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

Abstract

We investigated the intracellular signaling of OX40, a member of the tumor necrosis factor receptor family. Activation of NF-B 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 glutathione S-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-B activation. The introduction of the dominant negative mutants of TRAF2 and TRAF5 into HSB-2-OX40 cells suppressed NF-B 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-B activation. These results indicate that the NF-B 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 ϩ T cells and some human T cell leukemia virus type I (HTLV-I) 1 -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 (1). Molecular cloning of its cDNA (1-4) revealed that OX40 is a member of the nerve growth factor receptor/tumor necrosis factor receptor (NGF-R/TNF-R) superfamily which is now known to include low affinity nerve growth factor receptor (p75 NGF-R), tumor necrosis factor receptors (p50/55 TNF-R1 and p75/80 TNF-R2), lymphotoxin-␤ receptor, Fas antigen (CD95/APO-1), CD40, CD30, CD27, and 4-1 BB (5, 6). 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 p40 tax of HTLV-I (7-9). As expected, the deduced amino acid sequence of gp34 revealed that it is a member of the TNF family. Gp34 has been reported to be expressed on some HTLV-I-infected cell lines such as Hut 102 and MT-2 (10), human umbilical vein endothelial cells (11), and stimulated B lymphoblastoid cell line MSAB (12).Since its first description, OX40 has been known to transmit costimulatory signals to T cells. Recent studies w...

Modulation of life and death by the TNF receptor superfamily

“…The 62 kDa TRAF-3 protein, like TRAF-2, is expressed in virtually all tissues examined to date. Despite the fact that receptor-protein interaction occurs via a CD40 sub-domain which shares a low degree of homology with regions within TNF-RI, Fas, and the p75 subunit of NGFR, an overall a nity for the 62 amino acid intracellular region of CD40 presumably accounts for a more speci®c involvement in CD40L (Cheng et al, 1995;Hu et al, 1995;Mosialos et al, 1995;Rothe et al, 1995;Sato et al, 1995), and possibly LTb (Mosialos et al, 1995;Force et al, 1997) and OX-40 ligand-induced signal transduction pathways (Arch and Thompson, 1998;Kawamata et al, 1998).…”

Cutting Edge: Molecular Mechanisms of Synergy Between CD40 and the B Cell Antigen Receptor: Role for TNF Receptor-Associated Factor 2 in Receptor Interaction

“…Therefore, TRAF3 binding in the absence of TRAF2 may play a negative role in the synergy between BCR and CD40. Our prior studies and others have suggested a negative role for TRAF3 in signaling by CD40 (25), OX-40 (32,33), and CD27 (34). Thus, TRAF2 may inhibit TRAF3 or other molecule(s) from exerting a negative effect in synergy.…”

TNF Receptor-Associated Factor (TRAF) Signaling Network in CD4⁺ T-Lymphocytes

“…TRAF5 is recruited to CD27 (Akiba et al 1998), CD30 (Aizawa et al 1997), GITR (Esparza et al 2006), HVEM (Hsu et al 1997;Marsters et al 1997), and OX40 (Kawamata et al 1998). Traf5 −/− CD4 + T cells have impaired GITR cosignaling and defective GITR-mediated canonical NF-κB, p38, and ERK activation (Esparza et al 2006).…”

“…TRAF2 is recruited to 4-1BB (Arch and Thompson 1998;Jang et al 1998;Saoulli et al 1998), CD27 (Akiba et al 1998;Gravestein et al 1998;Yamamoto et al 1998), CD30 (Gedrich et al 1996;Lee et al 1996;Harlin et al 2002), DR3 (Chinnaiyan et al 1996;Pobezinskaya et al 2011), GITR (Kwon et al 1999;Esparza and Arch 2005), HVEM (Hsu et al 1997;Marsters et al 1997;Cheung et al 2009), OX40 (Arch and Thompson 1998;Kawamata et al 1998), and TNFR2 (Rothe et al 1994).…”

“…TRAF1 is recruited to 4-1BB (Arch and Thompson 1998;Jang et al 1998;Saoulli et al 1998), CD30 (Gedrich et al 1996;Lee et al 1996), GITR (Kwon et al 1999), HVEM (Marsters et al 1997), OX40 (Kawamata et al 1998), and TNFR2 (Rothe et al 1994). …”