1,25 Dihydroxyvitamin D3 is required for growth-independent expression of alkaline phosphatase in cultured rat osteoblasts

Summary Osteoblastic cells were isolated from periosteum-stripped parietal bones of neonatal rat calvaria, seeded at low density (5,000 cells/35 mm of Falcon dish), and cultured for 6 days in BGJ medium supplemented with 20% of vitamin D-depleted FCS or vitamin D and calcium-depleted FCS, with daily addition of 1,25 dihydroxyvitamin D₃ (10⁻⁹ M) or 24,25-dihydroxyvitamin D₃ (10⁻⁹ M). Plating efficiency, clonal growth (number and size distribution of the colonies formed), and the alkaline phosphatase phenotype were evaluated on days 2 and 6 of culture. (1) Culture for 6 days in media not supplemented with 1,25(OH)₂D₃ led to a significant (P<0.001) loss of the alkaline phosphatase phenotype of the osteoblastic cells; the loss was greater in proliferating cells than in nonproliferating ones and occurred in both 0.12 mM or 1.1 mM ionized calcium concentrations. (2) Daily addition of 1,25(OH)₂D₃ (10⁻⁹ M) but not 24,25(OH)₂D₃ maintained the basal percentage of Alk Pase positive cell units in nonproliferating cells and significantly reduced the loss of this phenotype in proliferating colonies. (3) This effect did not stem from an action of the hormone on cell growth. 1,25(OH)₂D₃ was also found to enhance the adhesiveness of the seeded osteoblasts, irrespective of the medium calcium concentration.