Signaling via a CD27-TRAF2-SHP-1 axis during naive T cell activation promotes memory-associated gene regulatory networks.

The interaction of the tumor necrosis factor receptor (TNFR) family member CD27 on naive CD8+ T (Tn) cells with homotrimeric CD70 on antigen-presenting cells (APCs) is necessary for T cell memory fate determination. Here, we examined CD27 signaling during Tn cell activation and differentiation. In conjunction with T cell receptor (TCR) stimulation, ligation of CD27 by a synthetic trimeric CD70 ligand triggered CD27 internalization and degradation, suggesting active regulation of this signaling axis. Internalized CD27 recruited the signaling adaptor TRAF2 and the phosphatase SHP-1, thereby modulating TCR and CD28 signals. CD27-mediated modulation of TCR signals promoted transcription factor circuits that induced memory rather than effector associated gene programs, which are induced by CD28 costimulation. CD27-costimulated chimeric antigen receptor (CAR)-engineered T cells exhibited improved tumor control compared with CD28-costimulated CAR-T cells. Thus, CD27 signaling during Tn cell activation promotes memory properties with relevance to T cell immunotherapy.

MST1 Negatively Regulates TNFα-Induced NF-κB Signaling through Modulating LUBAC Activity.

The nuclear factor (NF)-κB pathway plays a central role in inflammatory and immune responses, with aberrant activation of NF-κB signaling being implicated in various human disorders. Here, we show that mammalian ste20-like kinase 1 (MST1) is a previously unrecognized component of the tumor necrosis factor α (TNFα) receptor 1 signaling complex (TNF-RSC) and attenuates TNFα-induced NF-κB signaling. Genetic ablation of MST1 in mouse embryonic fibroblasts and bone marrow-derived macrophages potentiated the TNFα-induced increase in IκB kinase (IKK) activity, as well as the expression of NF-κB target genes. TNFα induced the recruitment of MST1 to TNF-RSC and its interaction with HOIP, the catalytic component of the E3 ligase linear ubiquitin assembly complex (LUBAC). Furthermore, MST1 activated in response to TNFα stimulation mediates the phosphorylation of HOIP and thereby inhibited LUBAC-dependent linear ubiquitination of NEMO/IKKγ. Together, our findings suggest that MST1 negatively regulates TNFα-induced NF-κB signaling by targeting LUBAC.

MAVS forms functional prion-like aggregates to activate and propagate antiviral innate immune response.

In response to viral infection, RIG-I-like RNA helicases bind to viral RNA and activate the mitochondrial protein MAVS, which in turn activates the transcription factors IRF3 and NF-κB to induce type I interferons. [corrected] We have previously shown that RIG-I binds to unanchored lysine-63 (K63) polyubiquitin chains and that this binding is important for MAVS activation; however, the mechanism underlying MAVS activation is not understood. Here, we show that viral infection induces the formation of very large MAVS aggregates, which potently activate IRF3 in the cytosol. We find that a fraction of recombinant MAVS protein forms fibrils that are capable of activating IRF3. Remarkably, the MAVS fibrils behave like prions and effectively convert endogenous MAVS into functional aggregates. We also show that, in the presence of K63 ubiquitin chains, RIG-I catalyzes the conversion of MAVS on the mitochondrial membrane to prion-like aggregates. These results suggest that a prion-like conformational switch of MAVS activates and propagates the antiviral signaling cascade.

Modulating TRADD to restore cellular homeostasis and inhibit apoptosis.

Cell death in human diseases is often a consequence of disrupted cellular homeostasis. If cell death is prevented without restoring cellular homeostasis, it may lead to a persistent dysfunctional and pathological state. Although mechanisms of cell death have been thoroughly investigated1-3, it remains unclear how homeostasis can be restored after inhibition of cell death. Here we identify TRADD4-6, an adaptor protein, as a direct regulator of both cellular homeostasis and apoptosis. TRADD modulates cellular homeostasis by inhibiting K63-linked ubiquitination of beclin 1 mediated by TRAF2, cIAP1 and cIAP2, thereby reducing autophagy. TRADD deficiency inhibits RIPK1-dependent extrinsic apoptosis and proteasomal stress-induced intrinsic apoptosis. We also show that the small molecules ICCB-19 and Apt-1 bind to a pocket on the N-terminal TRAF2-binding domain of TRADD (TRADD-N), which interacts with the C-terminal domain (TRADD-C) and TRAF2 to modulate the ubiquitination of RIPK1 and beclin 1. Inhibition of TRADD by ICCB-19 or Apt-1 blocks apoptosis and restores cellular homeostasis by activating autophagy in cells with accumulated mutant tau, α-synuclein, or huntingtin. Treatment with Apt-1 restored proteostasis and inhibited cell death in a mouse model of proteinopathy induced by mutant tau(P301S). We conclude that pharmacological targeting of TRADD may represent a promising strategy for inhibiting cell death and restoring homeostasis to treat human diseases.

ZFAND1 Recruits p97 and the 26S Proteasome to Promote the Clearance of Arsenite-Induced Stress Granules.

Stress granules (SGs) are cytoplasmic assemblies of mRNPs stalled in translation initiation. They are induced by various stress conditions, including exposure to the environmental toxin and carcinogen arsenic. While perturbed SG turnover is linked to the pathogenesis of neurodegenerative diseases, the molecular mechanisms underlying SG formation and turnover are still poorly understood. Here, we show that ZFAND1 is an evolutionarily conserved regulator of SG clearance. ZFAND1 interacts with two key factors of protein degradation, the 26S proteasome and the ubiquitin-selective segregase p97, and recruits them to arsenite-induced SGs. In the absence of ZFAND1, SGs lack the 26S proteasome and p97, accumulate defective ribosomal products, and persist after arsenite removal, indicating their transformation into aberrant, disease-linked SGs. Accordingly, ZFAND1 depletion is epistatic to the expression of pathogenic mutant p97 with respect to SG clearance, suggesting that ZFAND1 function is relevant to the multisystem degenerative disorder IBMPFD/ALS.

TRAF2 decrease promotes the TGF-ß-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-ß (TGF-ß). The decisive role of TGF-ß in the development of MAFLD-HCC was confirmed through the specific depletion of TGF-ß receptor II gene in the hepatocytes (Tgfbr2ΔHep) of mice. In TRAF2-/- cells treated with TGF-ß, 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-ß, 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-ß 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-ß-mTORC1 pathway by facilitating AXIN1-mediated Smad7 degradation.

Celastrol-Induced Nur77 Interaction with TRAF2 Alleviates Inflammation by Promoting Mitochondrial Ubiquitination and Autophagy.

Mitochondria play an integral role in cell death, autophagy, immunity, and inflammation. We previously showed that Nur77, an orphan nuclear receptor, induces apoptosis by targeting mitochondria. Here, we report that celastrol, a potent anti-inflammatory pentacyclic triterpene, binds Nur77 to inhibit inflammation and induce autophagy in a Nur77-dependent manner. Celastrol promotes Nur77 translocation from the nucleus to mitochondria, where it interacts with tumor necrosis factor receptor-associated factor 2 (TRAF2), a scaffold protein and E3 ubiquitin ligase important for inflammatory signaling. The interaction is mediated by an LxxLL motif in TRAF2 and results not only in the inhibition of TRAF2 ubiquitination but also in Lys63-linked Nur77 ubiquitination. Under inflammatory conditions, ubiquitinated Nur77 resides at mitochondria, rendering them sensitive to autophagy, an event involving Nur77 interaction with p62/SQSTM1. Together, our results identify Nur77 as a critical intracellular target for celastrol and unravel a mechanism of Nur77-dependent clearance of inflamed mitochondria to alleviate inflammation.

TNIK depletion induces inflammation and apoptosis in injured renal proximal tubule epithelial cells.

In the aftermath of acute kidney injury (AKI), surviving proximal tubule epithelia repopulate injured tubules to promote repair. However, a portion of cells fail to repair [termed failed-repair proximal tubule cells (FR-PTCs)] and exert ongoing proinflammatory and profibrotic effects. To better understand the molecular drivers of the FR-PTC state, we reanalyzed a mouse ischemia-reperfusion injury single-nucleus RNA-sequencing (snRNA-seq) atlas to identify Traf2 and Nck interacting kinase (Tnik) to be exclusively expressed in FR-PTCs but not in healthy or acutely injured proximal tubules after AKI (2 and 6 wk) in mice. We confirmed expression of Tnik protein in injured mouse and human tissues by immunofluorescence. Then, to determine the functional role of Tnik in FR-PTCs, we depleted TNIK with siRNA in two human renal proximal tubule epithelial cell lines (primary and immortalized hRPTECs) and analyzed each by bulk RNA-sequencing. Pathway analysis revealed significant upregulation of inflammatory signaling pathways, whereas pathways associated with differentiated proximal tubules such as organic acid transport were significantly downregulated. TNIK gene knockdown drove reduced cell viability and increased apoptosis, including differentially expressed poly(ADP-ribose) polymerase (PARP) family members, cleaved PARP-1 fragments, and increased annexin V binding to phosphatidylserine. Together, these results indicate that Tnik upregulation in FR-PTCs acts in a compensatory fashion to suppress inflammation and promote proximal tubule epithelial cell survival after injury. Modulating TNIK activity may represent a prorepair therapeutic strategy after AKI.NEW & NOTEWORTHY The molecular drivers of successful and failed repair in the proximal tubule after acute kidney injury (AKI) are incompletely understood. We identified Traf2 and Nck interacting kinase (Tnik) to be exclusively expressed in failed-repair proximal tubule cells after AKI. We tested the effect of siTNIK depletion in two proximal tubule cell lines followed by bulk RNA-sequencing analysis. Our results indicate that TNIK acts to suppress inflammatory signaling and apoptosis in injured renal proximal tubule epithelial cells to promote cell survival.

Functional analyses of TRAF6 gene in Argopecten scallops.

The tumor necrosis factor (TNF) receptor-associated factor (TRAF) family has been reported to be involved in many immune pathways. In a previous study, we identified 5 TRAF genes, including TRAF2, 3, 4, 6, and 7, in the bay scallop (Argopecten irradians, Air) and the Peruvian scallop (Argopecten purpuratus, Apu). Since TRAF6 is a key molecular link in the TNF superfamily, we conducted a series of studies targeting the TRAF6 gene in the Air and Apu scallops as well as their hybrid progeny, Aip (Air ♀ × Apu ♂) and Api (Apu ♀ × Air ♂). Subcellular localization assay showed that the Air-, Aip-, and Api-TRAF6 were widely distributed in the cytoplasm of the human embryonic kidney cell line (HEK293T). Additionally, dual-luciferase reporter assay revealed that among TRAF3, TRAF4, and TRAF6, only the overexpression of TRAF6 significantly activated NF-κB activity in the HEK293T cells in a dose-dependent manner. These results suggest a crucial role of TRAF6 in the immune response in Argopecten scallops. To investigate the specific immune mechanism of TRAF6 in Argopecten scallops, we conducted TRAF6 knockdown using RNA interference. Transcriptomic analyses of the TRAF6 RNAi and control groups identified 1194, 2403, and 1099 differentially expressed genes (DEGs) in the Air, Aip, and Api scallops, respectively. KEGG enrichment analyses revealed that these DEGs were primarily enriched in transport and catabolism, amino acid metabolism, peroxisome, lysosome, and phagosome pathways. Expression profiles of 28 key DEGs were confirmed by qRT-PCR assays. The results of this study may provide insights into the immune mechanisms of TRAF in Argopecten scallops and ultimately benefit scallop breeding.

B(a)P induces ovarian granulosa cell apoptosis via TRAF2-NFκB-Caspase1 axis during early pregnancy.

Benzo(a)pyrene [B(a)P] is an environmental endocrine disruptor with reproductive toxicity. The corpus luteum (CL) of the ovary plays an important role in embryo implantation and pregnancy maintenance. Our previous studies have shown that B(a)P exposure affects embryo implantation and endometrial decidualization in mouse, but its effects and mechanisms on CL function remain unclear. In this study, we explore the mechanism of ovarian toxicity of B(a)P using a pregnant mouse model and an in vitro model of human ovarian granulosa cells (GCs) KGN. Pregnant mice were gavaged with corn oil or 0.2 mg/kg.bw B(a)P from pregnant day 1 (D1) to D7, while KGN cells were treated with DMSO, 1.0IU/mL hCG, or 1.0IU/mL hCG plus benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE), a B(a)P metabolite. Our findings revealed that B(a)P exposure damaged embryo implantation and reduced estrogen and progesterone levels in early pregnant mice. Additionally, in vitro, BPDE impaired luteinization in KGN cells. We observed that B(a)P/BPDE promoted oxidative stress (OS) and inflammation, leading to apoptosis rather than pyroptosis in ovaries and luteinized KGN cells. This apoptotic response was mediated by the activation of inflammatory Caspase1 through the cleavage of BID. Furthermore, B(a)P/BPDE inhibited TRAF2 expression and suppressed NFκB signaling pathway activation. The administration of VX-765 to inhibit the Caspase1 activation, over-expression of TRAF2 using TRAF2-pcDNA3.1 (+) plasmid, and BetA-induced activation of NFκB signaling pathway successfully alleviated BPDE-induced apoptosis and cellular dysfunction in luteinized KGN cells. These findings were further confirmed in the KGN cell treated with H2O2 and NAC. In conclusion, this study elucidated that B(a)P/BPDE induces apoptosis rather than pyroptosis in GCs via TRAF2-NFκB-Caspase1 during early pregnancy, and highlighting OS as the primary contributor to B(a)P/BPDE-induced ovarian toxicity. Our results unveil a novel role of TRAF2-NFκB-Caspase1 in B(a)P-induced apoptosis and broaden the understanding of mechanisms underlying unexplained luteal phase deficiency.

NUMA1 modulates apoptosis of esophageal squamous cell carcinoma cells through regulating ASK1-JNK signaling pathway.

Esophageal squamous cell carcinoma (ESCC) is a common malignancy worldwide with a low survival rate due to a lack of therapeutic targets. Here, our results showed that nuclear mitotic apparatus protein 1 (NUMA1) transcript and protein levels are significantly upregulated in ESCC patient samples and its high expression predicated poor prognosis. Knock-down of NUMA1 promoted cell apoptosis and suppressed cell proliferation and colony formation. By using cell-derived xenograft (CDX) and patient-derived xenograft (PDX) mice models, we found silencing the NUMA1 expression suppressed tumor progression. In addition, conditional knocking-out of NUMA1 reduced 4NQO-induced carcinogenesis in mice esophagus, which further confirmed the oncogenic role of NUMA1 in ESCC. Mechanistically, from the immunoprecipitation assay we revealed that NUMA1 interacted with GSTP1 and TRAF2, promoted the association of TRAF2 with GSTP1 while inhibited the interaction of TRAF2 and ASK1, thus to regulate sustained activation of JNK. In summary, our findings suggest that NUMA1 plays an important role during ESCC progression and it functions through regulating ASK1-MKK4-SAPK/JNK signaling pathway.

SGK1 negatively regulates inflammatory immune responses and protects against alveolar bone loss through modulation of TRAF3 activity.

Serum- and glucocorticoid-regulated kinase 1 (SGK1) is a serine/threonine kinase that plays important roles in the cellular stress response. While SGK1 has been reported to restrain inflammatory immune responses, the molecular mechanisms involved remain elusive, especially in oral bacteria-induced inflammatory milieu. Here, we found that SGK1 curtails Porphyromonas gingivalis-induced inflammatory responses through maintaining levels of tumor necrosis factor receptor-associated factor (TRAF) 3, thereby suppressing NF-κB signaling. Specifically, SGK1 inhibition significantly enhances production of proinflammatory cytokines, including tumor necrosis factor α, interleukin (IL)-6, IL-1ß, and IL-8 in P. gingivalis-stimulated innate immune cells. The results were confirmed with siRNA and LysM-Cre-mediated SGK1 KO mice. Moreover, SGK1 deletion robustly increased NF-κB activity and c-Jun expression but failed to alter the activation of mitogen-activated protein kinase signaling pathways. Further mechanistic data revealed that SGK1 deletion elevates TRAF2 phosphorylation, leading to TRAF3 degradation in a proteasome-dependent manner. Importantly, siRNA-mediated traf3 silencing or c-Jun overexpression mimics the effect of SGK1 inhibition on P. gingivalis-induced inflammatory cytokines and NF-κB activation. In addition, using a P. gingivalis infection-induced periodontal bone loss model, we found that SGK1 inhibition modulates TRAF3 and c-Jun expression, aggravates inflammatory responses in gingival tissues, and exacerbates alveolar bone loss. Altogether, we demonstrated for the first time that SGK1 acts as a rheostat to limit P. gingivalis-induced inflammatory immune responses and mapped out a novel SGK1-TRAF2/3-c-Jun-NF-κB signaling axis. These findings provide novel insights into the anti-inflammatory molecular mechanisms of SGK1 and suggest novel interventional targets to inflammatory diseases relevant beyond the oral cavity.

The natural compound Sanggenon C inhibits PRRSV infection by regulating the TRAF2/NF-κB signalling pathway.

Porcine reproductive and respiratory syndrome (PRRS) is a serious infectious disease and one of the major causes of death in the global pig industry. PRRS virus (PRRSV) strains have complex and diverse genetic characteristics and cross-protection between strains is low, which complicates vaccine selection; thus, the current vaccination strategy has been greatly compromised. Therefore, it is necessary to identify effective natural compounds for the clinical treatment of PRRS. A small molecule library composed of 720 natural compounds was screened in vitro, and we found that Sanggenon C (SC) was amongst the most effective natural compound inhibitors of PRRSV infection. Compared with ribavirin, SC more significantly inhibited PRRSV infection at both the gene and protein levels and reduced the viral titres and levels of protein expression and inflammatory cytokine secretion to more effectively protect cells from PRRSV infection and damage. Mechanistically, SC inhibits activation of the NF-κB signalling pathway by promoting TRAF2 expression, thereby reducing PRRSV replication. In conclusion, by screening natural compounds, we found that SC suppresses PRRSV infection by regulating the TRAF2/NF-κB signalling pathway. This study contributes to a deeper understanding of the therapeutic targets and pathogenesis of PRRSV infection. More importantly, our results demonstrate that SC has potential as a candidate for the treatment of PRRS.

Diosmetin inhibits the growth and invasion of gastric cancer by interfering with M2 phenotype macrophage polarization.

Overturning M2 phenotype macrophage polarization is a promising therapeutic strategy for gastric cancer (GC). Diosmetin (DIO) is a natural flavonoid with antitumor effect. The aim of this study was to investigate the effect of DIO on polarization of M2 phenotype macrophages in GC. THP-1 cells were induced to M2 phenotype macrophages and co-cultured with AGS cells. The effects of DIO were determined by flow cytometry, qRT-PCR, CCK-8, Transwell, and western blot. To explore the mechanisms, THP-1 cells were transfected with adenoviral vectors containing tumor necrosis factor receptor-associated factor 2 (TRAF2) or si-TRAF2. DIO (0, 5, 10, and 20 µM) restrained the M2 phenotype macrophage polarization. In addition, DIO (20 µM) reversed the increased viability and invasion of AGS cells induced by the co-culture of M2 macrophages. Mechanistically, TRAF2 knockdown inhibited the effect of M2 phenotype macrophages on AGS cells' growth and invasion. Furthermore, DIO (20 µM) was found to decrease TRAF2/NF-κB activity in GC cells. However, TRAF2 overexpressed reversed the inhibitory effect of DIO on the co-culture system. The in vivo study confirmed that DIO treatment (50 mg/kg) could repress the growth of GC. DIO treatment markedly reduced the expressions of Ki-67 and N-cadherin, and decreased the protein levels of TRAF2 and p-NF-κB/NF-κB. In conclusion, DIO inhibited the growth and invasion of GC cells by interfering with M2 phenotype macrophage polarization through repression of the TRAF2/NF-κB signaling pathway.

TRAF2 and OTUD7B govern a ubiquitin-dependent switch that regulates mTORC2 signalling.

The mechanistic target of rapamycin (mTOR) has a key role in the integration of various physiological stimuli to regulate several cell growth and metabolic pathways. mTOR primarily functions as a catalytic subunit in two structurally related but functionally distinct multi-component kinase complexes, mTOR complex 1 (mTORC1) and mTORC2 (refs 1, 2). Dysregulation of mTOR signalling is associated with a variety of human diseases, including metabolic disorders and cancer. Thus, both mTORC1 and mTORC2 kinase activity is tightly controlled in cells. mTORC1 is activated by both nutrients and growth factors, whereas mTORC2 responds primarily to extracellular cues such as growth-factor-triggered activation of PI3K signalling. Although both mTOR and GßL (also known as MLST8) assemble into mTORC1 and mTORC2 (refs 11, 12, 13, 14, 15), it remains largely unclear what drives the dynamic assembly of these two functionally distinct complexes. Here we show, in humans and mice, that the K63-linked polyubiquitination status of GßL dictates the homeostasis of mTORC2 formation and activation. Mechanistically, the TRAF2 E3 ubiquitin ligase promotes K63-linked polyubiquitination of GßL, which disrupts its interaction with the unique mTORC2 component SIN1 (refs 12, 13, 14) to favour mTORC1 formation. By contrast, the OTUD7B deubiquitinase removes polyubiquitin chains from GßL to promote GßL interaction with SIN1, facilitating mTORC2 formation in response to various growth signals. Moreover, loss of critical ubiquitination residues in GßL, by either K305R/K313R mutations or a melanoma-associated GßL(ΔW297) truncation, leads to elevated mTORC2 formation, which facilitates tumorigenesis, in part by activating AKT oncogenic signalling. In support of a physiologically pivotal role for OTUD7B in the activation of mTORC2/AKT signalling, genetic deletion of Otud7b in mice suppresses Akt activation and Kras-driven lung tumorigenesis in vivo. Collectively, our study reveals a GßL-ubiquitination-dependent switch that fine-tunes the dynamic organization and activation of the mTORC2 kinase under both physiological and pathological conditions.

Co-ordinated control of the ADP-heptose/ALPK1 signalling network by the E3 ligases TRAF6, TRAF2/c-IAP1 and LUBAC.

ADP-heptose activates the protein kinase ALPK1 triggering TIFA phosphorylation at Thr9, the recruitment of TRAF6 and the subsequent production of inflammatory mediators. Here, we demonstrate that ADP-heptose also stimulates the formation of Lys63- and Met1-linked ubiquitin chains to activate the TAK1 and canonical IKK complexes, respectively. We further show that the E3 ligases TRAF6 and c-IAP1 operate redundantly to generate the Lys63-linked ubiquitin chains required for pathway activation, which we demonstrate are attached to TRAF6, TRAF2 and c-IAP1, and that c-IAP1 is recruited to TIFA by TRAF2. ADP-heptose also induces activation of the kinase TBK1 by a TAK1-independent mechanism, which require TRAF2 and TRAF6. We establish that ALPK1 phosphorylates TIFA directly at Thr177 as well as Thr9 in vitro. Thr177 is located within the TRAF6-binding motif and its mutation to Asp prevents TRAF6 but not TRAF2 binding, indicating a role in restricting ADP-heptose signalling. We conclude that ADP-heptose signalling is controlled by the combined actions of TRAF2/c-IAP1 and TRAF6.

Persistent Lipid Accumulation Leads to Persistent Exacerbation of Endoplasmic Reticulum Stress and Inflammation in Progressive NASH via the IRE1α/TRAF2 Complex.

Non-alcoholic steatohepatitis (NASH) is a metabolic disorder that often leads to other severe liver diseases, yet treatment options are limited. Endoplasmic reticulum (ER) stress is an important pathogenetic mechanism of NASH and plays a key role in tandem steatosis as well as liver inflammation. This study aims to develop a progressive NASH model through sustained lipid accumulation and to elucidate its molecular mechanism through IRE1α/TRAF2 complex. Male SD rats were fed a high-fat diet (HFD) for 4, 8, and 12 weeks to induce progressive NASH. MRNA sequencing and PPI analysis were used to screen core genes. Transmission electron microscopy, immunofluorescence staining, ELISA, qRT-PCR, and Western blotting were used at each time point to compare differences between each index of progressive NASH at 4, 8, and 12 weeks. Sustained lipid accumulation led to structural disruption of the ER, a reduction in ER number, and an increase of lipid droplet aggregation in hepatocytes. Persistent lipid accumulation led to a persistent increase in mRNA and protein expression of the IRE1α/TRAF2 complex, IKK/IκB/NF-κB signaling pathway and ASK1/JNK1 signaling pathway, and TNF-α, IL-1ß, and IL-6 also continued to increase. Persistent lipid accumulation led to a persistent exacerbation of ER stress and inflammation in progressive NASH via the IRE1α/TRAF2 complex.

TSG-6 inhibits hypertrophic scar fibroblast proliferation by regulating IRE1α/TRAF2/NF-κB signalling.

TNF-stimulated gene (TSG-6) was reported to suppress hypertrophic scar (HS) formation in a rabbit ear model, and the overexpression of TSG-6 in human HS fibroblasts (HSFs) was found to induce their apoptotic death. The molecular basis for these findings, however, remains to be clarified. HSFs were subjected to TSG-6 treatment. Treatment with TSG-6 significantly suppressed HSF proliferation and induced them to undergo apoptosis. Moreover, TSG-6 exposure led to reductions in collagen I, collagen III, and α-SMA mRNA and protein levels, with a corresponding drop in proliferating cell nuclear antigen (PCNA) expression indicative of impaired proliferative activity. Endoplasmic reticulum (ER) stress was also suppressed in these HSFs as demonstrated by decreases in Bip and p-IRE1α expression, downstream inositol requiring enzyme 1 alpha (IRE1α) -Tumor necrosis factor receptor associated factor 2 (TRAF2) pathway signalling was inhibited and treated cells failed to induce NF-κB, TNF-α, IL-1ß, and IL-6 expression. Overall, ER stress was found to trigger inflammatory activity in HSFs via the IRE1α-TRAF2 axis, as confirmed with the specific inhibitor of IRE1α STF083010. Additionally, the effects of TSG-6 on apoptosis, collagen I, collagen III, α-SMA, and PCNA of HSFs were reversed by the IRE1α activator thapsigargin (TG). These data suggest that TSG-6 administration can effectively suppress the proliferation of HSFs in part via the inhibition of IRE1α-mediated ER stress-induced inflammation (IRE1α/TRAF2/NF-κB signalling).

[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.

TNF receptor-associated factor 3 restrains B-cell receptor signaling in normal and malignant B cells.

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.