TRANSCRIPTIONAL REGULATION OF CRH GENE EXPRESSION

哺乳动物的应激反应在很大程度上是由 下丘脑-垂体-肾上腺(HPA)轴。关键的下丘脑释放 这个轴上的因子是促肾上腺皮质激素释放激素(CRH)。CRH 促肾上腺皮质激素(ACTH)的合成和分泌 脑下垂体前叶，进而刺激脑血管生成和释放 肾上腺皮质中的糖皮质激素。糖皮质激素然后调节 身体对压力的适应性反应。除了它在 下丘脑，CRH在大脑的许多其他区域产生， 外周，它似乎在适应性的协调中起作用 回应。因此，CRH的生产和释放代表着关键的控制级别 人类对外界刺激做出生理反应的能力可以 受到监管。 促肾上腺皮质激素释放激素的分泌调节已被广泛研究，但 负责激活或抑制表达的细胞机制 对CRH基因的了解仍然很少。这个项目的总体目标是 建议定义涉及到的分子机制 大鼠促肾上腺皮质激素释放激素基因的转录调控 类固醇和第二信使调节通路。先验基因 转移实验表明，crh基因受cAMP和cAMP的共同调控。 和糖皮质激素。该基因的糖皮质激素调节是 尤其耐人寻味，因为CRH基因似乎是不同的 受不同细胞类型的糖皮质激素调节。这部小说 糖皮质激素对CRH表达的调节将在 本提案具体侧重于：1)本地化和 阳性和/或基因的特征(通过定点突变) 用基因转移的方法治疗糖皮质激素阴性反应元件(S) 在培养的细胞系和原代培养物中；以及2)鉴定 用DNase I确定纯化的糖皮质激素受体的DNA结合部位 二甲基硫酸酯保护和干扰试验。然而， 转录因子经常相互作用，以中介独特的调节 任何特定的基因，所以糖皮质激素对CRH表达的调节 不能独立于涉及的其他监管机制而进行研究 在CRH的表达中。因此，我们将继续本地化其他客户代理 调控大鼠促肾上腺皮质激素释放激素基因的调控元件 5‘侧翼序列中DNA蛋白相互作用的特征 基因表达采用体外和体内生化方法。所获得的知识 这些研究将极大地增加我们对分子的理解 参与大鼠CRH基因转录调控的机制，以及 将使我们能够更好地理解这一重要的 体内的神经内分泌肽，

The mammalian stress response is mediated in large part by the hypothalamic-pituitary-adrenal (HPA) axis. The key hypothalamic releasing factor in this axis is corticotropin releasing hormone (CRH). CRH stimulates synthesis and secretion of adrenocorticotropin (ACTH) from the anterior pituitary which in turn stimulates the production and release of glucocorticoids from the adrenal cortex. Glucocorticoids then mediate the body's adaptive response to stress. In addition to its role in the hypothalamus, CRH is produced in many other regions of the brain and periphery where it appears to function in the coordination of adaptive responses. Thus, CRH production and release represent key control levels at which man's ability to respond physiologically to external stimuli can be regulated. The regulation of CRH secretion has been extensively studied, but the cellular mechanisms responsible for activating or repressing the expression of the CRH gene are still poorly understood. The overall goal of this proposal is to define the molecular mechanisms involved in the transcriptional regulation of the rat CRH gene, focusing specifically on the steroid and second messenger regulation pathways. Previous gene transfer experiments have shown that the CRH gene is regulated by both cAMP and glucocorticoids. The glucocorticoid regulation of this gene is especially intriguing, since the CRH gene appears to be differentially regulated by glucocorticoids in different cell types. This novel glucocorticoid regulation of CRH expression will be carefully examined in the present proposal by focusing specifically on: 1) the localization and characterization (by site-directed mutagenesis) of the positive and/or negative glucocorticoid responsive element(s) using gene transfer methods in cultured cell lines and primary cultures; and 2) the identification of DNA-binding sites for the purified glucocorticoid receptor using DNase I and dimethylsulfate protection and interference assays. However, transcription factors often interact to mediate the unique regulation of any specific gene, so the regulation of CRH expression by glucocorticoids cannot be studied independently of the other regulatory mechanisms involved in CRH expression. Therefore, we will continue to localize other cisacting control elements involved in regulation of the rat CRH gene and further characterize the DNAprotein interactions in the 5' flanking sequence of the gene using in vitro and in vivo biochemical methods. The knowledge gained from these studies will greatly increase our understanding of the molecular mechanisms involved in transcriptional control of the rat CRH gene, and will allow us to better understand the complex regulation of this important neuroendocrine peptide in vivo,