Synthesis of calbindin-D28K during mineralization in human bone marrow stromal cells

1α,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.