Molecular aspects of vitamin D metabolism and action

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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