Exploring the mechanism of Nav1.3 in the ION-CCI rat model based on the TLR4/TRAF6/NF-κB pathway.

BACKGROUND: Trigeminal neuralgia (TN) is a common and difficult-to-treat neuropathic pain disorder in clinical practice. Previous studies have shown that Toll-like receptor 4 (TLR4) modulates the activation of the NF-κB pathway to affect neuropathic pain in rats. Voltage-gated sodium channels (VGSCs) are known to play an important role in neuropathic pain electrical activity. OBJECTIVE: To investigate whether TLR4 can regulate Nav1.3 through the TRAF6/NF-κB p65 pathway after infraorbital nerve chronic constriction injury (ION-CCI). STUDY DESIGN: ION-CCI modeling was performed on SD (Sprague Dawley) rats. To verify the success of the modeling, we need to detect the mechanical pain threshold and ATF3. Then, detecting the expression of TLR4, TRAF6, NF-κB p65, p-p65, and Nav1.3 in rat TG. Subsequently, investigate the role of TLR4/TRAF6/NF-κB pathway in ION-CCI model by intrathecal injections of LPS-rs (TLR4 antagonist), C25-140 (TRAF6 inhibitor), and PDTC (NF-κB p65 inhibitor). RESULTS: ION-CCI surgery decreased the mechanical pain threshold of rats and increased the expression of ATF3, TLR4, TRAF6, NF-κB p-p65 and Nav1.3, but there was no difference in NF-κB p65 expression. After inject antagonist or inhibitor of the TLR4/TRAF6/NF-κB pathway, the expression of Nav1.3 was decreased and mechanical pain threshold was increased. CONCLUSION: In the rat model of ION-CCI, TLR4 in the rat trigeminal ganglion regulates Nav1.3 through the TRAF6/NF-κB p65 pathway, and TLR4 antagonist alleviates neuropathic pain in ION-CCI rats.

Involvement of tumor necrosis factor receptor-associated factor 6 (TRAF6) in NF-κB activation and antiviral immunity: Molecular and functional characterization of TRAF6 in red-spotted grouper (Epinephelus akaara).

Tumor necrosis factor receptor-associated factor 6 (TRAF6) is a member of the TRAF family of adaptor proteins involved in the signal transduction pathways of both TNF receptor and interleukin-1 receptor/Toll-like receptor superfamilies. In this study, red-spotted grouper (Epinephelus akaara) TRAF6 (EaTraf6) was identified and characterized. The open reading frame of EaTraf6, 1713 bp in length, encodes a putative protein of 570 amino acids and has a predicted molecular weight and theoretical isoelectric point of 64.11 kDa and 6.07, respectively. EaTraf6 protein contains an N-terminal RING-type zinc finger domain, two TRAF-type zinc finger domains, a coiled-coil region (zf-TRAF), and a conserved C-terminal meprin and TRAF homology (MATH) domain. EaTraf6 shared the highest amino acid sequence identity with its ortholog from Epinephelus coioides, and phylogenetic analysis showed all fish TRAF6s clustered together and apart from other species. qRT-PCR results revealed that EaTraf6 was ubiquitously expressed in all examined tissues, with the highest level detected in the blood. In the immune challenge, EaTraf6 exhibited modulated mRNA expression levels in the blood and spleen. The subcellular localization analysis revealed that the EaTraf6 protein was predominantly present in the cytoplasm; however, it could translocate into the nucleus following poly (I:C) stimulation. The antiviral function of EaTraf6 was confirmed by analyzing the expression of host antiviral genes and viral genomic RNA during viral hemorrhagic septicemia virus infection. Additionally, luciferase reporter assay results indicated that EaTraf6 is involved in the activation of the NF-κB signaling pathway upon poly (I:C) stimulation. Finally, the effect of EaTraf6 on cytokine gene expression and its role in regulating macrophage M1 polarization were demonstrated. Collectively, these findings suggest that EaTraf6 is a crucial immune-related gene that significantly contributes to antiviral functions and regulation of NF-κB activity in the red-spotted grouper.

Dahuang Lingxian Prescription Regulates Effect of TAK1 and TRAF6 Interaction and Co-localization on Inflammatory Response of Bile Duct Cells / 中国实验方剂学杂志

ObjectiveTo observe the repair effect of Dahuanglingxian prescription （DHLX） on bile duct epithelial cells of rats. To explore whether its mechanism of action is to adjust the mutual binding of transforming growth factor -β （TGF-β） activated kinase 1（TAK1） and tumor necrosis factor receptor-associated factor 6 （TRAF6）， and regulate the activation of the nuclear transcription factor -κB （NF-κB）/mitogen-activated protein kinase （MAPK） signaling pathway. MethodThe 20 SD rats were randomly divided into normal group and DHLX group， 10 rats in each group， were given saline and DHLX （320 mg·kg-1·d-1） for 8 days， to prepare normal serum and DHLX serum. Biliary epithelial cells were extracted from normal SD rats and divided into 9 groups： Normal group， model group （20 mg·L-1）， LPS+DHLX group （20 mg·L-1+10% DHLX）， LPS+PDTC group （20 mg·L-1+200 μmol·L-1）， LPS+SB203580 group （20 mg·L-1+0.5 μmol·L-1）， LPS+PDTC+SB203580 group （20 mg·L-1+200 μmol·L-1+0.5 μmol·L-1）， LPS+PDTC+DHLX group （20 mg·L-1+200 μmol·L-1+10% DHLX serum）， LPS+SB203580+DHLX group （20 mg·L-1+0.5 μmol·L-1+10% DHLX serum）， LPS+PDTC+SB203580 +DHLX group （20 mg·L-1+200 μmol·L-1+0.5 μmol·L-1+10% DHLX serum）. The microscopic observation of morphological changes in each group of cells after drug intervention. Enzyme-linked immunosorbent assay（ELISA） was used to detect the expression of （IL）-1β and IL-6 in each group of cells. Western blot detected the expression levels of TAK1 and TRAF6 proteins in each group of cells， Co-IP detected the interaction between TAK1 and TRAF6， and further observed the distribution and co-localization of TAK1 and TRAF6 using Laser confocal microscope. ResultAfter the action of LPS， the cell synapses are reduced， the cell body becomes significantly rounded and smaller， but the cell morphology of each group tends to be normal after medication. Compared with normal group， the expression levels of IL-1β and IL-6 in model group were significantly increased （P<0.05）， while the expression level of TAK1 was decreased while the expression level of TRAF6 was increased （P<0.05）. The content of TAK1-TRAF6 protein complex showed a decreasing trend， and the two proteins co-located in the cytoplasm. Compared with model group， the expression levels of IL-1β and IL-6 in LPS+DHLX group were significantly decreased （P<0.05）， the expression level of TAK1 was increased and the expression level of TRAF6 was decreased （P<0.05）， the content of TAK1-TRAF6 protein complex was significantly increased （P<0.01）， and the two proteins were significantly co-located in cytoplasm. Compared with LPS+DHLX group， the expression levels of IL-1β and IL-6 in other groups were significantly decreased （P<0.05，P<0.01）. TAK1-TRAF6 protein complex content in each group was significantly decreased after pathway blocker intervention （P<0.05）， while TAK1-TRAF6 protein complex content in each group was significantly increased after pathway blocker combined with DHLX intervention （P<0.05）. Co-localization of the TAK1-TRAF6 in cytoplasm was not obvious. ConclusionIn the LPS-induced inflammatory response of bile duct cells， the binding of TAK1 and TRAF6 showed a weakening trend， but DHLX could reverse the phenomenon， we think the mechanism of action may be related to promoting the mutual binding of TAK1 and TARF6 to inhibit the activation of the NF-κB/MAPK signaling pathway.

The extreme C-terminus of IRAK2 assures full TRAF6 ubiquitination and optimal TLR signaling.

Macrophages are fundamental for initiation, maintenance, and resolution of inflammation. They can be activated by 'Toll-like receptor' (TLR) engagement, which initiates critical pathways to fight infections. 'Interleukin receptor-associated kinase 2' (IRAK2) is part of the membrane-proximal Myddosome formed at IL-1R/TLRs, but utility and regulation of IRAK2 within is not completely understood. In this study, we addressed the importance of the evolutionary conserved extreme C-terminus of IRAK2 in TLR signaling. The last 55 amino acids lack any known functional domain. The C-terminus deletion mutant IRAK2Δ55 was hypofunctional and disabled to conduct TLR4-inducible NF-κB and ERK2 activation. Accordingly, it could neither fully support subsequent CD40 cell surface expression nor IL-6 and nitric oxide release. Interestingly, IRAK2Δ55 was still capable to bind to 'tumor necrosis factor receptor-associated factor 6' (TRAF6), which is requisite to activate TRAF6 as an E3-ubiquitin ligase for further downstream signaling. However, IRAK-dependent auto-ubiquitination of TRAF6 was impaired, when IRAK2Δ55 was bound. Thus, the conserved last 55 amino acids enable IRAK2 to sustain an optimal TLR response. This knowledge might spark ideas how overshooting inflammatory responses could be modified without blocking the entire immune response.

Molecular characterization and expression analysis of tumor necrosis factor receptor-associated factor 6 (traf6) like gene involved in antibacterial innate immune of fresh water crayfish, Procambarus clarkii.

In invertebrates, innate immunity was the crucial defending pattern against pathogenic microorganisms. For the past few years, Toll or Toll like receptors (TLRs) signaling pathway was studied extensively in crustaceans. Among the components of Toll or Toll like receptors (TLRs) signaling pathway, tumor necrosis factor receptor-associated factor 6 (TRAF6) acted as an important cytoplasmic adaptor, which was conserved from Drosophila to human. In this study, a new traf6 like gene was cloned from hepatopancreas of P. clarkii. After challenged respectively by S. aureus or E. ictaluri, the expression profiles were studied. And the results showed that the mRNA transcript of Pc-traf6 like gene was up-regulated significantly in the hemocytes, hepatopancreas, gills, and intestine of crayfish. After Pc-traf6 like gene was knocked down, the expression levels of transcription factor (Dorsal) and some crucial immunity effectors (ALF 3, Lysozyme 1, Lectin 1, and Crustin 2) in TLRs signaling pathway were dramatically suppressed. Simultaneously, the survival rate of crayfish challenged respectively by S. aureus or E. ictaluri was significantly decreased in RNAi assay. All these results indicated that Pc-traf6 like gene played an important role in regulating the expression of downstream effectors in the TLRs signaling pathway of crayfish.

H4 Receptor Inhibits Lipopolysaccharide-induced NF-κB Activation by Interacting with Tumor Necrosis Factor Receptor-Associated Factor 6.

Microglia, the resident immune cells of the central nervous system (CNS), are activated at the beginning of the inflammatory response and induce detrimental neuroinflammation by producing excessive pro-inflammatory cytokines. Nuclear factor kappa B (NF-κB) signaling facilitates the onset of microglia activation. However, the molecular mechanisms underlying the negative regulation of NF-κB remain to be fully elucidated. In the present study, our results indicated that H4R expression increased in a rat model of lipopolysaccharide (LPS)-induced CNS inflammation. Knockdown of H4R in microglia HAPI cells enhanced the production of cytokines following LPS stimulation. Co-immunoprecipitation experiments further revealed an interaction between H4R and tumor necrosis factor receptor-associated factor 6 (TRAF6) in microglia, which was verified both in vivo and in vitro. Our experimental results support our hypothesis that H4R interacts with TRAF6 to inhibit the release of inflammatory cytokines in LPS-induced microglia cells by decreasing TRAF6-mediated ubiquitination of K63. These findings provide theoretical and experimental evidence regarding the role of H4R in the microglia inflammatory response, which may aid in the development of novel treatments for inflammation.

A TRAF6 genetic variant is associated with low bone mineral density in rheumatoid arthritis.

OBJECTIVES: This study was aimed to investigate the association of the single nucleotide polymorphism of tumor necrosis factor receptor associated factor 6 (TRAF6), rs540386, with low bone mineral density (BMD) among patients with rheumatoid arthritis (RA). METHODS: TRAF6 rs540386 genotyping was performed by mutagenically separated PCR in a cohort of 188 (23 men, 165 women, median age, 56.2 years) adult RA patients and 224 age and gender-matched controls. BMD was measured using dual-energy X-ray absorptiometry (DXA) (Lunar Prodigy advance scans, GE Healthcare, USA). RESULTS: Among the RA patients, 64 (55 women, 9 men) had low BMD comprising of 57 patients with osteoporosis and 7 with osteopenia. Whereas TRAF6 rs540386 was not associated with RA susceptibility, it was however found to be a risk factor for reduced lumbar spine Z-score in the recessive model (OR = 3.34, 95% CI = (1.01-11.00), p = 0.038). This association was confirmed further in the multivariate logistic regression analysis taking into account several potential confounding factors (OR = 3.34 (1.01-11.00), p = 0.048). In addition, mean total femur Z-score was found to be reduced in TT patients when compared to CC + CT patients (- 1.30 ± 1.32 versus - 0.60 ± 1.05, p = 0.034). No association between TRAF6 rs540386 and local bone damage was observed. CONCLUSIONS: This study for the first time ever demonstrated an association between a genetic variant of TRAF6 and low BMD among patients with RA. Further investigations are needed to elucidate the exact role of this variant.

Forkhead box p3 controls progression of oral lichen planus by regulating microRNA-146a.

Oral lichen planus (OLP) is a severe T cell-mediated disorder of the mucosa, which causes chronic inflammation. Forkhead box P3 (Foxp3) regulates the immune response and plays an important role in immunological diseases. The current study aimed to determine the role of Foxp3 and microRNA (miR)-146a in OLP. Western blot analysis and a quantitative real-time polymerase chain reaction assay showed that the expression of Foxp3 and miR-146a was upregulated in OLP tissues and in lipopolysaccharide (LPS)-incubated HaCaT cells, compared with controls. Foxp3 inhibition significantly decreased miR-146a expression, ameliorated LPS stimulation by decreased cell proliferation, and apoptosis in LPS-incubated HaCaT cells as compared with the LPS group. Cotransfection of Foxp3 small interfering RNA and miR-146a mimics elevated cell proliferation and apoptosis compared with the Foxp3 small interfering RNA group. In addition, miR-146a overexpression upregulated, whereas miR-146a inhibition downregulated, the proliferation and apoptosis of LPS-incubated HaCaT cells. The target gene of miR--146a, tumor necrosis factor receptor-associated factor 6 (TRAF6), was predicted by bioinformatics software and identified by the luciferase reporter assay. Furthermore, Foxp3/miR-146a elevated T regulatory cells and regulated TRAF6 expression in CD4+ T cells that were isolated from peripheral blood of patients with OLP. In conclusion, our study suggests that Foxp3 and miR-146a regulate the progression of OLP by negatively regulating TRAF6, which may provide a promising therapeutic target for OLP treatment.

Inhibition of TRAF6 alleviates choroidal neovascularization in vivo.

Choroidal neovascularization (CNV) is a type of wet age-related macular degeneration (AMD) which is a major cause of blindness in elder patients. Tumor necrosis factor receptor-associated factor 6 (TRAF6) promotes tumor angiogenesis via upregulating the expression of hypoxia-inducible factor 1α (HIF-1α) and vascular endothelial growth factor (VEGF). Additionally, TRAF6 facilitates the inflammatory response in macrophages and microglia. Here, using mouse laser-induced CNV model and TRAF6 siRNA, the study shows that TRAF6 is a critical player in CNV. The expression of TRAF6, HIF-1α, and VEGF increased in the model. TFAF6 siRNA intravitreal (IVT) injection inhibited CNV formation, as well as expression of HIF-1α and VEGF, activation of macrophages and microglia. Together, our data suggest that TFAF6 inhibition reduces CNV formation via down-regulating expression of HIF-1α and VEGF and activation of macrophages and microglia, demonstrating the unique advantages of TRAF6 inhibition in the alleviation of AMD.

Identification of tumor necrosis factor receptor-associated factor 6 in the pearl mussel Hyriopsis cumingii and its involvement in innate immunity and pearl sac formation.

Tumor necrosis factor receptor-associated factor 6 (TRAF6) acts as a central intracellular signal adapter molecule that mediates the tumor necrosis factor receptor superfamily and the interleukin-1 receptor/Toll-like receptor family in vertebrates and invertebrates. In the present study, HcTRAF6, a molluscan homologue of TRAF6 from Hyriopsis cumingii, has been cloned and identified. The entire open reading frame of HcTRAF6 was found to comprise a 1965-bp region that encodes a predicted protein of 654 amino acids, which contains conserved characteristic domains including a RING domain, two TRAF-type zinc finger domains, a typical coiled coil and the MATH domain. Phylogenetic analysis revealed that HcTRAF6 was aggregated closely with CsTRAF6 from Cyclina sinensis in the invertebrate cluster of mollusks. Further, qRT-PCR analysis showed that HcTRAF6 mRNA was extensively distributed in mussel tissues with a high expression in gills. After immune stimulation with Aeromonas hydrophila and lipopolysaccharides, the transcription of HcTRAF6 was obviously induced in the gills and hemocytes. In addition, significant fluctuation in HcTRAF6 expression was observed in the pearl sac, gills and hemocytes after mantle implantation. These findings confirmed its role in the alloimmune response. Dual-luciferase reporter assay showed that over-expression of HcTRAF6 could enhance the activity of the NF-κB reporter in a dose-dependent manner. Further, the RNA interference showed that the up-regulation of antimicrobial peptides in anti-bacterial infection was strongly suppressed in HcTRAF6-silenced mussels and that depletion of HcTRAF inhibited the elimination of A. hydrophila. All these findings together prove that HcTRAF6 functions as an efficient regulator in innate immune mechanisms against invading pathogens and the alloimmune mechanism after mantle implantation in H. cumingii.

Molecular cloning and functional analysis of tumor necrosis factor receptor-associated factor 6 (TRAF6) in Crossastrea gigas.

Tumor necrosis factor receptor-associated factor 6 (TRAF6) has been demonstrated to be a key signaling molecule involved in adaptive and innate immunity. In this study, we obtained the full length CgTRAF6 cDNA and analyzed the characteristics of the ORF and the peptide sequence in Crassostrea gigas. The deduced protein sequence of CgTRAF6 includes a conserved C-terminal TRAF domain following the RING and the zinc finger domain. The TRAF domain is composed of coiled-coil TRAF-N and MATH (meprin and TRAF-C homology) subdomains. Furthermore, phylogenetic analysis revealed that CgTRAF6 is clustered together with other members TRAF6 family and is placed in a sub-cluster singly which had a close relationship with Drosophila melanogaster. Expression analysis of CgTRAF6 indicated its constitutive expression in all tissues including mantle, adductor muscle, digestive tract, gonads, heart, gill, and hemocyte. Immune challenge with Vibrio alginolyticus and poly I:C resulted in significant up-regulation of CgTRAF6 expression. Dual-luciferase reporter assays showed that CgTRAF6 could activate both pNF-κB-Luc and pISRE-Luc expression, suggesting CgTRAF6 is potentially involved in NF-κB and the interferon signaling pathway. Furthermore, RNAi mediated knockdown of CgTRAF6 resulted in the down-regulation of several putative anti-viral signaling (IRF) and effector (PKR & Viperin) molecules coding genes, 7 days post-injection. These results collectively indicate that CgTRAF6 is a member of TRAF6 sub-family and is potentially involved in immune defense system against invading bacteria and viruses in Crassostrea gigas.

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

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

Molecular genomic- and transcriptional-aspects of a teleost TRAF6 homolog: Possible involvement in immune responses of Oplegnathus fasciatus against pathogens.

Tumor necrosis factor receptor (TNFR)-associated factor 6 (TRAF6) is a crucial docking molecule for TNFR superfamily and Interleukin-1 receptor/Toll-like receptor (IL-1R/TLR) superfamily. As an adaptor protein in pathogen-induced signaling cascades, TRAF6 modulates both adaptive- and innate-immunity. In order to understand the immune responses of teleost TRAF6, Oplegnathus fasciatus TRAF6-like gene (OfTRAF6) was identified and characterized. Genomic length of OfTRAF6 (4 kb), obtained by means of a genomic BAC library, spanned seven exons which represented a putative coding sequence of 1716 bp and encoded 571 amino acids (aa) with an estimated molecular weight of 64 kDa. This putative protein demonstrated the classical tetra-domain architecture composed of a zinc finger RING-type profile, two zinc finger TRAF-type profiles, a coiled-coil region and a MATH domain. While the sequence similarity with human TRAF6 was 66.5%, OfTRAF6 shared a higher overall similarity with teleost homologs (∼75-92%). Phylogeny of TRAF-family was examined and TRAF6-subfamily appeared to be the precursor of other subfamilies. In addition, the clustering pattern confirmed that OfTRAF6 is a novel member of TRAF6subfamily. Based on comparative genomic analysis, we found that vertebrate TRAF6 exhibits two distinct structures in teleost and tetrapod lineages. An intron-loss event has probably occurred in TRAF6 gene during the evolution of tetrapods from teleosts. Inspection of putative OfTRAF6 promoter revealed the presence of several immune responsive transcription factor binding sites. Real-time qPCR assay detected OfTRAF6 transcripts in eleven juvenile fish tissues with higher levels in peripheral blood cells followed by liver. Putative role of OfTRAF6 in response to flagellin, LPS, poly I:C, pathogenic bacteria (Edwardsiella tarda and Streptococcus iniae) and rock bream iridovirus (RBIV) was profiled in different tissues and OfTRAF6 revealed up-regulated transcript levels. Altogether, these findings implicate that OfTRAF6 is not only involved in flagellin-induced signaling cascade, but also contributes to the antibacterial- and antiviral-responses.