TNF receptor-associated factor (TRAF) 3 is an intracellular protein that belongs to the TRAF family of molecules. The characteristic feature of TRAFs is a domain structure that includes (except TRAF1) Zn-binding RING and Finger domains, coiled-coil domains (TRAF-N) that promote multimer formation, and (except TRAF7) a TRAF-C domain important for interaction with cellular receptors and regulation of signaling pathways (Xie 2013). TRAF3 has many cell type- and context-specific roles, primarily as an adaptor protein with a large variety of binding partners.
Early attempts to characterize TRAF3 in vivo functions were hindered by the lack of useful animal models, as global deletion of TRAF3 in mice leads to hypoglycemia, leukopenia, and early postnatal lethality (Xu et al. 1996). Alternative models to study TRAF molecules relied on exogenous overexpression in epithelial and fibroblast cell lines, with NF-κB activation as the principal measurement. In this setting, TRAF2 and TRAF6 promote robust activation of canonical NF-κB, but TRAF3 does not. First identified by its association with the CD40 receptor, TRAF3 was initially designated as CD40-binding protein. CD40 belongs to the TNF receptor superfamily (TNFR-SF) that also includes BAFF receptor (BAFF-R). Subsequent studies revealed TRAF3 as a negative regulator of signaling through CD40 and BAFF-R (Hildebrand et al. 2011).
In Vivo Biological Roles of TRAF3
Technological advances allowed conditional deletion of Traf3 in mice, with Cre-loxP recombination permitting investigation of cell type-specific roles of TRAF3. The study of mice with B-cell-specific deletion of TRAF3 (Traf3flox/flox x CD19-Cre) revealed that TRAF3 is an important negative regulator of B-cell survival (Gardam et al. 2008; Xie et al. 2011b). These mice exhibit a profound increase in B-cell numbers in secondary lymphoid organs and B-cell infiltration into the parenchyma. This phenotype is explained by increased B-cell survival in vitro and in vivo without any changes in B-cell proliferation. Increase in B-cell survival is accompanied by elevated serum immunoglobulins, including autoantibodies, and immune complex deposition in the kidney (Xie et al. 2007). TRAF3 deficiency also leads to NIK-dependent increase in glucose utilization, and glucose is required for the enhanced survival phenotype (Mambetsariev et al. 2016b). Aged mice lacking TRAF3 in B cells also have higher propensity for developing B-cell malignancies, possibly because their survival advantage allows accumulation of additional mutations (Moore et al. 2012). In further support of TRAF3 as a tumor suppressor in B cells, TRAF3-inactivating mutations have been reported in a number of human B-cell malignancies (Moore et al. 2015).
T-cell-specific deletion of TRAF3 in Traf3flox/flox mice was achieved by breeding them to CD4-Cre mice. Unlike B cells, T cells lacking TRAF3 have no increase in survival, and numbers of mature CD4 and CD8 T cells in vivo are normal. However, T-cell responses to infection and immunization are severely blunted in the absence of TRAF3. Consistent with these functional defects, mechanistic studies showed TRAF3 association with the T-cell receptor (TCR) complex and defective early TCR signaling in TRAF3-deficient T cells (Xie et al. 2011a). In addition to inhibiting TCR signaling and T-cell responsiveness, loss of TRAF3 in T cells has additional important biological consequences. Development of central memory CD8 T cells and invariant natural killer (iNK) T cells is markedly inhibited in the absence of TRAF3. In contrast, T-cell TRAF3 deficiency results in increased regulatory CD4 T-cell (Treg) development in the thymus without changes in Treg survival or proliferation (Yi et al. 2015). These findings establish multifaceted roles for TRAF3 in T-cell development and function that are specific to this cell type.
Similarly to T cells, survival of myeloid cells is normal in the absence of TRAF3, but their function is also affected in a context-dependent manner. In its pro-inflammatory role, TRAF3 is required for type I interferon (IFN) induction in response to innate Toll-like receptor (TLR) or retinoic acid-inducible gene 1 (RIG-I) stimulation in macrophages and dendritic cells. Inflammasome activation and IL-1β release in response to RNA viruses are inhibited in the absence of TRAF3. In contrast, TRAF3 inhibits MyD88-dependent induction of pro-inflammatory cytokines in response to TLR stimulation. Possibly due to associated chronic inflammation, mice lacking TRAF3 in myeloid cells develop tumors of multiple precursor cell origins. In the context of obesity, myeloid TRAF3 has a detrimental role by promoting inflammation in the liver and insulin resistance. These contrasting roles of TRAF3 in myeloid cells show that its regulatory influence is highly context dependent for distinct receptors in the same cell type (Lalani et al. 2015).
Recently, a novel role for TRAF3 emerged in liver disease. Deletion of TRAF3 in hepatocytes of mice attenuates high-fat diet-induced steatosis, obesity, and type II diabetes, while its overexpression has the opposite effect. These findings suggest a potential role of TRAF3 in regulating function of nonimmune cells in a disease setting.
Study of TRAF3 in different cell types revealed many cell type-specific roles for this versatile protein. Mechanistic insight into how TRAF3 regulates cellular function is important for our understanding of disease pathophysiology and may open novel and exciting avenues for treatment. TRAF3 also exhibits receptor-specific roles, which are discussed below.
TRAF3 as a Regulator of TNFR-SF Receptors
TRAF3 is an important adaptor protein regulating intracellular signaling through cell surface receptors. It was first identified via its association with the cytoplasmic domain of TNFR-SF member CD40. CD40 binds to TRAF3 via its canonical TRAF-binding motif (PVQETL). BAFF-R binds TRAF3 via a similar PVPAT motif. TRAF3 binds to many additional members of this receptor family including TACI, BCMA, LT-βR, CD27, TNFR2, CD30, 4-1BB, OX-40, and GITR (Hildebrand et al. 2011; Yi et al. 2015). TRAF3 acts as a regulator of signaling through TNFR-SF receptors using multiple mechanisms.
In resting cells, cytoplasmic TRAF3 associates with NF-κB-inducing kinase (NIK) and recruits TRAF2 and cIAP to form an ubiquitin ligase complex, leading to NIK K48-linked poly-ubiquitination and degradation via the proteasome (Fig. 1). When the ligand binds a receptor such as BAFF-R or CD40, the NIK targeting complex localizes to the receptor, leading to K63-linked ubiquitination of TRAF2 and cIAP. These events switch targeting of K48 ubiquitination from NIK to TRAF2 and TRAF3. When these TRAFs are subsequently degraded, NIK is allowed to accumulate and activate downstream signaling pathways. The major such pathway studied to date is noncanonical NF-κB2 activation, which occurs when NIK phosphorylates the kinase IκB kinase α (IKKα), which in turn phosphorylates the transcription factor precursor p100. Subsequent partial proteasomal cleavage of p100 generates p52, which forms a transcription factor complex with RelB that translocates to the nucleus to promote many context-specific outcomes (Hildebrand et al. 2011). This has been most studied in B cells, where NIK and NF-κB2 are important for development, homeostasis, and survival (Brightbill et al. 2015). TNFR-SF receptors BAFF-R and CD40 transduce pro-survival signals in B cells that include NF-κB2. Consistent with this, hyper-surviving TRAF3-deficient B cells have constitutive NF-κB2 activation (Xie et al. 2007). However, TRAF3-deficient T cells, macrophages, and dendritic cells also exhibit constitutive NF-κB2 activation, but their survival is not altered (Xie et al. 2011a). In T cells, NIK promotes T-cell effector function, in response to TNFR-SF member OX40 (Murray et al. 2011). The functional significance of TRAF3 degradation and NF-κB2 activation through different TNFR-SF receptors in various cell types remains an intriguing avenue of future research.
TRAF3 also regulates additional signaling pathways activated by TNFR-SF receptors. TRAF3 inhibits CD40-mediated activation of the mitogen-activated protein kinases (MAPK), c-jun kinase (JNK), and p38 (Xie et al. 2004). However, activation of these pathways through the BAFF-R is not affected by TRAF3, which may be due to differences in TRAF-binding motifs of the two receptors (Hildebrand et al. 2011). TRAF3 is recruited to the CD40 receptor and undergoes K63-linked poly-ubiquitination within minutes of CD40 stimulation, an event mediated by the ubiquitin ligase NEDD4. This ubiquitination is required for CD40-mediated activation of Akt kinase and downstream isotype switching in B cells (Fang et al. 2014). This novel mechanism suggests that TRAF3 can also positively regulate signaling through TNFR-SF receptors.
TRAF3 also associates with a viral mimic of CD40. Latent membrane protein 1 (LMP1) is an oncoprotein encoded by the ubiquitous herpesvirus Epstein-Barr virus (EBV). LMP1 promotes abnormal survival and malignant transformation of B cells. However, in sharp contrast to CD40, LMP1 binding of TRAF3 does not promote its degradation, and LMP1 requires TRAF3 for activation of downstream signaling pathways (Graham et al. 2010). LMP1 also binds TRAF3 more tightly than CD40 and has a different TRAF-binding motif of PQQATD. One potential mechanism by which TRAF3 mediates LMP1 signaling is by recruiting TRAF5, which is largely dispensable for CD40 but critical for LMP1 signaling (Hildebrand et al., 2011).
TRAF3 as a Regulator of Cytokine Receptors
Interleukin-2 (IL-2) is a versatile cytokine important for immune cell function, including CD4- and CD8-positive T-cell responses. Tregs also require IL-2 for their differentiation and homeostasis, because IL-2 drives transition of Treg precursor cells to mature Tregs (Boyman and Sprent 2012). Interestingly, mice lacking TRAF3 in T cells exhibit an increase in thymic-derived Tregs. It was subsequently discovered that TRAF3-deficient T cells have enhanced IL-2-induced STAT5 phosphorylation without any increase in IL-2 receptor expression. TRAF3 associates with JAKs 1 and 3 after stimulation with IL-2 and recruits T-cell protein tyrosine phosphatase (TCPTP) to the IL-2 receptor complex. TCPTP dephosphorylates JAK/STAT proteins, inhibiting downstream signaling (Fig. 1). Increased signaling through IL-2 enhances the transition to mature Tregs in the thymus. Thus, T-cell TRAF3 regulates the size of the Treg population, by restraining Treg precursor sensitivity to IL-2 (Yi et al. 2015).
IL-6 was first identified as a T-cell released factor that promotes B-cell differentiation to antibody-producing plasma cells. It has been implicated in numerous inflammatory diseases and B-cell malignancies, including multiple myeloma. Similarly to IL-2, it signals through a receptor complex that induces JAK/STAT phosphorylation (Calabrese and Rose-John 2014). B cells deficient in TRAF3 also have altered responsiveness to IL-6, leading to increased downstream phosphorylation of JAK1 and STAT3. In a mechanism analogous to T-cell IL-2 signaling, TRAF3 recruits the protein tyrosine phosphatase, non-receptor type 22 (PTPN22) to inhibit signaling through the IL-6 receptor (Fig. 1). Interestingly, B-cell-specific TRAF3 deletion results in an IL-6-dependent increase in plasma cells, as does PTPN22 deficiency. Thus, TRAF3, by recruiting PTPN22, restrains IL-6 sensitivity and B-cell maturation to plasma cells (Lin et al. 2015). The importance of TRAF3-mediated regulation of IL-6 receptor in the context of human disease is yet to be explored, but it should be noted that a human PTPN22 mutation, R620W, fails to bind TRAF3 and is associated with increased propensity to several autoimmune diseases (Wang et al. 2013).
IL-17 is secreted mainly by Th17 cells to promote inflammation. It has been implicated in multiple autoimmune diseases, including multiple sclerosis. Unlike IL-2 and IL-6, IL-17 signals through a receptor that does not activate JAK/STAT molecules. IL-17R recruits the scaffolding protein Act1 and TRAFs 6, 2, and 5 to promote downstream signaling. TRAF3 negatively regulates IL-17R signaling by inhibiting receptor recruitment of Act1 and TRAF6. TRAF3 inhibits IL-17-mediated cytokine production and protects mice from experimental autoimmune encephalomyelitis (Gaffen et al. 2014). TRAF3 regulates signaling through multiple cytokine receptors directly, but there is evidence that TRAF3-mediated regulation of cytokine responsiveness may also be more nuanced. T-cell TRAF3 deficiency results in a tenfold decrease in splenic iNKT cells. IL-15 is an important cytokine that promotes iNKT cell development. In contrast to mechanisms described above, IL-15 receptor signaling is not regulated directly by TRAF3. Blunted TCR activation in the absence of TRAF3 leads to suboptimal induction of the transcription factor Tbet in early stages of iNKT development. The IL-15 receptor CD122 is a transcriptional target of Tbet, and its expression is reduced in the absence of TRAF3 as iNKT cells mature. With blunted responsiveness to IL-15, iNKT cells are not generated. Hence, TRAF3 is required for iNKT cells to develop by positively regulating IL-15 receptor expression (Yi et al. 2015).
TRAF3 as a Regulator of Innate Immune Receptors
Toll-like receptors (TLRs) are germline-encoded molecules that recognize pathogen-associated molecular patterns such as LPS and bacterial/viral nucleic acids. Typically, TLRs signal through two adaptor proteins, MyD88 and TIR-domain-containing adapter-inducing interferon-β (TRIF). MyD88 promotes activation of multiple transcription factors, including canonical NF-κB1, to induce expression of pro-inflammatory cytokines. TRIF recruits two kinases, TANK-binding kinase 1 (TBK1) and IKK that phosphorylate IFN regulatory factors (IRFs) to drive Type I IFN production (Kawai and Akira 2011). TRAF3 plays multiple context specific roles as both a positive and negative regulator of TLR signaling.
TRAF3 deficiency in dendritic cells and macrophages inhibits Type I IFN production in response to TLR stimulation. Upon receptor engagement, TRAF3 is recruited to the receptor and associates with TRIF where it undergoes K63-linked ubiquitination (Fig. 1). TRAF3 couples TLR signaling to the kinases TBK1 and IKK, which phosphorylate IRF3 and IRF7. Phosphorylated IRFs translocate to the nucleus and induce expression of Type I IFN. Similarly, other innate receptors such as RIG-I-like receptor also require TRAF3 for activation of downstream effector kinases needed for Type I IFN induction. TRAF3 is also required for MyD88-dependent type I IFN induction, assembling a complex with MyD88, IL-1 receptor-associated kinase (IRAK), and IKK molecules at the receptor to promote phosphorylation and activation of IRF7. Independent of proximal receptor signaling, NF-κB2 activation in the absence of TRAF3 may also impair Type I IFN expression through epigenetic mechanisms (Lalani et al. 2015; Xie 2013). Translational relevance of TRAF3-mediated regulation of Type I IFN manifested itself in a patient with a rare autosomal dominant mutation in a zinc-finger domain of TRAF3. The patient had a pediatric history of herpes simplex viral encephalitis. The R118W TRAF3 mutation expressed by the patient results in less protein production, without a change in mRNA. This leads to impaired TLR-mediated induction of type I IFN, highlighting the role of TRAF3 in the antiviral response (Perez de Diego et al. 2010). Interestingly, the impact of the mutant TRAF3 deficiency upon the T-cell responses of this patient was not addressed.
In addition to its role in promoting Type I IFN production in myeloid cells, TRAF3 is also an important anti-inflammatory factor in innate immunity. TRAF3 deficiency renders dendritic cells, macrophages, and B cells hyperresponsive to TLR stimulation. Myeloid-specific TRAF3 deletion is sufficient to increase mouse serum IL-6 and IL-12 while decreasing IL-10, leading to a pro-inflammatory homeostatic shift. This manifests itself in chronic inflammation and generation of tumors from multiple cell origins suggesting impaired tumor surveillance (Lalani et al. 2015). Interestingly, in B cells, TRAF3 deficiency not only promotes NF-κB1 activation and cytokine induction but also leads to enhanced Ig isotype switching and type I IFN activation in response to TLR stimuli (Xie et al. 2011b). Though the mechanism of this TLR hyperresponsiveness is not fully understood, TLR-mediated MAPK activation is unaltered, and this phenomenon is independent of NF-κB2 (Lalani et al. 2015; Xie et al. 2011b). One proposed mechanism is that TRAF3 constitutively binds transcription factors c-Rel and IRF5 which are downstream of TLR signaling. Similarly to NIK, TRAF3 recruits TRAF2 and c-IAP1/2 to these molecules and promotes their ubiquitination and degradation. In the absence of TRAF3, higher protein levels of IRF5 and c-Rel allow for a greater induction of pro-inflammatory cytokines (Jin et al. 2015). These findings suggest that TRAF3 may serve as a rheostat in regulating cell responsiveness to TLR stimulation.
TRAF3 as a Regulator of T-Cell Receptor Signaling
The T-cell antigen receptor (TCR) recognizes antigen presented by MHC molecules on antigen-presenting cells. Stimulation through the TCR engages a complex signaling cascade that leads to T-cell clonal expansion, proliferation, and effector function (Brownlie and Zamoyska 2013). TRAF3-deficient T cells mount impaired responses to primary infection and show blunted proliferation and cytokine production in response to TCR stimulation. iNKT cell development is also affected as reviewed above. In proximal TCR signaling, there is a decrease in phosphorylation of Zap70, linker for activation of T cells (LAT), phospholipase C (PLC)-γ, and extracellular signal-regulated kinase (ERK), key signaling molecules downstream of the TCR. Immunoprecipitation of the TCR complex shows TRAF3 association that requires stimulation through both CD3 and CD28 (Fig. 1) (Xie et al. 2011a).
The mechanism by which TRAF3 enhances TCR signaling is not well understood. Recent evidence shows that T-cell TRAF3 binds to two proteins that negatively regulate TCR activation, C-terminal Src kinase (Csk) and PTPN22. Following TCR activation, TRAF3 promotes Csk and PTPN22 shuttling from the membrane to the cytoplasm where the inhibitors cannot access their targets (Wallis et al. 2015). The role of TRAF3 in regulating localization of other proteins involved in TCR signaling is part of ongoing work. Mechanistic characterization of TRAF3 as a regulator of TCR signaling will have important implications for T-cell function in human disease.
TRAF3 as a Nuclear Protein
Though TRAF3 has been extensively studied as a cytoplasmic protein, histological analysis of TRAF3 expression shortly after its discovery showed nuclear localization in B cells and neurons (Krajewski et al. 1997). In human endothelial cells, TRAF3 localizes to the nucleus and inhibits CD40-mediated activation of transcription factor AP-1 (Urbich et al. 2001). More recent work revealed that in B cells, TRAF3 is a resident nuclear protein and has a functional nuclear localization signal in its TRAF-C domain. Once in the nucleus, it associates with the transcription factor cAMP response element-binding protein (CREB) and inhibits its stability through a mechanism analogous to TRAF3 NIK regulation. Loss of TRAF3 in B cells results in CREB protein elevation and induction of the pro-survival Bcl-2 family protein myeloid cell leukemia 1 (Mcl-1). CREB inhibition attenuates survival of TRAF3-deficient B cells (Mambetsariev et al. 2016a).
In addition to its role as an adaptor protein, TRAF3 contains Zn finger domains that give it the potential to associate with DNA and regulate transcription. In neuronal and human B cell lines, TRAF3 is found in chromatin extracts. In Neuro2a cells, stimulation through CD40 promotes TRAF3 accumulation in the nucleus and binding to the promoter of the gene expressing intercellular adhesion molecule 1 (Icam-1), complexed with TRAF2 and RNA polymerase (El Hokayem et al. 2017). These findings suggest that TRAF3, together with other TRAFs, may regulate transcription of genes directly. Emerging evidence for nuclear TRAF3 further expands the regulatory potential of this versatile protein. Context- and cell-specific roles of TRAF3 in the nucleus provide for an exciting avenue of future research.
TRAF3 is a versatile adaptor protein with many cell type- and context-specific roles. It regulates signaling pathways utilized by multiple surface receptors. These receptors include members of the TNFR-SF, cytokine receptors, innate immune receptors, and the T-cell antigen receptor. TRAF3-mediated signaling events have important implications for many diseases involving immune and nonimmune cells. Mechanistic understanding of the role of TRAF3 in different cell types has the potential to uncover novel therapeutic targets. TRAF3 is a negative regulator of noncanonical NF-κB2 activation, by promoting poly-ubiquitination and degradation of NIK. It also regulates stability of the transcription factors CREB, c-Rel, and IRF5 via an analogous mechanism. In the context of cytokine receptors, TRAF3 recruits phosphatases that act as negative regulators of downstream signaling. In contrast, TRAF3 is required for optimal activation of the TCR. Toll-like receptor signaling is both inhibited and potentiated by TRAF3, depending upon context. Though our understanding of TRAF3 function has expanded substantially in recent years, important questions remain. TRAF3 is involved in multiple signaling pathways, but how these signals are integrated is not well characterized. TRAF3 also localizes to multiple cellular compartments, but how its trafficking is regulated is not known. TRAF3 localization to the nucleus and association with DNA suggests that it has potential to regulate gene expression directly and identifying those regulatory targets is an exciting avenue for future research. Identifying novel pathways involving TRAF3 will uncover valuable insights into mechanisms of cell function and disease pathogenesis.
- El Hokayem J, Brittain GC 4th, Nawaz Z, Bethea JR. Tumor Necrosis Factor Receptor Associated Factors (TRAFs) 2 and 3 form a transcriptional complex with Phosho-RNA polymerase II and p65 in CD40 ligand activated Neuro2a cells. Mol Neurobiol. 2017:54(2):1301–13. doi:10.1007/s12035-016-9742-4.PubMedCrossRefPubMedCentralGoogle Scholar
- Lalani AI, Luo C, Han Y, Xie P. TRAF3: a novel tumor suppressor gene in macrophages. Macrophage (Houst). 2015;2:e1009.Google Scholar
- Perez de Diego R, Sancho-Shimizu V, Lorenzo L, Puel A, Plancoulaine S, Picard C, Herman M, Cardon A, Durandy A, Bustamante J, et al. Human TRAF3 adaptor molecule deficiency leads to impaired TLR3 response and susceptibility to herpes simplex encephalitis. Immunity. 2010;33:400–11.PubMedCrossRefGoogle Scholar
- Wallis A, Yi ZA, Bishop G. TRAF3 regulates CSK and PTPN22 to enhance T cell receptor signaling. J Immunol. 2015;194.Google Scholar