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

1,25(OH)2D 生成的分子调控

• 批准号: 2906285
• 负责人: ANTHONY A PORTALE
• 金额: $ 25.17万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-08-01 至 2003-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2906285
• 关键词: 1,25 dihydroxycholecalciferol; cytochrome P450; dietary calcium; enzyme mechanism; gene expression; genetic regulatory element; genetic transcription; hormone regulation /control mechanism; immunocytochemistry; in situ hybridization; intermolecular interaction; laboratory mouse; nuclear runoff assay; nutrition related tag; parathyroid hormones; phosphorus; polymerase chain reaction; protein localization; renal tubular transport; steroid hormone biosynthesis; tissue /cell culture; transfection; vitamin metabolism; western blottings

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（根据申请人的摘要改编）：总体目标 该建议的是对分子的详细理解 调节1,25-二羟基维生素D（1,25d）的合成。 虽然 这种类固醇激素在钙代谢中起着至关重要的作用 生长和组织分化，对分子知之甚少 调节其合成的机制。 关键定量调节 1,25d的一步是其1α-羟基化。 内源性前体，25-羟基维坦（25（OH）D），由酶催化 25（OH）D-1Alpha-羟化酶（1-霍斯）。 1-鹰骨是线粒体 细胞色素P450酶类似于肾上腺中类固醇的酶 和性腺。 Portale博士的实验室最近克隆了cDNA和基因 对于人类1alpha-羟化酶，指定为P450C1。 他们现在 建议研究1,25D生产调节的分子机制， 特别是PTH，磷和1,25D如何调节的合成 1,25d。 1,25D的产生在急性和慢性肾脏中无序 失败，X连锁的下磷酸rik，常染色体隐性维生素D 依赖的rick型1型，肾脏范科尼综合征，并且年龄高。 提出的对1-牛酶生理调节的研究 分子水平将为后续研究提供基础 通过改变该酶调节的潜在分子机制 通过衰老和肾脏疾病：1）他们将在啮齿动物P450C1和 引起对人P450C1蛋白的抗体； 2）他们将检查荷尔蒙 体内小鼠的P450C1 mRNA和蛋白质丰度调节 在体外分离的小鼠近端小管，并确定诱导的变化是否变化 由转录事件介导。 3）使用永生的人 近端小管细胞，他们将研究转录调节 启动子/记者结构的功能测定法，并将检查 通过班次分析，相关区域中的蛋白质/DNA相互作用， 紫外线交联，西南印迹以本地化特定的顺式元素 以及它们的同源DNA结合蛋白； 4）他们将确定 肾脏中P450C1 mRNA和蛋白质的组织分布，并将定位 P4501大鼠肾单位段中的P4501基因表达。