Journal of Cellular Biochemistry 88:545–556 (2003)

Raf-Independent and MEKK1-Dependent Activationof NF-kB by Hydrogen Peroxide in 70Z/3 pre-BLymphocyte Tumor Cells

Michael Lee* and Woo Suk Koh

Korea Institute of Toxicology, Korea Research Institute of Chemical Technology, P.O. Box 107,Yusong, Daejeon 305-600, Republic of Korea

Abstract We have previously demonstrated that hydrogen peroxide (H2O2) treatment of murine 70Z/3 pre-Blymphocytes inhibits the immune response to lipopolysaccharide by attenuating signaling through c-Jun N-terminalkinase (JNK) activation. In the present study, we further examined the signaling intermediates responsible forimmunosuppression by H2O2, focusing on NF-kB, a dimeric transcription factor whose activation is implicated in anumber of immune response. Treatment of 70Z/3 pre-B cells with H2O2 caused activation of NF-kB in the nuclei bydetection of NF-kB specific DNA binding, concomitant with phosphorylation of IkBa. H2O2 stimulation of NF-kBoccurred within 20 min of treatment, reached maximum level at 60 min, and sustained for 2 h or more. Especially, MEK1may contribute to H2O2-induced NF-kB activation as shown in the inhibition of NF-kB binding activity by the MEK1 in-hibitor, PD 98059, and H2O2-induced MEK1 activation. However, H2O2 exhibited no effect on the activity of Raf-1kinase, which was an upstream activator of MEK1. Furthermore, B-58l and a-hydroxyfarnesylphosphonic acid, twoinhibitors of Ras, did not block NF-kB activation. In addition, the transient transfection of a dominant negative Ras(RasN17) construct showed a negligible inhibitory effect on the activation of NF-kB byH2O2. Instead, treatment of 70Z/3cellswithH2O2 resulted in the activation ofMAPKkinase kinase 1 (MEKK1) aswell as JNK. Therefore, our data suggest thatH2O2 regulates the activity of NF-kB by MEK1 activation through MEKK1-dependent but Ras/Raf-independentmechanism. J. Cell. Biochem. 88: 545–556, 2003. � 2003 Wiley-Liss, Inc.

Key words: hydrogen peroxide; NF-kB; MEKK1; Raf; IkB kinase

Activation of the transcription factor, NF-kBhas been shown to be a key component ofimmune response [Ghosh et al., 1998]. Ourprevious results demonstrated that paclitaxel-induced immune suppression was associatedwith NF-kB activation via conventional PKCisotypes in lipopolysaccharide-stimulated 70Z/3pre-B lymphocyte tumor cells. [Lee and Jeon,2001]. NF-kB is composed of two subunits ofp50 and 65 kDa, which is present constitutivelyin the cytoplasm in an inactive state by in-hibitory proteins, IkB [Baldwin, 1996]. Upon

activation, IkB undergoes phosphorylation anddegradation, and theNF-kBheterodimer trans-locates into the nucleus, where it binds to DNAand activates transcription [Rice and Ernst,1993]. Recently, it has been widely known thata high molecular weight IkB kinase (IKK)complex, consisting of IKK-a, IKK-b, NF-kB-inducing kinase (NIK) and two adaptor or scaf-old proteins is involved in IkB phosphorylation[Woronicz et al., 1997].

Independent studies have also demonstrat-ed that MAPK kinase kinase 1 (MEKK1),which acts as an upstreamactivator of c-JunN-terminal kinase (JNK), activates IKK acti-vity [Lee et al., 1997]. MEKK1 has been mainlyimplicated in the activation of the JNK and theextracellular signal-regulated protein kinase(ERK) pathways based on its ability to acti-vate these kinases when overexpressed [Lange-Carter et al., 1993; Minden et al., 1994]. Mostattention has focused on JNK, in part becauseMEKK1 can induce apoptosis under certaincircumstances and JNK has been implicated in

� 2003 Wiley-Liss, Inc.

*Correspondence to: Michael Lee, Korea Institute ofToxicology, Korea Research Institute of Chemical Technol-ogy, P.O. Box 107, Yusong, Daejeon 305-600, Republic ofKorea. E-mail: mikelee@kitox.re.kr

Received 3 September 2002; Accepted 5 September 2002

DOI 10.1002/jcb.10376

this process, and becauseRaf isoforms appear tobe the major kinase for the ERK pathway[Dentet al., 1992; Kyriakis et al., 1992]. EachMAPK kinase (MAPKK), however, can be acti-vated by more than one MAPK kinase kinase(MEKK), increasing the complexity and diver-sity of MAPK signalling. In particular, activa-tion of ERK by growth factors depends on theRaf-1, but other MEKKs may activate ERK inresponse to pro-inflammatory stimuli [ChangandKarin, 2001].Also, the stimuli by serumandlysophosphatidic acid activate JNK and ERKat least in part through MEKK1 [Yujiri et al.,1998].

Reactive oxygen species (ROS) such as hydro-gen peroxide (H2O2) exert multiple regulatoryeffects on cell behavior and function as a secondmessenger [Finkel, 1998]. Especially, many celltypes produce H2O2 in response to a variety ofgrowth factor such as platelet-derived growthfactor (PDGF) and epidermal growth factor(EGF) [Sundaresan et al., 1995; Bae et al.,1997]. In addition, it has been demonstratedthat H2O2 has been known to stimulate phos-phorylation of the three identifiedMAPkinases,ERK, JNK, and p38 [Guyton et al., 1996; Iraniet al., 1997]. However, apart from functioningas second oxygen messenger of signal trans-duction, reactive oxygen species exert directtoxic effects on cell behavior. Especially, humanlymphocytes are quite susceptible to oxidativestress [El-Hag et al., 1986], although reactiveoxygen species can act as a trigger for lympho-cyte activation [Rosa, 1997]. Dumont et al.reported that H2O2 elicited apoptosis throughactivation of caspase-3 and NF-kB followedby elevated expression of p53 in human T cellline CEM C7 [Dumont et al., 1999]. Our pre-vious reports also demonstrated that H2O2

inhibited the immune response to lipopolysac-charide by attenuating signaling through JNKactivation in 70Z/3 pre-B cells [Lee and Yea,2000].

Here we show that H2O2 may induce immu-nomodulatory effects through the activation ofNF-kB in a Ras/Raf-independent, MEKK1-dependent manner that involves degradationof IkBa in 70Z/3 pre-B lymphocytes.

MATERIALS AND METHODS

Materials

Anti-Raf, anti-IKKa, anti-IKKb, and anti-MEKK1 were obtained from Santa Cruz

Biotechnology (Santa Cruz, CA). The phospho-specific antibodies were obtained from CellSignaling Technology (Beverly, MA). ProteinA-agarose was from Roche Molecular Bio-chemicals (Indianapolis, IN). B-581 and a-hydroxyfarnesylphosphonic acid were fromBiomol (Plymouth meeting, PA). Dulbecco’smodified Eagle’s medium (DMEM), fetal calfserum (FCS), and DMRIE-C reagent were pur-chased from Life Technologies, Inc. (Rockville,MD). Reagents for SDS–polyacrylamide gelelectrophoresis (SDS–PAGE) were from Bio-Rad (Hercules, CA). [g-32P]ATP (3,000Ci/mmol)was purchased from New England Nuclear(Boston, MA). GF 109203X, Go 6976, PD98059, and SB 203580 were purchased fromCalbiochem (San Diego, CA). H2O2 was ob-tained from Sigma (St. Louis, MO).

Mammalian Cell Cultureand Transient Transfection

The murine pre-B cell line, 70Z/3 (ATCCTIB 152), was obtained from theAmerican TypeCulture Collection (Manassas, VA). The cellswere cultured in RPMI 1640 medium supple-mentedwith100units ofpenicillin/ml, 100unitsof streptomycin/ml, 2mML-glutamine, and 10%FCS. 70Z/3 pre-B cells were transiently trans-fected with a dominant negative Ras cons-truct using DMRIE-C reagent (GIBCO-BRL).All assays were performed within 48 h aftertransfection, and the experiments were repeat-ed at least twice.

Electrophoretic Mobility Shift Assay (EMSA)

Nuclear extracts were prepared as previouslydescribed, and gel mobility shift assays wereperformed [Lee and Yang, 2000]. Briefly, 5 mgof nuclear extracts were incubated with 2 mgof poly (dI-dC) (Sigma) and 32P-end-labeledDNA probe (double-stranded synthetic 26-bpoligonucleotides GATCTCAGAGGGGACTTT-CCGAAGAGA containing the kB enhancer ofimmunoglobulin k light chain gene). The iden-tity of the shifted bands was confirmed bycompetitionwithunlabeled oligomer containingNF-kB site.

In Vitro Raf-1 Kinase Assay

The cell lysates were prepared as pre-viously described [Lee and Jeon, 2001]. Im-munoprecipitation was performed on the wholecell lysates using anti-Raf and protein A-agarose beads. After incubation for 2 h at 48C,

546 Lee and Koh

immunoprecipitates were washed twice withice-cold lysis buffer. After washing with kinasebuffer (20 mM Tris, pH 7.4, 20 mM NaCl,1mMDTT, 10mMMgCl2), Raf-1 kinase activitywas assayed by phosphorylation of the recom-binant MEK1 in the presence of PD 98059,which was used to inhibit the autokinase acti-vity of MEK1. The samples were resolved bySDS–PAGE, and phosphoproteins visualized byautoradiography.

IkB Kinase (IKK) Assay

For IKK assay, both anti-IKKa and IKKbantibodieswereused to immunoprecipitatebothkinases. After immunoprecipitation, kinase as-say was performed as described elsewhere [Leeand Jeon, 2001]. Briefly, the kinase reactionwas performed in 30 ml of kinase buffer (20 mMHEPES, pH7.8, 10mMMgCl2, 100 mMNa3VO4,20 mM b-glycerophosphate, 2 mM DTT, 50 mMNaCl) for30minat308Cin thepresenceof 10mMATP-10 mCi of [g-32P]ATP (10 Ci/mmol) (NENLife Science Products) and 500 ng of the sub-strate GST-IkBa (Santa Cruz Biotechnology).The reactions were terminated with 4� Laem-mli sample buffer. Proteins were analyzed on12.5% SDS–polyacrylamide gels, dried, andvisualized by autoradiography.

Immunoblot Analysis

Once 70Z/3 pre-B cells reached subconflu-ence, the cells were incubated for additionaltimes in the presence of H2O2. The cells werewashed twice with ice-cold phosphate-bufferedsaline (PBS), harvested by resuspension of thecell pellet in lysis buffer (20 mM Tris, pH 8.0,150 mM NaCl, 1% Triton X-100, 2 mM EDTA,containing 1 mM phenylmethylsulfonyl fluor-ide, 20 mg/ml aprotinin, 10 mg/ml leupeptin,20 mM b-glycerophosphate, and 2 mM sodiumfluoride). Cell lysates were clarified by centri-fugation at 15,000�g for 10 min at 48C, andtheir protein concentrations were determinedwith the aid of a BCA protein assay reagent kit(Pierce; Rockford, IL). For immunoblotting, celllysates were denatured in Laemmli samplebuffer, and resolved by SDS–polyacrylamidegel. The proteins were transferred to nitrocel-lulose, and immunoblot analysis was performedusing specific antibodies as indicated. Immunecomplexes on nitrocellulose were detected byenzyme-linked chemiluminescence (AmershamPharmacia Inc., Piscataway, NJ).

RESULTS

H2O2-Induced NF-kB Activation

In agreement with other reports [True et al.,2000] suggesting that reactive oxygen inter-mediates play a critical role in activation ofNF-kB induced by TNF and other agents,H2O2 increased NF-kB binding activity in nuc-lear extracts of 70Z/3 pre-B cells. Identity ofthe shifted bands was confirmed by competi-tion with unlabeled oligomer containing NF-kBsite. The kinetics of NF-kB activation in 70Z/3cells following H2O2 exposure were shown inFigure 1A. H2O2-induced NF-kB activation oc-curred within 20 min of treatment, reachedmaximum level at 60 min, and sustained for 2 hor more. DNA binding activity of NF-kB wasgradually increased at 100–300 mM H2O2 andsharply decreased at 1 mM (Fig. 1B).

Effect of H2O2 on IkB Kinaseand IkBa Phosphorylation

The event that occurs during activation ofNF-kB is the phosphorylation of IkB that serveto target it for ubiquitination and degradation.Therefore, we determined IkB phosphoryla-tion using a phospho-specific anti-IkBa anti-body that detects IkBa only when activated byphosphorylation at Ser-32. Treatment of cellswith H2O2 led to an increase of phosphorylatedIkBa reaching a plateau between 5 and 30 min(Fig. 2A). Next, to determine whether IkBaphosphorylationbyH2O2 results fromupstreamIKKcomplex,which phosphorylate the residuesin IkB molecule [Karin, 1999; Israel, 2000], wedirectly measured IKK complex kinase activityby immunoprecipitating both IKKa and IKKbfrom 70Z/3 cells that are treated with H2O2. Asshown in Figure 2B, H2O2 elicited a slight andtransient IKK activation.

Involvement of MEK1/ERK Pathway inH2O2-Induced NF-kB Activation

To further explore the mechanism responsi-ble for H2O2-induced NF-kB activation, severalinhibitors of tyrosine kinase and PKC wereutilized (Fig. 3). A conventional PKC inhibitor,Go 6976 partially inhibited H2O2-induced NF-kB activation. Although GF 109203X inhibitsconventional PKCs as well as novel PKCs, itis unlikely to be effective as much as Go 6976in the inhibition of conventional PKCs. Noapparent inhibition in H2O2-induced NF-kBactivation was observed with either genistein

Mechanism of H2O2-Induced NF-kB Activation 547

(tyrosine kinase inhibitor) or GF 109203X (panPKC inhibitor), which were pretreated the cellsfor 2 h before H2O2 treatment. On the otherhand, since previous reports showed that MAPkinase pathway is important in the activation ofNF-kB [Nakano et al., 1998], we also investi-gated the effect of PD 98059 (MEK inhibitor),or SB 203580 (p38 kinase inhibitor) on NF-kB activation by H2O2. PD 98059 markedly

inhibited H2O2-induced NF-kB activation sug-gesting a critical role forMEK1.U0126, anotherselective MEK inhibitor, also effectively reduc-ed the activation of NF-kB (data not shown). Incontrast, SB 203580 enhanced rather thaninhibited H2O2-induced NF-kB activation. Thisfinding is consistent with the reported resultsthat SB203580 enhanced NF-kB transcrip-tional activity [Birkenkamp et al., 2000].

Fig. 1. NF-kB activation by hydrogen peroxide (H2O2). Murine70Z/3 pre-B cells were treated with 300 mM H2O2 for theindicated time (A), or at indicated concentrations for 30 min(B). Nuclear extracts were prepared, incubated with a 32P-labeledkBprobe, andanalyzedbyEMSA.ThepositionsofNF-kBcomplexes are shown. Identity of the shifted bands was

confirmed by competition with unlabeled oligomer containingNF-kB site. The upper panel presented is representative of threeindependent experiments. The lower panel represents dataquantitated by densitometry, expressed as fold stimulation. Thevalues are the mean of three separate experiments with error barrepresenting the standard deviations (*P< 0.05).

548 Lee and Koh

H2O2 Induces NF-kB Activation ThroughMEK1/ERK Signaling Independent of Ras-Raf

Since Ras/Raf signaling has been shown to beupstream activators of MEK1/ERK pathway,we further investigated the involvement of Ras/Raf in the activation of NF-kB in 70Z/3 cellsexposed toH2O2. In this study, 70Z/3 pre-B cellswere transiently transfected with a dominantnegative Ras construct (RasN17), which blocksendogenous Ras function interfering with up-stream activation of Ras proteins [Chen et al.,1994]. As previously reported [Pahan et al.,2000], the expression of a dominant negativemutant of Ras demonstrated inhibitory effectson NF-kB activation induced by lipopolysac-charide used as a positive control, indicatingthat the transfected cells expressed RasN17proteins sufficient for inhibiting Ras function.However, transient transfection of RasN17 hadlittle inhibitory effect on H2O2-induced NF-kBactivation (Fig. 4A). In addition, B-581 and a-hydroxyfarnesylphosphonic acid, which blockthe Ras localization and function by inhibiting

farnesyltransferase activity, showed no inhibi-tory effect on H2O2-induced NF-kB activa-tion (Fig. 4B). Moreover, contrary to the ideathat Raf-1 is involved in NF-kB activation[Flory et al., 1998], H2O2 did not activate Raf-1detectably (Fig. 4C). LPS, used as a positivecontrol, demonstrated activating effects onRaf-1. Since the electrophoretic mobility ofRaf-1 has been known to be an adequate moni-tor for the activation state of Raf-1, we alsoinvestigated themobility shift of the C-terminalfragment of Raf-1 (RfIII) expressed in 70Z/3cells. It has been previously reported thatthis 33 kDa RfIII exhibited serum- and phor-bol ester-induced shift in gel mobility thatmimicked the shift observed with full-lengthRaf-1 [Olah et al., 1995]. A significant shiftin electrophoretic mobility was observed withRfIII after LPS treatment of the overexpressorcells. However, H2O2 showed no effect on RfIIImobility shift (Fig. 4C). These results implythat MEK1/ERK signaling, which is associat-ed with H2O2-induced NF-kB activation, isdistinct from Ras/Raf.

Fig. 2. Effect ofH2O2on IkB-a phosphorylation.A: 70Z/3pre-Bcells were treated with 300 mMH2O2 for the indicated time, andthe whole cell lysates were analyzed by Western blot. To detectH2O2-induced IkB phosphorylation, it was used a polyclonalphospho-specific anti-IkB-a antibody which detects IkB-a onlywhen activated by phosphorylation at Ser-32 (upper panel).In parallel experiments, immunoblot analysis was also per-formedusing anti-IkB-a to show similar amounts of proteins in alllanes (lower panel). B: 70Z/3 pre-B cells were treated as indi-cated above. Both anti-IKKa and IKKb antibodies were used to

immunoprecipitate both kinases from the cell lysates. The immu-noprecipitates of IKK complex were analyzed in the in vitrokinase assay using GST-IkBa as a substrate (upper panel). Equalamounts of the immunoprecipitated kinase complex present ineach lane were confirmed by immunoblotting for IKKa (lowerpanel). Numbers listed below each band indicate value quanti-fiedbydensitometry of immunoblots, expressedas fold change inphosphorylation, in which the phosphorylation observed inunstimulated cells was defined as 1. The results presented arerepresentative of three independent experiments.

Mechanism of H2O2-Induced NF-kB Activation 549

Fig. 3. MEK/ERK pathway is required for NF-kBactivation. 70Z/3 pre-B cells were pretreated witheither GF 109203X (1 mM), Go 6976 (1 mM), PD98059 (50 mM), SB 203580 (30 mM), or genistein(60 mM) for 2 h before stimulation with H2O2 for30 min. EMSA was performed on nuclear extracts.Identity of the shifted bands was confirmed bycompetition with unlabeled oligomer containingNF-kB site. Results of one representative experimentof three are shown in the upper panel. The lowerpanel represents data quantitated by densitometry,expressed as fold stimulation. The values are themean of three separate experiments with error barrepresenting the standard deviations (*P< 0.05).

Fig. 4. Ras/Raf pathway is not involved in H2O2-induced NF-kB activation. A: 70Z/3 pre-B cells were transfected with controlvector or dominant negativeRas (RasN17). After 48h, transfectedcells were treated with either LPS or H2O2 for 30 min. Nuclearextracts were prepared, incubated with a 32P-labeled kB probe,and analyzed by EMSA. B: 70Z/3 pre-B cells were pretreated (þ)or not (�) with either B-581 (50 mM) or a-hydroxyfarnesylpho-sphonic acid (10 mM) for 24 h before treatment with H2O2 for30 min. EMSA was performed on nuclear extracts. The lowerpanels of (A) and (B) represent data quantitated by densitometry,expressed as fold stimulation. The values are the mean of threeseparate experiments with error bar representing the standarddeviations (*P< 0.05). C: 70Z/3 pre-B cells were incubated inthe presence of 0.5% serum for 24 h to minimize serum effect onRaf-1 kinase and then treated with either 300 mM H2O2 for theindicated time, or 1mg/ml LPS for 20min. For in vitro Raf-1 kinase

assays, Raf-1 immunoprecipitates were incubated in kinasebuffer containing10mCiof [g-32P]ATP in thepresenceofMEK1asa substrate. Samples were resolved by electrophoresis on 10%SDS–PAGE, and the radioactivity incorporated into the 32P-labeled MEK-1 protein was determined by autoradiography.In themiddle panel, immunoprecipitateswere prepared in paral-lel with those assayed for kinase activity, separated by SDS–PAGE, transferred to filters, and probedwith anti-Raf antibody. Inthe bottom panel, 70Z/3 cells expressing C-terminal Raffragment (RfIII) were incubated in the presence of 0.5% serumfor 24 h prior to treatment with either vehicle, H2O2 (300 mM,30 min), or LPS (1 mg/ml, 20 min). Proteins were separatedby electrophoresis on SDS–polyacrylamide gels (12.5%). Theelectrophoretic mobility of RfIII was examined by immunoblotanalysis using the anti-Raf antibody. The results presented arerepresentative of three independent experiments.

550 Lee and Koh

Fig. 4.

Mechanism of H2O2-Induced NF-kB Activation 551

MEKK1 Pathway is Required for HydrogenPeroxide-Induced NF-kB Activation

Since MEKK1 is implicated in the activationof IKK [Lee et al., 1997], the effect of H2O2 onMEKK1 activation was measured in 70Z/3 pre-B lymphocytes. We directly measured MEKK1activity by immunoprecipitation with an anti-body against MEKK1, followed by in vitro auto-phosphorylation assay. Despite few publisheddirect demonstration, it is assumed that auto-activation of MEKK1 via autophosphorylationof Thr-575 might be an immediate response toinitial kinase activation [Deak and Templeton,1997]. The autophosphorylation ofMEKK1wasfirst observed 10 min after treatment of 70Z/3pre-B cells with H2O2, reaching a maximum at30min (Fig. 5A). The activation of JNK,which isa downstream effector of MEKK1, was alsodetectable in cells treated with H2O2 (Fig. 5B).Additionally, SEK1 and MEK1 activation wasestimated by measuring the level of phosphor-ylation in whole cell lysates using an antibodyraised against phosphorylated peptides thatcorrespond to the active forms of SEK1 andMEK1. Surprisingly, H2O2 appeared to haveno effect on SEK1 phosphorylation (Fig. 5C)despite that SEK1 is a downstream kinase ofMEKK1. Rather, treatment of pre-B cells withH2O2 substantially increased MEK1 phosphor-ylation. Moreover, the kinetics of H2O2-inducedMEK1phosphorylationwaswell correlatedwiththose of NF-kB binding activity. Phosphoryla-tion of MEK1 was first observed 20 min aftertreatment of 70Z/3 pre-B cells with H2O2 andsustained for 2 h or more (Fig. 5D). Whenwestern blotting was performed, no changein the level of MEK1 protein was observedthroughout the treatment period. Thus, thesedata suggest that MEK1 rather than SEK1 isinvolved in H2O2-induced activation of NF-kBthat is dependent of MEKK1.

DISCUSSION

In the present study, we showed that H2O2

wasable to stimulate the translocation ofNF-kBto the nucleus in 70Z/3 pre-B cells. However,contrary to other reports [Bowie and O’Neill,2000] that NF-kB activation by H2O2 was slow(120 min) and transient, H2O2-induced NF-kBactivation occurred within 20 min of treatment,reached maximum level at 60 min, and sus-tained for 2 h or more. In addition, treatment ofcells with H2O2 led to an increase of phosphory-

lated IkBa.Moreover, the present study showedthat H2O2 elicited a slight and transient IKKactivation. The results suggested by others[Jaspers et al., 2001], in which treatment withH2O2 increased IKK activity, concomitant withphosphorylation and ubiquitination of IkBa,showed consistency with our results.

MEKK1, which acts as an upstream activa-tor of JNK, is implicated in the activation ofIKK [Lee et al., 1997; Nakano et al., 1998].In this study, we found that H2O2 increasedthe autophosphorylation of MEKK1 as well asphosphorylation of JNK. Despite few publish-ed direct demonstration, it is assumed thatautoactivation of MEKK1 via autophospho-rylation of Thr-575 might be an immediateresponse to initial kinase activation [Deak andTempleton, 1997]. The kinetics ofH2O2-inducedMEKK1 phosphorylation was well correlatedwith those of NF-kB binding activity, suggest-ing that MEKK1 positively regulate NF-kBactivation in pre-B cells exposed to H2O2.However, H2O2 appeared to have no effect onSEK1 phosphorylation despite that the activa-tion signal of MEKK1 passes through SEK1.Instead, MEK1, which is an upstream activatorof ERK, was phosphorylated by H2O2 treat-ment. This result suggests that MEK1 maycontribute to H2O2-mediated NF-kB activationas shown in the inhibition of H2O2-induced NF-kBbinding activity byMEK inhibitor, PD 98059(Fig. 3), although Janssen-Heininger et al.[1999] showed the dose-dependent activationof NF-kB after exposure to PD 98059. Incontrast, Raf-1, which has been implicatedin the activation of MEK1, is unlikely to beinvolved in H2O2-induced NF-kB activation be-cause H2O2 exhibited no effect on Raf-1 kinaseactivity. This finding would be consistentwith the reported results that Raf-1 did notphosphorylate a purified GST-IkB—a fusionprotein [Hambleton et al., 1995]. In addition,a dominant negative Ras (RasN17) showednegligible inhibitory effects on H2O2-inducedNF-kB activation implying a role for Ras/Rafsignaling far more distal to NF-kB activationby H2O2, although several observations havedemonstrated that Ras is an important sen-sor of redox stress [Lander et al., 1995]. Thus,H2O2-mediated MEKK1 activation seems tobe attributable to the activation of MEK1/ERK pathway. In fact, the catalytic domain ofMEKK1 activates MEK1 by phosphorylatingit on the same sites that Raf-1 phosphorylates,

552 Lee and Koh

Fig. 5. The activation of multiple pathways including MEKK1/JNK and MEK1 by H2O2. A: Murine 70Z/3 pre-B cells weretreated with 300 mM H2O2 for the indicated time, and in vitroautokinase assays were performed on the immunoprecipitatedMEKK1 proteins to determine MEKK1 autophosphorylation. Theautophosphorylation of MEKK1 is indicated by arrow. B–D:H2O2-induced phosphorylations of JNK, SEK1, and MEK1 wereinvestigated using antibodies that detect kinases only whenactivated by phosphorylation. Murine 70Z/3 pre-B cells werestimulated with 300 mM H2O2 for the indicated time, and thewhole cell lysates were analyzed by Western blot. The phos-phorylated forms of JNK (B), SEK1 (C), and MEK1 (D) were

detected with immunoblotting using anti-phospho-JNK (Thr183/Tyr185), anti-phospho-SEK1 (Thr261) and anti-phospho-MEK1(Ser217/221) antibodies. The same blot was stripped and thenreprobed with anti-JNK, anti-SEK1, and anti-MEK1 antibodies toshow similar expression level of each protein in all lanes. In allexperiments, 70Z/3 pre-B cellswere incubated in the presence of0.5% serum for 24 h prior to H2O2 treatment to minimize serumeffect.Numbers listedbeloweachband indicate valuequantifiedby densitometry of immunoblots, expressed as fold change inphosphorylation, in which the phosphorylation observed in un-stimulated cells was defined as 1. Results of one representativeexperiment of three are shown.

Mechanism of H2O2-Induced NF-kB Activation 553

and MEKK1 activates ERK directly throughMEK1 [Gardner et al., 1994; Karandikaret al., 2000]. INF-a was also known to regulatethe activity of the MEK/ERK pathway througha Ras/Raf-independent mechanism [Romerioet al., 2000]. On the other hand, it has beenrecently reported that JNK could be activat-ed by a constitutively active MEK1 besidesSEK1 [Bagowski et al., 2001]. However, in ourstudy,MEK1didnotphosphorylateJNKinvitro(data not shown), suggesting that an indirect-

pathway may connect the MEK1/ERK cascadeto the JNK cascade. Therefore, our examinationof MAPK pathways revealed that MEKK1-dependent MEK1/ERK and JNK signaling arerequired for H2O2-induced NF-kB activation,such as okadaic acid-induced IL-6 gene expres-sion, which is mediated through the ERK1/2and JNK pathway-dependent activation of NF-kB transcriptional capacity [Tuyt et al., 1999].

Since redox signaling has been shown to acti-vate NF-kB by pathway that does not involvephosphorylation or degradation of IkBa [Imbertet al., 1996; Flohe et al., 1997],we also examinedan alternative pathway to NF-kB activation.An alternative route to NF-kB activation, in-dependent of IkB phosphorylation, involvesthe kinase Tpl-2/Cot (tumour progression locus2 kinase) which can phosphorylate p105, lead-ing to its processing into p50. In addition, Tpl-2phosphorylates and activates both MEK-1 andSEK-1 in vitro [Hagemann et al., 1999]. How-ever, the Cot response by H2O2 was not de-tectable in 70Z/3 pre-B cells (data not shown).

Many results have demonstrated a criticalrole for the PKC isoforms in theNF-kBpathwayat the level of IKK activation and IkB degrada-tion [Lallena et al., 1999; Trushin et al., 1999].In this study, a highly selective cell-permeableconventional PKC inhibitor, Go 6976 partiallyinhibited H2O2-induced NF-kB activation. Con-versely, treatment of cells with GF 109203X,which inhibits the conventional and novel iso-forms, had little or no effect on H2O2-inducedNF-kB activation. Although GF 109203X inhi-bits conventional PKCs in addition to novelPKCs, it is unlikely to be effective asmuch asGo6976 in the inhibition of conventional PKCs.Therefore, we can not exclude the possibilitythat conventional PKCs play, in part, an impor-tant role in theH2O2-inducedNF-kBactivation.However, Kaul et al. reported that conventionalPKC activity may modulate basal NF-kB activ-ity but does not participate in H2O2-stimulated

NF-kB activation [Kaul et al., 1998], implyingthat inhibitory effect of Go 6976 is due to theinhibition of basal NF-kB activity.

Originally, it has been known that NF-kBactivation suppresses the signals for cell death.However, Lin et al. [1999] reported the para-doxical role of NF-kB in apoptosis, function-ing as both a pro-apoptotic and anti-apoptoticregulatory factor within a single cell type. Ourresults lead to conclusion that Ras/Raf-independent, MEKK1-dependent MEK1 andJNK activation caused NF-kB activation, re-sulting in immune suppression in 70Z/3 pre-Bcells. Further experiments are required to de-termine if the dominant negative mutant ofMEKK1 prevents H2O2 from activating MEK1and NF-kB.

REFERENCES

Bae YS, Kang SW, Seo MS, Baines IC, Tekle E, Chock PB,Rhee SG. 1997. Epidermal growth factor (EGF)-inducedgeneration of hydrogen peroxide. Role in EGF receptor-mediated tyrosine phosphorylation. J Biol Chem 272:217–221.

Bagowski CP, Xiong W, Ferrell JE. 2001. c-Jun N-terminalkinase activation in Xenopus laevis eggs and embryos. Apossible non-genomic role for the JNK signaling path-way. J Biol Chem 276:1459–1465.

Baldwin JAS. 1996. The NF-kB and IkB proteins: New dis-coveries and insights. Annu Rev Immunol 14:649–683.

Birkenkamp KU, Tuyt LML, Lummen C, Wierenga ATJ,Kruijer W, Vellenga E. 2000. The p38 MAP kinaseinhibitor SB203580 enhances nuclear factor-kappa Btranscriptional activity by a non-specific effect upon theERK pathway. Br J Pharmacol 131:99–107.

Bowie A, O’Neill LAJ. 2000. Oxidative stress and nuclearfactor-kB activation. Biochem Pharmacol 59:13–23.

Chang L, Karin M. 2001. Mammalian MAP kinase sig-nalling cascades. Nature 410:37–40.

Chen S-Y, Huff SY, Lai C-C, Der CJ, Powers S. 1994. Ras-G15A protein shares highly similar dominant-negativebiological properties with Ras-17N and forms a stable,guanine-nucleotide resistant complex with CDC25 ex-change factor. Oncogene 9:2691–2698.

Deak JC, Templeton DJ. 1997. Regulation of the activity ofMEK kinase 1 (MEKK1) by autophosphorylation withinthe kinase activation domain. Biochem J 322:185–192.

Dent P, Haser W, Haystead TAJ, Vincent LA, Roberts TM,Sturgill TW. 1992. Activation of mitogen-activated pro-tein kinase kinase by v-Raf in NIH 3T3 cells and in vitro.Science 257:1404–1407.

Dumont A, Hehner SP, Hofmann TG, Ueffing M, Droge W,Schmitz ML. 1999. Hydrogen peroxide-induced apoptosisis CD95-independent, requires the release of mitochon-dria-derived reactive oxygen species and the activation ofNF-kB. Oncogene 18:747–757.

El-Hag A, Lipsky PE, Bennett M, Clark RA. 1986.Immunomodulation by neutrophil myeloperoxidase andhydrogen peroxide: Differential susceptibility of humanlymphocyte functions. J Immunol 136:3420–3426.

554 Lee and Koh

Finkel T. 1998. Oxygen radicals and signaling. Curr OpinCell Biol 10:248–253.

Flohe L, Brigelius-Flohe R, Saliou C, Traber MG, Packer L.1997. Redox regulation of NF-kB activation. Free RadicBiol Med 22:1115–1126.

Flory E, Weber CK, Chen P, Hoffmeyer A, Jassoy C,Rapp UR. 1998. Plasma membrane-targeted Raf kinaseactivates NF-kB and human immunodeficiency virustype 1 replication in T lymphocytes. J Virol 72:2788–2794.

Gardner AM, Vaillancourt RR, Lange-Carter CA,Johnson GL. 1994. MEK-1 phosphorylation by MEKkinase, Raf, and mitogen-activated protein kinase:Analysis of phosphopeptides and regulation of activity.Mol Cell Biol 5:193–201.

Ghosh S, MayMJ, Kopp EB. 1998. NF-kB and Rel proteins:Evolutionarily conserved mediators of immune res-ponses. Annu Rev Immunol 16:225–260.

Guyton KZ, Liu Y, Gorospe M, Xu Q, Holbrook NJ. 1996.Activation of mitogen-activated protein kinase by H2O2.Role in cell survival following oxidant injury. J Biol Chem271:4138–4142.

Hagemann D, Tropmair J, Rapp UR. 1999. Cot protoonco-protein activates the dual specificity kinases MEK-1 andSEK-1 and induces differentiation of PC12 cells. Onco-gene 18:1391–1400.

Hambleton J, McMahon M, DeFranco AL. 1995. Activationof Raf-1 and mitogen-activated protein kinase in murinemacrophages partially mimics lipopolysaccharide-induced signaling events. J Exp Med 182:147–154.

Imbert V, Rupec RA, Livolsi A, Pahl HL, Traenckner EB,Mueller-Dieckmann C, Farahifar D, Rossi B, Auberger P,Baeuerle PA, Peyron JF. 1996. Tyrosine phosphorylationof IkBa activates NFkB without proteolytic degradationof IkBa. Cell 86:787–798.

Irani K, Xia Y, Zweier JL, Sollott SJ, Der CJ, Fearon ER,Sundaresan M, Finkel T, Goldschmidt-Clermont PJ.1997. Mitogenic signaling mediated by oxidants in Ras-transformed fibroblasts. Science 275:1649–1652.

Israel A. 2000. The IKK complex: An integrator of all signalsthat activate NF-kB? Trends Cell Biol 10:129–133.

Janssen-Heininger YNW, Macara I, Mossman BT. 1999.Cooperativity between oxidants and tumor necrosis fac-tor in the activation of nuclear factor (NF)-kB: Require-ment of Ras/mitogen-activated protein kinases in theactivation of NF-kB by oxidants. Am J Respir Cell MolBiol 20:942–952.

Jaspers I, Zhang W, Fraser A, Samet JM, Reed W. 2001.Hydrogen peroxide has opposing effects on IKK activityand IkBa breakdown in airway epithelial cells. Am JRespir Cell Mol Biol 24:769–777.

Karandikar M, Xu S, Cobb MH. 2000. MEKK1 binds Raf-1and the ERK2 cascade components. J Biol Chem 275:40120–40127.

Karin M. 1999. The beginning of the end: IkB kinase (IKK)and NF-kB activation. J Biol Chem 274:27339–27342.

Kaul N, Gopalakrishna R, Gundimeda U, Choi J, FormanHJ. 1998. Role of protein kinase C in basal and hydrogenperoxide-stimulated NF-kB activation in the murinemacrophage J774A.1 cell line. Arch Biochem Biophys350:79–86.

Kyriakis JM, AppH, Zhang X-F, Banerjee P, BrautiganDL,Rapp UR, Avruch J. 1992. Raf-1 activates MAP kinase-kinase. Nature 358:417–421.

Lallena M-J, Diaz-Meco MT, Bren G, Paya CV, Moscat J.1999. Activation of IkB kinase b by protein kinase Cisoforms. Mol Cell Biol 19:2180–2188.

Lander HM, Ogiste JS, Teng KK, Novogrodsky A. 1995.p21ras as a common signaling target of reactive freeradicals and cellular redox stress. J Biol Chem 270:21195–21198.

Lange-Carter CA, Pleiman CM, Gardner AM, Blumer KJ,Johnson GL. 1993. A divergence in the MAP kinaseregulatory network defined by MEK kinase and Raf.Science 260:315–319.

Lee M, Jeon YJ. 2001. Paclitaxel-induced immune suppres-sion is associated with NF-kB activation via conventionalPKC isotypes in lipopolysaccharide-stimulated 70Z/3 pre-B lymphocyte tumor cells. Mol Pharmacol 59:248–253.

Lee M, Yang KH. 2000. 2-acetylaminofluorene suppressesimmune response through the inhibition of nuclearfactor-kB activation during the early stage of B cell deve-lopment. Toxicol Lett 114:173–180.

Lee M, Yea SS. 2000. Hydrogen peroxide inhibits theimmune response to lipopolysaccharide by attenuatingsignaling through c-Jun N-terminal kinase and p38 asso-ciated with protein kinase C. Immunopharmacology 48:165–172.

Lee FS, Hagler J, Zhijian J, Chen ZJ, Maniatis T. 1997.Activation of the IkB kinase complex by MEKK1, akinase of the JNK pathway. Cell 88:213–222.

Lin B, Williams-Skipp C, Tao Y, Schleicher MS, Cano LL,Duke RC, Scheinman RI. 1999. NF-kB functions a both apro-apoptotic and anti-apoptotic regulatory factor withina single cell type. Cell Death Differ 6:570–582.

Minden A, Lin A, McMahonM, Lange-Carter C, Derijard B,Davis RJ, Johnson GL, Karin M. 1994. Differentialactivation of ERK and JNK mitogen-activated proteinkinases by Raf-1 and MEKK. Science 266:1719–1723.

Nakano H, Shindo M, Sakon S, Nishinaka S, Mihara M,Yagita H, Okumura K. 1998. Differential regulation ofIkB kinase a and b by two upstream kinases, NF-kB-inducing kinase and mitogen-activated protein kinase/ERK kinase kinase-1. Proc Natl Acad Sci USA 95:3537–3542.

Olah Z, Ferrier A, Lehel C, Anderson WB. 1995. The33-kDa C-terminal domain of Raf-1 protein kinase exhi-bits a Ras-independent serum- and phorbol ester-inducedshift in gel mobility. Biochem Biophys Res Commun214:340–347.

Pahan K, Liu X, McKinney MJ, Wood C, Sheikh FG,Raymond JR. 2000. Expression of a dominant-negativemutant of p21(ras) inhibits induction of nitric oxidesynthase and activation of nuclear factor-kB in primaryastrocytes. J Neurochem 74:2288–2295.

Rice NR, Ernst MK. 1993. In vivo control of NF-kBactivation by IkB-a. EMBO J 12:4685–4695.

Romerio F, Riva A, Zella D. 2000. Interferone-a2b reducesphosphorylation and activity of MEK and ERK througha Ras/Raf-independent mechanism. Br J Cancer 83:532–538.

Rosa LF. 1997. Effect of adrenaline on lymphocyte meta-bolism and function. A mechanism involving cAMP andhydrogen peroxide. Cell Biochem Funct 15:103–112.

Sundaresan M, Yu ZX, Ferrans VJ, Irani K, Finkel T. 1995.Requirement for generation of H2O2 for platelet-derivedgrowth factor signal transduction. Science 270:296–299.

Mechanism of H2O2-Induced NF-kB Activation 555

True AL, Rahman A, Malik AB. 2000. Activation of NF-kBinduced by H2O2 and TNF-a and its effects on ICAM-1expression in endothelial cells. Am J Physiol Lung CellMol Physiol 279:L302–L311.

Trushin SA, Pennington KN, Algeciras-Schimnich A,Paya CV. 1999. Protein kinase C and calcineurinsynergize to activate IkB kinase and NF-kB in T lym-phocytes. J Biol Chem 274:22923–22931.

Tuyt LML, Dokter WHA, Birkenkamp K, Koopmans SB,Lummen C, Kruijer W, Vellenga E. 1999. Extracellular-

regulated kinase 1/2, Jun N-terminal kinase, and c-Junare involved in NF-kB-dependent IL-6 expression inhuman monocytes. J Immunol 162:4893–4902.

Woronicz JD, Gao X, Cao Z, Rothe M, Goeddel DV. 1997.IkB kinase-b: NF-kB activation and complex formationwith IkB kinase-a and NIK. Science 278:866–870.

Yujiri T, Sather S, Fanger GR, Johnson GL. 1998. Role ofMEKK1 in cell survival and activation of JNK and ERKpathways defined by targeted gene disruption. Science282:1911–1914.

556 Lee and Koh