Vitamin D Hormone Signaling in Bone Mineral Homeostasis

这个项目的目标是描述肾脏维生素D激素的分子作用机制， 1，25-二羟基维生素D3(1，25(OH)2D3)，调节骨矿离子，预防疾病，如 软骨病/软骨病和骨质疏松。要检验的假设是1，25(OH)2D3发挥其作用 通过核维生素D受体(VDR)的连接来支持矿化的骨骼，这反过来又招募 其维甲酸X受体(RXR)异二聚体伙伴识别维生素D反应元件(VDREs) 骨相关靶基因启动子，1，25(OH)2D3-VDR-RXR吸引共调节蛋白/酶 通过染色质重塑和与RNA连接来抑制或诱导DNA转录的复合体 聚合酶II.培养的1，25(OH)2D3靶细胞将被用作特定目的设计的模型系统 阐明VDR在1，25(OH)2D3信号转导中的作用。目标1将确定1，25(OH)2D3-VDR 通过影响Wnt/LRP5/p-catenin调控成骨细胞发育/骨形成 BMP/SMAD/Runx2信号级联，并可能通过控制一个新的 成骨细胞中公认的磷酸调节系统，FGF23和PHEX。《目标2》将探讨 1，25(OH)2D3-VdR促进小肠和肾脏钙磷转运 上皮钙转运蛋白1(TRPV6)和钠-磷酸协同转运蛋白2c表达增强 (Npt2c)。方法学将包括基因组学和染色质免疫沉淀(芯片)分析。 扫描候选VDRE，加上DNA凝胶迁移率漂移、实时聚合酶链式反应和启动子解剖来识别 VDR控制的基因。将1，25(OH)2D3与其超活性类似物进行比较，芯片分析将评估完好无损 细胞VDR-RXR-VDRE结合，并表征转录共调节子的顺序招募 差异调控骨矿靶基因的表达。最后，在AIM 3中将使用芯片显示器 为了揭示新的上游VDR控制的基因，如果任何被提议的VDR靶点出现为 二次或三次通过调节反式因子诱导/抑制。建议的重要意义 研究表明，1，25(OH)2D3维持正常骨骼的确切分子机制 矿化作用以及在骨骼中表达的新的维生素D调节基因的相对重要性(目的 1)和小肠和肾脏(目标2)，不被理解；理解这些途径应该 增强我们开发维生素D模拟物以预防和治疗骨质疏松症的能力。因此，尽管 公认的维生素D在促进钙吸收和骨重建方面的相关性 骨质疏松的发展以及由此导致的脊柱、髋部和手腕骨折，通过这种方式 维生素D荷尔蒙实现这一有益的效果还没有完全阐明。定义 维生素D增强和保护矿化骨骼的分子途径可能不仅揭示了 骨合成代谢1，25(OH)2D3类似物预防和治疗骨质疏松症的新治疗策略，但 还可以揭示维生素D在结肠、皮肤、乳房和其他部位的抗癌作用

The goal of this project is to characterize the molecular mechanism of action of the renal vitamin D hormone, 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), in regulating bone mineral ions to prevent diseases such as rickets/osteomalacia and osteoporosis. The hypothesis to be tested is that 1,25(OH)2D3 exerts its actions to support the mineralized skeleton via liganding of the nuclear vitamin D receptor (VDR), which in turn recruits its retinoid X receptor (RXR)heterodimeric partner to recognize vitamin D responsive elements (VDREs) in bone relevant target gene promoters, with 1,25(OH)2D3-VDR-RXR attracting comodulator protein/enzyme complexes that either repress or induce DNA transcription by chromatin remodeling and linkage to RNA polymerase II. Cultured 1,25(OH)2D3target cells will be used as model systems for Specific Aims designed to elucidate the VDR-mediated events in signaling by 1,25(OH)2D3. Aim 1 will determine if 1,25(OH)2D3-VDR modulates osteoblast development/bone formation by impacting the Wnt/LRP5/p-catenin and BMP/SMAD/Runx2 signal cascades, and possibly limits bone overmineralization by controlling a newly recognized phosphate regulatory system, FGF23 and PHEX, in osteoblasts. Aim 2 will probe the role of 1,25(OH)2D3-VDR to stimulate calcium and phosphate translocation in small intestine and kidney through the enhanced expression of epithelial calcium transporter 1 (TRPV6) and sodium-phosphate cotransporter 2c (Npt2c), respectively. Methodology will include genomics and chromatin immunoprecipitation (ChIP) assays to scan for candidate VDREs, plus DNA gel mobility shift, real time PCR and promoter dissection to identify VDR-controlled genes. Comparing 1,25(OH)2D3 to its superactive analogs, ChIP assays will assess intact cell VDR-RXR-VDRE binding and characterize the sequential recruitment of transcriptional comodulators that differentially control the expression of bone mineral target genes. Finally, in Aim 3 ChIP display will be used to reveal novel upstream VDR-controlled genes should any of the proposed VDR targets emerge as secondarily or tertiarily induced/repressed via mediating transfactors. The significance of the proposed studies is that the precise molecular mechanisms whereby 1,25(OH)2D3 maintains proper bone mineralization, as well as the relative importance of novel vitamin D-regulated genes expressed in bone (Aim 1) and small intestine and kidney (Aim 2), are not understood; comprehending these pathways should enhance our ability to develop vitamin D mimetics to prevent and treat osteopenic disorders. Thus, despite the recognized relevance of vitamin D in promoting calcium absorption and bone remodeling to preclude the development of osteoporosis and resulting fractures of the spine, hip and wrist, the manner through which the vitamin D hormone accomplishes this beneficial effect has not been fully elucidated. Defining the molecular pathway of vitamin D action to enhance and preserve the mineralized skeleton may not only reveal new therapeutic strategies to prevent and treat osteoporosis with bone anabolic 1,25(OH)2D3 analogs, but could also shed light on the novel anticancer effects of vitamin D at sites such as the colon, skin, breast and