Tumor Necrosis Factor Receptor-associated Factor 6 Ubiquitination Research Articles

Astragaloside IV relieves passive heymann nephritis and podocyte injury by suppressing the TRAF6/NF-κb axis

The pathogenesis of membranous nephropathy (MN) involves podocyte injury that is attributed to inflammatory responses induced by local immune deposits. Astragaloside IV (AS-IV) is known for its robust anti-inflammatory properties. Here, we investigated the effects of AS-IV on passive Heymann nephritis (PHN) rats and TNF-α-induced podocytes to determine the underlying molecular mechanisms of MN. Serum biochemical parameters, 24-h urine protein excretion and renal histopathology were evaluated in PHN and control rats. The expression of tumor necrosis factor receptor associated factor 6 (TRAF6), the phosphorylation of nuclear factor kappa B (p-NF-κB), the expression of associated proinflammatory cytokines (TNF-α, IL-6 and IL-1β) and the ubiquitination of TRAF6 were measured in PHN rats and TNF-α-induced podocytes. We detected a marked increase in mRNA expression of TNF-α, IL-6 and IL-1β and in the protein abundance of p-NF-κB and TRAF6 within the renal tissues of PHN rats and TNF-α-induced podocytes. Conversely, there was a reduction in the K48-linked ubiquitination of TRAF6. Additionally, AS-IV was effective in ameliorating serum creatinine, proteinuria, and renal histopathology in PHN rats. This effect was concomitant with the suppression of NF-κB pathway activation and decreased expression of TNF-α, IL-6, IL-1β and TRAF6. AS-IV decreased TRAF6 levels by promoting K48-linked ubiquitin conjugation to TRAF6, which triggered ubiquitin-mediated degradation. In summary, AS-IV averted renal impairment in PHN rats and TNF-α-induced podocytes, likely by modulating the inflammatory response through the TRAF6/NF-κB axis. Targeting TRAF6 holds therapeutic promise for managing MN.

Rnd3 suppresses endothelial cell pyroptosis in atherosclerosis through regulation of ubiquitination of TRAF6.

As the main pathological basis for various cardiovascular and cerebrovascular diseases, atherosclerosis has become one of the leading causes of death and disability worldwide. Emerging evidence has suggested that Rho GTPase Rnd3 plays an indisputable role in cardiovascular diseases, although its function in atherosclerosis remains unclear. Here, we found a significant correlation between Rnd3 and pyroptosis of aortic endothelial cells (ECs). ApoeKO mice were utilized as a model for atherosclerosis. Endothelium-specific transgenic mice were employed to disrupt the expression level of Rnd3 in vivo. Mechanistic investigation of the impact of Rnd3 on endothelial cell pyroptosis was carried out using liquid chromatography tandem mass spectrometry (LC-MS/MS), co-immunoprecipitation (Co-IP) assays, and molecular docking. Evidence from gain-of-function and loss-of-function studies denoted a protective role for Rnd3 against ECs pyroptosis. Downregulation of Rnd3 sensitized ECs to pyroptosis under oxidized low density lipoprotein (oxLDL) challenge and exacerbated atherosclerosis, while overexpression of Rnd3 effectively prevented these effects. LC-MS/MS, Co-IP assay, and molecular docking revealed that Rnd3 negatively regulated pyroptosis signaling by direct interaction with the ring finger domain of tumor necrosis factor receptor-associated factor 6 (TRAF6). This leads to the suppression of K63-linked TRAF6 ubiquitination and the promotion of K48-linked TRAF6 ubiquitination, inhibiting the activation of NF-κB and promoting the degradation of TRAF6. Moreover, TRAF6 knockdown countered Rnd3 knockout-evoked exacerbation of EC pyroptosis in vivo and vitro. These findings establish a critical functional connection between Rnd3 and the TRAF6/NF-κB/NLRP3 signaling pathway in ECs, indicating the essential role of Rnd3 in preventing pyroptosis of ECs.

MiRNA-Induced Downregulation of IPMK in Macrophages Mediates Lipopolysaccharide-Triggered TLR4 Signaling.

Inositol polyphosphate multikinase (IPMK) is a pleiotropic enzyme responsible for the production of inositol polyphosphates and phosphoinositide. IPMK in macrophages was identified as a key factor for the full activation of the Toll-like receptor 4 (TLR4) signaling pathway and inflammation by directly interacting with tumor necrosis factor receptor-associated factor 6 (TRAF6). Here, dynamic changes of IPMK levels in lipopolysaccharide (LPS)-stimulated macrophages and their functional significance were investigated. Both the mRNA and protein levels of IPMK were acutely decreased in mouse and human macrophages when cells were stimulated with LPS for between 1 and 6 h. Analysis of the 3' untranslated region (UTR) of mouse IPMK mRNA revealed a highly conserved binding site for miR-181c. Transfection of miR-181c mimics into RAW 264.7 macrophages led to decreased IPMK 3'UTR-luciferase reporter activity and lowered endogenous IPMK levels. When the genomic deletion of a 33-bp fragment containing a putative miR-181c-binding site was introduced within the IPMK 3'UTR of RAW 264.7 macrophages (264.7Δ3'UTR), LPS-triggered downregulation of IPMK levels was prevented. LPS treatment in 264.7Δ3'UTR macrophages decreased TLR4-induced signaling and the expression of proinflammatory cytokines. In response to LPS stimulation, K63-linked ubiquitination of TRAF6 was impaired in 264.7Δ3'UTR macrophages, suggesting an action of IPMK in the suppression of TRAF6 activation. Therefore, our findings reveal that LPS-mediated suppression of IPMK regulates the full activation of TLR4 signaling and inflammation in macrophages.

TRAF6 regulates the signaling pathway influencing colorectal cancer function through ubiquitination mechanisms.

Tumor necrosis factor receptor‐associated factor‐6 (TRAF6) is a ubiquitin E3 ligase. TRAF6 plays an important role in tumor invasion and metastasis. However, the specific mechanism by which TRAF6 promotes colorectal cancer (CRC) metastasis is incompletely understood. This study aimed to determine whether TRAF6 affects the LPS‐NF‐κB‐VEGF‐C signaling pathway through ubiquitination, which plays a role in colorectal cancer metastasis. Here, our results showed that TRAF6 affected lymphangiogenesis through the LPS‐NF‐κB‐VEGF‐C signaling pathway. Using ubiquitination experiments, we found that TRAF6 was mainly ubiquitinated with the K63‐linked chains, and LPS promoted ubiquitination of TRAF6 and K63‐linked chains. More importantly, TRAF6 124mut is the main ubiquitination site of TRAF6 interacting with K63‐linked chains. TRAF6 affected the migration, invasion, and lymphatic metastasis of colorectal cancer through its ubiquitination. In subcutaneous xenograft models, TRAF6 124mut inhibited tumor growth. In conclusion, our results provide new insight for studying the mechanism of lymphangiogenesis in colorectal cancer to promote cancer metastasis, which may provide new ideas for tumor immunotherapy.

Novel clerodane-type diterpenoid Cintelactone A suppresses lipopolysaccharide -induced inflammation by promoting ubiquitination, proteasomal degradation of TRAF6

Cellular inflammation is the underlying cause of several diseases and development of a safe and effective anti-inflammatory drug is need-of-the hour for treatment of diseases like lung inflammation. Callicarpa integerrima Champ. is a well-known herbal medicine with hemostatic and anti-inflammatory functions. However, the exact ingredient exhibiting anti-inflammatory activity in C. integerrima Champ. is largely unknown. Here, we first isolated, purified and characterized a novel clerodane-type diterpenoid Cintelactone A (CA) from C. integerrima Champ. We demonstrated that CA could significantly inhibit lipopolysaccharide (LPS)-induced pro-inflammatory cytokines and mediators production both in mouse peritoneal macrophages and THP1 cells. Consistently, CA also relieved inflammation and reduced LPS-induced lung injury in mice. We systematically elucidated the mechanism of action as well. CA interacted with Arg78 of tumor necrosis factor receptor-associated factor 6 (TRAF6) by hydrogen bonding. It further promoted the K48-linked ubiquitination and proteasomal degradation of TRAF6, and suppressed the activation of NF-κB and MAPKs signaling pathways. Collectively, our study reveals that new clerodane-type diterpenoid CA suppresses LPS-induced inflammation by promoting TRAF6 degradation, suggesting that CA as the potential therapeutic candidate for the treatment of inflammation associated diseases.

P22077 inhibits LPS-induced inflammatory response by promoting K48-linked ubiquitination and degradation of TRAF6.

Inflammation is a biological process associated with multiple human disorders such as autoimmune diseases and metabolic diseases. Therefore, alleviation of inflammation is important for disease prevention or treatment. Recently, deubiquitinating enzymes (DUBs), especially ubiquitin specific protease-7 (USP7) attracts increasing attention as a potential drug target for inflammation. As an inhibitor of USP7, P22077 has been used to study the roles of USP7 in inflammatory response and neuroblastoma growth. However, the role and precise mechanism of P22077 in anti-inflammatory is still indistinct. In this study, we demonstrated that P22077 could attenuate the release of pro-inflammatory factors including TNF-α, IL-1β, IL-6 and NO, suppress mRNA expression of COX-2 and iNOS, and inhibit activation of NF-κB and MAPKs signaling pathways in Raw264.7 cells and mouse peritoneal macrophages after LPS stimulation. In vivo study showed that P22077 could relieve inflammatory response and reduce the lung injury in C57BL/6 mice with LPS-induced endotoxemia. Mechanically, P22077 might play an anti-inflammatory role by promoting tumor necrosis factor receptor-associated factor 6 (TRAF6) degradation via K48-linked polyubiquitination. These findings provide a rationale for the role of the P22077 in anti-inflammatory pathway and the promising clinical application of P22077 to treat inflammatory diseases.

Pellino1 regulates neuropathic pain as well as microglial activation through the regulation of MAPK/NF-\u03baB signaling in the spinal cord

BackgroundSpinal cord microglia plays a crucial role in the pathogenesis of neuropathic pain. However, the mechanisms underlying spinal microglial activation during neuropathic pain remain incompletely determined. Here, we investigated the role of Pellino1 (Peli1) and its interplay with spinal microglial activation in neuropathic pain.MethodsIn this study, we examined the effects of Peli1 on pain hypersensitivity and spinal microglial activation after chronic constriction injury (CCI) of the sciatic nerve in mice. The molecular mechanisms involved in Peli1-mediated hyperalgesia were determined by western blot, immunofluorescence, quantitative polymerase chain reaction (qPCR), and enzyme-linked immunosorbent assay (ELISA). We utilized immunoprecipitation to examine the ubiquitination of tumor necrosis factor receptor-associated factor 6 (TRAF6) following CCI. In addition, we explored the effect of Peli1 on BV2 microglial cells in response to lipopolysaccharide (LPS) challenge.ResultsWe found that CCI induced a significant increase in the levels of Peli1, which was present in the great majority of microglia in the spinal dorsal horn. Our results showed that spinal Peli1 contributed to the induction and maintenance of CCI-induced neuropathic pain. The biochemical data revealed that CCI-induced Peli1 in the spinal cord significantly increased mitogen-activated protein kinase (MAPK) phosphorylation, activated nuclear factor kappa B (NF-κB), and enhanced the production of proinflammatory cytokines, accompanied by spinal microglial activation. Peli1 additionally was able to promote K63-linked ubiquitination of TRAF6 in the ipsilateral spinal cord following CCI. Furthermore, we demonstrated that Peli1 in microglial cells significantly enhanced inflammatory reactions after LPS treatment.ConclusionThese results suggest that the upregulation of spinal Peli1 is essential for the pathogenesis of neuropathic pain via Peli1-dependent mobilization of spinal cord microglia, activation of MAPK/NF-κB signaling, and production of proinflammatory cytokines. Modulation of Peli1 may serve as a potential approach for the treatment of neuropathic pain.

The deubiquitinase OTUB1 augments NF-\u03baB-dependent immune responses in dendritic cells in infection and inflammation by stabilizing UBC13

Dendritic cells (DCs) are indispensable for defense against pathogens but may also contribute to immunopathology. Activation of DCs upon the sensing of pathogens by Toll-like receptors (TLRs) is largely mediated by pattern recognition receptor/nuclear factor-κB (NF-κB) signaling and depends on the appropriate ubiquitination of the respective signaling molecules. However, the ubiquitinating and deubiquitinating enzymes involved and their interactions are only incompletely understood. Here, we reveal that the deubiquitinase OTU domain, ubiquitin aldehyde binding 1 (OTUB1) is upregulated in DCs upon murine Toxoplasmagondii infection and lipopolysaccharide challenge. Stimulation of DCs with the TLR11/12 ligand T. gondii profilin and the TLR4 ligand lipopolysaccharide induced an increase in NF-κB activation in OTUB1-competent cells, resulting in elevated interleukin-6 (IL-6), IL-12, and tumor necrosis factor (TNF) production, which was also observed upon the specific stimulation of TLR2, TLR3, TLR7, and TLR9. Mechanistically, OTUB1 promoted NF-κB activity in DCs by K48-linked deubiquitination and stabilization of the E2-conjugating enzyme UBC13, resulting in increased K63-linked ubiquitination of IRAK1 (IL-1 receptor-associated kinase 1) and TRAF6 (TNF receptor-associated factor 6). Consequently, DC-specific deletion of OTUB1 impaired the production of cytokines, in particular IL-12, by DCs over the first 2 days of T. gondii infection, resulting in the diminished production of protective interferon-γ (IFN-γ) by natural killer cells, impaired control of parasite replication, and, finally, death from chronic T.encephalitis, all of which could be prevented by low-dose IL-12 treatment in the first 3 days of infection. In contrast, impaired OTUB1-deficient DC activation and cytokine production by OTUB1-deficient DCs protected mice from lipopolysaccharide-induced immunopathology. Collectively, these findings identify OTUB1 as a potent novel regulator of DCs during infectious and inflammatory diseases.

OTUD4 alleviates hepatic ischemia-reperfusion injury by suppressing the K63-linked ubiquitination of TRAF6

Hepatic ischemia-reperfusion (IR) injury can cause serious liver damage, leading to liver dysfunction after liver surgery, which is associated with NF-κB-mediated inflammation. The K63-linked auto-polyubiquitination of tumor necrosis factor receptor-associated factor 6 (TRAF6) is essential for the activation of NF-κB. Here, we found that OTU domain-containing protein 4 (OTUD4), a deubiquitinating enzyme (DUB), interacts with TRAF6 and decreases the K63 auto-polyubiquitination of TRAF6. In addition, the data showed that NF-κB activation was impaired and inflammatory factor levels were reduced after overexpressing OTUD4 in a hypoxia/reoxygenation (HR) model and a hepatic IR model. Additionally, the liver inflammatory response and tissue damage were ameliorated in mice overexpressing OTUD4.Taken together, these results show that OTUD4 can negatively regulate NF-κB activation by suppressing the K63-linked ubiquitination of TRAF6, thus alleviating hepatic ischemia-reperfusion injury.

TRAF6-TAK1 signaling drives Th9 differentiation

Abstract Naive CD4+ T cells differentiate into different subsets depending upon milieu of cytokine exposure. Interleukin-9 (IL-9)-producing CD4+ helper T cells (Th9 cells) are induced from naive CD4+ T cells by transforming growth factor-β (TGF-β) together with interleukin-4 (IL-4) during stimulation of the T cell antigen receptor (TCR) in vitro. The master regulator of Th9 cells as well as the molecular mechanisms by which TGF-β signals and IL-4 signals co-operatively drive the differentiation of Th9 cells are not fully understood. Previously, our group has reported that inhibitor of DNA-binding 3 (Id3), a transcription factor which inhibits DNA-binding of basic helix-loop-helix protein, such as E2A, controls the TGF-β-mediated differentiation of Th9 cells. We found that this process required activation of the kinase transforming growth factor beta-activated kinase 1 (TAK1). In addition, RNA-seq revealed that tumor necrosis factor receptor-associated factor 6 (TRAF6) was highly up-regulated by TGF-β together with IL-4 in CD4+ T cells. Since TRAF6 is one of the TAK-1 activator, we hypothesized TRAF6 is also involved in the differentiation of Th9 cells. We found that the expression and ubiquitination of TRAF6 was upregulated under the Th9 differentiation condition and it resulted in activation of TAK1. These regulations were attenuated both in TGF-β and Smad3 deficient mice, suggesting TRAF6-TAK1 signaling is involved in TGF-β-mediated Th9 differentiation.

Bortezomib enhances radiosensitivity in oral cancer through inducing autophagy-mediated TRAF6 oncoprotein degradation

BackgroundOral squamous cell carcinoma (OSCC) is a malignant tumor that may occur anywhere within the oral cavity. The survival rate of OSCC patients has not improved over the past decades due to its heterogeneous etiology, genetic aberrations, and treatment outcomes. We investigated the role of tumor necrosis factor receptor-associated factor 6 (TRAF6) in OSCC cells treated with bortezomib (a proteasome inhibitor) combined with irradiation (IR) treatment.MethodsThe effects of combined treatment in OSCC cells were investigated using assays of cell viability, autophagy, apoptosis, western blotting, and immunofluorescence staining. The ubiquitination of proteins was analyzed by immunoprecipitation. Stable knockdown of TRAF6 in OSCC cells was constructed with lentivirus. The xenograft murine models were used to observe tumor growth.ResultsWe found synergistic effects of bortezomib and IR on the viability of human oral cancer cells. The combination of bortezomib and IR treatment induced autophagic cell death. Furthermore, bortezomib inhibited IR-induced TRAF6 ubiquitination and inhibited TRAF6-mediated Akt activation. Bortezomib reduced TRAF6 protein expression through autophagy-mediated lysosomal degradation. TRAF6 played an oncogenic role in tumorigenesis of human oral cancer cells and oral tumor growth was suppressed by bortezomib and IR treatment. In addition, OSCC patients with expression of TRAF6 showed a trend towards poorer cancer-specific survival when compared with patients without TRAF6 expression.ConclusionsA combination of a proteasome inhibitor, IR treatment and TRAF6 inhibition could be a novel therapeutic strategy in OSCC.

The Ubiquitin E3 Ligase TRAF6 Exacerbates Ischemic Stroke by Ubiquitinating and Activating Rac1.

Stroke is one of the leading causes of morbidity and mortality worldwide. Inflammation, oxidative stress, apoptosis, and excitotoxicity contribute to neuronal death during ischemic stroke; however, the mechanisms underlying these complicated pathophysiological processes remain to be fully elucidated. Here, we found that the expression of tumor necrosis factor receptor-associated factor 6 (TRAF6) was markedly increased after cerebral ischemia/reperfusion (I/R) in mice. TRAF6 ablation in male mice decreased the infarct volume and neurological deficit scores and decreased proinflammatory signaling, oxidative stress, and neuronal death after cerebral I/R, whereas transgenic overexpression of TRAF6 in male mice exhibited the opposite effects. Mechanistically, we demonstrated that TRAF6 induced Rac1 activation and consequently promoted I/R injury by directly binding and ubiquitinating Rac1. Either functionally mutating the TRAF6 ubiquitination site on Rac1 or inactivating Rac1 with a specific inhibitor reversed the deleterious effects of TRAF6 overexpression during I/R injury. In conclusion, our study demonstrated that TRAF6 is a key promoter of ischemic signaling cascades and neuronal death after cerebral I/R injury. Therefore, the TRAF6/Rac1 pathway might be a promising target to attenuate cerebral I/R injury.SIGNIFICANCE STATEMENT Stroke is one of the most severe and devastating neurological diseases globally. The complicated pathophysiological processes restrict the translation of potential therapeutic targets into medicine. Further elucidating the molecular mechanisms underlying cerebral ischemia/reperfusion injury may open a new window for pharmacological interventions to promote recovery from stroke. Our study revealed that ischemia-induced tumor necrosis factor receptor-associated factor 6 (TRAF6) upregulation binds and ubiquitinates Rac1 directly, which promotes neuron death through neuroinflammation and neuro-oxidative signals. Therefore, precisely targeting the TRAF6-Rac1 axis may provide a novel therapeutic strategy for stroke recovery.

Tumor Necrosis Factor Receptor-Associated Factor 6 (TRAF6) Mediates Ubiquitination-Dependent STAT3 Activation upon Salmonella enterica Serovar Typhimurium Infection.

Salmonella enterica serovar Typhimurium can inject effector proteins into host cells via type III secretion systems (T3SSs). These effector proteins modulate a variety of host transcriptional responses to facilitate bacterial growth and survival. Here we show that infection of host cells with S Typhimurium specifically induces the ubiquitination of tumor necrosis factor receptor-associated factor 6 (TRAF6). This TRAF6 ubiquitination is triggered by the Salmonella pathogenicity island 1 (SPI-1) T3SS effectors SopB and SopE2. We also demonstrate that TRAF6 is involved in the SopB/SopE2-induced phosphorylation of signal transducer and activator of transcription 3 (STAT3), a signaling event conducive to the intracellular growth of S Typhimurium. Specifically, TRAF6 mediates lysine-63 ubiquitination within the Src homology 2 (SH2) domain of STAT3, which is an essential step for STAT3 membrane recruitment and subsequent phosphorylation in response to S Typhimurium infection. TRAF6 ubiquitination participates in STAT3 phosphorylation rather than serving as only a hallmark of E3 ubiquitin ligase activation. Our results reveal a novel strategy in which S Typhimurium T3SS effectors broaden their functions through the activation of host proteins in a ubiquitination-dependent manner to manipulate host cells into becoming a Salmonella-friendly zone.

Trim13 Potentiates Toll-Like Receptor 2-Mediated Nuclear Factor κB Activation via K29-Linked Polyubiquitination of Tumor Necrosis Factor Receptor-Associated Factor 6.

Ubiquitination is a versatile post-translational modification involved in nuclear factor-κB (NF-κB) activation of Toll-like receptor (TLR) signaling. Here, we demonstrated that Trim13, an E3 ubiquitin ligase, is up-regulated in macrophages upon stimulation with TLR2 ligand. Knockdown of Trim13 attenuated TLR2-mediated production of cytokines/chemokines and formation of foam cells as well as activation of NF-κB. Trim13 interacts with tumor necrosis factor receptor-associated factor 6 (TRAF6) and potentiates NF-κB activity via ubiquitination of TRAF6. Overexpression of inactive mutant (C10/13A) or really interesting new gene (RING) deletion mutant of Trim13 did not potentiate ubiquitination of TRAF6 or activation of NF-κB. These results suggest that the effects of Trim13 are dependent on its E3 ligase activity. Trim13 used K29-linked polyubiquitin chains for TRAF6 ubiquitination to promote NF-κB activity and thus potentiated activation of TLR2-mediated immune responses. Our data identify Trim13 as a positive regulator of NF-κB activation and suggest that K29-linked polyubiquitination is a specific ubiquitin-linked pattern involved in the control of TLR2 signaling.

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.

SiRNA-induced TRAF6 knockdown promotes the apoptosis and inhibits the invasion of human lung cancer SPC-A1 cells.

Tumor necrosis factor receptor-associated factor 6 (TRAF6) has been found to be involved in multiple cancers. However, the effect of small interfering RNA (siRNA)-induced knockdown of TRAF6 on the biological behaviors of cancer cells remains unknown. Thus, the present study aimed to investigate the effect of siRNA-induced knockdown of TRAF6 on the biological behaviors of human lung cancer SPC-A1 cells. The expression of TRAF6 was determined in human lung adenocarcinoma A549, non-small cell lung cancer H1650, human airway epithelial Calu-3 and human lung cancer SPC-A1 cell lines using quantitative RT-PCR (qRT-PCR) and western blotting at the transcriptional and translational levels. TRAF6 expression was knocked down in the SPC-A1 cells using an siRNA technique, and the effects of TRAF6 knockdown on NF-κB activity, cell proliferation, apoptosis, cell cycle, invasion and migration of the SPC-A1 cells were determined using electrophoretic mobility shift assay (EMSA), cell proliferation assay, flow cytometry, Transwell invasion assay and scratch wound assay. In addition, the protein expression of CD24, CXCR4, MMP1, MMP2, MMP9, TWIST, TIMP-2 and Slug was quantified using western blotting assay. Western blotting and qRT-PCR assays showed upregulation of TRAF6 at both the translational and transcriptional levels in the Calu-3 and SPC-A1 cells, and K63-linked ubiquitination of TRAF6 and constitutive NF-κB activation were detected in the SPC-A1 cells. Knockdown of TRAF6 inhibited the migration and invasion and promoted the apoptosis of the SPC-A1 cells, but had little effect on cell proliferation and the cell cycle. In addition, siRNA-induced TRAF6 knockdown caused a marked reduction in the protein expression of CD24 and CXCR4, but had little effect on MMP-1, MMP-2, MMP-9, Twist, TIMP-2 or Slug expression. The present study demonstrated that TRAF6 is upregulated in human lung cancer cells, and siRNA-induced TRAF6 knockdown inhibits the invasion of lung cancer cells and promotes apoptosis. It is suggested that TRAF6 may be a promising target for the therapy of lung cancer.

Phosphoinositide-dependent kinase-1 inhibits TRAF6 ubiquitination by interrupting the formation of TAK1–TAB2 complex in TLR4 signaling

Phosphoinositide-dependent protein kinase 1 (PDK1) plays a key role in the phosphoinositide 3-kinase (PI3K)–PDK1–Akt pathway that induces cell survival and cardiovascular protections through anti-apoptosis, vasodilation, anti-inflammation, and anti-oxidative stress activities. Although several reports have proposed the negative role of PDK1 in Toll-like receptor 4 (TLR4) signaling, the molecular mechanism is still unknown. Here we show that PDK1 inhibits tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF6) ubiquitination by interrupting the complex between transforming growth factor beta-activated kinase 1 (TAK1) and TAK1 binding protein 2 (TAB2), which negatively regulates TAK1 activity. The overexpression of PDK1 in 293/TLR4 cells resulted in suppressions of nuclear factor kappa B (NF-κB) activation and production of proinflammatory cytokines including interleukin (IL)-6 and TNF-α in response to lipopolysaccharide stimulation. Conversely, THP-1 human monocytes transiently cultured in low glucose medium displayed down-regulated PDK1 expression, and significantly enhanced TLR4-mediated signaling for the activation of NF-κB, demonstrating a negative role of PDK1. Biochemical studies revealed that PDK1 significantly interacted with TAK1, resulting in the inhibition of the association of TAB2 with TAK1, which led to the attenuation of TRAF6 ubiquitination. Moreover, PDK1-knockdown THP-1 cells displayed enhancement of downstream signals, activation of NF-κB, and increased production of pro-inflammatory cytokines IL-6, IL-1β, and TNF-α, which potentially led to the up-regulation of NF-κB-dependent genes in response to TLR4 stimulation. Collectively, the results demonstrate that PDK1 inhibits the formation of the TAK1–TAB2–TRAF6 complex and leads to the inhibition of TRAF6 ubiquitination, which negatively regulates the TLR4-mediated signaling for NF-κB activation.

Cylindromatosis (CYLD) inhibits Streptococcus pneumonia-induced plasminogen activator inhibitor-1 expression via interacting with TRAF-6

Streptococcus pneumoniae (S. p) remains one of the foremost causes of community-acquired pneumonia. Recent studies have shown that S. p lung infection is associated with plasminogen activator inhibitor-1 (PAI-1) expression, which inhibits acute lung injury. Such effects by S. p were negatively regulated by cylindromatosis (CYLD). The current study explored the underlying mechanisms. We showed that S. p-induced PAI-1 expression requires tumor necrosis factor receptor-associated factor 6 (TRAF-6) signaling. Si-RNA-mediated knockdown of TRAF-6 remarkably inhibited S. p-induced PAI-1 expression. Reversely, over-expression of wild type (wt-) TRAF-6 further potentiated PAI-1 expression in S. p-treated cells. We provided evidences to support that CYLD-mediated anti-PAI-1 activity might be through direct regulation of TRAF-6. Our results from co-immunoprecipitation (co-IP) and confocal microscopy assays confirmed a direct association between the CYLD and TRAF-6 in A549 cells. Over-expression of wt-CYLD remarkably inhibited TRAF-6 ubiquitination and subsequent PAI-1 expression. Introducing a mutated CYLD, on the other hand, enhanced TRAF-6 ubiquitination and PAI-1 expression. Together, these results indicate that TRAF-6 mediates S. p-induced PAI-1 expression, and CYLD inhibits PAI-1 expression probably through deubiquitinating TRAF-6. The current study provided molecular insights of CYLD-mediated activities in S. p-induced PAI-1 expression and possible acute lung injury.

TRAF6 is functional in inhibition of TLR4-mediated NF-κB activation by resveratrol

Resveratrol was suggested to inhibit Toll-like receptor (TLR)4-mediated activation of nuclear factor-κB (NF-κB) and Toll/interleukin-1 receptor domain-containing adaptor inducing interferon-β (TRIF)–(TANK)-binding kinase 1, but the myeloid differentiation primary response gene 88–tumor necrosis factor receptor-associated factor 6 (TRAF6) pathway is not involved in this effect. However, involvement of TRAF6 in this process is still elusive since cross talk between TRIF and TRAF6 has been reported in lipopolysaccharide (LPS)-induced signaling. Using RAW 264.7 macrophages, we determined the effect of resveratrol on LPS-induced TRAF6 expression, ubiquitination as well as activation of mitogen-activated protein (MAP) kinases and Akt in order to elucidate its involvement in TLR4 signaling. LPS-induced transient elevation in TRAF6 mRNA and protein expressions is suppressed by resveratrol. LPS induces the ubiquitination of TRAF6, which has been reported to be essential for Akt activation and for transforming growth factor-β activated kinase-1–NAP kinase kinase 6 (MKK6)-mediated p38 and c-Jun N-terminal kinase (JNK) activation. We found that resveratrol diminishes the effect of LPS on TRAF6 ubiquitination and activation of JNK and p38 MAP kinases, while it has no effect on the activation of extracellular-signal-regulated kinase (ERK)1/2. The effect of resveratrol on MAP kinase inhibition is significant since TRAF6 activation was reported to induce activation of JNK and p38 MAP kinase while not affecting ERK1/2. Moreover, Akt was identified previously as a direct target of TRAF6, and we found that, similarly to MAPKs, phosphorylation pattern of Akt followed the activation of TRAF6, and it was inhibited by resveratrol at all time points. Here, we provide the first evidence that resveratrol, by suppressing LPS-induced TRAF6 expression and ubiquitination, attenuates the LPS-induced TLR4–TRAF6, MAP kinase and Akt pathways that can be significant in its anti-inflammatory effects.

Mechanism of TRAF6 Intranuclear Translocation

Proper cellular response to external signals requires exact spatiotemporal positioning of intracellular components. Adaptor molecules are used to assemble protein complexes to facilitate signaling pathways. We studied the interplay between two adaptor molecules, tumor necrosis factor receptor associated factor‐6 (TRAF6) and Syntenin (Syn‐1). TRAF6 transduces signaling for the TNF and the Toll‐like/Interleukin receptor families. Many studies focused on cytoplasmic TRAF6, but few report on the inhibition of TRAF6 and the mechanisms by which it enters the nucleus. We find that overexpression of Syn‐1 not only attenuates TRAF6 signaling, but causes nuclear localization in both HEK293 and RAW246.7 cells. TRAF6 is an E3‐ligase whose activity we previously reported is maintained by auto‐ubiquitination and an open conformation. Knockdown of Syn‐1 potentiates TRAF6 activation and ubiquitination. Syn‐1 exhibits reduced effect on TRAF6 mutants that cannot be ubiquitinated. This implies that Syn‐1 binds to TRAF6 in a closed state. Protein domain deletions in both TRAF6 and Syn‐1 show that the full‐length molecules are not necessary for interaction. Mutagenesis or deletion of a newly identified TRAF Interaction motif (TIM) in Syn‐1 reveals differences in how Syn‐1 affects TRAF6 signaling and localization. Syn‐1 also localizes to the nucleus when overexpressed with either wild type or a constitutively active TRAF6. Lastly, Syn‐1 binds to the transcription factor Sox4 resulting in Sox4‐Syn‐1 relocalization to the nucleus. We have observed that overexpression of Sox4 also induces TRAF6 nuclear translocation, providing a mechanism for TRAF6 entry into the nucleus.