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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2008;28:878.)
© 2008 American Heart Association, Inc.

Cell Biology and Signaling

AP-1–Dependent Transcriptional Regulation of NADPH Oxidase in Human Aortic Smooth Muscle Cells

Role of p22^phox Subunit

Adrian Manea; Simona A. Manea; Anca V. Gafencu; Monica Raicu; Maya Simionescu

From the Institute of Cellular Biology and Pathology ‘Nicolae Simionescu’, Bucharest, Romania.

Correspondence to Maya Simionescu, PhD, Institute of Cellular Biology and Pathology ‘Nicolae Simionescu’, 8 B.P. Hasdeu Street, Bucharest, P.O. Box 35-14, Romania. E-mail maya.simionescu{at}icbp.ro

Objective— NADPH oxidase (NADPHox) is the major source of reactive oxygen species in vascular diseases; the mechanisms of enzyme activation are not completely elucidated. AP-1 controls the expression of many genes linked to vascular smooth muscle cells (SMCs) dysfunction. In this study we searched for the role of AP-1 in the regulation of NADPHox expression and function in human aortic SMCs exposed to proinflammatory conditions.

Methods and Results— Cultured SMCs were exposed to either angiotensin II (Ang II) or tumor necrosis factor (TNF)-. The lucigenin-enhanced chemiluminescence assay and real-time polymerase chain reaction analysis revealed that AP-1 and mitogen-activated protein kinase inhibitors reduced both Ang II or TNF--dependent upregulation of NADPHox activity and mRNA expression (NOX1, NOX4, p67^phox, p47^phox, p22^phox). Inhibitors of AP-1 significantly diminished the Ang II or TNF--stimulated p22^phox promoter activity and protein level. Transient overexpression of c-Jun/c-Fos upregulated p22^phox promoter activity. Transcription factor pull-down assay and chromatin immunoprecipitation demonstrated the physical interaction of c-Jun protein with predicted AP-1–binding sites in the p22^phox gene promoter.

Conclusions— In SMCs exposed to Ang II or TNF-, inhibition of AP-1–related pathways reduces NADPHox expression and the O₂^– production. The physical interaction of AP-1 with p22^phox gene promoter facilitates NADPHox regulation.

Key Words: NADPH oxidase • AP-1 • hypertension • atherosclerosis

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