MOLECULAR REGULATION OF 1,25(OH)2D PRODUCTION

描述(改编自申请人的摘要)：总体目标 这一提议的关键是对分子的详细理解 1，25-二羟基维生素D(1，25D)合成的调控。虽然 这种类固醇激素在骨骼的钙代谢中起着至关重要的作用 生长和组织分化，对这种分子知之甚少 其合成的调控机制。关键的量化监管 合成1，25D的步骤是它的1α-羟基化反应。 内源性前体25-羟基维生素D(25(OH)D)，由该酶催化 25(OH)D-1α-羟基酶(1-OHase)。1-OHase是一种线粒体 细胞色素P450酶类似于肾上腺中的类固醇合成酶 还有性腺。波塔尔博士的实验室最近克隆了该基因的cDNA和基因 对于人类1α-羟基酶，命名为P450c1。他们现在 建议研究1，25D产生的分子调控机制， 特别是甲状旁腺素、磷和1，25D是如何调节合成的 1，25D。急、慢性肾脏1，25D产生紊乱 衰竭、X连锁低磷血症、常染色体隐性维生素D 依赖型软骨病1型，肾Fanconi综合征，年龄较大。 1-OHase生理调节的研究进展 分子水平将为后续研究提供基础。 该酶调节改变的潜在分子机制 由于衰老和肾脏疾病：1)他们将克隆啮齿动物P450c1和 提高人类P450c1蛋白的抗体；2)他们将检查荷尔蒙 P450c1基因在小鼠体内和体内蛋白丰度的调节 体外分离小鼠近端小管，并确定是否诱导改变 都是由转录事件调节的。3)使用永生的人类 近端小管细胞，他们将研究转录调控使用 启动子/报告基因结构的功能分析，并将检查 通过带移分析相关区域的蛋白质/DNA相互作用， 紫外光交联和西南印迹定位特定顺式元素 和他们的同源DNA结合蛋白；以及4)他们将决定 P450c1 mRNA和蛋白在肾脏中的组织分布及定位 P4501基因在显微解剖大鼠肾单位节段中的表达。

DESCRIPTION (Adapted from the Applicant's Abstract): The overall objective of this proposal is to gain a detailed understanding of the molecular regulation of the synthesis of 1,25-dihydroxyvitamin D (1,25D). Although this steroid hormone plays a crucial role in calcium metabolism, bone growth, and tissue differentiation, little is known about the molecular mechanisms of regulation of its synthesis. The key quantitative regulatory step in the synthesis of 1,25D is its 1alpha-hydroxylation from its endogenous precursor, 25-hydroxyvitaminD (25(OH)D), catalyzed by the enzyme 25(OH)D-1alpha-hydroxylase (1-OHase). The 1-OHase is a mitochondrial cytochrome P450 enzyme similar to the steroidogenic enzymes in the adrenal and gonad. Dr. Portale's laboratory has recently cloned the cDNA and gene for the human 1alpha-hydroxylase enzyme, designated P450c1. They now propose to study the molecular mechanisms of regulation of 1,25D production, and specifically how PTH, phosphorus, and 1,25D regulate the synthesis of 1,25D. Production of 1,25D is disordered in acute and chronic renal failure, X-linked hypophosphatemic rickets, autosomal recessive vitamin D dependent rickets Type 1, renal Fanconi syndrome, and with advanced age. The proposed studies of the physiologic regulation of the 1-OHase at the molecular level will provide the basis for subsequent studies of the potential molecular mechanisms by which regulation of this enzyme is altered by aging and renal disease: 1) They will clone a cDNA for rodent P450c1 and raise antibodies to human P450c1 protein; 2) They will examine hormonal regulation of P450c1 mRNA and protein abundance in mice in vivo and in isolated mouse proximal tubules, in vitro, and determine if induced changes are mediated by transcriptional events. 3) Using immortalized human proximal tubule cells, they will study transcriptional regulation using functional assays of promoter/reporter constructs, and will examine protein/DNA interactions in the relevant regions by bandshift assays, UV-crosslinking, and Southwestern blotting to localize specific cis-elements and their cognate DNA binding proteins; and 4) They will determine the tissue distribution of P450c1 mRNA and protein in kidney and will localize P4501 gene expression in microdissected rat nephron segments.