REGULATION OF POMC GENE EXPRESSION IN PITUITARY

该计划的目标是阐明和表征细胞 以及负责CRH介导的 垂体前叶促肾上腺皮质激素细胞中POMC基因的表达。 这 我们和其他人以前的工作支持这一建议， 钙离子和cAMP在CRH信号中的作用 转导和我们最近发现的新的顺式作用元件 POMC启动子（-700/+63）对CRH的应答。 这些研究报告将 在良好建立的AtT 20肿瘤细胞系统中进行。 ATT20 细胞系已被广泛用于POMC基因的研究 表达，是理想的生化实验。 第一个目的是阐明钙和cAMP在 介导CRH刺激的信号转导，导致转录 在细胞核中激活。 细胞内钙将通过 显微荧光测量并与POMC水平进行比较 通过初级转录物溶液测量的转录 杂交/核酸酶保护和连续转录测定。 两 将讨论一些基本问题。a）是基础细胞内Ca++ 足够的是CRH/cAMP刺激的POMC所需的升高， c-fos基因表达B）Cd ~（++）抑制细胞增殖的机制是什么？ 基础和CRH激活POMC转录和c-fos参与？ 第二个目标将详细描述主要的顺式作用元件 （启动子区234/-133）负责介导CRH活化， 以及受信号转导调节的转录因子 在这个节目的第一部分调查的事件。 四个问题将 被解决。a）基础和CRH诱导因子的测定 （c-fos？）与主要（-234/-133）CRH调节POMC相互作用 启动子元件B）功能性解剖-234/133区域， 特异性启动子突变体，以确定 CRH转录调控的基础顺式元件和调节顺式元件 应答在异源TK/CAT启动子/报告系统中。C.完全 描述了由-171/-160元件组成的转录复合物 以及与之结合的蛋白质在调节CRH中的作用 方面的影响. d）-234/-133启动子元件中的突变是否引起 重大职能变化（目标2b）在以下方面具有类似的效果： 整个同源POMC启动子 除了加深我们对这一规则的理解外， 重要的神经内分泌基因，这些研究也将提高我们的 关于基因的分子和细胞机制的知识， 由细胞内钙离子调节，这个问题目前还很难解决， 明白

The goal of this program is to elucidate and characterize the cellular and molecular mechanisms responsible for the CRH mediated activation of POMC gene expression in the anterior pituitary corticotroph. This proposal is supported by our and others previous work showing the Importance of calcium as well as cAMP in mediating CRH signal transduction and our recent Identification of novel cis-acting elements in the POMC promoter (-700/+63) responding to CRH. These studies will be performed in the well established AtT20 tumor cell system. The AtT20 cell line has been utilized extensively for investigating POMC gene expression and Is Ideal for the proposed biochemical experiments. The first aim concerns elucidating the exact roles of calcium and cAMP In mediating CRH-stimulated signal transduction resulting in transcriptional activation in the nucleus. Intracellular calcium will be quantitated by microfluorescence measurements and compared to levels of POMC transcription measured by primary transcript solution hybridization/nuclease protection and run-on transcription assays. Two basic questions will be addressed. a) Is basal intracellular Ca++ sufficient of is an elevation required for CRH/cAMP stimulated POMC and c-fos gene expression? b) What is the mechanism of Cd++ inhibition of basal and CRH activated POMC transcription and is c-fos involved? The second aim will characterize in detail the major cis-acting elements (promoter region 234/-133) responsible for mediating CRH activation as well as the transacting factors regulated by the signal transduction events investigated in the first part of this program. Four issues will be addressed. a) Determination of the basal and CRH induced factors (c-fos?) which interact with the major (-234/-133) CRH regulatory POMC promoter element. b) Functionally dissect the -234/133 region with specific promoter mutants to determine the relative contribution between basal and regulated cis-elements in mediating the CRH transcriptional response In a heterologous TK/CAT promoter/reporter system. c) Fully delineate the transcriptional complex consisting of the -171/-160 element and the protein(s) that bind to it for their role in regulating CRH effects. d) Do mutations in the -234/-133 promoter element which cause major functional changes (aim 2b) have similar effects in the context of the entire homologous POMC promoter? In addition to furthering our understanding of the regulation of this important neuroendocrine gene, these studies will also enhance our knowledge about the molecular and cellular mechanisms by which genes are regulated by intracellular calcium ion, an issue that Is currently poorly understood.