Premium
Role of Egr‐1 in cAMP‐dependent protein kinase regulation of the phenylethanolamine N ‐methyltransferase gene
Author(s) -
Tai T. C.,
Morita Kyoji,
Wong Dona L.
Publication year - 2001
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1046/j.1471-4159.2001.00189.x
Subject(s) - forskolin , phenylethanolamine , transfection , protein kinase a , microbiology and biotechnology , reporter gene , creb , promoter , activator (genetics) , biology , regulation of gene expression , response element , cyclic amp response element binding protein , chemistry , gene expression , gene , transcription factor , kinase , cell culture , biochemistry , enzyme , genetics , tyrosine hydroxylase
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 μ m ) 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 ≥ 60 bp and < 893 bp of PNMT promoter demonstrated that the cAMP‐responsive element(s) lay between < 392 bp and ≥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.