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Hydrogen Peroxide Signaling through Tumor Necrosis Factor Receptor 1 Leads to Selective Activation of c-Jun N-terminal Kinase

2003; Elsevier BV; Volume: 278; Issue: 45 Linguagem: Inglês

10.1074/jbc.m308487200

1083-351X

Cristen Pantano, Punya Shrivastava, Brian McElhinney, Yvonne M. W. Janssen‐Heininger,

Bioactive Natural Diterpenoids Research

Binding of tumor necrosis factor-α (TNFα) to its receptor, TNF-R1, results in the activation of inhibitor of κB kinase (IKK) and c-Jun N-terminal kinase (JNK) pathways that are coordinately regulated and important in survival and death. We demonstrated previously that in response to hydrogen peroxide (H2O2), the ability of TNFα to activate IKK in mouse lung epithelial cells (C10) was inhibited and that H2O2 alone was sufficient to activate JNK and induce cell death. In the current study, we investigated the involvement of TNF-R1 in H2O2-induced JNK activation. In lung fibroblasts from TNF-R1-deficient mice the ability of H2O2 to activate JNK was inhibited compared with fibroblasts from control mice. Additionally, in C10 cells expressing a mutant form of TNF-R1, H2O2-induced JNK activation was also inhibited. Immunoprecipitation of TNF-R1 revealed that in response to H2O2, the adapter proteins, TRADD and TRAF2, and JNK were recruited to the receptor. However, expression of the adaptor protein RIP, which is essential for IKK activation by TNFα, was decreased in cells exposed to H2O2, and its chaperone Hsp90 was cleaved. Furthermore, data demonstrating that expression of TRAF2 was not affected by H2O2 and that overexpression of TRAF2 was sufficient to activate JNK provide an explanation for the inability of H2O2 to activate IKK and for the selective activation of JNK by H2O2. Our data demonstrate that oxidative stress interferes with IKK activation while promoting JNK signaling, creating a signaling imbalance that may favor apoptosis. Binding of tumor necrosis factor-α (TNFα) to its receptor, TNF-R1, results in the activation of inhibitor of κB kinase (IKK) and c-Jun N-terminal kinase (JNK) pathways that are coordinately regulated and important in survival and death. We demonstrated previously that in response to hydrogen peroxide (H2O2), the ability of TNFα to activate IKK in mouse lung epithelial cells (C10) was inhibited and that H2O2 alone was sufficient to activate JNK and induce cell death. In the current study, we investigated the involvement of TNF-R1 in H2O2-induced JNK activation. In lung fibroblasts from TNF-R1-deficient mice the ability of H2O2 to activate JNK was inhibited compared with fibroblasts from control mice. Additionally, in C10 cells expressing a mutant form of TNF-R1, H2O2-induced JNK activation was also inhibited. Immunoprecipitation of TNF-R1 revealed that in response to H2O2, the adapter proteins, TRADD and TRAF2, and JNK were recruited to the receptor. However, expression of the adaptor protein RIP, which is essential for IKK activation by TNFα, was decreased in cells exposed to H2O2, and its chaperone Hsp90 was cleaved. Furthermore, data demonstrating that expression of TRAF2 was not affected by H2O2 and that overexpression of TRAF2 was sufficient to activate JNK provide an explanation for the inability of H2O2 to activate IKK and for the selective activation of JNK by H2O2. Our data demonstrate that oxidative stress interferes with IKK activation while promoting JNK signaling, creating a signaling imbalance that may favor apoptosis. The lung is an important target for oxidant injury as a consequence of direct inhalation of oxidants or as a result of the production of oxidants during inflammation (1Janssen Y.M. Matalon S. Mossman B.T. Am. J. Physiol. 1997; 273: L789-L796PubMed Google Scholar, 2Janssen-Heininger Y.M. Poynter M.E. Baeuerle P.A. Free Radic. Biol. Med. 2000; 28: 1317-1327Crossref PubMed Scopus (604) Google Scholar). Although oxidants contribute to tissue damage, these species are also formed in virtually every cell type and are an integral part of normal cell function (3Finkel T. Curr. Opin. Cell Biol. 2003; 15: 247-254Crossref PubMed Scopus (1212) Google Scholar). Oxidants are required for proliferation (4Arbiser J.L. Petros J. Klafter R. Govindajaran B. McLaughlin E.R. Brown L.F. Cohen C. Moses M. Kilroy S. Arnold R.S. Lambeth J.D. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 715-720Crossref PubMed Scopus (400) Google Scholar, 5Arnold 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 (421) Google Scholar, 6Moore K.A. Sethi R. Doanes A.M. Johnson T.M. Pracyk J.B. Kirby M. Irani K. Goldschmidt-Clermont P.J. Finkel T. Biochem. J. 1997; 326: 17-20Crossref PubMed Scopus (69) Google Scholar), changes in cellular shape (7Nimnual A.S. Taylor L.J. Bar-Sagi D. Nat. Cell Biol. 2003; 5: 236-241Crossref PubMed Scopus (426) Google Scholar) and are involved in transcriptional regulation (2Janssen-Heininger Y.M. Poynter M.E. Baeuerle P.A. Free Radic. Biol. Med. 2000; 28: 1317-1327Crossref PubMed Scopus (604) Google Scholar). Despite the emerging role of redox signaling in cellular physiology, the exact mechanisms by which oxidants act as signaling molecules are under intense investigation, and many critical targets remain enigmatic. Oxidants have been demonstrated to regulate the activation of c-Jun N-terminal kinase (JNK), 1The abbreviations used are: JNK, c-Jun N-terminal kinase; IκB, inhibitor of κB; IKK, inhibitor of κB kinase; TNFα, tumor necrosis factor α; TNF-R1, TNF receptor 1; TRADD, TNF-R1-associated death domain; TRAF2, TNF-R-associated factor 2; DD, death domain; RIP, receptor-interacting protein; PBS, phosphate-buffered saline; HA, hemagglutinin; GST, glutathione S-transferase; TBS, Tris-buffered saline; GA, geldanamycin; CA, constitutively active.1The abbreviations used are: JNK, c-Jun N-terminal kinase; IκB, inhibitor of κB; IKK, inhibitor of κB kinase; TNFα, tumor necrosis factor α; TNF-R1, TNF receptor 1; TRADD, TNF-R1-associated death domain; TRAF2, TNF-R-associated factor 2; DD, death domain; RIP, receptor-interacting protein; PBS, phosphate-buffered saline; HA, hemagglutinin; GST, glutathione S-transferase; TBS, Tris-buffered saline; GA, geldanamycin; CA, constitutively active. as well as the transcription factor NF-κB (2Janssen-Heininger Y.M. Poynter M.E. Baeuerle P.A. Free Radic. Biol. Med. 2000; 28: 1317-1327Crossref PubMed Scopus (604) Google Scholar). JNK is a member of the family of mitogen-activated protein kinases, which is well known to be activated by oxidants and a variety of other stresses in many cell types, including lung epithelial cells (1Janssen Y.M. Matalon S. Mossman B.T. Am. J. Physiol. 1997; 273: L789-L796PubMed Google Scholar, 8Korn S.H. Wouters E.F. Vos N. Janssen-Heininger Y.M. J. Biol. Chem. 2001; 276: 35693-35700Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar, 9Amdjadi K. Sefton B.M. J. Biol. Chem. 2000; 275: 22520-22525Abstract Full Text Full Text PDF PubMed Scopus (5) Google Scholar). The contribution of JNK to many phenotypic outcomes, including survival (10Davis R.J. Cell. 2000; 103: 239-252Abstract Full Text Full Text PDF PubMed Scopus (3611) Google Scholar, 11Sabapathy K. Jochum W. Hochedlinger K. Chang L. Karin M. Wagner E.F. Mech. 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Cell. 2003; 11: 1479-1489Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar). In this regard, oxidant-induced JNK activation has been linked to apoptosis (16Chen K. Vita J.A. Berk B.C. Keaney Jr., J.F. J. Biol. Chem. 2001; 276: 16045-16050Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar, 17Aoki H. Kang P.M. Hampe J. Yoshimura K. Noma T. Matsuzaki M. Izumo S. J. Biol. Chem. 2002; 277: 10244-10250Abstract Full Text Full Text PDF PubMed Scopus (294) Google Scholar). Although oxidants have also been implicated in the activation of the transcription factor NF-κB, currently a number of significant controversies exist around the role of redox events in NF-κB activation (18Li N. Karin M. FASEB J. 1999; 13: 1137-1143Crossref PubMed Scopus (780) Google Scholar, 19Hayakawa M. Miyashita H. Sakamoto I. Kitagawa M. Tanaka H. Yasuda H. Karin M. Kikugawa K. EMBO J. 2003; 22: 3356-3366Crossref PubMed Scopus (363) Google Scholar). Under basal conditions NF-κB is sequestered in the cytoplasm through binding to the inhibitor of κB (IκB). Upon phosphorylation of IκB by IκB kinase (IKK), IκB is rapidly degraded via the 26 S proteasome, allowing NF-κB to translocate to the nucleus and activate the transcription of over 100 genes, including genes critical to cell survival (20Barinaga M. Science. 1996; 274: 724Crossref PubMed Scopus (59) Google Scholar, 21Wang C.Y. Mayo M.W. Korneluk R.G. Goeddel D.V. Baldwin Jr., A.S. Science. 1998; 281: 1680-1683Crossref PubMed Scopus (2566) Google Scholar, 22Baud V. Karin M. Trends Cell Biol. 2001; 11: 372-377Abstract Full Text Full Text PDF PubMed Scopus (1367) Google Scholar). The signaling events that are required for the activation of JNK and NF-κB have been investigated in great detail using the ligand tumor necrosis factor-α (TNFα). TNFα binds as a trimer to three TNF-R1 monomers causing aggregation of the intracellular death domains. The death domain (DD) containing-protein, TRADD, binds directly to the DD of TNF-R1 and then recruits the adaptor molecule (23Hsu H. Xiong J. Goeddel D.V. Cell. 1995; 81: 495-504Abstract Full Text PDF PubMed Scopus (1738) Google Scholar), TRAF2. TRAF2 is essential for JNK activation as well as the recruitment of the IKK complex to the receptor (24Devin A. Cook A. Lin Y. Rodriguez Y. Kelliher M. Liu Z. Immunity. 2000; 12: 419-429Abstract Full Text Full Text PDF PubMed Scopus (356) Google Scholar). Receptor-interacting protein (RIP) is recruited to TNF-R1 through interaction with TRADD and is essential for the activation of IKK (24Devin A. Cook A. Lin Y. Rodriguez Y. Kelliher M. Liu Z. Immunity. 2000; 12: 419-429Abstract Full Text Full Text PDF PubMed Scopus (356) Google Scholar). Additionally, TRADD can directly interact with FADD, which is involved in executing caspase-dependent cell death. Recently it has been demonstrated that activation of IKK and JNK following stimulation of TNF-R1 is coordinately regulated and that the activation of NF-κB regulates the extent and duration of JNK activation. Transcription of NF-κB-driven anti-apoptotic genes such as X-IAP is critical in preventing sustained activation of JNK and also in promoting survival (25Tang G. Minemoto Y. Dibling B. Purcell N.H. Li Z. Karin M. Lin A. Nature. 2001; 414: 313-317Crossref PubMed Scopus (660) Google Scholar, 26Tang F. Tang G. Xiang J. Dai Q. Rosner M.R. Lin A. Mol. Cell Biol. 2002; 22: 8571-8579Crossref PubMed Scopus (121) Google Scholar). Additionally, transient activation of JNK can also mediate survival signaling via JunD, which collaborates with NF-κB to increase the expression of the survival gene cIAP-2 (13Lamb J.A. Ventura J.J. Hess P. Flavell R.A. Davis R.J. Mol. Cell. 2003; 11: 1479-1489Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar). Conversely, when NF-κB-dependent gene transcription is prevented, JNK activation is prolonged allowing the execution of TNF-induced apoptosis (25Tang G. Minemoto Y. Dibling B. Purcell N.H. Li Z. Karin M. Lin A. Nature. 2001; 414: 313-317Crossref PubMed Scopus (660) Google Scholar, 26Tang F. Tang G. Xiang J. Dai Q. Rosner M.R. Lin A. Mol. Cell Biol. 2002; 22: 8571-8579Crossref PubMed Scopus (121) Google Scholar). Previously we demonstrated that the oxidant hydrogen peroxide (H2O2) inhibits IKK, activates JNK, and causes apoptosis (1Janssen Y.M. Matalon S. Mossman B.T. Am. J. Physiol. 1997; 273: L789-L796PubMed Google Scholar, 8Korn S.H. Wouters E.F. Vos N. Janssen-Heininger Y.M. J. Biol. Chem. 2001; 276: 35693-35700Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar). In the present study we have investigated the role of TNF-R1 in the activation of JNK by H2O2. We demonstrate here that H2O2 signals to JNK via TNF-R1 and TRAF2 and that IKK and NF-κB activation are prevented as a result of degradation of the adaptor protein, RIP, and cleavage of its chaperone, Hsp90. Cell Culture and Reagents—A line of spontaneously transformed mouse alveolar type II epithelial cells (C10) was propagated in CRML-1066 medium containing 50 units/ml penicillin, 50 μg/ml streptomycin, 2 mm l-glutamine, and 10% fetal bovine serum, all from Invitrogen. Murine recombinant TNFα was purchased from Calbiochem. The JNK, IKK, TRAF2, TRADD and Hsp90 antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA), and TNF-R1 antibody was obtained from R&D Systems. Primary lung fibroblasts were isolated from C57BL/6 or TNF-R1–/– mice (Jackson Laboratories, Bar Harbor, ME) by mincing lungs and propagation of explants in Dulbecco's modified Eagle's medium/F12 medium containing 50 units/ml penicillin, 50 μg/ml streptomycin, 2 mm l-glutamine, and 10% fetal bovine serum, all from Invitrogen. p60Δ cytoplasmic domain plasmid was provided by Dr. Michael Lenardo (National Institutes of Health, Bethesda, MD) and Flag-TRAF2 by Dr. Brian Seed (Massachusetts General Hospital, Boston, MA). NF-κB-luciferase and IκB-SR plasmids were provided by Dr. Patrick Baeuerle (Micromet, Martinsreid, Germany). CA-IKK was a kind gift from Dr. Michael Karin (University of California, San Diego), and glutathione S-transferase (GST)-IκB was provided by Dr. Rosa Ten (Mayo Clinic, Rochester, MN). Kinase Assays—Cells were exposed to the test agents and, at the indicated times, transferred to ice, washed once with cold PBS, and lysed as described elsewhere (8Korn S.H. Wouters E.F. Vos N. Janssen-Heininger Y.M. J. Biol. Chem. 2001; 276: 35693-35700Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar). Lysates were cleared by centrifugation at 14,000 rpm, 4 °C for 10 min. Protein concentrations were determined, and IKK or JNK was immunoprecipitated from 200 μg of protein with an IKKγ, JNK, or HA antibody (Santa Cruz) at 4 °C for 1.5 h using protein G-agarose beads (Invitrogen). For TNF-R1 immunoprecipitation and JNK kinase assay, TNF-R1 was immunoprecipitated from 700 μg of protein using an anti-TNF-R1 antibody from R&D Systems. Precipitates were washed twice with lysis buffer and once with kinase buffer (8Korn S.H. Wouters E.F. Vos N. Janssen-Heininger Y.M. J. Biol. Chem. 2001; 276: 35693-35700Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar). The kinase reaction was performed using 1 μg of GST-IκBα or GST-c-Jun as a substrate and 5 μCi of [γ-32P]adenosine triphosphate at 30 °C for 30 min. Reactions were stopped by the addition of 2× Laemmli sample buffer. Samples were boiled and stored at –20 °C. Proteins were separated on a 15% polyacrylamide gel. Gels were dried and examined by autoradiography. Transfection—Cells were transfected using LipofectAMINE-Plus (Invitrogen) according to the manufacturer's directions. Immunoprecipitation and Western Blotting—A fraction of lysates for analysis of adaptor protein expression or co-immunoprecipitation was added to 2× Laemmli sample buffer, boiled, and loaded on a 10% polyacrylamide gel. Proteins were transferred to nitrocellulose (Schleicher & Shuell), and membranes were subsequently blocked in 5% milk in tris-buffered saline (TBS). Levels of HA were detected with a monoclonal antibody (12CA5, Roche Applied Science). All other proteins were detected with antibodies from Santa Cruz according to the following protocol. Membranes blocked overnight in TBS/milk were washed two times for 15 min in TBS containing 0.05% Tween 20 and incubated with the primary antibody for 1 h at 4 °C. Membranes were washed three times for 20 min in TBS/Tween 20 and incubated with a peroxidase-conjugated secondary antibody (Vector Laboratories, Burlingame, CA) for 1 h at room temperature. After a 30-min wash with TBS/Tween 20, conjugated peroxidase was detected by ECL according to the manufacturer's instructions (Amersham Biosciences). For immunoprecipitations, cells were grown to confluence in 100-mm dishes, washed three times with PBS, and treated with test agents in PBS. Cells were lysed in immunoprecipitation buffer (50 mm Hepes, pH 7.4, 250 mm NaCl, 0.1% Nonidet P-40, 5 mm EDTA. 0.5 mm phenylmethylsulfonyl fluoride, 1% aprotinin), and lysates were incubated with TNF-R1 antibody for 2 h with rocking at 4 °C. After incubation with antibody, protein G-agarose beads (Invitrogen) were added for 1 h. Precipitates were washed three times with lysis buffer, and 2× Laemmli was added followed by boiling, loading onto a 10% polyacrylamide gel, and Western analysis. Assessment of NF-κB Transcriptional Activity—C10 cells were transiently transfected with a 6 κB-tk-luc plasmid containing 6 NFκB DNA elements and either TRAF2 or pCDNA3. Cells were treated with 1 ng/ml TNF for 4 h. Cells were lysed in Luciferase Assay Lysis Buffer (Promega, Madison, WI), and a luciferase assay was performed as previously described (8Korn S.H. Wouters E.F. Vos N. Janssen-Heininger Y.M. J. Biol. Chem. 2001; 276: 35693-35700Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar). H2O2 Activates JNK in a TNF-R1-dependent Manner—We demonstrated previously that H2O2 causes activation of JNK in lung epithelial cells while inhibiting TNFα-induced activation of IKK (8Korn S.H. Wouters E.F. Vos N. Janssen-Heininger Y.M. J. Biol. Chem. 2001; 276: 35693-35700Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar). To directly demonstrate that H2O2-induced JNK signaling is mediated by TNF-R1, lung fibroblasts were isolated from TNF-R1–/– mice, and JNK activation was assessed. The results shown in Fig. 1A demonstrate that the ability of H2O2 to activate JNK was substantially decreased in TNF-R1–/– cells compared with wild type controls at time points ranging from 15 min to 2 h. As expected, TNFα-induced activation of JNK was also abrogated in TNF-R1–/– fibroblasts. Additionally, overexpression of a truncated form of TNF-R1 (p60ΔCD), which lacks the intracellular death domain in C10 cells also resulted in inhibition of the TNFα- or H2O2-induced activation of JNK (Fig. 1B). These results demonstrate that H2O2-induced activation of JNK requires TNF-R1. Activation of JNK and IKK following TNF-R1 activation is coordinately regulated; the duration of JNK activation depends on the activity of NF-κB-dependent gene products such as XIAP, which associates with TNF-R1 to repress sustained JNK activity (25Tang G. Minemoto Y. Dibling B. Purcell N.H. Li Z. Karin M. Lin A. Nature. 2001; 414: 313-317Crossref PubMed Scopus (660) Google Scholar, 26Tang F. Tang G. Xiang J. Dai Q. Rosner M.R. Lin A. Mol. Cell Biol. 2002; 22: 8571-8579Crossref PubMed Scopus (121) Google Scholar). In agreement with these findings, inhibition of NF-κB activity via the expression of a dominant acting version of IκB, IκB-SR, led to an augmented and sustained JNK activity profile after exposure to TNFα, illustrating the repression of JNK by NF-κB activation downstream of TNF-R1 in lung epithelial cells (Fig. 2A). To further support a role of TNF-R1 in JNK activation by H2O2, we addressed whether JNK activation by H2O2 could also be regulated by the NF-κB pathway. We therefore expressed a constitutively active version of IKKβ (CA-IKKβ), and assessed the ability of H2O2 or TNFα to activate JNK. Compared with vector controls, CA-IKKβ-expressing cells displayed attenuated JNK activation following treatment with H2O2 or TNFα (Fig. 2B), illustrating that JNK activation by H2O2 is also subject to negative regulation by NF-κB, analogous to JNK activation by TNFα. H2O2 Causes Recruitment of Adapter Proteins to TNF-R1— TNF-R1 is catalytically inactive and requires the recruitment of adaptor proteins to its intracellular domain in order to transmit signals (28Tartaglia L.A. Ayres T.M. Wong G.H. Goeddel D.V. Cell. 1993; 74: 845-853Abstract Full Text PDF PubMed Scopus (1165) Google Scholar). Upon ligand binding, the intracellular domain aggregates and the DD-containing protein, TRADD, is recruited to and binds to the DD of the receptor (23Hsu H. Xiong J. Goeddel D.V. Cell. 1995; 81: 495-504Abstract Full Text PDF PubMed Scopus (1738) Google Scholar). TRADD forms a platform for TRAF2 and RIP (29Rothe M. Wong S.C. Henzel W.J. Goeddel D.V. Cell. 1994; 78: 681-692Abstract Full Text PDF PubMed Scopus (929) Google Scholar), which are required for JNK activation and the recruitment and activation of IKK (24Devin A. Cook A. Lin Y. Rodriguez Y. Kelliher M. Liu Z. Immunity. 2000; 12: 419-429Abstract Full Text Full Text PDF PubMed Scopus (356) Google Scholar). We therefore investigated whether H2O2 also causes recruitment of TRADD and TRAF2 to TNF-R1. Endogenous TNF-R1 was immunoprecipitated, and TRADD and TRAF2 co-immunoprecipitation was evaluated by Western blotting. As shown in Fig. 3A, TRADD co-precipitated with TNF-R1 in unstimulated cells and increased in response to TNFα or H2O2. Furthermore, in samples from TNFα- or H2O2-exposed cells post-immunoprecipitation, TRADD levels were decreased (Fig. 3B). TRAF2 also co-immunoprecipitated with TNF-R1 in control cells, increased following exposure to TNFα or H2O2 (Fig. 3C), and decreased in the post-immunoprecipitate lysates (Fig. 3D). Because it has been demonstrated that IKK is recruited to TNF-R1 (24Devin A. Cook A. Lin Y. Rodriguez Y. Kelliher M. Liu Z. Immunity. 2000; 12: 419-429Abstract Full Text Full Text PDF PubMed Scopus (356) Google Scholar), we next assessed whether JNK could also be recruited to TNF-R1 after incubation with TNFα or H2O2. As shown in Fig. 3E, JNK activity was present in the immunoprecipitated TNF-R1 and increased after exposure to TNFα or H2O2. Collectively, our findings suggest that TRADD, TRAF2, and JNK are associated with TNF-R1 in response to H2O2, and they support the role of TNF-R1 in the activation of JNK by H2O2. H2O2 Causes Degradation of RIP and Cleavage of Hsp90 — Despite the dependence of TNF-R1 in the H2O2-induced activation of JNK, H2O2 does not activate IKK or NF-κB (8Korn S.H. Wouters E.F. Vos N. Janssen-Heininger Y.M. J. Biol. Chem. 2001; 276: 35693-35700Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar). The activation of IKK by TNFα requires the recruitment of RIP and Hsp90 (30Chen G. Cao P. Goeddel D.V. Mol. Cell. 2002; 9: 401-410Abstract Full Text Full Text PDF PubMed Scopus (333) Google Scholar) to the TNF-R1 DD. Hsp90 and RIP form a complex; destabilization of Hsp90 leads to RIP degradation and consequently prevents the activation of IKK and NF-κB (31Lewis J. Devin A. Miller A. Lin Y. Rodriguez Y. Neckers L. Liu Z.G. J. Biol. Chem. 2000; 275: 10519-10526Abstract Full Text Full Text PDF PubMed Scopus (294) Google Scholar). We therefore investigated whether the lack of IKK activation in cells treated with H2O2 was due to destabilization of RIP and/or Hsp90 and assessed the levels of TRAF2, RIP, and Hsp90 by Western blot analysis. As seen in Fig. 4A, TRAF2 levels were not affected by H2O2 or TNFα in a time frame of up to 2 h. Evaluation of Hsp90 revealed that a lower molecular weight species of Hsp90 was present after treatment with H2O2 for 2 h (Fig. 4B) and first appeared after 30 min of exposure (data not shown). This Hsp90 cleavage product did not appear in cells treated with TNFα (Fig. 4B). In agreement with these findings, RIP levels were markedly decreased in cells treated with H2O2 for 2 h, whereas TNFα exposure did not affect RIP expression compared with sham controls (Fig. 4C, and data not shown). Because RIP and Hsp90 are required for IKK activation, their destabilization by H2O2 provides a plausible explanation for the inability of H2O2 to activate IKK. Overexpression of TRAF2 Activates JNK and Inhibits the Ability of TNFα to Activate IKK and NF-κB—The results presented above demonstrate that in response to H2O2, TRADD and TRAF2 are recruited to the TNF-R1, whereas RIP and Hsp90, which are essential for IKK activation, are destabilized. The absence of RIP at TNF-R1 has been demonstrated to enhance recruitment of TRADD and TRAF2, suggesting a competition for binding at TNF-R1 between TRADD/TRAF2 and RIP (24Devin A. Cook A. Lin Y. Rodriguez Y. Kelliher M. Liu Z. Immunity. 2000; 12: 419-429Abstract Full Text Full Text PDF PubMed Scopus (356) Google Scholar). Therefore, an enhanced presence of TRAF2 at TNF-R1 in response to H2O2 may sustain JNK activation while inhibiting IKK. To confirm that the effects of H2O2 in C10 cells can be mimicked by TRAF2 accumulation at the receptor, we overexpressed wild type TRAF2 and analyzed the activity of IKK, NF-κB, and JNK. TRAF2 overexpression was sufficient to inhibit the ability of TNFα to activate IKK and NF-κB (Fig. 5, A and B). Moreover, TRAF2 overexpression also led to activation of JNK under base-line conditions (Fig. 5C). Lastly, we determined whether H2O2-induced degradation of RIP and cleavage of Hsp90 would alter JNK and IKK activation in response to TNFα. As expected, pretreatment of C10 cells with H2O2 for 2 h, the time point associated with RIP degradation and Hsp90 cleavage (Fig. 4), led to a complete inhibition of IKK activation by TNFα. Although under these conditions JNK activation by H2O2 was no longer observed, pretreatment with H2O2 enhanced JNK activation in response to TNFα (Fig. 6). These data confirm that H2O2 causes a signaling imbalance at TNF-R1, leading to preferential activation of JNK, while inhibiting IKK.Fig. 6H2O2 disrupts TNFα-dependent signaling. Cells were exposed to 500 μm H2O2 for 2 h and then to 10 ng/ml TNFα for an additional 5 min (top panel) or 15 min (middle panel) for the assessment of IKK or JNK, respectively, via in vitro kinase assays. The bottom panel demonstrates JNK levels, as a loading control.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Although it has been established that H2O2 is a signaling molecule that can activate JNK (8Korn S.H. Wouters E.F. Vos N. 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Sustained JNK activation by H2O2 is not observed in response to TNFα because of the NF-κB-dependent expression of anti-apoptotic genes, which are recruited to TNF-R1 and inhibit prolonged JNK activity and TNFα-induced apoptosis (21Wang C.Y. Mayo M.W. Korneluk R.G. Goeddel D.V. Baldwin Jr., A.S. Science. 1998; 281: 1680-1683Crossref PubMed Scopus (2566) Google Scholar, 26Tang F. Tang G. Xiang J. Dai Q. Rosner M.R. Lin A. Mol. Cell Biol. 2002; 22: 8571-8579Crossref PubMed Scopus (121) Google Scholar). Conversely, overexpression of a constitutively active form of IKK resulted in an attenuation of H2O2-induced JNK activation, demonstrating that JNK activation by H2O2 is also subject to negative feedback regulation by NF-κB. It will be of interest to determine whether diminution of JNK activation as a result of NF-κB activation also represses oxidant-induced apoptosis, analogous to observations with TNFα. Although we recently demonstrated that H2O2 is capable of directly oxidizing IKK leading to its inactivation (8Korn S.H. Wouters E.F. Vos N. Janssen-Heininger Y.M. J. Biol. Chem. 2001; 276: 35693-35700Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar), in the present study we have demonstrated that H2O2 can also inhibit TNFα-induced NF-κB activation upstream by interfering with the formation of the TNF-R1 signaling complex. It is plausible that H2O2 can affect binding of TNFα to its receptor or can promote its shedding. Recent observations demonstrate that redox events affect the binding of TNFα to TNF-R1 (19Hayakawa M. Miyashita H. Sakamoto I. Kitagawa M. Tanaka H. Yasuda H. Karin M. Kikugawa K. EMBO J. 2003; 22: 3356-3366Crossref PubMed Scopus (363) Google Scholar), and H2O2-induced shedding of soluble TNF-R1 has been demonstrated in lung epithelial cells (42Hino T. Nakamura H. Abe S. Saito H. Inage M. Terashita K. Kato S. Tomoike H. Am. J. Respir. Cell Mol. Biol. 1999; 20: 122-128Crossref PubMed Scopus (28) Google Scholar). However, it is unlikely that these events have contributed to our present observations, as we have demonstrated that JNK activation by TNFα is enhanced in cells pretreated with H2O2 (Fig. 6), illustrating that TNFα is still able to induce signaling. The sites of H2O2-induced oxidation relevant to adaptor protein recruitment and degradation remain elusive. Cysteines present in the extracellular domain of TNF-R1 occur as disulfide bridges (43Banner D.W. D'Arcy A. Janes W. Gentz R. Schoenfeld H.J. Broger C. Loetscher H. Lesslauer W. Cell. 1993; 73: 431-445Abstract Full Text PDF PubMed Scopus (978) Google Scholar) and therefore are unlikely targets for oxidation by H2O2. However, cysteines contained in the intracellular DD may be prone to oxidation by H2O2 and could promote receptor clustering and adaptor protein recruitment. Furthermore, oxidant-induced dissociation of thioredoxin from apoptosis signal-regulating kinase-1 promotes its activation and binding to TRAF2 (44Liu H. Nishitoh H. Ichijo H. Kyriakis J.M. Mol. Cell Biol. 2000; 20: 2198-2208Crossref PubMed Scopus (445) Google Scholar), providing an additional explanation by which H2O2 promotes TNF-R1-dependent signaling to JNK. It is not known at this time whether H2O2 acting through TNF-R1 is ligand-independent; however failure to activate IKK strongly suggests that H2O2 affects TNF-R1 function in a manner distinct from TNFα. The H2O2-induced signaling imbalance at TNF-R1 may have important ramifications for the cellular fate. It is well known that oxidant-induced JNK activation can mediate apoptosis (16Chen K. Vita J.A. Berk B.C. Keaney Jr., J.F. J. Biol. Chem. 2001; 276: 16045-16050Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar, 17Aoki H. Kang P.M. Hampe J. Yoshimura K. Noma T. Matsuzaki M. Izumo S. J. Biol. Chem. 2002; 277: 10244-10250Abstract Full Text Full Text PDF PubMed Scopus (294) Google Scholar). It is of interest to note that TNFα itself can alter the redox potential of the cell and uses oxidants to signal (for review, see Ref. 27Garg A.K. Aggarwal B.B. Mol. Immunol. 2002; 39: 509-517Crossref PubMed Scopus (201) Google Scholar). It is conceivable that the extent of oxidant production, including H2O2, dictates whether TNF-R1 activation by TNFα will lead to cell death or survival. Our present findings point to a putative proapoptotic role for oxidants under inflammatory conditions by causing direct signaling through TNF-R1, in addition to promoting TNFα-induced apoptosis via c-Jun N-terminal kinase. We thank Drs. Michael Karin, Patrick Baeuerle, Michael Lenardo, Brian Seed, and Rosa Ten for providing the various plasmid constructs and Amy Guala for establishing cultures of primary lung fibroblasts.