Regulation of Human Corticotropin- Releasing Hormone Gene Expression by 3′,5′-Cyclic Adenosine Monophosphate in a Transformed Mouse Corticotroph Cell Line

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Abstract
In order to characterize potential mechanisms regulating the expression of the human CRH (hCRH) gene, an intact genomic fragment including 5''-flanking sequence of the hCRH gene was stably transfected into the mouse cortocotroph At2T20 cell line. The exogenous hCRH gene was expressed at a high frequency with accurate and efficient trascription in transformed cells. Northern blot analysis revealed a single species of CRH mRNA of 1.6 kilobases which was identical in size to human placental CRH mRNA. S1 analysis demonstrated a single cap site in both placenta and transformed AtT20 cells, corresponding to a site 23 base pairs downstream of the TATA box. Treatment with 8-bromo cAMP and phorbol ester resulted in a dose-dependent increase in CRH secretion during a 1-h incubation. Treatment with forskolin or 8-bromo-cAMP also produced a dose-dependent 4- to 10-fold increase in CRH mRNA levels, which was rapid (1 h) and sustained (6, 12, 24, and 48 h). These effects in a well characterized continuous cell culture system demonstrate pretranslational regulation of CRH expression by a cAMP-dependent pathway.

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