REGULATION OF POMC GENE EXPRESSION IN PITUITARY

这个项目的目标是阐明和描述细胞的特征 CRH介导的细胞活化的分子机制 POMC基因在垂体前叶促肾上腺皮质激素中的表达这 提案得到了我们和其他人的支持，之前的工作表明 钙离子和cAMP在介导CRH信号中的重要性 新的顺式作用元件的转导和我们的最新鉴定 在对CRH反应的POMC启动子(-700/+63)中。这些研究将是 在成熟的AtT20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，是建议的生化实验的理想选择。 第一个目标是阐明钙和cAMP在体内的确切作用。 介导CRH刺激的信号转导导致转录 核内的激活。细胞内钙将通过以下方式定量 微量荧光测量及其与POMC水平的比较 用初级转录溶液测定转录 杂交/核酸酶保护和连续转录检测。二 基本问题将会得到解答。A)是基础细胞内钙离子 足够的是CRH/cAMP刺激的POMC和 C-fos基因表达？B)Cd++抑制的机制是什么？ 基础和CRH激活POMC转录，c-fos是否参与？ 第二个目标将详细描述顺式作用的主要要素。 (启动子区域234/-133)负责介导CRH的激活，如 以及由信号转导调节的交易因子 本节目第一部分调查的事件。将有四个问题 被称呼为。A)确定基础因素和CRH诱导因素 (C-FOS？)它们与主要的(-234/-133)CRH调节POMC相互作用 启动子元素。B)对-234/133区域进行功能剖析 确定特定启动子突变体对 调节CRH转录的基础和调控顺式元件 在异源TK/CAT启动子/报告系统中的反应。C)充分 描绘由-171/-160元件组成的转录复合体 以及与之结合的蛋白质(S)，因为它们在调节CRH方面发挥了作用 效果。D)-234/-133启动子元件发生突变，导致 重大职能变化(目标2b)在以下背景下具有类似的影响 整个同源POMC启动子？ 除了加深我们对这一规则的理解之外 重要的神经内分泌基因，这些研究也将增强我们的 关于基因形成的分子和细胞机制的知识 受细胞内钙离子的调节，这是一个目前很差的问题 明白了。

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.