Vitamin A Is a Negative Regulator of Osteoblast Mineralization

Thomas Lind,Håkan Melhus,Gunnar Pejler,Göran Andersson,Anders Sundqvist,Annica Jacobson,Lijuan Hu,Luc Malaval

doi:10.1371/journal.pone.0082388

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.

Highlights

Excessive vitamin A intake is a risk factor for fracture in humans and the vitamin is the only known compound that can induce spontaneous fractures of long bones in animals [1,2,3,4]
A high dietary vitamin A intake leads to a reduced mineral apposition rate To control for indirect effects of vitamin A on appetite and general growth, we applied pair-feeding, i. e. the control rats were fed the same amount of food as that consumed by the vitamin A group
These results indicate that a high vitamin A intake has direct and inhibiting effects on bone formation

Summary

Introduction

Excessive vitamin A (retinol) intake is a risk factor for fracture in humans and the vitamin is the only known compound that can induce spontaneous fractures of long bones in animals [1,2,3,4]. Studies in rodents have shown that these spontaneous fractures are caused by a reduced bone diameter, whereas there is little or no effect on bone mineral density [5]. This bone thinning, in turn, appears to be caused by increased periosteal bone resorption and reduced diaphyseal radial growth [6,7]. CYP26B1 expression has been shown to be increased by RA and reduced by a pan-RAR antagonist, indicating that this gene is a direct target of RA [10,11,12]. Human null and hypomorphic mutations in this major regulator of RA concentration in osteoblastic cells, CYP26B1, lead to severe skeletal anomalies, demonstrating the importance of strict regulation of intracellular RA levels for human bone health [13]

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