The effects of vitamin D on osteoblast mineralization are well documented. Reports of the effects of vitamin D on osteoclasts, however, are conflicting, showing both inhibition and stimulation. Finding that resorbing osteoclasts in human bone express vitamin D receptor (VDR), we examined their response to different concentrations of 25-hydroxy vitamin D3 [25(OH)D3] (100 or 500 nmol·L−1) and 1,25-dihydroxy vitamin D3 [1,25(OH)2D3] (0.1 or 0.5 nmol·L−1) metabolites in cell cultures. Specifically, CD14+ monocytes were cultured in charcoal-stripped serum in the presence of receptor activator of nuclear factor kappa-B ligand (RANKL) and macrophage colony-stimulating factor (M-CSF). Tartrate-resistant acid phosphatase (TRAP) histochemical staining assays and dentine resorption analysis were used to identify the size and number of osteoclast cells, number of nuclei per cell and resorption activity. The expression of VDR was detected in human bone tissue (ex vivo) by immunohistochemistry and in vitro cell cultures by western blotting. Quantitative reverse transcription-PCR (qRT-PCR) was used to determine the level of expression of vitamin D-related genes in response to vitamin D metabolites. VDR-related genes during osteoclastogenesis, shown by qRT-PCR, was stimulated in response to 500 nmol·L−1 of 25(OH)D3 and 0.1–0.5 nmol·L−1 of 1,25(OH)2D3, upregulating cytochrome P450 family 27 subfamily B member 1 (CYP27B1) and cytochrome P450 family 24 subfamily A member 1 (CYP24A1). Osteoclast fusion transcripts transmembrane 7 subfamily member 4 (tm7sf4) and nuclear factor of activated T-cell cytoplasmic 1 (nfatc1) where downregulated in response to vitamin D metabolites. Osteoclast number and resorption activity were also increased. Both 25(OH)D3 and 1,25(OH)2D3 reduced osteoclast size and number when co-treated with RANKL and M-CSF. The evidence for VDR expression in resorbing osteoclasts in vivo and low-dose effects of 1,25(OH)2D3 on osteoclasts in vitro may therefore provide insight into the effects of clinical vitamin D treatments, further providing a counterpoint to the high-dose effects reported from in vitro experiments.
Dynamic regulation of the mitochondrial network by mitofusins (MFNs) modulates energy production, cell survival, and many intracellular signaling events, including calcium handling. However, the relative importance of specific mitochondrial functions and their dependence on MFNs vary greatly among cell types. Osteoclasts have many mitochondria, and increased mitochondrial biogenesis and oxidative phosphorylation enhance bone resorption, but little is known about the mitochondrial network or MFNs in osteoclasts. Because expression of each MFN isoform increases with osteoclastogenesis, we conditionally deleted MFN1 and MFN2 (double conditional KO (dcKO)) in murine osteoclast precursors, finding that this increased bone mass in young female mice and abolished osteoclast precursor differentiation into mature osteoclasts in vitro. Defective osteoclastogenesis was reversed by overexpression of MFN2 but not MFN1; therefore, we generated mice lacking only MFN2 in osteoclasts. MFN2-deficient female mice had increased bone mass at 1 year and resistance to Receptor Activator of NF-κB Ligand (RANKL)-induced osteolysis at 8 weeks. To explore whether MFN-mediated tethering or mitophagy is important for osteoclastogenesis, we overexpressed MFN2 variants defective in either function in dcKO precursors and found that, although mitophagy was dispensable for differentiation, tethering was required. Because the master osteoclastogenic transcriptional regulator nuclear factor of activated T cells 1 (NFATc1) is calcium-regulated, we assessed calcium release from the endoplasmic reticulum and store-operated calcium entry and found that the latter was blunted in dcKO cells. Restored osteoclast differentiation by expression of intact MFN2 or the mitophagy-defective variant was associated with normalization of store-operated calcium entry and NFATc1 levels, indicating that MFN2 controls mitochondrion–endoplasmic reticulum tethering in osteoclasts.
Alternative NF‐κB signaling promotes osteoclastogenesis and pathological bone loss, but the effect of sex on phenotype has not been explored. We disrupted alternative NF‐κB signaling by deletion of upstream kinase NF‐κB‐inducing kinase (NIK) or NF‐κB subunit RelB and found that both NIK‐deficient and RelB‐deficient female mice possessed more than twofold higher trabecular bone mass compared to controls, whereas no differences were observed in males. In vitro, RelB‐deficient precursors from female mice showed a more severe osteoclast (OC) differentiation defect than male, while WT had no sex bias. Next, we asked whether pharmacologic activation of alternative NF‐κB by inhibitor of apoptosis (IAP) antagonist BV6 has sex‐dependent effects on bone. Unlike male mice that lost bone, female mice on BV6 for 4 weeks showed no changes in either trabecular bone mass or OC number. Because estrogen generally suppresses NF‐κB, we hypothesized that estrogen protects bone from BV6 effects in vivo. Thus, we performed ovariectomy or sham surgery in female mice, then treated with BV6 or vehicle for 4 weeks. Although ovariectomy caused bone loss, BV6 did not have any additional impact, suggesting that direct estrogen effects do not cause resistance to BV6 in vivo. The osteopenic effects of IAP antagonists in males may have implications for their use in cancer therapy. © 2018 The Authors. JBMR Plus published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research.
Despite differences in the phamacokinetics of 25-hydroxycholecalciferol (25(OH)D3) and 25-hydroxyergocalciferol (25(OH)D2) in man, the effects of these and their 1α-hydroxylated forms (1,25(OH)2D3 and 1,25(OH)2D2) on cellular activity of vitamin D-responsive cells have hardly been compared. We studied differences in the effects of these metabolites on cell number, gene transcription, protein expression and mineralisation of cultured human bone marrow-derived stromal cells (hBMSC) and rapidly mineralising mouse 2T3 osteoblasts. 50–1000 nM 25(OH) and 0.05–10 nM 1,25(OH)2 metabolites were used. At high concentrations, 25(OH)D2/D3 and 1,25(OH)2D2/D3 suppressed cell number in both human and mouse cells. The suppression was greater with cholecalciferol (D3) metabolites than with those of ergocalciferol (D2). In both cell types, 25(OH)D2 and 25(OH)D3 increased the expression of osteopontin, osteocalcin, collagen-1, receptor activator of nuclear factor kappa-B ligand, vitamin D receptor, CYP24A1 and CYP27B1 genes. Whereas there was little or no difference between the effects of 25(OH)D2 and 25(OH)D3 in hBMSCs, differences were observed in the magnitude of the effects of these metabolites on the expression of most studied genes in 2T3 cells. Alkaline phosphatase (ALP) activity was increased by 25(OH)D2/D3 and 1,25(OH)2D2/D3 in hBMSC and 2T3 cells, and the increase was greater with the D3 metabolites at high concentrations. In hBMSCs, mineralisation was also increased by 25(OH)D2/D3 and 1,25(OH)2D2/D3 at high concentrations, with D3 metabolites exerting a greater influence. In 2T3 cells, the effects of these compounds on mineralisation were stimulatory at low concentrations and inhibitory when high concentrations were used. The suppression at high concentrations was greater with the D3 metabolites. These findings suggest that there are differences in the effects of 25-hydroxy and 1α,25(OH)2 metabolites of D3 and D2 on human preosteoblasts and mouse osteoblasts, with the D3 metabolites being more potent in suppressing cell number, increasing ALP activity and influencing mineralisation.
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