ACTH INDUCTION OF CYTOCHROME P450 IN ADRENAL CELLS

肾上腺细胞中细胞色素 P450 的 ACTH 诱导

• 批准号: 3228766
• 负责人: MICHAEL R WATERMAN
• 金额: $ 22.71万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-07-18 至 1994-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3228766
• 关键词: DNA footprinting; adrenal ferredoxin; adrenal glands; adrenocorticotropic hormone; chemical binding; cow; cyclic AMP; cytochrome P450; developmental genetics; embryo /fetus; enzyme induction /repression; female; gel mobility shift assay; genetic transcription; genetic translation; genomic imprinting; hormone regulation /control mechanism; human tissue; laboratory rat; molecular cloning; oxygenases; plasmids; polymerase chain reaction; protein kinase A; steroid biosynthesis; tissue /cell culture; transcription factor; transfection

Normal reproduction and the maintenance of life in all mammalian species requires complex patterns of steroidogenic activities. In this renewal application it is proposed to elucidate in biochemical terms the multifactorial regulation of steroid hydroxylase gene expression in the adrenal cortex. During the past granting period we have investigated three levels of regulation of expression of these genes (maintenance of optimal steroidogenic capacity regulated in part by ACTH via cAMP, tissue-specific, and developmental) using cDNA probes specific for the various steroid hydroxylase mRNAs. In the coming granting period we propose to establish at the transcriptional level, the trans-acting factors and cis-regulatory sequences involved this complex pattern of regulation of steroid hydroxylase gene expression. Utilizing genes encoding human and bovine P-450 17 alpha human and bovine P-450scc' human P-450C21 and bovine adrenodoxin we will identify adrenal nuclear proteins which transcription of these genes in response to cAMP (so-called SHIPs) and the cis-sequences to which they bind. A combination of gel retardation, DNA footprinting, CAT activities and in vitro transcription assays will be used to identify these factors followed by their purification and characterization. In addition we will identify similar factors which are required for the fetal imprinting of steroid hydroxylase genes in the fetal bovine adrenal cortex using the same techniques. Also we will determine the molecular basis of the cell specific regulation of P-450 17 alpha in bovine fasiculata-reticularis compared to zona glomerulose as well as the reason for the absence of expression of P-450 17 alpha in rat adrenal cortex. Likewise the cell specific factors which regulate adrenodoxin gene expression in adrenal cortex, liver and kidney will be identified. Finally utilizing mutant regulatory subunits of cAMP-dependent protein kinase we will establish whether this enzyme plays a role in steroid hydroxylase gene expression. In summary, during the proposed 5 years we will identify, characterize and compare trans-acting factors which regulate adrenal steroid hydroxylase gene expression at the hormone mediated, tissue-specific and developmental levels. The results of these studies will permit assessment of both the complexity and the relatedness of these processes. Furthermore they will show the way to a new set of genes, those encoding trans-acting factors which regulate steroid hydroxylase gene expression.

正常繁殖和所有哺乳动物的生命维持 物种需要复杂的类固醇活性模式。 在 该更新应用是为了在生化中阐明 术语类固醇羟化酶基因的多因素调节 在肾上腺皮质中的表达。 在过去的授予期内 我们研究了三个级别的调节表达的调节 这些基因（维持最佳类固醇生成能力 部分由ACTH通过CAMP，组织特异性和 发育性）使用针对各种类固醇特异的cDNA探针 羟化酶mRNA。 在未来的授予期内，我们建议 在转录级别建立跨性因素 和顺式调节序列涉及这种复杂的模式 调节类固醇羟化酶基因表达。 利用基因 编码人和牛P-450 17 Alpha人和牛P-450SCC' 人P-450C21和牛肾上腺素毒素我们将识别肾上腺 这些基因转录的核蛋白响应于 营地（所谓的船只）及其束缚的顺式序列。 凝胶延迟，DNA足迹，猫活动的组合 并且将使用体外转录分析来识别这些 遵循其纯化和表征的因素。 在 此外，我们将确定类似的因素 类固醇羟化酶基因的胎儿印迹 牛肾上腺皮质使用相同的技术。 我们也会 确定细胞特异性调节的分子基础 与Zona相比 肾小球以及缺乏表达的原因 P-450 17大鼠肾上腺皮质中的α。 同样的细胞特异性 调节肾上腺中肾上腺毒素基因表达的因素 将确定皮质，肝脏和肾脏。 最后利用 依赖CAMP的蛋白激酶的突变调节亚基我们将 确定该酶是否在类固醇羟化酶中起作用 基因表达。 总而言之，在拟议的5年中，我们将 识别，表征和比较跨性别因素 调节激素上的肾上腺类固醇羟化酶基因表达 介导的，组织特异性和发育水平。 结果 这些研究将允许评估复杂性和 这些过程的相关性。 此外，他们将向 转向新基因的方式，那些编码反式作用因素的方法 调节类固醇羟化酶基因表达。