NEGATIVE GLUCOCORTICOID REGULATION OF CYCLIC ADENOSINE 3',5'-MONOPHOSPHATE-STIMULATED CORTICOTROPIN-RELEASING HORMONE-REPORTER EXPRESSION IN ATT-20 CELLS

The negative glucocorticoid regulation of CRH gene expression is a cri tical control element in the hypothalamic-pituitary-adrenal axis. In t his study, the molecular mechanisms mediating the glucocorticoid repre ssion of cAMP-induced CRH-reporter expression in AtT-20 cells have bee n examined, In these cells, dexamethasone decreases forskolin-induced expression of CRH-reporter activity in a dose-dependent manner. This r epression is mediated by the glucocorticoid receptor (GR) and does not require ongoing protein synthesis, Several binding sites for the GR D NA-binding domain were identified within the CRH 5'-flanking and 5'-un translated regions utilizing in vitro DNase I protection assays. These sites were independently mutated and/or deleted. Functional studies i n transfected cells suggest that none of the protected DNA sequences m ediate the glucocorticoid regulation and that the regulatory element(s ) mediating negative glucocorticoid regulation is contained within the CRH DNA sequences from -248 to +4 bp relative to the major transcript ion initiation site, To further localize the DNA sequence(s) responsiv e to glucocorticoids, DNA fragments containing various amounts of huma n CRH 5'flanking sequences were inserted 5' to the SV40 promoter. An 1 8-bp DNA fragment containing the CRH cAMP-responsive element is suffic ient to confer both positive cAMP regulation and glucocorticoid repres sion of cAMP-stimulated expression to the SV40 promoter. These results suggest that glucocorticoid repression of forskolin-activated CRH-rep orter expression in AtT-20 cells occurs via interference with the cAMP -mediated activation of gene expression, possibly via direct or indire ct interactions between the GR and the cAMP-responsive element-binding proteins.