... (more)

lymph nodes, mammary glands, skin and the central nervous system. It is a member of a group of six closely related TRAF proteins, which serve as adapter molecules, coupling the TNF receptor (TNFR) superfamily to intracellular signaling events. Among the TRAF proteins, TRAF6 is unique in that, in addition to mediating TNFR family signaling, it is also essential for signaling downstream of an unrelated family of receptors, the interleukin-1 (IL-1) receptor/Toll-like receptor (IL-1R/TLR) superfamily. Gene targeting experiments have identified several indispensable physiological functions of TRAF6, and structural and biochemical studies have revealed the potential mechanisms of its action. By virtue of its many signaling roles, TRAF6 represents an important target in the regulation of many disease processes, including immunity, inflammation and osteoporosis.

... (more)

cer has not been extensively investigated. This study analyzed the effects of TRAF6 on the proliferation, apoptosis, cell cycle, migration, and invasion capability of lung cancer cell lines, as well as the potential molecular mechanisms involved.
METHODS: To address the expression of TRAF6 in lung cancer cells, four lung cancer cell lines (A549, H1650, SPC-A-1 and Calu-3) were assayed to determine the expression of TRAF6 protein by Western blot and TRAF6 mRNA via qRT-PCR. Moreover, siRNA targeting TRAF6 was introduced into SPC-A-1 and Calu-3 cells. Nuclear factor-¿B (NF-¿B) DNA-binding activity, apoptosis rates, cell proliferation, cell cycle, migration, and invasion were determined by electrophoretic mobility shift assay, flow cytometry, MTS assay, flow cytometry, scratch test, and transwell chamber assay, respectively. Western blot analysis was also performed to evaluate the expression of the following proteins through K63-ubiquitination: P65, CD24 and CXCR4. Whole-genome sequencing analysis was conducted using a second-generation sequencer in SPC-A-1 cells.
RESULTS: TRAF6 was highly up-expressed in SPC-A-1 and Calu-3 cell lines than the other two cells, which also showed K63-ubiquitinization in TRAF6. However, constitutive activation of NF-¿B was observed only in SPC-A-1 lung cancer cells. Downregulation of TRAF6 suppressed the NF-¿B activation, cell migration, and invasion but promoted the cell apoptosis of SPC-A-1 cells. Markedly decreased expression of CD24 and CXCR4 was observed in SPC-A-1 cells transfected by TRAF6 siRNA. Nevertheless, TRAF6 downregulation did not affect the proliferation and cell cycle of SPC-A-1 cells. Additionally, TRAF6 regulation did not affect the proliferation, apoptosis, cell cycle, migration, and invasion of Calu-3 cells. No mutations and no changes in gene copy numbers of TRAF6 were found by whole-exome sequencing of SPC-A-1 cells.
CONCLUSIONS: TRAF6 may be involved in cell migration, invasion, and apoptosis of SPC-A-1 cells, possibly through regulating the NF-¿B-CD24/CXCR4 pathway.¿.

... (more)

n can be stimulated by TNF-alpha in a time-dependent manner in mouse VSMCs. Knockdown of TRAF6 by siTRAF6 significantly attenuated agonist-CD137mAb induced increase of NF-kappaB p65 and NFATc1 in VSMCs. Pretreatment with a NF-kappaB 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-kappaB p65 pathways in VSMCs. These findings provide a novel target for treatment of atherosclerosis.

... (more)

viously described as a key component of epigenetic signaling machinery. We found that in response to microbial stimuli, MYSM1 accumulated in the cytoplasm where it interacted with and inactivated TRAF3 and TRAF6 complexes to terminate PRR pathways for pro-inflammatory and type I interferon responses. Consequently, Mysm1 deficiency in mice resulted in hyper-inflammation and enhanced viral clearance but also susceptibility to septic shock. We identified two motifs in MYSM1 that were essential for innate immune suppression: the SWIRM domain that interacted with TRAF3 and TRAF6 and the metalloproteinase domain that removed K63 polyubiquitins. This study identifies MYSM1 as a key negative regulator of the innate immune system that guards against an overzealous self-destructive immune response.

... (more)

actor 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 K d 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.

... (more)

ate immune and inflammatory responses induced by LPS. This study examined whether miR-146a will have a protective effect on sepsis-induced cardiac dysfunction. Lentivirus-expressing miR-146a (LmiR-146a) or lentivirus-expressing scrambled miR (LmiR-control) was delivered into the myocardium via the right carotid artery. Seven days after transfection, mice were subjected to cecal ligation and puncture (CLP). Untransfected mice were also subjected to CLP-induced sepsis. Cardiac function was examined by echocardiography before and 6 h after CLP. In vitro studies showed that increased miR-146a levels suppress LPS-induced IκBα phosphorylation and inflammatory cytokine production in both H9C2 cardiomyocytes and J774 macrophages. In vivo transfection of LmiR-146a attenuated sepsis-induced cardiac dysfunction. The values for percent ejection fraction and percent fractional shortening in LmiR-146a-transfected CLP mice were significantly greater than in untransfected CLP control. LmiR-146a transfection prevented sepsis-induced NF-κB activity, suppressed IRAK and TRAF6 expression in the myocardium, and attenuated sepsis-induced inflammatory cytokine production in both plasma and peritoneal fluid. In addition, LmiR-146a transfection decreased sepsis-induced infiltration of neutrophils and macrophages into the myocardium. LmiR-146a can also transfect macrophages in the periphery. We conclude that miR-146a attenuates sepsis-induced cardiac dysfunction by preventing NF-κB activation, inflammatory cell infiltration, and inflammatory cytokine production via targeting of IRAK and TRAF6 in both cardiomyocytes and inflammatory monocytic cells.

... (more)

re, we found that DAT inhibited nuclear factor-kappaB (NF-kappaB) signaling and induced apoptosis in primary effusion lymphoma (PEL), a subtype of non-Hodgkin's B-cell lymphoma caused by Kaposi's sarcoma-associated herpesvirus (KSHV). We examined the cytotoxic effects of DAS, DAD and DAT on PEL cells. DAT significantly reduced the viability of PEL cells compared with uninfected B-lymphoma cells, and induced the apoptosis of PEL cells by activating caspase-9. DAT induced stabilization of IkappaBalpha, and suppressed NF-kappaB transcriptional activity in PEL cells. We examined the mechanism underlying DAT-mediated IkappaBalpha stabilization. The results indicated that DAT stabilized IkappaBalpha by inhibiting the phosphorylation of IkappaBalpha by the IkappaB kinase (IKK) complex. Furthermore, DAT induced proteasomal degradation of TRAF6, and DAT suppressed IKKbeta-phosphorylation through downregulation of TRAF6. It is known that activation of NF-kappaB is essential for survival of PEL cells. In fact, the NF-kappaB inhibitor BAY11-7082 induced apoptosis in PEL cells. In addition, DAT suppressed the production of progeny virus from PEL cells. The administration of DAT suppressed the development of PEL cells and ascites in SCID mice xenografted with PEL cells. These findings provide evidence that DAT has antitumor activity against PEL cells in vitro and in vivo, suggesting it to be a novel therapeutic agent for the treatment of PEL.

... (more)

of a new missense mutation of NEMO (A323P) which causes a severe form of incontinentia pigmenti (OMIM#308300), an inherited disease characterized predominantly by skin inflammation. The A323P mutation was found to impair TNF-, IL-1-, LPS- and PMA/ionomycin-induced NF-kappaB activation, as well as to disrupt TRAF6-dependent NEMO polyubiquitination, due to a defective NEMO/TRAF6 interaction. Mutagenesis identified the affected ubiquitination sites as three lysine residues located in the vicinity of A323. Unexpectedly, these lysines were ubiquitinated together with two previously identified lysines not connected to TRAF6. Mutation of all these ubiquitination sites severely impaired NF-kappaB activation induced by stimulation with IL-1, LPS, Nod2/RICK or serum/LPA. In contrast, mutation at all of these sites had only a limited effect on stimulation by TNF. These findings indicate that post-translational modification of NEMO through K63-linked polyubiquitination is a key event in IKK activation and that perturbation of this step may cause human pathophysiology.

... (more)

s factor receptor-associated factor 6 (TRAF6), known as a key adaptor protein downstream of TLR4, is involved in the inflammatory response by activating multiple apoptotic signaling pathways. However, mechanisms of the suppressor of cytokine signaling-1 (SOCS-1) in regulating cell inflammation and apoptosis are still obscure.
OBJECTIVES: To investigate the TLR4-TRAF6 signaling pathway in intestinal ischemia and reperfusion injury, as well as SOCS-1 expression after ischemic preconditioning in the rat intestine.
METHODS: The small bowel ischemia, ischemia-reperfusion, and preconditioning models were induced using ligation of the superior mesenteric artery in male Sprague-Dawley rats; then, the mRNA and protein levels of TLR4, TRAF6, and SOCS-1 were analyzed using real-time PCR, Western blot, and immunohistochemistry, respectively.
RESULTS: The expression of TLR4 and TRAF6 was gradually increased with increasing intestinal ischemia duration, but increased substantially after ischemia-reperfusion injury. After ischemic preconditioning, TLR4 and TRAF6 expressions decreased; however, expression of SOCS-1 and the TLR4-TRAF6 pathway inhibitor was increased.
CONCLUSION: These data show that ischemic preconditioning may induce the activation of SOCS-1 to inhibit the TLR4-TRAF6 signaling pathway, thereby playing a protective role in ischemia-reperfusion injury.

... (more)

d Ki-67 index in 147 human glioma specimens, but positively correlated with patients' survival. Furthermore, two distinct subgroups of patients with grade I-IV gliomas with different prognoses were identified according to miR-146b-5p expression in our specimens. Cox regression showed that miR-146b-5p was an independent predictor for patients' survival. Overexpression of miR-146b-5p dramatically suppressed glioma cell proliferation and induced apoptosis. Mechanistically, we validated TRAF6 as a direct functional target of miR-146b-5p and found that miR-146b-5p overexpression significantly decreased phosphorylated TAK1 and IkappaBalpha, the pivotal downstream effectors of TRAF6. Moreover, TRAF6 expression was positively correlated with glioma grades and Ki-67 index but inversely correlated with miR-146b-5p expression and predicted poor prognosis of glioma patients. In glioblastoma cell lines, silencing of TRAF6 could mimic the anti-tumor effect of miR-146b-5p. Our findings identify miR-146b-5p as a tumor suppressor and novel prognostic biomarker of gliomas, and suggest miR-146b-5p and TRAF6 as potential therapeutic candidates for malignant gliomas.

... (more)

s long been used in clinical practice for cardioprotection. This study aimed to investigate whether the effects of exosomes derived from nicorandil pretreated MSC (MSCNIC-exo) could be enhanced in facilitating cardiac repair after acute myocardial infarction (AMI).
METHODS: MSCNIC-exo and MSC-exo were collected and injected into the border zone of infarcted hearts 30 minutes after coronary ligation in rats. Macrophage polarization was detected 3 days post-infarction, cardiac function as well as histological pathology were measured on the 28th day after AMI. Macrophages were separated from the bone marrow of rats for in vitro model. Exosomal miRNA sequencing was conducted to identify differentially expressed miRNAs between MSCNIC-exo and MSC-exo. MiRNA mimics and inhibitors were transfected to MSCs or macrophages to explore the specific mechanism.
RESULTS: Compared to MSC-exo, MSCNIC-exo showed superior therapeutic effects on cardiac functional and structural recovery after AMI and markedly elevated the ratio of CD68+ CD206+/ CD68+cells in infarcted hearts 3 days post-infarction. The notable ability of MSCNIC-exo to promote macrophage M2 polarization was also confirmed in vitro. Exosomal miRNA sequencing and both in vivo and in vitro experiments identified and verified that miR-125a-5p was an effector of the roles of MSCNIC-exo in vivo and in vitro. Furthermore, we found miR-125a-5p promoted macrophage M2 polarization by inhibiting TRAF6/IRF5 signaling pathway.
CONCLUSION: This study suggested that MSCNIC-exo could markedly facilitate cardiac repair post-infarction by promoting macrophage M2 polarization by upregulating miR-125a-5p targeting TRAF6/IRF5 signaling pathway, which has great potential for clinical translation.

... (more)

e. Here, using public data from the International Inflammatory Bowel Disease Genetics Consortium (IIBDGC) on the most recent genome-wide association studies (GWAS) for ulcerative colitis (UC) and Crohn's disease (CD), we found that genetic variants of the NUR77 gene are associated with increased risk for both UC and CD. Accordingly, Nur77 expression was significantly reduced in colon tissues from patients with UC or CD and mice treated with DSS. Nur77 deficiency increased the susceptibility of mice to DSS-induced experimental colitis and prevented intestinal recovery, whereas treatment with cytosporone B (Csn-B), an agonist for Nur77, significantly attenuated excessive inflammatory response in the DSS-induced colitis mouse model. Mechanistically, NUR77 acts as a negative regulator of TLR-IL-1R signalling by interacting with TRAF6. This interaction prevented auto-ubiquitination and oligomerization of TRAF6 and subsequently inhibited NF-kappaB activation and pro-inflammatory cytokine production. Taken together, our GWAS-based analysis and in vitro and in vivo studies have demonstrated that Nur77 is an important regulator of TRAF6/TLR-IL-1R-initiated inflammatory signalling, and loss of Nur77 may contribute to the development of IBD, suggesting Nur77 as a potential target for the prevention and treatment of IBD.

... (more)

rts 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-kappaB) activation and production of proinflammatory cytokines including interleukin (IL)-6 and TNF-alpha 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-kappaB, 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-kappaB, and increased production of pro-inflammatory cytokines IL-6, IL-1beta, and TNF-alpha, which potentially led to the up-regulation of NF-kappaB-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-kappaB activation.

... (more)

ctivation of NF-kappaB and interferon production. TRIM25 is required for the full activation of NF-kappaB at the downstream of MAVS, while it is not involved in IRF3 nuclear translocation. Mechanical studies showed that TRIM25 is involved in TRAF6-mediated NF-kappaB activation. These collectively indicate that TRIM25 plays an additional role in RIG-I/MDA5 signaling other than RIG-I ubiquitination via activation of NF-kappaB.

... (more)

dai virus (SeV)-induced activation of the IFN-beta promoter. Knockdown of endogenous RNF166, but not other RNFs, inhibited the IFN-beta production induced by SeV and encephalomyocarditis virus. RNF166 interacted with TRAF3 and TRAF6. SeV-induced ubiquitination of TRAF3 and TRAF6 was suppressed when endogenous RNF166 rather than RNF114/138 was knocked down. These findings suggest that RNF166 positively regulates RNA virus-triggered IFN-beta production by enhancing the ubiquitination of TRAF3 and TRAF6.

... (more)

ctions to facilitate K63-polyubiquitination of TRAF6 and thereby mediates nerve growth factor-induced activation of the NF-kappaB pathway. In brain of p62 knock-out mice we did not recover polyubiquitinated TRAF6. The UBA domain binds polyubiquitin chains and deletion of p62-UBA domain or mutation of F406V within the ubiquitin binding pocket of the UBA domain abolished TRAF6 polyubiquitination. Likewise, deletion of p62 N-terminal dimerization domain or the TRAF6 binding site had similar effects on both polyubiquitination and oligomerization of TRAF6. Nerve growth factor treatment of PC12 cells induced TRAF6 polyubiquitination along with formation of a p62-TRAF6-IKKbeta-PKC iota signal complex, while inhibition of the p62/TRAF6 interaction had an opposite effect. These results provide evidence for a mechanism whereby p62 serves to regulate the NF-kappaB pathway.

... (more)

activation of TGF-beta. However, the detailed mechanisms underlying the activation of these signaling pathways are not entirely known. Herein, we hypothesized that Ang II could promote the differentiation of CSCs into cardiac muscle-like cells by non-canonical TGF-beta/TRAF6 signaling pathway, and sought to test the hypothesis. C-kit(+) CSCs were isolated from neonatal Sprague Dawley (SD) rats, and their c-kit status was confirmed with immunofluorescence staining. A TGF-beta type I receptor inhibitor (SB431542) was used to inhibit SMAD2/3 phosphorylation. The small interfering RNA (siRNA)-mediated knockdown of TRAF6 was used to investigate the role of TRAF6 in TGF-beta signaling. Rescue of TRAF6 siRNA transfected cells with a 3'UTR-deleted siRNA insensitive construct was performed to rule out any off-target effects of the siRNA. TRAF6 dominant-negative (TRAF6DN) vector was constructed and used to infect c-kit(+) CSCs. Our results showed that the increase in JNK and p38 activation by Ang-II was blocked by siRNA. After transfection by TRAF6-siRNA or Ad-TRAF6, the cardiac specific markers and Wnt signaling proteins were tested by Western blotting. Physical interactions between TRAF6 and TGF-beta receptors were studied by co-immunoprecipitation. Forced expression of TRAF6 enhanced the expression of cTnT and Cx-43 but inhibited the expression of Wnt3a.Our data suggested that TRAF6 mediated Ang II-induced differential responses in c-kit(+) CSCs via the non-canonical TGF-beta signaling pathway.

... (more)

in which TGFbeta type I receptor (TbetaRI) is activated by the E3 ligase TNF-receptor-associated factor 6 (TRAF6) via the Lys63-linked polyubiquitination of TbetaRI. TRAF6 also contributes to activation of TNF-alpha-converting enzyme and presenilin-1, resulting in the proteolytic cleavage of TbetaRI and releasing the intracellular domain of TbetaRI, which is translocated to the nucleus to promote tumor invasiveness. In this report, we provide evidence that Lys178 of TbetaRI is polyubiquitinated by TRAF6. Moreover, our data suggest that TRAF6-mediated Lys63-linked ubiquitination of the TbetaRI intracellular domain is a prerequisite for TGFbeta regulation of mRNA for cyclin D1 (CCND1), expression, as well as for the regulation of other genes controlling the cell cycle, differentiation, and invasiveness of prostate cancer cells.

... (more)

phenotypes, whereas the pentose phosphate pathway plays an essential role in cell proliferation through the production of dihydronicotinamide adenine dinucleotide phosphate. OBJECTIVE: To identify the relationship between dihydronicotinamide adenine dinucleotide phosphate production and SM22alpha activity in the development and progression of vascular diseases. METHODS AND RESULTS: We showed that the expression and activity of glucose-6-phosphate dehydrogenase (G6PD) are promoted in platelet-derived growth factor (PDGF)-BB-induced proliferative VSMCs. PDGF-BB induced G6PD membrane translocation and activation in an SM22alpha K21 ubiquitination-dependent manner. Specifically, the ubiquitinated SM22alpha interacted with G6PD and mediated G6PD membrane translocation. Furthermore, we found that tumor necrosis factor receptor-associated factor (TRAF) 6 mediated SM22alpha K21 ubiquitination in a K63-linked manner on PDGF-BB stimulation. Knockdown of TRAF6 decreased the membrane translocation and activity of G6PD, in parallel with reduced SM22alpha K21 ubiquitination. Elevated levels of activated G6PD consequent to PDGF-BB induction led to increased dihydronicotinamide adenine dinucleotide phosphate generation through stimulation of the pentose phosphate pathway, which enhanced VSMC viability and reduced apoptosis in vivo and in vitro via glutathione homeostasis. CONCLUSIONS: We provide evidence that TRAF6-induced SM22alpha ubiquitination maintains VSMC survival through increased G6PD activity and dihydronicotinamide adenine dinucleotide phosphate production. The TRAF6-SM22alpha-G6PD pathway is a novel mechanism underlying the association between glucose metabolism and VSMC survival, which is beneficial for vascular repair after injury but facilitates atherosclerotic plaque stability.

... (more)

tor-associated factor 6 (TRAF6) levels by ELISA in serum of 36 HCV viremia patients and 42 age and gender matched healthy controls.
RESULTS: miRNA146a expression was significantly higher in HCV patients with a best cut off value 1.63 to discriminate between HCV patients and healthy controls. Meanwhile, it was negatively correlated to IRAK1 and TRAF6 levels and positively correlated to viral load in HCV patients.
CONCLUSIONS: miRNA146a has a potential role in HCV infection and viral replication through IRAK1 and TRAF6. It can also serve as a new screening method for HCV.