ACTH INDUCTION OF CYTOCHROME P450 IN ADRENAL CELLS

所有哺乳动物的正常繁殖和生命的维持 物种需要复杂的类固醇生成活性模式。 在 这一更新的应用，它被提出来阐明在生物化学 类固醇羟化酶基因的多因子调控 在肾上腺皮质的表达。 在过去的资助期内 我们已经研究了三个水平的表达调控， 这些基因（维持最佳类固醇生成能力 部分由ACTH通过cAMP调节，组织特异性， 发育）使用特异于各种类固醇的cDNA探针 羟化酶mRNA。 在未来的拨款期内，我们建议 在转录水平上建立反式作用因子 而顺式调控序列涉及这种复杂的 类固醇羟化酶基因表达的调控。 利用基因 编码人和牛P-450的17 α人和牛P-450 我们将鉴定人P-450 C21和牛肾上腺素还蛋白 核蛋白，这些基因的转录响应于 cAMP（所谓的SHIP）及其结合的顺式序列。 凝胶阻滞、DNA足迹、CAT活性的组合 体外转录分析将用于鉴定这些 因子，然后对其进行纯化和表征。 在 此外，我们将确定所需的类似因素 胎儿类固醇羟化酶基因的胎儿印记 牛肾上腺皮质用同样的技术。 我们也将 确定细胞特异性调节的分子基础， 牛网状束膜与牛结肠中P-450 17 α的比较 肾小球以及缺乏表达的原因， 大鼠肾上腺皮质中的P-450 17 α。 同样，细胞特异性 肾上腺皮质中肾上腺氧还蛋白基因表达的调控因素 将识别皮质、肝脏和肾脏。 最后利用 突变的cAMP依赖性蛋白激酶调节亚基，我们将 确定这种酶是否在类固醇羟化酶中起作用 基因表达。 总之，在拟议的五年内，我们将 识别、表征和比较反式作用因子， 调节肾上腺类固醇羟化酶基因表达的激素 介导的，组织特异性和发育水平。 结果 这些研究将允许评估的复杂性和 这些过程的关联性。 此外，他们将展示 一组新的基因，那些编码反式作用因子的基因， 其调节类固醇羟化酶基因表达。

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.