PACAP‐regulated phenylethanolamineN‐methyltransferase gene expression

J. Neurochem.(2010)115, 1195–1205.AbstractPituitary adenylate cyclase activating polypeptide (PACAP) induces the proximal −893 bp of rat phenylethanolamineN‐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.