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 the wild-type rat PNMT promoter-luciferase reporter gene construct pGL3RP893 into PC12 cells. Forskolin treatment (10 microM) of the transfected cells for 3--6 h maximally induced luciferase threefold. Induction by forskolin was mimicked by the cAMP analog, 8-Br-cAMP, and prevented in PC12 cells pretreated 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 constructs harboring > or = 60 bp and < 893 bp of PNMT promoter demonstrated that the cAMP-responsive element(s) lay between < 392 bp and > or =60 bp. Within this region of the promoter lies a functional binding element for Egr-1, a transcriptional activator of the PNMT gene. Forskolin treatment of PC12 cells also rapidly increased nuclear levels 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 decreased forskolin activation of the PNMT promoter. These findings demonstrate that the rat PNMT gene promoter can be activated via the cAMP-PKA signal transduction pathway, mediated by the immediate early gene transcription factor, Egr-1.
Department of Psychiatry, Harvard Medical School, Laboratory of Molecular and Developmental Neurobiology, McLean Hospital, Belmont, Massachusetts 02478, USA.
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Tai
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Morita
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Wong
D015124:8-Bromo Cyclic Adenosine Monophosphate / Q000494:pharmacology; D000818:Animals; D005576:Colforsin / Q000494:pharmacology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors; D004268:DNA-Binding Proteins / Q000378:metabolism*; D051766:Early Growth Response Protein 1; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D017930:Genes, Reporter; D017874:Immediate-Early Proteins / Q000378:metabolism; D007546:Isoquinolines / Q000494:pharmacology; D007700:Kinetics; D008156:Luciferases / Q000235:genetics; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D016716:PC12 Cells; D010625:Phenylethanolamine N-Methyltransferase / Q000235:genetics*; D011401:Promoter Regions, Genetic / Q000187:drug effects; D051381:Rats; D015398:Signal Transduction; D013449:Sulfonamides; D014157:Transcription Factors / Q000378:metabolism*; D014162:Transfection; D001616:beta-Galactosidase / Q000235:genetics
D004268:DNA-Binding Proteins; D051766:Early Growth Response Protein 1; C486110:Egr1 protein, rat; D017874:Immediate-Early Proteins; D007546:Isoquinolines; D013449:Sulfonamides; D014157:Transcription Factors; D005576:Colforsin; D015124:8-Bromo Cyclic Adenosine Monophosphate; D008156:Luciferases; D010625:Phenylethanolamine N-Methyltransferase; D017868:Cyclic AMP-Dependent Protein Kinases; D020928:Mitogen-Activated Protein Kinases; D001616:beta-Galactosidase; C063509:N-(2-(4-bromocinnamylamino)ethyl)-5-isoquinolinesulfonamide