Abstract
Genetic hypercalciuric stone-forming (GHS) rats have increased intestinal Ca absorption, decreased renal tubule Ca reabsorption and low bone mass, all of which are mediated at least in part by elevated tissue levels of the vitamin D receptor (VDR). Both 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) and bone morphogenetic protein 2 (BMP2) are critical for normal maintenance of bone metabolism and bone formation, respectively. The complex nature of bone cell regulation suggests a potential interaction of these two important regulators in GHS rats. In the present study, BMP2 expression is suppressed by the VDR-1,25(OH)2D3 complex in Bone Marrow Stromal Cells (BMSCs) from GHS and SD rat and in UMR-106 cell line. We used chromatin immunoprecipitation (ChIP) assays to identify VDR binding to only one of several potential binding sites within the BMP2 promoter regions. This negative region also mediates suppressor reporter gene activity. The molecular mechanisms underlying the down-regulation of BMP2 by 1,25(OH)2D3 were studied in vitro in BMSCs and UMR-106 cells using the DNA methyltransferase inhibitor 5-aza-2′-deoxycytidine (DAC) and the histone deacetylase inhibitor trichostatin A (TSA). Both DAC and TSA activate BMP2 expression in combination with 1,25(OH)2D3. Bisulfite DNA pyrosequencing reveals 1,25(OH)2D3 to completely hypermethylate a single CpG site in the same BMP2 promoter region identified by the ChIP and reporter gene assays. ChIP assays also show that 1,25(OH)2D3 can increase the repressive histone mark H3K9me2 and reduce the acetylation of histone H3 at the same BMP2 promoter region. Taken together, our results indicate that 1,25(OH)2D3 binding to VDR down-regulates BMP2 gene expression in BMSCs and osteoblast-like UMR-106 cells by binding to the BMP2 promoter region. The mechanism of this 1,25(OH)2D3-induced transcriptional repression of BMP2 involves DNA methylation and histone modification. The study provides novel evidence that 1,25(OH)2D3 represses bone formation through down-regulating BMP2 expression both in vivo and in vitro.
Highlights
Hypercalciuria in both Genetic Hypercalciuric Stone-forming (GHS) rats and patients with idiopathic hypercalciuria (IH), arises from intestinal Ca hyperabsorption, increased bone resorption, and decreased renal tubule Ca reabsorption [1,2,3,4]
vitamin D receptor (VDR) Levels and bone morphogenetic protein 2 (BMP2) Expression in GHS and SD Rats VDR levels are higher in Bone Marrow Stromal Cells (BMSCs) from GHS rats compared to SD rats (Figure 1A)
Pathologic changes in calcium metabolism including increased calcium transport across intestine, bone and kidney which characterize IH are found in GHS rats [7,38,39]
Summary
Hypercalciuria in both Genetic Hypercalciuric Stone-forming (GHS) rats and patients with idiopathic hypercalciuria (IH), arises from intestinal Ca hyperabsorption, increased bone resorption, and decreased renal tubule Ca reabsorption [1,2,3,4]. Low bone mass is found in both GHS rats [5] and patients with IH [6,7], but the pathogenesis of the bone disease is incompletely known. BMP2 is essential for bone formation [12], and BMP2 gene expression is lower in bone cells, kidney and intestine of GHS rats. We hypothesize that VDR down-regulates BMP2 expression which in turn contributes to low bone mass in GHS rats
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