Regulation of human corticotropin-releasing hormone gene expression.

Abstract

The mammalian stress response is controlled, in part, by hypothalamic corticotropin-releasing hormone (CRH)-expressing neurons that integrate multiple neuronal and hormonal signals to elicit an appropriate cellular response. As part of the feedback control in the stress axis, glucocorticoids repress CRH gene expression to restore homeostasis. Paradoxically, in human placenta, CRH expression is positively regulated by glucocorticoids, suggesting that glucocorticoids differentially regulate CRH expression in a cell-specific fashion. In this thesis, transfection of CRH-reporter constructs into cultured cell lines and in vitro biochemical methods have been utilized to characterize the molecular mechanisms mediating regulation of CRH expression by cAMP, membrane depolarization and glucocorticoids. A cAMP responsive element (CRE) has been localized in the human CRH 5$\sp\prime$ flanking DNA which activates CRH-reporter activity. In PC-12 (rat pheochromocytoma) cells, membrane depolarization increased CRH-reporter expression via calcium influx in a cAMP-dependent manner. Heterologous promoter constructs demonstrated that the CRH CRE conferred both the positive cAMP and the synergistic cAMP/depolarization regulation, suggesting that these two regulatory pathways converge at the level of the CRE and the CRE-binding proteins. The differential regulation of the CRH gene by glucocorticoids was demonstrated in AtT-20 (mouse pituitary) and PC-12 cells. A CRH-reporter construct containing 663 bp of 5$\sp\prime$ flanking DNA demonstrated cAMP-induced activity in both cell lines; glucocorticoids repressed this cAMP-mediated activation in AtT-20 cells whereas a synergistic increase in cAMP-stimulated reporter activity was observed in PC-12 cells. The glucocorticoid receptor (GR) is required for this differential regulation and several in vitro GR binding sites were identified by DNase I protection analysis. However, independent mutation and/or deletion of these sites did not affect the glucocorticoid regulation in AtT-20 and PC-12 cells. A 42 bp fragment of the CRH gene that includes the CRE appears to be sufficient to confer negative glucocorticoid regulation to a heterologous promoter (SV40). Our studies therefore demonstrate that this 42 bp CRH DNA fragment including the CRE is a multifunctional DNA regulatory element capable of integrating multiple signals resulting in a coordinated transcriptional response.