Differentiation of heterogeneous phenotypes in human osteoblast cultures in response to 1,25-dihydroxyvitamin D3

1,25(OH)2D3 (1,25-dihydroxyvitamin D3) inhibits the cell proliferation of human osteoblast-like cell cultures, but stimulates the synthesis of two of the phenotypic markers of the osteoblast, alkaline phosphatase and osteocalcin. It is not known whether all cells which synthesize alkaline phosphatase also synthesize osteocalcin in response to 1,25(OH)2D3. In this study we addressed this question by examining the response of human osteoblast-like cell cultures to 1,25(OH)2D3, using concurrent histochemical and immunochemical staining for alkaline phosphatase and osteocalcin, respectively. The cells were grown in the presence or absence of 1,25(OH)2D3 (10(-9) M) for 48 h. Co-localisation of osteocalcin and alkaline phosphatase in osteoblast-like cell cultures showed that not all cells which synthesize osteocalcin (about 9%) in response to 1,25(OH)2D3 synthesize alkaline phosphatase (about 24%) and vice versa. There was also a proportion of osteoblast-like cells which produce both osteocalcin and alkaline phosphatase simultaneously (about 12%). These findings suggest that during differentiation of bone-derived cells in cultures, in response to 1,25(OH)2D3, heterogeneous phenotypes with respect to expression of alkaline phosphatase and osteocalcin appear.