Faucheux C, Bareille R, Amedee J
INSERM U 443, Université Victor Ségalen Bordeaux 2, 146 rue Léo-Saignat, 33076 Bordeaux cedex, France.
Biochem J. 1998 Aug 1;333 ( Pt 3)(Pt 3):817-23. doi: 10.1042/bj3330817.
1alpha,25-Dihydroxyvitamin D3 [1,25(OH)2D3] is known to modulate Ca2+ metabolism in several cell types. Vitamin-D-dependent calcium binding proteins such as calbindin-D28K (28 kDa calcium binding proteins) have been shown to be regulated by 1,25(OH)2D3 but the mechanisms controlling calbindin synthesis are still poorly understood in human osteoblast cell culture models. The human bone marrow stromal cells (HBMSC) described in this paper developed a calcified matrix, expressed osteocalcin (OC), osteopontin (OP) and responded to 1,25(OH)2D3. The expression of vitamin D receptor mRNA was demonstrated by reverse transcription-PCR. Calbindin-D28K protein was identified only in cells arising from the sixth subculture, which exhibited a calcified matrix and all of the osteoblastic markers, e.g. OC and OP. It was demonstrated by dot-immunodetection using immunological probes, and by in situ hybridization using labelled cDNA probes. Moreover, vitamin D3 enhanced calbindin-D28K synthesis as well as OC synthesis and alkaline phosphatase activity. Uptake of 45Ca induced into the matrix by 1,25(OH)2D3 supports the hypothesis that the calcium-enriched matrix could trap calbindin-D proteins. In conclusion, the studies in vitro described in the present paper indicate, for the first time, a possible role of calbindin-D28K in mineralized matrix formation in HBMSC.
已知1α,25 - 二羟基维生素D3 [1,25(OH)2D3]可调节多种细胞类型中的钙代谢。维生素D依赖性钙结合蛋白，如钙结合蛋白-D28K（28 kDa钙结合蛋白），已被证明受1,25(OH)2D3调节，但在人成骨细胞培养模型中，控制钙结合蛋白合成的机制仍知之甚少。本文所述的人骨髓基质细胞（HBMSC）形成了钙化基质，表达骨钙素（OC）、骨桥蛋白（OP），并对1,25(OH)2D3有反应。通过逆转录 - PCR证明了维生素D受体mRNA的表达。仅在第六代传代产生的细胞中鉴定出钙结合蛋白-D28K蛋白，这些细胞表现出钙化基质和所有成骨细胞标志物，如OC和OP。通过使用免疫探针的斑点免疫检测和使用标记cDNA探针的原位杂交证明了这一点。此外，维生素D3增强了钙结合蛋白-D28K的合成以及OC的合成和碱性磷酸酶活性。1,25(OH)2D3诱导45Ca进入基质支持了富含钙的基质可能捕获钙结合蛋白-D蛋白的假说。总之，本文所述的体外研究首次表明钙结合蛋白-D28K在HBMSC矿化基质形成中可能发挥的作用。
