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

1,25(OH)2D 生成的分子调控

基本信息

批准号：
6523726
负责人：
ANTHONY A PORTALE
金额：
$ 27.42万
依托单位：
UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
依托单位国家：
美国
项目类别：
财政年份：
1998
资助国家：
美国
起止时间：
1998-08-01 至 2005-07-31
项目状态：
已结题

项目摘要

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.

描述（改编自申请人的摘要）：总体目标该提案的目的是为了详细了解分子 1,25-二羟基维生素 D (1,25D) 合成的调节。虽然这种类固醇激素在钙代谢、骨骼中起着至关重要的作用生长和组织分化，人们对分子机制知之甚少。其合成的调节机制。关键量化监管 1,25D 合成的步骤是其 1α-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-1α-羟化酶（1-OHase）。 1-OHase 是线粒体细胞色素 P450 酶与肾上腺中的类固醇生成酶相似和性腺。 Portale博士的实验室最近克隆了cDNA和基因为人类 1α-羟化酶，指定为 P450c1。他们现在提议研究1,25D生产调节的分子机制，具体来说，PTH、磷和 1,25D 如何调节 1,25D。急性和慢性肾病中 1,25D 的产生紊乱衰竭、X连锁低磷血症性佝偻病、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，且与高龄有关。 1-OHase 生理调节的拟议研究为后续研究提供分子水平的基础改变这种酶的调节的潜在分子机制通过衰老和肾脏疾病：1）他们将克隆啮齿动物 P450c1 的 cDNA 和产生针对人 P450c1 蛋白的抗体； 2）他们会检查荷尔蒙小鼠体内和体内 P450c1 mRNA 和蛋白质丰度的调节体外分离小鼠近端小管，并确定是否诱导变化由转录事件介导。 3）使用永生人类近端小管细胞，他们将使用以下方法研究转录调控启动子/报告基因构建体的功能测定，并将检查通过带移分析检测相关区域的蛋白质/DNA 相互作用，紫外线交联和西南印迹法定位特定的顺式元件及其同源 DNA 结合蛋白； 4) 他们将确定 P450c1 mRNA 和蛋白质在肾脏中的组织分布并将定位 P4501 基因在显微解剖的大鼠肾单位节段中的表达。