Evidence that both 1α,25‐dihydroxyvitamin D₃ and 24‐hydroxylated D₃ enhance human osteoblast differentiation and mineralization

Abstract

Vitamin D plays a 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 1alpha,25-(OH)2D3 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 1alpha,25-(OH)2D3 and the 24-hydroxylated metabolites 24R,25-(OH)2D3 and 1alpha,24R,25-(OH)3D3 induced gene transcription. All metabolites dose-dependently increased alkaline phosphatase (ALP) activity and osteocalcin (OC) production (protein and RNA), and directly enhanced mineralization. 1Alpha,24R,25-(OH)3D3 stimulated ALP activity and OC production most potently, while for mineralization it was equipotent to 1alpha,25-(OH)2D3. The nuclear VDR antagonist ZK159222 almost completely blocked the effects of all metabolites. Interestingly, 1beta,25-(OH)2D3, an inhibitor of membrane effects of 1alpha,25-(OH)2D3 in the intestine, induced gene transcription and increased ALP activity, OC expression and mineralization. In conclusion, not only 1alpha,25-(OH)2D3, but also the presumed 24-hydroxylated "degradation" products stimulate differentiation of human osteoblasts. 1Alpha,25-(OH)2D3 as well as the 24-hydroxylated metabolites directly enhance mineralization, with the nuclear VDR playing a central role. The intestinal antagonist 1beta,25-(OH)2D3 acts in bone as an agonist and directly stimulates mineralization in a nuclear VDR-dependent way.

24,25-Dihydroxyvitamin D3 cooperates with a stable, fluoromethylene LPA receptor agonist to secure human (MG63) osteoblast maturation

“…The application of ZK159222 was able to prevent the large increase in ALP for FHBP and 24R,25D co-treated cells. Our findings therefore provide further evidence in support of a biological action of 24R,25D via the nuclear VDR, evidence echoing the earlier findings by van Driel and colleagues for human foetal osteoblasts [12] in that ZK159222 was able to neutralise the effects of 24R,25D for these cells. These findings share significant overlap with 1,25D and LCA [39] and are also in agreement with the research described by others that the additional, hydroxylated, vitamin D3 metabolites can evoke VDR-mediated responses in target cells.…”

“…Despite the wealth of literature reporting on the effects of 1,25D for human osteoblasts (hOBs) only a handful of studies which describe the actions of 24R,25D for these cells have been forthcoming [7][8][9][10][11][12]. What remains to be determined is whether 24R,25D can promote hOB maturation when co-administered with agents known to synergistically co-operate with 1,25D; it is becoming clear that 1,25D often needs to interact with other factors to prosecute the desired response in target cells [13].…”

Effects of Vitamin D, K1, and K2 Supplementation on Bone Formation by Osteoblasts In Vitro: A Meta-analysis

“…The non-directional test is more conservative than the CI test and is therefore more likely to result in nonsignificance when the sample size is small and the variance is large [40]. Simultaneously, the confidence interval test is more affected by outliers than the robust non-directional test [41] and could lead to nonsignificance when there are outliers present. Altogether, this could indicate why the results of both statistical tests did not agree with each other in every scenario.…”

Vitamin D in cancer chemoprevention

“…Recent studies suggest that the anti-apoptotic effects of 1,25(OH) 2 D 3 on osteoblasts and osteocytes are mediated by Src, PI3K, and JNK kinases (Gocek & Studzinski, 2009). The association of VDR with other proteins appears to be important in Vit D-induced osteoblast differentiation (Gocek & Studzinski, 2009;van Driel et al, 2006;Woeckel et al, 2013). 1,25(OH) 2 D 3 may also regulate keratinocytes differentiation by increasing intracellular calcium levels through the induction of the expression of calcium receptor (CaR) and phospholipase C (PLC) which are critical for calcium to stimulate keratinocyte differentiation (Bikle, 2012).…”