The expression of the NADPH oxidase subunit p22phox is regulated by a redox-sensitive pathway in endothelial cells.
Endothelial dysfunction is characterized by increased levels of reactive oxygen species (ROS) and a prothrombotic state. The mechanisms linking thrombosis to ROS production in the endothelium are not well understood. We investigated the role of thrombin in regulating NADPH oxidase-dependent ROS production and expression of its subunit p22phox in the endothelial cell line EaHy926. Thrombin elicited a biphasic increase in ROS generation peaking within 15 min, but also at 3 h. The delayed response was accompanied by increased p22phox mRNA and protein expression. Two-photon confocal laser microscopy showed colocalization between p22phox and ROS production. Antioxidant treatment with vitamin C or diphenyleneiodonium abrogated thrombin-induced ROS production and p22phox expression, whereas H2O2 elevated ROS production and p22phox levels. Both responses were dependent on p38 MAP kinase and phosphatidylinositol-3-kinase (PI3 kinase)/Akt. Finally, p22phox was required for thrombin- or H2O2-stimulated proliferation. These data show that thrombin rapidly increases ROS production in endothelial cells, resulting, via activation of p38 MAP kinase and PI3 kinase/Akt, in upregulation of p22phox accompanied by a delayed increase in ROS generation and enhanced proliferation. These findings suggest a positive feedback mechanism whereby ROS, possibly generated by the NADPH oxidase, lead to elevated levels of p22phox and, thus, sustained ROS generation as is observed in endothelial dysfunction.
Djordjevic
Talija
T
Experimental Pediatric Cardiology, Clinic for Pediatric Cardiology and Congenital Heart Diseases, German Heart Center Munich at the Technical University Munich, Lazarettstrasse 36, D-80636 Munich, Germany.
Pogrebniak
Alexej
A
BelAiba
Rachida S
RS
Bonello
Steve
S
Wotzlaw
Christoph
C
Acker
Helmut
H
Hess
John
J
Görlach
Agnes
A
D015153:Blotting, Western; D002460:Cell Line; D042783:Endothelial Cells / Q000378:metabolism*; D015971:Gene Expression Regulation, Enzymologic; D006801:Humans; D006825:Hybridomas; D006861:Hydrogen Peroxide / Q000494:pharmacology; D026901:Membrane Transport Proteins / Q000096:biosynthesis*; D018613:Microscopy, Confocal; D019255:NADPH Oxidases / Q000096:biosynthesis* / Q000378:metabolism*; D010084:Oxidation-Reduction; D010750:Phosphoproteins / Q000096:biosynthesis*; D011494:Protein Kinases / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism*; D013917:Thrombin / Q000502:physiology*; D015854:Up-Regulation
D026901:Membrane Transport Proteins; D010750:Phosphoproteins; D017382:Reactive Oxygen Species; D006861:Hydrogen Peroxide; D019255:NADPH Oxidases; C086986:CYBA protein, human; D011494:Protein Kinases; D013917:Thrombin