title: Composite glucocorticoid regulation at a functionally defined negative glucocorticoid response element of the human corticotropin-releasing hormone gene.
abstract: Glucocorticoid-dependent negative feedback of the hypothalamic-pituitary-adrenal axis is mediated in part through direct inhibition of hypothalamic CRH gene transcription. In the present study, we sought to further localize and characterize glucocorticoid receptor (GR) and AP-1 interactions at a functionally defined negative glucocorticoid response element (nGRE) of the CRH promoter. Transient transfection studies in mouse corticotroph AtT-20 cells demonstrated that internal deletion of the nGRE (-278 to -249 nucleotides) within the context of 1 kb of the intact CRH promoter resulted in decreased 8-BrcAMP stimulation and glucocorticoid-dependent repression of CRH promoter activity. The nGRE conferred transcriptional activation by both cAMP and overexpressed c-jun or c-fos AP-1 nucleoproteins as well as specific glucocorticoid-dependent repression to a heterologous promoter. A similar profile of regulation was observed for the composite GRE derived from mouse proliferin promoter. The CRH nGRE was clearly distinct from the consensus cAMP response element (CRE) at -224 nucleotides, which increased basal activity and cAMP responsiveness of a heterologous promoter but did not confer glucocorticoid-dependent repression. High-affinity binding sites for both GR and AP-1 nucleoproteins were identified at adjacent elements within the nGRE. Mutations that disrupted either GR or AP-1 binding activity were associated with loss of glucocorticoid-dependent repression. These results are consistent with a composite mechanism of glucocorticoid-dependent repression involving direct DNA binding of GR and AP-1 nucleoproteins at discrete adjacent sites within the CRH promoter.
authors.affiliation: Department of Medicine, Albuquerque Veterans Administration Medical Center, New Mexico 87108, USA.
authors.forename: S P
authors.initials: SP
authors.lastname: Malkoski
authors.forename: R I
authors.initials: RI
authors.lastname: Dorin
mesh_terms: D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D017124:Conserved Sequence; D003346:Corticotropin-Releasing Hormone / Q000235:genetics* / Q000378:metabolism; D000242:Cyclic AMP / Q000378:metabolism; D017362:Cyclic AMP Response Element-Binding Protein / Q000235:genetics / Q000378:metabolism; D016762:Genes, fos; D016758:Genes, jun; D005938:Glucocorticoids / Q000378:metabolism*; D006801:Humans; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D051379:Mice; D009154:Mutation; D010903:Pituitary Gland, Anterior / Q000166:cytology / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011965:Receptors, Glucocorticoid / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012045:Regulatory Sequences, Nucleic Acid; D020218:Response Elements / Q000502:physiology*; D017384:Sequence Deletion; D018808:Transcription Factor AP-1 / Q000378:metabolism; D014158:Transcription, Genetic
chemical_list: D017362:Cyclic AMP Response Element-Binding Protein; D005938:Glucocorticoids; D011965:Receptors, Glucocorticoid; D011994:Recombinant Proteins; D018808:Transcription Factor AP-1; D003346:Corticotropin-Releasing Hormone; D000242:Cyclic AMP; D008156:Luciferases