Characterization of the roles of TNF receptor-associated factor 6 in CD40-mediated B lymphocyte effector functions

Signaling through CD40 in B cells leads to B cell proliferation, Ig and IL-6 secretion, isotype switching, and up-regulation of surface molecules. TNF receptor-associated factor (TRAF) proteins associate with the cytoplasmic tail of CD40 and act as adapter molecules. Of the six TRAFs identified to date, TRAFs 2, 3, 5, and 6 are reported to associate directly with the cytoplasmic tail of CD40, but previous studies have principally examined transient overexpression of TRAF6 in cells that do not normally express CD40. Thus, we examined the role of TRAF6 in CD40-mediated B lymphocyte effector functions using two approaches. We produced and stably expressed in mouse B cell lines a human CD40 molecule with two cytoplasmic domain point mutations (hCD40EEAA); this mutant fails to bind TRAF6, while showing normal association with TRAFs 2 and 3. We also inducibly expressed in B cells a transfected "dominant-negative" TRAF6 molecule which contains only the C-terminal TRAF-binding domain of TRAF6. Using both molecules, we found that TRAF6 association with CD40 is important for CD40-induced IL-6 and Ig secretion, and that TRAF6 mediates its effects on CD40-stimulated Ig secretion principally through its effects on IL-6 production by the B cell. TRAF6 association with CD40 was also found to be important for B7-1 up-regulation, but not for up-regulation of other surface molecules. Interestingly, however, although we could show TRAF6-dependent CD40-mediated activation of NF-kappaB in 293 kidney epithelial cells, no such effect was seen in B cells, suggesting that TRAF6 has cell-type-specific functions.     

Tumor necrosis factor receptor-associated factor (TRAF) 1 regulates CD40-induced TRAF2-mediated NF-kappaB activation

To investigate CD40 signaling complex formation in living cells, we used green fluorescent protein (GFP)-tagged CD40 signaling intermediates and confocal life imaging. The majority of cytoplasmic TRAF2-GFP and, to a lesser extent, TRAF3-GFP, but not TRAF1-GFP or TRAF4-GFP, translocated into CD40 signaling complexes within a few minutes after CD40 triggering with the CD40 ligand. The inhibitor of apoptosis proteins cIAP1 and cIAP2 were also recruited by TRAF2 to sites of CD40 signaling. An excess of TRAF2 allowed recruitment of TRAF1-GFP to sites of CD40 signaling, whereas an excess of TRAF1 abrogated the interaction of TRAF2 and CD40. Overexpression of TRAF1, however, had no effect on the interaction of TRADD and TRAF2, known to be important for tumor necrosis factor receptor 1 (TNF-R1)-mediated NF-kappaB activation. Accordingly, TRAF1 inhibited CD40-dependent but not TNF-R1-dependent NF-kappaB activation. Moreover, down-regulation of TRAF1 with small interfering RNAs enhanced CD40/CD40 ligand-induced NF-kappaB activation but showed no effect on TNF signaling. Because of the trimeric organization of TRAF proteins, we propose that the stoichiometry of TRAF1-TRAF2 heteromeric complexes ((TRAF2)2-TRAF1 versus TRAF2-(TRAF1)2) determines their capability to mediate CD40 signaling but has no major effect on TNF signaling.     

Differential requirements for tumor necrosis factor receptor-associated factor family proteins in CD40-mediated induction of NF-kappaB and Jun N-terminal kinase activation.

CD40 is a member of the tumor necrosis factor receptor family that mediates a number of important signaling events in B-lymphocytes and some other types of cells through interaction of its cytoplasmic (ct) domain with tumor necrosis factor receptor-associated factor (TRAF) proteins. Alanine substitution and truncation mutants of the human CD40ct domain were generated, revealing residues critical for binding TRAF2, TRAF3, or both of these proteins. In contrast to TRAF2 and TRAF3, direct binding of TRAF1, TRAF4, TRAF5, or TRAF6 to CD40 was not detected. However, TRAF5 could be recruited to wild-type CD40 in a TRAF3-dependent manner but not to a CD40 mutant (Q263A) that selectively fails to bind TRAF3. CD40 mutants with impaired binding to TRAF2, TRAF3, or both of these proteins completely retained the ability to activate NF-kappaB and Jun N-terminal kinase (JNK), implying that CD40 can stimulate TRAF2- and TRAF3-independent pathways for NF-kappaB and JNK activation. A carboxyl-truncation mutant of CD40 lacking the last 32 amino acids required for TRAF2 and TRAF3 binding, CD40(Delta32), mediated NF-kappaB induction through a mechanism that was suppressible by co-expression of TRAF6(DeltaN), a dominant-negative version of TRAF6, but not by TRAF2(DeltaN), implying that while TRAF6 does not directly bind CD40, it can participate in CD40 signaling. In contrast, TRAF6(DeltaN) did not impair JNK activation by CD40(Delta32). Taken together, these findings reveal redundancy in the involvement of TRAF family proteins in CD40-mediated NF-kappaB induction and suggest that the membrane-proximal region of CD40 may stimulate the JNK pathway through a TRAF-independent mechanism.     

A novel mechanism of CD40-induced apoptosis of carcinoma cells involving TRAF3 and JNK/AP-1 activation

Membrane-presented CD40 agonists can induce apoptosis in carcinoma, but not normal homologous epithelial cells, whereas soluble agonists are growth inhibitory but not proapoptotic unless protein synthesis is blocked. Here we demonstrate that membrane-presented CD40 ligand (CD154) (mCD40L), but not soluble agonists, triggers cell death in malignant human urothelial cells via a direct mechanism involving rapid upregulation of TNFR-associated factor (TRAF)3 protein, without concomitant upregulation of TRAF3 mRNA, followed by activation of the c-Jun N-terminal kinase (JNK)/activator protein-1 (AP-1) pathway and induction of the caspase-9/caspase-3-associated intrinsic apoptotic machinery. TRAF3 knockdown abrogated JNK/AP-1 activation and prevented CD40-mediated apoptosis, whereas restoration of CD40 expression in CD40-negative carcinoma cells restored apoptotic susceptibility via the TRAF3/AP-1-dependent mechanism. In normal human urothelial cells, mCD40L did not trigger apoptosis, but induced rapid downregulation of TRAF2 and 3, thereby paralleling the situation in B-lymphocytes. Thus, TRAF3 stabilization, JNK activation and caspase-9 induction define a novel pathway of CD40-mediated apoptosis in carcinoma cells.     

Role of TNF receptor-associated factor 2 in the activation of IgM secretion by CD40 and CD120b

TNFR-associated factors (TRAFs) participate in the signaling of many TNFR family members, including CD40, CD120a (TNFR1), and CD120b (TNFR2). Previously, we found that a dominant-negative TRAF2 molecule inhibits CD40-mediated Ab secretion by the mouse B cell line CH12.LX. However, disruption of the TRAF2 binding site in the cytoplasmic domain of CD40 does not diminish the ability of CD40 to stimulate Ab secretion, nor is this mutation able to circumvent the inhibition of Ab secretion by dominant-negative TRAF2. Here we demonstrate that CD40-induced TNF stimulates IgM production through CD120b and that CD120b signaling is required for optimal CD40-induced IgM secretion. Furthermore, although both CD40 and CD120b can bind TRAF2, TRAF2-dependent CD40 signals cannot substitute for TRAF2-dependent CD120b signals in the activation of IgM secretion. Our results indicate a potentially important role for CD120b in the activation of IgM secretion and that TRAF2 is used by CD40 and CD120b in distinct ways.     

Tumor necrosis factor receptor-associated factor 2 (TRAF2)-deficient B lymphocytes reveal novel roles for TRAF2 in CD40 signaling

CD40 function is initiated by tumor necrosis factor (TNF) receptor-associated factor (TRAF) adapter proteins, which play important roles in signaling by numerous receptors. Characterizing roles of individual TRAFs has been hampered by limitations of available experimental models and the poor viability of most TRAF-deficient mice. Here, B cell lines made deficient in TRAF2 using a novel homologous recombination system reveal new roles for TRAF2. We demonstrate that TRAF2 participates in synergy between CD40 and B cell antigen receptor signals, and in CD40-mediated, TNF-dependent IgM production. We also find that TRAF2 participates in the degradation of TRAF3 associated with CD40 signaling, a role that may limit inhibitory actions of TRAF3. Finally, we show that TRAF2 and TRAF6 have overlapping functions in CD40-mediated NF-kappaB activation and CD80 up-regulation. These findings demonstrate previously unappreciated roles for TRAF2 in signaling by TNF receptor family members, using an approach that facilitates the analysis of genes critical to the viability of whole organisms.     

TRAF7 potentiates MEKK3-induced AP1 and CHOP activation and induces apoptosis

The tumor necrosis factor receptor-associated factor (TRAF) protein family members are critically involved in activation of NF-kappaB, JNK, and p38 activation triggered by tumor necrosis factor (TNF) receptor family members and toll/interleukin-1 receptor (TIR)-containing receptors. TRAF proteins (except for TRAF1) contain an N-terminal RING finger domain that is essential for their functions. In this report, we identified a protein designated as TRAF7, which contains a RING finger domain and a zinc finger domain that are mostly conserved with those of TRAFs. TRAF7 also contains seven WD40 repeats at its C terminus. TRAF7 specifically interacted with MEKK3 and potentiated MEKK3-mediated AP1 and CHOP activation. Depletion of TRAF7 by antisense RNA inhibited MEKK3-mediated AP1 and CHOP activation. Moreover, overexpression of TRAF7 induced caspase-dependent apoptosis. Domain mapping experiments indicated that TRAF7 potentiated MEKK3-mediated AP1 and CHOP activation and induced apoptosis through distinct domains. Our studies identified a novel TRAF family member that is involved in MEKK3 signaling and apoptosis.     

Molecular mechanisms of TNFR-associated factor 6 (TRAF6) utilization by the oncogenic viral mimic of CD40, latent membrane protein 1 (LMP1)

Latent membrane protein 1 (LMP1), encoded by Epstein-Barr virus, is required for EBV-mediated B cell transformation and plays a significant role in the development of posttransplant B cell lymphomas. LMP1 has also been implicated in exacerbation of autoimmune diseases such as systemic lupus erythematosus. LMP1 is a constitutively active functional mimic of the tumor necrosis factor receptor superfamily member CD40, utilizing tumor necrosis factor receptor-associated factor (TRAF) adaptor proteins to induce signaling. However, LMP1-mediated B cell activation is amplified and sustained compared with CD40. We have previously shown that LMP1 and CD40 use TRAFs 1, 2, 3, and 5 differently. TRAF6 is important for CD40 signaling, but the role of TRAF6 in LMP1 signaling in B cells is not clear. Although TRAF6 binds directly to CD40, TRAF6 interaction with LMP1 in B cells has not been characterized. Here we tested the hypothesis that TRAF6 is a critical regulator of LMP1 signaling in B cells, either as part of a receptor-associated complex and/or as a cytoplasmic adaptor protein. Using TRAF6-deficient B cells, we determined that TRAF6 was critical for LMP1-mediated B cell activation. Although CD40-mediated TRAF6-dependent signaling does not require the TRAF6 receptor-binding domain, we found that LMP1 signaling required the presence of this domain. Furthermore, TRAF6 was recruited to the LMP1 signaling complex via the TRAF1/2/3/5 binding site within the cytoplasmic domain of LMP1.     

CD40 is essential in the upregulation of TRAF proteins and NF-kappaB-dependent proinflammatory gene expression after arterial injury

Despite extensive investigations, restenosis, which is characterized primarily by neointima formation, remains an unsolved clinical problem after vascular interventions. A recent study has shown that CD40 signaling through TNF receptor associated factor 6 (TRAF6) plays a key role in neointima formation after carotid artery injury; however, underlying mechanisms are not clearly elucidated. Because neointima formation may vary significantly depending on the type of injury, we first assessed the effect of CD40 deficiency on neointima formation in 2 injury models, carotid artery ligation and femoral artery denudation injury. Compared with wild-type mice, CD40 deficiency significantly reduced neointima formation and lumen stenosis in two different models. Further, we investigated the mechanism by which CD40 signaling affects neointima formation after arterial injury. In wild-type mice, the expression levels of CD40, several TRAF proteins, including TRAF1, TRAF2, TRAF3, TRAF5, and TRAF6, as well as total NF-kB p65 and phospho-NF-kB p65, in the carotid artery were markedly upregulated within 3-7 days after carotid ligation. Deficiency of CD40 abolished the injury-induced upregulation of TRAFs including TRAF6 and NF-kB-p65 in the injured vessel wall. Further, CD40(-/-) mice showed a significant decrease in the recruitment of neutrophils (at 3, 7d) and macrophages (at 7, 21d) into injured artery; this effect was most likely attributed to inhibition of NF-kB activation and marked downregulation of NF-kB-related gene expression, including cytokines (TNFα, IL-1β, IL-6), chemokines (MCP-1), and adhesion molecules (ICAM-1, VCAM-1). Moreover, neutrophil recruitment in a model of thioglycollate-induced peritonitis is impaired in CD40-deficient mice. In vitro data revealed that CD40 deficiency blocked CD40L-induced NF-kB p65 nuclear translocation in leukocytes. Altogether, our data identified for the first time that CD40 is essential in the upregulation of TRAF6, NF-kB activation, and NF-kB-dependent proinflammatory genes in vivo. Our findings firmly established the role for CD40 in neointima formation in 2 distinct injury models.     

Roles of the Kinase TAK1 in TRAF6-Dependent Signaling by CD40 and Its Oncogenic Viral Mimic, LMP1

The Epstein-Barr virus (EBV)-encoded protein latent membrane protein 1 (LMP1) is essential for EBV-mediated B cell transformation and plays a critical role in the development of post-transplant B cell lymphomas. LMP1 also contributes to the exacerbation of autoimmune diseases such as systemic lupus erythematosus (SLE). LMP1 is a functional mimic of the tumor necrosis factor receptor (TNFR) superfamily member CD40, and relies on TNFR-associated factor (TRAF) adaptor proteins to mediate signaling. However, LMP1 activation signals to the B cell are amplified and sustained compared to CD40 signals. We previously demonstrated that LMP1 and CD40 use TRAF molecules differently. Although associating with CD40 and LMP1 via separate mechanisms, TRAF6 plays a significant role in signal transduction by both. It is unknown whether TRAF6 mediates CD40 versus LMP1 functions via distinct or shared pathways. In this study, we tested the hypothesis that TRAF6 uses the kinase TAK1 to trigger important signaling pathways following both CD40 and LMP1 stimulation. We determined that TAK1 was required for JNK activation and interleukin-6 (IL-6) production mediated by CD40 and LMP1, in both mouse and human B cells. Additionally, TRAF3 negatively regulated TRAF6-dependent, CD40-mediated TAK1 activation by limiting TRAF6 recruitment. This mode of regulation was not observed for LMP1 and may contribute to the dysregulation of LMP1 compared to CD40 signals.     

TRAF3 forms heterotrimers with TRAF2 and modulates its ability to mediate NF-{kappa}B activation

FRET experiments utilizing confocal microscopy or flow cytometry assessed homo- and heterotrimeric association of human tumor necrosis factor receptor-associated factors (TRAF) in living cells. Following transfection of HeLa cells with plasmids expressing CFP- or YFP-TRAF fusion proteins, constitutive homotypic association of TRAF2, -3, and -5 was observed, as well as heterotypic association of TRAF1-TRAF2 and TRAF3-TRAF5. A novel heterotypic association between TRAF2 and -3 was detected and confirmed by immunoprecipitation in Ramos B cells that constitutively express both TRAF2 and -3. Experiments employing deletion mutants of TRAF2 and TRAF3 revealed that this heterotypic interaction minimally involved the TRAF-C domain of TRAF3 as well as the TRAF-N domain and zinc fingers 4 and 5 of TRAF2. A novel flow cytometric FRET analysis utilizing a two-step approach to achieve linked FRET from CFP to YFP to HcRed established that TRAF2 and -3 constitutively form homo- and heterotrimers. The functional importance of TRAF2-TRAF3 heterotrimerization was demonstrated by the finding that TRAF3 inhibited spontaneous NF-kappaB, but not AP-1, activation induced by TRAF2. Ligation of CD40 on Ramos B cells by recombinant CD154 caused TRAF2 and TRAF3 to dissociate, whereas overexpression of TRAF3 in Ramos B cells inhibited CD154-induced TRAF2-mediated activation of NF-kappaB. Together, these results reveal a novel association between TRAF2 and TRAF3 that is mediated by unique portions of each protein and that specifically regulates activation of NF-kappaB, but not AP-1.     

A novel mechanism for TNFR-associated factor 6-dependent CD40 signaling

Members of the TNFR family play critical roles in the regulation of the immune system. One member of the family critical for efficient activation of T-dependent humoral immune responses is CD40, a cell surface protein expressed by B cells and other APC. The cytoplasmic domain of CD40 interacts with several members of the TNFR-associated factor (TRAF) family, which link CD40 to intracellular signaling pathways. TRAF2 and 6 appear to play particularly important roles in CD40 signaling. Previous studies suggest that the two molecules have certain overlapping roles in signaling, but that unique roles for each molecule also exist. To better define the roles of TRAF2 and TRAF6 in CD40 signaling, we used somatic cell gene targeting to generate TRAF-deficient mouse B cell lines. A20.2J cells deficient in TRAF6 exhibit marked defects in CD40-mediated JNK activation and the up-regulation of CD80. Our previous experiments with TRAF2-deficient B cell lines suggest that TRAF6 and TRAF2 may have redundant roles in CD40-mediated NF-kappaB activation. Consistent with this hypothesis, we found CD40-mediated activation of NF-kappaB intact in TRAF6-deficient cells and defective in cells lacking both TRAF2 and TRAF6. Interestingly, we found that TRAF6 mutants defective in CD40 binding were able to restore CD40-mediated JNK activation and CD80 up-regulation in TRAF6-deficient cells, indicating that TRAF6 may be able to contribute to certain CD40 signals without directly binding CD40.