Vitamin A Is a Negative Regulator of Osteoblast Mineralization

Abstract

An excessive intake of vitamin A has been associated with an increased risk of fractures in humans. In animals, a high vitamin A intake leads to a reduction of long bone diameter and spontaneous fractures. Studies in rodents indicate that the bone thinning is due to increased periosteal bone resorption and reduced radial growth. Whether the latter is a consequence of direct effects on bone or indirect effects on appetite and general growth is unknown. In this study we therefore used pair-feeding and dynamic histomorphometry to investigate the direct effect of a high intake of vitamin A on bone formation in rats. Although there were no differences in body weight or femur length compared to controls, there was an approximately halved bone formation and mineral apposition rate at the femur diaphysis of rats fed vitamin A. To try to clarify the mechanism(s) behind this reduction, we treated primary human osteoblasts and a murine preosteoblastic cell line (MC3T3-E1) with the active metabolite of vitamin A; retinoic acid (RA), a retinoic acid receptor (RAR) antagonist (AGN194310), and a Cyp26 inhibitor (R115866) which blocks endogenous RA catabolism. We found that RA, via RARs, suppressed in vitro mineralization. This was independent of a negative effect on osteoblast proliferation. Alkaline phosphatase and bone gamma carboxyglutamate protein (Bglap, Osteocalcin) were drastically reduced in RA treated cells and RA also reduced the protein levels of Runx2 and Osterix, key transcription factors for progression to a mature osteoblast. Normal osteoblast differentiation involved up regulation of Cyp26b1, the major enzyme responsible for RA degradation, suggesting that a drop in RA signaling is required for osteogenesis analogous to what has been found for chondrogenesis. In addition, RA decreased Phex, an osteoblast/osteocyte protein necessary for mineralization. Taken together, our data indicate that vitamin A is a negative regulator of osteoblast mineralization.

Immediate effects of retinoic acid on gene expression in primary murine osteoblasts

“…Importantly, however, non-decalcified histology revealed that this dietary induction of hypervitaminosis A did not impair bone matrix mineralization, but caused specific alterations in bone cell differentiation, as the number of osteoclasts per bone surface was significantly increased by the high-retinol diet, while the number of osteoblasts was decreased. These data are in full agreement with a recently performed study where the authors supplemented the diet with even higher concentrations of retinol and observed a negative impact on the bone formation rate [48]. We next addressed the question of whether retinoic acid would directly affect gene expression in primary osteoblasts.…”

“…Collectively, our findings raise the possibility that the influence of retinoids on osteoblastogenesis and bone remodeling are attributable to a few specific changes in gene expression, and that the previously reported long-term effects on osteoblast proliferation and expression of differentiation markers have to be considered as secondary events [5,7,[9][10][11][12][13]48].…”

“…It was therefore conceivable to speculate that the effects of retinoids on osteoclasts are mediated indirectly, possibly through osteoblasts, representing the major source of the pro-osteoclastogenic cytokine Rankl and its decoy receptor Opg [45][46][47]. That osteoblasts respond to retinol has been shown in various studies, most of them demonstrating a negative effect of retinoids on proliferation and/or differentiation [9][10][11][12]48]. In contrast, it was also reported that RA promotes expression and activity of alkaline phosphatase in a mesenchymal cell line, and that this positive influence on osteoblast differentiation was enhanced by BMP signaling or over-expression of the transcriptional activators Runx2 and Msx2 [49].…”

“…We next addressed the question of whether retinoic acid would directly affect gene expression in primary osteoblasts. In fact, although it is well-established that ATRA and 9-cis-RA act as ligands of RAR/RXR transcription factors, most studies aiming at the identification of RA-responsive genes in osteoblasts have analyzed RAdependent expression of selected genes after long-term administration [5,7,[9][10][11]48]. Therefore, our major aim was to identify gene expression after short-term ATRA administration in an unbiased approach, i.e.…”

Osteoblast de- and redifferentiation is controlled by a dynamic response to retinoic acid during zebrafish fin regeneration

“…Retinoic acid (RA) has been shown to play multiple roles in bone development and repair by exerting pleiotropic effects on cells of the chondroblast, osteoblast and osteoclast lineages (Adams et al, 2007;Allen et al, 2002;Conaway et al, 2013;Dranse et al, 2011;Koyama et al, 1999;Laue et al, 2008Laue et al, , 2011Li et al, 2010;Lie and Moren, 2012;Lind et al, 2013;Nallamshetty et al, 2013;Song et al, 2005;Spoorendonk et al, 2008;Weston et al, 2003;Williams et al, 2009). Although partially conflicting results were reported, the general consensus for osteoblastogenesis is that RA signaling restricts osteoblast differentiation but promotes subsequent bone matrix synthesis by mature osteoblasts.…”