Corticotrope responsiveness to glucocorticoids is modulated via rapid CRF-mediated induction of the proto-oncogene c-fos

Abstract

Although corticotropin releasing factor (CRF) and glucocorticoid hormones (GC) act directly at the level on the anterior pituitary corticotrope cell to stimulate (CRF) or inhibit (GC) pro-opiomelanocortin (POMC) expression, the actions of GC on POMC have been shown to be impaired if corticotrope cells are coincubated or preincubated with CRF. In the present study we have measured secreted beta-endorphin (βEP) and changes in the level of nuclear POMC hnRNA as an indirect measure of gene transcription to characterize the molecular mechanisms involved in the CRF-mediated inhibition of glucocorticoid action. In primary cultures of rat anterior pituitary cells either co-treated or pretreated with CRF, acute dexamethasone (DEX)-mediated inhibition of POMC hnRNA levels was impaired. In contrast, the ability of CRF to block glucocorticoid action was abolished if the cells were pretreated with the protein synthesis inhibitor puromycin. Since previous studies have demonstrated that components of the AP1 transcription factor can modulate glucocorticoid receptor activity in other systems, we examined the regulation of the proto-oncogenes c- fos and c- jun in response to CRF. Treatment of the corticotrope cell line (AtT-20) with CRF rapidly activated c- fos mRNA to levels 11–12-fold above control by 30 and 60 min, with no apparent elevation of c- jun mRNA levels. Pretreatment of AtT-20 cells with antisense c- fos oligonucleotides prevented CRF from blocking glucocorticoid inhibition of POMC hnRNA levels and βEP release. In addition, in AtT-20 cells acutely stimulated with CRF, the number of nuclear localized glucocorticoid receptor-ligand complexes was indistinguishable from control cells suggesting that CRF-mediated impairment of glucocorticoid function is independent of steroid binding and nuclear translocation. Taken together, these data strongly suggest that CRF-induced blockade of glucocorticoid action in corticotrope cells is dependent upon the rapid de novo synthesis of Fos. This may represent in part the cellular mechanism whereby in vivo CRF can not only stimulate the corticotrope POMC system, but can also potentiate the response by acute impairment of negative glucocorticoid feedback.