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

描述（改编自申请人的摘要）：总体目标 这一建议的目的是获得分子的详细了解， 调节1，25-二羟维生素D（1，25 D）的合成。 虽然 这种类固醇激素在钙代谢、骨骼 生长和组织分化，对分子生物学知之甚少。 其合成的调节机制。 关键的定量监管 1，25 D的合成步骤是其1 α-羟基化， 内源性前体，25-羟基维生素D（25（OH）D），由酶催化 25（OH）D-1 α-羟化酶（1-OHase）。 1-OH酶是一种线粒体酶， 细胞色素P450酶类似于肾上腺中的类固醇生成酶 和生殖腺 Portale博士的实验室最近克隆了cDNA和基因 人类1 α-羟化酶，命名为P450 c1。 他们现在 建议研究1，25 D生产调节的分子机制， 特别是PTH、磷和1，25 D如何调节 1，25D。 急性和慢性肾脏疾病中1，25 D的产生是紊乱的 衰竭，X连锁低磷血症佝偻病，常染色体隐性遗传维生素D 依赖性佝偻病1型，肾范可尼综合征，并与高龄。 对1-OHase的生理调节的研究， 分子水平将为后续研究提供基础。 改变这种酶调节的潜在分子机制 1）他们将克隆啮齿动物P450 c1的cDNA， 提高对人类P450 c1蛋白的抗体; 2）他们将检查激素 P450 c1 mRNA和蛋白丰度在小鼠体内和体内的调节 分离的小鼠近端小管，在体外，并确定是否诱导的变化 是由转录事件介导的。 3)使用永生化的人类 近曲小管细胞，他们将研究转录调控使用 启动子/报告基因构建体的功能测定，并将检查 通过带移测定在相关区域中的蛋白质/DNA相互作用， UV交联和Southwestern印迹定位特定的顺式元件 和他们的同源DNA结合蛋白;和4）他们将决定 P450 c1 mRNA和蛋白在肾脏中的组织分布及定位 P4501基因在大鼠肾单位中的表达。

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.