H2O2-dependent Activation of GCLC-ARE4 Reporter Occurs by Mitogen-activated Protein Kinase Pathways without Oxidation of Cellular Glutathione or Thioredoxin-1
2004; Elsevier BV; Volume: 279; Issue: 7 Linguagem: Inglês
10.1074/jbc.m307547200
ISSN1083-351X
AutoresYoung‐Mi Go, Jerry J. Gipp, Ríona Mulcahy, Dean P. Jones,
Tópico(s)Genomics, phytochemicals, and oxidative stress
ResumoThe gp91phox homologue Nox1 produces H2O2, which induces cell growth, transformation, and tumorigenicity. However, it has not been clear whether H2O2 effects are mediated indirectly via a generally oxidizing cellular environment or whether H2O2 more directly targets specific signaling pathways. Here, we investigated signaling by H2O2 induced by Nox1 overexpression using a luciferase reporter regulated by the antioxidant response element ARE4. Surprisingly, Nox1-derived H2O2 activated the reporter gene 15-fold with no effect on the redox state of the major thiol antioxidant substances, glutathione and thioredoxin. H2O2 signaling to ARE4 was mediated by activation of both the c-Jun N-terminal kinase and ERK1/2 pathways modulated by Ras. Thus, "redox signaling" resulting in kinase signaling pathways is distinct from "oxidative stress," and is mediated by discrete, localized redox circuitry. The gp91phox homologue Nox1 produces H2O2, which induces cell growth, transformation, and tumorigenicity. However, it has not been clear whether H2O2 effects are mediated indirectly via a generally oxidizing cellular environment or whether H2O2 more directly targets specific signaling pathways. Here, we investigated signaling by H2O2 induced by Nox1 overexpression using a luciferase reporter regulated by the antioxidant response element ARE4. Surprisingly, Nox1-derived H2O2 activated the reporter gene 15-fold with no effect on the redox state of the major thiol antioxidant substances, glutathione and thioredoxin. H2O2 signaling to ARE4 was mediated by activation of both the c-Jun N-terminal kinase and ERK1/2 pathways modulated by Ras. Thus, "redox signaling" resulting in kinase signaling pathways is distinct from "oxidative stress," and is mediated by discrete, localized redox circuitry. Reactive oxygen species (ROS 1The abbreviations used are: ROSreactive oxygen speciesMAPmitogen-activated proteinERKextracellular signal-regulated kinaseJNKc-Jun N-terminal kinaseAREantioxidant response elementGCLCglutamate-cysteine ligase, catalytic subunitHAhemagglutininHBSSHanks' balanced salt solutionDCFdichlorofluoresceinDTTdithiothreitolHGH2O2-generatingTrxthioredoxin.1The abbreviations used are: ROSreactive oxygen speciesMAPmitogen-activated proteinERKextracellular signal-regulated kinaseJNKc-Jun N-terminal kinaseAREantioxidant response elementGCLCglutamate-cysteine ligase, catalytic subunitHAhemagglutininHBSSHanks' balanced salt solutionDCFdichlorofluoresceinDTTdithiothreitolHGH2O2-generatingTrxthioredoxin.; H2O2, superoxide, and hydroxyl radical) are conventionally viewed as cytotoxic, causing oxidative damage to DNA, lipid, and proteins, and have been associated with aging, neurodegenerative disease, cancer, and cardiovascular disorders (1Griendling K.K. Alexander R.W. 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Consistent with more specificity, ROS are produced in response to growth factors and cytokines (8Bae Y.S. Kang S.W. Seo M.S. Baines I.C. Tekle E. Chock P.B. Rhee S.G. J. Biol. Chem. 1997; 272: 217-221Abstract Full Text Full Text PDF PubMed Scopus (1085) Google Scholar, 9Sundaresan M. Yu Z.X. Ferrans V.J. Irani K. Finkel T. Science. 1995; 270: 296-299Crossref PubMed Scopus (2285) Google Scholar). The discovery (10Cheng G. Cao Z. Xu X. van Meir E.G. Lambeth J.D. Gene (Amst.). 2001; 269: 131-140Crossref PubMed Scopus (691) Google Scholar, 11Lambeth J.D. Cheng G. Arnold R.S. Edens W.A. Trends Biochem. Sci. 2000; 25: 459-461Abstract Full Text Full Text PDF PubMed Scopus (328) Google Scholar, 12Suh Y.A. Arnold R.S. Lassegue B. Shi J. Xu X. Sorescu D. Chung A.B. Griendling K.K. Lambeth J.D. 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Kilroy S. Arnold R.S. Lambeth J.D. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 715-720Crossref PubMed Scopus (397) Google Scholar, 12Suh Y.A. Arnold R.S. Lassegue B. Shi J. Xu X. Sorescu D. Chung A.B. Griendling K.K. Lambeth J.D. Nature. 1999; 401: 79-82Crossref PubMed Scopus (1263) Google Scholar, 14Arnold R.S. Shi J. Murad E. Whalen A.M. Sun C.Q. Polavarapu R. Parthasarathy S. Petros J.A. Lambeth J.D. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 5550-5555Crossref PubMed Scopus (414) Google Scholar). The phenotype was reversed by coexpression of catalase, implicating H2O2 generated from Nox1 as a mediator of growth and angiogenesis. Two hypotheses apply to signaling roles for ROS. The prevailing view is that ROS induce a broad change in the intracellular redox environment, which should be reflected in oxidation of the major intracellular thiol buffering systems, glutathione and thioredoxin. Consistent with this, added H2O2, as well as redox-cycling drugs, oxidize glutathione and thioredoxin and create a generalized oxidative stress. An alternate hypothesis is that low level of ROS target specific signaling pathways and need not involve a generalized oxidative environment. Among the possible signaling pathways that might be regulated by ROS are the mitogen-activated protein (MAP) kinase pathways (15De Keulenaer G.W. Ushio-Fukai M. Yin Q. Chung A.B. Lyons P.R. Ishizaka N. Rengarajan K. Taylor W.R. Alexander R.W. Griendling K.K. Arterioscler. Thromb. Vasc. Biol. 2000; 20: 385-391Crossref PubMed Scopus (90) Google Scholar, 16Ramachandran A. Moellering D. Go Y.M. Shiva S. Levonen A.L. Jo H. Patel R.P. Parthasarathy S. Darley-Usmar V.M. Biol. Chem. 2002; 383: 693-701Crossref PubMed Scopus (34) Google Scholar, 17Ueda S. Masutani H. Nakamura H. Tanaka T. Ueno M. Yodoi J. Antioxid. Redox Signal. 2002; 4: 405-414Crossref PubMed Scopus (459) Google Scholar, 18Yoshizumi M. Tsuchiya K. Tamaki T. J. Med. Invest. 2001; 48: 11-24PubMed Google Scholar). In mammalian cells, these include the extracellular signal-regulated kinases (ERK1/2), c-Jun N-terminal kinase (JNK), and p38 kinase. The MAP kinase cascades are key signaling pathways that transduce and integrate numerous signals from the cell surface to the nucleus. MAP kinases can be activated by various external stimuli such as growth factors, ligands acting on GTP-binding protein-coupled receptors, and various stresses (heat-shock, radiation, etc.). The activated MAP kinases phosphorylate specific molecular targets such as transcription factors that can then regulate the expression of specific genes. Antioxidant response elements (AREs; a.k.a. electrophile response element) are found in the promoters of many of antioxidant and other detoxifying enzyme genes including GCLC (glutamate-cysteine ligase, catalytic subunit), glutathione S-transferase (GST), heme oxygenase-1, and NAD(P)H-quinol oxidoreductase (19Jaiswal A.K. Biochem. Pharmacol. 1994; 48: 439-444Crossref PubMed Scopus (223) Google Scholar, 20Wasserman W.W. Fahl W.E. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 5361-5366Crossref PubMed Scopus (630) Google Scholar). These elements respond to oxidants, including hydrogen peroxide (21Prestera T. Talalay P. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8965-8969Crossref PubMed Scopus (218) Google Scholar, 22Xie T. Belinsky M. Xu Y. Jaiswal A.K. J. Biol. Chem. 1995; 270: 6894-6900Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar), as well as a variety of electrophilic compounds. The ARE core sequence (GTGACNNNGC) contains an embedded sequence that shows similarity with the AP-1 binding site (T(G/T)ACTCA) (23Moinova H.R. Mulcahy R.T. J. Biol. Chem. 1998; 273: 14683-14689Abstract Full Text Full Text PDF PubMed Scopus (239) Google Scholar). The NF-E2-related factor, Nrf2, can bind to and activate ARE by complexing with AP-1-related proteins such as c-Jun, Jun-B, Jun-D, or c-Fos, while small Maf proteins (MafG and MafK) inhibit gene expression by complexing with Nrf2 (24Dhakshinamoorthy S. Jaiswal A.K. J. Biol. Chem. 2000; 275: 40134-40141Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar, 25Nguyen T. Huang H.C. Pickett C.B. J. Biol. Chem. 2000; 275: 15466-15473Abstract Full Text Full Text PDF PubMed Scopus (318) Google Scholar, 26Jeyapaul J. Jaiswal A.K. Biochem. Pharmacol. 2000; 59: 1433-1439Crossref PubMed Scopus (175) Google Scholar, 27Venugopal R. Jaiswal A.K. Oncogene. 1998; 17: 3145-3156Crossref PubMed Scopus (481) Google Scholar). Constitutive and β-naphthoflavone-induced expression of human GCLC gene is mediated by a distal ARE sequence, ARE4, in which an embedded AP-1 site is well conserved (28Wild A.C. Gipp J.J. Mulcahy T. Biochem. J. 1998; 332: 373-381Crossref PubMed Scopus (100) Google Scholar). In this study, we utilized ARE4 from the GCLC gene as a model system to investigate signal transduction systems that can mediate gene expression induced by ROS. We used NIH 3T3 cells with stable overexpression of Nox1 to provide increased H2O2 generation. We found that the low level of H2O2 generated by Nox1 expression in HG (H2O2-generating) cells did not result in detectable oxidation of either glutathione or thioredoxin. However, H2O2 activated transcription by triggering kinase cascades that lie upstream of transcription factors rather than by acting distally on ARE4-specific transcription factors themselves. Activation of ARE4 was signaled by both the ERK1/2 and JNK pathways modulated by H2O2-dependent Ras pathway. Therefore, these results show that H2O2-induced signaling occurs by mechanisms that are not associated with global oxidative stress as indicated by GSH and thioredoxin-1. Cell Culture, Transfection, and Treatments—NIH 3T3 cells stably transfected with either vector control (Cont1, Cont2), Nox1 (HG1, HG2, HG3), or Nox1 plus catalase (Cat1, Cat2) were maintained (37oC, 5% CO2) in a growth medium (Dulbecco's modified Eagle's medium containing 10% fetal calf serum). The plasmids used in this study are as follows: ARE4-Luc (the ARE4 region of GCLC inserted into a pT81 luciferase vector) (29Mulcahy R.T. Wartman M.A. Bailey H.H. Gipp J.J. J. Biol. Chem. 1997; 272: 7445-7454Abstract Full Text Full Text PDF PubMed Scopus (418) Google Scholar), pcDNA3.1 containing the lacZ gene (Invitrogen), human catalase, dominant negative mutant of H-Ras (N17-H-Ras), or dominant positive mutant of H-Ras (V12-H-Ras). Plasmids encoding hemagglutinin-tagged ERK2 (HA-ERK2) and HA-JNK1 were described (30Jo H. Sipos K. Go Y.M. Law R. Rong J. McDonald J.M. J. Biol. Chem. 1997; 272: 1395-1401Abstract Full Text Full Text PDF PubMed Scopus (234) Google Scholar). Dominant negative mutants of JNKK1 (JNKK1K179M) and MEK1 (MEK1K79M) were gifts of A. Lin (University of Chicago, IL) and K. L. Guan (University of Michigan), respectively. Endotoxin-free plasmids were prepared using a maxiprep kit following the manufacturer's instructions (Qiagen). For transient transfections, cells (4-5 × 105 cells/plate) were grown overnight in the growth medium and transfected using FuGENE 6 (Promega). The inhibitors U0126 and JNK inhibitor II were from Calbiochem. Glucose oxidase and N-acetylcysteine were from Sigma. Enzyme Activities and Protein Content—Luciferase activity was normalized for transfection efficiency by dividing by β-galactosidase activity. To initiate luciferase activity, cell lysate (20 μl) was added to 100 μl of reaction buffer (Promega) and luminescence recorded at 30 °C using a luminometer. β-Galactosidase activity was quantified by monitoring cleavage of o-nitrophenyl-β-d-galactopyranoside (31Rosenthal N. Methods Enzymol. 1987; 152: 704-720Crossref PubMed Scopus (401) Google Scholar). Protein was measured using the Bio-Rad DC kit. MAP Kinase Assays—ERK phosphorylation was examined by Western blotting using an antibody specific to the phosphorylated form of ERK (Cell Signaling Technology) and was quantified by densitometry of the autoradiogram. For immune complex assays, antibodies to JNK1 (Santa Cruz Biotechnology) or to p38 kinase were incubated with the soluble lysates (100 μg) for 1 h at 4 °C, followed by 1-h incubation with protein G-agarose beads. The precipitate was washed four times in lysis buffer (50 mm Hepes, 150 mm NaCl, 0.1 mm phenylmethylsulfonyl fluoride, 1.0 mm sodium vanadate, 1.0% Triton X-100, 10% glycerol) and once in buffer A (20 mm HEPES, pH 7.6, 20 mm MgCl2, 20 mm β-glycerophosphate, 20 mmp-nitrophenyl phosphate, 0.1 mm vanadate, and 2 mm dithiothreitol). The immune complexes were incubated (20 min, 30 °C) in 20 μl of buffer A containing either GST-cJun (5 μg each) or ATF2 (5 μg each) and 5 μCi of [γ-32P]ATP. The reaction products were resolved by SDS-PAGE, transferred to a polyvinylidene difluoride membrane, exposed to obtain an autoradiogram, and counted by scintillation counting to obtain amount of radioactivity in each band. Glutathione Analysis and Redox Potential (Eh) Calculation—GSH and GSSG were quantified as the S-carboxymethyl-N-dansyl derivatives by high performance liquid chromatography with fluorescence detection, expressed as concentrations based on cell volume. GSH and GSSG concentrations were used to calculate the steady-state redox potential values using the Nernst equation (32Jones D.P. Carlson J.L. Samiec P.S. Sternberg Jr., P. Mody Jr., V.C. Reed R.L. Brown L.A. Clin. Chim. Acta. 1998; 275: 175-184Crossref PubMed Scopus (428) Google Scholar,33Jones D.P. Methods Enzymol. 2002; 348: 93-112Crossref PubMed Scopus (616) Google Scholar). Redox State of Trx Using Redox Western Blot Analysis—Cells were treated for 1 h with H2O2, washed twice by phosphate-buffered saline, lysed, and carboxymethylated (10 mm iodoacetic acid, 50 mm Tris, pH 8.3, 3 mm EDTA, 6 m guanidine HCl, 0.5% Triton X-100, 30 min at 37 °C in the dark). As controls, cell lysate or purified thioredoxin was treated with H2O2 (0.5 mm, 5 min) or the reductant dithiothreitol (0.5 mm, 10 min) and then carboxymethylated. Excess reagents were removed using a Sephadex G-25 spin column. The redox state of Trx1 was determined by a Western blot method as described previously (34Fernando M.R. Nanri H. Yoshitake S. Nagata-Kuno K. Minakami S. Eur. J. Biochem. 1992; 209: 917-922Crossref PubMed Scopus (200) Google Scholar, 35Holmgren A. Bjornstedt M. Methods Enzymol. 1995; 252: 199-208Crossref PubMed Scopus (809) Google Scholar, 36Watson W.H. Pohl J. Montfort W.R. Stuchlik O. Reed M.S. Powis G. Jones D.P. J. Biol. Chem. 2003; 278: 33408-33415Abstract Full Text Full Text PDF PubMed Scopus (239) Google Scholar). Briefly, the electrophoresis was performed under native conditions to facilitate separation of the differently charged forms of Trx1. Derivatized proteins were separated by native polyacrylamide gel electrophoresis (PAGE). Gels were electroblotted to polyvinylidene difluoride membranes and probed with an antibody to Trx1 (American Diagnostica). Bands corresponding to Trx1 were visualized using chemiluminescent detection. The use of this method the redox forms of Trx1 can be separated as three different bands including a band with fully reduced Trx1, a band with the active site in the disulfide form (Cys-32 and Cys-35), and a band in which the protein has two disulfides, one at the active site and the other involving Cys-62 and Cys-69 (36Watson W.H. Pohl J. Montfort W.R. Stuchlik O. Reed M.S. Powis G. Jones D.P. J. Biol. Chem. 2003; 278: 33408-33415Abstract Full Text Full Text PDF PubMed Scopus (239) Google Scholar). Measurement of H2O2—Relative concentrations of intracellular H2O2 were determined as described previously (14Arnold R.S. Shi J. Murad E. Whalen A.M. Sun C.Q. Polavarapu R. Parthasarathy S. Petros J.A. Lambeth J.D. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 5550-5555Crossref PubMed Scopus (414) Google Scholar). Confluent cells in 100-mm dishes (5-6 × 106 cells) were washed with 6 ml of HBSS and released by using 0.25% trypsin (w/v), 1 mm EDTA, followed by the addition of 5% fetal bovine serum in HBSS. After pelleting, cells were resuspended in 5% fetal bovine serum in HBSS and counted. Dichlorofluorescin diacetate was added to a final concentration of 2 μm, and incubated for 1 h in the dark at room temperature. Dichlorofluorescein (DCF) fluorescence was determined by flow cytometry using a FACS Calibur BD Biosciences (excitation wavelength, 488 nm; emission wavelength, 515-545 nm) with 0.5 × 106 cells per 3 ml 5% fetal bovine serum in HBSS. Extracellular H2O2 was measured as described previously (14Arnold R.S. Shi J. Murad E. Whalen A.M. Sun C.Q. Polavarapu R. Parthasarathy S. Petros J.A. Lambeth J.D. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 5550-5555Crossref PubMed Scopus (414) Google Scholar). Immunofluorescence—Cells were fixed in 4% paraformaldehyde and 0.1% glutaraldehyde for 30 min, permeabilized in phosphate-buffered saline containing 0.1% Triton X-100 plus 50 mm lysine for 30 min, and blocked in phosphate-buffered saline containing 2% bovine serum albumin, 10% goat serum, and 0.1% Triton X-100 for 1 h. Cells were incubated with a polyclonal antibody against Nrf2 (Santa Cruz Biotechnology) or c-Jun (Cell Signaling Technology) overnight at 4 °C and with Cy3 conjugated goat anti-rabbit for 30 min. Washed cells were mounted with slow-Fade, and fluorescence was visualized using a fluorescence microscope (Olympus BX60). H2O2Generated by Nox1 Overexpression Stimulates ARE4 Reporter Activation—DCF fluorescence as an indicator for H2O2 production was examined (Fig. 1A). NIH 3T3 cells stably overexpressing Nox1 (HG1) have DCF fluorescence that is elevated 5-fold relative to control cells (Cont1) indicating that HG1 cells have continuously stimulated H2O2 production. Glucose/glucose oxidase, which continuously generate H2O2 without superoxide production, also elevated DCF fluorescence (Fig. 1A, 2-fold increases at 3 milliunits/ml glucose oxidase) in control cells. To explore H2O2 signaling, we used luciferase under the regulation of ARE4, the antioxidant response element from GCLC, which is known to respond to ROS (28Wild A.C. Gipp J.J. Mulcahy T. Biochem. J. 1998; 332: 373-381Crossref PubMed Scopus (100) Google Scholar, 29Mulcahy R.T. Wartman M.A. Bailey H.H. Gipp J.J. J. Biol. Chem. 1997; 272: 7445-7454Abstract Full Text Full Text PDF PubMed Scopus (418) Google Scholar). Luciferase activity was increased 8-fold in HG1 cells and 15-fold in another H2O2-generating cell line, HG2. Coexpression of the H2O2 scavenger, catalase (Fig. 1B), or addition of the chemical antioxidant N-acetylcysteine (Fig. 1C) significantly inhibited activation of luciferase, indicating that ARE4 activation in HG cells is H2O2-dependent. H2O2 production by glucose/glucose oxidase stimulated ARE4 activation in control (Cont1) cells as shown in Fig. 1D. Therefore, these results suggest that the ARE4 element is activated in response to continuously generated H2O2 and can be used to explore H2O2 signaling. Redox States of Glutathione Were Not Changed in Cells Generating H2O2by Overexpressing Nox1 or in Control Cells Generating H2O2by Glucose/Glucose Oxidase—To evaluate cellular redox states under conditions of H2O2-mediated signaling, we examined amounts of reduced (GSH) and oxidized (GSSG) glutathione and the calculated redox potential of the GSH/GSSG couple. HG1 cells had elevated H2O2 as shown in Fig. 1A; however, these cells showed no differences in the level of GSSG and GSH compared with control cells (Cont1) (Fig. 2A, bottom). The calculated glutathione redox potentials were not changed significantly (Fig. 2A, top). In contrast, exposure of control cells to a single dose of H2O2 (a concentration used in published experiments to induce oxidative stress, growth arrest, and apoptosis) increased glutathione disulfide, GSSG, with a corresponding 20 mV oxidation in redox potential (Cont1*). In addition, control cells generating H2O2 continuously by glucose/glucose oxidase as shown in Fig. 1D were also tested to examine redox states of glutathione. However, there was no detectable increase in glutathione redox states (Fig. 2B, top) and levels of GSH and GSSG (Fig. 2B, bottom) even at 5 milliunits of glucose oxidase per ml, which was the condition that stimulated ARE4 activation due to H2O2 production. Thus, H2O2-mediated signaling occurred without global oxidation as indicated by the GSH/GSSG system in cells. Redox State of Thioredoxin in H2O2-generating Cells—To further test the effect of H2O2 generation on global cellular redox state under conditions of redox signaling, we examined redox state of another major cellular thiol component, thioredoxin-1 (Trx1). Oxidation of human Trx1 can be measured using Redox Western blot analysis (36Watson W.H. Pohl J. Montfort W.R. Stuchlik O. Reed M.S. Powis G. Jones D.P. J. Biol. Chem. 2003; 278: 33408-33415Abstract Full Text Full Text PDF PubMed Scopus (239) Google Scholar) in which the mobility of Trx1 under native gel conditions is modified as a function of redox state (Fig. 3, as purified h-Trx1 ± H2O2). Changes in the redox state of Trx1 were not detected in cells generating H2O2 (HG1) compared with control (Cont1) (Fig. 3, top panel and bottom graph). Control and HG cells with mouse-derived thioredoxin were mostly in the reduced form (34Fernando M.R. Nanri H. Yoshitake S. Nagata-Kuno K. Minakami S. Eur. J. Biochem. 1992; 209: 917-922Crossref PubMed Scopus (200) Google Scholar, 37Watson W.H. Jones D.P. FEBS Lett. 2003; 543: 144-147Crossref PubMed Scopus (99) Google Scholar), and only the reduced (but not the oxidized) form of mouse Trx was visualized immunochemically. In both control (Cont1) and HG1 cells, addition of H2O2 to lysates resulted in loss of the reduced band (Fig. 3, top panel, lanes for lysates with 0.5 mm H2O2). Both control and HG cells contained reduced Trx (Fig. 3, top panel and bottom graph, lanes for cells with none), which decreased upon exposure of cells to 2 mm H2O2. The amount of reduced Trx visualized in each cell line was the same as that seen in an equivalent volume of cell lysate treated with reducing reagent, dithiothreitol (DTT). Thus, most or all of the Trx is in the reduced form regardless of H2O2 generation. As expected, however, HG1 compared with Cont1 showed a significant loss of reduced Trx when cells were treated with 2 mm of H2O2 (top panel and bottom graph). As a control for protein loading, cell lysates were analyzed to determine total amount of ERK1/2 using Western blot (Fig. 3, bottom panel). ERK1/2 expression was observed without any change in HG1 compared with control (Cont1) cells. Also, there was no detectable change in ERK1/2 level by treating cells with H2O2 or DTT as shown in Fig. 3 (bottom panel). These results together with the results of glutathione redox show that H2O2-dependent signaling mechanisms can occur without affecting glutathione and thioredoxin oxidation or redox potential. H2O2-induced Translocation of c-Jun but Not Nrf2 to the Nucleus—ARE4 activation is known to be regulated by transcription factor systems including Nrf2, KEAP1, c-Jun, and other binding proteins. Nrf2 translocates to the nucleus in response to oxidant stress and xenobiotics, and this is mediated by release from binding proteins in the cytosol (38Sekhar K.R. Spitz D.R. Harris S. Nguyen T.T. Meredith M.J. Holt J.T. Gius D. Marnett L.J. Summar M.L. Freeman M.L. Guis D. Free Radic. Biol. Med. 2002; 32: 650-662Crossref PubMed Scopus (64) Google Scholar). When H2O2-generating cells (HG1) were compared with control cells (Cont1) using immunofluorescence, there was no increase in translocation of Nrf2 to the nucleus, whereas that of c-Jun was observed (Fig. 4). Nrf2 was partially excluded from the nucleus in HG cells. Notably, H2O2 has been previously shown to induce translocation of c-Jun in oligodendrocytes (39Vollgraf U. Wegner M. Richter-Landsberg C. J. Neurochem. 1999; 73: 2501-2509Crossref PubMed Scopus (159) Google Scholar). These data are consistent with regulation of ARE4 by H2O2 being mediated in part by nuclear translocation of c-Jun. H2O2-dependent Activation of ARE4 Is Regulated by AP-1—To further examine the possible mechanisms for H2O2 signaling of ARE4, experiments were performed with ARE4 mutants. The ARE4 response element containing a consensus antioxidant response element also includes a binding region for the transcription factor, AP-1, which is a multimer containing c-Jun, c-Fos, and additional proteins. In general, activation of JNK results in activation of c-Jun, following by translocation of c-Jun to the nucleus. We tested whether H2O2-dependent ARE4 activation is associated with a transcription factor, AP-1. ARE4 consensus includes the AP1 binding region; thus, the effects of mutants were examined comparing with that of wild type ARE4 (Fig. 5). HG1 cells were transiently transfected with either empty vector, wild type of ARE4 (WT), or mutant (m1, m2, or m3) as described under "Experimental Procedures." Wild type of ARE4 activation was stimulated in HG1 compared with control cells (Cont1); however, its activation was significantly blocked by mutation obtained in the AP1 specific region (m1, m2) but not in non-AP1 site (m3) (Fig. 5). Together with the above results on Nrf2 translocation, these results indicate that ARE4 activation by H2O2 in HG cells is associated with the AP-1 transcription factor and not with Nrf2. H2O2Production in HG Cells Stimulates the Activation of ERK1/2 and JNK but Not p38 Kinase—To explore the mechanisms of H2O2-dependent ARE4 activation, we examined MAP kinase pathway as upstream signaling pathways in NIH 3T3 cells generating H2O2 (HG1, HG2) or control (Cont1, Cont2). ERK1/2 activity was stimulated ∼8-fold in H2O2-generating cells (Fig. 6A). Likewise, JNK activity was stimulated ∼2-3-fold, based on its ability to phosphorylate c-Jun (Fig. 6B). On the other hand, p38 kinase activity, which is readily activated ∼4-fold by anisomycine, was unaffected by H2O2 generation. Using Western blotting, there were no changes in the levels of protein expression of the MAP kinases in H2O2-generating compared with control cells (Fig. 6, A-C). The ERK1/2 Pathway Mediates H2O2-dependent Regulation of ARE4—ERK1/2 is activated by the upstream kinases MEK1/2. Using an in vitro kinase assay, H2O2-stimulated ERK1/2 activity was completely inhibited upon transfection with a dominant negative mutant of MEK1, MEK1K79M (Fig. 7A, upper panel, bar graph). Similarly, treatment with the MEK inhibitor, U0126, inhibited H2O2-dependent ERK1/2 phosphorylation in a dose-dependent manner (Fig. 7B, upper panel, bar graph). H2O2-dependent ARE4 activation was partially inhibited in parallel with inhibition of H2O2-dependent ERK1/2 activation by both these treatments. Thus, H2O2 regulates ARE4-dependent transcription partly via activation of the ERK1/2 pathway. H2O2-dependent Regulation of ARE4 Is Mediated in Part by the JNK Pathway—Because H2O2 stimulates JNK activation, we examined whether ARE4 is also coregulated by JNK activation. A dominant negative mutant of JNKK1, JNKK1K179M, transfected into HG1 cells resulted in a dose-dependent decrease in JNK activity (Fig. 8A, top panel, middle bar graph), with p
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