Central Role of the Scaffold Protein Tumor Necrosis Factor Receptor-associated Factor 2 in Regulating Endoplasmic Reticulum Stress-induced Apoptosis

Abstract

The endoplasmic reticulum represents the quality control site of the cell for folding and assembly of cargo proteins. A variety of conditions can alter the ability of the endoplasmic reticulum (ER) to properly fold proteins, thus resulting in ER stress. Cells respond to ER stress by activating different signal transduction pathways leading to increased transcription of chaperone genes, decreased protein synthesis, and eventually to apoptosis. In the present paper we analyzed the role that the adaptor protein tumor necrosis factor-receptor associated factor 2 (TRAF2) plays in regulating cellular responses to apoptotic stimuli from the endoplasmic reticulum. Mouse embryonic fibroblasts derived from TRAF2-/- mice were more susceptible to apoptosis induced by ER stress than the wild type counterpart. This increased susceptibility to ER stress-induced apoptosis was because of an increased accumulation of reactive oxygen species following ER stress, and was abolished by the use of antioxidant. In addition, we demonstrated that the NF-κB pathway protects cells from ER stress-induced apoptosis, controlling ROS accumulation. Our results underscore the involvement of TRAF2 in regulating ER stress responses and the role of NF-κB in protecting cells from ER stress-induced apoptosis. The endoplasmic reticulum represents the quality control site of the cell for folding and assembly of cargo proteins. A variety of conditions can alter the ability of the endoplasmic reticulum (ER) to properly fold proteins, thus resulting in ER stress. Cells respond to ER stress by activating different signal transduction pathways leading to increased transcription of chaperone genes, decreased protein synthesis, and eventually to apoptosis. In the present paper we analyzed the role that the adaptor protein tumor necrosis factor-receptor associated factor 2 (TRAF2) plays in regulating cellular responses to apoptotic stimuli from the endoplasmic reticulum. Mouse embryonic fibroblasts derived from TRAF2-/- mice were more susceptible to apoptosis induced by ER stress than the wild type counterpart. This increased susceptibility to ER stress-induced apoptosis was because of an increased accumulation of reactive oxygen species following ER stress, and was abolished by the use of antioxidant. In addition, we demonstrated that the NF-κB pathway protects cells from ER stress-induced apoptosis, controlling ROS accumulation. Our results underscore the involvement of TRAF2 in regulating ER stress responses and the role of NF-κB in protecting cells from ER stress-induced apoptosis. In eukaryotic cells, proteins must be correctly folded and assembled before to transit to intracellular organelles and the cell surface (1Berridge M.J. Biochem. J. 1995; 312: 1-11Crossref PubMed Scopus (1049) Google Scholar, 2Sambrook J.F. Cell. 1990; 61: 197-199Abstract Full Text PDF PubMed Scopus (238) Google Scholar). A number of cellular stress conditions can interfere with protein folding, leading to accumulation of unfolded or misfolded proteins in the endoplasmic reticulum (ER) 3The abbreviations used are: ER, endoplasmic reticulum; NF-κB, nuclear factor-κB; MEF, mouse embryonic fibroblast; TRAF, tumor necrosis factor receptor-associated factor; ROS, reactive oxygen species; JNK, c-Jun N-terminal kinase; SAPK, stress-activated protein kinase; TNF, tumor necrosis factor; WT, wild type; H2DCFDA, dichlorodihydrofluorescein diacetate; MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium; FL, full-length; NAC, N-acetyl-l-cysteine. lumen. The ER has evolved specific signaling pathway to deal with the potential danger represented by the misfolded proteins. This adaptive response is named unfolded protein response (3Kaufman R.J. Genes Dev. 1999; 13: 1211-1233Crossref PubMed Scopus (1934) Google Scholar). Activation of unfolded protein response results in attenuation of protein synthesis, and up-regulation of genes encoding chaperones that facilitate the protein folding process in the ER. Thus, unfolded protein response reduces accumulation and aggregation of malfolded proteins, giving the cell the possibility of correcting the environment inside the ER (3Kaufman R.J. Genes Dev. 1999; 13: 1211-1233Crossref PubMed Scopus (1934) Google Scholar, 4Sitia R. Braakman I. Nature. 2003; 426: 891-894Crossref PubMed Scopus (575) Google Scholar). However, if the damage is too strong and homeostasis cannot be restored, the mammalian unfolded protein response initiates apoptosis. In mammalian cells, three transmembrane proteins Ire1α (5Tirasophon W. Welihinda A.A. Kaufman R.J. Genes Dev. 1998; 12: 1812-1824Crossref PubMed Scopus (746) Google Scholar), Ire1β (6Wang X.Z. Harding H.P. Zhang Y. Jolicoeur E.M. Kuroda M. Ron D. EMBO J. 1998; 17: 5708-5717Crossref PubMed Scopus (658) Google Scholar), and PERK (7Shi Y. Vattem K.M. Sood R. An J. Liang J. Stramm L. Wek R.C. Mol. Cell. Biol. 1998; 18: 7499-7509Crossref PubMed Google Scholar) act as ER stress sensor proteins and play important roles in transducing the stress signals initiated by the accumulation of malfolded proteins from the ER to the cytoplasm and nucleus. Ire1s and PERK are kept in an inactive state through association of their N-terminal lumen domain with the chaperone BiP. Following accumulation of malfolded proteins in the lumen of the ER, BiP dissociates to bind the malfolded proteins and Ire1s and PERK undergo oligomerization and transphosphorylation within their cytoplasmic kinase domains (8Bertolotti A. Zhang Y. Hendershot L.M. Harding H.P. Ron D. Nat. Cell. Biol. 2000; 2: 326-332Crossref PubMed Scopus (2104) Google Scholar, 9Breckenridge D.G. Germain M. Mathai J.P. Nguyen M. Shore G.C. Oncogene. 2003; 22: 8608-8618Crossref PubMed Scopus (652) Google Scholar). Other stress response pathways are activated following ER stress, such as the JNK/SAPK and NF-κB pathways (10Urano F. Wang X.Z. Bertolotti A. Zhang Y. Chung P. Harding H.P. Ron D. Science. 2000; 287: 664-666Crossref PubMed Scopus (2328) Google Scholar, 11Leonardi A. Vito P. Mauro C. Pacifico F. Ulianich L. Consiglio E. Formisano S. Di Jeso B. Endocrinology. 2002; 143: 2169-2177Crossref PubMed Scopus (29) Google Scholar). Activation of these pathways following ER stress is mediated by the physical and functional interaction of Ire1α and TRAF2 (10Urano F. Wang X.Z. Bertolotti A. Zhang Y. Chung P. Harding H.P. Ron D. Science. 2000; 287: 664-666Crossref PubMed Scopus (2328) Google Scholar). The central role played by TRAF2 in mediating cellular response to ER stress has been proposed based upon the observation that ectopic expression of a dominant negative mutant of TRAF2 lacking the N terminus Ring finger domain, blocks ER stress-induced NF-κB and JNK/SAPK activation, and that mouse embryonic fibroblast derived from TRAF2 knock-out mice failed to activate NF-κB following ER stress (10Urano F. Wang X.Z. Bertolotti A. Zhang Y. Chung P. Harding H.P. Ron D. Science. 2000; 287: 664-666Crossref PubMed Scopus (2328) Google Scholar, 11Leonardi A. Vito P. Mauro C. Pacifico F. Ulianich L. Consiglio E. Formisano S. Di Jeso B. Endocrinology. 2002; 143: 2169-2177Crossref PubMed Scopus (29) Google Scholar). TRAF2 was initially identified as a TNF receptor 2 interacting protein (13Rothe M. Wong S.C. Henzel W.J. Goeddel D.V. Cell. 1994; 78: 681-692Abstract Full Text PDF PubMed Scopus (932) Google Scholar). Interestingly, TRAF2-deficient MEFs are very sensitive to cell death induced by TNF and other members of the TNF receptor family (14Yeh W.C. Shahinian A. Speiser D. Kraunus J. Billia F. Wakeham A. de la Pompa J.L. Ferrick D. Hum B. Iscove N. Ohashi P. Rothe M. Goeddel D.V. Mak T.W. Immunity. 1997; 7: 715-725Abstract Full Text Full Text PDF PubMed Scopus (712) Google Scholar, 15Lee S.Y. Reichlin A. Santana A. Sokol K.A. Nussenzweig M.C. Choi Y. Immunity. 1997; 7: 703-713Abstract Full Text Full Text PDF PubMed Scopus (405) Google Scholar). At least part of the anti-apoptotic effect of TRAF2 can be explained by its function as a mediator of NF-κB activation, thus leading to NF-κB-dependent expression of anti-apoptotic genes. The anti-apoptotic activity of NF-κB also involves inhibition of the JNK cascade via at least two distinct mechanisms: through GADD45-β-mediated blockade of MKK7 and interference with ROS production (16Papa S. Zazzeroni F. Bubici C. Jayawardena S. Alvarez K. Matsuda S. Nguyen D.U. Pham C.G. Nelsbach A.H. Melis T. De Smaele E. Tang W.J. D'Adamio L. Franzoso G. Nat. Cell. Biol. 2004; 6: 146-153Crossref PubMed Scopus (302) Google Scholar, 17Pham C.G. Bubici C. Zazzeroni F. Papa S. Jones J. Alvarez K. Jayawardena S. De Smaele E. Cong R. Beaumont C. Torti F.M. Torti S.V. Franzoso G. Cell. 2004; 119: 529-542Abstract Full Text Full Text PDF PubMed Scopus (536) Google Scholar). It is well known that ROS or oxidative stress plays an important role in various physiological and pathological processes such as aging, inflammation, and neurodegenerative diseases (18Bray T.M. Proc. Soc. Exp. Biol. Med. 1999; 222: 195Crossref PubMed Scopus (30) Google Scholar, 19Forsberg L. deFaire U. Morgenstern R. Arch. Biochem. Biophys. 2001; 389: 84-93Crossref PubMed Scopus (263) Google Scholar, 20Finkel T. Curr. Opin. Cell Biol. 2003; 15: 247-254Crossref PubMed Scopus (1222) Google Scholar). Recently, it has been demonstrated that accumulation of misfolded protein within the lumen of the ER causes accumulation of ROS and cell death (21Haynes C.M. Titus E.A. Cooper A.A. Mol. Cell. 2004; 15: 767-776Abstract Full Text Full Text PDF PubMed Scopus (513) Google Scholar). However, it is currently unknown whether some of the key molecules involved in ER stress response, such as TRAF2, are involved in modulation of ROS and induction of apoptosis. Here we use MEFs derived from TRAF2 knock-out mice to study the role of TRAF2 in the regulation of pro-survival or pro-apoptotic pathways following ER stress. Cell Culture and Biological Reagents—Wild type (WT) and TRAF2-/- murine embryonic fibroblasts (MEFs) were provided by Drs. T. W. Mak and W. C. Yeh (14Yeh W.C. Shahinian A. Speiser D. Kraunus J. Billia F. Wakeham A. de la Pompa J.L. Ferrick D. Hum B. Iscove N. Ohashi P. Rothe M. Goeddel D.V. Mak T.W. Immunity. 1997; 7: 715-725Abstract Full Text Full Text PDF PubMed Scopus (712) Google Scholar). WT and JNK1/2-/- and WT and-/- MEFs were provided by Dr. R. Davis and Dr. G. Franzoso, respectively (22Tournier C. Hess P. Yang D.D. Xu J. Turner T.K. Nimnual A. Bar-Sagi D. Jones S.N. Flavell R.A. Davis R.J. Science. 2000; 288: 870-874Crossref PubMed Scopus (1542) Google Scholar, 23De Smaele E. Zazzeroni F. Papa S. Nguyen D.U. Jin R. Jones J. Cong R. Franzoso G. Nature. 2001; 414: 308-313Crossref PubMed Scopus (657) Google Scholar). Cells were cultured in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% fetal calf serum, 100 units/ml penicillin, and 100 μg/ml streptomycin. Thapsigargin was from Calbiochem and used at 5–50 nm; tunicamycin was purchased from Roche and used at 50–150 ng/ml. Dichlorodihydrofluorescein diacetate (H2DCFDA) (Calbiochem) was dissolved in Me2SO and used at 5 μm; l-NAC was dissolved in sterile water and used at 5 mm. Anti-TRAF2, anti-IκBα, and anti-JNK antibodies were purchased from Santa Cruz Biotechnology. The TRAF2 full-length expression vector was previously described (24Leonardi A. Ellinger-Ziegelbauer H. Franzoso G. Brown K. Siebenlist U. J. Biol. Chem. 2000; 275: 271-278Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). Western Blot Analysis—Subconfluent monolayer of murine embryonic fibroblasts were washed with phosphate-buffered saline and then lysed in a lysis buffer containing 20 mm HEPES, pH 7.5, 150 mm NaCl, 10% glycerol, 1% Triton X-100, supplemented with a mixture of protease inhibitors (Roche). Equal amounts of total proteins (50 μg) were resolved by SDS-polyacrylamide gels. Separated proteins were transferred to polyvinylidene difluoride membranes (Millipore, Bedford, MA) at 4 °C for Western blot analysis. Filters were blocked for 1 h at room temperature with 10% nonfat dry milk in TBS-T buffer (10 mm Tris-HCl, pH 8, 150 mm NaCl, 0.1% Tween 20). Then, filters were probed with specific antibodies in the same buffer for 14–16 h at 4 °C. After TBS-T washing to remove excess primary antibodies, the blots were incubated in horseradish peroxidase-coupled secondary antibody for 1 h followed by enhanced chemiluminescence detection of the proteins with Hyper-film ECL detection (Amersham Biosciences). Luciferase Assay—For luciferase assay, WT, TRAF2-/-, and TRAF2FL MEFs (4 × 105 cells per well) were seeded in 6-well (35 mm) plates. After 12 h cells were transfected with 0.5 μg of Ig-κB-LUC reporter gene plasmid using Lipofectamine. Cells were stimulated with thapsigargin or tunicamycin for 8 h, and reporter gene activity was determined by the luciferase assay system (Promega). A pRSV-β-galactosidase vector (0.2 μg) was used to normalize for transfection efficiencies. Retroviral Infection—Full-length hemagglutinin-tagged TRAF2 was subcloned into the retroviral expression vector pBMN by standard cloning techniques. pBMN vector was then transfected in a packaging cell line using Lipofectamine. 48 h after transfection, the viral supernatants were supplemented with Polybrene (9 mg/ml) and filtered through a 0.45-mm filter. TRAF2-/- fibroblasts (1 × 106) were incubated with viral supernatants for 48 The expression of protein was by Western blot total cell using ER and of × were seeded in and incubated for or 48 h at °C with different of thapsigargin or Cell was using and an to the (Promega). Cell death was by the cell membranes with or to I. G. M.C. F. C. J. PubMed Scopus Google Scholar). were analyzed by using a with were of at least three of ROS oxygen species were with into the cells it is into a by is to in the of intracellular Cells were with tunicamycin or thapsigargin for or 48 h, and incubated at °C for in the of in cells were used as After cells were washed with phosphate-buffered in phosphate-buffered and analyzed by using a Cell Biosciences). were used for the kinase assay that was at °C for with 2 μg of of in a total of 20 of kinase buffer mm HEPES, pH mm mm and The was The was by in and the were resolved by proteins were by of TRAF2-/- MEFs to ER is a protein that signals from and the ER (10Urano F. Wang X.Z. Bertolotti A. Zhang Y. Chung P. Harding H.P. Ron D. Science. 2000; 287: 664-666Crossref PubMed Scopus (2328) Google Scholar, 11Leonardi A. Vito P. Mauro C. Pacifico F. Ulianich L. Consiglio E. Formisano S. Di Jeso B. Endocrinology. 2002; 143: 2169-2177Crossref PubMed Scopus (29) Google Scholar, H. H. J. Cell 2002; PubMed Google Scholar). the role of TRAF2 in apoptosis induced by ER stress, we MEFs derived from TRAF2-/- mice and WT MEFs with of thapsigargin and ER stress by and After a some were In WT MEFs an of cellular stress response, TRAF2-/- MEFs and these were of apoptosis, we WT and TRAF2-/- in with thapsigargin or tunicamycin a in apoptosis in TRAF2-/- MEFs in WT The to apoptosis in TRAF2-/- MEFs was because of an of these cells, that of TRAF2 cell TRAF2-/- MEFs the same susceptibility as WT MEFs to and cell death results a specific role for TRAF2 in signals from the ER. ROS in TRAF2-/- stress has been to oxidative stress and apoptosis (21Haynes C.M. Titus E.A. Cooper A.A. Mol. Cell. 2004; 15: 767-776Abstract Full Text Full Text PDF PubMed Scopus (513) Google Scholar). to some the the increased susceptibility to ER stress-induced apoptosis, we ROS production in WT and TRAF2-/- in with thapsigargin or tunicamycin an in ROS production in TRAF2-/- MEFs in of these cells with TRAF2 blocked ROS accumulation following with thapsigargin and tunicamycin A and whether the increased production of ROS was for the susceptibility of TRAF2-/- MEFs to ER stress-induced apoptosis, TRAF2-/- MEFs were with thapsigargin or tunicamycin in the of different and 48 h cell was by assay and the ROS by in abolished ROS accumulation and these cells from apoptosis. results were by using as Interestingly, also the of WT MEFs and TRAF2-/- MEFs apoptosis following with tunicamycin and thapsigargin were by and results demonstrated that susceptibility of TRAF2-/- MEFs to ER stress-induced apoptosis was because of increased accumulation of It is that in TRAF2-/- cells, of ROS and apoptosis were in the of ER and and production with endoplasmic reticulum stress-induced apoptosis. WT, TRAF2-/-, and TRAF2FL MEFs were with Me2SO 150 tunicamycin or 20 thapsigargin for 48 h, in the or of or ROS production was by after with Cell was by are from three was by the NF-κB Activation Cells from ER the central role played by TRAF2 to correctly signal of NF-κB and JNK from ER, we of these pathways control ROS accumulation and cells from ER stress-induced apoptosis. MEFs derived from knock-out and knock-out mice were with thapsigargin or tunicamycin in the or of in MEFs very of ROS following with thapsigargin and with decreased ROS accumulation by In JNK1/2-/- MEFs an accumulation of ROS to WT MEFs is in the susceptibility of and JNK1/2-/- MEFs to apoptosis induced by thapsigargin or tunicamycin in the or of in MEFs were susceptible to apoptosis with WT and with increased cell JNK1/2-/- MEFs susceptibility to ER stress-induced cell as with WT results that NF-κB protects cells from ER stress-induced apoptosis by controlling ROS accumulation. the the NF-κB and the JNK pathways after ER stress, we of pathways in WT, TRAF2-/-, and TRAF2FL with tunicamycin of NF-κB in WT and TRAF2FL as demonstrated by the of the and by the increased activity of a luciferase reporter gene In the of TRAF2 it was to of The of NF-κB was functional as demonstrated by the of the a known gene of NF-κB Activation of JNK in WT and TRAF2FL MEFs stimulated with tunicamycin was after and decreased with JNK In in TRAF2-/- of JNK was h after and for to 12 This of JNK was by This that TRAF2 was to activate JNK after ER stress, and that the increased of ROS in the of TRAF2 the of This is in with that after TNF the of JNK TRAF2 and that the of JNK ROS S. M. T. T. Y. H. K. T. H. EMBO J. 2003; 22: PubMed Scopus Google Scholar). these results that following ER stress, the of NF-κB was for from apoptosis by ROS and controlling JNK The endoplasmic reticulum is the site for protein and folding, and also as a cellular site for that interfere with protein folding or to ER stress and eventually cell of apoptosis induced by death and is well the by ER stress apoptosis is In the present we present a central role played by TRAF2 in regulation of pro-apoptotic and anti-apoptotic pathways initiated at the ER. that TRAF2-/- MEFs increased susceptibility to ER stress-induced apoptosis. This increased susceptibility to ER stress-induced apoptosis was because of accumulation of ROS following ER stress, and was abolished by the use of such as In addition, we demonstrated that NF-κB was protecting cells from ER stress-induced apoptosis by controlling ROS accumulation. TRAF2 has been demonstrated to be involved in signaling from endoplasmic reticulum to with (10Urano F. Wang X.Z. Bertolotti A. Zhang Y. Chung P. Harding H.P. Ron D. Science. 2000; 287: 664-666Crossref PubMed Scopus (2328) Google Scholar), of the ER transmembrane proteins involved in signals from the ER. TRAF2 of the JNK/SAPK and the NF-κB pathways following ER stress (10Urano F. Wang X.Z. Bertolotti A. Zhang Y. Chung P. Harding H.P. Ron D. Science. 2000; 287: 664-666Crossref PubMed Scopus (2328) Google Scholar, 11Leonardi A. Vito P. Mauro C. Pacifico F. Ulianich L. Consiglio E. Formisano S. Di Jeso B. Endocrinology. 2002; 143: 2169-2177Crossref PubMed Scopus (29) Google Scholar). This is of TNF in TRAF2 of the NF-κB pathway and pro-apoptotic JNK and the of the cell be determined by these NF-κB its anti-apoptotic activity by function N. T. EMBO J. 1998; 17: PubMed Scopus Google Scholar, P. N. K. C. S. G. R. M. A. Nature. PubMed Scopus Google Scholar, M. K. A. J. C. Zhang M. R. M. Cell. 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The of ROS in regulating of JNK following TNF receptor has been in a NF-κB cell S. M. T. T. Y. H. K. T. H. EMBO J. 2003; 22: PubMed Scopus Google Scholar). NF-κB the ROS accumulation in JNK and cell Our and that a also for the ER. In induction of ER stress causes of NF-κB and In the of TRAF2 or the NF-κB pathway is and the JNK activity is leading to cell ROS JNK ROS JNK by at least two different mechanisms: by and protein kinase H. S. S. L. H. M. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar) and activating the protein K. A. T. H. K. K. K. T. H. EMBO 2001; 2: PubMed Scopus Google Scholar). This kinase be activated via ROS and TRAF2 and has been demonstrated to be for cell death after ER stress, at least in cells K. A. T. H. K. K. K. T. H. EMBO 2001; 2: PubMed Scopus Google Scholar, H. A. K. K. K. K. A. H. Genes Dev. 2002; PubMed Scopus Google Scholar). It be that after ER stress and in the of a functional NF-κB activation, is activated by the increased of ROS and JNK and cell it is from results that the of a functional NF-κB is for increased induction of ROS following ER stress, the by NF-κB function is It has been demonstrated that NF-κB expression of an involved in and of ROS accumulation C.G. Bubici C. Zazzeroni F. Papa S. Jones J. Alvarez K. Jayawardena S. De Smaele E. Cong R. Beaumont C. Torti F.M. Torti S.V. Franzoso G. Cell. 2004; 119: 529-542Abstract Full Text Full Text PDF PubMed Scopus (536) Google Scholar). However, it is that in to up-regulation of genes involved in of the ROS, NF-κB also control transcription of genes that production of Our results the central role played by TRAF2 in regulating of NF-κB following ER stress, and also the functional of Recently, it has been demonstrated that in to the NF-κB activation, leading to of NF-κB following ER stress following ER stress, of eukaryotic factor 2 of the leading to of NF-κB J. Zhang Y. D. Kaufman R.J. N. Harding H.P. Ron D. Mol. Cell. Biol. 2004; PubMed Scopus Google Scholar). The two of of NF-κB following ER stress, the and the eukaryotic factor are It is that to activate NF-κB upon ER stress. However, the of the eukaryotic factor 2 and NF-κB is the functional of NF-κB to be at least in In cells lacking TRAF2 or functional NF-κB undergo cell death after ER stress. In in the present study we for the that the adaptor protein TRAF2 plays a central role in regulating signaling from the ER and that the of NF-κB, mediated by TRAF2, protects cells from ER stress-induced apoptosis. TRAF2 and NF-κB be potential to control ER stress-induced apoptosis. Dr. for ROS