Protein kinase C-dependent regulation of the human AT1 promoter in vascular smooth muscle cells.
The expression level of angiotensin II (ANG II) type 1 receptors (AT1) determines the magnitude of ANG II signaling in vascular smooth muscle cells (VSMC). AT1 mRNA expression in cultured bovine VSMC increased twofold after 8 h of protein kinase C (PKC) activation with phorbol 12-myristate 13-acetate (PMA), whereas stimulation with forskolin did not alter the AT1 mRNA level. The expression of AT1 promoter/exon 1 [-513/+92 base pairs (bp)] luciferase constructs transfected into VSMC increased 2.4-fold with PMA stimulation. In-gel kinase assays demonstrated rapid phosphorylation of mitogen-activating protein kinases (MAPK) ERK1 and ERK2 by PMA. Electrophoretic gel mobility shift assays showed sequence-specific binding of nuclear proteins from PMA-activated VSMC, identified as activator protein 1 (AP-1) complex in competition assays, to a radiolabeled AT1-promoter fragment (-368/-399 bp). Recombinant AP-1 binds in a sequence-specific manner to the -386/-399-bp region. Site-specific mutagenesis destroying the AP-1 site, the adjacent polyoma enhancer activator 3 element, or both sites simultaneously indicated that both sites together are necessary and sufficient to control basal and PMA-induced activation of the human AT1 promoter in transfected VSMC. The capability of the phorbol ester PMA to activate the human AT1 promoter in VSMC via an AP-1 element suggests a prominent role for PKC/MAPK and Ets proteins in AT1 regulation.
Holzmeister
J
J
Department of Internal Medicine, Cardiology, Virchow Klinikum of the Humboldt University, Berlin, Germany.
Graf
K
K
Warnecke
C
C
Fleck
E
E
Regitz-Zagrosek
V
V
D000818:Animals; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism; D002417:Cattle; D002478:Cells, Cultured; D017868:Cyclic AMP-Dependent Protein Kinases / Q000494:pharmacology; D006367:HeLa Cells; D006801:Humans; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000187:drug effects* / Q000502:physiology*; D016297:Mutagenesis, Site-Directed; D011401:Promoter Regions, Genetic / Q000187:drug effects*; D011493:Protein Kinase C / Q000494:pharmacology*; D012333:RNA, Messenger / Q000378:metabolism; D011945:Receptors, Angiotensin / Q000235:genetics* / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D018808:Transcription Factor AP-1 / Q000378:metabolism
D012333:RNA, Messenger; D011945:Receptors, Angiotensin; D018808:Transcription Factor AP-1; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D013755:Tetradecanoylphorbol Acetate