1α，25-二羟维生素D3对BMP2基因表达的调控及其机制研究
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摘要
研究背景
维生素D3是目前临床上治疗骨质疏松的常用药物,它在体内通过羟化生成活性代谢产物1,25(OH)2D3(1a,25-dihydroxyvitamin D3)而发挥生理作用。1,25(OH)2D3是一种具有多种生物效应的激素前体,其主要是通过与靶细胞内的维生素D受体(Vitamin D receptor, VDR)结合后调节各种基因的表达,从而发挥其生理效应。它具有促进肠道钙和磷的吸收,促进肾小管对钙、磷的重吸收的作用。它一方面可以协同甲状旁腺激素刺激骨组织脱钙,提高血钙、磷浓度；另一方面也可以抑制甲状旁腺激素的合成释放,从而抑制甲状旁腺激素的骨溶解作用,使骨丢失减少。但是目前关于1,25(OH)2D3对骨形成及成骨细胞作用的报道仍很不一致。部分研究认为1,25(OH)2D3可诱导成骨细胞的分化成熟,对骨组织的形成有直接的促进作用。而另外一些研究则认为1,25(OH)2D3对成骨细胞有明显的抑制作用,直接抑制骨形成。因此,1,25(OH)2D3对骨形成的作用及机制仍需要进一步的研究。
骨形态发生蛋白(Bone morphogenetic protein, BMP)是一组分泌性的多功能蛋白质,除BMP1外均属于TGF-β(转化生长因子-p)超家族成员,在体内可以通过旁分泌和自分泌的形式诱导骨的形成,是目前公认最强的骨诱导因子。BMP2是其中研究和应用最广泛的成骨生长因子之一,它可以在体外诱导间充质干细胞向成骨细胞分化,体内诱导骨形成。BMP2在启动和调节骨形成中起着关键的作用,因此任何影响BMP2基因表达的因素都可能会影响骨形成。
若能阐明1,25(OH)2D3与BMP2基因表达的关系,对进一步理解1,25(OH)2D3在骨形成中的作用会有很大的帮助,目前文献中尚未有此类报道。
遗传性高钙尿(Genetic hypercalciuric stone-forming, GHS)大鼠是研究特发性高钙尿(Idiopathic hypercalciuria, IH)患者的理想动物模型,它们都表现为小肠的钙吸收增加,肾脏的尿钙重吸收减少,骨密度减低。既往的研究表明GHS大鼠中肠钙吸收的增加,尿钙重吸收的减少与组织中VDR水平的增加有很大的关系。但是GHS大鼠中骨密度减低的原因仍不是十分清楚。本研究旨在通过探寻GHS大鼠中VDR与BMP2的关系来分析这种动物模型中骨密度减低的可能原因,并进一步探讨1,25(OH)2D3对BMP2基因表达的调控及可能的机制,从而进一步分析1,25(OH)2D3在骨形成中的作用及机制。
目的：
1、研究GHS大鼠和正常SD大鼠相同组织中维生素D受体蛋白和BMP2mRNA的基础表达水平并进行比较,分析VDR和BMP2可能存在的联系。
2、体外培养GHS大鼠和SD大鼠的骨髓基质干细胞,以及UMR-106细胞,研究1,25(OH)2D3对BMP2mRNA表达的影响。
3、研究1,25(OH)2D3调控BMP2基因转录表达的作用机制。
4、探讨DNA甲基化和组蛋白修饰在1,25(OH)2D3调控BMP2基因表达中的作用。
方法：
1、选取成年GHS大鼠和SD大鼠,断颈处死,无菌条件下取出股骨和胫骨,抽取骨髓,应用红细胞裂解法及直接贴壁法获取并体外培养骨髓基质干细胞,无菌条件下取出肾脏及小肠,置入液氮中快速冷冻。应用蛋白质印迹(Western blot)法检测GHS和SD大鼠骨髓基质干细胞、肾脏和小肠中的维生素D受体蛋白的水平。应用实时定量聚合酶链反应(Quantitative real-time polymerase chain reaction, qRT-PCR)法检测GHS和SD大鼠骨髓基质干细胞、肾脏和小肠中的BMP2mRNA的表达。
2、分别应用1,25(OH)2D3处理体外培养的GHS大鼠和SD大鼠的骨髓基质干细胞和UMR-106细胞,应用qRT-PCR法检测细胞中BMP2mRNA表达的变化。
3、应用软件分析BMP2基因转录调控区内可能的VDR结合位点,根据分析结果的基因序列,利用引物设计软件Primer Premier5.0辅助设计相应的扩增引物。
4、应用染色质免疫沉淀技术(Chromatin Immuno-Precipitation, ChIP)检测VDR与BMP2转录调控区DNA的结合,应用方法3中获得的扩增引物对ChIP产物进行PCR扩增,扩增产物进行凝胶电泳分析。
5、克隆方法4中获得的BMP2转录调控区内VDR结合位点的基因片段,构建含此基因片段的荧光素酶基因表达载体,通过荧光素酶报告基因检测系统分析此片段的转录活性。
6、DNA甲基转移酶抑制剂5-aza-2'-deoxycytidine (DAC)单独或联合1,25(OH)2D3处理体外培养的骨髓基质干细胞和UMR-106细胞,应用qRT-PCR检测BMP2mRNA的表达变化。组蛋白去乙酰酶抑制剂trichostatin A (TSA)单独或联合1,25(OH)2D3处理细胞,应用qRT-PCR检测BMP2mRNA的表达变化。
7、1,25(OH)2D3处理UMR-106细胞后提取基因组DNA,应用重亚硫酸盐焦磷酸测序法(Bisulfite pyrosequencing)检测BMP2基因转录调控区CpG位点的甲基化状态的改变。
8、1,25(OH)2D3处理UMR-106细胞后,应用染色质免疫沉淀技术检测BMP2转录调控区H3K9甲基化、组蛋白H3乙酰化程度的改变。
结果：
1、GHS大鼠骨髓基质干细胞、肾脏和小肠中的维生素D受体蛋白的水平较正常SD大鼠明显增加,BMP2mRNA的表达较正常SD大鼠明显降低。
2、以10-8mol/L的1,25(OH)2D3分别处理体外培养的SD大鼠和GHS大鼠骨髓基质干细胞6h、12h和24h,与空白对照组比较,BMP2mRNA的表达均明显降低。以10-8mol/L的1,25(OH)2D3分别处理培养的UMR-106细胞1h、6h、12h、24h和48h,与空白对照组比较,BMP2mRNA的表达均明显降低。以不同浓度的1,25(OH)2D3分别处理体外培养的GHS大鼠和SD大鼠的骨髓基质干细胞和UMR-106细胞24h,与空白对照组比较,BMP2mRNA的表达均明显降低。
3、软件分析了包括BMP2基因及其侧翼序列在内的共计28,545bp的一段基因序列,共获得8个可能存在的VDR结合位点(Region A-H)。
4、以VDR抗体进行ChIP实验,将ChIP产物PCR扩增后,进行凝胶电泳分析显示VDR能结合BMP2转录调控区的Region C片段。
5、成功构建了pGL3-Region C-promoter荧光素酶报告基因质粒,将其转染入UMR-106细胞,结果显示构建的pGL3-Region C-promoter荧光素酶报告基因与空白对照的PGL3-promoter相比,表达活性显著降低。1,25(OH)2D3的处理,使报告基因的荧光素酶表达活性进一步显著降低。
6、不同浓度(0.5,1,2umol/L)的DAC处理来源于GHS大鼠的骨髓基质干细胞及UMR-106细胞后,均显著上调BMP2mRNA的表达,仅有0.5umol/L的DAC上调来源于SD大鼠的骨髓基质干细胞中BMP2mRNA的表达。但是当与1,25(OH)2D3联合使用时,与单独1,25(OH)2D3处理组相比,较高浓度(1,2umol/L)的DAC均能显著上调三种细胞中BMP2mRNA的表达。
7、不同浓度(20,100,500nmol/L)的TSA处理来源于SD大鼠的骨髓基质干细胞后,显著上调BMP2mRNA的表达,而仅有20nmol/L的TSA上调来源于GHS大鼠的骨髓基质干细胞中BMP2mRNA的表达。当与1,25(OH)2D3联合使用处理来源于SD和GHS大鼠的骨髓基质干细胞,以及UMR-106细胞时,与单独1,25(OH)2D3处理组相比,较高浓度(100,500nmol/L)的TSA均能显著上调BMP2mRNA的表达。
8、1,25(OH)2D3处理后,重亚硫酸盐测序显示BMP2基因转录调控区RegionC区域的一个CpG位点完全甲基化,而对照组同一CpG位点仍呈去甲基化状态。
9、染色质免疫沉淀技术检测显示1,25(OH)2D3处理UMR-106细胞后,提高了BMP2转录调控区Region C区域H3K9甲基化的程度,显著降低了BMP2转录调控区Region C区域组蛋白H3乙酰化的程度。
结论：
1、在GHS大鼠的相同组织中,与正常SD大鼠比较,VDR水平升高而BMP2mRNA的表达降低。
2、在体外培养条件下,1,25(OH)2D3可明显抑制骨髓基质干细胞及类成骨细胞中BMP2mRNA的表达。3、1,25(OH)2D3可通过与VDR的复合,进一步结合BMP2转录调控区的Region C区域,从而抑制BMP2基因的转录表达。
4、DNA甲基化和组蛋白修饰协同参与1,25(OH)2D3调控BMP2基因的转录表达过程。
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-106cells
Conclusions:
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.
维生素D3是目前临床上治疗骨质疏松的常用药物,它在体内通过羟化生成活性代谢产物1,25(OH)2D3(1a,25-dihydroxyvitamin D3)而发挥生理作用。1,25(OH)2D3是一种具有多种生物效应的激素前体,其主要是通过与靶细胞内的维生素D受体(Vitamin D receptor, VDR)结合后调节各种基因的表达,从而发挥其生理效应。它具有促进肠道钙和磷的吸收,促进肾小管对钙、磷的重吸收的作用。它一方面可以协同甲状旁腺激素刺激骨组织脱钙,提高血钙、磷浓度；另一方面也可以抑制甲状旁腺激素的合成释放,从而抑制甲状旁腺激素的骨溶解作用,使骨丢失减少。但是目前关于1,25(OH)2D3对骨形成及成骨细胞作用的报道仍很不一致。部分研究认为1,25(OH)2D3可诱导成骨细胞的分化成熟,对骨组织的形成有直接的促进作用。而另外一些研究则认为1,25(OH)2D3对成骨细胞有明显的抑制作用,直接抑制骨形成。因此,1,25(OH)2D3对骨形成的作用及机制仍需要进一步的研究。
骨形态发生蛋白(Bone morphogenetic protein, BMP)是一组分泌性的多功能蛋白质,除BMP1外均属于TGF-β(转化生长因子-p)超家族成员,在体内可以通过旁分泌和自分泌的形式诱导骨的形成,是目前公认最强的骨诱导因子。BMP2是其中研究和应用最广泛的成骨生长因子之一,它可以在体外诱导间充质干细胞向成骨细胞分化,体内诱导骨形成。BMP2在启动和调节骨形成中起着关键的作用,因此任何影响BMP2基因表达的因素都可能会影响骨形成。
若能阐明1,25(OH)2D3与BMP2基因表达的关系,对进一步理解1,25(OH)2D3在骨形成中的作用会有很大的帮助,目前文献中尚未有此类报道。
遗传性高钙尿(Genetic hypercalciuric stone-forming, GHS)大鼠是研究特发性高钙尿(Idiopathic hypercalciuria, IH)患者的理想动物模型,它们都表现为小肠的钙吸收增加,肾脏的尿钙重吸收减少,骨密度减低。既往的研究表明GHS大鼠中肠钙吸收的增加,尿钙重吸收的减少与组织中VDR水平的增加有很大的关系。但是GHS大鼠中骨密度减低的原因仍不是十分清楚。本研究旨在通过探寻GHS大鼠中VDR与BMP2的关系来分析这种动物模型中骨密度减低的可能原因,并进一步探讨1,25(OH)2D3对BMP2基因表达的调控及可能的机制,从而进一步分析1,25(OH)2D3在骨形成中的作用及机制。
目的：
1、研究GHS大鼠和正常SD大鼠相同组织中维生素D受体蛋白和BMP2mRNA的基础表达水平并进行比较,分析VDR和BMP2可能存在的联系。
2、体外培养GHS大鼠和SD大鼠的骨髓基质干细胞,以及UMR-106细胞,研究1,25(OH)2D3对BMP2mRNA表达的影响。
3、研究1,25(OH)2D3调控BMP2基因转录表达的作用机制。
4、探讨DNA甲基化和组蛋白修饰在1,25(OH)2D3调控BMP2基因表达中的作用。
方法：
1、选取成年GHS大鼠和SD大鼠,断颈处死,无菌条件下取出股骨和胫骨,抽取骨髓,应用红细胞裂解法及直接贴壁法获取并体外培养骨髓基质干细胞,无菌条件下取出肾脏及小肠,置入液氮中快速冷冻。应用蛋白质印迹(Western blot)法检测GHS和SD大鼠骨髓基质干细胞、肾脏和小肠中的维生素D受体蛋白的水平。应用实时定量聚合酶链反应(Quantitative real-time polymerase chain reaction, qRT-PCR)法检测GHS和SD大鼠骨髓基质干细胞、肾脏和小肠中的BMP2mRNA的表达。
2、分别应用1,25(OH)2D3处理体外培养的GHS大鼠和SD大鼠的骨髓基质干细胞和UMR-106细胞,应用qRT-PCR法检测细胞中BMP2mRNA表达的变化。
3、应用软件分析BMP2基因转录调控区内可能的VDR结合位点,根据分析结果的基因序列,利用引物设计软件Primer Premier5.0辅助设计相应的扩增引物。
4、应用染色质免疫沉淀技术(Chromatin Immuno-Precipitation, ChIP)检测VDR与BMP2转录调控区DNA的结合,应用方法3中获得的扩增引物对ChIP产物进行PCR扩增,扩增产物进行凝胶电泳分析。
5、克隆方法4中获得的BMP2转录调控区内VDR结合位点的基因片段,构建含此基因片段的荧光素酶基因表达载体,通过荧光素酶报告基因检测系统分析此片段的转录活性。
6、DNA甲基转移酶抑制剂5-aza-2'-deoxycytidine (DAC)单独或联合1,25(OH)2D3处理体外培养的骨髓基质干细胞和UMR-106细胞,应用qRT-PCR检测BMP2mRNA的表达变化。组蛋白去乙酰酶抑制剂trichostatin A (TSA)单独或联合1,25(OH)2D3处理细胞,应用qRT-PCR检测BMP2mRNA的表达变化。
7、1,25(OH)2D3处理UMR-106细胞后提取基因组DNA,应用重亚硫酸盐焦磷酸测序法(Bisulfite pyrosequencing)检测BMP2基因转录调控区CpG位点的甲基化状态的改变。
8、1,25(OH)2D3处理UMR-106细胞后,应用染色质免疫沉淀技术检测BMP2转录调控区H3K9甲基化、组蛋白H3乙酰化程度的改变。
结果：
1、GHS大鼠骨髓基质干细胞、肾脏和小肠中的维生素D受体蛋白的水平较正常SD大鼠明显增加,BMP2mRNA的表达较正常SD大鼠明显降低。
2、以10-8mol/L的1,25(OH)2D3分别处理体外培养的SD大鼠和GHS大鼠骨髓基质干细胞6h、12h和24h,与空白对照组比较,BMP2mRNA的表达均明显降低。以10-8mol/L的1,25(OH)2D3分别处理培养的UMR-106细胞1h、6h、12h、24h和48h,与空白对照组比较,BMP2mRNA的表达均明显降低。以不同浓度的1,25(OH)2D3分别处理体外培养的GHS大鼠和SD大鼠的骨髓基质干细胞和UMR-106细胞24h,与空白对照组比较,BMP2mRNA的表达均明显降低。
3、软件分析了包括BMP2基因及其侧翼序列在内的共计28,545bp的一段基因序列,共获得8个可能存在的VDR结合位点(Region A-H)。
4、以VDR抗体进行ChIP实验,将ChIP产物PCR扩增后,进行凝胶电泳分析显示VDR能结合BMP2转录调控区的Region C片段。
5、成功构建了pGL3-Region C-promoter荧光素酶报告基因质粒,将其转染入UMR-106细胞,结果显示构建的pGL3-Region C-promoter荧光素酶报告基因与空白对照的PGL3-promoter相比,表达活性显著降低。1,25(OH)2D3的处理,使报告基因的荧光素酶表达活性进一步显著降低。
6、不同浓度(0.5,1,2umol/L)的DAC处理来源于GHS大鼠的骨髓基质干细胞及UMR-106细胞后,均显著上调BMP2mRNA的表达,仅有0.5umol/L的DAC上调来源于SD大鼠的骨髓基质干细胞中BMP2mRNA的表达。但是当与1,25(OH)2D3联合使用时,与单独1,25(OH)2D3处理组相比,较高浓度(1,2umol/L)的DAC均能显著上调三种细胞中BMP2mRNA的表达。
7、不同浓度(20,100,500nmol/L)的TSA处理来源于SD大鼠的骨髓基质干细胞后,显著上调BMP2mRNA的表达,而仅有20nmol/L的TSA上调来源于GHS大鼠的骨髓基质干细胞中BMP2mRNA的表达。当与1,25(OH)2D3联合使用处理来源于SD和GHS大鼠的骨髓基质干细胞,以及UMR-106细胞时,与单独1,25(OH)2D3处理组相比,较高浓度(100,500nmol/L)的TSA均能显著上调BMP2mRNA的表达。
8、1,25(OH)2D3处理后,重亚硫酸盐测序显示BMP2基因转录调控区RegionC区域的一个CpG位点完全甲基化,而对照组同一CpG位点仍呈去甲基化状态。
9、染色质免疫沉淀技术检测显示1,25(OH)2D3处理UMR-106细胞后,提高了BMP2转录调控区Region C区域H3K9甲基化的程度,显著降低了BMP2转录调控区Region C区域组蛋白H3乙酰化的程度。
结论：
1、在GHS大鼠的相同组织中,与正常SD大鼠比较,VDR水平升高而BMP2mRNA的表达降低。
2、在体外培养条件下,1,25(OH)2D3可明显抑制骨髓基质干细胞及类成骨细胞中BMP2mRNA的表达。3、1,25(OH)2D3可通过与VDR的复合,进一步结合BMP2转录调控区的Region C区域,从而抑制BMP2基因的转录表达。
4、DNA甲基化和组蛋白修饰协同参与1,25(OH)2D3调控BMP2基因的转录表达过程。
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-106cells
Conclusions:
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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