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

Abstract

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 Rac1 (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-kappaB), increased activity of caspase-3, and markedly augmented endothelial cell susceptibility to TNF-induced apoptosis. Direct inhibition of NF-kappaB through adenoviral expression of the super repressor form of inhibitor of kappaBalpha (I-kappaB 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 Rac1 is an important regulator of TNF-induced ROS production in endothelial cells. Moreover, they suggest that Rac1-dependent ROS, directly or indirectly, lead to protection against TNF-induced death, whereas mitochondrial-derived ROS promote TNF-induced apoptosis.

The biphasic redox sensing of SENP3 accounts for the HIF-1 transcriptional activity shift by oxidative stress

“…The dual or biphasic regulatory effects of ROS have been reported for many years [2,6,[41][42][43] . For instance, ROS can mediate phenotypes ranging from survival and growth to apoptosis in vascular endothelial and smooth muscle cells, which may be considered both physiological and pathophysiological [41] .…”

Cytotoxic Necrotizing Factor 1 Prevents Apoptosis via the Akt/IκB Kinase Pathway: Role of Nuclear Factor-κB and Bcl-2

“…Rho GTPases have a role in a number of cellular processes such as cytoskeleton remodeling, transcriptional activation, growth control, metastasis, development, and apoptosis. In particular, several groups have indicated Rac as either positive or negative player in the apoptotic program, depending on the cell line investigated and on the stimulus applied (Nishida et al, 1999;Embade et al, 2000;Deshpande et al, 2000;Pervaiz et al, 2001;Harrington et al, 2002;Murga et al, 2002;Zhang et al, 2004). However, the majority of the reports ascribe a protective role to active Rac1.…”

Pro-inflammatory cytokines increase reactive oxygen species through mitochondria and NADPH oxidase in cultured RPE cells

“…Intracellular ROS Production-Intracellular ROS production by human RPE cells in response to TNF-α, IL-1β, or IFN-γ treatment was measured by a cell-based fluorometric assay based on deacetylation and oxidation of non-fluorescent reduced CM-H 2 DCFDA (Molecular Probes) into fluorescent CM-DCF. The reliability and specificity of the probe have been established in various cells including cultured human RPE cells (Deshpande et al, 2000;Nishikawa et al, 2000;Touyz et al, 2001;Yamagishi et al, 2001;Higgins et al, 2003;Li et al, 2003;Fukami et al, 2004;Hermann et al, 2004;Hwang et al, 2004;Kannan et al, 2004;Shanker et al, 2004;Kim et al, 2005). RPE cells were seeded into 96-well plates pre-coated with poly-D-lysine (Sigma-Aldrich) and cultured for 1 day or 7days.…”