LncRNA promoted inflammatory response in ischemic heart failure through regulation of miR-455-3p/TRAF6 axis.

OBJECTIVES: Ischemic heart failure (IHF) is the most common cause of death globally. Growing evidence shows abnormal expression of long non-coding RNAs in heart failure patients. This study aims to investigate the effect of sex-determining region Y-box 2 (SOX2) overlapping transcript (SOX2-OT) on the regulation of the inflammatory response in ischemic heart failure. METHODS: IHF rat and oxygen and glucose deprivation (OGD) cell models were established. qRT-PCR was employed to investigate the expression of SOX2-OT. ELISA, western blot and cell viability/apoptosis assays were performed to assess the effects of SOX2-OT. Online software program was used to identify miRNAs that target SOX2-OT, followed by validation using RNA pull-down. Potential targets of miRNAs were searched, and examined by immunoblotting, qRT-PCR and luciferase reporter assay. RESULTS: SOX2-OT was up-regulated in IHF and OGD. Knockdown of SOX2-OT promoted cell proliferation, decreased apoptosis rate and cell oxidative damage, and ameliorated inflammatory response. SOX2-OT contains binding sites for miR-455-3p, miR-5586-3p and miR-1252-5p. RNA pull-down confirmed the binding ability between SOX2-OT and miR-455-3p. TRAF6 is a direct target of miR-455-3p. Moreover, the regulatory activity of SOX2-OT on inflammatory response was partially through its negative regulation of miR-455-3p, which directly regulates TRAF6. Down-regulation of SOX2-OT improved myocardial dysfunction in IHF rat. CONCLUSIONS: Our results reveal that SOX2-OT may be a driver of IHF through repression of miR-455-3p, and miR-455-3p alleviates IHF by targeting TRAF6. Therefore, SOX2-OT/miR-455-3p/TRAF6 may be a potential target for advanced therapeutic strategy for IHF.

Hepatocyte TNF Receptor-Associated Factor 6 Aggravates Hepatic Inflammation and Fibrosis by Promoting Lysine 6-Linked Polyubiquitination of Apoptosis Signal-Regulating Kinase 1.

Activation of apoptosis signal-regulating kinase 1 (ASK1) is a key driving force of the progression of nonalcoholic steatohepatitis (NASH) and represents an attractive therapeutic target for NASH treatment. However, the molecular and cellular mechanisms underlying ASK1 activation in the pathogenesis of NASH remain incompletely understood. In this study, our data unequivocally indicated that hyperactivated ASK1 in hepatocytes is a potent inducer of hepatic stellate cell (HSC) activation by promoting the production of hepatocyte-derived factors. Our previous serial studies have shown that the ubiquitination system plays a key role in regulating ASK1 activity during NASH progression. Here, we further demonstrated that tumor necrosis factor receptor-associated factor 6 (TRAF6) promotes lysine 6 (Lys6)-linked polyubiquitination and subsequent activation of ASK1 to trigger the release of robust proinflammatory and profibrotic factors in hepatocytes, which, in turn, drive HSC activation and hepatic fibrosis. Consistent with the in vitro findings, diet-induced liver inflammation and fibrosis were substantially attenuated in Traf6+/- mice, whereas hepatic TRAF6 overexpression exacerbated these abnormalities. Mechanistically, Lys6-linked ubiquitination of ASK1 by TRAF6 facilitates the dissociation of thioredoxin from ASK1 and N-terminal dimerization of ASK1, resulting in the boosted activation of ASK1-c-Jun N-terminal kinase 1/2 (JNK1/2)-mitogen-activated protein kinase 14(p38) signaling cascade in hepatocytes. Conclusion: These results suggest that Lys6-linked polyubiquitination of ASK1 by TRAF6 represents a mechanism underlying ASK1 activation in hepatocytes and a key driving force of proinflammatory and profibrogenic responses in NASH. Thus, inhibiting Lys6-linked polyubiquitination of ASK1 may serve as a potential therapeutic target for NASH treatment.

Tumor Necrosis Factor Receptor-Associated Factor 6 Promotes Hepatocarcinogenesis by Interacting With Histone Deacetylase 3 to Enhance c-Myc Gene Expression and Protein Stability.

The oncogene c-Myc is aberrantly expressed and plays a key role in malignant transformation and progression of hepatocellular carcinoma (HCC). Here, we report that c-Myc is significantly up-regulated by tumor necrosis factor receptor-associated factor 6 (TRAF6), an E3 ubiquitin ligase, in hepatocarcinogenesis. High TRAF6 expression in clinical HCC samples correlates with poor prognosis, and the loss of one copy of the Traf6 gene in Traf6+/- mice significantly impairs liver tumorigenesis. Mechanistically, TRAF6 first interacts with and ubiquitinates histone deacetylase 3 (HDAC3) with K63-linked ubiquitin chains, which leads to the dissociation of HDAC3 from the c-Myc promoter and subsequent acetylation of histone H3 at K9, thereby epigenetically enhancing the mRNA expression of c-Myc. Second, the K63-linked ubiquitination of HDAC3 impairs the HDAC3 interaction with c-Myc and promotes c-Myc protein acetylation, which thereby enhances c-Myc protein stability by inhibiting carboxyl terminus of heat shock cognate 70-kDa-interacting protein-mediated c-Myc ubiquitination and degradation. Importantly, TRAF6/HDAC3/c-Myc signaling is also primed in hepatitis B virus-transgenic mice, unveiling a critical role for a mechanism in inflammation-cancer transition. In clinical specimens, TRAF6 positively correlates with c-Myc at both the mRNA and protein levels, and high TRAF6 and c-Myc expression is associated with an unfavorable prognosis, suggesting that TRAF6 collaborates with c-Myc to promote human hepatocarcinogenesis. Consistently, curbing c-Myc expression by inhibition of TRAF6 activity with a TRAF6 inhibitor peptide or the silencing of c-Myc by small interfering RNA significantly suppressed tumor growth in mice. Conclusion: These findings demonstrate the oncogenic potential of TRAF6 during hepatocarcinogenesis by modulating TRAF6/HDAC3/c-Myc signaling, with potential implications for HCC therapy.

TRAF6 promotes the progression and growth of colorectal cancer through nuclear shuttle regulation NF-kB/c-jun signaling pathway.

AIMS: TRAF6 is an intracellular signal adapter molecule plays a significant role in tumor development. However, the specific mechanism causes and promotes of colorectal cancer keep largely unknown. Therefore, we sought to investigate the roles and the molecular mechanisms of TRAF6 in regulation colorectal cancer. MATERIAL AND METHODS: The immunohistochemistry analyzed the expression of TRAF6 in colorectal cancer samples and analyzed the effects of expression of TRAF6 on the prognosis in colorectal cancer. The roles of TRAF6 in regulating colorectal cancer cell proliferation, colony formation, cell migration, cell wound healing and cell invasion were evaluated in vitro. Animal studies were performed to investigate the effects of TRAF6 on tumor growth. mRNA abundance of key genes was analyzed via qPCR. Protein level of TRAF6 and NF-κB/AP-1 signaling pathways was examined by Western blot. Luciferase reporter and Immunofluorescence assays were used to identify the activities NF-κB/AP-1 signaling pathways. KEY FINDINGS: TRAF6 high expression in colorectal cancer tissues. And colorectal cancer patients with high expression of TRAF6 had a poor survival rate. TRAF6 knockdown can inhibit proliferation, migration, and invasion of colorectal cancer cells in vitro and in vivo experiments. TRAF6 activates the TRAF6-NF-κB/AP-1 signaling pathway by entering the nucleus, causing biobehavioral changes in colorectal cancer cells. SIGNIFICANCE: TRAF6 plays a vital role in the progression of colorectal cancer. What's more, research elucidating the biological mechanisms of TRAF6 can treated as potential therapeutic target for colorectal cancer.

TRAF6 directs FOXP3 localization and facilitates regulatory T-cell function through K63-linked ubiquitination.

Regulatory T cells (Tregs) are crucial mediators of immune control. The characteristic gene expression and suppressive functions of Tregs depend considerably on the stable expression and activity of the transcription factor FOXP3. Transcriptional regulation of the Foxp3 gene has been studied in depth, but both the expression and function of this factor are also modulated at the protein level. However, the molecular players involved in posttranslational FOXP3 regulation are just beginning to be elucidated. Here, we found that TRAF6-deficient Tregs were dysfunctional in vivo; mice with Treg-restricted deletion of TRAF6 were resistant to implanted tumors and displayed enhanced anti-tumor immunity. We further determined that FOXP3 undergoes K63-linked ubiquitination at lysine 262 mediated by the E3 ligase TRAF6. In the absence of TRAF6 activity or upon mutation of the ubiquitination site, FOXP3 displayed aberrant, perinuclear accumulation and disrupted regulatory function. Thus, K63-linked ubiquitination by TRAF6 ensures proper localization of FOXP3 and facilitates the transcription factor's gene-regulating activity in Tregs. These results implicate TRAF6 as a key posttranslational, Treg-stabilizing regulator that may be targeted in novel tolerance-breaking therapies.

Macrophage CD40 signaling drives experimental autoimmune encephalomyelitis.

The costimulatory CD40L-CD40 dyad plays a major role in multiple sclerosis (MS). CD40 is highly expressed on MHCII+ B cells, dendritic cells and macrophages in human MS lesions. Here we investigated the role of the CD40 downstream signaling intermediates TNF receptor-associated factor 2 (TRAF2) and TRAF6 in MHCII+ cells in experimental autoimmune encephalomyelitis (EAE). Both MHCII-CD40-Traf2-/- and MHCII-CD40-Traf6-/- mice showed a reduction in clinical signs of EAE and prevented demyelination. However, only MHCII-CD40-Traf6-/- mice displayed a decrease in myeloid and lymphoid cell infiltration into the CNS that was accompanied by reduced levels of TNF-α, IL-6 and IFN-γ. As CD40-TRAF6 interactions predominantly occur in macrophages, we subjected CD40flfl LysMcre mice to EAE. This myeloid-specific deletion of CD40 resulted in a significant reduction in EAE severity, reduced CNS inflammation and demyelination. In conclusion, the CD40-TRAF6 signaling pathway in MHCII+ cells plays a key role in neuroinflammation and demyelination during EAE. Concomitant with the fact that CD40-TRAF6 interactions are predominant in macrophages, depletion of myeloid CD40 also reduces neuroinflammation. CD40-TRAF6 interactions thus represent a promising therapeutic target for MS. © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Macrophage subpopulations and their impact on chronic allograft rejection versus graft acceptance in a mouse heart transplant model.

Macrophages infiltrating the allografts are heterogeneous, consisting of proinflammatory (M1 cells) as well as antiinflammatory and fibrogenic phenotypes (M2 cells); they affect transplantation outcomes via diverse mechanisms. Here we found that macrophage polarization into M1 and M2 subsets was critically dependent on tumor necrosis factor receptor-associated factor 6 (TRAF6) and mammalian target of rapamycin (mTOR), respectively. In a heart transplant model we showed that macrophage-specific deletion of TRAF6 (LysMCre Traf6 fl/fl ) or mTOR (LysMCre Mtorfl/fl ) did not affect acute allograft rejection. However, treatment of LysMCre Mtorfl/fl recipients with CTLA4-Ig induced long-term allograft survival (>100 days) without histological signs of chronic rejection, whereas the similarly treated LysMCre Traf6 fl/fl recipients developed severe transplant vasculopathy (chronic rejection). The presentation of chronic rejection in CTLA4-Ig-treated LysMCre Traf6 fl/fl mice was similar to that of CTLA4-Ig-treated wild-type B6 recipients. Mechanistically, we found that the graft-infiltrating macrophages in LysMCre Mtorfl/fl recipients expressed high levels of PD-L1, and that PD-L1 blockade readily induced rejection of otherwise survival grafts in the LysMCre Mtorfl/fl recipients. Our findings demonstrate that targeting mTOR-dependent M2 cells is critical for preventing chronic allograft rejection, and that graft survival under such conditions is dependent on the PD-1/PD-L1 coinhibitory pathway.

Tumor necrosis factor receptor-associated factor 6 as a nuclear factor kappa B-modulating therapeutic target in cardiovascular diseases: at the heart of it all.

Inflammatory and immune signaling has been documented as a root cause of many cardiovascular pathologies. In this review, we explore the emerging role of tumor necrosis factor receptor-associated factor 6 (TRAF6)-nuclear factor kappa B (NF-κB) signaling axis in atherosclerosis, ischemic heart disease, pathologic cardiac hypertrophy or heart failure, myocarditis, and sepsis-induced cardiomyopathy. We discuss the current understanding of cardiac inflammation in heart disease, present the TRAF6 signaling axis in the heart, then summarize what is known about TRAF6 in pathophysiology of heart disease including proof-of-concept studies that identify the utility of blocking TRAF6 to attenuate cardiac dysfunction, which suggests that TRAF6 is a novel, druggable target in treating cardiovascular disease incurred by inflammatory processes.

Regulation of RANKL-induced osteoclastogenesis by RING finger protein RNF114.

Normal bone remodeling is a continuous process orchestrated by bone-resorbing osteoclasts and bone-forming osteoblasts, which an imbalance in bone remodeling results in metabolic bone diseases. RANKL, a member of the TNF cytokine family, functions as a key stimulator for osteoclast differentiation and maturation. Here, we report that RNF114, previously identified as a psoriasis susceptibility gene, plays a regulatory role in the RANKL/RANK/TRAF6 signaling pathway that mediates osteoclastogenesis. Our results demonstrated that RNF114 expression was significantly down-regulated in mouse osteoclast precursor cells undergoing RANKL-induced osteoclast differentiation. RNF114 knockout did not affect development or viability of the subpopulation of bone marrow macrophages capable of differentiating into osteoclasts in culture. However, in the presence of RANKL, RNF114 knockout bone marrow macrophages exhibited enhanced cell proliferation and augmented osteoclast differentiation, as shown by an increased expression of mature osteoclast markers, increased osteoclastic TRAP activity and bone resorption. Conversely, ectopic expression of RNF114 inhibited CTSK expression, TRAP activity, and bone resorption in RANKL-treated pre-osteoclasts. RNF114 also suppressed RANKL-activated NFATc1 expression and NFAT-regulated promoter activity. RNF114 suppressed TRAF6-, but not TAK1/TAB2-mediated NF-κB activation downstream of RANKL/RANK. In particular, TRAF6 protein levels were down-regulated by RNF114, possibly via K48-mediated proteasome-dependent degradation. These data suggested that RNF114's inhibitory effect on RANKL-stimulated osteoclastogenesis was mediated by blocking RANK/TRAF6/NF-κB signal transduction. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:159-166, 2018.

MicroRNA-126 Regulates Inflammatory Cytokine Secretion in Human Gingival Fibroblasts Under High Glucose via Targeting Tumor Necrosis Factor Receptor Associated Factor 6.

BACKGROUND: MicroRNAs (miRs) play a crucial role in inflammatory diseases, including periodontitis. Meanwhile, miRs act as biomarkers for predicting diabetes mellitus (DM). However, the regulatory mechanism of miR-126 on development of periodontitis in patients with DM still remains unclear. METHODS: Human gingival fibroblasts were cultured with low (5.5 mmol/L), medium (15 mmol/L), and high (25 mmol/L) glucose, respectively. Expressions of miR-126, tumor necrosis factor (TNF) receptor associated factor (TRAF) 6, and related cytokines were analyzed by real-time polymerase chain reaction (PCR). After transfection with miR-126 mimic, PCR and western blot were performed to detect level of TRAF6, and luciferase reporter assay confirmed if TRAF6 is the direct target of miR-126. Production of cytokines was measured using enzyme-linked immunosorbent assay. RESULTS: Increased glucose significantly suppressed miR-126 expression in human gingival fibroblasts (P <0.05). Also, high glucose increased TRAF6, interleukin (IL)-6, TNF-α, and chemical chemokine ligand (CCL) 2 levels, whereas it decreased IL-10 level. MiR-126 mimic significantly decreased TRAF6 mRNA and protein levels under high glucose (P <0.05). Also, miR-126 directly targeted TRAF6 through binding to its 3' untranslated region in human gingival fibroblasts. Overexpression of miR-126 significantly abrogated high glucose-induced secretion of proinflammatory cytokines such as IL-6, TNF-α, and CCL2 and promoted production of IL-10. CONCLUSION: These data suggest that miR-126 inhibits inflammation of human gingival fibroblasts under high glucose through targeting TRAF6, which may be a potential therapeutic target for periodontitis concomitant with DM.

TRAF6 regulates satellite stem cell self-renewal and function during regenerative myogenesis.

Satellite cells are a stem cell population within adult muscle and are responsible for myofiber regeneration upon injury. Satellite cell dysfunction has been shown to underlie the loss of skeletal muscle mass in many acquired and genetic muscle disorders. The transcription factor paired box-protein-7 (PAX7) is indispensable for supplementing the reservoir of satellite cells and driving regeneration in normal and diseased muscle. TNF receptor-associated factor 6 (TRAF6) is an adaptor protein and an E3 ubiquitin ligase that mediates the activation of multiple cell signaling pathways in a context-dependent manner. Here, we demonstrated that TRAF6-mediated signaling is critical for homeostasis of satellite cells and their function during regenerative myogenesis. Selective deletion of Traf6 in satellite cells of adult mice led to profound muscle regeneration defects and dramatically reduced levels of PAX7 and late myogenesis markers. TRAF6 was required for the activation of MAPKs ERK1/2 and JNK1/2, which in turn activated the transcription factor c-JUN, which binds the Pax7 promoter and augments Pax7 expression. Moreover, TRAF6/c-JUN signaling repressed the levels of the microRNAs miR-1 and miR-206, which promote differentiation, to maintain PAX7 levels in satellite cells. We also determined that satellite cell-specific deletion of Traf6 exaggerates the dystrophic phenotype in the mdx (a mouse model of Duchenne muscular dystrophy) mouse by blunting the regeneration of injured myofibers. Collectively, our study reveals an essential role for TRAF6 in satellite stem cell function.

CD137 Regulates NFATc1 Expression in Mouse VSMCs through TRAF6/NF-κB p65 Signaling Pathway.

Our previous study proved that CD137-CD137L interaction can regulate the expression of NFATc1. Here, we investigated whether CD137 signaling regulates the expression of NFATc1 in mice VSMCs through TRAF6/NF-κB p65 pathway. Data shows that the CD137 expression can be stimulated by TNF-α in a time-dependent manner in mouse VSMCs. Knockdown of TRAF6 by siTRAF6 significantly attenuated agonist-CD137mAb induced increase of NF-κB p65 and NFATc1 in VSMCs. Pretreatment with a NF-κB inhibitor PDTC for 30 min inhibited the expression of p-p65 in both cytoplasm and nucleus in VSMCs. Thus, the protein level of NFATc1 can be suppressed through inhibition of p-p65. Finally, we also show that the levels of IL-2 and IL-6 can be increased by agonist-CD137 stimulation and decreased when NFATc1 was suppressed. Our data suggest that activated CD137 signaling regulates the expression of NFATc1 and its downstream factors through TRAF6/NF-κB p65 pathways in VSMCs. These findings provide a novel target for treatment of atherosclerosis.

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

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. CONCLUSIONS: TRAF6 may be involved in cell migration, invasion, and apoptosis of SPC-A-1 cells, possibly through regulating the NF-қB-CD24/CXCR4 pathway.
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Loss of Tifab, a del(5q) MDS gene, alters hematopoiesis through derepression of Toll-like receptor-TRAF6 signaling.

TRAF-interacting protein with forkhead-associated domain B (TIFAB) is a haploinsufficient gene in del(5q) myelodysplastic syndrome (MDS). Deletion of Tifab results in progressive bone marrow (BM) and blood defects, including skewed hematopoietic stem/progenitor cell (HSPC) proportions and altered myeloid differentiation. A subset of mice transplanted with Tifab knockout (KO) HSPCs develop a BM failure with neutrophil dysplasia and cytopenia. In competitive transplants, Tifab KO HSPCs are out-competed by wild-type (WT) cells, suggesting a cell-intrinsic defect. Gene expression analysis of Tifab KO HSPCs identified dysregulation of immune-related signatures, and hypersensitivity to TLR4 stimulation. TIFAB forms a complex with TRAF6, a mediator of immune signaling, and reduces TRAF6 protein stability by a lysosome-dependent mechanism. In contrast, TIFAB loss increases TRAF6 protein and the dynamic range of TLR4 signaling, contributing to ineffective hematopoiesis. Moreover, combined deletion of TIFAB and miR-146a, two genes associated with del(5q) MDS/AML, results in a cooperative increase in TRAF6 expression and hematopoietic dysfunction. Re-expression of TIFAB in del(5q) MDS/AML cells results in attenuated TLR4 signaling and reduced viability. These findings underscore the importance of efficient regulation of innate immune/TRAF6 signaling within HSPCs by TIFAB, and its cooperation with miR-146a as it relates to the pathogenesis of hematopoietic malignancies, such as del(5q) MDS/AML.

Glucocorticoid-induced tumor necrosis factor receptor-related protein co-stimulation facilitates tumor regression by inducing IL-9-producing helper T cells.

T cell stimulation via glucocorticoid-induced tumor necrosis factor receptor (TNFR)-related protein (GITR) elicits antitumor activity in various tumor models; however, the underlying mechanism of action remains unclear. Here we demonstrate a crucial role for interleukin (IL)-9 in antitumor immunity generated by the GITR agonistic antibody DTA-1. IL-4 receptor knockout (Il4ra(-/-)) mice, which have reduced expression of IL-9, were resistant to tumor growth inhibition by DTA-1. Notably, neutralization of IL-9 considerably impaired tumor rejection induced by DTA-1. In particular, DTA-1-induced IL-9 promoted tumor-specific cytotoxic T lymphocyte (CTL) responses by enhancing the function of dendritic cells in vivo. Furthermore, GITR signaling enhanced the differentiation of IL-9-producing CD4(+) T-helper (TH9) cells in a TNFR-associated factor 6 (TRAF6)- and NF-κB-dependent manner and inhibited the generation of induced regulatory T cells in vitro. Our findings demonstrate that GITR co-stimulation mediates antitumor immunity by promoting TH9 cell differentiation and enhancing CTL responses and thus provide a mechanism of action for GITR agonist-mediated cancer immunotherapies.

Interleukin-1 Acts via the JNK-2 Signaling Pathway to Induce Aggrecan Degradation by Human Chondrocytes.

OBJECTIVE: Aggrecan enables articular cartilage to bear load and resist compression. Aggrecan loss occurs early in osteoarthritis and rheumatoid arthritis and can be induced by inflammatory cytokines such as interleukin-1 (IL-1). IL-1 induces cleavage of specific aggrecans characteristic of the ADAMTS proteinases. The aim of this study was to identify the intracellular signaling pathways by which IL-1 causes aggrecan degradation by human chondrocytes and to investigate how aggrecanase activity is controlled by chondrocytes. METHODS: We developed a cell-based assay combining small interfering RNA (siRNA)-induced knockdown with aggrecan degradation assays. Human articular chondrocytes were overlaid with bovine aggrecan after transfection with siRNAs against molecules of the IL-1 signaling pathway. After IL-1 stimulation, released aggrecan fragments were detected with AGEG and ARGS neoepitope antibodies. Aggrecanase activity and tissue inhibitor of metalloproteinases 3 levels were measured by enzyme-linked immunosorbent assay. Low-density lipoprotein receptor-related protein 1 (LRP-1) shedding was analyzed by Western blotting. RESULTS: ADAMTS-5 is a major aggrecanase in human chondrocytes, regulating aggrecan degradation in response to IL-1. The tumor necrosis factor receptor-associated 6 (TRAF-6)/transforming growth factor ß-activated kinase 1 (TAK-1)/MKK-4 signaling axis is essential for IL-1-induced aggrecan degradation, while NF-κB is not. Of the 3 MAPKs (ERK, p38, and JNK), only JNK-2 showed a significant role in aggrecan degradation. Chondrocytes constitutively secreted aggrecanase, which was continuously endocytosed by LRP-1, keeping the extracellular level of aggrecanase low. IL-1 induced aggrecanase activity in the medium in a JNK-2-dependent manner, possibly by reducing aggrecanase endocytosis, because IL-1 caused JNK-2-dependent shedding of LRP-1. CONCLUSION: The signaling axis TRAF-6/TAK-1/MKK-4/JNK-2 mediates IL-1-induced aggrecanolysis. The level of aggrecanase is controlled by its endocytosis, which may be reduced upon IL-1 stimulation because of LRP-1 shedding.

The TLR signaling adaptor TRAM interacts with TRAF6 to mediate activation of the inflammatory response by TLR4.

TLRs act as sentinels in professional immune cells to detect and initiate the innate immune response to pathogen challenge. TLR4 is a widely expressed TLR, responsible for initiating potent immune responses to LPS. TRAM acts to bridge TLR4 with TRIF, orchestrating the inflammatory response to pathogen challenge. We have identified a putative TRAF6-binding motif in TRAM that could mediate a novel signaling function for TRAM in TLR4 signaling. TRAM and TRAF6 association was confirmed by immunoprecipitation of endogenous, ectopically expressed and recombinant proteins, which was ablated upon mutation of a key Glu residue in TRAM (TRAM E183A). TRAF6 and TRAM were observed colocalizing using confocal microscopy following ectopic expression in cells and the ability of TRAM and TRAM E183A to activate luciferase-linked reporter assays was determined in HEK293 and TRAF6-deficient cells. Importantly, TRAM-deficient macrophages reconstituted with TRAM E183A display significantly reduced inflammatory TNF-α, IL-6, and RANTES protein production compared with WT TRAM. These results demonstrate a novel role for TRAM in TLR4-mediated signaling in regulating inflammatory responses via its interaction with TRAF6, distinct from its role as a bridging adaptor between TLR4 and TRIF.

Serum amyloid A induces interleukin-33 expression through an IRF7-dependent pathway.

Interleukin-33 (IL-33), an IL-1 family cytokine and nuclear alarmin, is constitutively expressed in epithelial barrier tissues and human blood vessels. However, little is known about the induced expression of IL-33 in monocytes and macrophages, which are major cytokine-producing cells of the innate immune system. Here, we report the induction of IL33 expression in both human monocytes and mouse macrophages from C57BL/6 mice by the acute-phase protein serum amyloid A (SAA). SAA-induced transcriptional activation of the Il33 gene, resulting in nuclear accumulation of the IL-33 protein. TLR2, one of the SAA receptors, was primarily responsible for the induction of IL-33. Progressive deletion of the human IL-33 promoter led to the identification of two potential binding sites for interferon regulatory factor 7 (IRF7), one of which (-277/-257) was found to be important for SAA-stimulated IL-33 promoter activity. IRF7 was recruited to the IL-33 promoter upon SAA stimulation, and silencing IRF7 expression in THP-1 cells abrogated SAA-induced Il33 expression. SAA also promoted an interaction between TNF receptor-associated factor 6 and IRF7. Taken together, these results identify IRF7 as a critical transcription factor for SAA-induced Il33 expression in monocytes and macrophages.

MicroRNA-125b protects against myocardial ischaemia/reperfusion injury via targeting p53-mediated apoptotic signalling and TRAF6.

AIMS: The present study examined the role of microRNA-125b (miR-125b) in myocardial ischaemia/reperfusion (I/R) injury. We constructed lentivirus-expressing miR-125b (LmiR-125b) and developed transgenic mice with overexpression of miR-125b. METHODS AND RESULTS: LmiR-125b was transfected into mouse hearts through the right common carotid artery. Lentivirus vector (LmiR-Con) served as vector control. Untreated mice served as I/R control. Sham operation served as sham control. Seven days after transfection, the hearts were subjected to ischaemia (45 min) followed by reperfusion (4 h). Myocardial infarct size was analysed by 2,3,5-triphenyltetrazolium chloride staining. In separate experiments, hearts were subjected to ischaemia (45 min) followed by reperfusion for up to 7 days. Cardiac function was measured by echocardiography before, as well as 3 and 7 days after myocardial I/R. Increased expression of miR-125b significantly decreased I/R-induced myocardial infarct size by 60% and prevented I/R-induced decreases in ejection fraction (EF%) and fractional shortening (%FS). Transgenic mice with overexpression of miR-125b also showed the protection against myocardial I/R injury. Increased expression of miR-125b attenuated I/R-induced myocardial apoptosis and caspase-3/7 and -8 activities. Western blot showed that increased expression of miR-125b suppresses p53 and Bak1 expression in the myocardium. In addition, transfection of LmiR-125b decreased the levels of TNF receptor-associated factor 6 (TRAF6) and prevented I/R-induced NF-κB activation. CONCLUSION: miR-125 protects the myocardium from I/R injury by preventing p53-mediated apoptotic signalling and suppressing TRAF6-mediated NF-κB activation.

MiR-125a TNF receptor-associated factor 6 to inhibit osteoclastogenesis.

MicroRNAs (miRNAs) play important roles in osteoclastogenesis and bone resorption. In the present study, we found that miR-125a was dramatically down-regulated during macrophage colony stimulating factor (M-CSF) and receptor activator of nuclear factor-κB ligand (RANKL) induced osteoclastogenesis of circulating CD14+ peripheral blood mononuclear cells (PBMCs). Overexpression of miR-125a in CD14+ PBMCs inhibited osteoclastogenesis, while inhibition of miR-125a promoted osteoclastogenesis. TNF receptor-associated factor 6 (TRAF6), a transduction factor for RANKL/RANK/NFATc1 signal, was confirmed to be a target of miR-125a. EMSA and ChIP assays confirmed that NFATc1 bound to the promoter of the miR-125a. Overexpression of NFATc1 inhibited miR-125a transcription, and block of NFATc1 expression attenuated RANKL-regulated miR-125a transcription. Here, we reported that miR-125a played a biological function in osteoclastogenesis through a novel TRAF6/ NFATc1/miR-125a regulatory feedback loop. It suggests that regulation of miR-125a expression may be a potential strategy for ameliorating metabolic disease.