Study of the role of TRAF6 on differentiation and activation of the osteoclast.

TRAF6对破骨细胞分化和活化作用的研究。

• 批准号: 13470304
• 负责人: OHNISHI Isao
• 金额: $ 6.02万
• 依托单位: The University of Tokyo
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 2001
• 资助国家: 日本
• 起止时间: 2001 至 2003
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-13470304/
• 关键词: Osteoclast; RANKL; TRAFG; IL-1; IL-1

Signals emanating from the receptor for interleukin-1 (IL-1) and receptor activator of NF kappa B ligand (RANKL) stimulate transcription factors AP-1 through mitogen-activated protein kinase (MAPK) activation and NF kappa B through I kappa B kinase (IKK) activation. These kinases are thought to be activated by tumor necrosis factor receptor-associated factor 6 (TRAF6). However, molecular mechanisms by which TRAFG activates various downstream kinases remain to be elucidated. We identified functional domains of TRAF6 under physiological conditions established by appropriate expression of TRAF6 mutants in TRAF6-deficient cells. The RING finger domain is not required for differentiation of splenocytes to multinuclear osteoclasts, but is essential for osteoclast maturation. Thus, TRAF6 plays essential roles in both the differentiation and maturation of osteoclasts by activating various kinases via its multiple domains. We next examined the relationship between IL-1/TRAF6-dependent and c-Src-mediated pathways in the activation of osteoclast-like cells (prefusion cells (pOCs); multinucleated cells) formed in the murine eoculture system. In normal pOCs treated with IL-1, anti-TRAF6 antibodies coprecipitate p130(Cas), protein tyrosine kinase 2, and c-Src. In Src-deficient pOCs, this molecular complex was not detected, suggesting that c-Src is required for formation of the TRAFG, p130(Cas), and protein tyrosine kinase 2 complex. Moreover, an immunocytochemical analysis revealed that in osteoclast-Like multinucleated cells, IL-1 induced redistribution of TRAF6 to actin ring structures formed at the cell periphery, where TRAF6 also colocalized with c-Src. Taken together, these data suggest that IL-I signals feed into the tyrosine kinase pathways through a TRAF6-Src molecular complex, which regulates the cytoskeletal reorganization essential for osteoclast activation.

从受体发出的白介素-1（IL-1）和NF Kappa B配体（RANKL）受体激活剂发出的信号通过丝裂原激活的蛋白激酶（MAPK）激活刺激转录因子AP-1，而NF Kappa B通过I KAPPA B KINase（IKK）激活。这些激酶被认为是通过肿瘤坏死因子受体相关因子6（TRAF6）激活的。然而，TRAFG激活各种下游激酶的分子机制仍有待阐明。我们在TRAF6缺陷型细胞中适当表达TRAF6突变体确定的生理条件下确定了TRAF6的功能结构域。将脾细胞分化为多核破骨细胞的环状不是必需的，但对于破骨细胞的成熟至关重要。因此，TRAF6通过通过其多个域激活各种激酶来激活整骨细胞的分化和成熟。接下来，我们研究了在鼠电子培养系统中形成的骨细胞样细胞（预灌注细胞（POC）；多核细胞）激活中IL-1/TRAF6依赖性和C-SRC介导的途径之间的关系。在用IL-1处理的正常POC中，抗TRAF6抗体共沉淀P130（CAS），蛋白酪氨酸激酶2和C-SRC。在缺乏SRC的POC中，未检测到该分子复合物，这表明C-SRC是形成TRAFG，P130（CAS）和蛋白酪氨酸激酶2复合物所必需的。此外，一种免疫细胞化学分析表明，在破骨细胞样的多核细胞中，IL-1诱导TRAF6的重新分布到细胞周围形成的肌动蛋白环结构，其中TRAF6也与C-SRC共定位。综上所述，这些数据表明IL-I信号通过TRAF6-SRC分子复合物进食酪氨酸激酶途径，该复合物调节了整骨细胞激活必不可少的细胞骨架重组。

项目成果

Yamamoto A, Fukuda A, Seto H, Miyazaki T, Kadono Y, Sawada Y, Nakamura I, Katagiri H, Asano T, Tanaka Y, Oda H, Nakamura K, Tanaka S: "Suppression of arthritic bone destruction by adenovirus-mediated dominant negative ras gene transfer to synoviocytes and

Yamamoto A、Fukuda A、Seto H、Miyazaki T、Kadono Y、Sawada Y、Nakamura I、Katagiri H、Asano T、Tanaka Y、Oda H、Nakamura K、Tanaka S：“腺病毒介导的显性抑制关节炎骨破坏

Tanaka, S., Nakamura, I., Inoue, J., Oda, H., Nakamura, K.: "Signal transduction pathways regulating osteoclast differentiation and function."J Bone Miner Metab.. 21. 123-133 (2003)

Tanaka, S.、Nakamura, I.、Inoue, J.、Oda, H.、Nakamura, K.：“调节破骨细胞分化和功能的信号转导途径。”J Bone Miner Metab.. 21. 123-133 (2003)

Akiyama T, Bouillet P, Miyazaki T, Kadono Y, Chikuda H, Chung U, Fukuda A, Hikita A, Seto H, Okada T, Inaba T, Sanjay A, Baron R, Kawaguchi H, Oda H, Nakamura K, Strasser A, Tanaka S.: "Regulation of Osteoclast Apoptosis by Ubiquitination of Proapoptotic

Akiyama T、Bouillet P、Miyazaki T、Kadono Y、Chikuda H、Chung U、Fukuda A、Hikita A、Seto H、Okada T、Inaba T、Sanjay A、Baron R、Kawaguchi H、Oda H、Nakamura K、Strasser A

Kobayashi, N., Kadono, Y., Naito, A., Matsumoto, K., Yamamoto, T., Tanaka, S., Inoue, J.: "Segregation of TRAF6-mediated signaling pathways clarifies its role in osteoclastogenesis."EMBO J. 11. 1271-1280 (2001)

Kobayashi, N.、Kadono, Y.、Naito, A.、Matsumoto, K.、Yamamoto, T.、Tanaka, S.、Inoue, J.：“TRAF6 介导的信号通路的分离阐明了其在破骨细胞生成中的作用。”

Kobayashi, N., Kadono, Y., Naito, A., Matsumoto, K., Yamamoto, T., Tanaka, S., Inoue, J: "Segregation of TRAF6-mediated signaling pathways clarifies its role in osteoclastogenesis."Embo J. 20. 1271-1280 (2001)

Kobayashi, N.、Kadono, Y.、Naito, A.、Matsumoto, K.、Yamamoto, T.、Tanaka, S.、Inoue, J：“TRAF6 介导的信号通路的分离阐明了其在破骨细胞生成中的作用。”Embo