Molecular aspects of vitamin D metabolism and action

维生素 D 代谢和作用的分子方面

• 批准号: 04404072
• 负责人: SUDA Tatsuo
• 金额: $ 17.28万
• 依托单位: Showa University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (A)
• 财政年份: 1992
• 资助国家: 日本
• 起止时间: 1992 至 1993
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-04404072/
• 关键词: 1alpha, 25(OH)_2D_3; calcium absorption; bone resorption; osteoclast; osteoblast; gp130; interleukin 6; interleukin 11; 骨吸収; 副甲状腺ホルモン; インターロイキン6; インターロイキン11; ビタミンD; カルシウム; ビタミンDレセプター; 1α,25(OH)_2D_3; 24,25(OH)_2D_3; 骨

25-Hydroxyvitamin D_3 [25(OH)D_3] is metabolized primarily in the kidney to either 1alpha, 25(OH)_2D_3 [1alpha, 25(OH)_2D_3] or 24,25-dihydroxyvitamin D_3 [24,25(OH)_2D_3] by the mitochndrial enzymes 1alpha-hydroxylase and 24-hydrxylase. In order to investigate the regulation of vitamin D metabolism, we have partially purified the 25(OH)D_3 1alpha-hydroxylase from the kidney. The chick mitochondrial cytochrome P-450, 1alpha, 25(OH)_2D_3 24-hydroxylase was partially purified sequential polyethylene glycol precipitation, aminopentyl-Sepharose 4B, CM-cellulose, and hydroxyapatite chromatography. The turnover rate of the final preparation, when reconstituted with NADPH, adrenodoxin, and adrenodoxin reductase, was more than 4. The urified protein was finally aplied to SDS-PAGE under a reducing condition. The proteins on the gels were stained with Coomassie brilliant blue and transferred onto Immobilon by electroblotting, and their N-terminal sequences were determined using a protein sequenc … More er.It has been reported that soluble interleukin-6 receptor (sIL-6R) is detected in the serum of healthy individuals and its level is increased in patients with multiple myeloma and huma immunodeficiency virus infection. Although several reports have suggested that sIL-6R potentiates IL-6 action, its hysilogical role remains unclear. In this study, we examined the role of sIL-6R on osteoclast formation by IL-6, using a coculture of mouse osteoblasts and bone marrow cells. Neither recombinant muse IL-6 (mIL-6) nor mouse sIL-6R (smIL-6) induced osteoclast-like multinucleated cell (MNC) formation when they were added separately. In cntrast, simultaneous treatment with mIL-6 and smIL-6R strikingly induced MNC formation. These MNCs satisfied major criteria of authentic osteoclasts, such as tartrateresistant acid phosphatase (TRAP) activity, calcitonin receptors, and pit formation n dentine slices. The MNC formation induced by mIL-6 and smIL-6R was dose-dependently inhibited by adding mnoclonal anti-mouse IL-6R antibody. It is likely that osteoblasts and osteoclast progenitors are capable of transducing a signal from a complex of IL-6 and sIL-6R through gp130, even though they may have no or a very small number of IL-6Rs. Factors such as IL-11, oncotatin M, and leukemia inhibitory factor, which are known to exert their functons through gp130 (the signaltransducing chain of IL-6R), also induced MNC formation in our coculture system. These results suggest that increased circulating or locally produced SIL-6R induces osteoclast formation in the presence of IL-6 mediated by a mechanissm involving gp130. This may play an important physiological or pathological role in conditions associated with increased osteoclastic bone resorption. Less

25-羟基维生素 D_3 [25(OH)D_3] 主要在肾脏中通过线粒体酶代谢为 1alpha, 25(OH)_2D_3 [1alpha, 25(OH)_2D_3] 或 24,25-二羟基维生素 D_3 [24,25(OH)_2D_3] 1α-羟化酶和24-羟化酶。为了研究维生素D代谢的调节，我们从肾脏中部分纯化了25(OH)D_3 1α-羟化酶。鸡线粒体细胞色素 P-450, 1α, 25(OH)_2D_3 24-羟化酶通过顺序聚乙二醇沉淀、氨基戊基-Sepharose 4B、CM-纤维素和羟基磷灰石层析进行部分纯化。当用NADPH、肾上腺素还蛋白和肾上腺素还素还原酶重构时，最终制剂的周转率大于4。纯化的蛋白质最终在还原条件下应用于SDS-PAGE。凝胶上的蛋白质用考马斯亮蓝染色，并通过电印迹转移到 Immobilon 上，并使用蛋白质序列测定其 N 端序列……更多呃。据报道，在健康个体的血清中检测到可溶性白细胞介素 6 受体 (sIL-6R)，其水平在多发性骨髓瘤和人免疫缺陷病毒感染患者中升高。尽管一些报告表明 sIL-6R 增强 IL-6 的作用，但其生理学作用仍不清楚。在这项研究中，我们通过小鼠成骨细胞和骨髓细胞的共培养，研究了 sIL-6R 对 IL-6 破骨细胞形成的作用。重组 muse IL-6 (mIL-6) 和小鼠 sIL-6R (smIL-6) 单独添加时均不会诱导破骨细胞样多核细胞 (MNC) 形成。相反，同时用mIL-6和smIL-6R处理显着诱导MNC形成。这些跨国公司满足真正破骨细胞的主要标准，例如抗酒石酸酸性磷酸酶（TRAP）活性、降钙素受体和牙本质切片中的小凹形成。通过添加单克隆抗小鼠IL-6R抗体，mIL-6和smIL-6R诱导的MNC形成受到剂量依赖性抑制。成骨细胞和破骨细胞祖细胞很可能能够通过 gp130 转导来自 IL-6 和 sIL-6R 复合物的信号，即使它们可能没有或具有极少量的 IL-6R。 IL-11、Oncotatin M 和白血病抑制因子等因子已知通过 gp130（IL-6R 的信号转导链）发挥其功能，也在我们的共培养系统中诱导 MNC 形成。这些结果表明，循环或局部产生的 SIL-6R 增加会在 IL-6 存在的情况下诱导破骨细胞形成，这是由涉及 gp130 的机制介导的。这可能在与破骨细胞骨吸收增加相关的病症中发挥重要的生理或病理作用。较少的

项目成果

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TAMURA, T.et al.：“胰淀素在骨中的作用机制。”

Jin,C.H.et al.: "1alpha,25-Dihydroxyvitamin D_3 regulation in vivoproduction of the third component of complement (C3) in bone." Endocrinology. 131. 2468-2475 (1992)

Jin,C.H.等人：“1α,25-二羟基维生素 D_3 调节骨中补体第三种成分 (C3) 的体内生成。”

Kozono,T.et al.: "A chimeric analog of human and salmon calcitonin eliminates antigenicity and reduces gastrointestinal disturbances." Endocrinology. 131. 2885-2890 (1992)

Kozono,T.等人：“人类和鲑鱼降钙素的嵌合类似物消除了抗原性并减少胃肠道紊乱。”

Suda. T.: "Role of vitamin D in bone resorption." J. Cell. Biochem.49. 53-58 (1992)

苏达。

Tamura,T.et al.: "A new resorption assay with mouse osteoclast-like multinucleated cells formed in vitro." J.Bone Miner.Res.8. 953-960 (1993)

Tamura,T.等人：“一种利用体外形成的小鼠破骨细胞样多核细胞进行的新吸收测定。”