AP-1-dependent transcriptional regulation of NADPH oxidase in human aortic smooth muscle cells: role of p22phox subunit.
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.


RESULTS
Cultured SMCs were exposed to either angiotensin II (Ang II) or tumor necrosis factor (TNF)-alpha. 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-alpha-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-alpha-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-alpha, inhibition of AP-1-related pathways reduces NADPHox expression and the O(2)(-) production. The physical interaction of AP-1 with p22(phox) gene promoter facilitates NADPHox regulation.
Manea
Adrian
A
Institute of Cellular Biology and Pathology Nicolae Simionescu, 8 B.P. Hasdeu Street, Bucharest, P.O. Box 35-14, Romania.
Manea
Simona A
SA
Gafencu
Anca V
AV
Raicu
Monica
M
Simionescu
Maya
M
D000804:Angiotensin II / Q000494:pharmacology; D001013:Aorta, Thoracic / Q000378:metabolism* / Q000473:pathology; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects / Q000502:physiology*; D006801:Humans; D009131:Muscle, Smooth, Vascular / Q000378:metabolism* / Q000473:pathology; D000074624:NADPH Oxidase 1; D000074663:NADPH Oxidase 4; D019255:NADPH Oxidases / Q000235:genetics / Q000378:metabolism*; D010100:Oxygen / Q000378:metabolism; D010750:Phosphoproteins / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D018808:Transcription Factor AP-1 / Q000378:metabolism*; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
D010750:Phosphoproteins; D012333:RNA, Messenger; D018808:Transcription Factor AP-1; D014409:Tumor Necrosis Factor-alpha; C079789:neutrophil cytosol factor 67K; D000804:Angiotensin II; D000074624:NADPH Oxidase 1; D000074663:NADPH Oxidase 4; D019255:NADPH Oxidases; C402799:NOX1 protein, human; C498910:NOX4 protein, human; C086986:CYBA protein, human; C060505:neutrophil cytosolic factor 1; D010100:Oxygen