PACAP‐regulated phenylethanolamine N‐methyltransferase gene expression

• Published in: Journal of neurochemistry 2010-12, Vol.115 (5), p.1195-1205
• Main Authors: Tai, Tze Chun, Wong‐Faull, David C., Claycomb, Robert, Aborn, Jennifer L., Wong, Dona Lee
• Format: Article
• Language: English
• Subjects: Adenylate cyclase; Animals; AP‐2; Binding sites; Biological and medical sciences; Carrier Proteins - genetics; Carrier Proteins - metabolism; Cellular biology; cyclic AMP; Drug Interactions; EGR-1 protein; Egr‐1; Electrophoretic Mobility Shift Assay - methods; Enzyme Activation - drug effects; Fundamental and applied biological sciences. Psychology; Gene deletion; Gene expression; Gene Expression Regulation, Enzymologic - drug effects; Membrane Proteins - genetics; Membrane Proteins - metabolism; Molecular and cellular biology; Molecular genetics; Mutation; Neurochemistry; Neurotransmitter Agents - pharmacology; PACAP; PC12 Cells; Phenylethanolamine N-methyltransferase; Phenylethanolamine N-Methyltransferase - genetics; Phenylethanolamine N-Methyltransferase - metabolism; Pheochromocytoma cells; Phospholipase C; Pituitary adenylate cyclase-activating polypeptide; Pituitary Adenylate Cyclase-Activating Polypeptide - pharmacology; PKA; PLC; PNMT gene; Promoter Regions, Genetic - drug effects; Promoters; Proteins; Rats; RNA, Messenger - metabolism; RNA, Small Interfering - pharmacology; Rodents; Signal Transduction - drug effects; siRNA; Sp1 protein; Transcription Factor AP-2 - genetics; Transcription Factor AP-2 - metabolism; Transcription factors; Transcription. Transcription factor. Splicing. Rna processing; Transfection - methods
• ISSN: 0022-3042; 1471-4159
• DOI: 10.1111/j.1471-4159.2010.07005.x

J. Neurochem. (2010) 115, 1195–1205. Pituitary adenylate cyclase activating polypeptide (PACAP) induces the proximal −893 bp of rat phenylethanolamine N‐methyltransferase (PNMT) gene promoter in PC12 cells via PACAP type I receptors. Deletion mutation analysis suggested that the initial −392 bp of promoter, containing early growth response protein (Egr‐1), specificity protein 1 (Sp1) and activator protein 2 (AP‐2) binding sites (−165, −168 and −103 bp, respectively), was sufficient for PACAP activation. Egr‐1 and AP‐2 involvement was supported by PACAP induction of their mRNA and protein. Mutation of the Egr‐1, Sp1 and AP‐2 elements showed that the Egr‐1 site was essential for PACAP stimulation. Mutation of the −103 bp AP‐2 site partially reduced PACAP activation of the promoter. Mutation of two upstream AP‐2 sites at −573 and −650 bp, separately or in tandem, also prevented promoter induction by PACAP. siRNA knock‐down of Egr‐1 and AP‐2 suppressed promoter activation for the −893 bp construct. Egr‐1 siRNA knock‐down also eliminated the residual activation observed for the −103 bp AP‐2 mutant construct, suggesting that Egr‐1 and AP‐2 through respective −165 and −650/−573/−103 bp sites cooperatively stimulate the promoter. PACAP responses appear orchestrated through cAMP‐protein kinase A and phospholipase C signaling as MDL12,330A, H89 and U73122, respectively, inhibited promoter induction by PACAP and reduced PACAP‐stimulation of Egr‐1, AP‐2 and PNMT mRNA and protein and Egr‐1 and AP‐2 protein/DNA complex formation. Findings are the first to show that PACAP stimulates PNMT promoter‐driven gene expression via PACAP type I receptors and cAMP‐protein kinase A and phospholipase C signaling, recruiting Egr‐1 and AP‐2 as cooperative regulators, and the first to associate the transcription factor AP‐2 to PACAP‐mediated gene induction.