TRAF6 directs FOXP3 localization and facilitates regulatory T-cell function through K63-linked ubiquitination.

Regulatory T cells (Tregs) are crucial mediators of immune control. The characteristic gene expression and suppressive functions of Tregs depend considerably on the stable expression and activity of the transcription factor FOXP3. Transcriptional regulation of the Foxp3 gene has been studied in depth, but both the expression and function of this factor are also modulated at the protein level. However, the molecular players involved in posttranslational FOXP3 regulation are just beginning to be elucidated. Here, we found that TRAF6-deficient Tregs were dysfunctional in vivo; mice with Treg-restricted deletion of TRAF6 were resistant to implanted tumors and displayed enhanced anti-tumor immunity. We further determined that FOXP3 undergoes K63-linked ubiquitination at lysine 262 mediated by the E3 ligase TRAF6. In the absence of TRAF6 activity or upon mutation of the ubiquitination site, FOXP3 displayed aberrant, perinuclear accumulation and disrupted regulatory function. Thus, K63-linked ubiquitination by TRAF6 ensures proper localization of FOXP3 and facilitates the transcription factor's gene-regulating activity in Tregs. These results implicate TRAF6 as a key posttranslational, Treg-stabilizing regulator that may be targeted in novel tolerance-breaking therapies.

Hepatocyte TNF Receptor-Associated Factor 6 Aggravates Hepatic Inflammation and Fibrosis by Promoting Lysine 6-Linked Polyubiquitination of Apoptosis Signal-Regulating Kinase 1.

Activation of apoptosis signal-regulating kinase 1 (ASK1) is a key driving force of the progression of nonalcoholic steatohepatitis (NASH) and represents an attractive therapeutic target for NASH treatment. However, the molecular and cellular mechanisms underlying ASK1 activation in the pathogenesis of NASH remain incompletely understood. In this study, our data unequivocally indicated that hyperactivated ASK1 in hepatocytes is a potent inducer of hepatic stellate cell (HSC) activation by promoting the production of hepatocyte-derived factors. Our previous serial studies have shown that the ubiquitination system plays a key role in regulating ASK1 activity during NASH progression. Here, we further demonstrated that tumor necrosis factor receptor-associated factor 6 (TRAF6) promotes lysine 6 (Lys6)-linked polyubiquitination and subsequent activation of ASK1 to trigger the release of robust proinflammatory and profibrotic factors in hepatocytes, which, in turn, drive HSC activation and hepatic fibrosis. Consistent with the in vitro findings, diet-induced liver inflammation and fibrosis were substantially attenuated in Traf6+/- mice, whereas hepatic TRAF6 overexpression exacerbated these abnormalities. Mechanistically, Lys6-linked ubiquitination of ASK1 by TRAF6 facilitates the dissociation of thioredoxin from ASK1 and N-terminal dimerization of ASK1, resulting in the boosted activation of ASK1-c-Jun N-terminal kinase 1/2 (JNK1/2)-mitogen-activated protein kinase 14(p38) signaling cascade in hepatocytes. Conclusion: These results suggest that Lys6-linked polyubiquitination of ASK1 by TRAF6 represents a mechanism underlying ASK1 activation in hepatocytes and a key driving force of proinflammatory and profibrogenic responses in NASH. Thus, inhibiting Lys6-linked polyubiquitination of ASK1 may serve as a potential therapeutic target for NASH treatment.

Tumor Necrosis Factor Receptor-Associated Factor 6 Promotes Hepatocarcinogenesis by Interacting With Histone Deacetylase 3 to Enhance c-Myc Gene Expression and Protein Stability.

The oncogene c-Myc is aberrantly expressed and plays a key role in malignant transformation and progression of hepatocellular carcinoma (HCC). Here, we report that c-Myc is significantly up-regulated by tumor necrosis factor receptor-associated factor 6 (TRAF6), an E3 ubiquitin ligase, in hepatocarcinogenesis. High TRAF6 expression in clinical HCC samples correlates with poor prognosis, and the loss of one copy of the Traf6 gene in Traf6+/- mice significantly impairs liver tumorigenesis. Mechanistically, TRAF6 first interacts with and ubiquitinates histone deacetylase 3 (HDAC3) with K63-linked ubiquitin chains, which leads to the dissociation of HDAC3 from the c-Myc promoter and subsequent acetylation of histone H3 at K9, thereby epigenetically enhancing the mRNA expression of c-Myc. Second, the K63-linked ubiquitination of HDAC3 impairs the HDAC3 interaction with c-Myc and promotes c-Myc protein acetylation, which thereby enhances c-Myc protein stability by inhibiting carboxyl terminus of heat shock cognate 70-kDa-interacting protein-mediated c-Myc ubiquitination and degradation. Importantly, TRAF6/HDAC3/c-Myc signaling is also primed in hepatitis B virus-transgenic mice, unveiling a critical role for a mechanism in inflammation-cancer transition. In clinical specimens, TRAF6 positively correlates with c-Myc at both the mRNA and protein levels, and high TRAF6 and c-Myc expression is associated with an unfavorable prognosis, suggesting that TRAF6 collaborates with c-Myc to promote human hepatocarcinogenesis. Consistently, curbing c-Myc expression by inhibition of TRAF6 activity with a TRAF6 inhibitor peptide or the silencing of c-Myc by small interfering RNA significantly suppressed tumor growth in mice. Conclusion: These findings demonstrate the oncogenic potential of TRAF6 during hepatocarcinogenesis by modulating TRAF6/HDAC3/c-Myc signaling, with potential implications for HCC therapy.

LncRNA promoted inflammatory response in ischemic heart failure through regulation of miR-455-3p/TRAF6 axis.

OBJECTIVES: Ischemic heart failure (IHF) is the most common cause of death globally. Growing evidence shows abnormal expression of long non-coding RNAs in heart failure patients. This study aims to investigate the effect of sex-determining region Y-box 2 (SOX2) overlapping transcript (SOX2-OT) on the regulation of the inflammatory response in ischemic heart failure. METHODS: IHF rat and oxygen and glucose deprivation (OGD) cell models were established. qRT-PCR was employed to investigate the expression of SOX2-OT. ELISA, western blot and cell viability/apoptosis assays were performed to assess the effects of SOX2-OT. Online software program was used to identify miRNAs that target SOX2-OT, followed by validation using RNA pull-down. Potential targets of miRNAs were searched, and examined by immunoblotting, qRT-PCR and luciferase reporter assay. RESULTS: SOX2-OT was up-regulated in IHF and OGD. Knockdown of SOX2-OT promoted cell proliferation, decreased apoptosis rate and cell oxidative damage, and ameliorated inflammatory response. SOX2-OT contains binding sites for miR-455-3p, miR-5586-3p and miR-1252-5p. RNA pull-down confirmed the binding ability between SOX2-OT and miR-455-3p. TRAF6 is a direct target of miR-455-3p. Moreover, the regulatory activity of SOX2-OT on inflammatory response was partially through its negative regulation of miR-455-3p, which directly regulates TRAF6. Down-regulation of SOX2-OT improved myocardial dysfunction in IHF rat. CONCLUSIONS: Our results reveal that SOX2-OT may be a driver of IHF through repression of miR-455-3p, and miR-455-3p alleviates IHF by targeting TRAF6. Therefore, SOX2-OT/miR-455-3p/TRAF6 may be a potential target for advanced therapeutic strategy for IHF.

Macrophage CD40 signaling drives experimental autoimmune encephalomyelitis.

The costimulatory CD40L-CD40 dyad plays a major role in multiple sclerosis (MS). CD40 is highly expressed on MHCII+ B cells, dendritic cells and macrophages in human MS lesions. Here we investigated the role of the CD40 downstream signaling intermediates TNF receptor-associated factor 2 (TRAF2) and TRAF6 in MHCII+ cells in experimental autoimmune encephalomyelitis (EAE). Both MHCII-CD40-Traf2-/- and MHCII-CD40-Traf6-/- mice showed a reduction in clinical signs of EAE and prevented demyelination. However, only MHCII-CD40-Traf6-/- mice displayed a decrease in myeloid and lymphoid cell infiltration into the CNS that was accompanied by reduced levels of TNF-α, IL-6 and IFN-γ. As CD40-TRAF6 interactions predominantly occur in macrophages, we subjected CD40flfl LysMcre mice to EAE. This myeloid-specific deletion of CD40 resulted in a significant reduction in EAE severity, reduced CNS inflammation and demyelination. In conclusion, the CD40-TRAF6 signaling pathway in MHCII+ cells plays a key role in neuroinflammation and demyelination during EAE. Concomitant with the fact that CD40-TRAF6 interactions are predominant in macrophages, depletion of myeloid CD40 also reduces neuroinflammation. CD40-TRAF6 interactions thus represent a promising therapeutic target for MS. © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Macrophage subpopulations and their impact on chronic allograft rejection versus graft acceptance in a mouse heart transplant model.

Macrophages infiltrating the allografts are heterogeneous, consisting of proinflammatory (M1 cells) as well as antiinflammatory and fibrogenic phenotypes (M2 cells); they affect transplantation outcomes via diverse mechanisms. Here we found that macrophage polarization into M1 and M2 subsets was critically dependent on tumor necrosis factor receptor-associated factor 6 (TRAF6) and mammalian target of rapamycin (mTOR), respectively. In a heart transplant model we showed that macrophage-specific deletion of TRAF6 (LysMCre Traf6 fl/fl ) or mTOR (LysMCre Mtorfl/fl ) did not affect acute allograft rejection. However, treatment of LysMCre Mtorfl/fl recipients with CTLA4-Ig induced long-term allograft survival (>100 days) without histological signs of chronic rejection, whereas the similarly treated LysMCre Traf6 fl/fl recipients developed severe transplant vasculopathy (chronic rejection). The presentation of chronic rejection in CTLA4-Ig-treated LysMCre Traf6 fl/fl mice was similar to that of CTLA4-Ig-treated wild-type B6 recipients. Mechanistically, we found that the graft-infiltrating macrophages in LysMCre Mtorfl/fl recipients expressed high levels of PD-L1, and that PD-L1 blockade readily induced rejection of otherwise survival grafts in the LysMCre Mtorfl/fl recipients. Our findings demonstrate that targeting mTOR-dependent M2 cells is critical for preventing chronic allograft rejection, and that graft survival under such conditions is dependent on the PD-1/PD-L1 coinhibitory pathway.

Serum amyloid A induces interleukin-33 expression through an IRF7-dependent pathway.

Interleukin-33 (IL-33), an IL-1 family cytokine and nuclear alarmin, is constitutively expressed in epithelial barrier tissues and human blood vessels. However, little is known about the induced expression of IL-33 in monocytes and macrophages, which are major cytokine-producing cells of the innate immune system. Here, we report the induction of IL33 expression in both human monocytes and mouse macrophages from C57BL/6 mice by the acute-phase protein serum amyloid A (SAA). SAA-induced transcriptional activation of the Il33 gene, resulting in nuclear accumulation of the IL-33 protein. TLR2, one of the SAA receptors, was primarily responsible for the induction of IL-33. Progressive deletion of the human IL-33 promoter led to the identification of two potential binding sites for interferon regulatory factor 7 (IRF7), one of which (-277/-257) was found to be important for SAA-stimulated IL-33 promoter activity. IRF7 was recruited to the IL-33 promoter upon SAA stimulation, and silencing IRF7 expression in THP-1 cells abrogated SAA-induced Il33 expression. SAA also promoted an interaction between TNF receptor-associated factor 6 and IRF7. Taken together, these results identify IRF7 as a critical transcription factor for SAA-induced Il33 expression in monocytes and macrophages.

TNFR-associated factor 6 regulates TCR signaling via interaction with and modification of LAT adapter.

TNFR-associated factor (TRAF)6 is an essential ubiquitin E3 ligase in immune responses, but its function in adaptive immunity is not well understood. In this study, we show that TRAF6 is recruited to the peripheral ring of the T cell immunological synapse in Jurkat T cells or human primary CD4(+) T cells conjugated with staphylococcal enterotoxin E-pulsed B cells. This recruitment depends on TRAF6 interacting with linker for activation of T cells (LAT) via its TRAF domain. Although LAT was indispensable for TCR/CD28-induced TRAF6 ubiquitination and its ligase activity, RNA interference-induced TRAF6 knockdown in T cells decreased TCR/CD28-induced LAT ubiquitination, tyrosine phosphorylation, and association with tyrosine kinase ZAP70. Overexpression of TRAF6 or its catalytically inactive form C70A promoted and decreased, respectively, LAT tyrosine phosphorylation upon stimulation. Moreover, LAT was ubiquitinated at Lys(88) by TRAF6 via K63-linked chain. In addition, TRAF6 was required for and synergized with LAT to promote the TCR/CD28-induced activation of NFAT. These results reveal a novel function and mechanism of TRAF6 action in the TCR-LAT signaling pathway distinct from its role in TCR-induced NF-κB activation, indicating that LAT also plays an adapter role in TCR/CD28-induced activation of TRAF6.

MiR-125a TNF receptor-associated factor 6 to inhibit osteoclastogenesis.

MicroRNAs (miRNAs) play important roles in osteoclastogenesis and bone resorption. In the present study, we found that miR-125a was dramatically down-regulated during macrophage colony stimulating factor (M-CSF) and receptor activator of nuclear factor-κB ligand (RANKL) induced osteoclastogenesis of circulating CD14+ peripheral blood mononuclear cells (PBMCs). Overexpression of miR-125a in CD14+ PBMCs inhibited osteoclastogenesis, while inhibition of miR-125a promoted osteoclastogenesis. TNF receptor-associated factor 6 (TRAF6), a transduction factor for RANKL/RANK/NFATc1 signal, was confirmed to be a target of miR-125a. EMSA and ChIP assays confirmed that NFATc1 bound to the promoter of the miR-125a. Overexpression of NFATc1 inhibited miR-125a transcription, and block of NFATc1 expression attenuated RANKL-regulated miR-125a transcription. Here, we reported that miR-125a played a biological function in osteoclastogenesis through a novel TRAF6/ NFATc1/miR-125a regulatory feedback loop. It suggests that regulation of miR-125a expression may be a potential strategy for ameliorating metabolic disease.

CD137 Regulates NFATc1 Expression in Mouse VSMCs through TRAF6/NF-κB p65 Signaling Pathway.

Our previous study proved that CD137-CD137L interaction can regulate the expression of NFATc1. Here, we investigated whether CD137 signaling regulates the expression of NFATc1 in mice VSMCs through TRAF6/NF-κB p65 pathway. Data shows that the CD137 expression can be stimulated by TNF-α in a time-dependent manner in mouse VSMCs. Knockdown of TRAF6 by siTRAF6 significantly attenuated agonist-CD137mAb induced increase of NF-κB p65 and NFATc1 in VSMCs. Pretreatment with a NF-κB inhibitor PDTC for 30 min inhibited the expression of p-p65 in both cytoplasm and nucleus in VSMCs. Thus, the protein level of NFATc1 can be suppressed through inhibition of p-p65. Finally, we also show that the levels of IL-2 and IL-6 can be increased by agonist-CD137 stimulation and decreased when NFATc1 was suppressed. Our data suggest that activated CD137 signaling regulates the expression of NFATc1 and its downstream factors through TRAF6/NF-κB p65 pathways in VSMCs. These findings provide a novel target for treatment of atherosclerosis.

Conditional targeting of tumor necrosis factor receptor-associated factor 6 reveals opposing functions of Toll-like receptor signaling in endothelial and myeloid cells in a mouse model of atherosclerosis.

BACKGROUND: Previous studies implicated Toll-like receptor signaling as a critical pathogenic pathway in atherosclerosis, but the cell-specific mechanisms by which Toll-like receptors act to control atherosclerotic plaque development remain poorly understood. METHODS AND RESULTS: To study the cell-specific role of tumor necrosis factor receptor-associated factor 6 (TRAF6) in atherosclerosis, we generated ApoE(-/-) mice with endothelial cell- or myeloid cell-specific TRAF6 deficiency using Cre/LoxP-mediated gene targeting. Endothelial TRAF6 deficiency reduced atherosclerosis in female ApoE(-/-) mice by inhibiting nuclear factor-κB-dependent proinflammatory gene expression and monocyte adhesion to endothelial cells. In contrast, myeloid cell-specific TRAF6 deficiency caused exacerbated atherosclerosis, with larger plaques containing more necrotic areas in both male and female ApoE(-/-) mice. TRAF6-deficient macrophages showed impaired expression of the antiinflammatory and atheroprotective cytokine interleukin-10, elevated endoplasmic reticulum stress, increased sensitivity to oxidized low-density lipoprotein-induced apoptosis, and reduced capacity to clear apoptotic cells. Thus, the reduced antiinflammatory properties, coupled with increased sensitivity to apoptosis and impaired efferocytosis capacity of TRAF6-deficient macrophages, result in exacerbated atherosclerosis development in TRAF6(MYKO)/ApoE(-/-) mice. CONCLUSION: Toll-like receptor-mediated TRAF6 signaling acts in endothelial cells to promote atherosclerosis but displays atheroprotective, antiinflammatory and prosurvival functions in myeloid cells.

TNF receptor-associated factor 6 regulates proliferation, apoptosis, and invasion of glioma cells.

Tumor necrosis factor receptor-associated factor 6 (TRAF6), which plays an important role in inflammation and immune response, is an essential adaptor protein for the NF-κB (nuclear factor κB) signaling pathway. Recent studies have shown that TRAF6 played an important role in tumorigenesis and invasion by suppressing NF-κB activation. However, up to now, the biologic role of TRAF6 in glioma has still remained unknown. To address the expression of TRAF6 in glioma cells, four glioma cell lines (U251, U-87MG, LN-18, and U373) and a non-cancerous human glial cell line SVG p12 were used to explore the protein expression of TRAF6 by Western blot. Our results indicated that TRAF6 expression was upregulated in human glioma cell lines, especially in metastatic cell lines. To investigate the role of TRAF6 in cell proliferation, apoptosis, invasion, and migration of glioma, we generated human glioma U-87MG cell lines in which TRAF6 was either overexpressed or depleted. Subsequently, the effects of TRAF6 on cell viability, cell cycle distribution, apoptosis, invasion, and migration in U-87MG cells were determined with 3-(4,5-dimethylthiazol-2-yl) 2,5-diphenyl tetrazolium bromide (MTT) assay, flow cytometry analysis, transwell invasion assay, and wound-healing assay. The results showed that knockdown of TRAF6 could decrease cell viability, suppress cell proliferation, invasion and migration, and promote cell apoptosis, whereas overexpression of TRAF6 displayed the opposite effects. In addition, the effects of TRAF6 on the expression of phosphor-NF-κB (p-p65), cyclin D1, caspase 3, and MMP-9 were also probed. Knockdown of TRAF6 could lower the expression of p-p65, cyclin D1, and MMP-9, and raise the expression of caspase 3. All these results suggested that TRAF6 might be involved in the potentiation of growth, proliferation, invasion, and migration of U-87MG cell, as well as inhibition of apoptosis of U-87MG cell by abrogating activation of NF-κB.

Crucial role of CD40 signaling in vascular wall cells in neointimal formation and vascular remodeling after vascular interventions.

OBJECTIVE: It has been shown that CD40-TRAF6 axis in leukocytes plays a significant role in neointimal formation after carotid ligation. Because CD40 and TRAF6 are expressed not only in leukocytes but also in vascular cells, we examined the role of CD40 contributed by vascular wall cells in neointimal formation after carotid ligation in an atherogenic environment. METHODS AND RESULTS: Both CD40 and TRAF6 in medial smooth muscle cells (SMCs) was upregulated significantly at 3 days and more prominently at 7 days after injury in wildtype mice, but the TRAF6 upregulation was abolished in CD40(-/-) mice. In vitro, TRAF6 expression was induced by cytokines (tumor necrosis factor -α, interleukin-1ß) via a NF-κB-dependent manner in wildtype SMCs, but this induction was blocked in CD40-deficient SMCs. Bone marrow chimeras revealed a comparable reduction in neointimal formation and lumen stenosis in mice lacking either vascular wall- or bone marrow-associated CD40. Lacking vascular wall-associated CD40 resulted in a significant reduction in monocyte/macrophage accumulation, NF-κB activation, and multiple proinflammatory mediators (ICAM-1, VCAM-1, MCP-1, MMP-9, tissue factor). In vitro data confirmed that CD40 deficiency or TRAF6 knockdown suppressed CD40L-induced proinflammatory phenotype of SMCs by inhibition of NF-κB activation. Moreover, both in vivo and in vitro data showed that CD40 deficiency prevented injury-induced SMC apoptosis but did not affect SMC proliferation and migration. CONCLUSIONS: CD40 signaling through TRAF6 in vascular SMCs seems to be centrally involved in neointimal formation in a NF-κB-dependent manner. Modulating CD40 signaling on local vascular wall may become a new therapeutic target against vascular restenosis.

TRAF6 is a T cell-intrinsic negative regulator required for the maintenance of immune homeostasis.

TRAF6 has a key role in the regulation of innate immune responses by mediating signals from both TNF receptor and interleukin-1 receptor/Toll-like receptor superfamilies. Here we show that T cell-specific deletion of TRAF6 unexpectedly results in multiorgan inflammatory disease. TRAF6-deficient T cells exhibit hyperactivation of the phosphatidylinositol 3-kinase (PI3K)-Akt pathway compared with wild-type T cells and, as a result, become resistant to suppression by CD4+ CD25+ regulatory T cells. These data identify a previously unrecognized role for TRAF6 in the maintenance of peripheral tolerance, and suggest the presence of a T cell-intrinsic control mechanism to render responder T cells susceptible to tolerizing signals.

De novo whole transcriptome analysis of the fish louse, Argulus siamensis: first molecular insights into characterization of Toll downstream signalling molecules of crustaceans.

Argulus siamensis is a major ectoparasitic pathogen of freshwater fish capable of causing substantial economic loss. None of the available control measures have been able to address the problem of argulosis resourcefully. To combat this pathogen effectively, it is necessary to have a comprehensive understanding of its life processes with information on various genes involved. The transcriptome studies can generate introductory information about genes participating in physiological processes of the parasite which could be targeted for their control. In this study, the transcriptome sequencing of A. siamensis was performed on Illumina HiSeq 2000 platform which generated 75,126,957 high quality reads. A total of 46,352 transcript contigs were assembled with average length of 1211bp and N50 length of 2302bp. In total, 19,290 CDS including 184 novel CDS and 59,019 open reading frames (ORFs) were identified from the assembled contigs. Gene ontology and Kyoto Encylopedia of Genes and Genomes pathway analysis were performed to classify contigs into their functional categories and regulation pathways. Additionally, 1171 simple sequence repeats were identified from the assembled contigs. Further, twelve contigs with high similarity with downstream molecules of the mammalian toll like receptor (TLR) pathway were validated by their inductive expressions in response to lipopolysaccharide (LPS) of Gram negative bacteria, Escherichia coli and Gram positive bacteria, Staphylococcus aureus. The transcriptome of an ectoparasite A. siamensis was sequenced, assembled, annotated, and the downstream signalling molecules of Toll pathway characterized. The transcriptome data generated will facilitate studies on functional genomics that will subsequently be applied for vaccine development and other control strategies against the parasite.

Adenosine A1 receptor regulates osteoclast formation by altering TRAF6/TAK1 signaling.

Adenosine is an endogenous nucleoside that modulates many physiological processes through four receptor subtypes (A(1), A(2a), A(2b), A(3)). Previous work from our laboratory has uncovered a critical role for adenosine A(1) receptor (A(1) R) in osteoclastogenesis both in vivo and in vitro. Our current work focuses on understanding the details of how A(1) R modulates the receptor activator of NF-κB ligand (RANKL)-induced signaling in osteoclastogenesis. Osteoclasts were generated from mouse bone marrow precursors in the presence of RANKL and macrophage-colony stimulating factor. A pharmacological antagonist of A(1) R (DPCPX) inhibited RANKL-induced osteoclast differentiation, including osteoclast-specific genes (Acp5, MMP9, ß(3) Integrin, α(v) Integrin, and CTSK) and osteoclast-specific transcription factors such as c-fos and nuclear factor of activated T cells cytoplasmic 1 (NFATc1) expression in a dose-dependent manner. DPCPX also inhibited RANKL-induced activation of NF-κB and JNK/c-Jun but had little effect on other mitogen-activated protein kinases (p38 and Erk). Finally, immunoprecipitation analysis showed that blockade of A(1)R resulted in disruption of the association of tumor necrosis factor receptor-associated factor 6 (TRAF6) and transforming growth factor-ß-activated kinase 1 (TAK1), a signaling event that is important for activation of NF-κB and JNK, suggesting the participation of adenosine/A(1)R in early signaling of RANKL. Collectively, these data demonstrated an important role of adenosine, through A(1)R in RANKL-induced osteoclastogenesis.

A novel TNF receptor-associated factor 6 binding domain mediates NF-kappa B signaling by the common cytokine receptor beta subunit.

GM-CSF, IL-3, and IL-5 are proinflammatory cytokines that control the production and function of myeloid and lymphoid cells. Their receptors are composed of a ligand-specific alpha subunit and a shared common signal-transducing beta subunit (beta common receptor or GM-CSFR beta [beta(c)]). The pleiotropic nature of biologic outcomes mediated by beta(c) and the presence of large, uncharacterized regions of its cytoplasmic domain suggest that much remains to be learned about its downstream signaling pathways. Although some previous work has attempted to link beta(c) with NF-kappaB activation, a definitive mechanism that mediates this pathway has not been described and, to date, it has not been clear whether the receptor can directly activate NF-kappaB. We demonstrate that NF-kappaB activation by beta(c) is dependent on TNFR-associated factor 6 (TRAF6) and that association of TRAF6 with beta(c) requires a consensus-binding motif found in other molecules known to interact with TRAF6. Furthermore, point mutation of this motif abrogated the ability of beta(c) to mediate NF-kappaB activation and reduced the viability of an IL-3-dependent hematopoietic cell line. Because this receptor plays a key role in hematopoiesis and the beta(c) cytoplasmic domain identified in this work mediates hematopoietic cell viability, this new pathway is likely to contribute to immune cell biology. This work is significant because it is the first description of a TRAF6-dependent signaling pathway associated with a type I cytokine receptor. It also suggests that TRAF6, a mediator of TNFR and TLR signaling, may be a common signaling intermediate in diverse cytokine receptor systems.

IL-17 signaling for mRNA stabilization does not require TNF receptor-associated factor 6.

IL-17 alone is a relatively weak inducer of gene expression, but cooperates with other cytokines, including TNF-alpha, to generate a strong response in part via prolongation of mRNA t(1/2). Because TNFR-associated factor 6 (TRAF6) has been reported to be essential for signaling by IL-17, we examined its involvement in IL-17-mediated mRNA stabilization. Although overexpression of TRAF6 in HeLa cells activates NF-kappaB, it does not stabilize transfected KC mRNA. Furthermore, a dominant-negative TRAF6 abrogates NF-kappaB activation, but does not block IL-17-induced chemokine mRNA stabilization. IL-17 can stabilize KC and MIP-2 mRNAs comparably in TNF-alpha-treated mouse embryo fibroblasts from TRAF6(+/+) and TRAF6(-/-) mice. TRAF6 is known to couple upstream signals with activation of p38 MAPK and mitogen activated protein kinase activated protein kinase 2, both of which have been shown to be important for Toll/IL-1R-mediated mRNA stabilization in various cell types. Inhibition of p38 MAPK, however, does not block IL-17-induced KC mRNA stabilization, and IL-17 can stabilize KC mRNA equally in mouse embryo fibroblasts from both wild-type and mitogen activated protein kinase activated protein kinase 2/3 doubly-deficient mice. Finally, IL-17 can amplify the levels of multiple TNF-alpha-stimulated mRNAs in wild-type and TRAF6-deficient cells, but not in cells from Act1(-/-) mice. Collectively, these findings demonstrate the existence of a TRAF6/p38 MAPK-independent pathway that couples the IL-17R with enhanced mRNA stability. Because the most potent effects of IL-17 on gene expression are obtained in cooperation with other cytokines such as TNF-alpha, these findings suggest that this pathway is a major contributing mechanism for response to IL-17.

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.

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.