Molecular aspects of vitamin D metabolism and action

25-羟基维生素D_3[25(OH)D_3]主要在肾脏中被线粒体酶1α-羟基酶和24-羟基酶代谢为1α，25(OH)_2D_3[1α，25(OH)_2D_3]或24，25-二羟基维生素D_3[24，25(OH)_2D_3]。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)D31α-羟基酶。鸡线粒体细胞色素P-450，1α，25(OH)_2D_3 24-羟基酶经聚乙二醇沉淀、氨基戊基-Sepharose4B、CM-纤维素柱层析和羟基磷灰石层析得到部分纯化。最终产物经NADPH、肾上腺素和肾上腺素还原酶重组后，转化率大于4。纯化后的蛋白在还原条件下进行SDS-PAGE分析。凝胶上的蛋白质用考马斯亮蓝染色，通过电印迹转移到固定化蛋白上，并用蛋白质序列…测定其N末端序列此外，已有报道在健康人血清中检测到可溶性白介素6受体(sIL-6R)，在多发性骨髓瘤和人类免疫缺陷病毒感染患者中sIL-6R水平升高。尽管一些报道表明sIL-6R增强了IL-6的作用，但其生理作用尚不清楚。在这项研究中，我们利用小鼠成骨细胞和骨髓细胞的共同培养，研究了sIL-6R在IL-6诱导的破骨细胞形成中的作用。重组小鼠IL-6(mIL-6)和小鼠sIL-6R(sIL-6)单独作用均不能诱导破骨细胞样多核细胞(MNC)的形成。相反，mIL-6和SMIL-6R联合作用可显著诱导MNC的形成。这些MNC符合真实破骨细胞的主要标准，如抗酒石酸酸性磷酸酶(TRAP)活性、降钙素受体和牙本质切片上的凹坑形成。加入抗小鼠IL-6R单抗可剂量依赖性地抑制mIL-6和SMIL-6R诱导的MNC形成。成骨细胞和破骨细胞前体细胞可能能够通过gp130传递IL-6和sIL-6R复合体的信号，即使它们可能没有IL-6Rs或数量很少。IL-11、抑癌素M和白血病抑制因子等已知通过IL-6R的信号转导链gp130发挥作用的因子也在我们的共培养体系中诱导了单核细胞的形成。这些结果表明，增加循环或局部产生的sIL-6R在IL-6存在的情况下诱导破骨细胞的形成，这是由涉及gp130的机制介导的。这可能在与破骨细胞性骨吸收增加相关的条件下发挥重要的生理或病理作用。较少

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