Abstract
The small GTPase rac1 controls actin redistribution to membrane ruffles in fibroblasts and other cell types, as well as the activation of the NADPH oxidase in phagocytes. We explored the possibility that these two processes could be related. We used a replication-deficient adenoviral vector to overexpress the constitutively active form of rac1, rac V12 , in human and mouse aortic endothelial cells. We show here that, in addition to membrane ruffle formation, rac V12 induced an increase in the total amount of F-actin within endothelial cells. Concurrently, rac V12 -overexpressing cells produced significantly higher amounts of free radicals, as detected by the fluorescent probe 5-(and-6)-chloromethyl-2′,7′-dichloro-dihydrofluorescein diacetate, than cells infected with a control virus encoding the bacterial β-galactosidase (Ad-βGal). To assess the specific role of superoxide in rac V12 -induced actin reorganization, we co-expressed the human enzyme Cu,Zn-superoxide dismutase (SOD), by means of another adenoviral vector construct. Overexpressed SOD reduced the concentration of superoxide detected in Ad-rac V12 -transfected cells and reversed the effects of Ad-rac V12 on the content of filamentous actin. MnTMPyP, an SOD mimetic, as well as the antioxidant N-acetyl cysteine, had similar effects, in that they reduced not only the free radicals production, but also ruffle formation and the concentration of F-actin within rac V12 -overexpressing endothelial cells. Our data support the hypothesis that superoxide is one of the important mediators acting downstream of rac1 on the pathway of actin cytoskeleton remodeling in endothelial cells.
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