussion & Conclusion
The present study showed that nine PPR proteins are required for RNA editing at all 12 known
sites in P. patens. This is the first evidence of identification of a complete set of PPR editing factors
involved in all RNA editing events in plant organelles. Our functional analysis of the DYW domainstrongly suggested that the DYW domain is essential for
RNA editing. Taken together with all observations, we
propose a model of RNA editing in plant organelles (Fig.
1). The PPR motif tract recognizes the target editing site
and the DYW domain catalyzes C-to-U editing reaction.
To prove this model, further genetic and biochemical
experiments need to progress using the model plant, P.
patens.

- 44 -

References
1. Sugita, M., Ichinose, M., Ide, M. and Sugita, C. Architecture of the PPR gene family in the moss
Physcomitrella patens. RNA Biology, 10 (9), 1439-1445 (2013). doi:10.4161/rna.24772.
2. Ichinose, M., Sugita, C., Yagi, Y., Nakamura, T. and Sugita, M. Two DYW subclass PPR proteins
are involved in RNA editing of ccmFc and atp9 transcripts in the moss Physcomitrella patens:
First complete set of PPR editing factors in plant mitochondria. Plant & Cell Physiology 54 (11),1907-1916 (2013). doi: 10.1093/pcp/pct132

一般の皆様へ
植物のミトコンドリアと葉緑体で mRNA の特定のシチジン（C）がウリジン（U）に変換される「RNA編集」という奇妙な現象が起ることが知られています。しかし、C から U への RNA 編集の生物学的な意義や分子メカニズムについてはほとんど分かっていないのが現状です。本研究では新規のRNA 編集因子を３種同定するとともに、RNA 編集因子の DYW ドメインが C から U への RNA 編
集の酵素反応を担っている可能性が高いことを初めて明らかにしました。これらの成果を基盤として、今後はエネルギー生産工場である植物オルガネラの機能と RNA 編集との関係を分子レベルで解析していく予定です。

- 45 -

Clarification of novel mechanism ofcatecholamine biosynthesis by ERK5
Yutaro Obara
Yamagata University, School of Medicine
obaray@med.id.yamagata-u.ac.jp

Abstract
Previously, we demonstrated that extracellular signal-regulated kinase 5 (ERK5) was responsible
for neurite outgrowth and tyrosine hydroxylase (TH) expression in PC12 cells. In the present
study, we identified p36 as an ERK5-dependent and ERK1/2-independent gene and reported thenovel role of the ERK5/p36 signaling in the catecholamine biosynthesis. p36 was induced by nerve
growth factor in time- and concentration-dependent manners. TH protein levels were reduced by
p36 knockdown with no changes in the mRNA levels. Ubiquitination of TH was enhanced andcatecholamine biosynthesis was consistently reduced by p36 knockdown. ERK5 induces p36 gene
expression during neural differentiation, which is responsible for catecholamine biosynthesis.
Key words : ERK5, p36, catecholamine, tyrosine hydroxylase, NGF

Introduction
Extracellular signal-regulated kinase 5 (ERK5) is a member of mitogen-activated protein kinase(MAPK) family and strongly activated by neurotrophic factors such as nerve growth factor (NGF).
Previously, we showed ERK5 was involved in morphological and functional differentiation ofneuronal cells induced by NGF(1-2). However, the detailed mechanism of neuronal differentiation by
ERK5 still remains unclear. In this study, we attempted to clarify the mechanism of biosynthesis ofcatecholamines as neurotransmitters by ERK5 in detail.

Results
We newly cloned PC12 cells which stably expressed ERK5 shRNA. In response to NGF (100 ng/
ml, 5 min), both ERK5 and ERK1/2 were phosphorylated in PC12 cells transfected with empty
vector. However, only ERK1/2 phosphorylation was induced by NGF in PC12 cells where ERK5
expression is knocked-down by ERK5 shRNA. When these cells were cultured in the presenceor absence of NGF (100 ng/ml) for a day, tyrosine hydroxylase (TH) expression was selectively
abolished by ERK5 knockdown in both basal and NGF-stimulated states. We also examined neurite
outgrowth in these ERK5 shRNA-expressing cells and observed that the NGF-induced neurite
outgrowth was significantly blocked by ERK5-knockdown.
We next attempted to examine the role of ERK5 in primary cultured sympathetic neurons.
Superior cervical ganglia containing abundant sympathetic neurons was incubated in the presenceor absence of U0126 (20 μM) or BIX02189 (3, 30 μM). NGF (2 ng/ml) promoted axon elongation- 46 -

at approximately 30 μm/h. BIX02189 completely blocked axon elongation whereas U0126 did
not cause a significant inhibition. Next, we examined the involvement of ERK5 in TH protein
expression in dissociated sympathetic neurons. Four days after ERK5 shRNA was transfected into
the sympathetic neurons, TH protein expression was significantly reduced.
We performed microarray analysis using ERK5-specific inhibitor, BIX02189 (30 μM) and ERK1/2specific inhibitor, U0126 (30 μM) to identify genes that were induced by ERK5 specifically, andidentified 46 ERK5-specifc genes. Among them, we focused on p36 and attempted to clarify therole of ERK5/p36 signaling in the TH expression and catecholamine biosynthesis during neuraldifferentiation because that of p36 in neurons is completely unknown.
PC12 cells were stimulated with NGF (100 ng/ml) for 0.5-6 h or NGF (1-100 ng/ml) for 2 h, thenp36 gene expression was examined by RT-PCR. p36 gene expression pattern showed time- andconcentration-dependency. PC12 cells were stimulated with NGF (100 ng/ml) for 2 h in the presenceor absence of U0126 (30 μM) and BIX02189 (30 μM). Consistent with the microarray results, p36
gene expression by NGF was significantly blocked by BIX02189, but not U0126.
To investigate whether p36 is involved in TH expression, p36 expression was knocked-down
by siRNA, and then TH expression levels were examined in PC12 cells. p36 siRNA blocked TH
expression in basal state (decreased by 30.3% compared with control siRNA) and PC12 cells
differentiated by NGF (100 ng/ml, a day) (decreased by 61.7% of the TH levels up-regulated
by NGF). Tyrosine hydroxylase gene expression was also examined by RT-PCR. In PC12 cells
transfected with p36 siRNA, the p36 gene expression induced by NGF (100 ng/ml, 2 and 4 h) was
largely blocked, but TH gene expression levels in the same samples were not altered.
PC12 cells were transfected with p36 siRNA, then incubated with NGF (100 ng/ml) for 24 h.
Six hours before the terminating the reaction, proteasome inhibitor, MG132 (1 μM) was added to
cultures. After immunoprecipitation of TH, ubiquitinated TH protein was examined by Western
blotting. Ubiquitination of TH was promoted in p36 siRNA-treated cells, suggesting that p36 blocks
ubiquitination of TH protein.
The content of total catecholamines in PC12 cells including dopamine, noradrenalin and adrenalin
was measured by the ethylenediamine condensation method. The cells were incubated with orwithout NGF (100 ng/ml) for a day, then catecholamine levels within the cells were measured. NGF
facilitated the catecholamine biosynthesis in control PC12 cells, but catecholamine biosynthesis
was totally inhibited in PC12 cells that stably expressed ERK5 shRNA, which is consistent with
the TH protein levels. Furthermore, PC12 cells were transfected with control siRNA or p36 siRNAand incubated with NGF (100 ng/ml) for a day, then catecholamines contained in these cells were
measured. The catecholamine biosynthesis was inhibited by p36 siRNA, which is consistent withthe reduced TH protein levels.

- 47 -

Discussion & Conclusion
These results suggest that p36 induced by ERK5 stabilizes TH protein by blocking TH
ubiquitination. Hence, ERK5 regulates TH levels in two ways, 1) TH gene expression by activatingtranscription factors and 2) TH protein stabilization by inducing p36. Our findings that ERK5-p36
signaling regulates catecholamine biosynthesis by the mechanisms described above are novel, andthis signaling cascade may be involved in catecholamine-related diseases including hypertension,
Parkinson’s disease, depression, schizophrenia and sleep problems. We are currently examining the
involvements of ERK5 in hypertension caused by pheochromocytoma and Parkinson’s disease using
human pathological samples, and determine whether ERK5-selective blockers or activators areuseful for the treatment of these diseases.

References
1 Obara et al., J Biol Chem 284, 23564-23573, 2009.
2 Obara et al., Mol Pharmacol 77, 10-16, 2010

一般の皆様へ
私たちは以前に MAPK ファミリーである ERK5 が神経突起の伸展と（カテコラミンの生合成酵素の一つである）チロシン水酸化酵素の発現に必須であることを明らかにした。本研究課題では、
ERK5 シグナルによって遺伝子発現が上昇した p36 が、チロシン水酸化酵素のユビキチン化を抑制することによってチロシン水酸化酵素タンパク質を安定化し、カテコラミンの生合成を促進していることを明らかにした。

- 48 -

Assessing the biological significance and function ofneuron-specific imprinted genes in brain plasticity
Shin-ichi Horike
Advanced Science Research Center, Kanazawa University
sihorike@staff.kanazawa-u.ac.jp

Abstract
In order to understand the biological significance of genomic imprinting, we crossed heterozygous
PWS-IC Hs females with heterozygous PWS-ICΔ35kb males. As expected, paternally expressed
genes such as Snrpn, Ipw are ectopically expressed from maternal alleles in the imprint-reverse
mice. Unfortunately, most of these mice were dead within postnatal 7days. Therefore, we could not
perform their behavior analysis. Although some pups are alive, they seem to be smaller than wild
type mice. In this mouse, expression of paternally expressed genes Magel2, Ndn was disrupted. This
result shows that complete loss of IC function on the Hs allele.
Key words : Imprinting, Epigenetics, methylation, neuronal development

Introduction
Genomic imprinting is an epigenetic phenomenon by which certain genes can be expressed ina parent-of-origin-specific manner. Although more than 80 imprinted genes have been isolated
from the human and mouse genomes, their biological significance remain unknown. In this study,
we tried to create the imprint-reverse mouse to understand the biological significance of genomic
imprinting.

Results
This study was done in collaboration with Dr. Resnick (University of Florida). He has previously
created two type of transgenic mice, which are the PWS-IC Δ35kb and the PWS-IC Hs mice, inorder to evaluate the role of IC (Imprinting center) in Prader-Willi/Angelman syndrome (PWS/AS) locus. Mice (the PWS-IC Δ35kb) with a 35kb larger deletion involving the putative PWSIC lack expression of paternally expressed genes Magel2, Ndn, Snrpn, Ipw, and manifest several
phenotypes common to PWS infants. In contrast, mice inheriting the human PWS-IC paternally(the PWS-IC Hs) lack expression of paternally expressed genes Magel2, Ndn and shows postnatal
lethality and growth deficiency. In this mouse, Dr.Resnick has placed the murine PWS/AS region
under the control of a human PWS-IC. Interestingly, he found that the human PWS-IC (Hs)acquired methylation in the female mouse germline and the human PWS-IC (Hs) fails to maintainmethylation in somatic cells. Therefore, I suggested that the Snrpn gene would be ectopically
expressed from maternal allele when the human PWS-IC (Hs) is inherited from mother. In orderto detect gene expression from the maternal chromosome following maternal inheritance of thePWS-IC Hs allele (PWS-ICHs/+), I took advantage of PWS-ICΔ35kb mice (PWS-IC+/del) which have

- 49 -

no paternal gene expression. Females heterozygous for the PWS-ICHs allele were bred with carrier
males for the PWS-ICdel allele. Following maternal inheritance onto a null paternal background(PWS-ICHs/del), the expression pattern observed was identical to that observed following paternal
inheritance of the PWS-IC Hs allele (PWS-IC+/Hs). Expression levels of Snrpn, MBII-13, MBII-85,
MBII-52 and Ube3a-as were comparable to normal paternal levels of gene expression. In contrast,
expression of paternally expressed genes Magel2, Ndn was disrupted. In this study, I tried to create
the imprint–reverse mice to breed the PWS-ICΔ35kb male mouse (PWS-IC+/del) with the PWS-IC
Hs female mouse (PWS-ICHs/+). Most of these imprint–reverse mice were dead within postnatal
7days. Although some pups are alive, they seem to be smaller than wild type mice. Therefore, wehypothesized that the loss of IC function seen in human PWS-IC allele shows that the sequences
and trans-acting factors involved in activation of Magel2, Ndn have diverged. Recently, despite thefact that PWS-IC is known to be necessary for the coordinately controlled gene expression of PWS/
AS locus, the mechanism by which it act and how it influence to the several protein-coding genes
over long distance, is unknown. In order to define the precise long-range gene regulation of PWS/
AS region, I examined DNA-FISH analyses on the paternal or maternal human chromosome 15
and PWS-IC deleted human chromosome 15, which were established by our human chromosome
engineering techniques. Our FISH analyses showed that active paternal MAGEL2 locus was
looping out from its chromosome territory, but inactive maternal locus was retained in the insideof its chromosome territory. Interestingly, this spatial genome positioning of MAGEL2 locus wasabolished in the paternal PWS-IC deleted chromosome without altered cis interaction between
PWS-IC and MAGEL2 locus. Thus, the sequences and trans-acting factors involved in activationof Magel2, Ndn have diverged between human and mouse. Unfortunately, we could not performthe behavior analysis of the imprint-reverse mice, although we planned to perform their behavior
analysis to examine the biological significance of genomic imprinting. However, this study showsa model where PWS/AS locus is regulated epigenetically at the level of both inter- and intrachromosomal associations and that this spatial organization within a chromosome territory is
regulated by PWS-IC over long distance. These results provide new insights into the imprinted gene
regulation.

Discussion & Conclusion
There are a number of hypotheses to explain why imprinting happens in mammals. One of these is
the genetic conflict hypothesis. Until now, over 80 imprinted genes are identified and most of them
are involved in growth and metabolism. In general, paternal expressed gene favors the productionof larger offspring, and maternal expressed gene favors smaller offspring. Because often maternallyand paternally expressed genes work in the same growth pathways, this conflict of interest sets
up an epigenetic battle between mother and father. However, some of the imprinted genes suchas Snrpn, Ube3A are involved in brain development. Their biological significance of genomic
imprinting remains unknown. Therefore, we tried to create the imprint-reverse mice. Unfortunately,
we could not perform the behavior analysis of the imprint-reverse mice to examine the biological- 50 -

significance of genomic imprinting in brain development. In near future, we will try to examine thebiological significance of genomic imprinting in brain development.

References
1. Johnstone KA, DuBose AJ, Futtner CR, Elmore MD, Brannan CI, Resnick JL. (2006) “humanimprinting centre demonstrates conserved acquisition but diverged maintenance of imprinting ina mouse model for Angelman syndrome imprinting defects.” Hum Mol Genet. 15(3):393-404.
2. Yang T, Adamson TE, Resnick JL, Leff S, Wevrick R, Francke U, Jenkins NA, Copeland NG,
Brannan CI (1998) “A mouse model for Prader-Willi syndrome imprinting-centre mutations.” Nat
Genet. 19(1):25-31.

一般の皆様へ
私たちの細胞の中には，母親と父親から受け継がれてきた染色体が各々１本ずつ存在します。基
本的には，同じ機能を持っているのですが，ごく一部の遺伝子は，受け継がれてきた親由来に依存して機能するものが存在します。機能的に同じ遺伝子であるのに，なぜ故に親由来に依存しているのかは未だ明らかにされておりません。本研究では，それらの遺伝子の親由来を逆転させたマウスを樹立することで，その生物学的意味に迫ろうと考えました。本研究成果では，最終的な答えは出ておりませんが，今後この研究を進展させていくことでその答えを見出そうと考えております。

- 51 -

Intracellular logistics regulated by LIS1 and lissencephaly
Masami Yamada
Graduate School of Medicine, Osaka-City University
my26@med.osaka-cu.ac.p

Abstract
Cytoplasmic dynein involves in the movement of a wide range of highly regulated intracellulartransport towards the minus ends of microtubules. LIS1 forms an idling complex with dynein, andkinesin-1 transports a LIS1-dynein-tubulin complex to plus ends of microtubules via mNUDC11,4.
Rab6a releases LIS1 from this idling complex, allowing dynein to initiate retrograde movement8.
However, the regulatory mechanism underlying release of dynactin bound cargoes from dynein
motor remains largely unknown. Here, we report that ADP-ribosylation factor-like 3 (Arl3) anddynein light chain, LC8 coordinately induce dissociation of dynein and dynactin.
Key words : Cytoplasmic dynein, Arl-3(ADP-ribosylation factor-like3), DYNLL1/LC8Introduction
Dynactin was first identified as a cytosolic activity that allowed cytoplasmic dynein to drive
long-range movements of membrane vesicles on microtubules in vitro. Indeed, the main functionof dynactin is to facilitate the attachment of cytoplasmic dynein to its cargo, and can function as
an adaptor for dynein motor activity. One of the challenges now is to address the machinery that
regulates unloadings of cargoes from dynein. Thus, we have investigated the regulatory factors
underlying release of dynactin from dynein motor using a candidate gene approach and a genetical
approach.

Results
We previously demonstrated that Rab6a is an essential factor to activate idling dynein and to loadcargo at the plus-end of microtubules8. We assumed that other small GTPases may be involved in
the unloading process. Members of ADP-ribosylation factor (Arf) family, including the Arf-like(Arl) proteins regulate membrane traffic and organelle structure. Therefore, we focused on Arf/Arl
family proteins and examined whether they were able to dissociate dynactin from dynein. On theother hand, LC8 family members of dynein light chains are highly conserved ubiquitous eukaryotic
homodimer proteins that interact with dynein, and are involved in diverse biological functions.
We first performed microtubule pull-down assays to examine the effects of Arf/Arl family andLC8 on dynein binding to dynactin. Dynactin by itself is capable of direct binding to microtubules.
In addition, cytoplasmic dynein binds dynactin through a direct interaction between the dyneinintermediate chains (DICs) and dynactin. Therefore, we examined whether Arf/Arl family and LC8
are able to release dynein from microtubule bound dynactin under a high ATP condition, in which
dynein carried the low-affinity states with microtubules. Addition of the GTP-bound Arl3(Q71L)- 52 -

mutant or LC8 significantly released dynein from microtubules, respectively. The GDP-bound Arl3
mutant, Arl3(T31N) did not display any releasing properties. Importantly, Arl3(Q71L) and LC8enhanced the dissociation between dynein and dynactin. In contrast, Arl3(Q71L) and/or LC8 did notdisplay any obvious effects on the interaction of dynactin and microtubules. LC8 binds dynein andmay promote the assembly of the dynein motor complex, while the target molecule of Arl3 remains
unknown. To examine whether Arl3 binds either dynein or dynactin, we performed pull-down assay
using Arl3(Q71L) conjugated beads, which revealed that dynactin specifically precipitated with
Arl3(Q71L) conjugated beads, while Arl3(T31N) did not display obvious interactions with either
dynein or dynactin. We conclude that dynactin is a target molecule of Arl3.
We next performed immunoprecipitation assays using cell lysates from MEF cells after
adding Arl3(Q71L) and LC8. We precipitated dynein complexes by an anti-DIC, and found thatdynactin co-precipitated with dynein. Importantly, the amount of dynactin co-precipitation was
significantly decreased in the presence of either Arl3(Q71L) or LC8. Microtubule pull-down assays
and immunoprecipitation revealed that the GTP bound mutant, Arl3(Q71L) and LC8 facilitateddetachment of dynactin from dynein.
To obtain further compelling evidence forArl3(Q71L) or LC8 mediated dissociation of
dynactin, we performed in vitro single molecule
assays using purified dynein, dynactin, as well
as recombinant proteins of Arl3(Q71L) and LC8(Figure 1). To visualize the release of dynactin,
we used Q-dots as extremely photostable fluorescent probes. We initially made moving complexesof dynein and dynactin, and then added either Arl3(Q71L) or LC8. Arl3(Q71L) formed a transient
triple complex and co-migrated, which was followed by the dissociation of a dynactin-Arl3(Q71L)
complex, resulting in dynein moving alone (Figure 1a). On the other hand, LC8 also made atransient triple complex, co-migrated and released dynactin, whereas LC8 co-migrated as a complex
with dynein (Figure 1b).
To explore in vivo interaction of Arl3(Q71L)
and LC8 with dynactin and dynein respectively,
we applied TIRF microscopy to trace their
movement in MEF cells (Figure 2). Dynactin is
an adapter that allows dynein to bind cargo, andis required for dynein activation. Dynein-bounddynactin is transported to the minus end of
microtubules. Indeed, EGFP-dynactin exhibiteda punctate appearance in the perinuclear region.
Importantly, mCherry-Arl3(Q71L) displayed
clear co-migration with EGFP-dynactin (Figure 2a). We next applied TIRF microscopy to trace
EGFP-DIC1 and mCherry-LC8. EGFP-DIC1 and mCherry-LC8 displayed co-migration to the- 53 -

perinuclear region, suggesting they are moving to
the minus-end of microtubules (Figure 2b). Thus, weconcluded that Arl3(Q71L) and LC8 bind dynactin anddynein, respectively in vivo.
Finally, we performed siRNA mediated Arl3 andLC8 knockdown experiments. Knockdown of Arl3
and/or LC8 induced an abnormal localization ofdynein, dynactin and related organelles (Figure 3).
Our findings uncovered the surprising functional
relevance of GTP-bound Arl3 and LC8 for theunloading regulation of dynactin bound cargo from dynein motor.

References
1. Masami Yamada, Shiori Toba, Yuko Yoshida, Koji Haratani, Daisuke Mori, Yoshihisa Yano,
Yuko Mimori-Kiyosue, Takeshi Nakamura, Kyoko Itoh, Shinji Fushiki, Mitsutoshi Setou,
Anthony Wynshaw-Boris, Takayuki Torisawa, Yoko Y. Toyoshima and Shinji Hirotsune;
LIS1 and NDEL1 coordinate the plus-end-directed transport of cytoplasmic dynein.
The EMBO Journal. 2008; 27(19):2471-2483.
2. Daisuke Mori, Masami Yamada, Yuko Mimori-Kiyosue, Yasuhiro Shirai, Atsushi Suzuki, Shigeo
Ohno, Hideaki Saya, Anthony Wynshaw-Boris and Shinji Hirotsune;
An essential role of the aPKC-Aurora A-NDEL1 pathway on neurite elongation via modulation of
microtubule dynamics.
Nature Cell Biology (article). 2009; 11(9):1057-1068.
3. Masami Yamada, Yuko Yoshida, Daisuke Mori, Takako Takitoh, Mineko Kengaku, Hiroki
Umeshima, Keizo Takao, Tsuyoshi Miyakawa, Makoto Sato, Hiroyuki Sorimachi, Anthony
Wynshaw-Boris and Shinji Hirotsune;
Inhibition of calpain increases LIS1 and partially rescues in vivo phenotypes in a mouse model oflissencephaly.
Nature Medicine (article). 2009; 15(10):1202-1207.
4. Masami Yamada, Shiori Toba, Takako Takitoh, Yuko Yoshida, Daisuke Mori, Takeshi Nakamura,
Atsuko H. Iwane, Toshio Yanagida, Hiroshi Imai, Li-yuan Yu-Lee, Trina Schroer, Anthony
Wynshaw-Boris and Shinji Hirotsune;mNUDC is required for plus-end directed transport of cytoplasmic dynein and dynactins bykinesin-1.
The EMBO Journal.2010; 29(3):517-531.
5. Takayuki Torisawa, Akiko Nakayama, Ken’ya Furuta, Masami Yamada, Shinji Hirotsune andYoko Y. Toyoshima;
Functional dissection on LIS1 and NDEL1 towards understanding the molecular mechanism ofcytoplasmic dynein regulation.
Journal of Biological Chemistry.2010; 286:1959-1965.
6. Masami Yamada, Shinji Hirotsune and Anthony Wynshaw-Boris;
A novel strategy for therapeutic intervention for the genetic disease: Preventing proteolytic
cleavage using small chemical compound.
The International Journal of Biochemistry and Cell Biology. 2010; 42(9):1401-1407.
7. Shiori Toba, Yasuhisa Tamura, Kanako Kumamoto, Masami Yamada, Keizo Takao, Satoko
Hattori, Tsuyoshi Miyakawa, Yosky Kataoka, Mitsuyoshi Azuma, Kiyoshi Hayasaka, Masano
Amamoto, Keiko Tominaga, Anthony Wynshaw-Boris, Mitsuhiro Kato and Shinji Hirotsune;
Post-natal therapeutic intervention for lissencephaly using a blood-brain-barrier permeablecalpain inhibitor, SNJ1945.
Scientific Reports(article). 2013; doi:10.1038/srep 01224.

- 54 -

8. Masami Yamada, Kanako Kumamoto, Shintaro Mikuni, Yoshiyuki Arai, Masataka Kinjo,
Takeharu Nagai, Yoshikazu Tsukasaki, Tomonobu M. Watanabe, Mitsuru Fukui, Mingyue Jin,
Shiori Toba and Shinji Hirotsune;
Rab6a releases LIS1 from a dynein idling complex and activates dynein for retrograde movement.
Nature Communications(article). 2013; doi:10.1038/ncomms3033.
9. Masami Yamada, Mingyue Jin, Yoshiyuki Arai, Takeharu Nagai and Shinji Hirotsune:
A regulatory mechanism of cargo unloading: Arl3 and LC8 coordinately induce dissociation of
the dynein-dynactin interaction.
Under revision in Nature Communications

一般の皆様へ
滑脳症は、脳の「しわ」が無くなる先天性神経疾患の一つであり、臨床的には重度の精神遅滞、運動失調、てんかん発作等を主な症状とする。発症の原因は、染色体 17 番目にある Lis1 遺伝子の変異あるいは欠損によるものである。この LIS1 の機能不全が、微小管モーターである細胞質ダイニンによる輸送システムの異常から神経細胞の遊走の異常を引き起こすことで疾患の発症に至ると考えられる。本研究課題は、神経細胞の中心体近傍に於ける細胞質ダイニンへの積み荷・積み降ろしの分子メカニズムを明らかにすることで、「細胞内物流の攪乱・破綻」から「神経細胞遊走の欠如」、さらには滑脳症発症に至るメカニズムの解明を目指した。

- 55 -

Elucidation of molecular functions of Imp3 in
osteosarcoma progression, and development of novel anti-Imp3
targeted therapeutic approach
Takatsune Shimizu
Hoshi University
t-shimizu@hoshi.ac.jp

Abstract
Osteosarcoma (OS) is a malignant tumor of bone and therapeutic target molecules that confer
malignant properties on OS have not been clarified. Using a newly established mouse OS model,
we identified Igf2 mRNA-binding protein3 (Igf2bp3; also called Imp3) as a critical regulator
responsible for an increased tumorigenic capacity in vivo. Imp3 confers the ability of anchorageindependent growth, loss of contact inhibition, and resistance to anoikis. Such malignant properties
caused by Imp3 are not attributable to the action of Igf2 alone. Our results indicate that Imp3 as apotential therapeutic target for this malignancy.
Key words : osteosarcoma, IGF2 mRNA binding protein 3 (IMP3)

Introduction
We previously established mouse osteosarcoma (OS) cells (designated as AX cells) which
generate tumors in syngeneic mice with pathophysiology mimicking human disease. Notably,
AX cells constantly acquire an increased tumorigenic capacity during tumor development andserial transplantation markedly shortens the disease course. Through comparative gene expression
analyses, we picked up Imp3 as one of the responsive molecules. Imp3 is primarily expressed during
embryogenesis and whose aberrant expression correlates with poor prognosis in many malignancies.
In the present study, we tried to clarify the molecular functions of Imp3 in malignant phenotype ofOS.

Results
We previously established mouse OS cells (AX cells) which develop tumors similar to humanpathological structures. Notably, secondary transplantation of AXT cells, originated from AXderived tumors, much shortened the disease course compared to primary inoculation, suggestingthat AX cells acquired higher tumorigenic ability in vivo. To identify the molecular events
underlying this phenomenon, we performed gene expression profiling of these cells. We extracted
candidate molecules according to the following criteria: The expression level (1) is low in normal
tissue; (2) is associated with poor prognosis in various human cancers; and (3) is directly regulate
tumorigenic activity in vivo by forced expression in AX cells and its depletion in AXT cells. Then
Imp3 meets all these criteria.
The expression of Imp3 is predominantly limited during embryogenesis and in various tumors.

- 56 -

Imp3 is thus considered an oncofetal protein. When we examined the expression level of IMP3
in human OS, immunohistochemical analyses of a tissue array containing 40 human OS samples
revealed that IMP3 was highly expressed in 90% of the specimens. Therefore, deregulation of IMP3
expression frequently occurs also in human OS.
The analysis using single-cell cloning indicated that AX clones that originally exhibit low level of
Imp3 expression in vitro can become cells that express Imp3 at high level during tumor formations.
Since treatment of epigenetic modification agents (5AzaD and TSA) in AX cells significantly upregulated Imp3 expression, suggesting the involvement of the epigenetic regulation.
We next evaluated whether forced expression or knockdown of Imp3 might affect the tumorigenic
activity of AX cells or AXT cells, respectively. Firstly, we generated AX cells that stably
overexpress Imp3 by retroviral gene transfer. Whereas Imp3 overexpressed AX cells and control
cells exhibited similar growth rate under normal culture conditions, the proliferation rate of Imp3
overexpressed cells was markedly higher than that of the control cells under nonadherent conditions.
Examination of tumorigenicity showed that the tumors generated by Imp3 overexpressed cells being
significantly larger than those formed by control cells. Next we knocked down Imp3 expressionin AXT cells by shRNAs. Whereas knockdown of Imp3 scarcely affected cell proliferation undernormal culture conditions, the growth rate of Imp3-depleted AXT cells was markedly reduced
compared to that of control cells under nonadherent conditions. We next investigated anchorageindependent survival of control and Imp3-depleted AXT cells. Flow cytometric analysis of cells
stained with annexin V and propidium iodide (PI) revealed that the size of the double-negative(viable) population was significantly smaller for Imp3-depleted cells than for control cells under
nonadherent conditions. These findings suggest that the up-regulation of Imp3 expression in tumor
cells contributes to their escape from anoikis. In addition, whereas control cells continued to growpast confluence, resulting in high saturation density, Imp3-depleted AXT cells exhibited contact
inhibition and lower saturation density. Moreover, knockdown of Imp3 drastically attenuated tumor
generations from AXT cells in vivo. Collectively, aberrant Imp3 expression in tumor cells promotes
anchorage-independent growth and loss of contact inhibition and directly confers high tumorigenic
activity in vivo.
Imp3 is an RNA binding protein and implicated in the regulation of target mRNAs. We further
examined its localization by centrifugation of cell lysates on a sucrose gradient. Imp3 was found toco-localize largely with rpS6 in the gradient fractions, suggesting that it localizes to ribosomes andpolysomes and involves in translational process.
Igf2 mRNA has been implicated as a main target of Imp3. We therefore examined whether theeffects of Imp3 on cell behavior observed in this study might be mediated by Igf2. However,supplement of Igf2 failed to increase the proliferation rate of AX cells to the level apparent for AXT
cells. All mice injected subcutaneously with Igf2-knocked down AXT cells by shRNAs generated
OS tumors, and Igf2 expression did not correlate with tumor weight. These results suggest that theeffects of Imp3 are not attributable to the augmentation of Igf2 signaling alone.

- 57 -

Discussion & Conclusion
We identified Imp3 as a critical molecule that affords malignant characteristics in AX cells andinvolves in the conversion into AXT cells in vivo. Both gain and loss of function of Imp3 in OS cells
revealed that Imp3 confers the ability of anchorage-independent growth, loss of contact inhibition,and resistance to anoikis, all of which lead to the tumorgenic activity. The effects of Imp3 have beenreported to be mediated through translational activation of Igf2. However, our findings indicatethat the malignant properties conferred by Imp3 are not attributable to the action of Igf2 alone. Our
preliminary data of RNA immunoprecipitation assays accompanied by gene expression profiling(RIP-CHIP) suggested that Imp3 can bind plenty of mRNAs in OS cells and regulate various
kinds of molecules at the protein level. Recent accumulated evidence indicates that overexpressionof IMP3 can become a significant indicator of metastasis and prognosis in several malignancies.
Therefore, further clarification of upstream and downstream molecular events of IMP3 can bringabout potential therapeutic strategies for OS as well as other tumor types.

References
1. Ueki A, Shimizu T, Masuda K, Yamaguchi S, Ishikawa T, Sugihara E, Onishi N, Kuninaka S,
Miyoshi K, Muto A, Toyama Y, Banno K, Aoki D, Saya H. Up-regulation of Imp3 confers in vivo
tumorigenicity on murine osteosarcoma cells. Plos One 7, e50621 (2012)
2. Shimizu T, Ishikawa T, Sugihara E, Kuninaka S, Miyamoto T, Mabuchi Y, Matsuzaki Y, Tsunoda
T, Miya F, Morioka H, Nakayama R, Kobayashi E, Toyama Y, Kawai A, Ichikawa H, Hasegawa
T, Okada S, Ito T, Ikeda Y, Suda T, Saya H. c-MYC overexpression with loss of Ink4a/Arf
transforms bone marrow stromal cells into osteosarcoma accompanied by loss of adipogenesis.
Oncogene 29,5687-99 (2010)

一般の皆様へ
骨肉腫は若年者に多く発症する難治性の悪性腫瘍です。新規治療法の開発が急務であり、私達は独自に樹立したマウス骨肉腫モデルを用いて克服に取り組んでいます。この度、骨肉腫悪性化に重要な因子として mRNA に結合するタンパク「IMP3」を絞り込みました。解析の結果、癌の生体内での進展に必要なイベントに関わることが明らかとなりました。IMP3 は近年、癌の予後不良因子である、との報告が相次いでおります。IMP3 が制御する分子、更には IMP3 の発現を制御する機構の解明
は癌の根本治療法の開発に繋がると考えられ、私達は更なる解析を進めております。

- 58 -

Development of metal nanoparticles for organic synthesis
aiming a substitution and reduction of rare metals
Mitsuhiro ARISAWA
Osaka University
arisaw@phs.osaka-u.ac.jp

Abstract
We have developed a sulfur-modified gold-supported palladium material (SAPd) with palladium,Pd, nanoparticles on its surface; it is a recyclable, low-leaching Pd catalyst. We also succeeded using
SAPd, the first examples of Pd-nanoparticle-catalyzed unactivated C(sp3)–H bond functionalizationof amides, using 8-aminoquinoline as a directing group, to yield ethynylated products, and Pdnanoparticle-catalyzed 1,7-Pd migration reaction for the synthesis of benzotriazoles via C-H bondactivation. Furthermore, through detailed analysis of SAPd structure, we have developed sulfurmodified gold-supported nickel material (SANi).
Key words : Metal nanoparticle, Palladium, Nickel, Carbon-hydrogen bond activation, Heterocycles

Introduction
Cross-coupling reactions with an organometallic catalyst are a key for the synthesis offunctional molecules such as pharmaceuticals, agricultural chemicals, solar cells, and organic
electroluminescent displays. Palladium-based catalysts, in particular, show remarkable performancedue to their high efficiency. Metal cross-coupling reactions generally require the use of an excellent
ligand. Cross-coupling reactions of metals, including these Pd-coupling reactions, have evolved withligand development.
The use of ligands is, however, associated with some problems, such as the need for productpurification. Furthermore, phosphorous ligands are easily oxidized and inert reaction conditions are
generally required. Therefore, we hypothesized that these problems would be resolved if the metal
cross-coupling could proceed without the use of a ligand, allowing for functional molecules to besynthesized easily and in high purity.

Results
We have developed a sulfur-modified gold-supported palladium material (SAPd) with palladium,Pd, nanoparticles on its surface; it is a recyclable, low-leaching Pd catalyst for ligand-free SuzukiMiyaura coupling and ligand-free Buchwald-Hartwig reaction.
Two independent experiments using transmission electron microscopy (TEM) and/or X-ray
absorption fine structure (XAFS) analysis provided complementary structural information of
SAPd. The effective catalysts in SAPd are the Pd nanoparticles, which allow for high chemicaltransformations due to their large surface area. The conventional nanoparticles used in carbon-- 59 -

carbon coupling reactions are usually formed by a metal salt reduction and are generally stabilized
by polymeric molecules, tetraalkylammonium salt, and ionic liquids. The use of an appropriate
stabilizing agent is therefore critical not only to obtain nanoparticles of a suitable size to form highly
active catalysts, but also to stabilize the surface so that leaching is minimized and nanoparticle
aggregations are prevented. Therefore, the above results are important toward the assertion that
sulfur-modified gold provides another example of an appropriate support for self-assembled multilayers of Pd nanoparticles (5 nm) in high density. Based on this valuable structural information of
SAPd, we have successfully developed sulfur-modified gold-supported nickel material (SANi),
which is a recyclable, low-leaching Ni catalyst for ligand-free Kumada-Tamao coupling and ligandfree Negishi coupling.
We also developed a new strategy for Pdnanoparticle, NP, -catalyzed direct ethynylation ofaliphatic carboxylic acid derivatives via C(sp3)–
H bond functionalization at the β positions ofamides, with 8-aminoquinoline as a directing
group. We obtained moderate yields of the desiredalkynylated products, but yields calculated based on recovered amides were almost the same as
those obtained under homogeneous conditions. Because of the low leaching properties of SAuPd,
the catalyst could be recycled more than 10 times. This protocol shows that Pd NPs are effective
catalysts for C(sp3)–H bond activation.
Furthermore, we have developed a new strategy for the synthesis of benzotriazoles using a Pd-NPcatalyzed 1,7-Pd migration reaction, which proceeded via the activation and functionalization of aC(sp2)-H bond. It is noteworthy that this reaction required the addition of an oxidant and provided
good to excellent yields of the desired 1-aryl benzotriazole products. Because this procedure
involves the unique combination of Pd-NPs with a hypervalent iodine reagent, it could be used inseveral other systems for the activation of C(sp2)-H bonds, which could lead to the development of
new methods in synthesis.

Discussion & Conclusion
We have developed a sulfur-modified gold-supported palladium material (SAPd) with palladium,Pd, nanoparticles on its surface; it is a recyclable, low-leaching Pd catalyst for ligand-free SuzukiMiyaura coupling and ligand-free Buchwald-Hartwig reaction. We also succeeded using SAPd, thefirst examples of Pd-nanoparticle-catalyzed ligand-free unactivated C(sp3)–H bond functionalizationof amides, using 8-aminoquinoline as a directing group, to yield ethynylated products, and Pdnanoparticle-catalyzed ligand-free 1,7-Pd migration reaction for the synthesis of benzotriazoles
via C-H bond activation. Furthermore, through detailed analysis of SAPd structure using TEM
and XAFS, we have developed sulfur-modified gold-supported nickel material (SANi), which is a
recyclable, low-leaching Ni catalyst for ligand-free Kumada-Tamao coupling and ligand-free Negishi
coupling.

- 60 -

References
1. “Palladium-Nanoparticle-Catalyzed 1,7-Palladium Migration Involving C-H Activation,
Followed by Intramolecular Amination: Regioselective Synthesis of N1-Arylbenzotriazoles andan Evaluation of Their Inhibitory Activity Towards Indoleamine 2,3-Dioxygenase” Koji Takagi,
Mohammad Al-Amin, Naoyuki Hoshiya, Johan Wouters, Hiroshi Sugimoto, Yoshitsugu Shiro,
Hayato Fukuda, Satoshi Shuto, Mitsuhiro Arisawa, J. Org. Chem. 2014, in press.
2. “Palladium-Nanoparticle-Catalyzed Direct Ethynylation of Aliphatic Carboxylic Acid Derivatives
via C(sp3)–H Bond Functionalization” Mohammad Al-Amin, Mitsuhiro Arisawa, Satoshi Shuto,
Yusuke Ano, Mamoru Tobisu and Naoto Chatani, Adv. Synth. Catal. 2014, 356, 1631-1637.
3. “Ligand-free Suzuki-Miyaura Coupling with Sulfur-modified Gold-supported Palladium in
the Synthesis of a Conformationally-Restricted Cyclopropane Compound Library with ThreeDimensional Diversity” Mitsuhiro Arisawa, Takatoshi Sato, Naoyuki Hoshiya, Mohammad AlAmin, Yuji Kogami and Satoshi Shuto, ACS Combi. Sci. 2014, 16, 215-220.
4. “Development of Second Generation Gold-Supported Palladium Material with Low-Leaching
and Recyclable Characteristics in Aromatic Amination” Mohammad Al-Amin, Satoshi Arai,
Naoyuki Hoshiya, Tetsuo Honma, Yusuke Tamenori, Takatoshi Sato, Mami Yokoyama, Akira
Ishii, Masashi Takeuchi, Tomohiro Maruko, Satoshi Shuto, Mitsuhiro Arisawa, J. Org. Chem.
2013, 78, 7575-7581.

一般の皆様へものづくり国ニッポンが他の国よりも有利に且つ効率よく機能性分子（医薬品・農薬・太陽電池・液晶等）を製造することが出来る、新しい製造技術—金属ナノ粒子触媒を開発しました。即ち、２０１０年にノーベル化学賞が与えられたパラジウムカップリングでは、これまでリガンドの添加が必須と考えられてきましたが、金属ナノ粒子触媒はこれらリガンドの添加を必要とせず、目的とする化合物を安価かつ迅速に多検体製造・精製することが出来、生命科学の親展に寄与することが可能です。

- 61 -

Epigenetic regulation by nuclear pore complex in cancer progression
Richard Wong
Department of Biology, Institute of Science and Enginnering, Kanazawa University
rwong@staff.kanazawa-u.ac.jp

Abstract
The nuclear pore channel is made by 30 different proteins creating octagonal symmetry, selectively
facilitating the transports of proteins and RNAs during interphase. Recently, we found that
nucleoporins Rae1 and Nup98 contributed to leukemogenesis. At this moment we entirely do notknow why several nucleoporins often found abnormality in leukemia patients. We hypothesis that
nucleoporins may contribute to epigenetic mechanism in leukemogenesis. Nups can regulate criticaltranscription factors nuclear trafficking and mitotic functions. In this grant, we have generated
Nup98-JARID1A transgenic mice to investigate epigenetic mechanism in carcinogenesis by
nucleoporins-histone modifiers orchestration.
Key words : Nuclear pore complex, Rae1 Nup98, Nup98-JARID1A

Introduction
Chromosomal translocation in leukemia patients are observed to occur in several NPC proteins,
including NUP98 and NUP214/CAN gene loci. NUP98 is translocated to more than 20 different
partners, including not only homeodomain transcription factors, (most commonly HOXA9) but also
other proteins such as topoisomerase I (TOP1) and JARID1A. Recently, we showed that NUP98
binding partner, Rae1 (RNA export factor 1) involved in the development of NUP98-HOXA9
leukemogenesis (Funasaka et al., 2011 Cell Cycle). We imagine that nucleoporins might contributeto epigenetic mechanism in leukemogenesis and NPC/Nups can regulate critical transcription
factors nuclear trafficking and mitotic functions. We want to investigate epigenetic mechanism incarcinogenesis through a novel approach of nucleoporins-histone modifiers orchestration.

Results
A cellular model for Nup98-homeodomain fusion induced leukemogenesis cites aberrant
transcriptional activation of novel target genes as the basis for cellular transformation, but there isinsufficient direct clinical evidence for this hypothesis. On the other hand, Rae1, a bona fide binding
partner of Nup98, has been indicated as abnormal in breast and lung cancer patients. We and others
demonstrated that Rae1 is critical in the maintenance of spindle bipolarity: depleted Rae1 leading tochromosome instability and multipolar spindles, caused aneuploidy, which is often found in various
cancers. An attractive possibility for Nup98 fusion-mediated leukemogenesis is that functional
Rae1 is reduced in Nup98 leukemogenic fusion cells. This would enhance supernumerous spindle
pole formation/multiple centrosomes, which would enhance tumorigenesis. This aspect of Nup98-- 62 -

mediated leukemogenesis has not been explored at all.
First, to verify the effect of Nup98 or Nup98-HOXA9
overexpression on Rae1 distribution and expression, we
generated visible GFP-tag constructs, GFP-HA-Nup98 and
GFP-HA-Nup98-HOXA9 and transfected them into HeLa
cells (Figure 1). We found that Rae1 levels were robustly
reduced in highly expressing GFP-Nup98-HOXA9 HeLa cells
and the human breast adenocarcinoma cell line, MCF7 andmore significantly, the leukemia cell line, K562 comparing toa much lesser extent in GFPNu98 cells. Besides those highly
transfected GFP-Nup98-HOXA9 or GFP-Nup98 cells were
elevated chromosome segregation defects significantly. We
further quantitated (relative %) the relative Rae1 fluorescenceintensity, we found that RAE1 was significantly also reduced (p < 0.05) in GFP-Nup98-HOXA9 but
not in GFP-Nup98-transfected HeLa and MCF7 cells. Taken together, these results suggest that Rae1
expression and localization patterns are critical to Nup98‑HOXA9 leukemogenesis (Funasaka et al.
2011).
Next, to test our speculation that the reductionin Ra1 in Nup98-HOXA9 fusions is critical toleukemogenesis, we investigated NUP98-HOXA9expressing transgenic mice. The histologicalexamination of leukemic Nup98-HOXA9 transgenic
animals (n = 12) revealed that megakaryocytic
cells were frequently found in the spleen; thiswas seldom obser ved in WT lit ter mates andnon-leukemic Nup98-HOXA9 transgenic mice
(Figure. 2). Consistently, we found that Rae1 andendogeneous Nup98 expression was reduced in those multinucleated megakaryocytic spleen cells
of NUP98-HOXA9 transgenic mice (three independent transgenic lines), but not in WT mice byimmunoblotting, in triplicate experiments. The same membrane
was re-probed with β-actin as a loading control (Figure. 2).
Interestingly, low RAE1 expression was also found in the
spleen of NUP98‑HOXA9 transgenic mice compared to the WT
and especially in those multinucleated megakaryocytic spleencells that were observed only in NUP98-HOXA9 leukemictransgenic mice by confocal microscopy(Figure 2)(Funasaka et
al. 2011).
We want to elucidate the transcriptional role of nucleoporins
in tumorigenesis and leukemic stem cells formation, we plan to- 63 -

compare the difference between the difference of Nup98 fusions, HOX genes (Nup98-HOXA9) andthose fusions with PHD domain (Nup98-JARID1A) (Figure2). We have generated Nup98-JARID1A
transgenic mice (Figure 3).
Since we found that Rae1 is reduced in Nup98-HOXA9 Tg leukemogenic mice. We areinvestigating Rae1 localization and expression in another Nup98 fusion transgenic Nup98-JARID1A
mice that we generated recently in our laboratory.

Discussion & Conclusion
Our study demonstrates that reduced the expression level of endogenous Nup98 protein inmegakaryocytic Nup98-HOXA9 transgenic mice (Figure 2) and NUP98-HOXA9 mRNA in thebone marrow of an AML patient (1). Because of the heterozygous nature of Nup98 translocationsin leukemia patients and the lack of detectable fusion protein at the NPC, the NPC in thesepatients is presumably deficient in Nup98. Such a 30–50% reduction in Nup98 protein levels could
seriously affect the normal function of Rae1 and/or Nup98. In fact, our data support this notion. In
particular, in wholly depleted Nup98detailed in vitro, rodent and human clinical data, we proposeda tentative model (negative feedback loop) to explain how Rae1 contributed in Nup98 fusionmediated leukemogenesis. We suggest that overexpression of the Nup98 leukemogenic fusion
protein (e.g.Nup98-HOXA9) causes a reduction in endogenous Nup98. Nup98 reduction also leads
to a disruption in Rae1 localization and function during interphase and cell division. We found thatRae1 depletion led to aneuploidy and caused spindle and chromosome segregation defects and theformation of multiple centrosomes, thereby activating leukemogenesis. Recently, we have generated
another Nup98 fusion transgenic mice, we will test Rae1 localization and expression in Nup98JARID1A mice.

References
Funasaka T, Nakano H, Wu Y, Hashizume C, Gu L, Nakamura T, Wang W, Zhou P, Moore M,
Sato H and Wong RW (2011) RNA export factor RAE1 contributes to NUP98-HOXA9-mediatedleukemogenesis.
Cell Cycle 10(9):1456-1467.

一般の皆様へ
急性骨髄性白血病患者の白血病細胞では、核膜孔複合体タンパク質 NUP98 の遺伝子領域において高頻度に染色体転座がみられる。転座した NUP98 遺伝子とキメラ遺伝子を形成する遺伝子は20 種類以上が知られているが、本研究では中でも NUP98 とヒストン脱アセチル化酵素 JARID1A とのキメラ遺伝子 Nup98-JARID1A に注目し、動物核膜孔複合体タンパク質－ヒストン複合体が制御する癌化におけるエピジェネティック制御機構を解析した。本研究で白血病発症モデル動物として
Nup98-JARID1A トランスジェニックマウスを樹立できたため、今後は生体内における癌化におけるエピジェネティック制御機構を解明することで、白血病治療薬の開発に発展できると期待している。

- 64 -

Artificial Regulation of Protein Degradation by the Proteasome
INOBE, Tomonao
Frontier Research Core for Life Sciences, University of Toyama
inobe@ctg.u-toyama.ac.jp

Abstract
Proteins degradation by the proteasome are involved in the many cellular processes. Thus theregulation of proteasomal degradation is critically important for cellular activities. Here we reveals
that new cellular strategies to enhance or inhibit proteasome-mediated protein degradation by
regulating the unstructured region of the substrate protein. These strategies provide useful ways toselectively control the protein degradation in the cell.
Key words : Proteasome, Protein degradation, Unstructured region

Introduction
Effective proteolysis of a folded protein by the proteasome requires the presence of an unstructured
region in the substrate, in addition to a polyubiquitin chain. However, not all unstructured regions
can promote degradation as the degradation initiation site. We have characterized the nature of
unstructured initiation region for efficient degradation. These results indicate that the property of
the unstructured initiation site determines the protein’s fate (Inobe, T. and Matouschek, A. Curr.
Opin. Struct. Biol. 2014). Here, we revealed new cellular strategies to regulating proteasomemediated protein degradation by regulating the unstructured initiation site of the substrate protein.

Results
First, we investigated whether degradation of the substrate protein could be inhibited by small
molecules that bind to the unstructured initiation site of the substrate. To achieve this, weconstructed model substrates whose unstructured initiation site contains a tetracysteine motif,
CCPGCC (TC-motif ). The tetracysteine motif is specifically recognized by the biarsenicalfluorescent reagents, FlAsH or ReAsH. This model substrate could escape degradation in thepresence of FlAsH/ReAsH, whereas the unstructured initiation site without FlAsH/ReAsH wasrecognized and degraded by the proteasome. The degradation of the tetracysteine substrate is also
inhibited by FlAsH/ReAsH in the cultured cell HEK293T. We further tested whether this method isapplicable in vivo. We constructed the engineered yeast cells by introducing the plasmid expressing
the TC-motif containing model substrate constructed from the gene product of URA3 into the yeast
lacking URA3 gene and cultivated them in SC media lacking uracil in the presence or absence of
ReAsH. In the presence of ReAsH, we observed the growth of the engineered yeast cells, indicatingthat ReAsH binding to the unstructured region of the model substrate inhibits the degradationof the model substrate, increases its concentration inside the cells and promotes the growth of- 65 -

the engineered yeast cells. In addition to the artificial tetracysteine model substrate, inhibitionof the proteasomal degradation by the unstructured region ligand is observed in intrinsicallydisordered proteins (IDPs). The model substrate containing an IDP protein, mutant staphylococal
nuclease (SNase), as an unstructured initiation site was degraded by the proteasome, but it escaped
degradation when mutant SNase moiety bind to a nucleotide inhibitor, prAp. This result indicatesthat the proteasomal degradation of many other IDPs must be regulated by their binding partners.
Next, we developed adapter proteins that deliver the protein containing the effective unstructured
initiation site to the proteasome. Our previous work showed that unstructured initiation siteand polyubiquitin chain can be separated into two different subunits that form a larger protein
complex, indicating that subunit with polyubiquitin chain can work as a adapter proteins for the
degradation of the subunit with unstructured region (Prakash, S. and Inobe, T. et al Nature Chem.
Biol. 2009). Here, we designed the proteasome adaptor protein for the mutant huntingtin protein(HTT), which contains an abnormally expanded polyQ tract. Adapter proteins were constructedby fusing the polyQ-binding peptide (QBP1), which binds specifically to an expanded polyQ
tract, but not to a short polyQ motif, to proteasome-binding domains such as polyubiquitin (Ub).
Although N-terminally truncated mutant HTT proteins (tNHTT) was barely degraded without theubiquitination, we demonstrated that the Ub-QBP1 adapter selectively delivered non-ubiquitinatedtNHTT to the proteasome and greatly enhanced the degradation of tNHTT by the proteasome in thein vitro degradation assay using purified components. Furthermore, we observed that the expressionof the Ub-QBP1 adapter in Neuro2a cell with an inducible tNHTT-EGFP expression reduced thenumber of nuclear deposits which are mainly composed of aggregation of tNHTT-EGFP relative tothe expression of the adapters lacking either Ub or QBP1 by inducing the proteasome degradation.
Therefore, such clearance by adapter protein could provide treatment options of for PolyQ diseases.

Discussion & Conclusion
Our experiments suggest that degradation of many cellular proteins are either inhibited or induced
by strategies described above. Actually the degradation of the transcription factor Sox4 is inhibitedby interaction with its binding partner Syntenin on the unstructured degron of Sox2 (Beekman, JM.
et al. Oncogene, 2011). On the other hand, it was reported that viral proteins E7 delivers the tumor
suppressor Rb to the proteasome and induce the degradation (Gonzalez, SL et al., J. Virol. 2001).
Therefore, using similar induction and inhibition methods of the proteasome-mediated degradation,
we can artificially control the degradation of specific cellular protein. Such degradation control may
result in the artificial regulation of the cellular concentration of specific proteins.

References
1. Inobe, T. and A. Matouschek (2014). “Paradigms of protein degradation by the proteasome.” Curr
Opin Struct Biol 24C: 156-164.
2. Prakash, S., et al. (2009). “Substrate selection by the proteasome during degradation of protein
complexes.” Nat Chem Biol 5(1): 29-36.

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3. Beekman, J. M., et al. (2011). “Syntenin-mediated regulation of Sox4 proteasomal degradation
modulates transcriptional output.” Oncogene.
4. Gonzalez, S. L., et al. (2001). “Degradation of the retinoblastoma tumor suppressor by the humanpapillomavirus type 16 E7 oncoprotein is important for functional inactivation and is separablefrom proteasomal degradation of E7.” J Virol 75(16): 7583-7591.

一般の皆様へ
我々は細胞内の蛋白質の分解を担うプロテアソームの厳密なターゲット蛋白質認識メカニズムを利用して、蛋白質濃度の人工的な制御方法の開発に取り組んでいる。特にプロテアソーム分解のシグナ
ルとしてポリユビキチン鎖以上の重要な役割を担う変性領域に注目して、分解制御方法の開発を行っている。この方法により細胞内の蛋白質濃度の調整が可能になれば、病気の治療にも貢献できるのではないかと期待している。

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Regulatory mechanism of mucosal healing by DNA methylation
Hiroyuki TEZUKA
Tokyo Medical and Dental University
tezco.bre@mri.tmd.ac.jp

Abstract
In this study, we tried to clarify the mechanism of mucosal healing in colitis. Here we found that
DNA methyltransferase-3a (Dnmt3a) was predominantly expressed in the crypts of the colon understeady-state conditions. During the colonic inflammation, the expression of Dnmt3a was reduced inprogressive phase, and was restored in recovery phase. However, using conditional knockout mice,the deficiency of Dnmt3a in epithelial cells did not affect in the mucosal healing of the colitis. These
results suggested that Dnmt3a is not essential for the mucosal healing.
Key words : DNA methltransferase, Inflammatory bowel disease, mucosal healing

Introduction
Inflammatory bowel disease (IBD) is a chronic relapsing inflammatory disorder, and is classified
into Crohn’s disease (CD) and ulcerative colitis (UC)1. However, the mechanism by which mucosal
healing is induced in recovery phase is unknown. In this context, in has been shown that Dnmts are
susceptible gene loci for the IBD2. In addition, it was recently reported that Dnmt3a is essential forthe differentiation of hematopoietic stem cells3. Based on these findings, we postulate that Dnmt3a
is important for the mucosal healing in the recovery phase. In this study, we tried to clarify the roleof Dnmt3a in the mucosal healing of IBD.

Results
We first examined the expression of Dnmts, including de novo Dnmt3a and Dnmt3b, in theintestine under steady-state conditions. Dnmt3a was predominantly expressed in epithelial cellsof colonic crypts including stem cells. In contrast, Dnmt3b was not expressed in the crypts. To
determine the role of Dnmt3a in the crypts, we applied dextran sodium sulfate (DSS)-induced
colitis, an acute IBD model. C57BL/6J mice were administered 2% DSS for 7 days and then replaced
to water for 5 days. Intestinal epithelial cell-expression of Dnmt3a was much reduced during the
progression of colitis. Interestingly, the expression of Dnmt3a was restored in the recovery phaseand the Dnmt3a+ epithelial cells expressed Ki67, a marker for proliferating cells. In contrast, theexpression of Dnmt3b was not detected in both progressive and recovery phases of the colitis.
To address the role of Dnmt3a in the intestinal homeostasis, we generated conditional knockout
mice with Villin-Cre-mediated epithelial-specific deletion of Dnmt3a (Dnmt3a IECKO) and its
littermate control (Dnmt3aflox/flox) mice. These mice developed normally, and did not show any signof disease. The frequency of Treg, Th17, and IgA+ plasma cells in the intestinal lamina propria wasunaffected in Dnmt3aIECKO mice under steady-state conditions. Consistent with these results, the- 68 -

level of IgA in the sera and intestinal contents was unaffected in Dnmt3aIECKO mice. In addition,
the frequency of myeloid cells including dendritic cells and macrophages in the intestine was also
normal.
To determine whether Dnmt3a is essential for the recovery phase of colitis, we used DSS to induce
acute colitis in Dnmt3aIECKO and Dnmt3aflox/flox mice. In the progressive phase of the colitis, there
were no differences in the diarrhea, rectal bleeding, shortened colon length, reduced body weight,
and infiltration of myeloid cells between these mice. Unfortunately, in the recovery phase, there
were no differences in remodeling of epithelial layer between these mice. The expression of Dnmt3b
was not induced in colitic Dnmt3aIECKO mice. Similar results were also obtained in Dnmt3bIECKO
mice.

Discussion & Conclusion
DNA methylation is catalyzed by three active forms of Dnmt, namely Dnmt1, Dnmt3a, andDnmt3b1. Of these, Dnmt3a and Dnmt3b are responsible for establishment of new methylation
pattern in genomic DNA1. In this context, we found that Dnmt3a is predominantly expressed in
the crypts, suggesting that Dnmt3a is involved in the generation and differentiation of epithelial
cells. However, the mucosal healing was unaffected in deficiency of Dnmt3a in epithelial cells,
and epithelial cells-expression of Dnmt3b was not induced during the colitis in Dnmt3aIECKO mice.
These results suggested that Dnmt3a is dispensable for the mucosal healing and Dnmt3b do notcompensate functionally for Dnmt3a. In this regard, Dnmt3a and Dnmt3b are important risk fociassociated with CD and UC, respectively. Because DSS-induced colitis is a model resembling UC,
we could not find any differences in Dnmt3aIECKO mice. To clear this problem, we are planning to do
an experiment using a model resembling CD (e.g. T cell-transfer model).

References
1 Cho, J.H., The genetics and immunopathogenesis of inflammatory bowel disease. Nature Review
Immunology 8 : 458-466 (2008).
2 Low, D., et al., DNA methylation in inflammatory bowel disease and beyond. World Journal of
Gastroenterology 19 : 5238-5249 (2013).
3 Challen, G.A., et al., Dnmt3a is essential for hematopoietic stem cell differentiation. Nature
Genetics 44 : 23-31 (2011).

一般の皆様へ
本研究課題では、炎症性腸疾患 ( クローン病や潰瘍性大腸炎 ) の寛解期における粘膜治癒機構を明らかにすることを目的とする。この目的のため、申請者は同疾患の感受性遺伝子の一つである
DNA メチル化酵素 Dnmt3a に着目した。その結果、Dnmt3a はマウス大腸陰窩で恒常的に発現していることや、同発現は薬剤誘導性大腸炎の病態形成期では減少し、寛解期では回復することを見出した。これらの結果から、Dnmt3a は粘膜治癒に重要であると考え、上皮細胞のみ Dnmt3a を欠損するマウスに大腸炎を誘導した。その結果、Dnmt3a は粘膜治癒に必須ではないことが判明した。
今後は、上記とは異なる腸炎モデルを用いて検討する予定である。

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Actin Cytoskeleton Structure Regulation by TRIOBP,
a novel gene responsible to human deafness
Shin-ichiro Kitajiri
Department of Otolaryngology, Kyoto University Hospitalkitajiri@ent.kuhp.kyoto-u.ac.jp

Abstract
We performed functional analyses on each isoform of TRIOBP, a novel actin-bundling protein anda responsible gene of hereditary hearing loss in humans. Our findings included the identificationof sites associated with actin bundling, TRIOBP oligomer formation, isoform-specific functions,and intracellular protein-protein interactions, as well as showing the effect of TRIOBP on receptor
localization in stereocilia.
Key words : hearing loss, stereocilia, actin

Introduction
We identified TRIOBP mutations based on human hearing loss genealogy. TRIOBP is a novel
actin-bundling protein that is localized to the rootlets of hair cell stereocilia. Of the three TRIOBP
isoforms (T1/4/5), T4 and/or T5 are important for hearing, as knockout (KO) of either gene in miceleads to a failure in rootlet formation, resulting in deafness. But the function of each isoform andthe domains they use to bundle actin are unknown. Thus, in this study, we aimed to address thesequestions.

Results(1) Solubilization conditions analysis using a fluorescence size exclusion chromatography
We successfully solubilized each TRIOBP
isoform with some detergents. In addition,
we found that T1, T4, and T5 formed homoor hetero- oligomers under the solubilization
conditions. (Right Fig.)(2) Biochemical analysis of the interactions between each isoform/fragment and actin
We sought to identify the domain related to bundling in T4. We found that two specific repetitive
T4 sequences (R1 and R2) had actin-bundling ability. Further, we showed that each sequence binds
to actin in a different manner (ionic and hydrophobic binding).
(3) Generation and analysis of isoform-specific KO mice
We generated and then analyzed T1 and T5 isoform-specific KO mice. The T5-specific KO
mouse has been shown to have severe hearing loss with rootlets hypoplasia. We also generated thetransgenic mice expressing a GFP fusion T4. In vivo localization and turnover analyses were madepossible by using the mice.

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(4) Gene transfer analysis in an inner ear organ culture system
We have succeeded in producing adenovirus vectors to transfer T5 for rescue assay.
(5) Identification of molecules that cooperate with TRIOBPT1 directly interacts with the GEF molecule “Trio (340 kDa)”, which controls actin dynamics.
Thus, a high probability exists that T1 forms complexes with other molecules in the inner ear. To
test this, immunoprecipitation was performed on inner ear tissue, using a T1 antibody. Various
actin-binding proteins, cytoskeleton-associated molecules, and second messengers were identified.
We also showed that the proteins that were normally localized to the stereocilia tips became
localized basally within the stereocilia of the inner ear in T4/T5dKO mice.

Discussion & Conclusion(1) TRIOBP is thought to form homo- or hetero-oligomer to bundle actin, as well as to form andmaintain the rootlets.
(2) We predict that the mechanism is as follows: new actin is captured by ionic binding of R1,
thickening the bundle, and then the each actin within the bundle is strengthened by hydrophobic
binding of R2.
(3) T5-specific knockout mice did not exhibit deafness, but did display severe hearing loss.
Phenotypic analysis and comparison of T4/5 knockout mice will clarify the functional differences
and interactions between T5 and T4.
(4) Gene transfer into inner ear hair cells is known to be difficult, but is feasible and highly efficientby using the adenoviral vectors generated in the present study. This will facilitate future
cytological analyses of inner ear hair cells.
(5) In T4/5 knockout mice, we not only found molecules that bind directly to TRIOBP, but alsochanges in the distribution of proteins that localize in the stereocilia. This shows the potential of
TRIOBP as a gatekeeper in selecting the molecules that localize to stereocilia tips.

References
1. Kumeta, M. ;Gilmore, J. L. ;Umeshima, H. ;Ishikawa, M. ;Kitajiri, S. ;Horigome, T. ;Kengaku,
M. ;Takeyasu, K, Caprice/MISP is a novel F-actin bundling protein critical for actin-based
cytoskeletal reorganizations, Genes Cells. 2014 Apr;19(4):338-49
2. Bao J, Bielski E, Bachhawat A, Taha D, Gunther LK, Thirumurugan K, Sakamoto T, Kitajiri S,
R1 motif is the major actin-binding domain of TRIOBP-4, Biochemistry. 2013 Aug 6;52(31):525664.
3. Tomohito Higashi, Shinsaku Tokuda, Shin-ichiro Kitajiri, Sayuri Masuda, Hiroki Nakamura,
Yukako Oda, Mikio Furuse, Analysis of the ‘angulin’ proteins LSR, ILDR1 and ILDR2-tricellulin recruitment, epithelial barrier function and implication in deafness pathogenesis J Cell
Sci. 2013 Feb 15;126(Pt 4):966-977.

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一般の皆様へ
難聴の有病率は約 20％で、もっとも頻度の高い身体障害である。しかし聴覚受容の分子メカニズ
ムには未だ不明な点が多い。内耳で音を感知するのは不動毛とよばれる構造で、これには細胞質へ伸びるアクチン束の「根」が存在する。われわれは、ヒト遺伝性難聴の原因遺伝子であり、アクチンを束ねて根を形成する (Trio Binding Protein) TRIOBP という分子を同定し、その機能解析を進めている。ノックアウトマウスを使った解析から TRIOBP は根の形成に必須で、無くなると毛の剛性が低くなり、聾や難聴になることが分かった。現在、引き続きその詳細なメカニズムを解析している。内耳障害の多くは不動毛の変性により始まると考えられており、その変性を防ぐことは、難聴の治療、予防に繋がると期待できる。

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Study of chromosome instability caused by
the impairment of centrosome maturation
Yuko Ozaki
Research Institute for Radiation Biology & Medicine, Hiroshima University
yuko-ozaki@hiroshima-u.ac.jp

Abstract
We isolated a gene encoding a centrosome protein, Miki (mitotic kinetics regulator) from 7q21.3
sub-band as a candidate responsible gene for monosomy 7, which is frequently detected in myeloid
leukemia. Miki localizes to the Golgi apparatus and, in late G2 to prophase, Miki is poly(ADPribosyl)ated by tankyrase-1. PARsylated Miki then translocated to mitotic centrosomes and plays apivotal role in centrosome maturation. Miki-downregulation in cancer cells by Miki-specific RNAi
inhibited centrosome maturation resulting in severe chromosome misalignment and prometaphasearrest. As a result, these mitotic impairment induced abnormal nuclear morphology such as bi-,tri- or multiple-nuclei with or without small nuclei and resulted in a wide variation of chromosome
number.
Key words : Chromosome instability / Leukemia / Centrosome maturation

Introduction
We previously identified candidate myeloid tumor suppressor genes (SAMD9, SAMD9L, and Miki)in the 7q21.3 sub-band1). We found that haploinsufficiency of SAMD9L and/or SAMD9 gene(s)contributes to myeloid transformation 2) and Miki plays a pivotal role in centrosome maturation3).
Miki localizes to the Golgi apparatus and, in late G2 to prophase, Miki is poly(ADP-ribosyl)atedby tankyrase-1 and then translocated to mitotic centrosomes. Miki-specific RNAi-treated cells
induced severe chromosome misalignment, which resulted in subsequent initiation of apoptosis andthe accumulation of cells containing abnormal nuclear morphology resulting in a wide variationof chromosome number. Here we demonstrate that low levels of Miki induced abnormal mitosis
directly cause chromosome instability that could determine the fate of cancer patients.

Results
We previously selected two short interfering (si) RNAs (siRNA#79 and #80 that effectively
downregulate Miki transcripts in HeLa cells. Upon treatment of HeLa cells with siRNA#80(100nM) for 48 hours, we frequently observed severe chromosome misalignment such as widely andirregularly scattered chromosomes and prometaphase arrest.
To investigate mechanism(s) through which Miki downregulation causes these mitotic impairment,
we monitored the movement of lagging chromosomes in pseudometaphase relative to those innormal late prometaphase. In control siRNA-treated U2OS cells expressing H2B-GFP, all lagging- 73 -

chromosomes in late prometaphase moved toward the metaphase plate at a velocity greaterthan 1 μm/min, typically 2 to 5 μm/min. In contrast, nearly half of the lagging chromosomes in
pseudometaphase cells resulting from siRNA#80 treatment moved less than 1 μm/min during
the 15 minutes observation time. Chromosomes may move slowly due to insufficient microtubuletension at kinetochores caused by unfixed spindle poles resulting in loss of robustness of mitotic
microtubules.
These findings suggest that Miki downregulation impairs critical centrosome function(s) suchas microtubule nucleation. As demonstrated previously by others 4), accumulation of the EB1
microtubule tip-binding protein at centrosomes indicates active microtubule nucleation duringmitosis. Treatment of HeLa cells with siRNA#80 induced mitotic cells with a pseudometaphase
phenotype with markedly reduced centrosomal EB1 signals.
Reduced microtubule nucleation in response to Miki downregulation was also demonstrated in
microtubule regrowth assays. Mitotic spindles degraded completely by simultaneous treatment ofcells with nocodazole and cold. The cells were then cultured in nocodazole-free medium at 37℃ .
Within five minutes, control siRNA-treated mitotic cells showed small asters (< 5 μm) forming at
centrosomes, then, eight minutes later, large asters (> 5 μm) were observed in 50% of mitotic cells,
and numbers increased to nearly 100% within 15 minutes. In contrast, Miki-specific siRNA-treatedmitotic cells had weaker γ-tubulin signals in centrosomes than cells in interphase, frequently formed
multiple small asters, and only 16% of mitotic cells showed large asters after 15 minutes.
The above findings imply that Miki downregulation might impair the γ-tubulin ring complex(γ-TuRC) localizing to the pericentriolar matrix (PCM) that scaffolds microtubule nucleation sites
at mitotic centrosomes. Major components of γ-TuRC accumulate in mitotic centrosomes during
the short period from late G2 to prometaphase known as ‘centrosome maturation’. For example,
the intensity of γ-tubulin immunostaining in prometaphase centrosomes of HeLa cells treatedwith control siRNA increased several-fold relative to those at interphase. Similarly, other major
components of γ-TuRC such as GCP2, Kendrin/Pericentrin and CG-NAP/AKAP450 accumulated
in mitotic centrosomes.5) These results suggested that insufficient centrosome maturation andmicrotubule nucleation by downregulation of Miki induced mitotic impairment such as severe
chromosome misalignment and prometaphase arrest.
As a results, these mitotic impairment induced abnormal nuclear morphology such as bi-, tri- or
multiple-nuclei with or without small nuclei and resulted in a wide variation of chromosome number.
Cell lines harboring monosomy 7 such as F-36P and MDS-L expressed Miki at barely detectablelevels. Giemsa staining of these cells showed abnormal mitosis and nuclear abnormalities similar
to those in HeLa cells treated with siRNA#80. Conversely, Miki protein levels were increased inthese cells using a Miki-expressing pantropic retrovirus (MSCV-Miki/ires-CD8). Immunostaining
revealed virtually no Miki signal during mitosis in uninfected or control virus-infected cells. Using
immunostaining, Miki was detected at the spindles in 3 to 10% of mitotic cells, and chromosomealignment also recovered in Miki-positive cells. In addition, multiple small nuclei as a result of
PCDC (premature chromosome decondensation : chromosomes decondense before segregation- 74 -

that results in the formation of small nuclei) were observed in the bone marrow pictures of myeloid
leukemia patients associated with monosomy 7 at high frequency. These results suggested thatchromosomal instability due to Miki-downregulation causes aneuploidy that subsequently induces
progression of myeloid leukemia.

Discussion & Conclusion
Chromosome scattering and pseudometaphase phenotypes are seen routinely in certain cancer
cells, but the molecular mechanisms underlying these abnormalities are largely unknown. As wepreviously reported (Asou et al., 2009), low Miki expression levels in MDS cells relate to so-called
colchicine mitosis (Chromosome scattering and pseudometaphase) and subsequent abnormal nuclear
morphology (bi-, tri- or multiple-nuclei with micronuclei) that characterize MDS. Intriguingly, theCG-NAP gene (1.2 Mb centromeric to Miki in band 7q21) also maps to a region frequently deleted
in MDS; approximately 20% of MDS patients lose one allele of both Miki and CG-NAP genes. This
indicates that loss of chromosome arm 7q results in low expression of two crucial factors in thetankyrase-1/Miki-dependent system for centrosome maturation, and that this may profoundly impair
progression of prometaphase. Detailed mechanisms through which impairment of the tankyrase-1/
Miki-dependent system perturbs cancer cell mitosis are now under investigation in our laboratory,
because abnormal mitosis directly causes chromosome instability that could determine the fate ofcancer patients.

References
1 Asou, H., Matsui, H., Ozaki, Y., Nagamachi, A., Nakamura, M., Aki, D. & Inaba T. :Identification of a common microdeletion cluster in 7q21.3 subband among patients with myeloid
leukemia and myelodysplastic syndrome, Biochem.Biophys.Res.Commun., 383:245-251, 2009.
2 Nagamachi,A., Matsui, H., Asou, H., Ozaki, Y., Aki, D., Kanai, A., Takubo, K., Suda, T.,
Nakamura, T., Wolff, L., Honda, H. & Inaba, T. : Haploinsufficiency of SAMD9L, an Endosome
Fusion Facilitator, Causes Myeloid Malignancies in Mice Mimicking Human Diseases with
Monosomy 7. Cancer Cell, 24:305–317, 2013
3 Ozaki, Y., Matsui, H., Asou, H., Nagamachi, A., Aki, D., Honda, H., Yasunaga, S., Takihara, Y.,
Yamamoto, T., Izumi, S., Ohsugi, M. & Inaba T. : Poly-ADP ribosylation of Miki by tankyrase-1
promotes centrosome maturation. Molecular Cell, 47:694-706, 2012.
4 Vaughan, K.T. : TIP maker and TIP marker; EB1 as a master controller of microtubule plus ends.
J Cell Biol, 171:197-200, 2005.
5 Takahashi, M., Yamagiwa, A., Nishimura, T., Mukai, H. & Yoshitaka Ono. : Centrosomal
proteins CG-NAP and kendrin provide microtubule nucleation sites by anchoring gamma-tubulin
ring complex, Molecular biology of the cell, 13:3235-3245, 2002.

一般の皆様へ
多くのがんにおいて、染色体の数の異常、欠失など様々な染色体の構造異常が知られていたが、近年の技術の進歩により、白血病だけではなく、多くの固形腫瘍でも染色体の数や構造の異常が発見された。これを称して「染色体不安定性」と呼ぶが、今ではそれががんの代名詞にまでなっており、発がんの原因として重要であると考えられている。本研究は、がん細胞の染色体不安定性メカニズムを明らかにし、がんの新規治療法を開発することを最終目標とした基礎医学研究である。

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Elucidation of the mechanisms by which impaired clearance of
apoptotic cells results in severe inflammation
Hiroyasu Nakano
Juntendo University Graduate School of Medicine
hiroyasu.nakano@med.toho-u.ac.jp

Abstract
Using a murine model, in which Kupffer cells or infiltrated monocytes were depleted, we foundthat monocytes, but not Kupffer cells, play a crucial role in suppression of inflammation after acuteliver injury. Moreover, taken that depletion of monocytes enhanced inflammation associated withcell death, our model might be useful to amplify and identify signals released from dying cells thatare involved in inflammation.
Key words : Apoptosis, monocytes, TNFα, inflammation

Introduction
Dying cells are rapidly engulfed by phagocytes to maintain tissue homeostasis. Impairmentof elimination of dead cells might elicit persistent release of cytoplasmic contents and nuclear
DNAs from dead cells, resulting in aberrant inflammation. Several lines of evidence strongly
suggest that impairment of dying cells results in the development of autoimmune diseases. Kupffer
cells are dominant phagocytes in the liver and considered to play a crucial role in elimination of
microorganisms delivered from intestines. However, it is unclear whether Kupffer cells play a majorrole in eliminating dying hepatocytes after liver injury.

Results
We found that hepatocyte-specific cFlip-deficient (cFlipF/F;Alb-Cre) mice were highly susceptible
to TNFα-induced hepatitis and completely recovered from hepatitis at 24 hours after injection. UsingcFlipF/F;Alb-Cre mice, we first tested whether Kupffer cells plays a crucial role in elimination of dying
hepatocytes after acute liver injury. Notably, depletion of Kupffer cells with clodronate liposomes
did not impair elimination of dying hepatocytes, or enhance inflammation after TNFα injection.
To deplete infiltrating monocytes and granulocytes from peripheral blood, we next reconstituted
cFlipF/F;Alb-Cre mice with bone marrow (BM) cells from human diphtheria toxin receptor (hDTR)
transgenic mice, in which expression of hDTR gene was controlled by the lysozyme M (LysM) genepromoter. Since murine cells were not susceptible to DT-induced cytotoxicity, only hDTR-positive
monocytes and granulocytes were eliminated after DT injection. We found that TNFα-induced
hepatitis was significantly exacerbated in DT-treated cFlipF/F;Alb-Cre mice reconstituted with
hDTR-positive BM cells, and the mice succumbed within 2-3 hours after injection. Moreover, serum
concentrations of TNFα and interleukin-6 were significantly elevated in these mice, suggesting- 76 -

that signals released from dying cells were significantly amplified, and hepatitis and inflammation
were exacerbated. Furthermore, combined depletion of Kupffer cells and monocytes along with
granulocytes did not suppress TNFα-induced IL-6 production, suggesting that danger-associatedmolecular patterns (DAMPs) released from dying hepatocytes might act on cells other than Kupffer
cells to produce large amounts of IL-6.

Discussion & Conclusion
In the present study, we showed that cFlipF/F;Alb-Cre mice reconstituted with bone marrow cells of
hDTR transgenic mice revealed an unexpected role for monocytes in suppression of inflammation
after acute liver injury. Moreover, Kupffer cells do not play a major role in elimination of dying
hepatocytes, or respond to DAMPs after acute liver injury. Furthermore, taken that depletion of
infiltrated monocytes enhanced inflammation after acute liver injury, our model might be useful toamplify and identify signals released from dying cells that are involved in inflammation.

References
1. Piao, X., S. Komazawa-Sakon, T. Nishina, M. Koike, J.H. Piao, H. Ehlken, H. Kurihara,
M. Hara, N. Van Rooijen, G. Schutz, M. Ohmuraya, Y. Uchiyama, H. Yagita, K. Okumura, Y.W.
He, and H. Nakano. 2012. c-FLIP Maintains Tissue Homeostasis by Preventing Apoptosis andProgrammed Necrosis. Sci Signal 5:ra93.
2. Shindo, R., H. Kakehashi, K. Okumura, Y. Kumagai, and H. Nakano. 2013. Critical contributionof oxidative stress to TNFalpha-induced necroptosis downstream of RIPK1 activation. Biochem
Biophys Res Commun 436:212-216.

一般の皆様へ肝臓でのアポトーシス亢進マウスを用いた解析から、死んだ肝細胞の除去や細胞死後の炎症の収束には、肝臓に存在する主な食細胞と考えられてきたクッパー細胞は必須でないことが明らかとなった。また、骨髄由来の単球や好中球を薬剤投与により選択的に除去できるマウスの骨髄を移入した実験から、肝炎に伴う炎症の収束にはこれらの細胞が重要な役割を果たしている事を明らかにした。
現在，そのメカニズムの解析を行っているところであるが、我々のモデルは肝死細胞から放出されるシグナルを増幅させることができる事から、このマウスを用いることにより、これまで同定の困難であった死細胞放出因子を同定する上で有用なモデルである可能性が示された。

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Molecular mechanisms for the production and maintenance ofproliferative cells in the subventricular zone.
Hidenori Tabata
Department of Molecular Neurobiology, Institute for Developmental Research,
Aichi Human Service Centertabata@inst-hsc.jp

Abstract
Humans have acquired huge brains during evolution and that is attribute to expansion of asecondary proliferative zone, subventricular zone (SVZ) in fetal stages. In mice, the productionof proliferative cells in the SVZ from the lateral ventricular zone (VZ) is relatively high to thedorsomedial VZ. To identify genes responsible for primate cortical expansion, we compared theexpression profiles between the lateral and dorosomedial VZ in mice, and identified 29 lateral highand 9 medial high genes. We then performed bioinformatics evolutionary analyses between primatesand non-primates, and found the significantly weaker negative selection pressures on 3 genes, andstronger one on 3 genes in primate lineages than in non-primate lineages.
Key words : neural stem cells, human evolution, notch signaling, selection bias,
subventricular zone (SVZ)

Introduction
During neocortical development, excitatory neurons are produced from apical progenitors in theventricular zone (VZ) or from basal progenitors in the subventricular zone (SVZ). In primates
including humans, the number of basal progenitors dramatically increased, and this is thought tounderlie the acquisition of a huge cortex during evolution (Smart et al., 2002; Fietz et al., 2010;
Hansen et al., 2010). We previously reported that basal progenitor production rate against postmitotic
neurons from the lateral cortical VZ is higher than that in the dorsomedial VZ in mice (Tabata et al.,2009; 2012). Taking advantage of this difference, we conducted screening for molecules regulating
the basal progenitor production rate, and estimated the selection pressures during primate evolution
on candidate genes (Tabata et al., 2013).

Results
We have observed the migratory difference between direct progenies of the VZ cells andproliferative cells within the SVZ. The former finishes their final division in the VZ and stays there
for about 10 hours, and then, they transform into multipolar cells and accumulate just above the VZ.
On the other hand, the proliferative cells in the SVZ exit the VZ earlier than the former with somaltranslocation mode (Tabata et al., 2009). They differ the timing of VZ exit, we therefore called
these population slowly exiting population (SEP) and rapidly exiting population (REP), respectively.

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Because the ratio of the production of REP to that of SEP from the VZ is high in the lateral VZ and
low in the dorsomedial VZ (Tabata et al., 2009), we compared the gene expression profiles between
the lateral and dorsomedial VZ to screen for candidate genes regulating the REP/SEP production
ratio in mouse cortex. We first introduced a GFP expression vector into the lateral or dorsomedial
VZ at embryonic day (E) 15, and GFP-positive cells were collected by FACS 18 h later. We extracted
RNA from the GFP-positive cells and performed transcriptome analyses using DNA microarrays.
As a result, we identified 396 and 80 probe sets with high lateral and high dorsomedial expression
patterns, respectively. We then obtained the gene ontologies (GOs) for the probe sets and narrowed
down the candidates to those with a GO related to development or the cell cycle. We also selectedtranscription factors to obtain potential key factors for the REP/SEP production ratio. Using theseprocedures, we identified 62 and 23 genes with higher expressions in the lateral and dorsomedial
VZ regions, respectively. Next, we examined the expression profiles of these 85 candidate genes
using an in-situ hybridization database, the Brain Gene Expression Map (Magdaleno et al., 2006), or
by performing in situ hybridization experiment. Overall, we identified 29 and 9 genes expressed ina lateral-high to medial-low or a medial-high to lateral-low gradient pattern in the VZ, respectively.
We then performed bioinformatics evolutionary analyses for these 38 genes. The evolutionarypressures on the candidate genes were estimated using evolutionary models, which are based on thesynonymous and non-synonymous base substitution rates (dN/dS) of the coding DNA sequences. To
estimate the dN/dS ratios, we first investigated the one-to-one correspondence of candidate genes
among 13 mammalian species and 1 reptile, the Anole lizard. We excluded 3 candidate genes thatwere found to have undergone gene loss or gain events when mice and humans were compared.
To figure out evolutionary pressures of the remaining 35 candidate genes, we applied the “two-ratio”
evolutionary model to the candidate genes. The likelihood ratio test, which compares the “one-ratio”
and “two-ratio” evolutionary models, suggested that the dN/dS ratios of 3 genes (Jag1, Ntrk2, andPmp22) were significantly lower in primate lineages than in non-primate lineages (P < 0.05). These
results indicated that the negative selection for Jag1, Ntrk2, and Pmp22 in the primate lineages wasstronger than that in the non-primate lineages, implying that the molecular functions of these three
genes are likely to be more essential in primates than in non-primates. In contrast, the dN/dS ratiosof 3 other genes (Megf11, Dmrt3, and Cntn3) were significantly higher in the primate lineages,
compared with those in the non-primate lineages (P < 0.05).
Next, we performed additional evolutionary analyses for Megf11, Dmrt3, and Cntn3. dN/dS ratios
higher than 1.0 are indicative of adaptive molecular evolution (Hughes and Nei, 1988; Messierand Stewart, 1997; Zhang, 2003). Although the dN/dS ratios averaged over the whole amino acidresidues of these 3 gene products were less than 1.0, the branch-site models for Megf11 suggested
that 7 residues were under positive selection in primate lineages. Arg630 is harbored in the laminintype EGF-like domain, which mediates cell adhesion, growth, migration and differentiation. These
results suggested that Megf11 underwent adaptive molecular evolution in primate lineages.

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Discussion & Conclusion
In this study, we screened for genes that were expressed in a lateral-medial gradient pattern inthe mouse VZ to identify candidate molecules that were potentially involved in the expansion of
the cerebral cortex during primate evolution. We then further performed detailed evolutionary
analyses of the resulting candidate genes. As a result, significant changes in evolutionary pressure
between primate and non-primate lineages were found for 6 genes. These genes are known/suggested to be involved in cell proliferation, differentiation, and adhesion and might affect the
REP/SEP production ratio in mice. The significance of Notch signaling in the proliferation of bRG
cells has been previously reported (Hansen et al., 2010). Jag1 is a conventional ligand of Notch
receptors, and therefore an important candidate that might have evolved to increase the REP/SEP
production ratio in primates. Megf11 is a member of the MEGF family. Although MEGF family
proteins were initially shown to mediate engulfment as a binding protein for cell corpses (Zhou etal., 2001; Wu et al., 2009), a later study demonstrated that MEGF family proteins also act as a Notch
signaling modifier (Krivtsov et al., 2007). The role of the genes identified here in mice and whether
evolutionary changes in these genes were involved in the expansion of the OSVZ in primates should
be clarified in future studies.

References
1. Fietz SA, Kelava I, Vogt J, Wilsch-Bräuninger M, Stenzel D, Fish JL, Corbeil D, Riehn A,
Distler W, Nitsch R, Huttner WB (2010) OSVZ progenitors of human and ferret neocortex are
epithelial-like and expand by integrin signaling. Nat Neurosci 13:690–699.
2. Hansen DV, Lui JH, Parker PRL, Kriegstein AR (2010) Neurogenic radial glia in the outersubventricular zone of human neocortex. Nature 464:554–561.
3. Hughes AL, Nei M (1988) Pattern of nucleotide substitution at major histocompatibility complex
class I loci reveals overdominant selection. Nature 335:167–170.
4. Krivtsov AV, Rozov FN, Zinovyeva MV, Hendrikx PJ, Jiang Y, Visser JWM, Belyavsky AV(2007) Jedi—a novel transmembrane protein expressed in early hematopoietic cells. J Cell
Biochem 101:767–784.
5. Magdaleno S, Jensen P, Brumwell CL, Seal A, Lehman K, Asbury A, Cheung T, Cornelius T,
Batten DM, Eden C, Norland SM, Rice DS, Dosooye N, Shakya S, Mehta P, Curran T (2006)
BGEM: an in situ hybridization database of gene expression in the embryonic and adult mousenervous system. PLoS Biol 4:e86.
6. Messier W, Stewart CB (1997) Episodic adaptive evolution of primate lysozymes. Nature
385:151–154.
7. Smart IHM, Dehay C, Giroud P, Berland M, Kennedy H (2002) Unique morphological featuresof the proliferative zones and postmitotic compartments of the neural epithelium giving rise tostriate and extrastriate cortex in the monkey. Cereb Cortex 12:37–53.
8. Tabata H, Hachiya T, Nagata K-I, Sakakibara Y, Nakajima K (2013) Screening for candidate
genes involved in the production of mouse subventricular zone proliferative cells and an
estimation of their changes in evolutionary pressure during primate evolution. Frontiers in
Neuroanatomy 7.
9. Tabata H, Kanatani S, Nakajima K (2009) Differences of migratory behavior between directprogeny of apical progenitors and basal progenitors in the developing cerebral cortex. Cereb
Cortex 19:2092–2105.
10. Tabata H, Yoshinaga S, Nakajima K (2012) Cytoarchitecture of mouse and human subventricularzone in developing cerebral neocortex. Exp Brain Res 216:161–168.

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11. Wu H-H, Bellmunt E, Scheib JL, Venegas V, Burkert C, Reichardt LF, Zhou Z, Fariñas I, Carter
BD (2009) Glial precursors clear sensory neuron corpses during development via Jedi-1, an
engulfment receptor. Nat Neurosci 12:1534–1541.
12. Zhang J (2003) Evolution of the human ASPM gene, a major determinant of brain size. Genetics
165:2063–2070.
13. Zhou Z, Hartwieg E, Horvitz HR (2001) CED-1 Is a Transmembrane Receptor that Mediates
Cell Corpse Engulfment in C. elegans. Cell 104:43–56.

一般の皆様へ
哺乳類における大脳皮質神経細胞は脳室帯、および脳室下帯で産生されるが、霊長類では脳室下帯が著しく発達し、膨大な数の神経細胞が生み出される。マウスにも脳室下帯での神経細胞産生が少ないながら認められ、大脳外側では内側に比して多い。マウス胎仔脳におけるこのような偏りが生じるメカニズムを解明することで、ヒトが進化過程で巨大脳を獲得した理由に迫れる可能性がある。
本研究課題でいくつかの有望な候補分子を得た。今後、これらの遺伝子の機能解析を通じて、ヒトの高次脳機能の源泉を探り、さらにはその機能不全による精神疾患の理解につなげたい。

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Role of the VIPLa-mediated glycoprotein trafficking
in locomotion in zebrafish
Kazuhide Asakawa
National Institute of Genetics
kasakawa@nig.ac.jp

Abstract
We identified a zebrafish VIPL mutant that displayed defective locomotion. By using calcium
imaging, we found that the Mauthner cell, a brainstem reticulospinal neuron that triggers theescape behavior, was not activated in response to touch sensation in the VIPL mutant animals. Our
results suggest that VIPL is necessary for the activation of the brainstem reticulospinal neuron invertebrates.
Key words : VIPL, calcium imaging, reticulospinal neuron, Mauthner cell, locomotion, zebrafish

Introduction
Brainstem reticulospinal neurons (RSNs) serve as the major descending motor control system invertebrates. However, the cellular and molecular mechanisms of brainstem-spinal cord locomotor
circuit formation have remained largely elusive. In the present study, we studied the function ofzebrafish VIPLa, which is a member of the evolutionarily conserved L-type lectin, in locomotion.

Results
In order to identify molecules that are involved in formation of locomotor circuit, we performeda transposon-based genetic screen for zebrafish mutants that displayed an altered locomotor
pattern. We identified a recessive mutant that failed to initiate the escape swimming in the larvalstages. Molecular characterization of the transposon insertion revealed that the locomotor deficit
was caused by the disruption of VIPLa/lman2la (VIPLa, hereafter) gene, which encodes an
evolutionarily conserved L-type lectin. By performing immunofluorescence analysis, we foundthat VIPLa protein is expressed in the whole body and, at subcellular level, localized strongly
to the Golgi-apparatus and weakly to the cytoplasm, consistent with the role of human VIPL inglycoprotein-trafficking in cultured cells (Neve, 2003).
To understand the neural basis of the locomotor deficits in the VIPLa mutant, we first investigatedby calcium imaging the function of the Mauthner cell, a bilateral pair of RSNs in zebrafish,
which is activated prior to fast escape response. To label the Mauthner cell with the calcium
indicator GCaMP, we crossed the enhancer trap line for Mauthenr cell (hspGFFDMC130A) with
UAS:GCaMP line and obtained the hspGFFDMC130A;UAS:GCaMP double transgenic animals.
Tactile stimulation was applied in the head region by the piezo-assisted needle, and the GCaMP
fluorescence was monitored before and after tactile stimulation by using the two-photon microscopy.

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From the calcium imaging experiments, we found that, in the VIPLa homozygous larvae, the touchevoked calcium transient was significantly reduced in the Mauthner cell, suggesting that the VIPLa
mutant is defective in the Mauthner cell activation.
The Mauthner cell is one of the direct synaptic targets of the trigeminal sensory (Tg, hereafter)
neurons, which perceive touch sensation in the head and convey sensory information to thecentral nervous system (Kimmel, 1990). Therefore, the defect in Mauthner cell activation could
be caused either by an impaired function of the Tg neuron or a defect in the Mauthner cell itself.
To discriminate these two possibilities, we investigated the activity of Tg neurons in VIPLa
mutants. To monitor the calcium transients in the central axons of Tg neurons with GCaMP, we
generated vglut2a-GFF line, which labels Tg neurons with Gal4. We crossed vglut2a-GFF line with
UAS:GCaMP line and observed GCaMP signal in the central axon of Tg neurons in the vglut2aGFF;UAS:GCaMP double transgenic animals. Upon tactile stimulation in the head, a prominentcalcium transient was evoked in the central axon of Tg neurons at the rhombomere (r, hereafter) 6
level. Therefore, we used the GCaMP fluorescence in the r6 as readout of Tg neuron activity. We
found that an increase of GCaMP signal was observed in r6 in the VIPLa homozygous animals at
a similar level to that observed in VIPLa heterozygous siblings, suggesting that Tg neurons can be
activated in the VIPLa homozygous animals.
Altogether, above results suggest that the locomotor deficit of VIPLa mutants is due to anabnormality in the Mauthner cell itself, rather than in the touch receptor neurons.

Discussion & Conclusion
Our results suggest that VIPLa is necessary for the activation of the Mauthner cell, and imply that
VIPLa-mediated glycoprotein trafficking plays a critical role in activation of RSN in vertebrates.
We have previously shown that the ablation of the Mauthner cell reduces the escape response rate
down to 30 % (escape initiation/stimulation). The fact that the VIPLa mutants did not initiate escapeat all in response to tactile stimulation suggests that the defect is not solely due to the Mauthnercell but also to other RSNs involved in the escape initiation. Calcium imaging of much wider areaof the brainstem will reveal other RSNs affected by the VIPLa mutation. As the touch receptor Tg
neurons use glutamate for neural transmission, our results predict that VIPLa-midiated glycoprotein
trafficking may be involved in glutamatergic neurotransmission and/or the following action potential
generation in the Mauthner cell. Identification of cargo glycolprotein(s) for VIPLa lection will reveal
the molecular role of VIPL in these processes.

References
1. Neve EP, Svensson K, Fuxe J, Pettersson RF. VIPL, a VIP36-like membrane protein with aputative function in the export of glycoproteins from the endoplasmic reticulum. Experimental
Cell Research, 288, 70-83 2003
2. Kimmel CB, Hatta K, Metcalfe WK. Early axonal contacts during development of an identified
dendrite in the brain of the zebrafish. Neuron, 4, 535-545, 1990

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一般の皆様へ
我々は、熱帯魚ゼブラフィッシュをモデルに用いて、身体の動きに異常を示す変異体を探索しました。その結果、魚類からヒトまで保存された VIPLa 遺伝子の機能が異常になった変異体では、網様体脊髄路ニューロン（RS ニューロン）と呼ばれる脳のニューロンの活動が著しく低下し、ゼブラフィ
ッシュが泳げなくなることを発見しました。VIPLa は、糖鎖が付加されたタンパク質を細胞内の適切な場所に輸送する役目を担っています。これらのことから、RS ニューロンの活動に必要であり、尚かつ VIPLa の輸送制御を受ける、未知の糖鎖タンパク質の存在が示唆されます。

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Explorative study on lysine demethylase inhibitors
by rational drug design
Takayoshi Suzuki
Kyoto Prefectural University of Medicine
suzukit@koto.kpu-m.ac.jp

Abstract
We have identified novel lysine-specific demethylase 1 inhibitors and Jumonji C domaincontaining histone demethylase inhibitors by rational drug design. These inhibitors showedcancer cell growth-inhibitory activity, suggesting the possibility of the histone lysine demethylase
inhibitors as anticancer agents.
Key words : Epigenetics, Lysine Demethylase, Inhibitor, Anticancer agent

Introduction
Two classes of lysine demethylases (KDMs) have been identified since 2004. One class includes
lysine-specific demethylase 1 (LSD1, also known as KDM1A) and LSD2 (also known as KDM1B),
which are flavin-dependent amine oxidase domain-containing enzymes. The other class comprises
the recently discovered Jumonji domain-containing protein (JMJD) histone demethylases, which
are Fe(II) and α-ketoglutarate-dependent enzymes. As there is increasing evidence that KDMs areassociated with various disease states, they have emerged as attractive targets for the developmentof new therapeutic drugs. In this work, we attempted to identify KDM inhibitors by rational drug
design and demonstrate their possibility as anticancer agents.

Results
1) Discovery of LSD1-selective inhibitors1,2)
Phenylcyclopropylamine (PCPA) is a well-known irreversible LSD1 inhibitor, but its potency andselectivity versus monoamine oxidases (MAOs; also FAD-dependent enzymes) are inadequate.
We discovered a series of LSD1 inactivators designed on the basis of the concept that LSD1 could
be potently and selectively inactivated by delivering PCPA directly to the enzyme’s active site.
This was achieved by conjugating the PCPA moiety to an LSD1 substrate moiety, which serves asa carrier targeting LSD1. As proof of concept, we initially designed and prepared a peptide, which
utilizes histone H3-21 peptide, a substrate of LSD1, as a carrier for PCPA. This peptide potently
inhibited LSD1. Mechanistic studies by means of kinetic analysis and mass spectrometry showedthat the peptide binds to the LSD1 active site via the H3-21 peptide moiety, and then the enzyme
is inactivated by FAD-PCPA adduct formation (as in the case of PCPA itself), with release of thecarrier peptide. Next, the peptide carrier was replaced with various small-molecular structures.
Among the resulting small molecules, NCD-25 and NCD-38 showed at least 60-fold more potent- 85 -

LSD1-inhibitory activity (comparable to that of the peptide inactivator) than PCPA itself in enzyme
assays, without inhibiting MAOs. While peptide 1a was only weakly active in cell-based assays,
NCD-25 and NCD-38 strongly inhibited growth of HeLa and SH-SY5Y cancer cells.
2) Discovery of JMJD inhibitors3
Several JMJDs such as KDM2, KDM4, KDM5, and KDM7 have been implicated in tumorigenesis.
For example, it has been reported that KDM7B (also known as JHDM1F, PHF8, KIAA1111) is
associated with proliferation of prostate cancer cells and osteosarcoma cells. Therefore, KDM2/7subfamily inhibitors are of interest, both as tools for probing the biological functions of KDM2/7
subfamily and also as candidate anticancer agents. On the basis of the crystal structure of KDM7B,
we designed and prepared a series of hydroxamate analogues bearing an alkyl chain. Enzyme assays
revealed that NCDM-64 potently inhibits KDM2A, KDM7A, and KDM7B, with IC50s of 6.8, 0.2,
and 1.2 μM, respectively. While inhibitors of KDM4s did not show any effect on cancer cells tested,
the KDM2/7-subfamily inhibitor NCDM-64 exerted antiproliferative activity. We also examined
whether NCDM-64 down-regulates the expression of E2F1, whose gene expression has beenreported to be regulated by KDM7B, in HeLa cells by quantitative RT-PCR. As a result, NCDM-64
significantly decreases the mRNA level of E2F1 at 80 μM in which the growth of HeLa cells wasaffected. These data suggest that the KDM7B-mediated regulation of E2F1 gene expression may be
one of the mechanisms of growth regulation in some cancer cells. In addition, we also investigatedthe effect of NCDM-64 on cell cycle progression by FACS analysis. HeLa cells and KYSE150 cells
incubated with 10 or 100 μM of NCDM-64 for 24 h showed a dose-dependent reduction in G2-M
phase, whereas there was a dose-dependent increase in G0-G1 phase. These results revealed that
HeLa cells and KYSE150 cells cultured with compound 9 arrested in the G0/G1 phase of the cell
cycle, which is consistent with the down-regulation of E2F1 by NCDM-64. These data indicate thepotential for KDM2/7 inhibitors as anticancer agents.

Discussion & Conclusion
We have designed and synthesized a series of novel LSD1 inactivators based on our new conceptof LSD1-targeted PCPA delivery. We tested this concept with a peptide and confirmed that it does
indeed inhibit LSD1 selectively and efficiently by delivering PCPA directly to the LSD1 active site.
The enzyme is then inactivated by FAD-PCPA adduct formation, as in the case of PCPA itself, withrelease of the carrier peptide. We also demonstrated that this strategy could be applied to the designof nonpeptide, small-molecule LSD1 inactivators, and identified NCD-25 and NCD-38 that show
potent anticancer effects. We also have identified a novel KDM2/7 subfamily inhibitor NCDM-64
by structure-based drug design, which should be useful as a lead structure in the development of
more potent and selective KDM2/7 inhibitors. Such inhibitors are candidates for anticancer agents
as well as tools for studying the biological roles of KDM2/7 subfamily in cells.

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References
1. “Lysine-Specific Demethylase 1-Selective Inactivators: Protein-Targeted Drug Delivery
Mechanism“ Daisuke Ogasawara, Yukihiro Itoh, Hiroki Tsumoto, Taeko Kakizawa, Koshiki
Mino, Kiyoshi Fukuhara, Hidehiko Nakagawa, Makoto Hasegawa, Ryuzo Sasaki, Tamio
Mizukami, Naoki Miyata, and Takayoshi Suzuki. Angew. Chem. Int. Ed. 2013, 52, 8620–8624.
2. “Synthesis, LSD1 Inhibitory Activity, and LSD1 Binding Model of Optically Pure Lysine-PCPA
Conjugates” Yukihiro Itoh, Daisuke Ogasawara, Yosuke Ota, Tamio Mizukami, and Takayoshi
Suzuki. Comput. Struct. Biotechnol. J. 2014, 9, e201402002.
3. “Identification of the KDM2/7 Histone Lysine Demethylase Subfamily Inhibitor and its
Antiproliferative Activity” Takayoshi Suzuki, Hiroki Ozasa, Yukihiro Itoh, Peng Zhan, Hideyuki
Sawada, Koshiki Mino, Louise Walport, Rei Ohkubo, Akane Kawamura, Masato Yonezawa,
Yuichi Tsukada, Anthony Tumber, Hidehiko Nakagawa, Makoto Hasegawa, Ryuzo Sasaki, Tamio
Mizukami, Christopher J. Schofield, and Naoki Miyata. J. Med. Chem. 2013, 56, 7222–7231.

一般の皆様へ
リシン脱メチル化酵素である LSD1 や JMJD は、がんの増殖の原因であることが分かっています。
したがって、LSD1 阻害剤や JMJD 阻害剤は、新規抗がん剤候補化合物として期待されています。今回、弱い LSD1 阻害薬「トラニルシプロミン」を LSD1 だけに運び込むドラッグデリバリー型 LSD1
阻害剤を設計し、合成した結果、強い抗がん活性を示す LSD1 特異的阻害剤を見出しました。また、
JMJD の構造情報を基にしたドラッグデザインにより、がん細胞増殖阻害活性を示す JMJD 阻害剤も見出しました。これらのリシン脱メチル化酵素阻害剤を基にした新規抗がん剤の開発が期待されます。

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Comprehensive proteomic analysis of molecules that are targeted forcancer prognosis or therapy
Kiyotsugu Yoshida
Jikei University School of Medicine
kyoshida@jikei.ac.jp

Abstract
The proteomic analysis is a powerful tool for identification of unknown molecules that play pivotal
roles in carcinogenesis. By using 2DICAL technology, we have performed proteomic analysis oftumor suppressor p53-related genes from cancer cells.
Key Words : Proteome, 2DICAL

Introduction
Abnormal genetic alterations in tumor suppressor gene p53 are frequently identified in thehuman cancer. The tumor suppressor p53 is stabilized and activated by a variety of cellularstresses including radiation, oxidative stresses and DNA damaging agents. The p53 functions in
the cellular response to stress by inducing apoptosis, cell cycle arrest, DNA repair, senescence,or differentiation. Available evidences suggest that promoter selectivity of p53 is regulated by its
post translational modifications. For instance, the phosphorylations at Ser15 and Ser20 influencebinding of p53 to promote cell cycle arrest and DNA repair. Additionally, the phosphorylation at
Ser46 is required to induce p53-dependent apoptosis and appears to be critical for the induction of
pro-apoptotic genes (Oda et al., 2000; Yoshida et al., 2006; Taira et al., 2007). Number of reports
has brought into the questions of requirement of Ser46 phosphorylation in cellular apoptosis. The
objective of current study is to investigate the pro-apoptotic genes induced by Ser46 phosphorylationof p53 in human cancer cell line, using 2DICAL-based proteomic analysis.

Results
By comprehensive expression analysis, we have identified amphiregulin (AREG) as a new p53responsive gene in a Ser46 phosphorylation-specific manner (Taira et al., 2014). Subsequentstudies indicated that AREG is targeted to the nucleus and induces apoptosis in response to DNA
damage. To determine the functional significance of AREG on apoptosis, we searched AREGbinding proteins by 2DICAL-based proteomic analysis. By 2DICAL analysis, the 79 peaks were
statistically significant. The MS peaks were subjected to MS/MS analysis to identify amino acidsequences. Mascot search showed that DDX5 was identified as a novel AREG binding protein. We
then confirmed that nuclear AREG interacts with DDX5 upon genotoxic stress. Previous study
has shown that DDX5 engages micro-RNA processing, especially among the converting step fromprimary miRNAs to precursor miRNA. DDX5 regulates miRNAs (i.e., miR-15a) biogenesis in- 88 -

response to DNA damage. Based on these findings, we hypothesized that AREG regulates tumor
suppressive miRNAs processing via DDX5 interaction. We finally found that AREG regulates
precursor microRNA processing (i.e., miR-15a) with DDX5 to reduce the expression of antiapoptotic protein Bcl-2. These findings collectively support a novel mechanism in which theinduction of AREG by Ser46-phosphorylated p53 is required for the microRNA biogenesis in theapoptotic response to DNA damage.

Discussion & Conclusion
The tumor suppressive function of p53 is tightly regulated by its post-translational modifications.
Although Ser46 phosphorylation is a critical modification for apoptosis induction, a molecularmechanism by which Ser46-phosphorylated p53 induces apoptosis remains unclear. Here we clarify
that AREG is specifically induced in a Ser46 phosphorylation-specific manner. Notably, AREG
co-localizes with DDX5 in the nucleus and regulates tumor suppressive microRNA biogenesis in
response to DNA damage. These findings support a novel model in which Ser46-phosphorylated p53
orchestrates tumor suppressive microRNA expression in the apoptotic response to DNA damage.

References
1. Oda K, Arakawa H, Tanaka T, Matsuda K, Tanikawa C, Mori T, Nishimori H, Tamai K, Tokino
T, Nakamura Y, Taya Y. p53AIP1, a potential mediator of p53-dependent apoptosis, and its
regulation by Ser-46-phosphorylated p53. Cell 102:849-52 (2000)
2. Taira N, Nihira K, Yamaguchi T, Miki Y, Yoshida K. DYRK2 is targeted to the nucleus andcontrols p53 via Ser46 phosphorylation in the apoptotic response to DNA damage. Mol. Cell
25:725-738 (2007)
3. Taira N, Yamaguchi T, Kimura J, Lu Z-G, Fukuda S, Higashiyama S, Ono M, Yoshida K.
Induction of amphiregulin by p53 promotes apoptosis via control of microRNA biogenesis in
response to DNA damage. Proc. Natl. Acad. Sci. USA 111:717-722 (2014)
4. Yoshida K, Liu H, Miki Y. Protein kinase C delta regulates Ser46 phosphorylation of p53 tumor
suppressor in the apoptotic response to DNA damage. J. Biol. Chem. 281:5734-5740 (2006)

一般の皆様へ
現在我が国では二人に一人の方ががんになり、三人に一人の方ががんで亡くなっています。がんを克服することは、現代医療における最大の課題となっています。本研究において、がん抑制遺伝子である p53 ががん細胞を死滅させるアポトーシスと呼ばれる仕組みを明らかにしました。p53 の活性化によってある種のサイトカインが産生され、アポトーシスに関わる遺伝子の発現を調節することでこの細胞死が実行されていたのです。この発見を応用することで、新たな治療法開発に繋がることが期
待されています。

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Molecular mechanism of Derlin family-mediatednovel endoplasmic reticulum quality control system
Hideki Nishitoh
Division of Biochemistry and Molecular Biology, Department of Medical Sciences,
University of Miyazaki
nishitoh@med.miyazaki-u.ac.jp

Abstract
The accumulation of misfolded proteins disturbs the function of the endoplasmic reticulum (ER),resulting numerous conformational diseases. Cells possess ER quality control systems to adapt to
ER stress and maintain their function. Translational attenuation is general mechanism to restore thefolding capacity, however, not all ER protein synthesis is arrested during ER stress. Here we foundthat Derlin family proteins, which are components of ER-associated degradation, promote to reroutethe specific ER proteins, but not ER chaperones (e.g. BiP, PDI and GRP94), from the translocon to
the proteasome through the interaction with signal recognition particle (SRP). p97 AAA ATPaseand BAG6, which are known to contribute to transport the ERAD substrates to the proteasome after
retrotranslocation, hand over the rerouted proteins from Derlin family proteins to the proteasome.
Moreover, during acute ER stress, most of newly synthesized endogenous transthyretin proteins
are rerouted to the cytosol without cleavage of signal peptide, resulting in degradation by ubiquitin
proteasome system. These findings demonstrate that Derlin family proteins-mediated substratespecific rerouting of the ER proteins contributes to maintain the ER homeostasis through theeffective degradation of ER proteins without translocation.
Key Words : ER stress, Derlin family protein, ER quality control

Introduction
Secretory and transmembrane proteins translocate into the endoplasmic reticulum (ER) througheither co-translational or post-translational approaches. Correctly folded proteins exit the ER and
are either targeted to the membrane or released from the cell surface through the secretory pathway.
Alterations in ER homeostasis cause accumulation of unfolded/misfolded proteins in the ER andtrigger ER stress. To maintain ER homeostasis, mammalian cells possess a highly conserved
signaling pathway, termed the unfolded protein response (UPR), which is mediated by three typesof ER transmembrane receptors, PERK, ATF6 and IRE1. A large number of groups have described
the contribution of the UPR dysfunction or the UPR itself to pathogenic mechanisms of a varietyof human diseases (e.g. neurodegenerative diseases, metabolic diseases, inflammatory diseases,
diabetes mellitus, cancers, virus infections and so on).

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Results
Derlin family proteins are components of the ER AD machiner y and are required for
retrotranslocation of unfolded/misfolded proteins from the ER to the cytosol. Mammalian
Derlin family proteins, which consist of Derlin-1, Derlin-2 and Derlin-3, form homo- or heterooligomers and interact with many ERAD components, such as HRD1, SEL1L, Herp, VIMP and
p97 AAA ATPase. Although Derlin family proteins and Der1 (yeast homolog of mammalian
Derlin family) have been reported to be potential retrotranslocon candidates, the components of theretrotranslocon complex remain controversial. Derlin family proteins have been also demonstrated
to be inactive members of the rhomboid family of intramembrane proteases and to play essential
roles in retrotranslocation through conserved functional domains other than the catalytic domain.
A couple years ago, we have identified Derlin-1 as a molecular target of mutant SOD1, which is a
cause of familial amyotrophic lateral sclerosis (ALS), in the pathogenesis of motor neuron disease1.
Interaction between mutant SOD1 and Derlin-1 triggers prolonged ER stress through the disruptionof ERAD and eventually contributes to motor neuron death. Moreover, wild-type SOD1 has afunction as a molecular switch that activates the physiological ER stress response. The dissociationof zinc from SOD1 during zinc depletion induces a mutant-like conformational change in wildtype SOD1, resulting in the reversible interaction with Derlin-1 and thereby inducing the UPR.
The UPR triggered by the zinc-depletion-induced SOD1-Derlin-1 interaction aids in restoring cellhomeostasis through attenuation of protein synthesis, induction of ER chaperones and facilitationof zinc incorporation via an induction of zinc transporter2. However, physiological role of Derlinfamily proteins in the ER quality control remains controversial. In this study, we investigated therole of Derlin family proteins in the maintenance of ER homeostasis. ER stress promotes reroutingof specific ER proteins, but not BiP, GRP94 and protein folding disulfide isomerase (PDI), to thecytosol without cleavage of signal peptide. Rerouting of signal peptide-uncleaved ER proteins isfacilitated by exogenous expression of Derlin family proteins and is mitigated by deletion of Derlinfamily proteins. Moreover, Derlin family proteins interact not only with ER pQC substrates but
also with SRP and SR under the condition of ER stress and proteasome inhibition. Collectively,
these findings demonstrate the novel molecular mechanism of ER pQC that Derlin family proteins
capture the specific ER proteins from the RNC-SRP complex and reroute ER pQC substrates fromthe translocation pathway to the degradation pathway without cleavage of their signal peptides,
resulting in maintenance of ER homeostasis by increasing the ER folding capacity.

Discussion & Conclusion
Our findings demonstrated a novel mechanism of ER pQC, which controls protein load into the
ER through Derlin family proteins-mediated rerouting, p97- and BAG6-mediated transport andproteasomal degradation of specific ER proteins (not including ER chaperones, e.g. BiP, GRP94 and
PDI). Further studies should clarify the precise mechanisms underlying ER pQC for understanding
and identification of therapeutic target for the ER stress-related diseases.

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References
1. Fujisawa T., Homma K., Yamaguchi N., Kadowaki H., Tsuburaya N., Naguro I., Matsuzawa A.,
Takeda K., Takahashi Y., Goto J., Tsuji S., Nishitoh H., Ichijo H.
A novel monoclonal antibody reveals a conformational alteration shared by amyotrophic lateralsclerosis-linked SOD1 mutants. Ann. Neurol. 72:739–749 (2012)
2. Homma K., Fujisawa T., Tsuburaya N., Yamaguchi N., Kadowaki H., Takeda K., Nishitoh H.,
Matsuzawa A., Naguro I., Ichijo, H.
SOD1 as a molecular switch for initiating the homeostatic ER stress response under zinc
deficiency. Mol. Cell 52:75-86 (2013)

一般の皆様へ
小胞体は分泌タンパク質や膜タンパク質といった多くのタンパク質が折り畳まれ立体構造をとる場である。しかし、様々なストレスや遺伝子変異などによりこれらのタンパク質は正常に折り畳まれず、
異常タンパク質となって小胞体内に蓄積し小胞体の機能を阻害する。さらにこの蓄積が重篤化すると、糖尿病や神経変性疾患などの様々な疾患に関連する細胞死が引き起こされる。真核生物ではこのような小胞体ストレス状況から細胞を守るために、高度に保存された以下の小胞体品質管理システムが存在する。（１）小胞体に送り込まれる新規合成タンパク質を減らして小胞体の過剰な負荷を回避するための翻訳抑制機構、（２）タンパク質の折り畳み効率を上げるための小胞体シャペロンの転写活
性化、（３）折り畳み異常タンパク質の蓄積を減少させるための ER からの分解機構。それらに加え
近年、新規小胞体品質管理システムとして ER 近傍での翻訳時分解が報告された。ER 翻訳時分解とは小胞体ストレス誘導時にさらなる負荷を避けるために、分泌タンパク質や膜タンパク質の新生ポリペプチド鎖が小胞体への移行を阻害され、行き場を失った新生ポリペプチド鎖がプロテアソームによって速やかに分解される機構である。これまでその分子機構はほとんど明らかになっておらず、我々はその解明を試みた。その結果、小胞体ストレスによって誘導され小胞体関連分解に関与する小胞
体膜タンパク質の過剰発現により新生ポリペプチド鎖の小胞体への移行の阻害がみられた。さらに、
小胞体ストレスにより小胞体膜タンパク質と新生ポリペプチド鎖複合体の会合がみられた。これらのことから、小胞体膜タンパク質は ER 翻訳時分解機構の最初の引き金として機能する可能性が示唆された。このような分子メカニズムは、小胞体ストレスが関与する多くの疾患の克服につながると期待される。

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Development of NO donor drug based on furoxan structure
Ryosuke Matsubara
Kobe University
matsubara.ryosuke@people.kobe-u.ac.jp

Abstract
Furoxan is a relatively new class of nitric oxide (NO) donor and is thought of as a potential core
structure of a drug. For the purpose of development of NO donor drug with furoxan framework, wehave searched for the efficient synthetic ways of furoxans and its applications.
Key Words : furoxan, nitric oxide, drug

Introduction
Furoxan (1,2,5-oxadiazole 2-oxide) is a relatively new class of nitric oxide (NO) donor and isthought of as a potential core structure of a drug. However, the development of NO donor drug
based on the furoxan structure has been impeded. One of the reasons for it is thought to be the lackof general synthetic methods for furoxans. There are only sporadic reports on the substitution ofsubstituents in the 3- and 4-positions of furoxans after the formation of furoxan ring skeleton. Thatthe mechanism with which furoxans generates NO has not been thoroughly investigated makes
difficult the rational design of furoxan-based NO donor. We therefore decided to develop in thisproject the general synthetic methods for furoxans and establish the basis for the development of
NO-donor drug.

Results
We started the investigation of the furoxan formation with the alkenes and nitrosyl cation sources.
To our delight, we found that the combination of aryl-substituted olefins (styrene derivatives) andnitrosonium salt gave the desired product furoxan in good yields. Conventionally, sodium nitrite
and Brønsted acid such as acetic acid are used for the synthesis of furoxan. However, the yields are
generally medium to low and are variable depending on the literature. The yields in our reactions
are consistently good and various furoxans can be synthesized. The regioselectivity, the ratio of
3-aryl- and 4-arylfuroxan, is generally high in favor of 4-arylfuroxan.

In order to reveal the origin of high regioselectivity, the reactions of the styrenes with different sizeof substituents were performed.
The effect of water in the reaction was also examined by the deliberate addition of water.

- 93 -

All the results obtained suggested that the reaction of the sytrenes with nitrosonium
tetrafluoroborate proceeds not via the radical species.
(Manuscript under preparation for submission)

Discussion & Conclusion
We have developed the efficient synthetic methods for furoxans. Various styrenes reacted withnitrosonium salt in the developed conditions, affording the corresponding furoxans in good to highyields with high selectivity. In contrast to the conventional methods using the acidic media, thedeveloped method uses basic media and therefore complementary to the conventional methods.
The developed method may be useful for the synthesis of the furoxans bearing the acid-sensitive
functional groups.
Since the efficient synthetic methods for furoxans have been established, we are now synthesizing
various types of furoxans to be subjected to the NO-releasing assay. Our current research focuses oninvestigating the NO-releasing mechanism from the furoxans.

References
R. Matsubara, Y. Saeki, J. Li, K. Eda “Synthesis of Furoxans from Styrenes under Basic or
Neutral Conditions” Synthesis 45, 1524-1528 (2013).

一般の皆様へ
一酸化窒素は、生体内で様々な役割を担っている重要な分子です。一酸化窒素を好きな時に好きなだけ放出するような薬剤は、様々な病気に対する効果が期待できます。そのような薬剤を一酸化窒素ドナーと呼びます。私たちのグループでは、フロキサンという分子構造を有する新しい一酸化窒
素ドナーの開発を行っております。今回のプロジェクトではその効率的な合成法の開発を行いました。
今まで合成が困難であった分子を合成できるようになり、今後は医薬品となりうる分子の創製を目指していきたいと考えています。

- 94 -

Development of Novel Genetic Screening Assay using
Next-Generation Sequencer and Clinical Application of
Personalized Medicine in Inherited Arrhythmia Syndromes
Hiroshi Watanabe
Department of Cardiovascular Biology and Medicine
Hiroshi7@med.niigata-u.ac.jp

Abstract
There are various inherited arrhythmia syndromes and several causative genes have been identified
during last two decades. However, the frequency of identification of genetic causes is not enough inpatients with inherited arrhythmia syndromes. Recent development of next generation sequencing
methods allows markedly high throughput and in this study, we developed the custom assay toscreen a large number of candidate arrhythmia susceptibility genes with next generation sequencing
technique. We performed genetic screening using our next generation sequencing assay in 35
probands with inherited arrhythmia syndromes, and identified novel candidate genes indicating
usefulness of the assay to identify causative genes.
Key Words : genetics, heart disease, arrhythmia, personalized medicine

Introduction
There are various inherited arrhythmia syndromes such as long QT syndrome, Brugada syndrome,
catecholaminergic polymorphic ventricular tachycardia, and progressive cardiac conduction disease.1
During last two decades, several causative genes have been identified in inherited arrhythmia
syndromes. However, the frequency of identification of genetic causes is not enough in patients with
inherited arrhythmia syndromes. For example, pathogenic mutations are identified in only up to
30% patients with Brugada syndrome.2 Recent development of next-generation sequencing allows
markedly high throughput,3, 4 and the aim of this study was to apply the sequencing technique into
genetic screening for inherited arrhythmia syndromes.

Results
This study included 35 probands who had various arrhythmia syndromes including long QT
syndrome, short QT syndrome, Brugada syndrome, progressive cardiac conduction disease, sinus
node dysfunction, and atrial fibrillation. All patients who participated in this study gave written
informed consent prior to genetic and clinical investigations in accordance with the standards of the
Declaration of Helsinki and the local ethics committees.
We selected 240 candidate arrhythmia susceptibility genes that are expressed in heart and that havebeen suggested to have a role in cardiac electrophysiology. Targeted exon capture was performed forthe 240 candidate arrhythmia susceptibility genes using the SureSelect Target Enrichment System- 95 -

according to the manufacturer’s suggestions (Agilent Technologies, Inc. CA). The captured DNA
was sequenced on the Genome Analyzer IIx platform (Illumina Inc. CA) with paired-end reads of
101 base pairs (bp) for insert libraries consisting of 150-200 bp fragments. On average for targeted
capture sequencing, 1.1 Gbp of short read sequence data were generated and 98.9% were mapped to
the reference human genome. Captured DNA was sequenced on Illumina HiSeq2000 (performed at
RIKEN) platforms.
An average of 6.4 Gbp of short read sequence data were generated with 98.6% mapped successfully
to the reference human genome and 66-fold average coverage for all capture exons. Sequence reads
were mapped to the human reference genome (GRCh37) using the Burrows-Wheeler Aligner (BWA:
version 0.6.1). Possible duplicate reads were removed using SAM tools and custom software leaving
an average of 0.8 Gb and 5.5 Gb for targeted capture and exome sequencing, respectively. Morethan 93% of targeted regions were covered by at least 10 reads. After filtering by pair mapping
distance, mapping uniqueness and orientation between paired reads, the mapping result files wereconverted into the pileup format using SAM tools. Variant calling was conducted in part on thebasis of published methods. We, further, used the following quality control filters: (i) alignments
near putative in/dels were refined using GATK, and (ii) a strand bias filter excluded variants whosealternative allele was preferentially found on one of the two available read orientations at the site.
Variants found in dbSNP Build 137, 1000 Genomes, or Exome Variant Server (EVS) databases
were excluded from further analyses. Synonymous and intronic (other than canonical splice sites)
variants were also excluded. Three other exome databases (RIKEN database of 731 non-cardiac
disease Japanese exomes, Human Genetic Variation Browser (HGVD) database including exome
data obtained from 1208 Japanese subjects and the Institute of Human Genetics Helmholtz Zentrum
München database of >3,000 exomes of European ancestry) were also queried for the candidate
mutations.
In 35 patients with various inherited arrhythmia syndromes, all of the patients with long QT
syndrome, short QT syndrome, Brugada syndrome, progressive cardiac conduction disease, sinus
node dysfunction, and/or and atrial fibrillation had multiple rare variants in candidate arrhythmia
susceptibility genes. In order to identify novel genes that can cause inherited arrhythmia syndrome
with a mutation in a single gene, we selected genes from those in which we identified rare variants,
and screened for additional patients that were affected by same arrhythmia syndrome, in which theoriginal probands were affected. We are now planning to conduct functional analysis in order toelucidate the mechanisms by which the novel genes cause arrhythmia phenotypes.

Discussion & Conclusion
Although many causative genes for inherited arrhythmia syndromes have been identified during
last two decades, the frequency of positive results of genetic screening is not satisfied and further
identification of causative genes is needed.5-7 However, it has become difficult to identify novel
causative genes for inherited arrhythmia syndromes because of low throughput of conventional- 96 -

genetic screening methods such as Sanger sequencing methods. Recent development of next
generation sequencing methods allows markedly high throughput and in this study, we developed
the custom assay to screen a large number of candidate arrhythmia susceptibility genes with next
generation sequencing technique.3, 4 We performed genetic screening using our next generation
sequencing assay in 35 probands who were affected by various inherited arrhythmia syndromes, andidentified novel candidate genes of inherited arrhythmia syndromes. In conclusion, our custom next
generation sequencing assay may be useful to identify causative genes in patients with inheritedarrhythmia syndromes.

References
1. Priori SG, Wilde AA, Horie M, Cho Y, Behr ER, Berul C, Blom N, Brugada J, Chiang CE,
Huikuri H, Kannankeril P, Krahn A, Leenhardt A, Moss A, Schwartz PJ, Shimizu W, Tomaselli
G, Tracy C. Executive summary: Hrs/ehra/aphrs expert consensus statement on the diagnosis
and management of patients with inherited primary arrhythmia syndromes. Heart Rhythm. 2013
2. Veerakul G, Nademanee K. Brugada syndrome. Circ J. 2012;76:2713-2722
3. Mahida S, Hogarth AJ, Cowan C, Tayebjee MH, Graham LN, Pepper CB. Genetics of congenitaland drug-induced long qt syndromes: Current evidence and future research perspectives.
Journal of interventional cardiac electrophysiology : an international journal of arrhythmias
and pacing. 2013;37:9-19
4. Brion M, Quintela I, Sobrino B, Torres M, Allegue C, Carracedo A. New technologies in
the genetic approach to sudden cardiac death in the young. Forensic science international.
2010;203:15-24
5. Antzelevitch C. J wave syndromes: Molecular and cellular mechanisms.
Journal of electrocardiology. 2013
6. Berne P, Brugada J. Brugada syndrome 2012. Circ J. 2012;76:1563-1571
7. Schwartz PJ, Ackerman MJ, George AL, Jr., Wilde AA. Impact of genetics on the clinicalmanagement of channelopathies. J Am Coll Cardiol. 2013;62:169-180

一般の皆様へ
心臓に器質的な異常を伴わない、不整脈症候群は遺伝性疾患であることが知られています。現在
までに、不整脈症候群の原因となる遺伝子が多く見つかっていますが、不整脈の種類によっては大部分の症例で原因となる遺伝子が見つかりません。このため本研究では、最近開発された極めて高速な遺伝子検査法の遺伝性不整脈への応用を目的としました。我々は、遺伝性不整脈の症例において本法を用いて遺伝子検査を行い、新たな原因遺伝子の候補を複数見つけ、このような方法が有用な新しい遺伝子検査であることが示唆されました。

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Functional Regulation of Nicotinic Acetylcholine Receptors
by a Novel Family of Endogenous Neurotoxin-like Molecules
Hidemi Misawa
Department of Pharmacology, Faculty of Pharmacy, Keio University
misawa-hd@pha.keio.ac.jp

Abstract
We have identified novel snake/frog toxin-like nAChRs-binding peptides that may serve asbalancers of neuronal activity in the adult nervous system. Genome database screening andphylogenic tree analysis detected 31 proteins with the conserved three-finger domain of Ly-6/
neurotoxin superfamily (Ly6SF). Biochemical and histological analyses suggested some of the
Ly6SF proteins bind or co-localize with alpha4beta2 nicotinic acetylcholine receptors. Theseobservations suggest that the Ly6SF genes extracted from the genome database are potentiallyinvolved in higher-order brain functions through nAChR regulation.
Key Words : nicotinic acetylcholine receptor, prototoxin, allosteric modulator

Introduction
The Ly-6/neurotoxin superfamily (Ly6SF) is a group of protein containing the characteristic
pattern with four to five disulfide bonds that are essential to build the unique three-dimensional
structure so-called three-finger domain (TFD). The Ly6SF members consist of either secreted
proteins or membrane-attached proteins containing a GPI-anchor sequence. The TFD was first
identified in the alpha-bungarotoxin, a snake neurotoxin with potent antagonistic activity onto
nicotinic acetylcholine receptors (nAChR). Accumulated evidence has suggested that nAChRs
are functionally regulated by endogenous snake/frog toxin-like nAChRs-binding peptides, called
prototoxins, which were encoded in the human genome.

Results
Recently it has been reported that some endogenously expressed Ly6SF proteins (such as Lynx1,
Lynx2, SLURP-1 and SLURP-2) modulate nAChR function, either as positive allosteric modulators
or as antagonists. To explore novel endogenous nAChR modulators, we tried to identify TFDcontaining proteins from the genome database, and found that there are 31 members of this family
protein common to both human and mouse. In order to narrow down these candidate genes tonovel Ly6SF members that may serve as balancers of neuronal activity in the adult nervous system,functional relationship analyses among these 31 proteins were conducted by the phylogenic tree
prediction method (MEGA5 and ClustalX2.0). The result showed that LyPD2, LyPD6 and PSCA
were identified as possible novel nAChR modulators. We also conducted comprehensive qRT-PCR
analyses to compare each Ly6SF mRNA distribution in various organs or cells, and found that these- 98 -mRNA species were expressed in different cell types. Followings were identified as brain-enriched
Ly6SF molecles: Ly6E, Ly6H, Lynx1, Lynx2, LyPD6 and LyPD7.
Next we analyzed biochemical protein-protein interaction between each Ly6SF protein andalpha4beta2 nAChR. Each Ly6SF proteins were expressed in HEK293 cells together withalpha4beta2 nAChR, and protein-protein interaction was analyzed by co-immunoprecipitation assay.
Along with Lynx1, an already known (and also well-characterized) alpha4beta2 nAChR antagonist,
LyPD2, LyPD6 and PSCA were found to be co-immunoprecipitated with alpha4beta2 nAChRs.
Furthermore, the histological localization analyses of these Ly6SF molecules in the mouse brain
by in-situ hybridization revealed that some Ly6SF genes were specifically expressed in a neuronal
subpopulation in the central nervous system, e.g. the pyramidal cell layer in the hippocampus or
cerebral corte.
Lastly, we tried to establish a whole-cell patch-clamp assay system to directly analyze the effectof Ly6SF on nAChR activity. We established alpha4beta2 nAChR-TRPV1 (delta35)-expressing
HEK293 cells, in which cells ACh-evoked currents could be reliably recorded. The cell line will beof use to directly characterize Ly6SF-nAChR physiological interactions.
These observations suggest that the Ly6SF genes extracted from the genome database arepotentially involved in higher-order brain functions through nAChR regulation.

Discussion & Conclusion
Cholinergic hypoactivity in the central nervous system is known to induce attention deficit
and cognitive impairment observed in various brain diseases such as Alzheimer’s disease andschizophrenia. nAChR is involved in these important processes by augmenting the release ofvarious neurotransmitters. nAChR is also suggested to have strong analgesic and anti-inflammatory
roles. Therefore development of novel nAChR modulators will hold promise to cure thesedevastating conditions. We have identified several candidate Ly6SF molecules that share the same
three-dimensional structures (TFD) as that of snake/frog neurotoxins. By analyzing detailed modeof action between the Ly6SF molecules and nAChR, we are aiming at developing novel drugs thataugment the endogenous cholinergic tone in a spatially and temporally restricted manner.

References
1. Fujii, T., Horiguchi, K., Sunaga, H., Moriwaki, Y., Misawa, H., Kasahara, T., Tsuji, S. and
Kawashima, K. SLURP-1, an endogenous alpha7 nicotinic acetylcholine receptor allostericligand, is expressed in CD205(+) dendritic cells in human tonsils and potentiates lymphocytic
cholinergic activity. J. Neuroimmunol., 267, 43-49 (2014)
2. Narumoto, O., Niikura, Y., Ishii, S., Morihara, H., Okashiro, S., Nakahari, T., Nakano,T., Matsumura, H., Shimamoto, C., Moriwaki, Y., Misawa, H., Yamashita, N., Nagase, T.,
Kawashima, K. and Yamashita, N. Effect of secreted lymphocyte antigen-6/urokinase-type
plasminogen activator receptor-related pepetide-1 (SLURP-1) on airway epithelial cells. Biochem.
Biophys. Res. Commun., 438, 175-179 (2013)
3. Kawashima, K., Fujii, T., Moriwaki, Y. and Misawa, H. Critical roles of acetylcholine and themuscarinic and nicotinic acetylcholine receptors in the regulation of immune function. Life Sci.,
91, 1027-1032 (2012)- 99 -

4. Swamynathan, S., Buela, K-A., Kinchington, P., Lathrop, K. L., Misawa, H., Hendricks, R.
L. and Swamynathan, S. K. Klf4 regulates the expression of Slurp1, which functions as an
immunomodulatory peptide in the mouse cornea. Invest. Ophtalmol. Vis. Sci., 53, 8433-8446(2012)
5. Kawashima, K., Fujii, T., Moriwaki, Y. Misawa, H. and Horiguchi K. Reconciling neuronallyand nonneuronally derived acetylcholine in the regulation of immune function. Ann. N.Y. Acad.
Sci., 1261, 7-17 (2012)

一般の皆様へ
毒蛇に噛まれたらひとたまりもありません。蛇毒にはニコチン性アセチルコリン受容体 (nAChR) に結合して筋肉の働きを麻痺させるタンパク質が含まれています。最近の研究によると、ヒトの体内にもこの蛇毒と似た構造をもつタンパク質（プロトトキシン）があり、nAChR の機能を調節していることが分かってきました。不思議なことです。一方で、nAChR は脳にも存在し、記憶や学習といった高次機能にも大切であることが分かっています。アルツハイマー病や統合失調症では、この nAChRの働きに異常が見られます。我々は、新たなプロトトキシンの発見と解析を通して、これらの病気の治療薬の創製を目指しています。

- 100 -

Development of orange-colored bimolecular fluorescencecomplementation (BiFC) assay
Yutaka Kodama
Center for Bioscience Research and Education, Utsunomiya University
kodama@cc.utsunomiya-u.ac.jp

Abstract
Bimolecular fluorescence complementation (BiFC) assay is a fluorescent protein-based method
to visualize protein-protein interactions in living cells. This study’s purpose is a development oforange-colored BiFC method as a new BiFC system.
Key Words : BiFC, bimolecular fluorescence complementation, fluorescent protein, protein-protein
interaction, visualization.

Introduction
Protein-protein interactions play important roles in various biological processes. To analyze
protein-protein interactions, many strategies have been employed so far. For example, yeast two
hybrid, in vitro pull-down, in vivo co-immunoprecipitation, fluorescence resonance energy transfer,bioluminescence resonance energy transfer and bimolecular fluorescence complementation (BiFC)
assays are often utilized. Among those, BiFC assay, which is based on splitting a fluorescentprotein, has been widely accepted over the past 10 years.

Results
The BiFC technology is based on structural complementation between two non-f luorescentN-terminal and C-terminal fragments from a fluorescent protein (Hu et al. 2002). Fluorescentprotein consists of antiparallel 11 β-strands with an α-helix inside and several short helicalstructures. The β-strands form a β-barrel structure, and the chromophore, located on the insideof the β-barrel structure, is chemically formed by specific three residues (Tsien 1998). For BiFC
analysis, several studies have demonstrated that fluorescent proteins can be split at some loops orwithin a β-stand (Kodama and Hu 2012). The resulting two non-fluorescent fragments are fused tointerest of proteins that may interact. If the two proteins of interest interact, the two non-fluorescentfragments are brought into close proximity to be reconstituted into an intact fluorescent protein,
giving rising fluorescence (Kodama and Hu 2012). The complemented fluorescence could beobserved under any fluorescence microscope, and the fluorescence images could be acquired for a
detailed analysis.
To date, many fluorescent proteins have been subjected to BiFC assay (Kodama and Hu 2012).
Based on previous studies, it is likely that physicochemical feature of BiFC resembles to that of thematerial fluorescent protein; for example, excitation and emission spectrum of EYFP-based BiFC

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are identical to that of EYFP. Resent development of BiFC techniques could cover a broad range ofspectra, and over 10 fluorescent proteins have been utilized to the BiFC system (Kodama and Hu
2012). Depending on research interest, BiFC users could choose appropriate system from the BiFC
color palette.
In this study, orange-colored BiFC system has been developed to produce a new colored BiFC
system. To develop orange-colored BiFC system, orange fluorescent proteins were utilized asmaterials, and new vector was designed and constructed for expression of BiFC complex in plant
cells. To compare fluorescence intensities of fluorescent proteins and the BiFCs, protoplast cells
were isolated from Arabidopsis thaliana plants and fluorescent proteins or BiFC fragments weretransiently introduced into the protoplasts via polyethylene glycol method.
[1] Design and construction of new expression vector
To efficiently develop a new BiFC system, we designed and constructed a new transient
expression vector for plant cell. The vector was optimized to the Gateway cloning system,
invented and commercialized by Invitrogen (USA). In the expression vector, gene for fluorescentprotein or BiFC fragment can be controlled by a 35S promoter from the cauliflower mosaic virus
and a terminator of nopaline synthase from Agrobacterium tumefaciens.
[2] Selection of an orange fluorescent protein
To select appropriate orange f luorescent protein for the new BiFC system, we comparedcharacteristics of several orange fluorescent proteins in Arabidopsis thaliana protoplast cells.
After the experiments in the protoplasts with the expression vector and the polyethylene glycol
method, we identified an orange fluorescent protein preferred to an orange-colored BiFC system.
[3] Development of orange-colored BiFC systems
Using an orange fluorescent protein selected above, we have developed several types of BiFC
system. For example, we tried to split 3 different sites within the loop structures of the orange
fluorescent protein. Subsequently we compared some characteristics of the BiFC systems. For
example, background fluorescence mediated by a self-assembly between BiFC fragments wascompared by using fluorescent microscopy and the ImageJ software with an original macroprogram. During this study supported by the Novartis Foundation, we identified an orangecolored BiFC system with a low background fluorescence signal.

Discussion & Conclusion
To date, over 10 BiFC systems have been developed for visualization of protein-protein interaction
in living cells. For example, blue, cyan, green, yellow and red colors have been reported. The color
palette has visualized not only single protein-protein interaction, but also multiple protein-protein
interactions (Kodama and Wada 2009). In this study, an orange fluorescent protein-based BiFC
system was successfully developed, and thereby new color could be added to the BiFC color palette.
Because the system seems to be used in various biological materials such as plants, mammalians
and microorganisms, it will strongly support many studies in various research fields.

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References
1. Hu CD, Chinenov Y and Kerppola TK (2002) Visualization of Interactions among bZIP and Rel
Family Proteins in Living Cells Using Bimolecular Fluorescence Complementation. Mol Cell
9:789-798.
2. Tsien RY (1998) The green fluorescent protein. Annu Rev Biochem 67:509-544.
3. Kodama Y and Hu CD (2012) Bimolecular fluorescence complementation (BiFC): A 5-year
update and future perspectives. Biotechniques, 53:285-298.
4. Kodama Y & Wada M (2009) Simultaneous visualization of two protein complexes in a single
plant cell by using multicolor fluorescence complementation analysis. Plant Mol Biol 70:211-217.

一般の皆様へ細胞の中では、無数のタンパク質がお互いに引っ付いたり離れたり（タンパク質間相互作用）しています。タンパク質間相互作用は、生物が生きる上で大切な働きをするため、これを解析することはとても重要なことです。タンパク質間相互作用を解析する様々な技術の中でも、最近は、視覚的に見えるようにする技術が注目されています。とくに、二分子蛍光補完（BiFC）法と呼ばれる技術は、その簡便さから多くの研究分野で利用されており、様々なタンパク質間相互作用が可視化されてきました。本研究課題は、新しい BiFC 法の開発研究であり、細胞内で起こるタンパク質間相互作用ネット
ワークの全貌解明に貢献します。

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Regulation of polarized secretion of Wnt
by post-translational modification in epithelial cells
Akira Kikuchi
Graduate School of Medicine, Osaka University
akikuchi@molbiobc.med.osaka-u.ac.jp

Abstract
We found that Wnt3a and Wnt5a were secreted basolatelaly in polarized cells, whereas Wnt11
was apically. The glycan attached to Wnt11 was required for its apical secretion and the basolateralsecretion of Wnt3a and Wnt5a depended on clathrin and AP-1. Their receptors also showed thepolarized localization to the appropriate membranes so that the corresponding Wnt signal isactivated efficiently. However, the receptors were loaded on distinct vesicles from Wnts. Basolateralsecretion, but not apical secretion, of Wnt5a was required for the formation of the apico-basolateralpolarity. Thus, the polarized sorting Wnts and receptors might be regulated by distinct mechanisms
and play important roles in epithelial morophogenesis.
Key Words : Wnt, polarity, glycosylation, lipidation, epithelial cells

Introduction
Wnts are the large family of secreted molecules that are important for developmental processes anddefective Wnt signaling in postnatal life causes human diseases (1). Great advances have been madein understanding the mechanisms by which Wnt activates the signaling pathways and regulates
cellular functions. In contrast, how Wnts and their receptors are expressed and trafficked remains
unclear. The sorting process into the apical and basal regions occurs at the level of the Trans-Golgi
network by incorporation of apical and basolateral proteins into distinct vesicles (2). We attempted
to clarify the mechanisms underlying the polarized trafficking of Wnts and their receptors.

Results(1) The post-translational modification of Wnts.
Glycosylation of proteins is important for their folding and sorting (3). Wnts have been shown
to be glycoproteins, but their glycan profiles have never been analyzed, because purificationof enough amounts Wnts proteins to analyze glycosylation was hard. We tried to identify
oligosaccharides attached to purified Wnts by mass-spectrometry. Wnt11 was modified withcomplex-(Asn40) and high-mannose-(Asn90 and Asn300) type glycans, Wnt3a was modified
with high-mannose-type glycans (Asn87 and Asn298), and Wnt5a was modified with highmannose-(Asn114 and Asn326) and hybrid-(Asn312) type glycans (4 and this study).
Wnt3a are also modified with palmitoleic acid post-translationally at Ser209 (5). Wnt11 and
Wnt5a were also found to be modified with palmitoleic acid at conserved Ser215 and Ser244,- 104 -

respectively (4 and this study). This palmitoleic acid modification was essential for the secretionof Wnt11 and Wnt5a, because their serine mutants was accumulated in the cells and neversecreted.
(2) Polarized trafficking of Wnts and their receptors.
Directional secretion of Wnts was examined in two-dimensionally polarized MDCK
cells. Wnt11 was secreted apically, whereas the majority of Wnt3a and Wnt5a was secreted
basolaterally, suggesting polarized secretion of Wnts. Wnt11 was observed in the vicinity ofapical lumen of ureteric buds in embryonic kidney, supporting apical secretion of endogenous
Wnt11 (4).
An Wnt receptor consists of one seven-transmembrane protein (Fz) and one singletransmembrane protein (LRP6, Ror2, and Ryk). A combination of two different receptors
recognizes one Wnt, although the mechanism is not clear. The trafficking of Wnt receptors
were also examined in polarized MDCK cells. Fz2 was localized to the basolateral membrane
mainly, and Fz7 was localized to both membranes evenly. LRP6 and Ror2 were trafficked to thebasolateral membranes, whereas Ryk was to both membranes. When polarized MDCK cells
were stimulated with Wnts, the signaling of Wnt3a and Wnt5a was activated more efficiently
at the basolateral membranes than the apical membranes. The activity of Wnt3a signaling wasassessed by LRP6 phosphorylation, and that of Wnt5a was measured by Rac activation. These
results suggested that an appropriate combination of Wnt ligands and receptors is trafficked tothe same destination.
(3) Mechanisms of apical and basolateral sorting of Wnts and receptors.
Wntless (Wls) recognizes palmitoleic acid of Wnts and is essential for the secretion of Wnts
(6). Therefore, knockdown of Wls suppressed the secretion of Wnts. Glycosylation processing at
Asn40 and galectin-3 were necessary for the apical secretion of Wnt11, while clathrin and adaptor
protein-1 (AP-1) were required for the basolateral secretion of Wnt3a and Wnt5a. By the additionof the complex-type glycan to the N-terminal region, Wnt3a and Wnt5a were secreted apically,
supporting that the importance of the complex-type glycan at the N-terminal region for the apicalsecretion of Wnts. The basolateral sorting of LRP6 and Ror2 also required clathrin and AP-1, but
not Wls.
(4) Involvement of apical lumen formation by Wnt5a basolateral secretion.
Rat intestinal epithelial cells (IEC6) express several Wnts, including Wnt5a, and developed asingle cell polarization and established cysts with apico-basal polarization. Knockdown of Wnt5a
in IEC6 cysts impaired the apico-basolateral polarization (7). In addition, knockdown of Wnt5ain MDCK cysts also impaired the formation of apical lumen formation, and basolateral, but notapical, secretion of Wnt5a rescued the phenotypes.

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Discussion & Conclusion
We have demonstrated the glycan profiles of Wnts for the first time. Among 19 Wnt family
members there are conserved Asn residues, and they were found to be modified with the mannosetype glycans. Our results showed that Wnts and their receptors are trafficked by different vesicles
through distinct mechanisms. Consistent with the sorting mechanisms, the addition of the complextype glycan to Wnt3a and Wnt5a made them been secreted apically (4 and this study), suggesting thedevelopment of the artificial manipulation of directional secretion of Wnts.
It has been reported that Fz2/LRP6 and Fz5/LRP6 mediate Wnt3a signaling, that Fz2/Ror2 andFz5/Ror2 mediate Wnt5a signaling, and that Fz7/Ryk mediates Wnt11 signaling. Consistently,
Wnt3a and Wnt5a activated their signaling mostly when they stimulated the basolateral membranes
when their specific receptors are located. What is the physiological role of polarized secretionof Wnts in epithelial cell? In IEC6 and MDCK cysts, basal secretion of Wnt5a and its efficientsignaling at the basolateral membranes due to the localization of its receptors would play importantroles in the formation of the apico-basolateral polarity. Therefore, the magnitude of Wnt signaling iscorrelated with the localization of Wnt ligands and receptors.

References(1) Kikuchi, A., Yamamoto, H., Sato, A., and Matsumoto, S. (2011). New insights into themechanism of Wnt signaling pathway activation. Int. Rev. Cell Mol. Biol. 291, 21-71.
(2) Rodriguez-Boulan, E., Kreitzer, G., and Musch, A. (2005). Organization of vesicular trafficking
in epithelia. Nat. Rev. Mol. Cell Biol. 6, 233-247.
(3) Vagin, O., Kraut, J.A., and Sachs, G. (2009). Role of N-glycosylation in trafficking of apicalmembrane proteins in epithelia. Am. J. Physiol. Renal Physiol. 296, F459-469.
(4) Yamamoto, H., Awada, C., Hanaki, H., Sakane, H., Tsujimoto, I., Takahashi, Y., Takao, T.,
and Kikuchi, A. (2013). The apical and basolateral secretion of Wnt11 and Wnt3a in polarized
epithelial cells is regulated by different mechanisms. J. Cell Sci. 126, 2931-2943(5) Takada, R., Satomi, Y., Kurata, T., Ueno, N., Norioka, S., Kondoh, H., Takao, T., and Takada, S.
(2006). Monounsaturated fatty acid modification of Wnt protein: its role in Wnt secretion. Dev.
Cell 11, 791-801.
(6) Port, F., and Basler, K. (2010). Wnt trafficking: new insights into Wnt maturation, secretion andspreading. Traffic 11, 1265-1271.
(7) Gon, H., Fumoto, K., Ku, Y., Matsumoto, S., and Kikuchi, A. (2013). Wnt5a signaling promotes
apical and basolateral polarization of single epithelial cells. Mol. Biol. Cell 24, 3764-3774.

一般の皆様へWnt は重要な細胞増殖分化因子であるために、これまでＷｎｔがどのようにして細胞機構を制御するかという点について精力的に解析され、Wnt シグナルの制御機構が明らかになってきました。しかし、Wnt がどのように分泌され、その方向性に意味があるのかは明らかにされてきませんでした。今回私達は上皮細胞において、Wnt の種類毎に異なる機構で適切な方向に分泌されることを明らかにしました。さらに、その方向性が上皮細胞の性状を決定するのに重要であることも示しました。これらの研究成果は、Wnt シグナルを標的とした再生研究や疾患治療研究に貢献することが期待されます。

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Synthetic studies on the virulent cell wall lipid of
Mycobacterium tuberculosis
Seijiro Hosokawa
Waseda University
seijiro@waseda.jp

Abstract
Stereoselective alkylation and acylation of the E,E-vinylketene silyl N,O-acetal possessing achiral auxiliary has been achieved. These reactions promote the rapid synthesis of polyketides. The
structure of methyl-branched alkyl chain found in PDIM, a virulent cell wall lipid of Mycobacterium
tuberculosis, has been constructed by the alkylation and the formal synthesis of khafrefungin has
been accomplished by the acylation reaction.
Key Words : Remote asymmetric induction, stereoselective reduction

Introduction
Tuberculosis has been a problem disease all over the world. PDIM, one of the cell wall lipid of
Mycobacterium tuberculosis, was reported as the virulent component, and latterly found in BCG
Tokyo 172 substrain. Therefore, PDIM is expected to be a vaccine component, although little
amount can be obtained from M. tuberculosis. To supply the cell wall lipid enough for researchers
we started the synthetic studies on PDIM. PDIM is composed of two tetramethyl substituted
saturated acids (mycocerosic acid) and phithiocerol. Recently, we have established a methodology toconstruct methyl-branched alkyl chain, which is applicable to the synthesis of PDIM.

Results
Mycocerosic acid, a component of PDIM, is a tetramethyl substituted saturated acid (eq. 1). We
have already established the methodology to construct reduced propionates (trimethyl substituted- 107 -

saturated acids) by the vinylogous Mukaiyama aldol reaction using the chiral silyl dienol ether 1 (theremote stereoinduction reaction) and subsequent regio- and stereoselective reduction reactions (eq.
2).
In order to synthesize mycocerosic acid, the tetramethyl substituted saturated acid, we haveestablished the remote-stereoinductive alkylation reaction (eq. 3). The reaction between the silyl
dienol ether 1 and allyliodide 5 in the presence of silver cation gave 7 stereoselectively. Birch
reduction of α,β-unsaturated imide 7 gave a new stereogenic center at C2 position in accompany
with removal of Bn group. The OH-group directing hydrogenation using rhodium(I) catalyst gave astereogenic center at C6 position stereoselectively. Thus, the tetramethyl substituted saturated acidderivative 8 has been synthesized. Studies on scope and limitation of the alkylation reaction using
the silyl dienol ether 1 and transformation of 8 to mycocerosic acid is in progress.

As analogy to the alkylation reaction (eq. 4), we also found that the silyl dienol ether 1 received theacylation reaction with acid anhydride in the presence of SnCl4 (eq. 5). The reaction proceeded in themanner of the remote stereoinduction to give ketone 12 as a single isomer. Both of these reactions
are called as 1,6-stereoinduction reaction because the original stereogenic center of the silyl dienol
ether 1 controlled the stereochemistry of the γ position of the product, of which stereogenic centers
directed as 1,6-positions.

The acylation product 12 features the ketone group which is possible to prolong the propionate
chain. The reaction was applied to the formal synthesis of khafrefungin, a potent antifungal agent(Scheme 1).
The synthesis started from the selective cross aldol condensation reaction with decanal 13 andpropanal. Treatment of the mixture of aldehydes and piperidine in the presence of AcOH gave
2-methyl-2-dodecenal 14 in 89% yield. The resulting unsaturated aldehyde 14 was submitted to
the vinylogous Mukaiyama aldol reaction using the silyl dienol ether 1 (the remote stereoinduction
reaction) to give anti adduct as the single isomer. The resulting anti adduct was converted to thesilyl ether 15, which received the hydrogenation using Adam’s catalyst to give 12S isomer 16 as the
major compound of a separable mixture (dr = 6:1, isolated yield of 16: 60%). DIBAL-H reduction
gave aldehyde 17 directly. On the other hand, ent-12 was prepared under the optimized conditions
by using vinylketene silyl N,O-acetal ent-1. The adduct ent-12 was coupled with unsaturated- 108 -

aldehyde 17 by the aldol condensation using SnCl4 and triethylamine to yield α,β,γ,δ-unsaturated
ketone 18 directly. Hydrolysis of imide 18 gave the carboxylic acid 19, of which spectral data wereconsistent with those of 19 previously synthesized by our group. Thus, the formal synthesis ofkhafrefungin has been accomplished.
Additionally, we synthesized the C4-epimer of 19 (20) to prove stability of the stereochemistryof C4 position in our route. Acylation adduct 12 was coupled with aldehyde 17 under the same
conditions used in the coupling ent-12 and 17. The adduct (the C4-epimer of 18) was exposed tohydrolysis conditions mentioned in Scheme 1 to afford carboxylic acid 20 (C4-epimer of 19). These
compound gave different 1H NMR spectra, of which did not include the stereoisomers. Thus, weconcluded that the C4 position was stable in our synthetic route of carboxylic acid 19.

Therefore, by using remote-stereoinductive acylation we have achieved the formal synthesis ofcarboxylic acid of khafrefungin (19) in only seven steps (the previous shortest synthesis: 14 steps).

Discussion & Conclusion
Mycocerosic acid, a component of PDIM, and khafrefungin belong to polypropionate which isfound widespread in natural product. The alkylation reaction of the E,E-vinylketene silyl N,Oacetal would be a powerful tool to construct the multiply methyl-branched saturated acids (reduced
polypropionates). The combination of the alkylation and stereoselective reductions might givethe reduced polypropionates quickly. On the other hand, the acylation reaction is found to be a
powerful tool to construct oxygenated polypropionates. The acylation product was additionally- 109 -

prolonged by using the α position of the ketone. The α,β,γ,δ-unsaturated moiety of the carboxylic
acid of khafrefungin has been constructed by one pot aldol reaction-dehydration sequence using theacylation adduct and the α,β-unsaturated aldehyde. Finally, the carboxylic acid of khafrefungin was
synthesized in only seven steps by using the reduction methodology and the acylation reduction.

References
Yuta Takahashi, Maiko Otsuka, Mio Harachi, Yuki Mukaeda, Seijiro Hosokawa, Stereoselective
Acylation of the E,E-Vinylketene Silyl N,O-Acetal and Its Application to the Synthesis ofKhafrefungin, manuscript in preparation.

一般の皆様へ
本研究は、有用な生物活性物質を手早く合成するための化学反応の研究と、それを利用したワクチンの合成研究です。今回、新しい結核ワクチンの元になると考えられる物質 PDIM の骨格を合成するための新しいタイプの反応（遠隔不斉誘導型アルキル化反応）を発見しました。また、その類縁反応として遠隔不斉誘導型アシル化反応も発見し、その反応を使って抗生物質カフレフンジンの短工程合成に成功しました。

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Synthesis of anticancer anthracycline antibiotics
having aza-spiro structure
Mitsuru Kitamura
Kyushu Institute of Technology
kita@che.kyutech.ac.jp

Abstract
A synthetic study of anticancer anthracycline antibiotics, kosinostatin was examined. Synthesisof key intermediate lactone, which corresponds to the BCDE ring fragment, was accomplished byapplying our recently developed alkoxycarbonylmethylation of diazonaphthoquinone. The stereo
selective construction of D ring was succeeded by trifluoroacetic acid-mediated cyclization of the
3,4-epoxycylohexanecarboxylic acid derivative.
Key Words : anticancer, antibiotics, quinocycline, kosinostatin

Introduction
The quinocycline antibiotics constitute a class of anthracycline natural products, and show
several antibiotic and anticancer activities.1 Quinocyclines have a unique structure consistingof a tetrahydronaphthacenequinone core (ABCD rings) having 3-chiral centers (C7, C9, C10)
connecting the unusual pyrrolopyrrole (FG rings) via N,O-acetal, and a sugar. Stimulated by theunique structure and its remarkable bioactivities, we started to synthesize one of the quinocycline
antibiotics, kosinostatin,1 and succeeded to synthesize the BCDE ring.

Results
Scheme 1 outlines our retrosynthesis plan. Aglycone 1 is simplified to tetracyclic lactone 3 byassuming the Diels–Alder reaction with diene 2 and nucleophilic addition of vinylamidine moiety
4. Three carbon chiral centers in D rings of lactone 3 were assumed to be constructed by theoxidative cyclization of alkenyl carboxylic acid; therefore, ester 5 was set as its precursor. The
D ring in 5 was expected to be constructed by olefin metathesis of diene 6, which, in turn, could
be derived from 1-naphthalenol 8 by the ortho-alkoxycarbonylmethylation and the successive

Scheme 1. Retrosynthesis of kosinostatin aglycone 1.

- 111 -

introduction of a methallyl group at the activated methylene position in 7.
Wittig–Horner reaction of aldehyde 9 with phosphonate 10 gave the corresponding unsaturated
ester, whose ester was hydrolyzed to give unsaturated acid 11 (Scheme 2). Treatment of 11 withacetic anhydride in the presence of sodium acetate gave naphthyl acetate, which was hydrolyzed togive naphthol 8b. Diazo-transfer reaction for 8b by 2-azido-1,3-dimethylimidazolinium chloride(ADMC, 12) formed by the reaction of 2-chloro-1,3-dimethylimidazolinium chloride (DMC) andsodium azide smoothly proceeded to afford corresponding diazonaphthoquinone 13.2 Rhodiumcatalyzed cyclization of 13 with ketene silyl acetal followed by a ring opening reaction successfully
proceeded to afford alkoxycarbonylmethylated naphthalene 7b in 36% yield in 3 steps from
1-naphthol 8b. Introduction of a methallyl group to the active methylene part in 7b proceeded toafford diene 6b, whose intramolecular olefin metathesis proceeded to afford DEF ring fragment 5.
Next we tried the transformation of 5 to lactone 3. Several reports have appeared about oxidative
cyclization of cyclohex-3-encarbonic acid to the corresponding hydroxy lactone via epoxide.
However, epoxidation of the alkene in the D ring of ester 5 or the corresponding carboxylic acid 15
was unsuccessful because of over oxidation of aromatic rings. Selective oxidation of the electronrich B ring in 5 was performed by ceric ammonium nitrate (CAN) to give quinone 16, whose
D ring’s epoxidation was accomplished by the treatment of m-CPBA to afford 17 as a ca 1/1
diastereomer mixture. The products were rather unstable, and the crude material was treated withtrifluoroacetic acid to obtain lactone 3. The structure/stereochemistry of 3 was identified by NMR
experiments (1H NMR, NOE). That is, the 1H NMR signal of Ha of the hydroxyl group was observed
as a doublet (J = 7.5 Hz) at 6.50 ppm in DMSO-d6, which was eliminated by the addition of D2O,
and NOE was detected between Ha and H b (2.14 ppm). These data suggested that the product from
17 was not δ-lactone but γ-lactone, and oxygen functional groups attached at C8 and C9 in D ringare placed in the anti position, as shown for compound 3.

Scheme 2. Synthesis of BCDE ring fragment 3. a) 10 (1,2 equiv.), NaH (3 equiv.), THF, –78℃ → rt, 3.5 h(75%). b) 10 M KOH aq. n-Bu4NBr (0.1 equiv.), 1,4-dioxane, 90℃, 24 h. c) NaOAc (2 equiv.), Ac2O, 140℃,
7 h. d) K2CO3, MeOH, 1,4-dioxane, rt, 4.5 h (3 steps 42%). e) DMC (5 equiv.), NaN3 (5 equiv.), CH3CN,–25℃, 1.5 h: then 8b, Et3N (2.5 equiv.), THF, –25℃ → rt, 3 h (89%). f) CH2=C(OTBS)(Ot-Bu) (2 equiv.),
1 mol% Rh2(OAc)4, 40℃, 1 h (45%). g) n-Bu4NF (2 equiv.), MeI (10 equiv.), Na2S2O4 (0.1 equiv.), THF, rt,
1.5 h (91%). h) CH2=C(CH3)CH2Br, NaH, 45℃, 3 h (91%). i) 5 mol% Grubbs 2nd cat., toluene, 80℃, 12 h(82%). j) CF3CO2H, CH2Cl2, rt, 1.5 h (quant.). k) Ce(NH4)2(NO3)6, CH3CN, H2O, 0℃, 10 min. l) m-CPBA,NaHCO3, CH2Cl2, rt, 2.5 h. m) CF3CO2H, CH2Cl2, 0℃, 40 min (3 steps 31%).

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Discussion & Conclusion
Toward the synthesis of kosinostatin, we synthesized the key intermediate lactone 3, whichcorresponds to the BCDE ring fragment. The precursor BCD ring fragment 5 was synthesized
via efficient application of our developed alkoxycarbonylmethylation of diazonaphthoquinone.
That is, poly-substituted naphthol 8b reacted with ADMC 12 efficiently to give corresponding
diazo-transfer product, diazonaphthoquinone 13 in high yield. 13 reacted with ketene silyl
acetal in the presence of Rh catalyst to give naphthofuran derivative 14, which was converted toalkoxycarbonylmethylated naphthalene 7b by treating F– in the presence of MeI. Currently, we are
studying the construction of A and FG rings, aiming at the synthesis of aglycone 1.

References
1. Furumai, T.; Igarashi, Y.; Higuchi, H.; Saito, N.; Oki, T. J. Antibiotics 2002, 55, 128, andreferences cited therein.
2. Kitamura, M.; Araki, K.; Matsuzaki, H.; Okauchi, T. Eur. J. Org. Chem. 2013. 5045.
3. Kitamura, M.; Tashiro, N.; Sakata, R.; Okauchi, T. Synlett 2010, 2503.

一般の皆様へがんは日本人の死亡原因の第一位の疾患であり，抗がん剤の開発は社会的要請が極めて高い。これまで，抗がん剤として臨床使用が承認されている治療薬の半数以上が天然由来の低分子であり，天然物はがん治療薬のシードとして非常に有用である。2002 年，アントラキノンの骨格に，他の化合物では見られないピロロピロリジン骨格が L 字型にスピロ結合したユニークな構造の抗がん活性を有する抗生物質コシノスタチンが単離された。本研究はコシノスタチンの全合成を試み，それを通じて種々の誘導体合成を行い，構造活性相関からがん治療薬を開発することを最終的な目標とする。
本助成期間においては，通常フォトレジストにしか利用されていないジアゾナフトキノンを鍵中間体として，コシノスタチンの母核 B-E 環の合成に成功した。

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Genetic and Epigenetic Landscape of Endothelial Cells Differentiation
Reveal the Transcriptional Factors Network
Yasuharu Kanki
The University of Tokyokanki@lsbm.org

Abstract
In this report, we analyzed the endothelial cell differentiation system from mouse ES cells by using
two comprehensive analyses ChIP-seqs and micorarrays.
From these experiments we found out H3K4me3 have strongly correlation with gene expression
profiles. Moreover, we discovered some bivalent genes such as Etv2, Gata2, Sox18 which are
upregulated at early time points and supposed to be candidates for master regulators of endothelial
cells differentiation. These findings have suggested that determination of cell fate is based on not
only master transcription factors but also epigenetic histone modifications.
Key Words : Vascular endothelial cells, histone modification, transcription factors

Introduction
Until now, many studies of vascular development have consisted of gene knockout and knockdown
experiments using mice and zebrafish(1). Although these works led to the new discoveries of vasculardevelopment in mammals, they could not sufficiently identify the precise molecular cascade on thevascular endothelial cell (ECs) differentiation. Therefore, our aim is to establish the more efficientand practical ECs differentiation system from embryonic stem (ES) or induced pluripotent stem
(iPS) cells. On this purpose, we systemically analyze transcriptome and epigenetic status during
ECs differentiation in detail.

Results
1. Transcriptome analysis during vascular endothelial cells differentiation
Firstly, we changed culture medium without LIF (Leukemia Inhibitory Factor) for 96 hours inorder to differentiate from mouse ES cells, then Flk (VEGF, vascular endothelial cell growth
factors, receptor 2) -positive mesoderm cells are sorted by MACS using anti-Flk antibody. If
these cells are stimulated by VEGF (50 ng/ml), cells have commitment to ECs (2). On the other
hand, cells differentiate to smooth muscle cells (SMCs) without VEGF stimuli.
To elucidate comprehensive gene expression profiles during ECs or SMCs differentiation, we
performed sequential microarrays 6,12,24 and 48 hours after with or without VEGF stimulation.
As a result, after 6 hours with VEGF stimulation, Tal1 and Etv2 are induced, which areconsistent of other reports(3). In addition, 12 hours after VEGF stimulation Gata2, Sox7, Sox18
and Fli1 were induced. These genes are members of transcription factors. In contrast, Pecam1
and VE-Cadherin, which are endothelial cell specific markers, were induced after 48 hours when

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these cells were considered to become endothelial cells.
These results suggested that specific transcription factors in nuclei are firstly induced, thenspecific cell surface markers are induced when undifferentiated cells have commitment tospecific-lineages.
2. Epigenetic landscape during ECs differentiation
Next, in order to depict epigenetic landscape about vascular development, we conducted ChIPseq (Chromatin immunoprecipitation with next generation sequencing) using H3K4me3 andH3K27me3 specific antibodies and FAIRE (Formaldehyde-Assisted Isolation of Regulatory
Elements) -seq for open chromatin regions. In general, H3K4me3 or H3K27me3 modifications
have correlation with active or repressive gene expression, respectively. In addition, double
positive genes (both H3K4me3 and H3K27me3) are called as bivalent genes, which tend to bemaster regulators for specific-lineage differentiation(4). From sequential ChIP-seq data, we foundout some bivalent genes under VEGF condition such as Etv2, Gata2, and Sox18, which are upregulated at early time points.
Next, to classify the histone modification
p a t t e r n s , we s el e c t e d a b o u t 2 0 0 V E G F
responsive genes (over 2 fold expression levelwith VEGF compared to the condition without
VEGF). Then we clustered based on tag counts
obtained from ChIP-seq for each gene (fig.1).
As shown in figure 1, genes belonging to thecluster 3 are VEGF-induced bivalent genes,
including Gata2, Sox17, Sox18, Tal1 and Sox7.
These cluster genes are transcription factors andhave specific histone modification patterns. Firstly these genes are repressed with H3K27me3
modification before VEGF stimulation. Secondly, after 6 hours VEGF stimulation, H3K27me3
signal didn’t reduce so much and almost no signals of H3K4me3 are identified. Third, after 48
hours VEGF stimulation, strong H3K4me3 signals are detected, which contributed to be bivalentstatus.
Finally, to test whether above bivalent genes (Gata2, Fli1, Sox7 and Sox18) have important roles
on vascular ECs differentiation, we transfected si-RNA and investigated the effect of efficiency
about ECs differentiation. As a result, Gata2 knockdown significantly reduced the expression ofECs specific marker genes and knockdown of 4 transcription factors lead to over 60 % reduction(fig.2). Especially, we have reported GATA2 is indispensable for the maintenance of matured
ECs specificity(5), so, taken together we can think that Gata2 is important transcription factor forboth differentiation and maintenance of vascular ECs.

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Discussion & Conclusion
In this repor t, we demonstrated powerful tools todetermine the candidates of master transcription factors
about specific lineage cell differentiation.
Until now, although transcription factor Etv2 has beenreported to be important for ECs differentiation during
vasculogenesis (6) , precise epigenetic mechanism andcombination of other transcription factor network havebeen largely unknown.
Transcription factor Gata2 belongs to be an evolutionallyconserved zinc finger protein family. In addition, it has been already generally appreciated frommany works that Gata2 is important for the maintenance and proliferation of hematopoietic stem
cells and megakaryocytes(7). In this report, we demonstrated that Gata2 is also critical for ECs
differentiation and we can speculate cell fate between blood cells and endothelial cells depends on
the partner proteins of Gata2.
Recently, we have reported that GATA2 has interaction with Ets family protein and AP1 in the
differentiated human dermal microvascular endothelial cells from ChIP-seq data(5). Taken together,
it is possible that the differentiation and maintenance of ECs are dependent of protein-protein
interaction between Gata2 and Ets or Sox family transcription factors.
In future, further analyses such as co-immunoprecipitation or mass spectrometry during ECs
differentiation would give insights into precise mechanism of ECs specificity.

References
1 De Val, S. Black, B. L. Dev Cell. 2009 Feb;16(2):180-95.
2 Yamashita, J. Itoh, H. Hirashima, M. Ogawa, M. Nishikawa, S. Yurugi, T. Naito, M. Nakao, K.
Nishikawa, S. Nature. 2000 Nov 2;408(6808):92-6.
3 Bloor, A. J. Sanchez, M. J. Green, A. R. Gottgens, B. J Hematother Stem Cell Res. 2002
Apr;11(2):195-206.
4 Bernstein, B. E. Mikkelsen, T. S. Xie, X. Kamal, M. Huebert, D. J. Cuff, J. Fry, B. Meissner, A.
Wernig, M. Plath, K. Jaenisch, R. Wagschal, A. Feil, R. Schreiber, S. L. Lander, E. S. Cell. 2006
Apr 21;125(2):315-26.
5 Kanki, Y. Kohro, T. Jiang, S. Tsutsumi, S. Mimura, I. Suehiro, J. Wada, Y. Ohta, Y. Ihara, S.
Iwanari, H. Naito, M. Hamakubo, T. Aburatani, H. Kodama, T. Minami, T. EMBO J. 2011 Jun
10;30(13):2582-95.
6 Kataoka, H. Hayashi, M. Nakagawa, R. Tanaka, Y. Izumi, N. Nishikawa, S. Jakt, M. L. Tarui, H.
Nishikawa, S. Blood. 2011 Dec 22;118(26):6975-86.
7 Tsai, F. Y. Keller, G. Kuo, F. C. Weiss, M. Chen, J. Rosenblatt, M. Alt, F. W. Orkin, S. H. Nature.
1994 Sep 15;371(6494):221-6.

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一般の皆様へ本研究では、再生医療の鍵を握る血管内皮細胞に焦点をあて、その分化機構を転写因子とエピゲ
ノム修飾因子の視点から解析し、新たな分化制御因子を発見するのが目的であった。高効率の分化系を用いて、マイクロアレイと ChIP-seq の結果を統合し、分化の初期に細胞の運命を決定づける因子として、Gata2, Fli1, Sox7, Sox18 の 4 つを抽出した。この 4 因子で例えば iPS 細胞からの内皮分化が可能であるかどうか、今後の検討課題である。

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Essential roles of class II PI3-kinase C2α
in vascular formation and homeostasis.
Kazuaki Yoshioka
Department of Physiology, Kanazawa University School of Medicine
yoshioka@med.kanazawa-u.ac.jp

Abstract
We generated class II PI3K-C2α knockout (KO) mice, which were embryonic lethal due to severe
defects in angiogenesis. Inducible endothelial cell (EC)-specific C2α deleted mice exhibited
vascular barrier dysfunction. C2α KO mice were much more sensitive to challenge with ananaphylaxis mediator platelet-activating factor with increased lethality and chronic infusion ofangiotensin II with the formation of dissecting aneurysms. In EC, siRNA-mediated C2α knockdown
induced decreased PI(3)P+-endosomes, impaired endosomal trafficking, and defective delivery of
VE-cadherin to adherens junction. C2α knockdown also impeded cell signaling including VEGF
receptor-2 and S1P1 receptor internalization and Rho activation on the endosomes. Thus, our data
disclose the novel crucial functions of PI3K-C2α in barrier integrity and vascular formation andrepresents a new therapeutic target for vascular diseases.
Key Words : PI3 kinase, angiogenesis, dissecting aneurysm, vascular permeability, VE-cadherin

Introduction
BVascular barrier function, which is structurally supported mainly by the adherens junction
comprising VE-cadherin, maintains low vascular permeability in healthy vasculatures. The
assembly of the adherens junction is tightly controlled by intracellular signaling molecules including
Rho GTPases. Phosphatidylinositol (PI) 3-kinase (PI3K) family regulates diverse cellular functions;while class I PI3Ks and class III Vps34 are well-characterized, the physiological roles of PI3K class
II, which comprises C2α, C2β and C2γ and exclusively produces PI(3)P, remain largely unknown.

Results
The permeability of a monolayer of C2α-depleted HUVEC was increased under both resting and
VEGF-A-stimulated conditions compared with that of control
HUVEC (P < 0.05). VEGF-A induced a greater increase inleakage of intravenously administered Evans blue dye in theskin of C2α+/– mice (P < 0.05). We studied the vascular responses
to hyperpermeability- and inf lammation-eliciting insults inglobal C2α+/– and wild-type mice. Intravenous (i.v.) injectionof a low-dose of platelet-activating factor (PAF), a mediator ofanaphylactic shock, did not affect survival in wild-type mice- 118 -

whereas it induced death in all C2α+/– littermates within 40 min with increases in hematocrit andEvans blue leakage in the lung. A higher dose of PAF, which induced death only in a portion (about30%) of wild-type mice, caused more rapid death of all C2α+/– mice. PAF-induced increases inplasma histamine and IL-4, anaphylactic mediators, were similar in wild-type and C2α+/– mice,suggesting that C2α deficiency primarily impairs endothelial function, resulting in vascular barrier
disruption (Fig.1)1,3.
In C2α+/– mice, chronic Ang II infusion induced
more robust hyperpermeability in the aorta andcoronary vessels compared with that in wild-type
littermates. En face immunostaining using VEcadherin-specific antibody of the aorta showed
disorganization of adherens junctions in C2α+/– mice
given Ang II. Concomitantly, a higher incidence ofaortic aneurysms with dissection, resultant ruptureand death was observed in C2α+/– mice comparedwith wild-type littermates (aneurysm occurrence;
23/48 C2α +/– mice vs. 5/44 wild-type mice) (P <0.01, Fig.2). Conditional EC-specific deletion of C2αalso resulted in a higher occurrence of dissecting
aneurysms compared with control mice (aneurysm
occurrence; 5/20 C2αiΔEC mice vs. 1/13 control mice)(P < 0.01). There was no difference in blood pressure between C2α-deleted and control mice. Thus,
the aortic wall of C2α-deficient mice seems to be fragile compared with that of WT mice3.
AngII infusion stimulated MMP-2 and MMP-9 activities, which are implicated in the formation ofaneurysms, in the aortic tissue, with a greater increase in C2α+/– mice compared with wild-type mice
(P < 0.05). Immunostaining using Mac3-specific antibody, a macrophage marker, revealed a greaternumber of infiltrating macrophages, which are the major source of MMPs, mainly in the adventitia
of C2α+/– mice compared with wild-type mice.
In sc-siRNA-treated control HUVEC that were transfected with the PI(3)P-specific probe mRFPtagged 2×FYVE domain, the mRFP-2×FYVE signal was mainly localized in endosomes. C2αdepletion markedly reduced the number of mRFP-2×FYVE+-vesicles (P < 0.01), whereas PI3K
p110α- or Vps34-depletion did not alter the number of mRFP-2×FYVE+-vesicles. Thus, C2α has asignificant contribution to PI(3)P accumulation in the endosomal compartment.
In C2α-depleted, but not p110α- or Vps34-depleted, EC, the trafficking of VE-cadherin between
the TGN and the intercellular junctions at the plasma membrane was disrupted. Because C2α isnecessary for endosomal trafficking and endosomes are now recognized to be involved in theactivation of signaling molecules including Rho GTPases, we explored how C2α regulates RhoA
activation in cells by adopting a fluorescence resonance energy transfer (FRET) imaging technique.
RhoA was activated in both the intracellular vesicular compartment and the plasma membrane, with- 119 -

intense signals at cell-cell contacts in HUVEC. A
substantial portion of the intracellular FRET signal
coincided with 2×FYVE signals. C2α knockdown
inhibited the R hoA-FRET signal in both theendosomes and the plasma membrane, indicatingthat C2α is necessary for RhoA activation in PI(3)P-enriched endosomes and the plasma membrane(Fig. 3)2,3.
We further studied the role of C2α in VEGF
receptor inter nalization, which could be an
event upstream of RhoA activation. VEGF-A
stimulated the internalization of VEGFR2 into
the 2×FYVE+-vesicular compartment, which wasdampened by C2α depletion. VEGF-A stimulationinduced phosphor ylation of VEGFR 2 at theplasma membrane in HUVEC at 2 min, and most
phosphorylated VEGFR2 was re-distributed in
the intracellular compartment, mainly in EEA1+endosomes, at 30 min. C2α depletion did not inhibit
the initial VEGFR2 phosphorylation in the plasma membrane at the early time point but inhibitedthe internalization of phosphorylated VEGFR2.

Discussion & Conclusion
Our data show that C2α is essential for maintaining barrier integrity in quiescent vessels as
evidenced by its protective role against VEGF-A-induced hyperpermeability. In anaphylaxis, a
50% reduction in C2α expression markedly increased mortality. In chronic vascular injury inducedby Ang II infusion, disruption of the vascular wall occurs, leading to fatal dissecting aneurysm
formation. C2α also likely plays a role in maintaining EC survival and vascular stability in quiescent
vessels. Collectively, our data are consistent with the notion that a normal level of C2α expression isessential for the maintenance of vascular
integrity in quiescent vasculature aswell as in neovesssel formation. Theseobservations point to the possibility
that C2α may become a new therapeutictarget for vascular diseases caused bybarrier disruption (Fig. 4).

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References
1 Cui H, Okamoto Y, Yoshioka K, Du W, Takuwa N, Zhang W, Asano M, Shibamoto T and
Takuwa Y. (2013) Sphingosine-1-phosphate receptor-2 protects against anaphylactic shock
through suppression of eNOS in mice. J. Allergy Clin. Immunol. 132(5), 1205-1214.
2 Biswas K, Yoshioka K, Asanuma K, Okamoto Y, Takuwa N, Sasaki T, and Takuwa Y. (2013)
Essential role of class II PI3K-C2α in sphingosine-1-phosphate receptor-1 mediated signaling andmigration in endothelial cells. J. Biol. Chem. 288(4) 2325-2339.
3 Yoshioka K, Yoshida K, Cui H, Wakayama T, Takuwa N, Okamoto Y, Du W, Qi X, Asanuma
K, Sugihara K, Aki S, Miyazawa H, Biswas K, Nagakura C, Ueno M, Iseki S, Schwartz RJ,
Okamoto H, Sasaki T, Matsui O, Asano M, Adams RH, Takakura N, and Takuwa Y. (2012)
Endothelial PI3K-C2α, a class II PI3K, has an essential role in angiogenesis and vascular barrier
function. Nature Med. 18(10) 1560-1569.
4 Bruce CR, Risis S, Babb JR, Yang C, Kowalski GM, Selathurai A, Lee-Young RS, Weir JM,
Yoshioka K, Takuwa Y, Meikle PJ, Pitson SM and Febbraio MA, Overexpression of sphingosine
kinase 1 prevents ceramide accumulation and ameliorates muscle insulin resistance in high fatfed mice. (2012) Diabetes, 61, 3148-3155.
5 Fujii M, Inoki I, Saga M, Morikawa N, Arakawa K, Inaba S, Yoshioka K, Konoshita T, Miyamori
I., Aldosterone inhibits endothelial morphogenesis and angiogenesis through the downregulationof vascular endothelial growth factor receptor-2 expression subsequent to peroxisome proliferatoractivated receptor gamma. (2012) J. Steroid Biochem. Mol. Biol. 129 (3-5), 145-152.

一般の皆様へ
今回私達は、これまで全くと言っていいほど生理的役割が不明であったクラス IIα 型 PI3 キナーゼ
酵素（PI3K-C2α）の遺伝子欠損マウスを用いた実験から、発生期の血管新生、生後の病的血管新生（虚血、腫瘍）及び血管恒常性維持に PI3K-C2α が重要な役割を果たすことを明らかにしました。
PI3K-C2α 欠損マウスでは、アンジオテンシン II 投与に対する解離性大動脈瘤形成の発症率上昇を引き起こしました。したがって、PI3K-C2α は血管形成と血管障壁の健全性において重要な役割を担っていて、血管系疾患の新しい治療標的となる事が期待されます。

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Mechanisms of Eph family receptor-mediated regulationof bone mineralization
Koichi Matsuo
School of Medicine, Keio University
matsuo@z7.keio.jp

Abstract
Our data reveal that lack or inhibition of EphA2 tyrosine kinase receptor enhances osteoblastic
bone mineralization in mice and in bone organ culture. Significantly, inhibition of EphA2 signaling
induces secretion of large matrix vesicles.
Key Words : bone, osteoblast, mineralization, matrix vesicle

Introduction
Bone matrix consists of mineral and organic components and contributes to mechanical strengthand mineral metabolism. Although osteoblasts are cells responsible for bone formation, how they
control mineralization is still largely unknown. In this study, we assessed the function of EphA2
receptor tyrosine kinase (Irie et al, 2009; Otaki and Matsuo, 2012) on osteoblasts in the process ofbone mineralization.

Results
Lack of EphA2 results in hypermineralization in culture and in mice.
In cell culture, loss or inhibition of EphA2 enhanced osteoblast differentiation and calcium
deposition by mature osteoblasts. In bone organ culture, treatment with EphA2 inhibitors
enhanced calcium uptake from the culture medium into bone and increased bone mineral density.
Furthermore, mice lacking EphA2 showed elevated bone mass and hypermineralization.
Lack of EphA2 enlarges matrix vesicles, exptracellular vesicles containing calcium phosphate.
To determine cellular mechanisms underlying hypermineralization, we examined osteoblasts andmineralizing bone matrix using scanning electron microscopy of adult trabecular bone. Various
sizes of matrix vesicle-like vesicles were identified in the extracellular space. Interestingly, larger
mineralized nodules were more readily observed in Epha2-/- than in wild-type mice. To reveal
the source of these large mineralized nodules we examined cultured osteoblasts by scanningelectron microscopy. Consistent with previous reports about matrix vesicles (Xiao et al., 2007),the diameter of the vesicles produced by differentiated wild-type osteoblasts was approximately
100 nm, and we did not observe such vesicles on undifferentiated osteoblasts. Strikingly, vesicles
produced by differentiated Epha2-/- osteoblasts were larger than those produced by wild-type
controls. The percentage of vesicles greater than 250 nm in diameter was significantly higher in
Epha2-/- osteoblasts compared with wild-type controls. Energy dispersive X-ray spectrometry
detected calcium and phosphorus elements in vesicles produced by both wild-type and Epha2-/-- 122 -

mature osteoblasts, suggesting that these vesicles are matrix vesicles or mineralized nodules. For
simplicity, we call these calcium phosphate-containing vesicles collectively as matrix vesicles in thisstudy. Moreover, large matrix vesicles were also observed on differentiated osteoblasts treated withcompound 1 [2-hydroxy-4-(2,5-dimethyl-1H-pyrrol-1-yl)benzoic acid] or compound 2 [2-hydroxy-5(2,5-dimethyl-1H-pyrrol-1-yl)benzoic acid] for the last three days of osteoblastogenic culture. These
data suggest that loss or inhibition of EphA2 receptors promotes enlargement of matrix vesicles andconsequently enhances mineral deposition by osteoblasts.
EphA2 inhibition increases the proportion of large matrix vesicles showing high alkaline
phosphatase activity
To more closely examine their physical and biochemical properties, we isolated matrix vesicles
from mature osteoblast cultures treated with or without compound 1 or compound 2. As expected,
scanning electron microscopy analysis revealed that matrix vesicles isolated from mature osteoblast
cultures not treated with inhibitors were approximately 100 nm in diameter, and those particles
were absent in immature osteoblast cultures. Flow cytometry analysis show that such microvesicles(100 nm – 1 μm) are beyond the size resolution of “forward scatter”, but that “side scatter” is amore sensitive means of detecting vesicles of this size (Dragovic et al., 2011). We confirmed thesize of isolated matrix vesicles using reference beads in diameter 100 nm for the flow cytometry
analysis. Mature osteoblasts cultured in the presence of compound 2 for three days produced ahigher percentage of matrix vesicles with high side scatter. matrix vesicle side scatter was positively
correlated with alkaline phosphatase activity in matrix vesicles from mature osteoblast cultures bothin the presence or absence of compound 2. Remarkably, the population of large-sized matrix vesicles
derived from compound 2-treated osteoblasts showed high alkaline phosphatase activity, suggestingthat increased size and alkaline phosphatase activity of matrix vesicles due to EphA2 inhibition
underlies enhanced bone mineralization.

Discussion & Conclusion
A highly significant finding reported here using scanning electron microscopy and f low
cytometry is that matrix vesicles derived from EphA2-deficient mature osteoblasts or from wildtype osteoblasts treated with EphA2 inhibitors are larger than controls. Furthermore, larger matrix
vesicles contain higher alkaline phosphatase activity, which explains enhanced mineralization.
Since EphA2 protein was present in isolated matrix vesicles, it will be of interest to determine
whether EphA2 functions in matrix vesicles, or directly regulates the membrane extrusion of“budding matrix vesicles”. There are reports that ephrins and Ephs regulate endocytosis (Pitulescu
and Adams, 2010) and membrane protrusion in response to chemoattractive factors in invading
cancer cells (Hiramoto-Yamaki et al., 2010). Therefore, EphA2 might regulate the structure of themembrane facing the bone surface, vesicle protrusion, and/or mineralization activity of released
matrix vesicles.

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References
1. Dragovic, R.A., C. Gardiner, A.S. Brooks, D.S. Tannetta, D.J. Ferguson, P. Hole, B. Carr, C.W.
Redman, A.L. Harris, P.J. Dobson, P. Harrison, and I.L. Sargent. 2011. Sizing and phenotyping ofcellulars vesicles using Nanoparticle Tracking Analysis. Nanomedicine.
2. Hiramoto-Yamaki, N., S. Takeuchi, S. Ueda, K. Harada, S. Fujimoto, M. Negishi, and H. Katoh.
2010. Ephexin4 and EphA2 mediate cell migration through a RhoG-dependent mechanism. J Cell
Biol 190:461-477.
3. Irie, N., Y. Takada, Y. Watanabe, Y. Matsuzaki, C. Naruse, M. Asano, Y. Iwakura, T. Suda, andK. Matsuo. 2009. Bidirectional signaling through ephrinA2-EphA2 enhances osteoclastogenesis
and suppresses osteoblastogenesis. J Biol Chem 284:14637-14644.
4. Matsuo, K. and Otaki, N. (2012) Bone cell interactions through Eph-ephrin: Bone modeling,
remodeling and associated diseases. Cell Adh. Migr. 6, 148-156.
5. Xiao, Z., C.E. Camalier, K. Nagashima, K.C. Chan, D.A. Lucas, M.J. de la Cruz, M. Gignac,
S. Lockett, H.J. Issaq, T.D. Veenstra, T.P. Conrads, and G.R. Beck, Jr. 2007. Analysis of theextracellular matrix vesicle proteome in mineralizing osteoblasts. J Cell Physiol 210:325-335.
6. Zhao, C., N. Irie, Y. Takada, K. Shimoda, T. Miyamoto, T. Nishiwaki, T. Suda, and K. Matsuo.
2006. Bidirectional ephrinB2-EphB4 signaling controls bone homeostasis. Cell Metab 4:111-121.

一般の皆様へ
骨は生命の基盤であり、力学的強度を持ち、ミネラルの代謝を担っています。骨を作る細胞は、骨芽細胞と呼ばれ、コラーゲン線維とミネラル成分とを分泌します。この骨が硬くなる「石灰化」の過程がどのように調節されているかについては不明の点が多いです。本研究では、チロシンキナーゼ型受容体のひとつ EphA2 を欠損するマウスや、EphA2 に対する阻害剤を加えた骨芽細胞では、ミネ
ラル成分を含む基質小胞の作用で、石灰化が促進することを示すデータが得られました。

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III.
Reports from the Recipients of Grants
for International Meetings- 125 -

Joint meeting :The 76th Annual Meeting of Japanese Society of Interferon and Cytokine Research
The 21th International Symposium on Molecular and Cell Biology of Macrophages
1. Representative
Akihiko Yoshimura, PhD
Professor Keio University School of Medicine
2. Opening period and Place
5/20-5/21, 2013
Toshi Center Hotel, Tokyo
3. Number of participants / Number of participating countries and areas
250 individuals from 8 countries
4. Total cost
￥10,620,000JPY
5. Main use of subsidy
Traveling costs for invited guest speakers and part of the room renting costs
6. Result and Impression
The theme of the meeting was “Cross-talk between innate and adaptive immunity: network
among macrophages, dendritic cells and helper T cells”. This was the second joint meeting andin this meeting we have more participants and posters than in the last one. We had more than 20
lectures and 65 posters. Participants discussed important findings on cross-talk among players
in innate and acquired immunity, and showed successful clinical applications of interferons andcytokines. We also selected young investigators for their excellent presentation and gave themprizes. Undoubtedly this meeting was so successful and we believe all participants enjoyedscience and developed interdisciplinary interactions and friendship.
7. Additional description
I would like to express our sincere thanks to The NOVARTIS Foundation for the financial
supports to our meeting.

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The 3rd International Moyamoya Meeting
1. Representative
Kiyohiro Houkin, MD
2. Opening period and Place
July 12th-13th , 2013
Sapporo, Japan
3. Number of participants / Number of participating countries and areas
100 participants from 5 countries
4. Total cost
￥800,0000JPY
5. Main use of subsidy
Honorarium for invited speakers
6. Result and Impression
Every participant brought data regarding the most recent advancement in research of theirrespective field.

This biannual meeting enabled us confirming our progress and guided our

future direction of the research.
7. Additional description
The proposal of international cooperative study has been brought to host university (Hokkaido
University) soon after this meeting. This is also one important aspect to hold an international
meeting.

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The 4th International Symposium on Dynamics of Mitochondria (DynaMito2013)
1. Representative
Koji Okamoto
Associate Professor, Graduate School of Frontier Biosciences, Osaka University
DynaMito2013, Chair
2. Opening period and PlaceOct 28 (Mon) – Nov 1 (Fri), 2013
Okinawa Zanpamisaki Royal Hotel, Okinawa, Japan
3. Number of participants / Number of participating countries and areas
Number of participants: 220
Number of participating countries and areas: 22
4. Total cost
￥25,070,000JPY
5. Main use of subsidy
Conference expenses
6. Result and Impression
Mitochondria are the power plants that produce most of ATP in eukaryotic cells. Studies in
the last decade have revealed that the energy-converting organelles frequently fuse, divide,and move, and that these dynamic behaviors are critical for shaping themselves and controlling
their quality. In addition, mitochondrial dynamics plays vital roles in metabolism, cell death,signaling, development, differentiation, and innate immunity. Strikingly, defects in mitochondrial
dynamics are associated with various human disorders such as neurodegeneration, diabetes,
cancer metastasis, and ageing, raising the possibility that mitochondrial regulators may serve
as novel targets for therapeutic approaches. DynaMito2013 provided an international platform
focusing on mitochondrial dynamics thereby allowing us to share and discuss new data towards
understanding of mitochondrial function, physiology, and pathogenesis.
7. Additional description
We had 62 graduate student attending DynaMito2013 including those giving highly intensive
oral and poster presentations and being awarded for Young Investigator Scholarships, Poster
Presentations, and Best Questions.

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The 8th International Conference on Cutting-Edge Organic Chemistry in Asia
The 4th New Phase International Conference on Cutting-Edge Organic Chemistry in Asia

1. Representative
Michio Murata (Ph.D)
Professor, Department of Chemistry, Osaka University
Atsushi Nishida (Ph.D)
Professor, Department of Pharmaceutical Science, Chiba University
2. Opening period and Place
November 25-28th, 2013
Osaka International Convention Center, Nakanoshima 5-3-51, Kita-ku, Osaka 530-0005
Rihga Royal Hotel Osaka, Nakanoshima 5-3-68, Kita-ku, Osaka 530-0005
3. Number of participants / Number of participating countries and areas
Participants; 205(China, Hong Kong, Japan, Korea, Malaysia, Philippine, Singapore, Thailand, Taiwan)
4. Total cost
￥20,657,200JPY
5. Main use of subsidy
Conference room charge
6. Result and Impression
During the conference periods, 1 key note lecture, 27 oral presentations and 127 poster
presentations had been held. Because all presenters are nominated by Coordinators of eachcountry, the scientific sessions covered broad aria of organic chemistry with high level of science.
Our social events, friendship dinners on day1 and 2 were very efficient to make an opportunity
to develop deep and warm personal relationship with each other from different countries. Total
83 lectureship awardees were nominated by the Coordinators of each country, who will join
lectureship tour in the selected countries.

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7. Additional description
The next ICCEOCA conference will be held in Malaysia, in 2014.

- 130 -

“New Perspectives of Molecular Imaging on Alzheimer’s disease”:
A Satellite Symposium of the 87th Annual Meeting of The Japanese Pharmacological Society

1. Representative
Kazuhiko Yanai
Professor, Department of Pharmacology, Tohoku University School of Medicine
2. Opening period and PlaceMarch 21, 2014
Tohoku University Centennial Hall, Sendai, Japan
3. Number of participants / Number of participating countries and areas
100 participants from 4 countries (Japan, Australia, France, Cuba)
4. Total cost
￥2,500,000JPY
5. Main use of subsidy
Facility rental fee and poster printing fee
6. Result and Impression
The 87th Annual Meeting of the Japanese Pharmacological Society was held in March 19-21 in
Sendai (Tohoku University Centennial Hall and Sendai International Center). In this meeting, we
held total 40 symposia that focused on various research themes including translational research,
imaging, regenerative medicine and structural biology. In this satellite symposium, 2 overseas
and 2 Japanese speakers introduced recent progress in the development of molecular imaging
biomarker in Alzheimer’s disease and its clinical application for early detection of disease-related
pathology in the brain. There was active discussion among the participants. They seem to besatisfied with the contents of this symposium.
7. Additional description
We have a plan to publish the contents of this symposium as a review article for the Journal of
Pharmacological Sciences.

- 131 -

27th Grant Report (FY2013)
The foundation has been conducting public interest activities such as research grant, meeting
grant and international exchange programs since its establishment on Sep. 4, 1987 in Japan underauthorization of the Ministry of Education, Science, Sports and Culture, followed by a transition toa public interest incorporated foundation on Apr. 1, 2012. The grants conducted in FY 2013 are as
follows.
27th Novartis Research Grant: 42 Researchers (JPY 1 mil.),		 Subtotal JPY 42 mil.
Research Meeting Grant:

5 Meetings (JPY 0.4 mil.),		

		

Subtotal JPY 2 mil.

Total

JPY 44 mil.

27th Novartis Research Grant (FY2013)
The grant is to aim supporting creative research in Japan in the field of bio, life science and relevant
chemistry. The 42 grantees are as follows.
#

Name

Institution
Tokyo Metropolitan
1 Kouji Hirota
University, Graduate Schoolof Science and Engineering
Ube National College ofDepartment of
2 Katsuya Shimabukuro Technology,
Chemical and BiologicalEngineering

Title

Research Project

Professor

Multipathway analysis of DNA repair usingchemical genetics approach

Lecturer

Nano correlative atomic force and electron
microscopy toward application to complex
biological systems

3 Takashi Okamoto

Nagoya City University,
Graduate School of Medical
Sciences

Professor

4 Masanobu Kawai

Osaka Medical Center
and Research Institute forMaternal and Child Health

Chief
Scientist

Nagoya City University,
Graduate School of Medical
Sciences
Hokkaido University,
Research Faculty ofAgriculture
Nagasaki University,
Graduate School of
Biomedical Sciences
Tokyo Medical and Dental
University, Medical ResearchInstitute

Assistant
Professor

Identification of interacting partner forthe p65 subunit of inducible transcription
factor NF-κB and development of novel
therapy using protein-protein interaction as
the direct target
Characterization of the role of brown
adipose tissue (BAT) dysfunction in thedevelopment of osteoporosis: Implicationof BAT transplantation as a strategy for thetreatment of osteoporosis
Termination of neuronal migration
regulated by morphological control of
migrating neurons in the postnatal brain

Lecturer

Molecular mechanism of salicylic acidinduced resistance against viruses in plants

Professor

Elucidation of phospho-signaling regulating
mitochondrial stress response

Associate
Professor

Molecular mechanism of malignant tumor
formation during chronic hypoxia

5 Masato Sawada
6 Kenji Nakahara
7 Kohsuke Takeda
8 Koh Nakayama
9 Tsukasa Okiyoneda

Kwansei Gakuin University,
Department of Bioscience

10 Takumi Watanabe

Institute of Microbial
Chemistry

11 Koji Hirano

Osaka University, Graduate
School of Engineering

12 Tomomi Kiyomitsu

Nagoya University, Graduate
School of Science

Identification of ubiquitin proteaseresponsible for peripheral protein quality
control
Development of novel leads based on
catalytic asymmetric synthesis: WecA
Chief
for anti-XDR-TB agent andResearcher inhibitors
cancer-stroma interaction disruptor for
anticancer agent
Discoveryof New Drug Candidates Based
Assistant
Development of Site-selective Direct
Professor on
Functionalization of Pyridones
Asymmetric Membrane
Assistant Research onCriticalfor Symmetric Cell
Professor Elongation
Division
Associate
Professor- 132 -#Name

13 Koichi Homma
14 Takuro Niidome

Institution
Teikyo University, Faculty of
Pharmaceutical Sciences
Kumamoto University,
Graduate School of Scienceand Technology

Title
Professor

Research Project”Memory priming” that determines thestart of the critical period for learning

Professor

Development of transdermal vaccination
system controlled by near infrared light

15 Shizue Ohsawa

Kyoto University, Graduate
School of Biostudies

Associate
Professor

16 Ken Natsuga

Hokkaido University
Hospital
Osaka University, Graduate
School of Dentistry

Assistant
Professor
Assistant
Professor

18 Ryo Nasu

Kyoto Prefectural Universityof Medicine

Assistant
Professor

19 Tetsufumi Ito
20 Yuichi Wakana

University of Fukui, Facultyof Medical Sciences
Tokyo University of
Pharmacy and Life Sciences

Assistant
Professor
Assistant
Professor

21 Beate Heissig

The University of Tokyo, The
Institute of Medical Science

Associate
Professor

22 Noriyuki Ouchi

Nagoya University, Graduate
School of Medicine

Professor

23 Koji Tamada

Yamaguchi University,
Graduate School of Medicine

Professor

24 Kiyotake Suenaga

Keio University, Faculty of
Science and Technology
National Institute ofAdvanced Industrial Scienceand Technology
Toho University, School of
Medicine
Kyushu University, Graduate
School of Engineering
Kumamoto University,
Graduate School of Science
Technology

Associate
Professor

Dissecting the mechanism of cellularsenescence that regulates tumor
microenvironment
Research on physiological and pathologicalroles of skin microbiome
The role of sialidase in Group B
Streptococcus pathogenesis
Development of novel treatment strategies
targeting the R-spondin-LGR5 axis of brain
tumor stem cells
Elucidating how the auditory information
is integrated by morphological techniques
CARTS-mediated protein transport fromthe Golgi complex to the cell surface
Membrane type-1 matrix metalloproteinasein multiple myeloma: a novel modulator of
the microenvironment
Research on the role of the novel
adipocytokine in heart disease
Development of cancer immunotherapy by
gene-modified T cells harboring efficient invivo survival potential
The apoptosis-inducing mechanism ofbiselyngbyaside, a marine macrolide

SeniorScientist

Functional analysis of Wnt3 factor
regulating adult stem cells

Associate
Professor

Okazaki Institute forIntegrative Bioscience

Professor

Functional analysis of a novel suppressive
factor at fertilization
Development of tubular construct based on
heparin-collagen conjugate
Analysis of non-coding RNA-Proteinsinteraction regulating sister chromatid
cohesion
Research on the molecular mechanism
underlying activation of purinergic
P2Y6 receptors and its application to thetreatment of heart failure

17 Masaya Yamaguchi

25 Tomoko Kuwabara
26 Naofumi Miwa
27 Hiroyuki Ijima
28 Takashi Ideue

29 Motohiro Nishida

30 Takeshi Takarada
31 Kohki Kawane
32 Yoshinori Fukui
33 Kyoji Horie
34 Asako Sugimoto
35 Masashi Toyoda
36 Tsuyoshi Miyakawa

Kanazawa University,
Institute of Medical,
Pharmaceutical and Health
Sciences
Kyoto University, Graduate
School of Medicine
Kyushu University, Medical
Institute of Bioregulation
Nara Medical University,
School of Medicine
Tohoku University, Graduate
School of Life Sciences
Tokyo Metropolitan Instituteof Gerontrogy
Fujita Health University,
Institute for Comprehensive
Medical Science

Professor
Assistant
Professor

Assistant
Professor

Potential of mesenchymal stem cell as drug
targetAssistant
Professor

The molecular mechanism of celldelamination, a novel mode of cell deathElucidation of the novel signal network that
regulates trafficking of secretory lysosomes
Genetic analyses of regulatory network ofpluripotency in embryonic stem cells
Analysis of the centrosome-independent
assembly of female meiotic spindles
Research on glycoconjugate regulation instem cell homeostasis
Identification of schizophrenia-likephenotypes by temporal regulation in
calcineurin function in mice
Synthesis of carbon-dioxide-derived novel
environment-conscious amphiphilic graft
copolymers and construction of theirmolecular assemblies
The Identification of Exosomal Micro
RNA Specific for the Recurrence of
Hepatocellular Carcinoma

Professor
Professor
Professor
Theme
LeaderProfessor

37 Satoshi Honda

Tokyo University of Science,
Department of Industrial
Chemistry

Assistant
Professor

38 Keishi Sugimachi

Kyushu University Beppu
Hospital

Associate
Professor- 133 -

#Name

39 Shinya Ohta
40 Kenji Kurokawa

Institution
Kochi University, Centre forInnovative and Translational
Medicine
Nagasaki International
University, Faculty of
Pharmaceutical Sciences

University of
41 Yoshitaka Hamashima Shizuoka,School of
Pharmaceutical Sciences

42 Yuuki Obata

Title
Assistant
Professor

Research ProjectProteomics analysis of subdomain specificPTM (Post translational modification)
proteome in the mitotic chromosome

Associate
Professor

Research on post-translational modification
process of bacterial lipoproteins

Professor

Tokyo University of Science,
Research Institute for
Biomedical Sciences

Assistant
Professor

Development of a novel catalytic
asymmetric method for the incorporationof a biologically significant trifluoromethyl
group
Spatiotemporal analysis of oncogenic
signaling by Kit tyrosine kinase 〜
Mechanism of autonomous proliferation inmastocytosis and gastrointestinal stromal
tumor (GIST) 〜

FY 2013 Research Meeting Grant
The grant is to aim supporting international research meetings in Japan in the field of bio, life
science and relevant chemistry. The 5 grantees are as follows.
Date(Place)Institution / Title

Name

Joint meeting of The 76th Annual
Meeting of Japanese Society ofand Cytokine Research and
1 Interferon
The 21th International Symposium
on Molecular and Cell Biology of
Macrophages

2013.5.20-21(Tokyo)Keio University, Schoolof Medicine / Professor

Akihiko Yoshimura

2 3rd International Moyamoya Meeting2013.7.12-13(Sapporo)

International Symposium on
3 4th
Dynamics of Mitochondria

2013.10.28-11.1(Okinawa)

8th International Conference on
4 The
Cutting-Edge Organic Chemistry in Asia

2013.11.25-28(Osaka)Hokkaido Univerisity,
Graduate School of
Medicine / Professor
Osaka University,
Graduate School of
Frontier Biosciences /Associate Professor
Osaka University,
Graduate School of
Science / Professor"New Perspectives of Molecular Imaging
on Neurodegenerative disorders" : A
5 Satellite Symposium of the 87th Annual
Meeting of the Japanese PharmacologicalSociety

2014.3.19-20(Sendai)Tohoku University,
School of Medicine /Professor#

Meeting- 134 -

Kiyohiro Houkin

Koji Okamoto

Michio Murata

Kazuhiko Yanai

第 27 期（2013 年度）助成事業報告
当財団は、文部大臣の認可を得て 1987 年 9 月 4 日に設立されて以来、研究助成を中心とした公益事業を行って来ました。2012 年 4 月 1 日には、制度改革に伴い、公益財団法人へ移行しております。2013 年度は、下記の総額 4,400 万円の助成事業を実施しました。
第 27 回ノバルティス研究奨励金
研究集会助成42 件 （1 件 100 万円）4,200 万円
5 件 （1 件 40 万円） 200 万円
総額 4,400 万円

第 27 回ノバルティス研究奨励金 (2013 年度 )この事業は、生物・生命科学および関連する化学の領域において、我が国で行われる創造的な研究の助成を目的としています。2013 年度は 42 件の助成を行いました。
（受付順 ・ 敬称略、 所属 ・ 職位は申請時、 贈呈額は 1 件 100 万円）
No

氏名

所属

職位

研究課題
化学遺伝学アプローチによる DNA 修復
1 廣 田 耕 志 首都大学東京理工学研究科
教授
マルチパスウェー解析
宇部工業高等専門学校
複雑な生命系に利用できるナノ相関顕微
2 島袋 勝弥
講師
物質工学科
鏡の開発
誘導型転写因子 NF-κB の p65 サブユニッ
名古屋市立大学大学院
ト蛋白と相互作用する宿主因子の同定と、
3 岡 本
尚
教授
医学研究科
蛋白分子間相互作用 (PPI) を標的とする悪性腫瘍に対する新たな治療戦略
褐色脂肪組織機能不全に伴う骨量減少分
大阪府立母子保健総合医療セ
主任
4 川井 正信
子機構の解明 ： 骨粗鬆症新規治療戦略とンター研究所
研究員
しての褐色脂肪細胞移植の有効性の検討
名古屋市立大学大学院
特任 Sema3E-PlexinD1 シグナルによる新生
5 澤田 雅人
医学研究科
助教 ニューロンの移動停止位置の制御機構
サリチル酸で誘導される植物のウイルス抵
6 中 原 健 二 北海道大学大学院農学研究院 講師
抗性の分子機構
ミトコンドリアのストレス応答を担うリン酸化7 武 田 弘 資 長崎大学薬学部教授
シグナルの解明
慢性的な低酸素環境が引き起こすがん
東京医科歯科大学
8 中 山
恒
准教授
悪性化の分子機構の解明
難治疾患研究所
形質膜タンパク質品質管理に関わるユビ
9 沖 米 田 司 関西学院大学理工学部准教授
キチンプロテアーゼの同定
触媒的不斉合成を基軸とした新しい医薬
微生物化学研究会
主席 リードの創製 ： WecA を阻害する抗超多10 渡 辺匠
微生物化学研究所
研究員 剤耐性結核剤， およびがん - 間質相互作用に働く抗がん剤
ピリドン類の位置選択的直接官能基化法
11 平 野 康 次 大阪大学大学院工学研究科
助教の開発に基づく医薬品候補化合物の創出
対称分裂を保障する非対称な細胞膜伸
12 清 光 智 美 名古屋大学大学院理学研究科
助教
長メカニズムの研究
学習能力を賦与する脳内分子機構 ： メモ
13 本 間 光 一 帝京大学薬学部教授
リープライミング- 135 -

No
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37

38

氏名

所属

職位

研究課題
近赤外光でコントロールする経皮ワクチン
新 留 琢 郎 熊本大学大学院自然科学研究科 教授
システムの開発
がん微小環境を制御する細胞老化の分
大澤 志津江 京都大学大学院生命科学研究科 講師
子基盤の解明皮膚マイクロビオームの生理学的 ・ 病理
夏 賀
健 北海道大学病院皮膚科
助教
学的意義の解明
シアル酸分 解酵素が B 群レンサ球菌の山 口 雅 也 大阪大学大学院歯学研究科
助教
病原性に果たす役割の解析
京都府立医科大学大学院
脳腫瘍幹細胞の R-spondin-LGR5 軸を
那 須
亮
助教
医学研究科
標的とした腫瘍根絶技術の開発
伊 藤 哲 史 福井大学医学部
助教 かたちから探る聴覚情報統合のしくみ
新規輸送キャリアー （CARTS） によるゴル
若 菜 裕 一 東京薬科大学生命科学部
助教
ジ体から細胞膜へのタンパク質輸送機構
多発性骨髄腫の微小環境における膜型
ベアーテ ハイジッヒ 東京大学医科学研究所准教授
マトリクスメタロプロテアーゼに関する研究
寄附講 新規アディポサイトカインの心臓病におけ
大 内 乗 有 名古屋大学大学院医学系研究科
座教授 る役割の解明
効率的な体内生存能を有する遺伝子改
玉 田 耕 治 山口大学大学院医学系研究科教授
変 T 細胞を用いたがん免疫療法の開発
海洋産マクロリド、 ビセリングビアサイドのア
末 永 聖 武 慶應義塾大学理工学部准教授
ポトーシス誘導機構
産業技術総合研究所
主任 成体幹細胞を制御する Wnt3 因子の機能桑原 知子
幹細胞工学研究センター
研究員 解析
三 輪 尚 史 東邦大学医学部
准教授 新規受精調節因子の機能解析
ヘパリン ‐ コラーゲンコンジュゲートを用い
井 嶋 博 之 九州大学大学院工学研究院
教授
た移植用管腔構造体の開発
姉妹染色体動態を制御するノンコーディン
井 手 上 賢 熊本大学大学院自然科学研究科 助教
グ RNA- タンパク間相互作用の解析
岡崎統合バイオサイエンスセンター
プリン作動性 P2Y6 受容体の作動機構の教授
西田 基宏
兼生理学研究所
解析と心不全治療への応用
間葉系幹細胞の創薬ターゲットとしての可宝 田 剛 志 金沢大学医薬保健研究域薬学系 助教
能性
特定 新たな細胞死様式である細胞脱落の分
川 根 公 樹 京都大学大学院医学研究科
助教 子機構の解明
分泌型リソソームの小胞輸送を制御する
福 井 宣 規 九州大学生体防御医学研究所教授
新規シグナルネットワークの解明
ES 細胞多能性制御ネットワークの遺伝学
堀 江 恭 二 奈良県立医科大学医学部
教授
的解析
東北大学大学院
卵細胞における中心体非依存的な紡錘杉本 亜砂子
教授
生命科学研究科
体構築メカニズムの解明
東京都健康長寿医療センター
研究 幹細胞の恒常性を制御する糖鎖探索とそ
豊田 雅士
研究所
副部長 の機能解析
藤田保健衛生大学
時期特異的なカルシニューリン機能制御教授
宮 川剛
総合医科学研究所
がもたらす統合失調症様表現型の同定
二酸化炭素を原料とする新規環境調和
本 多
智 東京理科大学工学部
助教 型両親媒性グラフト共重合体による分子集合体の構築
肝細胞癌再発を制御するエクソソーム内
診療准
杉 町 圭 史 九州大学病院別府病院外科
マイクロ RNA の同定と革新的な分子標的
教授
治療の開発

- 136 -

No

氏名39 太 田
40 黒 川41 濱 島42 小 幡所属

職位
研究課題
テニュア 分裂期染色体におけるサブドメイン特異トラック 的タンパク質翻訳後修飾のプロテオミクス
信 哉 高知大学教育研究部
特任助教 解析
細菌リポタンパク質の翻訳後修飾過程の健 児 長崎国際大学薬学部准教授
解明
医薬品合成を指向した触媒的不斉トリフ
義 隆 静岡県立大学薬学部教授
ルオロメチル化の開発研究
Kit チロシンキナーゼの細胞内ダイナミク
スと腫瘍化シグナリング 〜 マスト細胞腫
裕 希 東京理科大学生命医科学研究所 助教
瘍および消化管間質腫瘍の自律増殖機構の解明 〜2013 年度 研究集会助成
この事業は、生物・生命化学および関連する化学の領域において、我が国で開催される国際色豊かな研究集会の助成を目的としています。2013 年度は 5 件の助成を行いました。
（受付順・敬称略、所属・職位は申請時、贈呈額は 1 件 40 万円）
No

研究集会名

開催日
( 開催地 )

1

第 78 回日本インターフェロン ・サイトカイン学会 ・ 第 21 回マクロファージ分子細胞生物学国際シンポジウム ・ 合同学術集会

2013.5.20-21( 東京 )

慶應義塾大学医学部 ・ 教授 吉 村昭彦

2

第 3 回国際もやもや病会議

2013.7.12-13( 札幌 )

北海道大学大学院医学研
究科 ・ 教授

宝金清博

3

第 4 回ミトコンドリア ・ ダイナミクス
国際会議

2013.10.28-11.1 大阪大学大学院生命機能( 沖縄 )
研究科 ・ 教授

岡本

浩二

4

第 8 回アジア最先端有機化学国際会議

2013.11.25-28( 大阪 )

大阪大学大学院理学研究
科 ・ 教授村田道雄

5「神経変性疾患に関する分子イ
メージングの新しい展開」 第 87回日本薬理学会年会サテライト国
際シンポジウム2014.3.19-20( 仙台 )

東北大学大学院医学系研
究科 ・ 教授

谷内一彦- 137 -

所属・職位

氏名

27th Financial Report

Balance Sheet

As of March 31, 2014
Account

Movement of Net Assets(Unit: JP Yen)

From April 1st, 2013 to March 31, 2014
(Unit: JP Yen)

AmountAccount

I Assets

I General Net Assets Changes

1. Current Assets

1. Ordinary Income & Expenditure

Current Assets Total

35,743,523

2. Fixed Assets(1) Basic Fund
Basic Fund Total

1,100,000,000(2) Other Long-term Assets
Other Long-term Assets Total
Fixed Assets Total
Assets Total

Amount(1) Ordinary Income
Donation

40,050,000

Ordinary Income Total

62,650,150(2) Ordinary Expenditure
Project Expenses

54,198,562

Grant Expense(44,000,000)

1,180,014,182

Novartis Research Grant

42,000,000

1,215,757,705

Research Meeting Grant

2,000,000

80,014,182

II Liabilities

Administrative Expense

3,626,679

1. Current Liabilities

Ordinary Expenditure Total

57,825,241

42,057,874

Ordinary Balance of Current Period

4,824,909

42,057,874

2. Nonrecurring Profit & Loss

Current Liabilities Total
Liabilities Total
III Equity (Net Assets)

Nonrecurring Balance of Current Period

01. Designated Net Assets

General Net Assets Ending Balance

173,699,831

Designated Net Assets Total

1,000,000,000(Amount appropriating to Basic Fund)(1,000,000,000)

2. General Net Assets(Amount appropriating to Basic Fund)

173,699,831(100,000,000)

Equity Total (Net Assets)

1,173,699,831

Liabilities & Equity Total

1,215,757,705- 138 -II Designated Net Assets Changes
Designated Net Assets Change0

Designated Net Assets Ending Balance 1,000,000,000
III Net Assets Balance Ending Balance

1,173,699,831

第 27 期（2013 年度）財務報告

貸借対照表

正味財産増減計算書

2014 年 3 月 31 日現在（単位：円）

科

目

金

額

科

Ⅰ資産の部

目

決算額

Ⅰ一般正味財産増減の部

１． 流動資産
流動資産合計2013 年 4 月 1 日～ 2014 年 3 月 31 日
（単位：円）

１． 経常増減の部
35,743,523(1) 経常収益

２． 固定資産受取寄付金

40,050,000(1) 基本財産

経常収益計

62,650,150

基本財産合計1,100,000,000(2) その他固定資産
その他固定資産合計
固定資産合計
資産合計

事業費

54,198,562

支払助成金

44,000,000

1,180,014,182

ノバルティス研究奨励金

42,000,000

1,215,757,705

研究集会助成金

2,000,000

80,014,182

管理費

3,626,679

経常費用計

57,825,241

42,057,874

当期経常増減額

4,824,909

42,057,874

2． 経常外増減の部

Ⅱ負債の部
１． 流動負債
流動負債合計負債合計(2) 経常費用

当期経常外増減額

Ⅲ正味財産の部

一般正味財産期末残高

１． 指定正味財産0173,699,831

指定正味財産合計

1,000,000,000（うち基本財産への充当額）(1,000,000,000)

当期指定正味財産増減額

0173,699,831

指定正味財産期末残高

1,000,000,000２．一般正味財産（うち基本財産への充当額）
正味財産合計
負債及び正味財産合計(100,000,000)
1,173,699,831
1,215,757,705- 139 -

Ⅱ指定正味財産増減の部

Ⅲ正味財産期末残高

1,173,699,831

List of Board Members[Board of Trustees]

5 trustees, 2 auditors

As of Oct. 1, 2014

Post

Name

Chairman

Akimichi KANEKO

Dean, Professor, MD, Graduate School of Health Science, Kio
University; Emeritus Professor, Keio University

Shigetaka ASANO

Visiting Professor, MD, School of Medicine, Kobe University;
Emeritus Professor, University of Tokyo

Masao ENDOH

Emeritus Professor, MD, Yamagata University

Michael FERRIS

President, Novartis Holding Japan K.K.;
Director, Novartis Pharma K.K.

Toshio SUDA

Professor, MD, Keio University, School of Medicine

Tokuzo NAKAJIMA

Certified Public Accountant

Masanori FUSE

Department Head, Region Finance, Novartis Pharma K.K.

Trustee

Auditor[Board of Councilors]
Post

Title

10 councilors

Name

Chairman Tsuneyoshi KUROIWA

As of Oct. 1, 2014

Title
Member of the Japan Academy;
Emeritus Professor, University of Tokyo

Norio AKAIKE

Director, Kumamoto Health Science University;
Emeritus Professor, Kyushu University

Max M. BURGER

Novartis Science Board;
Professor, MD, University of Basel

Tohru HIROSE

Director, Division Head Japan Development,
Novartis Pharma K.K.

Hiroyuki KAWASHIMA

Former Professor, Graduate School of Medical & Dental Sciences,
Niigata University

Councilor Shigeo KOYASU

Acting Director, RIKEN Center for Integrative Medical Sciences

Tadanori MAYUMIEmeritus Professor, Osaka University

Miwako MORI

Professor, Health Sciences University of Hokkaido;
Emeritus Professor, Hokkaido University

Akihiko NAKANOProfessor, University of Tokyo, Science Department; Team
Leader, RIKEN (Institute of Physical & Chemical Research)

Masakatsu SHIBASAKI

Director, Microbial Chemistry Research Center, Microbial
Chemistry Research Foundation- 140 -

[Grantee Selection Committee] 20 members
Post

Name

Chairman Takeo KISHIMOTO
Tomoko BETSUYAKUAs of Oct. 1, 2014

Title
Visiting Professor, Science and Education Center,
Ochanomizu University
Professor, MD, School of Medicine, Keio University

MD, Graduate School of Medicine,
Masanori HATAKEYAMA Professor,
University of Tokyo
Hidenori ICHIJO

Professor, Dentist, Graduate School of Pharmaceutical Sciences,
University. of Tokyo

Nobuya INAGAKI

Professor, MD, Graduate School of Medicine, Kyoto University

Motomu KANAI

Masabumi MINAMI

Professor, Graduate School of Pharmaceutical Sciences,
University of Tokyo
Professor, Graduate School of Frontier Sciences,
University of Tokyo
Professor, Graduate School of Pharmaceutical Sciences,
Hokkaido University

Tetsuji MIURA

Professor, MD, Sapporo Medical University

Toyoaki MUROHARA

Professor, MD, Graduate School of Medicine, Nagoya University

Junichi NABEKURA

Professor, MD, National Institute for Physiological Sciences

Hiroyuki NAKAMURA

Shinichi OKA

Professor, Chemical Resources Laboratory,
Tokyo Institute of Technology
Professor, MD, Graduate School of Medical and Dental Sciences,
Tokyo Medical and Dental University
Director, MD, Aids Clinical Center,
National Center for Global Health and Medicine

Makoto SASAKI

Professor, Graduate School of Life Sciences, Tohoku University

Toshiharu SHIKANAI

Professor, Graduate School of Science, Kyoto University

Hiroshi TAKAYANAGI

Professor, MD, Graduate School of Medicine,
University of Tokyo

Hiroyuki TSUTSUIProfessor, MD, School of Medicine, Hokkaido University

Akihiro UMEZAWA

Director, MD,
National Institute for Child Health and Development

Masato YASUIProfessor, MD, School of Medicine, Keio University

Shigeyuki KAWANO

MemberYoshihiro OGAWA- 141 -

公益財団法人ノバルティス科学振興財団

役 員 名 簿2014 年 10 月 1 日現在 ( 順不同、 敬称略 )
職

名

氏

名

現

就任年月日

常勤･非常勤

畿央大学大学院健康科学研究科長･教授
慶應義塾大学名誉教授

2012 年 4 月 1 日

非常勤

神戸大学大学院医学系研究科客員教授
東京大学名誉教授

2012 年 4 月 1 日

非常勤

遠 藤 政 夫 山形大学名誉教授

2012 年 4 月 1 日

非常勤

須 田 年 生 慶應義塾大学医学部教授

2012 年 4 月 1 日

非常勤代表理事 金 子 章 道
浅野 茂隆

理

監

職

事

事

マイケル・フェリス

ノバルティスホールディングジャパン㈱代表取締役社長、ノバルティス ファーマ㈱取締役

2014 年 6 月 6 日

非常勤

中嶋 德三

中嶋德三公認会計士事務所
公認会計士

2012 年 4 月 1 日

非常勤2012 年 4 月 1 日

非常勤

布施

正 則 ノバルティス ファーマ㈱経理・財務統括部長

評議員名簿2014 年 10 月 1 日現在 ( 順不同、敬称略 )
職

名

氏

名

就任年月日

常勤･非常勤日本学士院会員
東京大学名誉教授

2012年 4月1日

非常勤

熊本保健科学大学理事 ・ 研究顧問
九州大学名誉教授

2012年 4月1日

非常勤

川島 博行 元新潟大学大学院医歯学総合研究科教授

2012年 4月1日

非常勤

小安 重夫

理化学研究所統合生命医科学研究センター長代行

2012年 4月1日

非常勤

柴崎 正勝

公益財団法人微生物化学研究会
微生物化学研究所長

2012年 4月1日

非常勤

中野 明彦 東京大学大学院理学系研究科教授

2012年 4月1日

非常勤

廣 瀬

2014 年9月9日

非常勤マックス ・ ノバルティス サイエンスボード
ブルガー バーゼル大学教授

2012年 4月1日

非常勤

眞弓 忠範 大阪大学名誉教授

2012年 4月1日

非常勤

2012年 4月1日

非常勤

評議員長 黒岩 常祥
赤池 紀扶

評議員

現

職

徹 ﾉﾊﾞﾙﾃｨｽ ﾌｧｰﾏ ( 株 ) 取締役開発本部長

森 美和子

北海道医療大学客員教授
北海道大学名誉教授- 142 -

選考委員名簿2014 年 10 月 1 日現在 ( 順不同、敬称略 )
職 名

氏 名

現

選考委員長 岸 本 健 雄

職

就任年月日

お茶の水女子大学
2011年 6月17日
サイエンス＆エデュケーションセンター客員教授

常勤･非常勤非常勤一 條 秀 憲 東京大学大学院薬学系研究科教授

2011年 6月17日

非常勤

稲 垣 暢 也 京都大学大学院医学研究科教授

2012年 6月15日

非常勤

梅澤 明弘

国立成育医療研究センター
再生医療センター長

2012年 6月15日

非常勤

岡国立国際医療センター
エイズ治療 ・ 研究開発センター長

2011年 6月17日

非常勤

東京医科歯科大学大学院
医歯学総合研究科教授

2014 年 6月6日

非常勤2014 年 6月6日

非常勤

河 野 重 行 東京大学大学院新領域創成科学研究科教授 2013 年 6月14日

非常勤

佐 々 木 誠 東北大学大学院生命科学研究科教授

2012年 6月15日

非常勤

2011年 6月17日

非常勤

広 東京大学大学院医学系研究科教授

2011年 6月17日

非常勤

筒 井 裕 之 北海道大学大学院医学研究科教授

2012年 6月15日

非常勤

中 村 浩 之 東京工業大学資源化学研究所教授

2013 年 6月14日

非常勤

鍋 倉 淳 一 自然科学研究機構生理学研究所教授

2013 年 6月14日

非常勤

畠 山 昌 則 東京大学大学院医学系研究科教授

2013 年 6月14日

非常勤

別 役 智 子 慶應義塾大学医学部教授

2013 年 6月14日

非常勤

三 浦 哲 嗣 札幌医科大学医学部教授

2013 年 6月14日

非常勤

南2011年 6月17日

非常勤室 原 豊 明 名古屋大学大学院医学系研究科教授

2011年 6月17日

非常勤

安 井 正 人 慶應義塾大学医学部教授

2013 年 6月14日

非常勤

慎 一

小川 佳宏
金 井

求 東京大学大学院薬学系研究科教授

選考委員 鹿 内 利 治 京都大学大学院理学研究科教授
高 柳

雅 文 北海道大学大学院薬学研究院教授- 143 -

事務局便りご寄附のお願い
当財団は、 自然科学における創造的な研究の奨励等を行うことにより、 学術の振興を図り、 国民の健康と福祉の向上に寄与することを目的に公益事業を行っております。
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ご寄附は、 随時受付けております。
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事務局より
本年度もお陰様で、 財団年報を発行できることとなりました。 これも偏に、 助成を受けられた皆様および財団関係者の皆様のご尽力の賜物と心より感謝申し上げます。
1987 年 9 月の財団設立以来、 助成件数は総数で 1,552 件、 総額 18 億 5 千万円余りに達しました。
事務局は、 今後とも財団の設立目的である学術の進展に寄与するべく、 研究助成を中心とした公益事業に邁進して参ります。
引き続きご指導、 ご支援の程よろしくお願い申し上げます。

事務局長 松田光陽〒 106-0031 東京都港区西麻布 4-16-13
西麻布 28 森ビル 2F
Tel:03-5464-1460 Fax:03-5467-3055
E- メール ： novartisfound.japan@novartis.com
ホームページ :http://www.novartisfound.or.jp- 144 -

- 145 -