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,
