Inhibition of corticotrophin-releasing hormone transcription by inducible cAMP-early repressor in the hypothalamic cell line, 4B.

We have shown recently that the rapid decline in corticotrophin-releasing hormone (CRH) transcription following activation by stress is associated with induction and binding to the CRH promoter of the repressor isoforms of cAMP responsive element modulator (CREM), inducible cAMP early repressor (ICER). The ability of ICER to inhibit CRH transcription was examined in the hypothalamic cell line, 4B, which expresses CRH. Co-transfection of the inhibitory isoforms of CREM, ICER I and II and CREMbeta, and CRH promoter-luciferase constructs in 4B cells blunted basal and forskolin-stimulated CRH promoter activity, an effect which was abolished by mutation of the CRE of the CRH promoter. Western blot analyses and electromobility gel-shift and super-shift showed increases in endogenous ICER after 3 h of incubation with forskolin. Consistent with an inhibitory effect of CREM on CRH transcription, chromatin immunoprecipitation assays in cells transfected with ICER I revealed recruitment of CREM by the CRH promoter in conjunction with decreases in Pol II association. The study shows that generation of ICER following prolonged stimulation with forskolin, or transfection of an ICER expression vector in hypothalamic cell lines expressing CRH, is associated with CREM binding to the CRH promoter and transcriptional repression. The data support the hypothesis that induction of repressor isoforms of CREM is part of an intracellular feedback mechanism contributing to the termination of CRH transcription during stimulation.