TNF-alpha/cycloheximide-induced apoptosis in intestinal epithelial cells requires Rac1-regulated reactive oxygen species.
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.
Jin
Shi
S
Dept. of Physiology, Univ. of Tennessee Health Science Center, 894 Union Ave., Memphis, TN 38163, USA.
Ray
Ramesh M
RM
Johnson
Leonard R
LR
D000111:Acetylcysteine / Q000494:pharmacology; D000634:Aminoquinolines / Q000494:pharmacology; D000818:Animals; D000975:Antioxidants / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects*; D053148:Caspase 3 / Q000378:metabolism; D002460:Cell Line; D003513:Cycloheximide / Q000494:pharmacology*; D003600:Cytosol / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004847:Epithelial Cells / Q000187:drug effects* / Q000201:enzymology / Q000378:metabolism / Q000473:pathology; D006861:Hydrogen Peroxide / Q000494:pharmacology; D007413:Intestinal Mucosa / Q000187:drug effects* / Q000201:enzymology / Q000378:metabolism / Q000473:pathology; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism; D048055:Mitogen-Activated Protein Kinase 8 / Q000378:metabolism; D048056:Mitogen-Activated Protein Kinase 9 / Q000378:metabolism; D019255:NADPH Oxidases / Q000037:antagonists & inhibitors / Q000378:metabolism; D009861:Onium Compounds / Q000494:pharmacology; D016877:Oxidants / Q000494:pharmacology; D018384:Oxidative Stress / Q000187:drug effects*; D011743:Pyrimidines / Q000494:pharmacology; D051381:Rats; D017382:Reactive Oxygen Species / Q000378:metabolism*; D012402:Rotenone / Q000494:pharmacology; D015398:Signal Transduction / Q000187:drug effects; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism*; D014475:Uncoupling Agents / Q000494:pharmacology; D020830:rac1 GTP-Binding Protein / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*
D000634:Aminoquinolines; D000975:Antioxidants; D004791:Enzyme Inhibitors; C487513:NSC 23766; D009861:Onium Compounds; D016877:Oxidants; D011743:Pyrimidines; D017382:Reactive Oxygen Species; D014409:Tumor Necrosis Factor-alpha; D014475:Uncoupling Agents; D012402:Rotenone; C007517:diphenyleneiodonium; D003513:Cycloheximide; D006861:Hydrogen Peroxide; D019255:NADPH Oxidases; D048056:Mitogen-Activated Protein Kinase 9; D048055:Mitogen-Activated Protein Kinase 8; D053148:Caspase 3; C499134:Rac1 protein, rat; D020830:rac1 GTP-Binding Protein; D000111:Acetylcysteine