2024/02/01 更新

写真a

オカモト クニアキ
岡元 邦彰
OKAMOTO Kuniaki
所属
医歯薬学域 教授
職名
教授
外部リンク

学位

  • 博士(歯学) ( 九州大学 )

研究キーワード

  • 破骨細胞

  • 口腔がん

  • 細胞内輸送

研究分野

  • ライフサイエンス / 常態系口腔科学

所属学協会

  • 病態と治療におけるプロテアーゼインヒビター研究会

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  • 日本生化学会

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  • The molecular Biology Society of Japan

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  • 歯科基礎医学会

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  • 日本薬理学会

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  • 日本分子生物学会

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▼全件表示

 

論文

  • Novel mechanism of cisplatin resistance in head and neck squamous cell carcinoma involving extracellular vesicles and a copper transporter system. 国際誌

    Tatsuo Ogawa, Kisho Ono, Shoji Ryumon, Hotaka Kawai, Tomoya Nakamura, Koki Umemori, Kunihiro Yoshida, Hideka Kanemoto, Kyoichi Obata, Norie Yoshioka, Tatsuo Okui, Kuniaki Okamoto, Hitoshi Nagatsuka, Soichiro Ibaragi

    Head & neck   2024年1月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    BACKGROUND: Cisplatin (CDDP) plays a central role in chemotherapy for head and neck squamous cell carcinoma (HNSCC), but drug resistance in HNSCC chemotherapy remains a problem, and the mechanism of CDDP resistance is unclear. We investigated CDDP-resistance mechanisms mediated by extracellular vesicles (EVs) and ATPase copper transporting beta (ATP7B) in HNSCC. METHODS: We established CDDP-resistant sublines of HNSCC cells and verified their ATP7B expression. We used an EV secretion inhibitor (GW4869) and ATP7B short hairpin (sh)RNA transfection to examine the correlation between EV secretion and ATP7B expression. RESULTS: The CDDP-resistant HNSCC sublines showed decreased CDDP sensitivity and increased ATP7B expression. GW4869 suppressed ATP7B expression, and ATP7B shRNA transfection suppressed EV secretion. The suppressions of EV secretion and ATP7B expression both enhanced CDDP's cell-killing effect. CONCLUSIONS: EVs were involved in the ATP7B-mediated mechanism underlying CDDP resistance. Further clarification of the EV-induced CDDP-resistance mechanism may lead to novel therapeutic strategies for HNSCC.

    DOI: 10.1002/hed.27620

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  • Rab11 suppresses head and neck carcinoma by regulating EGFR and EpCAM exosome secretion. 査読 国際誌

    Kunihiro Yoshida, Kaung Htike, Takanori Eguchi, Hotaka Kawai, Htoo Shwe Eain, Manh Tien Tran, Chiharu Sogawa, Koki Umemori, Tatsuo Ogawa, Hideka Kanemoto, Kisho Ono, Hitoshi Nagatsuka, Akira Sasaki, Soichiro Ibaragi, Kuniaki Okamoto

    Journal of oral biosciences   2023年12月

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    担当区分:最終著者, 責任著者   記述言語:英語   掲載種別:研究論文(学術雑誌)  

    OBJECTIVES: Rab11(Rab11a and Rab11b) localizes primarily along recycling endosomes in cells and is involved in various intracellular trafficking processes, including membrane receptor recycling and secretion of exosomes or small extracellular vesicles (EVs). Although Rab11 is closely associated with the progression and metastasis of various cancer types, little is known about Rab11' role in head and neck squamous cell carcinoma (HNSCC). In this study, we investigated the roles of Rab11a and Rab11b in HNSCC. METHODS: The clinical significance of Rab11 expression in HNSCC was investigated using a public database and tissue microarray analysis. Stable cell lines with loss and gain of Rab11a or Rab11b were originally established to investigate their roles in the proliferative, migratory, and invasive capabilities of HNSCC cells. RESULTS: Database analysis revealed a significant association between Rab11b mRNA expression and a favorable patient survival rate in HNSCC. Tissue microarray analysis revealed that Rab11b expression was the highest in normal tissues and gradually decreased across the stages of HNSCC progression. Overexpression of Rab11a or Rab11b resulted in a decrease in epidermal growth factor receptor (EGFR), Epithelial cell adhesion molecule (EpCAM) exosome secretion, and the migratory and invasive potential of HNSCC cells. The knockdown of Rab11a or Rab11b increased EpCAM/CD9 exosome secretion in addition to the migratory and invasive potential of HNSCC cells. CONCLUSIONS: Rab11 suppresses HNSCC by regulating EGFR recycling and EpCAM exosome secretion in HNSCC cells. Our results indicate that Rab11b is a superior prognostic indicator of HNSCC and holds promise for developing novel therapeutic strategies.

    DOI: 10.1016/j.job.2023.11.007

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  • Abr, a Rho-regulating protein, modulates osteoclastogenesis by enhancing lamellipodia formation by interacting with poly(ADP-ribose) glycohydrolase. 査読 国際誌

    Fatima Farhana, Eiko Sakai, Yu Koyanagi, Yu Yamaguchi, Mohammad Ibtehaz Alam, Kuniaki Okamoto, Takayuki Tsukuba

    Molecular biology reports   2023年7月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    BACKGROUND: Osteoclasts are multinucleated bone-resorbing cells formed by the fusion of monocyte/macrophage lineage. During osteoclast differentiation, Rho GTPases are involved in various processes, including cell migration, adhesion, and polarity. However, the role of Rho-regulatory molecules in the regulation of osteoclast differentiation remains unclear. In this study, among these genes, we focused on active breakpoint cluster region-related (Abr) protein that is a multifunctional regulator of Rho GTPases. METHODS AND RESULTS: We examined using knockdown and overexpression experiments in RANKL-stimulated RAW-D macrophages whether Abr regulates osteoclast differentiation and cell morphology. We observed an increase in Abr expression during osteoclast differentiation and identified expression of a variant of the Abr gene in osteoclasts. Knockdown of Abr suppressed osteoclast differentiation and resorption. Abr knockdown markedly inhibited the expression of osteoclast markers, such as Nfatc1, c-fos, Src, and Ctsk in osteoclasts. Conversely, overexpression of Abr enhanced the formation of multinucleated osteoclasts, bone resorption activity, and osteoclast marker gene expression. Moreover, Abr overexpression accelerated lamellipodia formation and induced the formation of well-developed actin in osteoclasts. Importantly, the Abr protein interacted with poly(ADP-ribose) glycohydrolase (PARG) and Rho GTPases, including RhoA, Rac1/2/3, and Cdc42 in osteoclasts. CONCLUSIONS: Taken together, these results indicate that Abr modulates osteoclastogenesis by enhancing lamellipodia formation via its interaction with PARG.

    DOI: 10.1007/s11033-023-08690-0

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  • Rab44 Deficiency Induces Impaired Immune Responses to Nickel Allergy. 査読 国際誌

    Mayuko Noguromi, Yu Yamaguchi, Keiko Sato, Shun Oyakawa, Kuniaki Okamoto, Hiroshi Murata, Takayuki Tsukuba, Tomoko Kadowaki

    International journal of molecular sciences   24 ( 2 )   2023年1月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    Rab44 was recently identified as an atypical Rab GTPase that possesses EF-hand and coiled-coil domains at the N-terminus, and a Rab-GTPase domain at the C-terminus. Rab44 is highly expressed in immune-related cells such as mast cells, macrophages, osteoclasts, and granulocyte-lineage cells in the bone marrow. Therefore, it is speculated that Rab44 is involved in the inflammation and differentiation of immune cells. However, little is known about the role of Rab44 in inflammation. In this study, we showed that Rab44 was upregulated during the early phase of differentiation of M1- and M2-type macrophages. Rab44-deficient mice exhibited impaired tumor necrosis factor alpha and interleukin-10 production after lipopolysaccharide (LPS) stimulation. The number of granulocytes in Rab44-deficient mice was lower, but the lymphocyte count in Rab44-deficient mice was significantly higher than that in wild-type mice after LPS stimulation. Moreover, Rab44-deficient macrophages showed impaired nickel-induced toxicity, and Rab44-deficient mice showed impaired nickel-induced hypersensitivity. Upon nickel hypersensitivity induction, Rab44-deficient mice showed different frequencies of immune cells in the blood and ears. Thus, it is likely that Rab44 is implicated in immune cell differentiation and inflammation, and Rab44 deficiency induces impaired immune responses to nickel allergies.

    DOI: 10.3390/ijms24020994

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  • HSP90 drives the Rab11a‐mediated vesicular transport of the cell surface receptors in osteoclasts 査読 国際誌

    Manh Tien Tran, Yuka Okusha, Kaung Htike, Chiharu Sogawa, Takanori Eguchi, Tomoko Kadowaki, Eiko Sakai, Takayuki Tsukuba, Kuniaki Okamoto

    Cell Biochemistry and Function   40 ( 8 )   838 - 855   2022年12月

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    担当区分:最終著者, 責任著者   記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:Wiley  

    Rab11a, which ubiquitously localizes to early and recycling endosomes, is required for regulating the vesicular transport of cellular cargos. Interestingly, our previous study revealed that Rab11a served as a negative regulator of osteoclastogenesis by facilitating the lysosomal proteolysis of (1) colony-stimulating factor-1 (c-fms) receptor and (2) receptor activator of nuclear factor-κB (RANK) receptor, thereby resulting in inhibition of osteoclast (OC) differentiation, maturation, and bone-resorbing activity. However, the molecular mechanisms of how Rab11a negatively affected osteoclastogenesis were largely unknown. Heat shock protein (HSP90), including two isoforms HSP90α and HSP90β, necessitates the stability, maturation, and activity of a broad range of its clients, and is essentially required for a vast array of signal transduction pathways in nonstressful conditions. Furthermore, cumulative evidence suggests that HSP90 is a vital element of the vesicular transport network. Indeed, our recent study revealed that HSP90, a novel effector protein of Rab11b, modulated Rab11b-mediated osteoclastogenesis. In this study, we also found that Rab11a interacted with both HSP90α and HSP90β in OCs. Upon blockade of HSP90 ATPase activity by a specific inhibitor(17-allylamino-demethoxygeldanamycin), we showed that (1) the ATPase domain of HSP90 was a prerequisite for the interaction between HSP90 and Rab11a, and (2) the interaction of HSP90 to Rab11a sufficiently maintained the inhibitory effects of Rab11a on osteoclastogenesis. Altogether, our findings undoubtedly indicate a novel role of HSP90 in regulating Rab11a-mediated osteoclastogenesis.

    DOI: 10.1002/cbf.3745

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    その他リンク: https://onlinelibrary.wiley.com/doi/full-xml/10.1002/cbf.3745

  • Coronin 1CはGDP特異的Rab44エフェクターでありマクロファージの細胞運動を調節することで破骨細胞形成を制御する

    山口 優, 門脇 知子, 相原 希美, 大山 要, 岡元 邦彰, 坂井 詠子, 筑波 隆幸

    日本生化学会大会プログラム・講演要旨集   95回   1P - 175   2022年11月

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    記述言語:日本語   出版者・発行元:(公社)日本生化学会  

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  • HSP90 drives the Rab11a-Mediated Vesicular Transport of the Cell Surface Receptors in Osteoclasts 査読 国際共著

    Tran MT, Okusha Y, Htike K, Sogawa C, Eguchi E, Kadowaki T, Sakai E, Tsukuba T, Okamoto K.

    Cell Biochem Funct   40 ( 8 )   828 - 855   2022年9月

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    担当区分:最終著者, 責任著者   記述言語:英語   掲載種別:研究論文(学術雑誌)  

    DOI: 10.1002/cbf.3745.

  • 細胞外小胞に搭載されたムーンライティングMMPによるマトリックス制御と転移促進(Metalloproteinase on extracellular vesicles regulates ECM in tumor microenvironment and promotes invasion and metastasis)

    奥舎 有加, タハ・エマン, 陸 彦因, シータ・モナ, 河合 穂高, 十川 千春, 岡元 邦彰, 江口 傑徳

    日本癌学会総会記事   81回   P - 1118   2022年9月

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    記述言語:英語   出版者・発行元:(一社)日本癌学会  

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  • 高転移性癌細胞由来の細胞外小胞に搭載されたMMP3によるCtgf/Ccn2発現調節機能と癌転移促進(Extracellular vesicles enriched with moonlighting metalloproteinase are highly transmissive, Pro-tumorigenic, and trans-activates cellular communication network factor(CCN2/CTGF): CRISPR against cancer)

    奥舎 有加, 江口 傑徳, Tran Manh T., 十川 千春, 吉田 賀弥, 板垣 まみ, Taha Eman A., 小野 喜章, 青山 絵理子, 岡村 裕彦, 小崎 健一, Calderwood Stuart K., 滝川 正春, 岡元 邦彰

    Journal of Oral Biosciences Supplement   2022   35 - 35   2022年9月

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    記述言語:日本語   出版者・発行元:(一社)歯科基礎医学会  

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  • Coronin1C Is a GDP-Specific Rab44 Effector That Controls Osteoclast Formation by Regulating Cell Motility in Macrophages. 査読 国際誌

    Yu Yamaguchi, Tomoko Kadowaki, Nozomi Aibara, Kaname Ohyama, Kuniaki Okamoto, Eiko Sakai, Takayuki Tsukuba

    International journal of molecular sciences   23 ( 12 )   2022年6月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    Osteoclasts are multinucleated bone-resorbing cells that are formed by the fusion of macrophages. Recently, we identified Rab44, a large Rab GTPase, as an upregulated gene during osteoclast differentiation that negatively regulates osteoclast differentiation. However, the molecular mechanisms by which Rab44 negatively regulates osteoclast differentiation remain unknown. Here, we found that the GDP form of Rab44 interacted with the actin-binding protein, Coronin1C, in murine macrophages. Immunoprecipitation experiments revealed that the interaction of Rab44 and Coronin1C occurred in wild-type and a dominant-negative (DN) mutant of Rab44, but not in a constitutively active (CA) mutant of Rab44. Consistent with these findings, the expression of the CA mutant inhibited osteoclast differentiation, whereas that of the DN mutant enhanced this differentiation. Using a phase-contrast microscope, Coronin1C-knockdown osteoclasts apparently impaired multinuclear formation. Moreover, Coronin1C knockdown impaired the migration and chemotaxis of RAW-D macrophages. An in vivo experimental system demonstrated that Coronin1C knockdown suppresses osteoclastogenesis. Therefore, the decreased cell formation and fusion of Coronin1C-depleted osteoclasts might be due to the decreased migration of Coronin1C-knockdown macrophages. These results indicate that Coronin1C is a GDP-specific Rab44 effector that controls osteoclast formation by regulating cell motility in macrophages.

    DOI: 10.3390/ijms23126619

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  • Rab34 plays a critical role as a bidirectional regulator of osteoclastogenesis 査読

    Yunxia Feng, Manh Tien Tran, Yanyin Lu, Kaung Htike, Yuka Okusha, Chiharu Sogawa, Takanori Eguchi, Tomoko Kadowaki, Eiko Sakai, Takayuki Tsukuba, Kuniaki Okamoto

    Cell Biochemistry and Function   2022年3月

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    担当区分:最終著者, 責任著者   掲載種別:研究論文(学術雑誌)   出版者・発行元:Wiley  

    DOI: 10.1002/cbf.3691

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    その他リンク: https://onlinelibrary.wiley.com/doi/full-xml/10.1002/cbf.3691

  • 口腔癌のエクソソームを介した腫瘍進展機序の解明と新規治療戦略の開発に向けて 分子シャペロン搭載エクソソームの可能性

    小野 喜章, 江口 傑徳, 十川 千春, 奥舎 有加, 岡元 邦彰, 佐々木 朗

    口腔組織培養学会誌   31 ( 1 )   33 - 34   2022年3月

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    記述言語:日本語   出版者・発行元:日本口腔組織培養学会  

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  • Resident stroma-secreted chemokine CCL2 governs myeloid-derived suppressor cells in the tumor microenvironment 査読

    May Wathone Oo, Hotaka Kawai, Kiyofumi Takabatake, Shuta Tomida, Takanori Eguchi, Kisho Ono, Qiusheng Shan, Toshiaki Ohara, Saori Yoshida, Haruka Omori, Shintaro Sukegawa, Keisuke Nakano, Kuniaki Okamoto, Akira Sasaki, Hitoshi Nagatsuka

    JCI Insight   2021年12月

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    掲載種別:研究論文(学術雑誌)   出版者・発行元:American Society for Clinical Investigation  

    DOI: 10.1172/jci.insight.148960

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  • A novel role of HSP90 in regulating osteoclastogenesis by abrogating Rab11b-driven transport 査読

    Manh Tien Tran, Yuka Okusha, Yunxia Feng, Chiharu Sogawa, Takanori Eguchi, Tomoko Kadowaki, Eiko Sakai, Takayuki Tsukuba, Kuniaki Okamoto

    Biochimica et Biophysica Acta (BBA) - Molecular Cell Research   1868 ( 10 )   119096 - 119096   2021年9月

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    担当区分:最終著者, 責任著者   記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:Elsevier BV  

    Heat shock protein 90 (HSP90) is a highly conserved molecular chaperone that plays a pivotal role in folding, activating and assembling a variety of client proteins. In addition, HSP90 has recently emerged as a crucial regulator of vesicular transport of cellular proteins. In our previous study, we revealed Rab11b negatively regulated osteoclastogenesis by promoting the lysosomal proteolysis of c-fms and RANK surface receptors via the axis of early endosome-late endosome-lysosomes. In this study, using an in vitro model of osteoclasts differentiated from murine macrophage-like RAW-D cells, we revealed that Rab11b interacted with both HSP90 isoforms, HSP90 alpha (HSP90α) and HSP90 beta (HSP90β), suggesting that Rab11b is an HSP90 client. Using at specific blocker for HSP90 ATPase activity, 17-allylamino-demethoxygeldanamycin (17-AAG), we found that the HSP90 ATPase domain is indispensable for maintaining the interaction between HSP90 and Rab11b in osteoclasts. Nonetheless, its ATPase activity is not required for regulating the turnover of endogenous Rab11b. Interestingly, blocking the interaction between HSP90 and Rab11b by either HSP90-targeting small interfering RNA (siHSP90) or 17-AAG abrogated the inhibitory effects of Rab11b on osteoclastogenesis by suppressing the Rab11b-mediated transport of c-fms and RANK surface receptors to lysosomes via the axis of early endosome-late endosome-lysosomes, alleviating the Rab11b-mediated proteolysis of these surface receptors in osteoclasts. Based on our observations, we propose a HSP90/Rab11b-mediated regulatory mechanism for osteoclastogenesis by directly modulating the c-fms and RANK surface receptors in osteoclasts, thereby contributing to the maintenance of bone homeostasis.

    DOI: 10.1016/j.bbamcr.2021.119096

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  • Exosome-Based Molecular Transfer Activity of Macrophage-Like Cells Involves Viability of Oral Carcinoma Cells: Size Exclusion Chromatography and Concentration Filter Method 査読 国際誌

    Yanyin Lu, Takanori Eguchi, Chiharu Sogawa, Eman A. Taha, Manh Tien Tran, Toshiki Nara, Penggong Wei, Shiro Fukuoka, Takuya Miyawaki, Kuniaki Okamoto

    Cells   10 ( 6 )   1328 - 1328   2021年5月

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    担当区分:最終著者   記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:MDPI AG  

    Extracellular vesicles (EV) heterogeneity is a crucial issue in biology and medicine. In addition, tumor-associated macrophages are key components in cancer microenvironment and immunology. We developed a combination method of size exclusion chromatography and concentration filters (SEC-CF) and aimed to characterize different EV types by their size, cargo types, and functions. A human monocytic leukemia cell line THP-1 was differentiated to CD14-positive macrophage-like cells by stimulation with PMA (phorbol 12-myristate 13-acetate) but not M1 or M2 types. Using the SEC-CF method, the following five EV types were fractionated from the culture supernatant of macrophage-like cells: (i) rare large EVs (500–3000 nm) reminiscent of apoptosomes, (ii) EVs (100–500 nm) reminiscent of microvesicles (or microparticles), (iii) EVs (80–300 nm) containing CD9-positive large exosomes (EXO-L), (iv) EVs (20–200 nm) containing unidentified vesicles/particles, and (v) EVs (10–70 nm) containing CD63/HSP90-positive small exosomes (EXO-S) and particles. For a molecular transfer assay, we developed a THP-1-based stable cell line producing a GFP-fused palmitoylation signal (palmGFP) associated with the membrane. The THP1/palmGFP cells were differentiated into macrophages producing palmGFP-contained EVs. The macrophage/palmGFP-secreted EXO-S and EXO-L efficiently transferred the palmGFP to receiver human oral carcinoma cells (HSC-3/palmTomato), as compared to other EV types. In addition, the macrophage-secreted EXO-S and EXO-L significantly reduced the cell viability (ATP content) in oral carcinoma cells. Taken together, the SEC-CF method is useful for the purification of large and small exosomes with higher molecular transfer activities, enabling efficient molecular delivery to target cells.

    DOI: 10.3390/cells10061328

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  • Macrophage-Derived Small Exosomes: Efficient Transmission and Cytotoxicity to Cancer Cells 査読

    Yanyin Lu, Takanori Eguchi, Chiharu Sogawa, Eman A. Taha, Manh Tien Tran, Toshiki Nara, Penggong Wei, Shiro Fukuoka, Takuya Miyawaki, Kuniaki Okamoto

    2021年4月

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    担当区分:最終著者   出版者・発行元:MDPI AG  

    Tumor-associated macrophages are a key component in the tumor microenvironment, secreting extracellular vesicles (EVs) such as exosomes and other various factors for intercellular communication. However, macrophage-derived EVs heterogeneity and their cytotoxicity to cancer cells has not been well understood. Here, we aimed to separately isolate various types of macro-phage-EVs by size exclusion chromatography (SEC) method and investigate EV transmission and cytotoxicity to oral cancer cells. For fluorescence-labeling of cellular and EV membranes, palmitoylation signal-fused GFP and tdTomato were expressed in THP-1 monocytic cells and HSC-3 oral cancer cells, respectively. We found that fluorescence-labeled EVs secreted by macrophages were highly transmissive to oral cancer cells than those from parental monocytic cells. In a co-culture system and conditioned medium (CM), a macrophage-secreted unidentified factor was cytotoxic to oral cancer cells. We fractionated macrophage-derived EVs by the SEC method and performed western blotting to characterize various EV types. Three fractions were characterized: small exosomes (EXO-S: < 50 nm) fraction containing HSP90α, HSP90β, CD63 (EV marker) and β-actin; large exosomes (EXO-L: 50-200 nm) fraction containing CD9 (EV marker) and HSP90β; large EVs (100-500 nm) fraction. Notably, the macrophage-derived small exosomes fraction was cytotoxic to oral cancer cells, while large exosomes and large EVs were not. There-fore, it was implicated that macrophage-derived small exosomes are cytotoxic with high trans-mission potential to cancer cells.

    DOI: 10.20944/preprints202104.0464.v1

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  • Rab44 isoforms similarly promote lysosomal exocytosis, but exhibit differential localization in mast cells 査読

    Tomoko Kadowaki, Yu Yamaguchi, Kohei Ogawa, Mitsuko Tokuhisa, Kuniaki Okamoto, Takayuki Tsukuba

    FEBS Open Bio   11 ( 4 )   1165 - 1185   2021年4月

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    掲載種別:研究論文(学術雑誌)   出版者・発行元:Wiley  

    DOI: 10.1002/2211-5463.13133

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    その他リンク: https://onlinelibrary.wiley.com/doi/full-xml/10.1002/2211-5463.13133

  • Gel-Free 3D Tumoroids with Stem Cell Properties Modeling Drug Resistance to Cisplatin and Imatinib in Metastatic Colorectal Cancer. 査読 国際誌

    Chiharu Sogawa, Takanori Eguchi, Yuri Namba, Yuka Okusha, Eriko Aoyama, Kazumi Ohyama, Kuniaki Okamoto

    Cells   10 ( 2 )   2021年2月

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    担当区分:最終著者   記述言語:英語   掲載種別:研究論文(学術雑誌)  

    Researchers have developed several three-dimensional (3D) culture systems, including spheroids, organoids, and tumoroids with increased properties of cancer stem cells (CSCs), also called cancer-initiating cells (CICs). Drug resistance is a crucial issue involving recurrence in cancer patients. Many studies on anti-cancer drugs have been reported using 2D culture systems, whereas 3D cultured tumoroids have many advantages for assessing drug sensitivity and resistance. Here, we aimed to investigate whether Cisplatin (a DNA crosslinker), Imatinib (a multiple tyrosine kinase inhibitor), and 5-Fluorouracil (5-FU: an antimetabolite) alter the tumoroid growth of metastatic colorectal cancer (mCRC). Gene expression signatures of highly metastatic aggregative CRC (LuM1 cells) vs. low-metastatic, non-aggregative CRC (Colon26 and NM11 cells) were analyzed using microarray. To establish a 3D culture-based multiplexing reporter assay system, LuM1 was stably transfected with the Mmp9 promoter-driven ZsGreen fluorescence reporter gene, which was designated as LuM1/m9 cells and cultured in NanoCulture Plate®, a gel-free 3D culture device. LuM1 cells highly expressed mRNA encoding ABCG2 (a drug resistance pump, i.e., CSC/CIC marker), other CSC/CIC markers (DLL1, EpCAM, podoplanin, STAT3/5), pluripotent stem cell markers (Sox4/7, N-myc, GATA3, Nanog), and metastatic markers (MMPs, Integrins, EGFR), compared to the other two cell types. Hoechst efflux stem cell-like side population was increased in LuM1 (7.8%) compared with Colon26 (2.9%), both of which were markedly reduced by verapamil treatment, an ABCG2 inhibitor. Smaller cell aggregates of LuM1 were more sensitive to Cisplatin (at 10 μM), whereas larger tumoroids with increased ABCG2 expression were insensitive. Notably, Cisplatin (2 μM) and Imatinib (10 μM) at low concentrations significantly promoted tumoroid formation (cell aggregation) and increased Mmp9 promoter activity in mCRC LuM1/m9, while not cytotoxic to them. On the other hand, 5-FU significantly inhibited tumoroid growth, although not completely. Thus, drug resistance in cancer with increased stem cell properties was modeled using the gel-free 3D cultured tumoroid system. The tumoroid culture is useful and easily accessible for the assessment of drug sensitivity and resistance.

    DOI: 10.3390/cells10020344

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  • Liquid-based 3D Cultured Tumoroids Modeling Resistance to Cisplatin and Imatinib in Metastatic Colorectal Cancer 査読

    Chiharu Sogawa, Takanori Eguchi, Yuri Namba, Eriko Aoyama, Kazumi Ohyama, Kuniaki Okamoto

    2020年12月

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    担当区分:最終著者   出版者・発行元:MDPI AG  

    Researchers have developed and used several three-dimensional (3D) culture systems, including spheroids, organoids, and tumoroids. Drug resistance is a crucial issue involving recurrence in cancer patients. Many studies on anticancer drugs have been done in 2D culture systems, where-as 3D cultured tumoroids have many advantages for assessing drug sensitivity and resistance. Here, we aim to investigate whether Cisplatin (a DNA crosslinker), Imatinib (a multiple tyro-sine kinase inhibitor), and 5-Fluorouracil (5-FU: an antimetabolite) alter tumoroid growth of metastatic colorectal cancer (mCRC). To establish a liquid-based 3D multiplexing reporter assay system, LuM1 (a murine mCRC cell line) was stably transfected with the Mmp9 promoter-driven ZsGreen reporter gene, which was designated as LuM1/m9 cells and cultured in NanoCulture Plate (NCP), a 3D culture device. The larger tumoroids were not sensitive to Cisplatin and ex-pressed ABCG2 (a marker of cancer stem cells, a.k.a. a drug efflux transporter), whereas smaller cell-aggregates were more sensitive to Cisplatin. Both Imatinib and Cisplatin significantly in-creased tumoroid growth (larger than 300 μm2) and Mmp9 promoter activity and were not cytotoxic to the mCRC tumoroids. On the other hand, 5-FU was cytotoxic to the tumoroids and significantly inhibited tumoroid growth, although not completely. Thus, platinum resistance and imatinib resistance in mCRC were modeled using the liquid-based 3D cultured tumoroid system. The tumoroid culture is useful and easily accessible for the assessment of drug sensitivity and resistance.

    DOI: 10.20944/preprints202012.0582.v1

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  • The Inhibitory Role of Rab11b in Osteoclastogenesis through Triggering Lysosome-Induced Degradation of c-Fms and RANK Surface Receptors 査読

    Manh Tien Tran, Yuka Okusha, Yunxia Feng, Masatoshi Morimatsu, Penggong Wei, Chiharu Sogawa, Takanori Eguchi, Tomoko Kadowaki, Eiko Sakai, Hirohiko Okamura, Keiji Naruse, Takayuki Tsukuba, Kuniaki Okamoto

    International Journal of Molecular Sciences   21 ( 24 )   9352 - 9352   2020年12月

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    担当区分:最終著者, 責任著者   掲載種別:研究論文(学術雑誌)   出版者・発行元:MDPI AG  

    Rab11b, abundantly enriched in endocytic recycling compartments, is required for the establishment of the machinery of vesicle trafficking. Yet, no report has so far characterized the biological function of Rab11b in osteoclastogenesis. Using in vitro model of osteoclasts differentiated from murine macrophages like RAW-D cells or bone marrow-derived macrophages, we elucidated that Rab11b served as an inhibitory regulator of osteoclast differentiation sequentially via (i) abolishing surface abundance of RANK and c-Fms receptors; and (ii) attenuating nuclear factor of activated T-cells c1 (NFATc-1) upstream signaling cascades, following RANKL stimulation. Rab11b was localized in early and late endosomes, Golgi complex, and endoplasmic reticulum; moreover, its overexpression enlarged early and late endosomes. Upon inhibition of lysosomal function by a specific blocker, chloroquine (CLQ), we comprehensively clarified a novel function of lysosomes on mediating proteolytic degradation of c-Fms and RANK surface receptors, drastically ameliorated by Rab11b overexpression in RAW-D cell-derived osteoclasts. These findings highlight the key role of Rab11b as an inhibitor of osteoclastogenesis by directing the transport of c-Fms and RANK surface receptors to lysosomes for degradation via the axis of early endosomes-late endosomes-lysosomes, thereby contributing towards the systemic equilibrium of the bone resorption phase.

    DOI: 10.3390/ijms21249352

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  • Rab11A Functions as a Negative Regulator of Osteoclastogenesis through Dictating Lysosome-Induced Proteolysis of c-fms and RANK Surface Receptors 査読

    Yuka Okusha, Manh Tien Tran, Mami Itagaki, Chiharu Sogawa, Takanori Eguchi, Tatsuo Okui, Tomoko Kadowaki, Eiko Sakai, Takayuki Tsukuba, Kuniaki Okamoto

    Cells   9 ( 11 )   2384 - 2384   2020年10月

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    担当区分:最終著者, 責任著者   掲載種別:研究論文(学術雑誌)   出版者・発行元:MDPI AG  

    Osteoclast differentiation and activity are controlled by two essential cytokines, macrophage colony-stimulating factor (M-CSF) and the receptor activator of nuclear factor-κB ligand (RANKL). Rab11A GTPase, belonging to Rab11 subfamily representing the largest branch of Ras superfamily of small GTPases, has been identified as one of the crucial regulators of cell surface receptor recycling. Nevertheless, the regulatory role of Rab11A in osteoclast differentiation has been completely unknown. In this study, we found that Rab11A was strongly upregulated at a late stage of osteoclast differentiation derived from bone marrow-derived macrophages (BMMs) or RAW-D murine osteoclast precursor cells. Rab11A silencing promoted osteoclast formation and significantly increased the surface levels of c-fms and receptor activator of nuclear factor-κB (RANK) while its overexpression attenuated osteoclast formation and the surface levels of c-fms and RANK. Using immunocytochemical staining for tracking Rab11A vesicular localization, we observed that Rab11A was localized in early and late endosomes, but not lysosomes. Intriguingly, Rab11A overexpression caused the enhancement of fluorescent intensity and size-based enlargement of early endosomes. Besides, Rab11A overexpression promoted lysosomal activity via elevating the endogenous levels of a specific lysosomal protein, LAMP1, and two key lysosomal enzymes, cathepsins B and D in osteoclasts. More importantly, inhibition of the lysosomal activity by chloroquine, we found that the endogenous levels of c-fms and RANK proteins were enhanced in osteoclasts. From these observations, we suggest a novel function of Rab11A as a negative regulator of osteoclastogenesis mainly through (i) abolishing the surface abundance of c-fms and RANK receptors, and (ii) upregulating lysosomal activity, subsequently augmenting the degradation of c-fms and RANK receptors, probably via the axis of early endosomes–late endosomes–lysosomes in osteoclasts.

    DOI: 10.3390/cells9112384

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  • 発生と疾患にみる新たな細胞間コミュニケーション オルガノイドと細胞外小胞を癌研究に応用する

    江口 傑徳, 十川 千春, 岡元 邦彰

    Journal of Oral Biosciences Supplement   2020   118 - 118   2020年9月

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    記述言語:日本語   出版者・発行元:(一社)歯科基礎医学会  

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  • Outer membrane vesicles of Porphyromonas gingivalis attenuate insulin sensitivity by delivering gingipains to the liver 査読

    Mariko Seyama, Kaya Yoshida, Kayo Yoshida, Natsumi Fujiwara, Kisho Ono, Takanori Eguchi, Hotaka Kawai, Jiajie Guo, Yao Weng, Yuan Haoze, Kenta Uchibe, Mika Ikegame, Akira Sasaki, Hitoshi Nagatsuka, Kuniaki Okamoto, Hirohiko Okamura, Kazumi Ozaki

    Biochimica et Biophysica Acta - Molecular Basis of Disease   1866 ( 6 )   2020年6月

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    掲載種別:研究論文(学術雑誌)  

    Outer membrane vesicles (OMVs) are nanosized particles derived from the outer membrane of gram-negative bacteria. Oral bacterium Porphyromonas gingivalis (Pg) is known to be a major pathogen of periodontitis that contributes to the progression of periodontal disease by releasing OMVs. The effect of Pg OMVs on systemic diseases is still unknown. To verify whether Pg OMVs affect the progress of diabetes mellitus, we analyzed the cargo proteins of vesicles and evaluated their effect on hepatic glucose metabolism. Here, we show that Pg OMVs were equipped with Pg-derived proteases gingipains and translocated to the liver in mice. In these mice, the hepatic glycogen synthesis in response to insulin was decreased, and thus high blood glucose levels were maintained. Pg OMVs also attenuated the insulin-induced Akt/glycogen synthase kinase-3 β (GSK-3β) signaling in a gingipain-dependent fashion in hepatic HepG2 cells. These results suggest that the delivery of gingipains mediated by Pg OMV elicits changes in glucose metabolisms in the liver and contributes to the progression of diabetes mellitus.

    DOI: 10.1016/j.bbadis.2020.165731

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  • Dimethyl fumarate prevents osteoclastogenesis by decreasing NFATc1 expression, inhibiting of erk and p38 MAPK phosphorylation, and suppressing of HMGB1 release 査読

    Tsuyoshi Nishioku, Momomi Kawamoto, Ryuya Okizono, Eiko Sakai, Kuniaki Okamoto, Takayuki Tsukuba

    Biochemical and Biophysical Research Communications   2020年6月

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    掲載種別:研究論文(学術雑誌)   出版者・発行元:Elsevier BV  

    DOI: 10.1016/j.bbrc.2020.05.088

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  • Knockout of MMP3 Weakens Solid Tumor Organoids and Cancer Extracellular Vesicles 査読

    Eman Taha, Chiharu Sogawa, Yuka Okusha, Hotaka Kawai, May Oo, Abdellatif Elseoudi, Yanyin Lu, Hitoshi Nagatsuka, Satoshi Kubota, Ayano Satoh, Kuniaki Okamoto, Takanori Eguchi

    Cancers   12 ( 5 )   1260 - 1260   2020年5月

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    掲載種別:研究論文(学術雑誌)   出版者・発行元:MDPI AG  

    The tumor organoid (tumoroid) model in three-dimensional (3D) culture systems has been developed to reflect more closely the in vivo tumors than 2D-cultured tumor cells. Notably, extracellular vesicles (EVs) are efficiently collectible from the culture supernatant of gel-free tumoroids. Matrix metalloproteinase (MMP) 3 is a multi-functional factor playing crucial roles in tumor progression. However, roles of MMP3 within tumor growth and EVs have not unveiled. Here, we investigated the protumorigenic roles of MMP3 on integrities of tumoroids and EVs. We generated MMP3-knockout (KO) cells using the CRISPR/Cas9 system from rapidly metastatic LuM1 tumor cells. Moreover, we established fluorescent cell lines with palmitoylation signal-fused fluorescent proteins (tdTomato and enhanced GFP). Then we confirmed the exchange of EVs between cellular populations and tumoroids. LuM1-tumoroids released large EVs (200–1000 nm) and small EVs (50–200 nm) while the knockout of MMP3 resulted in the additional release of broken EVs from tumoroids. The loss of MMP3 led to a significant reduction in tumoroid size and the development of the necrotic area within tumoroids. MMP3 and CD9 (a category-1 EV marker tetraspanin protein) were significantly down-regulated in MMP3-KO cells and their EV fraction. Moreover, CD63, another member of the tetraspanin family, was significantly reduced only in the EVs fractions of the MMP3-KO cells compared to their counterpart. These weakened phenotypes of MMP3-KO were markedly rescued by the addition of MMP3-rich EVs or conditioned medium (CM) collected from LuM1-tumoroids, which caused a dramatic rise in the expression of MMP3, CD9, and Ki-67 (a marker of proliferating cells) in the MMP3-null/CD9-low tumoroids. Notably, MMP3 enriched in tumoroids-derived EVs and CM deeply penetrated recipient MMP3-KO tumoroids, resulting in a remarkable enlargement of solid tumoroids, while MMP3-null EVs did not. These data demonstrate that EVs can mediate molecular transfer of MMP3, resulting in increasing the proliferation and tumorigenesis, indicating crucial roles of MMP3 in tumor progression.

    DOI: 10.3390/cancers12051260

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  • The large GTPase Rab44 regulates granule exocytosis in mast cells and IgE-mediated anaphylaxis. 査読 国際誌

    Tomoko Kadowaki, Yu Yamaguchi, Mizuho A Kido, Takaya Abe, Kohei Ogawa, Mitsuko Tokuhisa, Weiqi Gao, Kuniaki Okamoto, Hiroshi Kiyonari, Takayuki Tsukuba

    Cellular & molecular immunology   17 ( 12 )   1287 - 1289   2020年4月

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  • Extracellular vesicles enriched with moonlighting metalloproteinase are highly transmissive, pro-tumorigenic, and trans-activates cellular communication network factor (Ccn2/ctgf): Crispr against cancer 査読

    Yuka Okusha, Takanori Eguchi, Manh T. Tran, Chiharu Sogawa, Kaya Yoshida, Mami Itagaki, Eman A. Taha, Kisho Ono, Eriko Aoyama, Hirohiko Okamura, Ken Ichi Kozaki, Stuart K. Calderwood, Masaharu Takigawa, Kuniaki Okamoto

    Cancers   12 ( 4 )   2020年4月

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    掲載種別:研究論文(学術雑誌)  

    © 2020 by the authors. Licensee MDPI, Basel, Switzerland. Matrix metalloproteinase 3 (MMP3) plays multiple roles in extracellular proteolysis as well as intracellular transcription, prompting a new definition of moonlighting metalloproteinase (MMP), according to a definition of protein moonlighting (or gene sharing), a phenomenon by which a protein can perform more than one function. Indeed, connective tissue growth factor (CTGF, aka cellular communication network factor 2 (CCN2)) is transcriptionally induced as well as cleaved by MMP3. Moreover, several members of the MMP family have been found within tumor-derived extracellular vesicles (EVs). We here investigated the roles of MMP3-rich EVs in tumor progression, molecular transmission, and gene regulation. EVs derived from a rapidly metastatic cancer cell line (LuM1) were enriched in MMP3 and a C-terminal half fragment of CCN2/CTGF. MMP3-rich, LuM1-derived EVs were disseminated to multiple organs through body fluid and were pro-tumorigenic in an allograft mouse model, which prompted us to define LuM1-EVs as oncosomes in the present study. Oncosome-derived MMP3 was transferred into recipient cell nuclei and thereby trans-activated the CCN2/CTGF promoter, and induced CCN2/CTGF production in vitro. TRENDIC and other cis-elements in the CCN2/CTGF promoter were essential for the oncosomal responsivity. The CRISPR/Cas9-mediated knockout of MMP3 showed significant anti-tumor effects such as the inhibition of migration and invasion of tumor cells, and a reduction in CCN2/CTGF promoter activity and fragmentations in vitro. A high expression level of MMP3 or CCN2/CTGF mRNA was prognostic and unfavorable in particular types of cancers including head and neck, lung, pancreatic, cervical, stomach, and urothelial cancers. These data newly demonstrate that oncogenic EVs-derived MMP is a transmissive trans-activator for the cellular communication network gene and promotes tumorigenesis at distant sites.

    DOI: 10.3390/cancers12040881

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  • Cell Stress Induced Stressome Release Including Damaged Membrane Vesicles and Extracellular HSP90 by Prostate Cancer Cells. 査読 国際誌

    Takanori Eguchi, Chiharu Sogawa, Kisho Ono, Masaki Matsumoto, Manh Tien Tran, Yuka Okusha, Benjamin J Lang, Kuniaki Okamoto, Stuart K Calderwood

    Cells   9 ( 3 )   2020年3月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    Tumor cells exhibit therapeutic stress resistance-associated secretory phenotype involving extracellular vesicles (EVs) such as oncosomes and heat shock proteins (HSPs). Such a secretory phenotype occurs in response to cell stress and cancer therapeutics. HSPs are stress-responsive molecular chaperones promoting proper protein folding, while also being released from cells with EVs as well as a soluble form known as alarmins. We have here investigated the secretory phenotype of castration-resistant prostate cancer (CRPC) cells using proteome analysis. We have also examined the roles of the key co-chaperone CDC37 in the release of EV proteins including CD9 and epithelial-to-mesenchymal transition (EMT), a key event in tumor progression. EVs derived from CRPC cells promoted EMT in normal prostate epithelial cells. Some HSP family members and their potential receptor CD91/LRP1 were enriched at high levels in CRPC cell-derived EVs among over 700 other protein types found by mass spectrometry. The small EVs (30-200 nm in size) were released even in a non-heated condition from the prostate cancer cells, whereas the EMT-coupled release of EVs (200-500 nm) and damaged membrane vesicles with associated HSP90α was increased after heat shock stress (HSS). GAPDH and lactate dehydrogenase, a marker of membrane leakage/damage, were also found in conditioned media upon HSS. During this stress response, the intracellular chaperone CDC37 was transcriptionally induced by heat shock factor 1 (HSF1), which activated the CDC37 core promoter, containing an interspecies conserved heat shock element. In contrast, knockdown of CDC37 decreased EMT-coupled release of CD9-containing vesicles. Triple siRNA targeting CDC37, HSP90α, and HSP90β was required for efficient reduction of this chaperone trio and to reduce tumorigenicity of the CRPC cells in vivo. Taken together, we define "stressome" as cellular stress-induced all secretion products, including EVs (200-500 nm), membrane-damaged vesicles and remnants, and extracellular HSP90 and GAPDH. Our data also indicated that CDC37 is crucial for the release of vesicular proteins and tumor progression in prostate cancer.

    DOI: 10.3390/cells9030755

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  • Antiparkinson Drug Benztropine Suppresses Tumor Growth, Circulating Tumor Cells, and Metastasis by Acting on SLC6A3/DAT and Reducing STAT3 査読

    Chiharu Sogawa, Takanori Eguchi, Manh Tien Tran, Masayuki Ishige, Kilian Trin, Yuka Okusha, Eman Ahmed Taha, Yanyin Lu, Hotaka Kawai, Norio Sogawa, Masaharu Takigawa, Stuart K. Calderwood, Kuniaki Okamoto, Ken-ichi Kozaki

    Cancers   12 ( 2 )   523 - 523   2020年2月

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    掲載種別:研究論文(学術雑誌)   出版者・発行元:MDPI AG  

    Tumor growth, progression, and therapy resistance are crucial factors in the prognosis of cancer. The properties of three-dimensional (3D) tumor-like organoids (tumoroids) more closely resemble in vivo tumors compared to two-dimensionally cultured cells and are therefore effectively used for assays and drug screening. We here established a repurposed drug for novel anticancer research and therapeutics using a 3D tumoroid-based screening system. We screened six pharmacologically active compounds by using an original tumoroid-based multiplex phenotypic screening system with a matrix metalloproteinase 9 (MMP9) promoter-driven fluorescence reporter for the evaluation of both tumoroid formation and progression. The antiparkinson drug benztropine was the most effective compound uncovered by the screen. Benztropine significantly inhibited in vitro tumoroid formation, cancer cell survival, and MMP9 promoter activity. Benztropine also reduced the activity of oncogenic signaling transducers and trans-activators for MMP9, including STAT3, NF-κB, and β-catenin, and the properties of cancer stem cells/cancer-initiating cells. Benztropine and GBR-12935 directly targeted the dopamine transporter DAT/SLC6A3, whose genetic alterations such as amplification were correlated with poor prognosis for cancer patients. Benztropine also inhibited the tumor growth, circulating tumor cell (CTC) number, and rate of metastasis in a tumor allograft model in mice. In conclusion, we propose the repurposing of benztropine for anticancer research and therapeutics that can suppress tumor progression, CTC, and metastasis of aggressive cancers by reducing key pro-tumorigenic factors.

    DOI: 10.3390/cancers12020523

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  • Extracellular Oncosomes Rich in Moonlighting Metalloproteinase (MMP3) Are Transmissive, Pro-Tumorigenic, and Induces Cellular Communication Network Factor 2 (CCN2/CTGF): CRISPR against Cancer 査読

    Yuka Okusha, Takanori Eguchi, Manh Tien Tran, Chiharu Sogawa, Kaya Yoshida, Mami Itagaki, Eman Ahmed Taha, Kisho Ono, Eriko Aoyama, Hirohiko Okamura, Ken-ichi Kozaki, Stuart K. Calderwood, Masaharu Takigawa, Kuniaki Okamoto

    Preprints   doi: 10.20944/preprints202002.0281.v1   2020年2月

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    担当区分:最終著者   記述言語:英語   掲載種別:研究論文(学術雑誌)  

    DOI: 10.20944/preprints202002.0281.v1

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  • CDC37 and HSP90 are Essential for Stressome Release and Tumor Progression in Resistant Prostate Cancer 査読

    Takanori Eguchi, Chiharu Sogawa, Kisho Ono, Masaki Matsumoto, Manh Tien Tran, Yuka Okusha, Benjamin Lang, Kuniaki Okamoto, Stuart Calderwood

    2020年2月

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    出版者・発行元:MDPI AG  

    Tumor cells exhibit a resistance-associated secretory phenotype involving extracellular vesicles (EVs) and heat shock proteins (HSPs). This response occurs in response to cell stress and cancer therapeutics. HSPs are stress-responsive molecular chaperones promoting proper protein folding, while also being released from cells with EVs as well as in free form as alarmins. We have here investigated the secretory phenotype of castration-resistant prostate cancer (CRPC) cells using proteome analysis. We have also examined the roles of the key co-chaperone CDC37 in stressome release, epithelial-to-mesenchymal transition (EMT), and tumor progression. A number of HSP family members and their common receptor CD91/LRP1 were enriched at high levels in CRPC cell-derived EVs among over 700 other protein species. The small EVs (30 to 200 nm in size, potentially exosomes) were released even in a non-heated condition from the prostate cancer cells, whereas EMT-coupled release of EVs (200 to 500 nm, likely ectosomes) with associated HSP90α was increased after heat shock stress (HSS). Lactate dehydrogenase, a marker of membrane leakage/damage of cells, was also released upon HSS from the prostate cancer cells. During this stress response, intracellular CDC37 was also transcriptionally inducible by heat shock factor 1, and knockdown of CDC37 decreased EMT-coupled release of EVs. Triple knockdown of CDC37, HSP90α, and HSP90β was required for efficient reduction of the chaperone trio and to reduce tumorigenicity of the CRPC cells in vivo. Taken together, the data indicated that CDC37 and HSP90 are essential for stressome release and for tumorigenesis in resistant cancer.

    DOI: 10.20944/preprints202002.0148.v1

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  • Triple knockdown of CDC37, HSP90-alpha and HSP90-beta diminishes extracellular vesicles-driven malignancy events and macrophage M2 polarization in oral cancer 査読

    Kisho Ono, Chiharu Sogawa, Hotaka Kawai, Manh Tien Tran, Eman A. Taha, Yanyin Lu, May Wathone Oo, Yuka Okusha, Hirohiko Okamura, Soichiro Ibaragi, Masaharu Takigawa, Ken-Ichi Kozaki, Hitoshi Nagatsuka, Akira Sasaki, Kuniaki Okamoto, Stuart K. Calderwood, Takanori Eguchi

    Journal of Extracellular Vesicles   9 ( 1 )   1769373 - 1769373   2020年1月

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    掲載種別:研究論文(学術雑誌)   出版者・発行元:Informa UK Limited  

    DOI: 10.1080/20013078.2020.1769373

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  • (-)-Epigallocatechin-3-gallate inhibits RANKL-induced osteoclastogenesis via downregulation of NFATc1 and suppression of HO-1-HMGB1-RAGE pathway. 査読

    Tsuyoshi Nishioku, Toshiki Kubo, Tsukushi Kamada, Kuniaki Okamoto, Takayuki Tsukuba, Takuhiro Uto, Yukihiro Shoyama

    Biomedical research (Tokyo, Japan)   41 ( 6 )   269 - 277   2020年

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    Osteoporosis disturbs the balance of bone metabolism, and excessive bone resorption causes a decrease in bone density, thus increasing the risk of fracture. (-)-Epigallocatechin-3-gallate (EGCG) is the most abundant catechin contained in green tea. EGCG has a variety of pharmacological activities. Recently, it was reported that EGCG inhibits osteoclast differentiation, but the details of the mechanism underlying the EGCG-mediated suppression of osteoclastogenesis are unknown. In this study, we investigated the effects of EGCG on several signaling pathways in osteoclastogenesis. EGCG suppressed the expression of the nuclear factor of activated T cells cytoplasmic-1 (NFATc1), the master regulator of osteoclastogenesis. EGCG decreased the expression of cathepsin K, c-Src, and ATP6V0d2 and suppressed bone resorption. We also found that EGCG upregulated heme oxygenase-1 (HO-1) and suppressed the extracellular release of high-mobility group box 1 (HMGB1). In addition, EGCG decreased the expression of the receptor for advanced glycation end products (RAGE), which is the receptor of HMGB1, in osteoclastogenesis. In summary, our study showed that EGCG could inhibit osteoclast differentiation through the downregulation of NFATc1 and the suppression of the HO-1-HMGB1-RAGE pathway. EGCG might have the potential to be a lead compound that suppresses bone resorption in the treatment of osteoporosis.

    DOI: 10.2220/biomedres.41.269

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  • Exosome Release of Drugs: Coupling with Epithelial-Mesenchymal Transition 査読

    Takanori Eguchi, Kisho Ono, Stuart Calderwood, Kuniaki Okamoto

    2019年12月

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    担当区分:最終著者   出版者・発行元:MDPI AG  

    Extracellular vesicles (EVs), such as exosomes or oncosomes are released with molecules unfavorable for survival from cells. In addition, accumulating evidence has shown that tumor cells often eject anti-cancer drugs such as chemotherapeutics and targeted drugs within EVs, a novel mechanism of drug resistance. The EV-releasing, drug resistance phenotype is often coupled with cellular dedifferentiation and transformation, cells undergoing epithelial-mesenchymal transition (EMT) and taking on a cancer stem cell phenotype. Recent studies have shown that the release of EVs is also involved in immunosuppression. The concept of the resistance-associated secretory phenotype (RASP) is reviewed herein.

    DOI: 10.20944/preprints201912.0386.v1

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  • オルガノイドを応用したドラッグリポジショニング開発

    十川 千春, 江口 傑徳, Tran Tien Manh, 石毛 真行, 河合 穂高, 奥舎 有加, 中野 敬介, 十川 紀夫, 小崎 健一, 岡元 邦彰

    岡山歯学会雑誌   38 ( 2 )   89 - 89   2019年12月

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    記述言語:日本語   出版者・発行元:岡山歯学会  

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  • 新しい腫瘍オルガノイド多元評価システムの開発

    十川 チハル, 江口 傑徳, 大山 和美, 奥舎 有加, 中野 敬介, 十川 紀夫, 岡元 邦彰

    Journal of Oral Biosciences Supplement   2019   165 - 165   2019年10月

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    記述言語:日本語   出版者・発行元:(一社)歯科基礎医学会  

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  • Tumor Angiogenic Inhibition Triggered Necrosis (TAITN) in Oral Cancer. 査読

    Yoshida S, Kawai H, Eguchi T, Sukegawa S, Oo MW, Anqi C, Takabatake K, Nakano K, Okamoto K, Nagatsuka H

    Cells   8 ( 7 )   2019年7月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

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  • KBTBD11, a novel BTB-Kelch protein, is a negative regulator of osteoclastogenesis through controlling Cullin3-mediated ubiquitination of NFATc1. 査読

    Narahara S, Sakai E, Kadowaki T, Yamaguchi Y, Narahara H, Okamoto K, Asahina I, Tsukuba T

    Scientific Report   9 ( 1 )   3523   2019年3月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

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  • A reporter system evaluates tumorigenesis, metastasis, β-catenin/MMP regulation, and druggability. 招待 査読

    Sogawa C, Eguchi T, C, i, auth, Corresponding author, Okusha Y, Ono K, Ohyama K, Iizuka M, Kawasaki R, Hamada Y, Takigawa M, Sogawa N, Okamoto K, Kozaki K

    Tissue Engineering Part A.   2019年2月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

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  • Nicotine promotes lymph node metastasis and cetuximab resistance in head and neck squamous cell carcinoma. 査読

    Shimizu R, Ibaragi S, Eguchi T, Kuwajima D, Kodama S, Nishioka T, Okui T, Obata K, Takabatake K, Kawai H, Ono K, Okamoto K, Nagatsuka H, Sasaki A

    International journal of oncology   54 ( 1 )   283 - 294   2019年1月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    DOI: 10.3892/ijo.2018.4631

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  • 脂質・コレステロール排出ポンプABCG1標的による腫瘍内エクソソーム蓄積および腫瘍縮小

    江口 傑徳, 十川 千春, 難波 友里, 奥舎 有加, 河合 穂高, 小野 喜章, 板垣 まみ, 村上 純, 大山 和美, 浅海 淳一, 岡元 邦彰

    日本薬理学会年会要旨集   92   2-YIA-26   2019年

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    記述言語:日本語   出版者・発行元:公益社団法人 日本薬理学会  

    The ATP-binding cassette transporter G1 (ABCG1) is a cholesterol lipid efflux pump whose role in tumor growth has been largely unknown. Our transcriptomics revealed that ABCG1 was powerfully expressed in rapidly metastatic, aggregative colon cancer cells, in all the ABC transporter family members. Coincidently, genetic amplification of ABCG1 is found in 10% to 35% of clinical samples of metastatic cancer cases. Expression of ABCG1 was further elevated in three-dimensional tumoroids (tumor organoids) within stemness-enhancing tumor milieu, whereas depletion of ABCG1 lowered cellular aggregation and tumoroid growth in vitro as well as hypoxia-inducible factor 1α in cancer cells around the central necrotic areas in tumors in vivo. Notably, depletion of ABCG1 triggered the intracellular accumulation of extracellular vesicles (EVs) and regression of tumoroids. Collectively, these data suggest that ABCG1 plays a crucial role in tumorigenesis in metastatic cancer and that depletion of ABCG1 triggers tumor regression with the accumulation of EVs, their derivatives and cargos, implicating a novel ABCG1-targeting therapeutic strategy by which redundant and toxic substances may be accumulated in tumors leading to their regression.

    DOI: 10.1254/jpssuppl.92.0_2-yia-26

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  • MMP9プロモーター活性を指標とした三次元培養システムを用いた抗がん剤スクリーニングによる薬剤再開発

    十川 千春, 江口 傑徳, 奥舎 有加, 大山 和美, 小野 喜章, 十川 紀夫, 岡元 邦彰

    日本薬理学会年会要旨集   92   1-P-096   2019年

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    記述言語:日本語   出版者・発行元:公益社団法人 日本薬理学会  

    Cancer is one of the most serious diseases all over the world, especially metastasis and drug resistance are leading causes of death. There is an urgent need to establish new strategies for drug discovery. Success in the drug discovery depends on the development of appropriate tumor models that correspond closely to native tumor situation. Matrix metalloproteinases (MMPs) represent the most prominent family of proteinases associated with tumorigenesis and are regulators of tumor milieu. The cancer stem cell model fits well with tumorigenesis, metastasis and drug resistance. We have shown that cancer cell aggregation led to hypoxic tumoroids with marked upregulation of reprogramming and stemness genes as increased cancer stem cell using a 3D culture system. In the present study, we established a novel MMP9 promoter-driven cell-based reporter system using a rapidly metastatic colon cancer cell in the 3D culture system that evaluates cancer stemness and invasiveness. We used a concept of drug repositioning-using known molecules for new indications. We selected several compounds with inhibition to both tumoroid formation and MMP9 promoter activity. One of the compounds inhibited primary tumor formation, invasion and metastasis.

    DOI: 10.1254/jpssuppl.92.0_1-p-096

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  • Regulatory Roles of HSP90-Rich Extracellular Vesicles 査読

    Takanori Eguchi, Kisho Ono, Kazumi Kawata, Kuniaki Okamoto, Stuart K. Calderwood

    Heat Shock Proteins   3 - 17   2019年

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    掲載種別:論文集(書籍)内論文   出版者・発行元:Springer International Publishing  

    DOI: 10.1007/978-3-030-23158-3_1

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  • 破骨細胞分化を制御する新規Rabタンパク質の同定と機能解析

    奥舎 有加, 板垣 まみ, 十川 千春, 江口 傑徳, 岡元 邦彰

    岡山歯学会雑誌   37 ( 2 )   82 - 82   2018年12月

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    記述言語:日本語   出版者・発行元:岡山歯学会  

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  • Carcinogenic epithelial-mesenchymal transition initiated by oral cancer exosomes is inhibited by anti-EGFR antibody cetuximab. 査読 国際誌

    Toshifumi Fujiwara, Takanori Eguchi, Chiharu Sogawa, Kisho Ono, Jun Murakami, Soichiro Ibaragi, Jun-Ichi Asaumi, Stuart K Calderwood, Kuniaki Okamoto, Ken-Ichi Kozaki

    Oral oncology   86   251 - 257   2018年11月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    Overexpression and increased signaling from the epidermal growth factor receptor (EGFR) often changes oral squamous cell carcinoma (OSCC) and thus EGFR is frequently targeted molecularly by the therapeutic antibody cetuximab. We assessed the roles of OSCC-derived extracellular vesicles (EVs), including exosomes in the trafficking of cetuximab and in epithelial-mesenchymal transition (EMT) of epithelial cells. OSCC cells abundantly expressed EGFR, which was secreted from cells with OSCC-EVs upon EGF stimulations. The OSCC-EGFR-EVs were then able to enter into and transform epithelial cells leading to increased mesenchymal traits with increased vimentin and spindle-like shapes. EGF priming of OSCC cells further increased this EMT-initiating effect of the OSCC-EVs. The internalization and pro-EMT effects of the OSCC-EVs were largely blocked by cetuximab. Thus, OSCC-derived EVs transform normal epithelial cells into a mesenchymal phenotype and anti-EGFR therapeutic antibody cetuximab inhibits such a carcinogenic effect of the OSCC-EVs.

    添付ファイル: 71. Fujiwara (Oral Oncol).pdf

    DOI: 10.1016/j.oraloncology.2018.09.030

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  • Depletion of Lipid Efflux Pump ABCG1 Triggers the Intracellular Accumulation of Extracellular Vesicles and Reduces Aggregation and Tumorigenesis of Metastatic Cancer Cells. 査読 国際誌

    難波 友里, 岡元 邦彰, その他

    Front Oncol.   8   376 - 376   2018年10月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    添付ファイル: 72. Namba (Frontiers in Oncol).pdf

    DOI: 10.3389/fonc.2018.00376

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  • Anti-EGFR antibody cetuximab is secreted by oral squamous cell carcinoma and alters EGF-driven mesenchymal transition. 査読 国際誌

    Toshifumi Fujiwara, Takanori Eguchi, Chiharu Sogawa, Kisho Ono, Jun Murakami, Soichiro Ibaragi, Jun-Ichi Asaumi, Kuniaki Okamoto, Stuart K Calderwood, Ken-Ichi Kozaki

    Biochemical and biophysical research communications   503 ( 3 )   1267 - 1272   2018年9月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    Genetic amplification, overexpression, and increased signaling from the epidermal growth factor receptor (EGFR) are often found in oral squamous cell carcinoma (OSCC) and thus EGFR is frequently targeted molecularly by the therapeutic antibody cetuximab. We assessed effects of cetuximab in control of EGF-driven malignant traits of OSCC cells. EGF stimulation promoted progression level of mesenchymal traits in OSCC cells, which were attenuated by cetuximab but incompletely. We pursued a potential mechanism underlying such incomplete attenuation of OSCC malignant traits. Cetuximab promoted secretion of EGFR-EVs by OSCC cells and failed to inhibit EGF-driven secretion of EGFR-EVs. Cetuximab was also found to be robustly secreted with the EGFR-EVs by the OSCC cells. Thus, EGF promotes the level of mesenchymal traits of OSCC cells and secretion of EGFR-EVs, which involve cetuximab resistance.

    添付ファイル: 69. Fujiwara (BBRC).pdf

    DOI: 10.1016/j.bbrc.2018.07.035

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  • HSP-enriched properties of extracellular vesicles involve survival of metastatic oral cancer cells. 査読 国際誌

    Kisho Ono, Takanori Eguchi, Chiharu Sogawa, Stuart K Calderwood, Junya Futagawa, Tomonari Kasai, Masaharu Seno, Kuniaki Okamoto, Akira Sasaki, Ken-Ichi Kozaki

    Journal of cellular biochemistry   119 ( 9 )   7350 - 7362   2018年9月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    Cancer cells often secrete extracellular vesicles (EVs) that carry heat shock proteins (HSPs) with roles in tumor progression. Oral squamous cell carcinoma (OSCC) belongs to head and neck cancers (HNC) whose lymph-node-metastases often lead to poor prognosis. We have examined the EV proteome of OSCC cells and found abundant secretion of HSP90-enriched EVs in lymph-node-metastatic OSCC cells. Double knockdown of HSP90α and HSP90β, using small interfering RNA significantly reduced the survival of the metastatic OSCC cells, although single knockdown of each HSP90 was ineffective. Elevated expression of these HSP90 family members was found to correlate with poor prognosis of HNC cases. Thus, elevated HSP90 levels in secreted vesicles are potential prognostic biomarkers and therapeutic targets in metastatic OSCC.

    添付ファイル: 68. Ono (J Cell Biochem).pdf

    DOI: 10.1002/jcb.27039

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  • 急速転移性癌細胞株由来細胞外小胞による破骨細胞分化および癌細胞浸潤の制御

    板垣 まみ, 十川 千春, 奥舎 有加, 江口 傑徳, 小野 喜章, 岡元 邦彰

    Journal of Oral Biosciences Supplement   2018   347 - 347   2018年9月

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    記述言語:日本語   出版者・発行元:(一社)歯科基礎医学会  

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  • 破骨細胞分化を負に制御するRabタンパク質の同定

    奥舎 有加, 板垣 まみ, 十川 千春, 江口 傑徳, 坂井 詠子, 筑波 隆幸, 岡元 邦彰

    Journal of Oral Biosciences Supplement   2018   219 - 219   2018年9月

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    記述言語:日本語   出版者・発行元:(一社)歯科基礎医学会  

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  • Rutaecarpine attenuates osteoclastogenesis by impairing macrophage colony stimulating factor and receptor activator of nuclear factor κ-B ligand-stimulated signalling pathways. 査読 国際誌

    Yutaka Fukuma, Eiko Sakai, Shunsuke Komaki, Kazuhisa Nishishita, Kuniaki Okamoto, Takayuki Tsukuba

    Clinical and experimental pharmacology & physiology   45 ( 8 )   863 - 865   2018年8月

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    記述言語:英語  

    Rutaecarpine is a major alkaloid isolated from Evodia rutaecarpa. Here, we investigated the effects of rutaecarpine on osteoclast differentiation induced by macrophage colony stimulating factor (M-CSF) and receptor activator of nuclear factor κ-B ligand (RANKL) in bone marrow-derived macrophages (BMMs). Treatment with rutaecarpine significantly inhibited osteoclastogenesis and prevented bone resorption of BMM-derived osteoclasts. Mechanistically, rutaecarpine decreased the protein level of nuclear factor of activated T cells cytoplasmic-1 (NFATc1) and the phosphorylation of other signalling pathways during the osteoclast differentiation. Thus, rutaecarpine may be useful as a therapeutic agent for the treatment of bone diseases.

    DOI: 10.1111/1440-1681.12941

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  • Organoids with cancer stem cell-like properties secrete exosomes and HSP90 in a 3D nanoenvironment 査読

    Takanori Eguchi, Chiharu Sogawa, Yuka Okusha, Kenta Uchibe, Ryosuke Iinuma, Kisho Ono, Keisuke Nakano, Jun Murakami, Manabu Itoh, Kazuya Arai, Toshifumi Fujiwara, Yuri Namba, Yoshiki Murata, Kazumi Ohyama, Manami Shimomura, Hirohiko Okamura, Masaharu Takigawa, Tetsuya Nakatsura, Ken-ichi Kozaki, Kuniaki Okamoto, Stuart K. Calderwood

    PLoS ONE   13 ( 2 )   2018年2月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:Public Library of Science  

    Ability to form cellular aggregations such as tumorspheres and spheroids have been used as a morphological marker of malignant cancer cells and in particular cancer stem cells (CSC). However, the common definition of the types of cellular aggregation formed by cancer cells has not been available. We examined morphologies of 67 cell lines cultured on three dimensional morphology enhancing NanoCulture Plates (NCP) and classified the types of cellular aggregates that form. Among the 67 cell lines, 49 cell lines formed spheres or spheroids, 8 cell lines formed grape-like aggregation (GLA), 8 cell lines formed other types of aggregation, and 3 cell lines formed monolayer sheets. Seven GLA-forming cell lines were derived from adenocarcinoma among the 8 lines. A neuroendocrine adenocarcinoma cell line PC-3 formed asymmetric GLA with ductal structures on the NCPs and rapidly growing asymmetric tumors that metastasized to lymph nodes in immunocompromised mice. In contrast, another adenocarcinoma cell line DU-145 formed spheroids in vitro and spheroid-like tumors in vivo that did not metastasize to lymph nodes until day 50 after transplantation. Culture in the 3D nanoenvironment and in a defined stem cell medium enabled the neuroendocrine adenocarcinoma cells to form slowly growing large organoids that expressed multiple stem cell markers, neuroendocrine markers, intercellular adhesion molecules, and oncogenes in vitro. In contrast, the more commonly used 2D serum-contained environment reduced intercellular adhesion and induced mesenchymal transition and promoted rapid growth of the cells. In addition, the 3D stemness nanoenvironment promoted secretion of HSP90 and EpCAM-exosomes, a marker of CSC phenotype, from the neuroendocrine organoids. These findings indicate that the NCP-based 3D environment enables cells to form stem cell tumoroids with multipotency and model more accurately the in vivo tumor status at the levels of morphology and gene expression.

    DOI: 10.1371/journal.pone.0191109

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  • Actin binding LIM 1 (abLIM1) negatively controls osteoclastogenesis by regulating cell migration and fusion. 査読 国際誌

    Haruna Narahara, Eiko Sakai, Yu Yamaguchi, Shun Narahara, Mayumi Iwatake, Kuniaki Okamoto, Noriaki Yoshida, Takayuki Tsukuba

    Journal of cellular physiology   234 ( 1 )   486 - 499   2018年1月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    Actin binding LIM 1 (abLIM1) is a cytoskeletal actin-binding protein that has been implicated in interactions between actin filaments and cytoplasmic targets. Previous biochemical and cytochemical studies have shown that abLIM1 interacts and co-localizes with F-actin in the retina and muscle. However, whether abLIM1 regulates osteoclast differentiation has not yet been elucidated. In this study, we examined the role of abLIM1 in osteoclast differentiation and function. We found that abLIM1 expression was upregulated during receptor activator of nuclear factor kappa-B ligand (RANKL)-induced osteoclast differentiation, and that a novel transcript of abLIM1 was exclusively expressed in osteoclasts. Overexpression of abLIM1 in the murine monocytic cell line, RAW-D suppressed osteoclast differentiation and decreased expression of several osteoclast-marker genes. By contrast, small interfering RNA-induced knockdown of abLIM1 enhanced the formation of multinucleated osteoclasts and markedly increased the expression of the osteoclast-marker genes. Mechanistically, abLIM1 regulated the localization of tubulin, migration, and fusion in osteoclasts. Thus, these results indicate that abLIM1 negatively controls osteoclast differentiation by regulating cell migration and fusion mediated via actin formation.

    添付ファイル: 65. Narahara(J Cell Physiol).pdf

    DOI: 10.1002/jcp.26605

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  • Dihydroartemisinin represses osteoclastogenesis of bone marrow macrophages through reduced NFATc1 expression and impaired phosphorylation of IκBα. 査読

    小巻 俊介, 岡元 邦彰, その他

    Biomedical Research (Tokyo)   39 ( 4 )   169 - 177   2018年

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    添付ファイル: 70. Komaki (Biomed Res).pdf

    DOI: 10.2220/biomedres.39.169

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  • The intranuclear PEX domain of MMP involves proliferation, migration, and metastasis of aggressive adenocarcinoma cells. 査読 国際誌

    奥舎 有加, 岡元 邦彰, その他

    J Cell Biochem.   119 ( 9 )   7363 - 7376   2018年

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    添付ファイル: 67. Okusha (J Cell Biochem).pdf

    DOI: 10.1002/jcb.27040

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  • Rutaecarpine attenuates osteoclastogenesis by impairing M-CSF and RANKL-stimulated signaling pathways. 査読

    福間 裕, 岡元 邦彰, その他

    Clin Exp Pharmacol Physiol.   2018年

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    添付ファイル: 66. Fukuma (Clin Exp Pharmacol Physiol.pdf

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  • Transcription factor EB (TFEB) influences invasion and migration in oral squamous cell carcinomas. 査読 国際誌

    坂元 裕, 岡元 邦彰, その他

    Oral Dis.   24 ( 5 )   741 - 748   2018年

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    添付ファイル: 63. Sakamoto (Oral Dis).pdf

    DOI: 10.1111/odi.12826

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  • 扁平上皮癌細胞の増殖能・浸潤能・転移能における転写因子TFEBの役割

    坂元 裕, 山下 健太郎, 岡元 邦彰, 門脇 知子, 梅田 正博, 筑波 隆幸

    生命科学系学会合同年次大会   2017年度   [1P - 1012]   2017年12月

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    記述言語:日本語   出版者・発行元:生命科学系学会合同年次大会運営事務局  

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  • 異なる転移能を有する口腔扁平上皮癌細胞由来エクソソームに含まれるプロテオームの特性

    小野 喜章, 江口 傑徳, 十川 千春, 村上 純, 藤原 敏史, 笠井 智成, 妹尾 昌治, 佐々木 朗, 小崎 健一, 岡元 邦彰

    生命科学系学会合同年次大会   2017年度   [1LBA - 052]   2017年12月

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    記述言語:日本語   出版者・発行元:生命科学系学会合同年次大会運営事務局  

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  • プロテアーゼ活性を持たないプロテアーゼの炎症および癌における役割

    江口 傑徳, 奥舎 有加, Calderwood Stuart, 岡元 邦彰

    生命科学系学会合同年次大会   2017年度   [2LBA - 028]   2017年12月

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    記述言語:日本語   出版者・発行元:生命科学系学会合同年次大会運営事務局  

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  • 薬剤耐性遺伝子を標的としたがん幹細胞制御

    難波 友里, 江口 傑徳, 十川 千春, 奥舎 有加, 村上 純, 浅海 淳一, 岡元 邦彰, 小崎 健一

    生命科学系学会合同年次大会   2017年度   [1LBA - 050]   2017年12月

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  • Keap1遺伝子欠損はNrf2の活性化を介して破骨細胞分化を抑制する

    坂井 詠子, 福間 裕, 西下 一久, 岡元 邦彰, 筑波 隆幸

    生命科学系学会合同年次大会   2017年度   [1P - 0588]   2017年12月

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  • The Rho-specific guanine nucleotide exchange factor Plekhg5 modulates cell polarity, adhesion, migration, and podosome organization in macrophages and osteoclasts. 査読 国際誌

    Mayumi Iwatake, Kazuhisa Nishishita, Kuniaki Okamoto, Takayuki Tsukuba

    Experimental cell research   359 ( 2 )   415 - 430   2017年10月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    Osteoclasts are multinucleated bone-resorbing cells that are formed by fusion of monocyte/macrophage lineage. Osteoclasts and macrophages generate podosomes that are actin-based dynamic organelles implicated in cell adhesion, spreading, migration, and degradation. However, the detailed mechanisms of podosome organization remain unknown. Here, we identified the Rho-specific guanine-nucleotide exchange factor (Rho-GEF) Plekhg5 as an up-regulated gene during differentiation of osteoclasts from macrophages. Knockdown of Plekhg5 with small interfering RNA in both macrophages and osteoclasts induced larger cell formation with impaired cell polarity and resulted in an elongated and flattened shape. In macrophages, Plekhg5 depletion enhanced random migration, but impaired directional migration, adhesion, and matrix degradation. Plekhg5 in osteoclasts affected random migration, podosome organization, and bone resorption. Plekhg5 depletion affected signaling and localization of several Rho downstream effectors. In fact, end-binding protein 1 (EB1), cofilin and vinculin were abnormally localized in Plekhg5-depleted cells, and mDia1 and LIM kinase (LIMK)1 were upregulated in Plekhg5-depleted cells compared with control cells. However, overexpression of Plekhg5 in macrophages induced an increase in its mRNA level, but failed to increase the protein level, indicating that overexpressed Plekhg5 was degraded in macrophages but not HEK293T cells. Thus, Plekhg5 affects cell polarity, migration, adhesion, degradation, and podosome organization in macrophages and osteoclasts.

    添付ファイル: 62. Iwatake (Exp Cell Res).pdf

    DOI: 10.1016/j.yexcr.2017.08.025

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  • Effects of deficiency of Kelch-like ECH-associated protein 1 on skeletal organization: a mechanism for diminished nuclear factor of activated T cells cytoplasmic 1 during osteoclastogenesis 査読

    Eiko Sakai, Masanobu Morita, Masahiro Ohuchi, Mizuho A. Kido, Yutaka Fukuma, Kazuhisa Nishishita, Kuniaki Okamoto, Ken Itoh, Masayuki Yamamoto, Takayuki Tsukuba

    FASEB JOURNAL   31 ( 9 )   4011 - 4022   2017年9月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:FEDERATION AMER SOC EXP BIOL  

    Kelch-like ECH-associated protein 1 (Keap1) binds to nuclear factor E2 p45-related factor 2 (Nrf2), a transcription factor for antioxidant enzymes, to suppress Nrf2 activation. The role of oxidative stress in many diseases supports the possibility that processes that are associated with Nrf2 activation might offer therapeutic potential. Nrf2 deficiency induces osteoclastogenesis, which is responsible for bone loss, by activating receptor activator of NF-kappa B ligand (RANKL)-mediated signaling; however, the effects of Keap1 deficiency remain unclear. By using Keap1-deficient newborn mice, we observed that talus and calcaneus bone formation was partially retarded and that osteoclast number was reduced in vivo without severe gross abnormalities. In addition, Keap1-deficient macrophages were unable to differentiate into osteoclasts in vitro via attenuation of RANKL-mediated signaling and expression of nuclear factor of activated T cells cytoplasmic 1 (NFATc1), a key transcription factor that is involved in osteoclastogenesis. Furthermore, Keap1 deficiency up-regulated the expression of Mafb, a negative regulator of NFATc1. RANKL-induced mitochondrial gene expression is required for down-regulation of IFN regulatory factor 8 (IRF-8), a negative transcriptional regulator of NFATc1. Our results indicate that Keap1 deficiency down-regulated peroxisome proliferator-activated receptor-gamma coactivator 1 beta and mitochondrial gene expression and up-regulated Irf8 expression. These results suggest that the Keap1/Nrf2 axis plays a critical role in NFATc1 expression and osteoclastogenic progression.

    添付ファイル: 60. Sakai (FASEB ).pdf

    DOI: 10.1096/fj.201700177R

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  • Mmp遺伝子を標的とし、高転移性癌細胞の生存と転移を抑制する

    奥舎 有加, 江口 傑徳, 十川 千春, 中野 敬介, 岡元 邦彰, 小崎 健一

    Journal of Oral Biosciences Supplement   2017   378 - 378   2017年9月

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  • 核内タンパク質HMGM1 ヘムオキシゲナーゼ1の発現抑制はカスパーゼ3の活性化、HMGB1の細胞外遊離と破骨細胞分化に必要である

    坂井 詠子, 岡元 邦彰, 筑波 隆幸

    Journal of Oral Biosciences Supplement   2017   124 - 124   2017年9月

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  • Actin-Binding LIM protein 1は破骨細胞における細胞骨格形成と細胞機能を制御する

    楢原 春菜, 坂井 詠子, 岡元 邦彰, 筑波 隆幸, 吉田 教明

    Journal of Oral Biosciences Supplement   2017   280 - 280   2017年9月

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  • Keap1遺伝子欠損はIRF-8とMafBの発現上昇を介して破骨細胞分化を抑制する

    坂井 詠子, 城戸 瑞穂, 福間 裕, 西下 一久, 岡元 邦彰, 筑波 隆幸

    Journal of Oral Biosciences Supplement   2017   241 - 241   2017年9月

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    記述言語:日本語   出版者・発行元:(一社)歯科基礎医学会  

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  • Rab44, a novel large Rab GTPase, negatively regulates osteoclast differentiation by modulating intracellular carcium levels followd by NFATc1 activation. 査読

    山口 優, 岡元 邦彰, その他

    Cell Mol Life Sci.   75   33 - 48   2017年8月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    添付ファイル: 61. Yamaguchi (Cell Mol Life Sci).pdf

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  • The dental resin monomers HEMA and TEGDMA have inhibitory effects on osteoclast differentiation with low cytotoxicity 査読

    Hiroyuki Inamitsu, Kuniaki Okamoto, Eiko Sakai, Kazuhisa Nishishita, Hiroshi Murata, Takayuki Tsukuba

    JOURNAL OF APPLIED TOXICOLOGY   37 ( 7 )   817 - 824   2017年7月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:WILEY  

    The dental resin monomers 2-hydroxyethyl methacrylate (HEMA) and triethylene glycol dimethacrylate (TEGDMA) are released from the resin matrix due to unpolymerized monomers; once released, they influence various biological functions and the viability of cells in the oral environment. Although HEMA and TEGDMA have various effects on cells, including inflammation, inhibition of cell proliferation or differentiation, and apoptosis, the effects of these monomers on osteoclasts remain unknown. In this study, we investigated the effects of HEMA and TEGDMA on osteoclast differentiation of bone marrow-derived macrophages or murine monocytic cell line RAW-D. Both HEMA and TEGDMA inhibited osteoclast formation and their bone-resorbing activity at non-cytotoxic concentrations. Moreover, HEMA and TEGDMA decreased the expression of nuclear factor of activated T cells cytoplasmic-1 (NFATc1), a master regulator of osteoclast differentiation, and of osteoclast markers that are transcriptionally regulated by NFATc1, including Src and cathepsin K. Regarding their effects on signaling pathways involved in osteoclast differentiation, HEMA impaired the phosphorylation of extracellular signal-regulated kinase and Jun N-terminal kinase, whereas TEGDMA attenuated the phosphorylation of Akt and Jun N-terminal kinase. Thus, HEMA and TEGDMA inhibit osteoclast differentiation through different signaling pathways. This is the first report on the effects of the monomers HEMA and TEGDMA on osteoclasts. Copyright (C) 2017 John Wiley & Sons, Ltd.

    添付ファイル: 58. Inamitsu(J Appl Toxicol.pdf

    DOI: 10.1002/jat.3429

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  • New functions of lysosomes in bone cells 査読

    Takayuki Tsukuba, Eiko Sakai, Kazuhisa Nishishita, Tomoko Kadowaki, Kuniaki Okamoto

    Journal of Oral Biosciences   59 ( 2 )   92 - 95   2017年5月

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    担当区分:最終著者   記述言語:英語  

    © 2017 Japanese Association for Oral Biology Background Lysosomes are intracellular acidic organelles that contain approximately 50 hydrolases and 25 species of integral membrane proteins. Although lysosome-like specific compartments, termed lysosome-related organelles (LROs), are found in osteoclasts, their functions in these cells and lysosomal functions in osteoblasts remain to be elucidated. Highlight Recently, we found that expression of RAB27A is markedly increased during osteoclastic differentiation. RAB27A deficiency causes multinucleation and giant cell formation, characterized by abnormal transport of cell surface receptors and LROs into osteoclasts. Furthermore, we have shown that transcription factor EB (TFEB), a master regulator of lysosomal biogenesis, regulates osteoblastic differentiation. Overexpression of TFEB in preosteoblastic MC3T3-E1 cells enhances osteoblastic differentiation via decreased expression of activating transcription factor 4 (ATF4) and CCAAT/enhancer-binding protein homologous protein (CHOP). These results indicate that the expression of ATF4 and CHOP is essential for differentiation into osteoblasts. Conclusion RAB27A participates in bone resorption by LROs in osteoclasts. In addition, lysosomal biogenesis modulated by TFEB is necessary for osteoblastic differentiation.

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  • Cathepsin K modulates invasion, migration and adhesion of oral squamous cell carcinomas invitro 査読

    K. Yamashita, M. Iwatake, K. Okamoto, S-i Yamada, M. Umeda, T. Tsukuba

    ORAL DISEASES   23 ( 4 )   518 - 525   2017年5月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:WILEY  

    ObjectiveCathepsin K was initially discovered as an osteoclast-specific cysteine proteinase, but the enzyme is also expressed in various cancers including oral squamous cell carcinomas. This study aimed to clarify the function of cathepsin K in oral squamous cell carcinomas.
    Materials and MethodsExpression levels of cathepsin K were examined in six types of cell carcinomas. Carcinomas overexpressing cathepsin K were constructed. Effects of cathepsin K overexpression and treatment with odanacatib, a specific cathepsin K inhibitor, on cell invasion, migration and adhesion were analysed.
    ResultsDifferent levels of cathepsin K were expressed in carcinomas. Cathepsin K was predominantly localised in lysosomes. Cathepsin K overexpression impaired the proliferation of carcinomas. Invasion analysis showed that cathepsin K overexpression enhanced invasion and migration of carcinomas, whereas inhibition of cathepsin K by odanacatib caused the opposite effects in carcinomas. Cathepsin K overexpression also increased cell adhesion and slightly increased surface expression of the adhesion receptor CD29/integrin (1).
    ConclusionsThe enhanced invasion of carcinomas resulting from cathepsin K overexpression is probably due to the increased cell migration and adhesion. Thus, cathepsin K is implicated not only in protein degradation but also in invasion, migration and adhesion of oral squamous cell carcinomas.

    添付ファイル: 59. Yamashita (Oral Dis).pdf

    DOI: 10.1111/odi.12643

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  • Sanguiin H-6, a constituent of Rubus parvifolius L., inhibits receptor activator of nuclear factor-B ligand-induced osteoclastogenesis and bone resorption in vitro and prevents tumor necrosis factor-α-induced osteoclast formation in vivo. 査読

    坂井 詠子, 岡元 邦彰, その他

    Phytomedicine   23 ( 8 )   828 - 837   2016年7月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    添付ファイル: 57. Sakai (Phytomedicine) .pdf

    DOI: 10.1016/j.phymed.2016.04.002

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  • The Transcription Factor EB (TFEB) Regulates Osteoblast Differentiation Through ATF4/CHOP-Dependent Pathway 査読

    Erika Yoneshima, Kuniaki Okamoto, Eiko Sakai, Kazuhisa Nishishita, Noriaki Yoshida, Takayuki Tsukuba

    JOURNAL OF CELLULAR PHYSIOLOGY   231 ( 6 )   1321 - 1333   2016年6月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:WILEY-BLACKWELL  

    Osteoblasts are bone-forming cells that produce large amounts of collagen type I and various bone matrix proteins. Although osteoblast differentiation is highly regulated by various factors, it remains unknown whether lysosomes are directly involved in osteoblast differentiation. Here, we demonstrate the transcription factor EB (TFEB), a master regulator of lysosomal biogenesis, modulates osteoblast differentiation. The expression levels of TFEB as well as those of endosomal/lysosomal proteins were up-regulated during osteoblast differentiation using mouse osteoblastic MC3T3-E1 cells. By gene knockdown (KD) experiments with small interfering RNA (siRNA), TFEB depletion caused markedly reduced osteoblast differentiation as compared with the control cells. Conversely, overexpression (OE) of TFEB resulted in strikingly enhanced osteoblastogenesis compared to the control cells. By analysis of down-stream effector molecules, TFEB KD was found to cause marked up-regulation of activating transcription factor 4 (ATF4) and CCAAT/enhancer-binding protein homologous protein (CHOP), both of which are essential factors for osteoblastogenesis. In contrast, TFEB OE promoted osteoblast differentiation through reduced expression of ATF4 and CHOP without differentiation agents. Given the importance of ATF4 and CHOP in osteoblastogenesis, it is clear that the TFEB-regulated signaling pathway for osteoblast differentiation is involved in ATF4/CHOP-dependent signaling pathway. (c) 2015 Wiley Periodicals, Inc.

    添付ファイル: 56. Yoneshima (J Cellular Physiol).pdf

    DOI: 10.1002/jcp.25235

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  • Dual Effects of Liquiritigenin on the Proliferation of Bone Cells: Promotion of Osteoblast Differentiation and Inhibition of Osteoclast Differentiation 査読

    Kaho Uchino, Kuniaki Okamoto, Eiko Sakai, Erika Yoneshima, Mayumi Iwatake, Yutaka Fukuma, Kazuhisa Nishishita, Takayuki Tsukuba

    PHYTOTHERAPY RESEARCH   29 ( 11 )   1714 - 1721   2015年11月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:WILEY-BLACKWELL  

    Bone is constantly controlled by a balance between osteoblastic bone formation and osteoclastic bone resorption. Liquiritigenin is a plant-derived flavonoid and has various pharmacological effects, such as antioxidative, antitumor, and antiinflammatory effects. Here, we show that liquiritigenin has dual effects on the proliferation of bone cells, regarding the promotion of osteoblast differentiation and the inhibition of osteoclast differentiation. Liquiritigenin-treated murine osteoblastic MC3T3-E1 cells showed an increased alkaline phosphatase activity and enhanced phosphorylation of Smad1/5 compared with untreated cells. Moreover, liquiritigenin inhibited osteoclast differentiation, its bone-resorption activity through slightly decreased the phosphorylation of extracellular signal-regulated kinase, c-Jun N-terminal kinase, and inhibitor of nuclear factor kappa B; however, the phosphorylation of Akt and p38 slightly increased in bone marrow-derived osteoclasts. The expression levels of the osteoclast marker proteins nuclear factor of activated T-cell cytoplasmic-1, Src, and cathepsin K diminished. These results suggest that liquiritigenin may be useful as a therapeutic and/or preventive agent for osteoporosis or inflammatory bone diseases. Copyright (C) 2015 John Wiley & Sons, Ltd.

    添付ファイル: 55. Uchino (Phytother res).pdf

    DOI: 10.1002/ptr.5416

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  • Punicalagin attenuates osteoclast differentiation by impairing NFATc1 expression and blocking Akt- and JNK-dependent pathways 査読

    Mayumi Iwatake, Kuniaki Okamoto, Takashi Tanaka, Takayuki Tsukuba

    MOLECULAR AND CELLULAR BIOCHEMISTRY   407 ( 1-2 )   161 - 172   2015年9月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:SPRINGER  

    Punicalagin is a bioactive polyphenol that is classified as an ellagitannin. Although punicalagin has been shown to have various pharmacological effects, such as anti-oxidative, anti-inflammatory, and anti-tumor effects, no studies have reported the effects of punicalagin on osteoclasts (OCLs). In this study, we investigated the effects of punicalagin on OCL differentiation by receptor activator of nuclear factor kappa-B ligand in the murine monocytic RAW-D cell line and bone marrow-derived macrophages (BMMs). Treatment with punicalagin significantly inhibited OCL formation from RAW-D cells and BMMs and prevented bone resorption of BMM-derived OCLs. Moreover, punicalagin impaired multinucleation and actin-ring formation in OCLs, and decreased the protein levels of nuclear factor of activated T cells cytoplasmic-1 (NFATc1), which is a master regulator of OCL differentiation, and concomitantly reduced the expression levels of Src and cathepsin K, which are transcriptionally regulated by NFATc1. The effects of punicalagin on intracellular signaling during the OCL differentiation of BMMs indicated that punicalagin-treated OCLs displayed markedly reduced phosphorylation of Jun N-terminal kinase and Akt, and partially impaired phosphorylation of extracellular signal-regulated kinase, p38 mitogen-activated protein kinase, and inhibitor of nuclear factor kappa-B alpha compared with untreated OCLs. Thus, punicalagin may affect bone metabolism by inhibiting OCL differentiation.

    添付ファイル: 53. Iwatake (Mol Cell Biochem).pdf

    DOI: 10.1007/s11010-015-2466-3

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  • Cobalt protoporphyrin represses osteoclastogenesis through blocking multiple signaling pathways 査読

    Yuka Yashima, Kuniaki Okamoto, Eiko Sakai, Mayumi Iwatake, Yutaka Fukuma, Kazuhisa Nishishita, Takayuki Tsukuba

    BIOMETALS   28 ( 4 )   725 - 732   2015年8月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:SPRINGER  

    Cobalt protoporphyrin (CoPP) is a metallo-protoporphyrin that works as a powerful inducer of heme oxigenase-1 (HO-1) in various tissues and cells. Our recent studies have demonstrated that induction of HO-1 by several reagents inhibited differentiation and activation of osteoclasts (OCLs), which are multinucleated bone resorbing cells. However, the effects of CoPP on osteoclastogenesis remain to be elucidated. In this study, we report that CoPP inhibits receptor activator of nuclear factor kappa B ligand (RANKL)-induced OCL formation in a dose dependent manner. Importantly, CoPP had little cytotoxicity, but rather enhanced cell proliferation of OCLs. CoPP suppressed the protein levels of nuclear factor of activated T cells cytoplasmic-1 (NFATc1) as well as those of OCLs markers such as Src and cathepsin K, which are transcriptionally regulated by NFATc1 in mature OCLs. Western blot analyses also showed that CoPP abolished RANKL-stimulated phosphorylation of several major signaling pathways such as I kappa B, Akt, ERK, JNK and p38 MAPKs in OCL precursor cells. Thus, our results show that CoPP represses osteoclastogenesis through blocking multiple signaling pathways.

    添付ファイル: 51. Yashima (Biometals).pdf

    DOI: 10.1007/s10534-015-9861-9

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  • Cafestol has a weaker inhibitory effect on osteoclastogenesis than kahweol and promotes osteoblast differentiation 査読

    Yutaka Fukuma, Eiko Sakai, Kazuhisa Nishishita, Kuniaki Okamoto, Takayuki Tsukuba

    BIOFACTORS   41 ( 4 )   222 - 231   2015年7月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:WILEY-BLACKWELL  

    Bone homeostasis is regulated by a balance between osteoclast (OCL)-mediated bone resorption and osteoblast (OBL)-mediated bone formation. Thus, developing a compound that simultaneously inhibits OCL function and promotes OBL function would be useful as a new medical therapy for bone diseases. Here, we examined the effects of cafestol, a coffee diterpene, on the differentiation of OCLs and OBLs. Cafestol prevented OCL formation in a dose-dependent manner and suppressed the bone-resorbing activity of OCLs. Interestingly, the viability of OCLs treated with 10-50 mu M cafestol was significantly higher than that of untreated cells. At the molecular level, cafestol markedly decreased RANKL-induced phosphorylation of extracellular signal-regulated kinase (Erk) and inhibitor of nuclear factor kappa B alpha (IB). Compared to kahweol, another coffee-specific diterpene, the inhibitory effects of cafestol were milder on OCL differentiation, and cafestol and kahweol showed different characteristics in induction of the phase antioxidant enzymes and sensitivities in nuclear factor-erythroid 2-related factor 2 (Nrf2)-deficient BMMs. In addition to inhibiting OCLs, cafestol enhanced the differentiation of osteoblastic cells by increasing the mRNA levels of differentiation markers. Thus, cafestol inhibits OCL differentiation and promotes OBL differentiation, suggesting that cafestol may be a novel agent for bone diseases. (c) 2015 BioFactors, 41(4):222-231, 2015

    添付ファイル: 54. Fukuma (BioFactors).pdf

    DOI: 10.1002/biof.1218

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  • Castalagin Exerts Inhibitory Effects on Osteoclastogenesis Through Blocking a Broad Range of Signaling Pathways with Low Cytotoxicity 査読

    Mayumi Iwatake, Kuniaki Okamoto, Takashi Tanaka, Takayuki Tsukuba

    PHYTOTHERAPY RESEARCH   29 ( 6 )   917 - 924   2015年6月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:WILEY-BLACKWELL  

    Castalagin is a rare plant polyphenol that is classified as a hydrolyzable tannin. Although it has antioxidant, antitumorigenic, and leishmanicidal effects, the utility of castalagin against bone diseases remain to be elucidated. Here, we investigated the effects of castalagin on the differentiation of osteoclasts (OCLs), multinucleated bone-resorbing cells. After stimulation with receptor activator of nuclear factor kappa-B ligand (RANKL), the formation of OCLs from bone marrow-derived macrophages was significantly inhibited by castalagin even at 1M. However, castalagin displayed little cytotoxicity at a higher concentration of 50M. The effects of castalagin on intracellular signaling during OCL differentiation showed that castalagin suppresses RANKL-stimulated phosphorylation of major signaling pathways including protein kinase B (Akt), extracellular signal-regulated kinase, Jun N-terminal kinase, p38 mitogen-activated protein kinases, and inhibitor of nuclear factor kappa B alpha. Moreover, following castalagin treatment, the protein levels of nuclear factor of activated T-cells, cytoplasmic 1, a master regulator for OCL differentiation, and NF-B were decreased. Thus, castalagin exerts inhibitory effects on osteoclastogenesis through blockage of a broad range of signaling pathways, but has low cytotoxicity. Copyright (c) 2015 John Wiley & Sons, Ltd.

    添付ファイル: 50. Iwatake (Phytother res).pdf

    DOI: 10.1002/ptr.5333

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  • Rab27A Regulates Transport of Cell Surface Receptors Modulating Multinucleation and Lysosome-Related Organelles in Osteoclasts 査読

    Megumi Shimada-Sugawara, Eiko Sakai, Kuniaki Okamoto, Mitsunori Fukuda, Tetsuro Izumi, Noriaki Yoshida, Takayuki Tsukuba

    SCIENTIFIC REPORTS   5   2015年4月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:NATURE PUBLISHING GROUP  

    Rab27A regulates transport of lysosome-related organelles (LROs) and release of secretory granules in various types of cells. Here, we identified up-regulation of Rab27A during differentiation of osteoclasts (OCLs) from bone-marrow macrophages (BMMs), by DNA microarray analysis. Rab27A deficiency in OCLs, using small interfering RNA (siRNA) knockdown in RAW-D cell line or BMMs derived from ashen mice, which display genetic defects in Rab27A expression, induced multinucleated and giant cells. Upon stimulation with macrophage-colony stimulating factor (M-CSF) and receptor activator of nuclear factor kappa-B ligand (RANKL), essential cytokines for OCL differentiation, phosphorylation levels of extracellular signal-regulated kinase (Erk), proto-oncogene tyrosine-protein kinase (Src), and p-38 were slightly enhanced in ashen BMMs than in wild-type BMMs. The cell surface level of c-fms, an M-CSF receptor, was slightly higher in ashen BMMs than in wild-type BMMs, and down-regulation of RANK, a RANKL receptor, was delayed. In addition to receptors, OCLs derived from ashen mice exhibited aberrant actin ring formation, abnormal subcellular localization of lysosome-associated membrane protein (LAMP2) and cathepsin K (CTSK), and marked reduction in resorbing activity. Thus, these findings suggest that Rab27A regulates normal transport of cell surface receptors modulating multinucleation and LROs in OCLs.

    添付ファイル: 51. Shimada-Sugawara (Sci Rep).pdf

    DOI: 10.1038/srep09620

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  • Cathepsin E: An Aspartic Protease with Diverse Functions and Biomedical Implications 査読

    K. Yamamoto, K. Okamoto, T. Tsukuba

    Encyclopedia of Cell Biology   1   681 - 690   2015年1月

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    記述言語:英語   掲載種別:論文集(書籍)内論文   出版者・発行元:Elsevier Inc.  

    Aspartic peptidases are widely distributed among vertebrates, yeast, plants, fungi, bacteria, and viruses. Of these, the A1 family members, including cathepsin E and ß-secretase, are involved in specific and/or nonspecific degradation of proteins and peptides in intra- and/or extracellular spaces. In the last decade, cathepsin E and ß-secretase have received enormous interest because of their involvement in important biological processes and the close association of their abnormal expression and uncontrolled activity with human diseases. This review summarizes the current knowledge on biochemical properties and functions of cathepsin E and highlights the pathophysiological conditions caused by its deficiency.

    DOI: 10.1016/B978-0-12-394447-4.10078-1

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  • Defective adipose tissue development associated with hepatomegaly in cathepsin E-deficient mice fed a high-fat diet 査読

    Tomoko Kadowaki, Mizuho A. Kido, Junko Hatakeyama, Kuniaki Okamoto, Takayuki Tsukuba, Kenji Yamamoto

    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS   446 ( 1 )   212 - 217   2014年3月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:ACADEMIC PRESS INC ELSEVIER SCIENCE  

    Cathepsin E is an intracellular aspartic proteinase, which is predominantly distributed in immune-related and epithelial cells. However, the role of the enzyme in adipose tissues remains unknown. In this study, we investigated the characteristics of cathepsin E-deficient (CatE(-/-)) mice fed a high-fat diet (HFD), as a mouse model of obesity. HFD-fed CatE(-/-) mice displayed reduced body weight gain and defective development of white adipose tissue (WAT) and brown adipose tissue (BAT), compared with HFD-fed wild-type mice. Moreover, fat-induced CatE(-/-) mice showed abnormal lipid accumulation in non-adipose tissues characterized by hepatomegaly, which is probably due to defective adipose tissue development. Detailed pathological and biochemical analyses showed that hepatomegaly was accompanied by hepatic steatosis and hypercholesterolemia in HFD-induced CatE(-/-) mice. In fat-induced CatE(-/-) mice, the number of macrophages infiltrating into WAT was significantly lower than in fat-induced wild-type mice. Thus, the impaired adipose tissue development in HFD-induced CatE(-/-) mice was probably due to reduced infiltration of macrophages and may lead to hepatomegaly accompanied by hepatic steatosis and hypercholesterolemia. (C) 2014 Elsevier Inc. All rights reserved.

    添付ファイル: 47. Kadowaki (BBRC).pdf

    DOI: 10.1016/j.bbrc.2014.02.089

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  • Repression of cathepsin E expression increases the risk of mammary carcinogenesis and links to poor prognosis in breast cancer 査読

    Tomoyo Kawakubo, Atsushi Yasukochi, Tatsuya Toyama, Satoru Takahashi, Kuniaki Okamoto, Takayuki Tsukuba, Seiji Nakamura, Yasuhiko Ozaki, Koichi Nishigaki, Hiroko Yamashita, Kenji Yamamoto

    CARCINOGENESIS   35 ( 3 )   714 - 726   2014年3月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:OXFORD UNIV PRESS  

    Despite advances in detection and treatment for breast cancer (BC), recurrence and death rates remain unacceptably high. Therefore, more convenient diagnostic and prognostic methods still required to optimize treatments among the patients. Here, we report the clinical significance of the serum cathepsin E (CatE) activity as a novel prognostic marker for BC. Correlation analysis between the serum levels of CatE expression and clinicopathological parameters revealed that the activity levels, but not the protein levels, were negatively associated with the stages and progression of BC. Univariate and multivariate analyses demonstrated that the serum CatE activity was significantly correlated with favorable prognostic outcomes of the patients. The functional link of CatE expression to BC progression was further corroborated by in vivo and in vitro studies with mice exhibiting different levels of CatE expression. Multiparous CatE(/) mice spontaneously developed mammary tumors concomitant with morphological transformation and altered growth characteristics of the mammary glands. These alterations were associated in part with the induction of epithelialmesenchymal transition and the activation of -catenin-dependent pathway in mammary cells. Loss of CatE strongly induced the translocation and accumulation of Wnt5a in the nuclei, thereby leading to the aberrant trafficking, maturation and secretion of Wnt5a and the impaired signaling. The interaction of CatE and Wnt5a was verified by proximity ligation assay and by knockdown or restoration of CatE expression in the mammary cells. Consequently, our data demonstrate that CatE contributes to normal growth and development of mammary glands through proper trafficking and secretion of Wnt5a.

    添付ファイル: 47. Kawakubo (Carcinogenesis).pdf

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  • Inhibitory effects of tert-butylhydroquinone on osteoclast differentiation via up-regulation of heme oxygenase-1 and down-regulation of HMGB1 release and NFATc1 expression 査読

    Yu Yamaguchi, Eiko Sakai, Hiroshi Sakamoto, Reiko Fumimoto, Yutaka Fukuma, Kazuhisa Nishishita, Kuniaki Okamoto, Takayuki Tsukuba

    JOURNAL OF APPLIED TOXICOLOGY   34 ( 1 )   49 - 56   2014年1月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:WILEY-BLACKWELL  

    Osteoclasts (OCLs) are multinucleated bone-resorbing cells that are differentiated by receptor activator of nuclear factor kappa-B ligand (RANKL) and macrophage colony-stimulating factor (M-CSF). Our recent studies have shown that heme-oxygenase-1 (HO-1), a stress-induced cytoprotective enzyme, plays an important role in OCL differentiation, although the pharmacological significance of this effect remains unknown. In this study, we investigated the effects of tert-butylhydroquinone (tBHQ), a pharmacological HO-1 inducer, on in vitro differentiation of bone marrow-derived macrophages (BMMs) or murine monocytic cell line RAW-D into OCLs. tBHQ inhibited the formation and the bone-resorbing activity of OCLs. Moreover, tBHQ treatment decreased the expression of nuclear factor of activated T cells cytoplasmic-1 (NFATc1), a master regulator of OCL differentiation, and of OCL markers transcriptionally regulated by NFATc1, such as Src and cathepsin K. In addition, tBHQ impaired phosphorylation of extracellular signal-regulated kinase, p38 mitogen-activated protein kinase (MAPK), Jun N-terminal kinase, Akt, and inhibitor of nuclear factor kappa B alpha (I kappa B alpha). Finally, we show that tBHQ inhibited the release of high mobility group box 1 (HMGB1), a recently identified activator of OCL differentiation. Thus, tBHQ inhibits OCL differentiation through the HO-1/HMGB1 pathways. Copyright (c) 2012 John Wiley & Sons, Ltd.

    添付ファイル: 48. Yamaguchi (J Appl Toxicol).pdf

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  • Cobalt protoporphyrin prevents osteoclastogenesis via blocking of I kappa B phosphorylation 査読

    Yuka Yashima, Kuniaki Okamoto, Eiko Sakai, Kazuhisa Nishishita, Takayuki Tsukuba

    JOURNAL OF PHARMACOLOGICAL SCIENCES   124   232P - 232P   2014年

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    記述言語:英語   出版者・発行元:JAPANESE PHARMACOLOGICAL SOC  

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  • Cathepsin E Deficiency Impairs Autophagic Proteolysis in Macrophages 査読

    Takayuki Tsukuba, Michiyo Yanagawa, Tomoko Kadowaki, Ryosuke Takii, Yoshiko Okamoto, Eiko Sakai, Kuniaki Okamoto, Kenji Yamamoto

    PLOS ONE   8 ( 12 )   2013年12月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:PUBLIC LIBRARY SCIENCE  

    Cathepsin E is an endosomal aspartic proteinase that is predominantly expressed in immune-related cells. Recently, we showed that macrophages derived from cathepsin E-deficient (CatE(-/-)) mice display accumulation of lysosomal membrane proteins and abnormal membrane trafficking. In this study, we demonstrated that CatE(-/-) macrophages exhibit abnormalities in autophagy, a bulk degradation system for aggregated proteins and damaged organelles. CatE(-/-) macrophages showed increased accumulation of autophagy marker proteins such as LC3 and p62, and polyubiquitinated proteins. Cathepsin E deficiency also altered autophagy-related signaling pathways such as those mediated by the mammalian target of rapamycin (mTOR), Akt, and extracellular signal-related kinase (ERK). Furthermore, immunofluorescence microscopy analyses showed that LC3-positive vesicles were merged with acidic compartments in wild-type macrophages, but not in CatE(-/-) macrophages, indicating inhibition of fusion of autophagosome with lysosomes in CatE(-/-) cells. Delayed degradation of LC3 protein was also observed under starvation-induced conditions. Since the autophagy system is involved in the degradation of damaged mitochondria, we examined the accumulation of damaged mitochondria in CatE(-/-) macrophages. Several mitochondrial abnormalities such as decreased intracellular ATP levels, depolarized mitochondrial membrane potential, and decreased mitochondrial oxygen consumption were observed. Such mitochondrial dysfunction likely led to the accompanying oxidative stress. In fact, CatE(-/-) macrophages showed increased reactive oxygen species (ROS) production and up-regulation of oxidized peroxiredoxin-6, but decreased antioxidant glutathione. These results indicate that cathepsin E deficiency causes autophagy impairment concomitantly with increased aberrant mitochondria as well as increased oxidative stress.

    添付ファイル: 46. Tsukuba (Plosone).pdf

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  • Structural and phylogenetic comparison of napsin genes: The duplication, loss of function and human-specific pseudogenization of napsin B 査読

    Kazuhisa Nishishita, Eiko Sakai, Kuniaki Okamoto, Takayuki Tsukuba

    GENE   517 ( 2 )   147 - 157   2013年4月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:ELSEVIER SCIENCE BV  

    Aspartic proteinases form a widely distributed protein superfamily, including cathepsin D, cathepsin E, pepsins, renin, BACE and napsin. Human napsin genes are located on human chromosome 19q13, which comprises napsin A and napsin B. Napsin B has been annotated as a pseudogene because it lacks an in-frame stop codon; its nascent chains are cotranslationally degraded. Until recently, there have been no studies concerning the molecular evolution of the napsin protein family in the human genome. In the present study, we investigated the evolution and gene organization of the napsin protein family. Napsin B orthologs are primarily distributed in primates, while napsin A orthologs are the only napsin genes in other species. The corresponding regions of napsin B in the available sequences from primate species contain an in-frame stop codon at a position equivalent to that of human napsin A. In addition, a rare single-nucleotide polymorphism (SNP) that creates a proper stop codon in human napsin B was identified using HapMap populations. Recombinant protein expression and three-dimensional comparative modeling revealed that napsin B exhibits residual activity toward synthetic aspartic protease substrates compared with napsin A, presumably through a napsin B-specific Arg287 residue. Thus, napsin B was duplicated from napsin A during the early stages of primate evolution, and the subsequent loss of napsin B function during primate evolution reflected ongoing human-specific napsin B pseudogenization. (C) 2013 Elsevier B.V. All rights reserved.

    添付ファイル: 44. NIshishita (Gene).pdf

    DOI: 10.1016/j.gene.2013.01.013

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  • Fisetin inhibits osteoclastogenesis through prevention of RANKLInduced ROS production by Nrf2-mediated Up-regulation of Phase II antioxidant enzymes 査読

    Eiko Sakai, Megumi Shimada-Sugawara, Yu Yamaguchi, Hiroshi Sakamoto, Reiko Fumimoto, Yutaka Fukuma, Kazuhisa Nishishita, Kuniaki Okamoto, Takayuki Tsukuba

    Journal of Pharmacological Sciences   121 ( 4 )   288 - 298   2013年

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    Osteoclasts (OCLs) are multinucleated bone-resorbing cells that are differentiated by stimulation with receptor activator of nuclear factor kappa-B ligand (RANKL) and macrophage colony-stimulating factor. We recently demonstrated that regulation of heme-oxygenase 1 (HO-1), a stress-induced cytoprotective enzyme, also functions in OCL differentiation. In this study, we investigated effects of fisetin, a natural bioactive flavonoid that has been reported to induce HO-1 expression, on the differentiation of macrophages into OCLs. Fisetin inhibited the formation of OCLs in a dose-dependent manner and suppressed the bone-resorbing activity of OCLs. Moreover, fisetin-treated OCLs showed markedly decreased phosphorylation of extracellular signal-regulated kinase, Akt, and Jun N-terminal kinase, but fisetin did not inhibit p38 phosphorylation. Fisetin up-regulated mRNA expression of phase II antioxidant enzymes including HO-1 and interfered with RANKL-mediated reactive oxygen species (ROS) production. Studies with RNA interference showed that suppression of NF-E2-related factor 2 (Nrf2), a key transcription factor for phase II antioxidant enzymes, rescued fisetin-mediated inhibition of OCL differentiation. Furthermore, fisetin significantly decreased RANKL-induced nuclear translocation of cFos and nuclear factor of activated T cells cytoplasmic-1 (NFATc1), which is a transcription factor critical for osteoclastogenic gene regulation. Therefore, fisetin inhibits OCL differentiation through blocking RANKLmediated ROS production by Nrf2-mediated up-regulation of phase II antioxidant enzymes. © The Japanese Pharmacological Society.

    添付ファイル: 45. Sakai (J Pharmacol Sci).pdf

    DOI: 10.1254/jphs.12243FP

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  • Deltamethrin inhibits osteoclast differentiation via regulation of heme oxygenase-1 and NFATc1 査読

    Hiroshi Sakamoto, Eiko Sakai, Reiko Fumimoto, Yu Yamaguchi, Yutaka Fukuma, Kazuhisa Nishishita, Kuniaki Okamoto, Takayuki Tsukuba

    TOXICOLOGY IN VITRO   26 ( 6 )   817 - 822   2012年9月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:PERGAMON-ELSEVIER SCIENCE LTD  

    Deltamethrin is a widely used pyrethroid pesticide. Although the cytotoxicity of deltamethrin has been reported, especially in neuronal cells, there is no information concerning the effects of deltamethrin on osteoclasts (OCLs). In this study, we showed that deltamethrin inhibited OCL differentiation in vitro. The effects of deltamethrin on OCL differentiation by receptor activator of nuclear factor kappa-B ligand (RANKL) were investigated in bone marrow-derived macrophages (BMMs) or the murine monocytic cell line RAW-D. Treatment with deltamethrin inhibited OCL formation and bone resorption and up-regulated expression of heme oxygenase-1 (HO-1), an anti-oxidative stress enzyme. Deltamethrin also decreased the protein levels of nuclear factor of activated T cells cytoplasmic-1 (NFATc1), which is a master regulator for OCL differentiation, and concomitantly reduced the expression levels of Src and cathepsin K, which are transcriptionally regulated by NFATc1. The effects of deltamethrin on intracellular signaling during the OCL differentiation of BMMs indicated that deltamethrin-treated OCLs displayed impaired phosphorylation of extracellular signal-regulated kinase, p38 mitogen-activated protein kinase, Jun N-terminal kinase, and Akt, and slightly delayed phosphorylation of inhibitor of nuclear factor kappa B alpha (I kappa beta alpha) compared with untreated OCLs. Thus, deltamethrin possibly affects bone metabolism by inhibiting OCL differentiation. (c) 2012 Elsevier Ltd. All rights reserved.

    添付ファイル: 43. Sakamoto (Toxicol In Vitro).pdf

    DOI: 10.1016/j.tiv.2012.05.005

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  • The Coffee Diterpene Kahweol Prevents Osteoclastogenesis via Impairment of NFATc1 Expression and Blocking of Erk Phosphorylation 査読

    Reiko Fumimoto, Eiko Sakai, Yu Yamaguchi, Hiroshi Sakamoto, Yutaka Fukuma, Kazuhisa Nishishita, Kuniaki Okamoto, Takayuki Tsukuba

    JOURNAL OF PHARMACOLOGICAL SCIENCES   118 ( 4 )   479 - 486   2012年4月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:JAPANESE PHARMACOLOGICAL SOC  

    Osteoclasts (OCLs) are multinucleated bone resorbing cells whose differentiation is regulated by receptor activator of nuclear factor kappa-B ligand (RANKL) and macrophage colony-stimulating factor (M-CSF). It is known that inflammatory cytokines and oxidative stress stimulate differentiation of OCLs. Here we evaluated the effects of kahweol, a coffee-specific diterpene, which has been reported to possess anti-oxidant and anti-inflammatory properties, on the differentiation of bone marrow derived macrophages (BMMs) or murine monocytic cell line RAW-D cells into OCLs. Kahweol dose-dependently inhibited the formation of tartrate-resistant acid phosphatase staining positive OCLs from both BMMs and RAW-D cells. In addition, kahweol prevented the bone resorbing activity of OCLs. Kahweol completely abolished RANKL-stimulated phosphorylation of extracellular signal-regulated kinase and impaired phosphorylation of Akt. Moreover, the protein levels of nuclear factor of activated T cells cytoplasmic-1 (NFATc1), a master regulator for OCL differentiation; and OCL markers transcriptionally regulated by NFATc1 such as Src and cathepsin K were down-regulated by kahweol treatment. As one of the molecular mechanisms for the inhibitory effects of kahweol, we also showed that kahweol up-regulated heme oxygenase-1 and inhibited high mobility group box I release. Thus, kahweol in coffee is a useful constituent for inhibition of OCL differentiation.

    添付ファイル: 42. Fumimoto (J Pharmacol Sci).pdf

    DOI: 10.1254/jphs.11212FP

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  • Genetic backgrounds and redox conditions influence morphological characteristics and cell differentiation of osteoclasts in mice 査読

    Shun Narahara, Haruna Matsushima, Eiko Sakai, Yutaka Fukuma, Kazuhisa Nishishita, Kuniaki Okamoto, Takayuki Tsukuba

    CELL AND TISSUE RESEARCH   348 ( 1 )   81 - 94   2012年4月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:SPRINGER  

    Osteoclasts (OCLs) are multinucleated giant cells and are formed by the fusion of mononuclear progenitors of monocyte/macrophage lineage. It is known that macrophages derived from different genetic backgrounds exhibit quite distinct characteristics of immune responses. However, it is unknown whether OCLs from different genetic backgrounds show distinct characteristics. In this study, we showed that bone-marrow macrophages (BMMs) derived from C57BL/6, BALB/c and ddY mice exhibited considerably distinct morphological characteristics and cell differentiation into OCLs. The differentiation of BMMs into OCLs was comparatively quicker in the C57BL/6 and ddY mice, while that of BALB/c mice was rather slow. Morphologically, ddY OCLs showed a giant cell with a round shape, C57BL/6 OCLs were of a moderate size with many protrusions and BALB/c OCLs had the smallest size with fewer nuclei. The intracellular signaling of differentiation and expression levels of marker proteins of OCLs were different in the respective strains. Treatment of BMMs from the three different strains with the reducing agent N-acetylcysteine (NAC) or with the oxidation agent hydrogen peroxide (H2O2) induced changes in the shape and sizes of the cells and caused distinct patterns of cell differentiation and survival. Thus, genetic backgrounds and redox conditions regulate the morphological characteristics and cell differentiation of OCLs.

    添付ファイル: 41. Narahara (Cell Tissue Res).pdf

    DOI: 10.1007/s00441-012-1325-8

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  • Role of the transcription factor Sp1 in regulating the expression of the murine cathepsin E gene 査読

    Kuniaki Okamoto, Yoshiko Okamoto, Tomoyo Kawakubo, Jun-ichi Iwata, Yoshiyuki Yasuda, Takayuki Tsukuba, Kenji Yamamoto

    JOURNAL OF BIOCHEMISTRY   151 ( 3 )   263 - 272   2012年3月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:OXFORD UNIV PRESS  

    Cathepsin E (CE) is an intracellular aspartic proteinase that is exclusively expressed in cells of the gastrointestinal tracts, lymphoid tissues, urinary organs and red blood cells. However, the molecular mechanism by which CE is predominantly expressed in these cells remains unknown. Here, we report the identification of several transcription start sites of the CE gene and their regulatory factors in gastric adenosarcoma cells. We first identified several unique transcription start sites in mouse CE genes by an oligo cap method. Their analysis also revealed the existence of a non-coding region similar to 24-kb upstream of exon 1 in the CE gene and also the existence of two transcripts for CE. Luciferase analyses in upstream of exon 1 revealed that this site contained putative binding regions for the transcription factors Sp1, AP-1 and cEts-1 essential for the expression of CE gene. Moreover, electrophoretic mobility shift assays revealed that the protein-oligonucleotides complex of the Sp1 site were supershifted by an anti-Sp1 antibody. The chromatin immunoprecipitation assay showed that Sp1 bound to the CE promoter region. In addition, overexpression of the Sp1 protein increased the expression of the CE protein. Altogether, these results suggest that Sp1 binding plays a particularly important role in the regulation of CE gene expression.

    添付ファイル: 40. Okamoto (j Biochem).pdf

    DOI: 10.1093/jb/mvr135

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  • Suppression of RANKL-dependent heme oxygenase-1 is required for high mobility group box 1 release and osteoclastogenesis 査読

    Eiko Sakai, Megumi Shimada-Sugawara, Kazuhisa Nishishita, Yutaka Fukuma, Mariko Naito, Kuniaki Okamoto, Koji Nakayama, Takayuki Tsukuba

    JOURNAL OF CELLULAR BIOCHEMISTRY   113 ( 2 )   486 - 498   2012年2月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:WILEY-BLACKWELL  

    The differentiation of osteoclasts is regulated by several essential cytokines, such as receptor activator of nuclear factor ?B ligand (RANKL) and macrophage colony-stimulating factor. Recently, high mobility group box 1 (HMGB1), a chromatin protein, also has been identified as one of these osteoclast differentiation cytokines. However, the molecular mechanisms that control HMGB1 release from osteoclast precursor cells are not known. Here, we report that RANKL-induced suppression of heme oxygenase-1 (HO-1), a heme-degrading enzyme, promotes HMGB1 release during osteoclastogenesis. In contrast, induction of HO-1 with hemin or curcumin in bone marrow-derived macrophages or RAW-D murine osteoclast precursor cells inhibited osteoclastogenesis and suppressed HMGB1 release. Since an inhibitor for p38 mitogen-activated protein kinase (MAPK) prevented the RANKL-mediated HO-1 suppression and extracellular release of HMGB1, these effects were p38 MAPK-dependent. Moreover, suppression of HO-1 in RAW-D cells by RNA interference promoted the activation of caspase-3 and HMGB1 release, whereas overexpression of HO-1 inhibited caspase-3 activation as well as HMGB1 release. Furthermore, these effects were regulated by redox conditions since antioxidant N-acetylcysteine abolished the HO-1/HMGB1/caspase-3 axis. These results suggest that RANKL-dependent HO-1 suppression leads to caspase-3 activation and HMGB1 release during osteoclastogenesis. J. Cell. Biochem. 113: 486498, 2012. (C) 2011 Wiley Periodicals, Inc.

    DOI: 10.1002/jcb.23372

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  • Suppression of RANKL-dependent heme oxygenase-1 is required for high mobility group box 1 release and osteoclastogenesis 査読

    Eiko Sakai, Megumi Sugawara, Kazuhisa Nishishita, Yutaka Fukuma, Kuniaki Okamoto, Takayuki Tsukuba

    JOURNAL OF PHARMACOLOGICAL SCIENCES   118 ( 2 )   208P - 208P   2012年

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:JAPANESE PHARMACOLOGICAL SOC  

    添付ファイル: 39. Sakai (J Cell Biochem).pdf

    DOI: 10.1002/jcb.23372

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  • Cathepsin E is critical for proper trafficking of cell surface proteins 査読

    Takayuki Tsukuba, Kuniaki Okamoto, Kenji Yamamoto

    Journal of Oral Biosciences   54 ( 1 )   48 - 53   2012年

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    記述言語:英語   掲載種別:研究論文(国際会議プロシーディングス)   出版者・発行元:Japanese Association for Oral Biology  

    Cathepsin E is an intracellular aspartic proteinase of the pepsin superfamily, which is predominantly expressed in certain cell types, such as the immune-related cells and rapidly regenerating cells. Recent studies have demonstrated that loss of cathepsin E in mice impairs their normal immune responses. In antigen-presenting cells (APC) such as macrophages, dendritic cells, and microglia, cathepsin E is localized mainly in the endosomal component and regulates the nature and functions of these cells. Deficiency of cathepsin E in macrophages induces a novel form of lysosomal storage disorder manifesting the accumulation of major lysosomal membrane glycoproteins such as LAMP-1 and LAMP-2 and elevated lysosomal pH. Such alterations in these cells are linked to abnormal intracellular trafficking of secretory and cell surface proteins. In fact, cathepsin E deficiency leads to increased secretion of a variety of soluble lysosomal enzymes including cathepsins and glycosidases, into the culture medium. By contrast, the expression and localization of cell surface proteins including Toll-like receptors, chemotactic receptors, and cell-adhesion receptors, as well as LAMPs, is significantly decreased by cathepsin E deficiency. While these alterations are not observed with cathepsin E-deficient (CatE-/-) dendritic cells, the cell surface expression and localization of the costimulatory molecules CD86, CD80, and CD40 were significantly increased in these cells, indicating that cathepsin E differentially regulates the nature and function of these two APC. This review focuses on the emerging roles of cathepsin E in proper intracellular trafficking of both secretory and cell surface proteins in APC. © 2012 Japanese Association for Oral Biology.

    DOI: 10.1016/j.job.2011.10.001

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  • The role of cathepsin E in terminal differentiation of keratinocytes. 査読

    Kawakubo T, Yasukochi A, Okamoto K, Okamoto Y, Nakamura S, Yamamoto K

    Biological chemistry   392 ( 6 )   571 - 585   2011年4月

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    記述言語:英語   出版者・発行元:Walter de Gruyter GmbH  

    DOI: 10.1515/BC.2011.060

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  • Cathepsin E-deficient mice show increased susceptibility to bacterial infection associated with the decreased expression of multiple cell surface Toll-like receptors. 査読

    Tsukuba T, Yamamoto S, Yanagawa M, Okamoto K, Okamoto Y, Nakayama KI, Kadowaki T, Yamamoto K

    Journal of biochemistry   140 ( 1 )   57 - 66   2006年7月

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    記述言語:英語   出版者・発行元:Japanese Biochemical Society  

    DOI: 10.1093/jb/mvj132

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    その他リンク: http://search.jamas.or.jp/link/ui/2007054591

  • Mutational analysis of residues in two consensus motifs in the active sites of cathepsin E1 査読

    Jian Liu, Takayuki Tsukuba, Kuniaki Okamoto, Masamichi Ohishi, Kenji Yamamoto

    Journal of Biochemistry   132 ( 3 )   493 - 499   2002年

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    Cathepsin E, an intracellular aspartic proteinase of the pepsin family, is composed of two homologous domains, each containing the catalytic Asp residue in a consensus DTG motif. Here we examine the significance of residues in the motifs of rat cathepsin E by substitution of Asp98, Asp283, and Thr284 with other residues using site-directed mutagenesis. Each of the mutant proenzymes, as well as the wild-type protein, was found in culture media and cell extracts when heterologously expressed in human embryonic kidney 293T cells. The single mutants D98A, D283A, and D283E, and the double mutants D98A/D283A and D98E/D283E showed neither autocatalytic processing nor enzymatic activities under acidic conditions. However, the D98E and T284S mutants retained the ability to transform into the mature forms, although they exhibited only about 13 and 40% of the activity of the wild-type enzyme, respectively. The Km values of these two mutants were similar to those of the wild-type enzyme, but their kcat values were greatly decreased. The Ki values for pepstatin and the Ascaris pepsin inhibitor of the mutants and the wild-type enzyme were almost the same. The circular dichroism spectra of the two mutants were essentially the same as those of the wild-type enzyme at various pH values. These results indicate that (i) Asp98, Asp283, and Thr284 are indeed critical for catalysis, and (ii) the decrease in the catalytic activity of D98E and T284S mutants is brought about by an effect on the kinetic process from the enzyme-substrate complex to the release of the product. © 2002 Japanese Biochemical Society.

    DOI: 10.1093/oxfordjournals.jbchem.a003247

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  • Characterization of phagosomal subpopulations along endocytic routes in osteoclasts and macrophages. 査読

    Sakai E, Miyamoto H, Okamoto K, Kato Y, Yamamoto K, Sakai H

    J Biochem.   130 ( 6 )   823 - 831   2001年

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

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  • Design and synthesis of sensitive fluorogenic substrates specific for Lys-gingipain 査読

    Naoko Abe, Atsuyo Baba, Tomoko Kadowaki, Kuniaki Okamoto, Shinji Okazaki, Tetsuji Asao, Kenji Yamamoto

    Journal of Biochemistry   128 ( 5 )   877 - 881   2000年

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:Oxford University Press  

    Lys-gingipain (Kgp) is a major cysteine proteinase produced by the oral anaerobic bacterium Porphyromonas gingivalis, and has been implicated as a major pathogen in the development and progression of advanced adult periodontitis. This enzyme is believed to act as a major virulence factor of the disease, yet there exist no convenient and sensitive substrates for analyzing its biological activity. For a better understanding of the importance of this enzyme in the organism, there is an urgent need for specific substrates. Here we designed and synthesized two peptide 4-methyl-coumaryl-7-amides (MCA), carbobenzoxy (Z)-His-Glu-Lys-MCA, and Z-Glu-Lys-MCA, and tested their possible use as sensitive substrates for Kgp with limited specificity. Both substrates exhibited greater k(cat)/K(m) values than the best known Kgp substrates described so far. Both substrates were resistant to Arg-gingipain, another pathogenic cysteine proteinase from P. gingivalis, as well as trypsin and cathepsins B, L, and H. The levels of Kgp in various microorganisms and human cells were determined with Z-His-Glu-Lys-MCA. Little or no Kgp-like activity was detected in either other microorganisms or human cells tested. These results indicate that the present substrates are a valuable and fast tool for routine assays and for mechanistic studies on Kgp.

    DOI: 10.1093/oxfordjournals.jbchem.a022826

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  • Role of N-glycosylation in cathepsin E. A comparative study of cathepsin E with distinct N-linked oligosaccharides and its nonglycosylated mutant 査読

    Yoshiyuki Yasuda, Shinobu Ikeda, Hideaki Sakai, Takayuki Tsukuba, Kuniaki Okamoto, Kazuhisa Nishishita, Akifumi Akamine, Yuzo Kato, Kenji Yamamoto

    European Journal of Biochemistry   266 ( 2 )   383 - 391   1999年12月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    Cathepsin E (CE), a nonlysosomal, intracellular aspartic proteinase, exists in several molecular forms that are N-glycosylated with high-mannose and/or complex-type oligosaccharides. To investigate the role of N- glycosylation on the catalytic properties and molecular stability of CE, both natural and recombinant enzymes with distinct oligosaccharides were purified from different sources. An N-glycosylation minus mutant, that was constructed by site-directed mutagenesis (by changing asparagine residues to glutamine and aspartic acid residues at positions 73 and 305 in potential N- glycosylation sites of rat CE) and expressed in normal rat kidney cells, was also purified to homogeneity from the cell extracts. The kinetic parameters of the nonglycosylated mutant were found to be essentially equivalent to those of natural enzymes N-glycosylated with either high-mannose or complex- type oligosaccharides. In contrast, the nonglycosylated mutant showed lower pH and thermal stabilities than the glycosylated enzymes. The nonglycosylated mutant exhibited particular sensitivity to conversion to a monomeric form by 2-mercaptoethanol, as compared with those of the glycosylated enzymes. Further, the high-mannose-type enzymes were more sensitive to this agent than the complex-type proteins. A striking difference was found between the high- mannose and complex-type enzymes in terms of activation by ATP at a weakly acidic pH. At pH 5.5, the complex-type enzymes were stabilized by ATP to be restored to the virtual activity, whereas the high-mannose-type enzymes as well as the nonglycosylated mutant were not affected by ATP. These results suggest that N-glycosylation in CE is important for the maintenance of its- proper folding upon changes in temperature, pH and redox state, and that the complex-type oligosaccharides contribute to the completion of the tertiary structure to maintain its active conformation in the weakly acidic pH environments.

    DOI: 10.1046/j.1432-1327.1999.00863.x

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  • Genetic analyses of proteolysis, hemoglobin binding, and hemagglutination of Porphyromonas gingivalis: construction of mutants with rgpA , rgpB , kgp , and hagA .

    Shi Y, Ratnayake DB, Okamoto K, Abe N, Yamamoto K, Nakayama K

    Journal of Biological Chemistry   274 ( 25 )   17955 - 17960   1999年

  • Biochemical and functional properties of lysine-specific cysteine proteinase (Lys-Gingipain) as a virulence factor of Porphyromonas gingivalis in periodontal disease 査読

    Naoko Abe, Tomoko Kadowaki, Kuniaki Okamoto, Koji Nakayama, Masamichi Ohishi, Kenji Yamamoto

    Journal of Biochemistry   123 ( 2 )   305 - 312   1998年

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:Oxford University Press  

    The oral anaerobic bacterium Porphyromonas gingivalis has been implicated as a major etiologic agent of progressive periodontal disease. A novel lysine-specific cysteine proteinase, termed 'Lys-gingipain,' was purified from the culture supernatant of the Arg-gingipain-deficient mutant of P. gingivalis (KDP112) by a simple method including immunoaffinity chromatography. The purified enzyme was found to be composed of a single polypeptide of M(r) = 51,000. Analysis of the enzymatic properties revealed several distinctive features of this enzyme. The proteolytic activity was remarkably activated by thiol-reducing agents and inhibited by idoacetamide, idoacetic acid, and leupeptin. The enzyme was also inhibited by the chloromethyl ketones of tosyl-L-lysine and tosyl-L-phenylalanine. However, internal protease inhibitors, such as cystatins and α1-antichymotrypsin, had no effect on the activity, suggesting its resistance to normal host defense systems in vivo. Despite its narrow specificity for synthetic substrates containing Lys in the P1 site, the enzyme extensively degraded human type I collagen and immunoglobulins G and A (both serum and secretory types). Most important, the enzyme was able to disrupt the functions of polymorphonuclear leukocytes, as shown by its inhibitory effect on the generation of active oxygen species from the activated cells. These results suggest that Lys-gingipain, like Arg-gingipain, plays a crucial role as a virulence factor from P. gingivalis in the development of periodontal disease via the direct destruction of periodontal tissue components and the disruption of normal host defense mechanisms.

    DOI: 10.1093/oxfordjournals.jbchem.a021937

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  • Arg-gingipain acts as a major processing enzyme for various cell surface proteins in Porphyromonas gingivalis. 査読

    Kadowaki T, Nakayama K, Yoshimura F, Okamoto K, Abe N, Yamamoto K

    J. Biol. Chem.   273   29072 - 29076   1998年

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    DOI: 10.1074/jbc.273.44.29072

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  • Involvement of a Lysine-specific cysteine Proteinase in Hemoglobin Adsorption and Heme Accumulation by Porphyromonas gingivalis. 査読

    Okamoto K, Nakayama K, Kadowaki T, Abe N, Ratnayake D.B, Yamamoto K

    J. Biol. Chem.   273   21225 - 21231   1998年

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    DOI: 10.1074/jbc.273.33.21225

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  • Possible roles of N-linked oligosaccharides on cathepsin E 査読

    H Sakai, S Ikeda, K Nishishita, Y Kato, K Okamoto, T Tsukuba, K Yamamoto

    PROTEOLYSIS IN CELL FUNCTIONS   13   55 - 60   1997年

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    記述言語:英語   掲載種別:研究論文(国際会議プロシーディングス)   出版者・発行元:I O S PRESS  

    Cathepsin E (CE) is a glycosylated intracellular aspartic proteinase. In this study, the role of N-glycosylation in intracellular trafficking and stability of CE was investigated in human gastric adenocarcinoma cells expressing endogenous CE (AGS-1) and recombinant normal rat kidney (NRK) cells transfected with rat CE cDNA. When these cells were treated with tunicamycin, a patent inhibitor for N-glycosylation, the initially synthesized unglycosylated proCE with an apparent molecular mass of 43 kDa was rapidly disappeared from the cells. Since secretion into the medium was not observed, the unglycosylated CE was suggested to be degraded within these cells. The amino acid sequence predicted from the cDNA revealed that rat CE (RCE) contained two potential N-glycosylation sites in the NH2-terminal (Asn(73)-Phe-Thr(75)) and COOH-terminal (Asn(305)-Val-Tnr(307)) portions. To further elucidate the significance of N-glycosylation on the stability of CE, we constructed the N-glycosylation-deficient mutants of RCE by site-directed mutagenesis (Thr(75)-->Ala(75) or Asn(305)-->Gln(305)) and expressed in NRK cells. In contrast to unglycosylated RCE generated by tunicamycin treatment, each mutant was stably retained in the cells and converted into a fully catalytically active form by a brief acid treatment in a similar manner as the wild-type CE. Pulse-chase experiments with S-35.methionine also revealed that there was no significant difference in the half-lives between each mutant and the wild-type CE in the cells. The results suggest that the deficiency of either potential N-glycosylation site alone does not affect the intracellular stability of RCE.

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  • Biosynthesis and trafficking of cathepsin E 査読

    K Yamamoto, H Nakanishi, T Tsukuba, K Okamoto, H Sakai, K Nishishita, Y Kato

    PROTEOLYSIS IN CELL FUNCTIONS   13   215 - 222   1997年

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    記述言語:英語   掲載種別:研究論文(国際会議プロシーディングス)   出版者・発行元:I O S PRESS  

    Cathepsin E (CE) is an intracellular aspartic proteinase that exists mainly as a homodimer with a molecular mass of about 82 kDa and partly as a monomer of 42 kDa. In contrast to the lysosomal aspartic proteinase cathepsin D (CD), CE has a limited distribution in some animal species and is normally located in non-lysosomal compartments such as the plasma membrane, the endocytic organelles, and the endoplasmic reticulum (ER). The intracellular localization appears to be dependent on cell types expressing this enzyme. Our recent studies with pulse-chase kinetic analysis in Various cell types revealed that CE was initially synthesized as a larger precursor and underwent different proteolytic and glycolytic processing depending on cell types. This paper describes three different processing systems of CE in certain cell types. The biosynthesis and processing were studied by pulse-chase analysis with S-35-methionone and immunocytochemistry using specific antibodies to CE. In primary cultures of rat peritoneal macrophages and brain microglial cells, CE was first synthesized as a proenzyme with high-mannose-type oligosaccharide chains and subsequently processed to the mature form bearing complex-type oligosaccharides via the intermediate form within a 4 h chase period. The maturation of proCE in these cells was completely blocked by bafilomycin A1, a potent inhibitor of a vacuolar type of H+-ATPase, indicating that the maturation of CE precursor occurs in cellular acidic compartments. In rat thymocytes and mouse Friend erythroleukemia cells, the initially synthesized proCE was processed to the intermediate form within a 6 h chase period, but scarcely processed to the mature form even after a 48 h chase. However, the CE precursors in these cells became resistant to digestion by endo-beta-N-acetylglucosaminidase (Endo H) within several hours of synthesis. Interestingly, CE in thymocytes from dexamethasone-treated rats was found to be greatly processed to the mature form. In recombinant CHO and NRK cells expressing exogenous CE, the initially synthesized proenzyme was processed to the intermediate form, but not to the mature form, which did not undergo glycolytic processing.

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  • Cloning and sequencing of the gene encoding a novel lysine-specific cysteine proteinase (Lys-Gingipain) in porphyromonas gingivalis: Structural relationship with the arginine-specific cysteine proteinase (Arg-Gingipain) 査読

    Kuniaki Okamoto, Tomoko Kadowaki, Koji Nakayama, Kenji Yamamoto

    Journal of Biochemistry   120 ( 2 )   398 - 406   1996年

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:Oxford University Press  

    Lys-gingipain (KGP), so termed due to its peptide cleavage specificity for lysine residues, is a cysteine proteinase produced by the Gram-negative anaerobic bacterium Porphyromonas gingivalis. Mixed oligonucleotide primers designed from the NH2-terminal sequence of the purified enzyme were used to clone the KGP-encoding gene (kgp) from the organism. The nucleotide sequence of kgp had a 5,169-bp open reading frame encoding 1,723 amino acids with a calculated molecular mass of 218 kDa. As the extracellular mature enzyme had an apparent molecular mass of 51 kDa in gels, the precursor of KGP was found to comprise at least four domains, the signal peptide, the NH2-terminal prodomain, the mature proteinase domain, and the COOH-terminal hemagglutinin domain, and to be proteolytically processed during its transport. Importantly, the COOH-terminal region contained three direct repeats of two different amino acid sequences, LKWD(or E)AP and YTYTVYRDGTKI, and the subdomains located between the two repeats exhibited strong similarity to those of Arg-gingipain (RGP), another major cysteine proteinase produced by the organism and having cleavage specificity for arginine residues, although the arrangement of the subdomains was not necessarily identical in the two enzymes. Since the I(GP activity was greatly decreased in RGP-deficient mutants and since the most probable site of the propeptide cleavage was present in the homologous sequence highly susceptible to proteolysis by RGP, the precursor of KGP is likely to be processed by RGP to form the mature enzyme.

    DOI: 10.1093/oxfordjournals.jbchem.a021426

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  • Structure and function of a novel arginine-specific cysteine proteinase (argingipain) as a major periodontal pathogenic factor from Porphyromonas gingivalis 査読

    Kenji Yamamoto, Tomoko Kadowaki, Kuniaki Okamoto, Masahiro Yoneda, Koji Nakayama, Yoshio Misumi, Yukio Ikehara

    Advances in Experimental Medicine and Biology   389   33 - 42   1996年

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    Scopus

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  • Construction and characterization of arginine-specific cysteine proteinase(Arg-ginigpain)-deficient mutants of Porphyromonas gingivalis: evidence for singificant contribution of Arg-gingipain to virulence.

    Nakayama K, Kadowaki T, Okamoto K, Yamamoto K

    Journal of Biological Chemistry   270 ( 40 )   23619 - 23626   1995年

  • Structural characterization of argingipain, a novel arginine-specific cysteine proteinase as a major periodontal pathogenic factor from Porphyromonas gingivalis. 査読

    Okamoto K, Misumi Y, Kadowaki T, Yoneda M, Yamamoto K

    Arch. Biochem. Biophys.   316   917 - 925   1995年

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    DOI: 10.1006/abbi.1995.1123

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書籍等出版物

  • 第6版現代歯科薬理学

    岡元 邦彰( 担当: 分担執筆 ,  範囲: 脂質異常治療薬 痛風治療薬)

    医歯薬出版株式会社  2017年 

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  • 第5版現代歯科薬理学

    岡元 邦彰( 担当: 分担執筆 ,  範囲: 血液および造血臓器に作用する薬物 脂質異常治療薬 痛風治療薬)

    医歯薬出版株式会社  2012年 

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  • 「第4版現代歯科薬理学」

    岡元 邦彰( 担当: 分担執筆 ,  範囲: 止血に用いられる薬物 血液および造血臓器に作用する薬物)

    医歯薬出版株式会社  2005年 

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  • Arg-gingipain and Lys-gingipain: a novel class of cysteine proteinases.

    山本 健二, 岡元 邦彰, その他( 担当: 共著)

    Birkhäusen Verlag  1999年 

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  • Structural characterization of pathogenic cysteine proteinase from the oral bacterium Porphysromonas gingivalis

    IOS Press  1997年 

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  • Possible roles of N-linked oligosaccharides on cathepsin E

    IOS Press  1997年 

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  • Possible roles of N-linked oligosaccharides on cathepsin E

    IOS Press  1997年 

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  • Structural characterization of pathogenic cysteine proteinase from the oral bacterium Porphysromonas gingivalis

    IOS Press  1997年 

     詳細を見る

  • Biosynthesis and trafficking of cathepsin E

    IOS Press  1997年 

     詳細を見る

  • Biosynthesis and trafficking of cathepsin E

    IOS Press  1997年 

     詳細を見る

  • Biosynthesis and trafficking of cathepsin E

    IOS Press  1997年 

     詳細を見る

  • Structural characterization of pathogenic cysteine proteinase from the oral bacterium Porphysromonas gingivalis

    IOS Press  1997年 

     詳細を見る

  • Possible roles of N-linked oligosaccharides on cathepsin E

    IOS Press  1997年 

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▼全件表示

MISC

  • オルガノイドと細胞外小胞を癌研究に応用する

    江口傑徳, 江口傑徳, 十川千春, 岡元邦彰

    Journal of Oral Biosciences Supplement (Web)   2020   2020年

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  • 分子シャペロントリオによるエクソソーム制御,腫瘍悪性化およびマクロファージ分極について

    江口傑徳, 小野喜章, 小野喜章, 河合穂高, チャン チェンマン, 十川千春, 奥舎有加, 岡元邦彰

    日本臨床ストレス応答学会大会抄録集   14th   2019年

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  • 新しい腫瘍オルガノイド多元評価システムの開発

    十川チハル, 江口傑徳, 江口傑徳, 大山和美, 奥舎有加, 中野敬介, 十川紀夫, 岡元邦彰

    Journal of Oral Biosciences Supplement (Web)   2019   2019年

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  • ニコチンは口腔扁平上皮癌細胞のセツキシマブ耐性を促進する

    清水 理恵子, 伊原木 聰一郎, 江口 傑徳, 奥井 達雄, 高畠 清文, 河合 穂高, 小野 喜章, 岡元 邦彰, 長塚 仁, 佐々木 朗

    岡山歯学会雑誌   37 ( 2 )   80 - 81   2018年12月

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    記述言語:日本語   出版者・発行元:岡山歯学会  

    J-GLOBAL

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  • 口腔扁平上皮癌診断・治療における分子シャペロンHSP90含有細胞外小胞の可能性

    小野 喜章, 江口 傑徳, 十川 千春, 奥舎 有加, 河合 穂高, 中野 敬介, 佐々木 朗, 岡元 邦彰, 小崎 健一

    Journal of Oral Biosciences Supplement   2018   333 - 333   2018年9月

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    記述言語:日本語   出版者・発行元:(一社)歯科基礎医学会  

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  • 核内およびエクソソーム中に存在するMMPs/PEXの癌進展における役割とその抑制

    奥舎 有加, 江口 傑徳, 十川 千春, 小野 喜章, 奥井 達雄, 中野 敬介, 岡元 邦彰

    Journal of Oral Biosciences Supplement   2018   150 - 150   2018年9月

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    記述言語:日本語   出版者・発行元:(一社)歯科基礎医学会  

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  • 癌の治療抵抗性と転移におけるHSP90およびMMP3の役割

    江口 傑徳, 小野 喜章, 奥舎 有加, 十川 千春, 内部 健太, 中野 敬介, 奥井 達雄, 滝川 正春, 岡元 邦彰, カルダーウッド・スチュアート

    Journal of Oral Biosciences Supplement   2018   142 - 142   2018年9月

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    記述言語:日本語   出版者・発行元:(一社)歯科基礎医学会  

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  • 癌の治療抵抗性と転移におけるHSP90およびMMP3の役割

    江口傑徳, 江口傑徳, 小野喜章, 小野喜章, 奥舎有加, 十川千春, 内部健太, 中野敬介, 中野敬介, 奥井達雄, 滝川正春, 岡元邦彰, CALDERWOOD SK

    Journal of Oral Biosciences Supplement (Web)   2018   2018年

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  • 急速転移性癌細胞株由来細胞外小胞による破骨細胞分化および癌細胞浸潤の制御

    板垣まみ, 十川千春, 奥舎有加, 江口傑徳, 江口傑徳, 小野喜章, 岡元邦彰

    Journal of Oral Biosciences Supplement (Web)   2018   2018年

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  • 口腔扁平上皮癌診断・治療における分子シャペロンHSP90含有細胞外小胞の可能性

    小野喜章, 江口傑徳, 江口傑徳, 十川千春, 奥舎有加, 河合穂高, 中野敬介, 佐々木朗, 岡元邦彰, 小崎健一

    Journal of Oral Biosciences Supplement (Web)   2018   2018年

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  • 破骨細胞分化を負に制御するRabタンパク質の同定

    奥舎有加, 板垣まみ, 十川千春, 江口傑徳, 江口傑徳, 坂井詠子, 筑波隆幸, 岡元邦彰

    Journal of Oral Biosciences Supplement (Web)   2018   2018年

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  • 核内およびエクソソーム中に存在するMMPs/PEXの癌進展における役割とその抑制

    奥舎有加, 江口傑徳, 江口傑徳, 十川千春, 小野喜章, 小野喜章, 奥井達雄, 中野敬介, 岡元邦彰

    Journal of Oral Biosciences Supplement (Web)   2018   2018年

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  • 転移性の異なる細胞株を用いた核内MMP3の機能解析とその役割

    奥舎有加, 江口傑徳, 十川千春, 奥井達雄, 中野敬介, 小崎健一, 岡元邦彰

    日本薬理学会近畿部会プログラム・要旨集   133rd   2018年

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  • 新規高分子量Gタンパク質Rab44はCa2+流入及びNFATc1経路を介して破骨細胞分化を負に制御する

    山口優, 坂井詠子, 岡元邦彰, 鍜治屋浩, 岡部幸司, 門脇知子, 筑波隆幸

    日本生化学会大会(Web)   90th   2017年

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  • 破骨細胞分化を制御する新規Rabタンパク質の同定と解析

    山口優, 坂井詠子, 岡元邦彰, 門脇知子, 筑波隆幸

    日本病態プロテアーゼ学会学術集会プログラム抄録集   22nd   2017年

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  • 破骨細胞分化を負に制御する新規Kelchタンパク質の同定と機能解析

    楢原峻, 楢原峻, 坂井詠子, 山口優, 楢原春菜, 門脇知子, 岡元邦彰, 朝比奈泉, 筑波隆幸

    日本生化学会大会(Web)   90th   2017年

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  • 口腔扁平上皮癌細胞由来エクソソームに含まれる分子シャペロンについての検討

    小野喜章, 小野喜章, 江口傑徳, 十川千春, 村上純, 藤原敏史, 藤原敏史, 笠井智成, 妹尾昌治, 佐々木朗, 小崎健一, 岡元邦彰

    臨床ストレス応答学会大会抄録集   12th   2017年

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  • 癌や炎症性疾患において分子シャペロンHSPsが誘導される新たな経路:核内MMPとヘテロクロマチンタンパク質の相互作用

    江口傑徳, 江口傑徳, 奥舎有加, 小崎健一, 岡元邦彰, CALDERWOOD Stuart K

    臨床ストレス応答学会大会抄録集   12th   2017年

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  • Mmp遺伝子を標的とし,高転移性癌細胞の生存と転移を抑制する

    奥舎有加, 江口傑徳, 十川千春, 中野敬介, 岡元邦彰, 小崎健一

    Journal of Oral Biosciences Supplement (Web)   2017   2017年

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  • 転移性の異なる癌細胞株の網羅的遺伝子発現解析による浸潤・転移促進因子の探索と機能解明

    奥舎有加, 江口傑徳, 十川千春, 中野敬介, 小崎健一, 岡元邦彰

    硬組織再生生物学会学術大会・総会プログラム・抄録集   26th   2017年

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  • 歯科用モノマーによる破骨細胞分化に対する効果

    稲光 宏之, 岡元 邦彰, 坂井 詠子, 村田 比呂司, 筑波 隆幸

    Journal of Oral Biosciences Supplement   2014   180 - 180   2014年9月

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    記述言語:日本語   出版者・発行元:(一社)歯科基礎医学会  

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  • Chemical constituent of Sanguisorba officinalis inhibits osteoclastogenesis

    Eiko Sakai, Mayumi Iwatake, Yutaka Fukuma, Kazuhisa Nishishita, Kuniaki Okamoto, Takashi Tanaka, Takayuki Tsukuba

    JOURNAL OF PHARMACOLOGICAL SCIENCES   124   232P - 232P   2014年

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    記述言語:英語   掲載種別:研究発表ペーパー・要旨(国際会議)   出版者・発行元:JAPANESE PHARMACOLOGICAL SOC  

    Web of Science

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  • コバルトプロトポルフィリンの破骨細胞分化および活性化に対する影響

    八島由佳, 八島由佳, 岡元邦彰, 坂井詠子, 西下一久, 筑波隆幸

    Journal of Oral Biosciences Supplement (Web)   2014   ROMBUNNO.P2‐33 (WEB ONLY)   2014年

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    記述言語:日本語  

    J-GLOBAL

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  • Up-regulation mechanisms of endosomal/lysosomal proteins during osteoblast differentiation

    Erika Yoneshima, Kuniaki Okamoto, Eiko Sakai, Noriaki Yoshida, Takayuki Tsukuba

    JOURNAL OF PHARMACOLOGICAL SCIENCES   121   170P - 170P   2013年

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    記述言語:英語   掲載種別:研究発表ペーパー・要旨(国際会議)   出版者・発行元:JAPANESE PHARMACOLOGICAL SOC  

    Web of Science

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  • Cafestol prevents osteoclastogenesis via impairment of NFATc1 expression and blocking of Erk phosphorylation

    Yutaka Fukuma, Eiko Sakai, Megumi Sugawara, Erika Yoneshima, Kazuhisa Nishishita, Kuniaki Okamoto, Takayuki Tsukuba

    JOURNAL OF PHARMACOLOGICAL SCIENCES   121   249P - 249P   2013年

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    記述言語:英語   掲載種別:研究発表ペーパー・要旨(国際会議)   出版者・発行元:JAPANESE PHARMACOLOGICAL SOC  

    Web of Science

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  • Identification of two transcrips and in vivo promoter analysis for cathepsin E

    Kuniaki Okamoto, Yoshiko Okamoto, Eiko Sakai, Kazuhisa Nishishita, Kenji Yamamoto, Takayuki Tstikuba

    JOURNAL OF PHARMACOLOGICAL SCIENCES   118   259P - 259P   2012年

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    記述言語:英語   掲載種別:研究発表ペーパー・要旨(国際会議)   出版者・発行元:JAPANESE PHARMACOLOGICAL SOC  

    Web of Science

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  • Accumulation of NADPH oxidase (NOX2) and increased oxidative stress in cathepsin E-deficient macrophages

    Takayuki Tsukuba, Tomoko Kadowaki, Kuniaki Okamoto, Kenji Yamamoto

    JOURNAL OF PHARMACOLOGICAL SCIENCES   118   256P - 256P   2012年

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    記述言語:英語   掲載種別:研究発表ペーパー・要旨(国際会議)   出版者・発行元:JAPANESE PHARMACOLOGICAL SOC  

    Web of Science

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  • Fisetinl prevents osteoclastogenesis via impairment of NFATc1 expression and blocking of Erk phosphorylation

    Megumi Sugawara, Eiko Sakai, Kazuhisa Nishishita, Yutaka Fukuma, Kuniaki Okamoto, Noriaki Yoshida, Takayuki Tsukuba

    JOURNAL OF PHARMACOLOGICAL SCIENCES   118   164P - 164P   2012年

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    記述言語:英語   掲載種別:研究発表ペーパー・要旨(国際会議)   出版者・発行元:JAPANESE PHARMACOLOGICAL SOC  

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  • Receptor activator of NF-kappa B ligand dependent heme oxygenase-1 suppression is required for high mobility group box 1 release, caspase-3 activation, and subsequent osteoclastogenesis

    Eiko Sakai, Megumi Shimada, Kazuhisa Nishishita, Yutaka Fukuma, Kuniaki Okamoto, Takayuki Tsukuba

    JOURNAL OF PHARMACOLOGICAL SCIENCES   115   219P - 219P   2011年

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    記述言語:英語   掲載種別:研究発表ペーパー・要旨(国際会議)   出版者・発行元:JAPANESE PHARMACOLOGICAL SOC  

    Web of Science

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  • Nrf2 activators inhibit osteoclastogenesis induced by receptor activator of NF-kappa B ligand

    Reiko Fumimoto, Hiroshi Sakamoto, Yu Yamaguchi, Eiko Sakai, Kuniaki Okamoto, Takayuki Tsukuba

    JOURNAL OF PHARMACOLOGICAL SCIENCES   115   219P - 219P   2011年

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    記述言語:英語   掲載種別:研究発表ペーパー・要旨(国際会議)   出版者・発行元:JAPANESE PHARMACOLOGICAL SOC  

    Web of Science

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  • Autophagy impairment associated with increased aberrant mitochondria and oxidative stress in cathepsin E-deficient macrophages

    Takayuki Tsukuba, Michiyo Yanagawa, Tomoko Kadowaki, Yoshiko Okamoto, Kuniaki Okamoto, Kenji Yamamoto

    JOURNAL OF PHARMACOLOGICAL SCIENCES   112   216P - 216P   2010年

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    記述言語:英語   掲載種別:研究発表ペーパー・要旨(国際会議)   出版者・発行元:JAPANESE PHARMACOLOGICAL SOC  

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  • Transcription factor Sp1 regulates the expression of the murine cathepsin E gene in gastric adenosarcoma cells

    Kuniaki Okamoto, Yoshiko Okamoto, Tomoyo Kawakubo, Kenji Yamamoto, Takayuki Tsukuba

    JOURNAL OF PHARMACOLOGICAL SCIENCES   112   216P - 216P   2010年

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    記述言語:英語   掲載種別:研究発表ペーパー・要旨(国際会議)   出版者・発行元:JAPANESE PHARMACOLOGICAL SOC  

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  • Carbon dioxide laser irradiation stimulates mineralization in rat dental pulp cells

    Y. Yasuda, E. Ohtomo, T. Tsukuba, K. Okamoto, T. Saito

    INTERNATIONAL ENDODONTIC JOURNAL   42 ( 10 )   940 - 946   2009年10月

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    記述言語:英語   出版者・発行元:WILEY-BLACKWELL PUBLISHING, INC  

    P>Aim
    To examine the effect of carbon dioxide laser irradiation on mineralization in dental pulp cells.
    Methodology
    Rat dental pulp cells were irradiated with a carbon dioxide laser at 2 W output power for 20, 40 and 60 s, and were cultured in ascorbic acid and beta-glycerophosphate containing media. Cell viability was examined 24 h after laser irradiation by a modified MTT assay. Alizarin Red S staining was performed 10 days after laser irradiation. The amounts of secreted collagen from the cells after irradiation were quantified following Sirius Red staining. The expression levels of collagen type I and HSP47, collagen-binding stress protein, were analysed by real-time PCR. HSP47 protein expression was examined by Western blotting. Statistical analysis was performed using one-way analysis of variance (anova) followed by the Tukey's multiple comparison test.
    Results
    The cell viability was not affected by laser irradiation at 2 W for up to 40 s. However, it was significantly decreased by 20% at 60 s (P < 0.05). The amount of mineralization after 10 days of irradiation at 2 W for 40 s was significantly increased in comparison to the other conditions (P < 0.05). The extracellular collagen production was significantly increased by 73% on day 2 and 38% on day 4 after laser irradiation (P < 0.05). Although collagen type I gene expression was not changed by laser irradiation, HSP47 gene and protein expression was induced within 12 and 24 h, respectively.
    Conclusions
    These results suggested that carbon dioxide laser irradiation stimulated mineralization in dental pulp cells. The laser irradiation also increased HSP47 expression but not collagen gene expression.

    DOI: 10.1111/j.1365-2591.2009.01598.x

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  • Carbon dioxide laser irradiation stimulates mineralization in rat dental pulp cells

    Y. Yasuda, E. Ohtomo, T. Tsukuba, K. Okamoto, T. Saito

    INTERNATIONAL ENDODONTIC JOURNAL   42 ( 10 )   940 - 946   2009年10月

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    記述言語:英語   出版者・発行元:WILEY-BLACKWELL PUBLISHING, INC  

    P>Aim
    To examine the effect of carbon dioxide laser irradiation on mineralization in dental pulp cells.
    Methodology
    Rat dental pulp cells were irradiated with a carbon dioxide laser at 2 W output power for 20, 40 and 60 s, and were cultured in ascorbic acid and beta-glycerophosphate containing media. Cell viability was examined 24 h after laser irradiation by a modified MTT assay. Alizarin Red S staining was performed 10 days after laser irradiation. The amounts of secreted collagen from the cells after irradiation were quantified following Sirius Red staining. The expression levels of collagen type I and HSP47, collagen-binding stress protein, were analysed by real-time PCR. HSP47 protein expression was examined by Western blotting. Statistical analysis was performed using one-way analysis of variance (anova) followed by the Tukey's multiple comparison test.
    Results
    The cell viability was not affected by laser irradiation at 2 W for up to 40 s. However, it was significantly decreased by 20% at 60 s (P < 0.05). The amount of mineralization after 10 days of irradiation at 2 W for 40 s was significantly increased in comparison to the other conditions (P < 0.05). The extracellular collagen production was significantly increased by 73% on day 2 and 38% on day 4 after laser irradiation (P < 0.05). Although collagen type I gene expression was not changed by laser irradiation, HSP47 gene and protein expression was induced within 12 and 24 h, respectively.
    Conclusions
    These results suggested that carbon dioxide laser irradiation stimulated mineralization in dental pulp cells. The laser irradiation also increased HSP47 expression but not collagen gene expression.

    DOI: 10.1111/j.1365-2591.2009.01598.x

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  • Impaired chemotaxis and cell adhesion due to decrease in several cell-surface receptors in cathepsin E-deficient macrophages

    Takayuki Tsukuba, Michiyo Yanagawa, Kuniaki Okamoto, Yoshiko Okamoto, Yoshiyuki Yasuda, Keiichi I. Nakayama, Tomoko Kadowaki, Kenji Yamamoto

    JOURNAL OF BIOCHEMISTRY   145 ( 5 )   565 - 573   2009年5月

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    記述言語:英語   出版者・発行元:OXFORD UNIV PRESS  

    Cathepsin E is an endo-lysosomal aspartic proteinase exclusively present in immune system cells. Previous studies have shown that cathepsin E-deficient (CatE(/)) mice display aberrant immune responses such as atopic dermatitis and higher susceptibility to bacterial infection. However, the mechanisms underlying abnormal immune responses induced by cathepsin E deficiency are still unclear. In this study, we found that the cell-surface levels of chemotactic receptors, including chemokine receptor (CCR)-2 and N-formyl peptide receptors (FPRs), were clearly diminished in CatE(/)macrophages compared with those in wild-type cells. Consistently, chemotaxis of CatE(/)macrophages to MCP-1 and N-formyl-methionyl-leucyl-phenylalanine was also decreased. Similar to the chemotactic receptors, the surface expressions of the adhesion receptors CD18 (integrin (2)) and CD 29 (integrin (1)) in CatE(/) macrophages were significantly decreased, thereby reducing cell attachment of CatE(/) macrophages. These results indicate that the defects in chemotaxis and cell adhesion are likely to be involved in the imperfect function of CatE(/)macrophages.

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  • Impaired chemotaxis and cell adhesion due to decrease in several cell-surface receptors in cathepsin E-deficient macrophages

    Takayuki Tsukuba, Michiyo Yanagawa, Kuniaki Okamoto, Yoshiko Okamoto, Yoshiyuki Yasuda, Keiichi I. Nakayama, Tomoko Kadowaki, Kenji Yamamoto

    JOURNAL OF BIOCHEMISTRY   145 ( 5 )   565 - 573   2009年5月

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    記述言語:英語   出版者・発行元:OXFORD UNIV PRESS  

    Cathepsin E is an endo-lysosomal aspartic proteinase exclusively present in immune system cells. Previous studies have shown that cathepsin E-deficient (CatE(/)) mice display aberrant immune responses such as atopic dermatitis and higher susceptibility to bacterial infection. However, the mechanisms underlying abnormal immune responses induced by cathepsin E deficiency are still unclear. In this study, we found that the cell-surface levels of chemotactic receptors, including chemokine receptor (CCR)-2 and N-formyl peptide receptors (FPRs), were clearly diminished in CatE(/)macrophages compared with those in wild-type cells. Consistently, chemotaxis of CatE(/)macrophages to MCP-1 and N-formyl-methionyl-leucyl-phenylalanine was also decreased. Similar to the chemotactic receptors, the surface expressions of the adhesion receptors CD18 (integrin (2)) and CD 29 (integrin (1)) in CatE(/) macrophages were significantly decreased, thereby reducing cell attachment of CatE(/) macrophages. These results indicate that the defects in chemotaxis and cell adhesion are likely to be involved in the imperfect function of CatE(/)macrophages.

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  • カテプシンE欠損マクロファージにおけるオートファジーの低下とそれに伴うミトコンドリア機能異常と酸化ストレスの上昇

    筑波隆幸, 門脇知子, 坂井詠子, 岡元邦彰, 山本健二

    Journal of Oral Biosciences   51 ( Supplement )   2009年

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  • Determination of active site of lysine-specific cysteine proteinase (Lys-gingipain) by use of a Porphyromonas gingivalis plasmid system

    Yutaka Ishida, JinPing Hu, Eiko Sakai, Tomoko Kadowaki, Kenji Yamamoto, Takayuki Tsukuba, Yuzo Kato, Koji Nakayama, Kuniaki Okamoto

    ARCHIVES OF ORAL BIOLOGY   53 ( 6 )   538 - 544   2008年6月

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    記述言語:英語   出版者・発行元:PERGAMON-ELSEVIER SCIENCE LTD  

    Porphyromonas gingivalis, a major etiological bacterium of periodontal diseases, produces a unique lysine-specific cysteine proteinase (Lys-gingipain, Kgp) implicated in the virulence of this organism. Our observations show the expression of a catalytically active recombinant Kgp in a P. gingivalis Kgp-null mutant and the restoration of its functions by the use of a shuttle plasmid vector stable in P. gingivalis. The Kgp-expressing mutant exhibited a similar catalytic activity to that of the wild-type strain. This mutant also restored the ability to form black-pigmented colonies on blood agar plates and to generate a 19-kDa haemoglobin receptor protein responsible for haemoglobin binding. In order to establish the importance of the active-site Cys residue and elucidate its role in bacterial black pigmentation we constructed three Kgp mutants with changed potential active-site Cys residues. The cells expressing a single mutation (C476A) showed the high Kgp activity and the black pigmentation. In contrast, the cells expressing the single mutant (C477A) and the double mutant (C476A/C477A) exhibited neither Kgp activity nor black pigmentation. These results indicate that the 477th Cys residue is essential for both the Kgp activity and the black pigmentation of P. gingivalis. (C) 2008 Elsevier Ltd. All rights reserved.

    DOI: 10.1016/j.archoralbio.2008.01.004

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  • Determination of active site of lysine-specific cysteine proteinase (Lys-gingipain) by use of a Porphyromonas gingivalis plasmid system

    Yutaka Ishida, JinPing Hu, Eiko Sakai, Tomoko Kadowaki, Kenji Yamamoto, Takayuki Tsukuba, Yuzo Kato, Koji Nakayama, Kuniaki Okamoto

    ARCHIVES OF ORAL BIOLOGY   53 ( 6 )   538 - 544   2008年6月

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    記述言語:英語   出版者・発行元:PERGAMON-ELSEVIER SCIENCE LTD  

    Porphyromonas gingivalis, a major etiological bacterium of periodontal diseases, produces a unique lysine-specific cysteine proteinase (Lys-gingipain, Kgp) implicated in the virulence of this organism. Our observations show the expression of a catalytically active recombinant Kgp in a P. gingivalis Kgp-null mutant and the restoration of its functions by the use of a shuttle plasmid vector stable in P. gingivalis. The Kgp-expressing mutant exhibited a similar catalytic activity to that of the wild-type strain. This mutant also restored the ability to form black-pigmented colonies on blood agar plates and to generate a 19-kDa haemoglobin receptor protein responsible for haemoglobin binding. In order to establish the importance of the active-site Cys residue and elucidate its role in bacterial black pigmentation we constructed three Kgp mutants with changed potential active-site Cys residues. The cells expressing a single mutation (C476A) showed the high Kgp activity and the black pigmentation. In contrast, the cells expressing the single mutant (C477A) and the double mutant (C476A/C477A) exhibited neither Kgp activity nor black pigmentation. These results indicate that the 477th Cys residue is essential for both the Kgp activity and the black pigmentation of P. gingivalis. (C) 2008 Elsevier Ltd. All rights reserved.

    DOI: 10.1016/j.archoralbio.2008.01.004

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  • Dentin phosphophoryn promotes cellular migration of human dental pulp cells

    Yoshiyuki Yasuda, Masanobu Izumikawa, Kuniaki Okamoto, Takayuki Tsukuba, Takashi Saito

    JOURNAL OF ENDODONTICS   34 ( 5 )   575 - 578   2008年5月

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    記述言語:英語   出版者・発行元:ELSEVIER SCIENCE INC  

    Dentin phosphophoryn (DPP) is a dentin sialophosphoprotein gene product that has an RGD motif and repeat sequences of aspartic acid and phosphoserine. To date, the function of DPP in the early stage of reparative dentin formation still remains unclear. The objective of this study was to evaluate the effects of DPP on pulp cell migration and proliferation. DPP promoted cell migration in a concentration-dependent manner, thus increasing it by about 3-fold at 1000 ng/mL compared with the control, but it had no effect on cell proliferation. Dephosphorylated DPP also promoted cell migration, similarly to DPP. However, cell migration was significantly suppressed by the addition of alpha v beta 3 integrin antibody to the medium. Furthermore, porcine DPP-derived RGD peptide, but not its mutant RAD peptide, significantly promoted cell migration. These results indicated that the RGD motif of DPP plays an important role in the migration of human dental pulp cells.

    DOI: 10.1016/j.joen.2008.02.018

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  • Association of cathepsin E deficiency with the increased territorial aggressive response of mice

    Naoki Shigematsu, Takaichi Fukuda, Tsuneyuki Yamamoto, Tsuyoshi Nishioku, Taku Yamaguchi, Masaru Himeno, Keiichi I. Nakayama, Takayuki Tsukuba, Tomoko Kadowaki, Kuniaki Okamoto, Shun Higuchi, Kenji Yamamoto

    JOURNAL OF NEUROCHEMISTRY   105 ( 4 )   1394 - 1404   2008年5月

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    記述言語:英語   出版者・発行元:WILEY  

    Cathepsin E is an endolysosomal aspartic proteinase predominantly expressed in cells of the immune system, but physiological functions of this protein in the brain remains unclear. In this study, we investigate the behavioral effect of disrupting the gene encoding cathepsin E in mice. We found that the cathepsin E-deficient (CatE-/-) mice were behaviorally normal when housed communally, but they became more aggressive compared with the wild-type littermates when housed individually in a single cage. The increased aggressive response of CatE-/- mice was reduced to the level comparable to that seen for CatE+/+ mice by pretreatment with an NK-1-specific antagonist. Consistent with this, the neurotransmitter substance P (SP) level in affective brain areas including amygdala, hypothalamus, and periaqueductal gray was significantly increased in CatE-/- mice compared with CatE+/+ mice, indicating that the increased aggressive behavior of CatE-/- mice by isolation housing followed by territorial challenge is mainly because of the enhanced SP/NK-1 receptor signaling system. Double immunofluorescence microscopy also revealed the co-localization of SP with synaptophysin but not with microtubule-associated protein-2. Our data thus indicate that cathepsin E is associated with the SP/NK-1 receptor signaling system and thereby regulates the aggressive response of the animals to stressors such as territorial challenge.

    DOI: 10.1111/j.1471-4159.2008.05242.x

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  • Dentin phosphophoryn promotes cellular migration of human dental pulp cells

    Yoshiyuki Yasuda, Masanobu Izumikawa, Kuniaki Okamoto, Takayuki Tsukuba, Takashi Saito

    JOURNAL OF ENDODONTICS   34 ( 5 )   575 - 578   2008年5月

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    記述言語:英語   出版者・発行元:ELSEVIER SCIENCE INC  

    Dentin phosphophoryn (DPP) is a dentin sialophosphoprotein gene product that has an RGD motif and repeat sequences of aspartic acid and phosphoserine. To date, the function of DPP in the early stage of reparative dentin formation still remains unclear. The objective of this study was to evaluate the effects of DPP on pulp cell migration and proliferation. DPP promoted cell migration in a concentration-dependent manner, thus increasing it by about 3-fold at 1000 ng/mL compared with the control, but it had no effect on cell proliferation. Dephosphorylated DPP also promoted cell migration, similarly to DPP. However, cell migration was significantly suppressed by the addition of alpha v beta 3 integrin antibody to the medium. Furthermore, porcine DPP-derived RGD peptide, but not its mutant RAD peptide, significantly promoted cell migration. These results indicated that the RGD motif of DPP plays an important role in the migration of human dental pulp cells.

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  • Association of cathepsin E deficiency with the increased territorial aggressive response of mice

    Naoki Shigematsu, Takaichi Fukuda, Tsuneyuki Yamamoto, Tsuyoshi Nishioku, Taku Yamaguchi, Masaru Himeno, Keiichi I. Nakayama, Takayuki Tsukuba, Tomoko Kadowaki, Kuniaki Okamoto, Shun Higuchi, Kenji Yamamoto

    JOURNAL OF NEUROCHEMISTRY   105 ( 4 )   1394 - 1404   2008年5月

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    記述言語:英語   出版者・発行元:WILEY  

    Cathepsin E is an endolysosomal aspartic proteinase predominantly expressed in cells of the immune system, but physiological functions of this protein in the brain remains unclear. In this study, we investigate the behavioral effect of disrupting the gene encoding cathepsin E in mice. We found that the cathepsin E-deficient (CatE-/-) mice were behaviorally normal when housed communally, but they became more aggressive compared with the wild-type littermates when housed individually in a single cage. The increased aggressive response of CatE-/- mice was reduced to the level comparable to that seen for CatE+/+ mice by pretreatment with an NK-1-specific antagonist. Consistent with this, the neurotransmitter substance P (SP) level in affective brain areas including amygdala, hypothalamus, and periaqueductal gray was significantly increased in CatE-/- mice compared with CatE+/+ mice, indicating that the increased aggressive behavior of CatE-/- mice by isolation housing followed by territorial challenge is mainly because of the enhanced SP/NK-1 receptor signaling system. Double immunofluorescence microscopy also revealed the co-localization of SP with synaptophysin but not with microtubule-associated protein-2. Our data thus indicate that cathepsin E is associated with the SP/NK-1 receptor signaling system and thereby regulates the aggressive response of the animals to stressors such as territorial challenge.

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  • Berberine inhibits RANKL-induced osteoclast formation and survival through suppression of nuclear factor κB and Akt pathways

    Hu J-P, Nishishita K, Sakai E, Yoshida H, Kato Y, Tsukuba T, Okamoto K

    European Journal of Pharmacology   580 ( 1-2 )   70 - 79   2008年2月

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  • Berberine inhibits RANKL-induced osteoclast formation and survival through suppressing the NF-kappa B and Akt pathways

    Jin-Ping Hu, Kazuhisa Nishishita, Eiko Sakai, Hajime Yoshida, Yuzo Kato, Takayuki Tsukuba, Kuniaki Okamoto

    EUROPEAN JOURNAL OF PHARMACOLOGY   580 ( 1-2 )   70 - 79   2008年2月

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    記述言語:英語   出版者・発行元:ELSEVIER SCIENCE BV  

    Berberine, an isoquinoline alkaloid isolated from several medicinal plants, has been reported to possess anti-bacterial, anti-inflammatory and antitumor properties. Although berberine also inhibits osteoclastogenesis and bone resorption, the molecular machinery for its inhibitory effects remains unknown. This study focused on the suppressive effects of berberine on receptor activator of nuclear factor kappa B (NF-kappa B) ligand (RANKL)induced osteoclastogenesis and survival. Berberine inhibited RANKL-mediated osteoclast fort-nation and survival while having no cytotoxic effects on bone marrow macrophages or osteoblastic cells. Berberine attenuated RANKL-induced activation of NF-kappa B through inhibiting phosphorylation at the activation loop of I kappa B alpha kinase, phosphorylation and degradation of I kappa B alpha, and NF-kappa B p65 nuclear translocation. RANKL-induced Akt phosphorylation was strongly inhibited by berberine; however, neither monocyte/macrophage-colony stimulating factor (M-CSF)-induced nor insulin-induced Akt activation was inhibited by the drug. Under M-CSF- and RANKL-deprived condition, berberine increased the active form of caspase-3 in osteoclasts. By contrast, berberine did not potentiate the activation of caspase-3 in M-CSF-deprived bone marrow macrophages. These findings indicate that berberine inhibits osteoclast formation and survival through suppression of NF-kappa B and Akt activation and that both pathways in the osteoclast lineage are highly sensitive to berberine treatment. (C) 2007 Elsevier B.V. All rights reserved.

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  • カテプシンE欠損はオートファジーの低下とそれに伴うミトコンドリア機能異常と酸化ストレスを引き起こす。

    筑波隆幸, 柳川三千代, 門脇知子, 岡本美子, 岡元邦彰, 中山敬一, 山本健二

    生化学   2008年

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  • カテプシンE欠損マウスにおける高脂血症および脂肪肝の誘導

    門脇知子, 岡元邦彰, 山本健二, 筑波隆幸

    Journal of Oral Biosciences   50 ( Supplement )   2008年

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  • カテプシンE欠損による遊走能および細胞接着能の低下

    筑波隆幸, 柳川三千代, 岡元邦彰, 門脇知子, 山本健二

    Journal of Oral Biosciences   50 ( Supplement )   2008年

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  • Cathepsin E prevents tumor growth and metastasis by catalyzing the proteolytic release of soluble TRAIL from tumor cell surface

    Tomoyo Kawakubo, Kuniaki Okamoto, Jun-Ichi Lwata, Masashi Shin, Yoshiko Okamoto, Atsushi Yasukochi, Keiichi I. Nakayama, Tomoko Kadowaki, Takayuki Tsukuba, Kenji Yamamoto

    CANCER RESEARCH   67 ( 22 )   10869 - 10878   2007年11月

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    記述言語:英語   出版者・発行元:AMER ASSOC CANCER RESEARCH  

    The aspartic proteinase cathepsin E is expressed predominantly in cells of the immune system and highly secreted by activated phagocytes, and deficiency of cathepsin E in mice results in a phenotype affecting immune responses. However, because physiologic substrates for cathepsin E have not yet been identified, the relevance of these observations to the physiologic functions of this protein remains speculative. Here, we show that cathepsin E specifically induces growth arrest and apoptosis in human prostate carcinoma tumor cell lines without affecting normal cells by catalyzing the proteolytic release of soluble tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) from the cell surface. The antitumor activity of cathepsin E was corroborated by in vivo studies with mice bearing human and mouse tumor transplants. Administration of purified cathepsin E into human tumor xenografts in nude mice dose-dependently induced apoptosis in the tumor cells to inhibit tumor growth. The growth, viability, and metastasis of mouse B16 melanoma cells were also more profound in cathepsin E-deficient mice compared with those in the syngeneic wild-type and transgenic mice overexpressing cathepsin E. Taken together, the number of apoptotic tumor cells, as well as tumor-infiltrating activated macrophages, was apparently reduced in cathepsin E-deficient mice compared with those in the other two groups, implying the positive correlation of endogenous cathepsin E levels with the extent of tumor suppression in vivo. These results thus indicate thatcathepsinEplaysasubstantialroleinhostdefense against tumor cells through TRAIL-dependent apoptosis and/or tumor-associated macrophage-mediated cytotoxicity.

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  • Cathepsin E prevents tumor growth and metastasis by catalyzing the proteolytic release of soluble TRAIL from tumor cell surface

    Tomoyo Kawakubo, Kuniaki Okamoto, Jun-Ichi Lwata, Masashi Shin, Yoshiko Okamoto, Atsushi Yasukochi, Keiichi I. Nakayama, Tomoko Kadowaki, Takayuki Tsukuba, Kenji Yamamoto

    CANCER RESEARCH   67 ( 22 )   10869 - 10878   2007年11月

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    記述言語:英語   出版者・発行元:AMER ASSOC CANCER RESEARCH  

    The aspartic proteinase cathepsin E is expressed predominantly in cells of the immune system and highly secreted by activated phagocytes, and deficiency of cathepsin E in mice results in a phenotype affecting immune responses. However, because physiologic substrates for cathepsin E have not yet been identified, the relevance of these observations to the physiologic functions of this protein remains speculative. Here, we show that cathepsin E specifically induces growth arrest and apoptosis in human prostate carcinoma tumor cell lines without affecting normal cells by catalyzing the proteolytic release of soluble tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) from the cell surface. The antitumor activity of cathepsin E was corroborated by in vivo studies with mice bearing human and mouse tumor transplants. Administration of purified cathepsin E into human tumor xenografts in nude mice dose-dependently induced apoptosis in the tumor cells to inhibit tumor growth. The growth, viability, and metastasis of mouse B16 melanoma cells were also more profound in cathepsin E-deficient mice compared with those in the syngeneic wild-type and transgenic mice overexpressing cathepsin E. Taken together, the number of apoptotic tumor cells, as well as tumor-infiltrating activated macrophages, was apparently reduced in cathepsin E-deficient mice compared with those in the other two groups, implying the positive correlation of endogenous cathepsin E levels with the extent of tumor suppression in vivo. These results thus indicate thatcathepsinEplaysasubstantialroleinhostdefense against tumor cells through TRAIL-dependent apoptosis and/or tumor-associated macrophage-mediated cytotoxicity.

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  • Construction of recombinant hemagglutinin derived from the gingipain-encoding gene of Porphyromonas gingivalis, identification of its target protein on erythrocytes, and inhibition of hemagglutination by an interdomain regional peptide. 国際誌

    Eiko Sakai, Mariko Naito, Keiko Sato, Hitoshi Hotokezaka, Tomoko Kadowaki, Arihide Kamaguchi, Kenji Yamamoto, Kuniaki Okamoto, Koji Nakayama

    Journal of bacteriology   189 ( 11 )   3977 - 86   2007年6月

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    記述言語:英語  

    Porphyromonas gingivalis, an anaerobic gram-negative bacterium associated with chronic periodontitis, can agglutinate human erythrocytes. In general, hemagglutination can be considered the ability to adhere to host cells; however, P. gingivalis-mediated hemagglutination has special significance because heme markedly accelerates growth of this bacterium. Although a number of studies have indicated that a major hemagglutinin of P. gingivalis is intragenically encoded by rgpA, kgp, and hagA, direct evidence has not been obtained. We demonstrated in this study that recombinant HGP44(720-1081), a fully processed HGP44 domain protein, had hemagglutinating activity but that an unprocessed form, HGP44(720-1138), did not. A peptide corresponding to residues 1083 to 1102, which was included in HGP44(720-1138) but not in HGP44(720-1081), could bind HGP44(720-1081) in a dose-dependent manner and effectively inhibited HGP44(720-1081)-mediated hemagglutination, indicating that the interdomain regional amino acid sequence may function as an intramolecular suppressor of hemagglutinating activity. Analyses by solid-phase binding and chemical cross-linking suggested that HGP44 interacted with glycophorin A on the erythrocyte membrane. Glycophorin A and, more effectively, asialoglycophorin, which were added exogenously, inhibited HGP44(720-1081)-mediated hemagglutination. Treatment of erythrocytes with RgpB proteinase resulted in degradation of glycophorin A on the membrane and a decrease in HGP44(720-1081)-mediated hemagglutination. Surface plasmon resonance detection analysis revealed that HGP44(720-1081) could bind to asialoglycophorin with a dissociation constant of 3.0 x 10(-7) M. These results indicate that the target of HGP44 on the erythrocyte membrane appears to be glycophorin A.

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  • Construction of recombinant hemagglutinin derived from the gingipain-encoding gene of Porphyromonas gingivalis, identification of its target protein on erythrocytes, and inhibition of hemagglutination by an interdomain regional peptide. 国際誌

    Eiko Sakai, Mariko Naito, Keiko Sato, Hitoshi Hotokezaka, Tomoko Kadowaki, Arihide Kamaguchi, Kenji Yamamoto, Kuniaki Okamoto, Koji Nakayama

    Journal of bacteriology   189 ( 11 )   3977 - 86   2007年6月

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    記述言語:英語  

    Porphyromonas gingivalis, an anaerobic gram-negative bacterium associated with chronic periodontitis, can agglutinate human erythrocytes. In general, hemagglutination can be considered the ability to adhere to host cells; however, P. gingivalis-mediated hemagglutination has special significance because heme markedly accelerates growth of this bacterium. Although a number of studies have indicated that a major hemagglutinin of P. gingivalis is intragenically encoded by rgpA, kgp, and hagA, direct evidence has not been obtained. We demonstrated in this study that recombinant HGP44(720-1081), a fully processed HGP44 domain protein, had hemagglutinating activity but that an unprocessed form, HGP44(720-1138), did not. A peptide corresponding to residues 1083 to 1102, which was included in HGP44(720-1138) but not in HGP44(720-1081), could bind HGP44(720-1081) in a dose-dependent manner and effectively inhibited HGP44(720-1081)-mediated hemagglutination, indicating that the interdomain regional amino acid sequence may function as an intramolecular suppressor of hemagglutinating activity. Analyses by solid-phase binding and chemical cross-linking suggested that HGP44 interacted with glycophorin A on the erythrocyte membrane. Glycophorin A and, more effectively, asialoglycophorin, which were added exogenously, inhibited HGP44(720-1081)-mediated hemagglutination. Treatment of erythrocytes with RgpB proteinase resulted in degradation of glycophorin A on the membrane and a decrease in HGP44(720-1081)-mediated hemagglutination. Surface plasmon resonance detection analysis revealed that HGP44(720-1081) could bind to asialoglycophorin with a dissociation constant of 3.0 x 10(-7) M. These results indicate that the target of HGP44 on the erythrocyte membrane appears to be glycophorin A.

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  • Cathepsin E deficiency induces a novel form of lysosomal storage disorder showing the accumulation of lysosomal membrane sialoglycoproteins and the elevation of lysosomal pH in macrophages

    Michiyo Yanagawa, Takayuki Tsukuba, Tsuyoshi Nishioku, Yoshiko Okamoto, Kuniaki Okamoto, Ryosuke Takii, Yoshihiro Terada, Keiichi I. Nakayama, Tomoko Kadowaki, Kenji Yamamoto

    JOURNAL OF BIOLOGICAL CHEMISTRY   282 ( 3 )   1851 - 1862   2007年1月

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    記述言語:英語   出版者・発行元:AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

    Cathepsin E, an endolysosomal aspartic proteinase predominantly expressed in cells of the immune system, has an important role in immune responses. However, little is known about the precise roles of cathepsin E in this system. Here we report that cathepsin E deficiency (CatE(-/-)) leads to a novel form of lysosome storage disorder in macrophages, exhibiting the accumulation of the two major lysosomal membrane sialoglycoproteins LAMP-1 and LAMP-2 and the elevation of lysosomal pH. These striking features were also found in wild-type macrophages treated with pepstatin A and Ascaris inhibitor. Whereas there were no obvious differences in their expression, biosynthesis, and trafficking between wild-type and CatE(-/-) macrophages, the degradation rates of these two membrane proteins were apparently decreased as a result of cathepsin E deficiency. Because there was no difference in the vacuolar-type H+ -ATPase activity in both cell types, the elevated lysosomal pH in CatE(-/-) macrophages is most likely due to the accumulation of these lysosomal membrane glycoproteins highly modified with acidic monosaccharides, thereby leading to the disruption of non-proton factors controlling lysosomal pH. Furthermore, the selective degradation of LAMP-1 and LAMP-2, as well as LIMP-2, was also observed by treatment of the lysosomal membrane fraction isolated from wild-type macrophages with purified cathepsin E at pH 5. Our results thus suggest that cathepsin E is important for preventing the accumulation of these lysosomal membrane sialoglycoproteins that can induce a new form of lysosomal storage disorder.

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  • Cathepsin E deficiency induces a novel form of lysosomal storage disorder showing the accumulation of lysosomal membrane sialoglycoproteins and the elevation of lysosomal pH in macrophages

    Michiyo Yanagawa, Takayuki Tsukuba, Tsuyoshi Nishioku, Yoshiko Okamoto, Kuniaki Okamoto, Ryosuke Takii, Yoshihiro Terada, Keiichi I. Nakayama, Tomoko Kadowaki, Kenji Yamamoto

    JOURNAL OF BIOLOGICAL CHEMISTRY   282 ( 3 )   1851 - 1862   2007年1月

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    記述言語:英語   出版者・発行元:AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

    Cathepsin E, an endolysosomal aspartic proteinase predominantly expressed in cells of the immune system, has an important role in immune responses. However, little is known about the precise roles of cathepsin E in this system. Here we report that cathepsin E deficiency (CatE(-/-)) leads to a novel form of lysosome storage disorder in macrophages, exhibiting the accumulation of the two major lysosomal membrane sialoglycoproteins LAMP-1 and LAMP-2 and the elevation of lysosomal pH. These striking features were also found in wild-type macrophages treated with pepstatin A and Ascaris inhibitor. Whereas there were no obvious differences in their expression, biosynthesis, and trafficking between wild-type and CatE(-/-) macrophages, the degradation rates of these two membrane proteins were apparently decreased as a result of cathepsin E deficiency. Because there was no difference in the vacuolar-type H+ -ATPase activity in both cell types, the elevated lysosomal pH in CatE(-/-) macrophages is most likely due to the accumulation of these lysosomal membrane glycoproteins highly modified with acidic monosaccharides, thereby leading to the disruption of non-proton factors controlling lysosomal pH. Furthermore, the selective degradation of LAMP-1 and LAMP-2, as well as LIMP-2, was also observed by treatment of the lysosomal membrane fraction isolated from wild-type macrophages with purified cathepsin E at pH 5. Our results thus suggest that cathepsin E is important for preventing the accumulation of these lysosomal membrane sialoglycoproteins that can induce a new form of lysosomal storage disorder.

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  • Cathepsin E mediates membrane trafficking in macrophages

    Takayuki Tsukuba, Shinya Yamamoto, Michiyo Yanagawa, Yoshiko Okamoto, Kuniaki Okamoto, Kenji Yamamoto

    JOURNAL OF PHARMACOLOGICAL SCIENCES   103   214P - 214P   2007年

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    記述言語:英語   掲載種別:研究発表ペーパー・要旨(国際会議)   出版者・発行元:JAPANESE PHARMACOLOGICAL SOC  

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  • カテプシンE欠損によるミトコンドリア機能異常と酸化ストレス亢進

    筑波隆幸, 柳川三千代, 柳川三千代, 岡元邦彰, 門脇知子, 山本健二

    Journal of Oral Biosciences   49 ( Supplement )   2007年

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  • P.gingivalis感染とインスリン抵抗性に関する実験的解析

    門脇知子, 瀧井良祐, 筑波隆幸, 岡元邦彰, 山本健二

    Journal of Oral Biosciences   49 ( Supplement )   2007年

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  • カテプシンE欠損はマクロファージの膜蛋白質輸送異常とミトコンドリア機能異常を起こす

    筑波隆幸, 柳川三千代, 岡元邦彰, 岡本美子, 門脇知子, 山本健二

    生化学   2007年

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  • Pepstatin A, an aspartic proteinase inhibitor, suppresses RANKL-induced osteoclast differentiation. 国際誌

    Hajime Yoshida, Kuniaki Okamoto, Tsutomu Iwamoto, Eiko Sakai, Kazuhiro Kanaoka, Jin-Ping Hu, Mitsue Shibata, Hitoshi Hotokezaka, Kazuhisa Nishishita, Akio Mizuno, Yuzo Kato

    Journal of biochemistry   139 ( 3 )   583 - 90   2006年3月

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    記述言語:英語  

    Pepstatin A is well known to be an inhibitor of aspartic proteinases such as pepsin, cathepsins D and E. Except for its role as a proteinase inhibitor, however, the pharmacological action of pepstatin A upon cells remain unclear. In this study, we found that pepstatin A suppressed receptor activator of NF-kappaB ligand (RANKL)-induced osteoclast differentiation. Pepstatin A suppressed the formation of multinuclear osteoclasts dose-dependently. This inhibition of the formation only affected osteoclast cells, i.e., not osteoblast-like cells. Furthermore, pepstatin A also suppressed differentiation from pre-osteoclast cells to mononuclear osteoclast cells dose-dependently. This inhibition seems to be independent of the activities of proteinases such as cathepsin D, because the formation of osteoclasts was not suppressed with the concentration that inhibited the activity of cathepsin D. Cell signaling analysis indicated that the phosphorylation of ERK was inhibited in pepstatin A-treated cells, while the phosphorylation of IkappaB and Akt showed almost no change. Furthermore, pepstatin A decreased the expression of nuclear factor of activated T cells c1 (NFATc1). These results suggest that pepstatin A suppresses the differentiation of osteoclasts through the blockade of ERK signaling and the inhibition of NFATc1 expression.

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  • Pepstatin A, an aspartic proteinase inhibitor, suppresses RANKL-induced osteoclast differentiation. 国際誌

    Hajime Yoshida, Kuniaki Okamoto, Tsutomu Iwamoto, Eiko Sakai, Kazuhiro Kanaoka, Jin-Ping Hu, Mitsue Shibata, Hitoshi Hotokezaka, Kazuhisa Nishishita, Akio Mizuno, Yuzo Kato

    Journal of biochemistry   139 ( 3 )   583 - 90   2006年3月

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    記述言語:英語  

    Pepstatin A is well known to be an inhibitor of aspartic proteinases such as pepsin, cathepsins D and E. Except for its role as a proteinase inhibitor, however, the pharmacological action of pepstatin A upon cells remain unclear. In this study, we found that pepstatin A suppressed receptor activator of NF-kappaB ligand (RANKL)-induced osteoclast differentiation. Pepstatin A suppressed the formation of multinuclear osteoclasts dose-dependently. This inhibition of the formation only affected osteoclast cells, i.e., not osteoblast-like cells. Furthermore, pepstatin A also suppressed differentiation from pre-osteoclast cells to mononuclear osteoclast cells dose-dependently. This inhibition seems to be independent of the activities of proteinases such as cathepsin D, because the formation of osteoclasts was not suppressed with the concentration that inhibited the activity of cathepsin D. Cell signaling analysis indicated that the phosphorylation of ERK was inhibited in pepstatin A-treated cells, while the phosphorylation of IkappaB and Akt showed almost no change. Furthermore, pepstatin A decreased the expression of nuclear factor of activated T cells c1 (NFATc1). These results suggest that pepstatin A suppresses the differentiation of osteoclasts through the blockade of ERK signaling and the inhibition of NFATc1 expression.

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  • Characterization of rat cathepsin E and mutants with changed active-site residues and lacking propeptides and N-glycosylation, expressed in human embryonic kidney 293T cells

    T Tsukuba, S Ikeda, K Okamoto, Y Yasuda, E Sakai, T Kadowaki, H Sakai, K Yamamoto

    FEBS JOURNAL   273 ( 1 )   219 - 229   2006年1月

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    記述言語:英語   出版者・発行元:WILEY-BLACKWELL  

    To study the roles of the catalytic activity, propeptide, and N-glycosylation of the intracellular aspartic proteinase cathepsin E in biosynthesis, processing, and intracellular trafficking, we constructed various rat cathepsin E mutants in which active-site Asp residues were changed to Ala or which lacked propeptides and N-glycosylation. Wild-type cathepsin E expressed in human embryonic kidney 293T cells was mainly found in the LAMP-1-positive endosomal organelles, as determined by immunofluorescence microscopy. Consistently, pulse-chase analysis revealed that the initially synthesized pro-cathepsin E was processed to the mature enzyme within a 24 h chase. This process was completely inhibited by brefeldin A and bafilomycin A, indicating its transport from the endoplasmic reticulum (ER) to the endosomal acidic compartment. Mutants with Asp residues in the two active-site consensus motifs changed to Ala and lacking the propeptide (Leu23-Phe58) and the putative ER-retention sequence (Ser59-Asp98) were neither processed nor transported to the endosomal compartment. The mutant lacking the ER-retention sequence was rapidly degraded in the ER, indicating the importance of this sequence in correct folding. The single (N92Q or N324D) and double (N92Q/N324D) N-glycosylation-deficient mutants were neither processed into a mature form nor transported to the endosomal compartment, but were stably retained in the ER without degradation. These data indicate that the catalytic activity, propeptides, and N-glycosylation of this protein are all essential for its processing, maturation, and trafficking.

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  • Characterization of rat cathepsin E and mutants with changed active-site residues and lacking propeptides and N-glycosylation, expressed in human embryonic kidney 293T cells

    T Tsukuba, S Ikeda, K Okamoto, Y Yasuda, E Sakai, T Kadowaki, H Sakai, K Yamamoto

    FEBS JOURNAL   273 ( 1 )   219 - 229   2006年1月

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    記述言語:英語   出版者・発行元:WILEY-BLACKWELL  

    To study the roles of the catalytic activity, propeptide, and N-glycosylation of the intracellular aspartic proteinase cathepsin E in biosynthesis, processing, and intracellular trafficking, we constructed various rat cathepsin E mutants in which active-site Asp residues were changed to Ala or which lacked propeptides and N-glycosylation. Wild-type cathepsin E expressed in human embryonic kidney 293T cells was mainly found in the LAMP-1-positive endosomal organelles, as determined by immunofluorescence microscopy. Consistently, pulse-chase analysis revealed that the initially synthesized pro-cathepsin E was processed to the mature enzyme within a 24 h chase. This process was completely inhibited by brefeldin A and bafilomycin A, indicating its transport from the endoplasmic reticulum (ER) to the endosomal acidic compartment. Mutants with Asp residues in the two active-site consensus motifs changed to Ala and lacking the propeptide (Leu23-Phe58) and the putative ER-retention sequence (Ser59-Asp98) were neither processed nor transported to the endosomal compartment. The mutant lacking the ER-retention sequence was rapidly degraded in the ER, indicating the importance of this sequence in correct folding. The single (N92Q or N324D) and double (N92Q/N324D) N-glycosylation-deficient mutants were neither processed into a mature form nor transported to the endosomal compartment, but were stably retained in the ER without degradation. These data indicate that the catalytic activity, propeptides, and N-glycosylation of this protein are all essential for its processing, maturation, and trafficking.

    DOI: 10.1111/j.1742-4658.2005.05062.x

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  • Gingipains inactivate a cell surface ligand on Porphyromonas gingivalis that induces TLR2- and TLR4-independent signaling

    M Kishimoto, A Yoshimura, M Naito, K Okamoto, K Yamamoto, DT Golenbock, Y Hara, K Nakayama

    MICROBIOLOGY AND IMMUNOLOGY   50 ( 4 )   315 - 325   2006年

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    記述言語:英語   出版者・発行元:CENTER ACADEMIC PUBL JAPAN  

    Arginine-specific gingipain and lysine-specific gingipain are two major cysteine proteinases produced by Porphyromonas gingivalis. To clarify the role of gingipains in the interaction between P gingivalis and the innate immune system, CHO reporter cells expressing TLR2 or TLR4 were stimulated with wildtype or gingipain-deficient P gingivalis cells and activation of nuclear factor-kappa B in these cells was examined. While CHO/CD14 cells and 7.19 cells, an MD-2-defective mutant derived from CHO/CD14 cells, failed to respond to wild-type P gingivalis, they responded to gingipain-deficient P gingivalis. On the other hand, CHO/CD14/TLR2 cells responded to both wild-type and gingipain-deficient P gingivalis. These results suggested that gingipains have no effects on TLR2-dependent signaling from P gingivalis but have inhibitory effects on TLR2- and TLR4-independent signaling in CHO cells. Indeed, the activity of gingipain-deficient P gingivalis to induce the activation of 7.19 cells was diminished after treatment of the bacterial cells with gingipains. We next partially purified bacterial cell components activating 7.19 cells from gingipain-deficient P gingivalis. The activity of the partially purified components was diminished by treatment with heat or gingipains. It is also noteworthy that anti-CD14 mAb inhibited the activation of 7.19 cells induced by the partially purified components. These results indicated that the components of R gingivalis that were able to induce TLR2- and TLR4-independent signaling were inactivated by gingipains before being recognized by CD14. The inactivation of the components would be helpful for P gingivalis to escape from the innate immune system.

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  • Gingipains inactivate a cell surface ligand on Porphyromonas gingivalis that induces TLR2- and TLR4-independent signaling

    M Kishimoto, A Yoshimura, M Naito, K Okamoto, K Yamamoto, DT Golenbock, Y Hara, K Nakayama

    MICROBIOLOGY AND IMMUNOLOGY   50 ( 4 )   315 - 325   2006年

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    記述言語:英語   出版者・発行元:CENTER ACADEMIC PUBL JAPAN  

    Arginine-specific gingipain and lysine-specific gingipain are two major cysteine proteinases produced by Porphyromonas gingivalis. To clarify the role of gingipains in the interaction between P gingivalis and the innate immune system, CHO reporter cells expressing TLR2 or TLR4 were stimulated with wildtype or gingipain-deficient P gingivalis cells and activation of nuclear factor-kappa B in these cells was examined. While CHO/CD14 cells and 7.19 cells, an MD-2-defective mutant derived from CHO/CD14 cells, failed to respond to wild-type P gingivalis, they responded to gingipain-deficient P gingivalis. On the other hand, CHO/CD14/TLR2 cells responded to both wild-type and gingipain-deficient P gingivalis. These results suggested that gingipains have no effects on TLR2-dependent signaling from P gingivalis but have inhibitory effects on TLR2- and TLR4-independent signaling in CHO cells. Indeed, the activity of gingipain-deficient P gingivalis to induce the activation of 7.19 cells was diminished after treatment of the bacterial cells with gingipains. We next partially purified bacterial cell components activating 7.19 cells from gingipain-deficient P gingivalis. The activity of the partially purified components was diminished by treatment with heat or gingipains. It is also noteworthy that anti-CD14 mAb inhibited the activation of 7.19 cells induced by the partially purified components. These results indicated that the components of R gingivalis that were able to induce TLR2- and TLR4-independent signaling were inactivated by gingipains before being recognized by CD14. The inactivation of the components would be helpful for P gingivalis to escape from the innate immune system.

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  • Berberine inhibits RANKL-, TNF alpha-, LPS- and PGN-induced mature osteoclasts survival

    JP Hu, K Nishishita, E Sakai, K Okamoto, Y Kato

    JOURNAL OF PHARMACOLOGICAL SCIENCES   100   268P - 268P   2006年

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    記述言語:英語   掲載種別:研究発表ペーパー・要旨(国際会議)   出版者・発行元:JAPANESE PHARMACOLOGICAL SOC  

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  • Involvement of glycosphingolipids in LPS mediated survival of mature osteoclasts

    E Sakai, S Fukumoto, K Nishishita, JP Hu, K Okamoto, Y Kato

    JOURNAL OF PHARMACOLOGICAL SCIENCES   100   65P - 65P   2006年

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    記述言語:英語   掲載種別:研究発表ペーパー・要旨(国際会議)   出版者・発行元:JAPANESE PHARMACOLOGICAL SOC  

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  • P.gingivalis感染の生活習慣病増悪機序について

    門脇知子, 筑波隆幸, 瀧井良祐, 重松直樹, 岡元邦彰, 山本健二

    Journal of Oral Biosciences   48 ( Supplement )   2006年

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  • カテプシンE欠損マクロファージにおける膜タンパク質の輸送異常

    筑波隆幸, 山本晋也, 山本晋也, 柳川三千代, 柳川三千代, 岡元邦彰, 門脇知子, 山本健二

    Journal of Oral Biosciences   48 ( Supplement )   2006年

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  • The role of the cathepsin E propeptide in correct folding, maturation and sorting to the endosome

    Y Yasuda, T Tsukuba, K Okamoto, T Kadowaki, K Yamamoto

    JOURNAL OF BIOCHEMISTRY   138 ( 5 )   621 - 630   2005年11月

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    記述言語:英語   出版者・発行元:OXFORD UNIV PRESS  

    Cathepsin E (CE) is an endosomal aspartic proteinase of the A1 family that is highly homologous to the lysosomal aspartic proteinase cathepsin D (CD). Newly synthesized CE undergoes several proteolytic processing events to yield mature CE, from which the N-terminal propeptide usually comprising 39 amino acids is removed. To define the role of the propeptide of CE in its biosynthesis and processing, we constructed two fusion proteins using chimeric DNAs encoding the CE propeptide fused to the mature CD tagged with HA at the COOH terminus (termed ED-HA) and encoding the CD propeptide fused to the mature CE (termed DE). Pulse-chase analysis revealed that wild-type CE expressed in human embryonic kidney cells is autoproteolytically processed into mature CE within a 12-h chase, whereas the chimeric DE failed to be converted into mature CE even after a 24-h chase. The DE chimera was nevertheless capable of acid-dependent autoactivation in vitro to yield a catalytically active form, although its specificity constants (k(cat)/K(m)) were considerably high but less (35%) than those of the wild-type CE. By contrast, the chimeric ED-HA expressed in HeLa cells underwent neither processing into a catalytically active enzyme nor acid-dependent autoactivation in vitro. The ED-HA protein was less stable than wt-CD-HA, as determined on pulse-chase analysis and on trypsin digestion. These data indicate that the propeptide of CE is essential for the correct folding, maturation, and targeting of this protein to its final destination.

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  • The role of the cathepsin E propeptide in correct folding, maturation and sorting to the endosome

    Y Yasuda, T Tsukuba, K Okamoto, T Kadowaki, K Yamamoto

    JOURNAL OF BIOCHEMISTRY   138 ( 5 )   621 - 630   2005年11月

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    記述言語:英語   出版者・発行元:OXFORD UNIV PRESS  

    Cathepsin E (CE) is an endosomal aspartic proteinase of the A1 family that is highly homologous to the lysosomal aspartic proteinase cathepsin D (CD). Newly synthesized CE undergoes several proteolytic processing events to yield mature CE, from which the N-terminal propeptide usually comprising 39 amino acids is removed. To define the role of the propeptide of CE in its biosynthesis and processing, we constructed two fusion proteins using chimeric DNAs encoding the CE propeptide fused to the mature CD tagged with HA at the COOH terminus (termed ED-HA) and encoding the CD propeptide fused to the mature CE (termed DE). Pulse-chase analysis revealed that wild-type CE expressed in human embryonic kidney cells is autoproteolytically processed into mature CE within a 12-h chase, whereas the chimeric DE failed to be converted into mature CE even after a 24-h chase. The DE chimera was nevertheless capable of acid-dependent autoactivation in vitro to yield a catalytically active form, although its specificity constants (k(cat)/K(m)) were considerably high but less (35%) than those of the wild-type CE. By contrast, the chimeric ED-HA expressed in HeLa cells underwent neither processing into a catalytically active enzyme nor acid-dependent autoactivation in vitro. The ED-HA protein was less stable than wt-CD-HA, as determined on pulse-chase analysis and on trypsin digestion. These data indicate that the propeptide of CE is essential for the correct folding, maturation, and targeting of this protein to its final destination.

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  • Identification of a new membrane-associated protein that influences transport/maturation of gingipains and adhesins of Porphyromonas gingivalis

    K Sato, E Sakai, PD Veith, M Shoji, Y Kikuchi, H Yukitake, N Ohara, M Naito, K Okamoto, EC Reynolds, K Nakayama

    JOURNAL OF BIOLOGICAL CHEMISTRY   280 ( 10 )   8668 - 8677   2005年3月

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    記述言語:英語   出版者・発行元:AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

    The dual membrane envelopes of Gram-negative bacteria provide two barriers of unlike nature that regulate the transport of molecules into and out of organisms. Organisms have developed several systems for transport across the inner and outer membranes. The Gram-negative periodontopathogenic bacterium Porphyromonas gingivalis produces proteinase and adhesin complexes, gingipains/adhesins, on the cell surface and in the extracellular milieu as one of the major virulence factors. Gingipains and/or adhesins are encoded by kgp, rgpA, rgpB, and hagA on the chromosome. In this study, we isolated a P. gingivalis mutant (porT), which showed very weak activities of gingipains in the cell lysates and culture supernatants. Subcellular fractionation and immunoblot analysis demonstrated that precursor forms of gingipains and adhesins were accumulated in the periplasmic space of the porT mutant cells. Peptide mass fingerprinting and N-terminal amino acid sequencing of the precursor proteins and the kgp'-'rgpB chimera gene product in the porT mutant indicated that these proteins lacked the signal peptide regions, consistent with their accumulation in the periplasm. The PorT protein seemed to be membrane-associated and exposed to the periplasmic space, as revealed by subcellular fractionation and immunoblot analysis using anti-PorT antiserum. These results suggest that the membrane-associated protein PorT is essential for transport of the kgp, rgpA, rgpB, and hagA gene products across the outer membrane from the periplasm to the cell surface, where they are processed and matured.

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  • Identification of a new membrane-associated protein that influences transport/maturation of gingipains and adhesins of Porphyromonas gingivalis

    K Sato, E Sakai, PD Veith, M Shoji, Y Kikuchi, H Yukitake, N Ohara, M Naito, K Okamoto, EC Reynolds, K Nakayama

    JOURNAL OF BIOLOGICAL CHEMISTRY   280 ( 10 )   8668 - 8677   2005年3月

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    記述言語:英語   出版者・発行元:AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

    The dual membrane envelopes of Gram-negative bacteria provide two barriers of unlike nature that regulate the transport of molecules into and out of organisms. Organisms have developed several systems for transport across the inner and outer membranes. The Gram-negative periodontopathogenic bacterium Porphyromonas gingivalis produces proteinase and adhesin complexes, gingipains/adhesins, on the cell surface and in the extracellular milieu as one of the major virulence factors. Gingipains and/or adhesins are encoded by kgp, rgpA, rgpB, and hagA on the chromosome. In this study, we isolated a P. gingivalis mutant (porT), which showed very weak activities of gingipains in the cell lysates and culture supernatants. Subcellular fractionation and immunoblot analysis demonstrated that precursor forms of gingipains and adhesins were accumulated in the periplasmic space of the porT mutant cells. Peptide mass fingerprinting and N-terminal amino acid sequencing of the precursor proteins and the kgp'-'rgpB chimera gene product in the porT mutant indicated that these proteins lacked the signal peptide regions, consistent with their accumulation in the periplasm. The PorT protein seemed to be membrane-associated and exposed to the periplasmic space, as revealed by subcellular fractionation and immunoblot analysis using anti-PorT antiserum. These results suggest that the membrane-associated protein PorT is essential for transport of the kgp, rgpA, rgpB, and hagA gene products across the outer membrane from the periplasm to the cell surface, where they are processed and matured.

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  • Alteration of substance P levels in various regions of mouse brain caused by cathepsin E-deficiency

    N Shigematsu, T Nishioku, T Tsukuba, T Fukuda, T Yamamoto, T Yamaguchi, S Kohsaka, K Okamoto, Y Okamoto, Y Tanaka, M Himeno, S Higuchi, K Yamamoto

    JOURNAL OF PHARMACOLOGICAL SCIENCES   97   72P - 72P   2005年

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    記述言語:英語   掲載種別:研究発表ペーパー・要旨(国際会議)   出版者・発行元:JAPANESE PHARMACOLOGICAL SOC  

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  • Up-regulation, Enhanced Maturation, and Secretion of Cathepsin E in Mouse Macrophages Treated with Interferon-γ or Lipopolysaccharide

    Michiyo Yanagawa, Takayuki Tsukuba, Kuniaki Okamoto, Ryosuke Takii, Yoshihiro Terada, Tomoko Kadowaki, Kenji Yamamoto

    journal of oral biosciences   48 ( 3 )   218 - 225   2005年

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    記述言語:英語  

    Cathepsin E is an intracellular aspartic proteinase that is predominantly localized in the endosomal compartments of antigen presenting cells including macrophages. Here, we have investigated the expression of cathepsin E in mouse macrophages treated with interferon (IFN)-γ, lipopolysaccharide (LPS), interleukin (IL)-10, and IL-4. The mRNA levels of cathepsin E in macrophages stimulated with IFN-γ and LPS were increased, but conversely, that with IL-10 was decreased. However, upon stimulation with IFN-γ or LPS, the activity levels of cathepsin E in the activated cells were markedly decreased, but those in the culture media were increased. Immunoblot analysis revealed that procathepsin E in the cells was largely converted to the mature form in the cells upon stimulation with IFN-γ. In addition, the extracellular enzyme was reacted with antibodies to mature cathepsin E but not with antibodies for procathepsin E. By contrast, when macrophages were treated with IL-4, cathepsin E production was significantly decreased in both the cell and the medium. These results indicate that, upon stimulation with IFN-γ and LPS, cathepsin E is up-regulated in macrophages, which is accompanied by the enhanced maturation and secretion of the enzyme. Conversely, cathepsin E is down-regulated by treatment with IL-4 and IL-10. © 2006, Japanese Association for Oral Biology. All rights reserved.

    DOI: 10.2330/joralbiosci.48.218

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  • Up-regulation, Enhanced Maturation, and Secretion of Cathepsin E in Mouse Macrophages Treated with Interferon-γ or Lipopolysaccharide

    Michiyo Yanagawa, Takayuki Tsukuba, Kuniaki Okamoto, Ryosuke Takii, Yoshihiro Terada, Tomoko Kadowaki, Kenji Yamamoto

    journal of oral biosciences   48 ( 3 )   218 - 225   2005年

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    記述言語:英語  

    Cathepsin E is an intracellular aspartic proteinase that is predominantly localized in the endosomal compartments of antigen presenting cells including macrophages. Here, we have investigated the expression of cathepsin E in mouse macrophages treated with interferon (IFN)-γ, lipopolysaccharide (LPS), interleukin (IL)-10, and IL-4. The mRNA levels of cathepsin E in macrophages stimulated with IFN-γ and LPS were increased, but conversely, that with IL-10 was decreased. However, upon stimulation with IFN-γ or LPS, the activity levels of cathepsin E in the activated cells were markedly decreased, but those in the culture media were increased. Immunoblot analysis revealed that procathepsin E in the cells was largely converted to the mature form in the cells upon stimulation with IFN-γ. In addition, the extracellular enzyme was reacted with antibodies to mature cathepsin E but not with antibodies for procathepsin E. By contrast, when macrophages were treated with IL-4, cathepsin E production was significantly decreased in both the cell and the medium. These results indicate that, upon stimulation with IFN-γ and LPS, cathepsin E is up-regulated in macrophages, which is accompanied by the enhanced maturation and secretion of the enzyme. Conversely, cathepsin E is down-regulated by treatment with IL-4 and IL-10. © 2006, Japanese Association for Oral Biology. All rights reserved.

    DOI: 10.2330/joralbiosci.48.218

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  • Porphyromonas gingivalisの産生するシステインプロテアーゼ(Kgp)の活性中心の決定

    岡元邦彰, HU P J, 坂井詠子, 西下一久, 門脇知子, 山本健二, 中山浩次, 加藤有三

    Journal of Oral Biosciences   47 ( Supplement )   2005年

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  • Cathepsin E is important for endosomal/lysosomal systems in macrophages against a bacterial infection

    T Tsukuba, M Yanagawa, Y Okamoto, K Okamoto, K Yanamoto

    JOURNAL OF PHARMACOLOGICAL SCIENCES   97   78P - 78P   2005年

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    記述言語:英語   掲載種別:研究発表ペーパー・要旨(国際会議)   出版者・発行元:JAPANESE PHARMACOLOGICAL SOC  

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  • Glycosphingolipids regulate osteoclastgenesis mediated by lipopolysaccharide

    E Sakai, S Fukumoto, Y Narita, M Tanaka, M Tachi, H Hotokezaka, K Kanaoka, K Okamoto, Y Kato

    JOURNAL OF PHARMACOLOGICAL SCIENCES   97   178P - 178P   2005年

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    記述言語:英語   掲載種別:研究発表ペーパー・要旨(国際会議)   出版者・発行元:JAPANESE PHARMACOLOGICAL SOC  

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  • Roles of Arg- and Lys-gingipains in coaggregation of Porphyromonas gingivalis: identification of its responsible molecules in translation products of rgpA, kgp, and hagA genes

    N Abe, A Baba, R Takii, K Nakayama, A Kamaguchi, Y Shibata, Y Abiko, K Okamoto, T Kadowaki, K Yamamoto

    BIOLOGICAL CHEMISTRY   385 ( 11 )   1041 - 1047   2004年11月

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    記述言語:英語   出版者・発行元:WALTER DE GRUYTER & CO  

    Arg- (Rgp) and Lys-gingipains (Kgp) are two individual cysteine proteinases produced by Porphyromonas gingivalis, an oral anaerobic bacterium, and are implicated as major virulence factors in a wide range of pathologies of adult periodontitis. Coaggregation of this bacterium with other oral bacteria is an initial and critical step in infectious processes, yet the factors and mechanisms responsible for this process remain elusive. Here we show that the initial translation products of the rgpA, kgp and hemagglutinin hagA genes are responsible for coaggregation of P gingivalis and that the proteolytic activity of Rgp and Kgp is indispensable in this process. The rgpA rgpB kgp- and rgpA kgp hagA-deficient triple mutants exhibited no coaggregation activity with Actinomyces viscosus, whereas the kgp-null and rgpA rgpB-deficient double mutants significantly retained this activity. Consistently, the combined action of Rgp- and Kgp-specific inhibitors strongly inhibited the coaggregation activity of the bacterium, although single use of Rgp- or Kgp-specific inhibitor significantly retained this activity. We also demonstrate that the 47- and 43-kDa proteins produced from the translation products of the rgpA, kgp, and hagA genes by proteolytic activity of both Rgp and Kgp are responsible for the coaggregation of P. gingivalis.

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  • Roles of Arg- and Lys-gingipains in coaggregation of Porphyromonas gingivalis: identification of its responsible molecules in translation products of rgpA, kgp, and hagA genes

    N Abe, A Baba, R Takii, K Nakayama, A Kamaguchi, Y Shibata, Y Abiko, K Okamoto, T Kadowaki, K Yamamoto

    BIOLOGICAL CHEMISTRY   385 ( 11 )   1041 - 1047   2004年11月

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    記述言語:英語   出版者・発行元:WALTER DE GRUYTER & CO  

    Arg- (Rgp) and Lys-gingipains (Kgp) are two individual cysteine proteinases produced by Porphyromonas gingivalis, an oral anaerobic bacterium, and are implicated as major virulence factors in a wide range of pathologies of adult periodontitis. Coaggregation of this bacterium with other oral bacteria is an initial and critical step in infectious processes, yet the factors and mechanisms responsible for this process remain elusive. Here we show that the initial translation products of the rgpA, kgp and hemagglutinin hagA genes are responsible for coaggregation of P gingivalis and that the proteolytic activity of Rgp and Kgp is indispensable in this process. The rgpA rgpB kgp- and rgpA kgp hagA-deficient triple mutants exhibited no coaggregation activity with Actinomyces viscosus, whereas the kgp-null and rgpA rgpB-deficient double mutants significantly retained this activity. Consistently, the combined action of Rgp- and Kgp-specific inhibitors strongly inhibited the coaggregation activity of the bacterium, although single use of Rgp- or Kgp-specific inhibitor significantly retained this activity. We also demonstrate that the 47- and 43-kDa proteins produced from the translation products of the rgpA, kgp, and hagA genes by proteolytic activity of both Rgp and Kgp are responsible for the coaggregation of P. gingivalis.

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  • カテプシンEによる免疫アレルギー性疾患の制御機構

    筑波 隆幸, 柳川 三千代, 甲村 恵子, 梯 裕恵, 岡元 邦彰, 山本 健二

    Journal of oral biosciences   46 ( 5 )   474 - 474   2004年9月

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    記述言語:日本語   出版者・発行元:歯科基礎医学会  

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  • ベルベリンのマウス骨髄細胞からの破骨細胞形成への抑制作用

    胡 錦萍, 西下 一久, 柴田 光枝, 坂井 詠子, 金岡 和博, 吉田 一, 岡元 邦彰, 加藤 有三

    Journal of oral biosciences   46 ( 5 )   463 - 463   2004年9月

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  • KIAA0377遺伝子産物の破骨細胞様細胞形成におよぼす影響

    西下 一久, 石田 豊, 東洋 一, 岡元 邦彰, 加藤 有三

    Journal of oral biosciences   46 ( 5 )   465 - 465   2004年9月

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  • Laminin alpha 2 is essential for odontoblast differentiation regulating dentin sialoprotein expression

    K Yuasa, S Fukumoto, Y Kamasaki, A Yamada, E Fukumoto, K Kanaoka, K Saito, H Harada, E Arikawa-Hirasawa, Y Miyagoe-Suzuki, S Takeda, K Okamoto, Y Kato, T Fujiwara

    JOURNAL OF BIOLOGICAL CHEMISTRY   279 ( 11 )   10286 - 10292   2004年3月

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    記述言語:英語   出版者・発行元:AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

    Laminin alpha2 is subunit of laminin-2 (alpha2beta1gamma1), which is a major component of the muscle basement membrane. Although the laminin alpha2 chain is expressed in the early stage of dental mesenchyme development and localized in the tooth germ basement membrane, its expression pattern in the late stage of tooth germ development and molecular roles are not clearly understood. We analyzed the role of laminin alpha2 in tooth development by using targeted mice with a disrupted lama2 gene. Laminin alpha2 is expressed in dental mesenchymal cells, especially in odontoblasts and during the maturation stage of ameloblasts, but not in the pre-secretory or secretory stages of ameloblasts. Lama2 mutant mice have thin dentin and a widely opened dentinal tube, as compared with wildtype and heterozygote mice, which is similar to the phenotype of dentinogenesis imperfecta. During dentin formation, the expression of dentin sialoprotein, a marker of odontoblast differentiation, was found to be decreased in odontoblasts from mutant mice. Furthermore, in primary cultures of dental mesenchymal cells, dentin matrix protein, and dentin sialophosphoprotein, mRNA expression was increased in laminin-2 coated dishes but not in those coated with other matrices, fibronectin, or type I collagen. Our results suggest that laminin alpha2 is essential for odontoblast differentiation and regulates the expression of dentin matrix proteins.

    DOI: 10.1074/jbc.M310013200

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  • Laminin alpha 2 is essential for odontoblast differentiation regulating dentin sialoprotein expression

    K Yuasa, S Fukumoto, Y Kamasaki, A Yamada, E Fukumoto, K Kanaoka, K Saito, H Harada, E Arikawa-Hirasawa, Y Miyagoe-Suzuki, S Takeda, K Okamoto, Y Kato, T Fujiwara

    JOURNAL OF BIOLOGICAL CHEMISTRY   279 ( 11 )   10286 - 10292   2004年3月

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    記述言語:英語   出版者・発行元:AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

    Laminin alpha2 is subunit of laminin-2 (alpha2beta1gamma1), which is a major component of the muscle basement membrane. Although the laminin alpha2 chain is expressed in the early stage of dental mesenchyme development and localized in the tooth germ basement membrane, its expression pattern in the late stage of tooth germ development and molecular roles are not clearly understood. We analyzed the role of laminin alpha2 in tooth development by using targeted mice with a disrupted lama2 gene. Laminin alpha2 is expressed in dental mesenchymal cells, especially in odontoblasts and during the maturation stage of ameloblasts, but not in the pre-secretory or secretory stages of ameloblasts. Lama2 mutant mice have thin dentin and a widely opened dentinal tube, as compared with wildtype and heterozygote mice, which is similar to the phenotype of dentinogenesis imperfecta. During dentin formation, the expression of dentin sialoprotein, a marker of odontoblast differentiation, was found to be decreased in odontoblasts from mutant mice. Furthermore, in primary cultures of dental mesenchymal cells, dentin matrix protein, and dentin sialophosphoprotein, mRNA expression was increased in laminin-2 coated dishes but not in those coated with other matrices, fibronectin, or type I collagen. Our results suggest that laminin alpha2 is essential for odontoblast differentiation and regulates the expression of dentin matrix proteins.

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  • Cathepsin E deficiency and brain functions

    N Shigematsu, T Nishioku, T Tsukuba, S Kohsaka, K Okamoto, Y Okamoto, T Yamamoto, M Himeno, K Yamamoto

    JOURNAL OF PHARMACOLOGICAL SCIENCES   94   89P - 89P   2004年

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    記述言語:英語   掲載種別:研究発表ペーパー・要旨(国際会議)   出版者・発行元:JAPANESE PHARMACOLOGICAL SOC  

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  • Effect of Pepstatin A in osteoclast

    H Yoshida, K Okamoto, M Shibata, E Sakai, K Kanaoka, K Yuasa, A Mizuno, K Nishishita, Y Kato

    JOURNAL OF PHARMACOLOGICAL SCIENCES   94   188P - 188P   2004年

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    記述言語:英語   掲載種別:研究発表ペーパー・要旨(国際会議)   出版者・発行元:JAPANESE PHARMACOLOGICAL SOC  

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  • Glycosphingolipids regulate osteoclastgenesis mediated by receptor activator of NF-kB ligand and lipopolysaccharide

    E Sakai, S Fukumoto, H Hotokezaka, E Fukumoto, M Shibata, K Kanaoka, T Iwamoto, K Okamoto, Y Kato

    JOURNAL OF PHARMACOLOGICAL SCIENCES   94   184P - 184P   2004年

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    記述言語:英語   掲載種別:研究発表ペーパー・要旨(国際会議)   出版者・発行元:JAPANESE PHARMACOLOGICAL SOC  

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  • Association of cathepsin E deficiency with development of atopic dermatitis

    T Tsukuba, K Okamoto, Y Okamoto, M Yanagawa, K Kohmura, Y Yasuda, H Uchi, T Nakahara, M Furue, K Nakayama, T Kadowaki, K Yamamoto, KI Nakayama

    JOURNAL OF BIOCHEMISTRY   134 ( 6 )   893 - 902   2003年12月

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    記述言語:英語   出版者・発行元:OXFORD UNIV PRESS  

    Atopic dermatitis (AD) is a pruritic inflammatory skin diseases associated with a family history of atropy. Here we show that mice lacking the endolysosomal. aspartic proteinase cathepsin E spontaneously develop skin lesions similar to those of humans with AD when reared under conventional conditions but not under specific pathogen-free conditions. These mice showed the increase in the ratio of CD4(+)/CD8(+) T cells, the strong polarization of naive T cells to T helper 2 cells, and the systemic accumulation of IL-18 and IL-1beta accompanied by a marked increase in IL-4, IL-5, and IgE. The relative rates of degradation of IL-18 and IL-1beta were significantly lower in cathepsin E-deficient mice than wild-type mice. These results strongly suggest that the development of AD in cathepsin E-deficient mice is initiated by systemic accumulation of IL-18 and IL-1beta, mainly due to their reduced turnover rates. In addition, the reduced expression of cathepsin E was also observed in erythrocytes of both humans with AD and the AD mouse model NC/Nga. Cathepsin E deficiency might thus be responsible for the induction of AD in humans and mice.

    DOI: 10.1093/jh/mvg216

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  • Association of cathepsin E deficiency with development of atopic dermatitis

    T Tsukuba, K Okamoto, Y Okamoto, M Yanagawa, K Kohmura, Y Yasuda, H Uchi, T Nakahara, M Furue, K Nakayama, T Kadowaki, K Yamamoto, KI Nakayama

    JOURNAL OF BIOCHEMISTRY   134 ( 6 )   893 - 902   2003年12月

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    記述言語:英語   出版者・発行元:OXFORD UNIV PRESS  

    Atopic dermatitis (AD) is a pruritic inflammatory skin diseases associated with a family history of atropy. Here we show that mice lacking the endolysosomal. aspartic proteinase cathepsin E spontaneously develop skin lesions similar to those of humans with AD when reared under conventional conditions but not under specific pathogen-free conditions. These mice showed the increase in the ratio of CD4(+)/CD8(+) T cells, the strong polarization of naive T cells to T helper 2 cells, and the systemic accumulation of IL-18 and IL-1beta accompanied by a marked increase in IL-4, IL-5, and IgE. The relative rates of degradation of IL-18 and IL-1beta were significantly lower in cathepsin E-deficient mice than wild-type mice. These results strongly suggest that the development of AD in cathepsin E-deficient mice is initiated by systemic accumulation of IL-18 and IL-1beta, mainly due to their reduced turnover rates. In addition, the reduced expression of cathepsin E was also observed in erythrocytes of both humans with AD and the AD mouse model NC/Nga. Cathepsin E deficiency might thus be responsible for the induction of AD in humans and mice.

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  • ベルベリンによる破骨細胞分化因子(RANKL)細胞内シグナリング抑制効果

    胡 錦萍, 西下 一久, 柴田 光枝, 岡元 邦彰, 加藤 有三

    歯科基礎医学会雑誌   45 ( 5 )   374 - 374   2003年9月

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    記述言語:日本語   出版者・発行元:歯科基礎医学会  

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  • スフィンゴ糖脂質はRANKやc-Srcのラフトへの局在と破骨細胞形成を調節する

    坂井 詠子, 福本 敏, 金岡 和博, 柴田 光枝, 福本 恵美子, 岩本 勉, 岡元 邦彰, 加藤 有三

    歯科基礎医学会雑誌   45 ( 5 )   343 - 343   2003年9月

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    記述言語:日本語   出版者・発行元:歯科基礎医学会  

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  • ラミニンα2の歯の発生における役割

    湯浅 健司, 福本 敏, 釜崎 陽子, 山田 亜矢, 福本 恵美子, 斉藤 幹, 岡元 邦彰, 藤原 卓, 加藤 有三

    歯科基礎医学会雑誌   45 ( 5 )   290 - 290   2003年9月

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    記述言語:日本語   出版者・発行元:歯科基礎医学会  

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  • 口腔癌における破骨細胞分化因子の役割

    岩本 勉, 福本 敏, 柳本 惣市, 吉田 一, 岡元 邦彰, 吉冨 泉, 川崎 五郎, 加藤 有三, 水野 明夫

    歯科基礎医学会雑誌   45 ( 5 )   319 - 319   2003年9月

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    記述言語:日本語   出版者・発行元:歯科基礎医学会  

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  • Regulation of osteoclast survival and activation by glycosphingolipids.

    K. Kanaoka, S. Fukumoto, K. Yuasa, K. Okamoto, M. Shibata, E. Sakai, Y. Kato, T. Iwamoto, F. Hashimoto, N. Yoshida

    JOURNAL OF DENTAL RESEARCH   82   B358 - B358   2003年6月

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    記述言語:英語   掲載種別:研究発表ペーパー・要旨(国際会議)   出版者・発行元:INT AMER ASSOC DENTAL RESEARCHI A D R/A A D R  

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  • Subcellular localization of the receptor activator of NF-KappaB and SRC kinase in detergent insoluble fractions of osteoclasts

    E Sakai, S Fukumoto, K Kanaoka, M Shibata, T Iwamoto, E Fukumoto, K Okamoto, Y Kato

    BONE   32 ( 5 )   S155 - S155   2003年5月

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  • The regulation of bone resorption in tooth formation and eruption processes in mouse alveolar crest devoid of cathepsin K

    M Okaji, H Sakai, E Sakai, M Shibata, F Hashimoto, Y Kobayashi, N Yoshida, K Okamoto, K Yamamoto, Y Kato

    JOURNAL OF PHARMACOLOGICAL SCIENCES   91 ( 4 )   285 - 294   2003年4月

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    記述言語:英語   出版者・発行元:JAPANESE PHARMACOLOGICAL SOC  

    Osteoclastic bone resorption has recently been implicated in the tooth formation and eruption in alveolar bone. Cathepsin K (CK) is a cysteine proteinase expressed predominantly in osteoclasts and is believed to play a critical role in degradation of bone matrix proteins. Here we present evidence that the alveolar bone resorption is essential for the tooth formation and that eruption proceeds normally in CK-deficient (CK-/-) mice. Radiographic and histological analyses revealed that the alveolar bone from these animals had no significant abnormalities during the tooth development between 5 and 28 days after birth. The tooth crown was normally erupted through the alveolar bone layer at 28 days after birth. The number of tartrate-resistant acid phosphatase-positive multinuclear cells in the alveolar bone around the tooth germ was apparently increased in 5-day-old CK-/- mice compared with age-matched littermates. More important, however, the immunohistochemical localization of matrix metalloproteinase-9 (MMP-9) was clearly increased in the CK-/- osteoclasts. In contrast, no significant difference in the immunoreactivity for cathepsin D was observed between the CK-/- osteoclasts and the wild-type ones. These results indicate that CK-/- osteoclasts are fully differentiated and are capable of degrading the organic phase of alveolar bone during the tooth formation and eruption, which may result from the compensatory action by MMP-9 increasingly expressed in the osteoclasts.

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  • The regulation of bone resorption in tooth formation and eruption processes in mouse alveolar crest devoid of cathepsin K

    M Okaji, H Sakai, E Sakai, M Shibata, F Hashimoto, Y Kobayashi, N Yoshida, K Okamoto, K Yamamoto, Y Kato

    JOURNAL OF PHARMACOLOGICAL SCIENCES   91 ( 4 )   285 - 294   2003年4月

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    記述言語:英語   出版者・発行元:JAPANESE PHARMACOLOGICAL SOC  

    Osteoclastic bone resorption has recently been implicated in the tooth formation and eruption in alveolar bone. Cathepsin K (CK) is a cysteine proteinase expressed predominantly in osteoclasts and is believed to play a critical role in degradation of bone matrix proteins. Here we present evidence that the alveolar bone resorption is essential for the tooth formation and that eruption proceeds normally in CK-deficient (CK-/-) mice. Radiographic and histological analyses revealed that the alveolar bone from these animals had no significant abnormalities during the tooth development between 5 and 28 days after birth. The tooth crown was normally erupted through the alveolar bone layer at 28 days after birth. The number of tartrate-resistant acid phosphatase-positive multinuclear cells in the alveolar bone around the tooth germ was apparently increased in 5-day-old CK-/- mice compared with age-matched littermates. More important, however, the immunohistochemical localization of matrix metalloproteinase-9 (MMP-9) was clearly increased in the CK-/- osteoclasts. In contrast, no significant difference in the immunoreactivity for cathepsin D was observed between the CK-/- osteoclasts and the wild-type ones. These results indicate that CK-/- osteoclasts are fully differentiated and are capable of degrading the organic phase of alveolar bone during the tooth formation and eruption, which may result from the compensatory action by MMP-9 increasingly expressed in the osteoclasts.

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  • Disruption of structural and functional integrity of alpha(2)-macroglobulin by cathepsin E

    M Shibata, H Sakai, E Sakai, K Okamoto, K Nishishita, Y Yasuda, Y Kato, K Yamamoto

    EUROPEAN JOURNAL OF BIOCHEMISTRY   270 ( 6 )   1189 - 1198   2003年3月

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    記述言語:英語   出版者・発行元:BLACKWELL PUBLISHING LTD  

    alpha(2) -Macroglobulin (alpha2M) is an abundant glycoprotein with the intrinsic capacity for capturing diverse proteins for rapid delivery into cells. After internalization by the receptor- mediated endocytosis, alpha2M-protein complexes were rapidly degraded in the endolysosome system. Although this is an important pathway for clearance of both alpha2M and biological targets, little is known about the nature of alpha2M degradation in the endolysosome system. To investigate the possible involvement of intracellular aspartic proteinases in the disruption of structural and functional integrity of alpha2M in the endolysosome system, we examined the capacity of alpha2M for interacting with cathepsin E and cathepsin D under acidic conditions and the nature of its degradation. alpha2M was efficiently associated with cathepsin E under acidic conditions to form noncovalent complexes and rapidly degraded through the generation of three major proteins with apparent molecular masses of 90, 85 and 30 kDa. Parallel with this reaction, alpha2M resulted in the rapid loss of its antiproteolytic activity. Analysis of the N-terminal amino-acid sequences of these proteins revealed that alpha2M was selectively cleaved at the Phe811-Leu812 bond in about 100mer downstream of the bait region. In contrast, little change was observed for alpha2M treated by cathepsin D under the same conditions. Together, the synthetic SPAFLA peptide corresponding to the Ser808-Ala813 sequence of human alpha2M, which contains the cathepsin E-cleavage site, was selectively cleaved by cathepsin E, but not cathepsin D. These results suggest the possible involvement of cathepsin E in disruption of the structural and functional integrity of alpha2M in the endolysosome system.

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  • Disruption of structural and functional integrity of alpha(2)-macroglobulin by cathepsin E

    M Shibata, H Sakai, E Sakai, K Okamoto, K Nishishita, Y Yasuda, Y Kato, K Yamamoto

    EUROPEAN JOURNAL OF BIOCHEMISTRY   270 ( 6 )   1189 - 1198   2003年3月

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    記述言語:英語   出版者・発行元:BLACKWELL PUBLISHING LTD  

    alpha(2) -Macroglobulin (alpha2M) is an abundant glycoprotein with the intrinsic capacity for capturing diverse proteins for rapid delivery into cells. After internalization by the receptor- mediated endocytosis, alpha2M-protein complexes were rapidly degraded in the endolysosome system. Although this is an important pathway for clearance of both alpha2M and biological targets, little is known about the nature of alpha2M degradation in the endolysosome system. To investigate the possible involvement of intracellular aspartic proteinases in the disruption of structural and functional integrity of alpha2M in the endolysosome system, we examined the capacity of alpha2M for interacting with cathepsin E and cathepsin D under acidic conditions and the nature of its degradation. alpha2M was efficiently associated with cathepsin E under acidic conditions to form noncovalent complexes and rapidly degraded through the generation of three major proteins with apparent molecular masses of 90, 85 and 30 kDa. Parallel with this reaction, alpha2M resulted in the rapid loss of its antiproteolytic activity. Analysis of the N-terminal amino-acid sequences of these proteins revealed that alpha2M was selectively cleaved at the Phe811-Leu812 bond in about 100mer downstream of the bait region. In contrast, little change was observed for alpha2M treated by cathepsin D under the same conditions. Together, the synthetic SPAFLA peptide corresponding to the Ser808-Ala813 sequence of human alpha2M, which contains the cathepsin E-cleavage site, was selectively cleaved by cathepsin E, but not cathepsin D. These results suggest the possible involvement of cathepsin E in disruption of the structural and functional integrity of alpha2M in the endolysosome system.

    DOI: 10.1046/j.1432-1033.2003.03479.x

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  • Porphyromonas gingivalisの産生するリジン特異的システインプロテアーゼ発現系の確立

    岡元邦彰, 胡錦へい, 柴田光枝, 坂井詠子, 門脇知子, 庄子幹郎, 山本健二, 中山浩次, 加藤有三

    歯科基礎医学会雑誌   45 ( 5 )   2003年

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  • Cathepsin E and atopic dermatitis.

    T Tsukuba, K Okamoto, K Yamamoto

    JOURNAL OF PHARMACOLOGICAL SCIENCES   91   13P - 13P   2003年

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    記述言語:英語   掲載種別:研究発表ペーパー・要旨(国際会議)   出版者・発行元:JAPANESE PHARMACOLOGICAL SOC  

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  • Expression and subcellular localization of caveolin with Src kinase and RANK in osteoclasts.

    E Sakai, S Fukumoto, K Kanaoka, M Shibata, T Iwamoto, E Fukumoto, K Okamoto, Y Kato

    JOURNAL OF PHARMACOLOGICAL SCIENCES   91   71P - 71P   2003年

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    記述言語:英語   掲載種別:研究発表ペーパー・要旨(国際会議)   出版者・発行元:JAPANESE PHARMACOLOGICAL SOC  

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  • Purification and characterization of mouse soluble RANK expressed in a baculovirus system.

    T Imai, M Shibata, K Kanaoka, K Yuasa, A Mizuno, K Okamoto, Y Kato

    JOURNAL OF PHARMACOLOGICAL SCIENCES   91   239P - 239P   2003年

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    記述言語:英語   掲載種別:研究発表ペーパー・要旨(国際会議)   出版者・発行元:JAPANESE PHARMACOLOGICAL SOC  

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  • なぜ,カテプシンK欠損マウスにおいて歯は正常に萌出できるのか?

    岡地 雅代, 柴田 光枝, 坂井 詠子, 岡元 邦彰, 山本 健二

    歯科基礎医学会雑誌   44 ( 5 )   433 - 433   2002年9月

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    記述言語:日本語   出版者・発行元:歯科基礎医学会  

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  • Porphyromonas gingivalisによる赤血球凝集機構解明の試み

    坂井 詠子, 佛坂斉祉, 庄子 幹郎, 鎌口 有秀, 岡元 邦彰, 加藤 有三, 中山 浩次

    歯科基礎医学会雑誌   44 ( 5 )   466 - 466   2002年9月

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    記述言語:日本語   出版者・発行元:歯科基礎医学会  

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  • The role of propeptide of cathepsin E in its biosynthetic pathway

    Y Yasuda, K Kohmura, K Okamoto, T Tsukuba, K Yamamoto

    JAPANESE JOURNAL OF PHARMACOLOGY   88   183P - 183P   2002年

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    記述言語:英語   掲載種別:研究発表ペーパー・要旨(国際会議)   出版者・発行元:JAPANESE PHARMACOLOGICAL SOC  

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  • カテプシンD欠損マウスにおける長骨骨端線の消失

    岡元 邦彰, 前田 英史, 加藤 有三, 山本 健二

    歯科基礎医学会雑誌   43 ( 5 )   581 - 581   2001年8月

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    記述言語:日本語   出版者・発行元:歯科基礎医学会  

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  • 歯周病原性細菌の蛋白質分解酵素を介する生存戦略 (新世紀における蛋白質科学の進展) -- (第3部 生命現象の理解と医療・創薬に向けて)

    山本 健二, 馬場 貴代, 岡元 邦彰

    蛋白質核酸酵素   46 ( 11 )   1426 - 1427,1781〜1788   2001年8月

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    記述言語:日本語   出版者・発行元:共立出版  

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  • Pathophysiological roles of two types of gingipains in periodontal diseases

    K. Yamamoto, A. Baba, K. Okamoto, T. Kadowaki

    Tanpakushitsu kakusan koso. Protein, nucleic acid, enzyme   46 ( 11 Suppl )   1781 - 1788   2001年8月

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    掲載種別:書評論文,書評,文献紹介等  

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  • New functional aspects of cathepsin D and cathepsin E

    T Tsukuba, K Okamoto, Y Yasuda, W Morikawa, H Nakanishi, K Yamamoto

    MOLECULES AND CELLS   10 ( 6 )   601 - 611   2000年12月

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    記述言語:英語   掲載種別:書評論文,書評,文献紹介等   出版者・発行元:SPRINGER-VERLAG SINGAPORE PTE LTD  

    Cathepsin D (CD) and cathepsin E are representative lysosomal and nonlysosomal aspartic proteinases, respectively, and play an important role in the degradation of proteins, the generation of bioactive proteins, antigen processing, etc. Recently, several lines of evidence have suggested the involvement of these two enzymes in the execution of neuronal death pathways induced by aging, transient forebrain ischemia, and excessive stimulation of glutamate receptors with excitotoxins. CD has also been shown to mediate apoptosis induced by various stimuli and p53-dependent tumor suppression. To gain more insight into in vivo functions of CD, mice deficient in this enzyme were generated. The mutant animals showed a progressive atrophy of the intestinal mucosa, a massive destruction of lymphoid organs, and a profound accumulation of ceroid lipofuscin, and developed a phenotype resembling neuronal ceroid lipofucinosis, suggesting that CD is essential for proteolysis of proteins regulating cell growth and tissue homeostasis. It has also been shown that CD molecules secreted from human prostate carcinoma cells are responsible for the generation of angiostatin, a potent endogenous inhibitor of angiogenesis, suggesting its contribution to the prevention of tumor growth and angiogenesis-dependent growth of metastases. Interestingly, pro-CD from human breast carcinoma cells showed a significantly lower angiostatin-generating activity than that from prostate carcinoma cells. Since deglycosylated CD molecules from both carcinoma cells showed a low angiostatin-generating activity, this discrepancy appeared to be attributed to the difference in the carbohydrate structures of CD molecules between the two cell types and to contribute to their potency to prevent tumor growth and metastases.

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  • Porphyromonas gingivalis proteinases as virulence determinants in progression of periodontal diseases

    Tomoko Kadowaki, Koji Nakayama, Kuniaki Okamoto, Naoko Abe, Atsuyo Baba, Yixin Shi, Dinath B. Ratnayake, Kenji Yamamoto

    Journal of Biochemistry   128 ( 2 )   153 - 159   2000年8月

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    記述言語:英語   掲載種別:書評論文,書評,文献紹介等  

    Porphyromonas gingivalis, one of the major causative agents of periodontal diseases, produces large amounts of arginine- and lysine-specific cysteine proteinases in cell-associated and secretory forms, which are now referred to as Arg-gingipain (Rgp) and Lysgingipain (Kgp), respectively. A number of studies have revealed that these proteinases are closely associated with the periodontopathogenesis of this bacterium: destruction of periodontal connective tissues, disruption of host defense mechanisms, and development and maintenance of inflammation in periodontal pockets. With respect to the physiology of the bacterium, Rgp and Kgp are indispensable for it to obtain nutrients from the environment, since it cannot utilize saccharides as carbon/energy sources for growth and totally depends on peptides and amino acids that are provided from environmental proteins by Rgp and Kgp. Furthermore, proteolytic activities of Rgp and Kgp contribute to processing/maturation of various cell-surface proteins of P. gingivalis, such as fimA fimbrilin (a subunit of major fimbriae), 75-kDa protein (a subunit of minor fimbriae), hemagglutinins, and the hemoglobin receptor protein, which are important for the bacterium to colonize and proliferate in the gingival crevice and to invade the periodontium. These findings strongly indicate critical roles of Rgp and Kgp in the virulence of P. gingivalis.

    DOI: 10.1093/oxfordjournals.jbchem.a022735

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  • 歯周病原性細菌の産生するリジン特異的システインプロテアーゼの分布と機能解析

    安部直子, 門脇知子, 岡元邦彰, 岡崎真治, 浅尾哲次, 中山浩次, 山本健二

    歯科基礎医学会雑誌   42 ( 5 )   2000年

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  • 歯周病原性細菌のヘムおよびアミノ酸獲得機構

    山本健二, 岡元邦彰, 門脇知子, 安部直子, 馬場貴代, 中山浩次

    生化学   72 ( 8 )   2000年

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  • カテプシンDおよびEの性状解析に有用な新しい合成基質の作製

    安田 善之, 岡元 邦彰, 岡本 美子, 景山 節, 内山 安男, 木南 英紀, 山本 健二

    生化学   71 ( 8 )   867 - 867   1999年8月

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    記述言語:日本語   出版者・発行元:(公社)日本生化学会  

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  • 歯周病原性細菌Porphyromonas gingivalisが産生するシステインプロテアーゼの機能.

    山本 健二, 門脇 知子, 岡元 邦彰, 安部 直子, 中山 浩次

    炎症   18 ( 4 )   259 - 264   1998年

  • Structural and functional characterization of periodontal pathogenic cysteine proteinases from Porphyromonas gingivalis

    K. Yamamoto, T. Kadowaki, K. Okamoto, K. Nakayama

    Tanpakushitsu kakusan koso. Protein, nucleic acid, enzyme   42 ( 14 Suppl )   2425 - 2432   1997年10月

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  • Biological roles of a novel class of cysteine proteinases from Porphyromonas gingivalis in periodontal disease progression

    K Yamamoto, T Kadowaki, K Okamoto, N Abe, K Nakayama

    MEDICAL ASPECTS OF PROTEASES AND PROTEASE INHIBITORS   15   139 - 149   1997年

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    記述言語:英語   出版者・発行元:I O S PRESS  

    We have studied the role of a novel class of cysteine proteinases from the oral anaerobic bacterium Porphyromonas gingivalis in periodontal disease progression. Two unique cysteine proteinases, so termed Arg-gingipain (RGP) and Lys-gingipain (KGP) due to their peptide cleavage specificity at arginine and lysine residues, respectively, have been implicated as major virulence factors in periodontal disease. Both enzymes are shown to be involved in the direct destruction of periodontal tissues and the disruption of normal host defense mechanisms. These enzymes have also been implicated in the hemagglutinating activity of the organism. To determine the structuraI, genetic, and functional relationships between the two enzymes, we have cloned the RGP- and KGP-encoding genes and ultimately constructed the rgp- or kgp-deficient mutants from the organism. In the course of this study, RGP has been shown to be encoded by two separate genes (rgpA and rgpB) on the chromosome of the organism. Deduced amino acid sequences of both RGP- and KGP-encoding genes revealed that the precursors of both enzymes were composed of at least four domains: the signal peptide, the NH2-terminal prosequence, the proteinase domain, and the COOH-terminal hemagglutinin domain. Although both enzymes were apparently different in the amino acid sequences of the NH2-terminal prosequences and the proteinase domains, their COOH-terminal hemagglutinin domains exhibited significant similarity. Analysis of the rgpA rgpB double mutant confirmed that RGP was a major virulence factor of the organism. However, the disruption of the KGP gene did not affect either the bactericidal activity of polymorphonucler cells or the hemagglutinin activity of the organism.

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  • ラットカテプシンE分子のN-結合型糖鎖の役割

    池田 仁子, 西下 一久, 坂井 英昭, 岡元 邦彰, 筑波 隆幸, 加藤 有三, 山本 健二

    日本分子生物学会年会プログラム・講演要旨集   19   255 - 255   1996年8月

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  • Porphyromonas gingivalisアルギニン特異的システインプロテアーゼ(Arg-gingipain)による外膜タンパク質ならびに分泌タンパク質のプロセシング

    門脇知子, 中山浩次, 吉村文信, 岡元邦彰, 山本健二

    日本分子生物学会年会プログラム・講演要旨集   19th   1996年

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  • 歯周病と病原性プロテアーゼ

    山本健二, 門脇知子, 岡元邦彰

    モダンフィジシャン   16 ( 12 )   1996年

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  • Porphyromonas gingivalisの病原性プロテアーゼ遺伝子の構造比較

    岡元邦彰, 門脇知子, 中山浩次, 山本健二

    日本分子生物学会年会プログラム・講演要旨集   19th   1996年

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講演・口頭発表等

  • 新規腫瘍オルガノイド形成多元評価システムの薬剤スクリーニングへの応用

    十川千春, 江口傑徳, 石毛真行, 河合穂高, 奥舎有加, 中野敬介, 十川紀夫, 小崎健一, 岡元邦彰

    2019年12月15日 

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    開催年月日: 2019年12月15日

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  • Keap1遺伝子欠損はNrf2の活性化を介して破骨細胞分化を抑制する

    坂井詠子, 福間裕, 西下一久, 岡元邦彰, 筑波隆幸

    第40回日本分子生物学会年会/第90回日本生化学会大会  2017年12月 

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    開催年月日: 2017年12月6日 - 2017年12月9日

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  • 口腔扁平上皮癌由来エクソソームニ含まれるプロテオームの特性

    小野喜章, 江口傑徳, 十川千春, 村上純, 藤原敏史, 笠井智成, 妹尾昌治, 佐々木朗, 小崎健一, 岡元邦彰

    第40回日本分子生物学会年会/第90回日本生化学会大会  2017年12月 

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    開催年月日: 2017年12月6日 - 2017年12月9日

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  • Transcription factor Sp1 regulates the expression of the murine cathepsin E gene in gastric adenosarcoma cells

    2010年 

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  • Transcription factor Sp1 regulates the expression of the murine cathepsin E gene in gastric adenosarcoma cells

    第83回日本薬理学会年会  2010年 

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  • 破骨細胞分化における鉄代謝とミトコンドリアストレスの重要性

    第33回日本鉄バイオサイエンス学会学術集会  2009年 

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  • カテプシンE欠損によるオートファジー低下とそれに伴うミトコンドリア機能低下と酸化ストレスの増大

    第14回日本病態プロテアーゼ学会  2009年 

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  • IMPAIRMENT OF AUTOPHAGY ACCOMPANIED BY INCREASED ABERRANT MITOCHONDRIA AND OXIDATIVE STRESS IN CATHEPSIN E DEFICIENT MACROPHAGES

    2009年 

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  • カテプシンE欠損マクロファージにおけるオートファジーの低下とそれに伴うミトコンドリア機能異常と酸化ストレスの上昇

    第51回歯科基礎医学会学術大会  2009年 

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  • IMPAIRMENT OF AUTOPHAGY ACCOMPANIED BY INCREASED ABERRANT MITOCHONDRIA AND OXIDATIVE STRESS IN CATHEPSIN E DEFICIENT MACROPHAGES

    5th International Symposium on Autophagy  2009年 

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  • 鉄代謝を介した新規の破骨細胞分化機構

    第51回歯科基礎医学会学術大会  2009年 

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  • カテプシンE欠損はマクロファージの膜蛋白質輸送異常とミトコンドリア機能異常を起こす

    第31回日本分子生物学会/第81回日本生化学会合同大会  2008年 

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  • カテプシンE欠損による遊走能および細胞接着能の低下

    第50回歯科基礎医学会学術大会  2008年 

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  • カテプシンE遺伝子発現に関する転写因子Sp1

    第31回日本分子生物学会/第81回日本生化学会合同大会  2008年 

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  • カテプシンE欠損マウスにおける高脂血症および脂肪肝の誘導

    第50回歯科基礎医学会学術大会  2008年 

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  • カテプシンE遺伝子発現に関与する転写因子Sp1

    第50回歯科基礎医学会学術大会  2008年 

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  • RANKL誘導性破骨細胞分化における鉄結合蛋白の関与

    第50回歯科基礎医学会学術大会  2008年 

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  • 赤血球におけるカテプシンEの役割

    第49回歯科基礎医学会学術大会  2007年 

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  • P. gingivalis感染とインスリン抵抗性に関する実験的解析

    第49回歯科基礎医学会学術大会  2007年 

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  • ASSOCIATION OF CATHEPSIN E WITH HOST DEFFENCE AGAINST TUMOR CELLS

    5TH General Meeting of the International Proteolysis Society  2007年 

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  • 成熟破骨細胞の延命と脂質ラフト形成におけるリセドロネートおよびD-PDMPの作用

    第49回歯科基礎医学会学術大会  2007年 

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  • Cathepsin E mediates membrane trafficking in macrophages

    第80回日本薬理学会  2007年 

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  • カテプシンE欠損によるミトコンドリア機能異常と酸化ストレス亢進

    第49回歯科基礎医学会学術大会  2007年 

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  • ベルベリンによる破骨細胞生存の抑制機構の解明

    第49回歯科基礎医学会学術大会  2007年 

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  • Cathepsin E mediates membrane trafficking in macrophages

    2007年 

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  • CONTROL OF THE ENDOSOME/LYSOSOME SYSTEM OF MACROPHAGES BY CATHEPSIN E

    2007年 

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  • ASSOCIATION OF CATHEPSIN E WITH HOST DEFFENCE AGAINST TUMOR CELLS

    2007年 

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  • CONTROL OF THE ENDOSOME/LYSOSOME SYSTEM OF MACROPHAGES BY CATHEPSIN E

    5TH General Meeting of the International Proteolysis Society  2007年 

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  • Porphyromonas gingivalisの血球凝集および接着性におけるHGP44の役割とC末端ペプチドの阻害効果

    第48回歯科基礎医学会学術大会  2006年 

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  • P. gingivalis感染の生活習慣病増悪機序について

    第48回歯科基礎医学会学術大会  2006年 

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  • マウスカテプシンE遺伝子発現におけるSp1の関与

    日本分子生物学会2006フォーラム  2006年 

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  • カテプシンE欠損マクロファージにおける膜タンパク質の輸送異常

    第48回歯科基礎医学会学術大会  2006年 

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  • Berberine inhibits RANKL, TNFa, LPS and PGN-induced mature osteoclasts survival

    第79回日本薬理学会総会  2006年 

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  • Involvement of glycosphingolipids in LPS mediated survival of mature osteoclasts

    第79回日本薬理学会総会  2006年 

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  • A possible mechanism for cathepsin E-mediated tumor supression

    Satellite Meeting for the 20th IUBMB International Congress and 11th FAOBMB Congress  2006年 

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    会議種別:ポスター発表  

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  • Suppression of carcinogenesis and tumor growth by cathepsin E

    20th IUBMB International Congress of Biochemistry and Molecular Biology  2006年 

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    会議種別:ポスター発表  

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  • スフィンゴ糖脂質による破骨細胞内LPSシグナル伝達の制御

    第47回歯科基礎医学会学術大会  2005年 

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    会議種別:ポスター発表  

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  • ベルベリンの破骨細胞形成抑制作用のメカニズム検討

    第47回歯科基礎医学会学術大会  2005年 

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    会議種別:ポスター発表  

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  • Aberrant antigen processing in cathepsin E-deficient dendritic cells

    第78回日本生化学会  2005年 

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    会議種別:ポスター発表  

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  • 破骨細胞におけるスフィンゴ糖脂質の役割

    第47回歯科基礎医学会学術大会  2005年 

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  • Cathepsin E is important for endo/lysosomal systems in macrophages against a bacterial infection.

    第78回日本薬理学会総会  2005年 

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  • Alterration of substance P levels in various regions of mouse brain caused by cathepsin E-deficiency

    日本薬理学会総会  2005年 

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  • Porphyromonas gingivalisの産生するシステインプロテアーゼ(Kgp)の活性中心の決定

    第47回歯科基礎医学会学術大会  2005年 

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    会議種別:ポスター発表  

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  • Glycosphingolipids regulate osteoclastgenesis mediated by lipopolysaccharide

    第78回日本薬理学会総会  2005年 

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  • Berberine Inhibits RANKL, TNF-a, LPS and Peptidoglycan (PGN)-induced Fusion of Osteoclasts

    日中医学会  2005年 

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    会議種別:ポスター発表  

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  • カテプシンE欠損が及ぼすマクロファージの機能異常

    第58回日本薬理学会西南部会  2005年 

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  • ベルベリンによる破骨細胞形成抑制の分子機構

    第58回日本薬理学会西南部会  2005年 

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▼全件表示

Works(作品等)

  • 破骨細胞におけるカテプシンEの役割と創薬にむけた基質分子の探究

    2009年
    -
    2011年

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  • 骨疾患における創薬のターゲットとなりうるプロテアーゼの作用機序の解明

    2007年
    -
    2009年

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  • 歯周病原性細菌の産生するプロテアーゼの膜輸送システムの解明

    2005年

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受賞

  • 日本生化学会JB論文賞

    2004年  

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    受賞国:日本国

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共同研究・競争的資金等の研究

  • 難治性癌の細胞外小胞によるマクロファージハイジャックと転移ニッチの解明

    研究課題/領域番号:23H03310  2023年04月 - 2026年03月

    日本学術振興会  科学研究費助成事業  基盤研究(B)

    江口 傑徳, 河合 穂高, 高橋 賢, 冨樫 庸介, 岡元 邦彰

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    配分額:18720000円 ( 直接経費:14400000円 、 間接経費:4320000円 )

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  • 三次元腫瘍オルガノイド評価系により見出された新規癌転移抑制化合物の創薬展開

    研究課題/領域番号:20K09904  2020年04月 - 2023年03月

    日本学術振興会  科学研究費助成事業  基盤研究(C)

    十川 千春, 岡元 邦彰, 江口 傑徳, 河合 穂高, 青山 絵理子

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    配分額:4420000円 ( 直接経費:3400000円 、 間接経費:1020000円 )

    癌の悪性化、転移および再発を制御するには、分子機序の解明と有効な抗腫瘍薬の開発が不可欠である。近年、癌の悪性化には癌細胞を取りまく腫瘍微小環境が影響するといわれ、癌細胞のみならず腫瘍微小環境を制御する因子も標的分子の候補に入れた検討が必要である。本研究課題では、三次元腫瘍オルガノイド形成とMMP9発現をモニタリングすることによる、独自の多元薬物評価系によって見出したヒット化合物をもとに、新規癌転移抑制薬開発に向けた創薬展開を行うことを目的とする。
    令和3年度は、これまで得られたヒット化合物のさらなるブラッシュアップのため、腫瘍微小環境に対する薬剤の効果を評価可能な高次アッセイ系の構築を引き続き行った。エクソソームをはじめとする細胞外小胞(Extracellular Vesicles: EV)は腫瘍微小環境制御に関わるとされるが、免疫系細胞が分泌するEVの腫瘍細胞に対する影響について、昨年度確立した蛍光または発光モニタリングシステムを応用することにより検討した。マクロファージ様に分化させたTHP-1細胞から分泌されたEVをサイズ排除クロマトグラフィーを用いて粒子径により分画し、CD9陽性の大型EV(80-300 nm)とCD63/HSP90陽性小型EV(20-200 nm)を得た。蛍光標識したこれらのEVは口腔癌細胞HSC-3に効率的に取り込まれ、口腔癌細胞の生存率を大幅に低下させることが明らかとなった。
    以上のことから、マクロファージ様細胞におけるEVの分泌を促進する薬剤は抗がん作用を発揮する可能性が高く、腫瘍微小環境に対する薬剤の効果を検討する上で重要であると考えられた。

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  • MZF1誘導性の新規がん幹細胞ストレス耐性に関する検証

    研究課題/領域番号:17K11642  2017年04月 - 2021年03月

    日本学術振興会  科学研究費助成事業  基盤研究(C)

    江口 傑徳, 十川 千春, 奥舎 有加, 岡元 邦彰

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    配分額:4680000円 ( 直接経費:3600000円 、 間接経費:1080000円 )

    難治性癌は、細胞ストレスへの抵抗性を有す。本研究では(1)66種の細胞株、特に、転移性前立腺癌オルガノイドの癌幹細胞様性質と細胞外熱ショックタンパク質(eHSP)・エクソソーム分泌、(2)癌促進転写因子MZF1と腫瘍抑制転写因子SCAND1によるCDC37遺伝子の相反的制御機構、eHSP90・膜損傷エクソソーム含有ストレソーム、(3)転移性口腔癌細胞由来エクソソームのプロテオーム、eHSPのバイオマーカーとしての蓋然性、癌進展およびM2型マクロファージ等微小環境悪性化におけるエクソソーム機能、を解明し、(4)転移性癌治療戦略としてトリプルノックダウン法を開発した。

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  • 癌遺伝子プロモーター活性を指標とした新規スクリーニング系による既存薬再開発

    研究課題/領域番号:17K11643  2017年04月 - 2021年03月

    日本学術振興会  科学研究費助成事業  基盤研究(C)

    十川 千春, 江口 傑徳, 奥舎 有加, 大山 和美, 中野 敬介, 岡元 邦彰, 十川 紀夫

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    配分額:4550000円 ( 直接経費:3500000円 、 間接経費:1050000円 )

    本研究は、癌の転移および治療抵抗性を統合的に評価できる薬剤スクリーニングシステムの開発とそれを応用した既存薬再開発を目的とした。癌の浸潤・転移過程において高発現するMMP9遺伝子プロモーター活性を指標とし、転移性マウス大腸癌細胞を用いて、MMP9のプロモーター誘導性蛍光レポーター細胞を構築した。さらに生体内の腫瘍特性により近づけるために,三次元腫瘍オルガノイド(tumoroid)モニタリングを応用し、癌幹細胞性とMMP9レポーター活性とが同時に評価可能な薬物評価系を確立した。既存薬スクリーニングの結果、パーキンソン病治療薬であるベンツトロピンの抗腫瘍効果を見出した。

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  • 免疫系細胞の膜輸送を制御する新規分子の発見とその病態の解明

    研究課題/領域番号:17H04379  2017年04月 - 2020年03月

    日本学術振興会  科学研究費助成事業  基盤研究(B)

    筑波 隆幸, 坂井 詠子, 岡元 邦彰, 門脇 知子, 西下 一久

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    配分額:16900000円 ( 直接経費:13000000円 、 間接経費:3900000円 )

    我々は独自の研究から、破骨細胞の分化過程で増大する遺伝子Rab44を発見した。本研究では、Rab44の機能を特に免疫系細胞について明らかにした。まず分子メカニズムとして、Rab44欠損はリソソームからのカルシウム流入を増大させる事を見出した。次に組織分布を調べると、Rab44は骨髄細胞に高レベルで発現し、他の組織では低いレベルで発現することが分かった。さらにRab44遺伝子欠損マウスを作製し、アナフィラキシー反応を惹起すると同マウスは野生型マウスに比べ、アナフィラキシー症状が半分程度減少することが分かった。これらの結果から、Rab44はアレルギー反応を調節することが明らかとなった。

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  • フラボノイドをベースにした抗がん作用をもつサプリメントの開発

    2016年04月 - 2019年03月

    日本学術振興会科学研究費補助金  基盤研究 (C)(一般) 

    岡元 邦彰

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    担当区分:研究代表者  資金種別:競争的資金

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  • Keap1/Nrf2による骨代謝制御機構の解明とインシリコ創薬への応用

    研究課題/領域番号:16K11480  2016年04月 - 2019年03月

    日本学術振興会  科学研究費助成事業  基盤研究(C)

    坂井 詠子, 筑波 隆幸, 西下 一久, 岡元 邦彰

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    配分額:4810000円 ( 直接経費:3700000円 、 間接経費:1110000円 )

    研究代表者は酸化ストレスを減らすNrf2の活性化が破骨細胞分化を著しく抑制するだけでなく、骨芽細胞分化も阻害することを明らかにした。しかしながら予想に反して、骨格の顕著な異常は認められなかった。さらに、Keap1/Nrf2ダブルノックアウトマウスを作製し、Nrf2の恒常的な活性化がNFATc1抑制分子であるMafBの発現を上昇し、破骨細胞分化を負に制御していることを明らかにした。一方、1360種類の合成化合物および海洋微生物由来成分のうち、非常に低濃度で破骨細胞分化抑制効果を持つものや、あるいはNrf2を活性化するものを見出した。病態モデルマウスへの候補化合物の投与と病態の解析を行った。

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  • CD147シグナルを基盤とした関節リウマチの骨破壊分子機構

    研究課題/領域番号:16K08567  2016年04月 - 2019年03月

    日本学術振興会  科学研究費助成事業  基盤研究(C)

    西奥 剛, 岡元 邦彰, 筑波 隆幸

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    配分額:4680000円 ( 直接経費:3600000円 、 間接経費:1080000円 )

    関節リウマチにおいて発現増加するCD147が、破骨細胞のポドソームを介したアクチンリング形成を亢進させるという仮説について検討を行った。破骨細胞の分化において、CD147の発現は有意に増加し、細胞膜に局在していた。破骨細胞においてポドソームマーカーの発現とアクチンリングの形成を確認し、CD147はアクチンリングと共局在していた。アクチンリングによるシーリングに関与しているMT1-MMPの発現について検討し、破骨細胞において発現増加が見られた。しかし、CD147とMT1-MMTとの相互作用は確認できなかった。破骨細胞において発現増加するCD147はポドソームの形成に関与している可能性が示された。

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  • 歯周病細菌による肝臓内侵入・定着機構のバイオイメージングを用いた解析

    研究課題/領域番号:16K15790  2016年04月 - 2018年03月

    日本学術振興会  科学研究費助成事業  挑戦的萌芽研究

    筑波 隆幸, 坂井 詠子, 岡元 邦彰, 門脇 知子

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    配分額:3510000円 ( 直接経費:2700000円 、 間接経費:810000円 )

    歯周病細菌は口腔内の血流に乗って全身へ播種し、様々な全身疾患の憎悪に深く関与している。最近、歯周病細菌が非アルコール性脂肪性肝疾患の発症にも関与していることが報告されたが、詳細なメカニズムは不明である。本研究では、バイオイメージング解析により、歯周病菌が血流に乗ってどのように肝臓に定着・侵入して、どのような影響を及ぼすのかに関して、培養細胞を用いた変化を観察した。その結果歯周病細菌は主にオートファゴソームやリソソームに存在する事が明らかとなった。また細胞内に脂肪滴が存在すると同菌の感染初期での生存率が増大すること、さらに脂肪滴は菌排除のオートリソソーム形成に影響を及ぼすことも分かった。

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  • 破骨細胞におけるリソソーム分泌機構の制御因子の同定と分子メカニズムの解析

    研究課題/領域番号:16K11510  2016年04月 - 2018年03月

    日本学術振興会  科学研究費助成事業  基盤研究(C)

    西下 一久, 筑波 隆幸, 岡元 邦彰, 坂井 詠子

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    配分額:4810000円 ( 直接経費:3700000円 、 間接経費:1110000円 )

    1)マクロファージ及び破骨細胞Rab27A結合タンパク質の同定
    Rabタンパク質は通常、種々のエフェクタータンパク質と相互作用して働く。そのために結合タンパク質の同定は、rabタンパク質を理解するうえで必要不可欠である。さらに破骨細胞では、リソソームを分泌するという特徴があるにもかかわらず、破骨細胞におけるRab結合タンパク質はこれまで同定されていない。そこでマクロファージ及び破骨細胞におけるRab27A結合タンパク質を同定する方法として以下の方法を試みた。
    ①酵母ツーハイブリッドシステムを用いて相互作用する分子を分子生物学的に同定する方法。この方法は2つの対象タンパク質間の直接相互作用を試験するための、高感度な方法である。相互作用パートナーは、その後選択された酵母コロニーで対応するプラスミドをシークエンシングすることによって得ることができる。しかしこの方法では同定できなかった。
    ②Rab27A過剰発現細胞を用いて、Rab27Aプルダウンアッセイを行い、結合タンパク質をプロテオーム解析により同定する方法。恒常的に活性型であるドミナント・アクティブ型、恒常的に不活性型であるドミナント・ネガティブ型を発現させて、結合タンパク質の違いがあるかどうかを解析した。
    ③結合タンパク質がRab27Aのどの部位に結合するかを調べるために、部位特異的遺伝子変異体を用いて結合部位の同定を試みた。さらにこの実験系を用いて、結合するタンパク質を免疫沈降やプロテオーム解析により同定した。

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  • 中国に潜在する天然薬物の調査による新しい骨代謝治療薬の発掘

    研究課題/領域番号:15H05298  2015年04月 - 2018年03月

    日本学術振興会  科学研究費助成事業  基盤研究(B)

    筑波 隆幸, 坂井 詠子, 岡元 邦彰, 西下 一久, 胡 錦萍

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    配分額:15990000円 ( 直接経費:12300000円 、 間接経費:3690000円 )

    骨粗鬆症は高齢者の人口増加に伴い、日本での患者数は急増している。現在使用されている骨粗鬆症治療薬のビスフォスフォネートは顎骨壊死などの副作用が非常に強いため、毒性の少ない薬物の開発が急務である。本研究の目的は、天然物の宝庫であり長寿村として知られている中国・吉林省の天然薬物から、毒性の少ない骨代謝治療薬を発掘することである。種々の解析の結果、Rutaecarpine とDihydroartemisininが毒性が少なく、強い破骨細胞抑制効果を持つことが明らかとなった。さらにその詳細な分子メカニズムについても解析した。

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  • 骨吸収抑制効果を持つポリフェノールの画期的な同定システムの開発と応用

    研究課題/領域番号:26670906  2014年04月 - 2016年03月

    日本学術振興会  科学研究費助成事業  挑戦的萌芽研究

    筑波 隆幸, 岡元 邦彰, 坂井 詠子

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    配分額:3640000円 ( 直接経費:2800000円 、 間接経費:840000円 )

    骨の分解を行う破骨細胞の成熟過程には、ヘムオキシゲナーゼ1(HO-1)の発現減少が必須である。そのためにHO-1を発現誘導する抗酸化物を投与すると、破骨細胞の成熟・活性化を抑制できる事が考えられる。効率的にHO-1を誘導できる抗酸化物として、食物に含まれるポリフェノールが知られているが、未同定の物質が多く残されている。本研究では、破骨細胞を抑制できるポリフェノールを迅速かつ簡便に検出できるシステムを構築した。今後は同定されたポリフェノールに関して、骨吸収抑制メカニズムをさらに詳細に解明する。これらの結果を応用して、骨吸収を抑制するサプリメントや天然で副作用の少ない薬物の開発を試みる。

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  • 上皮性癌における細胞内プロテアーゼの新しい機能の解析

    研究課題/領域番号:25462920  2013年04月 - 2016年03月

    日本学術振興会  科学研究費助成事業  基盤研究(C)

    福間 裕, 筑波 隆幸, 岡元 邦彰, 西下 一久, 坂井 詠子

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    配分額:4940000円 ( 直接経費:3800000円 、 間接経費:1140000円 )

    これまで癌とプロテアーゼの研究は多くの報告がなされており、プロテアーゼは発癌を助長する「悪玉因子」として長い間認識されてきた。しかしながら、我々はエンドソーム・リソソーム性プロテアーゼであるカテプシンEが抗腫瘍効果を示す「善玉因子」として機能することを見出した。本研究ではカテプシンE欠損は乳腺細胞の癌化を促進することと、カテプシンE欠損による変動遺伝子群及びタンパク質の同定を試みた。その結果カテプシンE欠損乳腺細胞は正常細胞と比べてWnt5aタンパク質が著しく減少していた。従ってカテプシンE欠損により、Wnt-βカテニン経路のシグナルの異常が癌発症の原因となる可能性が示唆された。。

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  • Nrf2による破骨細胞制御機構の解明と分子標的に着目した天然物リガンドの探索

    研究課題/領域番号:25462892  2013年04月 - 2016年03月

    日本学術振興会  科学研究費助成事業  基盤研究(C)

    坂井 詠子, 筑波 隆幸, 岡元 邦彰, 西下 一久

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    配分額:5070000円 ( 直接経費:3900000円 、 間接経費:1170000円 )

    破骨細胞分化におけるNrf2/Keap1の役割を調べた。Keap1遺伝子欠損破骨細胞前駆細胞ではERK, p38MAPK, JNKのリン酸化阻害とNFATc1の発現抑制により破骨細胞分化が阻害された。一方、Nrf2遺伝子欠損破骨細胞前駆細胞では、ERK, p38 MAPK, JNKの活性化とNFATc1とc-Fosの核移行促進による破骨細胞分化促進が認められた。しかし大腿骨マイクロCTの結果や、大腿骨切片のヘマトキシリン・エオジン染色の結果からは、マウス間で顕著な差は認められなかった。さらにカフェストールとカウェオール、サンギンH-6と分解産物エラグ酸の破骨細胞分化阻害効果を比較した。

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  • 歯周病細菌の宿主細胞内輸送に注目した全身疾患発症メカニズムの解明

    研究課題/領域番号:25293383  2013年04月 - 2016年03月

    日本学術振興会  科学研究費助成事業  基盤研究(B)

    筑波 隆幸, 中山 浩次, 岡元 邦彰, 西下 一久, 坂井 詠子

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    配分額:17420000円 ( 直接経費:13400000円 、 間接経費:4020000円 )

    近年、歯周病細菌は非アルコール性脂肪性肝疾患(NAFLD)への関与が指摘されている。しかし同菌の肝細胞内における細胞内動態は明らかとなっていない。本研究ではヒト肝細胞株HepG2細胞を用いて脂肪肝モデルを作製し、歯周病細菌の細胞内動態を解析した。同菌をHepG2細胞に感染させると、同菌は細胞内の脂肪滴に存在しなかった。脂肪滴が存在するHepG2細胞では、感染後初期で細胞内での歯周病細菌の生存数が有意に多いことが明らかとなった。以上より、肝細胞内で脂肪滴が蓄積すると、歯周病細菌のオートファジー‐リソソームシステムによる排除を遅延させ、そのことがNAFLDの増悪因子となる可能性が示唆された。

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  • ステロイドによって誘発される膜タンパク質の細胞生物学的解析

    2012年04月 - 2015年03月

    日本学術振興会科学研究費補助金  基盤研究 (C)(一般) 

    岡元 邦彰

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    担当区分:研究代表者  資金種別:競争的資金

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  • プロテアーゼの機能異常による慢性炎症の発症機序の解明

    研究課題/領域番号:24592836  2012年04月 - 2015年03月

    日本学術振興会  科学研究費助成事業  基盤研究(C)

    西下 一久, 坂井 詠子, 岡元 邦彰, 筑波 隆幸, 福間 裕, 菅原 めぐみ

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    配分額:5330000円 ( 直接経費:4100000円 、 間接経費:1230000円 )

    アスパラギン酸プロテアーゼの比較遺伝学的解析と欠損マウス細胞の解析を行った。カテプシンE遺伝子を欠失している反芻動物等と、カテプシンEを発現している他の動物との生理学的相違点に着目すると、抗微生物ペプチド群の活性制御へのカテプシンEの関与が考えられた。生理的機能が明確でないナプシンでは、霊長類進化の過程で遺伝子重複、変異による酵素活性の減弱と偽遺伝子化が生じていると考えられた。カテプシンE欠損マウス由来マクロファージでオートファジーの低下と酸化ストレスの上昇が観察され、高脂肪食によって生じる欠損マウスの血中コレステロール高値と脂肪肝はマクロファージの白色脂肪への流入の減少との関連が示唆された。

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  • ライブイメージングによる歯周病細菌の心血管系組織定着機構の解明

    研究課題/領域番号:24659840  2012年04月 - 2014年03月

    日本学術振興会  科学研究費助成事業  挑戦的萌芽研究

    筑波 隆幸, 岡元 邦彰, 坂井 詠子

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    配分額:3770000円 ( 直接経費:2900000円 、 間接経費:870000円 )

    歯周病細菌であるP.gingivalisのライブイメージングについて、ヒト大動脈内皮細胞を用いて観察を行った。同菌は細胞内で6~8時間観察できたが、18時間から24時時間後には蛍光が検出できなくなった。この間にリソソームへ輸送され分解されたと考えられる。
    次に、マウスを用いてライブイメージングを行った。動脈分岐部や細い血管では、P.gingivalisが付着する様子が観察できた。さらに動脈硬化モデルであるApoE欠損マウスでは、P.gingivalisの血管への付着が増大したことから、血液中のコレステロールが細胞内への取り込みの際に、P.gingivalisと相互作用する可能性が推察された。

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  • 破骨細胞に関するカテプシンEの役割と創薬にむけた探求

    2011年04月 - 2012年03月

    日本学術振興会科学研究費補助金  基盤研究 (C)(一般) 

    岡元 邦彰

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    担当区分:研究代表者  資金種別:競争的資金

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  • 鉄を介した破骨細胞分化調節機構の分子メカニズムを解明する

    研究課題/領域番号:22592069  2010年 - 2012年

    日本学術振興会  科学研究費助成事業  基盤研究(C)

    坂井 詠子, 筑波 隆幸, 岡元 邦彰, 西下 一久

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    配分額:4550000円 ( 直接経費:3500000円 、 間接経費:1050000円 )

    ヘム分解酵素であるヘムオキシゲナーゼ1(HO-1) の発現低下がカスパーゼ3の活性化と HMGB1 の遊離を促進し破骨細胞分化を促進することを明らかにした。またHO-1の発現誘導作用のあるKahweolやFisetinなどのポリフェノールやtBHQが破骨細胞分化を抑制することを明らかにした。HO-1 の発現は転写因子Nrf2により調節されていることから、Nrf2ノックアウトマウスの骨髄細胞を用いて破骨細胞を形成させたところ、野生型マウスに比べ顕著な破骨細胞形成促進が見られた。

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  • 歯周病における上皮細胞の病態解析

    研究課題/領域番号:22592086  2010年 - 2012年

    日本学術振興会  科学研究費助成事業  基盤研究(C)

    福間 裕, 筑波 隆幸, 岡元 邦彰, 西下 一久, 坂井 詠子

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    配分額:4550000円 ( 直接経費:3500000円 、 間接経費:1050000円 )

    歯周病は、細菌感染による歯肉上皮細胞の破綻とそれに続く免疫担当細胞によって引き起こされる疾患である。しかし、上皮細胞の病態解析は免疫担当細胞の解析に比べ、殆ど行われていない。本研究では、ヒトのカテプシンCとLystと呼ばれるリソソーム機能に異常をきたすと、重度の歯周病を引き起こすことに着目した。カテプシンCノックアウトマウスおよびLyst遺伝子に変異があるベージュマウスを使用して、歯周病性細菌を感染させて、上皮細胞に関する生理学的・病理学的役割について解明を目指した。

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  • 骨疾患における創薬のターゲットとなりうるプロテアーゼの作用機序の解明

    2009年04月 - 2011年03月

    日本学術振興会科学研究費補助金  基盤研究 (C)(一般) 

    岡元 邦彰

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    担当区分:研究代表者  資金種別:競争的資金

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  • 破骨細胞におけるカテプシンEの役割と創薬にむけた基質分子の探究

    研究課題/領域番号:21592369  2009年 - 2011年

    日本学術振興会  科学研究費助成事業  基盤研究(C)

    岡元 邦彰, 筑波 隆幸, 西下 一久, 坂井 詠子

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    配分額:4680000円 ( 直接経費:3600000円 、 間接経費:1080000円 )

    破骨細胞への分化・活性化においてRANK(Receptor Activator of NF-κB)/ RANKL(RANK Ligand), OPG(Osteoprotegerin)の系とMacrophage colony stimulating factor(M-CSF)/ c-fmsの系は重要なシグナル伝達経路である。カテプシンE(CE)の欠損マウスは破骨細胞の形成において、野生型マウスと比較して有意な破骨細胞数の減少が認められたため、これらの経路を含めた解析を進めてきた。破骨細胞数の減少と一致して、リアルタイムPCRでは、M-CSFおよびRANKLの膜表面受容体c-fms、RANK、シグナル経路のc-fos、破骨細胞特異的マーカーであるカテプシンK、酒石酸耐性アルカリホスファターゼ、等には発現の減少傾向にはあるものの、統計学的に有意な差は認められなかった。また、破骨細胞前駆細胞の融合に関係しているといわれているDC-STAMPとOC-STAMPについても同様の結果であった。そこで、包括的に細胞内のmRNAの発現を調べるために、Microarray解析を行った。すると、いくつかの因子の上昇あるいは下降が認められたが、その中で、酸化ストレスに関する因子の上昇が認められた。そこで、RANKL添加前と添加後の過酸化水素水を測定したところ、CE欠損マウスにおいて過酸化水素水産生量の上昇が認められた。つまり、CE欠損マウスは何かの原因で酸化ストレスを受けている可能性が示唆される結果となった。また、オートファジー関連分子について解析を行ったところ、p62、LC3、ユビキチンが、細胞可溶画分から不溶画分へと移動した。この結果と、破骨細胞数の減少に酸化ストレスが関与している、という結果と考えあわせると、CE欠損マウスにおける破骨細胞数の減少はオートファジーが亢進し、破骨細胞のミトコンドリアにダメージが生じ、破骨細胞の形成に抑制が生じたことが示唆された。

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  • 破骨細胞におけるアスパラギン酸プロテアーゼの機能

    研究課題/領域番号:20592175  2008年 - 2010年

    日本学術振興会  科学研究費助成事業  基盤研究(C)

    西下 一久, 筑波 隆幸, 岡元 邦彰, 坂井 詠子

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    配分額:4680000円 ( 直接経費:3600000円 、 間接経費:1080000円 )

    酸性条件下で活性を発揮するアスパラギン酸プロテアーゼの破骨細胞分化における役割を探った。カテプシンE欠損マウス由来骨髄細胞における破骨細胞形成抑制はアポトーシス亢進により生じ、我々が以前報告したアスパラギン酸プロテアーゼ阻害剤による破骨細胞分化抑制時に観察されたRANKL下流のシグナリング抑制とは別の分子機構によることが示唆された。また、新規プロテアーゼであるナプシンの比較遺伝学的分析と生化学的解析を行い、破骨細胞分化への関与が示唆された。

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  • 歯周病原性細菌の産生するプロテアーゼの膜輸送システムの解明

    2007年04月 - 2009年03月

    日本学術振興会科学研究費補助金  基盤研究 (C)(一般) 

    岡元 邦彰

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    担当区分:研究代表者  資金種別:競争的資金

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  • 歯周病発症におけるオートファジー機能の解明と治療への応用

    研究課題/領域番号:21390496  2007年 - 2011年

    日本学術振興会  科学研究費助成事業  基盤研究(B)

    筑波 隆幸, 岡元 邦彰, 西下 一久, 坂井 詠子

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    配分額:18720000円 ( 直接経費:14400000円 、 間接経費:4320000円 )

    重度の歯周病を発症する遺伝子疾患の多くは、リソソーム関連遺伝子の機能異常である。しかし、その分子メカニズムは明らかになっていない。最近我々は、歯周病原性細菌であるPorphyromonas gingivalis(以下P. gingivalis)を細胞に感染させるとオートファジーが誘導され、排除される可能性があると想定している。本研究では、新しい生体防御システムとして明らかにされつつあるオートファジー機構について、特に歯科領域で重要な歯周病に関するメカニズムを解明することを目標にし、口腔疾患の新たな予防、診断、治療法の分子基盤の確立を目指す。

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  • 歯周病細菌アドヘジン蛋白とその内部領域ペプチドを利用する創薬にむけた基礎的研究

    研究課題/領域番号:19592151  2007年 - 2008年

    日本学術振興会  科学研究費助成事業  基盤研究(C)

    坂井 詠子, 中山 浩次, 岡元 邦彰, 内藤 真理子

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    配分額:3900000円 ( 直接経費:3000000円 、 間接経費:900000円 )

    歯周病原細菌Porphyromonas gingivalisのrgp A遺伝子産物であるHGP44のN末端アミノ酸を欠失させた変異蛋白を作成し、赤血球凝集活性を調べた。N末端20アミノ酸を欠失すると血球凝集活性が消失したため、この領域が活性中心である可能性が考えられた。シアル酸のはずれた糖蛋白と結合しやすく、また赤血球凝集の強さが動物種によって異なることからGalBl-4GlcNAcの糖鎖を介して糖蛋白と結合している可能性が示唆された。

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  • 歯周病原性細菌におけるタンパク質発現機構の確立

    2005年04月 - 2007年03月

    日本学術振興会科学研究費補助金  基盤研究 (C)(一般) 

    岡元 邦彰

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    担当区分:研究代表者  資金種別:競争的資金

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  • 歯周病に関わる因子における細胞生物学的研究

    2004年04月 - 2005年03月

    長崎大学  教育改善推進費 

    岡元 邦彰

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    担当区分:研究代表者  資金種別:競争的資金

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  • スフィンゴ糖脂質による破骨細胞の機能制御を解明する

    研究課題/領域番号:16591860  2004年 - 2005年

    日本学術振興会  科学研究費助成事業  基盤研究(C)

    坂井 詠子, 加藤 有三, 岡元 邦彰, 福本 敏

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    配分額:3500000円 ( 直接経費:3500000円 )

    細胞膜上には脂質ラフトと呼ばれるコレステロールとスフィンゴ糖脂質に富む場所が存在し、多くのシグナル分子が局在している。グラム陰性細菌の主要な構成成分であるリポポリサッカライド(LPS)は、骨吸収促進因子として知られており、TLR4やMyD88、TRAF6などの分子を介して破骨細胞を延命させる。本研究では、破骨細胞においてLPSを認識する自然免疫系のシグナル分子がラフトに存在するかを明らかにし、破骨細胞の延命とシグナル伝達におけるスフィンゴ糖脂質の役割を調べた。LPS存在下、破骨細胞はよく延命する。LPSと同時にGlcCer合成阻害剤であるPDMP添加により延命効果は阻害された。さらに、外からGM1などのスフィンゴ糖脂質を追加するとPDMPによる阻害効果はキャンセルされた。LPS刺激後経時的に破骨細胞を回収し、Triton不溶性画分をショ糖密度勾配超遠心とWestern blotting法を用いてシグナル分子の局在を解析した。刺激30分後には、ラフト分子であるGM1,Flotillin,SrcがTriton不溶性画分3に局在していた。またTLR4,CD14,MyD88,IRAK1/4,TRAF6などの自然免疫系のシグナル分子は、それまでばらばらに存在していたものが画分3に集積してくるのが確認された。興味深いことにPDMPを添加すると画分3への集積が見られなくなり、さらに外からGM1を追加すると、これら分子の画分3への集積が回復した。LPSはNF-κBを活性化させ破骨細胞を延命させる。LPS刺激30分後にはNF-κBp65subunitの核移行が蛍光免疫染色で観察された。さらにPDMP処理によりp65の核移行は抑制され、GM1の追加により核移行は回復した。以上より、破骨細胞の自然免疫システムにはラフトやスフィンゴ糖脂質が重要な働きをしていることが明らかとなった。

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  • 歯周病原性細菌におけるタンパク質の新規膜透過システムの解明

    研究課題/領域番号:16659516  2004年 - 2005年

    日本学術振興会  科学研究費助成事業  萌芽研究

    加藤 有三, 岡元 邦彰, 西下 一久

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    配分額:2800000円 ( 直接経費:2800000円 )

    歯周病原性細菌Porphyromonas gingivalis(P.g.)の産生する2つのシステインプロテアーゼRgpとKgpは歯周疾患の発症および進行に重要な役割をしていると考えられている。これら2つの酵素はプロテアーゼドメインと血球凝集素ドメインの2つの機能ドメインをもつ構造上類似した酵素である。我々は、前回P.g.内でのKgpの発現法を確立し、Kgpの活性中心は248番目と249番目のシステインが重要であることを明らかとした。今回はまず、非常に大きなC末端血球凝集素ドメインの役割を解析するために、C末端ドメインの長さの異なるプラスミドを作製した。野生型におけるKgpのC末端血球凝集素ドメインは3つのサブドメインに分けられるため、これらプラスミドを欠損変異体に発現させるKgpの長さはそれぞれのサブドメインを参考にした。昨年度のKgpの発現法をもとにP.g.内で安定に維持できるプラスミドpYKP028のKpnI部位に目的の長さのKgp遺伝子を挿入し、まず、大腸菌SM10λpirへ導入した。大腸菌での選別はアンピシリンで行った。これを接合伝達によりKgp欠損変異体へ導入した。ここでの選別はテトラサイクリンを用いた。現在、これらの変異体を解析中であるが、これらの変異体の中で、全遺伝子を含むプラスミドを導入した変異体は活性が非常に弱かった。もしかすると、C末端領域が非常に大きいため、発現効率が下がった可能性も考えられる。今後は、さらに構造解析を進め、血球凝集素ドメインのKgp輸送における役割、およびにプロドメインの役割についても解析を加えていきたい。

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  • 歯周病原性細菌の産生するプロテアーゼの高次構造と輸送機構に関する研究

    2003年04月 - 2005年03月

    日本学術振興会科学研究費補助金  基盤研究奨励研究A 

    岡元 邦彰

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    担当区分:研究代表者  資金種別:競争的資金

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  • 破骨細胞による骨吸収とアトピー性皮膚炎との因果関係における研究

    研究課題/領域番号:15390563  2003年 - 2005年

    日本学術振興会  科学研究費助成事業  基盤研究(B)

    加藤 有三, 岡元 邦彰, 西下 一久, 柴田 光枝, 坂井 詠子

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    配分額:14900000円 ( 直接経費:14900000円 )

    成熟型破骨細胞は、まず、血液幹細胞が、Macrophage-colony Stimulating Factor(M-CSF)の存在下、破骨細胞前駆細胞へと分化し、さらに、間葉系細胞からのReceptor Activator of NF-κB (RANK) Ligandが破骨細胞前駆細胞の膜表面に存在するRANKと結合することによって、シグナルが細胞内へ伝達され、細胞が互いに融合して多核の破骨細胞を形成する。その際、RANK ligandとそのおとり受容体としてのOsteoprotegerin(OPG)が破骨細胞への分化および活性化を制御している。本研究期間において、アスパラギン酸プロテアーゼの阻害剤であるペプスタチンAが破骨細胞形成を直接抑制していること、特に破骨細胞前駆細胞から単核の破骨細胞へ分化する段階を強く抑制していることが明らかとされた。この抑制は、カテプシンDの活性には依存していないことが示されたため、その作用メカニズムを調べるため、RANKの下流のシグナル分子のリン酸化の解析を行った。RANKL刺激によるIκB, Akt, p38のリン酸化には抑制は認められなかったが、ERKのリン酸化には抑制が認められた。また、最近破骨細胞の分化・活性化において、非常に注目を集めている分子nuclear factor of activated T cells c1(NFATc1)について解析を行った結果、タンパク質レベルでその発現が抑制されていることが明らかとなった。そこで、NFATc1のメッセージレベルでの解析をReal Time PCR法を用いて行ったところ、ペプスタチンAを作用させたものでは、有意にその発現量が減少していた。以上のことより、ペプスタチンAは破骨細胞分化・活性化を、ERKのリン酸化を抑制すること、NFATc1の発現量を抑制することにより抑制することが明らかとされた。

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  • 破骨細胞の多核化・活性化におけるアネキシンファミリーの機能解析

    研究課題/領域番号:14571765  2002年 - 2003年

    日本学術振興会  科学研究費助成事業  基盤研究(C)

    坂井 詠子, 柴田 光枝, 岡元 邦彰, 加藤 有三

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    配分額:3000000円 ( 直接経費:3000000円 )

    破骨細胞形成に関与する分子を明らかにする目的で、DNAイクロアレイの方法を用い、RANKL+M-CSF刺激で発現上昇してくるmRNAを調べたところ、アネキシン2,4,5が有意に上昇していた。確かに破骨細胞に、これらが蛋白として発現しているかを調べるため、特異抗体を用いて免疫染色を行った。アネキシン2,4,5全て単核破骨細胞にも多核破骨細胞にも存在し細胞全体に広く染色された。アネキシンの発現はM-CSF単独添加の場合でも認められ、特に破骨細胞特異的に発現しているわけではなかった。ある種の細胞ではアネキシンが細胞膜上の脂質ラフトに存在することが報告されている。破骨細胞における局在を調べるため、破骨細胞を1% Triton X-100で可溶化し、ショ糖密度勾配遠心法で分画を行った後、特異抗体でウェスタンブロッティングを行った。アネキシン2と4はTriton不溶性の軽い画分には存在せず、破骨細胞においてはラフト以外のところに局在したが、一方、RANKやc-Src, TRAF2,6などはラフトに局在した。ラフトを構成するスフィンゴ糖脂質の合成阻害剤D-PDMPを添加すると、RANKやc-Srcはラフトに局在できなくなり、それに伴い破骨細胞形成が顕著に抑制されたのに対し、ラフトに局在しないc-Fmsやアネキシンの局在は変化しなかった。外からスフィンゴ糖脂質を細胞培養に加えると、RANKやc-Srcはラフトに局在するようになり、それに伴い破骨細胞形成も回復するが、これらは脂質ラフトの形成が回復したためと思われる。成熟破骨細胞において特にRANKL/RANKを介したシグナル伝達は細胞膜上のラフトと呼ばれるミクロドメインで行われていることが本研究で明らかになったが、アネキシンは脂質ラフト以外のところに局在して何らかの機能を果たしていることが示唆された。

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  • 歯周病原性細菌における新規の膜結合型タンパク質の遺伝学的探求とその精製の試み

    研究課題/領域番号:14657479  2002年 - 2003年

    日本学術振興会  科学研究費助成事業  萌芽研究

    加藤 有三, 柴田 光枝, 岡元 邦彰

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    配分額:2700000円 ( 直接経費:2700000円 )

    歯周病原性細菌Porphyromonas gingivalis(P.g.)の産生する2つのシステインプロテアーゼRgpとKgpはプロテアーゼドメインと血球凝集素ドメインの2つの機能ドメインを持つ構造上類似した酵素である。プロテアーゼドメインの内膜から外膜への膜輸送機構にC末端側の血球凝集素ドメインが関与しているかどうかを調べるため、2つの酵素の内の一つKgpをKgp欠損変異体で発現させた。まず、Pg内で安定に存在するプラスミドpYKP028にKgp遺伝子を挿入し、Kgp欠損変異体KDP129へ導入した。このプラスミドにはテトラサイクリンの薬剤耐性遺伝子が含まれており、これにより変異体の選別を行った。発現の確認は合成基質を用いたKgp活性測定と抗体を用いたウエスタンブロット解析で行った。プロテアーゼの正確なC末端側が決定されていないため、分子量から換算したプロテアーゼドメインを導入した結果、その活性および発現は認められなかった。これが正確なC末端側ではなかったためかどうかは現在のところ不明であるが、血球凝集素ドメインの一部を含むコンストラクトではKgp活性および発現が認められたことから、プロテアーゼの発現に血球凝集素ドメインが必要である可能性が示唆された。この変異体はKgp欠損変異体が失った性質、すなわち黒色色素産生能などの回復も認められ、野生型とほぽ同等の性質を持つものと思われる。さらに、血球凝集素ドメインのどの部位が必須か等の解析を行う予定である。

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  • 歯周病制圧法の開発とその応用研究

    研究課題/領域番号:13357016  2001年 - 2004年

    日本学術振興会  科学研究費助成事業  基盤研究(A)

    山本 健二, 筑波 隆幸, 筑波 知子, 岡元 邦彰

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    配分額:50440000円 ( 直接経費:38800000円 、 間接経費:11640000円 )

    本研究では、両酵素群の遺伝子産物の大部分(80%以上)を占めると言われる菌体膜結合型酵素群の性状と機能を明らかにし、菌体外に分泌される可溶性単量体酵素群との相違点を明確にした。さらに、これまでの研究ならびに本研究を通じて明らかにされた両酵素群の機能を制御する目的で、それぞれの酵素に特異的な阻害剤の開発を行い、それらの歯周病における予防・治療への応用の可能性を探った。RgpおよびKgpは単量体として存在するばかりでなく、菌体表面で高分子複合体として存在することが報告されている。しかし、これらの膜結合複合体の分離精製の例はきわめて少なく、それらの構成や性状については未解明の部分が多く残されている。そこで筆者らは、膜結合複合体の分離精製を行い、その構造と性質を、それぞれの単量体分子と比較しながら、明らかにした。特記すべきは、膜結合型複合体は単量体分子に比べて、コラーゲンやエラスチンなどに対する分解活性が強く、宿主細胞に対する傷害作用強かった。さらに興味深いことは、膜結合型複合体はジンジバリス菌の宿主応答からの回避に強く寄与していることが示された。一方、両酵素に対する特異的な阻害剤はジンジバリス菌を不活性化し、歯周病の発症・進展を抑制することが期待されることから、両酵素に対する特異的阻害剤の開発を行った。Rgpに対する阻害剤KYT-1とKgpに対する阻害剤KYT-36はそれぞれに対して特異的であり、両者の併用によってジンジパリス菌の保有する病原性機能はほとんど抑制された。また、ジンジバリス菌の増殖・生存も両阻害剤の共存下で強く抑制されることがわかった。これらの結果は、RgpやKgpの遺伝子欠損変異株を用いた研究結果とよく一致しており、両酵素がジンジバリス菌のもつ生理的病理的機能の大部分を担っていることを強く示すとともに、KYT-1とKYT-36の併用によってジンジバリス菌のもつ病原性を強く阻害する可能性を強く示唆しており、今後歯周病やこれに関連する疾患の予防管理面に有効な手段となることが期待される。

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  • The role of proteinase in osteoclast formation and activation

    2001年

    Grant-in-Aid for Scientific Research 

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    資金種別:競争的資金

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  • 破骨細胞形成・活性化におけるプロテアーゼの役割

    2001年

    科学研究費補助金 

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    資金種別:競争的資金

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  • 神経系におけるオートファジー・リソソームシステム

    研究課題/領域番号:12146203  2000年 - 2004年

    日本学術振興会  科学研究費助成事業  特定領域研究

    山本 健二, 筑波 隆幸, 筑波 知子, 岡元 邦彰, 中西 博

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    配分額:31600000円 ( 直接経費:31600000円 )

    カテプシンEは抗原提示細胞やリンパ球、消化管上皮細胞等の免疫担当細胞に限局して存在していることから、生体防御系への関与が指摘されてきたが具体的な根拠は示されていない。そこで、本年度は、カテプシンEの生体防御系における役割を解明する目的で、カテプシンEノックアウトマウス(CatE-/-)由来のマクロファージの性状解析を行った。CatE^<-/->由来マクロファージでは、カテプシンDやBなどの可溶性リソソーム蛋白質の細胞外分泌が著明に亢進しているのに対し、それらとは生合成経路が異なるリソソーム膜糖蛋白質のLAMP-1,LAMP-2およびLGP85/LIMP-2に著明な蓄積が観察された。蛍光インディケーターを用いて酸性コンパートメントのpHを測定してみると、CatE-/-のpHは6.5となっており、正常細胞の5.3に比べて著しく上昇を示していた。これと関連して、endocytotic vesicle fusion eventsが障害されており、ファゴソーム膜蛋白質のNADPHオキシダーゼの活性増大が観察され、マクロファージが産生する活性酸素種の量も著明に増大していた。これと関連して、アネキシンIや酸化型ペロキシレドキシン6などの増加や還元型グルタチオンの減少などが観察され、CatE^<-/->由来マクロファージが強い酸化ストレスを受けていることが明らかにされた。カテプシンE欠損によるこうした変化はマクロファージのもつエンドリソソーム系の蛋白質分解能、殺菌能、細胞生存能などに著しい低下をもたらしていることが明らかにされ、カテプシンE欠損がマクロファージのもつ重要な種々の生理機能に著しい障害をもたらしているが示された。

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  • 歯周病におけるプロテアーゼ機能と生体反応に関する基礎的研究

    研究課題/領域番号:11307042  1999年 - 2001年

    日本学術振興会  科学研究費助成事業  基盤研究(A)

    山本 健二, 岡元 邦彰, 筑波 知子, 筑波 隆幸

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    配分額:36850000円 ( 直接経費:35200000円 、 間接経費:1650000円 )

    歯周病は歯槽骨を含む歯周組織の広範な破壊を特徴としており、宿主細胞ならびに口腔微生物由来のプロテアーゼの関与が指摘されている。本研究では、歯周病の主要な病原性細菌(ジンジバリス菌)の産生する2種類のシステインプロテアーゼRgpおよびKgpの生理的病理的機能、および宿主由来のアスパラギン酸プロテアーゼの新たな生理機能の解明を行った。本研究を通じて以下のことが明らかにされた。(1)RgpおよびKgpは、それぞれの遺伝子欠損変異体や特異的阻害剤を用いた解析から、ジンジバリス菌が歯周ポケット内で発育増殖するために不可欠なヘムやアミノ酸を獲得するために必須の成分であること、(2)RgpおよびKgpは、ジンジバリス菌が歯周ポケット内で発育増殖するための菌自身あるいは他菌との共凝集反応や宿主細胞や宿主蛋白質への接着反応に必須であること、(3)線維芽細胞や好中球、血管内皮細胞などの宿主細胞の傷害過程において中心的な役割を果たしていること、(4)宿主のリソソーム性アスパラギン酸プロテアーゼのカテプシンDの欠損は、失明と痙攣発作を誘発し、神経細胞内にオートファゴソームやオスミン酸親性の顆粒状沈殿物の蓄積を誘導し、神経性セロイド様リポフスチン蓄積症と類似した症状を発現さすこと、(5)カテプシンDの類似酵素カテプシンEは、マクロファージや脳内ミクログリアにおいて、外来抗原のプロセシング・提示機構で必須の役割を担っていること、(6)カテプシンEはインバリアント鎖のプロセシングには関与しないこと、等々である。これらの結果から、歯周病の発症進展において、RgpおよびKgpはジンジバリス菌の病原性因子の本体であることが実証され、新たな創薬ターゲットとなり得ることが示された。また、カテプシンDおよびカテプシンEが生体防御系において重要な機能を担っていることが明らかにされたことにより、これらの酵素の歯周病への関与が示唆され、これらの活性制御を目的とした創薬研究の重要性も示された。

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  • 遺伝子欠損変異体を用いた歯周病原性細菌の蛋白分解システムの解析

    研究課題/領域番号:11771140  1999年 - 2000年

    日本学術振興会  科学研究費助成事業  奨励研究(A)

    岡元 邦彰

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    配分額:2200000円 ( 直接経費:2200000円 )

    筆者らは、昨年よりP.gingivalisの産生する2つのシステインプロテアーゼ(RgpとKgp)の完全欠損変異体を用いて様々な解析を行い、これらの酵素の役割について明らかとしてきた。本年は、それら欠損変異体に加えて、活性部位指向型の合成ペプチドインヒビターを用いて、RgpとKgpの宿主細胞における影響について解析を行った。合成ペプチドインヒビターはRgp特異的なインヒビター(KYT-1)とKgp特異的インヒビター(KYT-36)を、口腔内における宿主由来の細胞としては歯肉線維芽細胞(Gin-1)を用いて、P.gingivalis野生株と各欠損変異体の培養上清の歯肉線維芽細胞における影響を調べた。まず、P.gingivalis野生株の培養上清と歯肉線維芽細胞とを培養したところ、計時的に歯肉線維芽細が紡錘形から円形に形態変化をおこしやがて浮遊してきた。RgpあるいはKgpが歯肉線維芽細の接着を破壊したものと考えられたため、次に、各欠損変異体の培養上清で同様の実験を行ったところ、Rgpの欠損変異体とRgp.Kgpの両酵素の欠損変異体において細胞の浮遊率の減少が認められた。すなわち、Rgpにより細胞の浮遊が惹起されている可能性が高いことが明らかとされた。ここで先に述べた合成ペプチドインヒビターを用いて同じ実験を行ったところ、KYT-1において歯肉線維芽細胞の浮遊化が抑制され、P.gingivalis培養上清未処理の細胞と同様な像を呈した。このことにより歯肉線維芽細胞の浮遊化にはRgpが関与していることが考えられた。そこで、細胞接着における因子のうちでRgpのターゲットとなる物質を同定するためにP.gingivalis処理と未処理の歯肉線維芽細胞でSDS電気泳動後CBB染色を行った。すると、およそ200KDaの部位に著しい違いを認めたため、この部位のN末端アミノ酸配列を調べたところ、フィブロネクチンであることがわかった。そこで、フィブロネクチンの抗体を用いてウエスタンブロット解析を行ったところ、明らかにフィブロネクチンの分解が生じていた。また、フィブロネクチンと細胞を接着しているインテグリンについても抗体を用いて解析を行ったところ、インテグリンについても一部のサブユニットがRgpのターゲットとなっていることが明らかとされた。以上のことより、P.gingivalisの培養上清の歯肉線維芽細胞への影響は、細胞接着因子と細胞外マトリックスをP.gingivalisの産生するプロテアーゼが破壊することにより細胞を浮遊化させ、さらには細胞死へと導くものと考えられた。これには、プロテアーゼ欠損変異体と合成インヒビターの実験よりKgpよりRgpが主に関与しているものと思われた。

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  • Study on transport of aspartic proteinase

    1993年

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    資金種別:競争的資金

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  • 歯周病原性細菌の産生するプロテアーゼに関する研究

    1993年

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    資金種別:競争的資金

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  • アスパラギン酸プロテアーゼの輸送機構に関する研究

    1993年

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    資金種別:競争的資金

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  • Study on proteinase from periodontal pathogen

    1993年

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    資金種別:競争的資金

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担当授業科目

  • ボランティア入門 (2023年度) 第4学期  - 金4,金5,金6,金7

  • 口腔機能再建学演習 (2023年度) 特別  - その他

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  • 薬理学各論1 (2023年度) 第1学期  - 水1,水2,水3

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  • 医療法学・社会福祉学 (2021年度) 特別  - その他

  • 口の機能と健康管理 (2021年度) 第2学期  - 木3~4

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  • 医療法学・社会福祉学 (2018年度) 特別  - その他

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社会貢献活動

  • 朝日医療大学校歯科衛生学科

    役割:講師

    2021年10月 - 現在

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  • 姫路歯科衛生専門学校

    役割:講師

    2018年12月 - 現在

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  • インターナショナル岡山歯科衛生士専門学校

    役割:講師

    2018年9月 - 現在

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  • 骨粗鬆症とその治療薬

    役割:講師

    岡山大学  2018年8月30日

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    種別:講演会

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  • 岡山大学歯学部紹介, 歯科薬理学講義, 歯科薬理学教室での研究内容

    役割:講師

    津山高等学校  2018年6月29日

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    種別:出前授業

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