Abstract
The most biologically active metabolite 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) has well known direct effects on osteoblast growth and differentiation in vitro. The precursor 25-hydroxyvitamin D3 (25(OH)D3) can affect osteoblast function via conversion to 1,25(OH)2D3, however, it is largely unknown whether 25(OH)D3 can affect primary osteoblast function on its own. Furthermore, 25(OH)D3 is not only converted to 1,25(OH)2D3, but also to 24R,25-dihydroxyvitamin D3 (24R,25(OH)2D3) which may have bioactivity as well. Therefore we used a primary human osteoblast model to examine whether 25(OH)D3 itself can affect osteoblast function using CYP27B1 silencing and to investigate whether 24R,25(OH)2D3 can affect osteoblast function. We showed that primary human osteoblasts responded to both 25(OH)D3 and 1,25(OH)2D3 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)2D3 and 24R,25(OH)2D3 dose-dependently. Silencing of CYP27B1 resulted in a decline of 1,25(OH)2D3 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)D3. We demonstrated that 24R,25(OH)2D3 increased mRNA levels of alkaline phosphatase, osteocalcin and osteopontin. In addition, 24R,25(OH)2D3 strongly increased CYP24 mRNA. In conclusion, the vitamin D metabolites 25(OH)D3, 1,25(OH)2D3 and 24R,25(OH)2D3 can affect osteoblast differentiation directly or indirectly. We showed that primary human osteoblasts not only respond to 1,25(OH)2D3, but also to 24R,25(OH)2D3 by enhancing osteoblast differentiation. This suggests that 25(OH)D3 can affect osteoblast differentiation via conversion to the active metabolite 1,25(OH)2D3, but also via conversion to 24R,25(OH)2D3. Whether 25(OH)D3 has direct actions on osteoblast function needs further investigation.
Highlights
Vitamin D deficiency, a common condition in the elderly population, has been associated to numerous skeletal health problems
The metabolite 25(OH)D3 is metabolized in the kidney by the enzyme 1a-hydroxylase (CYP27B1) into the biologically most active metabolite 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) [3], which is the classical pathway for vitamin D activation
Primary human osteoblasts were cultured in the presence of 1,25(OH)2D3 or 25(OH)D3 for 6 days to compare the effects of these metabolites on the proliferation
Summary
Vitamin D deficiency, a common condition in the elderly population, has been associated to numerous skeletal health problems. The metabolite 25(OH)D3 is metabolized in the kidney by the enzyme 1a-hydroxylase (CYP27B1) into the biologically most active metabolite 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) [3], which is the classical pathway for vitamin D activation. Both 25(OH)D3 and 1,25(OH)2D3 are metabolized by the enzyme 24-hydroxylase (CYP24), responsible for the first step in the inactivation process, to respectively 24R,25-dihydroxyvitamin D3 (24R,25(OH)2D3). Alternative pathways for vitamin D activation have been described, and one of such is the CYP11A1-mediated pathway [5] This pathway for activation of vitamin D has been demonstrated in placentas ex utero, adrenal glands ex vivo and in cultured epidermal keratinocytes and colonic Caco-2 cells [6,7]. Hydroxyvitamin D derivatives synthesized by the action of CYP11A1 act on the vitamin D receptor (VDR), and on the retinoic acid related receptors a and c (RORa and RORc) [8]
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