Tumor Necrosis Factor Receptor-associated Factor 6 Interaction Research Articles

IKK-mediated TRAF6 and RIPK1 interaction stifles cell death complex assembly leading to the suppression of TNF-α-induced cell death.

Tumor necrosis factor α (TNF-α) is a pro-inflammatory cytokine capable of inducing extrinsic apoptosis and necroptosis. Tumor necrosis factor receptor-associated factor 6 (TRAF6), an E3 ligase, is a member of the TRAF family of proteins, which mediates inflammatory signals by activating nuclear factor kappa B (NFкB) and mitogen-activated protein kinase (MAPK). Although the functions of TRAF6 have been identified, its role in TNF-α-induced cell death remains poorly understood. Here, we report that TRAF6 is a negative modulator of TNF-α-induced cell death but does not affect TNF-α-induced NFκB activation. TRAF6 deficiency accelerates both TNF-α-induced apoptosis and necroptosis; however, the acceleration can be reversed by reconstituting TRAF6 or TRAF6C70A, suggesting that E3 ligase activity is not required for this activity. Mechanistically, TRAF6 directly interacts with RIPK1 during TNF-α-induced cell death signaling, which prevents RIPK1 from interacting with components of the cell death complex such as itself, FADD or RIPK3. These processes suppress the assembly of the death complex. Notably, IKK was required for TRAF6 to interact with RIPK1. In vivo, Traf6-/- embryos exhibited higher levels of cell death in the liver but could be rescued by the simultaneous knockout of Tnf. Finally, TRAF6 knockdown xenografts were highly sensitive to necroptotic stimuli. We concluded that TRAF6 suppresses TNF-α-induced cell death in coordination with IKK complexes in vivo and in vitro by suppressing the assembly of cell death complex.

Inhibition of Neddylation Suppresses Osteoclast Differentiation and Function In Vitro and Alleviates Osteoporosis In Vivo.

Neddylation, or the covalent addition of NEDD8 to specific lysine residue of proteins, is a reversible posttranslational modification, which regulates numerous biological functions; however, its involvement and therapeutic significance in osteoporosis remains unknown. Our results revealed that during the soluble receptor activator of nuclear factor-κB ligand (sRANKL)-stimulated osteoclast differentiation, the neddylation and expression of UBA3, the NEDD8-activating enzyme (NAE) catalytic subunit, were dose- and time-dependently upregulated in RAW 264.7 macrophages. UBA3 knockdown for diminishing NAE activity or administering low doses of the NAE inhibitor MLN4924 significantly suppressed sRANKL-stimulated osteoclast differentiation and bone-resorbing activity in the macrophages by inhibiting sRANKL-stimulated neddylation and tumor necrosis factor receptor-associated factor 6 (TRAF6)-activated transforming growth factor-β-activated kinase 1 (TAK1) downstream signaling for diminishing nuclear factor-activated T cells c1 (NFATc1) expression. sRANKL enhanced the interaction of TRAF6 with the neddylated proteins and the polyubiquitination of TRAF6’s lysine 63, which activated TAK1 downstream signaling; however, this process was inhibited by MLN4924. MLN4924 significantly reduced osteoporosis in an ovariectomy- and sRANKL-induced osteoporosis mouse model in vivo. Our novel finding was that NAE-mediated neddylation participates in RANKL-activated TRAF6–TAK1–NFATc1 signaling during osteoclast differentiation and osteoporosis, suggesting that neddylation may be a new target for treating osteoporosis.

The Interaction of TRAF6 With Neuroplastin Promotes Spinogenesis During Early Neuronal Development.

Correct brain wiring depends on reliable synapse formation. Nevertheless, signaling codes promoting synaptogenesis are not fully understood. Here, we report a spinogenic mechanism that operates during neuronal development and is based on the interaction of tumor necrosis factor receptor-associated factor 6 (TRAF6) with the synaptic cell adhesion molecule neuroplastin. The interaction between these proteins was predicted in silico and verified by co-immunoprecipitation in extracts from rat brain and co-transfected HEK cells. Binding assays show physical interaction between neuroplastin’s C-terminus and the TRAF-C domain of TRAF6 with a Kd value of 88 μM. As the two proteins co-localize in primordial dendritic protrusions, we used young cultures of rat and mouse as well as neuroplastin-deficient mouse neurons and showed with mutagenesis, knock-down, and pharmacological blockade that TRAF6 is required by neuroplastin to promote early spinogenesis during in vitro days 6-9, but not later. Time-framed TRAF6 blockade during days 6–9 reduced mEPSC amplitude, number of postsynaptic sites, synapse density and neuronal activity as neurons mature. Our data unravel a new molecular liaison that may emerge during a specific window of the neuronal development to determine excitatory synapse density in the rodent brain.

Tumor necrosis factor receptor-associated factor 6 interaction with alpha-synuclein enhances cell death through the Nuclear Factor-kB pathway

BackgroundParkinson's disease (PD) is a neurodegenerative disease characterized by intracellular inclusions named Lewy bodies (LB), and alpha-synuclein (asyn) is the major component of these protein aggregates. The precise physiological and pathological roles of asyn are not fully understood. Nevertheless, asyn present in LB is ubiquitinated but fails to reach the 26S proteasome. The mutation A30 P is related to an aggressive and early-onset form of PD. Tumor necrosis factor receptor-associated factor 6 (TRAF6) is an E3 ubiquitin ligase, and it interacts and ubiquitinates the asyn in atypical chains (lysine K6, K27, K29, and K33). Methods: Here, we investigated the role of TRAF6 interaction with asyn and the involvement of nuclear factor κB (NF-κB), a key transcription factor in pro-inflammatory signaling pathway activation. Results and ConclusionWe demonstrated that TRAF6 binds to both WT and the mutant form A30 P asyn in an SH-SY5Y cell model. Additionally, the interaction between TRAF6 and WT asyn induced an increase in the activation of NF-κB, leading to changes in TNF, IL-1β and IL-10 levels and culminating in reduced cell viability. Interestingly, the activation of NF-κB and gene regulation were not found in A30 P asyn. These data point to a novel role of TRAF6 in the pathophysiology of PD.

Shikonin mitigates ovariectomy-induced bone loss and RANKL-induced osteoclastogenesis via TRAF6-mediated signaling pathways

BackgroundPostmenopausal osteoporosis results from estrogen withdrawal and is characterized mainly by bone resorption. Shikonin is a bioactive constitute of Chinese traditional herb which plays a role in antimicrobial and antitumor activities. The study was designed to investigate the role of shikonin on postmenopausal osteoporosis and explore its underlying mechanisms. MethodsImmunofluorescence staining was performed to evaluate the effects of shikonin on actin ring formation. The expression levels of the nuclear factor kappa-B (NF-κB) and mitogen-activated protein kinase (MAPK) pathway were determined by Western blot analysis. To determine whether shikonin influences the receptor activator of nuclear factor-κB ligand (RANKL)-induced association between receptor activator of NF-κB (RANK) and tumor necrosis factor receptor associated factor 6 (TRAF6), immunofluorescence staining and immunoprecipitation experiments were performed. During our validation model, histomorphometric examination and micro-computed tomography (CT) were conducted to assess the morphology of osteoporosis. ResultsShikonin prevented bone loss by inhibiting osteoclastogenesis in vitro and improving bone loss in ovariectomized mice in vivo. At the molecular level, Western blot analysis indicated that shikonin inhibited the phosphorylation of inhibitor of NF-κB (IκB), P50, P65, extracellular regulated protein kinases (ERK), c-Jun N-terminal kinase (JNK), and P38. Interaction of TRAF6 and RANK was prevented, and downstream MAPK and NF-κB signaling pathways were downregulated. ConclusionOsteoclastic bone resorption was reduced in the presence of shikonin in vitro and in vivo. Shikonin is a promising candidate for treatment of postmenopausal osteoporosis.

Effect of the SSeCKS–TRAF6 interaction on gastrodin-mediated protection against 2,3,7,8-tetrachlorodibenzo-p-dioxin-induced astrocyte activation and neuronal death

The ubiquitous environmental pollutant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) has been shown to trigger neurotoxicity. In this study, we investigated the protective effects of gastrodin on TCDD-induced neurotoxicity and the underlying molecular mechanisms. The results show that gastrodin decreased cell viability, tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) release, and inducible nitrix oxide synthase (iNOS) and glial fibrillary acidic protein (GFAP) expression in TCDD-treated C6 cells. TCDD stimulated NF-κB signalling activation, demonstrated by increased p-NF-κB expression and translocation of nuclear Factor kappa B (NF-κB) to the nucleus. TCDD did not affect TRAF6 protein expression but enhanced the attenuated the Src-suppressed-C Kinase Substrate (SSeCKS)–tumor necrosis factor receptor-associated factor 6 (TRAF6) interaction, thereby triggering NF-κB signalling activation. Gastrodin inhibited TCDD-induced NF-κB signalling activation by lessening the SSeCKS–TRAF6 interaction in vitro. Gastrodin attenuated SSeCKS–TRAF6 interaction in vivo and protected mice from NF-κB signalling activation following TCDD exposure. Finally, gastrodin blocked the apoptosis of PC12 neuronal cells induced by medium conditioned with TCDD-treated astrocytes. In summary, gastrodin inhibited TCDD-induced NF-κB signalling activation by lessening the SSeCKS–TRAF6 interaction, resulting in attenuated astrocyte activation and subsequent neuronal apoptosis. These findings will contribute to an improved understanding of TCDD-induced neurotoxicity and strategies to antagonise it using gastrodin.

Nedd8 modification of Cullin-5 regulates lipopolysaccharide-induced acute lung injury.

Lung infections are major causes of acute lung injury (ALI), with limited effective treatment available. Tumor necrosis factor receptor-associated factor 6 (TRAF6) is an essential adaptor regulating Toll-like receptors (TLRs). We recently identified Cullin-5 (Cul-5) as a prominent component in the regulation of TRAF6 polyubiquitination, but its physiological significance in ALI has not been explored. In this study, we investigated the potential role of Cul-5 in regulating ALI using mice receiving intratracheal instillation of LPS. We observed that Cul-5-deficient mice displayed reduced lung injury compared with wild-type mice as evidenced by histological analysis, alveolar neutrophil infiltration, and lung liquid accumulation. In addition, inflammatory cytokine expression in bronchoalveolar lavage fluid and lung tissue was also markedly reduced in LPS-treated Cul-5-deficient mice. Interestingly, intratracheal adoptive transfer of Cul-5+/- but not Cul-5+/+ macrophages attenuated neutrophil recruitment, alveolar inflammation, and loss of barrier function in LPS-challenged wild-type mice. Finally, we demonstrated that Cul-5 neddylation following LPS exposure induced Cul-5 and TRAF6 interaction and, thereby, TFAR6 polyubiquitination, leading to NF-κB activation and generation of proinflammatory cytokines. Our data show that neural precursor cell expressed developmentally downregulated protein 8 (Nedd8) modification of Cul-5 is required for its interaction with TRAF6 and activation of the TLR4-TRAF6 signaling pathway in LPS-induced ALI, a feature that may be explored for therapeutic intervention.

Interaction of Tumor Necrosis Factor Receptor-associated Factor 6 (TRAF6) and Vav3 in the Receptor Activator of Nuclear Factor κB (RANK) Signaling Complex Enhances Osteoclastogenesis

The signaling pathway downstream of stimulation of receptor activator of nuclear factor κB (RANK) by RANK ligand is crucial for osteoclastogenesis. RANK recruits TNF receptor-associated factor 6 (TRAF6) to TRAF6-binding sites (T6BSs) in the RANK cytoplasmic tail (RANKcyto) to trigger downstream osteoclastogenic signaling cascades. RANKcyto harbors an additional highly conserved domain (HCR) that also activates crucial signaling during RANK-mediated osteoclastogenesis. However, the functional cross-talk between T6BSs and the HCR in the RANK signaling complex remains unclear. To characterize the cross-talk between T6BSs and the HCR, we screened TRAF6-interacting proteins using a proteomics approach. We identified Vav3 as a novel TRAF6 binding partner and evaluated the functional importance of the TRAF6-Vav3 interaction in the RANK signaling complex. We demonstrated that the coiled-coil domain of TRAF6 interacts directly with the Dbl homology domain of Vav3 to form the RANK signaling complex independent of the TRAF6 ubiquitination pathway. TRAF6 is recruited to the RANKcyto mutant, which lacks T6BSs, via the Vav3 interaction; conversely, Vav3 is recruited to the RANKcyto mutant, which lacks the IVVY motif, via the TRAF6 interaction. Finally, we determined that the TRAF6-Vav3 interaction resulting from cross-talk between T6BSs and the IVVY motif in RANKcyto enhances downstream NF-κB, MAPK, and NFATc1 activation by further strengthening TRAF6 signaling, thereby inducing RANK-mediated osteoclastogenesis. Thus, Vav3 is a novel TRAF6 interaction partner that functions in the activation of cooperative signaling between T6BSs and the IVVY motif in the RANK signaling complex.

Higher TRAF6 and Fbxo 3 Expression Is With Progressive Multiple Myeloma and Blockage Of TRAF6 Inhibits Tumor Growth and Bone Resorption

Tumor necrosis factor receptor-associated factor 6 (TRAF6) has been implicated in regulating the NF-kB and JNK signal transduction pathways; and, thus, is likely to promote tumor cell proliferation and osteoclast formation. F-box proteins such as the E3 ligase component Fbxo3 and another F-box family member Fbxl 2, regulate TRAF protein signaling.First, we investigated TRAF6 expression in bone marrow mononuclear cells (BMMCs) from multiple myeloma (MM) patients with progressive disease or in remission and healthy subjects. The results showed higher TRAF6 protein expression among MM patients with progressive disease than among those in remission or healthy subjects. Notably, changes in TRAF6 protein expression in MM BMMCs were found to correlate with response of individual patients to treatment with the proteasome inhibitors bortezomib or carfilzomib. We have previously reported inhibition of MM cell proliferation and increase of apoptosis through regulation of the NF-kB and JNK pathways using a silencing TRAF6 C-domain mRNA construct. In this study, we cloned a 167 amino acid (in residues 333 to 508) portion of the TRAF6 dominant negative (TRAF6dn) and synthesized decoy peptides of the TRAF6 interaction domain with CD40 and the TRAF6-RANK binding domain. Decreases in cell proliferation and increase in cell apoptosis in MM BMMCs treated with TRAF6dn occurred in a concentrationdependent fashion. We also found TRAF6dn markedly inhibited osteoclast cell formation of monocytes induced by RANKL and mCSF in a dose-dependent manner. Next, we examined the effect of TRAF6dn on NF-kB and JNK by determining phosphorylation of JUN kinase kinase (JNKK), which activates the MAP kinase homologues SAPK and JNK in response to IL-1 receptor stimulation. MM BMMCs exposed to the TRAF6dn fragment or TRAF6 decoy peptide showed reduced phosphoNF-kB protein and phosphorylation of JNKK. These studies suggest that the TRAF6dn or combined TRAF6 decoy and CD40 decoy peptide may be excellent agents to block both MM cell growth and osteoclast formation in MM.We further investigated the protein expression of Fbxo 3, Fbxl 2 and TRAF6 in fresh BMMCs from MM patients with progressive disease or in remission. Results of Western blot analysis showed protein expression of Fbxo 3 and TRAF6 was increased and Fbxl 2 was decreased among patients with progressive disease compared to patients in remission.Thus, these results may offer a new mechanism through which MM tumor cells are regulated and provide a new therapeutic approach to treat MM and reduce the clinical consequences resulting from enhanced bone loss that commonly occur in these patients. Disclosures:No relevant conflicts of interest to declare.

Tumor Necrosis Factor Receptor-associated Factor 6 (TRAF6) Associates with Huntingtin Protein and Promotes Its Atypical Ubiquitination to Enhance Aggregate Formation

Huntington disease (HD) is a neurodegenerative disorder caused by an expansion of polyglutamines in the first exon of huntingtin (HTT), which confers aggregation-promoting properties to amino-terminal fragments of the protein (N-HTT). Mutant N-HTT aggregates are enriched for ubiquitin and contain ubiquitin E3 ligases, thus suggesting a role for ubiquitination in aggregate formation. Here, we report that tumor necrosis factor receptor-associated factor 6 (TRAF6) binds to WT and polyQ-expanded N-HTT in vitro as well as to endogenous full-length proteins in mouse and human brain in vivo. Endogenous TRAF6 is recruited to cellular inclusions formed by mutant N-HTT. Transient overexpression of TRAF6 promotes WT and mutant N-HTT atypical ubiquitination with Lys6, Lys27, and Lys29 linkage formation. Both interaction and ubiquitination seem to be independent from polyQ length. In cultured cells, TRAF6 enhances mutant N-HTT aggregate formation, whereas it has no effect on WT N-HTT protein localization. Mutant N-HTT inclusions are enriched for ubiquitin staining only when TRAF6 and Lys6, Lys27, and Lys29 ubiquitin mutants are expressed. Finally, we show that TRAF6 is up-regulated in post-mortem brains from HD patients where it is found in the insoluble fraction. These results suggest that TRAF6 atypical ubiquitination warrants investigation in HD pathogenesis.

Nuclear Tumor Necrosis Factor Receptor-associated Factor 6 in Lymphoid Cells Negatively Regulates c-Myb-mediated Transactivation through Small Ubiquitin-related Modifier-1 Modification

Tumor necrosis factor receptor-associated factor 6 (TRAF6) is an adaptor/scaffold protein that mediates several important signaling pathways, including the tumor necrosis factor-R:NF-kappaB pathway, involved in immune surveillance, inflammation, etc. Because most studies of TRAF6 function have focused primarily on its role as an adaptor molecule in signaling pathways in the cytoplasm, the potential functions of TRAF6 in other cellular compartments has not been previously investigated. Here, we demonstrate that TRAF6 resides not only in the cellular cytoplasm but is also found in the nuclei of both normal and malignant B lymphocytes. TRAF6 does not possess a nuclear localization signal but enters the nucleus through the nuclear pore complex containing RanGap1. Chromatin immunoprecipitation cloning experiments demonstrated that nuclear TRAF6 associates with c-Myb within the 5'-end of the c-Myb promoter. Further analysis showed that nuclear TRAF6 is modified by small ubiquitin-related modifier-1, interacts with histone deacetylase 1, and represses c-Myb-mediated transactivation. Thus, TRAF6 negatively regulates c-Myb through a novel repressor function in the nuclei of both normal and malignant B-lymphocytes that could represent a novel control mechanism that maintains cell homeostasis and immune surveillance.

Blockage of TRAF6 Signaling Inhibits Osteoclast Formation and Tumor Cell through Blocking the NF-kB and JNK Pathways in Multiple Myeloma.

We have recently shown that silencing of tumor necrosis factor receptor-associated factor 6 (TRAF6) with a C-terminal siRNA inhibits proliferation and increases apoptosis of multiple myeloma (MM) tumor cells. In addition, TRAF6 ubiquitin ligase is also essential for receptor activator of nuclear factor kappa B ligand (RANKL) signaling and osteoclast differentiation. Based on TRAF6, CD40, and RANKL sequences and the TRAF6 interaction domain with CD40 or RANKL resides between residues 333 to 508, we cloned a sequence representing a 167 amino acid sequence from this domain in order to produce a TRAF6 dominant negative fragment (TRAF6dn) from the NIH gene bank (U78798) into the PCRII-TOPO vector. Subsequently, we re-cloned this fragment into an expression vector (pLenti6.2-hTRAF6dn). Expression of the TRAF6 dominant negative peptide was confirmed by Western blot analysis. We used human MM monocytes isolated by anti-CD14 micro-bead affinity column from MM patients' peripheral blood (PB) or bone marrow (BM). In order to quantify osteoclast formation, the cells were fixed and stained for tartrate resistant acid phosphatase following seven days of culture. The BM and PB CD14+ cells were cultured on slide-culture dishes at a density of 2 × 105 cells per well. The cells infected with the pLenti6.2-hTRAF6dn or with the control vector, pLenti6.2/GW/EmGFP, were treated with 50ng/ml RANKL and 10ng/ml mCSF at the beginning of the culture period, and these factors were added again during a medium change after three days of incubation. We found that the TRAF6dn vector significantly inhibited osteoclast cell formation of CD14+ cells induced by RANKL and mCSF in a concentration dependent fashion compared with the control group. Furthermore, we examined c-Jun, a component of the transcription factor complex AP-1, which binds and activates transcription at TRE/AP-1 elements. The results showed that total endogenous c-Jun is reduced after TRAF6dn blocks TRAF6 signaling whereas cells infected with the control vector showed no changes in c-Jun. We further examined the effects of TRAF6dn on MM cell growth and apoptosis. Both tumor cells from fresh MM BM and the U266 and MM1s cell lines showed decreased cell proliferation and increased apoptosis in the presence of the TRAF6dn vector at 72 hours whereas the control vector had no effect on MM tumor cell growth or apoptosis. Furthermore, the TRAF6dn vector led to marked decreases in phospho-NF-kB protein levels compared to the control vector. Thus, we have demonstrated that inhibition of TRAF6 with a dominant negative construct both inhibits MM cell growth as well as osteoclast formation, and also reduces NF-kB activation and c-Jun levels which likely results in decreased activation of TRE/AP-1 elements. These studies suggest that the inhibition of TRAF6 may be an excellent therapeutic target for multiple myeloma since its inhibition results in suppression of tumor growth as well as osteoclast formation.

Identification of TIFA as an Adapter Protein That Links Tumor Necrosis Factor Receptor-associated Factor 6 (TRAF6) to Interleukin-1 (IL-1) Receptor-associated Kinase-1 (IRAK-1) in IL-1 Receptor Signaling

Tumor necrosis factor receptor-associated factor 6 (TRAF6) transduces signals from members of the Toll/interleukin-1 (IL-1) receptor family by interacting with IL-1 receptor-associated kinase-1 (IRAK-1) after IRAK-1 is released from the receptor-MyD88 complex upon IL-1 stimulation. However, the molecular mechanisms underlying regulation of the IRAK-1/TRAF6 interaction are largely unknown. We have identified TIFA, a TRAF-interacting protein with a forkhead-associated (FHA) domain. The FHA domain is a motif known to bind directly to phosphothreonine and phosphoserine. In transient transfection assays, TIFA activates NFkappaBeta and c-Jun amino-terminal kinase. However, TIFA carrying a mutation that abolishes TRAF6 binding or mutations in the FHA domain that are known to abolish FHA domain binding to phosphopeptide fails to activate NFkappaBeta and c-Jun amino-terminal kinase. TIFA, when overexpressed, binds both TRAF6 and IRAK-1 and significantly enhances the IRAK-1/TRAF6 interaction. Furthermore, analysis of endogenous proteins indicates that TIFA associates with TRAF6 constitutively, whereas it associates with IRAK-1 in an IL-1 stimulation-dependent manner in vivo. Thus, TIFA is likely to mediate IRAK-1/TRAF6 interaction upon IL-1 stimulation.