| Purpose of this study was to test the role of the small GTPase Rac1 in apoptosis in intestinal epithelial cells (IEC-6). Rac1 was activated within 30 min during tumor necrosis factor-alpha(TNF-alpha)/cycloheximide(CHX)-induced apoptosis as judged by the level of GTP-Rac1, the level of membrane-associated Rac1, and lamellipodia formation. Inhibition of Rac1, either by a specific inhibitor of Rac1, NSC23766, or expression of a dominant negative Rac1, protected cells from apoptosis by inhibiting caspase-3, -8, and -9 activities. Inhibition of Rac1 significantly prevented TNF-alpha/CHX-induced activation of c-Jun N-terminal kinases (JNK1/2), the major proapoptotic kinase, during apoptosis. Inhibition of Rac1 did not modulate TNF-alpha/CHX-induced activation of ERK1/2 and Akt, and this inhibition protected cells from apoptosis independently of these two prosurvival kinases. Although p38 MAPK was activated during apoptosis, activation of p38 MAPK was not required for apoptosis, and inhibition of Rac1 did not alter p38 MAPK activity. Moreover, TNF-alpha/CHX-induced production of mitochondrial reactive oxygen species (ROS), DNA fragmentation, and caspase-3 activity was dramatically attenuated by using an anti-oxidant, N-acetylcystein, or an inhibitor of complex I of mitochondrial electron transport chain, rotenone. Inhibition of Rac1 decreased mitochondrial permeability change and attenuated TNF-alpha/CHX-induced mitochondria-derived ROS production. In addition, inhibition of Rac1 prevented an early burst of ROS production, which was detected within 20--30 min during apoptosis, and this early burst of ROS was totally blocked by diphenyleneiodonium (DPI), a specific inhibitor of flavoprotein-containing membrane-associated oxidase. However, rotenone had no appreciable effect on this early burst of ROS production. In addition, DPI mimicked the effect of inhibition of Rac1 on TNF-alpha/CHX-induced DNA fragmentation, caspase-3 activity, and JNK1/2 activity. These data indicated that small GTPase Rac1 mediates proapoptotic JNK pathway via a DPI-sensitive membrane-associated oxidase in TNF-alpha/CHX-induced apoptosis. |
