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Am J Physiol Gastrointest Liver Physiol 294: G928-G937, 2008. First published January 24, 2008; doi:10.1152/ajpgi.00219.2007
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HORMONES AND SIGNALING

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

Shi Jin, Ramesh M. Ray, and Leonard R. Johnson

Department of Physiology, University of Tennessee Health Science Center, Memphis, Tennessee

Submitted 16 May 2007 ; accepted in final form 17 January 2008


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Previously we have shown that both Rac1 and c-Jun NH2-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 (H2O2)-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.

intestinal epithelial cells-6; N17Rac1; diphenyleneiodonium; JNK1/2; oxidative stress

REACTIVE OXYGEN SPECIES, (ROS), namely H2O2, O2bullet–, and OH–, are important mediators in cellular signal transduction cascades regulating proliferation (8), apoptosis (4, 23), and migration (28). Exposure to stimuli such as cytokines or growth factors increases intracellular ROS in a variety of cells including fibroblasts (52), endothelial cells (35), cardiac myocytes (48), and epithelial cells (5), suggesting that these highly reactive molecules play crucial roles in cellular signaling.

Tumor necrosis factor-{alpha} (TNF-{alpha}), a pleiotropic cytokine produced by many cells and originally identified by its cytotoxic effects, induces cell death in some types of cells, and it also elicits a wide range of physiological responses, such as inflammation, cell proliferation, and differentiation. Recent accumulating evidence has demonstrated that ROS are key mediators of many cellular responses to TNF-{alpha} such as apoptosis (1), transcriptional factor activation (15), calcium spark activation (7), and insulin signaling (20).

The small GTPase Rac1 has been established as an important mediator in regulation of cytoskeletal organization, cell migration, proliferation, and apoptosis (54). Rac1 is activated in response to TNF-{alpha} in a large body of cell lines including fibroblasts (49), endothelial cells (6, 30), and epithelial cells (21, 32). Recently, Rac1 has been implicated in the control of ROS production via activation of NADPH oxidase, lipooxygenase, and mitochondrial oxidant production (9).

Although TNF-{alpha} has been widely used as a potent apoptotic inducer, we have found that TNF-{alpha} alone cannot induce apoptosis in IEC-6 cells, and suppressing the synthesis of the short-lived antiapoptotic protein by cycloheximide (CHX) is required for TNF-{alpha}-induced apoptosis (2). Therefore, we used TNF-{alpha}/CHX to investigate the apoptotic signaling in intestinal epithelial cells (IEC). We have shown that both Rac1 and c-Jun NH2-terminal kinase (JNK) are key proapoptotic molecules in TNF-{alpha}/CHX-induced apoptosis in IEC-6 cells (2, 21, 38), whereas the role of ROS in TNF-{alpha}/CHX-induced apoptosis in IEC is unclear. Given this background, the present study tested the hypothesis that Rac1-mediated ROS production is required for TNF-{alpha}/CHX-induced apoptosis. We demonstrated that TNF-{alpha}/CHX-induced ROS production was required for apoptosis. Cellular enzymes, such as membrane associated-oxidase in addition to respiratory mitochondrial enzymes, were involved in ROS production induced by ligand (cytokine)-receptor interaction. Inhibition of Rac1 attenuated mitochondrial and cytoplasmic ROS production. The inhibitor of NADPH oxidase, diphenyleniodonium (DPI), mimicked the effect of inhibition of Rac1 on apoptosis. Taken together, these data indicate that TNF-{alpha}/CHX-induced apoptosis in IEC-6 cells requires generation of Rac1-regulated ROS.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Reagents. The IEC-6 cell line (ATCC CRL 1592) was obtained from the American Type Culture Collection (Manassas, VA) at passage 13. This cell line is derived from normal rat intestine and was developed and characterized by Quaroni et al. (36). IEC-6 cells are nontumorigenic, originate from intestinal crypt cells as judged by morphological and immunologic criteria, and retain the undifferentiated character of epithelial stem cells. Tests for mycoplasma were always negative. Cell cultureware was purchased from Corning Glass Works (Corning, NY). Cell culture medium, fetal bovine serum (FBS), dialyzed FBS (dFBS), trypsin/EDTA, antibiotics, and insulin were obtained from GIBCO (Grand Island, NY). Mammalian protein extraction reagent and the bicinchoninic acid protein assay reagent kit were purchased from Pierce (Rockford, IL). The enhanced chemiluminescence Western blot detection system was purchased from DuPont-New England Nuclear (Boston, MA). Caspase-3 substrate was purchased from Biomol Research Laboratories (Plymouth Meeting, PA). Rabbit anti-phospho-ERK1/2 antibody, rabbit anti-ERK1/2 antibody, mouse anti-phospho-JNK1/2 antibody, and rabbit anti-JNK1/2 antibody were purchased from Cell Signaling (Beverly, MA). Hydrogen peroxide (30%) was purchased from Fisher Scientific (Hampton, NH). TNF-{alpha} was obtained from Pharmingen International (San Diego, CA). The Cell Death Detection ELISA Plus kit was purchased from Roche Diagnostics (Indianapolis, IN). NSC23766 and rotenone (Rot) were purchased from Calbiochem (La Jolla, CA). Secondary antibodies conjugated to horseradish peroxidase, N-acetylcysteine (NAC), DPI, CHX, and dichlorodihydrofluorescein diacetate (DCFH-DA) were obtained from Sigma (St. Louis, MO). Dihydrorhodamine123 (DHR123), rhodamine123 (R123), and neutral red (NR) were purchased from Molecular Probes (Eugene, OR). All chemicals were of the highest purity commercially available.

Cell culture and transfection. The stock cell culture was maintained in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 5% heat-inactivated FBS, 10 µg/ml insulin, and 50 µg/ml gentamicin sulfate in T-150 flasks and incubated at 37°C in a humidified atmosphere of 90% air-10% CO2. Stock cells were passaged once weekly and fed three times per week, and passages 15–22 were used. During the experimental setup, cells were trypsinized with 0.05% trypsin and 0.53 mM EDTA and counted by using a Beckman Coulter Counter (model no. Z1). For the 4-day experimental setup, cells were grown in DMEM/5% dFBS to confluence for 3 days followed by serum deprivation for 24 h. IEC-6 cells were transfected with pMX-internal ribosome entry site (IRES)-green fluorescent protein (GFP)-N17-Rac1 (dominant negative) or pMX-IRES-GFP (vector). Stable clones were isolated and characterized as previously described (39).

Cellular viability assay. NR assay was used to evaluate the IEC-6 cell survival, as described previously (47). Briefly, after being treated with ROS inhibitors, IEC-6 cells were incubated with 50 µg/ml NR for 1 h. Subsequently, the monolayer was rinsed twice with Dulbecco's phosphate-buffered saline (DPBS), and the NR was extracted with 1% (vol/vol) acetic acid in a 50% (vol/vol) ethanol solution. Absorbance was read at 560 nm in a plate reader. The index of cellular viability was calculated as percentage of optical density with respect to untreated control cells.

Apoptosis assay. Cells were plated (day 0) in T-75 flasks at a density of 6.25 x 104 cells/cm2 in DMEM/dFBS with triplicate samples for each group. Cells were fed on day 2. On day 3, the cell culture medium was removed and replaced with serum-free medium. On day 4, TNF-{alpha} with CHX or H2O2 was added to the serum-free medium for 3 h. After various treatments, images were captured with a charge-coupled device camera. In some experiments, cells were incubated in the presence of the following inhibitors: NSC23766 (Rac1 inhibitor), NAC, Rot, or DPI. All inhibitors were also included in the medium during the 3-h exposure to TNF-{alpha}/CHX.

ROS detection. To determine the intracellular production of ROS, we used two different fluorogenic probes, DCFH-DA and DHR123. The following procedures were performed as described previously (1, 34). After various treatments, cells were incubated with DCFH-DA (10 µg/ml) or DHR123 (10 µM) for 30 min at 37°C. Some groups of cells were rapidly rinsed twice with DPBS and observed under a fluorescent microscopy. For quantitative measurement of ROS production, cells were rapidly rinsed twice with DPBS to remove the free probe, scraped in 750 µl of DPBS, and transferred into microcentrifuge tubes. The cells were pelleted at 2,000 g for 5 min at 4°C and resuspended in 0.5 ml of ice-cold DPBS. The cell suspensions were sonicated on ice for 15 s, clarified by centrifugation, and 100 µl of aliquots were dispensed into black 96-well plates in triplicate. The fluorescence in the supernatant was assessed with a plate reader (excitation 480 nm/emission 530 nm). The index of oxidation was calculated as percentage of fluorescence intensity compared with untreated control or vector cells.

Mitochondrial membrane potential measurement. Mitochondrial membrane potential ({Delta}{psi}m) was detected as described previously (17). Cells were grown for 4 days on six-well plates and treated with TNF-{alpha}/CHX for 1.5–2 h. Cells were washed with HBSS, covered with 2.5 µg/ml R123, and incubated at 37°C in a 5% CO2 incubator for 30 min. Cells were gently rinsed with HBSS and examined by fluorescent microscopy.

Quantitative DNA fragmentation ELISA. IEC-6 cells were harvested, lysed, and centrifuged to pellet nuclei. An aliquot of the supernatant was incubated with immunoreagents (anti-histone-biotin plus anti-DNA-peroxidase-conjugated antibody) in 96-well streptavidin-coated plates on a shaker. After samples were washed with incubation buffer, substrate buffer was added to each well, and absorbance was read at 405 nm in a microplate reader. Protein was determined by using the bicinchoninic acid method. DNA fragmentation was expressed as absorbance units per milligram protein per minute.

Caspase-3 activity assay. IEC-6 cells were harvested and washed with cold DPBS. The cell pellet was resuspended in ice-cold lysis buffer. After incubation on ice, the lysate was centrifuged at 10,000 g at 4°C for 10 min. The resulting supernatant was used for the measurement of caspase-3 activity. Each reaction (100 µl) contained 10 µl of cytosolic lysate, 70 µl of assay buffer, and 20 µl of 2 mM Ac-DEVD-p-nitroanilide, the substrate for caspase-3. The enzymatic reaction was carried out in 96-well plates at 37°C. Absorbance was read at 405 nm in a microplate reader. Protein was determined using the bicinchoninic acid method. Caspase activity was expressed as picomole p-NA released per milligram protein per minute.

Western blot analysis. IEC-6 cells were washed and lysed, and cell extracts were analyzed by Western blot with appropriate primary and secondary antibodies as described in our earlier publications (21, 39).

Statistics. Data are expressed as means ± SE. All experiments were repeated three times (n = 3). ANOVA with appropriate post hoc testing was used to determine the significance of the differences between the means of multiple treatments. P < 0.05 was regarded as statistically significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Oxidative stress and apoptosis in IEC-6 cells. Treatment with TNF-{alpha} results in an increase in ROS formation in endothelial cells, muscle cells, fibroblasts, and epithelial cells (7, 32, 52). In the present study we used DCFH-DA to measure the time course of TNF-{alpha}/CHX-induced ROS generation. The nonfluorescent DCFH-DA rapidly penetrates the cell membrane where it is deacetylated by intracellular esterases. A nonfluorescent compound, DCFH, is formed and is trapped in the cytosol where it is preferentially oxidized by hydrogen peroxide to the fluorescent DCF, which is retained within the cells and thus provides an index of overall cellular oxidation (42). As shown in Fig. 1A, bar 1, fluorescence intensity in cells challenged with 0.5 mM H2O2 for 15 min was significantly increased compared with that in the untreated control group, indicating the effectiveness of DCF to detect ROS in IEC-6 cells. After administration of TNF-{alpha}/CHX at various time intervals, significantly increased fluorescence intensity was observed within 20 min and persisted for 2 h without significant alteration (Fig. 1A, bars 3–8), suggesting that TNF-{alpha}/CHX induces oxidative stress in IEC-6 cells. Although DCF fluorescence is commonly used as an indicator of intracellular H2O2 levels, several studies indicate that DCF is an indicator of generalized oxidative stress rather than any particular ROS. Increased DCF fluorescence observed during apoptosis has been linked to increased cytochrome c release due to mitochondrial membrane permeability transition (18). We have shown that TNF-{alpha}/CHX leads to cytochrome c release via JNK activation and induces apoptosis in IEC-6 cells (2). Therefore, results presented in Fig. 1 indicate the cumulative effects of oxidative stress and cytochrome c release. Since H2O2 and TNF-{alpha}/CHX treatment generated similar levels of generalized oxidative stress, we examined apoptosis in response to different doses of H2O2. Cells were treated with H2O2 (100–1,000 µM) for 3 h, and DNA fragmentation was measured. H2O2 concentrations from 100- 250 µM showed a dose-dependent increase in apoptosis (Fig. 1B). However, H2O2 concentrations >250 µM caused necrosis within 1 h (data not shown).


Figure 1
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  		Fig. 1. Oxidative stress and apoptosis in intestinal epithelial cells (IEC)-6 cells. IEC-6 cells were grown as described in MATERIALS AND METHODS. A: oxidative stress in cells incubated with TNF-{alpha} (20 ng/ml) + cycloheximide (CHX) (25 µg/ml) for indicated time intervals was detected by dichlorodihydrofluorescein diacetate (DCFH-DA) as described in MATERIALS AND METHODS. One group of cells challenged with H2O2 (0.5 mM) for 15 min served as a positive control. Values are means ± SE of 3 observations. *P < 0.05, compared with untreated control group. B: cells were challenged with different concentrations of H2O2 for 3 h. DNA fragmentation was measured by ELISA as described in MATERIALS AND METHODS. Values are means ± SE of 3 observations. *P < 0.05, compared with corresponding control group. OD, optical density.

 
ROS inhibitors have no cytotoxic effects on IEC-6 cells. Our previous study showed that inhibition of Rac1 protects IEC-6 cells from apoptosis (21). In the present study we used several ROS inhibitors to test the hypothesis that Rac1-mediated ROS production regulates apoptosis. To assess whether these inhibitors have cytotoxic effects on IEC-6 cells, cell viability was measured using NR. Most living cells actively accumulate NR in lysosomes, a process that requires intact membranes and active metabolism. Failure to take up NR indicates that the cells have suffered damage. The dye taken up by the cell is subsequently extracted. The amount of NR taken into cells via endocytosis can be measured by optical density and is directly proportional to cell viability. As shown in Fig. 2, exposure to TNF-{alpha}/CHX or H2O2 significantly decreased cell viability; however, the viability of cells treated with Rac1 inhibitor (NSC23766) or ROS inhibitors (NAC, DPI, Rot) for 3 h was not different from that seen in untreated controls. These results suggest that the inhibitors had no cytotoxic effects on IEC-6 cells.


Figure 2
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  		Fig. 2. Effect of reactive oxygen species (ROS) inhibitors on cell viability. IEC-6 cells were grown as described in MATERIALS AND METHODS. Cells were treated with TNF-{alpha} (20 ng/ml) + CHX (25 µg/ml), NSC (NSC23766, 30 µM), N-acetylcysteine (NAC) (1 mM), diphenylene iodonium (DPI) (1 µM), rotenone (Rot) (1 µM) for 3 h, and H2O2 (1 mM) for 60 min. One group of cells served as a negative control (UT, untreated). After treatment, cell viability was measured by neutral red assay as described in MATERIALS AND METHODS. Values are means ± SE of 3 observations. *P < 0.05, compared with untreated control group.

 
Mitochondrial ROS is required for TNF-{alpha}/CHX-induced apoptosis. ROS is a well-known mediator of cell death (14, 44, 45, 56). It has been reported that electron leakage from the mitochondrial electron transport chain (ETC) is the primary source of ROS in response to TNF-{alpha} (46). Complex I, NADH oxidoreductase, and complex III, ubiquinol cytochrome c oxidoreductase, are the two major potential sites in the mitochondrial ETC for superoxide production (11); we used NAC or Rot to evaluate the role of mitochondrial ROS in apoptosis by DHR123 staining. DHR123 can easily cross the cell membrane and react with ROS in mitochondria to generate the positively charged R123 (26). NAC acts as both a precursor of reduced glutathione and a direct ROS scavenger (43). Rot, an inhibitor of complex I of the mitochondrial ETC, blocks the electron flow between mitochondrial complexes I and III and subsequently inhibits ROS production (48). As shown in Fig. 3A, bar 1, fluorescence intensity significantly increased in the group challenged with hydrogen peroxide for 15 min compared with that in the untreated group, suggesting the effectiveness of DHR123 for detection of ROS. After exposure to TNF-{alpha}/CHX for 90 min, the increased ROS production was significantly inhibited by NAC or Rot, suggesting the effectiveness of NAC and Rot on blocking mitochondrial ROS production (Fig. 3A, bars 2–7). Figure 3B demonstrates that a dramatic increase in DNA fragmentation in cells exposed to TNF-{alpha}/CHX was suppressed by NAC. A previous study has shown that treatment with Rot at a concentration of 1 µM for 24 h does not affect the viability of IEC-6 cells (29). As shown in Fig. 3C, TNF-{alpha}/CHX-induced DNA fragmentation was dramatically attenuated in the presence of Rot, suggesting that mitochondrial ROS formation is required for TNF-{alpha}/CHX-induced apoptosis. Based on our previous observations (21) and other studies (22, 27, 33) showing that JNK is a major apoptotic kinase and that ROS mediates JNK1/2 activation during apoptosis, we used Rot to test whether mitochondrial ROS regulates apoptosis via JNK1/2 activation. Rot treatment significantly prevented TNF-{alpha}/CHX-induced JNK1/2 phosphorylation but did not alter the phosphorylation of ERK1/2 (Fig. 4), suggesting that TNF-{alpha}/CHX-induced mitochondrial ROS production is required for JNK1/2 activation following treatment with TNF-{alpha}/CHX.


Figure 3
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  		Fig. 3. Effect of NAC or Rot on TNF-{alpha}/CHX-induced mitochondrial ROS production and DNA fragmentation. IEC-6 cells were grown as described in MATERIALS AND METHODS. A: cells were pretreated with NAC (0.5 mM) or Rot (0.5 µM) for 1 h and exposed to TNF-{alpha} (20 ng/ml) + CHX (25 µg/ml) for an additional 1.5 h. Mitochondrial ROS was measured by dihydrorhodamine123 (DHR123) as described in MATERIALS AND METHODS. One group of cells challenged with H2O2 (0.5 mM) for 15 min served as a positive control. Values are means ± SE of 3 observations. *P < 0.05, compared with corresponding control group. IEC-6 cells were grown as described in MATERIALS AND METHODS. B and C: cells were pretreated with NAC (0.5–2 mM, B), or Rot (0.5–2 µM, C) for 1 h followed by exposure to TNF-{alpha} (20 ng/ml) + CHX (25 µg/ml) for 3 h. DNA fragmentation was measured by ELISA as described in MATERIALS AND METHODS. Values are means ± SE of 3 observations. *P < 0.05, compared with corresponding control group.

 

Figure 4
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  		Fig. 4. Effect of Rot on TNF-{alpha}/CHX-induced phosphorylation of JNK1/2 and ERK1/2. IEC-6 cells were grown as described in MATERIALS AND METHODS. Cells were pretreated with or without Rot (1 µM) for 1 h and exposed to TNF-{alpha} (20 ng/ml) + CHX (25 µg/ml) for an additional 3 h. Phosphorylation and total JNK1/2 and ERK1/2 was determined by Western blot analysis as described in MATERIALS AND METHODS.

 
Rac1 inhibition prevents TNF-{alpha}/CHX-induced mitochondrial ROS. We evaluated the effect of inhibiting Rac1 on mitochondrial ROS production by using DHR123. There was a weak but homogeneous accumulation of R123 in the mitochondria of untreated cells due to basal ROS levels (Fig. 5Aa). The accumulation of R123 was strongly enhanced in cells exposed to TNF-{alpha}/CHX (Fig. 5Ab). The accumulation of R123 in cells exposed to NSC23766 alone (Fig. 5Ac) was similar to that of untreated cells. Rac1 inhibition largely prevented the TNF-{alpha}/CHX-induced accumulation of R123 (Fig. 5Ad). As shown in Fig. 5B, vector-transfected cells exposed to TNF-{alpha}/CHX for 90 min showed an increase in ROS formation compared with untreated cells. However, cells expressing dominant negative (DN)-Rac1 had significantly inhibited TNF-{alpha}/CHX-induced ROS production. These results suggest that the overproduction of mitochondrial ROS induced by TNF-{alpha}/CHX is mediated by Rac1 during apoptosis.


Figure 5
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  		Fig. 5. Effect of Rac1 inhibition on TNF-{alpha}/CHX-induced mitochondrial ROS production. IEC-6 cells were grown as described in MATERIALS AND METHODS. A: cells preincubated with or without NSC23766 (30 µM) were treated with TNF-{alpha} (20 ng/ml) + CHX (25 µg/ml) for 1.5 h. TNF-{alpha}/CHX-induced ROS production was detected by DHR123. a, control; b, cells exposed to TNF-{alpha}/CHX; c, cells incubated with NSC23766 alone; d, cells pretreated with NSC23766 and then exposed to TNF-{alpha}/CHX. B: IEC-6 cells transfected with empty vector or the dominant negative Rac1 mutant (DN-Rac1) were exposed to TNF-{alpha} (20 ng/ml) + CHX (25 µg/ml) for 1.5 h. Mitochondrial ROS was measured by DHR123 as described in MATERIALS AND METHODS. Values are means ± SE of 3 observations. *P < 0.05, compared with corresponding vector group.

 
Rac1 modulates extramitochondrial ROS formation during TNF-{alpha}/CHX-induced apoptosis. We tested whether TNF-{alpha}/CHX also induces oxidative stress from extramitochondrial site(s) and whether this oxidation phenomenon is Rac1 dependent. Interestingly, as shown in Fig. 6A, after administration of TNF-{alpha}/CHX for 30 min, cytosolic oxidative stress was significantly prevented in cells treated with the Rac1 inhibitor (NSC23766) and by DPI, a specific inhibitor of flavoprotein, a required component of membrane associated oxidases, such as the NADPH oxidase complex. However, Rot had no significant effect on the early burst of oxidative stress. TNF-{alpha}/CHX-induced oxidative stress was significantly inhibited in cells expressing DN-Rac1 (126 ± 1.3%) compared with vector-transfected cells (211 ± 2.0%). These data clearly suggest that mitochondrial oxidase is not involved in this event and that Rac1 mediates the generation of the relatively early burst of oxidative stress via a DPI-sensitive oxidase system. Unlike Rac1 inhibitor, NAC and DPI decreased the basal oxidative stress (Fig. 6A), suggesting that Rac1 does not modulate basal oxidative stress levels. We also studied the effect of NAC, NSC, and Rot on H2O2-induced apoptosis. Results in Fig. 6B show that NAC, a direct ROS scavenger, significantly decreased apoptosis. Rac1 inhibition by NSC23766 did not significantly prevent cell death after a challenge with H2O2, suggesting that Rac1 inhibition does not directly act to scavenge ROS and most likely interferes with the production of ROS. Interestingly, blocking electron transfer from mitochondria complex I to complex III by Rot significantly increased H2O2-induced apoptosis. These results imply that functional ETC is critical for the survival of the cells during oxidative stress and apoptosis induced by exogenous H2O2.


Figure 6
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  		Fig. 6. Effect of ROS inhibitors and inhibition of Rac1 on TNF-{alpha}/CHX-induced production of early burst of intracellular ROS and H2O2-induced apoptosis. IEC-6 cells were grown as described in MATERIALS AND METHODS. A: cells were preincubated with NSC (NSC23766, 30 µM), NAC (0.5 mM), DPI (0.5 µM), Rot (0.5 µM), and without inhibitors (UT, untreated) followed by exposure to TNF-{alpha} (20 ng/ml) + CHX (25 µg/ml) for 30 min. After treatment, ROS was measured by DCFH-DA as described in MATERIALS AND METHODS. Values are means ± SE of 3 observations. *P < 0.05, compared with corresponding group untreated with inhibitors (UT). B: cells were preincubated with NSC (NSC23766, 30 µM), NAC (0.5 mM), Rot (0.5 µM), and without inhibitors followed by exposure to H2O2 (250 µM) for 3 h. DNA fragmentation was measured with a colorimetric ELISA kit as described in MATERIALS AND METHODS. Values are means ± SE of 3 observations. *P < 0.05, compared with corresponding control group.

 
DPI prevents TNF-{alpha}/CHX-induced apoptosis. To evaluate the possible role of the cytosolic ROS in TNF-{alpha}/CHX-induced apoptosis, we analyzed the effect of DPI on apoptosis using morphology, DNA fragmentation, and JNK1/2 activation. Pretreatment with DPI for 1 h followed by TNF-{alpha}/CHX exposure for 3 h almost completely prevented the detachment of cells, and cultures retained the morphological features of untreated monolayers of IEC-6 cells (Fig. 7A). DPI significantly prevented TNF-{alpha}/CHX-induced DNA fragmentation (Fig. 7B). As expected, DPI significantly inhibited phosphorylation of JNK1/2 (Fig. 7C). Taken together, these data indicate that cytosolic ROS is required for TNF-{alpha}/CHX-induced apoptosis.


Figure 7
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  		Fig. 7. Effect of DPI on TNF-{alpha}/CHX-induced apoptosis. IEC-6 cells were grown as described in MATERIALS AND METHODS. Cells were preincubated with or without 0.5 µM DPI for 1 h followed by TNF-{alpha} (20 ng/ml) + CHX (25 µg/ml) for 3 h. A: apoptosis was measured by morphological analysis as described in MATERIALS AND METHODS. a, control; b, cells exposed to TNF-{alpha}/CHX for 3 h; c, cells exposed to DPI alone; d, cells pretreated with DPI and then exposed to TNF-{alpha}/CHX for 3 h. B: DNA fragmentation was measured with a colorimetric ELISA kit as described in MATERIALS AND METHODS. Values are means ± SE of 3 observations. *P < 0.05, compared with corresponding control group. C: JNK1/2 activation was determined by Western blot analysis.

 
Rac1 and cytosolic ROS-dependent changes in mitochondrial membrane potential. Growing evidence suggests that generation of ROS during apoptosis leads to mitochondrial damage (16, 41). Therefore, we examined mitochondrial membrane potential change ({Delta}{psi}m), an indicator of dysfunction of mitochondria, by using R123. Normal healthy mitochondria retain R123 with a characteristic granular localization until the change in membrane potential (Fig. 8A). As shown in Fig. 8E TNF-{alpha}/CHX treatment (1.5 h) caused a diffused localization of R123, suggesting a loss of {Delta}{psi}m. It should be noted that cellular distribution of R123 did not significantly change following TNF-{alpha}/CHX treatment in cells pretreated with NSC23766 (Rac1 inhibitor), DPI (NADPH oxidase inhibitor), or SP600125 (JNK1/2 inhibitor). These data indicate that a Rac1-cytosolic ROS-JNK pathway mediates mitochondrial membrane potential transition during apoptosis.


Figure 8
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  		Fig. 8. Effect of NSC23766, DPI, and SP600125 on TNF-{alpha}/CHX-induced mitochondrial membrane potential change. IEC-6 cells were grown as described in MATERIALS AND METHODS. Cells were preincubated with or without NSC23766 (NSC, 30 µM), DPI (0.5 µM), or SP600125 (25 µM), respectively, followed by TNF-{alpha} (20 ng/ml) + CHX (25 µg/ml) for 1.5–2 h. After treatment, mitochondrial membrane potential change was detected by R123. A, control; B, cells exposed to NSC23766 alone; C, cells exposed to DPI alone; D, cells exposed to SP600125 alone; E, cells exposed to TNF-{alpha}/CHX; F, cells pretreated with NSC23766 and then exposed to TNF-{alpha}/CHX; G, cells pretreated with DPI and then exposed to TNF-{alpha}/CHX; H, cells pretreated with SP600125 and then exposed to TNF-{alpha}/CHX.

 
Inhibition of Rac1, ROS, and JNK1/2 prevent TNF-{alpha}/CHX-induced caspase-3 activation. As shown in Fig. 9, various ROS inhibitors, NAC, Rot, and DPI, attenuated caspase-3 activation during apoptosis, which is similar to the effects of Rac1 and JNK inhibitors on caspase-3 activation, suggesting that inhibition of ROS prevents TNF-{alpha}/CHX-induced caspase-3 activation and thereby apoptosis.


Figure 9
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  		Fig. 9. Effect of inhibition of Rac1, ROS, and JNK on TNF-{alpha}/CHX-induced caspase-3 activation. IEC-6 cells were grown as described in MATERIALS AND METHODS. Cells were preincubated with or without NSC23766 (NSC, 30 µM), NAC (0.5 mM), DPI (0.5 µM), Rot (0.5 µM), or SP600125 (25 µM), respectively, followed by TNF-{alpha} (20 ng/ml) + CHX (25 µg/ml) for 3 h. After treatment, caspase-3 activity was measured. Values are means ± SE of 3 observations. *P < 0.05, compared with corresponding control group.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
ROS production mediated by the small GTPase Rac1 regulates cell proliferation, apoptosis, differentiation, and gene expression (19, 50). With respect to apoptosis, Rac1-dependent ROS production has varying effects, in some cases protecting cells from apoptosis (12), whereas many other studies have shown that Rac1 mediates apoptosis via NADPH oxidase-dependent ROS production (3, 10, 25). NADPH oxidase is a multicomponent enzyme complex present in the membranes of virtually all cells (55, 57). Activation of these systems depends on translocation of Rac from the cytoplasm to the cell membrane and activation of a flavin-dependent membrane-bound cytochrome, which is inhibited by DPI (31). Growing evidence suggests that Rac1 mediates ROS production not only by regulating the activity of NADPH oxidase but also by altering the function of mitochondria (13, 24, 37, 40, 51).

Our previous studies showed that Rac1 mediates TNF-{alpha}/CHX-induced apoptosis via JNK1/2 activation; however, the mechanism by which Rac1 activates JNK1/2 during apoptosis is still unclear. A number of reports show that TNF-{alpha}-induced ROS production and that an intracellular redox state modulate JNK activation (22, 27, 53). Our previous study showed that Rac1 and JNK1/2 are activated within 1 and 5 min, respectively, by TNF-{alpha}/CHX (21). The results of our previous and present study indicate that Rac1 increases ROS production through a DPI-sensitive oxidase system and activates JNK1/2. Rac1 is activated and accumulates in the membrane fraction after 10–30 min treatment with TNF-{alpha}/CHX (21). During the same time frame a strong burst of cytosolic ROS is observed (Fig. 1A). NSC or DPI attenuates this cytosolic ROS production, whereas Rot, a specific inhibitor of the mitochondrial ETC, has no appreciable effect (Fig. 6A). In addition, similar to the effect of Rac1 inhibition on apoptosis, DPI inhibits the TNF-{alpha}/CHX-induced JNK1/2 activation and apoptosis (Fig. 7). These data strongly suggest that, similarly to the NADPH oxidase system in phagocytic cells, an NADPH oxidase-like system functions as a ROS-generating system and is involved in TNF-{alpha}/CHX-induced JNK1/2 activation and apoptosis. We also used DHR123 to examine mitochondrial-derived ROS, which showed that living cells have strong R123 florescence after exposure to TNF-{alpha}/CHX for 90 min. Rot and Rac1 inhibition by NSC23766 or expression of dominant negative Rac1 prevented ROS production from mitochondrion (Figs. 3A and 5). Interestingly, similar to the effect of Rac1 inhibition on apoptosis, Rot inhibits DNA fragmentation (Fig. 3C), JNK activation (Fig. 4), and caspase-3 activation (Fig. 9) during apoptosis, suggesting that overproduction of mitochondrial ROS is required for TNF-{alpha}/CHX-induced JNK activation and apoptosis.

The exact species of ROS produced and the Rac1-regulated enzymes that produce ROS are uncharacterized at present. It should be noted that we have observed that H2O2-induced apoptosis is prevented by NAC, but not Rot and NSC (Fig. 6B), suggesting an important role for H2O2 and DPI-sensitive oxidase in downstream signaling during cytokine or oxidative stress-induced apoptosis. On the basis of our present findings and previous observations (summarized in Fig. 10), we conclude that TNF-{alpha}/CHX-induced apoptosis requires generation of Rac1-dependent ROS. Future directions will be the characterization of DPI-sensitive oxidase regulated by Rac1 and the mechanism by which ROS activates JNK1/2 during apoptosis in IEC.


Figure 10
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  		Fig. 10. Schematic representation of the Rac1-mediated ROS production in TNF-{alpha}/CHX-induced apoptosis in IEC-6 cells. Rac1 is activated during TNF-{alpha}-induced apoptosis. Activated Rac1 activates DPI-sensitive oxidase, leading to production of cytosolic ROS, which subsequently activates the JNK pathway. JNK results in mitochondrial dysfunction, additional ROS production, and release of cytochrome c. Caspase-3 is then activated, causing DNA fragmentation and apoptosis.

 

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This study was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-16505 and the Thomas A. Gerwin Endowment.


   ACKNOWLEDGMENTS
 
We sincerely acknowledge Mary Jane Viar for critically reading the manuscript and sincerely thank Mary Jane Viar, Dr. Rajivkumar J. Vaidya, Dr. Sujoy Bhattacharya, Dr. Wenlin Deng, Dr. Huazhang Guo, and Rebecca L. West for technical support. We thank Gregg Short for help in preparing the figures. We also appreciate Dr. Shyamali Basuroy, Dr. Kenneth E. Chapman, and Dr. Alok Tomar for helpful discussion and suggestions for this study.


   FOOTNOTES
 

Address for reprint requests and other correspondence: R. Ray, Dept. of Physiology, Univ. of Tennessee Health Science Center, 894 Union Ave., Memphis, TN 38163 (e-mail: rray{at}physio1.utmem.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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 RESULTS
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 REFERENCES
 

  1. Basuroy S, Bhattacharya S, Tcheranova D, Qu Y, Regan RF, Leffler CW, Parfenova H. HO-2 provides endogenous protection against oxidative stress and apoptosis caused by TNF-{alpha} in cerebral vascular endothelial cells. Am J Physiol Cell Physiol 291: C897–C908, 2006.[Abstract/Free Full Text]
  2. Bhattacharya S, Ray RM, Viar MJ, Johnson LR. Polyamines are required for activation of c-Jun NH2-terminal kinase and apoptosis in response to TNF-{alpha} in IEC-6 cells. Am J Physiol Gastrointest Liver Physiol 285: G980–G991, 2003.[Abstract/Free Full Text]
  3. Cacicedo JM, Benjachareowong S, Chou E, Ruderman NB, Ido Y. Palmitate-induced apoptosis in cultured bovine retinal pericytes: roles of NAD(P)H oxidase, oxidant stress, and ceramide. Diabetes 54: 1838–1845, 2005.[Abstract/Free Full Text]
  4. Carmona-Cuenca I, Herrera B, Ventura JJ, Roncero C, Fernandez M, Fabregat I. EGF blocks NADPH oxidase activation by TGF-beta in fetal rat hepatocytes, impairing oxidative stress, and cell death. J Cell Physiol 207: 322–330, 2006.[CrossRef][Web of Science][Medline]
  5. Chapman KE, Sinclair SE, Zhuang D, Hassid A, Desai LP, Waters CM. Cyclic mechanical strain increases reactive oxygen species production in pulmonary epithelial cells. Am J Physiol Lung Cell Mol Physiol 289: L834–L841, 2005.[Abstract/Free Full Text]
  6. Chen XL, Zhang Q, Zhao R, Medford RM. Superoxide, H2O2, and iron are required for TNF-{alpha}-induced MCP-1 gene expression in endothelial cells: role of Rac1 and NADPH oxidase. Am J Physiol Heart Circ Physiol 286: H1001–H1007, 2004.[Abstract/Free Full Text]
  7. Cheranov SY, Jaggar JH. TNF-{alpha} dilates cerebral arteries via NAD(P)H oxidase-dependent Ca2+ spark activation. Am J Physiol Cell Physiol 290: C964–C971, 2006.[Abstract/Free Full Text]
  8. Chess PR, O'Reilly MA, Sachs F, Finkelstein JN. Reactive oxidant and p42/44 MAP kinase signaling is necessary for mechanical strain-induced proliferation in pulmonary epithelial cells. J Appl Physiol 99: 1226–1232, 2005.[Abstract/Free Full Text]
  9. Chiarugi P, Cirri P. Redox regulation of protein tyrosine phosphatases during receptor tyrosine kinase signal transduction. Trends Biochem Sci 28: 509–514, 2003.[CrossRef][Web of Science][Medline]
  10. Chung YM, Bae YS, Lee SY. Molecular ordering of ROS production, mitochondrial changes, and caspase activation during sodium salicylate-induced apoptosis. Free Radic Biol Med 34: 434–442, 2003.[CrossRef][Web of Science][Medline]
  11. Curtin JF, Donovan M, Cotter TG. Regulation and measurement of oxidative stress in apoptosis. J Immunol Methods 265: 49–72, 2002.[CrossRef][Web of Science][Medline]
  12. Deshpande SS, Angkeow P, Huang J, Ozaki M, Irani K. Rac1 inhibits TNF-{alpha}-induced endothelial cell apoptosis: dual regulation by reactive oxygen species. FASEB J 14: 1705–1714, 2000.[Abstract/Free Full Text]
  13. Ding WX, Yin XM. Dissection of the multiple mechanisms of TNF-alpha-induced apoptosis in liver injury. J Cell Mol Med 8: 445–454, 2004.[Web of Science][Medline]
  14. Gil J, Almeida S, Oliveira CR, Rego AC. Cytosolic and mitochondrial ROS in staurosporine-induced retinal cell apoptosis. Free Radic Biol Med 35: 1500–1514, 2003.[CrossRef][Web of Science][Medline]
  15. Gloire G, Legrand-Poels S, Piette J. NF-kappaB activation by reactive oxygen species: fifteen years later. Biochem Pharmacol 72: 1493–1505, 2006.[CrossRef][Web of Science][Medline]
  16. Green DR. Apoptotic pathways: ten minutes to dead. Cell 121: 671–674, 2005.[CrossRef][Web of Science][Medline]
  17. Grouselle M, Tueux O, Dabadie P, Georgescaud D, Mazat JP. Effect of local anaesthetics on mitochondrial membrane potential in living cells. Biochem J 271: 269–272, 1990.[Web of Science][Medline]
  18. Halliwell B, Whiteman M. Measuring reactive species and oxidative damage in vivo and in cell culture: how should you do it and what do the results mean? Br J Pharmacol 142: 231–255, 2004.[CrossRef][Web of Science][Medline]
  19. Hordijk PL. Regulation of NADPH oxidases: the role of Rac proteins. Circ Res 98: 453–462, 2006.[Abstract/Free Full Text]
  20. Imoto K, Kukidome D, Nishikawa T, Matsuhisa T, Sonoda K, Fujisawa K, Yano M, Motoshima H, Taguchi T, Tsuruzoe K, Matsumura T, Ichijo H, Araki E. Impact of mitochondrial reactive oxygen species and apoptosis signal-regulating kinase 1 on insulin signaling. Diabetes 55: 1197–1204, 2006.[Abstract/Free Full Text]
  21. Jin S, Ray RM, Johnson LR. Rac1 mediates intestinal epithelial cell apoptosis via JNK. Am J Physiol Gastrointest Liver Physiol 291: G1137–G1147, 2006.[Abstract/Free Full Text]
  22. Kamata H, Honda S, Maeda S, Chang L, Hirata H, Karin M. Reactive oxygen species promote TNFalpha-induced death and sustained JNK activation by inhibiting MAP kinase phosphatases. Cell 120: 649–661, 2005.[CrossRef][Web of Science][Medline]
  23. Khanday FA, Yamamori T, Mattagajasingh I, Zhang Z, Bugayenko A, Naqvi A, Santhanam L, Nabi N, Kasuno K, Day BW, Irani K. Rac1 leads to phosphorylation-dependent increase in stability of the p66shc adaptor protein: role in Rac1-induced oxidative stress. Mol Biol Cell 17: 122–129, 2006.[Abstract/Free Full Text]
  24. Kimura S, Zhang GX, Nishiyama A, Shokoji T, Yao L, Fan YY, Rahman M, Suzuki T, Maeta H, Abe Y. Role of NAD(P)H oxidase- and mitochondria-derived reactive oxygen species in cardioprotection of ischemic reperfusion injury by angiotensin II. Hypertension 45: 860–866, 2005.[Abstract/Free Full Text]
  25. Lee YS, Kang YS, Lee JS, Nicolova S, Kim JA. Involvement of NADPH oxidase-mediated generation of reactive oxygen species in the apoptotic cell death by capsaicin in HepG2 human hepatoma cells. Free Radic Res 38: 405–412, 2004.[CrossRef][Web of Science][Medline]
  26. Lievre V, Becuwe P, Bianchi A, Bossenmeyer-Pourie C, Koziel V, Franck P, Nicolas MB, Dauca M, Vert P, Daval JL. Intracellular generation of free radicals and modifications of detoxifying enzymes in cultured neurons from the developing rat forebrain in response to transient hypoxia. Neuroscience 105: 287–297, 2001.[CrossRef][Web of Science][Medline]
  27. Nakano H, Nakajima A, Sakon-Komazawa S, Piao JH, Xue X, Okumura K. Reactive oxygen species mediate crosstalk between NF-kappaB and JNK. Cell Death Differ 13: 730–737, 2006.[CrossRef][Web of Science][Medline]
  28. Nimnual AS, Taylor LJ, Bar-Sagi D. Redox-dependent downregulation of Rho by Rac. Nat Cell Biol 5: 236–241, 2003.[CrossRef][Web of Science][Medline]
  29. Nishikawa M, Takeda K, Sato EF, Kuroki T, Inoue M. Nitric oxide regulates energy metabolism and Bcl-2 expression in intestinal epithelial cells. Am J Physiol Gastrointest Liver Physiol 274: G797–G801, 1998.[Abstract/Free Full Text]
  30. Papaharalambus C, Sajjad W, Syed A, Zhang C, Bergo MO, Alexander RW, Ahmad M. Tumor necrosis factor alpha stimulation of Rac1 activity. Role of isoprenylcysteine carboxylmethyltransferase. J Biol Chem 280: 18790–18796, 2005.[Abstract/Free Full Text]
  31. Papaiahgari S, Kleeberger SR, Cho HY, Kalvakolanu DV, Reddy SP. NADPH oxidase and ERK signaling regulates hyperoxia-induced Nrf2-ARE transcriptional response in pulmonary epithelial cells. J Biol Chem 279: 42302–42312, 2004.[Abstract/Free Full Text]
  32. Papakonstanti EA, Stournaras C. Tumor necrosis factor-alpha promotes survival of opossum kidney cells via Cdc42-induced phospholipase C-gamma1 activation and actin filament redistribution. Mol Biol Cell 15: 1273–1286, 2004.[Abstract/Free Full Text]
  33. Pham CG, Papa S, Bubici C, Zazzeroni F, Franzoso G. Oxygen JNKies: phosphatases overdose on ROS. Dev Cell 8: 452–454, 2005.[CrossRef][Web of Science][Medline]
  34. Qian W, Nishikawa M, Haque AM, Hirose M, Mashimo M, Sato E, Inoue M. Mitochondrial density determines the cellular sensitivity to cisplatin-induced cell death. Am J Physiol Cell Physiol 289: C1466–C1475, 2005.[Abstract/Free Full Text]
  35. Qian Y, Liu KJ, Chen Y, Flynn DC, Castranova V, Shi X. Cdc42 regulates arsenic-induced NADPH oxidase activation and cell migration through actin filament reorganization. J Biol Chem 280: 3875–3884, 2005.[Abstract/Free Full Text]
  36. Quaroni A, Wands J, Trelstad RL, Isselbacher KJ. Epithelioid cell cultures from rat small intestine. Characterization by morphologic and immunologic criteria. J Cell Biol 80: 248–265, 1979.[Abstract/Free Full Text]
  37. Radisky DC, Levy DD, Littlepage LE, Liu H, Nelson CM, Fata JE, Leake D, Godden EL, Albertson DG, Nieto MA, Werb Z, Bissell MJ. Rac1b and reactive oxygen species mediate MMP-3-induced EMT and genomic instability. Nature 436: 123–127, 2005.[CrossRef][Medline]
  38. Ray RM, Bhattacharya S, Johnson LR. Protein phosphatase 2A regulates apoptosis in intestinal epithelial cells. J Biol Chem 280: 31091–1100, 2005.[Abstract/Free Full Text]
  39. Ray RM, McCormack SA, Covington C, Viar MJ, Zheng Y, Johnson LR. The requirement for polyamines for intestinal epithelial cell migration is mediated through Rac1. J Biol Chem 278: 13039–13046, 2003.[Abstract/Free Full Text]
  40. Reinehr R, Becker S, Eberle A, Grether-Beck S, Haussinger D. Involvement of NADPH oxidase isoforms and Src family kinases in CD95-dependent hepatocyte apoptosis. J Biol Chem 280: 27179–27194, 2005.[Abstract/Free Full Text]
  41. Ricci JE, Gottlieb RA, Green DR. Caspase-mediated loss of mitochondrial function and generation of reactive oxygen species during apoptosis. J Cell Biol 160: 65–75, 2003.[Abstract/Free Full Text]
  42. Royall JA, Ischiropoulos H. Evaluation of 2',7'-dichlorofluorescin and dihydrorhodamine 123 as fluorescent probes for intracellular H2O2 in cultured endothelial cells. Arch Biochem Biophys 302: 348–355, 1993.[CrossRef][Web of Science][Medline]
  43. Sadowska AM, Manuel-Y-Keenoy B, De Backer WA. Antioxidant and anti-inflammatory efficacy of NAC in the treatment of COPD: discordant in vitro and in vivo dose-effects: a review. Pulm Pharmacol Ther 20: 9–22, 2007.[CrossRef][Web of Science][Medline]
  44. Sato T, Machida T, Takahashi S, Iyama S, Sato Y, Kuribayashi K, Takada K, Oku T, Kawano Y, Okamoto T, Takimoto R, Matsunaga T, Takayama T, Takahashi M, Kato J, Niitsu Y. Fas-mediated apoptosome formation is dependent on reactive oxygen species derived from mitochondrial permeability transition in Jurkat cells. J Immunol 173: 285–296, 2004.[Abstract/Free Full Text]
  45. Sawada M, Kiyono T, Nakashima S, Shinoda J, Naganawa T, Hara S, Iwama T, Sakai N. Molecular mechanisms of TNF-alpha-induced ceramide formation in human glioma cells: P53-mediated oxidant stress-dependent and -independent pathways. Cell Death Differ 11: 997–1008, 2004.[CrossRef][Web of Science][Medline]
  46. Schulze-Osthoff K, Beyaert R, Vandevoorde V, Haegeman G, Fiers W. Depletion of the mitochondrial electron transport abrogates the cytotoxic and gene-inductive effects of TNF. EMBO J 12: 3095–3104, 1993.[Web of Science][Medline]
  47. Shoji H, Oguchi S, Fujinaga S, Shinohara K, Kaneko K, Shimizu T, Yamashiro Y. Effects of human milk and spermine on hydrogen peroxide-induced oxidative damage in IEC-6 cells. J Pediatr Gastroenterol Nutr 41: 460–465, 2005.[CrossRef][Web of Science][Medline]
  48. Suematsu N, Tsutsui H, Wen J, Kang D, Ikeuchi M, Ide T, Hayashidani S, Shiomi T, Kubota T, Hamasaki N, Takeshita A. Oxidative stress mediates tumor necrosis factor-alpha-induced mitochondrial DNA damage and dysfunction in cardiac myocytes. Circulation 107: 1418–1423, 2003.[Abstract/Free Full Text]
  49. Sundaresan M, Yu ZX, Ferrans VJ, Sulciner DJ, Gutkind JS, Irani K, Goldschmidt-Clermont PJ, Finkel T. Regulation of reactive-oxygen-species generation in fibroblasts by Rac1. Biochem J 318: 379–382, 1996.[Web of Science][Medline]
  50. Suzukawa K, Miura K, Mitsushita J, Resau J, Hirose K, Crystal R, Kamata T. Nerve growth factor-induced neuronal differentiation requires generation of Rac1-regulated reactive oxygen species. J Biol Chem 275: 13175–13178, 2000.[Abstract/Free Full Text]
  51. Werner E, Werb Z. Integrins engage mitochondrial function for signal transduction by a mechanism dependent on Rho GTPases. J Cell Biol 158: 357–368, 2002.[Abstract/Free Full Text]
  52. Woo CH, Eom YW, Yoo MH, You HJ, Han HJ, Song WK, Yoo YJ, Chun JS, Kim JH. Tumor necrosis factor-alpha generates reactive oxygen species via a cytosolic phospholipase A2-linked cascade. J Biol Chem 275: 32357–32362, 2000.[Abstract/Free Full Text]
  53. Xu YC, Wu RF, Gu Y, Yang YS, Yang MC, Nwariaku FE, Terada LS. Involvement of TRAF4 in oxidative activation of c-Jun N-terminal kinase. J Biol Chem 277: 28051–28057, 2002.[Abstract/Free Full Text]
  54. Zhang B, Zhang Y, Shacter E. Rac1 inhibits apoptosis in human lymphoma cells by stimulating Bad phosphorylation on Ser-75. Mol Cell Biol 24: 6205–6214, 2004.[Abstract/Free Full Text]
  55. Zhang X, Shan P, Sasidhar M, Chupp GL, Flavell RA, Choi AM, Lee PJ. Reactive oxygen species and extracellular signal-regulated kinase 1/2 mitogen-activated protein kinase mediate hyperoxia-induced cell death in lung epithelium. Am J Respir Cell Mol Biol 28: 305–315, 2003.[Abstract/Free Full Text]
  56. Zhang Y, Wang H, Li J, Jimenez DA, Levitan ES, Aizenman E, Rosenberg PA. Peroxynitrite-induced neuronal apoptosis is mediated by intracellular zinc release and 12-lipooxygenase activation. J Neurosci 24: 10616–10627, 2004.[Abstract/Free Full Text]
  57. Zhao M, Wimmer A, Trieu K, Discipio RG, Schraufstatter IU. Arrestin regulates MAPK activation and prevents NADPH oxidase-dependent death of cells expressing CXCR2. J Biol Chem 279: 49259–49267, 2004.[Abstract/Free Full Text]




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