Cyclic adenosine 3′,5′-monophosphate responsive element binding protein phosphorylation is required but not sufficient for activation of corticotropin releasing hormone transcription

cAMP is a major regulator of CRH transcription. However, receptors activating CRH neurons (alpha-adrenergic and glutamatergic) do not signal through cAMP, suggesting that calcium phospholipid-dependent signaling synergizes with small elevations of intracellular cAMP. To test this hypothesis, we examined the relationship between activation of CRH transcription, cAMP production, and cAMP response element binding protein (CREB) phosphorylation in neuronal cultures treated with the adenylyl cyclase stimulator, forskolin, the phorbol ester, phorbol-12-myristate-13-acetate (PMA), or their combination. Forskolin, at threshold concentrations for cAMP production and CREB phosphorylation, induced CRH promoter-driven luciferase activity in 4B cells (EC(50) = 0.7 mu M) and CRH primary transcript in hypothalamic neurons (EC50 = 0.6 mu M). PMA alone failed to activate CRH transcription despite being as effective as forskolin in phosphorylating CREB (Ser133 and Ser121). Although PMA potentiated the effect of low forskolin concentrations on CRH transcription and CREB phosphorylation, there was no correlation between phosphorylated CREB levels and activation of CRH transcription. Similarly, the calcium/calmodulin-dependent kinase inhibitor, KN-93, enhanced PMA plus forskolin-stimulated CREB phosphorylation and inhibited CRH transcription. Suppression of CREB phosphorylation by the protein kinase A inhibitor, H89, or the CREB dominant negative, A-CREB, did not affect basal but blocked forskolin-stimulated transcription. This study shows that calcium phospholipid-dependent pathways potentiate the ability of small elevations of intracellular cAMP to activate CRH transcription, providing a mechanism by which non-cAMP-dependent regulators induce CRH gene expression. In addition, the data indicate that phosphorylated CREB is essential but not sufficient for activation of CRH transcription, suggesting that full promoter stimulation requires the interaction of phosphorylated CREB with a coactivator.