Role of Nox4 and Nox2 in hyperoxia-induced reactive oxygen species generation and migration of human lung endothelial cells.
In vascular endothelium, the major research focus has been on reactive oxygen species (ROS) derived from Nox2. The role of Nox4 in endothelial signal transduction, ROS production, and cytoskeletal reorganization is not well defined. In this study, we show that human pulmonary artery endothelial cells (HPAECs) and human lung microvascular endothelial cells (HLMVECs) express higher levels of Nox4 and p22(phox) compared to Nox1, Nox2, Nox3, or Nox5. Immunofluorescence microscopy and Western blot analysis revealed that Nox4 and p22(phox), but not Nox2 or p47(phox), are localized in nuclei of HPAECs. Further, knockdown of Nox4 with siRNA decreased Nox4 nuclear expression significantly. Exposure of HPAECs to hyperoxia (3-24 h) enhanced mRNA and protein expression of Nox4, and Nox4 siRNA decreased hyperoxia-induced ROS production. Interestingly, Nox4 or Nox2 knockdown with siRNA upregulated the mRNA and protein expression of the other, suggesting activation of compensatory mechanisms. A similar upregulation of Nox4 mRNA was observed in Nox2 2(-/-) ko mice. Downregulation of Nox4, or pretreatment with N-acetylcysteine, attenuated hyperoxia-induced cell migration and capillary tube formation, suggesting that ROS generated by Nox4 regulate endothelial cell motility. These results indicate that Nox4 and Nox2 play a physiological role in hyperoxia-induced ROS production and migration of ECs.
Pendyala
Srikanth
S
Department of Medicine, The University of Chicago, Chicago, Illinois 60637, USA.
Gorshkova
Irina A
IA
Usatyuk
Peter V
PV
He
Donghong
D
Pennathur
Arjun
A
Lambeth
J David
JD
Thannickal
Victor J
VJ
Natarajan
Viswanathan
V
D000818:Animals; D001483:Base Sequence; D002465:Cell Movement / Q000502:physiology*; D002478:Cells, Cultured; D017931:DNA Primers; D004730:Endothelium, Vascular / Q000166:cytology*; D055785:Gene Knockdown Techniques; D006801:Humans; D018496:Hyperoxia / Q000503:physiopathology*; D008168:Lung / Q000098:blood supply*; D008297:Male; D008562:Membrane Glycoproteins / Q000235:genetics / Q000502:physiology*; D051379:Mice; D018345:Mice, Knockout; D008856:Microscopy, Fluorescence; D000074662:NADPH Oxidase 2; D000074663:NADPH Oxidase 4; D019255:NADPH Oxidases / Q000235:genetics / Q000502:physiology*; D012333:RNA, Messenger / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics; D017382:Reactive Oxygen Species / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction
D017931:DNA Primers; D008562:Membrane Glycoproteins; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D017382:Reactive Oxygen Species; C056558:CYBB protein, human; D000074662:NADPH Oxidase 2; D000074663:NADPH Oxidase 4; D019255:NADPH Oxidases; C498910:NOX4 protein, human
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