Jin Shi, Ray Ramesh M, Johnson Leonard R
Dept. of Physiology, Univ. of Tennessee Health Science Center, 894 Union Ave., Memphis, TN 38163, USA.
Am J Physiol Gastrointest Liver Physiol. 2008 Apr;294(4):G928-37. doi: 10.1152/ajpgi.00219.2007. Epub 2008 Jan 24.
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.
此前我们已经表明，Rac1和c-Jun氨基末端激酶（JNK1/2）都是肿瘤坏死因子（TNF）-α/放线菌酮（CHX）诱导肠上皮细胞凋亡过程中的关键促凋亡分子，而活性氧（ROS）在凋亡中的作用尚不清楚。本研究检验了Rac1介导的ROS产生参与TNF-α诱导的凋亡这一假说。在本研究中，我们发现TNF-α/CHX诱导的ROS产生以及过氧化氢（H₂O₂）诱导的氧化应激会增加细胞凋亡。特异性抑制剂NSC23766对Rac1的抑制作用可阻止TNF-α诱导的ROS产生。抗氧化剂N-乙酰半胱氨酸（NAC）或线粒体电子传递链抑制剂鱼藤酮（Rot）可减弱线粒体ROS产生及细胞凋亡。Rot还可阻止凋亡过程中JNK1/2的激活。通过表达显性负性Rac1抑制Rac1，可减少TNF-α诱导的线粒体ROS产生。此外，Rac1抑制、二亚苯基碘鎓（DPI，一种NADPH氧化酶抑制剂）和NAC均可抑制TNF-α诱导的胞质ROS产生。另外，从形态学改变、DNA片段化和JNK1/2激活情况判断，DPI可抑制TNF-α诱导的细胞凋亡。线粒体膜电位变化依赖于Rac1或胞质ROS。最后，所有ROS抑制剂均抑制了caspase-3活性。因此，这些结果表明TNF-α诱导的凋亡需要肠上皮细胞中Rac1依赖性的ROS产生。
