Control of CCK gene transcription by PACAP in STC-1 cells

The mechanisms by which neuroendocrine stimulants regulate CCK gene transcr iption are unclear. We examined promoter activation by pituitary adenylate cyclase-activating polypeptide (PACAP), a known CCK secretagogue, in the en teroendocrine cell line STC-1. The promoter region from -70 to -87 bp, rela tive to the transcriptional start site, contains a composite calcium/cyclic AMP response element (CRE)/activator protein 1 (AP1) site that may bind CR E binding protein (CREB) and AP1. PACAP (with IBMX) stimulated expression o f an 87-bp construct 3.35 +/- 0.36-fold but had no effect on a 270 construc t. The effect was blocked by the protein kinase A inhibitor H-89 and by a d ominant-negative CREB plasmid. Mutation of the CRE/AP1 site to a canonical CRE site did not affect the response to PACAP, but mutation to a canonical AP1 site prevented it. CREB phosphorylation was increased after PACAP treat ment. Electrophoretic mobility shift assay and supershift analysis revealed that CREB and not AP1 bound to the CRE/AP1 site and that PACAP increased t he proportion of phosphorylated CREB that was bound. We conclude that PACAP increases CCK gene expression via a cAMP-mediated pathway involving CREB p hosphorylation by protein kinase A and activation of a composite CRE/AP1 si te.