Expression Of Tumor Necrosis Factor Receptor-associated Factor 6 Research Articles

Increased expression of microRNA-146a decreases myocardial ischaemia/reperfusion injury

We have reported that either toll-like receptor 4 deficiency (TLR4(-/-)) or TLR2 modulation protects against myocardial ischaemia/reperfusion (I/R) injury. The mechanisms involve attenuation of I/R-induced nuclear factor KappaB (NF-κB) activation. MicroRNA-146a (miR-146a) has been reported to target interleukin-1 receptor-associated kinase 1 (IRAK1) and tumor necrosis factor (TNF) receptor associated factor 6 (TRAF6), resulting in inhibiting NF-κB activation. This study examined the role of microRNA-146a in myocardial I/R injury. We constructed lentivirus expressing miR-146a (LmiR-146a). LmiR-146a was transfected into mouse hearts through the right common carotid artery. The lentivirus vector (LmiR-Con) served as vector control. Untransfected mice served as I/R control. Sham operation served as sham control. Seven days after transfection, the hearts were subjected to ischaemia (60 min) followed by reperfusion (4 h). Myocardial infarct size was analysed by triphenyltetrazolium chloride (TTC) staining. In separate experiments, the hearts were subjected to ischaemia (60 min) followed by reperfusion for up to 7 days. Cardiac function was measured by echocardiography prior to I/R, 3 and 7 days after myocardial I/R. LmiR-146a transfection significantly decreased I/R-induced myocardial infarct size by 55% and prevented I/R-induced decreases in ejection fraction (EF%) and fractional shortening (%FS). LmiR-146a transfection attenuated I/R-induced myocardial apoptosis and caspase-3/7 and -8 activities. LmiR-146a transfection suppresses IRAK1 and TRAF6 expression in the myocardium. In addition, transfection of LmiR-146a prevented I/R-induced NF-κB activation and inflammatory cytokine production. MicroRNA-146a protects the myocardium from I/R injury. The mechanisms may involve attenuation of NF-κB activation and inflammatory cytokine production by suppressing IRAK1 and TRAF6.

Effect of TRAF6 on the biological behavior of human lung adenocarcinoma cell

Tumor necrosis factor receptor-associated factor 6 (TRAF6) is a unique adaptor protein of the tumor necrosis factor receptor-associated factor family that mediates both tumor necrosis factor receptor and interleukin-1 receptor/Toll-like receptor signaling. A recent study showed that TRAF6 played an important role in tumorigenesis and invasion through activation of nuclear factor kappa B (NF-κB). However, the biological role of TRAF6 remains unknown in lung cancer up to now. To address the expression of TRAF6 in lung cancer cells, four lung cancer cell lines (A549, HCC827, NCI-H292, and 95-D) and human bronchial epithelial cells were used to detect the expression of TRAF6 protein by western blotting. Results indicated that TRAF6 displayed an upregulation in human lung cancer cell lines. To investigate the effects of TRAF6 on the biological behavior of human lung adenocarcinoma cell, we generated human lung adenocarcinoma A549 cell line in which TRAF6 was depleted. The results showed that downregulation of TRAF6 could decrease cell viability, suppress cell proliferation and invasion, and promote cell apoptosis. At the same time, we explored the effects of TRAF6 on the expression of the following proteins: phosphor-NF-κB (p-p65), cyclin D1, caspase-3, and matrix metalloproteinase 9 (MMP9). Downregulation of TRAF6 could decrease the expression of p-p65, cyclin D1, and MMP9 and increase the expression of caspase-3. All these results suggested that TRAF6 might be involved in the potentiation of growth, proliferation, and invasion of A549 cell line, as well as the inhibition of A549 cell apoptosis by the activation of NF-κB. To make a long story short, the overexpression of TRAF6 might be related to the tumorigenesis and invasion of lung cancer.

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.

TNF receptor-associated factor 6 in advanced non-small cell lung cancer: clinical and prognostic implications

Tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF6) represents a central point of convergence for the signal transduction by the TNFR and the IL-lR/TLR superfamilies. We conducted this retrospective clinical study focusing on TRAF6 expression associated with overall survival and chemotherapeutical sensitivity in a large population with advanced non-small cell lung cancer (NSCLC). A total of 324 patients with stage III and IV NSCLC were retrospectively enrolled. Immunohistochemistry was performed to evaluate the expression of TRAF6, apoptosis-related proteins Bcl-2, Bax, Fas, and FasL, as well as the density of CD8(+) and FOXP3(+) tumor infiltrating lymphocytes (TILs) in tumor microenvironment. A total of 193 carcinomas (59.6 %) were identified as high expression of TRAF6. TRAF6 expression was not significantly related with histology and clinic stage. No obvious correlation of TRAF6 expression with apoptosis-related protein and TILs density was identified. TRAF6 status was correlated inversely with response to chemotherapy in overall patients (response rates 24.9 and 32.8 %, for patients with high-TRAF6 and low-TRAF6 tumors, respectively, P = 0.039). However, multivariate logistic regression analysis could not identify TRAF6 status as an independent predictor for the response to chemotherapy in overall cohort (95 % CI: 0.91-3.32, P = 0.065). The overall survival was not significantly associated with TRAF6 expression (P = 0.616). Our results provide new insight for the biological properties and clinical relevance of the TRAF6 in NSCLC. TRAF6 is a promise target for therapeutic strategies against cancer.

TWEAK and TRAF6 regulate skeletal muscle atrophy

To discuss the roles and mechanisms of action of tumor necrosis factor-like weak inducer of apoptosis (TWEAK) and tumor necrosis factor receptor-associated factor 6 (TRAF6) in skeletal muscle atrophy. Proinflammatory cytokines are known to mediate muscle atrophy in many chronic disease states. However, their role in the loss of skeletal muscle mass in disuse conditions has just begun to be elucidated. Further, the initial signaling events leading to the activation of various catabolic pathways in skeletal muscle under different atrophic conditions are also less well understood. The TWEAK-Fn14 system has now been identified as a novel inducer of skeletal muscle wasting. Adult skeletal muscles express minimal levels of Fn14, the bona fide TWEAK receptor. Specific conditions of atrophy such as denervation, immobilization, or unloading rapidly induce the expression of Fn14 leading to TWEAK-induced activation of various proteolytic pathways in skeletal muscle. Recent studies have also demonstrated that the expression and activity of TRAF6 are increased in distinct models of muscle atrophy. Muscle-specific ablation of TRAF6 inhibits the induction of atrophy program in response to starvation, denervation, or cancer cachexia. Moreover, TWEAK also appears to activate some catabolic signaling through TRAF6-dependent mechanisms. Recent findings have uncovered TWEAK and TRAF6 as novel regulators of skeletal muscle atrophy. These proteins should potentially be used as molecular targets for prevention and/or treatment of muscular atrophy in future therapies.

Conditional deletion of tumor necrosis factor receptor-associated factor 6 (TRAF6) in medullary thymic epithelial cells leads to the development of classical autoimmune hepatitis in mice (123.40)

Abstract Autoimmune hepatitis (AIH) is an enigmatic disease in which loss of tolerance towards self-antigens is suspected among the causes; animal models are limited, however. TRAF6 is an important signaling adaptor protein that plays a critical role in T cell tolerance by regulating medullary thymic epithelial cell (mTEC) development. mTECs in turn regulate central tolerance by ectopically expressing self-antigens and eliminating autoreactive T cells during development. In this report, conditional knockout mice lacking TRAF6 expression in thymic epithelial cells (TRAF6ΔTEC) were generated using FoxN1-cre transgenic mice (FoxN1 expression is confined to mTECs). TRAF6 deletion inhibited mTEC development and the production of mature mTECs in the thymus. This correlated with spontaneous liver-specific autoimmunity and pathognomonic features of chronic autoimmune hepatitis including interphase and lobular hepatitis, T cell and plasma cell infiltration, elevated serum transaminases and IgG, and production of anti-nuclear autoantibodies against the livers of TRAF6ΔTEC animals. A significant increase in fibrosis was also detected in TRAF6ΔTEC mice compared to littermate controls. Our data indicate that mTECs play an intrinsic role in central tolerance, and their depletion leads to AIH. This model of spontaneous AIH represents an exciting new tool to investigate autoantigen-specific T and B cell responses and regulatory mechanisms underlying the disease.

NF-κB Is the Transcription Factor for FGF-2 That Causes Endothelial Mesenchymal Transformation in Cornea

To determine the role of nuclear factor-κB (NF-κB) during FGF-2-mediated endothelial mesenchymal transformation (EMT) in response to interleukin (IL)-1β stimulation in corneal endothelial cells (CECs). Expression and/or activation of IL-1 receptor-associated protein kinase (IRAK), TNF receptor-associated factor 6 (TRAF6), phosphatidylinositol 3-kinase (PI 3-kinase), IκB kinase (IKK), IκB, NF-κB, and FGF-2 were analyzed by immunoblot analysis. Cell proliferation was measured by 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) assay. NF-κB activity was measured by NF-κB ELISA kit, while binding of NF-κB to the promoter region of FGF-2 gene was determined by chromatin immunoprecipitation. Brief stimulation of CECs with IL-1β upregulated expression of IRAK and TRAF6 and activated PI 3-kinase; expression of IRAK and TRAF6 reached maximum within 60 minutes, after which the expression disappeared, while PI 3-kinase activity was observed up to 4 hours after IL-1β stimulation. Use of specific inhibitor to PI 3-kinase or IRAK demonstrated that IRAK activates PI 3-kinase, the signaling of which phosphorylates IKKα/β and degrades IκB, subsequently leading to activation of NF-κB. The induction of FGF-2 by IL-1β was completely blocked by inhibitors to NF-κB activation (sulfasalazine) or PI 3-kinase (LY294002), and both inhibitors greatly blocked cell proliferation of CECs. Chromatin immunoprecipitation further demonstrated that NF-κB is the transcription factor of FGF-2 as NF-κB binds the putative NF-κB binding site of the FGF-2 promoter. These data suggest that IL-1β signaling combines the canonical pathway and the PI 3-kinase signaling to upregulate FGF-2 production through NF-κB, which plays a key role as a transcription factor of FGF-2 gene.

Protection Against Titanium Particle-Induced Inflammatory Osteolysis by the Proteasome Inhibitor Bortezomib In Vivo

Wear particle-induced vascularized granulomatous inflammation and subsequent inflammatory osteolysis is the most common cause of aseptic loosening after total joint replacement (TJR); however, the precise mechanism by which this occurs is unclear. This study investigates the effects of the proteasome inhibitor bortezomib (Bzb) on the expression of key biochemical markers of bone metabolism and vascularised granulomatous tissues, such as receptor activator of nuclear factor-κB ligand (RANKL), osteoprotegerin (OPG), vascular endothelial growth factor (VEGF) and tumor necrosis factor receptor-associated factor 6 (TRAF6). In addition, the effect of Bzb on apoptosis of CD68+ cells was examined. A total of 32 female BALB/C mice were randomly divided into four groups. After implantation of calvaria bone from syngeneic littermates, titanium (Ti) particles were injected into established air pouches for all mice (excluding negative controls) to provoke inflammatory osteolysis. Subsequently, Bzb was administered at a ratio of 0, 0.1, or 0.5 mg/kg on day 1, 4, 8, and 11 post-surgery to alleviate this response. All of the air pouches were harvested 14 days after the surgical procedure and were processed for molecular and histological analysis. The results demonstrated that Ti injection elevated the expression of RANKL, OPG, VEGF, and TRAF6 at both the gene and protein levels, increased counts of infiltrated cells and thickness of air pouch membranes, and elevated the apoptosis index (AI) of CD68+ cells. Bzb treatment significantly improved Ti particle-induced implanted bone osteolysis, attenuated vascularised granulomatous tissues and elevated AI of CD68+ cells. Therefore, the proteasome pathway may represent an effective therapeutic target for the prevention and treatment of aseptic loosening.

Decrease of miR-146b-5p in Monocytes during Obesity Is Associated with Loss of the Anti-Inflammatory but Not Insulin Signaling Action of Adiponectin

BackgroundLow adiponectin, a well-recognized antidiabetic adipokine, has been associated with obesity-related inflammation, oxidative stress and insulin resistance. Globular adiponectin is an important regulator of the interleukin-1 receptor-associated kinase (IRAK)/NFκB pathway in monocytes of obese subjects. It protects against inflammation and oxidative stress by inducing IRAK3. microRNA (miR)-146b-5p inhibits NFκB-mediated inflammation by targeted repression of IRAK1 and TNF receptor-associated factor-6 (TRAF6). Therefore, we measured the expression of miR-146b-5p in monocytes of obese subjects. Because it was low we determined the involvement of this miR in the anti-inflammatory, antioxidative and insulin signaling action of globular adiponectin.MethodsmiR-146b-5p expression in monocytes of obese subjects was determined by qRT-PCR. The effect of miR-146b-5p silencing on molecular markers of inflammation, oxidative stress and insulin signaling and the association with globular adiponectin was assessed in human THP-1 monocytes.ResultsmiR-146b-5p was downregulated in monocytes of obese persons. Low globular adiponectin decreased miR-146b-5p and IRAK3 in THP-1 monocytes, associated with increased mitochondrial reactive oxygen species (ROS). Intracellular ROS and insulin receptor substrate-1 (IRS1) protein were unchanged. Silencing of miR-146b-5p with an antisense inhibitor resulted in increased expression of IRAK1 and TRAF6 leading to more NFκB p65 DNA binding activity and TNFα. As a response IRAK3 and IRS1 protein increased. Mitochondrial and intracellular ROS production did not increase despite more inflammation. In addition, exposure of miR-146b-5p-depleted THP-1 monocytes to high levels of globular adiponectin resulted in an increased production of TNFα and intracellular ROS. Still, they did not lose their potential to increase IRAK3 and IRS1 protein and to decrease mitochondrial ROS.ConclusionmiR-146b-5p, decreased in monocytes during obesity, is a major mediator of the anti-inflammatory action of globular adiponectin. It appears not to be involved in insulin signaling possibly by protective response of IRAK3 and lack of mitochondrial ROS production.

Upregulation of tumor necrosis factor receptor-associated factor 6 correlated with synovitis severity in rheumatoid arthritis

IntroductionRheumatoid arthritis (RA) is a chronic inflammatory disease leading to joint destruction and disability. Focal bone erosion is due to excess bone resorption of osteoclasts. Tumor necrosis factor receptor-associated factor 6 (TRAF6) is one of the critical mediators both in inflammatory signal pathway and differentiation and resorption activity of osteoclasts. Here we aimed to investigate TRAF6 expression in RA synovium and its correlation with histological synovitis severity and radiological joint destruction in RA.MethodsSynovitis score was determined in needle biopsied synovium from 44 patients with active RA. Synovium from nine patients with osteoarthritis (OA) and seven with orthopedic arthropathies (Orth.A) were enrolled as "less inflamed" disease controls. Serial sections were stained immunohistochemically for TRAF6 as well as CD68 (macrophage), CD3 (T cell), CD20 (B cell), CD38 (plasmocyte), CD79a (B lineage cells from pre-B cell to plasmocyte stage), and CD34 (endothelial cell). Double immunofluorescence staining of TRAF6 and CD68 were tested. Densities of positive staining cells were determined and correlated with histological disease activity (synovitis score) and radiographic joint destruction (Sharp score).ResultsTRAF6 expression was found in the intimal and subintimal area of RA synovium, with intense staining found in the endochylema and nucleus of intimal synoviocytes and subintimal inflammatory cells. Double immunofluorescence staining showed TRAF6 was expressed in most of the intimal cells and obviously expressed in CD68+ cells and some other CD68- cells in subintimal area. Synovial TRAF6 was significantly over-expressed in the RA group compared with the OA and Orth.A group (2.53 ± 0.94 vs. 0.72 ± 0.44 and 0.71 ± 0.49, P < 0.0001). Synovial TRAF6 expression in RA correlated significantly with synovitis score (r = 0.412, P = 0.006), as well as the inflammatory cell infiltration (r = 0.367, P = 0.014). Significant correlation was detected between synovial TRAF6 expression and intimal CD68+ cells, as well as the cell density of subintimal CD68+ cells, CD3+ cells, CD20+ cells, CD38+ cells, and CD79a+ cells (all P < 0.05).ConclusionsElevated synovial TRAF6 expression correlated with synovitis severity and CD68+ cell density in RA. It is, therefore, hypothesized that synovial TRAF6 is involved in the pathogenesis of synovial inflammation and osteoclast differentiation in RA.

TNF/TNFR signal transduction pathway-mediated anti-apoptosis and anti-inflammatory effects of sodium ferulate on IL-1β-induced rat osteoarthritis chondrocytes in vitro

IntroductionSodium ferulate (SF) is a natural component of traditional Chinese herbs. Our previous study shows that SF has a protective effect on osteoarthritis (OA). The objective of this study was to investigate the effect of SF on the TNF/TNF receptor (TNFR) signal transduction pathway of rat OA chondrocytes.MethodsPrimary rat articular chondrocytes were co-treated with IL-1β and SF. Chondrocyte apoptosis was assessed by fluorescein isothiocyanate-annexin V/propidium iodide assay. The PCR array was used to screen the expression of 84 key genes involved in apoptosis. The release of TNFα and prostaglandin E2 were analyzed by ELISA. Expressions of proteins were assessed by western blotting. The activity of NF-κB was determined by electrophoretic mobility shift assay (EMSA). Gene expression of inducible nitric oxide synthase (iNOS) was evaluated by real-time quantitative PCR. The nitric oxide content was measured with the Griess method.ResultsAfter treatment with SF, the apoptosis rate of chondrocytes significantly attenuated (P < 0.01). Results of the apoptosis PCR array suggested that mRNA expression of some core proteins in the TNF/TNFR pathway showed valuable regulation. The protein expressions of TNFα, TNFR-1, TNF receptor-associated death domain, caspase-8 and caspase-3 were prevented by SF in a concentration-dependent manner. SF also inhibited activities of caspase-8 and caspase-3 compared with the OA model control (P < 0.01). TNF receptor-associated factor-2 expression, phosphorylations of inhibitor of NF-κB kinase (IKK) subunits alpha and beta, and NF-κB inhibitor, alpha (IκBα) were all concentration-dependently suppressed by SF treatment. The results of EMSA showed that SF inhibited the activity of NF-κB. In addition, the expressions of cycloxygenase-2 and iNOS and the contents of prostaglandin E2 and NO were attenuated with the treatment of SF (P < 0.01).ConclusionSF has anti-apoptosis and anti-inflammatory effects on an OA model induced by IL-1β in vitro, which were due to inhibitory actions on the caspase-dependent apoptosis pathway and the IKK/NF-κB signal transduction pathway of the TNF/TNFR pathway.

Quercetin Triggers Apoptosis of Lipopolysaccharide (LPS)-induced Osteoclasts and Inhibits Bone Resorption in RAW264.7 Cells

Aims: Quercetin, a flavonoid present in vegetables, has anti-inflammatory properties and potential inhibitory effects on bone resorption. Up to date, the effect of quercetin on lipopolysaccharide (LPS)-induced osteoclastogenesis has not yet been reported. In the current study, we evaluated the effect of quercetin on LPS-induced osteoclast apoptosis and bone resorption. Methods: RAW264.7 cells were non-treated, treated with LPS alone, or treated with both LPS and quercetin. After treatment, the number of osteoclasts, cell viability, bone resorption and osteoclast apoptosis were measured. The expressions of osteoclast-related genes including tartrate-resistant acid phosphatase (TRAP), matrix metalloproteinase-9 (MMP9) and cathepsin K (CK) were determined by real-time quantitative polymerase chain reaction (qPCR). Protein levels of receptor activator of nuclear factor-ĸB (RANK), tumor necrosis factor receptor-associated factor 6 (TRAF6), cyclooxygenase-2 (COX-2), Bax, Bcl-2 and mitogenactivated protein kinases (MAPKs) were measured using Western blotting assays. The MAPK signaling pathway was blocked by pretreatment with MAPK inhibitors. Results: LPS directly promoted osteoclast differentiation of RAW264.7 cells and upregulated the protein expression of RANK, TRAF6 and COX-2; while quercetin significantly decreased the number of LPS-induced osteoclasts in a dose-dependent manner. None of the treatments increased cytotoxicity in RAW264.7 cells. Quercetin inhibited mRNA expressions of osteoclast-related genes and protein levels of RANK, TRAF6 and COX-2 in LPS-induced mature osteoclasts. Quercetin also induced apoptosis and inhibited bone resorptive activity in LPS-induced mature osteoclasts. Furthermore, quercetin promoted the apoptotic signaling pathway including increasing the phosphorylation of p38-MAPK, c-Jun N-terminal kinases/stress-activated protein kinases

Molecular characterization of toll-like receptor 2 (TLR2), analysis of its inductive expression and associated down-stream signaling molecules following ligands exposure and bacterial infection in the Indian major carp, rohu ( Labeo rohita)

Toll-like receptors (TLRs) are one of the key components of innate immunity. Among various TLR types, TLR2 is involved in recognizing specific microbial structures such as peptidoglycan (PGN), lipoteichoic acid (LTA), zymosan etc., and after binding them it triggers myeloid differentiation primary response gene 88 (MyD88)-dependent signaling pathway to induce various cytokines. In this report, TLR2 gene was cloned and characterized in rohu ( Labeo rohita), which is highly commercially important fish species in the farming-industry of Indian subcontinent. Full-length rohu TLR2 (rTLR2) cDNA comprised of 2691 bp with a single open reading frame (ORF) of 2379 bp encoding a polypeptide of 792 amino acids (aa) with an estimated molecular mass of 90.74 kDa. Structurally, it comprised of one leucine-rich repeat region (LRR) each at N-terminal (LRR-NT; 44–55 aa) and C-terminal (LRR-CT; 574–590 aa), 21 LRRs in between C and N-terminal, one trans-membrane (TM) domain (595–612 aa), and one TIR domain (645–790 aa). Phylogenetically, rohu TLR2 was closely related to common carp and exhibited significant similarity (93.1%) and identity (88.1%) in their amino acids. During embryogenesis, rTLR2 expression was detected as early as ∼7 h post fertilization indicating its importance in embryonic innate immune defense system in fish. Basal expression analysis of rTLR2 showed its constitutive expression in all the tissues examined, highest was in the spleen and the lowest was in the eye. Inductive expression of TLR2 was observed following zymosan, PGN and LTA exposure and Streptococcus uberis and Edwardsiella tarda infections. Expression of immunoregulatory cytokine interleukin (IL)-8, in various organs was significantly enhanced by ligands exposure and bacterial infections, and was correlated with inductive expression of TLR2. In vitro studies showed that PGN treatment induced TLR2, MyD88 and TRAF6 (TNF receptor associated factor 6) expression, NF-κB (nuclear factor kappa B) activation and IL-8 expression. Blocking NF-κB resulted in down-regulation of PGN mediated IL-8 expression indicating the involvement of NF-κB in IL-8 induction. Together, these findings highlighted the important role of TLR2 in immune surveillance of various organs, and in augmenting innate immunity in fish in response to pathogenic invasion. This study will be helpful in developing preventive measures against infectious diseases in fish.

TRAF6 and IRF7 Control HIV Replication in Macrophages

The innate immune system recognizes virus infection and evokes antiviral responses which include producing type I interferons (IFNs). The induction of IFN provides a crucial mechanism of antiviral defense by upregulating interferon-stimulated genes (ISGs) that restrict viral replication. ISGs inhibit the replication of many viruses by acting at different steps of their viral cycle. Specifically, IFN treatment prior to in vitro human immunodeficiency virus (HIV) infection stops or significantly delays HIV-1 production indicating that potent inhibitory factors are generated. We report that HIV-1 infection of primary human macrophages decreases tumor necrosis factor receptor-associated factor 6 (TRAF6) and virus-induced signaling adaptor (VISA) expression, which are both components of the IFN signaling pathway controlling viral replication. Knocking down the expression of TRAF6 in macrophages increased HIV-1 replication and augmented the expression of IRF7 but not IRF3. Suppressing VISA had no impact on viral replication. Overexpression of IRF7 resulted in enhanced viral replication while knocking down IRF7 expression in macrophages significantly reduced viral output. These findings are the first demonstration that TRAF6 can regulate HIV-1 production and furthermore that expression of IRF7 promotes HIV-1 replication.

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.

Cadmium induces differentiation of RAW264.7 cells into osteoclasts in the presence of RANKL

The mechanism of cadmium effects on bone is not fully understood. In this study, we investigated the effects of cadmium on osteoclasts differentiation and the probable mechanism. RAW264.7 cells were exposed to cadmium (0–60nmol/L) in the presence or absence of receptor-activated nuclear factor κ B ligand (RANKL) for 5days. Then, the viability, tartrate-resistant acid phosphatase (TRAP) activity and the formation of TRAP positive multinucleated osteoclasts were observed. Receptor activator of nuclear factor κ B (RANK), tumor necrosis factor receptor associated factor 6 (TRAF6), c-src, c-fos, fos-related antigen 1 (Fra1) expression were determined by reverse transcription polymerase chain reaction. Cadmium increased TRAP activity (20–40%) and TRAP positive cell formation in the presence of RANKL, but had no obvious influence on them without RANKL. RANK, TRAF6, Fra1, c-src and c-fos (at 15–30nmol/L) expression were enhanced (30–70%) by cadmium in the presence of RANKL, but cadmium had little influence on them in the absence of RANKL. This study demonstrated that cadmium could induce differentiation of osteoclasts precursor into osteoclasts in the presence of RANKL. Even though the changes of gene expression were small, RANKL/RANK and downstream genes may play an important role in cadmium effects on osteoclasts.

Proteasome inhibition aggravates tumor necrosis factor-mediated bone resorption in a mouse model of inflammatory arthritis

The proteasome inhibitor bortezomib has potent anti-myeloma and bone-protective activity. Recently, bortezomib was shown to directly inhibit osteoclastogenesis. The aim of this study was to analyze the influence and therapeutic effect of bortezomib in a mouse model of inflammatory arthritis. Heterozygous human tumor necrosis factor α (hTNFα)-transgenic mice and their wild-type (WT) littermates were intravenously injected with 0.75 mg/kg of bortezomib or phosphate buffered saline twice weekly. The mice were assessed for clinical signs of arthritis. After 6 weeks of treatment, mice were analyzed for synovial inflammation, cartilage damage, bone erosions, and systemic bone changes. Osteoclast precursors from WT and hTNF-transgenic mice were isolated from bone marrow, treated with bortezomib, and analyzed for osteoclast differentiation, bone resorption, and expression of osteoclast-specific genes as well as apoptosis and ubiquitination. Bortezomib-treated hTNF-transgenic mice showed moderately increased inflammatory activity and dramatically enhanced bone erosions associated with a significant increase in the number of synovial osteoclasts. Interestingly, bortezomib did not alter systemic bone turnover in either hTNF-transgenic mice or WT mice. In vitro, treatment with therapeutically relevant concentrations of bortezomib resulted in increased differentiation of monocytes into osteoclasts and more resorption pits. Molecularly, bortezomib increased the expression of TNF receptor-associated factor 6, c-Fos, and nuclear factor of activated T cells c1 in osteoclast precursors. In TNF-mediated bone destruction, bortezomib treatment increased synovial osteoclastogenesis and bone destruction. Hence, proteasome inhibition may have a direct bone-resorptive effect via stimulation of osteoclastogenesis during chronic arthritis.

Epigallocatechin-3-gallate suppresses the global interleukin-1beta-induced inflammatory response in human chondrocytes

IntroductionEpigallocatechin-3-gallate (EGCG) is a bioactive polyphenol of green tea and exerts potent anti-inflammatory effects by inhibiting signaling events and gene expression. Interleukin-1beta (IL-1β) is the principal cytokine linked to cartilage degradation in osteoarthritis (OA). The objective of this study was to evaluate the global effect of EGCG on IL-1β-induced expression of proteins associated with OA pathogenesis in human chondrocytes.MethodsPrimary OA chondrocytes were pretreated with EGCG (10 to 100 uM) and then stimulated with IL-1β (5 ng/ml) for 24 hours. Culture supernatants were incubated with cytokine antibody arrays and immunoreactive proteins (80 proteins) were visualized by enhanced chemiluminiscence. Effect of EGCG on IL-1β-induced expression of 18 selected genes was verified by Real time-PCR and effect on IL-6, IL-8 and tumor necrosis factor-alpha (TNF-α) production was determined using specific ELISAs. Western immunoblotting was used to analyze the effect of EGCG on the interleukin-1 receptor-associated kinase 1 (IRAK-1) and TNF receptor-associated factor 6 (TRAF-6) proteins in IL-1β-stimulated chondrocytes. The role of nuclear factor kappa-B (NF-κB) and mitogen activated protein kinases (MAPKs) in the regulation of selected genes and the mechanism involved in EGCG mediated modulation of these genes was determined by using specific inhibitors for NF- κB (MG132) and MAPKs (p38-MAPK, SB202190; JNK-MAPK, SP600125, ERK-MAPK, PD98059).ResultsOut of 80 proteins present on the array, constitutive expression of 14% proteins was altered by EGCG treatment. No significant stimulatory effect was observed on the proteins associated with cartilage anabolic response. Stimulation with IL-1β enhanced the expression of 29 proteins. Expression of all 29 proteins up-regulated by IL-1β was found to be suppressed by EGCG. EGCG also inhibited the expression of the signaling intermediate TRAF-6 at 50 and 100 uM concentrations (P < 0.05). Our results identified several new targets of EGCG, including epithelial neutrophil activating peptide-78 (ENA-78), granulocyte macrophage colony stimulation factor (GM-CSF), growth- related oncogene (GRO), GRO-α, IL-6, IL-8, monocyte chemotactic protein-1 (MCP-1), MCP-3, macrophage inflammatory protein-1beta (MIP-1β), granulocyte chemotactic protein-2 (GCP-2), MIP-3alpha, interferon-gamma-inducible protein-10 (IP-10), nucleosome assembly protein-2 (NAP-2) and leukemia inhibitory factor (LIF). The inhibitory effects of EGCG were mainly mediated by inhibiting the activation of NF-κB and c-Jun N-terminal Kinase (JNK)-MAPK in human chondrocytes.ConclusionsOur results suggest that the potential of EGCG in OA treatment/prevention may be related to its ability to globally suppress the inflammatory response in human chondrocytes. These results identify additional new targets of EGCG and advocate that EGCG may be a potent chondroprotective agent in OA.

Increased Expression of TNF Receptor-Associated Factor 6 After Rat Traumatic Brain Injury

TRAF6 (TNF receptor-associated factor 6), a member of tumor necrosis factor receptor-associated factors family was identified as a molecule that binds to the cytoplasmic domain of CD40. TRAF6 functions as an adaptor, positively regulating the NF-κB, JNK pathway. Additionally, some studies have reported that TRAF6 is required for apoptosis within the developing CNS and regulates cell fate decisions by inducing caspase 8-dependent apoptosis. However, its distribution and function in the central nervous system (CNS) lesion are not well understood. In this study, we performed an acute traumatic brain injury model in adult rats. And we mainly examined protein expression and cellular localization of TRAF6 during rat traumatic brain injury (TBI). Western blot analysis showed TRAF6 level significantly improved at 7 days after injury, and then declined during the following days. The protein expression of TRAF6 was further analysed by immunohistochemistry. In comparison to contralateral cerebral cortex, we observed a highly significant accumulation of TRAF6 at the ipsilateral brain. Immunofluorescence double-labeling showed that TRAF6 was co-expressed with NeuN and GFAP. Besides, co-localization of TRAF6/active caspase 3 and TRAF6/proliferating cell nuclear antigen (PCNA) were detected in NeuN and GFAP, respectively. We also examined the expression profiles of proliferating cell nuclear antigen (PCNA) and active caspase 3 whose changes were correlated with the expression profiles of TRAF6. In conclusion, this is the first description of TRAF6 expression in traumatic brains. Our data suggested that TRAF6 might play important roles in CNS pathophysiology after TBI.