研究者データベース

研究者情報

マスター

アカウント(マスター)

  • 氏名

    大場 雄介(オオバ ユウスケ), オオバ ユウスケ

所属(マスター)

  • 医学研究院 生理系部門 生理学分野

所属(マスター)

  • 医学研究院 生理系部門 生理学分野

独自項目

syllabus

  • 2020, 基本医学研究法Ⅱ, Basic Research Methods in Medical Sciences, 修士課程, 医学院, 生理学、蛍光イメージング、脳科学 Physiology, fluorescence bioimaging, brain science
  • 2020, 基本医学研究, Master's Thesis Research in Medical Sciences, 修士課程, 医学院, シグナル伝達、細胞機能、バイオイメージング、蛍光 signal transduction, cell function, bioimaging, fluorescence
  • 2020, 基本医学総論, Basic Principles of Medicine, 修士課程, 医学院, シグナル伝達、細胞生理、バイオイメージング、蛍光 signal transduction, cell function, bioimaging, fluorescence
  • 2020, ソフトマター医工学特論, Soft Matter Medical Engineering, 修士課程, 生命科学院, 基礎医学、再生医学、バイオマテリアル、がん生物学
  • 2020, 医学研究法Ⅱ, Research Methods in Medical Sciences Ⅱ, 博士後期課程, 医学研究科, 生理学、蛍光イメージング、脳科学
  • 2020, 医学研究法Ⅱ, Research Methods in Medical Sciences Ⅱ, 博士後期課程, 医学院, 生理学、蛍光イメージング、脳科学
  • 2020, 医学総論, Principles of Medicine, 博士後期課程, 医学院, シグナル伝達、細胞生理、バイオイメージング、蛍光
  • 2020, 基盤医学研究, Dissertation Research in Medical Sciences, 博士後期課程, 医学院, シグナル伝達、細胞機能、バイオイメージング、蛍光
  • 2020, 生理学Ⅰ, PhysiologyⅠ, 学士課程, 医学部, 呼吸、循環、血液、体液・電解質調節、腎機能、内分泌、代謝・消化吸収
  • 2020, 保健生理学, Physiology, 学士課程, 医学部, 細胞・組織生理、循環生理、呼吸生理、消化・吸収の生理、代謝・内分泌生理、外部環境からの防御、発生と老化、神経・筋生理
  • 2020, 生理学実習, Physiology Practice, 学士課程, 医学部, 循環、血液、呼吸、筋電図、心電図、脳波

timetable

  • 修士課程, 医学院, 2020, 研究発表技法Ⅰ
  • 修士課程, 医学院, 2020, 研究発表技法Ⅱ
  • 博士後期課程, 医学研究科, 2020, 研究発表技法Ⅰ
  • 博士後期課程, 医学研究科, 2020, 研究発表技法Ⅱ
  • 博士後期課程, 医学研究科, 2020, 基盤医学研究Ⅱ
  • 博士後期課程, 医学研究科, 2020, 基盤医学研究Ⅰ
  • 博士後期課程, 医学研究科, 2020, 医学総論
  • 博士後期課程, 医学研究科, 2020, 医学研究法Ⅲ
  • 博士後期課程, 医学院, 2020, 研究発表技法Ⅰ
  • 博士後期課程, 医学院, 2020, 研究発表技法Ⅱ

PositionHistory

  • 経営戦略室室員, 2017年10月26日, 2019年3月31日
  • 経営戦略室室員, 2019年4月1日, 2020年9月30日
  • 総長補佐, 2017年11月1日, 2019年3月31日
  • 総長補佐, 2017年4月1日, 2017年10月31日
  • 総長補佐, 2019年4月1日, 2020年9月30日
  • 研究戦略室室員, 2017年4月1日, 2019年3月31日
  • 研究戦略室室員, 2019年4月1日, 2020年9月30日
  • 研究戦略室室員, 2020年10月12日, 2022年3月31日

researchmap

プロフィール情報

学位

  • 医学博士(北海道大学)

プロフィール情報

  • 大場, オオバ
  • 雄介, ユウスケ
  • ID各種

    200901014201313812

対象リソース

業績リスト

研究キーワード

  • 細胞膜動態   原子間力顕微鏡   FRET   GFP   バイオイメージング   Ras   細胞内情報伝達   イメージング   環境適応   生細胞   BiFC   多分子複合体   シグナル伝達   Gタンパク質   分子動態   エンドサイトーシス   

研究分野

  • ライフサイエンス / 生理学
  • ライフサイエンス / 細胞生物学
  • ライフサイエンス / 実験病理学

経歴

  • 2006年04月 - 2012年10月 北海道大学大学院医学研究科 准教授
  • 2004年03月 - 2006年03月 東京大学大学院医学系研究科 助手
  • 2001年12月 - 2005年03月 科学技術振興機構 さきがけ 研究員(兼任)
  • 2001年01月 - 2004年03月 大阪大学微生物病研究所 助手
  • 1998年04月 - 2000年12月 国立国際医療センター研究所 流動研究員

学歴

  • 1996年04月 - 2000年03月   北海道大学   大学院医学研究科   病理系専攻
  •         - 1996年03月   北海道大学   医学部   医学科

受賞

  • 2013年06月 ドイツ 科学・イノベーション フォーラム 東京・在日ドイツ商工会議所 ドイツ・イノベーション・アワード 「ゴットフリード・ワグネル賞 2013」
     RETバイオセンサーによるCML分子標的薬治療効果予測法 
    受賞者: 大場 雄介

論文

  • Yasuto Takeuchi, Rika Narumi, Ryutaro Akiyama, Elisa Vitiello, Takanobu Shirai, Nobuyuki Tanimura, Keisuke Kuromiya, Susumu Ishikawa, Mihoko Kajita, Masazumi Tada, Yukinari Haraoka, Yuki Akieda, Tohru Ishitani, Yoichiro Fujioka, Yusuke Ohba, Sohei Yamada, Yoichiroh Hosokawa, Yusuke Toyama, Takaaki Matsui, Yasuyuki Fujita
    Current biology : CB 30 4 670 - 681 2020年02月24日 [査読有り][通常論文]
     
    When oncogenic transformation or apoptosis occurs within epithelia, the harmful or dead cells are apically extruded from tissues to maintain epithelial homeostasis. However, the underlying molecular mechanism still remains elusive. In this study, we first show, using mammalian cultured epithelial cells and zebrafish embryos, that prior to apical extrusion of RasV12-transformed cells, calcium wave occurs from the transformed cell and propagates across the surrounding cells. The calcium wave then triggers and facilitates the process of extrusion. IP3 receptor, gap junction, and mechanosensitive calcium channel TRPC1 are involved in calcium wave. Calcium wave induces the polarized movement of the surrounding cells toward the extruding transformed cells. Furthermore, calcium wave facilitates apical extrusion, at least partly, by inducing actin rearrangement in the surrounding cells. Moreover, comparable calcium propagation also promotes apical extrusion of apoptotic cells. Thus, calcium wave is an evolutionarily conserved, general regulatory mechanism of cell extrusion.
  • Sayaka Kashiwagi, Yoichiro Fujioka, Aya O Satoh, Aiko Yoshida, Mari Fujioka, Prabha Nepal, Atsushi Tsuzuki, Ozora Aoki, Sarad Paudel, Hitoshi Sasajima, Yusuke Ohba
    Cell structure and function 44 2 183 - 194 2019年12月26日 [査読有り][通常論文]
     
    The discovery of fluorescent proteins (FPs) has revolutionized cell biology. The fusion of targeting sequences to FPs enables the investigation of cellular organelles and their dynamics; however, occasionally, such fluorescent fusion proteins (FFPs) exhibit behavior different from that of the native proteins. Here, we constructed a color pallet comprising different organelle markers and found that FFPs targeted to the mitochondria were mislocalized when fused to certain types of FPs. Such FPs included several variants of Aequorea victoria green FP (avGFP) and a monomeric variant of the red FP. Because the FFPs that are mislocalized include FPs with faster maturing or folding mutations, the increase in the maturation rate is likely to prevent their expected localization. Indeed, when we reintroduced amino acid substitutions so that the FP sequences were equivalent to that of wild-type avGFP, FFP localization to the mitochondria was significantly enhanced. Moreover, similar amino acid substitutions improved the localization of mitochondria-targeted pHluorin, which is a pH-sensitive variant of GFP, and its capability to monitor pH changes in the mitochondrial matrix. Our findings demonstrate the importance of selecting FPs that maximize FFP function.Key words: fluorescent protein, organelle, fusion protein, mitochondria.
  • Sayaka Kashiwagi, Yoichiro Fujioka, Takeshi Kondo, Aya O Satoh, Aiko Yoshida, Mari Fujioka, Hitoshi Sasajima, Maho Amano, Takanori Teshima, Yusuke Ohba
    Cell structure and function 44 2 195 - 204 2019年12月26日 [査読有り][通常論文]
     
    The oncogenic tyrosine kinase BCR-ABL activates a variety of signaling pathways and plays a causative role in the pathogenesis of chronic myelogenous leukemia (CML); however, the subcellular distribution of this chimeric protein remains controversial. Here, we report that BCR-ABL is localized to stress granules and that its granular localization contributes to BCR-ABL-dependent leukemogenesis. BCR-ABL-positive granules were not colocalized with any markers for membrane-bound organelles but were colocalized with HSP90a, a component of RNA granules. The number of such granules increased with thapsigargin treatment, confirming that the granules were stress granules. Given that treatment with the ABL kinase inhibitor imatinib and elimination of the N-terminal region of BCR-ABL abolished granule formation, kinase activity and the coiled-coil domain are required for granule formation. Whereas wild-type BCR-ABL rescued the growth defect in IL-3-depleted Ba/F3 cells, mutant BCR-ABL lacking the N-terminal region failed to do so. Moreover, forced tetramerization of the N-terminus-deleted mutant could not restore the growth defect, indicating that granule formation, but not tetramerization, through its N-terminus is critical for BCR-ABL-dependent oncogenicity. Our findings together provide new insights into the pathogenesis of CML by BCR-ABL and open a window for developing novel therapeutic strategies for this disease.Key words: BCR-ABL, subcellular localization, stress granule.
  • Takeshi Kondo, Mari Fujioka, Shinichi Fujisawa, Kaori Sato, Masumi Tsuda, Takuto Miyagishima, Akio Mori, Hiroshi Iwasaki, Yasutaka Kakinoki, Satoshi Yamamoto, Yoshihito Haseyama, Seisho Ando, Motohiro Shindo, Shuichi Ota, Mitsutoshi Kurosawa, Yusuke Ohba, Takanori Teshima
    International journal of hematology 110 4 482 - 489 2019年10月 [査読有り][通常論文]
     
    Nilotinib is widely used for primary treatment of patients with chronic myelogenous leukemia (CML). We previously reported that use of an FRET-based drug sensitivity test at diagnosis efficiently predicts the response to treatment with imatinib or dasatinib. Here, we conducted a phase-II study to evaluate the efficacy and safety of nilotinib treatment and identify useful biomarkers, including results of the FRET-based drug sensitivity test, for predicting treatment response. Data from 42 patients were used in the analysis. Major molecular response (MMR), MR4, and MR4.5 rates at 12 months were 64.3, 42.9, and 28.6%, respectively. Grade 3/4 non-hematologic adverse events occurred in 11 patients (26.2%). The dose intensity of nilotinib (> 76.44%) and halving time (HT, < 13.312 days) were identified as significant factors for MMR at 12 months. However, when we focused on patients whose dose intensity of nilotinib was > 76.44%, the FRET-based drug sensitivity test became a predictive factor of MR4 achievement at 12 months. Our study reconfirmed the efficacy and safety of nilotinib treatment in CML patients. Moreover, our results suggest that the FRET-based drug sensitivity test is an independent predictor for achievement of MR4 in patients treated with a sufficient dose intensity of nilotinib.
  • Nako Maishi, Hiroshi Kikuchi, Masumi Sato, Hiroko Nagao-Kitamoto, Dorcas A Annan, Shogo Baba, Takayuki Hojo, Misa Yanagiya, Yusuke Ohba, Genichiro Ishii, Kenkichi Masutomi, Nobuo Shinohara, Yasuhiro Hida, Kyoko Hida
    International journal of molecular sciences 20 18 2019年09月17日 [査読有り][通常論文]
     
    Tumor angiogenesis research and antiangiogenic drug development make use of cultured endothelial cells (ECs) including the human microvascular ECs among others. However, it has been reported that tumor ECs (TECs) are different from normal ECs (NECs). To functionally validate antiangiogenic drugs, cultured TECs are indispensable tools, but are not commercially available. Primary human TECs are available only in small quantities from surgical specimens and have a short life span in vitro due to their cellular senescence. We established immortalized human TECs (h-imTECs) and their normal counterparts (h-imNECs) by infection with lentivirus producing simian virus 40 large T antigen and human telomerase reverse transcriptase to overcome the replication barriers. These ECs exhibited an extended life span and retained their characteristic endothelial morphology, expression of endothelial marker, and ability of tube formation. Furthermore, h-imTECs showed their specific characteristics as TECs, such as increased proliferation and upregulation of TEC markers. Treatment with bevacizumab, an antiangiogenic drug, dramatically decreased h-imTEC survival, whereas the same treatment failed to alter immortalized NEC survival. Hence, these h-imTECs could be a valuable tool for drug screening to develop novel therapeutic agents specific to TECs or functional biological assays in tumor angiogenesis research.
  • Yoichiro Fujioka, Aya O Satoh, Kosui Horiuchi, Mari Fujioka, Kaori Tsutsumi, Junko Sasaki, Prabha Nepal, Sayaka Kashiwagi, Sarad Paudel, Shinya Nishide, Asuka Nanbo, Takehiko Sasaki, Yusuke Ohba
    Cell structure and function 44 1 61 - 74 2019年04月25日 [査読有り][通常論文]
     
    Endocytosis mediates the internalization and ingestion of a variety of endogenous or exogenous substances, including virus particles, under the control of intracellular signaling pathways. We have previously reported that the complex formed between the small GTPase Ras and phosphoinositide 3-kinase (PI3K) translocates from the plasma membrane to endosomes, signaling from which thereby regulates clathrin-independent endocytosis, endosome maturation, influenza virus internalization, and infection. However, the molecular mechanism by which the Ras-PI3K complex is recruited to endosomes remains unclear. Here, we have identified the amino acid sequence responsible for endosomal localization of the Ras-PI3K complex. PI3K lacking this sequence failed to translocate to endosomes, and expression of the peptide comprising this PI3K-derived sequence inhibited clathrin-independent endocytosis, influenza virus internalization, and infection. Moreover, treatment of cells with this peptide in an arginine-rich, cell-penetrating form successfully suppressed influenza virus infection in vitro and ex vivo, making this peptide a potential therapeutic agent against influenza virus infection.Key words: signal transduction, endocytosis, endosome, imaging, influenza virus.
  • Kazuhito V Tabata, Yoshihiro Minagawa, Yuko Kawaguchi, Mana Ono, Yoshiki Moriizumi, Seiya Yamayoshi, Yoichiro Fujioka, Yusuke Ohba, Yoshihiro Kawaoka, Hiroyuki Noji
    Scientific reports 9 1 1067 - 1067 2019年01月31日 [査読有り][通常論文]
     
    There is large demand for a quantitative method for rapid and ultra-sensitive detection of the influenza virus. Here, we established a digital influenza virus counting (DIViC) method that can detect a single virion without antibody. In the assay, a virion is stochastically entrapped inside a femtoliter reactor array device for the fluorogenic assay of neuraminidase, and incubated for minutes. By analyzing 600,000 reactors, the practical limit of detection reached the order of 103 (PFU)/mL, only 10-times less sensitive than RT-PCR and more than 1000-times sensitive than commercial rapid test kits (RIDTs). Interestingly, neuraminidase activity differed among virions. The coefficient of variance was 30-40%, evidently broader than that of alkaline phosphatase measured as a model enzyme for comparison, suggesting the heterogeneity in size and integrity among influenza virus particles. Sensitivity to oseltamivir also differed between virions. We also tested DIViC using clinical gargle samples that imposes less burden for sampling while with less virus titre. The comparison with RIDTs showed that DIViC was largely superior to RIDTs in the sensitivity with the clinical samples although a few false-positive signals were observed in some clinical samples that remains as a technical challenge.
  • Asuka Nanbo, Yusuke Ohba
    The Journal of infectious diseases 218 suppl_5 S388-S396  2018年11月22日 [査読有り][通常論文]
     
    The Ebola virus-encoded major matrix protein VP40 traffics to the plasma membrane, which leads to the formation of filamentous viral particles and subsequent viral egress. However, the cellular machineries underlying this process are not fully understood. In the present study, we have assessed the role of host endocytic recycling in Ebola virus particle formation. We found that a small GTPase Rab11, which regulates recycling of molecules among the trans-Golgi network, recycling endosomes, and the plasma membrane, was incorporated in Ebola virus-like particles. Although Rab11 predominantly localized in the perinuclear region, it distributed diffusely in the cytoplasm and partly localized in the periphery of the cells transiently expressing VP40. In contrast, Rab11 exhibited a perinuclear distribution when 2 VP40 derivatives that lack ability to traffic to the plasma membrane were expressed. Finally, expression of a dominant-negative form of Rab11 or knockdown of Rab11 inhibited both VP40-induced clusters at the plasma membrane and release of viral-like particles. Taken together, our findings demonstrate that Ebola virus exploits host endocytic recycling machinery to facilitate the trafficking of VP40 to the cell surface and the subsequent release of viral-like particles for its establishment of efficient viral egress.
  • Nanbo A, Katano H, Kataoka M, Hoshina S, Sekizuka T, Kuroda M, Ohba Y
    Cancers 10 7 2018年07月19日 [査読有り][通常論文]
     
    Infection of Epstein⁻Barr virus (EBV), a ubiquitous human gamma herpesvirus, is associated with various malignancies in B lymphocytes and epithelial cells. EBV encodes 49 microRNAs in two separated regions, termed the BART and BHRF1 loci. Although accumulating evidence demonstrates that EBV infection regulates the profile of microRNAs in the cells, little is known about the microRNAs in exosomes released from infected cells. Here, we characterized the expression profile of intracellular and exosomal microRNAs in EBV-negative, and two related EBV-infected Burkitt lymphoma cell lines having type I and type III latency by next-generation sequencing. We found that the biogenesis of exosomes is upregulated in type III latently infected cells compared with EBV-negative and type I latently infected cells. We also observed that viral and several specific host microRNAs were predominantly incorporated in the exosomes released from the cells in type III latency. We confirmed that multiple viral microRNAs were transferred to the epithelial cells cocultured with EBV-infected B cells. Our findings indicate that EBV infection, in particular in type III latency, modulates the biogenesis of exosomes and the profile of exosomal microRNAs, potentially contributing to phenotypic changes in cells receiving these exosomes.
  • Takeshi Kondo, Mari Fujioka, Masumi Tsuda, Kazunori Murai, Kohei Yamaguchi, Takuto Miyagishima, Motohiro Shindo, Takahiro Nagashima, Kentaro Wakasa, Nozomu Fujimoto, Satoshi Yamamoto, Masakatsu Yonezumi, Souichi Saito, Shinji Sato, Kazuei Ogawa, Takaaki Chou, Reiko Watanabe, Yuichi Kato, Shuichiro Takahashi, Yoshiaki Okano, Joji Yamamoto, Masatsugu Ohta, Hiroaki Iijima, Koji Oba, Satoshi Kishino, Junichi Sakamoto, Yoji Ishida, Yusuke Ohba, Takanori Teshima
    Cancer science 109 7 2256 - 2265 2018年07月 [査読有り][通常論文]
     
    Tyrosine kinase inhibitors (TKI) are used for primary therapy in patients with newly diagnosed CML. However, a reliable method for optimal selection of a TKI from the viewpoint of drug sensitivity of CML cells has not been established. We have developed a FRET-based drug sensitivity test in which a CrkL-derived fluorescent biosensor efficiently quantifies the kinase activity of BCR-ABL of living cells and sensitively evaluates the inhibitory activity of a TKI against BCR-ABL. Here, we validated the utility of the FRET-based drug sensitivity test carried out at diagnosis for predicting the molecular efficacy. Sixty-two patients with newly diagnosed chronic phase CML were enrolled in this study and treated with dasatinib. Bone marrow cells at diagnosis were subjected to FRET analysis. The ΔFRET value was calculated by subtraction of FRET efficiency in the presence of dasatinib from that in the absence of dasatinib. Treatment response was evaluated every 3 months by the BCR-ABL1 International Scale. Based on the ΔFRET value and molecular response, a threshold of the ΔFRET value in the top 10% of FRET efficiency was set to 0.31. Patients with ΔFRET value ≥0.31 had significantly superior molecular responses (MMR at 6 and 9 months and both MR4 and MR4.5 at 6, 9, and 12 months) compared with the responses in patients with ΔFRET value <0.31. These results suggest that the FRET-based drug sensitivity test at diagnosis can predict early and deep molecular responses. This study is registered with UMIN Clinical Trials Registry (UMIN000006358).
  • Yoichiro Fujioka, Shinya Nishide, Toyoyuki Ose, Tadaki Suzuki, Izumi Kato, Hideo Fukuhara, Mari Fujioka, Kosui Horiuchi, Aya O Satoh, Prabha Nepal, Sayaka Kashiwagi, Jing Wang, Mika Horiguchi, Yuko Sato, Sarad Paudel, Asuka Nanbo, Tadaaki Miyazaki, Hideki Hasegawa, Katsumi Maenaka, Yusuke Ohba
    Cell host & microbe 23 6 809 - 818 2018年06月13日 [査読有り][通常論文]
     
    Influenza A virus (IAV) infection is initiated by the attachment of the viral glycoprotein hemagglutinin (HA) to sialic acid on the host cell surface. However, the sialic acid-containing receptor crucial for IAV infection has remained unidentified. Here, we show that HA binds to the voltage-dependent Ca2+ channel Cav1.2 to trigger intracellular Ca2+ oscillations and subsequent IAV entry and replication. IAV entry was inhibited by Ca2+ channel blockers (CCBs) or by knockdown of Cav1.2. The CCB diltiazem also inhibited virus replication in vivo. Reintroduction of wild-type but not the glycosylation-deficient mutants of Cav1.2 restored Ca2+ oscillations and virus infection in Cav1.2-depleted cells, demonstrating the significance of Cav1.2 sialylation. Taken together, we identify Cav1.2 as a sialylated host cell surface receptor that binds HA and is critical for IAV entry.
  • Shinya Kitamura, Teruki Yanagi, Yuka Inamura-Takashima, Keisuke Imafuku, Hiroo Hata, Yoichiro Fujioka, Yusuke Ohba, Hiroshi Shimizu
    Journal of Dermatological Science 90 2 213 - 216 2018年05月01日 [査読有り][通常論文]
  • Kazuhiro Yachi, Masumi Tsuda, Shinji Kohsaka, Lei Wang, Yoshitaka Oda, Satoshi Tanikawa, Yusuke Ohba, Shinya Tanaka
    Signal transduction and targeted therapy 3 33 - 33 2018年 [査読有り][通常論文]
     
    Glioblastoma is the most aggressive and invasive brain tumor and has a poor prognosis; elucidating the underlying molecular mechanisms is essential to select molecular targeted therapies. Here, we investigated the effect of microRNAs on the marked invasiveness of glioblastoma. U373 glioblastoma cells were infected with 140 different microRNAs from an OncomiR library, and the effects of the invasion-related microRNAs and targeted molecules were investigated after repeated Matrigel invasion assays. Screening of the OncomiR library identified miR-23a as a key regulator of glioblastoma invasion. In six glioblastoma cell lines, a positive correlation was detected between the expression levels of miR-23a and invasiveness. A luciferase reporter assay demonstrated that homeobox D10 (HOXD10) was a miR-23a-target molecule, which was verified by high scores from both the PicTar and miRanda algorithms. Forced expression of miR-23a induced expression of invasion-related molecules, including uPAR, RhoA, and RhoC, and altered expression of glial-mesenchymal transition markers such as Snail, Slug, MMP2, MMP9, MMP14, and E-cadherin; however, these changes in expression levels were reversed by HOXD10 overexpression. Thus, miR-23a significantly promoted invasion of glioblastoma cells with polarized formation of focal adhesions, while exogenous HOXD10 overexpression reversed these phenomena. Here, we identify miR-23a-regulated HOXD10 as a pivotal regulator of invasion in glioblastoma, providing a novel mechanism for the aggressive invasiveness of this tumor and providing insight into potential therapeutic targets.
  • Asuka Nanbo, Junki Maruyama, Masaki Imai, Michiko Ujie, Yoichiro Fujioka, Shinya Nishide, Ayato Takada, Yusuke Ohba, Yoshihiro Kawaoka
    PLoS pathogens 14 1 e1006848  2018年01月 [査読有り][通常論文]
     
    Cell surface receptors for phosphatidylserine contribute to the entry of Ebola virus (EBOV) particles, indicating that the presence of phosphatidylserine in the envelope of EBOV is important for the internalization of EBOV particles. Phosphatidylserine is typically distributed in the inner layer of the plasma membrane in normal cells. Progeny virions bud from the plasma membrane of infected cells, suggesting that phosphatidylserine is likely flipped to the outer leaflet of the plasma membrane in infected cells for EBOV virions to acquire it. Currently, the intracellular dynamics of phosphatidylserine during EBOV infection are poorly understood. Here, we explored the role of XK-related protein (Xkr) 8, which is a scramblase responsible for exposure of phosphatidylserine in the plasma membrane of apoptotic cells, to understand its significance in phosphatidylserine-dependent entry of EBOV. We found that Xkr8 and transiently expressed EBOV glycoprotein GP often co-localized in intracellular vesicles and the plasma membrane. We also found that co-expression of GP and viral major matrix protein VP40 promoted incorporation of Xkr8 into ebolavirus-like particles (VLPs) and exposure of phosphatidylserine on their surface, although only a limited amount of phosphatidylserine was exposed on the surface of the cells expressing GP and/or VP40. Downregulating Xkr8 or blocking caspase-mediated Xkr8 activation did not affect VLP production, but they reduced the amount of phosphatidylserine on the VLPs and their uptake in recipient cells. Taken together, our findings indicate that Xkr8 is trafficked to budding sites via GP-containing vesicles, is incorporated into VLPs, and then promote the entry of the released EBOV to cells in a phosphatidylserine-dependent manner.
  • Asuka Nanbo, Takeshi Noda, Yusuke Ohba
    Frontiers in microbiology 9 454 - 454 2018年 [査読有り][通常論文]
     
    Herpesvirus subfamilies typically acquire their final envelope in various cytoplasmic compartments such as the trans-Golgi network (TGN), and endosomes prior to their secretion into the extracellular space. However, the sites for the final envelopment of Epstein-Barr virus (EBV), a ubiquitous human gamma herpesvirus, are poorly understood. Here, we characterized the sites for the final envelopment of EBV in Burkitt's lymphoma cell lines induced into the lytic cycle by crosslinking cell surface IgG. Electron microscopy revealed the various stages of maturation and egress of progeny virions including mature EBV in irregular cytoplasmic vesicles. Immunofluorescence staining showed that gp350/220, the major EBV glycoprotein, and the viral capsid antigen, p18, efficiently colocalized with a cis-Golgi marker, GM130. gp350/220 partly colocalized with the TGN, which was distributed in a fragmented and dispersed pattern in the cells induced into the lytic cycle. In contrast, limited colocalization was observed between gp350/220 and endosomal markers, such as a multi-vesicular bodies marker, CD63, a recycling endosome marker, Rab11, and a regulatory secretion vesicles marker, Rab27a. Finally, we observed that treatment of cells with brefeldin A, an inhibitor of vesicle trafficking between the endoplasmic reticulum and Golgi apparatus, resulted in the perinuclear accumulation of gp350/220 and inhibition of its distribution to the plasma membrane. Brefeldin A also inhibited the release of infectious EBV. Taken together, our findings support a model in which EBV acquires its final envelope in intracellular compartments containing markers of Golgi apparatus, providing new insights into how EBV matures.
  • Nanbo A, Ohashi M, Yoshiyama H, Ohba Y
    Frontiers in microbiology 9 984 - 984 2018年 [査読有り][通常論文]
     
    Infection of Epstein-Barr virus (EBV), a ubiquitous human gamma herpesvirus, is closely linked to various lymphoid and epithelial malignancies. Previous studies demonstrated that the efficiency of EBV infection in epithelial cells is significantly enhanced by coculturing them with latently infected B cells relative to cell-free infection, suggesting that cell-to-cell contact-mediated viral transmission is the dominant mode of infection by EBV in epithelial cells. However, a detailed mechanism underlying this process has not been fully understood. In the present study, we assessed the role of transforming growth factor β (TGF-β), which is known to induce EBV's lytic cycle by upregulation of EBV's latent-lytic switch BZLF1 gene. We have found that 5 days of cocultivation facilitated cell-to-cell contact-mediated EBV transmission. Replication of EBV was induced in cocultured B cells both with and without a direct cell contact in a time-dependent manner. Treatment of a blocking antibody for TGF-β suppressed both induction of the lytic cycle in cocultured B cells and subsequent viral transmission. Cocultivation with epithelial cells facilitated expression of TGF-β receptors in B cells and increased their susceptibility to TGF-β. Finally, we confirmed the spontaneous secretion of TGF-β from epithelial cells, which was not affected by cell-contact. In contrast, the extracellular microvesicles, exosomes derived from cocultured cells partly contributed to cell-to-cell contact-mediated viral transmission. Taken together, our findings support a role for TGF-β derived from epithelial cells in efficient viral transmission, which fosters induction of the viral lytic cycle in the donor B cells.
  • Kon S, Ishibashi K, Katoh H, Kitamoto S, Shirai T, Tanaka S, Kajita M, Ishikawa S, Yamauchi H, Yako Y, Kamasaki T, Matsumoto T, Watanabe H, Egami R, Sasaki A, Nishikawa A, Kameda I, Maruyama T, Narumi R, Morita T, Sasaki Y, Enoki R, Honma S, Imamura H, Oshima M, Soga T, Miyazaki JI, Duchen MR, Nam JM, Onodera Y, Yoshioka S, Kikuta J, Ishii M, Imajo M, Nishida E, Fujioka Y, Ohba Y, Sato T, Fujita Y
    Nature cell biology 19 5 530 - + 2017年05月 [査読有り][通常論文]
     
    Recent studies have revealed that newly emerging transformed cells are often apically extruded from epithelial tissues. During this process, normal epithelial cells can recognize and actively eliminate transformed cells, a process called epithelial defence against cancer (EDAC). Here, we show that mitochondrial membrane potential is diminished in RasV12-transformed cells when they are surrounded by normal cells. In addition, glucose uptake is elevated, leading to higher lactate production. The mitochondrial dysfunction is driven by upregulation of pyruvate dehydrogenase kinase 4 (PDK4), which positively regulates elimination of RasV12-transformed cells. Furthermore, EDAC from the surrounding normal cells, involving filamin, drives the Warburg-effect-like metabolic alteration. Moreover, using a cell-competition mouse model, we demonstrate that PDK-mediated metabolic changes promote the elimination of RasV12-transformed cells from intestinal epithelia. These data indicate that non-cell-autonomous metabolic modulation is a crucial regulator for cell competition, shedding light on the unexplored events at the initial stage of carcinogenesis.
  • Sayaka Saitoh, Takeshi Maruyama, Yuta Yako, Mihoko Kajita, Yoichiro Fujioka, Yusuke Ohba, Nobuhiro Kasai, Natsu Sugama, Shunsuke Kon, Susumu Ishikawa, Takashi Hayashi, Tomohiro Yamazaki, Masazumi Tada, Yasuyuki Fujita
    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 114 12 E2327 - E2336 2017年03月 [査読有り][通常論文]
     
    Newly emerging transformed cells are often eliminated from epithelial tissues. Recent studies have revealed that this cancer-preventive process involves the interaction with the surrounding normal epithelial cells; however, the molecular mechanisms underlying this phenomenon remain largely unknown. In this study, using mammalian cell culture and zebrafish embryo systems, we have elucidated the functional involvement of endocytosis in the elimination of RasV12-transformed cells. First, we show that Rab5, a crucial regulator of endocytosis, is accumulated in RasV12-transformed cells that are surrounded by normal epithelial cells, which is accompanied by up-regulation of clathrin-dependent endocytosis. Addition of chlorpromazine or coexpression of a dominant-negative mutant of Rab5 suppresses apical extrusion of RasV12 cells from the epithelium. We also show in zebrafish embryos that Rab5 plays an important role in the elimination of transformed cells from the enveloping layer epithelium. In addition, Rab5-mediated endocytosis of E-cadherin is enhanced at the boundary between normal and RasV12 cells. Rab5 functions upstream of epithelial protein lost in neoplasm (EPLIN), which plays a positive role in apical extrusion of RasV12 cells by regulating protein kinase A. Furthermore, we have revealed that epithelial defense against cancer (EDAC) from normal epithelial cells substantially impacts on Rab5 accumulation in the neighboring transformed cells. This report demonstrates that Rab5-mediated endocytosis is a crucial regulator for the competitive interaction between normal and transformed epithelial cells in mammals.
  • Makoto Ibata, Junko Iwasaki, Yoichiro Fujioka, Koji Nakagawa, Stephanie Darmanin, Masahiro Onozawa, Daigo Hashimoto, Yusuke Ohba, Shigetsugu Hatakeyama, Takanori Teshima, Takeshi Kondo
    CANCER SCIENCE 108 2 200 - 207 2017年02月 [査読有り][通常論文]
     
    Fusion tyrosine kinases play a crucial role in the development of hematological malignancies. FIP1L1-PDGFRA is a leukemogenic fusion kinase that causes chronic eosinophilic leukemia. As a constitutively active kinase, FIP1L1-PDGFRA stimulates downstream signaling molecules, leading to cellular proliferation and the generation of an anti-apoptotic state. Contribution of the N-terminal FIP1L1 portion is necessary for FIP1L1-PDGFRA to exert its full transforming activity, but the underlying mechanisms have not been fully characterized. We identified PIAS1 as a FIP1L1-PDGFRA association molecule by yeast two-hybrid screening. Our analyses indicate that the FIP1L1 portion of FIP1L1-PDGFRA is required for efficient association with PIAS1. As a consequence of the association, FIP1L1-PDGFRA phosphorylates PIAS1. Moreover, the kinase activity of FIP1L1-PDGFRA stabilizes PIAS1. Therefore, PIAS1 is one of the downstream targets of FIP1L1-PDGFRA. Moreover, we found that PIAS1, as a SUMO E3 ligase, sumoylates and stabilizes FIP1L1-PDGFRA. In addition, suppression of PIAS1 activity by a knockdown experiment resulted in destabilization of FIP1L1-PDGFRA. Therefore, FIP1L1-PDGFRA and PIAS1 form a positive cross-talk through their enzymatic activities. Suppression of sumoylation by ginkgolic acid, a small molecule compound inhibiting a SUMO E1-activating enzyme, also destabilizes FIP1L1-PDGFRA, and while the tyrosine kinase inhibitor imatinib suppresses FIP1L1-PDGFRA-dependent cell growth, ginkgolic acid or siRNA of PIAS1 has a synergistic effect with imatinib. In conclusion, our results suggest that sumoylation by PIAS1 is a potential target in the treatment of FIP1L1-PDGFRA-positive chronic eosinophilic leukemia.
  • Mika Horiguchi, Mari Fujioka, Takeshi Kondo, Yoichiro Fujioka, Xinxin Li, Kosui Horiuchi, Aya O. Satoh, Prabha Nepal, Shinya Nishide, Asuka Nanbo, Takanori Teshima, Yusuke Ohba
    CELL STRUCTURE AND FUNCTION 42 1 15 - 26 2017年 [査読有り][通常論文]
     
    Although the co-development of companion diagnostics with molecular targeted drugs is desirable, truly efficient diagnostics are limited to diseases in which chromosomal translocations or overt mutations are clearly correlated with drug efficacy. Moreover, even for such diseases, few methods are available to predict whether drug administration is effective for each individual patient whose disease is expected to respond to the drug(s). We have previously developed a biosensor based on the principle of Forster resonance energy transfer to measure the activity of the tyrosine kinase BCR-ABL and its response to drug treatment in patient-derived chronic myeloid leukemia cells. The biosensor harbors CrkL, one of the major substrates of BCR-ABL, and is therefore named Pickles after phosphorylation indicator of CrkL en substrate. The efficacy of this technique as a clinical test has been demonstrated, but the number of cells available for analysis is limited in a case-dependent manner, owing to the cleavage of the biosensor in patient-derived leukemia cells. Here, we describe an improved biosensor with an amino acid substitution and a nuclear export signal being introduced. Of the two predicted cleavage positions in CrkL, the mutations inhibited one cleavage completely and the other cleavage partially, thus collectively increasing the number of cells available for drug evaluation. This improved version of the biosensor holds promise in the future development of companion diagnostics to predict responses to tyrosine kinase inhibitors in patients with chronic myeloid leukemia.
  • Mari Fujioka, Yumi Asano, Shigeyuki Nakada, Yusuke Ohba
    Methods in Molecular Biology 1555 513 - 534 2017年 [査読有り][通常論文]
     
    Fluorescent proteins (FPs) displaying distinct spectra have shed their light on a wide range of biological functions. Moreover, sophisticated biosensors engineered to contain single or multiple FPs, including Förster resonance energy transfer (FRET)-based biosensors, spatiotemporally reveal the molecular mechanisms underlying a variety of pathophysiological processes. However, their usefulness for applied life sciences has yet to be fully explored. Recently, our research group has begun to expand the potential of FPs from basic biological research to the clinic. Here, we describe a method to evaluate the responsiveness of leukemia cells from patients to tyrosine kinase inhibitors using a biosensor based on FP technology and the principle of FRET. Upon phosphorylation of the tyrosine residue of the biosensor, binding of the SH2 domain to phosphotyrosine induces conformational change of the biosensor and brings the donor and acceptor FPs into close proximity. Therefore, kinase activity and response to kinase inhibitors can be monitored by an increase and a decrease in FRET efficiency, respectively. As in basic research, this biosensor resolves hitherto arduous tasks and may provide innovative technological advances in clinical laboratory examinations. State-of-the-art detection devices that enable such innovation are also introduced.
  • Ohba Yusuke, Fujioka Yoichiro
    Journal of Oral Biosciences 58 4 113 - 119 2016年11月 [査読有り][通常論文]
  • Asuka Nanbo, Kunihiro Kachi, Hironori Yoshiyama, Yusuke Ohba
    JOURNAL OF GENERAL VIROLOGY 97 11 2989 - 3006 2016年11月 [査読有り][通常論文]
     
    Epstein-Barr virus (EBV) establishes a lifelong latent infection in B lymphocytes and often is found in epithelial cells. Several lines of evidence indicate that viral transmission mediated by cell-to-cell contact is the dominant mode of infection by EBV for epithelial cells. However, its detailed molecular mechanism has not been fully elucidated. We investigated the role of host membrane trafficking machinery in this process. We have found that adhesion molecules critical for this process are expressed in EBV-positive and -negative Burkitt's lymphoma (BL) cells and multiple epithelial cell lines. Treatment with blocking antibodies against beta 1 and beta 2 integrin families and their ligands suppressed EBV transmission in a dose-dependent manner. We also confirmed that adhesion molecules are upregulated in co-cultured BL cells. Immunofluorescence staining revealed that the intracellular adhesion molecule 1 (ICAM-1) distributed to the cell surface and partially co-localized with recycling endosomes in co-cultured BL cells. Moreover, cell-to-cell EBV transmission was inhibited upon blocking endocytic recycling by expression of a dominant-negative form of a small GTPase Rab11 or by knockdown of Rab11, supporting the notion that the endocytic pathway-dependent trafficking of ICAM-1 to the cell surface of BL cells contributes to viral transmission by stabilizing cell-to-cell contact between the donor cells and recipient cells. Finally, we demonstrated that co-cultivation upregulated clathrin-mediated endocytosis in the recipient cells, allowing EBV to be internalized. Taken together, our findings demonstrate that EBV exploits host endocytic machinery in both donor and recipient cells, a process which is facilitated by cell-to-cell contact, thereby promoting successful viral transmission.
  • Nako Maishi, Yusuke Ohba, Kosuke Akiyama, Noritaka Ohga, Jun-ichi Hamada, Hiroko Nagao-Kitamoto, Mohammad Towfik Alam, Kazuyuki Yamamoto, Taisuke Kawamoto, Nobuo Inoue, Akinobu Taketomi, Masanobu Shindoh, Yasuhiro Hida, Kyoko Hida
    SCIENTIFIC REPORTS 6 28039  2016年06月 [査読有り][通常論文]
     
    Tumour blood vessels are gateways for distant metastasis. Recent studies have revealed that tumour endothelial cells (TECs) demonstrate distinct phenotypes from their normal counterparts. We have demonstrated that features of TECs are different depending on tumour malignancy, suggesting that TECs communicate with surrounding tumour cells. However, the contribution of TECs to metastasis has not been elucidated. Here, we show that TECs actively promote tumour metastasis through a bidirectional interaction between tumour cells and TECs. Co-implantation of TECs isolated from highly metastatic tumours accelerated lung metastases of low metastatic tumours. Biglycan, a small leucine-rich repeat proteoglycan secreted from TECs, activated tumour cell migration via nuclear factor-kappa B and extracellular signal-regulated kinase 1/2. Biglycan expression was upregulated by DNA demethylation in TECs. Collectively, our results demonstrate that TECs are altered in their microenvironment and, in turn, instigate tumour cells to metastasize, which is a novel mechanism for tumour metastasis.
  • Ryu Okumura, Takashi Kurakawa, Takashi Nakano, Hisako Kayama, Makoto Kinoshita, Daisuke Motooka, Kazuyoshi Gotoh, Taishi Kimura, Naganori Kamiyama, Takashi Kusu, Yoshiyasu Ueda, Hong Wu, Hideki Iijima, Soumik Barman, Hideki Osawa, Hiroshi Matsuno, Junichi Nishimura, Yusuke Ohba, Shota Nakamura, Tetsuya Iida, Masahiro Yamamoto, Eiji Umemoto, Koichi Sano, Kiyoshi Takeda
    NATURE 532 7597 117 - + 2016年04月 [査読有り][通常論文]
     
    Colonic epithelial cells are covered by thick inner and outer mucus layers(1,2). The inner mucus layer is free of commensal microbiota, which contributes to the maintenance of gut homeostasis(3-6). In the small intestine, molecules critical for prevention of bacterial invasion into epithelia such as Paneth-cell-derived anti-microbial peptides and regenerating islet-derived 3 (RegIII) family proteins have been identified(7-11). Although there are mucus layers providing physical barriers against the large number of microbiota present in the large intestine, the mechanisms that separate bacteria and colonic epithelia are not fully elucidated. Here we show that Ly6/PLAUR domain containing 8 (Lypd8) protein prevents flagellated microbiota invading the colonic epithelia in mice. Lypd8, selectively expressed in epithelial cells at the uppermost layer of the large intestinal gland, was secreted into the lumen and bound flagellated bacteria including Proteus mirabilis. In the absence of Lypd8, bacteria were present in the inner mucus layer and many flagellated bacteria invaded epithelia. Lypd8(-/-) mice were highly sensitive to intestinal inflammation induced by dextran sulfate sodium (DSS). Antibiotic elimination of Gram-negative flagellated bacteria restored the bacterial-free state of the inner mucus layer and ameliorated DSS-induced intestinal inflammation in Lypd8(-/-) mice. Lypd8 bound to flagella and suppressed motility of flagellated bacteria. Thus, Lypd8 mediates segregation of intestinal bacteria and epithelial cells in the colon to preserve intestinal homeostasis.
  • Yamada T, Tsuda M, Wagatsuma T, Fujioka Y, Fujioka M, Satoh AO, Horiuchi K, Nishide S, Nanbo A, Totsuka Y, Haga H, Tanaka S, Shindoh M, Ohba Y
    Scientific reports 6 23545  2016年03月 [査読有り][通常論文]
     
    Cellular interactions with the extracellular matrix play critical roles in tumor progression. We previously reported that receptor activator of NF-kappa B ligand (RANKL) specifically facilitates head and neck squamous cell carcinoma (HNSCC) progression in vivo. Here, we report a novel role for RANKL in the regulation of cell adhesion. Among the major type I collagen receptors, integrin alpha 2 was significantly upregulated in RANKL-expressing cells, and its knockdown suppressed cell adhesion. The mRNA abundance of integrin alpha 2 positively correlated with that of RANKL in human HNSCC tissues. We also revealed that RANK-NF-kappa B signaling mediated integrin alpha 2 expression in an autocrine/paracrine manner. Interestingly, the amount of active integrin beta 1 on the cell surface was increased in RANKL-expressing cells through the upregulation of integrin alpha 2 and endocytosis. Moreover, the RANK-integrin alpha 2 pathway contributed to RANKL-dependent enhanced survival in a collagen gel and inhibited apoptosis in a xenograft model, demonstrating an important role for RANKL-mediated cell adhesion in three-dimensional environments.
  • Sayaka Yamamoto, Yuta Yako, Yoichiro Fujioka, Mihoko Kajita, Takeshi Kameyama, Shunsuke Kon, Susumu Ishikawa, Yusuke Ohba, Yusuke Ohno, Akio Kihara, Yasuyuki Fujita
    Molecular biology of the cell 27 3 491 - 9 2016年02月01日 [査読有り][通常論文]
     
    At the initial step of carcinogenesis, transformation occurs in single cells within epithelia, where the newly emerging transformed cells are surrounded by normal epithelial cells. A recent study revealed that normal epithelial cells have an ability to sense and actively eliminate the neighboring transformed cells, a process named epithelial defense against cancer (EDAC). However, the molecular mechanism of this tumor-suppressive activity is largely unknown. In this study, we investigated a role for the sphingosine-1-phosphate (S1P)-S1P receptor 2 (S1PR2) pathway in EDAC. First, we show that addition of the S1PR2 inhibitor significantly suppresses apical extrusion of RasV12-transformed cells that are surrounded by normal cells. In addition, knockdown of S1PR2 in normal cells induces the same effect, indicating that S1PR2 in the surrounding normal cells plays a positive role in the apical elimination of the transformed cells. Of importance, not endogenous S1P but exogenous S1P is involved in this process. By using FRET analyses, we demonstrate that S1PR2 mediates Rho activation in normal cells neighboring RasV12-transformed cells, thereby promoting accumulation of filamin, a crucial regulator of EDAC. Collectively these data indicate that S1P is a key extrinsic factor that affects the outcome of cell competition between normal and transformed epithelial cells.
  • Takayuki Inuzuka, Yoichiro Fujioka, Masumi Tsuda, Mari Fujioka, Aya O. Satoh, Kosui Horiuchi, Shinya Nishide, Asuka Nanbo, Shinya Tanaka, Yusuke Ohba
    SCIENTIFIC REPORTS 6 21613  2016年02月 [査読有り][通常論文]
     
    Angiotensin II (AII) type 2 receptor (AT2R) negatively regulates type 1 receptor (AT1R) signaling. However, the precise molecular mechanism of AT2R-mediated AT1R inhibition remains poorly understood. Here, we characterized the local and functional interaction of AT2R with AT1R. AT2R colocalized and formed a complex with AT1R at the plasma membrane, even in the absence of AII. Upon AII stimulation, the spatial arrangement of the complex was modulated, as confirmed by Forster resonance energy transfer (FRET) analysis, followed by AT2R internalization along with AT1R. AT2R internalization was specifically observed only in the presence of AT1R; AT2R alone could not be internalized. The AT1R-specific inhibitor losartan completely inhibited both the conformational change and the internalization of AT2R with AT1R, whereas the AT2R-specific inhibitor PD123319 partially hindered these phenomena, demonstrating that the activation of both receptors was indispensable for these effects. In addition, treatment with the protein kinase C (PKC) inhibitors inhibited the ligand-dependent accumulation of AT2R but not that of AT1R in the endosomes. A mutation in the putative phosphorylation sites of AT2R also abrogated the co-internalization of ATR2 with AT1R and the inhibitory effect of ATR2 on AT1R. These data suggest that AT2R inhibits ligand-induced AT1R signaling through the PKC-dependent pathway.
  • 腫瘍血管内皮細胞はbiglycanの分泌を介してがんの転移を促進する
    間石 奈湖, 大場 雄介, 秋山 廣輔, 大賀 則孝, 浜田 淳一, 北本 宗子[永尾], Mohammad Alam T., 進藤 正信, 樋田 泰浩, 樋田 京子
    日本癌学会総会記事 74回 E - 1030 2015年10月 [査読有り][通常論文]
  • Yoshinori Makino, Masumi Tsuda, Yusuke Ohba, Hiroshi Nishihara, Hirofumi Sawa, Kazuo Nagashima, Shinya Tanaka
    CELL COMMUNICATION AND SIGNALING 13 35  2015年07月 [査読有り][通常論文]
     
    Background: The complex of Dock180/ELMO1 that functions as a bipartite guanine nucleotide exchange factor for Rac is essential for diverse physiological and pathological processes of cells such as cell migration, phagocytosis, and invasion of cancer cells. Among the Src-family tyrosine kinases (SFKs), it has been reported that Hck directly phosphorylates ELMO1, regulating phagocytosis by promoting activation of Rac1; however, the involvement of other SFKs in ELMO1 phosphorylation has remained unknown. Here, we identified novel tyrosine (Y) residues of ELMO1 phosphorylated by SFKs, and examined the effects on Rac1 activity, cell adhesion, spreading, and cell motility on extracellular matrix (ECM). Results: In this study, we unveiled that Src and Fyn can induce tyrosine phosphorylation of ELMO1 in in vivo and in vitro phosphorylation assays. Mutational analyses identified both Y720 and Y724 residues of ELMO1 as Src-mediated phosphorylation sites, preferentially on Y724. Single substitution of Y724 to Phe abrogated Rac1 activation triggered by Src. To elucidate the biological function of pY724, we established NIH3T3 cells stably expressing wild-type ELMO1 or its Y724F mutant together with Dock180. Among them, Y724-deficient cells exhibited a depletion of Rac1 activity with diminished phosphorylation of ELMO1 even upon the ECM-stimulation. It is noteworthy that NIH3T3 cells with ELMO1 Y724F were strikingly defective to promote cell spreading on fibronectin-coated dish, concomitantly exhibiting immature assemblies of actin stress fibers and focal adhesions. Eventually, ELMO1 Y724F significantly impaired cell migration. Conclusion: These results define that Src-mediated Y724 phosphorylation in ELMO1 plays a critical role for cell spreading via activation of Rac1, leading to promotion of cell migration. As the overexpression and/or hyperactivation of Src have been shown in a wide variety of human cancers, Src-mediated phosphorylation of Y724 in ELMO1 may regulate cancer cell adhesion to the ECM, invasion into surrounding tissues, and subsequent distant metastasis.
  • Ryuji Matsumoto, Masumi Tsuda, Lei Wang, Nako Maishi, Takashige Abe, Taichi Kimura, Mishie Tanino, Hiroshi Nishihara, Kyoko Hida, Yusuke Ohba, Nobuo Shinohara, Katsuya Nonomura, Shinya Tanaka
    Cancer science 106 6 709 - 17 2015年06月 [査読有り][通常論文]
     
    We have previously reported that an adaptor protein CRK, including CRK-I and CRK-II, plays essential roles in the malignant potential of various aggressive human cancers, suggesting the validity of targeting CRK in molecular targeted therapy of a wide range of cancers. Nevertheless, the role of CRK in human bladder cancer with marked invasion, characterized by distant metastasis and poor prognosis, remains obscure. In the present study, immunohistochemistry indicated a striking enhancement of CRK-I/-II, but not CRK-like, in human bladder cancer tissues compared to normal urothelium. We established CRK-knockdown bladder cancer cells using 5637 and UM-UC-3, which showed a significant decline in cell migration, invasion, and proliferation. It is noteworthy that an elimination of CRK conferred suppressed phosphorylation of c-Met and the downstream scaffold protein Gab1 in a hepatocyte growth factor-dependent and -independent manner. In epithelial-mesenchymal transition-related molecules, E-cadherin was upregulated by CRK elimination, whereas N-cadherin, vimentin, and Zeb1 were downregulated. A similar effect was observed following treatment with c-Met inhibitor SU11274. Depletion of CRK significantly decreased cell proliferation of 5637 and UM-UC-3, consistent with reduced activity of ERK. An orthotopic xenograft model with bioluminescent imaging revealed that CRK knockdown significantly attenuated not only tumor volume but also the number of circulating tumor cells, resulted in a complete abrogation of metastasis. Taken together, this evidence uncovered essential roles of CRK in invasive bladder cancer through the hepatocyte growth factor/c-Met/CRK feedback loop for epithelial-mesenchymal transition induction. Thus, CRK might be a potent molecular target in bladder cancer, particularly for preventing metastasis, leading to the resolution of clinically longstanding critical issues.
  • Tadasuke Tsukiyama, Akimasa Fukui, Sayuri Terai, Yoichiro Fujioka, Keisuke Shinada, Hidehisa Takahashi, Terry P. Yamaguchi, Yusuke Ohba, Shigetsugu Hatakeyama
    MOLECULAR AND CELLULAR BIOLOGY 35 11 2007 - 2023 2015年06月 [査読有り][通常論文]
     
    Wnt signaling pathways are tightly regulated by ubiquitination, and dysregulation of these pathways promotes tumorigenesis. It has been reported that the ubiquitin ligase RNF43 plays an important role in frizzled-dependent regulation of the Wnt/beta-catenin pathway. Here, we show that RNF43 suppresses both Wnt/beta-catenin signaling and noncanonical Wnt signaling by distinct mechanisms. The suppression of Wnt/beta-catenin signaling requires interaction between the extracellular protease-associated (PA) domain and the cysteine-rich domain (CRD) of frizzled and the intracellular RING finger domain of RNF43. In contrast, these N-terminal domains of RNF43 are not required for inhibition of noncanonical Wnt signaling, but interaction between the C-terminal cytoplasmic region of RNF43 and the PDZ domain of dishevelled is essential for this suppression. We further show the mechanism by which missense mutations in the extracellular portion of RNF43 identified in patients with tumors activate Wnt/beta-catenin signaling. Missense mutations of RNF43 change their localization from the endosome to the endoplasmic reticulum (ER), resulting in the failure of frizzled-dependent suppression of Wnt/beta-catenin signaling. However, these mutants retain the ability to suppress noncanonical Wnt signaling, probably due to interaction with dishevelled. RNF43 is also one of the potential target genes of Wnt/beta-catenin signaling. Our results reveal the molecular role of RNF43 and provide an insight into tumorigenesis.
  • Yoichiro Fujioka, Asuka Nanbo, Shin-ya Nishide, Yusuke Ohba
    ANALYTICAL SCIENCES 31 4 267 - 274 2015年04月 [査読有り][通常論文]
     
    In response to extracellular stimuli, cells display a variety of behaviors, including proliferation, differentiation, morphological changes and migration. The analysis of the spatiotemporal regulation of signal transduction in living cells is needed for a better understanding of such behaviors, and such investigations have been greatly accelerated by the development of fluorescent protein-based biosensors. Currently, by using these biosensors a range of molecular actions, including lipid metabolism, protein activation, and ion dynamics, can be visualized in living cells. We recently reported that intracellular calcium, with its relevant downstream signaling pathways consisting of the small GTPase Ras and the lipid kinase phoshoinositide-3-kinase (PI3K), can be exploited in an efficient incorporation of influenza A viruses into host cells via endocytosis using a set of biosensors based on fluorescent proteins and the principle of Forster resonance energy transfer. Here, we focus this review on fluorescent protein-based biosensors that have been utilized in our recent research reports.
  • Kosuke Akiyama, Nako Maishi, Noritaka Ohga, Yasuhiro Hida, Yusuke Ohba, Mohammad Towfik Alam, Taisuke Kawamoto, Hitomi Ohmura, Kenji Yamada, Chisaho Torii, Masanobu Shindoh, Kyoko Hida
    AMERICAN JOURNAL OF PATHOLOGY 185 2 572 - 580 2015年02月 [査読有り][通常論文]
     
    Tumor angiogenesis plays an important role in tumor progression and metastasis. Tumor endothelial cells (TECs) are a therapeutic target of antiangiogenic chemotherapy that was recently developed and is currently being investigated in the clinic with promising results. Low-dose chemotherapy, which is the Long-term administration of relatively Low doses of chemotherapeutic agents, has been proposed for targeting tumor angiogenesis in various types of cancers. ALthough the efficacy of low-dose chemotherapy has been confirmed in several clinical models, some studies show insufficienttherapeutic effect for malignant cancers. As a possible mechanism of the treatment failure, it has been considered that tumor cells may acquire resistance to this therapy. However, drug resistance by TECs may also be due to another mechanism for resistance of tumor cells to low-dose chemotherapy. We reported elsewhere that TECs were resistant to the anticancer drug paclitaxel, which is a mitotic inhibitor, concomitant with P-glycoprotein up-regulation. Verapamil, a P-glycoprotein inhibitor, abrogated TEC resistance in vitro. Herein, we demonstrated that verapamil coadministration enhanced the effects of Low-dose paclitaxel concomitant with inhibiting tumor angiogenesis in a preclinical in vivo mouse melanoma xenograft model. Furthermore, verapamil coadministration reduced lung metastasis. These results suggest that inhibiting P-gLycoprotein in TECs may be a novel strategy for low-dose chemotherapy targeting TECs.
  • Fujioka Y, Ohba Y
    Seikagaku. The Journal of Japanese Biochemical Society 87 1 91 - 100 2015年02月 [査読有り][通常論文]
  • Mohamed Kamel Hassan, Hidemichi Watari, Takashi Mitamura, Zainab Mohamed, Sherif F El-Khamisy, Yusuke Ohba, Noriaki Sakuragi
    Oncoscience 2 3 294 - 308 2015年 [査読有り][通常論文]
     
    MicroRNAs (miRNAs) have been reported to regulate the development of chemoresistance in many tumors. Stathmin 1 (STMN1) is a microtubule-depolymerizing molecule, involved in chemo-response; however, the mechanism of its regulation is unknown. Herein, the immunohistochemical study indicated significant upregulation of the STMN1 in the ovarian cancer tissues defined as resistant tumors compared with those defined as responsive tumors. STMN1 level elevated in the chemoresistant ovarian cancer cells, KF-TX, compared with the parental, KF, ones. Targeting STMN1 by siRNA restored taxane-sensitivity of KF-TX cells. Screening miRNA profiles from KF/KF-TX cellular set followed by bioinformatics-based prediction, revealed that miR-31 could be a possible regulator of STMN1. Down-modulation of miR-31 was verified by quantitative RT-PCR in the cellular set used. Overexpression of miR-31 in KF-TX cells (KF-TX-miR-31) significantly restored chemo-response and reduced STMN1 expression as well. STMN1 reduction-associated cellular characteristics such as enhanced microtubule polymerization and stability, as indicated by acetylated tubulin quantification, confocal visualization, and G2 phase delay, were observed in KF-TX-miR-31 cells, indicating the functional reduction of STMN1. miR-31 suppressed the luciferase activity in reporter construct containing the STMN1 3'-untranslated region (3'-UTR), confirming that miR-31 directly targets STMN1. miR-31 has therapeutic potency when introduced into ovarian cancer, in combination with taxane.
  • Y. Fujioka, M. Tsuda, A. Nanbo, T. Hattori, J. Sasaki, T. Sasaki, T. Miyazaki, Y. Ohba
    MOLECULAR BIOLOGY OF THE CELL 25 2014年12月 [査読有り][通常論文]
  • Koji Terada, Takahiro Horinouchi, Yoichiro Fujioka, Tsunehito Higashi, Prabha Nepal, Mika Horiguchi, Sarita Karki, Chizuru Hatate, Akimasa Hoshi, Takuya Harada, Yosuke Mai, Yusuke Ohba, Soichi Miwa
    JOURNAL OF BIOLOGICAL CHEMISTRY 289 51 35283 - 35295 2014年12月 [査読有り][通常論文]
     
    Two types of G protein-coupled receptors for endothelin-1 (ET-1), ET type A receptor (ETAR) and ETBR, closely resemble each other, but upon ET-1 stimulation, they follow totally different intracellular trafficking pathways; ETAR is recycled back to plasma membrane, whereas ETBR is targeted to lysosome for degradation. However, the mechanisms for such different fates are unknown. Here we demonstrated that ETBR but not ETAR was ubiquitinated on the cell surface following ET-1 stimulation and that ETBR was internalized and degraded in lysosome more rapidly than ETAR. The mutant ETBR (designated 5KR mutant) in which 5 lysine residues in the C-tail were substituted to arginine was not ubiquitinated, and its rates of internalization and degradation after ET-1 stimulation became slower, being comparable with those of ETAR. Confocal microscopic study showed that following ET-1 stimulation, ETAR and 5KR mutant of ETBR were co-localized mainly with Rab11, a marker of recycling endosome, whereas ETBR was co-localized with Rab7, a marker of late endosome/lysosome. In the 5KR mutant, ET-1-induced ERK phosphorylation and an increase in the intracellular Ca2+ concentration upon repetitive ET-1 stimulation were larger. A series of ETBR mutants (designated 4KR mutant), in which either one of 5 arginine residues of the 5KR mutant was reverted to lysine, were normally ubiquitinated, internalized, and degraded, with ERK phosphorylation being normalized. These results demonstrate that agonist-induced ubiquitination at either lysine residue in the C-tail of ETBR but not ETAR switches intracellular trafficking from recycling to plasma membrane to targeting to lysosome, causing decreases in the cell surface level of ETBR and intracellular signaling.
  • Roshan Mahabir, Mishie Tanino, Aiman Elmansuri, Lei Wang, Taichi Kimura, Tamio Itoh, Yusuke Ohba, Hiroshi Nishihara, Hiroki Shirato, Masumi Tsuda, Shinya Tanaka
    NEURO-ONCOLOGY 16 5 671 - 685 2014年05月 [査読有り][通常論文]
     
    Ionizing irradiation is an effective treatment for malignant glioma (MG); however, a higher rate of recurrence with more aggressive phenotypes is a vital issue. Although epithelial-mesenchymal transition (EMT) is involved in irradiation-induced cancer progression, the role for such phenotypic transition in MG remains unknown. To investigate the mechanism of irradiation-dependent tumor progression in MG, we performed immunohistochemistry (IHC) and qRT-PCR using primary and recurrent MG specimens, MG cell lines, and primary culture cells of MG. siRNA technique was used for MG cell lines. In 22 cases of clinically recurrent MG, the expression of the mesenchymal markers vimentin and CD44 was found to be increased by IHC. In paired identical MG of 7 patients, the expression of collagen, MMPs, and YKL-40 were also elevated in the recurrent MGs, suggesting the The Cancer Genome Atlas-based mesenchymal subtype. Among EMT regulators, sustained elevation of Snail was observed in MG cells at 21 days after irradiation. Cells exhibited an upregulation of migration, invasion, numbers of focal adhesion, and MMP-2 production, and all of these mesenchymal features were abrogated by Snail knockdown. Intriguingly, phosphorylation of ERK1/2 and GSK-3 were increased after irradiation in a Snail-dependent manner, and TGF- was elevated in both fibroblasts and macrophages but not in MG cells after irradiation. It was noteworthy that irradiated cells also expressed stemness features such as SOX2 expression and tumor-forming potential in vivo. We here propose a novel concept of glial-mesenchymal transition after irradiation in which the sustained Snail expression plays an essential role.
  • Mohamed K. Hassan, Hidemichi Watari, Alaa-eldin Salah-eldin, Ahmed S. Sultan, Zainab Mohamed, Yoichiro Fujioka, Yusuke Ohba, Noriaki Sakuragi
    PLOS ONE 9 4 e94213  2014年04月 [査読有り][通常論文]
     
    This study describes the sensitization mechanism to thermal stress by histone deacetylase inhibitors (HDACIs) in lung cancer cells and shows that Ku70, based on its acetylation status, mediates the protection of lung cancer from hyperthermia (42.5 degrees C, 1-6 hrs). Ku70 regulates apoptosis by sequestering pro-apoptotic Bax. However, its role in thermal stress is not fully understood. The findings showed that, pre-treating lung cancer cells with HDACIs, nicotinamide (NM) or Trichostatin A (TsA) or both significantly enhanced hyperthermia-induced Bax-dependent apoptosis in PC-10 cells. We found that hyperthermia induces SirT-1, Sirtuin, upregulation but not HDAC6 or SirT-3, therefore transfection with dominant negative SirT-1 (Y/H) also eliminated the protection and resulted in more cell death by hyperthermia, in H1299 cells through Bax activation. Hyperthermia alone primed lung cancer cells to apoptosis without prominent death. After hyperthermia Bax was upregulated, Bcl-2 was downregulated, the Bax/Bcl-2 ratio was inversed and Bax/Bcl-2 heterodimer was dissociated. Although hyperthermia did not affect total Ku70 expression level, it stimulated Ku70 deacetylation, which in turn could bind more Bax in the PC-10 cells. These findings suggest an escape mechanism from hyperthermia-induced Bax activation. To verify the role of Ku70 in this protection mechanism, Ku70 was silenced by siRNA. Ku70 silencing significantly sensitized the lung cancer cells to hyperthermia. The Ku70 KD cells underwent cytotoxic G1 arrest and caspase-dependant apoptosis when compared to scrambled transfectants which showed only G2/M cytostatic arrest in the cell lines investigated, suggesting an additional cell cycle-dependent, novel, role of Ku70 in protection from hyperthermia. Taken together, our data show a Ku70-dependent protection mechanism from hyperthermia. Targeting Ku70 and/or its acetylation during hyperthermia may represent a promising therapeutic approach for lung cancer.
  • Mami Matsuda-Lennikov, Futoshi Suizu, Noriyuki Hirata, Manabu Hashimoto, Kohki Kimura, Tadashi Nagamine, Yoichiro Fujioka, Yusuke Ohba, Toshihiko Iwanaga, Masayuki Noguchi
    PLOS ONE 9 1 e79795  2014年01月 [査読有り][通常論文]
     
    Autophagy is an evolutionarily conserved mechanism for the gross disposal of intracellular proteins in mammalian cells and dysfunction in this pathway has been associated with human disease. Although the serine threonine kinase Akt is suggested to play a role in this process, little is known about the molecular mechanisms by which Akt induces autophagy. Using a yeast two-hybrid screen, Phafin2 (EAPF or PLEKHF2), a lysosomal protein with a unique structure of N-terminal PH (pleckstrin homology) domain and C-terminal FYVE (Fab 1, YOTB, Vac 1, and EEA1) domain was found to interact with Akt. A sucrose gradient fractionation experiment revealed that both Akt and Phafin2 co-existed in the same lysosome enriched fraction after autophagy induction. Confocal microscopic analysis and BiFC analysis demonstrated that both Akt and Phafin2 accumulate in the lysosome after induction of autophagy. BiFC analysis using PtdIns (3) P interaction defective mutant of Phafin2 demonstrated that lysosomal accumulation of the Akt-Phafin2 complex and subsequent induction of autophagy were lysosomal PtdIns (3) P dependent events. Furthermore, in murine macrophages, both Akt and Phafin2 were required for digestion of fluorescent bacteria and/or LPS-induced autophagy. Taken together, these findings establish that lysosomal accumulation of Akt and Phafin2 is a critical step in the induction of autophagy via an interaction with PtdIns (3)P.
  • Mohamed Hassan, Hidemichi Watari, Ali AbuAlmaaty, Yusuke Ohba, Noriaki Sakuragi
    BIOMED RESEARCH INTERNATIONAL 2014 150845  2014年 [査読有り][通常論文]
     
    Apoptosis is the programmed cell death which maintains the healthy survival/death balance in metazoan cells. Defect in apoptosis can cause cancer or autoimmunity, while enhanced apoptosis may cause degenerative diseases. The apoptotic signals contribute into safeguarding the genomic integrity while defective apoptosis may promote carcinogenesis. The apoptotic signals are complicated and they are regulated at several levels. The signals of carcinogenesis modulate the central control points of the apoptotic pathways, including inhibitor of apoptosis (IAP) proteins and FLICE-inhibitory protein (c-FLIP). The tumor cells may use some of several molecular mechanisms to suppress apoptosis and acquire resistance to apoptotic agents, for example, by the expression of antiapoptotic proteins such as Bcl-2 or by the downregulation or mutation of proapoptotic proteins such as BAX. In this review, we provide the main regulatory molecules that govern the main basic mechanisms, extrinsic and intrinsic, of apoptosis in normal cells. We discuss how carcinogenesis could be developed via defective apoptotic pathways or their convergence. We listed some molecules which could be targeted to stimulate apoptosis in different cancers. Together, we briefly discuss the development of some promising cancer treatment strategies which target apoptotic inhibitors including Bcl-2 family proteins, IAPs, and c-FLIP for apoptosis induction.
  • Hideo Negishi, Kosuke Matsuki, Nobuyasu Endo, Hana Sarashina, Shoji Miki, Atsushi Matsuda, Keiko Fukazawa, Naoko Taguchi-Atarashi, Hiroaki Ikushima, Hideyuki Yanai, Junko Nishio, Kenya Honda, Yoichiro Fujioka, Yusuke Ohba, Tetsuo Noda, Shun'ichiro Taniguchi, Eisuke Nishida, Yongliang Zhang, Hongbo Chi, Richard A. Flavell, Tadatsugu Taniguchi
    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 110 49 19884 - 19889 2013年12月 [査読有り][通常論文]
     
    A major function of innate immune receptors is to recognize pathogen-associated molecular patterns and then evoke immune responses appropriate to the nature of the invading pathogen(s). Because innate immune cells express various types of these receptors, distinct combinations of signaling pathways are activated in response to a given pathogen. Although the conventional wisdom is that these signaling pathways cooperate with one another to ensure an effective host response, a more nuanced view recognizes antagonism between the individual pathways, where the attenuation of a signaling pathway(s) by others may shape the immune response. In this study, we show that, on Listeria monocytogenes infection, Toll-like receptor-triggered MyD88 signaling pathways suppress type I IFN gene induction, which is detrimental to macrophage bactericidal activity. These pathways target and suppress the IFN regulatory factor 3 (IRF3) transcription factor that is activated by the stimulator of IFN genes-TANK-binding kinase-1 kinase pathway. We also provide evidence for the involvement of the MAPK phosphatase family members, which renders IRF3 hypophosphorylated on Toll-like receptor signaling by enhancing the formation of an MAPK phosphatase-IRF3-TANK-binding kinase-1 ternary complex. This study, therefore, reveals a hitherto unrecognized and important contribution of a beneficial innate signaling interference against bacterial infections.
  • Yoichiro Fujioka, Masumi Tsuda, Asuka Nanbo, Tomoe Hattori, Junko Sasaki, Takehiko Sasaki, Tadaaki Miyazaki, Yusuke Ohba
    NATURE COMMUNICATIONS 4 2763  2013年11月 [査読有り][通常論文]
     
    Various viruses enter host cells via endocytosis, but the molecular mechanisms underlying the specific internalization pathways remain unclear. Here we show that influenza A viruses (IAVs) enter cells via redundant pathways of clathrin-mediated and clathrin-independent endocytosis, with intracellular Ca2+ having a central role in regulation of both pathways by activating a signalling axis comprising RhoA, Rho-kinase, phosphatidylinositol 4-phosphate 5-kinase (PIP5K) and phospholipase C (PLC). IAV infection induces oscillations in the cytosolic Ca2+ concentration of host cells, the prevention of which markedly attenuates virus internalization and infection. The small GTPase RhoA is found both to function downstream of the virus-induced Ca2+ response and itself to induce Ca2+ oscillations in a manner dependent on Rho-kinase and subsequent PIP5K-PLC signalling. This signalling circuit regulates both clathrin-mediated and clathrin-independent endocytosis during virus infection and seems to constitute a key mechanism for regulation of IAV internalization and infection.
  • 血管内皮細胞によるがん転移促進(Endothelial cells promote tumor metastasis)
    間石 奈湖, 大場 雄介, 大賀 則孝, 秋山 廣輔, 山本 和幸, 浜田 淳一, 川本 泰輔, アラム・モハメド・トウフィック, 大村 瞳, 進藤 正信, 樋田 泰浩, 樋田 京子
    日本癌学会総会記事 72回 127 - 127 2013年10月 [査読有り][通常論文]
  • P-gp阻害剤はメトロノミックケモセラピーの血管新生阻害効果を強める(P-gp inhibitor enhance antiangiogenic activity in metronomic chemotherapy)
    秋山 廣輔, 大賀 則孝, 樋田 泰浩, 間石 奈湖, ムハンマド・トフィック, 川本 泰輔, 大村 瞳, 山田 健司, 鳥居 ちさほ, 進藤 正信, 大場 雄介, 樋田 京子
    日本癌学会総会記事 72回 127 - 127 2013年10月 [査読有り][通常論文]
  • Tomoe Hattori, Tomohiro Arikawa, Yoichiro Fujioka, Junki Maruyama, Yousuke Nakayama, Yusuke Ohba, Toshiro Niki, Tadaaki Miyazaki, Mitsuomi Hirashima, Hiroshi Kida
    JAPANESE JOURNAL OF VETERINARY RESEARCH 61 1-2 5 - 18 2013年05月 [査読有り][通常論文]
     
    Galectin-9 (Gal-9) inhibited the infection of H1N1, H3N2 and H5N1 influenza A viruses in vitro and in vivo. Fifty percent effective doses (ED50) of Gal-9 were 0.1-0.5 mu M depending on virus strains in the plaque reduction assay. Gal-9 but not Gal-1 bound to the virus particles of A/Puerto Rico/8/34 (H1N1) (PR/8), resulting in inhibition of virus attachment to the host cells. Lactose but not sucrose inhibited the binding of Gal-9 to the viruses. Endogenous Gal-9 expression was detected and increased with the course of infection with influenza A viruses in mice. Fifty percent of Gal-9-transgenic mice survived after the challenge with PR/8, while all of the wild-type mice died. Gal-9 treatment of mice affected diminishing influenza virus replication in the lungs, body weight loss and the expression level of inflammatory cytokines. Combined administration of Gal-9 and oseltamivir was more effective than the use of single compound in mouse model. The present results indicate that Gal-9 is a candidate compound for influenza A virus infection therapy.
  • Hiroyuki Nagai, Sayaka Yasuda, Yusuke Ohba, Mitsunori Fukuda, Takeshi Nakamura
    Journal of Biochemistry 153 3 283 - 288 2013年03月 [査読有り][通常論文]
     
    The importance of interconnective signalling networks between distinct GTPases and their regulators is being recognized. EPI64C/TBC1D10C/carabin, a haematopoietically enriched GTPase-activating protein (GAP) for Rab35, has been shown to exhibit RasGAP activity. Owing to the diverged Rab specificities among the EPI64 members (EPI64A-C) and the relatively weak sequence conservation between EPI64A/B and EPI64C in their catalytic TBC domains, it is difficult to predict whether EPI64A and B will also have RasGAP activities. Therefore, in this study, we examined the RasGAP activities of all three EPI64 subfamily members. We found that EPI64A-C exhibited in vivo GAP activities towards Ras using three independent methods, spectrofluorometry with Förster resonance energy transfer (FRET) sensors, the Bos' pull-down assay and time-lapse FRET imaging. EPI64A and B were predominantly localized at the periphery of COS-7 cells. In COS-7 cells, confocal FRET imaging showed that H-Ras activity was higher at the Golgi than at the plasma membrane. Thus, we propose that EPI64A and B, which are ubiquitously expressed members of the EPI64 subfamily, inactivate Ras and certain Rabs at the periphery of cells. © 2012 The Authors 2012.
  • Yusuke Ohba, Yoichiro Fujioka, Shigeyuki Nakada, Masumi Tsuda
    Progress in Molecular Biology and Translational Science 113 313 - 348 2013年 [査読有り][通常論文]
     
    Green fluorescent protein and its relatives have shed their light on a wide range of biological problems. To date, with a color palette consisting of fluorescent proteins with different spectra, researchers can "paint" living cells as they desire. Moreover, sophisticated biosensors engineered to contain single or multiple fluorescent proteins, including FRET-based biosensors, spatiotemporally unveil molecular mechanisms underlying physiological processes. Although such molecules have contributed considerably to basic research, their abilities to be used in applied life sciences have yet to be fully explored. Here, we review the molecular bases of fluorescent proteins and fluorescent protein-based biosensors and focus on approaches aimed at applying such proteins to the clinic. © 2013 Elsevier Inc.
  • Hiromichi Fujioka, Kenji Nakahara, Naoyuki Kotoku, Yusuke Ohba, Yasushi Nagatomi, Tsung-Lung Wang, Yoshinari Sawama, Kenichi Murai, Kie Hirano, Tomohiro Oki, Shintaro Wakamatsu, Yasuyuki Kita
    Chemistry - A European Journal 18 43 13861 - 13870 2012年10月22日 [査読有り][通常論文]
     
    A route for the asymmetric synthesis of (-)-stenine, a member of the Stemona alkaloid family used as folk medicine in Asian countries, is described. The key features of the sequence employed include stereoselective transformations on a cyclohexane ring controlled by a chiral auxiliary unit and an intramolecular Mitsunobu reaction to construct the perhydroindole ring system. By using an intermediate in the route to (-)-stenine, an asymmetric synthesis of 9a-epi-stenine was also executed. The C(9a) stereocenter in 9a-epi-stenine was installed by using a Staudinger/aza-Wittig reaction of a keto-azide precursor followed by reduction of the resulting imine. The results of this effort demonstrate the applicability of the chiral auxiliary based strategy to the preparation of naturally occurring alkaloids that contain highly functionalized cyclohexane cores. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
  • Ryuta Arai, Masumi Tsuda, Takuya Watanabe, Toyoyuki Ose, Chikashi Obuse, Katsumi Maenaka, Akio Minami, Yusuke Ohba
    EUROPEAN JOURNAL OF CANCER 48 15 2417 - 2430 2012年10月 [査読有り][通常論文]
     
    Synovial sarcoma is an obstinate, high-grade malignancy because of its modest responses to radiotherapy and chemotherapy; the identification of effective therapeutics for this sarcoma is therefore necessary. Inhibition of Src family kinases (SFKs) suppresses the proliferation of synovial sarcoma cells in vitro, as we have previously reported. In this study, to validate the efficacy of Src inhibition in vivo, we employed SU6656, which was originally identified as a specific SFK inhibitor. SU6656 treatment significantly impaired the growth of established, existing tumours formed by synovial sarcoma cells in mice. Tumour cell invasion into the surrounding tissues was also abolished by SU6656. It is noteworthy that SU6656 but not PP2 induced a defect in cleavage furrow formation during cytokinesis, resulting in G2/M accumulation and subsequent apoptosis. Intriguingly, SU6656 abrogated the catalytic activities of Aurora kinases and led to the down-regulation of phosphorylated histone H3 coincidently with p53 accumulation, as did the Aurora kinase inhibitor VX-680. Structural comparison indicated an extensive similarity between the catalytic domains of SFKs and Aurora kinases. The structural analysis also revealed the potential binding mode of SU6656 to the ATP-binding cleft of Aurora B via four hydrogen bonds. SU6656 prevented angiogenesis within the tumours by attenuating vascular endothelial growth factor (VEGF) production by tumour cells and the subsequent chemotaxis of endothelial cells; these effects were the result of the inhibition of SFKs but not Aurora kinases. Based on these results, we hereby report a novel property of SU6656 as a dual inhibitor of SFKs and Aurora kinases, the suppression of both of which effectively abrogates tumour development and the progression of synovial sarcoma in vivo. (C) 2011 Elsevier Ltd. All rights reserved.
  • Shigeki Chiba, Muhammad Baghdadi, Hisaya Akiba, Hironori Yoshiyama, Ichiro Kinoshita, Hirotoshi Dosaka-Akita, Yoichiro Fujioka, Yusuke Ohba, Jacob V. Gorman, John D. Colgan, Mitsuomi Hirashima, Toshimitsu Uede, Akinori Takaoka, Hideo Yagita, Masahisa Jinushi
    NATURE IMMUNOLOGY 13 9 832 - 842 2012年09月 [査読有り][通常論文]
     
    The mechanisms by which tumor microenvironments modulate nucleic acid mediated innate immunity remain unknown. Here we identify the receptor TIM-3 as key in circumventing the stimulatory effects of nucleic acids in tumor immunity. Tumor-associated dendritic cells (DCs) in mouse tumors and patients with cancer had high expression of TIM-3. DC-derived TIM-3 suppressed innate immune responses through the recognition of nucleic acids by Toll-like receptors and cytosolic sensors via a galectin-9-independent mechanism. In contrast, TIM-3 interacted with the alarmin HMGB1 to interfere with the recruitment of nucleic acids into DC endosomes and attenuated the therapeutic efficacy of DNA vaccination and chemotherapy by diminishing the immunogenicity of nucleic acids released from dying tumor cells. Our findings define a mechanism whereby tumor microenvironments suppress antitumor immunity mediated by nucleic acids.
  • がん転移における腫瘍血管内皮細胞の役割(The role of tumor endothelial cells in tumor metastasis)
    間石 奈湖, 大場 雄介, 大賀 則孝, 秋山 廣輔, 山本 和幸, 浜田 淳一, 川本 泰輔, 大澤 崇宏, 近藤 美弥子, 大村 瞳, 進藤 正信, 樋田 泰浩, 樋田 京子
    日本癌学会総会記事 71回 85 - 85 2012年08月 [査読有り][通常論文]
  • 大場雄介, 津田真寿美
    生化学 84 5 359 - 365 2012年05月25日 [査読無し][通常論文]
  • Yusuke Ohba, Masumi Tsuda
    Seikagaku 84 5 359 - 365 2012年 [査読有り][通常論文]
  • 近藤健, 金安顕子, 盛暁生, 入江達朗, 津田真寿美, 森岡正信, 今村雅寛, 大場雄介
    臨床血液 52 9 1133  2011年09月30日 [査読無し][通常論文]
  • Yukiko Usami, Taku Hatano, Satoshi Imai, Shin-ichiro Kubo, Shigeto Sato, Shinji Saiki, Yoichiro Fujioka, Yusuke Ohba, Fumiaki Sato, Manabu Funayama, Hiroto Eguchi, Kaori Shiba, Hiroyoshi Ariga, Jie Shen, Nobutaka Hattori
    NEUROBIOLOGY OF DISEASE 43 3 651 - 662 2011年09月 [査読有り][通常論文]
     
    Parkinson's disease (PD) is a neurodegenerative disorder caused by loss of dopaminergic neurons. Although many reports have suggested that genetic factors are implicated in the pathogenesis of PD, molecular mechanisms underlying selective dopaminergic neuronal degeneration remain unknown. DJ-1 is a causative gene for autosomal recessive form of PARK7-linked early-onset PD. A number of studies have demonstrated that exogenous DJ-1 localizes within mitochondria and the cytosol, and functions as a molecular chaperon, as a transcriptional regulator, and as a cell protective factor against oxidative stress. However, the precise subcellular localization and function of endogenous DJ-1 are not well known. The mechanisms by which mutations in DJ-1 contributes to neuronal degeneration also remain poorly understood. Here we show by immunocytochemistry that DJ-1 distributes to the cytosol and membranous structures in a punctate appearance in cultured cells and in primary neurons obtained from mouse brain. Interestingly. DJ-1 colocalizes with the Golgi apparatus proteins GM130 and the synaptic vesicle proteins such as synaptophysin and Rab3A. Forster resonance energy transfer analysis revealed that a small portion of DJ-1 interacts with synaptophysin in living cells. Although the wild-type DJ-1 protein directly associates with membranes without an intermediary protein, the pathogenic L166P mutation of DJ-1 exhibits less binding to synaptic vesicles. These results indicate that DJ-1 associates with membranous organelles including synaptic membranes to exhibit its normal function. (C) 2011 Elsevier Inc. All rights reserved.
  • 腫瘍血管内皮細胞とがん転移との相互作用解析(Analysis of interaction between tumor endothelial cells and tumor metastasis)
    間石 奈湖, 大賀 則孝, 樋田 泰浩, 大場 雄介, 浜田 淳一, 秋山 廣輔, 山本 和幸, 大澤 崇宏, 近藤 美弥子, 川本 泰輔, 進藤 正信, 井上 農夫男, 樋田 京子
    日本癌学会総会記事 70回 430 - 430 2011年09月 [査読有り][通常論文]
  • Ohba Y, Tsuda M
    Nihon yakurigaku zasshi. Folia pharmacologica Japonica 138 1 13 - 17 1 2011年07月 [査読有り][通常論文]
  • Tamaki Yamada, Masumi Tsuda, Tomomi Takahashi, Yasunori Totsuka, Masanobu Shindoh, Yusuke Ohba
    AMERICAN JOURNAL OF PATHOLOGY 178 6 2845 - 2856 2011年06月 [査読有り][通常論文]
     
    Recent findings have focused attention on the molecular consequences of the microenvironment in tumor progression, but events occurring in cancer cells themselves in response to their ambient conditions remain obscure. Here, we identify receptor activator of nuclear factor kappa B ligand (RANKL) as a microenvironment-specific factor essential for tumorigenesis in vivo, using head and neck squamous cell carcinoma (HNSCC) as a model. In human HNSCC tissues, RANKL is abundantly expressed, and its expression level correlates with the histological grade of differentiation. RANKL levels are significantly higher in poorly differentiated SCCs than in well or moderately differentiated SCCs. In contrast, all HNSCC cell lines tested displayed extremely low RANKL expression; however, RANKL is efficiently up-regulated when these cell lines are inoculated in the head and neck region of mice. RANKL expression is restored in a microenvironment-specific manner, and cannot be observed when the cells are inoculated in the hindlimbs. Forced expression of RANKL compensates for tumor growth in the hindlimb milieu, promotes epithelial mesenchymal transition, and induces tumor angiogenesis, in a manner independent of vascular endothelial growth factor (VEGF). These results implicate RANKL expression causatively in tumor growth and progression in HNSCC in vivo. RANKL may provide a novel functional marker for biological malignancy and a therapeutic target based on the specific nature of the microenvironment. (Am J Pathol 2011, 178:2845-2854. DOI: 10.1016/j.ajpath.2011.02.003)
  • Kensuke Tsushima, Tomoko Osawa, Hideyuki Yanai, Akira Nakajima, Akinori Takaoka, Ichiro Manabe, Yusuke Ohba, Yasushi Imai, Tadatsugu Taniguchi, Ryozo Nagai
    FASEB JOURNAL 25 5 1531 - 1543 2011年05月 [査読有り][通常論文]
     
    Hypertension is a typical modern lifestyle-related disease that is closely associated with the development of cardiovascular disorders. Elevation of angiotensin II (ANG II) is one of several critical factors for hypertension and heart failure; however, the mechanisms underlying the ANG II-mediated pathogenesis are still poorly understood. Here, we show that ANG II-mediated cardiac fibrosis, but not hypertrophy, is regulated by interferon regulatory factor 3 (IRF3), which until now has been exclusively studied in the innate immune system. In a ANG II-infusion mouse model (3.0 mg/kg/d), we compared IRF3-deficient mice (Irf3(-/-)/Bcl2l12(-/-)) with matched wild-type (WT) controls. The development of cardiac fibrosis [3.95 +/- 0.62% (WT) vs. 1.41 +/- 0.46% (Irf3(-/-)/Bcl2l12(-/-)); P < 0.01] and accompanied reduction in left ventricle end-diastolic dimension [2.89 +/- 0.10 mm (WT) vs. 3.51 +/- 0.15 mm (Irf3(-/-)/Bcl2l12(-/-)); P=0.012] are strongly suppressed in Irf3(-/-)/Bcl2l12(-/-) mice, whereas hypertrophy still develops. Further, we provide evidence for the activation of IRF3 by ANG II signaling in mouse cardiac fibroblasts. Unlike the activation of IRF3 by innate immune receptors, IRF3 activation by ANG II is unique in that it is activated through the canonical ERK signaling pathway. Thus, our present study reveals a hitherto unrecognized function of IRF3 in cardiac remodeling, providing new insight into the progression of hypertension-induced cardiac pathogenesis.-Tsushima, K., Osawa, T., Yanai, H., Nakajima, A., Takaoka, A., Manabe, I., Ohba, Y., Imai, Y., Taniguchi, T., Nagai, R. IRF3 regulates cardiac fibrosis but not hypertrophy in mice during angiotensin II-induced hypertension. FASEB J. 25, 1531-1543 (2011). www.fasebj.org
  • Sumio Hayakawa, Souichi Shiratori, Hiroaki Yamato, Takeshi Kameyama, Chihiro Kitatsuji, Fumi Kashigi, Showhey Goto, Shoichiro Kameoka, Daisuke Fujikura, Taisho Yamada, Tatsuaki Mizutani, Mika Kazumata, Maiko Sato, Junji Tanaka, Masahiro Asaka, Yusuke Ohba, Tadaaki Miyazaki, Masahiro Imamura, Akinori Takaoka
    NATURE IMMUNOLOGY 12 1 37 - U56 2011年01月 [査読無し][通常論文]
     
    The poly(ADP-ribose) polymerases (PARPs) participate in many biological and pathological processes. Here we report that the PARP-13 shorter isoform (ZAPS), rather than the full-length protein (ZAP), was selectively induced by 5'-triphosphate-modified RNA (3pRNA) and functioned as a potent stimulator of interferon responses in human cells mediated by the RNA helicase RIG-I. ZAPS associated with RIG-I to promote the oligomerization and ATPase activity of RIG-I, which led to robust activation of IRF3 and NF-kappa B transcription factors. Disruption of the gene encoding ZAPS resulted in impaired induction of interferon-alpha (IFN-alpha), IFN-beta and other cytokines after viral infection. These results indicate that ZAPS is a key regulator of RIG-I signaling during the innate antiviral immune response, which suggests its possible use as a therapeutic target for viral control.
  • Koji Atarashi, Takeshi Tanoue, Tatsuichiro Shima, Akemi Imaoka, Tomomi Kuwahara, Yoshika Momose, Genhong Cheng, Sho Yamasaki, Takashi Saito, Yusuke Ohba, Tadatsugu Taniguchi, Kiyoshi Takeda, Shohei Hori, Ivaylo I. Ivanov, Yoshinori Umesaki, Kikuji Itoh, Kenya Honda
    SCIENCE 331 6015 337 - 341 2011年01月 [査読有り][通常論文]
     
    CD4(+) T regulatory cells (T(regs)), which express the Foxp3 transcription factor, play a critical role in the maintenance of immune homeostasis. Here, we show that in mice, T(regs) were most abundant in the colonic mucosa. The spore-forming component of indigenous intestinal microbiota, particularly clusters IV and XIVa of the genus Clostridium, promoted T(reg) cell accumulation. Colonization of mice by a defined mix of Clostridium strains provided an environment rich in transforming growth factor-beta and affected Foxp3(+) T(reg) number and function in the colon. Oral inoculation of Clostridium during the early life of conventionally reared mice resulted in resistance to colitis and systemic immunoglobulin E responses in adult mice, suggesting a new therapeutic approach to autoimmunity and allergy.
  • Yoichiro Fujioka, Masumi Tsuda, Tomoe Hattori, Junko Sasaki, Takehiko Sasaki, Tadaaki Miyazaki, Yusuke Ohba
    PLOS ONE 6 1 e16324  2011年01月 [査読有り][通常論文]
     
    Background: Influenza virus infection causes highly contagious, severe respiratory disorders and gives rise to thousands of deaths every year; however, the efficacy of currently approved defense strategies, including vaccines and neuraminidase inhibitors, is limited because the virus frequently acquires resistance via antigen drift and reassortment. It is therefore important to establish a novel, effective therapeutic strategy that is effective irrespective of viral subtype. Methodology/Principal Findings: Here, we identify the Ras-phosphoinositide 3-kinase (PI3K) signaling pathway as a host-cell regulatory mechanism for influenza virus entry. The binding of Ras to PI3K is specifically involved in clathrinin-dependent endocytosis, endosomal maturation, and intracellular transport of viruses, which result in decreased infectious efficacy of different subtypes of influenza viruses in cells lacking the Ras-PI3K interaction. Moreover, influenza virus infection indeed triggered Ras activation and subsequent PI3K activation in early endosomes. Conclusions/Significance: Taken together, these results demonstrate that the Ras-PI3K signaling axis acts as a host-oriented mechanism for viral internalization. Given that virus incorporation is a process conserved among virus subtypes and species, this signaling pathway may provide a target for potent, well-tolerated prophylactics and therapeutics against a broad range of viruses.
  • Sumio Hayakawa, Souichi Shiratori, Hiroaki Yamato, Takeshi Kameyama, Chihiro Kitatsuji, Fumi Kashigi, Showhey Goto, Shoichiro Kameoka, Daisuke Fujikura, Taisho Yamada, Tatsuaki Mizutani, Mika Kazumata, Maiko Sato, Junji Tanaka, Masahiro Asaka, Yusuke Ohba, Tadaaki Miyazaki, Masahiro Imamura, Akinori Takaoka
    NATURE IMMUNOLOGY 12 1 37 - U56 2011年01月 [査読有り][通常論文]
     
    The poly(ADP-ribose) polymerases (PARPs) participate in many biological and pathological processes. Here we report that the PARP-13 shorter isoform (ZAPS), rather than the full-length protein (ZAP), was selectively induced by 5'-triphosphate-modified RNA (3pRNA) and functioned as a potent stimulator of interferon responses in human cells mediated by the RNA helicase RIG-I. ZAPS associated with RIG-I to promote the oligomerization and ATPase activity of RIG-I, which led to robust activation of IRF3 and NF-kappa B transcription factors. Disruption of the gene encoding ZAPS resulted in impaired induction of interferon-alpha (IFN-alpha), IFN-beta and other cytokines after viral infection. These results indicate that ZAPS is a key regulator of RIG-I signaling during the innate antiviral immune response, which suggests its possible use as a therapeutic target for viral control.
  • Atarashi Koji, Tanoue Takeshi, Shima Tatsuichiro, Imaoka Akemi, Kuwahara Tomomi, Momose Yoshika, Cheng Genhong, Yamasaki Sho, Saito Takashi, Ohba Yusuke, Taniguchi Tadatsugu, Takeda Kiyoshi, Hori Shohei, Ivanov Ivaylo I, Umesaki Yoshinori, Itoh Kikuji, Honda Kenya
    Science 1 - 5 2010年12月23日 [査読無し][通常論文]
     
    <p>CD4<sup>+</sup> T regulatory cells (Tregs), expressing the Foxp3 transcription factor, play a critical role in the maintenance of immune homeostasis. Here, we show that in mice, Tregs were most abundant in the colonic mucosa. The spore-forming component of indigenous intestinal microbiota-particularly clusters IV and XIVa of the genus Clostridium, promoted Treg cell accumulation. Colonization of mice by a defined mix of Clostridium strains provided an environment rich in transforming growth factor-β (TGF-β) and affected Foxp3<sup>+</sup> Treg number and function in the colon. Oral inoculation of Clostridium during the early life of conventionally reared mice resulted in resistance to colitis and systemic IgE responses in adult mice, suggesting a new therapeutic approach to autoimmunity and allergy.</p>
  • Yasuhiro Saito, Naoko Murata-Kamiya, Toshiya Hirayama, Yusuke Ohba, Masanori Hatakeyama
    JOURNAL OF EXPERIMENTAL MEDICINE 207 10 2157 - 2174 2010年09月 [査読有り][通常論文]
     
    The Helicobacter pylori CagA bacterial oncoprotein plays a critical role in gastric carcinogenesis. Upon delivery into epithelial cells, CagA causes loss of polarity and activates aberrant Erk signaling. We show that CagA-induced Erk activation results in senescence and mitogenesis in nonpolarized and polarized epithelial cells, respectively. In nonpolarized epithelial cells, Erk activation results in oncogenic stress, up-regulation of the p21(Waf1/Cip1) cyclin-dependent kinase inhibitor, and induction of senescence. In polarized epithelial cells, CagA-driven Erk signals prevent p21(Waf1/Cip1) expression by activating a guanine nucleotide exchange factor-H1-RhoA-RhoA-associated kinase-c-Myc pathway. The microRNAs miR-17 and miR-20a, induced by c-Myc, are needed to suppress p21(Waf1/Cip1) expression. CagA also drives an epithelial-mesenchymal transition in polarized epithelial cells. These findings suggest that CagA exploits a polarity-signaling pathway to induce oncogenesis.
  • Tatsuaki Mizutani, Takeshi Kondo, Stephanie Darmanin, Masumi Tsuda, Shinya Tanaka, Minoru Tobiume, Masahiro Asaka, Yusuke Ohba
    CLINICAL CANCER RESEARCH 16 15 3964 - 3975 2010年08月 [査読有り][通常論文]
     
    Purpose: To develop a novel diagnostic method for the assessment of drug efficacy in chronic myeloid leukemia (CML) patients individually, we generated a biosensor that enables the evaluation of BCR-ABL kinase activity in living cells using the principle of fluorescence resonance energy transfer (FRET). Experimental Design: To develop FRET-based biosensors, we used CrkL, the most characteristic substrate of BCR-ABL, and designed a protein in which CrkL is sandwiched between Venus, a variant of YFP, and enhanced cyan fluorescent protein, so that CrkL intramolecular binding of the SH2 domain to phosphorylated tyrosine (Y207) increases FRET efficiency. After evaluation of the properties of this biosensor by comparison with established methods including Western blotting and flow cytometry, BCR-ABL activity and its response to drugs were examined in CML patient cells. Results: After optimization, we obtained a biosensor that possesses higher sensitivity than that of established techniques with respect to measuring BCR-ABL activity and its suppression by imatinib. Thanks to its high sensitivity, this biosensor accurately gauges BCR-ABL activity in relatively small cell numbers and can also detect <1% minor drug-resistant populations within heterogeneous ones. We also noticed that this method enabled us to predict future onset of drug resistance as well as to monitor the disease status during imatinib therapy, using patient cells. Conclusion: In consideration of its quick and practical nature, this method is potentially a promising tool for the prediction of both current and future therapeutic responses in individual CML patients, which will be surely beneficial for both patients and clinicians. Clin Cancer Res; 16(15); 3964-75. (C) 2010 AACR.
  • 腫瘍血管内皮細胞と腫瘍細胞との相互作用解析(Analysis of interaction between tumor endothelial cells and tumor cells)
    間石 奈湖, 大賀 則孝, 秋山 廣輔, 北山 和子, 近藤 美弥子, 川本 泰輔, 大澤 崇宏, 山本 和幸, 樋田 泰浩, 大場 雄介, 進藤 正信, 樋田 京子
    日本癌学会総会記事 69回 103 - 103 2010年08月 [査読有り][通常論文]
  • Kaori Tsutsumi, Yoichiro Fujioka, Masumi Tsuda, Hideaki Kawaguchi, Yusuke Ohba
    CELLULAR SIGNALLING 21 11 1672 - 1679 2009年11月 [査読有り][通常論文]
     
    Recent studies indicate the importance of spatiotemporal regulation in the diversity and specificity of intracellular signaling. Here, we show that Ras-PI3K signaling plays an important role in the local regulation of phosphatidylinositol metabolism in the endosome through live-cell imaging by using a bimolecular fluorescence complementation technique, in which molecular interaction is indicated by fluorescence emission. Using several possible combinations of Ras and the Ras-binding domain, we identified an optimal set of probe molecules that yielded the most significant increase in fluorescence intensity between the active and inactive forms of Ras. This combination revealed that, among the Ras effectors tested, phosphatidy-linositol 3-kinase (PI3K) was specifically implicated in signaling in the endosome. We also found that full length PI3K was recruited to the endosome in EGF- and Ras-dependent manners, which appears to be essential for the activation of PI3K in this compartment. Taken together, these findings demonstrate that the spatiotemporal regulation of Ras-PI3K signaling may dictate the activation of PI3K and subsequent downstream signaling in the endosome. (C) 2009 Elsevier Inc. All rights reserved.
  • 津田 真寿美, 大場 雄介
    細胞 41 11 462 - 465 ニューサイエンス社 2009年10月 [査読無し][通常論文]
  • Takuya Watanabe, Masumi Tsuda, Shinya Tanaka, Yusuke Ohba, Hideaki Kawaguchi, Tokifumi Majima, Hirofumi Sawa, Akio Minami
    MOLECULAR CANCER RESEARCH 7 9 1582 - 1592 2009年09月 [査読有り][通常論文]
     
    The adaptor protein Crk mediates intracellular signaling related to cell motility and proliferation and is implicated in human tumorigenesis. The role of Crk in the growth of human sarcoma has remained unclear, however. The present study shows that Crk-induced activation of Src and subsequent signaling by p38 mitogen-activated protein kinase (MAPK) contribute to the enhanced proliferation of human synovial sarcoma cells. Depletion of Crk by RNA interference markedly inhibited proliferation of the synovial sarcoma cell lines HS-SYII, SYO-1, and Fuji as well as prevented anchorage-independent growth. Conversely, reconstitution with CrkII by authentic small interfering RNA-resistant Crk gene restored proliferation in Crk-silenced SYO-1 cells. Crk-depleted synovial sarcoma cells manifested enhanced transcriptional activity and expression of the p16(INK4A) gene, resulting in their accumulation in G, phase of the cell cycle. In response to hepatocyte growth factor stimulation, Crk prominently induced the tyrosine phosphorylation of Grb2-associated binder 1 through activation of Src and focal adhesion kinase, and the Src family kinase inhibitor PP2 almost completely inhibited the proliferation of SYO-1 cells. Crk also induced the phosphorylation of p38 MAPK, and SB203580, a p38 MAPK-specific inhibitor, increased expression of p16(INK4A) gene in SYO-1 cells. Furthermore, SB203580 or depletion of p38 MAPK by small interfering RNA suppressed both the phosphorylation of Akt triggered by hepatocyte growth factor and the proliferation of SYO-1 cells. These results suggest that Crk promotes proliferation of human synovial sarcoma cells through activation of Src and its downstream signaling by a novel p38 MAPK-Akt pathway, with these signaling molecules providing potent new targets for molecular therapeutics. (Mol Cancer Res 2009;7(9):1582-92)
  • Mayumi Umeda, Naoko Murata-Kamiya, Yasuhiro Saito, Yusuke Ohba, Masayuki Takahashi, Masanori Hatakeyama
    JOURNAL OF BIOLOGICAL CHEMISTRY 284 33 22166 - 22172 2009年08月 [査読有り][通常論文]
     
    Infection with cagA-positive Helicobacter pylori is the strongest risk factor for the development of gastric carcinoma. The cagA gene product CagA, which is delivered into gastric epithelial cells, specifically binds to and aberrantly activates SHP-2 oncoprotein. CagA also interacts with and inhibits partitioning-defective 1 (PAR1)/MARK kinase, which phosphorylates microtubule-associated proteins to destabilize microtubules and thereby causes epithelial polarity defects. In light of the notion that microtubules are not only required for polarity regulation but also essential for the formation of mitotic spindles, we hypothesized that CagA-mediated PAR1 inhibition also influences mitosis. Here, we investigated the effect of CagA on the progression of mitosis. In the presence of CagA, cells displayed a delay in the transition from prophase to metaphase. Furthermore, a fraction of the CagA-expressing cells showed spindle misorientation at the onset of anaphase, followed by chromosomal segregation with abnormal division axis. The effect of CagA on mitosis was abolished by elevated PAR1 expression. Conversely, inhibition of PAR1 kinase elicited mitotic delay similar to that induced by CagA. Thus, CagA-mediated inhibition of PAR1, which perturbs microtubule stability and thereby causes microtubule-based spindle dysfunction, is involved in the prophase/metaphase delay and subsequent spindle misorientation. Consequently, chronic exposure of cells to CagA induces chromosomal instability. Our findings reveal a bifunctional role of CagA as an oncoprotein: CagA elicits uncontrolled cell proliferation by aberrantly activating SHP-2 and at the same time induces chromosomal instability by perturbing the microtubule-based mitotic spindle. The dual function of CagA may cooperatively
  • Kahori Shiba, Takeo Arai, Shigeto Sato, Shin-ichiro Kubo, Yusuke Ohba, Yoshikuni Mizuno, Nobutaka Hattori
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 383 3 331 - 335 2009年06月 [査読有り][通常論文]
     
    Parkinson disease (PD) is the most common movement disorder and is characterized by dopaminergic dysfunction. The majority of PD cases are sporadic: however, the discovery of genes linked to rare familial forms of the disease has provided crucial insight into the molecular mechanisms of disease pathogenesis. Multiple genes mediating familial forms of Parkinson's disease (PD) have been identified, such as parkin (PARK2) and phosphatase and tensin homologue deleted on chromosome ten (PTEN)-induced putative kinase 1: PINK1 (PARK6). Here, we showed that Parkin directly interacts with PINK1, but did not bind to pathogenic PINK1 mutants. Parkin, but not its pathogenic mutants, stabilizes PINK1 by interfering with its degradation via the ubiquitin-mediated proteasomal pathway. In addition, the interaction between Parkin and PINK1 resulted in reciprocal reduction of their solubility. Our results indicate that Parkin regulates PINK1 stabilization via direct interaction with PINK1, and operates through a common pathway with PINK1 in the pathogenesis of early-onset PD. (C) 2009 Elsevier Inc. All rights reserved.
  • Yusuke Ohba, Mayuko Takatsuji, Kenji Nakahara, Hiromichi Fujioka, Yasuyuki Kita
    Chemistry - A European Journal 15 14 3526 - 3537 2009年03月23日 [査読有り][通常論文]
     
    We present the highly efficient reaction procedure of the macrolactonization method via ethoxyvinyl esters (EVEs). The following procedure was performed: 1)The EVE was prepared from hydroxycarboxylic acid and ethoxyacetylene in the presence of the Ru catalyst [RuCl 2(p-cymene)] 2 in acetone 2) after filtration of the Ru catalyst through a short-neutral SiO 2 pad and evaporation of acetone, the EVE formed was diluted in 1,2-dichloroethane (DCE) and the solution was slowly added by a syringe pump to the highly diluted DCE solution of pTsOH (10mol%) at 80°C. Varioussized lactones could be produced by the method described here. It is note worthy that the method can give 9- to 14-membered macrolactones in good yields. This macrolactonization method via EVEs is useful for acid-/base-sensitive substrates. Furthermore, it was found that EVE formation was possible without loosing activity of the Ru catalyst even for the compounds with nucleophilic amine functions. The characteristic feature of the method was exemplified by the reaction of the compound 14 with many functional groups. © 2009 Wiley-VCH Verlag GmbH & Co. KGaA.
  • Takayuki Inuzuka, Masumi Tsuda, Hideaki Kawaguchi, Yusuke Ohba
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 379 2 510 - 513 2009年02月 [査読有り][通常論文]
     
    We have recently reported that transcription factor 8 (TCF8) negatively regulates pathological angiogenesis by regulating endothelial invasiveness by acting as a transcriptional attenuator of matrix metalloproteinase 1. TCF8 also modulates cell-matrix and cell-cell adhesion; however molecular mechanism of this TCF8 function remains obscure. Here, we provide evidence that TCF8 activates R-Ras, another class of angiogenic regulator, to suppress angiogenesis by a mechanism other than a transcriptional attenuator. Tube formation by human umbilical vein endothelial cells (HUVECs) facilitated by TCF8 suppression was significantly inhibited by the expression of constitutive active mutant of R-Ras. When we examined the mRNA expression levels of R-Ras regulators, no significant changes were observed to explain the R-Ras activation by TCF8. Interestingly, we found that TCF8 bound to CalDAG-GEFIII, an R-Ras activator, in the cytosol, indicating that TCF8 emanates signaling for R-Ras activation from cytosol to regulate angiogenesis negatively. (C) 2008 Elsevier Inc. All rights reserved.
  • Takayuki Inuzuka, Masumi Tsuda, Shinya Tanaka, Hideaki Kawaguchi, Yujiro Higashi, Yusuke Ohba
    CANCER RESEARCH 69 4 1678 - 1684 2009年02月 [査読有り][通常論文]
     
    Angiogenesis is involved in various physiologic and pathological conditions, including tumor growth, and is tightly regulated by the orchestration of proangiogenic and antiangiogenic factors. Inhibition of vascular endothelial growth factor (VEGF), the best-established antiangiogenic treatment in cancer, has shown some effectiveness; however, the identification of novel regulators, whose function is independent of VEGF, is required to achieve better outcomes. Here, we show that transcription factor 8 (TCF8) is up-regulated in endothelial cells during angiogenesis, acting as a negative regulator. Furthermore, TCF8 is specifically expressed in the endothelium of tumor vessels. 1cf8-heterozygous knockout mice are more permissive than wildtype mice to the formation of tumor blood vessels in s.c. implanted melanoma, which seems to contribute to the more aggressive growth and the lung metastases of the tumor in mutant mice. Suppression of TCF8 facilitates angiogenesis in both in vitro and ex vivo models, and displays comprehensive cellular phenotypes, including enhanced cell invasion, impaired cell adhesion, and increased cell monolayer permeability due to, at least partly, MMPI overexpression, attenuation of focal adhesion formation, and insufficient VE-cadherin recruitment, respectively. Taken together, our findings define a novel, integral role for TCF8 in the regulation of pathologic angiogenesis, and propose TCF8 as a target for therapeutic intervention in cancer. [Cancer Res 2009;69(4):1678-84]
  • Kaori Tsutsumi, Masumi Tsuda, Natsuka Yazawa, Hirotaka Nakamura, Seiichiro Ishihara, Hisashi Haga, Motoaki Yasuda, Rie Yamazaki, Hiroki Shirato, Hideaki Kawaguchi, Takeshi Nishioka, Yusuke Ohba
    CELL STRUCTURE AND FUNCTION 34 2 89 - 96 2009年 [査読有り][通常論文]
     
    Radiotherapy is an important noninvasive treatment for many types of cancer. However, it has been reported that the proliferative, invasive, and metastatic capacities of tumor cells can be increased in the repopulated tumors that survive radiotherapy. We have previously established a radiation-surviving cell model for the human non-small cell lung cancer cell line H1299 by harvesting relic cells 14 days after irradiation (IR cells). Here, we report that cell invasion, cell migration, and cell adhesion are enhanced in these surviving cancer cells. The mRNA expression levels of matrix metalloproteinases (MMPs), including mmp1, mmp2, and mmp9, were upregulated in IR cells compared with parental cells. A gelatin zymogram, wound healing assay, and invasion assay showed increased MMP activity, cell motility, and invasiveness in IR cells, respectively. Moreover, IR cells adhered more tightly to collagen-coated dishes than parental cells. Consistently, paxillin, phosphorylated FAK, integrin beta 1, and vinculin were strongly localized at focal adhesions in IR cells, as visualized by immunofluorescence. In this report, we identify molecules responsible for the malignant properties of tumor cells that survive irradiation. These molecules may be important therapeutic targets for the control of repopulated tumors after radiotherapy.
  • Kayoko Takeda, Ichiro Kinoshita, Yasushi Shimizu, Yusuke Ohba, Tomoo Itoh, Yoshihiro Matsuno, Toshiaki Shichinohe, Hirotoshi Dosaka-Akita
    BMC CANCER 8 328  2008年11月 [査読有り][通常論文]
     
    Background: A recent study has shown that phosphorylated c-Jun (p-c-Jun) interacts with TCF4 to form a complex that cooperatively enhances their transcriptional activity in the presence of beta-Catenin, and that their interaction is critical for mouse intestinal tumorigenesis. To determine the significance of these three proteins in human colorectal tumors, we analyzed their nuclear expression by immunohistochemistry. Methods: we analyzed their nuclear expression by immunohistochemistry using paraffin-embedded specimens of 68 resected colorectal tumors, which consisted of 19 adenomas, 14 high-grade intraepithelial neoplasia (HGINs) and 35 adenocarcinomas. We also analyzed the expression of MMP7, which has functional AP-I and TCF binding sites in its promoter. Results: Expression of p-c-Jun, TCF4 and beta-Catenin were significantly higher in adenomas than in the adjacent normal epithelia. Expression of p-c-Jun and beta-Catenin in HGINs and adenocarcinomas were also significantly higher than in the adjacent normal epithelia. p-c-Jun expression, but not TCF4 and beta-Catenin, was higher in adenomas and HGINs than in adenocarcinomas, in which p-c-Jun expression was negatively correlated with pT stage progression. Furthermore, significant correlations of expression were observed between p-c-Jun and TCF4 ( r = 0.25, p = 0.04), TCF4 and beta-Catenin ( r = 0.30, p = 0.01), p-c-Jun and MMP7 ( r = 0.26, p = 0.03), and TCF4 and MMP7 ( r = 0.39, p = 0.0008), respectively. Conclusion: These results suggest that nuclear expression of p-c-Jun, TCF4 and beta-Catenin have important roles in human colorectal tumor development and that p-c-Jun may play a pivotal role in the earlier stages of tumor development.
  • 大場雄介, 津田真寿美
    実験医学 26 15 2506 - 2513 2008年09月15日 [査読無し][通常論文]
  • Hiromichi Fujioka, Yusuke Ohba, Hideki Hirose, Kenji Nakahara, Kenichi Murai, Yasuyuki Kita
    Tetrahedron 64 19 4233 - 4245 2008年05月05日 [査読有り][通常論文]
     
    A novel double intramolecular iodoetherification of σ-symmetric diene acetals from (R,R)-hydrobenzoin occurred in highly diastereoselective manners to give tetrahydrofuran moieties with multiple chiral centers in a one-pot operation. The chemoselective discrimination of the two iodomethyl functions in the products was attained in various reactions. The reaction was applied to the concise asymmetric syntheses of rubrenolide and rubrynolide, in which the unit from the chiral auxiliary worked as a template to achieve the chemoselectivity and as the protecting group of the hydroxyl function. © 2008 Elsevier Ltd. All rights reserved.
  • Tamaki Yamada, Masumi Tsuda, Yusuke Ohba, Hideaki Kawaguchi, Yasunori Totsuka, Masanobu Shindoh
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 368 3 575 - 581 2008年04月 [査読無し][通常論文]
     
    Parathyroid hormone-related protein (PTHrP) is detected in many aggressive tumors and involved in malignant conversion; however, the underlying mechanism remains obscure. Here, we identified PTHrP as a mediator of epidermal growth factor receptor (EGFR) signaling to promote the malignancies of oral cancers. PTHrP mRNA was abundantly expressed in most of the quiescent oral cancer cells, and was significantly upregulated by EGF stimulation via ERK and p38 MAPK. PTHrP silencing by RNA interference, as well as EGFR inhibitor AG1478 treatment, significantly suppressed cell proliferation, migration, and invasiveness. Furthermore, combined treatment of AG1478 and PTHrP knockdown achieved synergistic inhibition of malignant phenotypes. Recombinant PTHrP substantially promoted cell motility, and rescued the inhibition by PTHrP knockdown, suggesting the paracrine/autocrine function of PTHrP. These data indicate that PTHrP contributes to the malignancy of oral cancers downstream of EGFR signaling, and may thus provide a therapeutic target for oral cancer. (c) 2008 Elsevier Inc. All rights reserved.
  • Tamaki Yamada, Masumi Tsuda, Yusuke Ohba, Hideaki Kawaguchi, Yasunori Totsuka, Masanobu Shindoh
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 368 3 575 - 581 2008年04月 [査読有り][通常論文]
     
    Parathyroid hormone-related protein (PTHrP) is detected in many aggressive tumors and involved in malignant conversion; however, the underlying mechanism remains obscure. Here, we identified PTHrP as a mediator of epidermal growth factor receptor (EGFR) signaling to promote the malignancies of oral cancers. PTHrP mRNA was abundantly expressed in most of the quiescent oral cancer cells, and was significantly upregulated by EGF stimulation via ERK and p38 MAPK. PTHrP silencing by RNA interference, as well as EGFR inhibitor AG1478 treatment, significantly suppressed cell proliferation, migration, and invasiveness. Furthermore, combined treatment of AG1478 and PTHrP knockdown achieved synergistic inhibition of malignant phenotypes. Recombinant PTHrP substantially promoted cell motility, and rescued the inhibition by PTHrP knockdown, suggesting the paracrine/autocrine function of PTHrP. These data indicate that PTHrP contributes to the malignancy of oral cancers downstream of EGFR signaling, and may thus provide a therapeutic target for oral cancer. (c) 2008 Elsevier Inc. All rights reserved.
  • Tatsuaki Mizutani, Kohichiro Tsuji, Yasuhiro Ebihara, Shinsuke Taki, Yusuke Ohba, Tadatsugu Taniguchi, Kenya Honda
    EXPERIMENTAL HEMATOLOGY 36 3 255 - 264 2008年 [査読有り][通常論文]
     
    Objective. Erythrocyte production is tightly regulated by cytokines, particularly erythropoietin (EPO), which affects expansion and viability of erythroid lineage cells via induction of several factors, including Bcl2-like 1 (Bcl-XL). Because type I interferon (IFN) is known to inhibit erythropoiesis, we studied mice deficient in the gene for interferon regulatory factor 2 (IRF2), which functions as a negative regulator of type I IFN signaling, in the context of erythropoiesis regulation. Materials and Methods. We performed hematologic analyses and detected normocytic anemia in Irj2-deficient mice. Results. Assessment of the maturation of erythroid progenitors in Irf2-deficient bone marrow by flow cytometry revealed a decreased number of late erythroblasts accompanied by an increased number of early erythroid progenitors. Irf2-deficient mice manifested elevated serum EPO levels, decreased Bcl-XL expression levels and enhanced apoptosis of erythroblasts, which may account for the decreased number of late erythroblasts. We further assessed the role of IRF2 in the regulation of type I IFN signaling during erythropoiesis, and found that additional homozygous mutation of IFNAR1, a subunit of type I IFN receptor complex, led to rescue of the defect of erythropoiesis in Irf2-deficient mice. Conclusions. Impaired erythropoiesis in Irf2-deficient mice results from excessive type I IFN signaling, which inhibits Bcl-XL expression in erythroid lineage cells. Our present study provides a mechanistic understanding of the potential cross-talk between type I IFN and EPO signaling pathways during erythropoiesis and may offer therapeutic insights into anemia. (c) 2008 ISEH - Society for Hematology and Stem Cells. Published by Elsevier Inc.
  • Hiromichi Fujioka, Yusuke Ohba, Kenji Nakahara, Mayuko Takatsuji, Kenichi Murai, Motoki Ito, Yasuyuki Kita
    Organic Letters 9 26 5605 - 5608 2007年12月20日 [査読有り][通常論文]
     
    The synthesis of (+)-Sch 642305 is an example of chiral auxiliary multiuse methodology, which shows a new perspective for the synthesis of compounds with multiple asymmetric centers. Thus, (+)-Sch 642305 was concisely synthesized from the known compound. Every reaction is stereoselective, and the chiral nonracemic hydrobenzoin worked as chiral auxiliary for desymmetrization of diene, as a template for attaining regio- and stereoselective reactions, as an oxygen source at the C4-position, and as a protecting group of hydroxyl functions. Namely, the chiral auxiliary played a role in every step throughout the synthesis. Furthermore, the synthesis contains a new protocol for obtaining α′-alkylated enone compounds. © 2007 American Chemical Society.
  • Kenji Funami, Miwa Sasai, Yusuke Ohba, Hiroyuki Oshiumi, Tsukasa Seya, Misako Matsumoto
    JOURNAL OF IMMUNOLOGY 179 10 6867 - 6872 2007年11月 [査読有り][通常論文]
     
    TLR3 recognizes viral dsRNA and induces antiviral immune responses. TLR3-mediated cell activation relies on Toll/IL-1R (TIR) domain-containing adaptor molecule-1 (TICAM-1, also named TIR domain-containing adaptor inducing IFN-beta or TRIF), which recruits downstream signaling molecules to activate the transcription factors IFN regulatory factor 3 (IRF-3) and NF-kappa B. The mechanisms by which TICAM-1 is activated and transmits signals remain largely unknown. In this study we show that TICAM-1 alters its distribution profile from a diffuse cytoplasmic form to a speckle-like structure in response to dsRNA. The receptor-interacting protein 1 (RIP1), a crucial signaling molecule for TICAM-1-mediated NF-kappa B activation, accumulated in the TICAM-1 speckles. In addition, NF-kappa B-activating kinase-associated protein 1 (NAP1), a downstream molecule linking TICAM-1 and the IRF-3-activating kinase TBK1 (TANK-binding kinase 1), was also recruited to the TICAM-1 speckles. Notably, a transient colocalization of TICAM-1 and TLR3 was observed before the extensive formation of the TICAM-1 speckles. Thus, the spatiotemporal mobilization of TICAM-1 in response to dsRNA and the formation of the TICAM-1 speckles containing RIP1 and NAP1 are important for the activation of the TLR3-TICAM-1 pathway.
  • Kouichi Tabu, Yusuke Ohba, Tadaki Suzuki, Yoshinori Makino, Taichi Kimura, Akiko Ohnishi, Mieko Sakai, Takuya Watanabe, Shinya Tanaka, Hirofurni Sawa
    MOLECULAR CANCER RESEARCH 5 10 1099 - 1109 2007年10月 [査読無し][通常論文]
     
    The basic helix-loop-helix transcription factor, oligodendrocyte lineage transcription factor 2 (OLIG2), is specifically expressed in the developing and mature central nervous system and plays an important role in oligodendrogenesis from neural progenitors. It is also expressed in various types of glial tumors, but rarely in glioblastoma. Although we previously showed that OLIG2 expression inhibits glioma cell growth, its role in tumorigenesis remains incompletely understood. Here, we investigated the effect of OLIG2 expression on the migration of the human glioblastoma cell line U12-1. In these cells, OLIG2 expression is controlled by the Tet-off system. Induction of OLIG2 expression inhibited both the migration and invasiveness of U12-1 cells. OLIG2 expression also increased the activity of the GTPase RhoA as well as inducing the cells to form stress fibers and focal adhesions. Experiments using short interfering RNA against p27(KiP1) revealed that up-regulation of the p27(Kip1) protein was not essential for RhoA activation, rather it contributed independently to the decreased motility of OLIG2-expressing U12-1 cells. Alternatively, semiquantitative reverse transcription-PCR analysis revealed that mRNA expression of RhoGAP8, which regulates cell migration, was decreased by OLIG2 expression. Furthermore, expression of C3 transferase, which inhibits Rho via ADP ribosylation, attenuated the OLIG2-induced inhibition of cell motility. Imaging by fluorescence resonance energy transfer revealed that in U12-1 cells lacking OLIG2, the active form of RhoA was localized to protrusions of the cell membrane. In contrast, in OLIG2-expressing cells, it lined almost the entire plasma membrane. Thus, OLIG2 suppresses the motile phenotype of glioblastoma cells by activating RhoA.
  • Kouichi Tabu, Yusuke Ohba, Tadaki Suzuki, Yoshinori Makino, Taichi Kimura, Akiko Ohnishi, Mieko Sakai, Takuya Watanabe, Shinya Tanaka, Hirofurni Sawa
    MOLECULAR CANCER RESEARCH 5 10 1099 - 1109 2007年10月 [査読有り][通常論文]
     
    The basic helix-loop-helix transcription factor, oligodendrocyte lineage transcription factor 2 (OLIG2), is specifically expressed in the developing and mature central nervous system and plays an important role in oligodendrogenesis from neural progenitors. It is also expressed in various types of glial tumors, but rarely in glioblastoma. Although we previously showed that OLIG2 expression inhibits glioma cell growth, its role in tumorigenesis remains incompletely understood. Here, we investigated the effect of OLIG2 expression on the migration of the human glioblastoma cell line U12-1. In these cells, OLIG2 expression is controlled by the Tet-off system. Induction of OLIG2 expression inhibited both the migration and invasiveness of U12-1 cells. OLIG2 expression also increased the activity of the GTPase RhoA as well as inducing the cells to form stress fibers and focal adhesions. Experiments using short interfering RNA against p27(KiP1) revealed that up-regulation of the p27(Kip1) protein was not essential for RhoA activation, rather it contributed independently to the decreased motility of OLIG2-expressing U12-1 cells. Alternatively, semiquantitative reverse transcription-PCR analysis revealed that mRNA expression of RhoGAP8, which regulates cell migration, was decreased by OLIG2 expression. Furthermore, expression of C3 transferase, which inhibits Rho via ADP ribosylation, attenuated the OLIG2-induced inhibition of cell motility. Imaging by fluorescence resonance energy transfer revealed that in U12-1 cells lacking OLIG2, the active form of RhoA was localized to protrusions of the cell membrane. In contrast, in OLIG2-expressing cells, it lined almost the entire plasma membrane. Thus, OLIG2 suppresses the motile phenotype of glioblastoma cells by activating RhoA.
  • Yoichiro Fujioka, Maki Utsumi, Yusuke Ohba, Yuichiro Watanabe
    PLANT AND CELL PHYSIOLOGY 48 9 1243 - 1253 2007年09月 [査読無し][通常論文]
     
    There has been much recent research on the contribution of microRNA (miRNA) in plant organogenesis and hormone action. In plants, it has been reported that Dicer-like 1 (DCL1), HYPONASTIC LEAVES1 (HYL1). and SERRATE (SE) are involved in the production of miRNAs. The means by which miRNAs are processed and transported is. not well understood in detail, however. In this study, we investigated the intracellular localization and intermolecular interaction of these molecules using imaging techniques, including bimolecular fluorescence complementation and fluorescence resonance energy transfer techniques, making use of various enhanced fluorescent proteins. We found that DCL1, HYL1 and SE formed bodies which localized in the nuclei. We were also able to locate the miRNA primary transcript using an MS2-tagged method on these bodies. It appears very likely that the observed DCL1-HYL1-SE nuclear body is involved in miRNA production. Co-expression of SmD3 or SmB proteins revealed the localization of DCL1-HYL1-SE complexes in the SmD3/SmB nuclear bodies.
  • Yoichiro Fujioka, Maki Utsumi, Yusuke Ohba, Yuichiro Watanabe
    PLANT AND CELL PHYSIOLOGY 48 9 1243 - 1253 2007年09月 [査読有り][通常論文]
     
    There has been much recent research on the contribution of microRNA (miRNA) in plant organogenesis and hormone action. In plants, it has been reported that Dicer-like 1 (DCL1), HYPONASTIC LEAVES1 (HYL1). and SERRATE (SE) are involved in the production of miRNAs. The means by which miRNAs are processed and transported is. not well understood in detail, however. In this study, we investigated the intracellular localization and intermolecular interaction of these molecules using imaging techniques, including bimolecular fluorescence complementation and fluorescence resonance energy transfer techniques, making use of various enhanced fluorescent proteins. We found that DCL1, HYL1 and SE formed bodies which localized in the nuclei. We were also able to locate the miRNA primary transcript using an MS2-tagged method on these bodies. It appears very likely that the observed DCL1-HYL1-SE nuclear body is involved in miRNA production. Co-expression of SmD3 or SmB proteins revealed the localization of DCL1-HYL1-SE complexes in the SmD3/SmB nuclear bodies.
  • Akinori Takaoka, ZhiChao Wang, Myoung Kwon Choi, Hideyuki Yanai, Hideo Negishi, Tatsuma Ban, Yan Lu, Makoto Miyagishi, Tatsuhiko Kodama, Kenya Honda, Yusuke Ohba, Tadatsugu Taniguchi
    NATURE 448 7152 501 - U14 2007年07月 [査読無し][通常論文]
     
    Central to innate immunity is the sensing of pathogen-associated molecular patterns by cytosolic and membrane-associated receptors(1-4). In particular, DNA is a potent activator of immune responses during infection or tissue damage(5-7), and evidence indicates that, in addition to the membrane-associated Toll-like receptor 9, an unidentified cytosolic DNA sensor(s) can activate type I interferon (IFN) and other immune responses(8-10). Here we report on a candidate DNA sensor, previously named DLM-1 ( also called Z-DNA binding protein 1 (ZBP1))(11), for which biological function had remained unknown; we now propose the alternative name DAI (DNA-dependent activator of IFN-regulatory factors(12)). The artificial expression of otherwise IFN-inducible DAI (DLM-1/ ZBP1) in mouse fibroblasts selectively enhances the DNA-mediated induction of type I IFN and other genes involved in innate immunity. On the other hand, RNA interference of messenger RNA for DAI (DLM-1/ ZBP1) in cells inhibits this gene induction programme upon stimulation by DNA from various sources. Moreover, DAI (DLM-1/ZBP1) binds to double-stranded DNA and, by doing so, enhances its association with the IRF3 transcription factor and the TBK1 serine/threonine kinase. These observations underscore an integral role of DAI (DLM-1/ZBP1) in the DNA-mediated activation of innate immune responses, and may offer new insight into the signalling mechanisms underlying DNA- associated antimicrobial immunity and autoimmune disorders.
  • Yusuke Ohba, Yukiko Kanao, Nobuyoshi Morita, Eri Fujii, Mai Hohrai, Mayuko Takatsuji, Hideki Hirose, Daisaku Miura, Akihiro Watari, Masuo Yutsudo, Hanjun Zhao, Norikazu Yabuta, Akihiko Ito, Yasuyuki Kita, Hiroshi Nojima
    International Journal of Cancer 121 1 47 - 54 2007年07月01日 [査読有り][通常論文]
     
    We previously reported that overexpressing connexin 26 (Cx26) enhances the spontaneous metastasis of mouse BL6 melanoma cells. In contrast, daily intraperitoneal injections of an oleamide derivative named MI-18 potently inhibits the spontaneous metastasis of BL6 cells. In the present study, we chemically synthesized a novel oleamide derivative named MI-22 and found that it also efficiently suppressed the spontaneous metastasis of BL6 cells. Both MI-18 and MI-22 inhibited the gap junction-mediated intercellular communications (GJIC) that are formed between HeLa cells by the ectopic expression of the hCx26 and hCx32 human connexin subtypes however, they had no effect on GJIC mediated by hCx40, hCx43 or hCx45. Fluorescently labeled MI-18 primarily localized not only at plasma membrane but also at Golgi/endosome. This suggests that this oleamide derivative may also act on the Cx26 molecules that accumulate in the Golgi/endosome because of their overexpression. Notably, neither derivative had a cytotoxic effect on HeLa cells when they were added into the tissue culture medium. Taken together, we propose that the MI-18 and MI-22 oleamide derivatives may serve as prototypes for novel and clinically important anticancer drugs. © 2007 Wiley-Liss, Inc.
  • Yusuke Ohba, Yukiko Kanao, Mayuko Takatsuji, Motoki Ito, Norikazu Yabuta, Hiroshi Nojima, Yasuyuki Kita
    Tetrahedron 63 18 3754 - 3761 2007年04月30日 [査読有り][通常論文]
     
    Oleamide is an interesting compound, which shows various pharmacological activities including the inhibitory effect of gap junction formation. Recently, the studies of the gap junction have been some of the hot topics in biology and its inhibitors have become more useful tools [Cravatt, B. F. Garcia, O. P. Siuzdak, G. Gilula, N. B. Henriksen, S. J. Boger, D. L. Lerner, R. A. Science 1995, 268, 1506-1509 Cravatt, B. F. Lerner, R. A. Boger, D. L. J. Am. Chem. Soc. 1996, 118, 580-590 Guan, X Cravatt, B. F. Ehring, G. R. Hall, J. E. Boger, D. L. Lerner, R. A. Gilula, N. B. J. Cell Biol. 1997, 139, 1785-1792 Boger, D. L. Patterson, J. E. Guan, X. Cravatt, B. F. Lerner, R. A. Gilula, N. B. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 4810-4815 Ito, A. Morita, N. Miura, D. Koma, Y. Kataoka, T. R. Yamasaki, H. Kitamura, Y. Kita, Y. Nojima, H. Carcinogenesis 2004, 25, 2015-2022]. However, many reports suggest that the functionalizations of oleamide to retain its biological activity were difficult [Boger, D. L. Patterson, J. E. Guan, X. Cravatt, B. F. Lerner, R. A. Gilula, N. B. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 4810-4815 Ito, A. Morita, N. Miura, D. Koma, Y. Kataoka, T. R. Yamasaki, H. Kitamura, Y. Kita, Y. Nojima, H. Carcinogenesis 2004, 25, 2015-2022]. The synthesis of the functionalized oleamide derivatives, whose biological activity is not blocked, has become attractive in both the biological and chemical fields. Herein, by linking the fluorophore to the oleamide by alkyl chains, we synthesized the fluorescently tagged oleamide whose biological feature is similar to that of oleamide. Moreover, we also synthesized other bioactive derivatives tagged by other groups such as the sugars and biotin via alkyl chain linkers. © 2007 Elsevier Ltd. All rights reserved.
  • Hiromichi Fujioka, Kenichi Murai, Ozora Kubo, Yusuke Ohba, Yasuyuki Kita
    Organic Letters 9 9 1687 - 1690 2007年04月26日 [査読有り][通常論文]
     
    The novel three-component reaction of aromatic aldehydes, ethylenediamine, and β-keto esters is described. In this reaction, β-keto esters react at the γ-position which is generally unreactive to produce the seven-membered ring compounds. Products have secondary amines and β-enamino esters, which can serve in further functionalizations to produce molecular diversity. © 2007 American Chemical Society.
  • Hiromichi Fujioka, Kenichi Murai, Ozora Kubo, Yusuke Ohba, Yasuyuki Kita
    Tetrahedron 63 3 638 - 643 2007年01月15日 [査読有り][通常論文]
     
    Imidazolines were prepared in one-pot operation from aldehydes and diamines through oxidation of aminal intermediates by NBS. This method could be applied to various aromatic and aliphatic aldehydes and N-nonsubstituted and N-monosubstituted 1,2-diamines. Furthermore, it was found that CH2Cl2 could be altered to TBME, a more environmentally friendly solvent, in the reaction using N-nonsubstituted 1,2-diamines. The reaction conditions were very mild and chemoselective. © 2006.
  • Hiromichi Fujioka, Hideki Hirose, Yusuke Ohba, Kenichi Murai, Kenji Nakahara, Yasuyuki Kita
    Tetrahedron 63 3 625 - 637 2007年01月15日 [査読有り][通常論文]
     
    Cerium ammonium nitrate (CAN) removed hydroxyethyl units from 2-hydroxyethyl ethers and 2-hydroxyethyl amines to produce alcohols and amines in good yields. Especially, removal of the 2-hydroxyethyl ethers from C2-symmetric diols, chiral 2,3-butanediol and chiral hydrobenzoin, was very useful for asymmetric syntheses using C2-symmetric diols. The reactions using dual abilities of CAN, i.e., the ability for removal of the 2-hydroxyethyl unit and the ability for acetal hydrolysis by a single electron transfer, were also achieved successfully. The reaction conditions were very mild and efficient, and many functional groups, which can be affected under normal conditions, were unaffected during the reaction. © 2006.
  • Takashi Fukano, Asako Sawano, Yusuke Ohba, Michiyuki Matsuda, Atsushi Miyawaki
    CELL STRUCTURE AND FUNCTION 32 1 9 - 15 2007年 [査読無し][通常論文]
     
    Although the consequences of Ras activation have been studied extensively in the context of oncogenesis, its regulation in physiological modes of signal transduction is not well understood. A fluorescent indicator, Raichu- Ras, was fused to the C- terminal hypervariable regions of H- Ras and K- Ras to create indicators for Ras activation within caveolae/ rafts ( Raichu- tH) and non- raft domains ( Raichu- tK) of the plasma membrane, respectively. Raichu- tH was also found abundantly in endomembranes. To monitor Ras activation with high spatial resolution, it is imperative to observe sectioned images of the signals. We have developed a wide-field fluorescence microscope equipped with a digital micromirror device ( DMD) to acquire optically sectioned images using fringe projection. This system provides reliable signals from fluorescence resonance energy transfer ( FRET) between cyan and yellow mutants of green fluorescent protein. We have used this system to demonstrate that, upon stimulation with growth factors, the two indicators are activated in spatially and temporally unique patterns.
  • Spatiotemporal Mobilization of Toll/IL-1 Receptor Domain-Containing Adaptor Molecule-1 in Response to dsRNA.
    J. Immunol. 179 10 6867 - 6872 2007年 [査読無し][通常論文]
  • Takashi Fukano, Asako Sawano, Yusuke Ohba, Michiyuki Matsuda, Atsushi Miyawaki
    CELL STRUCTURE AND FUNCTION 32 1 9 - 15 2007年 [査読有り][通常論文]
     
    Although the consequences of Ras activation have been studied extensively in the context of oncogenesis, its regulation in physiological modes of signal transduction is not well understood. A fluorescent indicator, Raichu- Ras, was fused to the C- terminal hypervariable regions of H- Ras and K- Ras to create indicators for Ras activation within caveolae/ rafts ( Raichu- tH) and non- raft domains ( Raichu- tK) of the plasma membrane, respectively. Raichu- tH was also found abundantly in endomembranes. To monitor Ras activation with high spatial resolution, it is imperative to observe sectioned images of the signals. We have developed a wide-field fluorescence microscope equipped with a digital micromirror device ( DMD) to acquire optically sectioned images using fringe projection. This system provides reliable signals from fluorescence resonance energy transfer ( FRET) between cyan and yellow mutants of green fluorescent protein. We have used this system to demonstrate that, upon stimulation with growth factors, the two indicators are activated in spatially and temporally unique patterns.
  • Hideo Negishi, Yasuyuki Fujita, Hideyuki Yanai, Shinya Sakaguchi, Xinshou Ouyang, Masahiro Shinohara, Hiroshi Takayanagi, Yusuke Ohba, Tadatsugu Taniguchi, Kenya Honda
    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 103 41 15136 - 15141 2006年10月 [査読無し][通常論文]
     
    The recognition of microbial components by Toll-like receptors (TLRs) initiates signal transduction pathways, which trigger the expression of a series of target genes. It has been reported that TLR signaling is enhanced by cytokines such as IFN-gamma, but the mechanisms underlying this enhancement remain unclear. The MyD88 adaptor, which is essential for signaling by many TILRs, recruits members of the IFN regulatory factor (IRF) family of transcription factors, such as IRF5 and IRF7, to evoke the activation of TLR target genes. In this study we demonstrate that IRF1, which is induced by IFN-gamma, also interacts with and is activated by MyD88 upon TLR activation. We provide evidence that MyD88-associated IRF1 migrates into the nucleus more efficiently than non-MyD88-associated IRF1 and that this IRF1 selectively participates in the TLR-dependent gene induction program. The critical role of MyID88-clepenclent "IRF1 licensing" is underscored by the observation that the induction of a specific gene subset downstream of the TLR-MyD88 pathway, such as IFN-beta, inducible NO synthase, and IL-12p35, are impaired in Irf1-deficient cells. Thus, our present study places IRF1 as an additional member participating in MyD88 signaling and provides a mechanistic insight into the enhancement of the TILR-dependent gene induction program by IFN-gamma.
  • Tsukasa Shibue, Saori Suzuki, Hideaki Okamoto, Hiroki Yoshida, Yusuke Ohba, Akinori Takaoka, Tadatsugu Taniguchi
    EMBO JOURNAL 25 20 4952 - 4962 2006年10月 [査読無し][通常論文]
     
    The activation of tumor suppressor p53 induces apoptosis or cell cycle arrest depending on the state and type of cell, but it is not fully understood how these different responses are regulated. Here, we show that Puma and Noxa, the well- known p53-inducible proapoptotic members of the Bcl-2 family, differentially participate in dual pathways of the induction of apoptosis. In normal cells, Puma but not Noxa induces mitochondrial outer membrane permeabilization (MOMP), and this function is mediated in part by a pathway that involves calcium release from the endoplasmic reticulum (ER) and the subsequent caspase activation. However, upon E1A oncoprotein expression, cells also become susceptible to MOMP induction by Noxa, owing to their sensitization to the ER-independent pathway. These findings offer a new insight into differential cellular responses induced by p53, and may have therapeutic implications in cancer.
  • Hideo Negishi, Yasuyuki Fujita, Hideyuki Yanai, Shinya Sakaguchi, Xinshou Ouyang, Masahiro Shinohara, Hiroshi Takayanagi, Yusuke Ohba, Tadatsugu Taniguchi, Kenya Honda
    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 103 41 15136 - 15141 2006年10月 [査読有り][通常論文]
     
    The recognition of microbial components by Toll-like receptors (TLRs) initiates signal transduction pathways, which trigger the expression of a series of target genes. It has been reported that TLR signaling is enhanced by cytokines such as IFN-gamma, but the mechanisms underlying this enhancement remain unclear. The MyD88 adaptor, which is essential for signaling by many TILRs, recruits members of the IFN regulatory factor (IRF) family of transcription factors, such as IRF5 and IRF7, to evoke the activation of TLR target genes. In this study we demonstrate that IRF1, which is induced by IFN-gamma, also interacts with and is activated by MyD88 upon TLR activation. We provide evidence that MyD88-associated IRF1 migrates into the nucleus more efficiently than non-MyD88-associated IRF1 and that this IRF1 selectively participates in the TLR-dependent gene induction program. The critical role of MyID88-clepenclent "IRF1 licensing" is underscored by the observation that the induction of a specific gene subset downstream of the TLR-MyD88 pathway, such as IFN-beta, inducible NO synthase, and IL-12p35, are impaired in Irf1-deficient cells. Thus, our present study places IRF1 as an additional member participating in MyD88 signaling and provides a mechanistic insight into the enhancement of the TILR-dependent gene induction program by IFN-gamma.
  • Tsukasa Shibue, Saori Suzuki, Hideaki Okamoto, Hiroki Yoshida, Yusuke Ohba, Akinori Takaoka, Tadatsugu Taniguchi
    EMBO JOURNAL 25 20 4952 - 4962 2006年10月 [査読有り][通常論文]
     
    The activation of tumor suppressor p53 induces apoptosis or cell cycle arrest depending on the state and type of cell, but it is not fully understood how these different responses are regulated. Here, we show that Puma and Noxa, the well- known p53-inducible proapoptotic members of the Bcl-2 family, differentially participate in dual pathways of the induction of apoptosis. In normal cells, Puma but not Noxa induces mitochondrial outer membrane permeabilization (MOMP), and this function is mediated in part by a pathway that involves calcium release from the endoplasmic reticulum (ER) and the subsequent caspase activation. However, upon E1A oncoprotein expression, cells also become susceptible to MOMP induction by Noxa, owing to their sensitization to the ER-independent pathway. These findings offer a new insight into differential cellular responses induced by p53, and may have therapeutic implications in cancer.
  • Hiromichi Fujioka, Kenichi Murai, Yusuke Ohba, Hideki Hirose, Yasuyuki Kita
    Chemical Communications 8 832 - 834 2006年 [査読有り][通常論文]
     
    The reaction of cyclohexa-2,5-dienyl-1-methylaldehyde and optically pure 1,2-diaryl-1,2-diamine followed by intramolecular bromo-amination produced a one-pot discrimination of two olefins in the cyclohexane system, which was used for the asymmetric synthesis of (-)-γ-lycorane. © The Royal Society of Chemistry 2006.
  • H Negishi, Y Ohba, H Yanai, A Takaoka, K Honma, K Yui, T Matsuyama, T Taniguchi, K Honda
    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 102 44 15989 - 15994 2005年11月 [査読有り][通常論文]
     
    The recognition of microbial components by Toll-like receptors (TLRs) is an event central to the activation of innate and adaptive immune systems. TLR activation triggers the induction of downstream target genes, wherein the TLR-interacting adaptor molecule MyD88 recruits various signaling molecules and transcription factors. Two members of the IFN regulatory factor (IRF) family of transcription factors, IRF-5 and IRF-7, interact with MyD88 and induce proinflammatory cytokines and type I IFNs, respectively. Here, we show that IRF-4 also interacts with MyD88 and acts as a negative regulator of TLR signaling. IRF-4 mRNA is induced by TLR activation, and IRF-4 competes with IRF-5, but not with IRF-7, for MyD88 interaction. The TLR-dependent induction of proinflammatory cytokines is markedly enhanced in peritoneal macrophages from mice deficient in the Irf4 gene, whereas the induction is inhibited by the ectopic expression of IRF-4 in a macrophage cell line. The critical function of IRF-4 in TLR signaling in vivo is underscored by the observation that Irf4-deficient mice show hypersensitivity to DNA-induced shock, with elevated serum proinflammatory cytokine levels. This study may provide an insight into the complex regulatory mechanisms of MyD88 signaling by IRFs.
  • Hiromichi Fujioka, Naoyuki Kotoku, Yoshinari Sawama, Hidetoshi Kitagawa, Yusuke Ohba, Tsung-Lung Wang, Yasushi Nagatomi, Yasuyuki Kita
    Chemical and Pharmaceutical Bulletin 53 8 952 - 957 2005年08月 [査読有り][通常論文]
     
    A novel asymmetric synthesis of the cyclohexane derivative functionalized by some substituents has been developed from the diene acetals (1), prepared from the corresponding diene aldehyde and (+)-hydrobenzoin. The treatment of 1 with NBS in the presence of MeOCH2CH2OH predominantly afforded 2 in a stereoselective manner. Subsequent alkylation of the methoxyethoxy group produced the optically active cyclohexene compounds (3) in good yields. The stereoselective chemical modification of the remaining olefin in 3 was made by OsO4-oxidation. © 2005 Pharmaceutical Society of Japan.
  • Hiromichi Fujioka, Yusuke Ohba, Hideki Hirose, Kenichi Murai, Yasuyuki Kita
    Organic Letters 7 15 3303 - 3306 2005年07月21日 [査読有り][通常論文]
     
    (Chemical Equation Presented) CAN is a good reagent for the transformation of 2-hydroxyethyl ether units to alcohols. Significantly, many functional groups can tolerate the reaction conditions, although they do not survive under many previously reported removal conditions. The reaction mechanism is clarified. © 2005 American Chemical Society.
  • K Honda, H Yanai, H Negishi, M Asagiri, M Sato, T Mizutani, N Shimada, Y Ohba, A Takaoka, N Yoshida, T Taniguchi
    NATURE 434 7034 772 - 777 2005年04月 [査読無し][通常論文]
     
    The type-I interferon (IFN-alpha/beta) response is critical to immunity against viruses and can be triggered in many cell types by cytosolic detection of viral infection, or in differentiated plasmacytoid dendritic cells by the Toll-like receptor 9 (TLR9) subfamily, which generates signals via the adaptor MyD88 to elicit robust IFN induction(1-4). Using mice deficient in the Irf7 gene (Irf7(-/-) mice), we show that the transcription factor IRF-7 is essential for the induction of IFN-alpha/beta genes via the virus-activated, MyD88-independent pathway and the TLR-activated, MyD88-dependent pathway. Viral induction of MyD88-independent IFN-alpha/beta genes is severely impaired in Irf7(-/-) fibroblasts. Consistently, Irf7(-/-) mice are more vulnerable than Myd88(-/-) mice to viral infection, and this correlates with a marked decrease in serum IFN levels, indicating the importance of the IRF-7-dependent induction of systemic IFN responses for innate antiviral immunity. Furthermore, robust induction of IFN production by activation of the TLR9 subfamily in plasmacytoid dendritic cells is entirely dependent on IRF-7, and this MyD88-IRF-7 pathway governs the induction of CD8(+) T-cell responses. Thus, all elements of IFN responses, whether the systemic production of IFN in innate immunity or the local action of IFN from plasmacytoid dendritic cells in adaptive immunity, are under the control of IRF-7.
  • K Honda, Y Ohba, H Yanai, H Negishi, T Mizutani, A Takaoka, C Taya, T Taniguchi
    NATURE 434 7036 1035 - 1040 2005年04月 [査読無し][通常論文]
     
    Robust type-I interferon (IFN-alpha/beta) induction in plasmacytoid dendritic cells, through the activation of Toll-like receptor 9 (TLR9), constitutes a critical aspect of immunity(1-6). It is absolutely dependent on the transcription factor IRF-7, which interacts with and is activated by the adaptor MyD88 (ref. 7). How plasmacytoid dendritic cells, but not other cell types (such as conventional dendritic cells), are able to activate the MyD88 IRF-7-dependent IFN induction pathway remains unknown. Here we show that the spatiotemporal regulation of MyD88 IRF-7 signalling is critical for a high-level IFN induction in response to TLR9 activation. The IFN-inducing TLR9 ligand, A/D-type CpG oligodeoxynucleotide (CpG-A)(3,4,8-11), is retained for long periods in the endosomal vesicles of plasmacytoid dendritic cells, together with the MyD88-IRF-7 complex. However, in conventional dendritic cells, CpG-A is rapidly transferred to lysosomal vesicles. We further show that conventional dendritic cells can also mount a robust IFN induction if CpG-A is manipulated for endosomal retention using a cationic lipid. This strategy also allows us to demonstrate endosomal activation of the IFN pathway by the otherwise inactive TLR9 ligand B/K-type oligodeoxynucleotide (CpG-B)(3,4,8-12). Thus, our study offers insights into the regulation of TLR9 signalling in space, potentially suggesting a new avenue for therapeutic intervention.
  • K Honda, H Yanai, H Negishi, M Asagiri, M Sato, T Mizutani, N Shimada, Y Ohba, A Takaoka, N Yoshida, T Taniguchi
    NATURE 434 7034 772 - 777 2005年04月 [査読有り][通常論文]
     
    The type-I interferon (IFN-alpha/beta) response is critical to immunity against viruses and can be triggered in many cell types by cytosolic detection of viral infection, or in differentiated plasmacytoid dendritic cells by the Toll-like receptor 9 (TLR9) subfamily, which generates signals via the adaptor MyD88 to elicit robust IFN induction(1-4). Using mice deficient in the Irf7 gene (Irf7(-/-) mice), we show that the transcription factor IRF-7 is essential for the induction of IFN-alpha/beta genes via the virus-activated, MyD88-independent pathway and the TLR-activated, MyD88-dependent pathway. Viral induction of MyD88-independent IFN-alpha/beta genes is severely impaired in Irf7(-/-) fibroblasts. Consistently, Irf7(-/-) mice are more vulnerable than Myd88(-/-) mice to viral infection, and this correlates with a marked decrease in serum IFN levels, indicating the importance of the IRF-7-dependent induction of systemic IFN responses for innate antiviral immunity. Furthermore, robust induction of IFN production by activation of the TLR9 subfamily in plasmacytoid dendritic cells is entirely dependent on IRF-7, and this MyD88-IRF-7 pathway governs the induction of CD8(+) T-cell responses. Thus, all elements of IFN responses, whether the systemic production of IFN in innate immunity or the local action of IFN from plasmacytoid dendritic cells in adaptive immunity, are under the control of IRF-7.
  • K Honda, Y Ohba, H Yanai, H Negishi, T Mizutani, A Takaoka, C Taya, T Taniguchi
    NATURE 434 7036 1035 - 1040 2005年04月 [査読有り][通常論文]
     
    Robust type-I interferon (IFN-alpha/beta) induction in plasmacytoid dendritic cells, through the activation of Toll-like receptor 9 (TLR9), constitutes a critical aspect of immunity(1-6). It is absolutely dependent on the transcription factor IRF-7, which interacts with and is activated by the adaptor MyD88 (ref. 7). How plasmacytoid dendritic cells, but not other cell types (such as conventional dendritic cells), are able to activate the MyD88 IRF-7-dependent IFN induction pathway remains unknown. Here we show that the spatiotemporal regulation of MyD88 IRF-7 signalling is critical for a high-level IFN induction in response to TLR9 activation. The IFN-inducing TLR9 ligand, A/D-type CpG oligodeoxynucleotide (CpG-A)(3,4,8-11), is retained for long periods in the endosomal vesicles of plasmacytoid dendritic cells, together with the MyD88-IRF-7 complex. However, in conventional dendritic cells, CpG-A is rapidly transferred to lysosomal vesicles. We further show that conventional dendritic cells can also mount a robust IFN induction if CpG-A is manipulated for endosomal retention using a cationic lipid. This strategy also allows us to demonstrate endosomal activation of the IFN pathway by the otherwise inactive TLR9 ligand B/K-type oligodeoxynucleotide (CpG-B)(3,4,8-12). Thus, our study offers insights into the regulation of TLR9 signalling in space, potentially suggesting a new avenue for therapeutic intervention.
  • Hiromichi Fujioka, Kenichi Murai, Yusuke Ohba, Atsushi Hiramatsu, Yasuyuki Kita
    Tetrahedron Letters 46 13 2197 - 2199 2005年03月28日 [査読有り][通常論文]
     
    The reactions of various aldehydes and 1,2-diamines followed by NXS treatment proceed at 0°C-rt to give the corresponding dihydroimidazoles in high yields. The reaction is mild, and many functional groups such as halogens, nitriles, and esters can exist. © 2005 Elsevier Ltd. All rights reserved.
  • A Takaoka, H Yanai, S Kondo, G Duncan, H Negishi, T Mizutani, S Kano, K Honda, Y Ohba, TW Mak, T Taniguchi
    NATURE 434 7030 243 - 249 2005年03月 [査読無し][通常論文]
     
    The activation of Toll-like receptors (TLRs) is central to innate and adaptive immunity(1-3). All TLRs use the adaptor MyD88 for signalling(4), but the mechanisms underlying the MyD88-mediated gene induction programme are as yet not fully understood. Here, we demonstrate that the transcription factor IRF-5 is generally involved downstream of the TLR-MyD88 signalling pathway for gene induction of proinflammatory cytokines, such as interleukin-6 (IL-6), IL-12 and tumour-necrosis factor-alpha. In haematopoietic cells from mice deficient in the Irf5 gene (Irf5(-/-) mice), the induction of these cytokines by various TLR ligands is severely impaired, whereas interferon-alpha induction is normal. We also provide evidence that IRF-5 interacts with and is activated by MyD88 and TRAF6, and that TLR activation results in the nuclear translocation of IRF-5 to activate cytokine gene transcription. Consistently, Irf5(-/-) mice show resistance to lethal shock induced by either unmethylated DNA or lipopolysaccharide, which correlates with a marked decrease in the serum levels of proinflammatory cytokines. Thus, our study identifies IRF-5 as a new, principal downstream regulator of the TLR-MyD88 signalling pathway and a potential target of therapeutic intervention to control harmful immune responses.
  • A Takaoka, H Yanai, S Kondo, G Duncan, H Negishi, T Mizutani, S Kano, K Honda, Y Ohba, TW Mak, T Taniguchi
    NATURE 434 7030 243 - 249 2005年03月 [査読有り][通常論文]
     
    The activation of Toll-like receptors (TLRs) is central to innate and adaptive immunity(1-3). All TLRs use the adaptor MyD88 for signalling(4), but the mechanisms underlying the MyD88-mediated gene induction programme are as yet not fully understood. Here, we demonstrate that the transcription factor IRF-5 is generally involved downstream of the TLR-MyD88 signalling pathway for gene induction of proinflammatory cytokines, such as interleukin-6 (IL-6), IL-12 and tumour-necrosis factor-alpha. In haematopoietic cells from mice deficient in the Irf5 gene (Irf5(-/-) mice), the induction of these cytokines by various TLR ligands is severely impaired, whereas interferon-alpha induction is normal. We also provide evidence that IRF-5 interacts with and is activated by MyD88 and TRAF6, and that TLR activation results in the nuclear translocation of IRF-5 to activate cytokine gene transcription. Consistently, Irf5(-/-) mice show resistance to lethal shock induced by either unmethylated DNA or lipopolysaccharide, which correlates with a marked decrease in the serum levels of proinflammatory cytokines. Thus, our study identifies IRF-5 as a new, principal downstream regulator of the TLR-MyD88 signalling pathway and a potential target of therapeutic intervention to control harmful immune responses.
  • Hiromichi Fujioka, Yusuke Ohba, Hideki Hirose, Kenichi Murai, Yasuyuki Kita
    Angewandte Chemie - International Edition 44 5 734 - 737 2005年01月21日 [査読有り][通常論文]
     
    Tetrahydrofuran units with multiple chiral centers are formed in a highly diastereoselective manner through the one-pot double intramolecular haloetherification of σ-symmetric diene acetals (see scheme). The utility of the method was demonstrated by the short asymmetric synthesis of (+)-rubrenolide.
  • NEGISHI Hideo, OHBA Yusuke, YANAI Hideyuki, TAKAOKA Akinori, HONMA Kiri, YUI Katsuyuki, MATSUYAMA Toshifumi, TANIGUCHI Tadatsugu, HONDA Kenya
    Natl. Acad. Sci. U.S.A. 102 44 15989 - 15994 2005年 [査読無し][通常論文]
  • Hiromichi Fujioka, Tetsuya Fujita, Naoyuki Kotoku, Yusuke Ohba, Yasushi Nagatomi, Atsushi Hiramatsu, Yasuyuki Kita
    Chemistry - A European Journal 10 21 5386 - 5397 2004年10月25日 [査読有り][通常論文]
     
    A new chiral auxiliary, a 3-endo-phenyl norbornene aldehyde derivative, which is a crystalline, very stable, and easily handled, was developed for the desymmetrization of meso-1,3- and meso-1,4-diols. The key step of the method, an intramolecular bromoetherification, proceeded in a highly diastereoselective manner. A four-step sequence, 1) acetalization, 2) intramolecular bromoetherification followed by acid hydrolysis, 3) protection of the alcohol, and 4) retrobromoetherification, transformed the meso-diols into optically active derivatives. The 3-endo-phenyl norbornene aldehyde derivative was simultaneously reformed and could be used repeatedly. This is the first chemical example of a single auxiliary that is applicable for highly enantioselective desymmetrization of meso-1,3- and meso-1,4-diols to the best of our knowledge, this is the best chemical method available for the desymmetrization of meso-1,4-diols.
  • K Honda, H Yanai, T Mizutani, H Negishi, N Shimada, N Suzuki, Y Ohba, A Takaoka, WC Yeh, T Taniguchi
    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 101 43 15416 - 15421 2004年10月 [査読無し][通常論文]
     
    Toll-like receptor (TLR) activation is central to immunity, wherein the activation of the TLR9 subfamily members TLR9 and TLR7 results in the robust induction of type I IFNs (IFN-alpha/beta) by means of the MyD88 adaptor protein. However, it remains unknown how the TLR signal "input" can be processed through MyD88 to "output" the induction of the IFN genes. Here, we demonstrate that the transcription factor IRF-7 interacts with MyD88 to form a complex in the cytoplasm. We provide evidence that this complex also involves IRAK4 and TRAF6 and provides the foundation for the TLR9-dependent activation of the IFN genes. The complex defined in this study represents an example of how the coupling of the signaling adaptor and effector kinase molecules together with the transcription factor regulate the processing of an extracellular signal to evoke its versatile downstream transcriptional events in a cell. Thus, we propose that this molecular complex may function as a cytoplasmic transductional-transcriptional processor.
  • H Yoshizaki, Y Ohba, MC Parrini, NG Dulyaninova, AR Bresnick, N Mochizuki, M Matsuda
    JOURNAL OF BIOLOGICAL CHEMISTRY 279 43 44756 - 44762 2004年10月 [査読無し][通常論文]
     
    Rho family GTPases play pivotal roles in cytokinesis. By using probes based on the principle of fluorescence resonance energy transfer (FRET), we have shown that in HeLa cells RhoA activity increases with the progression of cytokinesis. Here we show that in Rat1A cells RhoA activity remained suppressed during most of the cytokinesis. Consistent with this observation, the expression of C3 toxin inhibited cytokinesis in HeLa cells but not in Rat1A cells. Furthermore, the expression of a dominant negative mutant of Ect2, a Rho GEF, or Y-27632, an inhibitor of the Rho-dependent kinase ROCK, inhibited cytokinesis in HeLa cells but not in Rat1A cells. In contrast to the activity of RhoA, the activity of Rac1 was suppressed during cytokinesis and started increasing at the plasma membrane of polar sides before the abscission of the daughter cells in both HeLa and Rat1A cells. This type of Rac1 suppression was shown to be essential for cytokinesis because a constitutively active mutant of Rac1 induced a multinucleated phenotype in both HeLa and Rat1A cells. Moreover, the involvement of MgcRacGAP/CYK-4 in this suppression of Rac1 during cytokinesis was shown by the use of a dominant negative mutant. Because ML-7, an inhibitor of myosin light chain kinase, delayed the cytokinesis of Rat1A cells and because Pak, a Rac1 effector, is known to suppress myosin light chain kinase, the suppression of the Rac1-Pak pathway by MgcRacGAP may play a pivotal role in the cytokinesis of Rat1A cells.
  • H Yoshizaki, Y Ohba, MC Parrini, NG Dulyaninova, AR Bresnick, N Mochizuki, M Matsuda
    JOURNAL OF BIOLOGICAL CHEMISTRY 279 43 44756 - 44762 2004年10月 [査読有り][通常論文]
     
    Rho family GTPases play pivotal roles in cytokinesis. By using probes based on the principle of fluorescence resonance energy transfer (FRET), we have shown that in HeLa cells RhoA activity increases with the progression of cytokinesis. Here we show that in Rat1A cells RhoA activity remained suppressed during most of the cytokinesis. Consistent with this observation, the expression of C3 toxin inhibited cytokinesis in HeLa cells but not in Rat1A cells. Furthermore, the expression of a dominant negative mutant of Ect2, a Rho GEF, or Y-27632, an inhibitor of the Rho-dependent kinase ROCK, inhibited cytokinesis in HeLa cells but not in Rat1A cells. In contrast to the activity of RhoA, the activity of Rac1 was suppressed during cytokinesis and started increasing at the plasma membrane of polar sides before the abscission of the daughter cells in both HeLa and Rat1A cells. This type of Rac1 suppression was shown to be essential for cytokinesis because a constitutively active mutant of Rac1 induced a multinucleated phenotype in both HeLa and Rat1A cells. Moreover, the involvement of MgcRacGAP/CYK-4 in this suppression of Rac1 during cytokinesis was shown by the use of a dominant negative mutant. Because ML-7, an inhibitor of myosin light chain kinase, delayed the cytokinesis of Rat1A cells and because Pak, a Rac1 effector, is known to suppress myosin light chain kinase, the suppression of the Rac1-Pak pathway by MgcRacGAP may play a pivotal role in the cytokinesis of Rat1A cells.
  • K Honda, H Yanai, T Mizutani, H Negishi, N Shimada, N Suzuki, Y Ohba, A Takaoka, WC Yeh, T Taniguchi
    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 101 43 15416 - 15421 2004年10月 [査読有り][通常論文]
     
    Toll-like receptor (TLR) activation is central to immunity, wherein the activation of the TLR9 subfamily members TLR9 and TLR7 results in the robust induction of type I IFNs (IFN-alpha/beta) by means of the MyD88 adaptor protein. However, it remains unknown how the TLR signal "input" can be processed through MyD88 to "output" the induction of the IFN genes. Here, we demonstrate that the transcription factor IRF-7 interacts with MyD88 to form a complex in the cytoplasm. We provide evidence that this complex also involves IRAK4 and TRAF6 and provides the foundation for the TLR9-dependent activation of the IFN genes. The complex defined in this study represents an example of how the coupling of the signaling adaptor and effector kinase molecules together with the transcription factor regulate the processing of an extracellular signal to evoke its versatile downstream transcriptional events in a cell. Thus, we propose that this molecular complex may function as a cytoplasmic transductional-transcriptional processor.
  • C Miyagi, S Yamashita, Y Ohba, H Yoshizaki, M Matsuda, T Hirano
    JOURNAL OF CELL BIOLOGY 166 7 975 - 981 2004年09月 [査読無し][通常論文]
     
    Zebrafish signal transducer and activator of transcription 3 (STAT3) controls the cell movements during gastrulation. Here, we show that noncell-autonomous activity of STAT3 signaling in gastrula organizer cells controls the polarity of neighboring cells through Dishevelled-RhoA signaling in the Wnt-planar cell polarity (WntPCP) pathway. In STAT3-depleted embryos, although all the known molecules in the Wnt-PCP pathway were expressed normally, the RhoA activity in lateral mesendodermal cells was down-regulated, resulting in severe cell polarization defects in convergence and extension movements identical to Strabismus-depleted embryos. Cell-autonomous activation of Wnt-PCP signaling by DeltaN-dishevelled rescued the defect in cell elongation, but not the orientation of lateral mesendodermal cells in STAT3-depleted embryos. The defect in the orientation could be rescued by transplantation of shield cells having noncellautonomous activity of STAT3 signaling. These results suggest that the cells undergoing convergence and extension movement may sense the gradient of signaling molecules, which are expressed in gastrula organizer by STAT3 and noncell-autonomously activate PCP signaling in neighboring cells during zebrafish gastrulation.
  • C Miyagi, S Yamashita, Y Ohba, H Yoshizaki, M Matsuda, T Hirano
    JOURNAL OF CELL BIOLOGY 166 7 975 - 981 2004年09月 [査読有り][通常論文]
     
    Zebrafish signal transducer and activator of transcription 3 (STAT3) controls the cell movements during gastrulation. Here, we show that noncell-autonomous activity of STAT3 signaling in gastrula organizer cells controls the polarity of neighboring cells through Dishevelled-RhoA signaling in the Wnt-planar cell polarity (WntPCP) pathway. In STAT3-depleted embryos, although all the known molecules in the Wnt-PCP pathway were expressed normally, the RhoA activity in lateral mesendodermal cells was down-regulated, resulting in severe cell polarization defects in convergence and extension movements identical to Strabismus-depleted embryos. Cell-autonomous activation of Wnt-PCP signaling by DeltaN-dishevelled rescued the defect in cell elongation, but not the orientation of lateral mesendodermal cells in STAT3-depleted embryos. The defect in the orientation could be rescued by transplantation of shield cells having noncellautonomous activity of STAT3 signaling. These results suggest that the cells undergoing convergence and extension movement may sense the gradient of signaling molecules, which are expressed in gastrula organizer by STAT3 and noncell-autonomously activate PCP signaling in neighboring cells during zebrafish gastrulation.
  • A Takaya, Y Ohba, K Kurokawa, M Matsuda
    MOLECULAR BIOLOGY OF THE CELL 15 6 2549 - 2557 2004年06月 [査読無し][通常論文]
     
    Ra1A, a member of the Ras-family GTPases, regulates various cellular functions such as filopodia formation, endocytosis, and exocytosis. On epidermal growth factor (EGF) stimulation, activated Ras recruits guanine nucleotide exchange factors (GEFs) for RalA, followed by RalA activation. By using fluorescence resonance energy transfer-based probes for RalA activity, we found that the EGF-induced RalA activation in Cos7 cells was restricted at the EGF-induced nascent lamellipodia, whereas under a similar condition both Ras activation and Ras-dependent translocation of Ral GEFs occurred more diffusely at the plasma membrane. This EGF-induced RalA activation was not observed when lamellipodial protrusion was suppressed by a dominant negative mutant of Rac1, a GTPase-activating protein for Cdc42, inhibitors of phosphatidylinositol 3-kinase, or inhibitors of actin polymerization. On the other hand, EGF-induced lamellipodial protrusion was inhibited by microinjection of the RalA-binding domains of Ra1BP1 and Sec5. Furthermore, we found that RalA activity was high at the lamellipodia of migrating Madin-Darby canine kidney cells and that the migration of Madin-Darby canine kidney cells was perturbed by the microinjection of RalBP1-RalA-binding domain. Thus, RalA activation is required for the induction of lamellipodia, and conversely, lamellipodial protrusion seems to be required for the RalA activation, suggesting the presence of a positive feedback loop between RalA activation and lamellipodial protrusion. Our observation also demonstrates that the spatial regulation of RalA is conducted by a mechanism distinct from the temporal regulation conducted by Ras-dependent plasma membrane recruitment of Ral guanine nucleotide exchange factors.
  • A Takaya, Y Ohba, K Kurokawa, M Matsuda
    MOLECULAR BIOLOGY OF THE CELL 15 6 2549 - 2557 2004年06月 [査読有り][通常論文]
     
    Ra1A, a member of the Ras-family GTPases, regulates various cellular functions such as filopodia formation, endocytosis, and exocytosis. On epidermal growth factor (EGF) stimulation, activated Ras recruits guanine nucleotide exchange factors (GEFs) for RalA, followed by RalA activation. By using fluorescence resonance energy transfer-based probes for RalA activity, we found that the EGF-induced RalA activation in Cos7 cells was restricted at the EGF-induced nascent lamellipodia, whereas under a similar condition both Ras activation and Ras-dependent translocation of Ral GEFs occurred more diffusely at the plasma membrane. This EGF-induced RalA activation was not observed when lamellipodial protrusion was suppressed by a dominant negative mutant of Rac1, a GTPase-activating protein for Cdc42, inhibitors of phosphatidylinositol 3-kinase, or inhibitors of actin polymerization. On the other hand, EGF-induced lamellipodial protrusion was inhibited by microinjection of the RalA-binding domains of Ra1BP1 and Sec5. Furthermore, we found that RalA activity was high at the lamellipodia of migrating Madin-Darby canine kidney cells and that the migration of Madin-Darby canine kidney cells was perturbed by the microinjection of RalBP1-RalA-binding domain. Thus, RalA activation is required for the induction of lamellipodia, and conversely, lamellipodial protrusion seems to be required for the RalA activation, suggesting the presence of a positive feedback loop between RalA activation and lamellipodial protrusion. Our observation also demonstrates that the spatial regulation of RalA is conducted by a mechanism distinct from the temporal regulation conducted by Ras-dependent plasma membrane recruitment of Ral guanine nucleotide exchange factors.
  • K Kurokawa, RE Itoh, H Yoshizaki, Y Ohba, T Nakamura, M Matsuda
    MOLECULAR BIOLOGY OF THE CELL 15 3 1003 - 1010 2004年03月 [査読無し][通常論文]
     
    A major function of Rho-family GTPases is to regulate the organization of the actin cytoskeleton; filopodia, lamellipodia, and stress fiber are regarded as typical phenotypes of the activated Cdc42, Rac, and Rho, respectively. Using probes based on fluorescent resonance energy transfer, we report on the spatiotemporal regulation of Rac1 and Cdc42 at lamellipodia and membrane ruffles. In epidermal growth factor (EGF)-stimulated Cos1 and A431 cells, both Rac1 and Cdc42 were activated diffusely at the plasma membrane, followed by lamellipodial protrusion and membrane ruffling. Although Rac1 activity subsided rapidly, Cdc42 activity was sustained at lamellipodia. A critical role of Cdc42 in these EGF-induced morphological changes was demonstrated as follows. First, phorbol 12-myristate 13-acetate, which activated Rac1 but not Cdc42, could not induce full-grown lamellipodia in Cos1 cells. Second, a GTPase-activating protein for Cdc42, KIAA1204/ CdGAP, inhibited lamellipodial protrusion and membrane ruffling without interfering with Rac1 activation. Third, expression of the Cdc42-binding domain of N-WASP inhibited the EGF-induced morphological changes. Therefore, Rac1 and Cdc42 seem to synergistically induce lamellipodia and membrane ruffles in EGF-stimulated Cos1 cells and A431 cells.
  • Y Ohba, M Matsuda
    SEIKAGAKU 76 1 16 - 28 2004年01月 [査読有り][通常論文]
  • Yasuyuki Kita, Junko Futamura, Yusuke Ohba, Yoshinari Sawama, Jnaneshwara K. Ganesh, Hiromichi Fujioka
    Journal of Organic Chemistry 68 15 5917 - 5924 2003年07月25日 [査読有り][通常論文]
     
    The Lewis acid treatment of 2,3-epoxysulfonates with 2,3-dialkyl substituents or 2-alkyl-3-aryl substituents produced the rearrangement products via C3-cleavage of the oxirane ring in high yields. On the other hand, 2-aryl-3-alkyl-2,3-epoxysulfonates produced the products via C2-cleavage of the oxirane ring. The sulfonyloxy groups of the α-sulfonyloxy ketones, having a chiral benzylic quaternary carbon center obtained by the rearrangement of 2-alkyl-3-aryl-2,3-epoxysulfonates, were reductively eliminated to give the ketones with a chiral benzylic quaternary carbon center. The method was applied to the formal synthesis of (-)-aphanorphine and total syntheses of (-)-α-herbertenol and (-)-herbertenediol.
  • H Yoshizaki, Y Ohba, K Kurokawa, RE Itoh, T Nakamura, N Mochizuki, K Nagashima, M Matsuda
    JOURNAL OF CELL BIOLOGY 162 2 223 - 232 2003年07月 [査読無し][通常論文]
     
    Rho-family GTPases regulate many cellular functions. To visualize the activity of Rho-family GTPases in living cells, we developed fluorescence resonance energy transfer (FRET)-based probes for Rac1 and Cdc42 previously (Itoh, R.E., K. Kurokawa, Y. Ohba, H. Yoshizaki, N. Mochizuki, and M. Matsuda. 2002. Mol. Cell. Biol. 22:6582-6591). Here, we added two types of probes for RhoA. One is to monitor the activity balance between guanine nucleotide exchange factors and GTPase-activating proteins, and another is to monitor the level of GTP-RhoA. Using these FRET probes, we imaged the activities of Rho-family GTPases during the cell division of HeLa cells. The activities of RhoA, Rac1, and Cdc42 were high at the plasma membrane in interphase, and decreased rapidly on entry into M phase. From after anaphase, the RhoA activity increased at the plasma membrane including cleavage furrow. Rac1 activity was suppressed at the spindle midzone and increased at the plasma membrane of polar sides after telophase. Cdc42 activity was suppressed at the plasma membrane and was high at the intracellular membrane compartments during cytokinesis. In conclusion, we could use the FRET-based probes to visualize the complex spatio-temporal regulation of Rho-family GTPases during cell division.
  • H Yoshizaki, Y Ohba, K Kurokawa, RE Itoh, T Nakamura, N Mochizuki, K Nagashima, M Matsuda
    JOURNAL OF CELL BIOLOGY 162 2 223 - 232 2003年07月 [査読有り][通常論文]
     
    Rho-family GTPases regulate many cellular functions. To visualize the activity of Rho-family GTPases in living cells, we developed fluorescence resonance energy transfer (FRET)-based probes for Rac1 and Cdc42 previously (Itoh, R.E., K. Kurokawa, Y. Ohba, H. Yoshizaki, N. Mochizuki, and M. Matsuda. 2002. Mol. Cell. Biol. 22:6582-6591). Here, we added two types of probes for RhoA. One is to monitor the activity balance between guanine nucleotide exchange factors and GTPase-activating proteins, and another is to monitor the level of GTP-RhoA. Using these FRET probes, we imaged the activities of Rho-family GTPases during the cell division of HeLa cells. The activities of RhoA, Rac1, and Cdc42 were high at the plasma membrane in interphase, and decreased rapidly on entry into M phase. From after anaphase, the RhoA activity increased at the plasma membrane including cleavage furrow. Rac1 activity was suppressed at the spindle midzone and increased at the plasma membrane of polar sides after telophase. Cdc42 activity was suppressed at the plasma membrane and was high at the intracellular membrane compartments during cytokinesis. In conclusion, we could use the FRET-based probes to visualize the complex spatio-temporal regulation of Rho-family GTPases during cell division.
  • Y Ohba, K Kurokawa, M Matsuda
    EMBO JOURNAL 22 4 859 - 869 2003年02月 [査読無し][通常論文]
     
    Epidermal growth factor (EGF) activates Ras and Rap1 at distinct intracellular regions. Here, we explored the mechanism underlying this phenomenon. We originally noticed that in cells expressing Epac, a cAMP-dependent Rap1 GEF (guanine nucleotide exchange factor), cAMP activated Rap1 at the perinuclear region, as did EGF. However, in cells expressing e-GRF, a recombinant cAMP-responsive Ras GEF, cAMP activated Ras at the peripheral plasma membrane. Based on the uniform cytoplasmic expression of Epac and e-GRF, GEF did not appear to account for the non-uniform increase in the activities of Ras and Rap1. In contrast, when we used probes with reduced sensitivity to GTPase-activating proteins (GAPs), both Ras and Rap1 appeared to be activated uniformly in the EGF-stimulated cells. Furthermore, we calculated the local rate constants of GEFs and GAPs from the video images of Ras activation and found that GAP activity was higher at the central plasma membrane than the periphery. Thus we propose that GAP primarily dictates the spatial regulation of Ras family G proteins, whereas GEF primarily determines the timing of Ras activation.
  • Y Ohba, K Kurokawa, M Matsuda
    EMBO JOURNAL 22 4 859 - 869 2003年02月 [査読有り][通常論文]
     
    Epidermal growth factor (EGF) activates Ras and Rap1 at distinct intracellular regions. Here, we explored the mechanism underlying this phenomenon. We originally noticed that in cells expressing Epac, a cAMP-dependent Rap1 GEF (guanine nucleotide exchange factor), cAMP activated Rap1 at the perinuclear region, as did EGF. However, in cells expressing e-GRF, a recombinant cAMP-responsive Ras GEF, cAMP activated Ras at the peripheral plasma membrane. Based on the uniform cytoplasmic expression of Epac and e-GRF, GEF did not appear to account for the non-uniform increase in the activities of Ras and Rap1. In contrast, when we used probes with reduced sensitivity to GTPase-activating proteins (GAPs), both Ras and Rap1 appeared to be activated uniformly in the EGF-stimulated cells. Furthermore, we calculated the local rate constants of GEFs and GAPs from the video images of Ras activation and found that GAP activity was higher at the central plasma membrane than the periphery. Thus we propose that GAP primarily dictates the spatial regulation of Ras family G proteins, whereas GEF primarily determines the timing of Ras activation.
  • Yasuyuki Kita, Junko Futamura, Yusuke Ohba, Yoshinari Sawama, Jnaneshuwara K. Ganesh, Hiromichi Fujioka
    Tetrahedron Letters 44 2 411 - 413 2003年01月06日 [査読有り][通常論文]
     
    The rearrangement of the optically active 3-aryl-2-methyl-2,3-epoxy tosylate (> 98% ee) afforded the α-keto tosylate with a chiral quaternary carbon center and without loss of chirality. Reductive removal of the tosyloxy group gave the keto compound with a chiral quaternary carbon center, which was converted to (-)-herbertenediol (> 98% ee). © 2002 Elsevier Science Ltd. All rights reserved.
  • A Sakakibara, Y Ohba, K Kurokawa, M Matsuda, S Hattori
    JOURNAL OF CELL SCIENCE 115 24 4915 - 4924 2002年12月 [査読無し][通常論文]
     
    Chat (Cas/HEF1-associated signal transducer) is a novel signaling molecule with an N-terminal SH2 domain and C-terminal Cas/HEF1 association domain that is implicated in the regulation of cell adhesion. The Cas/HEF1 association domain also shows sequence similarity with guanine nucleotide exchange factors for Ras family small GTPases. In this study, we found significant activation of Rap1 in Chat-overexpressing cells. Myr-Chat, a membrane-targeted form of Chat, activated Rap1 more efficiently. Interestingly, Chat and Cas synergistically activated Rap1. Certain Cas, Crk or C3G mutants suppressed Rap I activation by Chat. We also confirmed the ternary complex formation consisting of Chat, Cas and Crk. Thus, it is likely that Chat-induced Rap1 activation was mediated by upregulation of the Cas-Crk-C3G signaling pathway rather than direct guanine nucleotide exchange factor activity of Chat. We further demonstrated that Myr-Chat expression induced cell periphery spreading and cell shape branching and that this activity also depended on the Cas-Crk-C3G pathway and Rap1 activity. Moreover, expression of Myr-Chat enhanced integrin-mediated cell adhesion. Taken together we propose a novel role for the Chat-Cas complex in controlling cell adhesion via the activation of Rap1.
  • A Sakakibara, Y Ohba, K Kurokawa, M Matsuda, S Hattori
    JOURNAL OF CELL SCIENCE 115 24 4915 - 4924 2002年12月 [査読有り][通常論文]
     
    Chat (Cas/HEF1-associated signal transducer) is a novel signaling molecule with an N-terminal SH2 domain and C-terminal Cas/HEF1 association domain that is implicated in the regulation of cell adhesion. The Cas/HEF1 association domain also shows sequence similarity with guanine nucleotide exchange factors for Ras family small GTPases. In this study, we found significant activation of Rap1 in Chat-overexpressing cells. Myr-Chat, a membrane-targeted form of Chat, activated Rap1 more efficiently. Interestingly, Chat and Cas synergistically activated Rap1. Certain Cas, Crk or C3G mutants suppressed Rap I activation by Chat. We also confirmed the ternary complex formation consisting of Chat, Cas and Crk. Thus, it is likely that Chat-induced Rap1 activation was mediated by upregulation of the Cas-Crk-C3G signaling pathway rather than direct guanine nucleotide exchange factor activity of Chat. We further demonstrated that Myr-Chat expression induced cell periphery spreading and cell shape branching and that this activity also depended on the Cas-Crk-C3G pathway and Rap1 activity. Moreover, expression of Myr-Chat enhanced integrin-mediated cell adhesion. Taken together we propose a novel role for the Chat-Cas complex in controlling cell adhesion via the activation of Rap1.
  • RE Itoh, K Kurokawa, Y Ohba, H Yoshizaki, N Mochizuki, M Matsuda
    MOLECULAR AND CELLULAR BIOLOGY 22 18 6582 - 6591 2002年09月 [査読無し][通常論文]
     
    Rho family G proteins, including Rac and Cdc42, regulate a variety of cellular functions such as morphology, motility, and gene expression. We developed fluorescent resonance energy transfer-based probes which monitored the local balance between the activities of guanine nucleotide exchange factors and GTPase-activating proteins for Rac1 and Cdc42 at the membrane. These probes, named Raichu-Rac and Raichu-Cdc42, consisted of a Cdc42- and Rac-binding domain of Pak, Rac1 or Cdc42, a pair of green fluorescent protein mutants, and a CAAX box of Ki-Ras. With these probes, we video imaged the Rac and Cdc42 activities. In motile HT1080 cells, activities of both Rac and Cdc42 gradually increased toward the leading edge and decreased rapidly when cells changed direction. Under a higher magnification, we observed that Rac activity was highest immediately behind the leading edge, whereas Cdc42 activity was most prominent at the tip of the leading edge. Raichu-Rac and Raichu-Cdc42 were also applied to a rapid and simple assay for the analysis of putative guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs) in living cells. Among six putative GEFs and GAPS, we identified KIAA0362/DBS as a GEF for Rac and Cdc42, KIAA1256 as a GEF for Cdc42, KIAA0053 as a GAP for Rac and Cdc42, and KIAA1204 as a GAP for Cdc42. In conclusion, use of these single-molecule probes to determine Rac and Cdc42 activity will accelerate the analysis of the spatiotemporal regulation of Rac and Cdc42 in a living cell.
  • RE Itoh, K Kurokawa, Y Ohba, H Yoshizaki, N Mochizuki, M Matsuda
    MOLECULAR AND CELLULAR BIOLOGY 22 18 6582 - 6591 2002年09月 [査読有り][通常論文]
     
    Rho family G proteins, including Rac and Cdc42, regulate a variety of cellular functions such as morphology, motility, and gene expression. We developed fluorescent resonance energy transfer-based probes which monitored the local balance between the activities of guanine nucleotide exchange factors and GTPase-activating proteins for Rac1 and Cdc42 at the membrane. These probes, named Raichu-Rac and Raichu-Cdc42, consisted of a Cdc42- and Rac-binding domain of Pak, Rac1 or Cdc42, a pair of green fluorescent protein mutants, and a CAAX box of Ki-Ras. With these probes, we video imaged the Rac and Cdc42 activities. In motile HT1080 cells, activities of both Rac and Cdc42 gradually increased toward the leading edge and decreased rapidly when cells changed direction. Under a higher magnification, we observed that Rac activity was highest immediately behind the leading edge, whereas Cdc42 activity was most prominent at the tip of the leading edge. Raichu-Rac and Raichu-Cdc42 were also applied to a rapid and simple assay for the analysis of putative guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs) in living cells. Among six putative GEFs and GAPS, we identified KIAA0362/DBS as a GEF for Rac and Cdc42, KIAA1256 as a GEF for Cdc42, KIAA0053 as a GAP for Rac and Cdc42, and KIAA1204 as a GAP for Cdc42. In conclusion, use of these single-molecule probes to determine Rac and Cdc42 activity will accelerate the analysis of the spatiotemporal regulation of Rac and Cdc42 in a living cell.
  • K Kurokawa, N Mochizuki, Y Ohba, H Mizuno, A Miyawaki, M Matsuda
    JOURNAL OF BIOLOGICAL CHEMISTRY 276 33 31305 - 31310 2001年08月 [査読有り][通常論文]
     
    An adaptor protein, CrkII, which is involved in a variety of signaling cascades such as cell growth, migration, and apoptosis, becomes phosphorylated on Tyr.. upon stimulation. Here, we report on a fluorescent resonance energy transfer-based sensor, which consists of CrkII sandwiched with cyan- and yellow-emitting variants of green fluorescent protein. This protein enabled us to monitor rapid and transient phosphorylation of CrkII upon epidermal growth factor stimulation in a living cell. However, rapid diffusion of the probes prevented us from specifying where the phosphorylation started within the cell. To overcome this problem, we fused the CAAX box of Ki-Ras to the carboxyl terminus of this probe and restricted its localization mostly to the plasma membrane. With this modified probe, we found that epidermal growth factor-induced phosphorylation of CrkII was initiated at the peripheral plasma membrane, moving toward the center of the cell. Moreover, this CAAX box-fused probe showed improvement in sensitivity and time resolution of the monitoring of CrkII phosphorylation. Thus, this pair of CrkII probes visualizes dynamic changes in the total and local levels of the tyrosine phosphorylation of CrkII in a living cell.
  • Y Ohba, K Ikuta, A Ogura, J Matsuda, N Mochizuki, K Nagashima, K Kurokawa, BJ Mayer, K Maki, J Miyazaki, M Matsuda
    EMBO JOURNAL 20 13 3333 - 3341 2001年07月 [査読無し][通常論文]
     
    C3G is a guanine nucleotide exchange factor (GEF) for Rap1, and is activated via Crk adaptor protein. To understand the physiological role of C3G, we generated C3G knockout mice. C3G(-/-) homozygous mice died before embryonic day 7.5. The lethality was rescued by the expression of the human C3G transgene, which could be excised upon the expression of Cre recombinase, From the embryo of this mouse, we prepared fibroblast cell lines, MEF-hC3G. Expression of Cre abolished the expression of C3G in MEF-hC3G and inhibited cell adhesion-induced activation of Rap1. The Cre-expressing MEF-hC3G showed impaired cell adhesion, delayed cell spreading and accelerated cell migration. The accelerated cell migration was suppressed by the expression of active Rap1, Rap2 and R-Ras, Expression of Epac and CalDAG-GEFI, GEFs for Rap1, also suppressed the accelerated migration of the CSG-deficient cells. This observation indicated that Rap1 activation was sufficient to complement the C3G deficiency. In conclusion, C3G-dependent activation of Rap1 is required for adhesion and spreading of embryonic fibroblasts and for the early embryogenesis of the mouse.
  • N Mochizuki, S Yamashita, K Kurokawa, Y Ohba, T Nagai, A Miyawaki, M Matsuda
    NATURE 411 6841 1065 - 1068 2001年06月 [査読無し][通常論文]
     
    G proteins of the Ras family function as molecular switches in many signalling cascades(1-3); however, little is known about where they become activated in living cells. Here we use FRET (fluorescent resonance energy transfer)-based sensors to report on the spatio-temporal images of growth-factor-induced activation of Ras and Rap1. Epidermal growth factor activated Ras at the peripheral plasma membrane and Rap1 at the intracellular perinuclear region of COS-1 cells. In PC12 cells, nerve growth factor-induced activation of Ras was initiated at the plasma membrane and transmitted to the whole cell body. After three hours, high Ras activity was observed at the extending neurites. By using the FRAP (fluorescence recovery after photobleaching) technique, we found that Ras at the neurites turned over rapidly; therefore, the sustained Ras activity at neurites was due to high GTP/GDP exchange rate and/or low GTPase activity, but not to the retention of the active Ras. These observations may resolve long-standing questions as to how Ras and Rap1 induce different cellular responses(4) and how the signals for differentiation and survival are distinguished by neuronal cells(5).
  • S Yamashita, N Mochizuki, Y Ohba, M Tobiume, Y Okada, H Sawa, K Nagashima, M Matsuda
    JOURNAL OF BIOLOGICAL CHEMISTRY 275 33 25488 - 25493 2000年08月 [査読無し][通常論文]
     
    We characterized a novel guanine nucleotide exchange factor (GEF) for Ras family G proteins that is highly homologous to CalDAG-GEFI, a GEF for Rap1 and R-Ras, and to RasGRP/CalDAG-GEFII, a GEF for Res and R-Ras. This novel GEF, referred to as CalDAG-GEFIII, increased the GTP/GDP ratio of Ha-Ras, R-Ras, and Rap1 in 293T cells. CalDAG-GEFIII promoted the guanine nucleotide exchange of Ha-Ras, R-Ras, and Rap1 in vitro also, indicating that CalDAG-GEFIII exhibited the widest substrate specificity among the known GEFs for Res family G proteins. Expression of CalDAG-GEFIII was detected in the glial cells of the brain and the glomerular mesangial cells of the kidney by in. situ hybridization. CalDAG-GEFIII activated ERW MAPK most efficiently, followed by CalDAG-GEFII and CalDAG-GEFI in 293T cells. JNK activation was most prominent in cells expressing CalDAG-GEFII, followed by CalDAG-GEFIII and CaLDAG-GEFI. Expression of CalDAG-GEFIII induced neuronal differentiation of PC12 cells and anchorage-independent growth of Rat1A cells less efficiently than did CalDAG-GEFII. Thus, coactivation of Rap1 by CalDAG-GEFIII apparently attenuated Ras-MAPK-dependent neuronal differentiation and cellular transformation. Altogether, CalDAG-GEFIII activated a broad range of Res family Gr proteins and exhibited a biological activity different from that of either CalDAG-GEFI or CalDAG-GEFII.
  • Y Ohba, N Mochizuki, K Matsuo, S Yamashita, M Nakaya, Y Hashimoto, M Hamaguchi, T Kurata, K Nagashima, M Matsuda
    MOLECULAR AND CELLULAR BIOLOGY 20 16 6074 - 6083 2000年08月 [査読無し][通常論文]
     
    Rap2 is a member of the Ras family of GTPases and exhibits 60% identity to Rap1, but the function and regulation of Rap2 remain obscure. We found that, unlike the other Ras family proteins, the GTP-bound active form exceeded 50% of total Rap2 protein in adherent cells. Guanine nucleotide exchange factors (GEFs) for Rap1, C3G, Epac (or cyclic AMP [cAMP]-GEF), CalDAG-GEFI, PDZ-GEF1, and GFR efficiently increased the level of GTP-Rap2 both in 293T cells and in vitro. GTPase-activating proteins (GAPs) for Rap1, rap1GAPII and SPA-1, stimulated Rap2 GTPase, but with low efficiency. The half-life of GTP-Rap2 was significantly longer than that of GTP-Rap1 in 293T cells, indicating that low sensitivity to GAPs caused a high GTP/GDP ratio on Rap2. Rap2 bound to the Ras-binding domain of Raf and inhibited Ras dependent activation of Elk1 transcription factor, as did Rap1. The level of GTP-Rap2 in rat 3Y1 fibroblasts was decreased by the expression of v-Src, and expression of a GTPase-deficient Rap2 mutant inhibited v-Src-dependent transformation of 3Y1 cells. Altogether, Rap2 is regulated by a similar set of GEFs and GAPs as Rap1 and functions as a slowly responding molecular switch in the Rap1 signaling cascade.
  • Y Ohba, N Mochizuki, S Yamashita, AM Chan, JW Schrader, S Hattori, K Nagashima, M Matsuda
    JOURNAL OF BIOLOGICAL CHEMISTRY 275 26 20020 - 20026 2000年06月 [査読無し][通常論文]
     
    We studied the regulation of three closely related members of Pas family G proteins, R-Ras, TC21 (also known as R-Ras2), and M-Ras (R-Ras3). Guanine nucleotide exchange of R-Ras and TC21 was promoted by RasGRF, C3G, CalDAG-GEFI, CalDAG-GEFII (RasGRP), and CalDAG-GEFIII both in 293T cells and in vitro. By contrast, guanine nucleotide exchange of M-Ras was promoted by the guanine nucleotide exchange factors (GEFs) for the classical Pas (Ha-, K-, and N-), including mSos, RasGRF, CalDAG-GEFII, and CalDAG-GEFIII. GTPase-activating proteins (GAPs) for Pas, Gap1(m), p120 GAP, and NF-1 stimulated all of the R-Ras, TC21, and M-Ras proteins, whereas R-Ras GAP stimulated R-Ras and TC21 but not M-Ras. We did not find any remarkable difference in the subcellular localization of R-Ras, TC21, or M-Ras when these were expressed with a green fluorescent protein tag in 293T cells and MDCK cells. In conclusion, TC21 and R-Ras were regulated by the same GEFs and GAPs, whereas M-Ras was regulated as the classical Ras.
  • N Mochizuki, Y Ohba, S Kobayashi, N Otsuka, AM Graybiel, S Tanaka, M Matsuda
    JOURNAL OF BIOLOGICAL CHEMISTRY 275 17 12667 - 12671 2000年04月 [査読無し][通常論文]
     
    v-crk is an oncogene identified originally in CT10 chicken tumor virus. C3G, a guanine nucleotide exchange factor (GEF) for Rap1 and R-Ras, is postulated to transduce the oncogenic signal of v-Crk to c-Jun kinase (JNK). We have found that R-Ras, but not Rap1, mediates JNK activation by v-Crk in 293T and NIH 3T3 cells. Constitutively activated R-Ras, R-Ras(Val-38), but not Rap1(Val-12) activated JNK, as did the constitutively active H-Ras(Val-12) or Bac1(Val-12). v-Crk activation of JNK was inhibited by a dominant-negative mutant of R-Ras, R-Ras(Asn-43). JNK activation by R-Ras(Val-38) was inhibited by a dominant-negative mutant of mixed lineage kinase 3. Among six GEFs for Res-family G proteins, mSos1, Ras-GRF, C3G, CalDAG-GEFI, Ras-GRP/CalDAG-GEFII, and Epac/cAMP-GEFI, GEFs for either H-Ras or R-Ras activated JNK and c-Jun-dependent transcription. CalDAG-GEFI and Epac/cAMP-GEFI, both of which are GEFs specific for Rap1, did not activate JNK or c-Jun-dependent transcription. These results demonstrate that R-Ras, but not Rap1, is the downstream effector of C3G to stimulate JNK, Finally, we found that expression of the dominant-negative R-Ras mutant induced flat reversion of NIH 3T3 cells transformed by v-Crk, suggesting that R-Ras-dependent JNK activation is critical for the transformation by v-Crk.
  • N Kawano, Y Ohba, K Nagashima
    ACTA NEUROPATHOLOGICA 99 2 214 - 218 2000年02月 [査読無し][通常論文]
     
    A study was undertaken to determine the pathological significance of previously unrecognized intracytoplasmic eosinophilic inclusions (IEIs) in ependymoma. The study group consisted of 58 ependymomas, all of which were pathologically characterized and graded according to the 1993 WHO classification. Electron microscopic studies were performed in 16 cases. The study showed that 33 (57%) ependymomas had IEIs and that in 8 cases these were abundant. Round and eosinophilic, their sizes varied from 10 mu m to a tiny dot. Similar eosinophilic bodies were also observed between tumor cells. The inclusions were weakly PAS positive. On immunostains, IEIs were frequently positive for glial fibrillary acidic protein, less often for S-100 protein, and for epithelial membrane protein and CAM 5.2. They were negative for AE1/AE3, carcinoembryonic antigen and Ber-EP4. Ultrastructurally, IEIs represented intracytoplasmic lumens containing microvilli and cilia. These microlumina also frequently contained granule-tubular materials. With reference to tumor subtypes, IEIs occurred most frequently in ordinary and clear cell ependymomas. IEIs were also present in 4 of 6 anaplastic ependymomas studied. In conclusion, IEIs represent microlumina and occur in more than a half of ependymomas including malignant examples. Their finding is a helpful diagnostic feature of ependymoma.
  • A pair of fluorescent resonance energy transfer-based probes for tyrosine phosphorylation of the CrkII adaptor protein in vivo.
    J. Biol. Chem. 276 33 6074 - 6083 2000年 [査読無し][通常論文]
  • MOCHIZUKI N, OHBA Y, KIYOKAWA E, KURATA T, MURAKAMI T, OZAKI T, KITABATAKE A, NAGASHIMA K, MATSUDA M
    Nature 400 6747 891 - 894 1999年08月 [査読無し][通常論文]
     
    Many receptors for neuropeptides and hormones are coupled with the heterotrimeric G(i) protein, which activates the p42/44 mitogen-activated protein kinase (ERK/MAPE;) cascade through both the alpha- and beta gamma-subunits of G(i) (refs 1-3). The beta gamma-subunit activates the ERK/MAPK cascade through tyrosine kinase(4-6). Constitutively active G alpha(i2) (gip2) isolated from adrenal and ovarian tumours(7,8) transforms Rat-1 fibroblasts and also activates the ERK/MAPK cascade by an unknown mechanism(9,10). The ERK/MAPK pathway is activated by Ras, and is inhibited when the low-molecular-mass GTP-binding protein Rap1 antagonizes pas function(11). Here we show that a novel isoform of Rap1 GTPase-activating protein, called rap1GAPII, binds specifically to the alpha-subunits of the G(i) family of heterotrimeric G-proteins. Stimulation of the G(i)-coupled m2-muscarinic receptor translocates rap1GAPII from the cytosol to the membrane and decreases the amount of GTP-bound Rap1. This decrease in GTP-bound Rap1 activates ERK/MAPK. Thus, the alpha-subunit of G(i) activates the Ras-ERK/MAPK mitogenic pathway by membrane recruitment of rap1GAPII and reduction of GTP-bound Rap1.
  • Yusuke Ohba, Hiroaki Suzuki, Hiroaki Hiraga, Tomoo Ito, Hirofumi Sawa, Makoto Nagai, Shin-Ichi Satoh, Hiroyuki Iwaki, Kazuo Nagashima
    Pathology International 49 7 653 - 657 1999年 [査読無し][通常論文]
     
    Peritoneal sarcomatosis was found in a 53-year-old male who had a history of resection of clear cell sarcoma (CCS) of the right wrist 7 years previously. Both the previous wrist tumor and the peritoneal disseminants consisted of small, spindle-shaped cells occasionally containing melanophages. Histologic features, histochemical demonstration of argentaffin granules, immunohistochemical reaction with HMB 45, and the demonstration of a chimeric transcript of EWS-ATF-1 established the diagnosis of CCS in the peritoneal tumors. As far as we are aware, this is the first case of a peritoneal sarcomatosis associated with CCS.

MISC

  • 藤岡容一朗, 田端和仁, 田端和仁, 大場雄介, 野地博行, 野地博行 実験医学 35 (19) 3197‐3203 2017年12月01日 [査読無し][通常論文]
  • 大場雄介, 山田珠希, 山田珠希, 山田珠希, 津田真寿美, 藤岡容一朗, 藤岡真理, 堀内浩水, 堀口美香, 佐藤絢, ネパール ブラバ, 王せい, 柏木彩花, 西出真也, 南保明日香, 芳賀永, 田中伸哉, 進藤正信 日本生理学雑誌(Web) 79 (2) 49 (WEB ONLY) -49 2017年05月 [査読無し][通常論文]
  • インフルエンザウイルス細胞侵入において鍵となる宿主タンパク質の同定
    藤岡 容一朗, 西出 真也, 尾瀬 農之, 加藤 いづみ, 福原 秀雄, 藤岡 真理, 堀内 浩水, 佐藤 絢, Nepal Prabha, 柏木 彩花, Wang Jing, 堀口 美香, Paudel Sarad, 南保 明日香, 宮崎 忠昭, 前仲 勝実, 大場 雄介 日本細胞生物学会大会講演要旨集 69回 61 -61 2017年05月 [査読無し][通常論文]
  • 藤岡容一朗, 西出真也, 尾瀬農之, 加藤いづみ, 福原秀雄, 藤岡真理, 堀内浩水, 佐藤絢, NEPAL Prabha, 柏木彩花, WANG Jing, 堀口美香, PAUDEL Sarad, 南保明日香, 宮崎忠昭, 前仲勝実, 前仲勝実, 大場雄介 日本細胞生物学会大会(Web) 69th ROMBUNNO.T8‐04(P2‐028) (WEB ONLY) -61 2017年05月 [査読無し][通常論文]
  • Yusuke Ohba, Yoichiro Fujioka Journal of Oral Biosciences 58 (4) 113 -119 2016年11月01日 [査読無し][通常論文]
     
    Background Fluorescent proteins have continued to shed light on cell biology since the cDNA of wild type green fluorescent protein was first isolated. Nowadays, these remarkable proteins are useful tools, not only in basic research, but also in clinical medicine. Highlight By taking advantage of fluorescent protein-based technologies, we identified a signaling network critical for influenza virus internalization and infection. In addition, we developed a highly sensitive biosensor for monitoring kinase activity that utilizes energy transfer between fluorescent proteins. This has led to a high-performance clinical test that enables the prediction of future therapeutic responses and the risk of acquired drug resistance for each individual patient before beginning molecular target therapy. Conclusion Technologies that utilize fluorescent proteins, such as the biosensor presented here, should find increasing applications in clinical medicine.
  • CRKアダプター蛋白質はHGF/c-Metフィードバックループを介して膀胱癌のEMTと転移を誘導する
    王 磊, 松本 隆児, 津田 真寿美, 間石 奈湖, 安部 崇重, 木村 太一, 谷野 美智枝, 西原 広史, 樋田 京子, 大場 雄介, 篠原 信雄, 田中 伸哉 日本癌学会総会記事 74回 J -1142 2015年10月 [査読無し][通常論文]
  • N. Maishi, Y. Ohba, K. Akiyama, N. Ohga, J. I. Hamada, M. T. Alam, M. Shindoh, Y. Hida, K. Hida EUROPEAN JOURNAL OF CANCER 51 S20 -S20 2015年09月 [査読無し][通常論文]
  • Masayuki Noguchi, Futoshi Suizu, Mami Lennikov-Matsuda, Bala Jyoti, Yusuke Ohba, Noriyuki Hirata MOLECULAR CANCER THERAPEUTICS 14 (7) 2015年07月 [査読無し][通常論文]
  • T. Kondo, M. Ibata, J. Iwasaki, Y. Fujioka, K. Nakagawa, S. Darmanin, M. Onozawa, D. Hashimoto, Y. Ohba, S. Hatakeyama, T. Teshima HAEMATOLOGICA 100 327 -327 2015年06月 [査読無し][通常論文]
  • インフルエンザウイルスのCa2+シグナルを介した宿主細胞侵入機構
    藤岡 容一朗, 津田 真寿美, 南保 明日香, 服部 ともえ, 佐々木 純子, 佐々木 雄彦, 宮崎 忠昭, 大場 雄介 日本細胞生物学会大会講演要旨集 66回 103 -103 2014年05月 [査読無し][通常論文]
  • インフルエンザウイルスのCa2+シグナルを介した宿主細胞侵入機構
    藤岡 容一朗, 津田 真寿美, 南保 明日香, 服部 ともえ, 佐々木 純子, 佐々木 雄彦, 宮崎 忠昭, 大場 雄介 日本細胞生物学会大会講演要旨集 66回 146 -146 2014年05月 [査読無し][通常論文]
  • 間石奈湖, 大場雄介, 大賀則孝, 大賀則孝, 秋山廣輔, 浜田淳一, 山本和幸, 川本泰輔, 進藤正信, 樋田泰浩, 樋田京子 日本病理学会会誌 103 (1) 209 2014年03月26日 [査読無し][通常論文]
  • Ras-PI3Kシグナルが制御する外来因子取込み機構の解析
    藤岡 容一朗, 津田 真寿美, 服部 ともえ, 佐々木 純子, 佐々木 雄彦, 宮崎 忠昭, 大場 雄介 日本生理学雑誌 76 (2) 81 -81 2014年03月 [査読無し][通常論文]
  • 間石奈湖, 大場雄介, 樋田泰浩, 浜田淳一, 山本和幸, 大賀則孝, 秋山廣輔, 川本泰輔, 大澤崇宏, 近藤美弥子, 大村瞳, 井上農夫男, 進藤正信, 樋田京子 日本口腔科学会雑誌 63 (1) 135 2014年01月10日 [査読無し][通常論文]
  • Ras-PI3Kシグナルが制御する外来因子取込み機構の解析
    藤岡 容一朗, 津田 真寿美, 服部 ともえ, 佐々木 純子, 佐々木 雄彦, 大場 雄介 日本細胞生物学会大会講演要旨集 65回 153 -153 2013年05月 [査読無し][通常論文]
  • 間石奈湖, 大場雄介, 山本和幸, 大賀則孝, 浜田淳一, 秋山廣輔, 川本泰輔, 大澤崇宏, 大村瞳, 樋田泰浩, 進藤正信, 樋田京子 日本病理学会会誌 102 (1) 437 2013年04月26日 [査読無し][通常論文]
  • 新井 隆太, 渡部 琢哉, 三浪 明男, 津田 真寿美, 大場 雄介 北海道整形災害外科学会 54 (2) 250 2013年03月 [査読無し][通常論文]
  • 間石奈湖, 大場雄介, 大賀則孝, 秋山廣輔, 山本和幸, 浜田淳一, 川本泰輔, ALAM Mohammad T, 樋田泰浩, 樋田京子 日本血管生物医学会学術集会プログラム・抄録集 21st 134 2013年 [査読無し][通常論文]
  • 樋田京子, 大賀則孝, 秋山廣輔, 間石奈湖, 川本泰輔, 大場雄介, 樋田泰浩 がん研究分野の特性等を踏まえた支援活動公開シンポジウムプログラム・抄録集 平成24年度 32 2013年 [査読無し][通常論文]
  • 間石奈湖, 大場雄介, 大賀則孝, 秋山廣輔, 山本和幸, 浜田淳一, 川本泰輔, ALAM MOHAMMAD T, 大村瞳, 樋田泰浩, 樋田京子 日本がん転移学会学術集会・総会プログラム抄録集 22nd 79 2013年 [査読無し][通常論文]
  • 新井 隆太, 渡部 琢哉, 津田 真寿美, 大場 雄介, 三浪 明男 日本整形外科学会雑誌 86 (8) S1101 2012年08月 [査読無し][通常論文]
  • 白鳥 聡一, 早川 清雄, 大和 弘明, 亀山 武志, 北辻 千展, 樫木 芙美, 後藤 翔平, 亀岡 章一郎, 藤倉 大輔, 山田 大翔, 水谷 龍明, 数馬田 美香, 佐藤 麻衣子, 田中 淳司, 浅香 正博, 大場 雄介, 宮崎 忠昭, 今村 雅寛, 高岡 晃教 北海道醫學雜誌 = Acta medica Hokkaidonensia 87 (4) 2012年08月01日 [査読無し][通常論文]
  • 我妻孝則, 津田真寿美, 山田珠希, 藤岡容一朗, 芳賀永, 戸塚靖則, 進藤正信, 大場雄介 日本分子生物学会年会プログラム・要旨集(Web) 35th 3P-0363 (WEB ONLY) 2012年 [査読無し][通常論文]
  • 間石奈湖, 大場雄介, 山本和幸, 大賀則孝, 浜田淳一, 秋山廣輔, 川本泰輔, 大澤崇宏, 近藤美弥子, 大村瞳, 樋田泰浩, 樋田京子 日本がん転移学会学術集会・総会プログラム抄録集 21st 104 2012年 [査読無し][通常論文]
  • DJ-1はシナプス小胞に局在する
    波田野 琢, 宇佐見 由希子, 久保 紳一郎, 大場 雄介, 有賀 寛芳, 斉木 臣二, 佐藤 栄人, 服部 信孝 Dementia Japan 25 (3) 424 -424 2011年10月 [査読無し][通常論文]
  • SrcおよびAuroraキナーゼ2重阻害による滑膜肉腫の相乗的in vivo抗腫瘍効果(Dual inhibition of Src and Aurora kinases abrogates tumor growth, invasion, and angiogenesis of synovial sarcoma in vivo)
    津田 真寿美, 新井 隆太, 渡部 琢哉, 尾瀬 農之, 小布施 力史, 前仲 勝実, 三浪 明男, 大場 雄介 日本癌学会総会記事 70回 274 -274 2011年09月 [査読無し][通常論文]
  • 慢性骨髄白血病に対する分子標的治療薬の反応性・抵抗性判定試験(Seeing response and resistance to BCR-ABL inhibition in chronic myeloid leukemia)
    大場 雄介, 金安 顕子, 水谷 龍明, ダルマニン・ステファニー, 近藤 健, 津田 真寿美 日本癌学会総会記事 70回 242 -242 2011年09月 [査読無し][通常論文]
  • RANKLは口腔癌細胞のインテグリンα2の発現と細胞接着を亢進する(RANKL upregulates integrin α2 expression and cell adhesion in oral cancer cells)
    大場 雄介, 山田 珠希, 藤岡 容一朗, 甲斐原 拓真, 戸塚 泰則, 進藤 正信, 津田 真寿美 日本細胞生物学会大会講演要旨集 63回 156 -156 2011年05月 [査読無し][通常論文]
  • 津田 真寿美, 新井 隆太, 大場 雄介 日本細胞生物学会大会要旨集 63rd 159 2011年05月 [査読無し][通常論文]
  • 染色体・核・遺伝子発現・シグナル伝達 インフルエンザウイルスは、カルシウムシグナル伝達によって、Ras-PI3K-仲介エンドサイトーシスを活性化する(Signal transduction/Chromosome/Cell nucleus/Gene expression Influenza viruses activate Ras-PI3K-mediated endocytosis via calcium signaling)
    藤岡 容一朗, 津田 真寿美, 服部 ともえ, 佐々木 純子, 佐々木 雄彦, 宮崎 忠昭, 大場 雄介 日本細胞生物学会大会講演要旨集 63回 112 -112 2011年05月 [査読無し][通常論文]
  • Nako Maishi, Noritaka Ohga, Yasuhiro Hida, Yusuke Ohba, Taisuke Kawamoto, Kosuke Akiyama, Kazuko Kitayama, Miyako Kondoh, Takahiro Osawa, Kazuyuki Yamamoto, Nobuo Inoue, Masanobu Shindoh, Kyoko Hida CANCER RESEARCH 71 2011年04月 [査読無し][通常論文]
  • 新井 隆太, 津田 真寿美, 渡部 琢哉, 尾瀬 農之, 小布施 力史, 前仲 勝実, 三浪 明男, 大場 雄介 日本分子生物学会年会プログラム・要旨集(Web) 34th WEB ONLY 2P-0690 2011年 [査読無し][通常論文]
  • 亀山武志, 早川清雄, 白鳥聡一, 大和弘明, 北辻千展, 樫木芙美, 後藤翔平, 亀岡章一郎, 藤倉大輔, 水谷龍明, 数馬田美香, 佐藤麻衣子, 今村雅寛, 浅香正博, 大場雄介, 宮崎忠昭, 高岡晃教 日本ウイルス学会学術集会プログラム・抄録集 58th 220 2010年10月15日 [査読無し][通常論文]
  • RANKL発現は腫瘍形成とEMTを亢進する(RANKL expression promotes tumorigenesis and epithelial mesenchymal transition)
    山田 珠希, 津田 真寿美, 戸塚 靖則, 進藤 正信, 大場 雄介 日本癌学会総会記事 69回 494 -494 2010年08月 [査読無し][通常論文]
  • 新井 隆太, 渡部 琢哉, 津田 真寿美, 川口 秀明, 大場 雄介, 三浪 明男 日本整形外科学会雑誌 84 (8) S1244 2010年08月 [査読無し][通常論文]
  • RANKLは微小環境特異的に発現し細胞接着・EMT・腫瘍形成を促進する生体内特異的機能マーカーである(RANKJL expression specifically observed in vivo promotes cell adhesion, epithelial mesenchymal transition, and tumor progression)
    山田 珠希, 津田 真寿美, 進藤 正信, 戸塚 泰則, 大場 雄介 日本細胞生物学会大会講演要旨集 62回 196 -196 2010年05月 [査読無し][通常論文]
  • メンブレントラフィック Ras-PI3Kシグナル経路がエンドサイトーシスとインフルエンザ感染を調節する(Membrane traffic The Ras-PI3K signaling pathway mediates endocytosis and the incorporation of influenza viruses)
    藤岡 容一朗, 津田 真寿美, 服部 ともえ, 佐々木 純子, 佐々木 雄彦, 宮崎 忠昭, 大場 雄介 日本細胞生物学会大会講演要旨集 62回 117 -117 2010年05月 [査読無し][通常論文]
  • 生体内腫瘍形成における腫瘍微小環境誘発性のRANKL発現の必要性(Requirement for tumor microenvironment-induced RANKL expression in the tumorigenesis in vivo)
    山田 珠希, 津田 真寿美, 戸塚 靖則, 進藤 正信, 大場 雄介 日本癌学会総会記事 68回 47 -47 2009年08月 [査読無し][通常論文]
  • FRETバイオセンサーを用いたCML分子標的薬剤の薬効評価系の構築(A FRET-based biosensor for the evaluation of the efficacy of molecular targeted drugs for chronic myeloid leukemia)
    水谷 龍明, 近藤 健, ダルマニン・ステファニー, 津田 真寿美, 田中 伸哉, 浅香 正博, 大場 雄介 日本癌学会総会記事 68回 457 -457 2009年08月 [査読無し][通常論文]
  • 谷野美智枝, 王磊, 津田真寿美, 西原広史, 大場雄介, 矢野聖二, 曽根三郎, 田中伸哉 日本呼吸器学会雑誌 47 237 2009年05月10日 [査読無し][通常論文]
  • 山田珠希, 津田真寿美, 大場雄介, 川口秀明, 戸塚靖則, 進藤正信 日本病理学会会誌 98 (1) 250 -250 2009年03月20日 [査読無し][通常論文]
  • 水谷龍明, 近藤健, STEPHANIE Darmanin, 津田真寿美, 川口秀明, 浅香正博, 大場雄介 日本病理学会会誌 98 (1) 278 2009年03月20日 [査読無し][通常論文]
  • 新井 隆太, 津田 真寿美, 渡部 琢哉, 川口 秀明, 大場雄介, 三浪 明男 日本分子生物学会年会講演要旨集 32nd (Vol.1) 231 2009年 [査読無し][通常論文]
  • Takayuki Inuzuka, Masumi Tsuda, Shinya Tanaka, Yujiro Higashi, Hideaki Kawaguchi, Yusuke Ohba JOURNAL OF MOLECULAR AND CELLULAR CARDIOLOGY 45 S11 -S12 2008年10月 [査読無し][通常論文]
  • 山田珠希, 津田真寿美, 大場雄介, 川口秀明, 戸塚靖則, 進藤正信 補体シンポジウム講演集 45th 180 -180 2008年07月 [査読無し][通常論文]
  • 山田珠希, 津田真寿美, 大場雄介, 戸塚靖則, 進藤正信, 川口秀明 適応医学 12 (1) 25 -25 2008年05月20日 [査読無し][通常論文]
  • Kaori Tsutsumi, Masumi Tsuda, Hideaki Kawaguchi, Yusuke Ohba FASEB JOURNAL 22 2008年04月 [査読無し][通常論文]
  • 山田珠希, 津田真寿美, 大場雄介, 川口秀明, 戸塚靖則, 進藤正信 生化学 2P-1143 2008年 [査読無し][通常論文]
  • 水谷龍明, 近藤健, DARMANIN Stephanie, 津田真寿美, 甲斐原拓真, 川口秀明, 浅香正博, 大場雄介 生化学 4P-1171 2008年 [査読無し][通常論文]
  • Hiroshi Nojima, Yusuke Ohba, Yasuyuki Kita Current Drug Safety 2 (3) 204 -211 2007年09月 [査読無し][通常論文]
     
    Despite considerable research, metastasis remains a major challenge in the clinical management of cancer. Recent reports show that abnormally augmented expression of Cx26 is responsible for the enhanced spontaneous metastasis of mouse BL6 melanoma cells. The function of Cx26 appears to be responsible for this phenotype since exogenous expression of a dominant-negative form of Cx26 and oleamide derivatives called MI-18 and MI-22 that specifically inhibit Cx26-mediated gap junction-mediated intercellular communications (GJIC) prevent the spontaneous metastasis of BL6 cells. As expected from their structural similarity to oleic acid (the major component of olive oil), both MI-18 and MI-22 are safe drugs nonetheless, they are potent inhibitors of the spontaneous metastasis of BL6 mouse melanoma cells. Thus, they are a novel prototype of an anti-metastasis drug that has minimal side eifects. While the primary tumors do not necessarily show strong Cx26-immumostaining signals, pronounced Cx26 expression is detected in the highly invasive tumor regions it is also more frequently observed in metastasized tumors. Thus, Cx26 expression may be useful as a prognostic tool that can predict the existence of highly metastatic cancer cells in clinical samples. © 2007 Bentham Science Publishers Ltd.
  • EGFシグナル伝達はPTHrPの発現を誘導し、口腔癌細胞の悪性化に関与する(EGF signaling regulates PTHrP expression and malignancies in oral cancer cells)
    山田 珠希, 津田 真寿美, 大場 雄介, 戸塚 靖則, 進藤 正信 日本癌学会総会記事 66回 501 -501 2007年08月 [査読無し][通常論文]
  • 藤岡容一朗, 内海真希, 岩崎信太郎, 大場雄介, 渡辺雄一郎 日本RNA学会年会要旨集 9th 138 2007年07月28日 [査読無し][通常論文]
  • 水谷龍明, 津田真寿美, 川口秀明, 大場雄介 生化学 4P-0935 2007年 [査読無し][通常論文]
  • 高岡晃教, 柳井秀元, 本田賢也, 大場雄介, 谷口維紹 Mol Med 42 308 -317 2005年12月15日 [査読無し][通常論文]
  • 大場雄介, 本田賢也, 柳井秀元, 高岡晃教, 谷口維紹 日本癌学会学術総会記事 64th 523 2005年08月15日 [査読無し][通常論文]
  • 柳井秀元, 本田賢也, 大場雄介, 高岡晃教, 吉田進昭, 谷口維紹 日本癌学会学術総会記事 64th 523 2005年08月15日 [査読無し][通常論文]
  • 本田賢也, 柳井秀元, 水谷龍明, 根岸英雄, 大場雄介, 高岡晃教, 谷口維紹 日本分子生物学会年会プログラム・講演要旨集 27th 345 2004年11月25日 [査読無し][通常論文]
  • 柳井秀元, 本田賢也, 朝霧政挙, 根岸英雄, 水谷龍明, 島田直也, 吉田進昭, 大場雄介, 高岡晃教 日本分子生物学会年会プログラム・講演要旨集 27th 593 2004年11月25日 [査読無し][通常論文]
  • Akiyuki Takaya, Yusuke Ohba, Kazuo Kurokawa, Michiyuki Matsuda CELL STRUCTURE AND FUNCTION 29 57 -57 2004年05月 [査読無し][通常論文]
  • N Mochizuki, Y Ohba, K Nagashima, A Endo, M Matsuda CIRCULATION 102 (18) 353 -353 2000年10月 [査読無し][通常論文]

書籍等出版物

  • Oral Cancer
    IN TECH 2012年
  • Bio Science 新用語ライブラリー免疫
    羊土社 2000年

所属学協会

  • 日本生理学会   日本細胞生物学会   日本生化学会   日本癌学会   日本分子生物学会   

共同研究・競争的資金等の研究課題

  • 蛍光偏光を用いた等方性分解能顕微鏡の開発と細胞膜動態の可視化
    日本学術振興会:科学研究費助成事業 国際共同研究加速基金(国際共同研究強化(B))
    研究期間 : 2019年10月 -2022年03月 
    代表者 : 大場 雄介, 藤岡 容一朗, 佐藤 絢
  • 細胞膜動態の高分解能計測によるエンドサイトーシス超初期過程の分子基盤の解明
    日本学術振興会:科学研究費助成事業 挑戦的研究(萌芽)
    研究期間 : 2019年06月 -2021年03月 
    代表者 : 大場 雄介
  • インフルエンザウイルス感染におけるシンギュラリティ
    日本学術振興会:科学研究費助成事業 新学術領域研究(研究領域提案型)
    研究期間 : 2019年04月 -2021年03月 
    代表者 : 大場 雄介
  • エンドゾームとミトコンドリアの連携ゾーンの解析
    日本学術振興会:科学研究費助成事業 新学術領域研究(研究領域提案型)
    研究期間 : 2018年04月 -2020年03月 
    代表者 : 大場 雄介
     
    本研究は、低分子量GTP結合タンパク質Rasと、その標的因子の1つであるphosphoinositide 3-kinase (PI3K)が、他の標的因子とは異なり上皮増殖因子 (epidermal growth factor, EGF) 刺激依存的にエンドソームでRasと複合体を形成する分子メカニズムを解明することから開始した。 Rasの主な標的因子のRas-binding domain(RBD)のアミノ酸配列を比較にり、PI3K特異的配列を見出し、Ras-PI3K endosomal localization(RAPEL)配列と命名した。RAPEL結合因子を酵母ツーハイブリッド法で探索したところ、ミトコンドリア外膜タンパク質voltage-dependent anion channel 2(VDAC2)が同定された。VDAC2のノックダウンによりRas-PI3K複合体のエンドソーム局在、クラスリン非依存性エンドサイトーシス、エンドソームの成熟化が抑制された。 次に、ミトコンドリアタンパク質がいかにしてエンドサイトーシスの制御に関与するかを調べるため、生細胞内でミトコンドリアとエンドソームのダイナミクスを詳細に観察したところ、両者は一過性に近接していることが明らかになった。この近接は、コントロール細胞ではEGF刺激後で促進されたのに対し、VDAC2ノックダウン細胞では変化が認められなかった。さらに光遺伝学を用いてミトコンドリア―エンドソーム間相互作用を青色光により誘導する系を構築したところ、相互作用依存性にエンドソームの酸性化が促進された。したがって、エンドソームとミトコンドリア間の接触ゾーンの形成がエンドソームとの成熟化を促進すること、RAPELとVDAC2がゾーン形成のtetheringに関与することが示唆された。
  • エンドソームとミトコンドリアの物理的相互作用と機能連関による細胞機能の調節機構
    日本学術振興会:科学研究費助成事業 基盤研究(B)
    研究期間 : 2017年04月 -2020年03月 
    代表者 : 大場 雄介
     
    昨年度に引き続き、低分子量Gタンパク質Rasと標的分子複合体のうち、Ras-PI3K複合体のみがエンドソーム移行する分子メカニズムの解明を目指し研究を行った。標的因子のRas-binding domain(RBD)のアミノ酸配列解析により、PI3K-RBDに存在する特徴的な配列を同定し、この配列を欠損させるとRas-PI3Kのエンドソーム局在が抑制されたことから、Ras-PI3K endosomal localization domain(RAPEL domain)と命名した。RAPEL過剰発現によりエンドサイトーシスが抑制され、エンドサイトーシスを介して細胞に侵入するインフルエンザウイルスの感染も抑制された。さらに、RAPELと細胞膜透過性ペプチド(cell penetrating peptide, CPP)との誘導ペプチドを合成した。ペプチドを導入した細胞にインフルエンザウイルスを暴露したところ、感染抑制効果が認められた。さらに、ペプチドによる感染抑制効果はヒト呼吸上皮細胞を用いたin situでも認められた。以上より、RAPELはペプチド療法にも応用可能であることが示された。これらの結果は、学術雑誌に受理された。 一方、上記の結果は、RAPELに結合する因子がRas-PI3K複合体のエンドソーム移行とエンドサイトーシスを制御することを示唆するものである。質量分析法と酵母ツーハイブリッド法によるスクリーニングで得られたRAPEL結合因子計50個のうち、6因子についての解析が進んでいる。うち3因子の解析が終了し論文を作成・投稿準備中である。因子Aについては新規のオルガネラ間相互作用を担うタンパク質であることが判明した。因子Bはイノシトールリン脂質依存的にRas-PI3Kのエンドソーム局在の制御因子であることを突き止めた。因子Cについては上記二つの負の制御因子であった。
  • 細胞認証システムの開発に向けた細胞内座標軸の設定
    日本学術振興会:科学研究費助成事業 挑戦的研究(萌芽)
    研究期間 : 2017年06月 -2019年03月 
    代表者 : 大場 雄介
     
    本研究は、種々の細胞種や細胞の置かれている状態において、各細胞内小器官の配置に関するパラメータを網羅的に取得し、このパラメータをもとに細胞を認証するための「テンプレート」とそれを記述するための「座標軸」を設定することを目的として実施中である。 この目的のため、様々なオルガネラのマーカー分子を構築したところ、一部で目的とするオルガネラ以外に局在するものがあった。特に、エンドソームマーカーとミトコンドリアマーカーでは融合する蛍光タンパク質によりその程度の差が顕著であり、EGFPを融合させた場合は目的とするオルガネラにほぼ局在するのに対し、VenusやSECFPを融合させた際には多くが細胞質に局在した。これら蛍光タンパク質のアミノ酸配列比較から、VenusやSECFP等にはフォールディングを促進する変異が導入されていることが明らかとなった。そこで、フォールディング促進変異が導入されていない黄色蛍光タンパク質enhansced yellow fluorescent protein (EYFP) とmitoを融合させたところ、局在がEGFPと同様になった。以上から、蛍光タンパク質のフォールディング速度とミトコンドリアマーカーの局在とが密接に関わることが明らかとなった。 一般的に、遺伝子導入後の蛍光検出可能な時間が短いため、フォールディングが早い方が望ましい(高性能な蛍光タンパク質)とされているが、オルガネラマーカー作製においてはその限りではないことが明らかとなった。したがって、オルガネラマーカー作製の際には様々な蛍光タンパク質を試し、最適な蛍光タンパク質を選択することが重要であると考えられる。以上は、細胞内小器官の配置を正確に定量するために必須の知見である。これらを用いて細胞認証の「テンプレート」を構築中である。
  • 細胞競合現象の時空間的解析とその操作
    日本学術振興会:科学研究費助成事業 新学術領域研究(研究領域提案型)
    研究期間 : 2017年04月 -2019年03月 
    代表者 : 大場 雄介
     
    本研究は、光による細胞競合の誘導系を開発し、細胞競合の発生を時空間的に厳密に制御すること、同時に分子の活性化、分子間相互作用、イオン濃度変化等を蛍光バイオイメージングで観察し、winner、looser間での細胞競合が生じるための時空間的な分子動態を詳細に明らかにすることを目的に行っている。 昨年度までに、488 nmの光で分子間相互作用が誘導されるCry2-CIBの系を用いて、二つのRas活性化法(RasV12とSosを用いるもの)を構築した。この二つの系を用いて、RasV12の系のみで細胞競合が観察され、Sosによる内在性Rasの活性化では細胞競合が誘導されないことを見出した。この結果は、細胞競合において何がwinnerとlooserを決めるかという根本的課題に迫る緒になると期待された。 しかし、この系では青色光の刺激が必要なため、シグナル伝達研究に有効なフェルスター共鳴エネルギー移動(Foerster resonance energy transfer, FRET)や蛍光タンパク質再構成法(bimolecular fluorescence complementation, BiFC)等の青色光を励起光として用いるイメージング手法との併用が難しい。そこで、それらイメージング手法との併用を目指し、遠赤色光受容タンパク質phytochromeB(PhyB)とphytochrome interaction factor 3(PIF3)の結合を遠赤色光により誘導できるオプトジェネティクスの手法を取り入れた。これにより、遠赤色光照射によりSrcを細胞質から細胞膜あるいはエンドソームに移行させることに成功した。また、様々なオルガネラに局在するPhyBとPIF3を作製し、異種オルガネラ間相互作用を誘導する系も併せて開発した。
  • 日本学術振興会:科学研究費助成事業 挑戦的萌芽研究
    研究期間 : 2016年04月 -2018年03月 
    代表者 : 今居 譲, 井下 強, 大場 雄介, 荒野 拓
     
    若年性パーキンソン病原因遺伝子ParkinとPINK1は、それぞれユビキチンリガーゼとミトコンドリア局在性キナーゼをコードする。PINK1とParkinは協働してミトコンドリアの品質管理に関与すること、その活性の喪失でドーパミン神経変性が起こることが明らかになってきた。本研究ではPINK1-Parkinシグナルを活性化しミトコンドリアの機能を高める創薬を目的として実施した。 PINK1-Parkinシグナルをモニターする細胞ベースのレポーターアッセイ系を開発し、約31万の化合物ライブラリーをスクリーニングした。次にミトコンドリア膜電位や細胞生存性への影響を評価し、3つの化合物候補を得た。
  • エンドサイトーシスと外来因子取込を制御する細胞内シグナル伝達マシナリー(国際共同研究強化)
    日本学術振興会:科学研究費助成事業 国際共同研究加速基金(国際共同研究強化)
    研究期間 : 2016年 -2018年 
    代表者 : 大場 雄介
     
    3月~9月の期間米国海洋生物学研究所(Marine Biological Laboratory, MBL)に滞在し、全反射型蛍光偏光顕微鏡の基礎を学び、その構築法を習得した。既存の観察側の偏光分光に加え、励起側の偏光を制御する光学系を開発し、その光学系を実際に構築した。また、蛍光タンパク質を細胞膜との相対角度が常に一定になるように固定し、蛍光タンパク質の双極子の方向が細胞膜のトポロジーに対して一定に配向するバイオセンサーを構築した。このバイオセンサーを発現する細胞膜を上記蛍光偏光顕微鏡で観察したところ、細胞膜なダイナミックな微小な変形、エンドサイトーシスの初期の膜変形などを、蛍光強度の変化として捉えることが可能になった。これらの変化はすべてが光学顕微鏡の回折限界以下の領域で生じる現象であり、新しい超解像観察技術へと発展が期待できる。さらに、膜への局在化シグナルの一部に脂質認識部位を導入することで、膜の組成変化を蛍光偏光の変化として捉えるバイオセンサーも構築した。 帰国後、MBL訪問研究者と連絡を取りながら光学素子と部材の選定を行い、必要機器を入手した。その後、自らの手で全反射型蛍光偏光顕微鏡を研究室にセットアップし、稼働させた。2月にはMBLの受け入れ研究者を日本に招き、セットアップした顕微鏡をon siteで確認した。また、意見交換と今後の研究展開について協議し、新規蛍光偏光顕微鏡のプランを共同で立案した。現在はその新しい蛍光変更顕微鏡のセットアップを行っており、まもなく稼働予定である。
  • ウイルス感染コンピテンシーの不均一性を決定する宿主因子の同定
    日本学術振興会:科学研究費助成事業 新学術領域研究(研究領域提案型)
    研究期間 : 2015年04月 -2017年03月 
    代表者 : 大場 雄介
     
    本研究では、インフルエンザウイルスの感染コンピテンシーを規定する因子の同定をその機能解析を行うことを目的に研究を進めている。これまで我々は、インフルエンザウイルスの細胞内取込において、カルシウムシグナルが重要であることを報告しており、本研究では細胞膜に存在するカルシウム制御に関与する分子に着目して研究を行っている。昨年度までに、ある細胞膜タンパク質がその制御に重要であることを見出した。この因子のノックダウンおよび薬剤での阻害によって、インフルエンザウイルス感染時のカルシウム応答は完全に抑制され、またプラークアッセイを用いた実験においてもインフルエンザ感染を強く抑制した。また、この分子の発現量とウイルス感染効率に強い相関があったことから、当該因子がウイルスが宿主細胞への感染過程において重要な役割と担っているおり、ウイルス感染コンピテンシーの規定因子の1つであるという観点から研究を進めた。 この因子はウイルス粒子のHAタンパク質と結合することが昨年までに明らかとなっていたため、当該因子のC末端細胞表面部位に存在するシアル酸化の標的になるアスパラギンをグルタミンに置換した変異体を作製した。変異体とHAの結合は野生型のそれと比べて減少していた。また、当該因子をノックダウンした細胞に野生型を戻すと、細胞内カルシウムの上昇とインフルエンザウイルス粒子の取り込み、インフルエンザ感染の全てが回復したが、変異型の発現を戻しても回復効果は認められなかった。以上から、シアル酸を介した当該因子とウイルス粒子の結合が、ウイルス感染時のカルシウム動員と粒子の取り込み、その後の感染成立に重要であることが示された。本研究成果は学術雑誌に投稿中である。
  • 日本学術振興会:科学研究費助成事業 挑戦的萌芽研究
    研究期間 : 2015年04月 -2017年03月 
    代表者 : 大場 雄介, 南保 明日香, 西出 真也, 藤岡 容一朗, 藤岡 真理, 堀内 浩水, 佐藤 絢
     
    我々は低分子量Gタンパク質Rasと標的分子PI3K複合体がエンドソームに局在し、エンドサイトーシスとウイルス粒子取込みを制御することを報告した。しかし、Ras-PI3K複合体がエンドソームに局在する分子機構は不明である。本研究では上記現象の原因となるアミノ酸配列を同定した。またその結合タンパク質のスクリーニングによりミトコンドリアタンパク質を同定した。このミトコンドリアタンパク質のノックダウンはRas-PI3Kのエンドソーム移行、エンドサイトーシス、ミトコンドリア―エンドソーム相互作用を阻害したので、PI3Kとの結合がオルガネラ間相互作用を介してエンドサイトーシスを調節することが示唆された。
  • 日本学術振興会:科学研究費助成事業 基盤研究(B)
    研究期間 : 2014年04月 -2017年03月 
    代表者 : 大場 雄介, 南保 明日香, 西出 真也, 藤岡 容一朗, 藤岡 真理, 堀内 浩水, 佐藤 絢
     
    エンドゾームは自ら積極的にシグナルを発することで多様な機能を発揮するプラットフォームである。しかし、生きた細胞でのダイナミクスと細胞機能との関連の詳細は不明な点が多い。本研究では、アンギオテンシンII(AII)2型受容体(AT2R)が、1型受容体(AT1R)シグナル伝達を負に調節するためには、AT1RとAT2Rのヘテロ二量体化とそのエンドサイトーシスによる内在化、さらに複合体内の受容体間分子配向変化が重要であることを見出した。また、この機能には両方の受容体の活性化が不可欠であること、プロテインキナーゼC(PKC)によるAT2RのC末端のセリン残基のリン酸化が重要であることも明らかにした。
  • バイオイメージングによるウイルス感染と細胞応答の定量解析
    日本学術振興会:科学研究費助成事業 新学術領域研究(研究領域提案型)
    研究期間 : 2014年04月 -2016年03月 
    代表者 : 大場 雄介
     
    本研究においては、細胞への感染成立には、インフルエンザウイルス粒子が何個必要かという命題を解くために、研究を行っている。そのため、ウイルス粒子が細胞内侵入する際に重要なシグナル伝達経路の解析と、ウイルス粒子の精密計測により粒子数とその経路の関係性を検討した。シグナル伝達解析においては感染時のカルシウム上昇に着目し、その責任分子の機能解析を進めた。同定した責任分子は24回膜貫通型の膜タンパク質であり、生化学的解析を行うための可溶化が困難であった。種々の界面活性剤を用いて可溶化条件を検討したところ、凝集せずに単量体として可溶化することに成功した。この条件を用いることで、この責任分子とウイルス粒子の結合を介してカルシウム上昇が引き起こされることが明らかになった。 また、領域内の東京大学工学系研究科・野地先生、田端先生との共同研究により、用いる ウイルスサンプルの粒子数とMOIの関係を明らかにしたうえで、感染性の評価を行った。その結果、ウイルス感染効率はこれまで考えられていたMOIよりも、粒子数に依存することが明らかになった。また、粒子数が多い時と少ない時では異なる細胞の応答性が観察された。粒子数が多い時には感染にある種の正のフィードバックのようなブースト機構が存在すること、そのブースト機構にカルシウムが重要であることが解った。実際に高い複製が認められ、多くの粒子が取り込まれたと考えられる細胞には、上記の責任分子が多く発言していた。一方で、複製が盛んではない(0ではない)細胞においては上記の責任分子の発現量が低く、カルシウム応答も見られないことから、カルシウム非依存性のメカニズムによりウイルスが感染するものと考えられた。両者の境界は細胞1つあたりウイルス粒子20個程度であり、感染には数10個程度の粒子が必要であることが明らかになった。
  • 日本学術振興会:科学研究費助成事業 挑戦的萌芽研究
    研究期間 : 2014年04月 -2016年03月 
    代表者 : 秋田 弘俊, 大場 雄介, 木下 一郎
     
    EGFR活性測定FRETバイオセンサーとフローサイトメーターを用いて、3つの肺癌細胞株においてEGFR阻害薬耐性細胞を単離した。耐性細胞の遺伝子発現パターンをcDNAマイクロアレイで解析したところ、各細胞株由来耐性細胞において共通に発現変動する遺伝子が認められた。さらにアノテーション解析を行ったところ、耐性細胞で亢進しているシグナル経路として3経路、低下しているものとして5経路が同定された。共通して亢進している遺伝子群にABCトランスポーターが含まれていることから、これらの経路や遺伝子は耐性化に一定以上の役割を担っているものと期待される。
  • 日本学術振興会:科学研究費助成事業 基盤研究(C)
    研究期間 : 2013年04月 -2016年03月 
    代表者 : 近藤 健, 大場 雄介, 南 陽介
     
    本研究においてはチロシンキナーゼ阻害剤(TKI)では治癒困難なフィラデルフィア染色体陽性急性リンパ球性白血病(Ph+ALL)の治療抵抗性機序の克服を目的とした。FRETの原理を応用して確立したBCR-ABLキナーゼ活性を可視化する技術を用いて、Ph+ALL細胞群に存在するTKI抵抗性を示す細胞集団を同定しTKI抵抗性細胞群において発現亢進している遺伝子を同定した。タンパク修飾系の一つであるSUMO化酵素の発現亢進が観察され、BCR-ABLがSUMO化修飾を受ける事を見いだした。更に、SUMO化阻害剤はPh+ALLの細胞死誘導を引き起こす事を確認した。
  • 日本学術振興会:科学研究費助成事業 新学術領域研究(研究領域提案型)
    研究期間 : 2010年04月 -2016年03月 
    代表者 : 松田 道行, 清川 悦子, 平塚 拓也, 大場 雄介, 青木 一洋, 中村 岳史
     
    FRETバイオセンサーは生きた細胞で細胞内情報伝達分子の活性を可視化する。しかし、これまで、ほとんど2次元培養細胞でのみの使用にとどまっていた。本研究では、FRETバイオセンサーを3次元培養あるいは生きた組織で使う技術を開発することを目的とした。FRETバイオセンサー遺伝子はしばしば組み換えを起こすために安定発現できなかった。この問題を克服するために、トランスポゾンを用いた遺伝子導入法を使った。特にTol2を用いるとFRETバイオセンサーを高発現するトランスジェニックマウスが容易に作成できた。このFRETマウスを使うことで、様々な細胞内情報伝達分子の活性が生きた組織で観察できた。
  • 日本学術振興会:科学研究費助成事業 挑戦的萌芽研究
    研究期間 : 2013年04月 -2015年03月 
    代表者 : 大場 雄介
     
    フィラデルフィア染色体陽性白血病の発症原因は、染色体相互転座により形成される融合遺伝子産物BCR-ABLの恒常的チロシンキナーゼ活性であり、BCR-ABLの特異的チロシンキナーゼ阻害薬の導入によりその治療成績は劇的に向上した。しかし、治療開始時・途上を問わず薬剤耐性症例が認められるため、極少数存在する耐性細胞の検出を可能とする技術開発が望まれている。本研究では、我々が開発した薬剤耐性細胞検出技術を基に、FRETバイオセンサーの改良、検出法の開発と改良、解析法の改良により薬剤耐性細胞検出能を従前技術の100倍に向上することを目的とし、結果として162倍の高感度化を達成した。
  • 日本学術振興会:科学研究費助成事業 基盤研究(B)
    研究期間 : 2011年04月 -2014年03月 
    代表者 : 大場 雄介, 津田 真寿美
     
    ウイルス感染症は科学技術が進歩した現代でも人類最大の脅威の一つであり、2009年に世間を震撼させた新型インフルエンザパンデミックは記憶に新しい。我々はイメージングを用いたシグナル伝達研究の過程で、エンドゾームにおいてRasと結合したPI3Kが、エンドサイトーシス制御に重要な役割を担うことを明らかにした。本研究では、Ras-PI3Kシグナルがインフルエンザウイルス感染に重要なこと、また細胞内カルシウムがインフルエンザウイルス感染過程を制御するキーとなる分子であることを明らかにした。これらの成果の応用による宿主細胞因子を標的とする新概念のウイルス感染抑制法確立も期待される。
  • 日本学術振興会:科学研究費助成事業 新学術領域研究(研究領域提案型)
    研究期間 : 2011年04月 -2013年03月 
    代表者 : 大場 雄介
     
    本研究では、腫瘍微小環境を舞台にがん細胞と間質細胞等が演じるドラマを分子レベルで解明する実験系の提供を第一目的とし、昨年度から研究を実施している。大きく分けて以下の2つの研究項目に取り組み、複雑系システムを理解するために多次元イメージング系の基礎を確立した。 細胞の蛍光ラベル:様々な蛍光タンパク質により細胞内のオルガネラをマークする発現ベクターを構築した。蛍光タンパク質については、Sirius、CFP、YFP、tdTomato、TurboFP650、KeimaおよびEGFPの7色それぞれについて、核(ヒストン)、細胞膜 、細胞膜、アクチン細胞骨格、細胞接着斑、早期/後期エンドソーム、ミトコンドリア、微小管、小胞体、ゴルジ装置、リサイクリングエンドゾームのオルガネラ局在発現ベクターを完成させた (計84種)。 3次元培養系の確立:前項で樹立した多彩な蛍光タンパク質でラベルした細胞を用いて、腫瘍微小環境を模倣した3次元培養系を構築した。 この培養条件下において液性因子や基質種類・混合比を調整しながら細胞の挙動を顕微鏡観察し、そのダイナミクスを時空間的に解析した。実際、扁平上皮癌細胞を用いた癌組織の形成を試験管内で再構成することに成功するとともに、その過程においての細胞の挙動の観察にも成功した。また、以前我々が同定した扁平上皮癌細胞の悪性度制御因子をこの系に用いることにより、高分化型扁平上皮癌から低分化型の癌組織を誘導することにも成功した。このことは、本研究で確立した3次元培養系が生体内の癌組織を反映していることが証明されたことになり、今後腫瘍微小環境内での詳細な分子メカニズムの解明のための協力なツールになると期待できる。
  • 日本学術振興会:科学研究費助成事業 挑戦的萌芽研究
    研究期間 : 2011年 -2012年 
    代表者 : 大場 雄介
     
    フェルスター共鳴エネルギー移動の原理を応用したバイオセンサーを用い、Ph陽性急性リンパ球性白血病からイマチニブ耐性細胞を単離し、その細胞特性を解析した。耐性細胞群で活性化している24のシグナル伝達経路を同定し、メバロチン酸経路阻害が耐性細胞の再増殖抑制に有効であることを明らかにした。本成果によりスタチンの併用がPh陽性急性リンパ球性白血病やイマチニブ耐性慢性骨髄性白血病の治療における有効な候補となることが示された。
  • 日本学術振興会:科学研究費助成事業 基盤研究(A)
    研究期間 : 2008年 -2011年 
    代表者 : 戸塚 靖則, 進藤 正信, 東野 史裕, 樋田 京子, 北村 哲也, 大場 雄介, 松本 健一, 大廣 洋一
     
    我々は、癌の「間質」に存在する腫瘍血管内皮細胞が正常血管内皮細胞とは異なった遺伝子背景をもつことを明らかにしてきた。近年、上皮間葉移行が癌細胞の浸潤・転移に重要な役割を果たしていることが明らかになってきた。上皮性の口腔扁平上皮癌細胞と間葉系細胞の相互作用を検索することは、癌細胞のみならず腫瘍に栄養・酸素を供給することで腫瘍の生存や増殖に関わっている間質細胞をターゲットにした効率的な治療法の確立にも寄与することが期待される。HuRは通常、核に存在しているタンパクでAU-rich element(ARE)mRNAの安定性に関与している。我々は、ウイルス発癌系でARE mRNAがHuRを介して安定化され細胞の形質転換(癌化)に関与していることを明らかにした。口腔環境は唾液腺など全身の臓器の中で特異な器官を有している。我々は破骨細胞の誘導因子であるRANCLが口腔環境で発現が高いことをみいだした。さらにヌードマウスに口腔癌細胞を移植すると口腔に移植した癌細胞が有意に増大することを発見し、このような所見はRANKLが口腔癌のstem cellの上皮間葉移行を生じさせることで誘導されることを明らかにした。腫瘍の微小環境は癌細胞の増殖や浸潤転移と深く関わっている。我々は、腫瘍間質に存在する血管(腫瘍血管)を分離培養し、その生物学的特徴について検索してきた。その結果、炎症を惹起することでも知られているCox-2を阻害することで腫瘍血管新生が抑制されること、腫瘍血管はVEGFシグナルを介したMDR1の転写亢進により抗癌剤に対する抵抗性を獲得することなどが明らかになり、このような検索結果は腫瘍血管をターゲットにした分子標的治療へ応用する可能性が高まった。
  • 日本学術振興会:科学研究費助成事業 挑戦的萌芽研究
    研究期間 : 2009年 -2010年 
    代表者 : 大場 雄介
     
    前年度に引き続き口腔癌をモデルに癌細胞微小環境適応因子として同定したRANKLの機能解析を行りた。RANKLのmRNAレベルおよびタンパクレベルでの発現をRT-PCRと免疫染色で行ったところ、いずれのヒト口腔癌組織における発現が培養細胞に比べて高く、なかでも低分化型の組織での発現が高かった。各種口腔癌細胞株における低いRANKLの発現量は、樹立した元の腫瘍の組織型が低分化型であっても同様であったが、培養癌細胞をヌードマウスの口腔に移植したところ、RANKLの発現が亢進した。一方で同じ細胞株をマウスの下肢に移植したところ、口腔に移植して形成された腫瘍と比較して有意に小さく、RANKLの発現も低かった。すなわち生体内の周囲組織依存的に発現したRANKLが、腫瘍形成を制御することが明らかになった。さらにRANKLの腫瘍形成における役割を明らかにするために、RANKL過剰発現細胞を樹立しマウスの下肢に接種したところ、口腔環境同様に腫瘍の形成が認められた。また組織学的に、コントロール細胞で形成された腫瘍が高分化型扁平上皮癌の組織型を示したのに対し、RANKL発現細胞によって形成された腫瘍は低分化性であった。以上の結果から、RANKLは生体内特異的に発現し腫瘍形成を制御する環境適応因子であり、低分化型の癌すなわち悪性度の高い癌の形成に関与する因子であることが明らかとなった。またこの組織型の変化は上皮間葉転換EMTによることも解明した。 また、RANKLの下流で誘導される因子としてインテグリンα2を同定し、現在悪性度との関与を解析中である。興味深いことにインテグリンα2の発現上昇は活性化型インテグリンの細胞内輸送を亢進するというデータが得られている。近年、EMT等の癌細胞の形態変化と細胞内膜輸送の関連性が報告されつつあり、我々のデータは新たな分子メカニズムを提供する可能性が示唆される。
  • 日本学術振興会:科学研究費助成事業 基盤研究(B)
    研究期間 : 2008年 -2010年 
    代表者 : 大場 雄介, 津田 真寿美
     
    がん細胞の浸潤・転移や血管内皮細胞の血管新生動員に重要なプロセスである上皮間葉移行(epithelial-mesenchymal transition, EMT)を共通に制御する転写因子TCF8を同定し、TCF8がEMTを制御する分子メカニズムをがん細胞側、血管内皮細胞側のそれぞれで解明した。また、がん間質側の違いが、がんそのものの悪性度に寄与する新たなエビデンスを見出し、新しいがんの制御法につながる新たな知見を提供した。
  • 蛍光イメージング技術の臨床応用
    共同研究
    研究期間 : 2010年
  • Clinical application of fluorescence imaging
    Cooperative Research
    研究期間 : 2010年
  • 日本学術振興会:科学研究費助成事業 特定領域研究
    研究期間 : 2005年 -2009年 
    代表者 : 谷口 維紹, 高岡 晃教, 田村 智彦, 本田 賢也, 大場 雄介
     
    インターフェロン、IRFs、Noxa等の分子について機能解析を行った結果、これらの分子が細胞のがん化抑制やアポトーシス誘導、がん転移など多様なシグナル伝達系の制御に関与していた。また、TLRシグナルにおけるクロストークを見出した。さらに、自然免疫応答シグナル伝達系の解析から、DAIやHMGB1, 2, 3が核酸に対する免疫応答シグナルに重要な役割を果たす事を見出した。これらのシグナルは発がんやがんの増悪と密接に関連しており、我々の研究成果は新たながん治療の開発に繋がる分子基盤を提供するものであると考えられる。
  • 日本学術振興会:科学研究費助成事業 基盤研究(B)
    研究期間 : 2007年 -2008年 
    代表者 : 本田 賢也, 竹田 潔, 多屋 長治, 大場 雄介
     
    腸管樹状細胞を機能的に細分化し、シグナル伝達機構を明らかにし、炎症性腸疾患との関わりを検討することを目的として研究を推進した。新たにCD70が活性化型と抑制型樹状細胞を区別するバイオマーカーとして利用できることが明らかにした。CD70は消化管粘膜固有層特異的に存在する樹状細胞集団の一部において高発現しており、他の臓器にはそのような細胞を認めなかった。更にCD70陽性粘膜固有層樹状細胞は、細胞外ATPの受容体であるP2XやP2Y受容体を高発現しており、ATP刺激によって、共培養したナイーブCD4T細胞をIL17を高産生するTH17細胞へと分化させることも見出した。即ちCD70陽性粘膜固有層樹状細胞は"活性化型樹状細胞"であると考えられた。
  • 口腔がんの顎骨浸潤は抑制できるか?-EGFレセプターリン酸化阻害による効果-
    日本学術振興会:科学研究費助成事業 萌芽研究
    研究期間 : 2007年 -2008年 
    代表者 : 戸塚 靖則, 進藤 正信, 大場 雄介, 北村 哲也, 樋田 京子, 東野 史裕, 津田 真寿美
     
    口腔がんの多くは舌や歯肉などの口腔内組織に発症するが、これらの臓器・組織は解剖学的に顎骨に近接しており、腫瘍の増大により容易に顎骨に浸潤する。この場合は、腫瘍の根治性の点から、顎骨離断術や部分切断術を施行せざるをえず、顔面の審美性や機能性に多大な障害を生じ、治癒後の社会生活やQOLに大きな影響を及ぼす。それ故、腫瘍の顎骨浸潤を抑制することが可能となれば、口腔がん患者にどって大きな福音となり、その開発が真に望まれている。 口腔癌細胞の多くは、epidemal growth factor (EGF) receptorを高頻度に発現しており、また顎下腺並びに唾液中には高濃度のEGFが存在していることが明らかになっている。従って、口腔癌の微小環境中に存在するEGFが、癌細胞に対してパラクライン的に作用し、骨浸潤に関与する形質発現に重要な影響を及ぼしていることが示唆される。 口腔がん細胞株HSC2, HSC3, HSC4, Ca9.22, SASを用いてEGFRの発現をWestern Blotで検討したところ、全てのがん細胞株でEGFRの発現亢進がみられた。これらの細胞をEGFで刺激した際、EGFRのリン酸化の亢進およびMAPKであるERK1/2, p38.JNKの活性化がみられた。ERK1/2はEGF依存的にPTHrPを発現亢進した。HSC2細胞にPTHrPのsiRNAを導入することで細胞増殖、浸潤活性の抑制がみられた。 このような所見は、口腔がん細胞の顎骨浸潤にはEGF/EGFRによるPTHrPの発現が重要な役割を演じていることを示唆するものだった。
  • 日本学術振興会:科学研究費助成事業 特定領域研究
    研究期間 : 2007年 -2008年 
    代表者 : 大場 雄介
     
    レニン-アンギオテンシン系の研究は国内外を問わずもっともホットな領域の一つといえる。アンジオテンシンは二つのセルセンサーAT1とAT2を介してシグナルを伝えることが知られているが、AT1については既に多くの知見が得られているのに対し、AT2の機能は未知のままである。我々はAT2の発現がAT1シグナルに制御されているという基礎的データを得ており、細胞はAT1からAT2へセンサーをモーダルシフトすることにより、外界に存在するアンジオテンシンのセンシング機構を制御しているものと考えられる、その分子機序が解明されればインパクトは大きい。 本研究ではAT1がアンジオテンシンをセンスして惹起されるERKの活性化を指標にAT1とAT2の機能連関の解明を試みた。AT2はAT2に対し、ERKのリン酸化に関しては拮抗的に働くが、この作用は他のEGFやLPAによるERKのリン酸化に対しては認められないことから、シグナル伝達の極めて上流でクロストークしていることが示唆された。また、蛍光タグを付加したAT1とAT2を発現させ、蛍光顕微鏡で観察したところ、両者が共発現した場合には互いの局在を変化させ共局在することが明らかになった。そこで、2つのセルセンサーの結合を解析したところ、免疫沈降法および蛍光共鳴エネルギー移動(FRET) 法の双方でAT1とAT2のアンジオテンシン依存的な結合が認められた。特にFRETを用いたイメージングでは2つのセルセンサーが細胞膜上で結合した後に、エンドサイトーシスされ、エンドソーム上で結合することが、両者センサーからのシグナルクロストークに重要であることが明らかとなった。これらの現象には、双方の受容体にリガンドが結合することが必要であり、更にその下流に位置する因子として、あるセリンスレオニンキナーゼの必要性を同定した。
  • 日本学術振興会:科学研究費助成事業 萌芽研究
    研究期間 : 2007年 -2008年 
    代表者 : 大場 雄介
     
    生きた細胞において多分子複合体が、何時何処で形成されるかを追及することは、生命をシステムと理解し、正常状態や病態発症メカニズムを解明する上で重要であにも関わらず、この重要なメカニズムを検出する実験系そのものが現在のところ確立されていない。本研究課題では、生きた細胞における多分子複合体の検出系を確立し、新時代の細胞内情報伝達研究推進への足がかりを築くことを目的として研究を開始した。まず、淡色で分子間相互作用の観察を可能とする、蛍光蛋白質再構成(BiFC)法を用いて蛋白質間相互作用の動的観察系を世界に先がかけて開発した。この技術を用い、RasとPI3Kによる特異的なエンドゾーム上での結合を画像化し、その動きを追跡することに成功した。更にこの研究の過程で、実際にBiFCと他の蛍光蛋白質および、免疫染色を併用することにより、Ras、PI3Kの結合とその代謝産物PIP_3の産生、エンドゾームマーカーであるEEA1の局在からなる、合計3つの蛋白質と一つの脂質の共局在を可視化することに成功した。それらの分子メカニズムを詳細に解析する過程で、RasによるPI3Kの活性化には二つの細胞内シグナル伝達経路が存在し、ひとつはRas非依存性、もう一つはRas依存性であり、後者がエンドゾームにおけるPI3Kの活性化に必要であることも示した。PI3Kの活性化は細胞の生存に必須であることから、その機能阻害は細胞障害が懸念されるが、今回得られた結果は、PI3Kの活性を時空間的に変調することでその一部の機能を調節1可能であることを示唆しており、エンドサイトーシスを介した種々の物質の取り込みを制御する新たな手法の開発に発展すると期待される。
  • 腫瘍細胞ー微小環境相互作用の時空間ダイナミクス
    科学研究費補助金
    研究期間 : 2008年
  • Spatiotemporal dynamics of tumor cell-microenvironment interaction
    Grant-in-Aid for Scientific Research
    研究期間 : 2008年
  • 日本学術振興会:科学研究費助成事業 若手研究(A)
    研究期間 : 2005年 -2007年 
    代表者 : 大場 雄介
     
    1.癌における時空間シグナルネットワーク (1)口腔内は上皮細胞増殖因子(EGF)が多量に存在する環境であるため、口腔癌の悪性化にはEGF受容体下流の情報伝達経路が重要である。口腔癌細胞において、EGF受容体の下流で上皮小体ホルモン関連蛋白質(PTHrP)が誘導され、PTHrPが口腔癌細胞の細胞増殖、運動能及び浸潤能等の悪性度を制御していることを明らかにした。本研究成果はBiochem. Biophys. Res. Commun.誌に発表した。 (2)悪性膠芽腫細胞における浸潤能を制御する分子機構として、転写因子OLIG2の発現と低分子量G蛋白質Rhoの活性化、さらにRhoの活性化の局在との関連を明らかにした。本研究成果はMol. Cancer Res.誌に発表した。 2.免疫におけるシグナルネットワーク (1)核酸による自然免疫系発動に関する分子動態について明らかにした。DNAをセンスする細胞内因子DAIを同定し、DAIとB型DNAの直接結合を画像化することに成功した。また、その過程が培養細胞でのインターフェロン産生に重要であることを明らかにし、Natureに報告した。また、2重鎖RNAに応答するToll様受容体下流におけるアダプター分子TICAM1の細胞内動態について解析し、J. Immunolo.誌に報告した。 (2)インターフェロン産生における負の制御因子IRF2のノックアウトマウスを用い、過剰なインターフェロンシグナルによって生じる慢性貧血の分子機序を明らかにした。本研究成果は、Exp. Hematol.誌に発表した。 本研究課題3年間での成果について、英文学術雑誌に計13本の報告を行った。
  • 蛍光バイオイメージングを用いた細胞内情報伝達の時空間制御機構の解明
    科学研究費補助金
    研究期間 : 2006年
  • Spatiotemporal regulation of signal transduction pathways using fluorescence imaging
    Grant-in-Aid for Scientific Research
    研究期間 : 2006年
  • 日本学術振興会:科学研究費助成事業 若手研究(B)
    研究期間 : 2002年 -2003年 
    代表者 : 大場 雄介
     
    RalはRasファミリーGたんぱく質の一つで、Rasにより活性化される。最近、エンドサイトーシス、エキソサイトーシス、フィロポディアの形成に対するRalの関与が示唆されつつあるものの、詳細な分子機序は未知の点が多い。そこで生きた細胞内でRal活性化の時空間的な制御機構の解析を通じて、Ralの生理的機能を知るために、蛍光共鳴エネルギー移動(FRET)の原理を利用したモニター分子を作製し解析した。 モニター分子の構造はYFP、RalA、RalBP、CFP及び膜局在化配列からなる。この分子を発現する293T細胞可溶化液のFRET効率を蛍光分光光度計で測定したところ、GTP結合率とFRET効率が正の相関を示した。従って作製したモニター分子はRalの活性化をFRET効率の上昇として検出できることが確認できた。次にCOS1細胞において上皮細胞増殖因子(EGF)依存性のRalの活性化を多重蛍光タイムラプス顕微鏡で観察し、FRET効率を画像化して解析したところ、EGFによって生じる葉状仮足に限局してRalが活性化していた。この結果はRalの活生化に関し初めて得られた時空間情報である。 さらに、EGF刺激によるRal活性化の機構を詳細に検討した。Rasの優勢劣性型変異体を発現した細胞では、Ralの活性化が抑制されていることから、EGF依存性のRal活性化にはRasの活性化を必要とすることが確認された。しかし、Rasの活性化は葉状仮足で高いが、必ずしも葉状仮足に限局するわけではないので、葉状仮足の形成もまたRalの活性化に必要であることが示唆された。実際、優勢劣性型変異体のRacを導入して、EGF依存性葉状仮足形成を阻害すると、Ralの活性化は阻害された。すなわち、増殖因子依存性Ralの活性化は複数の経路によって制御されていることが明らかとなった。
  • 低分子量G蛋白が細胞膜で情報を受け取るメカニズムの可視化
    日本学術振興会:科学研究費助成事業 特定領域研究
    研究期間 : 1999年 -2003年 
    代表者 : 松田 道行, 黒川 量雄, 大場 雄介, 中村 岳史, 望月 直樹
     
    本研究の目的は、細胞内情報伝達の重要なキープレーヤーである低分子量G蛋白の活性化の様子を生細胞でリアルタイムにモニターするプローブを作成し、バーチャル細胞作成のために必要な、細胞内情報伝達の時空間パラメータを獲得することである。本年度は、K-Ras、N-Ras、R-Ras、RalA、RalBのプローブ開発を行った。K-Ras、N-Ras、RalAに関しては感度の高いプローブの開発に成功したが、R-RasおよびRalBのプローブは感度が低く、改良の余地を残している。また、既存のプローブの高感度化をYFPおよびCFPをより明るいものに置換することにより達成した。これにより、画像データのシグナルノイズ比を向上させることに成功した。さらに、共焦点レーザー顕微鏡を用いて、細胞内の膜画分と細胞膜画分とのシグナルを定量的に分ける手法を確立した。 一方、これら新規に開発したプローブを使って様々な生命現象の解明に取り組んだ。特に、これまで機能の不明であったRalAが、細胞増殖因子の刺激により葉状突起で限局して活性化されること、RalAの活性化が増殖因子依存性あるいは細胞運動時の葉状突起の形成に必要であることをさまざまな手法を用いて証明した。RalAは小胞の融合に関与するとうい他グループのデータとあわせて考えると、葉状突起の形成には、細胞骨格系と細胞膜系の双方のダイナミックな変化が必要であり、RhoファミリーG蛋白はその前者に、RalAはその後者に密接に関わることが示唆された。

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