Transcriptional Activation of Scavenger Receptor Expression in Human Smooth Muscle Cells Requires AP-1/c-Jun and C/EBPß

Both AP-1 Binding and JNK Activation Are Induced by Phorbol Esters and Oxidative Stress

From the Gladstone Institute of Cardiovascular Disease and Cardiovascular Research Institute (M.M., R.E.P.), and the Departments of Pediatrics (M.M.) and Pathology (R.E.P.), University of California, San Francisco, and the Department of Medicine (C.K.G.), University of California, San Diego.

Correspondence to Robert E. Pitas, PhD, Gladstone Institute of Cardiovascular Disease, PO Box 419100, San Francisco, CA 94141-9100. E-mail rpitas{at}gladstone.ucsf.edu

Abstract—Reactive oxygen species generated by treatment of smooth muscle cells (SMCs) with either phorbol 12-myristate 13-acetate or with the combination of H₂O₂ and vanadate strongly induce expression of the class A scavenger receptor (SR-A) gene. In the current studies, cis-acting elements in the proximal 245 bp of the SR-A promoter were shown to direct luciferase reporter expression in response to oxidative stress in both SMCs and macrophages. A composite activating protein-1 (AP-1)/ets binding element located between –67 and –50 bp relative to the transcriptional start site is critical for macrophage SR-A activity. Mutation of either the AP-1 or the ets component of this site also prevented promoter activity in SMCs. Mutation of a second site located between –44 and –21 bp, which we have identified as a CCAAT/enhancer binding protein (C/EBP) element, reduced the inducible activity of the promoter in SMCs by 50%, suggesting that combinatorial interactions between these sites are necessary for optimal gene induction. Interactions between SMC nuclear extracts and the SR-A promoter were analyzed by electrophoretic mobility shift assay. c-Jun/AP-1 binding activity, specific for the –67- to –50-bp site, was induced in SMCs by the same conditions that increased SR-A expression. Moreover, phorbol 12-myristate 13-acetate, H₂O₂, or the combination of H₂O₂ and sodium orthovanadate (vanadate) activated c-Jun–activating kinase. The binding activity within SMC extracts specific for the C/EBP site was shown to be C/EBPß in SMCs. Taken together, these findings demonstrate that reactive oxygen species regulate the interactions between c-Jun/AP-1 and C/EBPß in the SR-A promoter. Furthermore, induction of oxidative stress in THP-1 cells, with a combination of 10 µmol/L vanadate and 100 µmol/L H₂O₂, induced macrophage differentiation, adhesion, and SR activity. These data suggest that vascular oxidative stress may contribute to the induction of SR-A expression and thereby promote the uptake of oxidatively modified low density lipoprotein by both macrophage and SMCs to produce foam cells in atherosclerotic lesions.

Key Words: scavenger receptor • activating protein-1 • c-Jun–activating kinase • phorbol ester • oxidative stress

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