Protein kinase C-dependent regulation of the human AT1 promoter in vascular smooth muscle cells

• Published in: The American journal of physiology Vol. 273; no. 2 Pt 2; p. H655
• Main Authors: Holzmeister, J, Graf, K, Warnecke, C, Fleck, E, Regitz-Zagrosek, V
• Format: Journal Article
• Language: English
• Published: United States 01.08.1997
• Subjects: Animals; Calcium-Calmodulin-Dependent Protein Kinases - metabolism; Cattle; Cells, Cultured; Cyclic AMP-Dependent Protein Kinases - pharmacology; HeLa Cells; Humans; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinases; Muscle, Smooth, Vascular - cytology; Muscle, Smooth, Vascular - drug effects; Muscle, Smooth, Vascular - physiology; Mutagenesis, Site-Directed; Promoter Regions, Genetic - drug effects; Protein Kinase C - pharmacology; Receptors, Angiotensin - genetics; Receptors, Angiotensin - metabolism; RNA, Messenger - metabolism; Tetradecanoylphorbol Acetate - pharmacology; Transcription Factor AP-1 - metabolism
• ISSN: 0002-9513; 2163-5773
• DOI: 10.1152/ajpheart.1997.273.2.h655

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.