Tumor Necrosis Factor Receptor-associated Factor 6 mRNA Research Articles

Embryonic Lethal Abnormal Visual-Like Protein 1 Aggravates Caerulein-Induced AR42J Cell Injury and Macrophage M1 Polarization to Accelerate Acute Pancreatitis by Upregulating TRAF6.

Tumor necrosis factor receptor-associated factor 6 (TRAF6) has been found to promote the progression of acute pancreatitis (AP). However, its underlying molecular mechanisms in AP need to be further revealed. Caerulein-induced AR42J cells were used to construct AP cell models. Cell viability and apoptosis were measured by Cell Counting Kit 8 assay and flow cytometry. Levels of inflammatory factors and oxidative stress-related markers were assessed. The medium of AR42J cells was collected for coculturing RAW264.7 cells. Macrophage marker CD86+ cell rates were checked with flow cytometry. The levels of TRAF6, embryonic lethal abnormal visual-like protein 1 (ELAVL1), and inducible nitric oxide synthase (iNOS) were examined by Western blot or quantitative real-time polymerase chain reaction. RNA immunoprecipitation assay was performed to evaluate the interaction between ELAVL1 and TRAF6. TRAF6 mRNA stability was tested using actinomycin D treatment. Caerulein treatment suppressed viability, induced AR42J cell apoptosis, inflammation, oxidative stress, and accelerated macrophage M1 polarization. TRAF6 downregulation could alleviate caerulein-induced AR42J cell injury and macrophage M1 polarization. ELAVL1 interacted with TRAF6 to stabilize its expression. Meanwhile, ELAVL1 knockdown relieved caerulein-induced AR42J cell injury and macrophage M1 polarization, while these effects were abolished by TRAF6 overexpression. TRAF6, stabilized by ELAVL1, promoted caerulein-induced AR42J cell injury and macrophage M1 polarization, suggesting that it might accelerate AP9 progression.

Expression and Clinical Significance of miR-146a and Tumor Necrosis Factor Receptor-Associated Factor 6 (TRAF6) in Myasthenia Gravis Patient Serum.

Objective To investigate the expression and clinical significance of miR-146a and tumor necrosis factor receptor-associated factor 6 (TRAF6) in myasthenia gravis (MG) patient serum. Methods The serum of 52 patients with MG and 60 healthy individuals was collected in our hospital. The expression of miR-146a and TRAF6 in serum was measured by real-time PCR (RT-PCR). Comparison among serum miR-146a and TRAF6 mRNA group clinical characteristics and assorted expressions was done with the correlation among the two groups evaluated. Logistics regression was used to analyze the effect of miR-146a and TRAF6 mRNA on MG development with the ROC curve applied for an investigation into the diagnostic role of miR-146a and TRAF6 mRNA expression in MG development. Results miR-146a and TRAF6 mRNA were significantly increased in the patients with MG compared with the healthy controls. Significant differences were identified in respiratory muscle endurance, muscle weakness level, vital capacity, and maximal voluntary ventilation between the two groups. Additionally, correlation analysis has discovered a positive correlation between miR-146a and TRAF mRNA expression in patients with MG. miR-146a and TRAF6 mRNA are independent MG occurrence factors exhibited by multivariate analysis while areas under ROC curve (AUCs) of miR-146a and TRAF6 mRNA in MG diagnosis were established by ROC curve analysis with results being 0.782 and 0.703, correspondingly. Conclusion miR-146a and TRAF6 mRNA are highly expressed in MG patients and can affect MG occurrence. miR-146a is a suitable candidate marker for diagnosing MG.

Effect of moxibustion at "Zusanli"(ST36) and "Shenshu"(BL23) on miR-155-mediated TLR4/NF-κB signaling involving amelioration of synovitis in rheumatoid arthritis rats

To explore the effect of moxibustion at "Zusanli"(ST36) and "Shenshu"(BL23) on synovitis, and expressions of miR-155, Toll-like receptor 4 (TLR4), myeloid differentiation factor 88 (MyD88), interlukine（IL-1） receptor-associated kinase (IRAK1), tumor necrosis factor receptor-associated factor 6 (TRAF6), nuclear factor κB (NF-κB), IL-1β, tumor necrosis factor receptor (TNF)-α and IL-6 mRNA and protein of synovial membrane in rheumatoid arthritis (RA) rats, so as to explore its mechanism underlying improvement of RA. A total of 48 male Wistar rats were randomly divided into normal control, model, moxibustion and antagonist groups (n=12 rats in each group). The RA model was replicated by placing the rats in a wind, cold and wet environment and injection of Freund's complete adjuvant (CFA, 0.5 mL) into the right hindlimb foot plantar. Moxibustion was applied to bilateral ST36 and BL23 for 30 min, once daily for 21 consecutive days. Rats of the antagonist group was treated by injection of TLR4 antagonist (TAK-242, 1 mg/mL, 0.1 mg/kg) via tail vein, once per day for consecutive 21 d. The joint swelling degree (JSD) and arthritis index (AI, red swelling scale) were determined, and the expression levels of various indicators of miR-155, and TLR4, myeloid MyD88, IRAK1, TRAF6, NF-κB, IL-1β, TNF-α and IL-6 mRNA and protein were assayed by quantitative real time-PCR and Western blot, separately. Compared with the normal control group, the JSD and AI, and the expression levels of synovial miR-155, TLR4, MyD88, IRAK1, TRAF6, NF-κB, IL-1β, TNF-α and IL-6 mRNA and protein were significantly increased in the model group (P<0.01). Compared with the model group, the increased levels of JSD and AI, and the expression levels of synovial miR-155, TLR4, MyD88, IRAK1, TRAF6, NF-κB, IL-1β, TNF-α and IL-6 mRNA and protein were notably down-regulated in both moxibustion and antagonist groups (P<0.01). The effects of moxibustion were evidently superior to the antagonist in down-regulating the abovementioned indexes (P<0.01), except TLR4 mRNA and protein. Moxibustion at ST36 and BL23 can reduce the synovitis of RA rats, which is related to its effects in suppressing the expressions of miR-155, TLR4, MyD88, IRAK1, TRAF6, NF-κB, IL-1β, TNF-α and IL-6 mRNA and protein (i.e., inhibition of miR-155/TLR4/NF-κB signaling).

Smoothened Stabilizes and Protects TRAF6 from Proteosomal Degradation: A Novel Non-Canonical Role of Smoothened with Implications in Lymphoma Biology

Tumor necrosis factor receptor-associated factor 6 (TRAF6), an (K63) E3-ligase, plays a crucial role in many biological processes and its activity is relevant in the biology of multiple cancers including diffuse large B cell lymphoma (DLBCL). Although molecules that trigger TRAF6 activation have been defined, those that stabilize TRAF6 levels and/or enhance TRAF6 function remain largely unclear. Previously, we found that activation of smoothened (SMO) with recombinant Hedgehog (Hh) ligand increased the binding between SMO with TRAF6, as well as TRAF6 protein levels (Blood 2013; 121:4718-28). In addition, transient overexpression of SMO resulted in increased K63-Ub of both TRAF6 and NEMO indicating stabilization of these proteins resulting in NF-kB activation. This is relevant, as more recently we found that TRAF6 amplifies pAKT signaling in DLBCL and that TRAF6 is the dominant E3 ligase for the K63-Ub of AKT in DLBCL. Moreover, TRAF6 recruitment to the cell membrane, and stabilization of its ubiquitination profile are facilitated by SMO. SMO is a member of the Frizzled-class G-protein-coupled receptor (GPCRs) and is traditionally known for its role as signal transducer in canonical Hedgehog (Hh) signaling. These observations prompted us to investigate whether the ability of SMO to increase TRAF6 levels is limited to ligand induced signaling, whether it contributes to chemoresistance in DLBCL cells, and whether SMO directly participates in controlling TRAF6 levels.To confirm the regulatory role of SMO in the TRAF6/AKT axis in DLBCL cells (HBL1 and HT) and further outline the nature of the underlying regulation, we measured the impact of activation of the Hh pathway with recombinant Shh ligand on TRAF6 levels, with and without SMO knockdown or recombinant SMO overexpression. Canonical Hh signaling results in the activation of the GLI1 transcription factor and the subsequent elevation of GLI1 mRNA levels is an established indicator of activation of the Hh pathway. However, neither SMO activation nor the knockdown of GLI1 had a significant impact on TRAF6 mRNA levels. These findings indicate that TRAF6 is not transcriptionally regulated by SMO signaling through GLI1 (canonical Hh signaling). In contrast, overexpression of SMO or siRNA knockdown of SMO resulted in an increase or decrease of TRAF6 protein levels, respectively. Consistent with the decrease of AKT activation (pAKT T308 and S473) after TRAF6 knockdown, the increase in TRAF6 levels that follows SMO overexpression resulted in an increase in the levels of AKT phosphorylation. Altogether, these observations suggest a post-translational regulation of TRAF6 by SMO. Indeed, stable knockdown of SMO dramatically reduces the half-life of TRAF6 in both HBL1 and HT cells in the presence of cyclohexamide. Furthermore, overexpression of SMO increases K63-Ub of both TRAF6 and AKT. In contrast, the SMO induced decrease in K48-Ub occurred only for TRAF6 but not for AKT. These data link the SMO-stimulated activation of TRAF6 to the enhancement of AKT signaling and protection of TRAF6 from proteasomal degradation. Mechanistically, we found that SMO, through its C-terminal tail, stabilizes TRAF6 and protects TRAF6 from proteosomal degradation, an effect mediated by ubiquitin-specific protease-8 (USP8). Importantly, this functional link between SMO and TRAF6 is reflected in DLBCL patient samples where high expression of both molecules correlates with poor prognosis.Resistance to DXR is a serious challenge in the treatment of DLBCL, and activated AKT is known to contribute to DXR resistance in multiple cancers including DLBCL. We evaluated whether SMO and TRAF6 support resistance to DXR in DLBCL cell lines. We exposed HT and HBL1 cells as well as their counterparts with stable knockdown of TRAF6 or SMO to DXR for 96hrs. Cell viability after exposure to DXR was determined by an Annexin V and PI staining assay. Silencing SMO or TRAF6 dramatically decreased cell survival after treatment with DXR.In summary, we report that SMO is needed to facilitate and maintain TRAF6-dependent elevated pAKT levels in DLBCL cell lines of germinal (GC) and non-GC subtypes, and that the SMO/TRAF6 axis contributes to DXR resistance in DLBCL. Our study reveals a novel and potential central cell survival signaling mechanism in which SMO stabilizes and protects TRAF6 from proteosomal degradation. DisclosuresLossos:Affimed: Research Funding.

TRAF6 regulates the effects of polarized maturation of tolerability: Marrow-derived dendritic cells on collagen-induced arthritis in mice.

The study aimed to investigate the relationship between tumor necrosis factor receptor-associated factor 6 (TRAF6) and a differentially mature dendritic cell (mDC) in collagen-induced arthritis (CIA) mice and to determine whether or not TRAF6 regulates the activation of an immature dendritic cell (iDC) and inhibits iDC maturation to induce immune tolerance. The mouse bone marrow stem cells were induced with recombinant granulocyte-macrophage colony-stimulating factor (rmGM-CSF) and recombinant interleukin-4 (rmIL-4) to differentiate immature dendritic cells (DCs), which were divided into four groups with different maturation states: rmGM-CSF, rmIL-4; TNF-α; LPS; and FK506 group. The levels of the cell surfaces of CD80, CD86, and MHI-II were analyzed by flow cytometry to prove DCs at different levels of maturity. The expression of IL-12 in DCs at different maturation states was detected by enzyme-linked immunosorbent assay (ELISA). The expression of TRAF6 mRNA and protein in each group of DCs was detected by a reverse transcription-polymerase chain reaction (RT-PCR) and western blot analysis. The results revealed that the differentiation of bone marrow cells into iDCs was significantly induced by cytokines (rmGM-CSF, IL-4). CD80, CD86, MHC-II were expressed in the four groups, and the difference between them was statistically significant (P<0.05). A higher degree of DC differentiation led to a gradual increase of IL-12 secretion in the four groups. The difference was statistically significant (P<0.05) for this secretion (group D, 10,620.73±276.73 pg/ml). The expression levels of TRAF6 mRNA were significantly higher in group D than those in the other three groups (P<0.01). Although there was no significant difference in the expression levels of TRAF6 mRNA between groups B and C, the expression levels of TRAF6 mRNA between groups B and C were higher than those of the control group. The TRAF6 protein expression was higher in group D than that in the other three groups (P<0.01), and the difference was statistically significant. There was a statistically significant difference in the TRAF6 protein expression between group A and groups B and C, but the expression in group C was higher than that in group B (P<0.01). In conclusion, the expression of co-stimulatory molecules gradually increased in the DCs of different maturation states, and the expression of IL-12, TRAF6 mRNA, and TRAF6 protein positively correlated with the degree of DC maturation. TRAF6 is important in iDC polarity and maturation.

Expression and significance of TRAF6 in rectus abdominis of gastric cancer patients

Objective To research the expression of tumor necrosis factor receptor-associated factor 6 (TRAF6) in rectus abdominis of gastric cancer patients and abdominal benign disease patients, and discuss the relationship between nutritional status and prognosis of gastric cancer patients with TRAF6. Methods Biopsies of the rectus abdominis were obtained intraoperatively from 64 gastric cancer patients and 53 subjects undergoing surgery for benign abdominal diseases. The expression levels of TRAF6 mRNA and protein were assessed through quantitative real time reverse transcriptase PCR (RT-QPCR) and Western blotting, and the relationships between nutritional status and prognosis of gastric cancer patients and the expression status of TRAF6 were analyzed. Results TRAF6 mRNA was significantly upregulated in muscle of stage Ⅰ (2.080±0.611), stage Ⅱ (2.126±0.640), stage Ⅲ (3.127±0.953), stage Ⅳ (3.289±0.734) gastric cancer compared with the control group (1.249±0.723), t=4.107, P=0.001; t=4.128, P=0.000; t=8.741, P=0.000; t=9.208, P=0.000. TRAF6 protein was significantly upregulated in muscle of stage Ⅰ (0.198±0.042), stage Ⅱ (0.209±0.051), stage Ⅲ (0.295±0.072), stage Ⅳ (0.345±0.084) gastric cancer compared with the control group (0.088±0.013), t=5.203, P=0.000; t= 5.642, P=0.000; t=9.351, P=0.000; t=9.854, P=0.000. TRAF6 was upregulated in 67.2% (43/64) of gastric cancer patients. The expression of TRAF6 in muscles of gastric cancer was associated with the percent of weight loss (χ2=12.231, P=0.000), the level of serum albumin (χ2=22.808, P=0.000) and the survival rate (χ2=3.933, P=0.047). Conclusion TRAF6 is significantly upregulated in rectus abdominis of gastric cancer, suggesting that TRAF6 may play an important role in progression and prognosis of cancer cachexia, and its overexpression suggests unfavorable prognosis. Key words: Stomach neoplasms; Rectus abdominis; Cachexia; Tumor necrosis factor receptor-asso-ciated factor 6

Relationship between TRAF6 and deterioration of HCC: an immunohistochemical and in vitro study

ObjectiveTo explore the relationship between tumor necrosis factor receptor-associated factor 6 (TRAF6) and the clinicopathological features in HCC as well as its biological function.MethodsTotally, 412 liver tissues were collected, including 171 hepatocellular carcinoma (HCC) and their corresponding non-tumor tissues, 37 cirrhosis and 33 normal liver tissues. The expression of TRAF6 was assessed by immunohistochemistry. Then, analysis of the correlations between TRAF6 expression and clinicopathological parameters in HCC was conducted. Furtherer, in vitro experiments on HepG2 and Hep3B cells were performed to validate the biological function of TRAF6 on HCC cells. TRAF6 siRNA was transfected into HepG2 and Hep3B cell lines and TRAF6 expression was evaluated with RT-qPCR and western blot. The assays of cell viability, proliferation, apoptosis and caspase-3/7 activity were carried out to investigate the effects of TRAF6 on HCC cells with RNA interference. Cell viability was assessed with Cell Titer-Blue kit. Cell proliferation was tested with MTS kit. Cell apoptosis was checked through morphologic detection with fluorescence microscope, as well as caspase-3/7 activity was measured with fluorogenic substrate detection.ResultsThe positive expression rate of TRAF6 protein was 49.7 % in HCC, significantly higher than that of normal liver (12.1 %), cirrhosis (21.6 %) and adjacent non-cancerous tissues (36.3 %, all P < 0.05). Upregulated TRAF6 was detected in groups with metastasis (Z = −2.058, P = 0.04) and with low micro-vessel density (MVD) expression (Z = −2.813, P = 0.005). Spearman correlation analysis further showed that the expression of TRAF6 was positively correlated with distant metastasis (r = 0.158, P = 0.039) and negatively associated with MVD (r = −0.249, P = 0.004). Besides, knock-down of TRAF6 mRNA in HCC cell lines HepG2 and Hep3B both resulted in cell viability and proliferation inhibition, also cell apoptosis induction and caspase-3/7 activity activation.ConclusionsTRAF6 may contribute to metastasis and deterioration of the HCC via influencing cell growth and apoptosis. Thus, TRAF6 might become a predictive and therapeutic biomarker for HCC.

Effect of TRAF6 Downregulation on Malignant Biological Behavior of Lung Cancer Cell Lines

背景与目的已有的研究提示肿瘤坏死因子受体相关因子6(tumor necrosis factor receptor-associated factor 6, TRAF6)在肺癌中常常扩增，可能扮演癌基因角色，但TRAF6的确切作用尚未充分阐明。本研究探索TRAF6表达对肺癌细胞株的增殖、凋亡、细胞周期、迁移及侵袭能力的影响以及可能作用机制。方法选用A549、H1650、SPC-A-1以及Calu-3等四种肺癌细胞株，应用蛋白印迹、qRT-PCR检测其TRAF6蛋白及mRNA表达。SPC-A-1、Calu-3细胞转染TRAF6 siRNA，以EMSA方法检测不同处理组核因子-κB的DNA结合活性，MTS法检测细胞增殖，流式细胞仪PI染色检测细胞凋亡，流式细胞仪进行细胞周期测定，划痕实验及Transwell小室法检测细胞迁移及侵袭能力，并应用蛋白印迹检测泛素化抗体、p65、CD24、CXCR4等蛋白表达。SPC-A-1细胞提取DNA后，应用二代测序法进行全基因组测序。结果在四种细胞株中，SPC-A-1和Calu-3细胞TRAF6相对高表达，TRAF6发生自身K63-泛素化，但仅在SPC-A-1细胞中观察到核因子-κB组成性活化。转染TRAF6 siRNA后，SPC-A-1、Calu-3细胞TRAF6表达明显下调，与空白组及对照组相比，下调TRAF6表达可抑制SPC-A-1细胞核因子-κB活性、降低迁移及侵袭能力以及促进细胞凋亡，CD24和CXCR4的表达也明显下调，但对细胞增殖及细胞周期无明显影响。下调TRAF6表达对Calu-3细胞株的核因子-κB活性、细胞增殖、凋亡、细胞周期、迁移及侵袭能力等均无明显影响。未发现SPC-A-1细胞株TRAF6基因突变或拷贝数改变。结论下调TRAF6表达可抑制SPC-A-1细胞迁移及侵袭能力，促进细胞凋亡，并且TRAF6可能是通过调控核因子-κB-CD24/CXCR4信号通路参与调控肺癌侵袭、细胞凋亡。

MiR-146b attenuates high-fat diet-induced non-alcoholic steatohepatitis in mice.

Non-alcoholic fatty liver disease (NAFLD) is one of the most common chronic liver diseases worldwide. In this study, we investigated the role of miR-146b in the Toll-like receptor-4 signaling pathway and high-fat diet (HFD)-induced NASH in vivo and in vitro. The effect of miR-146b on the expression of IL-1 receptor-associated kinase 1 (IRAK1) and tumor necrosis factor receptor-associated factor 6 (TRAF6) in RAW264.7 cells and HepG2 was studied, and the effect of miR-146b on lipid accumulation in HepG2 was also studied in vitro. The levels of IRAK1, TRAF6, NF-κB, and pro-inflammatory cytokines, as well as the histologic features and lipid accumulation in the livers of HFD-induced non-alcoholic steatohepatitis (NASH) and an miR-146b-administered HFD mouse model, were studied in vivo. After miR-146b administration, TRAF6 and IRAK1 mRNA and protein levels in macrophages after lipopolysaccharide administration and in HepG2 cells after oleic acid (OA) administration were significantly decreased in 146b group compared with control group (P < 0.001). The lipid accumulation in HepG2 cells exposed to OA was also decreased by inactivation of IRAK1 and TRAF6, then downregulation of the downstream molecules (NF-κB) and upregulation of the tension homolog deleted on chromosome 10 (PTEN) level. In vivo, after administration of miR-146b, TRAF6 and IRAK1 mRNA and protein levels as well as TNF-α and IL-6 mRNA and protein levels were decreased, and hematoxylin and eosin staining showed that the 146b group had low average adipose cell cross-sectional areas compared with control group. MiR-146b ameliorated HFD-induced NASH by directly suppressing IRAK1 and TRAF6.

Effect of elemene on the expressions of TRAF6 and caspase-8 in H22 liver tumor tissues of mice

Objective To investigate the effect of elemene on mRNA expressions of tumor necrosis factor (TNF) receptor associated factor 6 (TRAF6) and caspase-8 in tumor tissues of mice bearing hepatoma H22. Methods Forty BALB/c mice models bearing hepatoma H22 were established by subcutaneous inoculating tumor cells. Forty BALB/c mice were randomly divided into 4 groups: model group, low- and high-elemene dosage groups, and cisplatin group.The tumors after executing mice were weighted. The mRNA expressions of TRAF6 and caspase-8 in tumor tissues were detected by quantitative real-time reverse-transcription polymerase chain reaction (RT-PCR). Results The dosage of elemene could inhibit tumor growth. The inhibition ratio of cancer in the low- and high-elemene dosage and cisplatin group was 24.2%, 27.4%, and 28.2%, respectively. It reduced significantly tumor weights(P<0.01). Compared to the model group, the expression level of TRAF6 mRNA on tumors was decreased significantly, while the expression level of caspase-8 mRNA was increased significantly in the other groups(P<0.05). Conclusions The present results indicated that molecular mechanism of inhibition of liver cancer growth treated by elemene might be through down-regulating mRNA expressions of TRAF6 and caspase-8, promoting tumor cells apoptosis, and achieving the anti-tumor effect. Key words: Oils, volatile/PD; Liver neoplasms, experimental/DT/PA/ME; Tumor necrosis factor receptor-associated peptides and proteins/ME; Caspase 8/ME

Effect of tumor necrosis factor receptor associated factor 6 gene silencing by small interfering RNA on proliferation of odontoblast cells

To investigate the effect of small interfering RNA (siRNA)-mediated inhibition of tumor necrosis factor receptor associated factor 6(TRAF6) gene on murine odontoblast-like cell line, MDPC-23 cell and the effect of TRAF6 on MDPC-23 cell proliferation. The vectors expressing siRNA against TRAF6 were constructed and introduced into MDPC-23 cell with lipofectin , and the cell line with stable expression of siRNA of TRAF6 was obtained by G418 screening and colony culture. Reverse transcription-PCR (RT-PCR) and Western blotting were performed to detect the expression of TRAF6. The proliferation of transfected MDPC-23 cell was investigated through methabenzthiazuron (MTT) and flow cytometry (FCM) assay. The positive single colony was screened out, and was found to express siRNA against TRAF6 effectively because both TRAF6 mRNA and protein relative expression were significantly decreased in the experimental group (pSUPER-TRAF6siRNA: mRNA 0.163 ± 0.008, protein 0.215 ± 0.006) compared with controls (pSUPER: mRNA 0.778 ± 0.017, protein 0.964 ± 0.007 (P < 0.001). The A value of treated pSUPER-TRAF6siRNA cells (3 d: 0.46 ± 0.03, 5 d: 1.35 ± 0.06) was increased compared with controls (P<0.01). The result of proliferation index (PrI) was also increased compared with controls [pSUPER- TRAF6siRNA: (24.1 ± 2.2)%; pSUPER (11.2 ± 1.0)%; control (10.5 ± 0.7)%, P < 0.01]. The transcription and expression of TRAF6 gene were inhibited. The proliferation ability was increased in MDPC-23 cells by the constructed pSUPER-TRAF6siRNA vector. It may further influence the formation and repair of dentin, and may be involved in the regulation of normal tooth eruption and process of dentin repair after injury.

Change of Toll-like receptors in children with acute Epstein-barr virus infection

Objective To investigate the possible role of Toll-like receptors (TLRs) in immunological pathogenesis of Epstein-barr virus(EBV) infection. Methods Fifteen children with acute EBV infection, 18 children with infectious mononucleosis (IM), and 25 age-matched healthy children were enrolled in the study.Reverse-transcription polymerase chain reaction and real-time PCR were used to evaluate the levels of TLR2, TLR3, TLR7, TLR9, myeloid differentiation factor 88 (MyD88), tumor necrosis factor receptor-associated factor 6 (TRAF6), TGF-β activated kinase 1 (TAK1), TIR domain-containing adaptor protein inducing interferon β (TRIF), tumor necrosis factor receptor-associated factor 3 (TRAF3), TBK1, IL-1β, TNF-α, IFN-α and IFN-β mRNA expression in peripheral blood mononuclear cells(PBMC). The plasma concentrations of cytokines such as IL-12 and interferon gamma (IFN-γ) were determined by enzyme-linked immunosorbent assay. Results 1.Compared with healthy control group, the expression levels of TLR2, TLR3, TLR7, TLR9, MyD88, TRAF6, TAK1, TRIF, TRAF3 and TBK1 mRNA were up-regulated significantly from the children with acute EBV infection and IM (all P 0.05).2.The levels of cytokines expression such as IL-1β, TNF- α, IFN-α and IFN-β in children with acute EBV infection were higher than those of the healthy control group (all P<0.05).3.The plasma concentrations of IL-12 and IFN-γ were up-regulated (all P<0.05) in children with acute EBV infection. Conclusion Imbalance of antiviral /inflammatory response resulting from the aberrant activation of TLRs may be one of the factors causing disturbed immunological function in vivo infected by EBV. Key words: Epstein-barr virus; Infectious mononucleosis; Toll-like receptor

Expression of TRAF6 and ubiquitin mRNA in skeletal muscle of gastric cancer patients

ObjectiveTo investigate the prognostic significance of tumor necrosis factor receptor (TNFR),-associated factor 6 (TRAF6),-and ubiquitin in gastric cancer patients.MethodsBiopsies of the rectus abdominis muscle were obtained intra operatively from 102 gastric cancer patients and 29 subjects undergoing surgery for benign abdominal diseases, and muscle TRAF6 and ubiquitin mRNA expression and proteasome proteolytic activities were assessed.ResultsTRAF6 was significantly upregulated in muscle of gastric cancer compared with the control muscles. TRAF6 was upregulated in 67.65% (69/102) muscle of gastric cancer. Over expression of TRAF6 in muscles of gastric cancer were associated with TNM stage, level of serum albumin and percent of weight loss. Ubiquitin was significantly upregulated in muscle of gastric cancer compared with the control muscles. Ubiquitin was upregulated in 58.82% (60/102) muscles of gastric cancer. Over expression of ubiquitin in muscles of gastric cancer were associated with TNM (Tumor-Node-Metastasis) stage and weight loss. There was significant relation between TRAF6 and ubiquitin expression.ConclusionsWe found a positive correlation between TRAF6 and ubiquitin expression, suggesting that TRAF6 may up regulates ubiquitin activity in cancer cachexia. While more investigations are required to understand its mechanisms of TRAF6 and ubiquitin in skeletal muscle. Correct the catabolic-anabolic imbalance is essential for the effective treatment of cancer cachexia.

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.

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.

Hyaluronan reduces inflammation in experimental arthritis by modulating TLR-2 and TLR-4 cartilage expression

Previous studies have reported that low molecular mass HA and highly polymerized HA respectively elicited pro- and anti-inflammatory responses by modulating the toll-like receptor 4 (TLR-4) and the TLR-2. The activation of TLR-4 and TLR-2 mediated by collagen-induced arthritis (CIA) induces the myeloid differentiation primary response protein (MyD88) and the tumor necrosis factor receptor-associated factor 6 (TRAF6), and ends with the liberation of NF-kB which, in turn, stimulates pro-inflammatory cytokine production. The aim of this study was to investigate the influence of high molecular weight HA at different concentrations on TLR-4 and TLR-2 modulation in CIA in mice. Arthritis was induced in mice via intradermal injection of an emulsion containing bovine type II collagen in complete Freund's adjuvant. Mice were treated with HA intraperitoneally daily for 30 days. CIA increased TLR-4, TLR-2, MyD88 and TRAF6 mRNA expression and the related protein in the cartilage of arthritic joints. High levels of both mRNA and related protein were also detected for tumor necrosis factor alpha (TNF-α), interleukin 1-beta (IL-1-β), interleukin-17 (IL-17), matrix metalloprotease-13 (MMP-13) and inducible nitric oxide synthase (iNOS) in the joint of arthritic mice. HA treatment significantly limited CIA incidence and decreased all the parameters up-regulated by CIA. The improvement of biochemical parameters was also supported by histological analysis, plasma and synovial fluid HA levels. These results suggest that the TLR-4 and TLR-2 play an important role in the arthritis mechanism and the interaction/block of HA at high molecular mass may reduce inflammation and cartilage injury.

Characterization of apoptosis and proliferation in esophageal carcinoma EC109 cells following siRNA-induced down-regulation of TRAF6

Tumor necrosis factor receptor-associated factor 6 (TRAF6) is an activator of the NF-κB transcription factor. NF-κB is involved in a variety of inflammatory, anti-apoptotic, and gene regulatory pathways and was recently found to be over-expressed in esophageal cancer cells. Here we investigated the function of TRAF6 in the esophageal cancer cell line EC109. siRNA targeting TRAF6 was introduced into EC109 cells and TRAF6 mRNA and protein levels were subsequently examined via RT-PCR and western blotting. Rates of apoptosis and cell proliferation were also measured using flow cytometry, ethynyl deoxyuridine (EdU), and CCK-8 (Cell Counting Kit-8) assays. The real-time PCR array was applied to profile the expression of TRAF6 related genes. TRAF6-siRNA reduced TRAF6 mRNA and protein expressions. NF-κB p65 protein expression was decreased in TRAF6-targeting siRNA-transfected cells compared to cells of the negative control. TRAF6-siRNA also significantly inhibited proliferation and enhanced apoptosis of EC109 cells. These studies suggested that TRAF6 was required for NF-κB activation in EC109 cells and it may be a good molecular target for suppressing the survival and proliferation of esophageal cancer cells.