Disruption of TIGAR-TAK1 alleviates immunopathology in a murine model of sepsis.

Macrophage-orchestrated inflammation contributes to multiple diseases including sepsis. However, the underlying mechanisms remain to be defined clearly. Here, we show that macrophage TP53-induced glycolysis and apoptosis regulator (TIGAR) is up-regulated in murine sepsis models. When myeloid Tigar is ablated, sepsis induced by either lipopolysaccharide treatment or cecal ligation puncture in male mice is attenuated via inflammation inhibition. Mechanistic characterizations indicate that TIGAR directly binds to transforming growth factor ß-activated kinase (TAK1) and promotes tumor necrosis factor receptor-associated factor 6-mediated ubiquitination and auto-phosphorylation of TAK1, in which residues 152-161 of TIGAR constitute crucial motif independent of its phosphatase activity. Interference with the binding of TIGAR to TAK1 by 5Z-7-oxozeaenol exhibits therapeutic effects in male murine model of sepsis. These findings demonstrate a non-canonical function of macrophage TIGAR in promoting inflammation, and confer a potential therapeutic target for sepsis by disruption of TIGAR-TAK1 interaction.

The potential role of SNHG16/ miRNA-146a/ TRAF6 signaling pathway in the protective effect of zoledronate against colorectal cancer and associated osteoporosis in mouse model.

Bone fracture as a consequence of colorectal cancer (CRC) and associated osteoporosis (OP) is considered a risk factor for increasing the mortality rate among CRC patients. SNHG16/ miRNA-146a/ TRAF6 signaling pathway is a substantial contributor to neoplastic evolution, progression, and metastasis. Here, we investigated the effect of zoledronate (ZOL) on the growth of CRC and associated OP in a mouse model. Thirty Balb/c mice were divided into Naïve, azoxymethane (AOM)/dextran sodium sulfate (DSS), and ZOL groups. Body weight and small nucleolar RNA host gene 16 (SNHG16) expression, microRNA-146a, and TRAF6 in bone, colon, and stool were investigated. Samples of colon and bone were collected and processed for light microscopic, immunohistochemical staining for cytokeratin 20 (CK20), nuclear protein Ki67 (pKi-67), and caudal type homeobox transcription factor 2 (CDx2) in colon and receptor activator of nuclear factor kB (RANK) and osteoprotegerin (OPG) in bone. A computerized tomography (CT) scan of the femur and tibia was studied. ZOL produced a significant decrease in the expression of SNHG16 and TRAF6 and an increase in miRNA-146a in the colon and bone. ZOL administration improved the histopathological changes in the colon, produced a significant decrease in CK20 and Ki-67, and increased CDx2 expressions. In bone, ZOL prevented osteoporotic changes and tumour cell invasion produced a significant decrease in RANK and an increase in OPG expressions, alongside improved bone mineral density in CT scans. ZOL could be a promising preventive therapy against colitis-induced cancer and associated OP via modulation expression of SNHG16, miRNA-146a, and TRAF6.

TRAF6 enhances PD-L1 expression through YAP1-TFCP2 signaling in melanoma.

Immunotherapy represented by programmed cell death protein 1 (PD-1)/programmed death ligand 1 (PD-L1) monoclonal antibodies has led tumor treatment into a new era. However, the low overall response rate and high incidence of drug resistance largely damage the clinical benefits of existing immune checkpoint therapies. Recent studies correlate the response to PD-1/PD-L1 blockade with PD-L1 expression levels in tumor cells. Hence, identifying molecular targets and pathways controlling PD-L1 protein expression and stability in tumor cells is a major priority. In this study, we performed a Stress and Proteostasis CRISPR interference screening to identify PD-L1 positive modulators. Here, we identified TRAF6 as a critical regulator of PD-L1 in melanoma cells. As a non-conventional E3 ubiquitin ligase, TRAF6 is inclined to catalyze the synthesis and linkage of lysine-63 (K63) ubiquitin which is related to the stabilization of substrate proteins. Our results showed that suppression of TRAF6 expression down-regulates PD-L1 expression on the membrane surface of melanoma cells. We then used in vitro and in vivo assays to investigate the biological function and mechanism of TRAF6 and its downstream YAP1/TFCP2 signaling in melanoma. TRAF6 stabilizes YAP1 by K63 poly-ubiquitination modification, subsequently promoting the formation of YAP1/TFCP2 transcriptional complex and PD-L1 transcription. Inhibition of TRAF6 by Bortezomib enhanced cytolytic activity of CD8+ T cells by reduction of endogenous PD-L1. Notably, Bortezomib enhances anti-tumor immunity to an extent comparable to anti-PD-1 therapies with no obvious toxicity. Our findings reveal the potential of inhibiting TRAF6 to stimulate internal anti-tumor immunological effect for TRAF6-PD-L1 overexpressing cancers.

TIFAB regulates the TIFA-TRAF6 signaling pathway involved in innate immunity by forming a heterodimer complex with TIFA.

Nuclear factor κB (NF-κB) is activated by various inflammatory and infectious molecules and is involved in immune responses. It has been elucidated that ADP-ß-D-manno-heptose (ADP-Hep), a metabolite in gram-negative bacteria, activates NF-κB through alpha-kinase 1 (ALPK1)-TIFA-TRAF6 signaling. ADP-Hep stimulates the kinase activity of ALPK1 for TIFA phosphorylation. Complex formation between phosphorylation-dependent TIFA oligomer and TRAF6 promotes the polyubiquitination of TRAF6 for NF-κB activation. TIFAB, a TIFA homolog lacking a phosphorylation site and a TRAF6 binding motif, is a negative regulator of TIFA-TRAF6 signaling and is implicated in myeloid diseases. TIFAB is indicated to regulate TIFA-TRAF6 signaling through interactions with TIFA and TRAF6; however, little is known about its biological function. We demonstrated that TIFAB forms a complex not with the TIFA dimer, an intrinsic form of TIFA involved in NF-κB activation, but with monomeric TIFA. The structural analysis of the TIFA/TIFAB complex and the biochemical and cell-based analyses showed that TIFAB forms a stable heterodimer with TIFA, inhibits TIFA dimer formation, and suppresses TIFA-TRAF6 signaling. The resultant TIFA/TIFAB complex is a "pseudo-TIFA dimer" lacking the phosphorylation site and TRAF6 binding motif in TIFAB and cannot form the orderly structure as proposed for the phosphorylated TIFA oligomer involved in NF-κB activation. This study elucidated the molecular and structural basis for the regulation of TIFA-TRAF6 signaling by TIFAB.

Gender-related variation expressions of neuroplastin TRAF6, GluA1, GABA(A) receptor, and PMCA in cortex, hippocampus, and brainstem in an experimental epilepsy model.

Epileptic seizures are seen as a result of changing excitability balance depending on the deterioration in synaptic plasticity in the brain. Neuroplastin, and its related molecules which are known to play a role in synaptic plasticity, neurotransmitter activities that provide balance of excitability and, different neurological diseases, have not been studied before in epilepsy. In this study, a total of 34 Sprague-Dawley male and female rats, 2 months old, weighing 250-300 g were used. The epilepsy model in rats was made via pentylenetetrazole (PTZ). After the completion of the experimental procedure, the brain tissue of the rats were taken and the histopathological changes in the hippocampus and cortex parts and the brain stem were investigated, as well as the immunoreactivity of the proteins related to the immunohistochemical methods. As a result of the histopathological evaluation, it was determined that neuron degeneration and the number of dilated blood vessels in the hippocampus, frontal cortex, and brain stem were higher in the PTZ status epilepticus (SE) groups than in the control groups. It was observed that neuroplastin and related proteins TNF receptor-associated factor 6 (TRAF6), Gamma amino butyric acid type A receptors [(GABA(A)], and plasma membrane Ca2+ ATPase (PMCA) protein immunoreactivity levels increased especially in the male hippocampus, and only AMPA receptor subunit type 1 (GluA1) immunoreactivity decreased, unlike other proteins. We believe this may be caused by a problem in the mechanisms regulating the interaction of neuroplastin and GluA1 and may cause problems in synaptic plasticity in the experimental epilepsy model. It may be useful to elucidate this mechanism and target GluA1 when determining treatment strategies.

Mechanism of miR-7 mediating TLR4/TRAF6/NF-κB inflammatory pathway in colorectal cancer.

This study is aimed at investigating the roles of Toll-like receptor 4 (TLR4) and microRNA-7 (miR-7) in colorectal cancer (CRC) development and progression. We assessed TLR4 and miR-7 expression in CRC cells and tissues using reverse transcription-quantitative polymerase chain reaction. The relationship between miR-7 and TLR4 was analyzed through dual luciferase reporter assays. MTT, wound healing, and cell invasion assays were conducted to examine the effects of TLR4 and miR-7 on CRC cell proliferation, migration, and invasion. Western blotting was used to explore the involvement of the TRAF6/NF-κB signaling pathway. miR-7 was underexpressed in CRC, while TLR4 levels were increased. miR-7 negatively regulated TLR4 expression and its knockdown enhanced CRC cell proliferation, migration, and invasion. TLR4 knockdown had the opposite effects. The TRAF6/NF-κB pathway was linked to TLR4's role in tumor progression. miR-7 might inhibit TRAF6/NF-κB target a signaling pathway of TLR4 and promote CRC occurrence. miR-7 may therefore be used as a sensitive biomarker in CRC patients.

Potential involvement of circulating exosomal miRNA-146a in disease activity and TRAF6 gene expression in juvenile proliferative lupus nephritis.

BACKGROUND: Juvenile SLE (JSLE) is a complex autoimmune disorder that predominantly affects children and adolescents with several unique challenges, and microRNA-146a (miRNA-146a) might be an interesting anti-inflammatory molecule. Because exosomes in the blood might protect miRNAs, the association between circulating exosomal miRNA-146a and lupus proinflammatory genes, such as IRAK1 and TRAF6, was studied in peripheral blood mononuclear cells from people with JSLE. METHODS: Blood samples from 12 patients were collected every 3 months until 1 year with the recorded disease activity, and quantitative real-time PCR was used to determine the circulating exosomal miRNA-146a and the gene expression (IRAK1 and TRAF6). RESULTS: The mean age was 12.60±0.43 years at diagnosis and all patients had a complete response at 12 months. According to the nanoparticle tracking analysis, the abundance of exosomes was significantly lower at 3, 6 and 12 months compared with 0 months, while the level of circulating exosomal miRNA-146a was significantly higher at 12 months than at diagnosis (p<0.001). There was a negative correlation between the level of circulating exosomal miRNA-146a expression and the level of TRAF6 mRNA (r=-0.30, p=0.049). Moreover, there were correlations between circulating exosomal miRNA-146a and disease severity such as SLE Disease Activity Index 2000 score, anti-double-stranded DNA antibody and proteinuria (urine protein-creatinine ratio), respectively. Therefore, increasing the level of circulating exosomal miRNA-146a, which might control TRAF6 mRNA expression, could have an effect on the production of inflammatory cytokines. CONCLUSION: This suggests that miRNA-146a might serve as a non-invasive biomarker to evaluate the response to treatment in patients with juvenile lupus nephritis.

Distinct cross talk of IL-17 & TGF-ß with the immature CD11c+ TRAF6(-/-) -null myeloid dendritic cell-derived osteoclast precursor (mDDOCp) may engage signaling toward an alternative pathway of osteoclastogenesis for arthritic bone loss in vivo.

BACKGROUND: Dendritic cells (DCs), though borne heterogeneous, are the most potent antigen-presenting cells, whose critical functions include triggering antigen-specific naïve T-cell responses and fine-tuning the innate versus adaptive immunity at the osteo-immune and/or mucosal mesenchyme interface. We previously reported that immature myeloid-CD11c+ DCs/mDCs may act like osteoclast (OC) precursors (OCp/mDDOCp) capable of developing into functional OCs via an alternative pathway of inflammation-induced osteoclastogenesis; however, what are their contribution and signaling interactions with key osteotropic cytokines (i.e., interleukin-17 [IL-17] and transforming growth factor-ß [TGF-ß]) to bearing such inflammatory bone loss in vivo remain unclear to date. METHODS: Herein, we employed mature adult bone marrow-reconstituted C57BL/6 TRAF6(-/-) -null chimeras without the classical monocyte/macrophage (Mo/Mϕ)-derived OCs to address their potential contribution to OCp/mDDOCp-mediated osteoclastogenesis in the chicken type-II-collagen (CC-II)-induced joint inflammation versus arthritic bone loss and parallel associations with the double-positive CD11c+ TRAP+ TRAF6-null(-/-)  DC-like OCs detected in vivo via the quantitative dual-immunohistochemistry and digital histomorphometry for analyses. RESULTS: The resulting findings revealed the unrecognized novel insight that (i) immature myeloid-CD11c+ TRAF6(-/-) TRAP+ DC-like OCs were involved, co-localized, and strongly associated with joint inflammation and bone loss, independent of the Mo/Mϕ-derived classical OCs, in CC-II-immunized TRAF6(-/-) -null chimeras, and (ii) the osteotropic IL-17 may engage distinct crosstalk with CD11c+ mDCs/mDDOCp before developing the CD11c+ TRAP+ TRAF6(-/-) OCs via a TGF-ß-dependent interaction toward inflammation-induced arthritic bone loss in vivo. CONCLUSION: These results confirm and substantiate the validity of TRAF6(-/-) -null chimeras to address the significance of immature mCD11c+ TRAP+ DC-like OCs/mDDOCp subset for an alternative pathway of arthritic bone loss in vivo. Such CD11c+ mDCs/mDDOCp-associated osteoclastogenesis through the step-wise twist-in-turns osteo-immune cross talks are thereby theme highlighted to depict a summative re-visitation proposed.

A TLR4/TRAF6-dependent signaling pathway mediates NCoR coactivator complex formation for inflammatory gene activation.

The nuclear receptor corepressor (NCoR) forms a complex with histone deacetylase 3 (HDAC3) that mediates repressive functions of unliganded nuclear receptors and other transcriptional repressors by deacetylation of histone substrates. Recent studies provide evidence that NCoR/HDAC3 complexes can also exert coactivator functions in brown adipocytes by deacetylating and activating PPARγ coactivator 1α (PGC1α) and that signaling via receptor activator of nuclear factor kappa-B (RANK) promotes the formation of a stable NCoR/HDAC3/PGC1ß complex that coactivates nuclear factor kappa-B (NFκB)- and activator protein 1 (AP-1)-dependent genes required for osteoclast differentiation. Here, we demonstrate that activation of Toll-like receptor (TLR) 4, but not TLR3, the interleukin 4 (IL4) receptor nor the Type I interferon receptor, also promotes assembly of an NCoR/HDAC3/PGC1ß coactivator complex. Receptor-specific utilization of TNF receptor-associated factor 6 (TRAF6) and downstream activation of extracellular signal-regulated kinase 1 (ERK1) and TANK-binding kinase 1 (TBK1) accounts for the common ability of RANK and TLR4 to drive assembly of an NCoR/HDAC3/PGC1ß complex in macrophages. ERK1, the p65 component of NFκB, and the p300 histone acetyltransferase (HAT) are also components of the induced complex and are associated with local histone acetylation and transcriptional activation of TLR4-dependent enhancers and promoters. These observations identify a TLR4/TRAF6-dependent signaling pathway that converts NCoR from a corepressor of nuclear receptors to a coactivator of NFκB and AP-1 that may be relevant to functions of NCoR in other developmental and homeostatic processes.

Enhanced NF-κB activation via HIV-1 Tat-TRAF6 cross-talk.

The Tat proteins of HIV-1 and simian immunodeficiency virus (SIV) are essential for activating viral transcription. In addition, Tat stimulates nuclear factor κB (NF-κB) signaling pathways to regulate viral gene expression although its molecular mechanism is unclear. Here, we report that Tat directly activates NF-κB through the interaction with TRAF6, which is an essential upstream signaling molecule of the canonical NF-κB pathway. This interaction increases TRAF6 oligomerization and auto-ubiquitination, as well as the synthesis of K63-linked polyubiquitin chains to further activate the NF-κB pathway and HIV-1 transcription. Moreover, ectopic expression of TRAF6 significantly activates HIV-1 transcription, whereas TRAF6 knockdown inhibits transcription. Furthermore, Tat-mediated activation of NF-κB through TRAF6 is conserved among HIV-1, HIV-2, and SIV isolates. Our study uncovers yet another mechanism by which HIV-1 subverts host transcriptional pathways to enhance its own transcription.

Influence of sex and liver cirrhosis on the expression of miR-146a-5p and its target genes, IRAK1 and TRAF6.

Long-term alcohol misuse triggers cellular adaptions in susceptible regions of the human brain, resulting in neurodegeneration, neuroinflammation and altered gene expression. Previous studies have identified ∼35 miRNAs, including miR-146a-5p, which are up-regulated in the frontal cortex of males with alcohol use disorder (AUD), but the influence of liver cirrhosis and sex is unknown. The expression of miR-146a-5p, IRAK1, and TRAF6 was measured in the prefrontal cortex of controls and individuals with AUD with and without cirrhosis of the liver. Further, individuals were genotyped for two SNPs, rs2910164 and rs57095329. The expression of miR-146a-5p was significantly different between sexes. In males the expression of miR-146a-5p was increased in individuals with AUD with and without liver cirrhosis compared with controls. In females miR-146a-5p expression was significantly lower in individuals with AUD compared with both controls and those with AUD and cirrhosis, suggesting that both the severity of alcohol misuse and the sex of the individual influences the expression of miR-146a-5p. The expression of TRAF6 was significantly lower in individuals with uncomplicated AUD compared with those with AUD and cirrhosis. The expression of IRAK1 did not differ between groups or sexes. There was no influence of genotype on expression. Increased expression of miR-146a-5p did not correlate with decreased IRAK1 or TRAF6 expression suggesting a loss of regulatory control of the TLR4 pathway. Understanding sex-specific differences in the regulation of gene expression in AUD is key to determine which inflammatory pathways could be targeted for therapeutic intervention.

MiR-31-5p regulates the neuroinflammatory response via TRAF6 in neuropathic pain.

BACKGROUND: Neuropathic pain is chronic pain and has few effective control strategies. Studies have demonstrated that microRNAs have functions in neuropathic pain. However, no study has been conducted to demonstrate the role and mechanism of microRNA (miR)-31-5p in neuropathic pain. Accordingly, this study sought to determine the pathological role of miR-31-5p in chronic constriction injury (CCI) -induced neuropathic pain mouse models. METHODS: We used CCI surgery to establish mouse neuropathic pain model. Behavioral tests were performed to evaluate pain sensitivity of mice. Expressions of miR-31-5p and inflammatory cytokines in dorsal root ganglion (DRG) were examined by polymerase chain reaction. Animals or cells were received with/without miR-31-5p mimic or inhibitor to investigate its role in neuropathic pain. The mechanism of miR-31-5p was assayed using western blotting, immunofluorescence staining and dual-luciferase reporter assay. RESULTS: We found that CCI led to a significant decrease in miR-31-5p levels. Knockout of miR-31-5p and administration of miPEP31 exacerbated pain in C57BL/6 mice. Meanwhile, miR-31-5p overexpression increased the paw withdrawal threshold and latency. TRAF6 is one of the target gene of miR-31-5p, which can trigger a complex inflammatory response. TRAF6 was associated with pain and that reducing the DRG expression of TRAF6 could alleviate pain. In addition, miR-31-5p overexpression inhibited the TRAF6 expression and reduced the neuroinflammatory response. CONCLUSIONS: All the results reveal that miR-31-5p could potentially alleviate pain in CCI mouse models by inhibiting the TRAF6 mediated neuroinflammatory response. MiR-31-5p upregulation is highlighted here as new target for CCI treatment.

Cornuside protects against ischemic stroke in rats by suppressing the IL-17F/TRAF6/NF-κB pathway via the brain-gut axis.

Ischemic stroke is a serious neurological disease with limited therapeutic options; thus, it is particularly important to find effective treatments. Restoration of gut microflora diversity is an important factor in the treatment of ischemic stroke, but the mechanism remains unclear. Cornuside is known for its unique anti-inflammatory and circulation-promoting effects; however, whether it can effectively treat ischemic stroke and its therapeutic mechanisms remain unknown. In this study, we used a rat middle cerebral artery occlusion-reperfusion model (MCAO/R) to mimic ischemic stroke in humans and to assess the cerebral protective effects of cornuside in rats with ischemic stroke. Using 16S rRNA sequencing and RNA sequencing, we explored the cornuside mechanism in the brain-gut axis that confers protection against ischemic stroke. In conclusion, cornuside can inhibit the IL-17F/TRAF6/NF-κB pathway by improving the dysregulation of intestinal microflora, and reduce intestinal inflammation and neuroinflammation, which treated ischemic stroke rats.

KNOCKDOWN OF CIRC_0114428 ALLEVIATES LPS-INDUCED HK2 CELL APOPTOSIS AND INFLAMMATION INJURY VIA TARGETING MIR-215-5P/TRAF6/NF-ΚB AXIS IN SEPTIC ACUTE KIDNEY INJURY.

ABSTRACT: Background: Sepsis is a systemic inflammatory disease that can cause multiple organ damage. Circular RNAs (circRNAs) have been reported to play a regulatory role in sepsis-induced acute kidney injury (AKI); however, the role of circ_0114428 has not been studied. Methods: In this study, HK2 cells were treated with different concentrations of LPS to induce cell damage, and then the expressions of circ_0114428, microRNA-215-5p (miR-215-5p), and tumor necrosis factor receptor-associated factor 6 (TRAF6) were detected by quantitative real-time polymerase chain reaction (qRT-PCR), and Western blot examined the Bax and cleaved-Caspase-3 proteins. Cell proliferation was detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and thymidine analog 5-ethynyl-2'-deoxyuridine (EdU) assay. In addition, cell apoptosis was detected by flow cytometry, and the levels of inflammatory factors were detected by enzyme-linked immunosorbent assay. Results: After LPS treatment with different concentrations, we found that LPS at 10 µg/mL had the best effect on HK2 cells. Circ_0114428 was highly expressed in sepsis-AKI patients and LPS-treated HK2 cells. Knockdown of circ_0114428 restored the effects of LPS treatment on proliferation, apoptosis, and inflammatory response of HK2 cells. MiR-215-5p was a target of circ_0114428, and TRAF6 was a downstream target of miR-215-5p. Circ_0114428 regulated TRAF6 expression by sponging miR-215-5p in LPS-treated HK2 cells. Circ_0114428 regulated LPS-induced NF-κB signaling in HK2 cells by targeting miR-215-5p/TRAF6 axis. Conclusion: Circ_0114428 knockdown abolished the cell proliferation, apoptosis, and inflammatory damage in LPS-induced HK2 cells by targeting miR-215-5p/TRAF6/NF-κB.

EFHD2 cooperates with E3 ubiquitin ligase Smurf1 to facilitate virus infection by promoting the degradation of TRAF6 in teleost fish.

The ubiquitin-proteasome system is one of the most important protein stability regulation systems. It can precisely regulate host immune responses by targeting signaling proteins. TRAF6 is a crucial E3 ubiquitin ligase in host antiviral signaling pathway. Here, we discovered that EF-hand domain-containing protein D2 (EFHD2) collaborated with the E3 ubiquitin ligase Smurf1 to potentiate the degradation of TRAF6, hence facilitating RNA virus Siniperca chuatsi rhabdovirus infection. The mechanism analysis revealed that EFHD2 interacted with Smurf1 and enhanced its protein stability by impairing K48-linked polyubiquitination of Smurf1, thereby promoting Smurf1-catalyzed degradation of TRAF6. This study initially demonstrated a novel mechanism by which viruses utilize host EFHD2 to achieve immune escape and provided a new perspective on the exploration of mammalian innate immunity.IMPORTANCEViruses induce host cells to activate several antiviral signaling pathways. TNF receptor-associated factor 6 (TRAF6) plays an essential role in these pathways. Numerous studies have been done on the mechanisms of TRAF6-mediated resistance to viral invasion. However, little is known about the strategies that viruses employ to antagonize TRAF6-mediated antiviral signaling pathway. Here, we discovered that EFHD2 functions as a host factor to promote viral replication. Mechanistically, EFHD2 potentiates Smurf1 to catalyze the ubiquitin-proteasomal degradation of TRAF6 by promoting the deubiquitination and stability of Smurf1, which in turn inhibits the production of proinflammatory cytokines and interferons. Our study also provides a new perspective on mammalian resistance to viral invasion.

Catalpol antagonizes LPS-mediated inflammation and promotes osteoblast differentiation through the miR-124-3p/DNMT3b/TRAF6 axis.

BACKGROUND: Dysregulated inflammation and osteoblast differentiation are implicated in osteoporosis. Exploring the activity of catalpol in inflammation and osteoblast differentiation deepens the understanding of osteoporosis pathogenesis. METHODS: LPS was used to treated hFOB1.19 cells to induce inflammation and repress osteoblast differentiation. FOB1.19 cells were induced in osteoblast differentiation medium and treated with LPS and catalpol. Cell viability was assessed using CCK-8. ALP and Alizarin red S staining were conducted for analyzing osteoblast differentiation. The levels of IL-1ß, TNF-α and IL-6 were examined by ELISA. The methylation of TRAF6 promoter was examined through MS-PCR. The binding of miR-124-3p to DNMT3b and DNMT3b to TRAF6 promoter was determined with dual luciferase reporter and ChIP assays. RESULTS: LPS enhanced secretion of inflammatory cytokines and suppressed osteoblast differentiation. MiR-124-3p and TRAF6 were upregulated and DNMT3b was downregulated in LPS-induced hFOB1.19 cells. Catalpol protected hFOB1.19 cells against LPS via inhibiting inflammation and promoting osteoblast differentiation. MiR-124-3p targeted DNMT3b, and its overexpression abrogated catalpol-mediated protection in LPS-treated hFOB1.19 cells. In addition, DNMT3b methylated TRAF6 promoter to restrain its expression. Catalpol exerted protective effects through suppression of the miR-124-3p/DNMT3b/TRAF6 axis in hFOB1.19 cells. CONCLUSION: Catalpol antagonizes LPS-mediated inflammation and suppressive osteoblast differentiation via controlling the miR-124-3p/DNMT3b/TRAF6 axis.

Pterostilbene alleviates abdominal aortic aneurysm via inhibiting macrophage pyroptosis by activating the miR-146a-5p/TRAF6 axis.

Pterostilbene (PTE), a natural stilbene found in blueberries and several varieties of grapes, has several pharmacological activities, including anti-inflammatory and antioxidative activities. However, its role in abdominal aortic aneurysm (AAA), which is a severe inflammatory vascular disease, remains incompletely understood. In this study, we investigated the protective effects of natural stilbene PTE on AAA formation and the underlying mechanism. Two AAA mouse models (Ang II-induced model and PPE-induced model) were used to examine the effect of PTE on AAA formation. We showed that PTE administration attenuated AAA formation in mice. Furthermore, we found that PTE significantly inhibited inflammatory responses in mouse aortas, as PTE suppressed macrophage pyroptosis and prevented macrophage infiltration in aortas, resulting in reduced expression of pro-inflammatory cytokines in aortas. We also observed similar results in LPS + ATP-treated Raw 264.7 cells (a macrophage cell line) and primary peritoneal macrophages in vitro. We showed that pretreatment with PTE restrained inflammatory responses in macrophages by inhibiting macrophage pyroptosis. Mechanistically, miR-146a-5p and TRAF6 interventions in vivo and in vitro were used to investigate the role of the miR-146a-5p/TRAF6 axis in the beneficial effect of PTE on macrophage pyroptosis and AAA. We found that PTE inhibited macrophage pyroptosis by miR-146a-5p-mediated suppression of downstream TRAF6 expression. Moreover, miR-146a-5p knockout or TRAF6 overexpression abrogated the protective effect of PTE on macrophage pyroptosis and AAA formation. These findings suggest that miR-146a-5p/TRAF6 axis activation by PTE protects against macrophage pyroptosis and AAA formation. PTE might be a promising agent for preventing inflammatory vascular diseases, including AAA.

Role of TRAF6 from obscure puffer (Takifugu obscurus) in immune response against Edwardsiella tarda infection.

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.

HIF1α/miR-146α/TRAF6/NF-κB axis modulates hepatic iron overload-induced inflammation.

Transfusional therapy is used to cure anemia but raises the risk of hepatic iron overload (IO), which triggers oxidative stress damage, inflammation, and failure even fibrosis. microRNAs play a vital role in developing hepatic diseases. This study presented the mechanism by which IO induce hepatic inflammation through microRNAs. In this study, microRNA expression profiling in the liver was observed after IO for 2 weeks, in which the target microRNA will be found. IO activating the miR-146α/TRAF6/NF-κB pathway was validated, and the molecular mechanism of the IO-induced decrease of miR-146α in the liver was studied in vivo and in vitro. The expression of TRAF6/NF-κB (p65)-dependent inflammatory factors increased, whereas the expression of miR-146α decreased during the IO-induced inflammatory response in the liver. The reduced expression of HNF4α caused by HIF1α and miR-34α may decrease the expression of miR-146α. Overexpression of miR-146α alleviated the hepatic inflammatory response caused by IO. Our findings indicate that miR-146α is a key factor in inducing hepatic IO inflammation, which will be another potential target to prevent IO-induced hepatic damage.

lncRNA-Gm5532 regulates osteoclast differentiation through the miR-125a-3p/TRAF6 axis.

Long noncoding RNAs (lncRNAs) are important regulators of bone metabolism. In this study, lncRNA microarray analysis was used to identify differentially expressed lncRNAs in differentiated osteoclasts. lncRNA-Gm5532 is highly expressed during osteoclast differentiation. lncRNA-Gm5532 knockdown impairs osteoclast formation and bone resorption. Mechanistic experiments show that lncRNA-Gm5532 functions as a competing endogenous RNA (ceRNA) and acts as a sponge for miR-125a-3p, which promotes TNF receptor-associated factor 6 (TRAF6) expression. miR-125a-3p mimics suppress osteoclast differentiation and TAK1/NF-κB/MAPK signaling. The miR-125a-3p inhibitor reverses the negative effects of siGm5532 on osteoclast differentiation. In summary, our study reveals that lncRNA-Gm5532 functions as an activator in osteoclast differentiation by targeting the miR-125a-3p/TRAF6 axis, making it a novel biomarker and potential therapeutic target for osteoporosis.