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 microM) 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 microM. 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 microM) 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.
Letton
R W
RW
Division of Nephrology, Bone and Mineral Metabolism, University of Kentucky Medical Center, Lexington 40536.
Fanti
P
P
Malluche
H H
HH
D015650:24,25-Dihydroxyvitamin D 3 / Q000378:metabolism; D015652:25-Hydroxyvitamin D 2 / Q000378:metabolism; D000818:Animals; D003994:Bucladesine / Q000494:pharmacology; D002112:Calcifediol / Q000378:metabolism*; D000001:Calcimycin / Q000494:pharmacology; D002117:Calcitriol / Q000378:metabolism; D002478:Cells, Cultured; D005576:Colforsin / Q000494:pharmacology; D008401:Gas Chromatography-Mass Spectrometry; D007213:Indomethacin / Q000494:pharmacology; D010006:Osteoblasts / Q000378:metabolism*; D011453:Prostaglandins / Q000494:pharmacology; D051381:Rats
D011453:Prostaglandins; D005576:Colforsin; D015652:25-Hydroxyvitamin D 2; D000001:Calcimycin; D015650:24,25-Dihydroxyvitamin D 3; D003994:Bucladesine; D002117:Calcitriol; D002112:Calcifediol; D007213:Indomethacin