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• W2014373584 abstract "LIGHT is a tumor necrosis factor (TNF) ligand superfamily member, which binds two known cellular receptors, lymphotoxin-β receptor (LTβR) and the herpesvirus entry mediator (HveA). LIGHT is a homotrimer that activates proapoptotic and integrin-inducing pathways. Receptor binding residues via LIGHT were identified by introducing point mutations in the A′ → A“ and D → E loops of LIGHT, which altered binding to LTβR and HveA. One mutant of LIGHT exhibits selective binding to HveA and is inactive triggering cell death in HT29.14s cells or induction of ICAM-1 in fibroblasts. Studies with HveA- or LTβR-specific antibodies further indicated that HveA does not contribute, either cooperatively or by direct signaling, to the death pathway activated by LIGHT. LTβR, not HveA, recruits TNF receptor-associated factor-3 (TRAF3), and LIGHT-induced death is blocked by a dominant negative TRAF3 mutant. Together, these results indicate that TRAF3 recruitment propagates death signals initiated by LIGHT-LTβR interaction and implicates a distinct biological role for LIGHT-HveA system. LIGHT is a tumor necrosis factor (TNF) ligand superfamily member, which binds two known cellular receptors, lymphotoxin-β receptor (LTβR) and the herpesvirus entry mediator (HveA). LIGHT is a homotrimer that activates proapoptotic and integrin-inducing pathways. Receptor binding residues via LIGHT were identified by introducing point mutations in the A′ → A“ and D → E loops of LIGHT, which altered binding to LTβR and HveA. One mutant of LIGHT exhibits selective binding to HveA and is inactive triggering cell death in HT29.14s cells or induction of ICAM-1 in fibroblasts. Studies with HveA- or LTβR-specific antibodies further indicated that HveA does not contribute, either cooperatively or by direct signaling, to the death pathway activated by LIGHT. LTβR, not HveA, recruits TNF receptor-associated factor-3 (TRAF3), and LIGHT-induced death is blocked by a dominant negative TRAF3 mutant. Together, these results indicate that TRAF3 recruitment propagates death signals initiated by LIGHT-LTβR interaction and implicates a distinct biological role for LIGHT-HveA system. tumor necrosis factor lymphotoxin LT-β receptor herpesvirus entry mediator intercellular adhesion molecule interferon-γ monoclonal antibody 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide normal human dermal fibroblast polyacrylamide gel electrophoresis polymerase chain reaction TNF receptor-associated factor TNF receptor nuclear factor κB Dulbecco's modified Eagle's medium phosphate-buffered saline enzyme-linked immunosorbent assay bovine serum albumin fast protein liquid chromatography bis[2-(succinimidooxycarbonyloxy)ethyl]sulfone Cytokines related to tumor necrosis factor (TNF)1 mediate developmental and effector functions of the innate and adaptive immune systems. Signaling by TNF-related cytokines is initiated by aggregation of specific cell surface receptors. TNF, lymphotoxin α (LTα), and LTβ and the recently identified protein LIGHT exhibit distinct but overlapping patterns of binding to four cognate cell surface receptors that together define a core group within the larger TNF superfamily. TNF and LTα are homotrimeric ligands that bind two receptors, TNFR1 (55–60 kDa; CD120a) and TNFR2 (75–80 kDa; CD120b) (1.Smith C.A. Farrah T. Goodwin R.G. Cell. 1994; 76: 959-962Abstract Full Text PDF PubMed Scopus (1835) Google Scholar). LTα also forms heterotrimers with LTβ (2.Browning J.L. Ngam-ek A. Lawton P. DeMarinis J. Tizard R. Chow E.P. Hession C. O'Brine-Greco B. Foley S.F. Ware C.F. Cell. 1993; 72: 847-856Abstract Full Text PDF PubMed Scopus (435) Google Scholar), where the predominant form expressed by activated T cells is LTα1β2, which specifically binds the LTβR (3.Crowe 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, 4.Ware C.F. VanArsdale T.L. Crowe P.D. Browning J.L. Griffiths G.M. Tschopp J. Pathways for Cytolysis. Springer-Verlag, Basel1995: 175-218Google Scholar). LIGHT engages the herpesvirus entry mediator, HveA (also known as HVEM) (5.Montgomery R.I. Warner M.S. Lum B. Spear P.G. Cell. 1996; 87: 427-436Abstract Full Text Full Text PDF PubMed Scopus (1002) Google Scholar, 6.Mauri D.N. Ebner R. Montgomery R.I. Kochel K.D. Cheung T.C., Yu, G.-L. Ruben S. Murphy M. Eisenbery R.J. Cohen G.H. Spear P.G. Ware C.F. Immunity. 1998; 8: 21-30Abstract Full Text Full Text PDF PubMed Scopus (645) Google Scholar). The shared receptor binding patterns among these cytokines are observed with LIGHT binding LTβR and HveA binding LTα but not TNF or LTαβ heterotrimer. Although the complexity of receptor cross-utilization suggests functional redundancy of these cytokines, gene deletion studies in mice have revealed unique and cooperating roles for the LTαβ and TNF ligand-receptor systems in the development and function of the immune system. The LTαβ-LTβR system is required for the formation of lymphoid tissue (lymph nodes and Peyer's patches) as well as the segregation of T and B-lymphocytes into distinct compartments in the spleen and the formation of germinal centers (7.Fu Y.-X. Chaplin D. Annu. Rev. Immunol. 1999; 17: 399-433Crossref PubMed Scopus (561) Google Scholar). Lymphoid tissue is largely unaffected by deletion of TNF, TNFR1, or TNFR2; however, TNF and TNFR1 are important for correct formation of germinal centers (8.Matsumoto M. Mariathasan S. Nahm M.H. Baranyay F. Preschon J.J. Chaplin D.D. Science. 1996; 271: 1289-1291Crossref PubMed Scopus (341) Google Scholar, 9.Neumann B. Luz A. Pfeffer K. Holzmann B. J. Exp. Med. 1996; 184: 259-264Crossref PubMed Scopus (160) Google Scholar, 10.Fu Y.-X. Molina H. Matsumoto M. Huang G. Min J. Chaplin D.D. J. Exp. Med. 1997; 185: 2111-2120Crossref PubMed Scopus (166) Google Scholar). Roles for LIGHT and HveA have not yet been revealed by gene deletion studies; however, phenotypic differences between LTα- and LTβ-deficient mice (11.Koni P.A. Sacca R. Lawton P. Browning J.L. Ruddle N.H. Flavell R.A. Immunity. 1997; 6: 491-500Abstract Full Text Full Text PDF PubMed Scopus (521) Google Scholar, 12.Alimzhanov M.B. Kuprash D.V. Kosco-Vilbois M.H. Luz A. Turetskaya R.L. Tarakhovsky A. Rajewsky K. Nedospasov S.A. Pfeffer K. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 9302-9307Crossref PubMed Scopus (318) Google Scholar) as well as LTα- and LTβR-deficient mice (13.Futterer A. Mink K. Luz A. Kosco-Vilbois M.H. Pfeffer K. Immunity. 1998; 9: 59-70Abstract Full Text Full Text PDF PubMed Scopus (623) Google Scholar) implicate LIGHT/HveA signaling in some aspects of lymphoid tissue organization. That LIGHT engages both HveA and LTβR raises the question of whether these receptors signal independently or cooperatively. LTβR stimulates expression of adhesion molecules (14.Hochman P.S. Majeau G.R. Mackay F. Browning J.L. J. Inflam. 1996; 46: 220-234Google Scholar, 15.Degli-Esposti M.A. Davis-Smith T. Din W.S. Smolak P. Goodwin R.G. Smith C.A. J. Immunol. 1997; 158: 1756-1762PubMed Google Scholar) and induces apoptosis in adenocarcinoma cell lines when bound by LTα1β2 (16.Browning J.L. Miatkowski K. Sizing I. Griffiths D.A. Zafari M. Benjamin C.D. Meier W. Mackay F. J. Exp. Med. 1996; 183: 867-878Crossref PubMed Scopus (135) Google Scholar). Unlike the death domain-containing TNFRs (e.g. Fas (17.Tartaglia L.A. Rothe M. Hu Y.F. Goeddel D.V. Cell. 1993; 73: 213-216Abstract Full Text PDF PubMed Scopus (303) Google Scholar, 18.Itoh N. Nagata S. J. Biol. Chem. 1993; 268: 10932-10937Abstract Full Text PDF PubMed Google Scholar)), which initiate direct activation of the caspases leading to rapid apoptosis, the LTβR induces a slow apoptotic death (16.Browning J.L. Miatkowski K. Sizing I. Griffiths D.A. Zafari M. Benjamin C.D. Meier W. Mackay F. J. Exp. Med. 1996; 183: 867-878Crossref PubMed Scopus (135) Google Scholar), similar to TNFR2 (19.Grell M. Douni E. Wajant H. Lohden M. Clauss M. Maxeiner B. Georgopoulos S. Lesslauer W. Kollias G. Pfizenmaier K. Cell. 1995; 83: 793-802Abstract Full Text PDF PubMed Scopus (1151) Google Scholar) and CD30 (20.Smith C.A. Gruss H.-J. Davis T. Anderson D. Farrah T. Baker E. Sutherland G.R. Brannan C.I. Copeland N.G. Jenkins N.A. Grabstein K.H. Gliniak B. McAlister I.B. Fanslow W. Alderson M. Falk B. Gimpel S. Gillis S. Din W.S. Goodwin R.G. Armitage R.J. Cell. 1993; 73: 1349-1360Abstract Full Text PDF PubMed Scopus (511) Google Scholar, 21.Lee S.Y. Park C.G. Choi Y. J. Exp. Med. 1996; 183: 669-674Crossref PubMed Scopus (153) Google Scholar). Recent evidence suggests that apoptosis mediated by CD40 and TNFR2 occurs indirectly through the induction of TNF and activation of the TNFR1 pathway (22.Grell, M., Zimmerman, G., Gottfried, E., Chen, C., Grunwald, U., Huang, D. C. S., Lee, Y. H. W., Durkop, H., Engelmann, H., Scheurich, P., Wajant, H., and Strasser, A. (1999) EMBO J. 3034–3043Google Scholar). LTβR and HveA signal via TRAF molecules, a family of six RING finger proteins that bind directly to cytosolic domains of these receptors, allowing the propagation of signals to downstream effectors (23.Rothe M. Sarma V. Dixit V.M. Goeddel D.V. Science. 1995; 269: 1424-1427Crossref PubMed Scopus (975) Google Scholar, 24.Arch R. Gedrich R. Thompson C. Genes Dev. 1998; 12: 2821-2830Crossref PubMed Scopus (512) Google Scholar). For example, TRAF2 and TRAF5 act as adapters for NIK, a kinase that activates the IκB kinases generating the transcriptionally active form of NF-κB (25.Wallach D. Varfolomeev E.E. Malinin N.L. Goltsev Y.V. Kovalenko A.V. Boldin M.P. Annu. Rev. Immunol. 1999; 17: 331-367Crossref PubMed Scopus (1123) Google Scholar, 26.Li Z.W. Chu W. Hu Y. Delhase M. Deerinck T. Ellisman M. Johnson R. Karin M. J. Exp. Med. 1999; 189: 1839-1845Crossref PubMed Scopus (815) Google Scholar, 27.Nakano H. Oshima H. Chung W. Williams-Abbott L. Ware C. Yagita H. Okumura K. J. Biol. Chem. 1996; 271: 14661-14664Abstract Full Text Full Text PDF PubMed Scopus (310) Google Scholar). TRAF3 is involved in the propagation of signals via the LTβR that activate cell death (28.VanArsdale 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 (156) Google Scholar, 29.Force 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, 30.Wu 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). HveA also binds TRAF2 and TRAF5, which do not induce apoptosis, but activate NF-κB and JNK/AP1 pathways (31.Marsters S.A. Ayres T.M. Skuatch M. Gray C.L. Rothe M.L. Ashkenazi A. J. Biol. Chem. 1997; 272: 14029-14032Abstract Full Text Full Text PDF PubMed Scopus (263) Google Scholar, 32.Hsu H. Solovyev I. Colobero A. Elliott R. Kelley M. Boyle W.J. J. Biol. Chem. 1997; 272: 13471-13474Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar). LIGHT induces growth arrest of HT29 cells (33.Harrop J.A. McDonnell P.C. Brigham-Burke M. Lyn S.D. Minton J. Tan K.B. Dede K. Spampanato J. Silverman C. Hensley P. DiPrinzio R. Emery J.G. Deen K. Eichman C. Chabot-Fletcher M. Truneh A. Young P.R. J. Biol. Chem. 1998; 273: 27548-27556Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar) and, as a transfected cDNA, inhibits growth of some tumors in mice (34.Zhai Y. Guo R. Hsu T.-L., Yu, G.-L. Ni J. Kwon B.S. Jiang G. 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), and it may serve a costimulatory role in lymphocyte activation (33.Harrop J.A. McDonnell P.C. Brigham-Burke M. Lyn S.D. Minton J. Tan K.B. Dede K. Spampanato J. Silverman C. Hensley P. DiPrinzio R. Emery J.G. Deen K. Eichman C. Chabot-Fletcher M. Truneh A. Young P.R. J. Biol. Chem. 1998; 273: 27548-27556Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar). However, the relative contribution of HveA and LTβR to these LIGHT-mediated effects has not been established. Here, we have generated mutants of LIGHT that discriminate between its two receptors and utilized receptor-specific antibodies to reveal an indispensable role of the LTβR, but not HveA, in the apoptotic and integrin-inducing effects of LIGHT. The HT29.14S cell line is a clone of the HT29 colon adenocarcinoma sensitive to the proapoptotic activity of TNF-related ligands (16.Browning J.L. Miatkowski K. Sizing I. Griffiths D.A. Zafari M. Benjamin C.D. Meier W. Mackay F. J. Exp. Med. 1996; 183: 867-878Crossref PubMed Scopus (135) Google Scholar). HT29.14S cells transduced with retroviral vectors that express the TRAF3 dominant negative mutant T3Δ1–339 or empty vector as a control were generated as previously described (29.Force 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). Human embryonic kidney (HEK293) cells and 293T cells were obtained from the American Type Culture Collection (ATCC; Manassas, VA) and cultured in DMEM containing 10% fetal bovine serum with glutamine and penicillin/streptomycin. Normal human dermal fibroblasts (NHDF) from neonatal foreskins were purchased from Clonetics (San Diego, CA) and grown in DMEM supplemented with 10% fetal bovine serum, insulin (5 μg/ml), and fibroblast growth factor (1 ng/ml) (Sigma). Recombinant human interferon-γ (IFN-γ) and TNF were gifts of Dr. J. Browning (Biogen, Inc., Cambridge, MA). Fusion proteins constructed with the Fc region of human IgG1 and human HveA (HveA-Fc) (6.Mauri D.N. Ebner R. Montgomery R.I. Kochel K.D. Cheung T.C., Yu, G.-L. Ruben S. Murphy M. Eisenbery R.J. Cohen G.H. Spear P.G. Ware C.F. Immunity. 1998; 8: 21-30Abstract Full Text Full Text PDF PubMed Scopus (645) Google Scholar), LTβR (LTβR-Fc) (3.Crowe 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), TNF-R1-Fc (35.Crowe P.D. VanArsdale T.L. Walter B.N. Dahms K.M. Ware C.F. J. Immunol. Methods. 1994; 168: 79-89Crossref PubMed Scopus (47) Google Scholar, 36.Brunner T. Mogil R.J. LaFace D. Yoo N.J. Mahboubi A. Echeverri F. Martin S.J. Force W.R. Lynch D.H. Ware C.F. Nature. 1995; 373: 441-444Crossref PubMed Scopus (1271) Google Scholar), and TRAIL R1-Fc (37.Schneider P. Thome M. Burns K. Bodmer J.-L. Hofmann K. Kataoka T. Holler N. Tschopp J. Immunity. 1997; 7: 831-836Abstract Full Text Full Text PDF PubMed Scopus (593) Google Scholar) (gift from J. Tschopp) were prepared as described previously. Human LTα1β2 was expressed in insect cells as described by Browning et al. (38.Browning J.L. Miatkowski K. Griffiths D.A. Bourdon P.R. Hession C. Ambrose C.M. Meier W. J. Biol. Chem. 1996; 271: 8618-8626Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar) and purified by ion exchange on a SP Hitrap column (Amersham Pharmacia Biotech) followed by affinity purification with LTβR-Fc coupled to Affi-Gel (Bio-Rad) and depletion of contaminating ligands (mainly LTα2β1) by TNFR1-Fc affinity matrix (39.Rooney I. Butrovich K. Ware C.F. Methods Enzymol. 2000; 322: 345-363Crossref PubMed Google Scholar). Antibodies to HveA or LTβR (28.VanArsdale 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 (156) Google Scholar) were produced by immunizing goats with HveA-Fc or LTβR-Fc fusion proteins as described previously (40.VanArsdale T.L. Ware C.F. J. Immunol. 1994; 153: 3043-3050PubMed Google Scholar). Mouse monoclonal anti-LTβR antibodies BDA8 (IgG1) and CDH10 (IgG1) were gifts of Dr. J. Browning (Biogen, Inc.) (41.Browning 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). FLAG epitope-specific mAb M2 (anti-FLAG) was purchased from Sigma. Anti-ICAM-1 (P2A4) mAb was obtained from Chemicon International, Inc. Mouse monoclonal anti-HveA antibodies CW1 and CW8 were produced in mice with purified ectodomain of HveA. Details concerning the properties of these antibodies will be presented elsewhere. 2J. C. Whitbeck, manuscript in preparation. Sheep anti-mouse IgG coupled to horseradish peroxidase was purchased from Amersham Pharmacia Biotech. Full-length LIGHT was cloned from activated II23.D7 T cell hybridoma cells by reverse transcriptase-PCR using the following primer: forward, 5′-TATAAGCTTGAGGTTGAAGGACCCAGG-3′; reverse, 5′-CAGGGATCCCTTCCTTCACACCATGAAAGC-3′ (6.Mauri D.N. Ebner R. Montgomery R.I. Kochel K.D. Cheung T.C., Yu, G.-L. Ruben S. Murphy M. Eisenbery R.J. Cohen G.H. Spear P.G. Ware C.F. Immunity. 1998; 8: 21-30Abstract Full Text Full Text PDF PubMed Scopus (645) Google Scholar). The LIGHT PCR product was subcloned into pCDNA3.1(+) to create pCDNA3.1-LIGHT. The extracellular domain (encoding Gly66 to Val240) was amplified from pCDNA3-LIGHT by PCR using the following primers: forward, 5′-GTAGGAGAGATGGTCACCCGCCT-3′; reverse, 5′-GGAACGCGAATTCCCACGTGTCAGACCCATGTCCAAT-3′. The amplified LIGHT product was digested with EcoRI and ligated into theSnaB1 and EcoRI sites of pCDNA3.1-VCAM-FLAG, which contains the VCAM1 signal sequence fused to the FLAG epitope, 5′ of the FLAG epitope. HEK293 cells were transfected by the calcium phosphate method, and stable clones were selected with G418 and screened for LIGHT production. LIGHTt66 was purified from culture supernatants of cells grown in DMEM containing 0.5% fetal bovine serum. LIGHTt66 was purified by ion exchange chromatography with an SP Hitrap column (Amersham Pharmacia Biotech) and affinity chromatography with anti-FLAG (M2) coupled to Affi-Gel (Bio-Rad). LIGHTt66 was eluted from the column using 20 mm glycine, 150 mm NaCl, pH 3.0, and pH-neutralized immediately by collection into 50 mm Tris, pH 7.4. Protein concentration was determined by amino acid analysis and absorbency at 280 nm. Primer-introduced sequence modification was used to generate soluble LIGHT with the following single amino acid substitutions: G119E, L120Q, Q117T, and Y173F. Briefly, internal primers were designed to introduce a restriction site at the mutation location. Forward and reverse primers containing the mutations were used in separate PCRs to amplify two regions of soluble LIGHT. Primers were as follows: Q117T, 5′-ACGCTGGGCCTGGCCTTCCTGA-3“ and 5′-ACTCTCCCATAACAGCGGCC-3′; G119E, 5′-GAGCTGGCCTTGCTGAGGGGCCT-3” and 5′-CAGCTGAGTCTCCCATAACA-3′; L120Q, 5′-CAGGCCTTCCTGAGGGGCCTCA-3′ and 5′-GCCCAGCTGAGTCTCCCATAA-3′; Y173F, 5′-TTCCCCGAGGAGCTGGAGCT-3′ and 5′-GCGGGGTGTGCGCTTGTAGA-3′. The PCR products were ligated at the primer-introduced restriction enzyme site to create soluble LIGHT starting at amino acid Gly66 and containing one of the 4-amino acid substitutions. The LIGHTt66 mutants were excised and ligated into VCAM-FLAG-pCDNA3.1. The VCAM FLAG-LIGHT mutant inserts were cloned into pCDNA3.1(+) (Invitrogen). All constructs were sequenced (ABI310 automated sequencer) for unambiguous verification of the mutation. LIGHTt66 mutants were produced by calcium phosphate transient transfection of 293T cells. Mutant proteins were purified from 100 ml of culture supernatant in a one-step immunoaffinity procedure using an affinity matrix of anti-FLAG antibody (M2). Protein-containing fractions were dialyzed against PBS and sterilized after dilution in medium. Soluble LIGHT was measured using a capture ELISA method. The capture molecule (HveA-Fc or LTβR-Fc) was coated on wells of a microtiter plate (150 ng/well in 50 μl of 150 mm NaCl, 20 mm Tris, pH 9.6) at 4 °C. After washing with PBS, 0.5% Tween 20, purified ligands diluted in PBS, 3% BSA were added to the wells and incubated for 1 h at room temperature. After washing, mouse mAb anti-FLAG (M2) (10 μg/ml in PBS/BSA) was added for 1 h at room temperature, washed, and incubated with goat anti-mouse horseradish peroxidase (1:1500) for 1 h at room temperature. Color was developed with 2,2′-azino-bis(3-ethylbenz-thiazoline-6-sulfonic acid) (Sigma), and the OD was measured at 415 nm in a SpectraMax plate reader (Molecular Devices, Inc., Sunnyvale, CA). LIGHT was cross-linked by the addition of bis[2-(succinimidooxycarbonyloxy)ethyl]sulfone (BSOCOES) (Pierce) at final concentrations of 5 mm or by the addition of glutaraldehyde (0.1%) for 30 min at 4 °C, and the reaction was stopped by the addition of Tris (20 mm, pH 8.0). After SDS-PAGE, proteins were transferred to polyvinylidene fluoride membrane (Immobilon-P, Millipore Corp., Bedford, MA). Blots were incubated in PBS, 0.1% Tween containing 10% milk protein, 0.2% sodium azide) for 30 min. The blot was incubated with anti-FLAG M2 (5 μg/ml) and washed, and then horseradish peroxidase-conjugated secondary antibody (sheep anti-mouse IgG; Amersham Pharmacia Biotech; 1:1000) was added for 1 h. The blot was developed using chemiluminescent detection reagents (Supersignal; Pierce). The molecular weight of native LIGHTt66 was analyzed by gel filtration on a Superose 12 column using a FPLC 500 system (Amersham Pharmacia Biotech) at a flow rate of 0.5 ml/min with collection of 0.5-ml fractions. LIGHT was detected by ELISA and by Western blot with anti-FLAG. The elution volumes of calibration proteins (blue dextran (2000 kDa), apoferitin (443 kDa), bovine serum albumin (66 kDa), carbonic anhydrase (29 kDa), and cytochrome c (12 kDa) were measured by absorbency at 280 nm. HT29.14s cells (5000/well) in DMEM were incubated at 37 °C with serial dilutions of LIGHTt66 and other cytokines in a total volume of 100 μl of DMEM in the presence or absence of 80 units/ml of human IFN-γ. After 72 h, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was added, and the plate was incubated for 4 h at 37 °C. The medium was then aspirated, and 100 μl of acidified 70% isopropyl alcohol was added to dissolve the formazan product. TheA 570 was measured in multiwell spectrophotometer (Spectra Max 250, Molecular Devices, Inc.). The data represent the mean ± S.D. of triplicate wells. LIGHTt66 was modeled using the SwissModel server, version 3.0 (available on the World Wide Web) using 12TUN.pdb and 1TUN.pdb as the templates (42.Guex N. Peitsch M.C. Electrophoresis. 1997; 18: 2714-2723Crossref PubMed Scopus (9503) Google Scholar) and visualized using Rasmol (version 2.6). Association and dissociation rates of the interaction of LIGHTt66 and mutants with human HveA-Fc and LTβR-Fc were determined by surface plasmon resonance using a BIA-core X (BIA-core Inc., Piscataway, NJ). The capture molecule (50 μg/ml) was coupled to a CM5 sensor chip by amine coupling at pH 5.0. The sensor surface was equilibrated with PBS (20 mm sodium phosphate, 150 mm NaCl, pH 7.4), and sensorgrams were collected at 25 °C and a flow rate of 5 μl/min. A 10-μl injection of LIGHTt66 or mutant proteins were passed over the sensor surface, and after the association phase, 800 s of dissociation data were collected. The sensor surface was regenerated after each cycle with a 10-μl pulse of 10 mm glycine pH 2.0. Sets of five analyte concentrations, 100–500 nm, were collected and analyzed by nonlinear regression using the BIAevaluation software (version 2.1). Association and dissociation data were fitted on the basis of the simple AB ↔ A + B model. HT29.14s cells or NHDF (DMEM, 3% BSA) were incubated with mouse monoclonal antibodies in a total volume of 50 μl for 30 min at 4 °C. Cells were stained with goat anti-mouse IgG coupled to R-phycoerythrin for 30 min at 4 °C, and 5 × 103 cells were analyzed using a FACScan flow cytometer (Becton Dickinson, Mountain View, CA). One day post-transfection, 293T cells were seeded in eight-well chamber slides (Lab-Tek) at 3 × 104 cells/well and cultured for 18–36 h at 37 °C. Cells were washed twice with PBS, fixed for 10 min at room temperature in freshly prepared 2% paraformaldhyde in PBS (pH 7.0), washed twice with PBS, and then permeabilized in methanol for 2 min at room temperature. Cells were washed in PBS and then blocked for 10 min at room temperature in PBS containing 3% BSA. Polyclonal goat anti-LTβR IgG (28.VanArsdale 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 (156) Google Scholar), diluted to a final concentration of 20 μg/ml, rat anti-HveA (1:500 dilution) and mouse anti-FLAG-M2 to detect TRAF3, were diluted in PBS containing 3% BSA and 0.2% Triton X-100 (PBS/BSA/Triton). Primary antibodies were added to the wells to a final volume of 120 μl/well and incubated in a humidified chamber at room temperature for 1 h. Wells were then washed three times in PBS/BSA/Triton buffer. FITC-conjugated donkey anti-mouse IgG (Jackson ImmunoResearch Laboratories), in combination with Texas Red-conjugated donkey anti-goat IgG (Jackson ImmunoResearch Laboratories) or donkey anti-rat IgG-Texas Red, was diluted to a final concentration of 1:200 in PBS/BSA/Triton in a final volume of 120 μl/well. Slides were incubated in a humidified chamber at room temperature in the dark for 1 h and then washed three times in PBS/BSA/Triton. The slides were mounted in 80% glycerol in PBS, sealed, and kept at 4 °C in the dark for 1–7 days before visualization. Cells were observed with a Bio-Rad MRC-1024 confocal microscope with a krypton/argon ion laser and a × 60 Nikon objective. Images were acquired using the LaserSharp operation system and were analyzed and manipulated in Adobe PhotoShop. Empty vector-transfected cells or cells stained with normal goat serum or mouse isotype-specific IgG were used for negative controls. Neither control exhibited background staining. Representative staining patterns were based on counting 200 cells. 293T cells individually transfected with FLAG-tagged TRAF2, -3, or -5 alone or co-transfected with either HveA or LTβR were washed once with PBS and then lysed with 600 μl of lysis buffer (50 mm Tris, pH 9, 5 mm EDTA, 150 mm NaCl, 0.5% Triton X-100, 5 μg/ml leupeptin, 5 μg/ml aprotinin, 1 mm phenylmethylsulfonyl fluoride) at 4 °C for 30 min. Insoluble material was removed by centrifugation at 14,000 rpm for 3 min. Goat anti-HveA or goat anti-LTβR was added to the lysates at a final concentration of 5 μg/ml, and then protein G-Sepharose beads (Amersham Pharmacia Biotech) were added and mixed at 4 °C for 1 h. The beads were washed, and proteins were solubilized in SDS-PAGE sample buffer. Whole cell lysates were prepared by direct extraction of cell pellets into heated SDS-PAGE sample buffer and clarification by centrifugation (1500 × g for 10 min). Proteins were separated by SDS-PAGE and analyzed by blotting. In order to investigate cellular responses initiated by LIGHT, a soluble form (LIGHTt66) was engineered by truncation of the N-terminal 65 amino acids, which removes the cytoplasmic and transmembrane domains. An N-terminal FLAG epitope was added for detection and purification of the cytokine. This construct was expressed in HEK293 cells and purified to homogeneity by ion exchange and immunoaffinity chromatography. The final yield of protein was 80%, and the purity was >99% (Fig.1 A). Soluble LIGHTt66 behaved as an oligomer in SDS-PAGE following treatment with bifunctional cross-linkers (Fig. 1 B). Additionally, LIGHTt66 eluted in native gel filtration chromatography as a single narrow peak with a molecular mass of ∼76 kDa, which is consistent with a stable trimeric structure, characteristic of this family of cytokines. LIGHT contains two cysteines in the extracellular domain at positions 154 and 187; however, no evidence was found that a disulfide bond was necessary for the formation or stability of the trimer. Purified LIGHTt66 was active at inducing the death of adenocarcinoma HT29.14s cells with comparable efficiency to LTα1β2 (Fig.2 A). Cytotoxicity was dependent on IFN-γ (Fig. 2 B), as is characteristic of this cell line for induction of apoptosis by LTα1β2, TNF, Fas ligand, and TRAIL, and was maximal after 72 h (data not shown). Over a range of experiments, 50% loss of cell viability was achieved with doses of 10–100 pm LIGHT. LIGHTt66-induced death was blocked in a dose-dependent manner by receptor, LTβR-Fc, or HveA-Fc; however, a combination of Fas-Fc, TNFR1-Fc, and TRAILR2-Fc did not inhibit death in this system (Fig. 2 C). This latter observation demonstrates specificity of the Fc fusion proteins but also indicates that the death inducing activity of LIGHT is not dependent on induction of a secondary mediator, such as TNF, which was recently shown to occur when CD40 or TNFR2 are activated to induce cell death (22.Grell, M., Zimmerman, G., Gottfried, E., Chen, C., Grunwald, U., Huang, D. C. S., Lee, Y. H. W., Durkop, H., Engelmann, H., Scheurich, P., Wajant, H., and Strasser, A. (1999) EMBO J. 3034–3043Google Scholar). A three-dimensional model of LIGHT based on the crystallographic structure of LTα and TNF was generated to predict residues that are likely to be involved in receptor binding (42.Guex N. Peitsch M.C. Electrophoresis. 1997; 18: 2714-2723Crossref PubMed Scopus (9503) Google Scholar) (Fig.3 A). The primary receptor-binding residues in LTα are located in the connecting loops of the A → A“ and the D → E β-strands, which are located on opposite sides of the LIGHT subunit. The receptor binding site, located at the cleft formed between adjacent subunits, is formed as a composite of residues contributed by the A → A” loop from one subunit and the D → E loop of the neighboring subunit. Residues within these loops are not conserved between LIGHT and LTα, although significant conservation is apparent" @default.
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• W2014373584 title "The Lymphotoxin-β Receptor Is Necessary and Sufficient for LIGHT-mediated Apoptosis of Tumor Cells" @default.
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