TNF-α/cycloheximide-induced apoptosis in intestinal epithelial cells requires Rac1-regulated reactive oxygen species

Abstract

Previously we have shown that both Rac1 and c-Jun NH(2)-terminal kinase (JNK1/2) are key proapoptotic molecules in tumor necrosis factor (TNF)-alpha/cycloheximide (CHX)-induced apoptosis in intestinal epithelial cells, whereas the role of reactive oxygen species (ROS) in apoptosis is unclear. The present studies tested the hypothesis that Rac1-mediated ROS production is involved in TNF-alpha-induced apoptosis. In this study, we showed that TNF-alpha/CHX-induced ROS production and hydrogen peroxide (H(2)O(2))-induced oxidative stress increased apoptosis. Inhibition of Rac1 by a specific inhibitor NSC23766 prevented TNF-alpha-induced ROS production. The antioxidant, N-acetylcysteine (NAC), or rotenone (Rot), the mitochondrial electron transport chain inhibitor, attenuated mitochondrial ROS production and apoptosis. Rot also prevented JNK1/2 activation during apoptosis. Inhibition of Rac1 by expression of dominant negative Rac1 decreased TNF-alpha-induced mitochondrial ROS production. Moreover, TNF-alpha-induced cytosolic ROS production was inhibited by Rac1 inhibition, diphenyleneiodonium (DPI, an inhibitor of NADPH oxidase), and NAC. In addition, DPI inhibited TNF-alpha-induced apoptosis as judged by morphological changes, DNA fragmentation, and JNK1/2 activation. Mitochondrial membrane potential change is Rac1 or cytosolic ROS dependent. Lastly, all ROS inhibitors inhibited caspase-3 activity. Thus these results indicate that TNF-alpha-induced apoptosis requires Rac1-dependent ROS production in intestinal epithelial cells.

Mitoneet mediates TNFα induced necroptosis promoted by fructose and ethanol exposure

“…Treatment of hepatocytes with TNFa in the presence of a translational inhibitor such as cycloheximide leads to apoptosis (Jin et al, 2008;Pajak et al, 2005). The externalization of phosphatidylserine on the outer leaflet of the plasma membrane is an early and consistent marker of apoptosis and occurs late in necroptosis, only after the integrity of the plasma membrane has been breached (Koul et al, 2006;Okamoto et al, 2002;Van Antwerp et al, 1996).…”

Klotho Gene Deficiency Causes Salt-Sensitive Hypertension via Monocyte Chemotactic Protein-1/CC Chemokine Receptor 2–Mediated Inflammation

“…Several studies indicated that some cytokines, such as TNF-a and IL-6, were capable of activating Rac1, a member of the Rho family of GTPases. 43,44 Rac1 has been proven to play a pivotal role in activation of the Sgk1 signaling by salt excess independent of aldosterone by promoting MR nuclear translocation. 45 Although the infiltration of inflammatory cells and the upregulation of the Sgk1-NCC signaling in kidneys of KL (+/2) mice were attenuated or abolished by INCB3284 (Figures 3-5), the elevated BP level in KL(+/2) mice was not decreased by INCB3284.…”

Nuclear Factor-κB Is a Critical Mediator of Ste20-Like Proline-/Alanine-Rich Kinase Regulation in Intestinal Inflammation

“…[72][73][74] On the other hand, the combination of TNF with other cytokine such as IFN, 75 or with protein synthesis inhibitor cycloheximide, 76 or tyrosine kinase inhibitor 73 can induce strong apoptosis. Furthermore, we performed Western blots on TNF-␣ treated Caco2-BBE cells with caspase3 antibody, we could not find the cleavage of caspase3 either (data not shown).…”