Abstract
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.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Animals
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Calcium / metabolism
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Cholecystokinin / genetics*
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Colforsin / pharmacology
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Cyclic AMP / metabolism
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Cyclic AMP Response Element-Binding Protein / genetics
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Cyclic AMP Response Element-Binding Protein / metabolism
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Cyclic AMP-Dependent Protein Kinases / antagonists & inhibitors
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Cyclic AMP-Dependent Protein Kinases / metabolism
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Electrophoresis
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Enzyme Inhibitors / pharmacology
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Gene Expression Regulation, Neoplastic / drug effects
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Gene Expression Regulation, Neoplastic / physiology
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Genes, Reporter
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Isoquinolines / pharmacology
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Luciferases / genetics
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Mice
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Mutagenesis / physiology
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Neuroendocrine Tumors
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Neuropeptides / genetics*
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Oligonucleotide Probes
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Phosphorylation
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Pituitary Adenylate Cyclase-Activating Polypeptide
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Promoter Regions, Genetic / physiology
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Rats
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Sulfonamides*
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Transcription Factor AP-1 / genetics
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Transcription Factor AP-1 / metabolism
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Transcription, Genetic / physiology*
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Tumor Cells, Cultured
Substances
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Adcyap1 protein, mouse
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Adcyap1 protein, rat
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Cyclic AMP Response Element-Binding Protein
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Enzyme Inhibitors
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Isoquinolines
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Neuropeptides
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Oligonucleotide Probes
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Pituitary Adenylate Cyclase-Activating Polypeptide
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Sulfonamides
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Transcription Factor AP-1
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Colforsin
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Cholecystokinin
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Cyclic AMP
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Luciferases
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Cyclic AMP-Dependent Protein Kinases
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N-(2-(4-bromocinnamylamino)ethyl)-5-isoquinolinesulfonamide
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Calcium