Human corticotropin releasing hormone (hCRH), a 41 amino acid peptide synthesized in the hypothalamus, is a major regulator of the hypothalamic-pituitary-adrenal axis and thus plays a critical role in the normal endocrine response to stress. Hypotension, hemorrhage, hypovolemia, and adrenalectomy all stimulate CRH secretion. In addition, abnormal CRH secretion may play a role in the etiology of several important health problems, including stress-induced hypothalamic amenorrhea, Cushing's disease, Nelson's disease, and suppression of the hypothalamic-pituitary- adrenal axis after glucocorticoid treatment. Despite the importance of normal CRH secretion for man's well-being, the regulation of CRH gene expression in the hypothalamus and other tissues is poorly understood. Thus, the overall goal of this project is to understand the molecular basis for the regulation of human CRH gene expression by hormonal and cell-specific factors. Initially, the mouse anterior pituitary cell line, AtT-20, permanently transfected with the hCRH gene will be used as a multihormonal model for hCRH gene regulation. Factors which alter CRH gene expression will be identified and their influence on the rate of hCRH gene transcription, the structure and stability of hCRH mRNA, as well as on the biosynthesis and secretion of hCRH peptide will be characterized. The inhibition of hCRH gene expression with glucocorticoids and the stimulation of hCRH gene expression via the cAMP-dependent pathway will be examined in depth. Expression of the hCRH gene in neural, fibroblast, kidney and other cell lines will be used to identify cell- specific alterations in hCRH gene expression. Specific nucleotide sequences within the hCRH gene responsible for its correct expression and regulation by hormonal and cell-specific factors, will be identified by combining mutation analysis of the hCRH promoter with transient expression in heterlogous cells. The interaction between putative negative and positive regulatory elements as well as their dependence on orientation and location will be studied. The results of these experiments should allow us to suggest a detailed model explaining the organization and regulated expression of the hCRH gene.
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