Protein kinase C-dependent regulation of the human AT(1) promoter in vascular smooth muscle cells

The expression level of angiotensin II (ANG II) type 1 receptors (AT(1)) determines the magnitude of ANG II signaling in vascular smooth muscle cells (VSMC). AT(1) 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 AT(1) mRNA level. The expression of AT(1) 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 AT(1)-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 AT(1) promoter in transfected VSMC. The capability of the phorbol ester PMA to activate the human AT(1) promoter in VSMC via an AP-1 element suggests a prominent role for PKC/MAPK and Ets proteins in AT(1) regulation.