Control of CCK gene transcription by PACAP in STC-1 cells.
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.
Deavall
D G
DG
Physiological Laboratory, University of Liverpool, Liverpool L69 3BX, United Kingdom.
Raychowdhury
R
R
Dockray
G J
GJ
Dimaline
R
R
D000818:Animals; D002118:Calcium / Q000378:metabolism; D002766:Cholecystokinin / Q000235:genetics*; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000378:metabolism; D017362:Cyclic AMP Response Element-Binding Protein / Q000235:genetics / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D004586:Electrophoresis; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects / Q000502:physiology; D017930:Genes, Reporter; D007546:Isoquinolines / Q000494:pharmacology; D008156:Luciferases / Q000235:genetics; D051379:Mice; D016296:Mutagenesis / Q000502:physiology; D018358:Neuroendocrine Tumors; D009479:Neuropeptides / Q000235:genetics*; D015345:Oligonucleotide Probes; D010766:Phosphorylation; D051219:Pituitary Adenylate Cyclase-Activating Polypeptide; D011401:Promoter Regions, Genetic / Q000502:physiology; D051381:Rats; D013449:Sulfonamides; D018808:Transcription Factor AP-1 / Q000235:genetics / Q000378:metabolism; D014158:Transcription, Genetic / Q000502:physiology*; D014407:Tumor Cells, Cultured
C495402:Adcyap1 protein, mouse; C495403:Adcyap1 protein, rat; D017362:Cyclic AMP Response Element-Binding Protein; D004791:Enzyme Inhibitors; D007546:Isoquinolines; D009479:Neuropeptides; D015345:Oligonucleotide Probes; D051219:Pituitary Adenylate Cyclase-Activating Polypeptide; D013449:Sulfonamides; D018808:Transcription Factor AP-1; D005576:Colforsin; D002766:Cholecystokinin; D000242:Cyclic AMP; D008156:Luciferases; D017868:Cyclic AMP-Dependent Protein Kinases; C063509:N-(2-(4-bromocinnamylamino)ethyl)-5-isoquinolinesulfonamide; D002118:Calcium