doi:10.1152/ajpgi.00219.2007 294:928-937, 2008. First published Jan 24, 2008;Am J Physiol Gastrointest Liver Physiol

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

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TNF-�/cycloheximide-induced apoptosis in intestinal epithelial cells requiresRac1-regulated reactive oxygen species

Shi Jin, Ramesh M. Ray, and Leonard R. JohnsonDepartment of Physiology, University of Tennessee Health Science Center, Memphis, Tennessee

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

Jin S, Ray RM, Johnson LR. TNF-�/cycloheximide-induced apop-tosis in intestinal epithelial cells requires Rac1-regulated reactive oxygenspecies. Am J Physiol Gastrointest Liver Physiol 294: G928–G937, 2008.First published January 24, 2008; doi:10.1152/ajpgi.00219.2007.—Pre-viously we have shown that both Rac1 and c-Jun NH2-terminal kinase(JNK1/2) are key proapoptotic molecules in tumor necrosis factor(TNF)-�/cycloheximide (CHX)-induced apoptosis in intestinal epi-thelial cells, whereas the role of reactive oxygen species (ROS) inapoptosis is unclear. The present studies tested the hypothesis thatRac1-mediated ROS production is involved in TNF-�-induced apop-tosis. In this study, we showed that TNF-�/CHX-induced ROS pro-duction and hydrogen peroxide (H2O2)-induced oxidative stress in-creased apoptosis. Inhibition of Rac1 by a specific inhibitorNSC23766 prevented TNF-�-induced ROS production. The antioxi-dant, N-acetylcysteine (NAC), or rotenone (Rot), the mitochondrialelectron transport chain inhibitor, attenuated mitochondrial ROS pro-duction and apoptosis. Rot also prevented JNK1/2 activation duringapoptosis. Inhibition of Rac1 by expression of dominant negativeRac1 decreased TNF-�-induced mitochondrial ROS production.Moreover, TNF-�-induced cytosolic ROS production was inhibitedby Rac1 inhibition, diphenyleneiodonium (DPI, an inhibitor ofNADPH oxidase), and NAC. In addition, DPI inhibited TNF-�-induced apoptosis as judged by morphological changes, DNA frag-mentation, and JNK1/2 activation. Mitochondrial membrane potentialchange is Rac1 or cytosolic ROS dependent. Lastly, all ROS inhibi-tors inhibited caspase-3 activity. Thus these results indicate thatTNF-�-induced apoptosis requires Rac1-dependent ROS productionin intestinal epithelial cells.

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

REACTIVE OXYGEN SPECIES, (ROS), namely H2O2, O2•�, and

OH�, are important mediators in cellular signal transductioncascades regulating proliferation (8), apoptosis (4, 23), andmigration (28). Exposure to stimuli such as cytokines orgrowth factors increases intracellular ROS in a variety of cellsincluding fibroblasts (52), endothelial cells (35), cardiac myo-cytes (48), and epithelial cells (5), suggesting that these highlyreactive molecules play crucial roles in cellular signaling.

Tumor necrosis factor-� (TNF-�), a pleiotropic cytokineproduced by many cells and originally identified by its cyto-toxic effects, induces cell death in some types of cells, and italso elicits a wide range of physiological responses, such asinflammation, cell proliferation, and differentiation. Recentaccumulating evidence has demonstrated that ROS are keymediators of many cellular responses to TNF-� such as apop-tosis (1), transcriptional factor activation (15), calcium sparkactivation (7), and insulin signaling (20).

The small GTPase Rac1 has been established as an impor-tant mediator in regulation of cytoskeletal organization, cellmigration, proliferation, and apoptosis (54). Rac1 is activatedin response to TNF-� in a large body of cell lines includingfibroblasts (49), endothelial cells (6, 30), and epithelial cells(21, 32). Recently, Rac1 has been implicated in the control ofROS production via activation of NADPH oxidase, lipooxy-genase, and mitochondrial oxidant production (9).

Although TNF-� has been widely used as a potent apoptoticinducer, we have found that TNF-� alone cannot induceapoptosis in IEC-6 cells, and suppressing the synthesis of theshort-lived antiapoptotic protein by cycloheximide (CHX) isrequired for TNF-�-induced apoptosis (2). Therefore, we usedTNF-�/CHX to investigate the apoptotic signaling in intestinalepithelial cells (IEC). We have shown that both Rac1 and c-JunNH2-terminal kinase (JNK) are key proapoptotic molecules inTNF-�/CHX-induced apoptosis in IEC-6 cells (2, 21, 38),whereas the role of ROS in TNF-�/CHX-induced apoptosis inIEC is unclear. Given this background, the present study testedthe hypothesis that Rac1-mediated ROS production is requiredfor TNF-�/CHX-induced apoptosis. We demonstrated thatTNF-�/CHX-induced ROS production was required for apop-tosis. Cellular enzymes, such as membrane associated-oxidasein addition to respiratory mitochondrial enzymes, were in-volved in ROS production induced by ligand (cytokine)-recep-tor interaction. Inhibition of Rac1 attenuated mitochondrial andcytoplasmic ROS production. The inhibitor of NADPH oxi-dase, diphenyleniodonium (DPI), mimicked the effect of inhi-bition of Rac1 on apoptosis. Taken together, these data indicatethat TNF-�/CHX-induced apoptosis in IEC-6 cells requiresgeneration of Rac1-regulated ROS.

MATERIALS AND METHODS

Reagents. The IEC-6 cell line (ATCC CRL 1592) was obtainedfrom the American Type Culture Collection (Manassas, VA) atpassage 13. This cell line is derived from normal rat intestine and wasdeveloped and characterized by Quaroni et al. (36). IEC-6 cells arenontumorigenic, originate from intestinal crypt cells as judged bymorphological and immunologic criteria, and retain the undifferenti-ated character of epithelial stem cells. Tests for mycoplasma werealways negative. Cell cultureware was purchased from Corning GlassWorks (Corning, NY). Cell culture medium, fetal bovine serum(FBS), dialyzed FBS (dFBS), trypsin/EDTA, antibiotics, and insulinwere obtained from GIBCO (Grand Island, NY). Mammalian proteinextraction reagent and the bicinchoninic acid protein assay reagent kitwere purchased from Pierce (Rockford, IL). The enhanced chemilu-minescence Western blot detection system was purchased fromDuPont-New England Nuclear (Boston, MA). Caspase-3 substrate

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

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

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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was purchased from Biomol Research Laboratories (Plymouth Meet-ing, PA). Rabbit anti-phospho-ERK1/2 antibody, rabbit anti-ERK1/2antibody, 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-� was obtained from Pharmingen International(San Diego, CA). The Cell Death Detection ELISA Plus kit waspurchased from Roche Diagnostics (Indianapolis, IN). NSC23766 androtenone (Rot) were purchased from Calbiochem (La Jolla, CA).Secondary antibodies conjugated to horseradish peroxidase, N-acetyl-cysteine (NAC), DPI, CHX, and dichlorodihydrofluorescein diacetate(DCFH-DA) were obtained from Sigma (St. Louis, MO). Dihydro-rhodamine123 (DHR123), rhodamine123 (R123), and neutral red(NR) were purchased from Molecular Probes (Eugene, OR). Allchemicals were of the highest purity commercially available.

Cell culture and transfection. The stock cell culture was maintainedin Dulbecco’s Modified Eagle’s Medium (DMEM) supplementedwith 5% heat-inactivated FBS, 10 �g/ml insulin, and 50 �g/mlgentamicin sulfate in T-150 flasks and incubated at 37°C in a humid-ified atmosphere of 90% air-10% CO2. Stock cells were passagedonce weekly and fed three times per week, and passages 15–22 wereused. During the experimental setup, cells were trypsinized with0.05% trypsin and 0.53 mM EDTA and counted by using a BeckmanCoulter Counter (model no. Z1). For the 4-day experimental setup,cells were grown in DMEM/5% dFBS to confluence for 3 daysfollowed by serum deprivation for 24 h. IEC-6 cells were transfectedwith pMX-internal ribosome entry site (IRES)-green fluorescent pro-tein (GFP)-N17-Rac1 (dominant negative) or pMX-IRES-GFP (vec-tor). Stable clones were isolated and characterized as previouslydescribed (39).

Cellular viability assay. NR assay was used to evaluate the IEC-6cell survival, as described previously (47). Briefly, after being treatedwith ROS inhibitors, IEC-6 cells were incubated with 50 �g/ml NR for1 h. Subsequently, the monolayer was rinsed twice with Dulbecco’sphosphate-buffered saline (DPBS), and the NR was extracted with 1%(vol/vol) acetic acid in a 50% (vol/vol) ethanol solution. Absorbancewas read at 560 nm in a plate reader. The index of cellular viabilitywas calculated as percentage of optical density with respect to un-treated control cells.

Apoptosis assay. Cells were plated (day 0) in T-75 flasks at adensity of 6.25 � 104 cells/cm2 in DMEM/dFBS with triplicatesamples for each group. Cells were fed on day 2. On day 3, the cellculture medium was removed and replaced with serum-free medium.On day 4, TNF-� with CHX or H2O2 was added to the serum-freemedium for 3 h. After various treatments, images were captured witha charge-coupled device camera. In some experiments, cells wereincubated in the presence of the following inhibitors: NSC23766(Rac1 inhibitor), NAC, Rot, or DPI. All inhibitors were also includedin the medium during the 3-h exposure to TNF-�/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 �g/ml) or DHR123 (10 �M) for 30 min at 37°C. Some groups ofcells were rapidly rinsed twice with DPBS and observed under afluorescent microscopy. For quantitative measurement of ROS pro-duction, cells were rapidly rinsed twice with DPBS to remove the freeprobe, scraped in 750 �l of DPBS, and transferred into microcentri-fuge tubes. The cells were pelleted at 2,000 g for 5 min at 4°C andresuspended in 0.5 ml of ice-cold DPBS. The cell suspensions weresonicated on ice for 15 s, clarified by centrifugation, and 100 �l ofaliquots were dispensed into black 96-well plates in triplicate. Thefluorescence in the supernatant was assessed with a plate reader(excitation 480 nm/emission 530 nm). The index of oxidation wascalculated as percentage of fluorescence intensity compared withuntreated control or vector cells.

Mitochondrial membrane potential measurement. Mitochondrialmembrane potential (��m) was detected as described previously (17).Cells were grown for 4 days on six-well plates and treated withTNF-�/CHX for 1.5–2 h. Cells were washed with HBSS, coveredwith 2.5 �g/ml R123, and incubated at 37°C in a 5% CO2 incubatorfor 30 min. Cells were gently rinsed with HBSS and examined byfluorescent microscopy.

Quantitative DNA fragmentation ELISA. IEC-6 cells were har-vested, lysed, and centrifuged to pellet nuclei. An aliquot of thesupernatant was incubated with immunoreagents (anti-histone-biotinplus anti-DNA-peroxidase-conjugated antibody) in 96-well streptavi-din-coated plates on a shaker. After samples were washed withincubation buffer, substrate buffer was added to each well, andabsorbance was read at 405 nm in a microplate reader. Protein wasdetermined by using the bicinchoninic acid method. DNA fragmen-tation was expressed as absorbance units per milligram protein perminute.

Caspase-3 activity assay. IEC-6 cells were harvested and washedwith cold DPBS. The cell pellet was resuspended in ice-cold lysisbuffer. After incubation on ice, the lysate was centrifuged at 10,000 gat 4°C for 10 min. The resulting supernatant was used for themeasurement of caspase-3 activity. Each reaction (100 �l) contained10 �l of cytosolic lysate, 70 �l of assay buffer, and 20 �l of 2 mMAc-DEVD-p-nitroanilide, the substrate for caspase-3. The enzymaticreaction was carried out in 96-well plates at 37°C. Absorbance wasread at 405 nm in a microplate reader. Protein was determined usingthe bicinchoninic acid method. Caspase activity was expressed aspicomole p-NA released per milligram protein per minute.

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

Statistics. Data are expressed as means � SE. All experiments wererepeated three times (n � 3). ANOVA with appropriate post hoctesting was used to determine the significance of the differencesbetween the means of multiple treatments. P 0.05 was regarded asstatistically significant.

RESULTS

Oxidative stress and apoptosis in IEC-6 cells. Treatmentwith TNF-� results in an increase in ROS formation in endo-thelial cells, muscle cells, fibroblasts, and epithelial cells (7,32, 52). In the present study we used DCFH-DA to measure thetime course of TNF-�/CHX-induced ROS generation. Thenonfluorescent DCFH-DA rapidly penetrates the cell mem-brane where it is deacetylated by intracellular esterases. Anonfluorescent compound, DCFH, is formed and is trapped inthe cytosol where it is preferentially oxidized by hydrogenperoxide to the fluorescent DCF, which is retained within thecells and thus provides an index of overall cellular oxidation(42). As shown in Fig. 1A, bar 1, fluorescence intensity in cellschallenged with 0.5 mM H2O2 for 15 min was significantlyincreased compared with that in the untreated control group,indicating the effectiveness of DCF to detect ROS in IEC-6cells. After administration of TNF-�/CHX at various timeintervals, significantly increased fluorescence intensity wasobserved within 20 min and persisted for 2 h without signifi-cant alteration (Fig. 1A, bars 3–8), suggesting that TNF-�/CHX induces oxidative stress in IEC-6 cells. Although DCFfluorescence is commonly used as an indicator of intracellularH2O2 levels, several studies indicate that DCF is an indicator ofgeneralized oxidative stress rather than any particular ROS.Increased DCF fluorescence observed during apoptosis hasbeen linked to increased cytochrome c release due to mito-chondrial membrane permeability transition (18). We have

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shown that TNF-�/CHX leads to cytochrome c release via JNKactivation and induces apoptosis in IEC-6 cells (2). Therefore,results presented in Fig. 1 indicate the cumulative effects ofoxidative stress and cytochrome c release. Since H2O2 andTNF-�/CHX treatment generated similar levels of generalizedoxidative stress, we examined apoptosis in response to differ-ent doses of H2O2. Cells were treated with H2O2 (100–1,000�M) for 3 h, and DNA fragmentation was measured. H2O2

concentrations from 100- 250 �M showed a dose-dependentincrease in apoptosis (Fig. 1B). However, H2O2 concentrations250 �M caused necrosis within 1 h (data not shown).

ROS inhibitors have no cytotoxic effects on IEC-6 cells. Ourprevious study showed that inhibition of Rac1 protects IEC-6cells from apoptosis (21). In the present study we used severalROS inhibitors to test the hypothesis that Rac1-mediated ROSproduction regulates apoptosis. To assess whether these inhib-itors have cytotoxic effects on IEC-6 cells, cell viability wasmeasured using NR. Most living cells actively accumulate NRin lysosomes, a process that requires intact membranes andactive metabolism. Failure to take up NR indicates that thecells have suffered damage. The dye taken up by the cell issubsequently extracted. The amount of NR taken into cells viaendocytosis can be measured by optical density and is directlyproportional to cell viability. As shown in Fig. 2, exposure toTNF-�/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 wasnot different from that seen in untreated controls. These resultssuggest that the inhibitors had no cytotoxic effects on IEC-6cells.

Mitochondrial ROS is required for TNF-�/CHX-inducedapoptosis. ROS is a well-known mediator of cell death (14, 44,45, 56). It has been reported that electron leakage from themitochondrial electron transport chain (ETC) is the primarysource of ROS in response to TNF-� (46). Complex I, NADH

Fig. 1. Oxidative stress and apoptosis in intestinal epithelial cells (IEC)-6cells. IEC-6 cells were grown as described in MATERIALS AND METHODS.A: oxidative stress in cells incubated with TNF-� (20 ng/ml) � cycloheximide(CHX) (25 �g/ml) for indicated time intervals was detected by dichlorodihy-drofluorescein diacetate (DCFH-DA) as described in MATERIALS AND METHODS.One group of cells challenged with H2O2 (0.5 mM) for 15 min served as apositive control. Values are means � SE of 3 observations. *P 0.05,compared with untreated control group. B: cells were challenged with differentconcentrations of H2O2 for 3 h. DNA fragmentation was measured by ELISA asdescribed in MATERIALS AND METHODS. Values are means � SE of 3 observations.*P 0.05, compared with corresponding control group. OD, optical density.

Fig. 2. Effect of reactive oxygen species (ROS) inhibitors on cell viability.IEC-6 cells were grown as described in MATERIALS AND METHODS. Cells weretreated with TNF-� (20 ng/ml) � CHX (25 �g/ml), NSC (NSC23766, 30 �M),N-acetylcysteine (NAC) (1 mM), diphenylene iodonium (DPI) (1 �M), rote-none (Rot) (1 �M) for 3 h, and H2O2 (1 mM) for 60 min. One group of cellsserved as a negative control (UT, untreated). After treatment, cell viability wasmeasured by neutral red assay as described in MATERIALS AND METHODS. Valuesare means � SE of 3 observations. *P 0.05, compared with untreated controlgroup.

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oxidoreductase, and complex III, ubiquinol cytochrome coxidoreductase, are the two major potential sites in the mito-chondrial ETC for superoxide production (11); we used NACor Rot to evaluate the role of mitochondrial ROS in apoptosisby DHR123 staining. DHR123 can easily cross the cell mem-brane and react with ROS in mitochondria to generate thepositively charged R123 (26). NAC acts as both a precursor ofreduced glutathione and a direct ROS scavenger (43). Rot, aninhibitor of complex I of the mitochondrial ETC, blocks theelectron flow between mitochondrial complexes I and III andsubsequently inhibits ROS production (48). As shown inFig. 3A, bar 1, fluorescence intensity significantly increased inthe group challenged with hydrogen peroxide for 15 min com-pared with that in the untreated group, suggesting the effec-tiveness of DHR123 for detection of ROS. After exposure toTNF-�/CHX for 90 min, the increased ROS production wassignificantly inhibited by NAC or Rot, suggesting the effec-tiveness of NAC and Rot on blocking mitochondrial ROSproduction (Fig. 3A, bars 2–7). Figure 3B demonstrates that adramatic increase in DNA fragmentation in cells exposed toTNF-�/CHX was suppressed by NAC. A previous study hasshown that treatment with Rot at a concentration of 1 �M for24 h does not affect the viability of IEC-6 cells (29). As shownin Fig. 3C, TNF-�/CHX-induced DNA fragmentation wasdramatically attenuated in the presence of Rot, suggesting thatmitochondrial ROS formation is required for TNF-�/CHX-induced apoptosis. Based on our previous observations (21)and other studies (22, 27, 33) showing that JNK is a majorapoptotic kinase and that ROS mediates JNK1/2 activationduring apoptosis, we used Rot to test whether mitochondrialROS regulates apoptosis via JNK1/2 activation. Rot treatmentsignificantly prevented TNF-�/CHX-induced JNK1/2 phos-phorylation but did not alter the phosphorylation of ERK1/2(Fig. 4), suggesting that TNF-�/CHX-induced mitochondrialROS production is required for JNK1/2 activation followingtreatment with TNF-�/CHX.

Rac1 inhibition prevents TNF-�/CHX-induced mitochon-drial ROS. We evaluated the effect of inhibiting Rac1 onmitochondrial ROS production by using DHR123. There was aweak but homogeneous accumulation of R123 in the mitochon-dria of untreated cells due to basal ROS levels (Fig. 5Aa). Theaccumulation of R123 was strongly enhanced in cells exposedto TNF-�/CHX (Fig. 5Ab). The accumulation of R123 in cellsexposed to NSC23766 alone (Fig. 5Ac) was similar to that ofuntreated cells. Rac1 inhibition largely prevented the TNF-�/CHX-induced accumulation of R123 (Fig. 5Ad). As shown inFig. 5B, vector-transfected cells exposed to TNF-�/CHX for 90min showed an increase in ROS formation compared withuntreated cells. However, cells expressing dominant negative(DN)-Rac1 had significantly inhibited TNF-�/CHX-inducedROS production. These results suggest that the overproductionof mitochondrial ROS induced by TNF-�/CHX is mediated byRac1 during apoptosis.

Rac1 modulates extramitochondrial ROS formation duringTNF-�/CHX-induced apoptosis. We tested whether TNF-�/CHX also induces oxidative stress from extramitochondrialsite(s) and whether this oxidation phenomenon is Rac1 depen-dent. Interestingly, as shown in Fig. 6A, after administration ofTNF-�/CHX for 30 min, cytosolic oxidative stress was signif-icantly prevented in cells treated with the Rac1 inhibitor(NSC23766) and by DPI, a specific inhibitor of flavoprotein, a

Fig. 3. Effect of NAC or Rot on TNF-�/CHX-induced mitochondrial ROSproduction and DNA fragmentation. IEC-6 cells were grown as described inMATERIALS AND METHODS. A: cells were pretreated with NAC (0.5 mM) or Rot(0.5 �M) for 1 h and exposed to TNF-� (20 ng/ml) � CHX (25 �g/ml) for anadditional 1.5 h. Mitochondrial ROS was measured by dihydrorhodamine123(DHR123) as described in MATERIALS AND METHODS. One group of cellschallenged with H2O2 (0.5 mM) for 15 min served as a positive control. Valuesare means � SE of 3 observations. *P 0.05, compared with correspondingcontrol 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 �M, C) for 1 h followed by exposure to TNF-� (20 ng/ml) � CHX (25�g/ml) for 3 h. DNA fragmentation was measured by ELISA as described inMATERIALS AND METHODS. Values are means � SE of 3 observations. *P 0.05, compared with corresponding control group.

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required component of membrane associated oxidases, such asthe NADPH oxidase complex. However, Rot had no significanteffect on the early burst of oxidative stress. TNF-�/CHX-induced oxidative stress was significantly inhibited in cellsexpressing DN-Rac1 (126 � 1.3%) compared with vector-transfected cells (211 � 2.0%). These data clearly suggest thatmitochondrial oxidase is not involved in this event and thatRac1 mediates the generation of the relatively early burst ofoxidative stress via a DPI-sensitive oxidase system. UnlikeRac1 inhibitor, NAC and DPI decreased the basal oxidativestress (Fig. 6A), suggesting that Rac1 does not modulate basaloxidative stress levels. We also studied the effect of NAC,NSC, and Rot on H2O2-induced apoptosis. Results in Fig. 6Bshow that NAC, a direct ROS scavenger, significantly de-creased apoptosis. Rac1 inhibition by NSC23766 did not sig-nificantly prevent cell death after a challenge with H2O2,

suggesting that Rac1 inhibition does not directly act to scav-enge ROS and most likely interferes with the production ofROS. Interestingly, blocking electron transfer from mitochon-dria complex I to complex III by Rot significantly increasedH2O2-induced apoptosis. These results imply that functionalETC is critical for the survival of the cells during oxidativestress and apoptosis induced by exogenous H2O2.

DPI prevents TNF-�/CHX-induced apoptosis. To evaluatethe possible role of the cytosolic ROS in TNF-�/CHX-inducedapoptosis, we analyzed the effect of DPI on apoptosis using

morphology, DNA fragmentation, and JNK1/2 activation. Pre-treatment with DPI for 1 h followed by TNF-�/CHX exposurefor 3 h almost completely prevented the detachment of cells,and cultures retained the morphological features of untreatedmonolayers of IEC-6 cells (Fig. 7A). DPI significantly pre-

Fig. 5. Effect of Rac1 inhibition on TNF-�/CHX-induced mitochondrial ROSproduction. IEC-6 cells were grown as described in MATERIALS AND METHODS.A: cells preincubated with or without NSC23766 (30 �M) were treated withTNF-� (20 ng/ml) � CHX (25 �g/ml) for 1.5 h. TNF-�/CHX-induced ROSproduction was detected by DHR123. a, control; b, cells exposed to TNF-�/CHX; c, cells incubated with NSC23766 alone; d, cells pretreated withNSC23766 and then exposed to TNF-�/CHX. B: IEC-6 cells transfected withempty vector or the dominant negative Rac1 mutant (DN-Rac1) were exposedto TNF-� (20 ng/ml) � CHX (25 �g/ml) for 1.5 h. Mitochondrial ROS wasmeasured by DHR123 as described in MATERIALS AND METHODS. Values aremeans � SE of 3 observations. *P 0.05, compared with correspondingvector group.

Fig. 4. Effect of Rot on TNF-�/CHX-induced phosphorylation of JNK1/2 andERK1/2. IEC-6 cells were grown as described in MATERIALS AND METHODS.Cells were pretreated with or without Rot (1 �M) for 1 h and exposed toTNF-� (20 ng/ml) � CHX (25 �g/ml) for an additional 3 h. Phosphorylationand total JNK1/2 and ERK1/2 was determined by Western blot analysis asdescribed in MATERIALS AND METHODS.

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vented TNF-�/CHX-induced DNA fragmentation (Fig. 7B). Asexpected, DPI significantly inhibited phosphorylation ofJNK1/2 (Fig. 7C). Taken together, these data indicate thatcytosolic ROS is required for TNF-�/CHX-induced apoptosis.

Rac1 and cytosolic ROS-dependent changes in mitochon-drial membrane potential. Growing evidence suggests thatgeneration of ROS during apoptosis leads to mitochondrialdamage (16, 41). Therefore, we examined mitochondrial mem-brane potential change (��m), an indicator of dysfunction ofmitochondria, by using R123. Normal healthy mitochondriaretain R123 with a characteristic granular localization until the

change in membrane potential (Fig. 8A). As shown in Fig. 8ETNF-�/CHX treatment (1.5 h) caused a diffused localization ofR123, suggesting a loss of ��m. It should be noted that cellulardistribution of R123 did not significantly change followingTNF-�/CHX treatment in cells pretreated with NSC23766

Fig. 7. Effect of DPI on TNF-�/CHX-induced apoptosis. IEC-6 cells weregrown as described in MATERIALS AND METHODS. Cells were preincubated withor without 0.5 �M DPI for 1 h followed by TNF-� (20 ng/ml) � CHX (25�g/ml) for 3 h. A: apoptosis was measured by morphological analysis asdescribed in MATERIALS AND METHODS. a, control; b, cells exposed to TNF-�/CHX for 3 h; c, cells exposed to DPI alone; d, cells pretreated with DPI andthen exposed to TNF-�/CHX for 3 h. B: DNA fragmentation was measuredwith a colorimetric ELISA kit as described in MATERIALS AND METHODS. Valuesare means � SE of 3 observations. *P 0.05, compared with correspondingcontrol group. C: JNK1/2 activation was determined by Western blot analysis.

Fig. 6. Effect of ROS inhibitors and inhibition of Rac1 on TNF-�/CHX-induced production of early burst of intracellular ROS and H2O2-inducedapoptosis. IEC-6 cells were grown as described in MATERIALS AND METHODS.A: cells were preincubated with NSC (NSC23766, 30 �M), NAC (0.5 mM),DPI (0.5 �M), Rot (0.5 �M), and without inhibitors (UT, untreated) followedby exposure to TNF-� (20 ng/ml) � CHX (25 �g/ml) for 30 min. Aftertreatment, ROS was measured by DCFH-DA as described in MATERIALS AND

METHODS. Values are means � SE of 3 observations. *P 0.05, comparedwith corresponding group untreated with inhibitors (UT). B: cells were prein-cubated with NSC (NSC23766, 30 �M), NAC (0.5 mM), Rot (0.5 �M), andwithout inhibitors followed by exposure to H2O2 (250 �M) for 3 h. DNAfragmentation was measured with a colorimetric ELISA kit as described inMATERIALS AND METHODS. Values are means � SE of 3 observations. *P 0.05, compared with corresponding control group.

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Fig. 8. Effect of NSC23766, DPI, and SP600125 on TNF-�/CHX-induced mitochondrial membrane potential change.IEC-6 cells were grown as described in MATERIALS AND

METHODS. Cells were preincubated with or withoutNSC23766 (NSC, 30 �M), DPI (0.5 �M), or SP600125 (25�M), respectively, followed by TNF-� (20 ng/ml) � CHX(25 �g/ml) for 1.5–2 h. After treatment, mitochondrial mem-brane potential change was detected by R123. A, control; B,cells exposed to NSC23766 alone; C, cells exposed to DPIalone; D, cells exposed to SP600125 alone; E, cells exposed toTNF-�/CHX; F, cells pretreated with NSC23766 and thenexposed to TNF-�/CHX; G, cells pretreated with DPI and thenexposed to TNF-�/CHX; H, cells pretreated with SP600125and then exposed to TNF-�/CHX.

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(Rac1 inhibitor), DPI (NADPH oxidase inhibitor), orSP600125 (JNK1/2 inhibitor). These data indicate that a Rac1-cytosolic ROS-JNK pathway mediates mitochondrial mem-brane potential transition during apoptosis.

Inhibition of Rac1, ROS, and JNK1/2 prevent TNF-�/CHX-induced caspase-3 activation. As shown in Fig. 9, various ROSinhibitors, NAC, Rot, and DPI, attenuated caspase-3 activationduring apoptosis, which is similar to the effects of Rac1 andJNK inhibitors on caspase-3 activation, suggesting that inhibi-tion of ROS prevents TNF-�/CHX-induced caspase-3 activa-tion and thereby apoptosis.

DISCUSSION

ROS production mediated by the small GTPase Rac1 regu-lates cell proliferation, apoptosis, differentiation, and geneexpression (19, 50). With respect to apoptosis, Rac1-dependentROS production has varying effects, in some cases protectingcells from apoptosis (12), whereas many other studies haveshown that Rac1 mediates apoptosis via NADPH oxidase-dependent ROS production (3, 10, 25). NADPH oxidase is amulticomponent enzyme complex present in the membranes ofvirtually all cells (55, 57). Activation of these systems dependson translocation of Rac from the cytoplasm to the cell mem-brane and activation of a flavin-dependent membrane-boundcytochrome, which is inhibited by DPI (31). Growing evidencesuggests that Rac1 mediates ROS production not only byregulating the activity of NADPH oxidase but also by alteringthe function of mitochondria (13, 24, 37, 40, 51).

Our previous studies showed that Rac1 mediates TNF-�/CHX-induced apoptosis via JNK1/2 activation; however, themechanism by which Rac1 activates JNK1/2 during apoptosisis still unclear. A number of reports show that TNF-�-inducedROS production and that an intracellular redox state modulateJNK activation (22, 27, 53). Our previous study showed thatRac1 and JNK1/2 are activated within 1 and 5 min, respec-tively, by TNF-�/CHX (21). The results of our previous and

present study indicate that Rac1 increases ROS productionthrough a DPI-sensitive oxidase system and activates JNK1/2.Rac1 is activated and accumulates in the membrane fractionafter 10 –30 min treatment with TNF-�/CHX (21). Duringthe same time frame a strong burst of cytosolic ROS isobserved (Fig. 1A). NSC or DPI attenuates this cytosolic ROSproduction, whereas Rot, a specific inhibitor of the mitochon-drial ETC, has no appreciable effect (Fig. 6A). In addition,similar to the effect of Rac1 inhibition on apoptosis, DPIinhibits the TNF-�/CHX-induced JNK1/2 activation and apop-tosis (Fig. 7). These data strongly suggest that, similarly to theNADPH oxidase system in phagocytic cells, an NADPH oxi-dase-like system functions as a ROS-generating system andis involved in TNF-�/CHX-induced JNK1/2 activation andapoptosis. We also used DHR123 to examine mitochondrial-derived ROS, which showed that living cells have strong R123florescence after exposure to TNF-�/CHX for 90 min. Rot andRac1 inhibition by NSC23766 or expression of dominantnegative Rac1 prevented ROS production from mitochondrion(Figs. 3A and 5). Interestingly, similar to the effect of Rac1inhibition on apoptosis, Rot inhibits DNA fragmentation (Fig.3C), JNK activation (Fig. 4), and caspase-3 activation (Fig. 9)during apoptosis, suggesting that overproduction of mitochon-drial ROS is required for TNF-�/CHX-induced JNK activationand apoptosis.

The exact species of ROS produced and the Rac1-regulatedenzymes that produce ROS are uncharacterized at present. Itshould be noted that we have observed that H2O2-inducedapoptosis is prevented by NAC, but not Rot and NSC (Fig. 6B),suggesting an important role for H2O2 and DPI-sensitive oxi-dase in downstream signaling during cytokine or oxidativestress-induced apoptosis. On the basis of our present findingsand previous observations (summarized in Fig. 10), we con-clude that TNF-�/CHX-induced apoptosis requires generation

Fig. 9. Effect of inhibition of Rac1, ROS, and JNK on TNF-�/CHX-inducedcaspase-3 activation. IEC-6 cells were grown as described in MATERIALS AND

METHODS. Cells were preincubated with or without NSC23766 (NSC, 30 �M),NAC (0.5 mM), DPI (0.5 �M), Rot (0.5 �M), or SP600125 (25 �M),respectively, followed by TNF-� (20 ng/ml) � CHX (25 �g/ml) for 3 h. Aftertreatment, caspase-3 activity was measured. Values are means � SE of 3observations. *P 0.05, compared with corresponding control group.

Fig. 10. Schematic representation of the Rac1-mediated ROS production inTNF-�/CHX-induced apoptosis in IEC-6 cells. Rac1 is activated duringTNF-�-induced apoptosis. Activated Rac1 activates DPI-sensitive oxidase,leading to production of cytosolic ROS, which subsequently activates the JNKpathway. JNK results in mitochondrial dysfunction, additional ROS produc-tion, and release of cytochrome c. Caspase-3 is then activated, causing DNAfragmentation and apoptosis.

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of Rac1-dependent ROS. Future directions will be the charac-terization of DPI-sensitive oxidase regulated by Rac1 and themechanism by which ROS activates JNK1/2 during apoptosisin IEC.

ACKNOWLEDGMENTS

We sincerely acknowledge Mary Jane Viar for critically reading themanuscript 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 thefigures. We also appreciate Dr. Shyamali Basuroy, Dr. Kenneth E. Chapman,and Dr. Alok Tomar for helpful discussion and suggestions for this study.

GRANTS

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

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