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 mus cle cells (VSMC). AT(1) mRNA expression in cultured bovine VSMC increa sed twofold after 8 h of protein kinase C (PKC) activation with phorbo l 12-myristate 13-acetate (PMA), whereas stimulation with forskolin di d not alter the AT(1) mRNA level. The expression of AT(1) promoter/exo n 1 [-513/+92 base pairs (bp)] luciferase constructs transfected into VSMC increased 2.4-fold with PMA stimulation. In-gel kinase assays dem onstrated 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-activat ed VSMC, identified as activator protein 1 (AP-1) complex in competiti on assays, to a radiolabeled AT(1)-promoter fragment (-368/-399 bp). R ecombinant AP-1 binds in a sequence-specific manner to the -386/-399-b p region. Site-specific mutagenesis destroying the AP-1 site, the adja cent polyoma enhancer activator 3 element, or both sites simultaneousl y indicated that both sites together are necessary and sufficient to c ontrol 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 promi nent role for PKC/MAPK and Ets proteins in AT(1) regulation.