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.

BACKGROUND CPNE1 plays a vital role in regulating cell differentiation. The clinical and biological values of CPNE1 in prostate cancer are still unclear. The aim of this study was to investigate the clinicopathological value of CPNE1 and the association of CPNE1 with TRAF2 expression in patients with prostate cancer. MATERIAL AND METHODS CPNE1 expression in prostate cancer was analyzed using Gene Expression Omnibus (GEO) databases. The Cancer Genome Atlas (TCGA) dataset was used to investigate the association of CPNE1 expression with TRAF2 expression in prostate cancer. The association of CPNE1 expression with recurrence-free survival in patients was also analyzed using the TCGA dataset. Immunohistochemistry assay was performed to examine CPNE1 expression in 65 normal prostate samples and 114 prostate cancer samples. The recurrence-free survival in patients was evaluated using Kaplan-Meier curves and log-rank test. In addition, multivariate and univariate analyses of prognostic factors were investigated by Cox regression. The effect of CPNE1 on TRAF2 expression was explored in human prostate cancer DU-145 cells. RESULTS Our results showed that expression level of CPNE1 is higher in prostate cancer than in normal prostate tissues (P=0.006). In the GSE35988 dataset, CPNE1 expression was found to be upregulated in castration-resistant prostate cancer compared with non-castration-resistant prostate cancer (P<0.001). Furthermore, we found that CPNE1 high expression was significantly related to tumor stage, Gleason score, and poorer biochemical recurrence-free survival in prostate cancer patients. Co-expression analysis of TCGA data showed that CPNE1 is significantly associated with TRAF2 expression. CPNE1 overexpression can upregulate TRAF2 expression in prostate cancer DU-145 cells as determined by Western blotting and immunofluorescence assays. CONCLUSIONS Overall, our findings suggest that CPNE1 is a valuable prognostic marker for evaluating recurrence-free survival and is positively related to TRAF2 expression in prostate cancer.

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.

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.

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.

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.

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.

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.

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.

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.

Tumor necrosis factor receptor-associated factor 2 (TRAF2) is a critical mediator of tumor necrosis factor-α (TNF-α) signaling. However, the regulatory mechanisms of TRAF2 are not fully understood. Here we show evidence that TRAF2 requires brefeldin A-inhibited guanine nucleotide-exchange factor 1 (BIG1) to be recruited into TNF receptor 1 (TNFR1) signaling complexes. In BIG1 knockdown cells, TNF-α-induced c-Jun N-terminal kinase (JNK) activation was attenuated and the sensitivity to TNF-α-induced apoptosis was increased. Since these trends correlated well with those of TRAF2 deficient cells as previously demonstrated, we tested whether BIG1 functions as an upstream regulator of TRAF2 in TNFR1 signaling. As expected, we found that knockdown of BIG1 suppressed TNF-α-dependent ubiquitination of TRAF2 that is required for JNK activation, and impaired the recruitment of TRAF2 to the TNFR1 signaling complex (complex I). Moreover, we found that the recruitment of TRAF2 to the death-inducing signaling complex termed complex II was also impaired in BIG1 knockdown cells. These results suggest that BIG1 is a key component of the machinery that drives TRAF2 to the signaling complexes formed after TNFR1 activation. Thus, our data demonstrate a novel and unexpected function of BIG1 that regulates TNFR1 signaling by targeting TRAF2.

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.

Tumor necrosis factor (TNF) signaling pathway plays crucial roles in the regulation of various immune responses. In the present study, a TNF signaling pathway related regulatory factor, TRAF and TNF receptor-associated protein (TTRAP), was firstly identified from the mollusk Zhikong scallop Chlamys farreri (designated as CfTTRAP). The full-length cDNA of CfTTRAP was of 2326bp, containing an open reading frame (ORF) of 1008 bp encoding a polypeptide of 335 amino acids with the predicted molecular weight of 38.4 kDa. There was an Exo_endo_phos domain in CfTTRAP, and it was well conserved when compared with other TTRAPs, especially the endonuclease activity related motifs. The recombinant protein of CfTTRAP exhibited prominent endonuclease activity to digest the genome DNA from C. farreri in the presence of Mg(2+), but it could not digest genome DNA of Escherichia coli and Bacillus subtilis, indicating CfTTRAP was a new member of Mg(2+)/Mn(2+)-dependent phosphodiesterase enzymes (MDP) superfamily. The mRNA transcripts of CfTTRAP were detected in all tested tissues of scallop, including muscle, mantle, gonad, gill, kidney and hemocytes. The expression level of CfTTRAP mRNA in hemocytes varied greatly after the stimulation of LPS, PGN or β-glucan. LPS induced significant down-regulation (P<0.05) of CfTTRAP mRNA expression, while PGN or β-glucan up-regulated the expression significantly (P<0.01), indicating that this regulatory factor was involved in modulating immune responses towards different stimulus. The present results provided new evidences for the potential roles of such molecule in C. farreri, and further confirmed the existence of TTRAP modulated TNF signaling pathway in mollusk.

Levels of tumor necrosis factor-alpha (TNF-alpha) are increased in the brain in Alzheimer's disease (AD). The TNF-alpha/TNF-R signaling pathways involve complex interactions between several proteins, including TNF-receptor-associated factor-2 (TRAF-2). We have examined the distribution and levels of TRAF-2 in AD and control brains and also whether single nucleotide polymorphisms (SNPs) in the TRAF-2 gene are associated with AD and influence TRAF-2 expression. Immunohistochemistry demonstrated TRAF-2 in AD and control cortex in neurons, within plaque-associated neurites and some neurofibrillary tangles. Western blots revealed a band of the expected apparent molecular mass (approximately 50kDa) for TRAF-2, in homogenates of AD and control cortex. RT-PCR showed the levels of TRAF-2 mRNA to be significantly higher in the frontal cortex of AD than control brains (p=0.015). TRAF-2 mRNA expression was not linked to any SNPs. The 3' UTR SNP (rs7852970) GG allele was significantly protective against AD (p=0.030). Our findings suggest that the TRAF-2 pathway is involved AD. The mechanisms are currently unclear and need further examination.

Tumor necrosis factor receptor-associated factor (TRAF)2 is a critical adaptor molecule for tumor necrosis factor (TNF) receptors in inflammatory and immune signaling. Upon receptor engagement, TRAF2 is recruited to CD40 and translocates to lipid rafts in a RING finger-dependent process, which enables the activation of downstream signaling cascades including c-Jun NH(2)-terminal kinase (JNK) and nuclear factor (NF)-kappaB. Although TRAF1 can displace TRAF2 and CD40 from raft fractions, it promotes the ability of TRAF2 activate signaling over a sustained period of time. Removal of the RING finger of TRAF2 prevents its translocation into detergent-insoluble complexes and renders it dominant negative for signaling. TRAF1(-/-) dendritic cells show attenuated responses to secondary stimulation by TRAF2-dependent factors and increased stimulus-dependent TRAF2 degradation. Replacement of the RING finger of TRAF2 with a raft-targeting signal restores JNK activation and association with the cyto-skeletal protein Filamin, but not NF-kappaB activation. These findings offer insights into the mechanism of TRAF2 signaling and identify a physiological role for TRAF1 as a regulator of the subcellular localization of TRAF2.

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.

BACKGROUND Inflammation and oxidative stress play important roles in the pathogenesis of acute kidney injury (AKI). TRAF6 functions as a signal transducer in the Toll-like receptor 4 signaling pathway. Several reports have previously implicated TRAF6 signaling in kidney pathology. Here, we investigated whether TRAF6 blockade can mitigate inflammatory responses and oxidative stress in AKI. MATERIAL AND METHODS C57BL/6 mice were injected with lipopolysaccharide (LPS, 15 mg/kg) to induce AKI. Double immunofluorescence staining of kidney tissues showed that TRAF6 was localized to renal tubular epithelial cells, and then a tubular epithelial cell line (NRK-52E) was used for in vitro analysis. TRAF6 was blocked in vitro using siRNA and in vivo using AAV2/2 shRNA. RESULTS The knockdown of TRAF6 in mice by AAV2-shTRAF6 significantly reduced renal inflammation, oxidative stress, apoptosis and kidney dysfunction in AKI. In vitro, silencing the expression of TRAF6 attenuated LPS(0.5 μg/mL)-induced inflammatory responses and oxidative stress and upregulated proapoptotic factors. Furthermore, the beneficial actions of TRAF6 blockade were closely associated with its ability to increase IkappaB-alpha and Nrf2. CONCLUSIONS Our findings provide direct evidence that TRAF6 mediates LPS-induced inflammation and oxidative stress, leading to renal dysfunction. We also show that TRAF6 inhibition is a potential therapeutic option to prevent AKI.