Expression Of Tumor Necrosis Factor Receptor-associated Factor 2 Research Articles

TRAF2 decrease promotes the TGF-β-mTORC1 signal in MAFLD-HCC through enhancing AXIN1-mediated Smad7 degradation.

According to recent research, metabolic-associated fatty liver disease (MAFLD) has emerged as an important underlying etiology of hepatocellular carcinoma (HCC). However, the molecular mechanism of MAFLD-HCC is still unclear. Tumor necrosis factor receptor-associated factor 2 (TRAF2) is the key molecule to mediate the signal of inflammatory NF-κB pathway. This study aims to investigate the potential dysregulation of TRAF2 and its biological function in MAFLD-HCC. Huh7 TRAF2-/- demonstrated increased tumor formation ability compared to huh7 TRAF2+/+ when stimulated with transforming growth factor-β (TGF-β). The decisive role of TGF-β in the development of MAFLD-HCC was confirmed through the specific depletion of TGF-β receptor II gene in the hepatocytes (Tgfbr2ΔHep) of mice. In TRAF2-/- cells treated with TGF-β, both the glycolysis rate and lipid synthesis were enhanced. We proved the signal of the mechanistic target of rapamycin complex 1 (mTORC1) could be activated in the presence of TGF-β, and was enhanced in TRAF2-/- cells. The coimmunoprecipitation (co-IP) experiments revealed that TRAF2 fortified the Smurf2-mediated ubiquitination degradation of AXIN1. Hence, TRAF2 depletion resulted in increased Smad7 degradation induced by AXIN1, thus promoting the TGF-β signal. We also discovered that PLX-4720 could bind with AXIN1 and restrained the tumor proliferation of TRAF2-/- in mice fed with high-fat diet (HFD). Our findings indicate that TRAF2 plays a significant role in the pathogenesis of MAFLD-HCC. The reduction of TRAF2 expression leads to the enhancement of the TGF-β-mTORC1 pathway by facilitating AXIN1-mediated Smad7 degradation.

Xinfeng capsule inhibits lncRNA NONHSAT227927.1/TRAF2 to alleviate NF-κB-p65-induced immuno-inflammation in ankylosing spondylitis

Ethnopharmacological relevanceAnkylosing spondylitis (AS) is a chronic rheumatic disease known for its insidious and refractory symptoms, primarily associated with immuno-inflammation in its early stages, that affects the self-perception of patients (SPP). The exploration of long noncoding RNA (lncRNA) in immuno-inflammation of AS has garnered considerable interest. Additionally, the effectiveness of traditional Chinese medicine Xinfeng Capsule (XFC) in mitigating immuno-inflammation in AS has also been observed. However, the specific mechanisms still need to be characterized. Aim of the studyThis study elucidated the mechanism of the lncRNA NONHSAT227927.1/TRAF2/NF-κB axis in the immuno-inflammation of AS and XFC in AS treatment. MethodsLncRNA NONHSAT227927.1 and mRNA expression were assessed utilizing real-time fluorescence quantitative PCR. Protein level was determined using Western blot, and cytokine expression was measured using ELISA. Furthermore, mass spectrometry was used to analyze the binding proteins of lncRNA and rescue experiments were conducted to validate the findings. Inconsistencies in clinical baseline data were addressed using propensity score matching. The association between the XFC effect and indicator changes was evaluated using the Apriori algorithm. ResultsThe study revealed a substantial elevation in the expression of lncRNA NONHSAT227927.1 and tumor necrosis factor receptor-associated factor 2 (TRAF2) in AS-peripheral blood mononuclear cells. Its expression was also notably reduced after XFC treatment. In addition to this, there was a positive correlation between lncRNA NONHSAT227927.1 and TRAF2 with clinical immuno-inflammatory indicators. On the other hand, they showed a negative association with the SPP indicators. In vitro experiments have demonstrated that lncRNA NONHSAT227927.1 activated the nuclear factor (NF)-κB-p65 pathway by promoting TRAF2 expression. This activation resulted in enhanced IL-6 and TNF-α levels and reduced IL-10 and IL-4 levels. Conversely, XFC decreased the expression of lncRNA NONHSAT227927.1 and TRAF2, inhibiting the stimulation of the NF-κB-p65 cascade and restoring balance to the cytokines. The association rule analysis results indicated a strong association between XFC and decreased levels of C-reactive protein, erythrocyte sedimentation rate, and immunoglobulin A. Furthermore, XFC was strongly associated with improved SPP indicators, including general health, vitality, mental health, and role-emotional. ConclusionsLncRNA NONHSAT227927.1 plays a pro-inflammatory role in AS. XFC treatment may reverse lncRNA NONHSAT227927.1 to suppress TRAF2-mediated NF-κB-p65 activation, which in turn suppresses immuno-inflammation and improves SPP, thereby making XFC a promising candidate for therapeutic applications in AS management.

TRAF2 inhibits senescence in hepatocellular carcinoma cells via regulating the ROMO1/ NAD+/SIRT3/SOD2 axis

The suppression of tumor proliferation via cellular senescence has emerged as a promising approach for anti-tumor therapy. Tumor necrosis factor receptor-associated factor 2 (TRAF2), an adaptor protein involved in the NF-κB signaling pathway and reactive oxygen species (ROS) production, has been implicated in hepatocellular carcinoma (HCC) proliferation. However, little is currently known about whether TRAF2 promotes HCC development by inhibiting cellular senescence. Replicative senescence model and IR-induced mouse model demonstrated that TRAF2 expression was decrease in senescence cells or liver tissues. Depletion of TRAF2 could inhibit proliferation and arrest the cell cycle via activating p53/p21WAF1 and p16INK4a/pRb signaling pathways in HCC cells and eventually lead to cellular senescence. Mechanistically, TRAF2 deficiency increased the expression of mitochondrial protein reactive oxygen species modulator 1 (ROMO1) and subsequently activated the NAD+/SIRT3/SOD2 pathway to promote the production of ROS and cause mitochondrial dysfunction, which eventually contributed to DNA damage response (DDR). Our findings demonstrate that TRAF2 deficiency inhibits the proliferation of HCC by promoting senescence. Therefore, targeting TRAF2 through various approaches holds therapeutic potential for treating HCC.

The TRAF2-p62 axis promotes proliferation and survival of liver cancer by activating mTORC1 pathway

TRAF2 (Tumor necrosis factor receptor-associated factor 2) is a dual function protein, acting as an adaptor protein and a ubiquitin E3 ligase, which plays an essential role in mediating the TNFα-NFκB signal pathway. Dysregulated expression of TRAF2 has been reported in a variety of human cancers. Whether and how TRAF2 regulates the growth of liver cancer cells remains elusive. The goal of this study is to investigate potential dysregulation of TRAF2 and its biological function in liver cancer, and to elucidate the underlying mechanism, leading to validation of TRAF2 as an attractive liver cancer target. Here, we reported TRAF2 is up-regulated in human liver cancer cell lines and tissues, and high TRAF2 expression is associated with a poor prognosis of HCC patients. Proteomics profiling along with Co-immunoprecipitation analysis revealed that p62 is a new substrate of TRAF2, which is subjected to TRAF2-induced polyubiquitination via the K63 linkage at the K420 residue. A strong negative correlation was found between the protein levels of p62 and TRAF2 in human HCC samples. TRAF2 depletion inhibited growth and survival of liver cancer cells both in vitro and in vivo by causing p62 accumulation, which is partially rescued by simultaneous p62 knockdown. Mechanistically, TRAF2-mediated p62 polyubiquitylation activates the mTORC1 by forming the p62-mTORC1-Rag complex, which facilitates the lysosome localization of mTORC1. TRAF2 depletion inhibited mTORC1 activity through the disruption of interaction between p62 and the mTORC1 complex. In conclusion, our study provides the proof-of-concept evidence that TRAF2 is a valid target for liver cancer.

TRAF2 as a key candidate gene in clinical hepatitis B-associated liver fibrosis.

Objectives: Approximately 240 million individuals are infected with chronic hepatitis B virus (HBV) worldwide. HBV infection can develop into liver fibrosis. The mechanism of HBV-related liver fibrosis has not been fully understood, and there are few effective treatment options. The goal of this study was to use transcriptomics in conjunction with experimental validation to identify new targets to treat HBV-related liver fibrosis. Methods: To identify differentially expressed genes (DEGs), five liver tissues were collected from both healthy individuals and patients with chronic hepatitis B. NovoMagic and Java GSEA were used to screen DEGs and key genes, respectively. Immunocell infiltration analysis of RNA-seq data was, and the results were confirmed by Western blotting (WB), real-time quantitative polymerase chain reaction (RT-qPCR), and immunohistochemistry. Results: We evaluated 1,105 genes with differential expression, and 462 and 643 genes showed down- and upregulation, respectively. The essential genes, such as tumor necrosis factor (TNF) receptor-associated factor-2 (TRAF2), were screened out of DEGs. TRAF2 expression was abnormally high in hepatic fibrosis in patients with hepatitis B compared with healthy controls. The degree of hepatic fibrosis and serum levels of glutamate transaminase (ALT), aspartate aminotransferase (AST), and total bilirubin (TBIL) were positively linked with TRAF2 expression. TRAF2 may be crucial in controlling T lymphocyte-mediated liver fibrosis. Conclusion: Our findings imply that TRAF2 is essential for HBV-induced liver fibrosis progression, and it may potentially be a promising target for the treatment of hepatic fibrosis in hepatitis B.

Etanercept as a TNF-alpha inhibitor depresses experimental retinal neovascularization.

The formation of retinal neovascularization (RNV) is the primary pathological process underlying retinopathy of prematurity (ROP). Previous studies have shown that inflammatory factors are related to the formation of RNV. Tumor necrosis factor-α (TNF-α), as an important factor in the inflammatory response, is involved in the regulation of RNV formation. However, the mechanism through which TNF-α inhibition reduces RNV formation is not fully clarified. Therefore, the purpose of this study was to explore the effect of etanercept, an inhibitor of TNF-α, on RNV, and its possible mechanism. In vivo, an oxygen-induced retinopathy (OIR) mouse model was used to determine the effect of etanercept on the formation of RNV by performing immunostaining. The effect of etanercept on tumor necrosis factor receptor-associated factor 2 (TRAF2), pro-angiogenic-related factors, and pro/anti-inflammatory factors in OIR mice was assessed by real-time PCR and Western blotting. In vitro, the effect of etanercept on TNF-α-induced human retinal microvascular endothelial cell tube formation was evaluated by tube formation assays, and the potential mechanism of etanercept was explored by Western blotting. In vivo, etanercept reduced the area of RNV and decreased the expression of TRAF2 in the OIR mouse model. Etanercept also suppressed the expression of several pro-angiogenic factors and regulated the pro/anti-inflammatory factors. In vitro, etanercept reduced endothelial cell tube formation by inhibiting activation of the NF-κB signaling pathway. Etanercept can regulate pro/anti-inflammatory factors and reduce the expression of pro-angiogenic factors by inhibiting NF-κB phosphorylation, thereby reducing RNV formation.

TRAF2 protects against cerebral ischemia-induced brain injury by suppressing necroptosis

Necroptosis contributes to ischemia-induced brain injury. Tumor necrosis factor (TNF) receptor associated factor 2 (TRAF2) has been reported to suppress necroptotic cell death under several pathological conditions. In this study, we investigated the role of TRAF2 in experimental stroke using a mouse middle cerebral artery occlusion (MCAO) model and in vitro cellular models. TRAF2 expression in the ischemic brain was assessed with western blot and real-time RT-PCR. Gene knockdown of TRAF2 by lentivirus was utilized to investigate the role of TRAF2 in stroke outcomes. The expression of TRAF2 was significantly induced in the ischemic brain at 24 h after reperfusion, and neurons and microglia were two of the cellular sources of TRAF2 induction. Striatal knockdown of TRAF2 increased infarction size, cell death, microglial activation and the expression of pro-inflammatory markers at 24 h after reperfusion. TRAF2 expression and necroptosis were induced in mouse primary microglia treated with conditioned medium collected from neurons subject to oxygen and glucose deprivation (OGD) and in TNFα-treated mouse hippocampal neuronal HT-22 cells in the presence of the pan-caspase inhibitor Z-VAD. In addition, TRAF2 knockdown exacerbated microglial cell death and neuronal cell death under these conditions. Moreover, pre-treatment with a specific necroptosis inhibitor necrostatin-1 (nec-1) suppressed the cell death exacerbated by TRAF2 knockdown in the brain following MCAO, indicating that TRAF2 impacted ischemic brain damage through necroptosis mechanism. Taken together, our results demonstrate that TRAF2 is a novel regulator of cerebral ischemic injury.

Downregulation of TNF-α/TNF-R1 Signals by AT-Lipoxin A4 May Be a Significant Mechanism of Attenuation in SAP-Associated Lung Injury.

Our previous studies verified the potent anti-inflammatory effects against severe acute pancreatitis (SAP) of AT-Lipoxin A4 and their analogues. However, the anti-inflammatory effects of AT-Lipoxin A4 on SAP-associated lung injury are not thoroughly known. We used western blot, polymerase chain reaction (PCR), and immunofluorescence to investigate the downregulation of TNF-α signals in cellular and animal models of SAP-associated lung injury following AT-Lipoxin A4 intervention. In vitro, we found that AT-Lipoxin A4 markedly suppressed protein expression in TNF-α signals in human pulmonary microvascular endothelial cell, such as tumor necrosis factor receptor-associated factor 2 (TRAF2), TNF-R1-associated death domain (TRADD), receptor-interacting protein (RIP), vascular cell adhesion molecule-1 (VCAM-1), and E-selectin. Moreover, AT-Lipoxin A4 inhibited downstream signals activated by TNF-α, including NF-κB/p65, JNK/MAPK, and ERK/MAPK. In vivo, AT-Lipoxin A4 significantly decreased pathological scores of the pancreas and lungs and the serum levels of IL-6 and TNF-α. Immunofluorescence, western blotting, and real-time PCR assay showed that AT-Lipoxin A4 significantly attenuated the expression of TNF-R1, TRADD, TRAF2, and RIP in the lungs of SAP rats. In addition, the activation of NF-κB was also downregulated by AT-Lipoxin A4 administration as compared with SAP rats. AT-Lipoxin A4 could inhibit the production of proinflammatory mediators and activation of TNF-α downstream signals such as NF-κB and MAPK. Downregulation of TNF-α signals by AT-Lipoxin A4 may be a significant mechanism in the attenuation of SAP-associated lung injury.

Up-regulation of TRAF2 inhibits chondrocytes apoptosis in lumbar facet joint osteoarthritis

Tumor necrosis factor receptor-associated factor 2 (TRAF2) has been demonstrated that it plays a significant role in cell death receptor signal transduction. The purpose of this study was to investigate the expression of TRAF2 and its possible role in FJOA. We observed an up-regulation of TRAF2 in FJOA by immunohistochemistry and reverse transcription polymerase chain reaction (RT-PCR) compared to normal tissues. In vitro, we used TNF-α to stimulate Human SW1353 chondrosarcoma cells to establish the chondrocytes injury model. Western blot analysis revealed significant expression of TRAF2 and cleaved caspase-3/8 in SW1353 cells. Co-localization of TRAF2/cleaved caspase-3/8 was detected in the cells injury model by double-labeling immunofluorescent staining. We demonstrated a possible anti-apoptotic effect of TRAF2 in chondrocyte apoptosis in FJOA by knockdown of its expression with siRNA. Moreover, TRAF2 knockdown was demonstrated to enhance TNF-α-induced apoptosis by flow cytometry assay. In conclusion, our results show that the up-regulation of TRAF2 may play an important role in the inhibition of chondrocyte apoptosis of FJOA.

High expression of tumor necrosis factor receptor-associated factor 2 promotes tumor metastasis and is associated with unfavorable prognosis in gastric cancer.

Tumor necrosis factor receptor-associated factor 2 (TRAF2) is a key effector in the activation of nuclear factor kappa B (NF-κB). Nevertheless, the role of TRAF2 in gastric tumorigenesis remains little defined. Immunohistochemistry was used to find the relationship between TRAF2 expression and clinicopathological characteristics of gastric cancer patients, and nomogram was applied to predict the overall survival of patients. Besides, we performed transwell assays to detect the function of TRAF2 in promoting metastasis and explored the correlations between TRAF2, NF-κB, and interleukin-8 (IL-8) in vitro. In addition, we examined the correlation between TRAF2 and tumor microenvironment by immunohistochemistry staining. In our study, we found that TRAF2 expression was markedly increased in gastric cancer tissues. High intratumoral TRAF2 staining, which was associated with tumor invasion and metastasis, was also an independent poor prognosticator for gastric cancer patients. In vitro studies revealed that TRAF2 enhanced NF-κB activation and subsequent IL-8 expression in gastric cancer cells. Inhibition of NF-κB or IL-8 signaling attenuated TRAF2-induced migration and invasion abilities. High TRAF2 expression was confirmed to be associated with both high intratumoral and serum levels of IL-8. In addition, TRAF2 expression was positively correlated with neutrophil and macrophage infiltration as well as microvessels formation in gastric cancer samples. These results suggest that TRAF2 functions as an important modulator in tumor metastasis and tumor microenvironment formation and is a novel independent prognostic factor of gastric cancer.

Up-regulation of TRAF2 Suppresses Neuronal Apoptosis after Rat Spinal Cord Injury.

Tumor necrosis factor receptor-associated factor 2 (TRAF2) for signal transduction of the cell death receptor is well established. However, the role of TRAF2 in spinal cord injury (SCI) remains unclear. In this study, we detected the dynamic change patterns of TRAF2 expression using an acute spinal cord contusion (SCC) model in adult rats. Western blot analysis and immunohistochemistry identified significant upregulation of TRAF2 after SCI. Double-immunofluorescent staining demonstrated that the upregulated TRAF2 was found predominantly in neurons. Moreover, colocalization of TRAF2 with active caspase-3/-8 was detected in NeuN-positive cells. In vitro, we analyzed the association of TRAF2 with active caspase-3/8 on PC12 cells by western blot analysis, which paralleled the in vivo data. Knockdown ofTRAF2 with siRNA demonstrated its probable anti-apoptotic role in the process of neuronal apoptosis after SCI. To summarize, we have revealed for the first time the temporal and spatial expression profile of TRAF2 in SCI. Our data suggest that upregulation of TRAF2 triggered by trauma plays an important role in suppressing neuronal apoptosis after SCI.

Expression and significance of inositol requiting enzyme1α and tumor necrosis factor receptor-associated factor 2 in rats’ intestinal mucosa with intestinal ischemia-reperfusion injury

Objective To investigate the expression of inositol requiting enzyme1α (IRE1α) and tumor necrosis factor receptor-associated factor 2(TRAF2) and its significance through establishing models of intestinal ischemia reperfusion injury (IIRI) in rats. Methods According to the random number table, 50 male SD rats were randomly divided into 2 groups: sham operation group (n=10) and ischemia reperfusion (I/R) group (n=40). Sham group animals underwent laparotomy.I/R group rats were subjected to occlusion of the superior mesenteric artery for 30 min; then the blood flow was restored.I/R group animals were divided into 4 subgroups: 2 h, 6 h, 12 h, 24 h according to the time of reperfusion.Eight rats were examined based on the number of live rats in each subgroup.The HE staining pathological changes in intestinal samples were observed by the light microscope.The small intestinal epithelial cell apoptosis index (AI) was detected by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL). The expression levels of intestinal tissues tumor necrosis factor α (TNF-α) and plasma intestinal fatty acid-binding protein (I-FABP) were detected by ELISA tests.Situ end labeling method was used to detect intestinal cell AI.Western blot was applied to investigate the expression of endoplasmic reticulum stress(ERS) proteins IRE1α, phosphorylation IRE1α(p-IRE1α) and TRAF2 in all group rats intestinal tissues. Results (1)The pathological changes showed that the intestinal injury of I/R groups was more severe than that of sham group, especially at 6 h. (2)Compared with sham group, the expression levels of TNF-α[sham group(16.41±4.44) ng/L, 2 h group: (79.71±8.20) ng/L, 6 h group: (131.70±11.59) ng/L, 12 h group: (94.23±7.66) ng/L, 24 h group: (69.78±9.58) ng/L], AI[sham group: (3.93±0.77)%, 2 h group: (16.24±1.97)%, 6 h group: (42.19±2.40)%, 12 h group: (37.79±2.34)%, 24 h goup: (10.38±1.46)%] and plasma I-FABP [sham group: (0.65±0.10)×103 ng/L, 2 h group: (1.47±0.10)×103 ng/L, 6 h group: (2.36±0.17) ×103 ng/L, 12 h group: (37.79±2.34) ×103 ng/L, 24 group: (1.41±0.09) ×103 ng/L] were higher (F=231.462, 149.032, 162.491, all P<0.01). (3)The expression of TRAF-2 protein and p-IRE1α/IRE1α could be up-regulated after IIRI (F=40.473, 59.59, P<0.01). The expression of these proteins was up-regulated 2 h after reperfusion, peaking at 6-12 h reperfusion, and then decreased at 24 h, and the variation tendencies of all groups were the same. Conclusions IIRI could induce ERS, activate IRE1α and up-regulate TRAF2. IRE1α/TRAF2 mediating ERS might be involved in regulating the cell inflammation, apoptosis and increasing intestinal permeability after IIRI. Key words: Intestinal ischemia reperfusion injury; Inositol requiting enzyme1α; Inflammatory; Apoptosis

Stress-induced RNASET2 overexpression mediates melanocyte apoptosis via the TRAF2 pathway in vitro

The recent genome-wide association study identified a link between vitiligo and genetic variants in the ribonuclease T2 (RNASET2) gene; however, the functional roles of RNASET2 in vitiligo pathogenesis or in melanocyte apoptosis have yet to be determined. The current study was designed to investigate the vitiligo-related expression pattern of RNASET2 and its molecular function involving apoptosis-related signaling proteins and pathways. The results showed overexpression of RNASET2 in epidermis specimens from 40 vitiligo patients compared with that from matched healthy controls. In addition, in vitro analyses indicated that overexpression of RNASET2 was inducible in cultured primary human melanocytes and keratinocytes by stress conditions, that is, exposure to UV irradiation, hydrogen peroxide, and inflammatory factors, respectively, and led to increased cell apoptosis via the tumor necrosis factor receptor-associated factor 2 (TRAF2)–caspases pathway through the physical interaction of RNASET2 with TRAF2. Thus, RNASET2 may contribute to vitiligo pathogenesis by inhibiting TRAF2 expression and, as such, RNASET2 may represent a potential therapeutic target of vitiligo.

TRAF2 is Upregulated in Relapsing-Remitting Multiple Sclerosis

Objective: To elucidate the role of tumor necrosis factor (TNF) receptor signal transduction in multiple sclerosis (MS). Methods: We performed a cross-sectional analysis of the gene expression of TRAF2 (TNF receptor-associated factor 2) and RIP (receptor-interacting protein) in peripheral blood leukocytes of 23 relapsing-remitting (RR), 19 secondary progressive (SP) and 12 primary progressive (PP) MS patients as well as of 29 healthy controls by quantitative RT-PCR. Additionally, we monitored a subgroup of 15 RRMS patients longitudinally every 3 months over a 9-month time period. Results: TRAF2 expression was significantly elevated in RRMS patients compared to the other disease courses (p < 0.005, respectively) and the control group (p < 0.009). RIP expression was significantly elevated in the patient groups compared to the healthy group (p_healthy-RR < 0.002; p_healthy-PP < 0.003; p_healthy-SP < 0.06). Neither variable changed over the 9-month time course. Conclusion: TRAF2 and RIP1 elevation in leukocytes might be interpreted as the molecular equivalent of an elevated general inflammatory activity in MS patients compared to healthy control persons. TRAF2 elevation in RRMS reinforces the concept that different pathophysiological and immunological processes sustain RRMS and SPMS or PPMS.

Myocardial Recovery Is Associated with Molecular Hysteresis in a Model of Conditional Transgenesis

Background: The determinants of cardiac reverse remodeling (myocardial recovery) are unknown. We showed that the deleterious effects of tumor necrosis factor (TNF) signaling on LV remodeling are mediated through the TNF receptor associated factor 2 (TRAF2) pathway. Methods: To evaluate the determinants of myocardial recovery, we generated bitransgenic mice (ttA-TRAF2) that do not express TRAF2 in the presence of doxycycline (Dox), but expresses TRAF2 without Dox. At 8 weeks, ttA-TRAF2 mice were randomized to Dox for 4 weeks, to swith off TRAF2 expression, or no Dox to allow for sustained TRAF2 expression.We analyzed morphometrics, 2-D echo, myocyte contractility, myocardial collagen content, and gene expression at 12 weeks.

Targeting of Tumor Necrosis Factor Receptor 1 to Low Density Plasma Membrane Domains in Human Endothelial Cells

TNFR1 (tumor necrosis factor receptor 1) localizes to caveolae of human endothelial-derived EA.hy926 cells. Transduced TNFR1 molecules lacking amino acid residues 229-244 (spanning the transmembrane/intercellular boundary) are expressed on the cell surface equivalently to full-length TNFR1 molecules but incompletely localize to caveolae. A peptide containing this sequence pulls down CAV-1 (caveolin-1) and TNFR1 from cell lysates but fails to do so following disruption of caveolae with methyl-beta-cyclodextrin. We previously reported that methyl-beta-cyclodextrin eliminates caveolae and blocks tumor necrosis factor (TNF)-induced internalization of TNFR1 but not TNF-induced activation of NF-kappaB in EA.hy926 cells. Both CAV-1 and FLOT-2 (flotillin-2), organizing proteins of caveolae and lipid rafts, respectively, associate with caveolae in EA.hy926 cells. Small interfering RNA-mediated knockdown of CAV-1 but not FLOT-2 strikingly reduces caveolae number. Both knockdowns reduce total TNFR1 protein expression, but neither prevents TNFR1 localization to low density membrane domains, TNF-induced internalization of TNFR1, or NF-kappaB activation by TNF. Both CAV-1 and FLOT-2 knockdowns reduce TNF-mediated activation of stress-activated protein kinase (SAPK). However, both knockdowns reduce expression of TRAF2 (TNF receptor-associated factor-2) protein, and small interfering RNA targeting of TRAF2 also selectively inhibits SAPK activation. We conclude that TNFR1 contains a membrane-proximal sequence that targets the receptor to caveolae/lipid rafts. Neither TNFR1 targeting to nor internalization from these low density membrane domains depends upon CAV-1 or FLOT-2. Furthermore, both NF-kappaB and SAPK activation appear independent of both TNFR1 localization to low density membrane domains and to TNF-induced receptor internalization.

TRAF2 Suppresses Basal IKK Activity in Resting Cells and TNFα Can Activate IKK in TRAF2 and TRAF5 Double Knockout Cells

Tumor necrosis factor receptor (TNFR)-associated factor 2 (TRAF2) and TRAF5 are adapter proteins involved in TNFα-induced activation of the c-Jun N-terminal kinase and nuclear factor κB (NF-κB) pathways. Currently, TNFα-induced NF-κB activation is believed to be impaired in TRAF2 and TRAF5 double knockout (T2/5 DKO) cells. Here, we report instead that T2/5 DKO cells exhibit high basal IκB kinase (IKK) activity and elevated expression of NF-κB-dependent genes in unstimulated conditions. Although TNFα-induced receptor-interacting protein 1 ubiquitination is indeed impaired in T2/5 DKO cells, TNFα stimulation further increases IKK activity in these cells, resulting in significantly elevated expression of NF-κB target genes to a level higher than that in wild-type cells. Inhibition of NIK in T2/5 DKO cells attenuates basal IKK activity and restores robust TNFα-induced IKK activation to a level comparable with that seen in wild-type cells. This suggests that TNFα can activate IKK in the absence of TRAF2 and TRAF5 expression and receptor-interacting protein 1 ubiquitination. In addition, both the basal and TNFα-induced expression of anti-apoptotic proteins are normal in T2/5 DKO cells, yet these DKO cells remain sensitive to TNFα-induced cell death, due to the impaired recruitment of anti-apoptotic proteins to the TNFR1 complex in the absence of TRAF2. Thus, our data demonstrate that TRAF2 negatively regulates basal IKK activity in resting cells and inhibits TNFα-induced cell death by recruiting anti-apoptotic proteins to the TNFR1 complex rather than by activating the NF-κB pathway.

Enhancive effect of N,N'-dinitrosopiperazine on inducing precancerous lesion on nasal and/or nasopharyngeal epithelia of TgN(p53mt-LMP1)/HT mice.

To investigate the enhancive effect of N,N'-dinitrosopiperazine (DNP) on induced carcinogenesis in nasal and/or nasopharyngeal epithelia among TgN(p53mt-LMP1)/HT transgenic mice to examine the underlying mechanism for the development of nasopharyngeal carcinoma (NPC). TgN(p53mt-LMP1)/HT transgenic mice and the same strain of C(57)BL/6J wild-type mice both at the age of 5 months were randomly divided into 2 groups in parallel, respectively, i.e., TgN(p53mt-LMP1)/HT cancerous lesion-inducing group (TI), TgN(p53mt-LMP1)/HT control group (TC), C57BL/6J cancerous lesion-inducing group (CI), and C57BL/6J control group (CC). TI and CI mice were treated only with DNP for 16 weeks, twice each week, while TC and CC mice were given the same volume of saline as controls. At the end of treatment, animals were sacrificed to collect epithelial tissue samples from nasal cavity and nasopharynx for pathohistological evaluation by haematoxylin and eosin (HE) staining and for determination on the expression of TRAF2, c-Jun, and p16 by immunohistochemistry. Atypical hyperplasia was more significant in the samples of TI than in those of TC, CI, and CC, with the rates of lesions being 90%, 10%, 0, and 0 (P<0.01) respectively, though DNP was used alone in a much shortened inducing period at less dosage and without the use of carcinogenic promoter 12-O-tetradecanoylphorbol-13-acetate as usual. The expressions of tumor necrosis factor (TNF) receptor-associated factor 2 (TRAF2) and c-Jun in these samples were significantly up-regulated in TI (P<0.01), while the expression of p16 was significantly lower in TI than in the other groups (P<0.01). TgN(p53mt-LMP1)/HT mice hold inherited constitutional defect in immune surveillance function, which can be aggravated by environmental carcinogens, such as DNP used even though in a much less strength. The enhanced carcinogenesis-inducing effect of DNP on TgN(p53mt-LMP1)/HT mice should be closely associated with abnormal signaling of activator protein-1 (AP-1) pathway, especially up-regulated expressions of TRAF2 and c-Jun, and down-regulated expression of p16.

Thioredoxin and TRAF Family Proteins Regulate Reactive Oxygen Species-Dependent Activation of ASK1 through Reciprocal Modulation of the N-Terminal Homophilic Interaction of ASK1

Apoptosis signal-regulating kinase 1 (ASK1), a member of the mitogen-activated protein kinase kinase kinase family, plays pivotal roles in reactive oxygen species (ROS)-induced cellular responses. In resting cells, endogenous ASK1 constitutively forms a homo-oligomerized but still inactive high-molecular-mass complex including thioredoxin (Trx), which we designated the ASK1 signalosome. Upon ROS stimulation, the ASK1 signalosome unbinds from Trx and forms a fully activated higher-molecular-mass complex, in part by recruitment of tumor necrosis factor receptor-associated factor 2 (TRAF2) and TRAF6. However, the precise mechanisms by which Trx inhibits and TRAF2 and TRAF6 activate ASK1 have not been elucidated fully. Here we demonstrate that the N-terminal homophilic interaction of ASK1 through the N-terminal coiled-coil domain is required for ROS-dependent activation of ASK1. Trx inhibited this interaction of ASK1, which was, however, enhanced by expression of TRAF2 or TRAF6 or by treatment of cells with H2O2. Furthermore, the H2O2-induced interaction was reduced by double knockdown of TRAF2 and TRAF6. These findings demonstrate that Trx, TRAF2, and TRAF6 regulate ASK1 activity by modulating N-terminal homophilic interaction of ASK1.

Decreased expressions of the TNF-alpha signaling adapters in peripheral blood mononuclear cells (PBMCs) are correlated with disease activity in patients with systemic lupus erythematosus

Tumor necrosis factor (TNF)-alpha is a pleiotropic cytokine. Systemic lupus erythematosus (SLE) is an autoimmune and inflammatory disease. However, until now, the expression and pathophysiological role of TNF adapters in SLE have been poorly understood. This study aims to investigate the expression of mRNA for the TNF adapter proteins including TNF receptor-associated death domain (TRADD) protein, Fas-associated death domain (FADD) protein, receptor-interacting protein 1 (RIP-1), and TNF receptor-associated factor-2 (TRAF-2) in peripheral blood mononuclear cells (PBMCs) from patients with SLE and to explore the relationship between the expression of these adapters and the SLE disease activity. PBMCs were isolated from the venous blood of 51 SLE patients and 17 healthy subjects. The expression of mRNA for TNF adapter molecules such as TRADD, FADD, RIP-1, and TRAF-2 in PBMCs were analyzed by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) and quantitative real-time RT-PCR. There were constitutive expressions of mRNA for TRADD, FADD, RIP-1, and TRAF-2 in PBMCs from healthy subjects. The expression of mRNA for all the adapter molecules significantly decreased in PBMCs from patients with SLE, which were 0.38-, 0.69-, 0.59-, and 0.55-fold, respectively, compared to those of control subjects (P < 0.05). The expression of Caspase 3 was significantly increased in SLE patients (P < 0.01); however, the expression of IL-1beta was not significantly different between SLE and control subjects. The expression of TRADD, FADD, RIP-1, and TRAF-2 in PBMCs from patients with SLE were negatively correlated with SLEDAI, the correlation coefficient of which was -0.285, -0.280, -0.307, and -0.298, respectively (P < 0.05). The expression of mRNA for TNF adapter molecules TRADD, FADD, RIP-1, and TRAF-2 decreased significantly in PBMCs from patients with SLE, and the expression of these adapters were negatively correlated with the SLE activity index. These abnormalities may be involved in the immunopathogenic injury mediated by the aberration TNF-alpha signaling pathway in SLE.