Evidence that both 1 alpha,25-dihydroxyvitamin D-3 and 24-hydroxylated D-3 enhance human osteoblast differentiation and mineralization

Vitamin D playsa major role in the regulation of mineral homeostasis and affects bone metabolism. So far, detailed knowledge on the vitamin D endocrine system in human bone cells is limited. Here we investigated the direct effects of 1 alpha,25-(OH)(2)D-3 on osteoblast differentiation and mineralization. Also, we studied the impact of 24-hydroxylation, generally considered as the first step in the degradation pathway of vitamin D, as well as the role of the nuclear and presumed membrane vitamin D receptor (VDR). For this we used a human osteoblast cell line (SV-HFO) that has the potency to differentiate during culture forming a mineralized extracellular matrix in a 3-week period. Transcriptional analyses demonstrated that both 1 alpha,25-(OH)(2)D-3 and the 24-hydroxylated metabolites 24R,25-(OH)(2)D-3 and 1 alpha,24R,25-(OH)(3)D-3 induced gene transcription. All metabolites dose-dependently increased alkaline phosphatase (ALP) activity and osteocalcin (OC) production (protein and RNA), and directly enhanced mineralization. 1 alpha,24R,25-(OH)(3)D-3 stimulated ALP activity and OC production most potently, while for mineralization it was equipotent to 1 alpha,25(OH)(2)D-3. The nuclear VDR antagonist ZK159222 almost completely blocked the effects of all metabolites. Interestingly, 1 beta,25-(OH)(2)D-3, an inhibitor of membrane effects of 1 alpha,25-(OH)(2)D-3 in the intestine, induced gene transcription and increased ALP activity, OC expression and mineralization. In conclusion, not only 1 alpha,25-(OH)(2)D-3, but also the presumed 24-hydroxylated "degradation" products stimulate differentiation of human osteoblasts. 1 alpha,25-(OH)(2)D-3 aswell as the 24-hydroxylated metabolites directly enhance mineralization, with the nuclear VDR playing a central role. The intestinal antagonist 1 beta,25-(OH)(2)D-3 acts in bone as an agonist and directly stimulates mineralization in a nuclear VDR-dependent way