Regulation of 25‐hydroxyvitamin D₃ metabolism in cultures of osteoblastic cells

This study was designed to investigate the mechanisms involved in the regulation of the conversion of 25‐hydroxyvitamin D₃ (25‐OHD₃) to 1,25‐dihydroxyvitamin D₃ [1,25‐(OH)₂D₃] and 24,25‐dihydroxyvitamin D₃ [24,25‐(OH)₂D₃] in primary cultures of osteoblastlike cells from neonatal mouse calvariae. These cells, when incubated with tritiated 25‐OHD₃ ([³H]25‐OHD₃), spontaneously synthesized [³H]24,25‐(OH)₂D₃ 20‐50 times more efficiently than [³H]1,25‐(OH)₂D₃ at a rate of conversion that was substrate dependent and linear from 1 to 36 h. Gas chromatography‐mass spectrometry verified the identity of the dihydroxylated metabolites. The calcium ionophore A23187 (5 μM) consistently stimulated the synthesis of 1,25‐(OH)₂D₃ while suppressing the production of 24,25‐(OH)₂D₃. This effect was sustained for 36 h and was dose dependent for concentrations from 0.05 to 10 μM. Furthermore, A23187 stimulated cAMP production and indomethacin (50 ng/ml) blocked the A23187‐induced production of cAMP and 1,25‐(OH)₂D₃ but had no effect on the suppression of 24,25‐(OH)₂D₃ by A23187. This led to other experiments to find out whether the stimulative effect of A23187 on 1,25‐(OH)₂D₃ synthesis is mediated by prostaglandins or cAMP, or both. PGE₂ (10⁻⁸‐10⁻⁶ M) increased the production of 1,25‐(OH)₂D₃ and of 24,25‐(OH)₂D₃. Forskolin (0.01‐10 μM) and dibutyryl cAMP (0.1‐10 mM) increased the production of both metabolites but to a lesser degree than PGE₂. These data suggest that osteoblastlike cells are stimulated by A23187 to increase the synthesis of 1,25‐(OH)₂D₃ through mechanisms involving prostaglandins and cAMP. The synthesis of 24,25‐(OH)₂D₃ is suppressed by A23187 through different mechanisms.