Primary Human Osteoblasts in Response to 25-Hydroxyvitamin D-3, 1,25-Dihydroxyvitamin D-3 and 24R, 25-Dihydroxyvitamin D-3

The most biologically active metabolite 1,25-dihydroxyvitamin D-3 (1,25(OH)(2)D-3) has well known direct effects on osteoblast growth and differentiation in vitro. The precursor 25-hydroxyvitamin D-3 (25(OH) D-3) can affect osteoblast function via conversion to 1,25(OH)(2)D-3, however, it is largely unknown whether 25(OH) D-3 can affect primary osteoblast function on its own. Furthermore, 25(OH) D-3 is not only converted to 1,25(OH)(2)D-3, but also to 24R, 25-dihydroxyvitamin D-3 (24R, 25(OH)(2)D-3) which may have bioactivity as well. Therefore we used a primary human osteoblast model to examine whether 25(OH) D-3 itself can affect osteoblast function using CYP27B1 silencing and to investigate whether 24R, 25(OH)(2)D-3 can affect osteoblast function. We showed that primary human osteoblasts responded to both 25(OH) D-3 and 1,25(OH)(2)D-3 by reducing their proliferation and enhancing their differentiation by the increase of alkaline phosphatase, osteocalcin and osteopontin expression. Osteoblasts expressed CYP27B1 and CYP24 and synthesized 1,25(OH)(2)D-3 and 24R, 25(OH)(2)D-3 dose-dependently. Silencing of CYP27B1 resulted in a decline of 1,25(OH)(2)D-3 synthesis, but we observed no significant differences in mRNA levels of differentiation markers in CYP27B1-silenced cells compared to control cells after treatment with 25(OH) D-3. We demonstrated that 24R, 25(OH)(2)D-3 increased mRNA levels of alkaline phosphatase, osteocalcin and osteopontin. In addition, 24R, 25(OH)(2)D-3 strongly increased CYP24 mRNA. In conclusion, the vitamin D metabolites 25(OH) D-3, 1,25(OH)(2)D-3 and 24R, 25(OH)(2)D-3 can affect osteoblast differentiation directly or indirectly. We showed that primary human osteoblasts not only respond to 1,25(OH)(2)D-3, but also to 24R, 25(OH)(2)D-3 by enhancing osteoblast differentiation. This suggests that 25(OH) D-3 can affect osteoblast differentiation via conversion to the active metabolite 1,25(OH)(2)D-3, but also via conversion to 24R, 25(OH)(2)D-3. Whether 25(OH) D-3 has direct actions on osteoblast function needs further investigation.