h-89 and Neuroendocrine-Tumors

The mechanisms by which neuroendocrine stimulants regulate CCK gene transcription are unclear. We examined promoter activation by pituitary adenylate cyclase-activating polypeptide (PACAP), a known CCK secretagogue, in the enteroendocrine cell line STC-1. The promoter region from -70 to -87 bp, relative to the transcriptional start site, contains a composite calcium/cyclic AMP response element (CRE)/activator protein 1 (AP1) site that may bind CRE binding protein (CREB) and AP1. PACAP (with IBMX) stimulated expression of an 87-bp construct 3.35+/-0.36-fold but had no effect on a -70 construct. The effect was blocked by the protein kinase A inhibitor H-89 and by a dominant-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 treatment. Electrophoretic mobility shift assay and supershift analysis revealed that CREB and not AP1 bound to the CRE/AP1 site and that PACAP increased the proportion of phosphorylated CREB that was bound. We conclude that PACAP increases CCK gene expression via a cAMP-mediated pathway involving CREB phosphorylation by protein kinase A and activation of a composite CRE/AP1 site.

Topics: Animals; Calcium; Cholecystokinin; Colforsin; Cyclic AMP; Cyclic AMP Response Element-Binding Protein; Cyclic AMP-Dependent Protein Kinases; Electrophoresis; Enzyme Inhibitors; Gene Expression Regulation, Neoplastic; Genes, Reporter; Isoquinolines; Luciferases; Mice; Mutagenesis; Neuroendocrine Tumors; Neuropeptides; Oligonucleotide Probes; Phosphorylation; Pituitary Adenylate Cyclase-Activating Polypeptide; Promoter Regions, Genetic; Rats; Sulfonamides; Transcription Factor AP-1; Transcription, Genetic; Tumor Cells, Cultured