Activation of OX40 Signal Transduction Pathways Leads to Tumor Necrosis Factor Receptor-associated Factor (TRAF) 2- and TRAF5-mediated NF-κB Activation

We investigated the intracellular signaling of OX40, a member of the tumor necrosis factor receptor family. Activation of NF-κB in OX40-transfected HSB-2 cells was detected by electrophoretic mobility shift assay within 30 min after the binding of the ligand gp34. In vitro binding experiments showed that tumor necrosis factor receptor-associated factor (TRAF) 1, TRAF2, TRAF3, and TRAF5 but not TRAF4 associated with glutathioneS-transferase-OX40 fusion protein. The cotransfection experiments using human embryo kidney cell derived HEK 293T cells showed that TRAF2, TRAF3, and TRAF5 associated with OX40 in vivo. Studies with OX40 deletion mutants demonstrated that the cytoplasmic portion consisting of amino acid sequence 256–263 (GGSFRTPI) was required for the association with TRAFs and NF-κB activation. The introduction of the dominant negative mutants of TRAF2 and TRAF5 into HSB-2-OX40 cells suppressed NF-κB activation in a dose-dependent manner. In addition, the introduction of TRAF3 together with the dominant negative mutants of TRAF2 or TRAF5 further reduced NF-κB activation. These results indicate that the NF-κB activation resulting from OX40 stimulation is mediated by both TRAF2 and TRAF5, and is likely to be negatively modulated by TRAF3. We investigated the intracellular signaling of OX40, a member of the tumor necrosis factor receptor family. Activation of NF-κB in OX40-transfected HSB-2 cells was detected by electrophoretic mobility shift assay within 30 min after the binding of the ligand gp34. In vitro binding experiments showed that tumor necrosis factor receptor-associated factor (TRAF) 1, TRAF2, TRAF3, and TRAF5 but not TRAF4 associated with glutathioneS-transferase-OX40 fusion protein. The cotransfection experiments using human embryo kidney cell derived HEK 293T cells showed that TRAF2, TRAF3, and TRAF5 associated with OX40 in vivo. Studies with OX40 deletion mutants demonstrated that the cytoplasmic portion consisting of amino acid sequence 256–263 (GGSFRTPI) was required for the association with TRAFs and NF-κB activation. The introduction of the dominant negative mutants of TRAF2 and TRAF5 into HSB-2-OX40 cells suppressed NF-κB activation in a dose-dependent manner. In addition, the introduction of TRAF3 together with the dominant negative mutants of TRAF2 or TRAF5 further reduced NF-κB activation. These results indicate that the NF-κB activation resulting from OX40 stimulation is mediated by both TRAF2 and TRAF5, and is likely to be negatively modulated by TRAF3. Human OX40 is a 50-kDa cell surface glycoprotein expressed primarily on activated CD4+ T cells and some human T cell leukemia virus type I (HTLV-I) 1The abbreviations used are: HTLV-I, human T cell leukemia virus type I; NGF, nerve growth factor; TNF, tumor necrosis factor; R, receptor; TRAF, tumor necrosis factor receptor-associated factor; RT, reverse transcriptase; PCR, polymerase chain reaction; HA, hemagglutinin; luc, luciferase; CMV, cytomegalovirus; HEK, human embryo kidney; EMSA, electromobility shift assay; PAGE, polyacrylamide gel electrophoresis; GST, glutathione S-transferase. -infected T cell lines, but not on resting peripheral T cells, peripheral B cells, or thymocytes. OX40 was originally described as a cell surface antigen on the activated rat T cells (1Paterson D.J. Jefferies W.A. Green J.R. Brandon M.R. Corthesy P. Puklavic M. Williams A.F. Mol. Immunol. 1987; 24: 1281-1290Crossref PubMed Scopus (404) Google Scholar). Molecular cloning of its cDNA (1Paterson D.J. 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Cell. 1994; 76: 959-962Abstract Full Text PDF PubMed Scopus (1839) Google Scholar, 6Baker S.T. Reddy E.P. Oncogene. 1996; 12: 1-9PubMed Google Scholar). All the members of this superfamily share a characteristic repeating cysteine-rich motif in the extracellular domain, which is believed to be related to their ability to interact with the TNF-related ligands. The diverse cellular responses such as cell growth, differentiation, and programmed cell death (apoptosis) are triggered by the interaction between the members of the NGF-R/TNF-R superfamily and their ligands. The ligand for human OX40 was also cloned and identified as previously reported gp34, a cell surface protein expressed on HTLV-I-infected T cell lines and subsequently demonstrated to be induced by transactivator p40tax of HTLV-I (7Baum P.R. Gayle III R.B. Ramsdell F. Srinivasan S. Sorensen R.A. Watson M.L. Seldin M.F. Baker E. Sutherland G.R. Clifford K.N. Alderson M.R. Goodwin R.G. Fanslow W.C. 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We recently reported that the OX40/gp34 system directly mediates the adhesion of activated or HTLV-I-transformed T cells to vascular endothelial cells (11Imura A. Hori T. Imada K. Ishikawa T. Tanaka Y. Maeda M. Imamura S. Uchiyama T. J. Exp. Med. 1996; 183: 2185-2195Crossref PubMed Scopus (270) Google Scholar, 13Imura A. Hori T. Imada K. Kawamata S. Tanaka Y. Imamura S. Uchiyama T. Blood. 1997; 89: 2951-2958Crossref PubMed Google Scholar). Furthermore, we examined the role of the OX40/gp34 system in the development of angitis related diseases such as systemic lupus erythematosus and erythema nodosum (14Matsumura Y. Imura A. Hori T. Uchiyama T. Imamura S. Arch. Dermatol. Res. 1997; 289: 653-656Crossref PubMed Scopus (28) Google Scholar). Although these observations have served to delineate OX40 as a multifunctional cell surface molecule, its physiological as well as pathophysiological significance in viral infection, inflammation (15Weinberg A.D. Bourdette D.N. Sullivan T.J. Lemon M. 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We that the OX40 stimulation to NF-κB activation. on the a physiological and pathophysiological significance of the OX40 signaling in activated T cells is on the cDNA sequence of human gp34, cDNA of the of gp34 was by reverse chain The into of K. and to The of was described previously (11Imura A. Hori T. Imada K. Ishikawa T. Tanaka Y. Maeda M. Imamura S. Uchiyama T. J. Exp. Med. 1996; 183: 2185-2195Crossref PubMed Scopus (270) Google Scholar). The for OX40 cytoplasmic deletion mutants by using as the and as the The into to 1, and The for the OX40 deletion mutants to the on the cDNA of TRAF2, TRAF5, and human TRAF3 and the of TRAFs by The the of TRAFs and from cell line TRAF2, and or human peripheral cells TRAF3 and used as the and the The into the or a of W. C. of and of to or The of TRAF2 and TRAF5 by with the the amino acid sequence of TRAF2 H. Shu H.-B. Pan M.-G. Goeddel D.V. Cell. 1996; 84: 299-308Abstract Full Text Full Text PDF PubMed Scopus (1738) Google Scholar) and of TRAF5 H. H. W. Ware H. K. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar), respectively, and into for gp34 was by the extracellular portion of gp34 to the signal sequence of OX40 the The into the (11Imura A. Hori T. Imada K. Ishikawa T. Tanaka Y. Maeda M. Imamura S. Uchiyama T. J. Exp. Med. 1996; 183: 2185-2195Crossref PubMed Scopus (270) Google Scholar) of K. of gp34 into cells by the B. A. U. S. A. 1987; 84: PubMed Scopus Google Scholar). The cells with with for and with for The and with to the The of cells with gp34 was to the cell for The of cells with was by the as and to the Human T cell line HSB-2 and cell line in with and Human embryo kidney cell line HEK 293T and with with The of OX40 or OX40 deletion mutants 1, or by or 1, or into HSB-2 cells by the The of gp34 by into cells by the The cells in for and in for The of OX40 or gp34 on the cells was examined with a to the The was into HSB-2 cells or cells to cells or cells. The cells in with The intracellular in HSB-2-OX40 cells examined after on the of cells. The of cells in the cells was by min for using reverse and cDNA from the cells. of cells in HSB-2-OX40 cells was detected the from HSB-2-OX40 cells or cells with cells or cells, or with or for the as described previously E. Matthias P. W. Res. PubMed Scopus Google Scholar). The from HSB-2-OX40 cells for 30 with antibody (11Imura A. Hori T. Imada K. Ishikawa T. Tanaka Y. Maeda M. Imamura S. Uchiyama T. J. Exp. Med. 1996; 183: 2185-2195Crossref PubMed Scopus (270) Google Scholar) or receptor chain antibody to with cells also of a binding for and or The binding assay was as described previously with a W.R. J. Biol. Chem. 1994; 269: Full Text PDF PubMed Google Scholar). The of the induced NF-κB was examined by shift assay with antibody or antibody of in of E. Matthias P. W. Res. PubMed Scopus Google Scholar) was with of for min to the binding of or with of or into HEK 293T by the M. Sarma V. Dixit V.M. Goeddel D.V. Science. 1995; 269: 1424-1427Crossref PubMed Scopus (980) Google Scholar). of the cells in the and The cell with protein and antibody (11Imura A. Hori T. Imada K. Ishikawa T. Tanaka Y. Maeda M. Imamura S. Uchiyama T. J. Exp. Med. 1996; 183: 2185-2195Crossref PubMed Scopus (270) Google Scholar). The by with gel and to (11Imura A. Hori T. Imada K. Ishikawa T. Tanaka Y. Maeda M. Imamura S. Uchiyama T. J. Exp. Med. 1996; 183: 2185-2195Crossref PubMed Scopus (270) Google Scholar) with TRAFs by system The was for the of OX40 by The cDNA of cytoplasmic portion of OX40 to amino or was into the with or as described previously PubMed Scopus Google Scholar). of or was into HEK 293T cells by the of the cell by the as for the in binding assay and with or by for to or fusion protein was by with gel and to with antibody to or was by with gel and to with antibody TRAFs by system In the studies of NF-κB activation by of of or into HSB-2-OX40 cells together with of J. W.C. Mol. Cell. Biol. 1996; PubMed Google Scholar) of W. and of G. Baltimore D. Genes Dev. 1996; 10: 963-973Crossref PubMed Scopus (265) Google Scholar) by the N. A. F. 1987; PubMed Scopus Google Scholar). The of was to by of the cells in of of cell from to the in with the using a of cell to as G. Baltimore D. Genes Dev. 1996; 10: 963-973Crossref PubMed Scopus (265) Google Scholar). The by the In the experiments of and NF-κB or was with of and of into HSB-2-OX40 cells or HSB-2 cells by the The of was to by of the cells with cells or cells for and to and as described Activation of NF-κB in HSB-2-OX40 cells was detected by the cells with cells but not with cells The activation was detected from 30 min to of not after ligand stimulation and by of HSB-2-OX40 cells with The of the with antibody or antibody the of consisting of and in activation. results the HSB-2-OX40 cells with but not with Furthermore, the studies with HSB-2-OX40 deletion mutants demonstrated that NF-κB activation was detected in cells and cells, but not cells after ligand These results that the cytoplasmic portion of OX40 consisting of the amino acid sequence 256–263 (GGSFRTPI) was required for the activation of NF-κB 1, and has been reported that TRAFs with the receptors of several members of the TNF-R family and initiate the signal transduction the ligand We examined the association of TRAFs with OX40 using and fusion protein. As in and but not associated with and expressed in T cells and with antibody not Furthermore, the studies with deletion mutants demonstrated that TRAF2, TRAF3, and TRAF5 associated with and not but not with not or in In the cytoplasmic portion of OX40 consisting of the amino acid sequence 256–263 (GGSFRTPI) was required for the association with TRAF2, TRAF3, and TRAF5 in We examined the association of TRAFs with OX40 in vivo. and with antibody with OX40 in HEK 293T cells, that these TRAFs with OX40 in and expressed in HEK 293T cells and with the association of or with OX40 in was not detected this not the ability of TRAFs to NF-κB activation in HSB-2-OX40 cells, with the into HSB-2-OX40 cells. The assay of the cell demonstrated that TRAF2 and TRAF5 but not TRAF3, or TRAF4 to NF-κB activation in HSB-2-OX40 cells Since TRAF2, TRAF3, and TRAF5 to with OX40 in we examined the of TRAF3 on NF-κB activation mediated by TRAF2 or TRAF5 in HSB-2-OX40 cells. As in the introduction of TRAF3 reduced the of NF-κB activation by TRAF2 or on this we further examined the of TRAF2, TRAF5, and TRAF3 in NF-κB activation resulting from OX40 stimulation by the dominant negative of TRAF2 TRAF5 or type TRAF3 into HSB-2-OX40 cells. The HSB-2-OX40 cells stimulated by cells. As in the introduction of TRAF2 or TRAF5 suppressed the in a manner. Furthermore, the introduction of both TRAF2 and TRAF5 suppressed the to the The introduction of TRAF3 together with TRAF2 or TRAF5 reduced NF-κB activation which that TRAF3 NF-κB activation negatively in OX40 In the we demonstrated that and NF-κB activation was induced by OX40 We have in the of human OX40 of (11Imura A. Hori T. Imada K. Ishikawa T. Tanaka Y. Maeda M. Imamura S. Uchiyama T. J. Exp. Med. 1996; 183: 2185-2195Crossref PubMed Scopus (270) Google Scholar) OX40 signaling with the second a system of OX40-transfected HSB-2 cells and human cells. The of HSB-2-OX40 cells from cells was and the of cells in the cells was to be by using We also the of cells with the in of the to the from the Although the signals triggered by gp34 by gp34, the experiments with gp34 the The studies with OX40 deletion mutants demonstrated that the cytoplasmic portion of OX40 consisting of the amino acid sequence 256–263 (GGSFRTPI) was required for association with TRAFs and NF-κB activation. is that the for protein is in this portion of amino acid U. Dürkop H. Schnittger S. Ringeling J. Eitelbach F. Hummel M. Fonatsch C. Stein H. Eur. J. Immunol. 1994; 24: 677-683Crossref PubMed Scopus (117) Google Scholar). In addition, studies showed the induction of by OX40 for which the portion was required not we that this portion of amino acid a of the that signal OX40 studies demonstrated that several members of TRAFs with the members of the TNF-R family and initiate signal For example, is associated with TRAF2, or TRAF3 (23Mosialos G. Birkenbach M. Yalamanchili R. VanArsdale T. Ware C. Kief E. Cell. 1995; 80: 389-399Abstract Full Text PDF PubMed Scopus (906) Google Scholar, 24Rothe M. Pan M.-G. Henzel W.J. Ayres T.M. Goeddel D.V. Cell. 1995; 83: 1243-1252Abstract Full Text PDF PubMed Scopus (1057) Google Scholar, 25Rothe M. Wong S.C. Henzel W.J. Goeddel D.V. Cell. 1994; 78: 681-692Abstract Full Text PDF PubMed Scopus (933) Google Scholar, 26Rothe M. Sarma V. Dixit V.M. Goeddel D.V. Science. 1995; 269: 1424-1427Crossref PubMed Scopus (980) Google Scholar). TNF-R1 is associated with TRAF2 H. Shu H.-B. Pan M.-G. Goeddel D.V. Cell. 1996; 84: 299-308Abstract Full Text Full Text PDF PubMed Scopus (1738) Google Scholar). is associated with TRAF2 M. Sarma V. Dixit V.M. Goeddel D.V. Science. 1995; 269: 1424-1427Crossref PubMed Scopus (980) Google Scholar, 27Cheng G. Baltimore D. Genes Dev. 1996; 10: 963-973Crossref PubMed Scopus (265) Google Scholar) or TRAF3 (23Mosialos G. Birkenbach M. Yalamanchili R. VanArsdale T. Ware C. Kief E. 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In some of these in the and have been described as Mol. Cell. Biol. 1997; PubMed Google Scholar, J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar, Y. S. Res. 1997; PubMed Scopus Google Scholar). the motif that with and TRAF2, but not with TRAF3, is not in OX40, the motif that with TRAF2 and is and OX40 the OX40 deletion HSB-2-OX40 cells to intracellular signaling leading to NF-κB activation ligand stimulation and was to with TRAFs in further to amino acid are for the association with TRAFs and NF-κB activation. indicate that TRAFs are to the receptor by the with the receptor-associated such as H. Shu H.-B. Pan M.-G. Goeddel D.V. Cell. 1996; 84: 299-308Abstract Full Text Full Text PDF PubMed Scopus (1738) Google Scholar) such as M. Pan M.-G. Henzel W.J. Ayres T.M. Goeddel D.V. Cell. 1995; 83: 1243-1252Abstract Full Text PDF PubMed Scopus (1057) Google Scholar) and Lee Y. J. Exp. Med. 1997; PubMed Scopus Google Scholar). that the binding and the binding affinity between TRAFs and the receptors are by such In of the members of the TNF-R TRAF2 TRAF5 are responsible for the activation of the of Mol. Cell. Biol. 1997; PubMed Google Scholar), TRAF3, or TRAF4 C. C. C. P. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar) has not been We demonstrated that both TRAF2 and TRAF5 mediated NF-κB activation in OX40 TRAF3 on NF-κB activation as previously reported in signaling Mol. Cell. Biol. 1997; PubMed Google Scholar). studies be to the role of TRAF3 in OX40 NF-κB has a to the of the signaling in the several members of the TNF-R family. studies of the after NF-κB activation as well as signaling pathways be of the to be to a of the diverse cellular responses triggered by the stimulation of the members of the TNF-R family. several reported that the activation of NF-κB A.A. Baltimore D. Science. 1996; PubMed Scopus Google Scholar, D.J. T. Green Science. 1996; PubMed Scopus Google Scholar, Science. 1996; PubMed Scopus Google Scholar), which to the role of the OX40/gp34 system in vivo. The of the OX40/gp34 system the family is its ability to adhesion between activated or HTLV-I-transformed T cells and endothelial cells. that the OX40 signaling in T cells is triggered by the interaction with endothelial cells of the activated T cells OX40 are NF-κB activation in T cells to from which results in the and of the or in the of T cell of cells in the We are to K. for and and W. C. for the and We also K. for and