[Immune Regulation by TNF Receptor-associated Factor 5].

The tumor necrosis factor receptor (TNFR)-associated factor (TRAF) family of molecules are intracellular adaptors that regulate cellular signaling through members of the TNFR and Toll-like receptor superfamily. Mammals have seven TRAF molecules numbered sequentially from TRAF1 to TRAF7. Although TRAF5 was identified as a potential regulator of TNFR superfamily members, the in vivo function of TRAF5 has not yet been fully elucidated. We identified an unconventional role of TRAF5 in interleukin-6 (IL-6) receptor signaling involving CD4+ T cells. Moreover, TRAF5 binds to the signal-transducing glycoprotein 130 (gp130) receptor for IL-6 and inhibits the activity of the janus kinase (JAK)-signal transducer and activator of transcription (STAT) signaling pathway. In addition, Traf5-deficient CD4+ T cells exhibit significantly enhanced IL-6-driven differentiation of T helper 17 (Th17) cells, which exacerbates neuroinflammation in experimental autoimmune encephalomyelitis. Furthermore, TRAF5 demonstrates a similar activity to gp130 for IL-27, another cytokine of the IL-6 family. Additionally, Traf5-deficient CD4+ T cells display significantly increased IL-27-mediated differentiation of Th1 cells, which increases footpad swelling in delayed-type hypersensitivity response. Thus, TRAF5 functions as a negative regulator of gp130 in CD4+ T cells. This review aimed to explain how TRAF5 controls the differentiation of CD4+ T cells and discuss how the expression of TRAF5 in T cells and other cell types can influence the development and progression of autoimmune and inflammatory diseases.

METTL3-mediated m6 A methylation of TRAF5 inhibits lung adenocarcinoma cell metastasis via activation of the PI3K/AKT/NF-κB signaling pathway.

Tumor necrosis factor receptor-associated factor 5 (TRAF5) has been implicated in the pathogenesis of human malignancies. This work aimed to clarify the role of TRAF5 in lung adenocarcinoma (LUAD) progression. Herein, we uncovered that TRAF5 level was reduced in LUAD tissues. Low TRAF5 expression correlated with dismal prognosis in LUAD patients. Moreover, upregulated TRAF5 impeded cell viability, migration, and invasion, induced apoptosis in vitro, as well as impaired tumorigenicity in vivo. However, depletion of TRAF5 revealed opposing results. Moreover, TRAF5 was identified as the downstream target of methyltransferase-like 3 (METTL3)-elicited N6 -methyladenosine (m6 A) modification. METTL3 stabilized TRAF5 mRNA and positively modulated TRAF5 level. Further, TRAF5 depletion relieved the repressive phenotype caused by METTL3 addition. In addition, it was manifested that the METTL3/TRAF5 axis served as an inhibitor in LUAD through the PI3K/AKT/Nuclear Factor-Kappa B (NF-κB) signaling. Collectively, we propose that METTL3-mediated TRAF5 m6 A modification exerted as a vital tumor inhibitory function in LUAD development. The METTL3/TRAF5 axis may be a critical effector of LUAD progression.

TRAF5 regulates intestinal mucosal Th1/Th17 cell immune responses via Runx1 in colitis mice.

Inflammatory bowel disease (IBD) is a chronic gastrointestinal inflammatory disease associated with CD4+ Th1 and Th17 cell immune responses. Tumour necrosis factor-associated factor 5 (TRAF5) deficiency has been shown to aggravate DSS-induced colitis. However, the potential role of TRAF5 in regulating CD4+ T cell immune responses in the pathogenesis of IBD remains unclear. TRAF5-/- CD4+ CD45RBhigh T cells and WT CD4+ CD45RBhigh T cells were transferred to Rag2-/- mice via intravenous (i.v.) tail injection, respectively, to establish a chronic colitis model. Adeno-associated virus (AAV)-mediated gene knockout technique was used to knock out runt-associated transcription factor 1 (Runx1) expression in vivo. Specific cytokines of Th1 and Th17 cells were detected by quantitative RT-PCR, immunohistochemistry, ELISA, and flow cytometry. In T-cell transfer colitis mice, the Rag2-/- mice reconstituted with TRAF5-/- CD4+ CD45RBhigh T cells showed more severe intestinal inflammation than the WT control group, which was characterised by increased expression of INF-γ, TNF-α, IL-17a. Furthermore, we found that the INF-γ+ CD4+ , IL17a+ CD4+ , and INF-γ+ IL17a+ CD4+ T cells in the intestinal mucosa of Rag2-/- mice reconstituted with TRAF5-/- CD4+ CD45RBhigh T cells were significantly higher than those of the WT control group by flow cytometry. Mechanistically, knockout Runx1 inhibited the differentiation of TRAF5-/- CD4+ T cells into Th1 and Th17 cells in the intestinal mucosa of T-cell transfer colitis mice. TRAF5 regulates Th1 and Th17 cell differentiation and immune response through Runx1 to participate in the pathogenesis of colitis. Thus targeting TRAF5 in CD4+ T cells may be a novel treatment for IBD.

[Effect and mechanism of Bovis Calculus on ulcerative colitis by inhibiting IL-17/IL-17RA/Act1 signaling pathway].

This study aimed to elucidate the effect and underlying mechanism of Bovis Calculus in the treatment of ulcerative colitis(UC) through network pharmacological prediction and animal experimental verification. Databases such as BATMAN-TCM were used to mine the potential targets of Bovis Calculus against UC, and the pathway enrichment analysis was conducted. Seventy healthy C57BL/6J mice were randomly divided into a blank group, a model group, a solvent model(2% polysorbate 80) group, a salazosulfapyridine(SASP, 0.40 g·kg~(-1)) group, and high-, medium-, and low-dose Bovis Calculus Sativus(BCS, 0.20, 0.10, and 0.05 g·kg~(-1)) groups according to the body weight. The UC model was established in mice by drinking 3% dextran sulfate sodium(DSS) solution for 7 days. The mice in the groups with drug intervention received corresponding drugs for 3 days before modeling by gavage, and continued to take drugs for 7 days while modeling(continuous administration for 10 days). During the experiment, the body weight of mice was observed, and the disease activity index(DAI) score was recorded. After 7 days of modeling, the colon length was mea-sured, and the pathological changes in colon tissues were observed by hematoxylin-eosin(HE) staining. The levels of tumor necrosis factor-α(TNF-α), interleukin-1ß(IL-1ß), interleukin-6(IL-6), and interleukin-17(IL-17) in colon tissues of mice were detected by enzyme-linked immunosorbent assay(ELISA). The mRNA expression of IL-17, IL-17RA, Act1, TRAF2, TRAF5, TNF-α, IL-6, IL-1ß, CXCL1, CXCL2, and CXCL10 was evaluated by real-time polymerase chain reaction(RT-PCR). The protein expression of IL-17, IL-17RA, Act1, p-p38 MAPK, and p-ERK1/2 was investigated by Western blot. The results of network pharmacological prediction showed that Bovis Calculus might play a therapeutic role through the IL-17 signaling pathway and the TNF signaling pathway. As revealed by the results of animal experiments, on the 10th day of drug administration, compared with the solvent model group, all the BCS groups showed significantly increased body weight, decreased DAI score, increased colon length, improved pathological damage of colon mucosa, and significantly inhibited expression of TNF-α,IL-6,IL-1ß, and IL-17 in colon tissues. The high-dose BCS(0.20 g·kg~(-1)) could significantly reduce the mRNA expression levels of IL-17, Act1, TRAF2, TRAF5, TNF-α, IL-6, IL-1ß, CXCL1, and CXCL2 in colon tissues of UC model mice, tend to down-regulate mRNA expression levels of IL-17RA and CXCL10, significantly inhibit the protein expression of IL-17RA,Act1,and p-ERK1/2, and tend to decrease the protein expression of IL-17 and p-p38 MAPK. This study, for the first time from the whole-organ-tissue-molecular level, reveals that BCS may reduce the expression of pro-inflammatory cytokines and chemokines by inhibiting the IL-17/IL-17RA/Act1 signaling pathway, thereby improving the inflammatory injury of colon tissues in DSS-induced UC mice and exerting the effect of clearing heat and removing toxins.

Identification of necroptosis-related gene TRAF5 as potential target of diagnosing atherosclerosis and assessing its stability.

BACKGROUND: Atherosclerosis (AS) is a leading cause of morbidity and mortality in older patients and features progressive formation of plaques in vascular tissues. With the progression of atherosclerosis, plaque rupture may occur and cause stroke, myocardial infarction, etc. Different forms of cell death promote the formation of a necrotic core of the plaque, leading to rupture. Necroptosis is a type of programmed cell death that contributes to the development of cardiovascular disease. However, the role of necroptosis in AS has not yet been investigated. METHODS: The Gene Expression Omnibus (GEO) database was used to obtain gene expression profiles. Differentially expressed genes (DEGs) and necroptosis gene sets were used to identify necroptosis-related differentially expressed genes (NRDEGs). The NRDEGs were used to construct a diagnostic model and were further screened using least absolute shrinkage selection operator (LASSO) regression and random forest (RF) analysis. The discriminatory capacity of the NRDEGs was evaluated using receiver operating characteristic (ROC) curves. Immune infiltration levels were estimated based on CIBERSORTx analysis. The GSE21545 dataset, containing survival information, was used to determine prognosis-associated genes. Univariate and multivariate Cox regression analyses combined with survival analysis determined gene prognostic values. RNA and protein levels were detected by RT-qPCR and western blotting in arteriosclerosis obliterans(ASO) and normal vascular tissues. Vascular smooth muscle cells (VSMCs) were treated with oxidized low-density lipoprotein (ox-LDL) to develop cell models of advanced AS. The effects of protein knockdown on necroptosis were assessed by western blotting and flow cytometry. EdU and Cell Counting Kit-8 assays were used to examine cell proliferation. RESULTS: TNF Receptor Associated Factor 5 (TRAF5) was identified as a diagnostic marker for AS based on the AUC value in both the GSE20129 and GSE43292 datasets. According to differential expression analysis, LASSO regression analysis, RF analysis, univariate analysis, multivariate analysis, and gene-level survival analysis, TRAF5 was markedly associated with necroptosis in AS. Silencing TRAF5 promotes necroptosis and attenuates the proliferation of ox-LDL-induced cell models of advanced AS. CONCLUSIONS: This study identified a diagnostic marker of necroptosis-related atherosclerosis, TRAF5, which can also be used to diagnose and assess atherosclerotic plaque stability. This novel finding has important implications in the diagnosis and assessment of plaque stability in atherosclerosis.

Silencing of TRAF5 enhances necroptosis in hepatocellular carcinoma by inhibiting LTBR-mediated NF-κB signaling.

Background: Hepatocellular carcinoma (HCC) is a common malignancy with poor prognosis and high mortality. This study aimed to explore the oncogenic mechanisms of TRAF5 in HCC and provide a novel therapeutic strategy for HCC. Methods: Human HCC cell lines (HepG2, HuH7, SMMC-LM3, and Hep3B), normal adult liver epithelial cells (THLE-2), and human embryonic kidney cells (HEK293T) were utilized. Cell transfection was performed for functional investigation. qRT-PCR and western blotting were used to detect mRNA expression of TRAF5, LTBR, and NF-κB and protein expression of TRAF5, p-RIP1(S166)/RIP1, p-MLKL(S345)/MLKL, LTBR, and p-NF-κB/NF-κB. Cell viability, proliferation, migration, and invasion were evaluated using CCK-8, colony formation, wound healing, and Transwell assays. Cell survival, necrosis, and apoptosis were assessed using flow cytometry and Hoechst 33342/PI double staining. Co-immunoprecipitation and immunofluorescence were performed to determine the interaction between TRAF5 and LTBR. A xenograft model was established to validate the role of TRAF5 in HCC. Results: TRAF5 knockdown inhibited HCC cell viability, colony formation, migration, invasion, and survival but enhanced necroptosis. Additionally, TRAF5 is correlated with LTBR and TRAF5 silencing down-regulated LTBR in HCC cells. LTBR knockdown inhibited HCC cell viability, while LTBR overexpression eliminated the effects of TRAF5 deficiency on inhibiting HCC cell proliferation, migration, invasion, and survival. LTBR overexpression abolished the promotive function of TRAF5 knockdown on cell necroptosis. LTBR overexpression undid the suppressive effect of TRAF5 knockdown on NF-κB signaling in HCC cells. Moreover, TRAF5 knockdown suppressed xenograft tumor growth, inhibited cell proliferation, and promoted tumor cell apoptosis. Conclusions: TRAF5 deficiency facilitates necroptosis in HCC by suppressing LTBR-mediated NF-κB signaling.

PCSK9 inhibitor attenuates atherosclerosis by regulating SNHG16/EZH2/TRAF5-mediated VSMC proliferation, migration, and foam cell formation.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibitor has been demonstrated to exert a great cardioprotection in cardiometabolic impairments, including atherosclerosis. However, its underlying mechanism remains not fully understood. This study focuses on uncovering the actions of PCSK9 inhibitor on the connection between atherosclerosis and vascular smooth muscle cell (VSMC) behaviors. qRT-PCR was utilized to detect the expression of SNHG16. Proliferation and migration of VSMC were characterized by Cell Counting Kit-8 and wound healing assays. The intracellular lipids and foam cell formation were assessed by Oil Red O staining, fluorescence image, and cholesterol quantification kit. Atherosclerosis in vivo was evaluated by imaging the atherosclerotic lesions, hematoxylin-eosin staining, Oil Red O staining and Masson staining. The interaction between SNHG16 with EZH2 and histone H3 lysine 27 trimethylation (H3K27me3) were investigated by fluorescence in situ hybridization, RNA immunoprecipitation, and chromatin immunoprecipitation assays. A ApoE-/- mice model was used to validate the role of PCSK9 inhibitor and SNHG16 for atherosclerosis. The protective regulation of PCSK9 inhibitor was observed both in high-fat diet (HFD)-fed mice and oxidized low-density lipoprotein (ox-LDL)-treated VSMC, as manifested in the decreased the atherosclerotic lesions in vivo, as well as the weakened cell proliferation, migration, and formation of foam cells in vitro. SNHG16 was identified to be a downstream effector of PCSK9 inhibitor-mediated biological functions, of which knockdown also significantly ox-LDL-treated VSMC proliferation, migration, and foam cell formation abilities. SNHG16 epigenetically suppressed TRAF5 via recruiting EZH2. Silencing of TRAF5 abolished the protective effects of SNHG16 knockdown on the pathogenesis of atherosclerosis. Collectively, PCSK9 inhibitor attenuated atherosclerosis by regulating SNHG16/EZH2/TRAF5 axis to impair the proliferation, migration, and foam cell formation of VSMC.

Effect and mechanism of Bovis Calculus on ulcerative colitis by inhibiting IL-17/IL-17RA/Act1 signaling pathway / 中国中药杂志

This study aimed to elucidate the effect and underlying mechanism of Bovis Calculus in the treatment of ulcerative colitis(UC) through network pharmacological prediction and animal experimental verification. Databases such as BATMAN-TCM were used to mine the potential targets of Bovis Calculus against UC, and the pathway enrichment analysis was conducted. Seventy healthy C57BL/6J mice were randomly divided into a blank group, a model group, a solvent model(2% polysorbate 80) group, a salazosulfapyridine(SASP, 0.40 g·kg~(-1)) group, and high-, medium-, and low-dose Bovis Calculus Sativus(BCS, 0.20, 0.10, and 0.05 g·kg~(-1)) groups according to the body weight. The UC model was established in mice by drinking 3% dextran sulfate sodium(DSS) solution for 7 days. The mice in the groups with drug intervention received corresponding drugs for 3 days before modeling by gavage, and continued to take drugs for 7 days while modeling(continuous administration for 10 days). During the experiment, the body weight of mice was observed, and the disease activity index(DAI) score was recorded. After 7 days of modeling, the colon length was mea-sured, and the pathological changes in colon tissues were observed by hematoxylin-eosin(HE) staining. The levels of tumor necrosis factor-α(TNF-α), interleukin-1β(IL-1β), interleukin-6(IL-6), and interleukin-17(IL-17) in colon tissues of mice were detected by enzyme-linked immunosorbent assay(ELISA). The mRNA expression of IL-17, IL-17RA, Act1, TRAF2, TRAF5, TNF-α, IL-6, IL-1β, CXCL1, CXCL2, and CXCL10 was evaluated by real-time polymerase chain reaction(RT-PCR). The protein expression of IL-17, IL-17RA, Act1, p-p38 MAPK, and p-ERK1/2 was investigated by Western blot. The results of network pharmacological prediction showed that Bovis Calculus might play a therapeutic role through the IL-17 signaling pathway and the TNF signaling pathway. As revealed by the results of animal experiments, on the 10th day of drug administration, compared with the solvent model group, all the BCS groups showed significantly increased body weight, decreased DAI score, increased colon length, improved pathological damage of colon mucosa, and significantly inhibited expression of TNF-α,IL-6,IL-1β, and IL-17 in colon tissues. The high-dose BCS(0.20 g·kg~(-1)) could significantly reduce the mRNA expression levels of IL-17, Act1, TRAF2, TRAF5, TNF-α, IL-6, IL-1β, CXCL1, and CXCL2 in colon tissues of UC model mice, tend to down-regulate mRNA expression levels of IL-17RA and CXCL10, significantly inhibit the protein expression of IL-17RA,Act1,and p-ERK1/2, and tend to decrease the protein expression of IL-17 and p-p38 MAPK. This study, for the first time from the whole-organ-tissue-molecular level, reveals that BCS may reduce the expression of pro-inflammatory cytokines and chemokines by inhibiting the IL-17/IL-17RA/Act1 signaling pathway, thereby improving the inflammatory injury of colon tissues in DSS-induced UC mice and exerting the effect of clearing heat and removing toxins.

Grouper TRIM23 exerts antiviral activity against iridovirus and nodavirus.

TRIM (tripartite motif) proteins have been demonstrated to exert critical roles in host defense against different microbial pathogens. Among them, TRIM23 acts as an important regulatory factor in antiviral immune and inflammatory responses, but the roles of fish TRIM23 against virus infection still remain largely unknown. Here, we investigated the characteristics of TRIM23 homolog from orange spotted grouper (Epinephelus coioides) (EcTRIM23). EcTRIM23 encoded a 580 amino acid peptide, which shared 93.1%, 89.73% and 86.36% identity with golden perch (Perca flavescens), zebrafish (Danio rerio) and human (Homo sapiens), respectively. The transcription levels of EcTRIM23 were significantly up-regulated in response to Singapore grouper iridovirus (SGIV) and red-spotted grouper nervous necrosis virus (RGNNV) infection. EcTRIM23 overexpression in vitro significantly inhibited RGNNV and SGIV replication, evidenced by the delayed cytopathic effect (CPE) progression and the decreased expression of viral core genes. EcTRIM23 significantly increased the expression levels of interferon (IFN) related signaling molecules and pro-inflammatory cytokines, as well as the promoter activities of IFN and NF-κB, suggesting that EcTRIM23 exerted antiviral function by positively regulating host IFN response. Exogenous EcTRIM23 exhibited either diffuse or aggregated localization in grouper cells. After co-transfection, TANK binding kinase 1 (TBK1), TNF receptor associated factor (TRAF) 3 and TRAF4, TRAF5 and TRAF6 were found to interact with EcTRIM23 in grouper cells. Moreover, these proteins could be recruited and co-localized with EcTRIM23 in vitro. Together, our results demonstrated that fish TRIM23 exerted antiviral activity against fish viruses by interacting with multiple host proteins to regulate immune responses.

TRAF5 splicing variants associate with TRAF3 and RIP1 in NF-κB and type I IFN signaling in large yellow croaker Larimichthys crocea.

As a member of the tumor necrosis factor receptor-associated factor (TRAF) family, TRAF5 acts as a crucial adaptor molecule and plays important roles in the host innate immune responses. In the present study, the typical form and a splicing variant of TRAF5, termed Lc-TRAF5_tv1 and Lc-TRAF5_tv2 were characterized in large yellow croaker (Larimichthys crocea). The putative Lc-TRAF5_tv1 protein is constituted of 577 aa, contains a RING finger domain, two zinc finger domains, a coiled-coil domain, and a MATH domain, whereas Lc-TRAF5_tv2 protein is constituted of 236 aa and only contains a RING finger domain due to a premature stop resulted from the intron retention. Subcellular localization analysis revealed that both of Lc-TRAF5_tv1 and Lc-TRAF5_tv2 were localized in the cytoplasm, with Lc-TRAF5_tv2 found to aggregate around the nucleus. It was revealed that Lc-TRAF5_tv1 mRNA was broadly expressed in examined organs/tissues and showed extremely higher level than that of Lc-TRAF5_tv2, and both of them could be up-regulated under poly I:C, LPS, PGN, and Pseudomonas plecoglossicida stimulations in vivo. Interestingly, overexpression of Lc-TRAF5_tv1 and Lc-TRAF5_tv2 could significantly induce NF-κB but not IFN1 activation, whereas co-expression of them remarkably induced IFN1 activation but impaired NF-κB activation. In addition, both Lc-TRAF5_tv1 and Lc-TRAF5_tv2 were associated with TRAF3 and RIP1 in IFN1 activation, whereas only Lc-TRAF5_tv1 cooperated with TRAF3 and RIP1 in NF-κB activation. These results collectively indicated that the splicing variant together with the typical form of TRAF5 function importantly in the regulation of host immune signaling in teleosts.

LncRNA HCG18 upregulates TRAF4/TRAF5 to facilitate proliferation, migration and EMT of epithelial ovarian cancer by targeting miR-29a/b.

BACKGROUND: Although long noncoding RNA HLA complex group 18 (lncRNA HCG18) has been suggested to regulate cell growth in several tumours, the function of HCG18 in epithelial ovarian cancer (EOC) and its mechanism are still unclear. METHODS: shRNAs were applied to reduce HCG18 and related genes. For overexpression of miRNA, a miRNA mimic was transfected into cells. Quantitative real-time PCR (qRT-PCR) was used to detect levels of HCG18, miR-29a/b, and mRNAs. MTT, colony formation, wound healing and Transwell assays were used to evaluate cell proliferation, migration and invasion, respectively. A luciferase reporter assay was utilized to evaluate NF-κB activity and the binding of miRNAs with HCG18 or TRAF4/5. BALB nude mice injected with cells stably expressing shHCG18 or shNC were used for in vivo modelling. Subcutaneous tumour growth was monitored in nude mice, and immunohistochemistry (IHC) was used to determine expression of the proliferation marker Ki67. RESULTS: Abnormal expression of HCG18 and miR-29a/b was observed in EOC tissues. Knockdown of HCG18 using shRNA inhibited proliferation, migration, EMT and the proinflammatory pathway in EOC cells. miR-29a/b mimics and TRAF4/5 knockdown exhibited effects similar to HCG18 knockdown. Further experiments suggested that HCG18 directly targets miR-29a/b and upregulates TRAF4/5 expression, which are inhibited by targeting miR-29a/b. Moreover, overexpression of TRAF4/5 antagonized the inhibitory effect of HCG18 knockdown, suggesting that they are involved in HCG18-mediated oncogenic effects. Silencing HCG18 reduced tumour size and levels of Ki67 and TRAF4/5 while increasing miR-29a/b levels in vivo. CONCLUSIONS: Taken together, our data revealed an oncogenic signalling pathway mediated by HCG18 in ovarian cell lines, which functions as a ceRNA of miR-29a/b and thus derepresses expression levels of TRAF4/5, facilitating NF-κB pathway-mediated promotion of EOC cell proliferation and migration.

TNF Receptor-Associated Factor 5 Limits IL-27 Receptor Signaling in CD4+ T Lymphocytes.

TNF receptor-associated factor 5 (TRAF5) restrains early signaling activity of the IL-6 receptor in naive CD4+ T cells by interacting with the shared gp130 chain, although TRAF5 was initially discovered as a cytoplasmic adaptor protein to activate signaling mediated by TNF receptor family molecules. This leads to the question of whether TRAF5 limits signaling via the receptor for IL-27, which is composed of gp130 and WSX-1. The aim of this study is to clarify the role of TRAF5 in IL-27 receptor signaling and to understand the differential role of TRAF5 on cytokine receptor signaling. We found that Traf5 -/- CD4+ T cells displayed significantly higher levels of phosphorylated STAT1 and STAT-regulated genes Socs3 and Tbx21, as early as 1 h after IL-27 exposure when compared with Traf5 +/+ CD4+ T cells. Upon IL-27 and TCR signals, the Traf5 deficiency significantly increased the induction of IL-10 and promoted the proliferation of CD4+ T cells. Traf5 -/- mice injected with IL-27 displayed significantly enhanced delayed-type hypersensitivity responses, demonstrating that TRAF5 works as a negative regulator for IL-27 receptor signaling. In contrast, IL-2 and proliferation mediated by glucocorticoid-induced TNF receptor-related protein (GITR) and TCR signals were significantly decreased in Traf5 -/- CD4+ T cells, confirming that TRAF5 works as a positive regulator for cosignaling via GITR. Collectively, our results demonstrate that TRAF5 reciprocally controls signals mediated by the IL-27 receptor and GITR in CD4+ T cells and suggest that the regulatory activity of TRAF5 in gp130 is distinct from that in TNF receptor family molecules in a T cell.

Bioinformatics Analysis of miRNAs Targeting TRAF5 in DLBCL Involving in NF-κB Signaling Pathway and Affecting the Apoptosis and Signal Transduction.

Background: Diffuse large B-cell lymphoma (DLBCL) is an aggressive B-cell lymphoma with high heterogeneity. There is an unmet need to investigate valid indicators for the diagnosis and therapy of DLBCL. Methods: GEO database was utilized to screen for differentially expressed genes (DEGs) and differential miRNAs in DLBCL tissues. The Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) were applied to analyse DEGs. Then multiple databases were searched for related miRNAs within DLBCL, TNF receptor-associated factor 5 (TRAF5) and NF-kappa B (NF-κB) signaling pathways. The KOBAS database was used to assist in the screening of miRNAs of interest and construct the regulatory network of miRNA-mRNA. Finally, the expression level and diagnostic performance of miRNAs were analyzed with GEO datasets, and DEGs were identified from the GEPIA database. Results: DEGs were significantly concentrated in the NF-κB signaling pathway and cytokine-cytokine receptor interaction, and involved in the process of immune response and protein binding. MiR-15a-5p, miR-147a, miR-192-5p, miR-197-3p, miR-532-5p, and miR-650 were revealed to be targeting TRAF5 and participating in NF-κB signaling pathway and might impact the apoptosis and signal transduction of DLBCL. In the GEPIA database, TRAF5 was significantly overexpressed in DLBCL. The expression of miR-197-3p was upregulated within GEO datasets, while the rest of the miRNAs were downregulated in DLBCL. Conclusions: Subsets of miRNAs may participate in the NF-κB signaling pathway by co-targeting TRAF5 and could be prospective biomarkers exploring the pathogenesis of DLBCL.

Downregulation of long noncoding RNA SNHG7 protects against inflammation and apoptosis in Parkinson's disease model by targeting the miR-425-5p/TRAF5/NF-κB axis.

Accumulated evidence has manifested that long noncoding RNA (lncRNA) is involved in the progress of Parkinson's disease (PD). SNHG7, a novel lncRNA, has been found to be involved in tumorigenesis. However, SNHG7 expression and its functional effects on PD remain uncharted. Rotenone (Rot) was adopted to construct PD models in Sprague-Dawley (SD) rats and SH-SY5Y cells, respectively. The expression levels of caspase 3, tyrosine hydroxylase (TH), ionized calcium-binding adapter molecule 1 (Iba1) in SD rat striatum were measured via immunohistochemistry and western blot. Additionally, the expressions of inflammatory cytokines (interleukin 1ß [IL-1ß], IL-6, tumor necrosis factor α) and oxidative stress factors (malondialdehyde, superoxide dismutase, and glutathione peroxidase) in the brain tissues were examined using real-time polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. Moreover, the protein levels of tumor necrosis factor receptor-associated factor (TRAF5), I-κB, nuclear factor-κB (NF-κB), HO-1, Nrf2 were detected via western blot. Bioinformatics was applied to predict the targeting relationship between SNHG7, miR-425-5p, and TRAF5. Dual-luciferase activity assay and RNA immunoprecipitation assays were conducted to verify their interactions. In comparison to healthy donors, SNHG7 was found upregulated while miR-425-5p expression was downregulated in PD patients. Functional experiments confirmed that SNHG7 downregulation or miR-425-5p overexpression attenuated neuronal apoptosis in the Rot-mediated PD model, TH-positive cell loss, and microglial activation by mitigating inflammation and oxidative stress. Mechanistically, SNHG7 served as a competitive endogenous RNA by sponging miR-425-5p and promoted TRAF5 mediated inflammation and oxidative stress. Inhibition of SNHG7 ameliorated neuronal apoptosis in PD through relieving miR-425-5p/TRAF5/NF-κB signaling pathway modulated inflammation and oxidative stress, and similar results were observed in the Rot-mediated rat model of PD.

Genetic Deficiency of TRAF5 Promotes Adipose Tissue Inflammation and Aggravates Diet-Induced Obesity in Mice.

Objective: The accumulation of inflammatory leukocytes is a prerequisite of adipose tissue inflammation during cardiometabolic disease. We previously reported that a genetic deficiency of the intracellular signaling adaptor TRAF5 (TNF [tumor necrosis factor] receptor-associated factor 5) accelerates atherosclerosis in mice by increasing inflammatory cell recruitment. Here, we tested the hypothesis that an impairment of TRAF5 signaling modulates adipose tissue inflammation and its metabolic complications in a model of diet-induced obesity in mice. Approach and Results: To induce diet-induced obesity and adipose tissue inflammation, wild-type or Traf5-/- mice consumed a high-fat diet for 18 weeks. Traf5-/- mice showed an increased weight gain, impaired insulin tolerance, and increased fasting blood glucose. Weight of livers and peripheral fat pads was increased in Traf5-/- mice, whereas lean tissue weight and growth were not affected. Flow cytometry of the stromal vascular fraction of visceral adipose tissue from Traf5-/- mice revealed an increase in cytotoxic T cells, CD11c+ macrophages, and increased gene expression of proinflammatory cytokines and chemokines. At the level of cell types, expression of TNF[alpha], MIP (macrophage inflammatory protein)-1[alpha], MCP (monocyte chemoattractant protein)-1, and RANTES (regulated on activation, normal T-cell expressed and secreted) was significantly upregulated in Traf5-deficient adipocytes but not in Traf5-deficient leukocytes from visceral adipose tissue. Finally, Traf5 expression was lower in adipocytes from obese patients and mice and recovered in adipose tissue of obese patients one year after bariatric surgery. Conclusions: We show that a genetic deficiency of TRAF5 in mice aggravates diet-induced obesity and its metabolic derangements by a proinflammatory response in adipocytes. Our data indicate that TRAF5 may promote anti-inflammatory and obesity-preventing signaling events in adipose tissue.

Grouper TRAF5 exerts negative regulation on antiviral immune response against iridovirus.

Tumor necrosis factor receptor-associated factor 5 (TRAF5) is an intracellular protein that binds to the cytoplasmic portion of tumor necrosis factor receptors and mediates the activation of downstream nuclear factor-kappa B (NF-κB), interferon regulatory factor 3, and mitogen activated protein kinase signaling pathways. Compared with other TRAF proteins, TRAF5 is largely unknown in teleosts. In the present study, a TRAF5 homologue (HgTRAF5) from the hybrid grouper (Epinephelus fuscoguttatus♂ × Epinephelus lanceolatus♀) was cloned and characterized. The open reading frame of HgTRAF5 consists of 1743 nucleotides encoding a 581 amino acid protein with a predicted molecular mass of 64.90 kDa. Similar to its mammalian counterpart, HgTRAF5 contains an N-terminal RING finger domain, a zinc finger domain, and a C-terminal TRAF domain, including a coiled-coil domain and a MATH domain. HgTRAF5 shares 99.83% identity with giant grouper (Epinephelus lanceolatus) TRAF5. Quantitative real-time PCR analysis indicated that HgTRAF5 mRNA was broadly expressed in all examined tissues. The expression of HgTRAF5 increased after Singapore grouper iridovirus (SGIV) infection in grouper spleen (GS) cells. Intracellular localization analysis demonstrated that the full-length HgTRAF5 protein mainly distributed in the cytoplasm. HgTRAF5 overexpression also promoted SGIV replication during viral infection in vitro. HgTRAF5 significantly promoted the activities of interferon-ß, interferon-sensitive response element, and NF-κB. Taken together, these results are important for a better understanding of the function of TRAF5 in fish and reveal its involvement in the host response to immune challenge by SGIV.

MicroRNA-29b-3p promotes 5-fluorouracil resistance via suppressing TRAF5-mediated necroptosis in human colorectal cancer.

Drug resistance in colorectal cancer is a great challenge in clinic. Elucidating the deep mechanism underlying drug resistance will bring much benefit to diagnosis, therapy and prognosis in patients with colorectal cancer. In this study, miR-29b-3p was shown to be involved in resistance to 5-fluorouracil (5-FU)-induced necroptosis of colorectal cancer. Further, miR-29b-3p was shown to target a regulatory subunit of necroptosis TRAF5. Rescue of TRAF5 could reverse the effect of miR-29b-3p on 5-FU-induced necroptosis, which was consistent with the role ofnecrostatin-1 (a specific necroptosis inhibitor). Then it was demonstrated that miR-29b-3p was positively correlated with chemo-resistance in colorectal cancer while TRAF5 negatively. In conclusion, it is deduced that miR-29b-3p/TRAF5 signaling axis plays critical role in drug resistance in chemotherapy for colorectal cancer patients by regulating necroptosis. The findings in this study provide us a new target for interfere therapy in colorectal cancer.

Single nucleotide polymorphisms of TRAF2 and TRAF5 gene in ankylosing spondylitis: a case-control study.

Objective To investigate the role of eight locus polymorphisms of tumor necrosis factor receptor-associated factor 2 (TRAF2) and TRAF5 gene and their interaction in the susceptibility to ankylosing spondylitis (AS) in Chinese Han population. Methods Eight single nucleotide polymorphisms (SNPs) of TRAF2 (rs3750511, rs10781522, rs17250673, rs59471504) and TRAF5 (rs6540679, rs12569232, rs4951523, rs7514863) gene were genotyped in 673 AS patients and 687 controls. Results The SNPs of TRAF2 and TRAF5 do not indicate a correlation with the susceptibility of AS in Chinese Han population. Genotype frequencies of rs3750511 were statistically significant in females between patients and controls. The allele frequencies of rs10781522 and genotype frequencies of rs3750511 were statistically significant between groups of different diseases activity. One three-locus model, TRAF2 (rs10781522, rs17250673) and TRAF5 (rs12569232), had a maximum testing accuracy of 52.67% and a maximum cross-validation consistency (10/10) that was significant at the level of P = 0.0001, after determined empirically by permutation testing. As to environmental variables, only marginal association was found between sleep quality and AS susceptibility. Conclusion TRAF2 rs3750511 polymorphism may be associated with the susceptibility and severity of AS. Besides, the interaction of TRAF2 and TRAF5 genes may be associated with AS susceptibility, but many open questions remain.

Association of methylation level and transcript level in TRAF5 gene with ankylosing spondylitis: a case-control study.

To explore the association between methylation level and transcript level of TNF receptor-associated factor 5 (TRAF5) gene with ankylosing spondylitis (AS) in Chinese Han population. Methylation and mRNA expression level of the TRAF5 gene were tested in 98 patients and 98 healthy controls. Among the 21 CpG sites, methylation levels at eight sites were significantly different between AS patients and healthy controls. However, only three sites remained significantly different after the correction by the Benjamini-Hochberg method. Compared with controls, the CpG island of TRAF5 gene promoter was highly methylated in AS patients, and the relative mRNA expression level of TRAF5 was significantly reduced in AS patients. And the mRNA level was negatively correlated with the methylation level of TRAF5 gene in AS patients (rs = -0.453, P < 0.001). Subgroup analyses indicated that there was no significant difference in the level of methylation between groups of different status of HLA-B27 and medications in AS patients. Multiple linear regression showed that disease-modifying antirheumatic drugs could reduce methylation levels of AS patients after adjusting for the effects of other drugs. In conclusion, the hypermethylation of the TRAF5 might contribute to the pathogenesis of AS, but many open questions remain.

Tumor-Associated Macrophages Promote Oxaliplatin Resistance via METTL3-Mediated m6A of TRAF5 and Necroptosis in Colorectal Cancer.

As a third-generation platinum drug, oxaliplatin (OX) is widely used as the first-line chemotherapeutic agent in the treatment of colorectal cancer (CRC). CRC cells acquire resistance to chemotherapy and develop resistance, which is a major challenge for the treatment of advanced CRC. Recent studies have suggested that the therapeutic resistance of tumors is affected by the tumor microenvironment (TME). As a critical role among TME, tumor-associated macrophages (TAMs) play an important role. However, their regulatory mechanism underlying the drug resistance in CRC remains largely unknown. In the present study, we found that the density of macrophages infiltrated into the CRC tissues from OX-resistant patients was significantly higher compared with the OX-sensitive patients. Interestingly, both the total N6-methyladenosine (m6A) RNA content and the expression of its critical methyltransferase METTL3 were increased in the CRC tissues from OX-resistant patients compared with the OX-sensitive patients. Furthermore, we demonstrated that the M2-polarized TAMs enabled the OX resistance via the elevation of METTL3-mediated m6A modification in cells. Through whole-genome CRISPR screening and further validation, we found that TRAF5 contributes to the METTL3-triggered OX resistance in CRC cells. This study unveiled that M2-TAMs were important mediators for the acquisition of OX resistance. Furthermore, we provided evidence that targeting of M2-TAMs and METTL3-mediated m6A modification might be a promising adjuvant therapeutic strategy for CRC patients, especially for OX-resistant CRC patients.