| Background:Vitamin D3is commonly used in the treatment of Osteoporosis. It is hydroxylated into active metabolites1,25(OH)2D3in the body, which plays multiple physiological function. It plays a key role in the regulation of bone metabolism.1,25(OH)2D3regulates gene expression in many tissues via binding to its corresponding intra-nuclear receptor, VDR, a member of the steroid hormone receptor superfamily. It can promote the intestinal absorption and the renal tubule reabsorption of calcium and phosphorus. On one hand, it can collaborate with parathyroid hormone to stimulate bone decalcification to improve blood calcium and phosphorus concentrations; On the other hand, it can also decrease the producing and releasing of parathyroid hormone, thus inhibiting the osteolysis effect of parathyroid hormone, which reduce the bone loss. But until now, the effects of1,25(OH)2D3on bone formation and osteoblast are still in controversy. Some studies reported that1,25(OH)2D3induced the proliferation and differentiation of osteoblast, and it directly up-regulated bone formation. While, others reported that1,25(OH)2D3inhibited the function of osteoblast and down-regulated bone formation. Thus, the detailed mechanisms of1,25(OH)2D3in bone formation need further study.Bone morphogenetic proteins (BMPs), a set of secretory multifunctional proteins, are belong to the transformation growth factor-β (TGF-β) superfamily except BMP1. It induced bone formation in vivo and is recognized as the strongest osteoinductive factor. BMP2, one of the most well characterized BMPs, is an osteogenic factor that stimulates osteoblast differentiation in vitro, as well as bone formation in vivo. It plays a central role in initiating and regulating bone formation, so any factors that regulate the expression of BMP2would be expected to influence bone formation.The interaction between1,25(OH)2D3and BMP2is not fully known, and further studies may help a lot to understand the effects of1,25(OH)2D3on bone formation.Genetic Hypercalciuric Stone-forming (GHS) rat is a good model to study human idiopathic hypercalciuria (IH), as both of them show increased intestinal Ca absorption, decreased renal Ca reabsorption and low bone mass. Previous studies have reported high levels of VDR in GHS rat can account for all of the changes in calcium metabolism that lead to hypercalciuria. But the pathogenesis of the low bone mass is incompletely known. This study aims to explore the relationship between VDR levels and BMP2expression to analyze the possible reasons of low bone mass in GHS rats. Further studies were done on the effects and mechanisms of1,25(OH)2D3on BMP2gene expression, which may help a lot to understand the effects1,25(OH)2D3on bone formation.Objectives:1. To compare vitamin D receptor (VDR) levels and BMP2mRNA expressions in the same tissues of GHS Rats and SD Rats, and analyze the potential relationship between them.2. To culture Bone marrow stromal cells (BMSCs) from GHS rats and SD rats, and UMR-106cell lines, and study the effects of1,25(OH)2D3on BMP2mRNA expression.3. To study the potential regulatory mechanism of1,25(OH)2D3on the BMP2gene expression.4. To investigate the effects of DNA methylation and histone modification in1,25(OH)2D3-induced transcriptional regulation of BMP2.Methods:1. Bone marrow was obtained from tibias and femurs of adult GHS rats and SD rats in aseptic condition. The BMSCs purified by red blood cell lysis buffer and direct adherent method, were cultured in vitro. Kidney and intestine were obtained in aseptic condition and were directly put into liquid nitrogen for rapid frozen. VDR levels in BMSCs, kidney and intestine from GHS and SD rats were determined by Western blot. BMP2mRNA expression in BMSCs, kidney and intestine from GHS and SD rats were determined by quantitative real time polymerase chain reaction (qRT-PCR).2.1,25(OH)2D3were used to treat BMSCs and UMR-106cells, and the changes of BMP2mRNA expression were determined by qRT-PCR.3. BMP2promoter regions were screened for putative VDR binding site. Corresponding primer pairs for detecting these loci were designed using Primer Premier5.0software.4. VDR binding to the BMP2promoter was identified by ChIP assay. The DNA fragments were subjected to PCR with the primer pairs designed in method3.5. DNA fragment containing VDR binding site in BMP2promoter was cloned to construct a luciferase vector. The activity of BMP2promoter was investigated by luciferase reporter assay.6. Treat BMSCs and UMR-106cells with the DNA methyltransferase inhibitor5-aza-2’-deoxycytidine (DAC) or together with1,25(OH)2D3, and investigate BMP2expression by qRT-PCR. Treat cells with histone deacetylase inhibitor trichostatin A (TSA) or together with1,25(OH)2D3, and investigate BMP2expression by qRT-PCR.7. Isolate genomic DNA from UMR-106cells incubated with1,25(OH)2D3or vehicle. Methylation status of CpG sites in the BMP2promoter region was examined by Bisulfite pyrosequencing.8. H3k9methylation status and H3acetylation status in the BMP2promoter region were examined using ChIP assay.Results:1. VDR levels are much higher in BMSCs, kidney and intestine from GHS rats compared to SD rats. While BMP2mRNA expressions are lower in BMSCs, kidney and intestine from GHS rats compared to SD rats.2. BMP2mRNA expression in BMSCs was significantly down-regulated in the presence of1,25(OH)2D3at6,12and24hr in SD and GHS rats. Using UMR-106cells cultured with1,25(OH)2D3over1-48hr created a time-dependent decrease in BMP2mRNA expression. Suppression of BMP2mRNA by1,25(OH)2D3was dose-dependent in BMSCs from SD and GHS rats, and UMR-106cells.3. The entire BMP2and its flanking regions (a total of28,545bp) were screened for putative VDR binding site and eight putative VDREs were identified. Seven primer pairs were designed to match the putative binding sites.4. Using the ChIP assay, significant binding of VDR to BMP2promoter region C was identified in cells incubated with1,25(OH)2D3. The other putative sites showed no binding to VDR.5. Activity of the Luciferase promoter of the constructed reporter gene reduced significantly comparing with the control. Following administration of1,25(OH)2D3, the Luciferase activity of the constructed reporter gene furtherly reduced significantly.6. DAC (0.5umol/L) upregulated BMP2expression in BMSCs from SD rats. Consistent up-regulation of BMP2expression occurred in BMSCs from GHS rats and UMR-106cells following DAC (0.5,1.0and2.0umol/L) administration. When incubated with1,25(OH)2D3, higher concentrations (1.0and2.0μM) of DAC induced BMP2expression in cells.7. Different concentrations of TSA (20,100,500nmol/L) up-regulated BMP2expression in BMSCs from SD rats and only20nM TSA increased BMP2expression in BMSCs from GHS rats. With1,25(OH)2D3, both100and500nM TSA up-regulated BMP2expression in cells.8. Bisulfite pyrosequencing demonstrate that one CpG site was completely (100%,25C/0T) methylated after incubation with1,25(OH)2D3. In the untreated control cell line, this CpG site was not methylated.9. ChIP assays demonstrate that administration with1,25(OH)2D3increased dimethylation of H3k9and decreased the acetylation of histone H3in the BMP2promoter region C in UMR-106cellsConclusions:1. VDR levels increase, while BMP2expressions decrease in the same tissues of GHS rats. 2.1,25(OH)2D3administration down-regulates BMP2mRNA expression in vitro.3.1,25(OH)2D3down-regulates BMP2gene transcriptional expression in osteoblast-like cells by binding to a VDR binding site in the BMP2promoter.4. Both DNA methylation and histone modification are involved in1,25(OH)2D3-induced transcriptional regulation of BMP2. |
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