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• W2008671935 abstract "LIGHT (homologous tolymphotoxins, shows inducible expression, and competes with herpes simplex virus glycoprotein D forherpesvirus entry mediator, a receptor expressed byT lymphocytes) is a member of the tumor necrosis factor superfamily that can interact with lymphotoxin-β receptor (LTβR), herpes virus entry mediator, and decoy receptor (DcR3). In our previous study, we showed that LIGHT is able to induce cell death via the non-death domain containing receptor LTβR to activate both caspase-dependent and caspase-independent pathway. In this study, a LIGHT mutein, LIGHT-R228E, was shown to exhibit similar binding specificity as wild type LIGHT to LTβR, but lose the ability to interact with herpes virus entry mediator. By using both LIGHT-R228E and agonistic anti-LTβR monoclonal antibody, we found that signaling triggered by LTβR alone is sufficient to activate both caspase-dependent and caspase-independent pathways. Cross-linking of LTβR is able to recruit TRAF3 and TRAF5 to activate ASK1, whereas its activity is inhibited by free radical scavenger carboxyfullerenes. The activation of ASK1 is independent of caspase-3 activation, and kinase-inactive ASK1-KE mutant can inhibit LTβR-mediated cell death. This suggests that ASK1 is one of the factors involved in the caspase-independent pathway of LTβR-induced cell death. LIGHT (homologous tolymphotoxins, shows inducible expression, and competes with herpes simplex virus glycoprotein D forherpesvirus entry mediator, a receptor expressed byT lymphocytes) is a member of the tumor necrosis factor superfamily that can interact with lymphotoxin-β receptor (LTβR), herpes virus entry mediator, and decoy receptor (DcR3). In our previous study, we showed that LIGHT is able to induce cell death via the non-death domain containing receptor LTβR to activate both caspase-dependent and caspase-independent pathway. In this study, a LIGHT mutein, LIGHT-R228E, was shown to exhibit similar binding specificity as wild type LIGHT to LTβR, but lose the ability to interact with herpes virus entry mediator. By using both LIGHT-R228E and agonistic anti-LTβR monoclonal antibody, we found that signaling triggered by LTβR alone is sufficient to activate both caspase-dependent and caspase-independent pathways. Cross-linking of LTβR is able to recruit TRAF3 and TRAF5 to activate ASK1, whereas its activity is inhibited by free radical scavenger carboxyfullerenes. The activation of ASK1 is independent of caspase-3 activation, and kinase-inactive ASK1-KE mutant can inhibit LTβR-mediated cell death. This suggests that ASK1 is one of the factors involved in the caspase-independent pathway of LTβR-induced cell death. lymphotoxin-β receptor apoptosis signal-regulating kinase 1 interferon-γ herpes virus entry mediator tumor necrosis factor receptor-associated factor reactive oxygen species carboxyfullerenes influenza hemagglutinin tumor necrosis factor receptor lymphotoxin-α mitogen-activated protein kinase/extracellular signal-regulated kinase kinase 5 c-Jun N-terminal kinase monoclonal antibody mouse embryonic fibroblast myelin basic protein 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide 5-bromo-4-chloro-3-indolyl-β-d-galatopyranoside 1,4-piperazinediethanesulfonic acid 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone human LTβR Lymphotoxin-β receptor (LTβR)1 is a member of the tumor necrosis factor receptor (TNFR) superfamily and is ubiquitously expressed on the surface of most cell types, except T and B lymphocytes (1Force W.R. Walter B.N. Hession C. Tizard R. Kozak C.A. Browning J.L. Ware C.F. J. Immunol. 1995; 155: 5280-5288PubMed Google Scholar, 2Ware C.F. VanArsdale T.L. Crowe P.D. Browning J.L. Curr. Top. Microbiol. Immunol. 1995; 198: 175-218Crossref PubMed Scopus (228) Google Scholar). It has been reported that LTβR interacts specifically with two ligands: lymphotoxin LTα1/β2 (3Browning J.L. Dougas I. Ngam-ek A. Bourdon P.R. Ehrenfels B.N. Miatkowski K. Zafari M. Yampaglia A.M. Lawton P. Meier W. J. Immunol. 1995; 154: 33-46PubMed Google Scholar, 4Crowe P.D. VanArsdale T.L. Walter B.N. Ware C.F. Hession C. Ehrenfels B. Browning J.L. Din W.S. Goodwin R.G. Smith C.A. Science. 1994; 264: 707-710Crossref PubMed Scopus (1) Google Scholar) and LIGHT (5Mauri D.N. Ebner R. Montgomery R.I. Kochel K.D. Cheung T.C. Yu G.L. Ruben S. Murphy M. Eisenberg R.J. Cohen G.H. Spear P.G. Ware C.F. Immunity. 1998; 8: 21-30Abstract Full Text Full Text PDF PubMed Scopus (642) Google Scholar, 6Zhai Y. Guo R. Hsu T.L. Yu G.L. Ni J. Kwon B.S. Jiang G.W. Lu J. Tan J. Ugustus M. Carter K. Rojas L. Zhu F. Lincoln C. Endress G. Xing L. Wang S. Oh K.O. Gentz R. Ruben S. Lippman M.E. Hsieh S.L. Yang D. J. Clin. Invest. 1998; 102: 1142-1151Crossref PubMed Scopus (234) Google Scholar). There is ample evidences to demonstrate that LTβR plays an essential role in the development of lymphoid organs. Lymphoid nodes are deficient in LTα gene-deleted (LTα−/−) mice (7De Togni P. Goellner J. Ruddle N.H. Streeter P.R. Fick A. Mariathasan S. Smith S.C. Carlson R. Shornick L.P. Strauss-Schoenberger J. Science. 1994; 264: 703-707Crossref PubMed Scopus (868) Google Scholar), and the impairment of lymph node development as well as the loss of splenic architecture was also observed in LTβ knockout mice (8Koni P.A. Flavell R.A. J. Exp. Med. 1998; 187: 1977-1983Crossref PubMed Scopus (60) Google Scholar). Furthermore, LTβR-deficient mice are shown to lack Peyer's patches, colon-associated lymphoid tissues, and all lymph nodes (9Futterer A. Mink K. Luz A. Kosco-Vilbois M.H. Pfeffer K. Immunity. 1998; 9: 59-70Abstract Full Text Full Text PDF PubMed Scopus (622) Google Scholar). Interestingly, the administration of agonistic antibody to LTβR can induce lymph node development in LTα−/−mice (10Rennert P.D. Browning J.L. Hochman P.S. Int. Immunol. 1997; 9: 1627-1639Crossref PubMed Scopus (89) Google Scholar). In addition to its role in lymphoid organ formation, LTβR is also involved in host immune responses to foreign antigens. Blockade of LTβR with LTβR-Fc not only prevents germinal center formation in spleen but also results in impaired IgG antibody responses to sheep red blood cells (11Mackay F. Majeau G.R. Lawton P. Hochman P.S. Browning J.L. Eur. J. Immunol. 1997; 27: 2033-2042Crossref PubMed Scopus (169) Google Scholar). Moreover, administration of LTβR-Fc is shown to enhance host survival after virus challenge (12Puglielli M.T. Browning J.L. Brewer A.W. Schreiber R.D. Shieh W.J. Altman J.D. Oldstone M.B. Zaki S.R. Ahmed R. Nat. Med. 1999; 5: 1370-1374Crossref PubMed Scopus (57) Google Scholar) and is effective in preventing the onset of Th2 cell-mediated colitis (13Dohi T. Rennert P.D. Fujihashi K. Kiyono H. Shirai Y. Kawamura Y.I. Browning J.L. McGhee J.R. J. Immunol. 2001; 167: 2781-2790Crossref PubMed Scopus (82) Google Scholar). The cytoplasmic domains of TNFR families function as docking sites for downstream signaling molecules. Signaling occurs mostly through two classes of cytoplasmic adaptor proteins: death domain-containing molecules and TNFR-associated factors (TRAFs). The death domain-containing molecules or TRAFs are recruited to the cytoplasmic domain of members of TNFR after engagement with ligands. The cytoplasmic domain of LTβR does not contain consensus sequences characteristic of death domain; thus, LTβR-transduced signaling is mainly mediated by TRAFs. TRAF molecules consist of amino-terminal RING finger domain, central zinc finger loop, and carboxyl-terminal TRAF domain. The TRAF domain mediates interactions between TRAF proteins and both their upstream and downstream effectors, whereas the RING finger domain is reported to be necessary for TRAF effector activation (14Wajant H. Henkler F. Scheurich P. Cell. Signal. 2001; 13: 389-400Crossref PubMed Scopus (305) Google Scholar). Among the six TRAF proteins, TRAF2, TRAF3, and TRAF5 are found to associate with LTβR (15Force W.R. Cheung T.C. Ware C.F. J. Biol. Chem. 1997; 272: 30835-30840Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar, 16Force W.R. Glass A.A. Benedict C.A. Cheung T.C. Lama J. Ware C.F. J. Biol. Chem. 2000; 275: 11121-11129Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar, 17Nakano H. Oshima H. Chung W. Williams-Abbott L. Ware C.F. Yagita H. Okumura K. J. Biol. Chem. 1996; 271: 14661-14664Abstract Full Text Full Text PDF PubMed Scopus (304) Google Scholar). Further study has indicated that TRAF3 plays an important role in mediating LTβR-induced cell death (15Force W.R. Cheung T.C. Ware C.F. J. Biol. Chem. 1997; 272: 30835-30840Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar, 16Force W.R. Glass A.A. Benedict C.A. Cheung T.C. Lama J. Ware C.F. J. Biol. Chem. 2000; 275: 11121-11129Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar,18VanArsdale T.L. VanArsdale S.L. Force W.R. Walter B.N. Mosialos G. Kieff E. Reed J.C. Ware C.F. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 2460-2465Crossref PubMed Scopus (154) Google Scholar, 19Wu M.Y. Wang P.Y. Han S.H. Hsieh S.L. J. Biol. Chem. 1999; 274: 11868-11873Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar), whereas TRAF2 and TRAF5 have been shown to be involved in the activation of NF-κB (17Nakano H. Oshima H. Chung W. Williams-Abbott L. Ware C.F. Yagita H. Okumura K. J. Biol. Chem. 1996; 271: 14661-14664Abstract Full Text Full Text PDF PubMed Scopus (304) Google Scholar). Moreover, two serine/threonine protein kinases (p50 and p80) are reported to be associated with cytoplasmic region of LTβR (20Wu M.Y. Hsu T.L. Lin W.W. Campbell R.D. Hsieh S.L. J. Biol. Chem. 1997; 272: 17154-17159Abstract Full Text Full Text PDF PubMed Scopus (9) Google Scholar), but their roles in LTβR-mediated signaling have not been elucidated yet. Apoptosis signal-regulating kinase 1 (ASK1), also called mitogen-activated protein kinase/extracellular signal-regulated kinase kinase 5 (MEKK5), can be activated in response to various stress signals, including genotoxic stress (21Chen Z. Seimiya H. Naito M. Mashima T. Kizaki A. Dan S. Imaizumi M. Ichijo H. Miyazono K. Tsuruo T. Oncogene. 1999; 18: 173-180Crossref PubMed Scopus (168) Google Scholar), oxidative stress, reactive oxygen species (ROS) (22Gotoh Y. Cooper J.A. J. Biol. Chem. 1998; 273: 17477-17482Abstract Full Text Full Text PDF PubMed Scopus (312) Google Scholar), and laminar flow (23Liu Y. Yin G. Surapisitchat J. Berk B.C. Min W. J. Clin. Invest. 2001; 107: 917-923Crossref PubMed Scopus (94) Google Scholar). Furthermore, the kinase-inactive mutant of ASK1 inhibits cell death induced by tumor necrosis factor, Fas ligation, anti-cancer drugs, or withdrawal of neurotrophic factors (21Chen Z. Seimiya H. Naito M. Mashima T. Kizaki A. Dan S. Imaizumi M. Ichijo H. Miyazono K. Tsuruo T. Oncogene. 1999; 18: 173-180Crossref PubMed Scopus (168) Google Scholar, 24Ichijo H. Nishida E. Irie K. ten Dijke P. Saitoh M. Moriguchi T. Takagi M. Matsumoto K. Miyazono K. Gotoh Y. Science. 1997; 275: 90-94Crossref PubMed Scopus (1989) Google Scholar, 25Chang H.Y. Nishitoh H. Yang X. Ichijo H. Baltimore D. Science. 1998; 281: 1860-1863Crossref PubMed Scopus (527) Google Scholar, 26Wang T.H. Popp D.M. Wang H.S. Saitoh M. Mural J.G. Henley D.C. Ichijo H. Wimalasena J. J. Biol. Chem. 1999; 274: 8208-8216Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar, 27Kanamoto T. Mota M. Takeda K. Rubin L.L. Miyazono K. Ichijo H. Bazenet C.E. Mol. Cell. Biol. 2000; 20: 196-204Crossref PubMed Scopus (152) Google Scholar). ASK1 functions as an upstream component of the kinase cascades and interacts with a variety of molecules involved in stress-induced signaling pathways (21Chen Z. Seimiya H. Naito M. Mashima T. Kizaki A. Dan S. Imaizumi M. Ichijo H. Miyazono K. Tsuruo T. Oncogene. 1999; 18: 173-180Crossref PubMed Scopus (168) Google Scholar, 24Ichijo H. Nishida E. Irie K. ten Dijke P. Saitoh M. Moriguchi T. Takagi M. Matsumoto K. Miyazono K. Gotoh Y. Science. 1997; 275: 90-94Crossref PubMed Scopus (1989) Google Scholar). ASK1 phosphorylates and activates MKK4/7, which then activates the c-Jun NH2-terminal protein kinases (JNKs), also known as the stress-activated protein kinases. JNK activation requires phosphorylation at a specific motif (TPY). Moreover, ASK1 phosphorylates and activates MKK3 and MKK6, leading to activation of the p38 mitogen-activated protein kinases (24Ichijo H. Nishida E. Irie K. ten Dijke P. Saitoh M. Moriguchi T. Takagi M. Matsumoto K. Miyazono K. Gotoh Y. Science. 1997; 275: 90-94Crossref PubMed Scopus (1989) Google Scholar, 28Tobiume K. Matsuzawa A. Takahashi T. Nishitoh H. Morita K. Takeda K. Minowa O. Miyazono K. Noda T. Ichijo H. EMBO Rep. 2001; 2: 222-228Crossref PubMed Scopus (983) Google Scholar, 29Wang X.S. Diener K. Jannuzzi D. Trollinger D. Tan T.H. Lichenstein H. Zukowski M. Yao Z. J. Biol. Chem. 1996; 271: 31607-31611Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). It has been reported that JNK and p38 activations are abolished inASK1 −/− embryonic fibroblasts (28Tobiume K. Matsuzawa A. Takahashi T. Nishitoh H. Morita K. Takeda K. Minowa O. Miyazono K. Noda T. Ichijo H. EMBO Rep. 2001; 2: 222-228Crossref PubMed Scopus (983) Google Scholar). Signaling mediated by death domain-containing receptors, such as TNFRI and Fas, could be inhibited efficiently by caspase inhibitors. However, caspase inhibitor has only a partial effect to prevent LIGHT/IFN-γ-induced cell death (30Chen M.C. Hsu T.L. Luh T.Y. Hsieh S.L. J. Biol. Chem. 2000; 275: 38794-38801Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). In contrast, free radical scavenger carboxyfullerenes (C60) can completely inhibit LIGHT/IFN-γ-induced cell death (30Chen M.C. Hsu T.L. Luh T.Y. Hsieh S.L. J. Biol. Chem. 2000; 275: 38794-38801Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar), indicating the important roles of ROS in LIGHT/IFN-γ-induced cell death (30Chen M.C. Hsu T.L. Luh T.Y. Hsieh S.L. J. Biol. Chem. 2000; 275: 38794-38801Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). Since ROS are key mediators to activate ASK1, which contributes to progression of cell death (22Gotoh Y. Cooper J.A. J. Biol. Chem. 1998; 273: 17477-17482Abstract Full Text Full Text PDF PubMed Scopus (312) Google Scholar, 31Adler V. Yin Z. Tew K.D. Ronai Z. Oncogene. 1999; 18: 6104-6111Crossref PubMed Scopus (584) Google Scholar), we investigated the role of ASK1 in LIGHT-LTβR-induced cell death. Here we report that activation of LTβR alone, without the necessity to trigger HVEM activation, by either agonistic anti-LTβR mAb or a LIGHT mutein (LIGHT-R228E) incapable of HEVM binding, could induce the production of free radicals and the activation of ASK1. Blockade of ASK1 activation by free radical scavenger C60 could inhibit LTβR-mediated cell death. Thus, in addition to caspase activation, the activation of ASK1 also contributes to LTβR-mediated apoptotic pathways. The human hepatoma cells (Hep3BT2), human cervical carcinoma cells (HeLa), human embryonic kidney (HEK293) cells, and traf knockout mouse embryonic fibroblasts (MEFs) were maintained in Dulbecco's modified Eagle's medium (Invitrogen), supplemented with 10% (v/v) heat-inactivated fetal bovine serum (Invitrogen) at 37 °C in 5% (v/v) CO2. Plasmids containing the hLTβR and hLTβR-CD proteins have been described (19Wu M.Y. Wang P.Y. Han S.H. Hsieh S.L. J. Biol. Chem. 1999; 274: 11868-11873Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). The hemagglutinin (HA)-tagged expression constructs of ASK1, catalytically inactive ASK1-KE-HA, were kindly provided by Dr. Wen-Chen Yeh (32Hoeflich K.P. Yeh W.C. Yao Z. Mak T.W. Woodgett J.R. Oncogene. 1999; 18: 5814-5820Crossref PubMed Scopus (101) Google Scholar). The dominant negative TRAF mutants were provided by Dr. Wen-Chen Yeh (TRAF2 mutant) and Dr. Bharat B. Aggarwal (TRAF3, -5, and -6 mutants) vector. All of the TRAF mutants contained the c-Myc tag except TRAF6 mutant. For DNA transfection, cells were plated and grown for 16 h and transfected with expression vectors by the calcium phosphate method or by using LipofectAMINETM (Invitrogen). Monoclonal antibodies were prepared by immunizing Balb/c mice with recombinant human lymphotoxin β receptor-Fc (hLTβR-Fc) protein (6Zhai Y. Guo R. Hsu T.L. Yu G.L. Ni J. Kwon B.S. Jiang G.W. Lu J. Tan J. Ugustus M. Carter K. Rojas L. Zhu F. Lincoln C. Endress G. Xing L. Wang S. Oh K.O. Gentz R. Ruben S. Lippman M.E. Hsieh S.L. Yang D. J. Clin. Invest. 1998; 102: 1142-1151Crossref PubMed Scopus (234) Google Scholar). Spleen cells were fused with NS-1 cells, and hybridomas were screened by enzyme-linked immunosorbent assay. Anti-hLTβR monoclonal antibodies were selected by their specific binding to hLTβR but not to the Fc portion of human IgG1. The cDNA of extracellular region of LIGHT was cloned into pIZ/V5-His-FLAG (Invitrogen). Substitution of Arg228 by glutamic acid was performed by overlap extension using polymerase chain reaction (33Saiki R.K. Scharf S. Faloona F. Mullis K.B. Horn G.T. Erlich H.A. Arnheim N. Science. 1985; 230: 1350-1354Crossref PubMed Scopus (6659) Google Scholar). The primers used for polymerase chain reaction were designed to introduce anXhoI site as described in the followings: 5′-GAGGATGGTACCCGGTCTTACTTC-3′ (sense) and 5′-GAGTCGAACCAGGCGTTCATC-3′ (antisense). The PCR products were ligated at the XhoI site of pIZ/V5-His-FLAG-LIGHT to create pIZ/V5-His-FLAG-LIGHT(R228E). The construct was autosequenced (MB Mission Biotech) for verification of the mutation. The pIZ/V5-His-FLAG-LIGHT(R228E) construct was transfected into Sf21 cells by LipofectinTM(Invitrogen). Stable transfectants were selected with 500 μg/ml Zeocin (Invitrogen). Protein was purified by agarose beads conjugated with anti-FLAG antibody (M2) and followed by dialysis in phosphate-buffered saline as described (30Chen M.C. Hsu T.L. Luh T.Y. Hsieh S.L. J. Biol. Chem. 2000; 275: 38794-38801Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). ASK1-KE DNA construct (a gift from Dr. Wen-Chen Yeh) was transfected into Hep3BT2 using LipofectAMINETM (Invitrogen) as suggested by the vendor. Stable transfectants were selected with G418 (800 μg/ml Geneticin; Sigma), followed by immunoblot analysis to confirm the expression of ASK1-KE. The expression of ASK1-HA and TAK1-HA was detected by using anti-HA mAb (clone 3F10; Roche Molecular Biochemicals) or anti-human ASK1 antibodies (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). The expression of c-Myc-tagged TRAF2-DN, TRAF3-DN, and TRAF5-DN was detected by using anti-c-Myc tag polyclonal antibody (Upstate Biotechnology, Inc.). Rabbit polyclonal antibody against TRAF6 was obtained from Santa Cruz Biotechnology. Recombinant human IFN-γ was purchased from Roche Molecular Biochemicals. Cell lysates were prepared by the addition of lysis buffer (50 mm Tris, pH 8.0, 150 mm NaCl, 1% (v/v) Nonidet P-40, 1 mmphenylmethylsulfonyl fluoride, 2 μg/ml leupeptin, and 2 μg/ml aprotonin). Equal amounts of protein were subjected to electrophoresis, transferred onto nitrocellulose membrane (Hybond-C extra, Amersham Biosciences), and reacted with appropriate antibodies in phosphate-buffered saline containing 5% nonfat dry milk, 0.02% Tween 20. Blots were then incubated with horseradish peroxidase-conjugated secondary antibodies and reacted with enhanced chemiluminescence reagents subsequently (Amersham Biosciences). Association and dissociation rates of the interaction of LIGHT or LIGHT-R228E with human LTβR-Fc or HVEM-Fc were determined by surface plasmon resonance using a BIAcore® 2000 biomolecular interaction analysis system (BIA-core Inc., Piscataway, NJ). The Fc fusion proteins (50 μg/ml) were coupled to a CM5 sensor chip by amine coupling at pH 7.0. The sensor surface was equilibrated with phosphate-buffered saline, and sensorgrams were collected at 25 °C and a flow rate at 30 μl/min. A 120-μl injection of LIGHT or LIGHT-R228E was passed over the sensor surface. After the association phase, 600 s of dissociation data were collected. The sensor surface was regenerated after each cycle with a 15-μl pulse of 10 mm glycine (pH 2.0) twice with a 30-s interval. Sets of eight analyte concentrations, 100–800 nm, were collected and analyzed. To measure the activity of ASK1 in cell extracts, the immune complex was incubated at 30 °C for 30 min with 2 μg of substrates (such as myelin basic protein (MBP)) in 30 μl of solution containing 20 mm Tris-HCl (pH 7.5)/10 mm MgCl2/0.5 μCi of [γ-32P]ATP. Reactions were stopped by the addition of Laemmli sample buffer. Samples were then fractionated by SDS-PAGE, and proteins were visualized by Coomassie Blue staining. Phosphorylated proteins were identified by autoradiography and quantified by a densitometer (Amersham Biosciences). Cell death induced by overexpression of LTβR was determined by β-galactosidase-based cell morphology assay, and the killing effect of LIGHT/IFN-γ treatment was detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. For the β-galactosidase-based cell morphology assay, HeLa cells were co-transfected with lacZexpression vector, pBKCMV-lacZ. After 24 h of transfection, cells were fixed and then were stained with 5-bromo-4-chloro-3-indolyl-β-d-galatopyranoside (X-gal) to determine the percentage of apoptotic cells as described previously (19Wu M.Y. Wang P.Y. Han S.H. Hsieh S.L. J. Biol. Chem. 1999; 274: 11868-11873Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). The survival rate of Hep3BT2 cells was determined by MTT assay. Briefly, cells were seeded in 96-well flat bottom plates at a density of 5 × 103 cells/well. After treatment, 10 μl of 5 mg/ml MTT per well was added and incubated at 37 °C for 4 h. Cells were then lysed by the addition of 50 μl of 10% SDS in 0.4n HCl per well and incubated at 37 °C for another 16 h. The optical density of each sample was determined by measuring the absorbance at 570 versus 650 nm using an enzyme-linked immunosorbent assay reader (TECAN; RainBow) (30Chen M.C. Hsu T.L. Luh T.Y. Hsieh S.L. J. Biol. Chem. 2000; 275: 38794-38801Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). Cytosolic extracts were prepared by freezing and thawing of cells in extraction buffer (50 mm PIPES-NaOH, pH 7.0, 50 mm KCl, 5 mm EGTA, 2 mm MgCl2, 1 mm dithiothreitol, 1 mm phenylmethylsulfonyl fluoride, 1 μg/ml leupeptin, 1 μg/ml pepstatin A) as described (34Enari M. Talanian R.V. Wong W.W. Nagata S. Nature. 1996; 380: 723-726Crossref PubMed Scopus (965) Google Scholar). Cell lysates (50 μg) were diluted with 500 μl of ICE standard buffer (100 mm HEPES-KOH buffer, pH 7.5, 10% sucrose, 0.1% CHAPS, 10 mm dithiothreitol, 0.1 mg/ml ovalbumin) and incubated at 30 °C for 60 min with 20 μm fluorescent substrates. Fluorescence intensity was measured using a fluorescence spectrophotometer (Hitachi F-4500) at an excitation wavelength of 325 nm and emission wavelength of 392 nm. Cross-linking of cell surface receptor by ligand or by agonistic antibodies can trigger signal transduction, and members of the TNFR superfamily are reported to be activated by agonistic antibodies, such as anti-human Fas antibody (CH11) and anti-mouse Fas antibody (Jo2). To study the signaling transduced by LTβR, monoclonal antibodies against human LTβR were raised. One of the selected clones, 31G4D8, is found to bind to LTβR specifically. Anti-LTβR mAb 31G4D8 does not have any cytotoxic effect to Hep3BT2 or HT29, which are sensitive to LIGHT/IFN-γ-mediated cell death. However, in conjunction with IFN-γ, 31G4D8 mAb is able to induce cell death with similar extent as that induced by wild type LIGHT (Fig. 1). This observation is in agreement with the previous observation that overexpression of LTβR is able to induce cell death (19Wu M.Y. Wang P.Y. Han S.H. Hsieh S.L. J. Biol. Chem. 1999; 274: 11868-11873Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar), and LIGHT mutein incapable of binding to LTβR loses its ability to induce cell death (35Rooney I.A. Butrovich K.D. Glass A.A. Borboroglu S. Benedict C.A. Whitbeck J.C. Cohen G.H. Eisenberg R.J. Ware C.F. J. Biol. Chem. 2000; 275: 14307-14315Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar). To further confirm this argument, we designed a recombinant LIGHT mutein to bind LTβR but not HVEM, using a strategy of molecular modeling. A three-dimensional model for the interaction of LIGHT and its receptors (LTβR, HVEM, and DcR3) was generated by homology modeling (Molecular Simulation Inc., San Diego, CA) based on the crystallographic complex structure of LTα and TNFRI (Protein Data Bank code 1TNR) (36Banner 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 (971) Google Scholar, 37Eck M.J. Sprang S.R. J. Biol. Chem. 1989; 264: 17595-17605Abstract Full Text PDF PubMed Google Scholar, 38Eck M.J. Ultsch M. Rinderknecht E. de Vos A.M. Sprang S.R. J. Biol. Chem. 1992; 267: 2119-2122Abstract Full Text PDF PubMed Google Scholar). Residues of the receptor-binding sites of this system, conventionally denoted as the A-R interaction domain and the A-S interaction domain, were identified. A few charge or polar residues were chosen for site-specific mutagenesis with the prediction that their mutations would, depending on the type of receptor, either enhance or interrupt receptor binding through altered electrostatic interactions. One of the LIGHT muteins that we have substantially characterized, the mutation at amino acid 228 from arginine to glutamic acid (LIGHT-R228E) at the A-R interaction domain (see the model in Table I), met the modeling objective of the present study.Table IKinetics of ligand binding to receptors determined by surface plasmon resonanceValues are the means of at least five measurements over a ligand concentration range of 100–800 nm. K a, association rate constant; K d, dissociation rate constant; K D, equilibrium dissociation constant (from K d /K a ). Under the table is a homology-derived model of LIGHT (blue) in complex with LTβR (two units, one in orange the other in green), showing both the A–R interface (blue-orange) and the A–S interface (blue-green). The two amino acids whose mutants exhibited selective binding of HVEM and not LTβR (glycine 119) (35Rooney I.A. Butrovich K.D. Glass A.A. Borboroglu S. Benedict C.A. Whitbeck J.C. Cohen G.H. Eisenberg R.J. Ware C.F. J. Biol. Chem. 2000; 275: 14307-14315Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar) and vice versa (arginine 228) (this work) are labeled (pink). Open table in a new tab Values are the means of at least five measurements over a ligand concentration range of 100–800 nm. K a, association rate constant; K d, dissociation rate constant; K D, equilibrium dissociation constant (from K d /K a ). Under the table is a homology-derived model of LIGHT (blue) in complex with LTβR (two units, one in orange the other in green), showing both the A–R interface (blue-orange) and the A–S interface (blue-green). The two amino acids whose mutants exhibited selective binding of HVEM and not LTβR (glycine 119) (35Rooney I.A. Butrovich K.D. Glass A.A. Borboroglu S. Benedict C.A. Whitbeck J.C. Cohen G.H. Eisenberg R.J. Ware C.F. J. Biol. Chem. 2000; 275: 14307-14315Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar) and vice versa (arginine 228) (this work) are labeled (pink). The association and dissociation rates of wild type LIGHT and LIGHT-R228E to LTβR and HVEM were determined by surface plasmon resonance. As shown in Fig. 2 and TableI, the binding affinity of wild type LIGHT to both HVEM (K D = 8.81 ± 3.2 nm) and LTβR (K D = 8.72 ± 3.21 nm) is similar, whereas the binding affinity of LIGHT-R228E to HVEM is almost undetectable, and its binding affinity to LTβR (K D = 77.8 ± 41 nm) is reduced from that of the wild type but is clearly evident (Fig. 2 B). The reduction in affinity of R228E for LTβR-Fc was due to a decrease in association rate and an increase in dissociation rate (Table I). The binding of LIGHT-R228E to LTβR and the lack of it to HVEM were further confirmed by a competition analysis using LTβR-Fc or HVEM-Fc to inhibit wild type LIGHT and LIGHT-R228E-mediated cell death (Fig.2 C). Namely, wild type LIGHT/IFN-γ-induced cell death could be blocked by either LTβR-Fc or HVEM-Fc in a dose-dependent manner (Fig. 2 C, upper panel), whereas LIGHT-R228E/IFN-γ-induced cell death was only blocked by LTβR-Fc and not by HVEM-Fc (Fig. 2C, lower panel). These observations provided direct evidence that the amino acid arginine 228 is essential for the interaction between LIGHT and HVEM, and LTβR alone is sufficient for LIGHT-mediated cell death. Oxidative stress was reported to disrupt the ASK1-thioredoxin complex and thereby to activate ASK1 (39Saitoh M. Nishitoh H. Fujii M. Takeda K. Tobiume K. Sawada Y. Kawabata M. Miyazono K. Ichijo H. EMBO J. 1998; 17: 2596-2606Crossref PubMed Scopus (2045) Google Scholar). It has been shown that ROS play essential roles in LIGHT/IFN-γ-induced cell death (30Chen M.C. Hsu T.L. Luh T.Y. Hsieh S.L. J. Biol. Chem. 2000; 275: 38794-38801Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar); thus, we ask whether signaling through LTβR alone is enough to activate ASK1 activation to induce cell death. To address this question, HeLa cells were transfected with HA-tagged ASK1, followed by incubation with agonistic 31G4D8 mAb (Fig. 3 A) or LIGHT-R228E (Fig. 3 B) to test their ability to activate HA-tagged ASK1 by in vitro kinase assay. As shown in Fig.3 A, a rapid increase of ASK1 activity was observed at 5 min after 31G4D8 treatment and observed to last for at least 60 min (Fig.3 A). LIGHT-R228E had a similar" @default.
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