VITAMIN D HORMONE MECHANISM OF ACTION IN CULTURED CELLS

描述：此应用程序建议设计实验，以表征 维生素D激素1，25-二羟基维生素D3的作用机制 (1，25(OH)2D3)，在调控相关基因的表达中 骨矿物质稳态的病理生理学。计划研究的重点 核维生素D受体(VDR)是维生素D受体超家族的一员 作为1，25(OH)2D3功能的类固醇/维甲酸/甲状腺激素受体 配体激活的转录因子与维甲酸X的协同作用 受体(RXR)。在第一个具体目标中，利用培养的人类细胞， 计划评估：1)L/S基因的功能意义 人维生素D受体(HVDR)基因3‘非翻译区的多态性，2) CDX-2位点在5‘-非翻译区的多态性，以及3)细胞特异性 由新基因选择性剪接引起的N-末端变异hVDR蛋白 5‘-启动子外显子。通过检测hVDR表达和异构体对 1，25(OH)2D3在各种细胞中的作用，可以洞察到 1，25(OH)2D3及其非经典生物活性的分子基础 药物类似物，如在上皮细胞分化中。第二个具体问题 目的是继续确定hVDR的位置和意义 磷酸化，主要是蛋白激酶A催化的翻译后 受体的修饰。第三个具体目标涉及到刻画 激素配体、靶基因启动子和VDR相互作用的作用 转导转录调控信号的蛋白质。受控 实验将包括一个超活性的20-epi-1，25(OH)2D3类似物和一个维甲酸 它与RXR以及衍生的自然启动子构建物特异性结合 从骨钙素和骨桥蛋白维生素D调节基因来评价 DNA序列对1，25(OH)2D3激素反应的影响构成要素 活性RXRs和VDR将通过定点突变产生 此外，受体配体和RXR伙伴所起的作用。最后， 应用建议从成骨细胞中克隆一种新的维生素D受体共调控因子(S) 利用噬菌体展示方法学构建文库。这些研究针对的是 VDR基因、异构体和磷酸化受体蛋白及其共同调控 合作伙伴，期望促进对分子的详细了解 1，25(OH)2D3激素作用的基础及其临床治疗意义 骨质疏松症等骨骼疾病。

DESCRIPTION: This application proposes experiments designed to characterize the mechanism of action of the vitamin D hormone, 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), in regulating the expression of genes relevant to the pathophysiology of bone mineral homeostasis. The focus of the planned studies is the nuclear vitamin D receptor (VDR), a member of the superfamily of steroid/retinoid/thyroid hormone receptors that functions as a 1,25(OH)2D3 ligand-activated transcription factor in cooperation with the retinoid X receptor (RXR). In the first Specific Aim, utilizing cultured human cells, the plan is to evaluate the functional significance of the: 1) L/S gene polymorphism in the 3'-untranslated region of the human VDR (hVDR) mRNA, 2) Cdx-2 site polymorphism in the 5'-untranslated region, and 3) cell specific N-terminal variant hVDR proteins resulting from alternative splicing of novel 5'-promoter exons. By examining the impact of hVDR expression and isoforms on 1,25(OH)2D3 action in a variety of cells, insight may be gained into the molecular basis for the nonclassical biological actions of 1,25(OH)2D3 and its drug analogs, such as in epithelial cell differentiation. The second Specific Aim is to continue to determine the sites and significance of hVDR phosphorylation, focusing on protein kinase A catalyzed posttranslational modification of the receptor. The third Specific Aim involves characterizing the role of hormonal ligands, target gene promoters, and VDR-interacting proteins in transducing the signal for transcriptional regulation. Controlled experiments will include a superactive 20-epi-1,25(OH)2D3 analog and a retinoid that binds specifically to RXR, as well as natural promoter constructs derived from the osteocalcin and osteopontin vitamin D regulated genes to evaluate the influence of DNA sequence on 1,25(OH)2D3 hormonal responses. Constitutively active RXRs and VDR will be generated by site-directed mutagenesis to dissect further the roles played by receptor ligands and the RXR partner. Finally, the application proposes to clone a novel VDR co-regulator(s) from osteoblast cDNA libraries utilizing phage display methodology. These studies, directed at the VDR gene, isoform and phosphorylated receptor proteins, and their co-regulatory partners, are expected to facilitate a detailed understanding of the molecular basis of 1,25(OH)2D3 hormone action, with therapeutic implications for clinical disorders of bone such as osteoporosis.