Multiple mechanisms for TRAF3-mediated regulation of the T cell costimulatory receptor GITR.

Tumor necrosis factor receptor (TNFR)-associated factor 3 (TRAF3) plays context-specific roles in multiple receptor-mediated signaling pathways in different cell types. Mice lacking TRAF3 in T cells display defective T-cell-mediated immune responses to immunization and infection and demonstrate defective early signaling via the TCR complex. However, the role of TRAF3 in the function of GITR/TNFRSF18, an important costimulatory member of the TNFR superfamily, is unclear. Here we investigated the impact of T cell TRAF3 status on both GITR expression and activation of specific kinases in the GITR signaling pathway in T cells. Our results indicate that TRAF3 negatively regulates GITR functions in several ways. First, expression of GITR protein was elevated in TRAF3-deficient T cells, resulting from both transcriptional and posttranslational regulation that led to greater GITR transcript levels, as well as enhanced GITR protein stability. TRAF3 associated with T cell GITR in a manner dependent upon GITR ligation. TRAF3 also inhibited several events of the GITR mediated early signaling cascade, in a manner independent of recruitment of phosphatases, a mechanism by which TRAF3 inhibits signaling through several other cytokine receptors. These results add new information to our understanding of GITR signaling and function in T cells, which is relevant to the potential use of GITR to enhance immune therapies.

TRAF3 and NBR1 both influence the effect of the disease-causing CYLD(Arg936X) mutation on NF-κB activity.

Recently described Hungarian and Anglo-Saxon pedigrees that are affected by CYLD cutaneous syndrome (syn: Brooke-Spiegler syndrome (BSS)) carry the same disease-causing mutation (c.2806C>T, p.Arg936X) of the cylindromatosis (CYLD) gene but exhibit striking phenotypic differences. Using whole exome sequencing, missense genetic variants of the TRAF3 and NBR1 genes were identified in the affected family members of the Hungarian pedigree that are not present in the Anglo-Saxon pedigree. This suggested that the affected proteins (TRAF3 and NBR1) are putative phenotype-modifying factors. An in vitro experimental system was set up to clarify how wild type and mutant TRAF3 and NBR1 modify the effect of CYLD on the NF-κB signal transduction pathway. Our study revealed that the combined expression of mutant CYLD(Arg936X) with TRAF3 and NBR1 caused increased NF-κB activity, regardless of the presence or absence of mutations in TRAF3 and NBR1. We concluded that increased expression levels of these proteins further strengthen the effect of the CYLD(Arg936X) mutation on NF-κB activity in HEK293 cells and may explain the phenotype-modifying effect of these genes in CYLD cutaneous syndrome. These results raise the potential that detecting the levels of TRAF3 and NBR1 might help explaining phenotypic differences and prognosis of CCS.

SHP-1 suppresses the antiviral innate immune response by targeting TRAF3.

Type I interferons play a pivotal role in innate immune response to virus infection. The protein tyrosine phosphatase SHP-1 was reported to function as a negative regulator of inflammatory cytokine production by inhibiting activation of NF-κB and MAPKs during bacterial infection, however, the role of SHP-1 in regulating type I interferons remains unknown. Here, we demonstrated that knockout or knockdown of SHP-1 in macrophages promoted both HSV-1- and VSV-induced antiviral immune response. Conversely, overexpression of SHP-1 in L929 cells suppressed the HSV-1- and VSV-induced immune response; suppression was directly dependent on phosphatase activity. We identified a direct interaction between SHP-1 and TRAF3; the association between these two proteins resulted in diminished recruitment of CK1ε to TRAF3 and inhibited its K63-linked ubiquitination; SHP-1 inhibited K63-linked ubiquitination of TRAF3 by promoting dephosphorylation at Tyr116 and Tyr446. Taken together, our results identify SHP-1 as a negative regulator of antiviral immunity and suggest that SHP-1 may be a target for intervention in acute virus infection.

TRAF3 Modulation: Novel Mechanism for the Anti-inflammatory Effects of the Vitamin D Receptor Agonist Paricalcitol in Renal Disease.

BACKGROUND: CKD leads to vitamin D deficiency. Treatment with vitamin D receptor agonists (VDRAs) may have nephroprotective and anti-inflammatory actions, but their mechanisms of action are poorly understood. METHODS: Modulation of the noncanonical NF-κB2 pathway and its component TNF receptor-associated factor 3 (TRAF3) by the VDRA paricalcitol was studied in PBMCs from patients with ESKD, cytokine-stimulated cells, and preclinical kidney injury models. RESULTS: In PBMCs isolated from patients with ESKD, TRAF3 protein levels were lower than in healthy controls. This finding was associated with evidence of noncanonical NF-κB2 activation and a proinflammatory state. However, PBMCs from patients with ESKD treated with paricalcitol did not exhibit these features. Experiments in cultured cells confirmed the link between TRAF3 and NF-κB2/inflammation. Decreased TRAF3 ubiquitination in K48-linked chains and cIAP1-TRAF3 interaction mediated the mechanisms of paricalcitol action.TRAF3 overexpression by CRISPR/Cas9 technology mimicked VDRA's effects. In a preclinical model of kidney injury, paricalcitol inhibited renal NF-κB2 activation and decreased renal inflammation. In VDR knockout mice with renal injury, paricalcitol prevented TRAF3 downregulation and NF-κB2-dependent gene upregulation, suggesting a VDR-independent anti-inflammatory effect of paricalcitol. CONCLUSIONS: These data suggest the anti-inflammatory actions of paricalcitol depend on TRAF3 modulation and subsequent inhibition of the noncanonical NF-κB2 pathway, identifying a novel mechanism for VDRA's effects. Circulating TRAF3 levels could be a biomarker of renal damage associated with the inflammatory state.

Human TRAF3 adaptor molecule deficiency leads to impaired Toll-like receptor 3 response and susceptibility to herpes simplex encephalitis.

Tumor necrosis factor (TNF) receptor-associated factor 3 (TRAF3) functions downstream of multiple TNF receptors and receptors that induce interferon-α (IFN-α), IFN-ß, and IFN-λ production, including Toll-like receptor 3 (TLR3), which is deficient in some patients with herpes simplex virus-1 encephalitis (HSE). Mice lacking TRAF3 die in the neonatal period, preventing direct investigation of the role of TRAF3 in immune responses and host defenses in vivo. Here, we report autosomal dominant, human TRAF3 deficiency in a young adult with a history of HSE in childhood. The TRAF3 mutant allele is loss-of-expression, loss-of-function, dominant-negative and associated with impaired, but not abolished, TRAF3-dependent responses upon stimulation of both TNF receptors and receptors that induce IFN production. TRAF3 deficiency is associated with a clinical phenotype limited to HSE resulting from the impairment of TLR3-dependent induction of IFN. Thus, TLR3-mediated immunity against primary infection by HSV-1 in the central nervous system is critically dependent on TRAF3.

Nuclear TRAF3 is a negative regulator of CREB in B cells.

The adaptor protein TNF receptor-associated factor 3 (TRAF3) regulates signaling through B-lymphocyte receptors, including CD40, BAFF receptor, and Toll-like receptors, and also plays a critical role inhibiting B-cell homoeostatic survival. Consistent with these findings, loss-of-function human TRAF3 mutations are common in B-cell cancers, particularly multiple myeloma and B-cell lymphoma. B cells of B-cell-specific TRAF3(-/-) mice (B-Traf3(-/-)) display remarkably enhanced survival compared with littermate control (WT) B cells. The mechanism for this abnormal homeostatic survival is poorly understood, a key knowledge gap in selecting optimal treatments for human B-cell cancers with TRAF3 deficiency. We show here for the first time to our knowledge that TRAF3 is a resident nuclear protein that associates with the transcriptional regulator cAMP response element binding protein (CREB) in both mouse and human B cells. The TRAF-C domain of TRAF3 was necessary and sufficient to localize TRAF3 to the nucleus via a functional nuclear localization signal. CREB protein was elevated in TRAF3(-/-) B cells, without change in mRNA, but with a decrease in CREB ubiquitination. CREB-mediated transcriptional activity was increased in TRAF3-deficient B cells. Consistent with these findings, Mcl-1, an antiapoptotic target of CREB-mediated transcription, was increased in the absence of TRAF3 and enhanced Mcl-1 was suppressed with CREB inhibition. TRAF3-deficient B cells were also preferentially sensitive to survival inhibition with pharmacologic CREB inhibitor. Our results identify a new mechanism by which nuclear TRAF3 regulates B-cell survival via inhibition of CREB stability, information highly relevant to the role of TRAF3 in B-cell malignancies.

TRAF3 delays cyst formation induced by NF-κB signaling.

This study aims to investigate the effects of TNF receptors associated factor 3 (TRAF3) on the signaling pathway and expression of downstream products of nuclear factor kappa B (NF-κB) in the epithelial cells of renal ducts in individuals with polycystic kidney disease (PKD). We observe the TRAF3 genic overexpression of the epithelial cells, which form a tubular branch structure, in polycystic kidneys and to explore the protective effect of TRAF3 on the cystogenesis and progression of PKD. Western blotting analysis was conducted to examine the signaling changes of NF-κB in PKD the epithelial cells and TRAF3 transgenic PKD epithelial cells. Changes in the downstream apoptosis factor and cell proliferation in PKD epithelial cells and TRAF3 transgenic PKD epithelial cells were detected. A three-dimensional matrigel culture experiment was performed to examine abnormal tubulomorphogenesis in vitro. The overexpression of TRAF3 significantly inhibited the signaling pathway of NF-κB in the PKD epithelial cells, downregulated the expression of downstream factors Bcl-2 and Bcl-xl, and significantly decreased cystic epithelial cell proliferation. Additional branch structures were observed in the PKD epithelial cells with a three-dimensional culture compared to wildtype cells. TRAF3 may likely induce apoptosis and resistance to proliferation and may be a new target to inhibit the cyst formation in PKD by regulating the NF-κB signaling pathway Bcl-2 and Bcl-xl activity. © 2017 IUBMB Life, 69(3):170-178, 2017.

Targeting TRAF3 signaling protects against hepatic ischemia/reperfusions injury.

BACKGROUND & AIMS: The hallmarks of hepatic ischemia/reperfusion (I/R) injury, a common clinical problem that occurs during liver surgical procedures, include severe cell death and inflammatory responses that contribute to early graft failure and a higher incidence of organ rejection. Unfortunately, effective therapeutic strategies are limited. Tumor necrosis factor receptor (TNFR)-associated factor (TRAF) 3 transduces apoptosis and/or inflammation-related signaling pathways to regulate cell survival and cytokine production. However, the role of TRAF3 in hepatic I/R-induced liver damage remains unknown. METHODS: Hepatocyte- or myeloid cell-specific TRAF3 knockdown or transgenic mice were subjected to an I/R model in vivo, and in vitro experiments were performed by treating primary hepatocytes from these mice with hypoxia/reoxygenation stimulation. The function of TRAF3 in I/R-induced liver damage and the potential underlying mechanisms were investigated through various phenotypic analyses and biological approaches. RESULTS: Hepatocyte-specific, but not myeloid cell-specific, TRAF3 deficiency reduced cell death, inflammatory cell infiltration, and cytokine production in both in vivo and in vitro hepatic I/R models, whereas hepatic TRAF3 overexpression resulted in the opposite effects. Mechanistically, TRAF3 directly binds to TAK1, which enhances the activation of the downstream NF-κB and JNK pathways. Importantly, inhibition of TAK1 almost completely reversed the TRAF3 overexpression-mediated exacerbation of I/R injury. CONCLUSIONS: TRAF3 is a novel hepatic I/R mediator that promotes liver damage and inflammation via TAK1-dependent activation of the JNK and NF-κB pathways. Inhibition of hepatic TRAF3 may represent a promising approach to protect the liver against I/R injury-related diseases.

Myeloid cell TRAF3 promotes metabolic inflammation, insulin resistance, and hepatic steatosis in obesity.

Myeloid cells, particularly macrophages, mediate metabolic inflammation, thus promoting insulin resistance and metabolic disease progression in obesity. Numerous cytokines, toxic metabolites, damage-associated molecular patterns, and pathogen-associated molecular patterns are involved in activating macrophages via their cognate receptors in obesity. TRAF3 (TNF receptor-associated factor 3) is a common signaling molecule for these ligands/receptors and negatively regulates the proinflammatory NF-κB and MAPK pathways, but its metabolic activity is unknown. We here show that myeloid cell TRAF3 is required for metabolic inflammation and metabolic disease progression in obesity. Myeloid cell-specific deletion of TRAF3 significantly attenuated insulin resistance, hyperglycemia, hyperinsulinemia, glucose intolerance, and hepatic steatosis in mice with either genetic (ob/ob) or high-fat diet (HFD)-induced obesity. Myeloid cell-specific deletion of TRAF3 had the opposite effects on metabolic inflammation between obese and lean mice. It decreased the expression of proinflammatory cytokines in the liver and adipose tissue of obese mice and largely prevented HFD-induced inflammation in these metabolic tissues; by contrast, in lean mice, it increased the expression of proinflammatory cytokines in the liver and adipose tissue. These data suggest that, in obesity progression, myeloid TRAF3 functionally switches its activity from anti-inflammatory to proinflammatory modes, thereby coupling overnutrition to metabolic inflammation, insulin resistance, and metabolic disease.

TNF receptor-associated factor 3 is required for T cell-mediated immunity and TCR/CD28 signaling.

We recently reported that TNFR-associated factor (TRAF)3, a ubiquitously expressed adaptor protein, promotes mature B cell apoptosis. However, the specific function of TRAF3 in T cells has remained unclear. In this article, we report the generation and characterization of T cell-specific TRAF3(-/-) mice, in which the traf3 gene was deleted from thymocytes and T cells. Ablation of TRAF3 in the T cell lineage did not affect CD4 or CD8 T cell populations in secondary lymphoid organs or the numbers or proportions of CD4(+),CD8(+) or double-positive or double-negative thymocytes, except that the T cell-specific TRAF3(-/-) mice had a 2-fold increase in FoxP3(+) T cells. In striking contrast to mice lacking TRAF3 in B cells, the T cell TRAF3-deficient mice exhibited defective IgG1 responses to a T-dependent Ag, as well as impaired T cell-mediated immunity to infection with Listeria monocytogenes. Surprisingly, we found that TRAF3 was recruited to the TCR/CD28 signaling complex upon costimulation and that TCR/CD28-mediated proximal and distal signaling events were compromised by TRAF3 deficiency. These findings provide insights into the roles played by TRAF3 in T cell activation and T cell-mediated immunity.

TRAF3 negatively regulates calcineurin-NFAT pathway by targeting calcineurin B subunit for degradation.

Calcineurin (CN) is the only serine/threonine specific protein phosphatase regulated by Ca(2+) /calmodulin (CaM), which is composed of catalytic A subunit (CNA) and regulatory B subunit (CNB). Tumor necrosis factor (TNF) receptor associated factor 3 (TRAF3) is an essential component in the Toll like receptors and TNF receptors (TNFRs) pathways. The TRAF domain of TRAF3 interacts with a large range of proteins, which share consensus sequences known as TRAF interacting motifs (TIMs). By sequence alignment, we identified two potential TIMs in CNB. However, the relation between TRAF3 and CN has not been reported before. To explore this, we highly expressed the former insoluble TRAF domain of TRAF3 in soluble form by using CaM fusion system for the first time. We demonstrated that the TRAF domain of TRAF3 interacted with CNB. On further investigation, over-expression of TRAF3 inhibited endogenous CN's activity, which decreased NFAT reporter activity and IL-2 production. Knock-down of TRAF3 partially enhanced CN's activity. The possible mechanism was that TRAF3 functioned as ubiquitin E3 ligase for CN and promoted its degradation.

Differential TRAF3 utilization by a variant human CD40 receptor with enhanced signaling.

CD40 is required for T cell-dependent humoral immunity, but it can also contribute to the pathogenesis of autoimmunity and B cell malignancy. The TNFR-associated factor (TRAF)2 and TRAF6 adaptor proteins are positive regulators of CD40 signaling required to activate downstream kinase cascades and transcription factors. In contrast, TRAF3 can serve as a negative regulator of CD40 signaling, and CD40 signals are amplified in TRAF3(-/-) B cells. We previously reported a gain-of-function polymorphism of the human CD40 receptor, hCD40-P227A, which signals in an amplified manner to B lymphocytes. In this study, we show that hCD40-P227A binds more TRAF3 and TRAF5, as well as certain associated proteins, than wild-type-CD40. Studies in TRAF-deficient B cell lines revealed that hCD40-P227A uses TRAF3 as a positive rather than negative regulator. Although located outside of any known TRAF binding sites, the P227A polymorphism can alter TRAF binding and dramatically changes the role played by TRAF3 in CD40 signaling.

Hantaan virus triggers TLR3-dependent innate immune responses.

Immediately after viral infection, innate responses including expression of IFN-alpha/beta and IFN-stimulated genes (ISGs) are elicited ubiquitously by recruitment of specific pathogen recognition receptors. The velocity to induce IFN-alpha/beta and ISGs in response to an infection is often decisive for virulence. Interestingly, in primary endothelial cells ISGs are induced later by hantaviruses pathogenic to humans than those considered to be nonpathogenic or of low virulence. Here we demonstrate that pathogenic Hantaan (HTNV) and putatively nonpathogenic Prospect Hill hantavirus (PHV) differentially activate innate responses in the established cell lines A549 and HuH7. STAT1alpha phosphorylation was detectable 3 h after PHV inoculation but not within the first 2 days after HTNV inoculation. The velocity to induce the ISGs MxA and ISG15 correlated inversely with amounts of virus produced. Moreover, expression of the inflammatory chemokine CCL5 was also induced differentially. Both hantaviruses induced innate responses via TRAF3 (TNF receptor-associated factor 3), and TLR3 was required for HTNV-induced expression of MxA, but not for the MxA induction triggered by PHV. Infection of RIG-I-deficient HuH7.5 cells revealed that RIG-I (retinoic acid receptor I) was not necessary for induction of innate responses by PHV. Taken together, these data suggest that HTNV and PHV elicit different signaling cascades that converge via TRAF3. Early induction of antiviral responses might contribute to efficient elimination of PHV. Subsequent to clearance of the infection, innate responses most likely cease; vice versa, retarded induction of antiviral responses could lead to increased HTNV replication and dissemination, which might cause a prolonged inflammatory response and might contribute to the in vivo virulence.

Genomic and functional uniqueness of the TNF receptor-associated factor gene family in amphioxus, the basal chordate.

The TNF-associated factor (TRAF) family, the crucial adaptor group in innate immune signaling, increased to 24 in amphioxus, the oldest lineage of the Chordata. To address how these expanded molecules evolved to adapt to the changing TRAF mediated signaling pathways, here we conducted genomic and functional comparisons of four distinct amphioxus TRAF groups with their human counterparts. We showed that lineage-specific duplication and rearrangement were responsible for the expansion of amphioxus TRAF1/2 and 3 lineages, whereas TRAF4 and 6 maintained a relatively stable genome and protein structure. Amphioxus TRAF1/2 and 3 molecules displayed various expression patterns in response to microbial infection, and some of them can attenuate the NF-kappaB activation mediated by human TRAF2 and 6. Amphioxus TRAF4 presented two unique functions: activation of the NF-kappaB pathway and involvement in somite formation. Although amphioxus TRAF6 was conserved in activating NF-kappaB pathway for antibacterial defense, the mechanism was not the same as that observed in humans. In summary, our findings reveal the evolutionary uniqueness of the TRAF family in this basal chordate, and suggest that genomic duplication and functional divergence of the TRAF family are important for the current form of the TRAF-mediated signaling pathways in humans.

Roles of the TRAF2/3 binding site in differential B cell signaling by CD40 and its viral oncogenic mimic, LMP1.

The EBV protein, latent membrane protein 1 (LMP1), is a functional mimic of the cellular receptor CD40, but signals to B lymphocytes in an amplified and sustained manner compared with CD40. LMP1 contributes to the development of B cell lymphoma in immunosuppressed patients, and may exacerbate flares of certain autoimmune diseases. The cytoplasmic domain of LMP1 binds the signaling adaptor TRAF2 with lower avidity than the cytoplasmic domain of CD40, and TRAF2 is needed for CD40-mediated degradation of TRAFs 2 and 3. LMP1 doesn't induce TRAF degradation, and employs TRAF3 as a positive mediator of cell signaling, whereas CD40 signals are inhibited by TRAF3. We thus tested the hypothesis that relative affinity for TRAF2, and/or distinct sequence differences in the TRAF2/3 binding sites of CD40 vs LMP1, controls the disparate ways in which CD40 and LMP1 use TRAFs 2 and 3, and their distinct signaling characteristics. CD40 and LMP1 mutants in which the TRAF binding site sequences were swapped were examined, testing TRAF binding and degradation, and induction of B cell activation. Results revealed that TRAF binding affinity and TRAF binding site sequence dictate a distinct subset of CD40 vs LMP1 signaling properties. Examination of TRAF binding, degradation, cytokine production, IgM secretion, and the activation of c-Jun kinase and NF-kappaB revealed that some events are dictated by TRAF binding site sequences, others are partially regulated, and still others are independent of the TRAF binding site sequence.

Control of canonical NF-kappaB activation through the NIK-IKK complex pathway.

Articles in recent years have described two separate and distinct NF-kappaB activation pathways that result in the differential activation of p50- or p52-containing NF-kappaB complexes. Studies examining tumor-necrosis factor receptor-associated factors (TRAFs) have identified positive roles for TRAF2, TRAF5, and TRAF6, but not TRAF3, in canonical (p50-dependent) NF-kappaB activation. Conversely, it recently was reported that TRAF3 functions as an essential negative regulator of the noncanonical (p52-dependent) NF-kappaB pathway. In this article, we provide evidence that TRAF3 potently suppresses canonical NF-kappaB activation and gene expression in vitro and in vivo. We also demonstrate that deregulation of the canonical NF-kappaB pathway in TRAF3-deficient cells results from accumulation of NF-kappaB-inducing kinase (NIK), the essential kinase mediating noncanonical NF-kappaB activation. Thus, our data demonstrate that inhibition of TRAF3 results in coordinated activation of both NF-kappaB activation pathways.

IRF7 activation by Epstein-Barr virus latent membrane protein 1 requires localization at activation sites and TRAF6, but not TRAF2 or TRAF3.

Epstein-Barr virus (EBV) latent infection membrane protein 1 (LMP1), a constitutively aggregated and activated pseudoreceptor, activates IFN regulatory factor 7 (IRF7) through RIP1. We now report that the LMP1 cytoplasmic carboxyl terminal amino acids 379-386 bound IRF7 and activated IRF7. IRF7 activation required TRAF6 and RIP1, but not TRAF2 or TRAF3. LMP1 Y(384)YD(386), which are required for TRADD and RIP1 binding and for NF-kappaB activation, were not required for IRF7 binding, but were required for IRF7 activation, implicating signaling through TRADD and RIP1 in IRF7 activation. Association with active LMP1 signaling complexes was also critical for IRF7 activation because (i) a dominant-negative IRF7 bound to LMP1, blocked IRF7 association and activation, but did not inhibit LMP1 induced NF-kappaB or TBK1 or Sendai virus-mediated IFN stimulated response element activation; and (ii) two different LMP1 transmembrane domain mutants, which fail to aggregate, each bound IRF7 and prevented LMP1 from binding and activating IRF7 in the same cell, but did not prevent NF-kappaB activation. Thus, efficient IRF7 activation required association with LMP1 CTAR2 in proximity to LMP1 CTAR2 mediated kinase activation sites.

Mitochondrial Fission Factor Is a Novel Interacting Protein of the Critical B Cell Survival Regulator TRAF3 in B Lymphocytes.

Proteins controlling mitochondrial fission have been recognized as essential regulators of mitochondrial functions, mitochondrial quality control and cell apoptosis. In the present study, we identified the critical B cell survival regulator TRAF3 as a novel binding partner of the key mitochondrial fission factor, MFF, in B lymphocytes. Elicited by our unexpected finding that the majority of cytoplasmic TRAF3 proteins were localized at the mitochondria in resting splenic B cells after ex vivo culture for 2 days, we found that TRAF3 specifically interacted with MFF as demonstrated by co-immunoprecipitation and GST pull-down assays. We further found that in the absence of stimulation, increased protein levels of mitochondrial TRAF3 were associated with altered mitochondrial morphology, decreased mitochondrial respiration, increased mitochondrial ROS production and membrane permeabilization, which eventually culminated in mitochondria-dependent apoptosis in resting B cells. Loss of TRAF3 had the opposite effects on the morphology and function of mitochondria as well as mitochondria-dependent apoptosis in resting B cells. Interestingly, co-expression of TRAF3 and MFF resulted in decreased phosphorylation and ubiquitination of MFF as well as decreased ubiquitination of TRAF3. Moreover, lentivirus-mediated overexpression of MFF restored mitochondria-dependent apoptosis in TRAF3-deficient malignant B cells. Taken together, our findings provide novel insights into the apoptosis-inducing mechanisms of TRAF3 in B cells: as a result of survival factor deprivation or under other types of stress, TRAF3 is mobilized to the mitochondria through its interaction with MFF, where it triggers mitochondria-dependent apoptosis. This new role of TRAF3 in controlling mitochondrial homeostasis might have key implications in TRAF3-mediated regulation of B cell transformation in different cellular contexts. Our findings also suggest that mitochondrial fission is an actionable therapeutic target in human B cell malignancies, including those with TRAF3 deletion or relevant mutations.

TNF-Receptor-Associated Factor 3 in Litopenaeus vannamei Restricts White Spot Syndrome Virus Infection Through the IRF-Vago Antiviral Pathway.

Tumor necrosis factor receptor (TNFR)-associated factors (TRAFs) are vital signaling adaptor proteins for the innate immune response and are involved in many important pathways, such as the NF-κB- and interferon regulatory factor (IRF)-activated signaling pathways. In this study, the TRAF3 ortholog from the shrimp Litopenaeus vannamei (LvTRAF3) was cloned and characterized. LvTRAF3 has a transcript of 3,865 bp, with an open reading frame (ORF) of 1,002 bp and encodes a polypeptide of 333 amino acids, including a conserved TRAF-C domain. The expression of LvTRAF3 in the intestine and hemocyte was up-regulated in response to poly (I:C) challenge and white spot syndrome virus (WSSV) infection. RNAi knockdown of LvTRAF3 in vivo significantly increased WSSV gene transcription, viral loads, and mortality in WSSV-infected shrimp. Next, we found that LvTRAF3 was not able to induce the activation of the NF-κB pathway, which was crucial for synthesis of antimicrobial peptides (AMPs), which mediate antiviral immunity. Specifically, in dual-luciferase reporter assays, LvTRAF3 could not activate several types of promoters with NF-κB binding sites, including those from WSSV genes (wsv069, wsv056, and wsv403), Drosophila AMPs or shrimp AMPs. Accordingly, the mRNA levels of shrimp AMPs did not significantly change when TRAF3 was knocked down during WSSV infection. Instead, we found that LvTRAF3 signaled through the IRF-Vago antiviral cascade. LvTRAF3 functioned upstream of LvIRF to regulate the expression of LvVago4 and LvVago5 during WSSV infection in vivo. Taken together, these data provide experimental evidence of the participation of LvTRAF3 in the host defense to WSSV through the activation of the IRF-Vago pathway but not the NF-κB pathway.

Autophagy mediated by JNK1 resists apoptosis through TRAF3 degradation in osteoclastogenesis.

RANKL induces osteoclastogenesis via JNK1 signal that exerts an anti-apoptotic effect during osteoclastogenesis. However, the classic downstream c-Jun/AP-1 pathway is not included in anti-apoptosis of JNK1. Thus, the detail mechanism underlying JNK1-resisted apoptosis remains unknown during RANKL-induced osteoclastogenesis. RANKL-induced autophagy results in the degradation of the osteoclastogenesis-inhibitor TRAF3, and TRAF3 is thought as a regulator of apoptosis. Given the key effect of JNK1 in mediating autophagy, our study aims to investigate the significance of TRAF3 in bridging JNK1-mediated autophagy and apoptotic resistance during osteoclastogenesis. In this study, by using Bone Marrow-derived macrophages (BMMs) as osteoclast precursors (OCPs), we found that RANKL-induced TRAF3 degradation was significantly suppressed by JNK inhibitor (SP600125), which was restored by overexpression of Beclin1 (key autophagic protein). Nevertheless, TRAF3 silencing partially reversed the reduced osteoclastogenesis under SP600125 intervention. Besides, OCP apoptosis was positively regulated by TRAF3 overexpression, regardless of the application of autophagy inhibitor or SP600125. Remarkably, the enhanced apoptosis caused by the pharmacological inhibition of Beclin1 was reversed by TRAF3 silencing. Together, these results suggest that JNK1-mediated autophagy could promote RANKL-induced osteoclastogenesis via enhancing TRAF3 degradation. Importantly, JNK1 could prevent OCP apoptosis through autophagy-TRAF3 signaling, which provides more potential targets for clinical treatment of pathological bone loss.