Role of Egr-1 in cAMP-dependent protein kinase regulation of the phenylethanolamine N-methyltransferase gene

The molecular mechanism by which cAMP activates the rat phenylethanolamine N-methyltransferase (PNMT) gene was examined by transient transfection of t he wild-type rat PNMT promoter-luciferase reporter gene construct pGL3RP893 into PC12 cells. Forskolin treatment (10 muM) of the transfected cells for 3-6 h maximally induced luciferase threefold. Induction by forskolin was m imicked by the cAMP analog, 8-Br-cAMP, and prevented in PC12 cells pretreat ed with the protein kinase A (PKA) inhibitor H-89 or co-transfected with an expression construct for PKI, a polypeptide inhibitor of PKA. Furthermore, forskolin did not activate the PNMT promoter when the 893 bp PNMT promoter -reporter gene construct was transfected into the PKA-deficient cell line. A126. Detailed examination of the forskolin responsiveness of PNMT construc ts harboring greater than or equal to 60 bp and < 893 bp of PNMT promoter d emonstrated that the cAMP-responsive element(s) lay between < 392 bp and gr eater than or equal to 60 bp. Within this region of the promoter lies a fun ctional binding element for Egr-1, a transcriptional activator of the PNMT gene. Forskolin treatment of PC12 cells also rapidly increased nuclear leve ls of Egr-1 and the catalytic subunit of PKA (PKA-C), with the rise in PKA- C preceding that of Egr-1. Mutation of the -165 bp Egr-1 site markedly decr eased forskolin activation of the PNMT promoter. These findings demonstrate that the rat PNMT gene promoter can be activated via the cAMP-PKA signal t ransduction pathway, mediated by the immediate early gene transcription fac tor, Egr-1.