TRAF3 has diverse signaling functions, which vary by cell type. Uniquely in B lymphocytes, TRAF3 inhibits homeostatic survival. Highlighting the role of TRAF3 as a tumor suppressor, loss-of-function TRAF3 mutations are associated with human B-cell malignancies, while B-cell-specific deletion of TRAF3 in mice leads to autoimmunity and lymphoma development. The role of TRAF3 in inhibiting noncanonical NF-κB activation, CD40 and BAFF-R signaling to B cells is well documented. In contrast, TRAF3 enhances many T-cell effector functions, through associating with and enhancing signaling by the T-cell receptor (TCR)-CD28 complex. The present study was designed to determine the role of TRAF3 in signaling via the B-cell antigen receptor (BCR). The BCR is crucial for antigen recognition, survival, proliferation, and antibody production, and defects in BCR signaling can promote abnormal survival of malignant B cells. Here, we show that TRAF3 is associated with both CD79B and the BCR-activated kinases Syk and Btk following BCR stimulation. BCR-induced phosphorylation of Syk and additional downstream kinases was increased in TRAF3-/- B cells, with regulation observed in both follicular and marginal zone B-cell subsets. BCR stimulation of TRAF3-/- B cells resulted in increased surface expression of MHC-II, CD80, and CD86 molecules. Interestingly, increased survival of TRAF3-/- primary B cells was resistant to inhibition of Btk, while TRAF3-deficient malignant B-cell lines showed enhanced sensitivity. TRAF3 serves to restrain normal and malignant BCR signaling, with important implications for its role in normal B-cell biology and abnormal survival of malignant B cells.

TNF receptor associated factors (TRAFs) are a family of proteins primarily involved in both adaptive and innate immunity. In this study, we identified a novel TRAF3 gene in Apostichopus japonicus by transcriptome sequencing and RACE approaches (designated as AjTRAF3). The full-length of AjTRAF3 was of 2796 bp including a 5' untranslated region (UTR) of 83 bp, a 3' UTR of 1066 bp and a putative open reading frame of 1647 bp encoding a polypeptide of 548 amino acid residues. The representative domains such as a RING finger domain (residues 54-96), two TRAF domains with zinc finger structure (residues 141-228), a coiled coil and a meprin and TRAF homology (MATH) domain (residues 396-522) were all detected in the deduced amino acids of AjTRAF3. AjTRAF3 was ubiquitously expressed in all examined tissues with predominant expression in the body wall and slightly weaker in intestine, respiratory tree, tube feet, coelomocytes and longitudinal muscle. Time-course expression analysis in coelomocytes revealed that AjTRAF3 was significantly depressed towards Vibrio splendidus infection with a 0.20-fold decrease at 12 h, compared to control levels. AjTRAF3 silencing could elevate intracellular ROS levels by 2.08-fold and 2.09-fold compared to each control group in vitro and in vivo, respectively. Taken together, all these results suggested that AjTRAF3 may play a crucial role in the processes of anti-bacteria response in sea cucumber through regulating ROS production.

The microRNA miR-29b-3p shows important roles in regulating apoptosis and inflammation. However, its effects on intestinal ischaemia/reperfusion (II/R) injury have not been reported. Here we have investigated the functions of miR-29b-3p on II/R injury on order to find drug targets to treat the injury.

Acute pancreatitis (AP) is an inflammatory disease with high morbidity and mortality. The regulation mechanism of miRNA is involved in the production and development of various diseases, but the regulation mechanism of miRNA in AP is still not fully elucidated. The expression of miR-339-3p was detected using quantitative real-time PCR. The levels of TNF-α, IL-1β, and IL-6 were detected using enzyme-linked immunosorbent assay. Cell apoptosis was measured using flow cytometry. The protein expressions of TNF receptor-associated factor 3 (TRAF3), Bcl-2, C-caspase 3, Bax, p-p38, and p38 were measured using western blot. Luciferase reporter assay and RNA immunoprecipitation assay were applied to ensure that miR-399-3p targeted TRAF3. Caerulein promoted the expression of TNF-α, IL-1β, and IL-6, enhanced the expression of C-caspase 3 and Bax while inhibited Bcl-2 protein expression. Meanwhile, caerulein also reduced the expression of miR-339-3p and induced the expression of TRAF3 in rat pancreatic acinar cells. miR-399-3p transfection inhibited the levels of TNF-α, IL-1β, and IL-6 and C-caspase 3 and Bax protein expression as well as suppressed cell apoptosis, while increased Bcl-2 protein expression in caerulein-induced AP. TRAF3 has been verified as a target of miR-339-3p. Interestingly, the reduction of miR-399-3p inhibited the p38 pathway, which was impaired by the upregulation of TRAF3. In addition, the suppression effects of miR-339-3p on cell inflammation and apoptosis in caerulein-induced AP were reversed by enhancing TRAF3 expression. In this study, in vitro model of AP was characterized by strong inflammation and cell apoptosis. We have first demonstrated the regulatory network of miR-339-3p and TRAF3. Overexpression of miR-339-3p inhibited cell inflammation and cell apoptosis in caerulein-induced AP through modulating TRAF3 expression via the p38 pathway, providing a new therapeutic target in the treatment of AP.

TCR signaling is a prerequisite for early stage development of invariant natural killer T (iNKT) cells, whereas IL-15 signaling is required for expansion and maturation at later stages. In this study, we show that TNF receptor associated factor 3 (TRAF3) plays a critical role in the transition between these two distinct signaling pathways and developmental stages. TRAF3-deficient iNKT cells in CD4(Cre)TRAF3(flox/flox) (T-TRAF3(-/-)) mice exhibit defective up-regulation of T-bet and CD122, two critical molecules for IL-15 signaling, and as a consequence, IL-15-mediated iNKT cell proliferation and survival are impaired. Consistently, development of iNKT cells in T-TRAF3(-/-) mice shows a major defect at developmental stages 2 and 3, but not stages 0 and 1. We further demonstrated that defective T-bet up-regulation occurring during the stage 1 to stage 2 transition results from reduced TCR signaling in TRAF3(-/-) iNKT cells. In addition, mature TRAF3(-/-) iNKT cells displayed defective cytokine responses upon TCR stimulation. Collectively, our results reveal that by modulating the relative strength of TCR signaling, TRAF3 is an important regulator of iNKT cell development and functions.

Tumor necrosis factor receptor (TNFR)-associated factors (TRAFs) are vital signaling adaptor proteins for the innate immune response and are involved in many important pathways, such as the NF-κB- and interferon regulatory factor (IRF)-activated signaling pathways. In this study, the TRAF3 ortholog from the shrimp Litopenaeus vannamei (LvTRAF3) was cloned and characterized. LvTRAF3 has a transcript of 3,865 bp, with an open reading frame (ORF) of 1,002 bp and encodes a polypeptide of 333 amino acids, including a conserved TRAF-C domain. The expression of LvTRAF3 in the intestine and hemocyte was up-regulated in response to poly (I:C) challenge and white spot syndrome virus (WSSV) infection. RNAi knockdown of LvTRAF3 in vivo significantly increased WSSV gene transcription, viral loads, and mortality in WSSV-infected shrimp. Next, we found that LvTRAF3 was not able to induce the activation of the NF-κB pathway, which was crucial for synthesis of antimicrobial peptides (AMPs), which mediate antiviral immunity. Specifically, in dual-luciferase reporter assays, LvTRAF3 could not activate several types of promoters with NF-κB binding sites, including those from WSSV genes (wsv069, wsv056, and wsv403), Drosophila AMPs or shrimp AMPs. Accordingly, the mRNA levels of shrimp AMPs did not significantly change when TRAF3 was knocked down during WSSV infection. Instead, we found that LvTRAF3 signaled through the IRF-Vago antiviral cascade. LvTRAF3 functioned upstream of LvIRF to regulate the expression of LvVago4 and LvVago5 during WSSV infection in vivo. Taken together, these data provide experimental evidence of the participation of LvTRAF3 in the host defense to WSSV through the activation of the IRF-Vago pathway but not the NF-κB pathway.

The transcription factor, runt-related transcription factor 2 (Runx2), plays a pivotal role in the differentiation of the mesenchymal stem cells to the osteochondroblast lineages. We found by the drug repositioning strategy that a proton pump inhibitor, lansoprazole, enhances nuclear accumulation of Runx2 and induces osteoblastogenesis of human mesenchymal stromal cells. Systemic administration of lansoprazole to a rat femoral fracture model increased osteoblastogenesis. Dissection of signaling pathways revealed that lansoprazole activates a noncanonical bone morphogenic protein (BMP)-transforming growth factor-beta (TGF-β) activated kinase-1 (TAK1)-p38 mitogen-activated protein kinase (MAPK) pathway. We found by in cellulo ubiquitination studies that lansoprazole enhances polyubiquitination of the TNF receptor-associated factor 6 (TRAF6) and by in vitro ubiquitination studies that the enhanced polyubiquitination of TRAF6 is attributed to the blocking of a deubiquitination enzyme, cylindromatosis (CYLD). Structural modeling and site-directed mutagenesis of CYLD demonstrated that lansoprazole tightly fits in a pocket of CYLD where the C-terminal tail of ubiquitin lies. Lansoprazole is a potential therapeutic agent for enhancing osteoblastic differentiation.

Influenza A virus non-structural protein 1 (NS1) antagonizes interferon response through diverse strategies, particularly by inhibiting the activation of interferon regulatory factor 3 (IRF3) and IFN-β transcription. However, the underlying mechanisms used by the NS1 C-terminal effector domain (ED) to inhibit the activation of IFN-β pathway are not well understood. In this study, we used influenza virus subtype of H5N1 to demonstrate that the NS1 C-terminal ED but not the N-terminal RNA-binding domain, binds TNF receptor-associated factor 3 (TRAF3). This results in an attenuation of the type I IFN signaling pathway. We found that the NS1 C-terminal ED (named NS1/126-225) inhibits the active caspase activation and recruitment domain-containing form of RIG-I [RIG-I(N)]-induced IFN-β reporter activity, the phosphorylation of IRF3, and the induction of IFN-β. Further analysis showed that NS1/126-225 binds to TRAF3 through the TRAF domain, subsequently decreasing TRAF3 K63-linked ubiquitination. NS1/126-225 binding also disrupted the formation of the mitochondrial antiviral signaling (MAVS)-TRAF3 complex, increasing the recruitment of IKKε to MAVS; ultimately shutting down the RIG-I(N)-mediated signal transduction and cellular antiviral responses. This attenuation of cellular antiviral responses leads to evasion of the innate immune response. Taken together, our findings offer an important insight into the interplay between the influenza virus and host innate immunity.

Pancreatic cancer (PC) is one of the worst prognostic cancers. Here, we probed the anti-cancer activity of wogonoside (Wog), a flavonoid isolated from Scutellaria baicalensis Georgi, on PC, as well as potential molecular mechanism.

Recent studies showed that overwhelming inflammatory response mediated by the toll-like receptor (TLR)-related pathway was important in the development of acute lung injury (ALI). The aim of this study was to determine whether common genetic variation in four genes of the TLR signaling pathway were associated with sepsis-induced ALI susceptibility and risk of death in Chinese Han population.

Wnt/β-catenin signaling is a well-established driver of colon cancer; however, a targeted therapeutic agent has not reached clinics yet. In the present study, we report that the natural compound liquidambaric acid (LDA) inhibits oncogenic Wnt/β-catenin signaling in vitro and in vivo through its direct target tumor necrosis factor receptor-associated factor 2 (TRAF2). Mechanistically, TRAF2 positively regulates Wnt signaling by interacting with the N-terminal of β-catenin via its TRAF-C domain; this interaction is disrupted in presence of LDA. Particularly, a TRAF2/β-catenin/TCF4/TNIK complex is present in colon cancer cells, where TRAF2 is indispensable for the complex formation, and TRAF2/β-catenin and β-catenin/TCF4 interactions are disrupted upon LDA treatment. Our findings not only highlight that TRAF2 is an oncogenic regulator of Wnt/β-catenin signaling and colon cancer but also provide a lead compound targeting TRAF2 for cancer therapy.

Postmenopausal osteoporosis is caused by the deficiency of estrogen, which breaks bone homeostasis and induces levels of pro-inflammatory cytokines. Muscone is a potent anti-inflammatory agent and is used to treat bone fracture in traditional Chinese medicine. However, its anti-osteoclastogenic effects remain unclear. For in vitro study, morphology tests of osteoclastogenesis were firstly performed. And then, factors in RANK-induced NF-κB and MAPK pathways were examined by RT-PCR and Western blot, and the binding of TNF receptor-associated factor (TRAF)6 to RANK was inspected by coimmunoprecipitation and immunofluorescence staining. For in vivo experiments, C57BL/6 ovariectomized (OVX) mice were used for detection, including H&E staining, TRAP staining, and micro CT. As a result, muscone reduced OVX-induced bone loss in mice and osteoclast differentiation in vitro, by inhibiting TRAF6 binding to RANK, and then suppressed NF-κB and MAPK signaling pathways. The expression of the downstream biomarkers was finally inhibited, including NFATc1, CTR, TRAP, cathepsin K, and MMP-9. The inflammatory factors, TNF-a and IL-6, were also reduced by muscone. Taken together, muscone inhibited the binding of TRAF6 to RANK induced by RANKL, thus blocking NF-kB and MAPK pathways, and down-regulating related gene expression. Finally, muscone inhibited osteoclastogenesis and osteoclast function by blocking RANK-TRAF6 binding, as well as downstream signaling pathways in vitro. Muscone also reduced ovariectomy-induced bone loss in vivo.

In the present study, the aim was to investigate the role of microRNA-1180 (miR-1180) in the growth and apoptosis of prostate cancer, as well as to identify its direct targets. Initially, reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was performed to examine the expression of miR-1180 in the prostate cancer tissues and adjacent normal prostate tissues of 30 patients, as well as in DU145 and RWPE-1 cells. Next, DU145 cells were transfected with miR-1180 mimics, and the expression levels of associated proteins were determined by western blot assay. In addition, the role of miR-1180 in the proliferation, apoptosis, invasion and migration of DU145 cells was investigated by MTT, flow cytometry, cell invasion and wound healing assays, respectively. A dual-luciferase reporter assay was also performed to examine whether TNF receptor associated factor 1 (TRAF1) and B-cell lymphoma-2-associated athanogene 2 (BAG2) are direct targets of miR-1180. It was observed that miR-1180 expression was significantly decreased in the prostate cancer tissues compared with the normal prostate tissues, and was also inhibited in DU145 cells compared with RWPE-1 cells. Furthermore, transient overexpression of miR-1180 inhibited the proliferation, migration and invasion, and promoted the apoptosis of DU145 cells, as well as alleviated expression of associated proteins. The dual-luciferase reporter assay confirmed that TRAF1 and BAG2 are direct targets of miR-1180. These results suggested that miR-1180 contributed to prostate cancer by targeting TRAF1/BAG2 and by nuclear factor-κB signaling pathway activation.

HIV-1 Nef is a virulence factor that plays multiple roles during HIV replication. Recently, it has been described that Nef intersects the CD40 signalling in macrophages, leading to modification in the pattern of secreted factors that appear able to recruit, activate and render T lymphocytes susceptible to HIV infection. The engagement of CD40 by CD40L induces the activation of different signalling cascades that require the recruitment of specific tumor necrosis factor receptor-associated factors (i.e. TRAFs). We hypothesized that TRAFs might be involved in the rapid activation of NF-κB, MAPKs and IRF-3 that were previously described in Nef-treated macrophages to induce the synthesis and secretion of proinflammatory cytokines, chemokines and IFNβ to activate STAT1, -2 and -3.

Toll-like receptors (TLRs) play a pivotal role in the defense against invading pathogens by detecting pathogen-associated molecular patterns (PAMPs). TLR4 recognizes lipopolysaccharides (LPS) in the cell walls of Gram-negative bacteria, resulting in the induction and secretion of proinflammatory cytokines such as TNF-α and IL-6. The WW domain containing E3 ubiquitin protein ligase 1 (WWP1) regulates a variety of cellular biological processes. Here, we investigated whether WWP1 acts as an E3 ubiquitin ligase in TLR-mediated inflammation.

To investigate the toxic effects of microRNA-27a on breast cancer cells through inositol-acquiring enzyme 1-TNF receptor-associated factor 2 inhibition by mepivacaine.

Endometritis is a female reproductive disease that affects the cattle industries development and microRNAs (miRNAs) play a pivotal role and critical regulators of the innate immune response in varieties of diseases. The present study intends to investigate the regulatory role of miR-24-3p in the innate immune response involved in endometritis and evaluate its therapeutic potential.

BACKGROUND The dramatic increase of intervertebral disc degeneration (IDD) is considered to be a major cause of discogenic low back pain. The current study focused on the regulatory function of microRNA-194 (miR-194) on lipopolysaccharide (LPS)-induced inflammatory response in nucleus pulposus (NP) cells. MATERIAL AND METHODS LPS was used to treat NP cells to induce inflammatory responses. MiRNA and gene expression were detected by quantitative PCR. Proteins and protein expression levels were detected by Western blot and ELISA kit. Dual luciferase reporter assay was applied to identify the correlation between an miR-194- and TNF receptor-associated factor 6 (TRAF6) and to test NF-κB activity. RESULTS MiR-194 expression was reduced in LPS-induced NP cells. Both miR-194 overexpression and miR-194 inhibitor could regulate extracellular matrix (ECM) genes expression (Aggrecan and collagen II), MMP3, MMP13, ADAMTS4, and ADAMTS5, as well as inflammatory cytokines-associated genes (TNF-α, IL-1, IL-6, PGE2). Through a further study of the molecular mechanism, miR-194 was proved to be involved in the regulation of TRAF6 and its downstream signal molecule, nuclear factor-kappa B (NF-κB). CONCLUSIONS Finding of our study suggest that miR-194 can inhibit LPS-induced inflammatory response in NP cells of the intervertebral disc (IVD) by targeting TRAF6, which may contribute development of IDD biological therapy.

The protozoan parasite Toxoplasma gondii secretes proteins from specialized organelles, the rhoptries, and dense granules, which are involved in the modulation of host cell processes. Dense granule protein GRA15 activates the nuclear factor kappa B (NF-κB) pathway, which plays an important role in cell death, innate immunity, and inflammation. Exactly how GRA15 activates the NF-κB pathway is unknown. Here we show that GRA15 interacts with tumor necrosis factor receptor-associated factors (TRAFs), which are adaptor proteins functioning upstream of the NF-κB transcription factor. We identified several TRAF binding sites in the GRA15 amino acid sequence and showed that these are involved in NF-κB activation. Furthermore, a TRAF2 knockout cell line has impaired GRA15-mediated NF-κB activation. Thus, we determined the mechanism for GRA15-dependent NF-κB activation.IMPORTANCE The parasite Toxoplasma can cause birth defects and severe disease in immunosuppressed patients. Strain differences in pathogenicity exist, and these differences are due to polymorphic effector proteins that Toxoplasma secretes into the host cell to coopt host cell functions. The effector protein GRA15 of some Toxoplasma strains activates the nuclear factor kappa B (NF-κB) pathway, which plays an important role in cell death, innate immunity, and inflammation. We show that GRA15 interacts with TNF receptor-associated factors (TRAFs), which are adaptor proteins functioning upstream of the NF-κB transcription factor. Deletion of TRAF-binding sites in GRA15 greatly reduces its ability to activate the NF-κB pathway, and TRAF2 knockout cells have impaired GRA15-mediated NF-κB activation. Thus, we determined the mechanism for GRA15-dependent NF-κB activation.

Hepatocellular carcinoma (HCC) is difficult to treat, and is the second leading cause of cancer-related death worldwide. This study aimed to examine whether combination of wogonin and artesunate exhibits synergistic anti-HCC effect. Our data show that the combination treatment exhibits synergistic effect in reducing HCC cell viability by increasing apoptosis as indicated by the elevated cleavage of caspase 8, 3 and PARP. Interestingly, PCR array and the subsequent studies indicate that the combination treatment significantly increases the expression of DNA-damage-inducible, alpha (GADD45A), tumor necrosis factor (TNFα) and TNF receptor-associated factor 3 (TRAF3). Knockdown of GADD45A, TNFα or TRAF3 abolishes the combination treatment-enhanced apoptosis and the synergistic effect in reducing HCC cell viability. In the HCC-bearing xenograft mouse models, although the combination treatment increases the activity of NFκB in the tumor tissues, it exhibits a more potent anti-HCC effect than the mono-treatment, which may due to the enhanced apoptosis as indicated by the increased expression of GADD45A, TNFα, TRAF3 and apoptotic markers. Our study clearly demonstrates that the combination of artesunate and wogonin exhibits synergistic anti-HCC effect, and support the further development of this combination as alternative therapeutics for HCC management.