Rac1‐dependent ROS production mediates TNF‐alpha‐induced apoptosis in intestinal epithelial cells

Abstract

Reactive oxygen species (ROS) play a key role in cellular signaling pathways regulating cell proliferation, motility and apoptosis. We have shown that the small GTPase Rac1 regulates apoptosis via c-Jun N-terminal kinase (JNK) activation in intestinal epithelial cells (IEC-6). However, the mechanism by which Rac1 regulates JNK-mediated-apoptosis is unclear. The current studies test the hypothesis that Rac1-mediated ROS production is involved in TNF-α-induced apoptosis. First, mitochondrial ROS production increased during TNF-α-induced apoptosis, as judged by the ROS probe dihydrorhodamine123 (DHR123). The anti-oxidant, N-Acetyl-L-cysteine (NAC, 0.1mM) and mitochondrial electron transport chain complex I inhibitor rotenone (0.5μM) dramatically attenuated apoptosis as judged by DNA fragmentation ELISA assay. Inhibition of Rac1 by a Rac1 antagonist, NSC23766 (30μM), decreased TNF-α-induced mitochondrial permeability change and mitochondria-derived ROS production. In addition, an early burst of cytosolic ROS was inhibited by NSC23766, dominant negative Rac1, diphenyleneiodonium (NADPH oxidase inhibitor, DPI) and NAC. DPI inhibited TNF-α-induced apoptosis as judged by morphological changes, DNA fragmentation and JNK1/2 activation. Moreover, all ROS inhibitors inhibited caspase-3 activity. Thus, these results indicate that a Rac1-dependent-NADPH-oxidase-like system is involved in the initiation of TNF-α-induced apoptosis in intestinal epithelial cells. Supported by Thomas A. Gerwin Endowment.