A role for NADPH oxidase 4 in the activation of vascular endothelial cells by oxidized phospholipids.
Previous studies from our group have demonstrated that oxidized 1-palmitoyl-2-arachidonyl-sn-glycerol-3-phosphocholine (Ox-PAPC) activates over 1000 genes in human aortic endothelial cells (HAECs). Prominent among these are genes regulating inflammation, cholesterol homeostasis, antioxidant enzymes, and the unfolded protein response. Previous studies from our lab and others suggested that transcriptional regulation by Ox-PAPC may be controlled, at least in part, by reactive oxygen species. We now present evidence that Ox-PAPC activation of NADPH oxidase 4 (NOX4) is responsible for the regulation of two of these important groups of genes: those controlling inflammation and those involved in sterol regulation. Our data demonstrate that Ox-PAPC increases reactive oxygen species formation in HAECs as seen by DCF fluorescence. NOX4 is the major molecule responsible for this increase because downregulation of NOX4 and its components (p22(phox) and rac1) blocked the Ox-PAPC effect. Our data show that Ox-PAPC did not change NOX4 transcription levels but did induce recruitment of rac1 to the membrane for NOX4 activation. We present evidence that vascular endothelial growth factor receptor 2 (VEGFR2) activation is responsible for rac1 recruitment to the membrane. Finally, we demonstrate that knockdown of NOX4 and its components rac1 and p22(phox) decreases Ox-PAPC induction of inflammatory and sterol regulatory genes, but does not affect Ox-PAPC transcriptional regulation of other genes for antioxidants and the unfolded protein response. In summary, we have identified a VEGFR2/NOX4 regulatory pathway by which Ox-PAPC controls important endothelial functions.
Department of Pathology, University of California at Los Angeles, 10833 Le Conte Avenue, Los Angeles, CA 90095, USA.
Sangderk
S
Lee
Nima M
NM
Gharavi
Henry
H
Honda
Irene
I
Chang
Brandon
B
Kim
Nelson
N
Jen
Rongsong
R
Li
Alejandro
A
Zimman
Judith A
JA
Berliner
D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D042783:Endothelial Cells / Q000187:drug effects* / Q000502:physiology; D004730:Endothelium, Vascular / Q000166:cytology; D006801:Humans; D000074663:NADPH Oxidase 4; D019255:NADPH Oxidases / Q000235:genetics / Q000378:metabolism*; D010084:Oxidation-Reduction; D010713:Phosphatidylcholines / Q000494:pharmacology*; D034741:RNA, Small Interfering / Q000235:genetics; D017382:Reactive Oxygen Species / Q000378:metabolism; D015398:Signal Transduction; D040301:Vascular Endothelial Growth Factor Receptor-2 / Q000378:metabolism; D020830:rac1 GTP-Binding Protein / Q000378:metabolism
D010713:Phosphatidylcholines; D034741:RNA, Small Interfering; D017382:Reactive Oxygen Species; C472349:oxidized-L-alpha-1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine; D000074663:NADPH Oxidase 4; D019255:NADPH Oxidases; C498910:NOX4 protein, human; C086986:CYBA protein, human; D040301:Vascular Endothelial Growth Factor Receptor-2; D020830:rac1 GTP-Binding Protein