Rac1 inhibits TNF-α-induced endothelial cell apoptosis:: dual regulation by reactive oxygen species

Reactive oxygen species (ROS) have been implicated as mediators of tumor necrosis factor-alpha (TNF) -induced apoptosis. In addition to leading to cell death, ROS can also promote cell growth and/or survival. We investigated these two roles of ROS in TNF-induced endothelial cell apoptosis. Human umbilical vein endothelial cells (HUVECs) stimulated with TNF produced an intracellular burst of ROS. Adenoviral-mediated gene transfer of a dominant negative form of the small GTPase Rad (Rac1N17) partially suppressed the TNF-induced oxidative burst without affecting TNF-induced mitochondrial ROS production. HUVECs were protected from TNF-induced apoptosis. Expression of Rac1N17 blocked TNF-induced activation of nuclear factor-kappa B (NF-kappa B), increased activity of caspase-3, and markedly augmented endothelial cell susceptibility to TNF-induced apoptosis. Direct inhibition of NF-kappa B through adenoviral expression of the super repressor form of inhibitor of kappa B alpha (I-kappa B S32/36A) also increased susceptibility of HUVECs to TNF-induced apoptosis. Rotenone, a mitochondrial electron transport chain inhibitor, suppressed TNF-induced mitochondrial ROS production, proteolytic cleavage of procaspase-3, and apoptosis. These findings show that Rad is an important regulator of TNF-induced ROS production in endothelial cells. Moreover, they suggest that Rad-dependent ROS, directly or indirectly, lead to protection against TNF-induced death, whereas mitochondrial-derived ROS promote TNF-induced apoptosis.