PACAP‐regulated phenylethanolamine N‐methyltransferase gene expression

Abstract

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.