1. The biological functions of cell adhesion inhuman tumor

Takeshi Ito, Yuki Kumagai, Zenichi Tanei, YumiTsuboi, Anri Saito, Shuejen Shiu, Hiroyuki Kogai,Ken Akashi, Atsushi Kanatani, Takehiro Tsuchiya,Masaru Kasai, Takuya Tabuchi, Tomoko Maru-yama, Hiromi Ichihara, Atsuko Nakamura, MikaSakurai-Yageta, Kaoru Kiguchi, Motoi Oba,1 andYoshinori Murakami; 1Research Institute of Mo-lecular and Cell Biology of Cancer, Showa Univer-sity

Disruption of cell adhesion is a critical step to in-vasion and metastasis of human cancer and theiracquired resistance to several anti-cancer and mo-lecular targeting drugs. CADM1/TSLC1 is an im-munoglobulin superfamily cell adhesion moleculeand acts as a tumor suppressor in various cancers.In order to understand possible cross-talk ofCADM1 with other oncogenic signals, we have ex-amined mutation of the KRAS2 and TP53 genesand promoter methylation of the CADM1 and 4.1Bgenes in the same series of human non-small celllung cancer (NSCLC) and have found that pro-moter methylation of the CADM1 and 4.1B genes

occurs independently of the EGFR or the KRAS2mutation in non-small cell lung cancer (1). We,then, investigated dynamic regulation of the CADM1-4.1B-MPP3 complex in mature cell adhesion byfluorescence recovery after photobleaching (FRAP)analysis. Traditional FRAP analysis were performedfor relatively short period of around 10min. On theother hand, thanks to recent advances in the sensi-tive laser detector systems, we examine FRAP ofCADM1 complex for longer period of 60 min andanalyze the recovery with exponential curve-fittingto distinguish the fractions with different diffusionconstants. This approach reveals that the fluores-cence recovery of CADM1 is fitted to a single expo-nential function with a time constant (tau) of ap-proximately 16 min, whereas 4.1B and MPP3 arefitted to a double exponential function with twotaus of approximately 40-60 sec and 16 min. Thelonger tau is similar to that of CADM1, suggestingthat 4.1B and MPP3 have two distinct fractions, oneforming a complex with CADM1 and the other pre-sent as a free pool. Fluorescence loss in pho-tobleaching analysis supports the presence of a freepool of these proteins near the plasma membrane.Furthermore, double exponential fitting makes itpossible to estimate the ratio of 4.1B and MPP3 pre-

Department of Cancer Biology

Division of Molecular Pathology人癌病因遺伝子分野

Professor Yoshinori Murakami, M.D., Ph.D.Senior Assistant Professor Daisuke Matsubara, M.D., Ph.D.Assistant Professor Takeharu Sakamoto, Ph.D.Assistant Professor Takeshi Ito, Ph.D.

教 授 医学博士 村 上 善 則講 師 医学博士 松 原 大 祐助 教 医学博士 坂 本 毅 治助 教 医学博士 伊 東 剛

Human cancers develop and progress toward malignancy through accumulation ofmultiple genetic and epigenetic alterations. Elucidation of these alterations is es-sential to provide molecular targets for prevention, diagnosis, and treatment ofcancer. Our current interest is to understand the roles of cell adhesion in cancerinvasion and metastasis. Genomic and epigenomic abnormalities involved in hu-man tumors, including adult T-cell leukemia, cholangiocarcinoma, lung, breast,head and neck and urological cancers, are also being investigated.

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sent as a free pool and as a complex with CADM1as approximately 3:2 and 3:1, respectively. Theseapproaches demonstrate a central role of CADM1in the stability of its complex formation with 4.1Band MPP3 and would provide more mechanistic as-pects of the dynamics of protein complexes in ran-dom processes (2). We have also demonstrated theimportance of immunoglubuin superfamily cell ad-hesion molecules other than CADM1 in collabora-tion with others (3). Additional roles of CADM1 inimmunological recognition by CD4＋ cytotoxic Tlymphocyte was also investigated in collaborationwith others (4).

2. Analysis of oncogenic function of CADM1 inadult T-cell leukemia (ATL) and small celllung cancer (SCLC)

Takeshi Ito, Yuki Kumagai, Daisuke Suzuki, Shin-taro Mori, Tomoko Maruyama, Akiko Nakamura,Akiteru Goto2 and Yoshinori Murakami; 2Depart-ment of Pathology, Akita University GraduateSchool of Medicine.

In contrast to a tumor suppressor function ofCADM1 in epithelial cancers, CADM1 is overex-pressed in adult T-cell leukemia (ATL) and smallcell lung cancer (SCLC), conferring an invasive phe-notype characteristic to ATL. To understand the on-cogenic functions of CADM1 in ATL and SCLC thatare distinct from tumor suppressor functions inmany epithelial cancers, structural and functionalfeatures of N -linked and O-linked glycosylation ofCADM1 protein are being examined using massspectrometry in collaboration with Shimadzu Co.Ltd.. To establish sensitive diagnostic tools of SCLCthrough detecting CADM1, monoclonal antibodiesagainst the fragment of CADM1 unique to ATL orSCLC are being generated and characterized in col-laboration with researchers in the Institute of Ad-vanced Science and Technology, the University ofTokyo. These antibodies would be useful to im-prove the FACS system to identify and characterizemalignant ATL cells, whose prototype has been es-tablished in the IMSUT Hospital. Novel detectionsystems of SCLC and possible therapeutic strategiesagainst ATL and SCLC are also being investigatedby the support of several grants of applied medicalsciences.

3. Analyses of genomic and epigenomic altera-tions of human cholangiocarcinoma and headand neck, lung, breast, and urological can-cers.

Ken Akashi, Hiroyuki Kogai, Hasaya Dockduang,Ayako Sato, Takeshi Ito, Kaoru Kiguchi andYoshinori Murakami.

To unveil the molecular mechanisms of multi-stage carcinogenesis, genetic and epigenetic altera-tions in key molecules in human tumorigenesiswere examined in various cancers. Molecular tar-geting therapy to specific genetic alterations has notbeen established in head and neck squamous cellcarcinoma (HNSCC) except for cetuximab treat-ment. To characterize alterations of actionable onco-genes in HNSCC, we examined the gain of copyand mutation of the MET gene in 54 JapaneseHNSCC by droplet digital PCR (ddPCR). Copygain of the MET was detected in 10 of 54 (19％)HNSCCs and more frequently observed in tumorsof the hypopharynx (4 of 12; 33％) or larynx (5 of13; 38％) than those of the oral cavity (1 of 21; 4％)or oropharynx (0 of 8; 0％), suggesting the existenceof site-specific features in the oncogenic mecha-nisms of HNSCCs. Copy gain of the MET was alsoobserved preferentially in older patients, althoughno correlation in other parameters, including clini-cal stages and overall or recurrence-free survival,was observed. These results suggest that copy gainof the MET could provide an indicator for treat-ment with tyrosine kinase inhibitors for MET in asubset of hypopharyngeal or laryngeal cancer (5).We also examined the copy number alteration(CNA), a new category of somatic alterations at thehighly polymorphic sites of more than 1 kb, calledcopy number variation (CNV), in breast cancer,CCA, HNSCC and bladder cancer by comprehen-sive approaches using CNA-specific arrays, weidentified and being characterized a number of can-didate genes showing gain or loss of gene copies ineach cancers. Cholangiocarcinoma is one of the rep-resentative cancers which are refractory to anytherapeutic approaches. Using tyrosine kinase arrayand STATs profiling, we have reported the pre-dominant activation of STAT3 in cholangiocarci-noma in collaboration with others (6).

4. Identification of a novel Mint3 binding protein.

Hiroki J. Nakaoka, Seiko Yoshino, Akane Kana-mori, Miho Ishiura, Masaya Misumi, Yukari Aso,Takeharu Sakamoto

Unlike most cells, cancer cells activate hypoxiainducible factor-1 (HIF-1) to use glycolysis even atnormal oxygen levels, or normoxia1. Therefore,HIF-1 is an attractive target in cancer therapy.However, the regulation of HIF-1 during normoxiais not well characterised, although Mint3 was re-cently found to activate HIF-1 in cancer cells andmacrophages by suppressing the HIF-1 inhibitor,factor inhibiting HIF-1 (FIH-1). Thus, we analysedMint3-binding proteins to investigate the mecha-nism by which Mint3 regulates HIF-1. Yeast two-hybrid screening using Mint3 as bait identified pro-tein X as a novel factor regulating HIF-1 activity via

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Mint3. Protein X bound to the phosphotyrosine-binding domain of Mint3, formed a ternary com-plex with Mint3 and FIH-1, and co-localised withMint3 at the Golgi apparatus. Depletion of proteinX decreased the expression of HIF-1 target genesand reduced glycolysis in normoxic cancer cells. In-hibition of protein X in cancer cells by expressingshRNAs reduced tumourigenicity (7, 8).

5. Molecular pathological analyses of humanlung cancer

Daisuke Matsubara, Zenichi Tanei, Zhang Wen-Jing Toshiro Niki3 and Yoshinori Murakami; 3De-partment of Integrative Pathology, Jichi MedicalUniversity.

Lung cancer is a leading cause of cancer death inJapan. Understanding the molecular pathological

characteristics of human lung cancer is prerequisiteto overcome this malignant cancer. We have beeninterested in the epithelial-mesenchymal transition(EMT) observed in malignant cancer cells with in-vasive and metastatic ability and have identifiedprotein arginine methyltransferase 5 (PRMT5) as acandidate histone methyltransferase gene whose ex-pression was correlated with EMT by microarrayanalysis. We have also reported previously thatZEB1, one of the master regulators of EMT, is an-other candidate gene involved in EMT of lung can-cer. Then, we extensively examined possible aberra-tions of the switch/sucrose non-fermenting (SWI/SNF) complex genes, including BRG1, BRM, BAF47,ARID1A, and ARID1B, in human NSCLC andshowed high incidence of altered expression ofthese genes in a subset of NSCLC, especially inlarge cell carcinomas and pleomorphic carcinomasin collaboration with others (9).

Publications

1. Kogai H, Kikuchi S, Obana T, Tsuboi Y, Maru-yama T, Sakurai-Yageta M, Asamura H, Kanai Y,Murakami Y. Promoter methylation of theCADM1 and 4.1B genes occurs independently ofthe EGFR or the KRAS2 mutation in non-smallcell lung cancer. J Cancer Therapy , 6, 273-285,2015.

2. Sakurai-Yageta M, Maruyama T, Suzuki T, Ichi-kawa K, Murakami Y. Dynamic Regulation of aCell Adhesion Protein Complex IncludingCADM1 by Combinatorial Analysis of FRAPwith Exponential Curve-fitting. PLoS ONE, 10,e0116637, 2015.

3. Fujiyuki T, Yoneda M, Amagai Y, Obayashi K,Ikeda F, Shoji K, Murakami Y, Sato H, Kai C. Ameasles virus selectively blind to signaling lym-phocytic activation molecule shows anti-tumoractivity against lung cancer cells. Oncotarget , 6,24895-24903, 2015.

4. Takeuchi A, Badr MESG, Miyauchi K, IshiharaC, Onishi R, Guo Z, Sasaki Y, Ike H, Takumi A,Tsuji NM, Murakami Y, Katakai T, Kubo M,Saito T. CRTAM determines the CD4＋ cytotoxicT lymphocyte lineage. J Exp Med, 213, 123-138,2015.

5. Akashi K, Ebihara Y, Omura G, Saito Y, YoshidaM, Ando M, Asakage T, Yamasoba T, MurakamiY. Frequent Copy Gain of the MET Gene in Hy-popharyngeal and Laryngeal Cancer in the Japa-nese Population. J Cancer Therapy, 6, 1093-1102,2015.

6. Dokduang H, Techasen A, Namwat N, Khun-tikeo N, Pairojkul C, Murakami Y, Loilome W,Yongvanit P. STATs profiling reveals predomi-nantly-activated STAT3 in cholangiocarcinomagenesis and progression. J Hepatobiliary PancreatSci. in press.

7. 坂本毅治，「MT1-MMP/Mint3によるHIF-1活性化メカニズム」，メディカルレビュー社 THE LUNGperspectives, vol. 23 No. 3，７９―８３，２０１５．

8. 坂本毅治，「生体における水酸化シグナルを介した低酸素応答」，羊土社 実験医学 ７月号，１７１９―１７２３，２０１５．

9. Yoshimoto T, Matsubara D, Nakano T, TamuraT, Endo S, Sugiyama Y, Niki T. Frequent loss ofthe expression of multiple subunits of the SWI/SNF complex in large cell carcinoma and pleo-morphic carcinoma of the lung. Pathol Int. 65,595-602, 2015.

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1. Molecular mechanism of the regulation of NF-B transcription factor

Takao Seki, Hironori Ohtsuki, Kota Sakane,Ginga Komatsu, Yuko Hata1, Masa-aki Oyama1,Jin Gohda2, Aya Kojima3, Toshifumi Akizawa4,Taishin Akiyama and Jun-ichiro Inoue: 1MedicalProteomics Laboratory, and 2Center for Asian In-fectious Diseases, IMSUT; 3Ritsumeikan Univer-sity; 4Setsunan University.

Transcription factor NF-B binds specifically to adecameric motif of nucleotide, B site, and activates

transcription. The activation of NF-B has beendemonstrated to be carried out post-translationallyupon extracellular stimuli through membrane re-ceptors such as members of the TLR/IL-1R familyand of TNFR superfamily. In canonical NF-B path-way, NF-B forms a complex with regulatory pro-tein, IB, and is sequestered in the cytoplasm priorto stimulation. Upon stimulation, IB is rapidlyphosphorylated on two specific serine residues byIB kinase (IKK) complex followed by lysine 48 (K48)-linked ubiquitination and proteasome-depend-ent degradation of IB. NF-B subsequently translo-cates to the nucleus to activate transcription of tar-

Department of Cancer Biology

Division of Cellular and Molecular Biology分子発癌分野

Professor Jun-ichiro Inoue, Ph.D.Associate Professor Taishin Akiyama, Ph.D.Assistant Professor Yuu Taguchi, Ph.D.Assistant Professor Yuri Shibata, Ph.D.

教 授 薬学博士 井 上 純一郎准教授 薬学博士 秋 山 泰 身助 教 医学博士 田 口 祐助 教 生命科学博士 柴 田 佑 里

Gene expression is largely regulated by signal transduction triggered by variousstimulations. Several lines of evidence indicate that genetic defects of moleculesinvolved in the signal transduction or the gene expression lead to abnormal celldifferentiation or tumor formation. Our goal is to understand the molecular mecha-nisms of disease pathogenesis and oncogenesis by elucidating normal regulationof intracellular signal transduction and gene expression involved in cell prolifera-tion and differentiation. We have identified and been interested in Tumor necrosisfactor receptor-associated factor 6 (TRAF6), which acts as an E3 ubiquitin ligaseto generate Lys63-linked polyubiquitin chains that are crucial for transducing sig-nals emanating from the TNFR superfamily or the TLR/IL-1R family leading to acti-vation of transcription factor NF-B and AP-1. By generating TRAF6-deficientmice, we found that TRAF6 is essential for osteoclastogenesis, immune self-toler-ance, lymph node organogenesis and formation of skin appendices. We are cur-rently focusing on molecular mechanisms underlying TRAF6-mediated activationof signal transduction pathways and how TRAF6 is involved in osteoclastogenesisand self-tolerance. In addition, NF-B is constitutively activated in various cancercells and this activation is likely involved in the malignancy of tumors. Thus, weare also investigating the molecular mechanisms of the constitutive activation ofNF-B and how this activation leads to the malignancy of breast cancers andadult T cell leukemia (ATL).

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get genes. This project is to identify molecules thatregulate signal from membrane receptors to NF-B/IB complex. We have previously identified up-stream activators of NF-B, tumor necrosis factorreceptor-associated factor (TRAF) 6. TRAF6 containsRING domain in the N-terminus and acts as an E3ubiquitin-ligase to catalyze the lysine 63 (K63)-linked polyubiquitination of several signaling mole-cules and TRAF6 itself. To understand the molecu-lar mechanisms of TRAF6-mediated NF-B activa-tion, we try to identify proteins that are ubiquiti-nated by TRAF6 upon stimulation. We took advan-tage of using the peptide that specifically bindsK63-linked polyubiquitin chain to purify such pro-teins. We have confirmed that the peptide-based af-finity column is useful for specific concentration ofrecombinant K63-linked polyubiquitin chain, sug-gesting that it also works for purification of theproteins of our interest. We are also interested innoncanonical NF-B pathway, which is crucial forimmunity by establishing lymphoid organogenesisand B-cell and dendritic cell (DC) maturation. RelBis a major NF-B subunit in the pathway. To eluci-date the mechanism of the RelB-mediated immunecell maturation, a precise understanding of the rela-tionship between cell maturation and RelB expres-sion and activation at the single-cell level is re-quired. Therefore, we generated knock-in mice ex-pressing a fusion protein between RelB and fluores-cent protein (RelB-Venus) from the Relb locus. TheRelbVenus/Venus mice developed without any abnormali-ties observed in the Relb－/－ mice, allowing us tomonitor RelB-Venus expression and nuclear local-ization as RelB expression and activation.RelbVenus/Venus DC analyses revealed that DCs consist ofRelB-, RelBlowand RelBhigh populations. The RelBhigh

population, which included mature DCs with pro-jections, displayed RelB nuclear localization,whereas RelB in the RelBlow population was in thecytoplasm. Although both the RelBlow and RelB－populations barely showed projections, MHC II andco-stimulatory molecule expression were higher inthe RelBlow than in the RelB－ splenic conventionalDCs. Taken together, our results identify the RelBlow

population as a possible novel intermediate matura-tion stage of cDCs and the RelbVenus/Venus mice as auseful tool to analyze the dynamic regulation of thenon-canonical NF-B pathway.

2. Analysis of the physiological role of p47

Xiao Han, Yuri Shibata, Masaaki Oyama1, HirokoKozuka-Hata1, Jin Gohda2 and Jun-ichiro Inoue

p47 (also known as NSFL1C) is originally identi-fied as a major cofactor of the cytosolic ATPase as-sociated with various cellular activities p97, and thep47/p97 complex is required for the reassembly ofGolgi stacks at the end of mitosis. We have previ-

ously reported that p47 targets polyubiquitinatedNEMO for lysosomal degradation, thereby nega-tively regulating NF-B activation. We also haveshown that the expression of p47 is reduced inadult T-cell leukemia (ATL) patient-derived celllines, in which NF-B is constitutively activated.Our results suggest that the altered p47 expressionmay trigger development of various cancers. Tofurther investigate the physiological significance ofp47, we tried to generate p47 knockout mice. Het-erozygous knockout mice were viable and fertile,while homozygous knockout mice exhibited embry-onic lethality. We then obtained p47-deficient MEFsby introducing Cre recombinase into p47fl/fl MEFs. p47-deficient MEFs exhibited defective Golgistucyutr. We also crossed p47-loxP-flanked micewith LysM-Cre knock-in mice to disrupt p47 spe-cifically in macrophages. We obtained p47-deficientmacrophages and are investigating the effect of p47deficiency on NF-B signaling pathway.

3. Molecular mechanism of HTLV-1 Tax-inducedIKK activation

Yuri Shibata, Masaaki Oyama1, Hiroko Kozuka-Hata1 and Jun-ichiro Inoue

Activation of NF-B by human T-cell leukemiavirus type 1 (HTLV-1) Tax is thought to be crucialin T-cell transformation and the onset of adult T-cell leukemia (ATL). Although it is well known thatTax interacts with NEMO and induces IKK activa-tion, the molecular mechanism of Tax-induced IKKactivation remains largely unknown. To elucidatethis mechanism, we established a cell-free assaysystem, in which the IKK complex can be activatedby adding recombinant Tax to cytosolic extracts.Whereas recombinant Tax induced activation of theIKK complex in cytosolic extracts, it failed to acti-vate the purified IKK complex. These results sug-gest that Tax requires intermediary molecules forIKK activation. To identify such molecules, we pu-rified the Tax-IKK complex and analyzed its inter-acting proteins by mass spectrometry. We identifieda novel Tax-IKK-interacting protein, whose knock-down resulted in decreased binding of Tax to theIKK complex. Correspondingly, knockdown of thisprotein inhibited the Tax-induced, but not the cy-tokine-induced IKK activation. Taken together, thenovel Tax-IKK-interacting protein functions as anadaptor protein required for full IKK activation in-duced by Tax.

4. Molecular mechanism of RANK signaling inosteoclastogenesis

Yuu Taguchi, Yo Yumiketa, Mizuki Yamaniha,Yui Iwamae, Kichi Chou, Youko Hirayama,Masaaki Oyama1, Hiroko Kozuka-Hata1, Jin

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Gohda2, and Jun-ichiro Inoue

Osteoclasts are large multinucleated cells whichhave ability to degrade bone matrixes, and play acrucial role in bone homeostasis in concert with os-teoblast which have function of bone formation. Asa result of excess formation or activation of osteo-clasts, pathological bone resorption is observed inpostmenopausal osteoporosis and rheumatoid ar-thritis. Therefore, elucidating the molecular mecha-nism of osteoclastogenesis is important for under-standing bone diseases and developing novelstrategies to treat such diseases.Osteoclasts are differentiated from hematopoietic

stem cells upon stimulation with cytokines, for ex-ample macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor-B(RANK) ligand (RANKL). It is known that activa-tion of signal transduction pathway emanatingfrom receptor RANK is essential for osteoclasto-genesis. This RANK signaling activates transcrip-tional factors, NF-B and AP-1, through the E3ubiquitin ligase TRAF6, and also induces PLC2-mediated Ca2＋ oscillation. These signals lead to theinduction of NFATc1 that is a master transcrip-tional factor in osteoclastogenesis. We have previ-ously identified a novel domain in the cytoplasmicregion of RANK, and named Highly Conserved do-main in RANK (HCR), which does not have anyhomology of amino-acid sequence with other pro-teins. The HCR acts as a platform for formation ofsignal complex including TRAF6, PLC2 and adap-tor protein Gab2. This formation is involved in sus-taining activation of RANK signaling, and is essen-tial for the NFATc1 induction and osteoclastogene-sis. To elucidate the other functions and the precisemolecular mechanism of HCR, we performed yeasttwo-hybrid screening to identify the interactingprotein to HCR. We are investigating whether somecandidate proteins associate to HCR in osteoclastprecursor cells and are involved in HCR functionsand osteoclastogenesis. Moreover, to find the novelmolecules involved in osteoclastogenesis, we inves-tigated the change of gene expression levels duringosteoclastogenesis by analyzing microarray data.Since three genes were selected as candidates, weare now investigating whether these genes are im-portant for osteoclastogenesis in vivo. Furthermore,we also tried to elucidate the precise molecularmechanism at the subsequent step of NFATc1 in-duction in osteoclastogenesis from the point ofview of TRAF6 functions, and revealed that TRAF6is involved in cell-cell fusion and actin ring forma-tion in addition to NFATc1 induction. We are cur-rently trying to identify the novel target proteinubiquitinated by TRAF6.

5. TRAF6 regulates pregnancy-induced mam-mary gland development and maintenance ofepithelial stem cells

Mizuki Yamamoto, and Jun-ichiro Inoue

Mammary gland development is characterized bythe unique process by which the epithelium in-vades the stroma. During puberty, tubule formationis coupled with branching morphogenesis which es-tablishes the basic arboreal network emanatingfrom the nipple. During pregnancy, the ductal cellsundergo rapid proliferation and form alveolarstructures within the branches for milk production.Upon weaning of the pups, lactation stops and themammary gland undergoes rapid involution. In theRANK signaling TRAF6 activates MAPK and ca-nonical NF-B independently with IKK-mediatednon-canonical NF-B pathway. Previously, it hasbeen reported that deficiency of RANK and itsdownstream kinase IKK inhibited pregnancy-in-duced alveolar formation via Ccnd1 expression.However the role of TRAF6 in mammary gland de-velopment remains unclear. To elucidate the role ofTRAF6, we first analyzed TRAF6-deficient mam-mary gland structure in virgin and pregnant miceand found that that TRAF6-deficiency, like RANK-deficiency, inhibited pregnancy-induced alveolarformation but did not lead to any morphologicaldefect in non-pregnant gland.

6. Catalytic subunits of the phosphatase cal-cineurin interact with NF-B-inducing kinase(NIK) and attenuate NIK-dependent gene ex-pression

Miho Shinzawa, Hiroyasu Konno, Junwen Qin,Nobuko Akiyama, Maki Miyauchi, Hiroyuki O-hashi5, Etsuko Miyamoto-Sato5.6, Hiroshi Yana-gawa7, Taishin Akiyama and Jun-ichiro Inoue:5Division of Interactome Medical Sciences, The In-stitute of Medical Science, The University of To-kyo, Minato-ku, Tokyo, Japan; 6Division of Mo-lecular Biology, Research Institute for BiomedicalSciences, Tokyo University of Science, Yamazaki,Noda-shi, Chiba, Japan; 7Department of Bi-osciences and Informatics, Faculty of Science andTechnology, Keio University, Yokohama, Japan

Nuclear factor (NF)-B-inducing kinase (NIK) is asignal transducer that regulates a wide variety ofimmune systems by activating NF-B pathways.Aberrant NIK activation reportedly provokes tumormalignancy. Therefore, a precise regulation of NIKactivity should be required. In this study, we ex-plored novel interacting proteins of NIK by in vitrovirus screening. We then identified the catalyticsubunit A isoform of serine/threonine phosphatasecalcineurin (CnA) as a novel NIK-interacting pro-

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tein. Co-immunoprecipitation experiments con-firmed the interaction of NIK with CnA in livingcells. In addition to CnA, calcineurin catalyticsubunit A isoform (CnA) also interacted withNIK. We further found that both the kinase domainand C-terminal region of NIK binds to CnA andCnA. Furthermore, the phosphatase domain ofCnA is necessary for the binding to NIK. Interest-ingly, TRAF3, a critical regulator of NIK activity,also interacts with CnA and CnA. Knockdown ofCnA and CnA significantly enhanced expressionof the NIK-dependent gene Spi-B and activation ofRelA and RelB induced by lymphotoxin- receptor(LtR) signaling. These data suggest that CnA andCnA attenuate NF-B activation pathway medi-ated by LtR-NIK signaling. Taken together, thisstudy suggest that CnA and CnA fine-tune NIKfunctions.

7. Hypergravity provokes a temporary reductionin CD4＋CD8＋ thymocyte number and a persis-tent decrease in medullary thymic epithelialcell frequency in mice

Ryosuke Tateishi, Nobuko Akiyama, Maki Miya-uchi, Riko Yoshinaga, Hiroki Sasanuma8, TakashiKudo9, Miki Shimbo9, Masahiro Shinohara10, KojiObata11, Jun-ichiro Inoue, Masaki Shirakawa12,Dai Shiba12, Hiroshi Asahara10, Nobuaki Yoshida8,Satoru Takahashi9, Hironobu Morita11, andTaishin Akiyama: 8Laboratory of DevelopmentalGenetics, Center for Experimental Medicine andSystems Biology, The University of Tokyo; 9De-partment of Anatomy and Embryology, Faculty ofMedicine, University of Tsukuba; 10Department ofSystems BioMedicine, Graduate School of Medicaland Dental Sciences, Tokyo Medical and Dental

University; 11Department of Physiology, Gifu Uni-versity Graduate School of Medicine; 12JapanAerospace Exploration Agency (JAXA), Tsukuba,Japan

Gravity change influences a wide variety of im-munological systems. In this study, we investigatedthe effects of hypergravity (2G) on thymic cells inmice. Exposure of mice to 2G for relatively shortperiod (3 days) decreased the frequency of CD4＋CD8＋ thymocytes (DP) and mature medullary thy-mic epithelial cells (mTECs). Moreover, keratin-5and keratin-8 double-positive (K5＋K8＋) TECs,which reportedly contain TEC progenitors, were in-creased. The reduction of DP was recovered by anexposure to 2G for 14 days. On the other hand, thereduction of mature mTECs and the increment ofK5＋K8＋ TEC persisted in this period of 2G-expo-sure. Intriguingly, a surgical deletion of the innerear's vestibular apparatus suppressed these hyper-gravity effects. Gene expression analysis by quanti-tative PCR analysis revealed that Aire and RANK,which play critical role in mTEC function and de-velopment, were up-regulated by the 3-day expo-sure and subsequently down-regulated by the 14-day exposure to 2G. Unexpectedly, a surgical lesionof the inner ear's vestibular apparatus did not affectthe change in mTEC gene expression. Overall, thisstudy suggests that exposure of mice to hypergrav-ity provokes a transient reduction of DP and along-lasting reduction of mature mTECs in a ves-tibular system-dependent manner. Moreover, the2G exposure causes an aberrant expression ofmTEC genes in the vestibular system-independentfashion. These data might provide insight on the ef-fect of gravity change on immune system duringspaceflight and living.

Publications

Blackburn, J., Kawasaki, K., Porntaveetus, T., Kawa-saki, M., Otsuka-Tanaka, Y., Miake, Y., Ota, M.S.,Watanabe, M., Hishinuma, M., Nomoto, T., Oom-men, S., Ghafoor, S., Harada, F., Nozawa-Inoue,K., Maeda, T., Peterková, R., Lesot, H., Inoue, J.,Akiyama, T., Schmidt-Ullrich, R., Liu, B., Hu, Y.,Page, A., Ramírez, A., Sharpe, P.T. and Ohazama,A.＊ Excess NF-B induces ectopic odontogenesisin embryonic incisor epithelium. J. Dent. Res. 94,121-128 (2015).

Ito-Kureha, T., Koshikawa, N., Yamamoto, M., Sem-ba, K., Yamaguchi, N., Yamamoto, T., Seiki, M.and Inoue, J.＊ Tropomodulin 1 expression drivenby NF-B enhances breast cancer growth. CancerRes. 75, 62-72 (2015).

Sato, Y., Goto, E., Shibata, Y., Kubota, Y., Yama-gata, A., Goto-Ito, S., Kubota, K., Inoue, J., Take-kawa, M., Tokunaga, F. and Fukai, S. Structures

of CYLD USP with Met1- or Lys63-linked diu-biquitin reveal mechanisms for dual specificity.Nat. Struct. Mol. Biol. 22, 222-229 (2015).

Gohda, J., Ma, Y., Huang, Y., Zhang, Y., Gu, L.,Han, Y., Li, T., Gao, G.F., Inoue, J., Iwamoto, A.and Ishida, T＊. HIV-1 replicates in human osteo-clasts and enhances their differentiation in vitro.Retrovirology 12:12 doi:10.1186/s12977-015-0139-7(2015).

Shinzawa. M., Konno, H., Qin, J., Akiyama, N.,Miyauchi, M., Ohashi, H., Miyamoto-Sato, E.,Yanagawa, H., Akiyama, T.＊ and Inoue, J.＊ Cata-lytic subunits of the phosphatase calcineurin in-teract with NF-B-inducing kinase (NIK) and at-tenuate NIK-dependent gene expression. Sci. Rep.5:10758. doi: 10.1038/srep10758 (2015).

Seki, T., Yamamoto, M., Taguchi, Y., Miyauchi, M.,Akiyama, N, Yamaguchi, N., Gohda, J., Akiyama,

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T. and Inoue, J.＊ Visualization of RelB expressionand activation at the single-cell level during den-dritic cell maturation in Relb-Venus knock-inmice. J. Biochem. 158, 485-495. doi: 10.1093/jb/mvv064 (2015).

Varney, M.E., Niederkorn, M., Konno, H., Matsu-mura, T., Gohda, J., Yoshida, N., Akiyama, T.,Christie, S., Fang, J., Miller, D., Jerez, A., Karsan,A., Maciejewski, J.P., Inoue, J. and Starczynowski,D.T.＊ Loss of Tifab , a del(5q) MDS gene, altershematopoiesis through derepression of Toll-likereceptor-TRAF6 signaling J. Exp. Med. 212, 1967-1985. doi: 10.1084/jem.20141898 (2015).

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1. Activation of the receptor tyrosine kinaseMuSK by the cytoplasmic protein Dok-7 inneuromuscular synaptogenesis.

Tezuka, T., Inoue-Yamauchi, A.1, Weatherbee,SD.2, Izawa, Y., Ueta, R., and Yamanashi, Y.: 1Me-morial Sloan Kettering Cancer Center. 2Depart-ment of Genetics, Yale University.

Protein-tyrosine kinases (PTKs) play crucial rolesin a variety of signaling pathways that regulateproliferation, differentiation, motility, and other ac-tivities of cells. Therefore, deregulated PTK signalsgive rise to a wide range of diseases such as neo-plastic disorders. To understand the molecularbases of PTK-mediated signaling pathways, weidentified Dok-1 as a common substrate of manyPTKs in 1997. Since then, the Dok-family has beenexpanded to seven members, Dok-1 to Dok-7,which share structural similarities characterized byNH2-terminal pleckstrin homology (PH) and phos-photyrosine binding (PTB) domains, followed bySrc homology 2 (SH2) target motifs in the COOH-terminal moiety, suggesting an adaptor function.Indeed, as described below, Dok-1 and Dok-2 re-

cruit p120 rasGAP upon tyrosine phosphorylationto suppress Ras-Erk signaling. However, we foundthat Dok-7 acts as an essential cytoplasmic activatorof the muscle-specific receptor tyrosine kinase(RTK) MuSK in the formation of the neuromuscularjunction (NMJ), providing a new insight into RTK-mediated signaling. It now seems possible that locallevels of cytoplasmic activators, like Dok-7, controlthe activity of RTKs in concert with their extracellu-lar ligands.The NMJ is a synapse between a motor neuron

and skeletal muscle, where the motor nerve termi-nal is apposed to the endplate (the region of synap-tic specialization on the muscle). The contraction ofskeletal muscle is controlled by the neurotransmit-ter acetylcholine (ACh), which is released from thepresynaptic motor nerve terminal. To achieve effi-cient neuromuscular transmission, acetylcholine re-ceptors (AChRs) must be densely clustered on thepostsynaptic muscle membrane of the NMJ. Failureof AChR clustering is associated with disorders ofneuromuscular transmission such as congenital my-asthenic syndromes and myasthenia gravis, whichare characterized by fatigable muscle weakness. Theformation of NMJs is orchestrated by MuSK and by

Department of Cancer Biology

Division of Genetics腫瘍抑制分野

Professor Yuji Yamanashi, Ph.D.Assistant Professor Tohru Tezuka, Ph.D.Assistant Professor Ryo Ueta, Ph.D.Assistant Professor Sumimasa Arimura, Ph.D.

教 授 理学博士 山 梨 裕 司助 教 理学博士 手 塚 徹助 教 博士（生命科学） 植 田 亮助 教 医学博士 有 村 純 暢

The major interest of this division is in molecular signals that regulate a variety ofcellular activities. Our aim is to address how dysregulated cellular signals give riseto neoplastic, immune, neural, metabolic, or developmental disorders. Our goal isto understand the molecular bases of tumorigenesis and the development of otherintractable diseases as a path toward uncovering therapeutic targets. Currently,we are investigating regulatory mechanisms in protein-tyrosine kinase (PTK)-medi-ated signaling pathways and their pathophysiological roles in tumorigenesis, me-tastasis, inflammation, and myasthenia.

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neural agrin, an extracellular activator of MuSK.However, experimentally when motor nerves areablated, AChRs form clusters in the correct, centralregion of muscle during embryogenesis in a MuSK-dependent process known as prepatterning of thereceptors. In addition, in vivo overexpression ofMuSK causes neuromuscular synapse formation inthe absence of agrin, suggesting that muscle-intrin-sic, cell-autonomous activation of MuSK may beadequate to trigger presynaptic and postsynapticdifferentiation in vivo. However, the mechanismsby which MuSK is activated independently of nerveand agrin had long been unclear.Because both MuSK and the adaptor-like cyto-

plasmic protein Dok-7 are localized to the postsyn-aptic region of NMJs, we previously examined theirinteraction and found that Dok-7 is an essential cy-toplasmic activator of MuSK. In addition, we foundthat Dok-7 directly interacts with the cytoplasmicportion of MuSK and activates the RTK, and thatneural agrin requires Dok-7 in order to activateMuSK. Indeed, in vivo overexpression of Dok-7 in-creased MuSK activation and promoted NMJ for-mation. Conversely, mice lacking Dok-7 formedneither NMJs nor AChR clusters. Interestingly, micelacking Lrp4, which forms a complex with MuSKand acts as an essential agrin-binding module, donot show MuSK-dependent AChR prepatterning orNMJ formation. This suggests that Lrp4 is requiredfor MuSK activation under physiological condi-tions, in contrast to our observation that Dok-7 canactivate MuSK in the absence of Lrp4 or its ligandagrin, at least in vitro. Thus, we examined the ef-fects of forced expression of Dok-7 in skeletal mus-cle on NMJ formation in the absence of Lrp4 andfound that it indeed induces MuSK activation inmice lacking Lrp4. However, the activation level ofMuSK was significantly lower in the absence thanin the presence of Lrp4. Together, these data indi-cate that Lrp4 is required for efficient activation ofMuSK by Dok-7 in the muscle. Since Lrp4 is alsoessential for presynaptic differentiation of motornerve terminals (Nature 489: 438-442, 2012), this ap-parent cooperation between Lrp4 and Dok-7 inMuSK activation may be complicated. We are in-vestigating mechanisms of this cooperation in vivo.

2. Agrin's role aside from MuSK activation in thepostnatal maintenance of NMJs.

Tezuka, T., Hoshi, T., Burgess, RW.1, Ueta, R.,and Yamanashi, Y.: 1The Jackson Laboratory.

Although NMJ formation requires agrin underphysiological conditions, it is dispensable for NMJformation experimentally in the absence of the neu-rotransmitter acetylcholine, which inhibits postsyn-aptic specialization. Thus, it was hypothesized thatMuSK needs agrin together with Lrp4 and Dok-7 to

achieve sufficient activation to surmount inhibitionby acetylcholine. To test this hypothesis, we exam-ined the effects of forced expression of Dok-7 inskeletal muscle on NMJ formation in the absence ofagrin and found that it indeed restores NMJ forma-tion in agrin-deficient embryos. However, theseNMJs rapidly disappeared after birth, whereas ex-ogenous Dok-7-mediated MuSK activation wasmaintained. These findings indicate that the MuSKactivator agrin plays another role essential for thepostnatal maintenance, but not for embryonic for-mation, of NMJs. Because a pathogenic mutation ofagrin in patients with congenital myasthenic syn-dromes (see below) did not show impaired abilityto activate MuSK at least in vitro (Am. J. Hum.Genet. , 85: 155-167, 2009), the novel role of agrinmay be relevant to pathogenicity of the mutation.We are investigating how agrin plays the role inthe postnatal maintenance of NMJs.

3. Pathophysiological mechanisms underlyingDOK7 myasthenia.

Arimura, S., Tezuka, T., Eguchi, T., Yoshimura,T.1, Motomura, M.2, Yoshida, N.3, Beeson, D.4, andYamanashi, Y.: 1Department of OccupationalTherapy, Nagasaki University School of HealthSciences. 2Department of Electrical and ElectronicsEngineering, Faculty of Engineering, Nagasaki In-stitute of Applied Science. 3Laboratory of Develop-mental Genetics, IMSUT. 4Weatherall Institute ofMolecular Medicine, University of Oxford.

As mentioned above, impaired clustering ofAChRs could underlie NMJ disorders, be they auto-immune (MuSK antibody-positive myasthenia gra-vis) or genetic (congenital myasthenic syndromes(CMS)) in origin. Therefore, our findings that Dok-7activates MuSK to cluster AChRs and to form NMJssuggested DOK7 as a candidate gene for mutationsassociated with CMS. Indeed, we previously dem-onstrated that biallelic mutations in DOK7 underliea major subgroup of CMS with predominantlyproximal muscle weakness that did not show tubu-lar aggregates on muscle biopsy but were found tohave normal AChR function despite abnormallysmall and simplified NMJs. We further demon-strated that several mutations, including one associ-ated with the majority of patients with the disease,impaired Dok-7's ability to activate MuSK. Thisnew disease entity is termed "DOK7 myasthenia."To investigate pathophysiological mechanisms

underlying DOK7 myasthenia, we establishedknock-in mice (Dok-7 KI mice) that have a muta-tion associated with the majority of patients withDOK7 myasthenia. As expected, Dok-7 KI miceshowed characteristic features of severe muscleweakness and died between postnatal day 13 and20. Furthermore, they showed abnormally small

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NMJs lacking postsynaptic folding, a pathologicalfeature seen in patients with DOK7 myasthenia.Consistent with this, Dok-7 KI mice exhibited de-creased MuSK activity in skeletal muscle, indicatingthat the Dok-7 KI mice develop defects similar tothose found in patients with DOK7 myasthenia, al-though the mice exhibit a more severe phenotype.We are investigating other defects in NMJ functionand detailed pathophysiology in the Dok-7 KI mice.

4. DOK7 gene therapy that enlarges NMJs.

Arimura, S., Okada, T.1, Miyoshi, S., Ueta, R.,Kito, M., Yukimura, R., Tezuka, T., Okada, H.1,Kasahara, Y.1, Chiyo, T.2, Yoshimura, T.3, Mo-tomura, M.4, Yoshida, N.5, Beeson, D.6, Takeda, S.2,and Yamanashi, Y.: 1Department of Biochemistryand Molecular Biology, Nippon Medical School. 2

Department of Molecular Therapy, National Insti-tute of Neuroscience. 3Department of OccupationalTherapy, Nagasaki University School of HealthSciences. 4Department of Electrical and ElectronicsEngineering, Faculty of Engineering, Nagasaki In-stitute of Applied Science. 5Laboratory of Develop-mental Genetics, IMSUT. 6Weatherall Institute ofMolecular Medicine, University of Oxford.

As mentioned above, DOK7 myasthenia is associ-ated with impaired NMJ formation due to de-creased ability of Dok-7 to activate MuSK in myo-tubes at least in part. Interestingly, in vivo overex-pression of Dok-7 increased MuSK activation andpromoted NMJ formation in the correct, central re-gion of the skeletal muscle. Because these geneti-cally manipulated mice did not show obvious de-fects in motor activity, overexpression of Dok-7 inthe skeletal muscle of patients with DOK7 myasthe-nia might ameliorate NMJ formation and muscleweakness. To test this possibility, we generated anAdeno-associated virus-based vector (AAV-D7),which strongly expressed human Dok-7 in myo-tubes and induced AChR cluster formation. Indeed,therapeutic administration of AAV-D7 to Dok-7 KImice described above resulted in enlargement ofNMJs and substantial increases in muscle strengthand life span. Furthermore, when applied to modelmice of another neuromuscular disorder, autosomaldominant Emery-Dreifuss muscular dystrophy,therapeutic administration of AAV-D7 likewise re-sulted in enlargement of NMJs as well as positiveeffects on motor activity and life span. These resultssuggest that therapies aimed at enlarging the NMJmay be useful for a range of neuromuscular disor-ders. We are investigating the effects of AAV-D7administration on other types of neuromusculardisease models. Because we used cytomegaloviruspromoter, which showed high efficacy but little tis-sue-specificity of Dok-7 expression, for AAV-D7 inthe above experiments, we are developing another

type of AAV-D7, which contains muscle-specificpromoter for the safer therapeutic approach.

5. Lrp4 antibodies in patients with myastheniagravis.

Tezuka, T., Motomura, M.1, Hoshi, T., andYamanashi, Y.: 1Department of Electrical andElectronics Engineering, Faculty of Engineering,Nagasaki Institute of Applied Science.

Myasthenia gravis (MG) is an autoimmune dis-ease of the NMJ. About 80％ of patients with gener-alized MG have AChR antibodies, the presence ofwhich is a causative factor for the disease, and avariable proportion of the remaining patients (0-50％ throughout the world) have MuSK antibodies.However, diagnosis and clinical management re-main complicated for patients who are negative forMuSK and AChR antibodies. Given the essentialroles and postsynaptic localization of Lrp4 in theNMJ, we hypothesized that Lrp4 autoantibodiesmight be a pathogenic factor in MG. To test thishypothesis, we developed a luminescence-basedmethod to efficiently detect serum autoantibodies toLrp4 in patients, and found that 9 patients werepositive for antibodies to the extracellular portionof Lrp4 from a cohort of 300 patients with AChRantibody-negative MG. 6 of these 9 patients withLrp4 antibody-positive MG were also negative forMuSK antibodies, and generalized MG was diag-nosed in all 9 patients, who showed severe limbmuscle weakness or progressive bulbar palsy orboth. Thymoma was not observed in any of thesepatients, unlike the situation in patients with AChRantibody-positive MG. Furthermore, we confirmedthat serum antibodies to Lrp4 recognize its nativeform and inhibit binding of Agrin to Lrp4, which iscrucial for NMJs. Also, we found that Lrp4 autoan-tibodies were predominantly comprised of IgG1, acomplement activator, implicating the potential forthese antibodies to cause complement-mediated im-pairment of NMJs. Together, our findings indicatethe involvement of Lrp4 antibodies in the patho-genesis of AChR antibody-negative MG. Followingthis study, two groups in Germany and USA re-ported respectively that about 50％ and 10％ of MGpatients, who were negative for both MuSK andAChR antibodies, were positive for antibodies toLrp4 (J. Neurol., 259: 427-435, 2012; Arch. Neurol., 69:445-451, 2012). Also it was reported that antibodiesto Lrp4 induced MG in model animals (J. Clin. In-vest., 123: 5190-5202, 2013). Given that Lrp4 anti-bodies are reported to be involved in amyotrophiclateral sclerosis (ALS) (Ann. Clin. Transl. Neurol. , 1:80-87, 2014), which is associated with NMJ defects,we have expanded our research area with regard toLrp4 antibodies from MG to neuromuscular disor-ders characterized by NMJ defects.

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6. Roles of Dok-1 to Dok-6.

Arimura, S., Waseda, M., Kajikawa, S., Hoshi, T.,Kanno, T., Jozawa, H., Oda, H.1, Shimura, E.2,Nakae, S.2, Ezura, Y.3, Taguchi, Y.4, Inoue, J.4,Noda, M.3, and Yamanashi, Y.: 1Department ofPathology, Tokyo Women's Medical University.2Laboratory of Systems Biology, IMSUT. 3Depart-ment of Molecular Pharmacology, Medical Re-search Institute, Tokyo Medical and Dental Uni-versity. 4Division of Cellular and Molecular Biol-ogy, IMSUT.

Dok-family proteins can be classified into threesubgroups based on their structural similarities andexpression patterns; namely, 1) Dok-1, -2, and -3,which are preferentially expressed in hematopoieticcells, 2) Dok-4, -5, and -6, which are preferentiallyexpressed in non-hematopoietic cells, and 3) Dok-7,which is preferentially expressed in muscle cells. Asmentioned above, Dok-1 and its closest paralog,Dok-2, recruit p120 rasGAP upon tyrosine phos-phorylation to suppress Ras-Erk signaling. Al-though Dok-3 does not bind with p120 rasGAP, italso inhibits Ras-Erk signaling. Consistently, wepreviously demonstrated that Dok-1, Dok-2 andDok-3 are key negative regulators of hematopoieticgrowth and survival signaling. For example, Dok-1,Dok-2, and Dok-3 cooperatively inhibit macrophageproliferation and Dok-1－/－Dok-2－/－Dok-3－/－ mice de-velop histiocytic sarcoma, an aggressive malignancyof macrophages. In addition, we found that theseDok proteins cooperatively play critical anti-inflam-matory roles in lung homeostasis. We are furtherinvestigating roles of Dok-1 to Dok-6, includingthose in tumor malignancy, inflammatory disor-ders, and other types of intractable diseases.

7. Omic analyses.

Tezuka, T., Eguchi, T., Honda, M., Ueta, R., Ari-mura, S., Miyoshi. S., Hoshi, T., Iemura, S.1, Na-tsume, T.2, Oyama, M.3, Sagara, H.3, and Ya-manashi, Y.: 1Translational Research Center,Fukushima Medical University. 2National Instituteof Advanced Science and Technology, MolecularProfiling Research Center for Drug Discovery.3Medical Proteomics Laboratory, IMSUT.

To gain insights into signaling mechanisms un-derlying a variety of physiological and pathophysi-

ological events, including NMJ formation, tumori-genesis, and tumor metastasis, we have performedproteomic and transcriptomic analyses. We are in-vestigating the roles of candidate proteins andgenes that appear to be involved in each of thesebiological events. In addition, we have prepared ex-perimental settings for other omic approaches suchas glycomic and metabolomic analyses.For instance, we previously performed mass

spectrometric analysis of Lrp4-binding proteins andfound the chaperon Mesdc2 as a candidate. Weconfirmed their binding in cells, and revealed thatMesdc2 bind selectively to the lower molecularmass form of Lrp4 (lower Lrp4) but not to the up-per, more glycosylated form (upper Lrp4). Al-though the Mesdc2 binds to lower Lrp4, forced ex-pression of Mesdc2 increased upper Lrp4, implyinga role for Mesdc2 in the Lrp4 glycosylation, whichmight facilitate the receptor's cell surface expres-sion. Indeed, we found that down regulation ofMesdc2 expression in cultured myotubes sup-pressed cell-surface expression of Lrp4, or upperLrp4 more specifically. Furthermore, down regula-tion of Mesdc2 also inhibited agrin-induced post-synaptic specialization in myotubes, which requiresbinding of Lrp4 to its extracellular ligand, the neu-ral agrin. Together, these findings demonstratedthat Mesdc2 plays a key role in Lrp4-dependentpostsynaptic specialization probably by promotingglycosylation and cell-surface expression of Lrp4 inmyotubes. We are investigating roles for glycosyla-tion of Lrp4 in its pleiotropic activities.

8. Screening of chemical compound and siRNAlibraries.

Ueta, R., Yukimura R., Waseda, M., Honda, M.,Kito M., Sasaki, Y., Nagatoishi, S.1, Tsumoto, K.1,and Yamanashi, Y.: 1Medical Proteomics Labora-tory, IMSUT.

In addition to the omic analyses described above,we performed high throughput screenings ofchemical compound and siRNA libraries, aiming tointervene in pathogenic signals or to gain insightsinto signaling mechanisms underlying a variety ofbiological events. We continue the ongoing screen-ings to collect appropriate hit compounds and can-didate genes that may regulate important signal-ings.

Publications

Ueta, R. and Yamanashi, Y. Molecular signalingand its pathogenic alterations in neuromuscularjunction formation and maintenance. In "Proteinmodifications in pathogenic dysregulation of sig-

naling". (Springer), 309-325, 2015.有村純暢，山梨裕司．神経筋接合部の形成不全を伴う神経筋疾患に対する新規治療概念の創出：Dok-7の発現増強による神経筋接合部形成増強治療．実

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験医学．羊土社．３３（４）：６０３―６０６，２０１５

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