LOCALIZATION OF A NEGATIVE GLUCOCORTICOID RESPONSE ELEMENT OF THE HUMAN CORTICOTROPIN-RELEASING HORMONE GENE

Corticotropin releasing hormone (CRH) plays a primary role in mediatin g suprapituitary activation of the hypothalamic-pituitary-adrenal axis and is an important physiologic target of negative feedback regulatio n by glucocorticoids. We sought to define cis-acting regions of the CR H promoter responsible for cAMP-dependent activation and glucocorticoi d-dependent repression of CRH promoter activity. In transiently transf ected AtT-20 cells, cAMP-dependent transcriptional activation was medi ated largely through a classical, consensus, cAMP-response element (CR E) at -224 bp. Dexamethasone (DEX) produced a specific 2-3-fold repres sion of cAMP-stimulated, but not basal, CRH promoter activity. Using a series of 5' nested deletions, dexamethasone-dependent repression of cAMP-stimulated CRH promoter activity was localized to promoter sequen ces between -278 and -249 bp. Specific, high-affinity binding of gluco corticoid receptor (GR) DNA-binding domain to this promoter region was observed using an eletrophoretic mobility shift assay (EMSA). We conc lude that (i) cAMP dependent activation of the CRH promoter is mediate d primarily by the CRE at -224 bp, (ii) glucocorticoid-dependent repre ssion is specific for the CRH promoter, and not a generalized effect o f glucocorticoid signaling or interference with the protein kinase A ( PKA) signaling pathway, (iii) a highly conserved region between -278 a nd -249 bp is critical for glucocorticoid dependent repression, and (i v) GR is capable of interacting directly with this functionally define d negative glucocorticoid response element of the CRH promoter. (C) 19 97 Elsevier Science Ireland Ltd.