Search

Tumor necrosis factor receptor‐associated factor 6 (TRAF6) is a vital molecule of inflammatory signaling pathways in innate immune response against pathogens. To elucidate its role in defense against Edwardsiella tarda infection in teleost fish, TRAF6 homologue was identified from obscure puffer (Takifugu obscurus) and functionally analyzed in this study. The obscure puffer TRAF6 (ToTRAF6) is a protein of 565 amino acids containing conserved RING domain, zinc finger‐TRAF and MATH_TRAF6 domain. ToTRAF6 mRNA distributed in various healthy tissues of obscure puffer and was upregulated in the immune related tissues after E. tarda infection. ToTRAF6 protein was localized in the cytoplasm and aggregate as dots around the nuclei in FHM cells. The overexpression of ToTRAF6 in FHM cells decreased the quantity of E. tarda and induced the significant upregulation of downstream MAPK signaling pathway genes. These data suggest that ToTRAF6 is a key molecule of MAPK signaling pathway in defense against E. tarda infection. [ABSTRACT FROM AUTHOR]

AIM: To explore the mechanism of Toll-like receptor 4(TLR4) in the pulmonary inflammation induced by influenza A virus (IAV) based on animal experiments of wild-type (WT) and TLR4 deletion type (TLR4del) C57BL/6 mice. METHODS: Two groups of mice, namely WT and TLR4del, were established. The mice in both groups were intranasally infected with IAV to construct pneumonia models, with 48 mice in each group. The animals were treated on days 0, 1, 3, 5, 7 and 9 after infection. The body mass, lung index, spleen index and thymus index were measured or calculated by routine methods. The pathological changes of the mouse lung tissues at different time points were observed by hematoxylin-eosin (HE) staining. The levels of tumor necrosis factor-α(TNF-α), interleukin-1β(IL-1β), IL-6 and IL-8 in the bronchoalveolar lavage fluid were detected by ELISA, in order to examine the secretion of inflammatory factors at different time points. The mRNA and protein expression levels of TLR4, myeloid differentiation factor 88(MyD88)and tumor necrosis factor receptor-associated factor 6(TRAF6) in the mouse lung tissues were detected by RT-qPCR, immunohistochemistry and Western blot, in order to further clarify the mechanism of TLR4 in pulmonary inflammation induced by IAV infection. RESULTS: Compared with WT group, the mice in TLR4del group showed significant increases in the body mass, spleen index and thymus index (P<0. 05), and a significant decrease in the lung index (P<0. 05) 3 d after IAV infection. At the same time, the levels of TNF-α, IL-1β, IL-6 and IL-8 in the bronchoalveolar lavage fluid were significantly decreased in TLR4del group (P<0. 05). The HE staining results indicated that TLR4 deletion could effectively attenuate the pathological damage in the lung tissues of IAV-infected mice. The results of RT-qPCR, immunohistochemistry and Western blot showed that, compared with WT group, the mRNA and protein expression levels of MyD88 and TRAF6 in the lung tissues of the mice in TLR4del group were significantly reduced 3 d after infection (P<0. 05). CONCLUSION: The injury mechanism of IAV-induced pulmonary inflammation in mice may be related to the TLR4-MyD88-TRAF6 signaling pathway mediated by TLR4. [ABSTRACT FROM AUTHOR]

Objective：To study the binding of exogenous tumor necrosis factor receptor⁃associated factor 6 (TRAF6) to Krüppel⁃like factors 5 (KLF5) as well as the pattern and site of KLF5 polyubiquitination by TRAF6 in HEK 293T (i.e. 293 T) cells. Methods：The 293T cells were co⁃transfected with Flag⁃TRAF6, HA⁃KLF5 and ubiquitin (Ub) expression plasmids, or shTRAF6 and TRAF6 C70A plasmids in different combinations for 48 h. Then, the binding of TRAF6 to KLF5 and KLF5 K48/K63⁃linked polyubiquitination by TRAF6 were detected using immunoprecipitation (IP) and immunoblotting (IB) assays. Moreover, the plasmids with all lysine mutation of KLF5 were constructed, and co⁃transfected with TRAF6 overexpression plasmids into 293T cells. Thereafter, the level of KLF5 K63⁃ linked polyubiquitination and the lysine (site) of KLF5 K63 ⁃linked polyubiquitination were measured or identified by IP and IB. Results：TRAF6 and KLF5 in 293T cells could bind with each other. The overexpression of TRAF6 up ⁃ regulated while the knockdown or activity deficiency of TRAF6 down⁃regulated the level of KLF5 K63⁃linked polyubiquitination. The site of KLF5 K63⁃ linked polyubiquitination was its K99 or K100 lysine. Conclusion：TRAF6 can interact with KLF5 and modify the K99 and K100 of KLF5 via K63⁃linked polyubiquitination. [ABSTRACT FROM AUTHOR]

Yiqiu Zhang,1,* Chenlu Song,1,* Wei Ni,1,2 Qing Pei,1 Caixia Wang,1 Youguo Ying,3 Min Yao1 1Department of Plastic and Reconstructive Surgery, Shanghai Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, People’s Republic of China; 2Wuhan National Laboratory for Optoelectronics, Huazhong University of Science and Technology, Wuhan, People’s Republic of China; 3Department of Intensive Care Unit, Shanghai Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, People’s Republic of China*These authors contributed equally to this workCorrespondence: Min Yao; Youguo Ying, Shanghai Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine, 639 Zhizaoju Road, Shanghai, 200011, People’s Republic of China, Email my058@vip.sina.com; yingyouguo@sina.comPurpose: Acute kidney injury (AKI) is one of the most severe complications of sepsis, the pathological features of which are excessive inflammation and programmed cell death of resident renal cells. Heat shock protein 70 (HSP70) is a critical stress protein for repressing inflammation, however, its role in AKI is not fully understood. The current study aimed to determine the protective effect of HSP70 on septic AKI and its underlying mechanisms.Methods: Hsp70.1 knockout and wildtype mice were used for creating sepsis model by cecal ligation and puncture (CLP). Renal function, histological changes, pro-inflammatory cytokines, and apoptosis were analyzed with H&E, PAS, ELISA, western-blot, and immunofluorescence. Moreover, the effects of HSP70 on renal proximal tubular epithelial (HK-2) cells with LPS were assessed by measuring the levels of nuclear factor kappa B (NF-κB) signaling and downstream cytokines, viability, and apoptosis using western-blot, qRT-PCR, flow-cytometry, and immunofluorescence. Immunoprecipitate and immunoblotting were used for determining the interaction of HSP70 with tumor necrosis factor receptor-associated factor 6 (TRAF6). Exogenous HSP70 was applied to further identify its biological significance at the cellular and animal level.Results: Hsp70.1 deficiency significantly aggravated renal dysfunction with increasing serum levels of BUN, SCr, kidney injury molecule-1 (KIM-1), and neutrophil gelatinase-associated lipocalin (NGAL), and shortened survival in CLP mice. Furthermore, hsp70.1 knockout caused renal-tissue structural damage, especially proximal tubular, and inflammatory cascade and increased apoptotic cells, along with elevated Bax, caspase 3 and cleaved caspase 3, as well as decreased Bcl2 in vivo and vitro. Significantly, HSP70 directly interacted with TRAF6 in HK-2 cells, leading to suppression of inflammatory response and apoptosis. Moreover, exogenous HSP70 alleviated renal damage, decreased apoptosis and elevated survival rate in septic AKI in vivo and vitro.Conclusion: Our findings demonstrated that HSP70 played a critical role in sepsis-induced AKI via interaction with TRAF6 and inhibiting inflammation and apoptosis.Keywords: heat shock protein 70, sepsis, acute kidney injury, tumor necrosis factor receptor-associated factor 6, nuclear factor kappa B, apoptosis

[INLINE:1] An enriched environment is used as a behavioral intervention therapy that applies sensory, motor, and social stimulation, and has been used in basic and clinical research of various neurological diseases. In this study, we established mouse models of photothrombotic stroke and, 24 hours later, raised them in a standard, enriched, or isolated environment for 4 weeks. Compared with the mice raised in a standard environment, the cognitive function of mice raised in an enriched environment was better and the pathological damage in the hippocampal CA1 region was remarkably alleviated. Furthermore, protein expression levels of tumor necrosis factor receptor-associated factor 6, nuclear factor κB p65, interleukin-6, and tumor necrosis factor α, and the mRNA expression level of tumor necrosis factor receptor-associated factor 6 were greatly lower, while the expression level of miR-146a-5p was higher. Compared with the mice raised in a standard environment, changes in these indices in mice raised in an isolated environment were opposite to mice raised in an enriched environment. These findings suggest that different living environments affect the hippocampal inflammatory response and cognitive function in a mouse model of stroke. An enriched environment can improve cognitive function following stroke through up-regulation of miR-146a-5p expression and a reduction in the inflammatory response.

Objective To investigate the relationships of the expression of serum microRNA-98-5p (miR-98-5p) and tumor necrosis factor receptor-associated factor 6 (TRAF6）with sepsis complicated with lung injury. Methods Totally 172 patients with sepsis (observation group), including 59 patients with lung injury and 113 patients without lung injury, and 56 healthy volunteers (control group) who underwent physical examination during the same period were selected. Peripheral venous blood was collected and centrifuged to obtain the serum. The expression of miR-98-5p and TRAF6 mRNA in serum was detected by RT-qPCR. The serum miR-98-5p and TRAF6 mRNA expression levels were compared between the two groups. The relationship between serum miR-98-5p expression and TRAF6 mRNA expression in sepsis patients was analyzed. Multivariate Logistic regression model was used to analyze the risk factors for sepsis complicated with lung injury. Receiver operating characteristic (ROC) curve was used to analyze the value of serum miR-98-5p and TRAF6 mRNA expression in predicting sepsis complicated with lung injury. Results The relative expression of serum miR-98-5p in the observation group was lower than that in the control group, and the serum TRAF6 mRNA expression was higher than that in the control group (both P<0. 05). Pearson correlation analysis showed that serum miR-98-5p expression in sepsis patients was negatively correlated with serum TRAF6 mRNA expression (r=-0. 768, P<0. 01）. Multi⁃ variate Logistic regression analysis showed that septic shock, prolonged intensive care unit time, prolonged mechanical ventilation time, increased blood lactate, increased sepsis-related organ failure assessment score, and increased serum TRAF6 mRNA expression were independent risk factors for sepsis complicated with lung injury, while increased serum miR-98-5p expression was an independent protective factor (all P<0. 05). ROC curve analysis showed that the area under the curve of serum miR-98-5p and TRAF6 mRNA expression combined in predicting sepsis complicated with lung injury was higher than that of serum miR-98-5p and TRAF6 mRNA expression alone (both P<0. 05). Conclusion The decreased expression of serum miR-98-5p and the increased expression of serum TRAF6 mRNA are closely related to sepsis complicated with lung injury, and can be used as biomarkers to predict sepsis complicated with lung injury. [ABSTRACT FROM AUTHOR]

Interferon regulatory factor 5 (IRF5) is critical for the regulation of immune and inflammatory responses in health and diseases. However, the presence of IRF5 in human apical periodontitis remains unknown. This study aimed to explore the expression and colocalization of IRF5 with tumor necrosis factor receptor–associated factor 6 (TRAF6) and AKT2 in human apical periodontitis. A total of 39 human periapical tissues, including healthy gingival tissues (n = 12), periapical granulomas (PGs, n = 13), and radicular cysts (RCs, n = 14), were used in this study. The inflammatory infiltrates of lesions were evaluated by hematoxylin-eosin staining. The expression of IRF5 was detected by immunohistochemistry. Double immunofluorescence assessment was performed to colocalize IRF5 with CD68, TRAF6, and AKT2, respectively. Data were analyzed using the Kruskal-Wallis test. Immunohistochemistry revealed significantly higher expressions of IRF5 in PGs and RCs than the healthy control group. IRF5-CD68 double-positive cells were more predominant in RCs and PGs than the healthy control group. Significant differences of the IRF5-TRAF6 and IRF5-AKT2 double-positive cells were detected in periapical lesions compared with the healthy control tissues. IRF5 was highly expressed in macrophages of human periapical tissues and was colocalized with TRAF6 or AKT2 in human periapical tissues. These findings may provide new clues for understanding the pathogenesis of periapical diseases. [ABSTRACT FROM AUTHOR]

Abstract Background In its RING domain, tumor necrosis factor receptor-associated factor 6 (TRAF6) has ubiquitin E3 ligase activity that facilitates the formation of lysine 63-linked polyubiquitin chains. This activity is required to activate nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) and plays an important role in the IκB kinase (IKK) complex. Methods An in vitro ubiquitination assay was used to establish whether c-Cbl could promote TRAF6 ubiquitination. We assessed direct binding and performed fine mapping between c-Cbl and TRAF6 based on the results of an immunoprecipitation assay with cultured 293 T cells. The luciferase reporter assay was applied to establish if c-Cbl-mediated ubiquitination affected NF-κB activation after stimulus from various TRAF-mediated signals: tumor necrosis factor-α (TNF-α), receptor activator of NF-κB ligand (RANKL), and interleukin-1β (IL-1β). An in vivo ubiquitination assay was performed using endogenous immunoprecipitation of TRAF6 in bone marrow macrophages (BMMs) and osteoclasts. Results Here, we report on a form of TRAF6 ubiquitination that is mediated by c-Cbl, leading to the formation of lysine 48-linked polyubiquitin chains. The NF-κB activity induced by RANKL and IL-1β treatment is inhibited when c-Cbl is overexpressed, while the NF-κB activity induced by TNFα treatment is not. c-Cbl inhibits NF-κB activity mediated by TRAF6, but not by TRAF2. These findings show that c-Cbl ubiquitin ligase activity is essential for TRAF6 ubiquitination and negative regulation of NF-κB activity. Fine mapping revealed that the proline-rich domain of c-Cbl is critical for interaction with TRAF6. Stimulation with RANKL or interferon-γ (IFN-γ) caused c-Cbl to bind to polyubiquitinated TRAF6. Conclusions These findings indicate that the interaction of TRAF6 with c-Cbl causes lysine 48-linked polyubiquitination for both negative feedback regulation and signaling cross-talk between RANKL and IFN-γ.

Tumor necrosis factor receptor-associated factor 6 (TRAF6), a regulator of NF-κB signaling, has been discovered recently to be probably related to osteoarthritis, while the function of TRAF6 in lumbar facet joint osteoarthritis(FJOA)still remains unknown. The aim of this study was to probe the specific function of TRAF6 in chondrocytes and its connection with the pathophysiology of FJOA. We found upregulation of TRAF6 in FJOA cartilage by western blot analysis. In vitro, we stimulated immortalized human chondrocytes by LPS to establish the cells apoptosis model. Western blot analysis demonstrated that levels of TRAF6 and cleaved caspase-3/8 in the chondrocyte injury model increased significantly. Knockdown of TRAF6 suppressed the expression of matrix metallopeptidase-13 (MMP-13) and interleukin-6 (IL-6) induced by LPS, and alleviated cell apoptosis. Meanwhile, western blot and immunofluorescent staining demonstrated that IκBα degradation and p65 nuclear transportation were also inhibited, revealing that knockdown of TRAF6 suppressed activation of the NF-κB pathway in LPS-induced chondrocytes apoptosis model. Collectively, our findings suggest that TRAF6 plays a crucial role in FJOA development by regulating NF-κB signaling pathway. Knockdown of TRAF6 may supply a potential therapeutic strategy for FJOA. [ABSTRACT FROM AUTHOR]

Background: Irritable bowel syndrome is one of the most common gastrointestinal disorders. It is featured by abdominal pain in conjunction with altered bowel habits. However, the pathophysiology of the syndrome remains largely unknown. Tumor necrosis factor receptor-associated factor 6 (TRAF6) has been reported to be involved in neuropathic pain. The aim of this study was to investigate roles and mechanisms of TRAF6 in the chronic visceral hypersensitivity. Methods: Visceral hypersensitivity was induced by neonatal colonic inflammation and was identified by colorectal distention. The protein level, RNA level, and cellular distribution of TRAF6 and its related molecules were detected with Western blot, quantitative polymerase chain reaction, and immunofluorescence. In vitro spinal cord slice recording technique was performed to determine the synaptic transmission activities. Results: Neonatal colonic inflammation rats displayed visceral hypersensitivity at the age of six weeks. The expression of TRAF6 was obviously upregulated in spinal cord dorsal horn of neonatal colonic inflammation rats at the age of six weeks. Immunofluorescence study showed that TRAF6 was dominantly expressed in spinal astrocytes. Intrathecal injection of TRAF6 small interfering RNA (siRNA) significantly reduced the amplitude of spontaneous excitatory postsynaptic currents at the spinal dorsal horn level. Furthermore, knockdown of TRAF6 led to a significant downregulation of cystathionine β synthetase expression in the spinal dorsal horn of neonatal colonic inflammation rats. Importantly, intrathecal injection of TRAF6 siRNA remarkably alleviated visceral hypersensitivity of neonatal colonic inflammation rats. Conclusions: Our results suggested that the upregulation of TRAF6 contributed to visceral pain hypersensitivity, which is likely mediated by regulating cystathionine β synthetase expression in the spinal dorsal horn. Our findings suggest that TRAF6 might act as a potential target for the treatment of chronic visceral pain in irritable bowel syndrome patients. [ABSTRACT FROM AUTHOR]

Abstract Myeloid differentiation factor 88 (MyD88) and tumor necrosis factor receptor-associated factor 6 (TRAF6) are two critical signal transducers in toll-like receptor (TLR) pathway. In the present study, we identified and characterized the homologues of MyD88 and TRAF6 in Qihe crucian carp Carassius auratus , termed as CaMyD88 and CaTRAF6 , respectively, and examined their roles during pathogenic infection. Full-length cDNA of CaMyD88 was 2463 bp, including a 191 bp 5′-untranslated region (UTR), a 1417 bp 3′-UTR, and an 855 bp open reading frame (ORF) encoding for a putative protein with 284 amino acids. Full-length cDNA of CaTRAF6 was identified to be 2555 bp, consisting of a 52 bp 5′-UTR, an 871 bp 3′-UTR, and a 1632 bp ORF encoding a protein of 543 amino acids. Deduced amino acid sequences of CaMyD88 and CaTRAF6 contained the typical domains (CaMyD88: death domain and TIR domain; CaTRAF6: one RING-type zinc finger domain, two TRAF-type zinc finger domains, one coiled-coil region, and one conserved C-terminal meprin and TRAF homology domain) as in other fish. Quantitative Real-Time PCR (qRT-PCR) analysis revealed that both CaMyD88 and CaTRAF6 were ubiquitously expressed throughout the development stages and appeared to be developmentally regulated. In addition, CaMyD88 and CaTRAF6 had a broadly distribution of expression in all examined eleven tissues of healthy fish, although the transcript levels varied among the different tissues. Moreover, it was found that mRNA expressions of CaMyD88 and CaTRAF6 were generally up-regulated after stimulation by polyI:C, flagellin, and Aeromonas hydrophila in spite of the down-regulation appeared at some time points or tissues. These results indicated that CaMyD88 and CaTRAF6 play the critical roles in the immune defense of Qihe crucian carp against pathogenic invasion. The present findings will provide the valuable information for understanding the innate immune responses of Qihe crucian carp and contribute to develop the preventive way against pathogens. Highlights • Full length DNA and cDNA of MyD88 and TRAF6 were respectively characterized in Carassius auratus. • Maternal CaMyD88 or CaTRAF6 mRNA was gradually consumed in early period of developmental embryos. • Target gene mRNA expressions show obvious tissue specificity. • Expression patterns of two genes show the significant responses to immune challenges. [ABSTRACT FROM AUTHOR]

Abstract TRAF6 and TAK1 are known to play important roles in vertebrate innate immunity as molecular bridge, linking upstream toll-like receptors (TLRs) with the downstream MAPK and NF-κB signalling pathways. However, their roles in TLR signalling pathway have yet to be fully described in fish. Here we identified genes encoding TRAF6 (OmTRAF6) and TAK1 (OmTAK1) from rainbow trout, Oncorhynchus mykiss , and examined their roles during pathogenic infections. The deduced amino acid sequences of OmTRAF6 and OmTAK1 contained the characteristic domains conserved in the TRAF and TAK1 families, respectively (OmTRAF6: RING, two TRAF-type zinc fingers, CCR and MATH domains; OmTAK1: STKc and CCR domains). In RTH-149 cells, the expression of OmTRAF6 and OmTAK1 was increased by stimulation with Edwardsiella tarda and LPS. Silencing of OmTRAF6 and OmTAK1 in RTH-149 cells negatively regulated the LPS-induced phosphorylation of p38 MAPK and JNK. TAK1 inhibitor (5z)-7-Oxozeaenol significantly decreased the LPS-induced activation of NF-κB in RTH-149 cells. In addition, silencing of OmTRAF6 and OmTAK1 significantly decreased the expression of MAPKs and NF-κB downstream target genes induced by LPS in RTH-149 cells. These findings suggest that OmTRAF6 and OmTAK1 might function like those of mammals to regulate bacteria-triggered signalling pathway in fish. Highlights • OmTRAF6 and OmTAK1 are conserved with their homologs from other vertebrates. • Expression of TRAF6 and TAK1 is increased by stimulation with E. tarda and LPS. • Silencing of TRAF6 and TAK1 blocks LPS-induced phosphorylation of p38 MAPK and JNK. • TAK1 is critical for the activation of NF-κB in LPS-stimulated RTH-149 cells. • TRAF6 and TAK1 might function to regulate bacteria-triggered signalling in fish. [ABSTRACT FROM AUTHOR]

Background: Despite its high prevalence, morbidity, and mortality, sepsis-associated encephalopathy (SAE) is still poorly understood. The aim of this prospective and observational study was to investigate the clinical significance of calcium-binding protein A8 (S100A8) in serum and tumor necrosis factor receptor-associated factor 6 (TRAF6) in peripheral blood mononuclear cells (PBMCs) in diagnosing SAE and predicting its prognosis. Methods: Data of septic patients were collected within 24 h after Intensive Care Unit admission from July 2014 to March 2015. Healthy medical personnel served as the control group. SAE was defined as cerebral dysfunction in the presence of sepsis that fulfilled the exclusion criteria. The biochemical indicators, Glasgow Coma Scale, Acute Physiology and Chronic Health Evaluation score II, TRAF6 in PBMC, serum S100A8, S100β, and neuron-specific enolase were evaluated in SAE patients afresh. TRAF6 and S100A8 were also measured in the control group. Results: Of the 57 enrolled patients, 29 were diagnosed with SAE. The S100A8 and TRAF6 concentrations in SAE patients were both significantly higher than that in no-encephalopathy (NE) patients, and higher in NE than that in controls (3.74 ± 3.13 vs. 1.08 ± 0.75 vs. 0.37 ± 0.14 ng/ml, P < 0.01; 3.18 ± 1.55 vs. 1.02 ± 0.63 vs. 0.47 ± 0.10, P < 0.01). S100A8 levels of 1.93 ng/ml were diagnostic of SAE with 92.90% specificity and 69.00% sensitivity in the receiver operating characteristic (ROC) curve, and the area under the curve was 0.86 (95% confidence interval [CI]: 0.76–0.95). TRAF6-relative levels of 1.44 were diagnostic of SAE with 85.70% specificity and 86.20% sensitivity, and the area under the curve was 0.94 (95% CI: 0.88–0.99). In addition, S100A8 levels of 2.41 ng/ml predicted 28-day mortality of SAE with 90.00% specificity and 73.70% sensitivity in the ROC curve, and the area under the curve was 0.88. TRAF6 relative levels of 2.94 predicted 28-day mortality of SAE with 80.00% specificity and 68.40% sensitivity, and the area under the curve was 0.77. Compared with TRAF6, the specificity of serum S100A8 in diagnosing SAE and predicting mortality was higher, although the sensitivity was low. In contrast, the TRAF6 had higher sensitivity for diagnosis. Conclusions: Peripheral blood levels of S100A8 and TRAF6 in SAE patients were elevated and might be related to the severity of SAE and predict the outcome of SAE. The efficacy and specificity of S100A8 for SAE diagnosis were superior, despite its weak sensitivity. S100A8 might be a better biomarker for diagnosis of SAE and predicting prognosis.

Background and objective It has been proven that tumor necrosis factor receptor-associated factor 6 (TRAF6) was a commonly amplified oncogene in lung cancer. However, the precise role of TRAF6 protein in lung cancer 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-қB (NF-қB) 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-қB was observed only in SPC-A-1 lung cancer cells. Downregulation of TRAF6 suppressed the NF-κB 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. Conclusion TRAF6 may be involved in cell migration, invasion, and apoptosis of SPC-A-1 cells, possibly through regulating the NF-қB-CD24/CXCR4 pathway.

Tumor necrosis factor receptor-associated factor 6 (TRAF6) is one of the key adapter molecules in Toll-like receptor signal transduction that triggers downstream cascades involved in innate immunity. Despite of the well study in vertebrates, there is few data ascribe to this TRAF member in invertebrates, especially in bivalves. In the present study, a novel TRAF6 homologue termed McTRAF6 was firstly characterized in Mytilus coruscus . Like its counterparts in mammals, McTRAF6 shared the domain topology containing one RING domain, two zinc finger domains, one coiled-coil region and a MATH domain. McTRAF6 transcripts predominantly expressed in gills, digestive glands and hemocytes in M. coruscus , and were significantly up-regulated in hemocytes after challenge with lipopolysaccharide (LPS) and polyinosine-polycytidylic acid (poly I:C). Further, the subcellular localization in cytoplasm and the activation of Nk-κB or ISRE luciferase reporter by overexpressed McTRAF6 were identified in HEK293T cells. These results collectively indicate that McTRAF6 is a member of TRAF6 subfamily and plays a potential role in immune defense system against pathogenic agents invasions in thick shell mussel. To our knowledge, this is the first report on component of TLR signaling pathway in thick shell mussel, providing further evidence for the existence of TLR pathway in M. coruscus and contribute to clarify the innate immune system of thick shell mussel. [ABSTRACT FROM AUTHOR]

Objective Tumor necrosis factor receptor-associated factor 6 (TRAF6) is an essential component of the signaling complex that mediates osteoclastogenesis. As an adaptor protein of E3 ligase function, TRAF6 regulates NF-κB signaling via TAK1 and I-κB kinase (IKK) activation. Here, we investigated novel mechanisms by which TRAF6 signaling is regulated under receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclastogenesis. Design A yeast two-hybrid screen system identified cellular factors that interact with TRAF6. The interactions were confirmed by glutathione S-transferase pull-down and co-immunoprecipitation assays, followed by immuno-blotting. The role of TRAF6 in bone growth and remodeling was determined by osteoclast differentiation and bone-resorption pit assays. Regulatory mechanisms were examined by co-immunoprecipitation, immuno-blotting, real-time polymerase chain reaction, and luciferase reporter assays. Results: We show that B-cell chronic lymphatic leukemia protein 3 (BCL3) interacts with TRAF6 through its ankyrin-repeat domain and inhibits osteoclastogenesis in bone marrow derived macrophages (BMDMs). Further, TRAF6 interacts with CYLD to mediate BCL3 deubiquitination, which facilitates the cytoplasmic accumulation of BCL3 and represses BCL3 and p50 complex-mediated cyclin D1 transcription. Conclusions TRAF6 promotes RANKL-induced osteoclastogenesis by regulating novel non-canonical NF-κB signaling via BCL3 deubiquitination, indicating that BCL3 provides valuable insights into bone loss-associated diseases. [ABSTRACT FROM AUTHOR]

Atorvastatin is a member of the statin class of drugs, which competitively inhibit the activity of 5-hydroxy-3-methylglutaryl-coenzyme A reductase. The aim of the present study was to assess whether atorvastatin may protect BV-2 microglia and hippocampal neurons against oxygen-glucose deprivation (OGD)-induced neuronal inflammatory injury and to determine the underlying mechanisms by which its effects are produced. Cell viability and apoptotic ability were assessed using an MTT assay and annexin V-fluorescein isothiocyanate/propidium iodide double staining followed by flow cytometry, respectively. The expression of inflammation and apoptosis-associated mRNAs and proteins were assessed using reverse transcription-quantitative polymerase chain reaction and western blotting, and the expression of inflammatory factors was determined using ELISA. The results of the current study revealed that atorvastatin treatment suppressed the viability of OGD BV-2 microglia and hippocampal neurons. Furthermore, atorvastatin treatment reduced the expression of proinflammatory factors in OGD BV-2 microglia. Additionally, it was demonstrated to downregulate the toll-like receptor 4 (TLR4)/tumor necrosis factor receptor-associated factor 6 (TRAF6)/nuclear factor-κB (NF-κB) pathway in OGD BV-2 microglia. Atorvastatin also inhibited the apoptosis of OGD hippocampal neurons by regulating the expression of apoptosis-associated proteins. It was concluded that atorvastatin treatment may protect BV-2 microglia and hippocampal neurons from OGD-induced neuronal inflammatory injury by suppressing the TLR4/TRAF6/NF-κB pathway. This may provide a potential strategy for the treatment of neuronal injury. [ABSTRACT FROM AUTHOR]

Background: Tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF6) has been proved to play an important role in tumorigenesis, invasion, and metastasis. However, its precise role salivary adenoid cystic carcinoma (SACC) has not been determined. The aim of this study was to explore the role of TRAF6 in SACC including invasion and metastasis of SACC cells.Materials and Methods: Immunohistochemistry and quantitative real-time PCR were performed in SACC tissues paired with their adjacent normal tissues to analyze the expression of TRAF6. Downstream proteins expression was explored when TRAF6 was knockdown by siRNA.Results: The results show that TRAF6 is upregulated in SACC samples, especially in SACC with metastasis, which is closely correlated with an aggressive phenotype (P = .0073) and shorter life survival span (P = .0061) in SACC patients. Knockdown of TRAF6 can attenuate the promotion effect of SACC cell invasion induced by TGF-β. Western blot results also showed that silencing TRAF6 expression can inhibit the activation of SMAD2, SMAD3, ERK, p38, and JNK induced by TGF-β in SACC cells.Conclusion: These data suggested that TRAF6 regulates TGF-β-mediated SACC progression through SMAD2/3-ERK-p38-JNK cascades. [ABSTRACT FROM AUTHOR]

Activation of peroxisome proliferator-activated receptor α (PPARα) by di-(2-ethylhexyl) phthalate (DEHP) has an anti-inflammatory effect. This study investigated the potential combined influence of PPARα, tumor necrosis factor α-induced protein 3 (TNFAIP3/A20), and tumor necrosis factor receptor-associated factor 6 (TRAF6) on interleukin (IL)-12p40 production by macrophages exposed to DEHP and stimulated with lipopolysaccharide (LPS). LPS upregulated IL-12p40 expression by granulocyte-macrophage colony-stimulating factor-dependent macrophages (on day 9 of culture), whereas adding DEHP to cultures significantly attenuated the response of IL-12p40 to LPS stimulation. PPARα protein was also reduced by DEHP. Interestingly, transfection of macrophages with small interfering RNA (siRNA) duplexes for PPARα, TNFAIP3/A20, or dual oxidase 2 restored the response of IL-12p40 protein to LPS stimulation in the presence of DEHP. siRNAs for various protein kinase Cs (PKCs) (α, β, γ, or δ) also restored IL-12p40 production by macrophages exposed to LPS and DEHP. While LPS upregulated both IL-12p40 and TNFAIP3/A20 production, adding DEHP to cultures dramatically reduced IL-12p40 and TNFAIP3/A20 levels. Silencing of PKCa reduced TNFAIP3/A20 production, whereas PKCg siRNA (but not PKCβ or δ siRNA) significantly increased TNFAIP3/A20. TRAF6 was also attenuated by macrophages with DEHP. The PPARα/TNFAIP3/TRAF6 axis may have an important role in the mechanism through which DEHP reduces IL-12p40 production by LPS-stimulated macrophages. [ABSTRACT FROM AUTHOR]

The present study aimed to investigate the effect of Toll‑like receptor 4 (TLR4) on SiHa human cervical cancer cells and its potential molecular biological mechanisms. The expression of TLR4 following treatment with lipopolysaccharide (LPS) in in SiHa cervical cancer cells was detected by quantitative polymerase chain reaction (qPCR). LPS‑induced cell proliferation and apoptosis were detected by MTT assay as well as staining with propidium iodide (PI) and Annexin V/PI double staining. qPCR was performed to analyze the expression levels of tumor necrosis factor receptor‑associated factor 6 (TRAF6) and transforming growth factor‑activated kinase 1 (TAK1) genes. Western blot analysis was performed to analyze the expression of myeloid differentiation 88 (MyD88), nuclear factor‑κB (NF‑κB), cyclin D1 and signal transducer and activator of transcription 3 (STAT3) proteins. In the present study, it was revealed that TLR4 expression in SiHa cervical cancer cells may be upregulated by LPS. Additionally, LPS was able to increase the proliferation of SiHa cells. However, LPS treatment did not have an effect on apoptosis of the cells. In addition, the MyD88‑TRAF6‑TAK1 and NF‑κB‑cyclin D1‑STAT3 signaling pathways were induced in SiHa cells by LPS. These results suggested the effect of LPS and TLR4 on proliferation of SiHa human cervical cancer cells via the MyD88‑TRAF6‑TAK1 and NF‑κB‑cyclin D1‑STAT3 signaling pathways. [ABSTRACT FROM AUTHOR]

Tumor necrosis factor receptor-associated factor 6 (TRAF6) is an adapter protein that triggers downstream cascades mediated by both TNFR and the interleukin-1 receptor/Toll-like receptor (IL-1R/TLR) superfamily. TRAF6 is involved in various biological processes, including innate and adaptive immunity. In the present study, a homolog of TRAF6 from Macrobrachium rosenbergii (MrTRAF6) was identified and characterized. The full-length cDNA of MrTRAF6 consisted of 2,114 nucleotides with an open reading frame (ORF) of 1,695 nucleotides encoding a 564-amino acid protein that contained a conserved TRAF family motif including two RING-type zinc fingers and a C-terminal meprin and TRAF homology (MATH) domain. The putative amino sequence of Mr TRAF6 shared 45.5–97.3% identity with TRAF6s from other crustacean species with the highest identity to Macrobrachium nipponense TRAF6. Phylogenetic analysis revealed that Mr TRAF6 was closely related to TRAF6 of invertebrates and clustered with crustaceans. According to gene expression analysis, the MrTRAF6 transcript demonstrated broad expression in all tissues tested, with the highest expression level in gill and the lowest in muscle tissues. Upon immune challenge with Aeromonas hydrophila , significant upregulation of MrTRAF6 expression was found in the gill, hepatopancreas, hemocyte, and muscle. Furthermore, an RNA interference assay showed that silencing MrTRAF6 by dsRNA could reduce the expression of mannose-binding lectin (MBL) and crustin, but no significant change was detected in anti-lipopolysaccharide factor 5 (ALF5) levels. In addition, the cumulative mortality rate of MrTRAF6 -silenced M. rosenbergii was significantly increased after A. hydrophila infection. These findings indicated that MrTRAF6 is involved in antibacterial activity and plays a critical role in the innate immune response of M. rosenbergii. • M. rosenbergii TRAF6 shared 45.5–97.3% identity with other crustacean species. • MrTRAF6 mRNA showed the highest expression level in gill and the lowest in muscle. • MrTRAF6 expression was induced in various organs by Aeromonas hydrophila infection. • Knockdown of MrTRAF6 increased the mortality rate after A. hydrophila infection. • MBL and crustin expressions were decreased in MrTRAF6 -silenced shrimp. [ABSTRACT FROM AUTHOR]

Ubiquitin-specific protease 14 (USP14), one of three proteasome-associated deubiquitinating enzymes, has multifunctional roles in cellular context. Here, we report a novel molecular mechanism and function of USP14 in regulating autophagy induction and nuclear factor-kappa B (NF-κB) activation induced by toll-like receptor (TLR) 4 (TLR4). USP14 interacted with tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF6) and interrupted the association of Beclin 1 with TRAF6, leading to inhibition of TRAF6-mediated ubiquitination of Beclin 1. Reduced expression of USP14 in USP14-knockdown (USP14KD) THP-1 cells enhanced autophagy induction upon TLR4 stimulation as shown by the increased conversion of cytosolic LC3-I to membrane-bound LC3-II. Moreover, USP14KD human breast carcinoma MDA-MB-231 cells and USP14KD human hepatic adenocarcinoma SK-HEP-1 cells showed increased cell migration and invasion, indicating that USP14 is negatively implicated in the cancer progression by the inhibition of autophagy induction. Furthermore, we found that USP14 interacted with TAK1-binding protein (TAB) 2 protein and induced deubiquitination of TAB 2, a key factor in the activation of NF-κB. Functionally, overexpression of USP14 suppressed TLR4-induced activation of NF-κB. In contrast, USP14KD THP-1 cells showed enhanced activation of NF-κB, NF-κB-dependent gene expression, and production of pro-inflammatory cytokines such as IL-6, IL-1β, and tumor necrosis factor-α. Taken together, our data demonstrate that USP14 can negatively regulate autophagy induction by inhibiting Beclin 1 ubiquitination, interrupting association between TRAF6 and Beclin 1, and affecting TLR4-induced activation of NF-κB through deubiquitination of TAB 2 protein.

Atherosclerosis (AS) is a chronic inflammation in response to lipid accumulation. Increasing evidence has demonstrated that numerous microRNAs (miRs) have critical roles in inflammatory responses. A previous study suggested that miR-146b-5p is possibly associated with AS; however, its exact role has remained largely elusive. The present study aimed to investigate the potential role of miR-146b-5p in AS and to explore the underlying mechanism. Fist, the levels of miR-146b-5p were determined in foam cells and clinical specimens from patients with AS by reverse-transcription quantitative PCR. The role of miR-146b-5p in AS was then investigated by using miR-146b-5p inhibitor. The results demonstrated that the expression levels of miR-146b-5p were elevated in the lesions of patients with AS. In addition, the levels of miR-146b-5p in THP-1 cells stimulated with phorbol 12-myristate 13-acetate (100 nM) to induce their differentiation into macrophages were dose- and time-dependently elevated by oxidized low-density lipoprotein treatment applied for inducing foam cell formation. miR-146b-5p was also revealed to directly target tumor necrosis factor receptor-associated factor 6 (TRAF6), which functions as a signal transducer in the nuclear factor-κB (NF-κB) pathway. Furthermore, the present study reported for the first time that miR-146b-5p inhibition promotes the inflammatory response and enhances lipid uptake during foam cell formation. In conclusion, miR-146b-5p inhibition promoted chronic inflammation and had a detrimental role during AS-associated foam cell formation by targeting TRAF6. [ABSTRACT FROM AUTHOR]

Tumor necrosis factor receptor-associated factor 6 (TRAF6) is an endogenous adaptor of innate and adaptive immune responses, and serves a crucial role in tumor necrosis factor receptor and toll-like/interleukin-1 receptor signaling. Although studies have demonstrated that TRAF6 has oncogenic activity, its potential contributions to breast cancer in human remains largely uninvestigated. The present study examined the expression levels and function of TRAF6 in breast carcinoma (n=32) and adjacent healthy (n=25) tissue samples. Compared with adjacent healthy tissues, TRAF6 protein expression levels were significantly upregulated in breast cancer tissues. Reverse transcription-quantitative polymerase chain reaction analysis revealed a significant upregulation of the cellular proliferative marker Ki-67 and proliferation cell nuclear antigen expression levels in breast carcinoma specimens. Furthermore, protein expression levels of the accessory molecule, transforming growth factor β-activated kinase 1 (TAK1), were significantly increased in breast cancer patients, as detected by western blot analysis. As determined by MTT assay, TRAF6 exerted profoundly proliferative effects in the MCF-7 breast cancer cell line; however, these detrimental effects were ameliorated by TAK1 inhibition. Notably, protein kinase B (AKT)/glycogen synthase kinase (GSK)3β phosphorylation levels were markedly upregulated in breast cancer samples, compared with adjacent healthy tissues. In conclusion, an altered TRAF6-TAK1 axis and its corresponding downstream AKT/GSK3β signaling molecules may contribute to breast cancer progression. Therefore, TRAF6 may represent a potential therapeutic target for the treatment of breast cancer. [ABSTRACT FROM AUTHOR]

Tumor necrosis factor receptor-associated factor 6 (TRAF6) is an important E3 ubiquitin ligase, which is key to immunity. TRAF6 has been implicated in the invasive growth and metastasis of various types of cancer, including squamous cell carcinoma, gastric cancer, myelodysplastic syndromes and acute myeloid leukemia. In the present study, associations between multiple myeloma (MM) and TRAF6, its downstream component nuclear factor-κB (NF-κB) and bone marrow stromal cells (MSC) were investigated. The TRAF6 protein expression levels of 18 patients were positively correlated with the protein levels of β-2 microglobulin (r2=0.3472; P=0.01) and negatively correlated with albumin protein levels (r2=0.5494; P=0.0004). In vitro expression of the TRAF6 protein, phosphorylated transcription factor p65 and phosphorylated p100 in myeloma cell lines was induced by MSCs from patients with MM. In addition, the in vitro expression of TRAF6 was associated with an enhanced proliferation rate of myeloma cells, which was blocked by silencing TRAF6 using small interfering RNA. Due to the association between the TRAF6-NF-κB signaling pathway in myeloma cells and MSCs, this signaling pathway may be a useful prognostic and therapeutic target in myeloma. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

TRAF6 is a member of TRAF family，which serves as an adapter coupling for the TNF receptor (TNFR) superfamily in intracellular signaling events． Moreover，TRAF6 is unique for signaling downstream of another receptor family，the interleukin-1 (IL-1) receptor /Toll-like receptor (IL-1R/ TLR) superfamily，which plays critical roles in inflammation and innate and adaptive immune responses． TRAF6 regulates inflammation and links immunity to bone metabolism． This paper reviews the current studies in TRAF6 on bone metabolism． [ABSTRACT FROM AUTHOR]

Background: The inflammatory immune response plays an important role in mesenteric ischemia and ischemia-reperfusion injury. Toll-like receptor 4 (TLR4) is a critical receptor in transduction of the inflammatory response and plays an important role in intestinal homeostasis. Tumor necrosis 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. [ABSTRACT FROM AUTHOR]

MicroRNAs (miRs) have been observed to be involved in the modulation of various physiopathological processes. However, the impacts of miRNAs on muscle atrophy have not been fully investigated. In the present study, the results demonstrated that miR-351 was differentially expressed in the tibialis anterior (TA) muscle at various times following sciatic nerve transection, and the time-dependent expression profile of miR-351 was inversely correlated with that of tumor necrosis factor receptor-associated factor 6 (TRAF6) at the mRNA and protein levels. The dual luciferase reporter assay indicated that miR-351 was able to significantly downregulate the expression levels of TRAF6 by directly targeting the 3'-untranslated region of TRAF6. Overexpression of miR-351 inhibited a significant decrease in the wet weight ratio or cross-sectional area of the TA muscle following sciatic nerve transection. Western blot analysis indicated that the protein expression levels of TRAF6, muscle ring-finger protein 1 (MuRF1) and muscle atrophy F-box (MAFBx) in denervated TA muscles were suppressed by overexpression of miR-351. These results demonstrate that miR-351 inhibits denervation-induced atrophy of TA muscles following sciatic nerve transection at least partially through negative regulation of TRAF6 as well as MuRF1 and MAFBx, the two downstream signaling molecules of TRAF6. [ABSTRACT FROM AUTHOR]

Protein kinase B (AKT), is a pivotal component of pathways associated with cell survival, metabolism, invasion and metastasis. AKT mediates anti-apoptotic and proliferative signaling in response to essential cytokines. Tumor necrosis factor receptor-associated factor (TRAF)6, an E3 ubiquitin ligase, has been shown to ubiquitylate, as well as activate AKT. The present study used computational methods to determine the relevant amino acid residues at the binding site of TRAF6 and selected small molecules, which may bind to TRAF6. An ex vivo assay was performed to determine their antitumor activities and the possible mechanism of action. Quinine, a natural alkaloid that is well-known for its therapeutic treatment of malaria, exhibited a distinct antiproliferative and pro-apoptotic effect in HeLa and A549 tumor cell lines via the inhibition of the antiapoptotic protein, B-cell lymphoma (BCL)-2, and activation of the pro-apoptotic factor, BCL-2-associated X protein. Quinine inhibited the lipopolysaccharide (LPS)-induced activation of AKT by inhibiting its phosphorylation at Thr-308 and Ser-473, and reversing LPS-induced proliferation. These results suggested that the inhibition of AKT activation via targeting of TRAF6 with quinine may be a viable anticancer therapeutic approach and a successful example of the alternative use of the original therapeutic properties of this well-known natural product. [ABSTRACT FROM AUTHOR]

The matrine-type alkaloid, oxymatrine inhibits hepatitis B virus (HBV) replication but very little is known about these effects in other matrine-type alkaloids, including sophoridine and sophocarpine. Therefore, we compared the in vitro anti-HBV effects of matrine, oxymatrine, sophocarpine, and sophoridine by treating an HBV-transfected cell line (HepG2.2.15) with 0.4–1.6 mM of the compounds for 24 or 72 h. The levels of the HBV surface antigen (HBsAg) and e antigen (HBeAg) in the culture medium, as well as the intracellular and extracellular HBV DNA levels, were determined. Metabolomic analysis and detection of the mRNA level of p38 mitogen-activated protein kinase (MAPK), tumor necrosis factor receptor-associated factor (TRAF) 6, extracellular signal-regulated kinase (ERK) 1, NOD-like receptor family pyrin domain containing 10 (NLRP10), and caspase-1 were conducted in sophoridine-treated HepG2.2.15 cells. HepG2.2.15 cell exposure to 0.4–1.6 mM sophocarpine or sophoridine for 24 h reduced the HBsAg level of the medium more effectively than exposure to matrine and oxymatrine did, and reduced the HBeAg levels more effectively than these compounds did at 1.6 mM. Sophoridine (0.4–1.6 mM) reduced the cell medium HBV DNA levels more than the same concentrations of matrine, oxymatrine, or sophocarpine did. After 72 h, 0.4 and 0.8 mM sophoridine reduced HBsAg and intracellular HBV DNA levels more potently than matrine, oxymatrine, or sophocarpine did. Furthermore, sophoridine (0.8 mM) potently reduced the cell medium HBeAg levels while the metabolomic analyses revealed that HepG2.2.15 cells exposed to 0.8 mM sophoridine for 72 h exhibited reduced cycloleucine and phytosphingosine levels. In addition, the mRNA expression analyses revealed that HepG2.2.15 cells exposed to 0.8 mM sophoridine showed reduced levels of p38 MAPK, TRAF6, ERK1, NLRP10, and caspase-1. Sophoridine produced more potent anti-HBV effects than matrine, oxymatrine, and sophocarpine did. These effects may be related to the sophoridine-mediated reduction of p38 MAPK and TRAF6 levels. [ABSTRACT FROM AUTHOR]

Introduction Enterococcus faecalis is a frequently isolated microorganism in persistent periapical lesion or secondary infection. However, no evidence has demonstrated that E. faecalis induced inflammation directly in the apical area. This study aimed to explore the mechanism of the inflammatory responses of human periodontal ligament fibroblasts (PDLs) to E. faecalis . Methods PDLs were stimulated with heat-killed E. faecalis (HKEF) or lipoteichoic acid from E. faecalis (LTA) with or without silencing of tumor necrosis factor receptor-associated factor 6 (TRAF6). The expressions of toll-like receptor 2/4, nucleotide-binding oligomerization domain 1/2, and TRAF6 were detected by using quantitative real-time polymerase chain reaction and Western blot. The secretions of proinflammatory cytokines, including interleukin-1β, interleukin-6, interleukin-8, and tumor necrosis factor-α, were determined in the cell supernatants with enzyme-linked immunosorbent assay. Results Both HKEF and LTA stimulated the expression of toll-like receptor 2 and TRAF6 in a time-dependent manner. The secretions of proinflammatory cytokines were also increased. After silencing TRAF6, the upregulations of proinflammatory cytokines induced by HKEF or LTA were attenuated. Conclusions TRAF6 plays a pivotal role in inflammation induced by E. faecalis or its LTA in PDLs. [ABSTRACT FROM AUTHOR]

Copyright of Chinese Journal of Lung Cancer is the property of Chinese Journal of Lung Cancer and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Premature ovarian failure (POF), an ovarian disorder of multifactorial origin, is defined as the occurrence of amenorrhea, hypergonadotropism and hypoestrogenism in females <40 years old. Apoptosis of ovarian granulosa cells is important in POF and understanding the regulatory mechanism underlying ovarian granulosa cell apoptosis may be beneficial for the management of POF. Increasing evidence suggests that microRNAs (miRs) have a regulatory function in oocyte maturation and ovarian follicular development. In the present study, the expression of miR-146a in plasma and ovarian granulosa cells obtained from patients with POF, its effect on the apoptosis of ovarian granulosa cells and the possible underlying mechanisms were examined. The present study demonstrated that compared with the control groups, the expression of miR-146a in the plasma and in ovarian granulosa cells of patients with POF was significantly upregulated. Furthermore, it was found that miR-146a simultaneously targeted interleukin-1 receptor-associated kinase (IRAK1) and tumor necrosis factor receptor-associated factor 6 (TRAF6), which regulated the activity of nuclear factor-κB and IκBα. In addition, the results demonstrated that inhibition of the caspase cascade by caspase inhibitors attenuated the effects of miR-146a on ovarian granulosa cell apoptosis. Taken together, these results suggest that miR-146a has an important promoting effect on the apoptosis of granulosa cells by targeting IRAK1 and TRAF6 via the caspase cascade pathway. These results may be useful for the management of POF. [ABSTRACT FROM AUTHOR]

The effect of TGF-β1 on CpG DNA-induced type I IFN production was examined by reconstituting a series of signaling molecules in TLR 3 signaling. TGF-β1 inhibited CpG DNA-induced IFN-α4 productivity in HeLa cells. Transfection of IFN regulatory factor (IRF)7 but not TNF receptor-associated factor (TRAF)6 and TRAF3 into cells triggered IFN-α4 productivity, and TGF-β1 inhibited IRF7-mediated type I IFN production in the presence of TRAF6. TGF-β1 induced ubiquitination of TRAF6, although CpG DNA did not induce it. Moreover, TGF-β1 accelerated the ubiquitination of TRAF6 in the presence of CpG DNA. TGF-β1 ubiquitinated TRAF6 at K63 but not K48. TGF-β1 also induced ubiquitination of IRF7. Further, TGF-β1 did not impair the interaction of IRF7 and TRAF6. CpG DNA induced the phosphorylation of IRF7 in the presence of TRAF6, whereas TGF-β1 inhibited the IRF7 phosphorylation. Blocking of TRAF6 ubiquitination abolished the inhibition of CpG DNA-induced type I IFN production by TGF-β. Taken together, TGF-β was suggested to inhibit CpG DNA-induced type I IFN production transcriptionally via ubiquitination of TRAF6. [ABSTRACT FROM AUTHOR]

Endothelial dysfunction induced by intermittent hypoxia (IH) participates in obstructive sleep apnea syndrome (OSAS)-associated cardiovascular disorders. Myeloid differentiation primary response 88 (MyD88) and tumor necrosis factor receptor-associated factor 6 (TRAF6) regulate numerous downstream adaptors like mitogen-activated protein kinases (MAPKs) and the subsequent oxidative stress and inflammatory responses. This study aimed to characterize the role of MyD88/TRAF6 in IH-treated cell function and its associated signaling. Human umbilical vein endothelial cells (HUVECs) were randomly exposed to IH or normoxia for 0, 2, 4 and 6 h. Western blotting was used to detect the expression pattern of target gene proteins [angiotensin 1 receptor (ATR), p-ERK1/2, p-p38MAPK, MyD88 and TRAF6], and the relationships among these target genes down-regulated by the corresponding inhibitors were studied. Finally, the influence of these target genes on proliferation of HUVECs was also assessed by EdU analysis. Protein levels of ATR, TRAF6 and p-ERK1/2 were increased after IH exposure, with a slight rise in MyD88 and a dynamic change in p-p38MAPK. The down-regulation of TRAF6 by siRNA reduced ERK1/2 phosphorylation during IH without any effects on ATR. Blockade of ATR with valsartan decreased TRAF6 and p-ERK1/2 protein expression after IH exposure. ERK1/2 inhibition with PD98059 suppressed only ATR expression. IH promoted HUVECs proliferation, which was significantly suppressed by the inhibition of TRAF6, ATR and ERK1/2. The findings demonstrate that TRAF6 regulates the proliferation of HUVECs exposed to short-term IH by modulating cell signaling involving ERK1/2 downstream of ATR. Targeting the ATR-TRAF6-p-ERK1/2 signaling pathway might be helpful in restoring endothelial function. [ABSTRACT FROM AUTHOR]

Amyloid beta (Aβ) protein is the primary proteinaceous deposit found in the brains of patients with Alzheimer's disease ( AD). Evidence suggests that Aβ plays a central role in the development of AD pathology. Here, we show in PC12 cells, Aβ impairs tropomyosin receptor kinase A (TrkA) ubiquitination, phosphorylation, and its association with p75 NTR, p62, and TRAF6 induced by nerve growth factor. The ubiquitination and tyrosine phosphorylation of TrkA was also found to be impaired in postmortem human AD hippocampus compared to control. Interestingly, the nitrotyrosylation of TrkA was increased in AD hippocampus and this explains why the phosphotyrosylation and ubiquitination of TrkA was impaired. In AD brain, the production of matrix metalloproteinase-7 ( MMP-7), which cleaves pro NGF, was reduced, thereby leading to the accumulation of pro- NGF and a decrease in the level of active NGF. TrkA signaling events, including Ras/MAPK and phosphatidylinositol 3-kinase ( PI3K)/Akt pathways, are deactivated with Aβ and in the human AD hippocampus. Findings show that Aβ blocks the TrkA ubiquitination and downstream signaling similar to AD hippocampus. [ABSTRACT FROM AUTHOR]

This study examined the mechanism of the inhibitory effect of parthenolide (PTL) on the activity of NF-κB in multiple myeloma (MM). Human multiple myeloma cell line RPMI 8226 cells were treated with or without different concentrations of PTL for various time periods, and then MTT assay was used to detect cell proliferation. Cell cycle and apoptosis were flow cytometrically detected. The level of protein ubiquitination was determined by using immunoprecipitation. Western blotting was employed to measure the level of total protein ubiquitination, the expression of IκB-α in cell plasma and the content of p65 in nucleus. The content of p65 in nucleus before and after PTL treatment was also examined with immunofluorescence. Exposure of RPMI 8226 cells to PTL attenuated the level of ubiquitinated Nemo, increased the expression of IκB-α and reduced the level of p65 in nucleus, finally leading to the decrease of the activity of NF-κB. PTL inhibited cell proliferation, induced apoptosis and blocked cell cycle. Furthermore, the levels of ubiquitinated tumor necrosis factor receptor-associated factor 6 (TRAF6) and total proteins were decreased after PTL treatment. By using Autodock software package, we predicted that PTL could bind to TRAF6 directly and tightly. Taken together, our findings suggest that PTL inhibits the activation of NF-κB signaling pathway via directly binding with TRAF6, thereby suppressing MM cell proliferation and inducing apoptosis. [ABSTRACT FROM AUTHOR]

Fibroblast-like synoviocytes (FLSs) have a pivotal role in the destruction of joints in rheumatoid arthritis (RA). Tumor necrosis factor receptor-associated factor 6 (TRAF6) is a critical mediator in the inflammatory pathway and of the activity of osteoclasts. The aim of the present study was to investigate whether TRAF6 is involved in the progression of RA in mouse collagen-induced arthritis (CIA) and human RA FLSs in vitro. In vivo mouse models were transfected with TRAF6 small interfering (si)RNA (siTRAF6) and TRAF6 inhibition was achieved in FLSs using an anti-TRAF6 monoclonal antibody in vitro in order to assess the effects of TRAF6 inhibition on the migration and invasion of FLSs. Inhibition of TRAF6 using mouse specific siTRAF6 reduced the severity of arthritis and joint inflammation. Serum anti-collagen II antibodies, matrix metalloproteinase (MMP)-1, MMP-3 and MMP-9 were also inhibited in CIA mice by siTRAF6. The levels of MMPs produced by IL-1β-stimulated human RA-FLSs were reduced by anti-TRAF6 monoclonal antibody. TRAF6 blockade significantly suppressed the IL-1β-stimulated migration and invasion of human RA-FLSs. These results support a role for TRAF6 in the pathogenesis of RA, and suggest that the TRAF6 blockade may be a potential strategy in the management of RA. [ABSTRACT FROM AUTHOR]

An enriched environment is used as a behavioral intervention therapy that applies sensory, motor, and social stimulation, and has been used in basic and clinical research of various neurological diseases. In this study, we established mouse models of photothrombotic stroke and, 24 hours later, raised them in a standard, enriched, or isolated environment for 4 weeks. Compared with the mice raised in a standard environment, the cognitive function of mice raised in an enriched environment was better and the pathological damage in the hippocampal CA1 region was remarkably alleviated. Furthermore, protein expression levels of tumor necrosis factor receptor-associated factor 6, nuclear factor κB p65, interleukin-6, and tumor necrosis factor α, and the mRNA expression level of tumor necrosis factor receptor-associated factor 6 were greatly lower, while the expression level of miR-146a-5p was higher. Compared with the mice raised in a standard environment, changes in these indices in mice raised in an isolated environment were opposite to mice raised in an enriched environment. These findings suggest that different living environments affect the hippocampal inflammatory response and cognitive function in a mouse model of stroke. An enriched environment can improve cognitive function following stroke through up-regulation of miR-146a-5p expression and a reduction in the inflammatory response., Competing Interests: None

Pseudomonas aeruginosa (PA)-induced keratitis is characterized by inflammatory epithelial edema, stromal infiltration, corneal ulceration and can lead to vision loss. The present study aimed to study the effect of ubiquitin-specific protease 22 (USP22) on PA-induced keratitis. Using RT-qPCR and western blotting, significantly increased expression of USP22 was identified in mouse corneas and cultured RAW264.7 cells following PA stimulation. In addition, the results of in vivo experiments, western blot assay and ELISA suggested that the silencing of USP22 attenuated disease progression, downregulated the NF-κB pathway and suppressed the expression of pro-inflammatory cytokines following PA stimulation. Notably, it was identified that the expression of tumor necrosis factor receptor-associated factor 6 (TRAF6) was decreased by silencing of USP22 and USP22 was found to remove lysine 48-linked poly-ubiquitination chains from TRAF6 to stabilize TRAF6 expression and these effects were clearly aggravated following PA infection. [ABSTRACT FROM AUTHOR]

Highlights: [•] The full-length cDNAs of MyD88 and TRAF6 were cloned from Apostichopus japonicus. [•] AjMyD88 and AjTRAF6 mRNA transcripts were constitutively expressed in all examined tissues. [•] AjMyD88 and AjTRAF6 could be up-regulated toward Vibrio splendidus exposure with larger amplitude and longer duration in AjTRAF6. [Copyright &y& Elsevier]

Summary: Objective: MiR-146a exerts negative control on inflammatory responses by suppressing cytokine-induced expression of interleukin-1 receptor-associated kinase-1 (IRAK1) and tumor necrosis factor receptor-associated factor 6 (TRAF6) by impairing NF-κB activity and inhibiting the expression of target genes. Recent study suggests that histone deacetylases (HDACs) are involved in the regulation of microRNA (miRNA) expression. Therefore, we determined whether HDAC inhibitors can increase miR-146a expression, thereby inhibiting interleukin-1β (IL-1β)-induced signaling in osteoarthritis fibroblast-like synoviocytes (OA-FLS). Method: MiRNA expression was analyzed using real-time PCR. IL-1β-induced downstream signals and cytokine expression were evaluated using Western blotting and ELISA. Transcription factors regulating promoter activation were identified using chromatin immunoprecipitation assays. Results: IL-1β treatment of OA-FLS induced a mild (1.7-fold) increase in miR-146a expression that was unable to appropriately downregulate IRAK1 and TRAF6 expression. HDAC inhibitors, SAHA (vorinostat), and LBH589 (panobinostat) significantly (6.1- and 5.4-fold) elevated miR-146a expression by increasing the binding of the transcription factor NF-κB to the miR-146a promoter, and negatively regulated IL-1β-induced IKK/IκB/p65 phosphorylation signaling and IL-6 secretion. The increase in miR-146a expression induced by the HDAC inhibitors was prevented by transfection of miR-146a inhibitor or HDAC1 (class I HDAC), HDAC4 (class IIa HDAC), and HDAC6 (class IIb HDAC) overexpression, suggesting that they were due to inhibition of HDAC activity. Conclusions: Our study demonstrated that HDAC inhibitor treatment in OA-FLS significantly increased miR-146a expression and mediated markedly negative regulation to inhibit IL-1β-induced signaling and cytokine secretion. Our results indicate the potential rationale of anti-inflammatory effects for HDAC inhibitors. [Copyright &y& Elsevier]

Increasing evidence highlights the multiple roles of microRNAs (miRs) in the tumorigenesis of colorectal cancer (CRC); however, the molecular mechanism, particularly the target of miR-146b-5p in CRC has not been fully elucidated. The present study aimed to elucidate the influence of miR-146b-5p via regulating tumor necrosis factor receptor-associated factor 6 (TRAF6) in CRC. The expression levels of miR-146b-5p and TRAF6 in CRC tissue and cells were determined by reverse transcription quantitative PCR and western blotting. Binding between miR-146b-5p and TRAF6 was examined using a dual luciferase reporter gene assay. The impact of miR-146b-5p and TRAF6 on proliferation and migration of CRC cells was determined using Cell Counting Kit-8 and Transwell assays, respectively. An animal model of CRC was established to determine the carcinogenic effect of the miR-146b-5p-TRAF6 axis. The results demonstrated that miR-146b-5p was highly expressed in CRC tissue samples compared with in normal adjacent tissue samples and in CRC cells compared with in the normal NCM460 cell line, whereas TRAF6 was expressed at low levels. Overexpression of miR-146b-5p decreased TRAF6 expression in CRC HT29 and SW620 cells. miR-146b-5p targeted and inhibited TRAF6 expression in CRC cells. Furthermore, transfection with a miR-146b-5p mimic promoted the proliferation, migration and invasion of CRC cells and tumor growth; however, these effects were abolished by TRAF6 overexpression. Transfection with a miR-146b-5p inhibitor suppressed the proliferation of CRC cells. Taken together, the results from the present study demonstrated that miR-146b-5p could enhance the initiation and tumorigenesis of CRC by targeting TRAF6. These results will help elucidate the mechanisms underlying CRC development and will facilitate the development of targeted therapy for CRC. [ABSTRACT FROM AUTHOR]

Abstract: TRAF6 is an E3 ubiquitin ligase that transduces signals from members of the TLR/IL-1R family. Multiple molecules have been found to associate with TRAF6 and exert their functions in this pathway. Herein, by yeast two-hybrid screen using TRAF6 as bait, we identified PP4 as a potential TRAF6-interacting protein. PP4 physically interacted with TRAF6 and was recruited to TLR4 complex upon LPS stimulation. PP4 negatively regulated LPS-induced and TRAF6-mediated NF-κB activation by inhibiting the ubiquitination of TRAF6. LPS stimulation also induced the expression of PP4. Taken together, our findings suggest that PP4 is a negative feedback regulator of LPS/TLR4 pathway. [Copyright &y& Elsevier]