Vitamin D Hormone Signaling in Bone Mineral Homeostasis

该项目的目标是描述肾脏维生素D激素作用的分子机制， 1，25-二羟维生素D3（1，25（OH）2D 3），调节骨矿物质离子，预防疾病， 佝偻病/骨软化症和骨质疏松症。待检验的假设是，1，25（OH）2D 3发挥其作用， 通过核维生素D受体（VDR）的配体支持矿化骨骼，从而招募 它的类维生素A X受体（RXR）异二聚体配偶体识别维生素D反应元件（VDRES）， 骨相关靶基因启动子，具有1，25（OH）2D 3-VDR-RXR吸引共调节蛋白/酶 通过染色质重塑和与RNA连接抑制或诱导DNA转录的复合物 聚合酶II。培养的1，25（OH）2D 3靶细胞将用作特定目的设计的模型系统 阐明VDR介导的1，25（OH）2D 3信号传导事件。目标1将确定1，25（OH）2D 3-VDR是否 通过影响Wnt/LRP 5/β-连环蛋白调节成骨细胞发育/骨形成， BMP/SMAD/Runx 2信号级联，并可能通过控制新的 公认的磷酸盐调节系统，FGF 23和PHEX在成骨细胞。目标2将探讨 1，25（OH）2D 3-VDR刺激小肠和肾脏中的钙和磷酸盐易位，通过 上皮钙转运蛋白1（TRPV 6）和磷酸钠协同转运蛋白2c的表达增强 （Npt2c）。方法学将包括基因组学和染色质免疫沉淀（ChIP）测定 扫描候选VDRE，加上DNA凝胶迁移率变化，真实的时间PCR和启动子切割，以确定 VDR控制的基因将1，25（OH）2D 3与其超活性类似物进行比较，ChIP测定将评估完整的 细胞VDR-RXR-VDRE结合，并表征转录共调节因子的顺序募集， 差异控制骨矿物质靶基因的表达。最后，在Aim 3中将使用ChIP显示器 以揭示新的上游VDR控制基因，如果任何提出的VDR靶点出现， 通过介导转因子二次或三次诱导/抑制。建议的意义 研究表明，1，25（OH）2D 3维持正常骨骼的精确分子机制 矿化，以及新的维生素D调节基因在骨中表达的相对重要性（目的 1)以及小肠和肾脏（目标2），尚不清楚;理解这些途径应 增强我们开发维生素D模拟物以预防和治疗骨质减少症的能力。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， 骨质疏松症的发展和导致骨折的脊柱，髋关节和手腕，通过这种方式， 维生素D激素实现这一有益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不仅揭示了 用骨合成代谢1，25（OH）2D 3类似物预防和治疗骨质疏松症的新治疗策略， 还可以揭示维生素D在结肠、皮肤、乳房和其他部位的新抗癌作用。

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