Overexpression Of A20 Research Articles

A20 Alleviates the Inflammatory Response in Bovine Endometrial Epithelial Cells by Promoting Autophagy.

Endometritis represents a prevalent condition in perinatal dairy cows. Bovine endometrial epithelial cells (BEECs), as the primary interface between cavity and the external environment, are particularly vulnerable to infection by pathogenic bacteria following parturition. A20 is essential for regulating inflammation and modulating immune responses. Nevertheless, the exact role of A20 in the BEECs in response to inflammatory response is not fully understood. An endometritis model infected by Escherichia coli (E. coli) in vivo and a BEECs inflammation model induced with lipopolysaccharide (LPS) in vitro were built to investigate the function and governing mechanisms of A20 in endometritis. The results showed that infection with E. coli resulted in endometrial damage, inflammatory cell infiltration, and upregulation of inflammatory factors in dairy cows. Furthermore, A20 expression was upregulated in the endometrium of cows with endometritis and in BEECs following LPS stimulation. A20 overexpression attenuated the level of proinflammatory cytokines in LPS-stimulated BEECs; conversely, A20 knockdown lead to an exacerbated response to LPS stimulation. The overexpression of A20 was shown to activate autophagy and suppress the NF-κB signaling pathway in LPS-stimulated BEECs. However, blocking autophagy with chloroquine notably attenuated the anti-inflammatory effect of A20, leading to the activation of the NF-κB signaling pathway. In summary, the study demonstrated that A20's suppression of inflammation in LPS-stimulated BEECs is associated with the activation of autophagy. Therefore, the A20 protein showed potential as a novel treatment focus for managing endometritis in dairy cows.

Defective Necroptosis Mediates Chemotherapy Resistance in AML

Acute myeloid leukemia (AML) is a hematopoietic malignancy that can affect individuals of any age and, if left untreated, is always fatal. Standard induction chemotherapy, known as “7+3,” using drugs like cytarabine (Ara-C) and an anthracycline (daunorubicin or idarubicin), is typically given to young and fit older patients. While this treatment regimen has shown positive outcomes in patients who respond to it, relapse and refractory disease remain the primary causes of death. Prognostic classification, which combines cytogenetic and mutational status, can estimate overall and event-free survival but is not effective in predicting induction failure. Despite decades of research and treatment with 7+3 chemotherapy, the reasons behind variable treatment responses in AML patients remain unclear, especially for those who experience induction failure. Understanding these mechanisms could aid in risk-adapted treatment decisions and the identification of new therapeutic targets. Recent research has investigated gene expression and signaling patterns in leukemic cells at the time of diagnosis to categorize patients according to their response to chemotherapy. Notably, increased NF-kB signaling at diagnosis has been linked to chemotherapy resistance. NF-kB is a family of transcription factors involved in regulating genes linked to cancer cell survival, proliferation, and therapy resistance. However, the exact mechanism by which NF-kB activation contributes to chemotherapy failure and resistance in AML is yet to be determined. Using publicly available datasets, we found that AML patients undergoing induction failure exhibited elevated expression of TNFAIP3/A20, which is driven by NF-kB signaling and leads to a worse prognosis for patients undergoing 7+3 chemotherapy. In experiments with primary AML samples, it was observed that A20-High AML are resistant to Doxorubicin treatment, whereas A20-Low AML samples are sensitive to Doxorubicin. Knockdown of A20 in A20-High AML samples resulted in increased cell sensitivity to Doxorubicin, suggesting that elevated A20 expression reduces AML cells' response to the Doxorubicin both in vitro and in vivo. To understand why A20 is overexpressed in AML, we investigated its requirement on AML cell function in various models, including human AML cell lines, patient-derived samples, and mouse models of AML. Deletion or knockdown of A20 led to reduced leukemic progenitor function, cell viability, and AML development in vitro and in vivo. A20 is a dual ubiquitin-editing protein that regulates signaling pathways by acting either as a deubiquitinase to modulate NF-kB signaling or as an E3 ligase to control necroptosis. Further analyses revealed that A20 prevents necroptosis by targeting the necroptosis effector, RIPK1, for degradation through the proteasome in AML. Preventing necroptosis, either pharmacologically or genetically, restored the viability and clonogenicity of A20-deficient AML cells in vitro and in vivo. Notably, Doxorubicin, but not AraC, induced necroptosis in AML cells, which is suppressed in leukemic clones expressing A20. Examination of single-cell RNA-sequencing data from AML patients before and after Doxorubicin treatment confirmed the persistence of A20-expressing clones during treatment. These findings demonstrate that NF-kB-driven overexpression of A20 contributes to failed induction chemotherapy in AML, highlighting the therapeutic potential of targeting an alternative cell death pathway in AML.

A20 interacts with mTORC2 to inhibit the mTORC2/Akt/Rac1 signaling axis in hepatocellular carcinoma cells.

A20 acts as a tumor suppressor in hepatocellular carcinoma, especially inhibiting metastasis of the malignant cells. However, the mechanisms whereby A20 plays the inhibitory roles are not understood completely. Rac1 signaling is essential for cell migration in hepatocellular carcinoma metastasis. Nevertheless, it is not known whether and how A20 inhibits Rac1 signaling to suppress the migration of hepatocellular carcinoma cell. Thereby, we analyzed the relationship between A20 and Rac1 activation, as well as the activity of Akt and mTORC2, two signaling components upstream of Rac1, using gain and loss of function experiments. We found that the overexpression of A20 repressed, while the knockdown or knockout of A20 promoted, the activation of Rac1, Akt and mTORC2 in hepatocellular carcinoma cells. Moreover, the inhibitory effect of A20 on the mTORC2/Akt/Rac1 signaling axis was due to the interaction between A20 and mTORC2 complex. The binding of A20 to mTORC2 was mediated by the ZnF7 domain of A20 and M1 ubiquitin chain in the mTORC2 complex. Furthermore, A20 inhibited metastasis of hepatocellular carcinoma cells via restraining mTORC2 in a hepatocellular carcinoma xenograft mouse model. These findings revealed the relationship between A20 and mTORC2, and explained the molecular mechanisms of A20 in inhibition of hepatocellular carcinoma metastasis.

TRPC6 inhibits renal tubular epithelial cell pyroptosis through regulating zinc influx and alleviates renal ischemia-reperfusion injury.

Canonical transient receptor potential-6 (TRPC6) has been reported to be involved in cell damage after ischemia/reperfusion (I/R) injury in target organs. While the effect and of TRPC6 on pyroptosis in renal I/R injury remain unclear. In our study, we first established the renal I/R mouse model and oxygen-glucose deprivation and re-oxygenation (OGD/R) cell model, and investigated the impacts of TRPC6 on the pyroptosis-related proteins using CCK-8, western blot, ELISA, and immunofluorescence probes. Besides, we also explored the mechanism of TRPC6 in pyroptosis of renal tubular epithelial cells through A20 knockdown or overexpression and zinc chloride (ZnCl2 ) or a zinc ion chelator (TPEN) treatment. Our results indicated that I/R injury could cause downregulation of TRPC6 both in vivo and in vitro. In the I/R injury murine model, TRPC6 inhibition exacerbated tissue damage and upregulated NLRP3, ASC, caspase-1, IL-18, and IL-1β, which could be alleviated by the administration of ZnCl2 . In the OGD/R cell model, inhibitor of TRPC6 (SAR7334) reduced zinc ion influx, aggravated cell death and upregulated pyroptosis-related protein. The pyroptosis phenotype also could be alleviated by ZnCl2 and intensified by TPEN. Overexpression of A20 reduced the expression of pyroptosis-related protein, increased cell viability in the sh-TRPC6 and TPEN-treated OGD/R cell models, while A20 deficiency impaired the protective effect of zinc ion. Therefore, our results indicate that TRPC6 could promote zinc ion influx in renal tubular epithelial cells, thereby upregulating intracellular A20, inhibiting the activation of inflammasome NLRP3, and ultimately attenuating renal I/R injury.

A20 attenuates pyroptosis and apoptosis in nucleus pulposus cells via promoting mitophagy and stabilizing mitochondrial dynamics.

A20 is an anti-inflammatory molecule in nucleus pulposus (NP) cells. The anti-inflammatory properties of A20 are mainly attributed to its ability to suppress the NF-κB pathway. However, A20 can protect cells from death independently of NF-κB regulation. This study aimed to investigate the effects of A20 on pyroptosis and apoptosis of NP cells induced by lipopolysaccharide (LPS). NP cells induced by LPS were used as an in vitro model of the inflammatory environment of the intervertebral disc. Pyroptosis, apoptosis, and mitophagy marker proteins were detected. Then, NP cells were transfected with A20 overexpressed lentivirus or A20-siRNA. Annexin V FITC/PI, Western blotting, and immunofluorescence assays were used to detect the apoptosis, pyroptosis, and mitophagy of NP cells. Furthermore, the expressions of A20, related proteins, and related inflammatory cytokines were detected by western blotting, and ELISA. Apoptosis and pyroptosis of NP cells increased gradually treated with LPS for 12h, 24h, and 48h. Differently, the level of mitophagy increased first and then decreased, and was the highest at LPS treatment for 12h. Overexpression or knockdown of A20 in NP cells revealed that A20 attenuated the pyroptosis, apoptosis, and production of inflammatory cytokines of NP cells induced by LPS, while A20 sponsored mitophagy, reduced ROS production and collapse of mitochondrial membrane potential (ΔΨm). Moreover, A20 also promoted mitochondrial dynamic homeostasis and attenuated LPS-induced excessive mitochondrial fission. Excitingly, inhibition of mitophagy attenuated the effect of A20 on the negative regulation of pyroptosis of NP cells induced by LPS. Pyroptosis was accompanied by a large release of inflammatory cytokines. Inhibition of pyroptosis also significantly reduced apoptosis of NP cells. Finally, The mitochondria-targeted active peptide SS-31 inhibited LPS-induced pyroptosis and ROS production in NP cells. Tosumup, A20 attenuates pyroptosis and apoptosis of NP cells via promoting mitophagy and stabilizing mitochondrial dynamics. Besides, A20 reduces LPS-induced NP cell apoptosis by inhibiting NLRP3 inflammasome-mediated pyroptosis. It provides theoretical support for the reduction of functional NP cell loss in the intervertebral disc through the gene-targeted intervention of A20.

A20 alleviated caspase-1-mediated pyroptosis and inflammation stimulated by Porphyromonas gingivalis lipopolysaccharide and nicotine through autophagy enhancement.

Periodontitis is the leading cause of tooth loss, and patients with smoking habits are at an increased risk of developing periodontitis. A20 (the tumor necrosis factor alpha-induced protein 3, TNFAIP3) is one of the key regulators of inflammation and cell death in numerous tissues. Emerging researches indicated A20 as a fundamental molecule in the periodontal tissue. This study was to evaluate the role of A20 against cell death and inflammation in periodontitis and to elucidate the underlying mechanisms. In our study, western blot, autophagy detection, and transmission electron microscopy showed that lipopolysaccharide from Porphyromonas gingivalis (Pg.LPS) and nicotine (NI) could enhance the activation of autophagy. Pg.LPS and NI induce the pyroptosis of human periodontal ligament cells (hPDLCs), as evidenced by the decrease of membrane integrity and the increase of NLRP3, GSDMD, GSDMD-N, caspase-1 activity, and the pro-inflammatory cytokines of IL-1β, IL-6, TNF-α. Further researches were focused on that A20, an ubiquitin-editing enzyme, was linked to hPDLCs pyroptosis. Overexpression or silencing A20 could diminish or aggravate pyroptosis in hPDLCs by the modulation of autophagy. The above results demonstrated that A20 dictated the cross-talk between pyroptosis and autophagy. Overexpression of A20 enhanced autophagy to reduce pyroptosis, and thus alleviating inflammation, suggesting that A20 may be a potent target in the treatment of periodontitis.

A20 Attenuates the Fibrotic Response in the Trabecular Meshwork.

Although the extracellular matrix (ECM) in trabecular meshwork (TM) cells is known to be important in intraocular pressure (IOP) regulation, the molecular mechanisms involved in generating a glaucomatous environment in the TM are not completely understood. Recently we identified a molecular pathway, transforming growth factor beta 2 (TGFβ2)–toll-like receptor 4 (TLR4) signaling crosstalk, as an important regulator of glaucomatous damage in the TM, which contributes to fibrosis. Here we evaluate a novel molecular target, A20, also known as tumor necrosis factor alpha-induced protein 3 (TNFAIP3), which may help to block pathological TGFβ2–TLR4 signaling. Primary human TM cells were analyzed for A20 message and for A20 and fibronectin protein expression after treatment with TGFβ2. A20 message increased when the TLR4 pathway was inhibited in TM cells. In addition, TGFβ2, a known inducer of fibrosis, increased fibronectin expression, while at the same time decreasing the expression of A20. We then overexpressed A20 in TM cells in order to test the effect on treatment with TGFβ2, lipopolysaccharide (LPS), or cellular fibronectin extra domain A (cFN-EDA). Importantly, overexpression of A20 rescued the fibrotic response when TM cells were treated with TGFβ2, LPS, or cFN-EDA. In situ hybridization was used to probe for A20 RNA expression in age-matched control (C57BL/6J) mice and mice that constitutively express the EDA isoform of fibronectin (B6.EDA+/+). In this novel mouse model of glaucoma, A20 RNA was increased versus age-matched control mice in a cyclic manner at 6 weeks and 1 year of age, but not at 8 months. Overall, these data suggest that A20 may work through a negative feedback mechanism attenuating the ability of TGFβ2–TLR4 signaling to induce fibrosis.

A20 Is Increased in Fetal Lung in a Sheep LPS Model of Chorioamnionitis.

Chorioamnionitis is associated with an increased risk of preterm birth and aggravates adverse outcomes such as BPD. Development of BPD is associated with chronic inflammatory reactions and oxidative stress in the airways which may be antenatally initiated by chorioamnionitis. A20 is an immunomodulatory protein involved in the negative feedback regulation of inflammatory reactions and is a possible regulator protein in oxidative stress reactions. The influence of chorioamnionitis on A20 gene regulation in the fetal lung is unknown. We characterized the influence of LPS and proinflammatory cytokines on A20 expression in human lung endothelial (HPMEC-ST1.6R) and epithelial (A549) cells in vitro by real-time PCR and/or western blotting and used a sheep model of LPS-induced chorioamnionitis for in vivo studies. To study the functional role of A20, endogenous A20 was overexpressed in HPMEC-ST1.6R and A549 cells. LPS induced proinflammatory cytokines in HPMEC-ST1.6R and A549 cells. Both LPS and/or proinflammatory cytokines elevated A20 at transcriptional and translational levels. Intra-amniotic LPS transiently increased IL-1β, IL-6, IL-8, and TNF-α mRNA levels in fetal lamb lungs, associated with an increase in A20 mRNA and protein levels. Overexpression of A20 reduced proinflammatory cytokines in vitro. Repeated LPS exposure induced LPS tolerance for proinflammatory cytokines and A20 in vitro and in vivo. Antenatal inflammation induced a transient increase in proinflammatory cytokines in the preterm fetal lung. The expression of proinflammatory cytokines increased expression of A20. Elevated A20 may have a protective role by downregulating chorioamnionitis-triggered fetal lung inflammation. A20 may be a novel target for pharmacological interventions to prevent chorioamnionitis-induced airway inflammation and lung damage, which can result in BPD later in life.

A20 (TNFAIP3) alleviates viral myocarditis through ADAR1/miR-1a-3p-dependent regulation

ObjectiveTo investigate the effect of A20 and how A20 is regulated in viral myocarditis (VMC).MethodsBABL/C mice, primary neonatal rat cardiomyocytes and H9c2 cells were infected with Coxsackie virus B3 (CVB3) to establish animal and cellular models of VMC. H&E staining revealed the pathologic condition of myocardium. ELISA measured the serum levels of creatine kinase, creatine kinase isoenzyme and cardiac troponin I. The effects of A20, miR-1a-3p and ADAR1 were investigated using gain and loss of function approaches. ELISA measured the levels of IL-6, IL-18 and TNF-α in serum or cell culture supernatant. TUNEL staining and flow cytometry assessed the apoptosis of myocardium and cardiomyocytes, respectively. RNA-binding protein immunoprecipitation and dual-luciferase reporter assays verified the binding between A20 and miR-1a-3p. Co-immunoprecipitation assay verified the binding between ADAR1 and Dicer.ResultsA20 was underexpressed and miR-1a-3p was overexpressed in the myocardium of VMC mice as well as in CVB3-infected cardiomyocytes. Overexpression of A20 suppressed cardiomyocyte inflammation and apoptosis in vivo and in vitro. miR-1a-3p promoted CVB3-induced inflammation and apoptosis in cardiomyocytes by binding to A20. The expression of miR-1a-3p was regulated by ADAR1. ADAR1 promoted the slicing of miR-1a-3p precursor by binding to Dicer.ConclusionA20, regulated by ADAR1/miR-1a-3p, suppresses inflammation and cardiomyocyte apoptosis in VMC.

The Disassociation of the A20/HSP90 Complex via Downregulation of HSP90 Restores the Effect of A20 Enhancing the Sensitivity of Hepatocellular Carcinoma Cells to Molecular Targeted Agents.

NF-κB (nuclear factor κB) is a regulator of hepatocellular cancer (HCC)-related inflammation and enhances HCC cells’ resistance to antitumor therapies by promoting cell survival and anti-apoptosis processes. In the present work, we demonstrate that A20, a dominant-negative regulator of NF-κB, forms a complex with HSP90 (heat-shock protein 90) and causes the disassociation of the A20/HSP90 complex via downregulation of HSP90. This process restores the antitumor activation of A20. In clinical specimens, the expression level of A20 did not relate with the outcome in patients receiving sorafenib; however, high levels of HSP90 were associated with poor outcomes in these patients. A20 interacted with and formed complexes with HSP90. Knockdown of HSP90 and treatment with an HSP90 inhibitor disassociated the A20/HSP90 complex. Overexpression of A20 alone did not affect HCC cells. Downregulation of HSP90 combined with A20 overexpression restored the effect of A20. Overexpression of A20 repressed the expression of pro-survival and anti-apoptosis-related factors and enhanced HCC cells’ sensitivity to sorafenib. These results suggest that interactions with HSP90 could be potential mechanisms of A20 inactivation and disassociation of the A20/HSP90 complex and could serve as a novel strategy for HCC treatment.

A novel missense mutation in TNFAIP3 causes haploinsufficiency of A20

A20, encoded by TNFAIP3, is an effective anti-inflammatory molecule that plays a crucial role in inhibiting NF-κB signal transmission and is linked to multiple inflammatory diseases. It has been reported that the haploinsufficiency of A20 (HA20) caused by multiple base mutations in TNFAIP3 shows early-onset spontaneous Behçet-like disease. However, the mechanisms by which A20 mutations involved in inflammatory disease are incompletely defined. Herein, we reported a novel TNFAIP3 (c.1804A > T, p.T602S) variation, which has not been reported before. Summarizing the patient's immunodeficiency phenotype, we aimed to delineate the underlying mechanism for regulation of inflammation and immunity. Candidate genes associated with the Behçet-like phenotypes of the patient were screened and identified by using whole-exome and sanger sequencing. Functional studies were performed in A20(c.1804A > T, p.T602S) patient-derived peripheral blood mononuclear cells (PBMCs) and THP-1 cell lines by lentivirus mediating stable over-expression of A20 and A20(c.1804A > T, p.T602S) to analyze the activity of NF-κB signaling pathway. The clinical manifestations in patients with syndrome are Behçet-like disorder, and sequencing revealed heterozygous mutation in TNFAIP3 (c.1804A > T, p.T602S). Functional tests found that the PBMCs of the patient and his family carrying this heterozygous variant stimulated by LPS, TNF-α, or IL-1β, increased the levels of inflammatory factors and induced over-activation of the canonical NF-κB signaling pathway. Similar results were also observed in the stable transduction THP-1 (A20, c.1804A＞T) cell line stimulated by LPS, TNF-α or IL-1β. The novel loss-of-function A20 variation (c.1804A > T, p.T602S) causes over-activation of the canonical NF-κB signaling pathway and fail to terminate NF-κB signaling in response to stimulation by inflammatory cytokines. The variation triggers a dominantly-inherited Behçet-like disorder caused by haploinsufficiency of the A20 protein. Identification of the novel A20 mutation attaches great importance to prenatal diagnosis and fetal therapeutic intervention, drastically reducing the risk of newborns suffering from HA20.

Overexpression of the ubiquitin-editing enzyme A20 in the brain lesions of Multiple Sclerosis patients: moving from systemic to central nervous system

Multiple Sclerosis (MS) is a chronic demyelinating disease of the central nervous system (CNS) in which inflammation plays a key pathological role. Recent evidences showed that systemic inflammation induces increasing cell infiltration within meninges and perivascular spaces in the brain parenchyma, triggering resident microglial and astrocytic activation. The anti-inflammatory enzyme A20, also named TNF associated protein 3 (TNFAIP3), is considered a central gatekeeper in inflammation and peripheral immune system regulation through the inhibition of NF-kB. The TNFAIP3 locus is genetically associated to MS and its transcripts is downregulated in blood cells in treatment-naïve MS patients. Recently, several evidences in mouse models have led to hypothesize a function of A20 also in the CNS. Thus, here we aimed to unveil a possible contribution of A20 to the CNS human MS pathology. By immunohistochemistry/immunofluorescence and biomolecular techniques on post-mortem brain tissue blocks obtained from control cases (CC) and progressive MS cases, we demonstrated that A20 is present in CC brain tissues in both white matter (WM) regions, mainly in few parenchymal astrocytes, and in grey matter (GM) areas, in some neuronal populations. Conversely, in MS brain tissues, we observed increased expression of A20 by perivascular infiltrating macrophages, resident-activated astrocytes, and microglia in all the active and chronic active WM lesions. A20 was highly expressed also in the majority of active cortical lesions compared to the neighboring areas of normal-appearing grey matter (NAGM) and control GM, par-ticularly by activated astrocytes. We demonstrated increased A20 expression in the active MS plaques, particularly in macrophages and resident astrocytes, suggesting a key role of this molecule in chronic inflammation.

WITHDRAWN: Kdm5a promotes B cell activation in systemic lupus erythematosus via downregulation of A20 by histone modification

Systemic lupus erythematosus (SLE) is a classic autoimmune connective tissue disease, which leads to multiple organ system injury. Tumor necrosis factor-induced protein 3 (TNFAIP3), generally called A20, has been documented to go together with the development of SLE. However, the role and mechanism of A20 in the progression of SLE are still unrevealed. In our study, A20 was downregulated in B cells from SLE patients and B cell responsiveness was significantly elevated in SLE patients. Overexpression of A20 restrained the proliferation and induced the apoptosis of B cells. Additionally, trimethylation of histone H3 Lysine 4 (H3K4me3) was decreased in the A20 promoter of SLE B cells. Lysine demethylase 5A (Kdm5a) was significantly increased in B cells from SLE patients and negatively correlated with A20 expression. Further, Kdm5a knockdown increased the H3K4me3 level and A20 expression. More importantly, Kdm5a promoted the proliferation and inhibited the apoptosis of B cells in SLE via downregulation of A20. In general, Kdm5a promoted the proliferation and inhibited the apoptosis of B cells in SLE via downregulation of A20 by decreasing H3K4me3 enrichment level in the A20 promoter, suggesting a novel mechanism underlying SLE progression, and providing a promising therapeutic target for SLE. AVAILABILITY OF DATA AND MATERIALS: All data generated or analyzed during this study are included in this published article and its additional files.

RNF31 mediated ubiquitination of A20 aggravates inflammation and hepatocyte apoptosis through the TLR4/MyD88/NF-κB signaling pathway

Inflammatory cytokine storm is one of the main pathogenesis of acute liver injury, and accumulating evidence suggests that the E3 ubiquitin ligase ring finger protein 31 (RNF31) plays an important regulatory role in the activation of inflammatory pathways. We found that RNF31 expression was up-regulated in lipopolysaccharide (LPS)-treated HL-7702 cells. Western blotting results showed decreased expression of RNF31 and total ubiquitinated proteins after transfection of si-RNF31. The results of MTT assay indicated that cell viability was enhanced. Flow cytometry analysis showed that cell apoptosis and ROS content was decreased, and ELISA assay results exhibited that the inflammatory factors secretion was reduced. Interestingly, A20 protein expression was inhibited as RNF31 expression was upregulated. On this basis, we performed co-immunoprecipitation assays and found that RNF31 could interact with A20. Actinomycin tracing and proteasome inhibition experiments showed that RNF31 degrades A20 through the proteasome pathway. Furthermore, overexpression of A20 enhanced cell viability, reduced apoptosis, and inhibited ROS generation and inflammatory factor secretion. Mechanistic studies revealed that RNF31 was able to degrade A20, which affected the inflammatory response and hepatocyte apoptosis mediated by the toll like receptor 4 (TLR4)/myeloid differentiation factor88 (MyD88)/nuclear transcription factor-κB (NF-κB) signaling pathway. Moreover, knockdown of RNF31 attenuated the inflammatory response induced by d-Gal/LPS in mice with acute liver injury. In conclusion, RNF31 degrades A20 by ubiquitination and activates the TLR4/MyD88/NF-κB signaling pathway to aggravate acute liver injury.

Simultaneous transduction of dendritic cells with A20 and BTLA genes stimulates the development of stable and efficient tolerogenic dendritic cells and induces regulatory T cells

BackgroundThis study investigated the potential of simultaneous overexpression of A20 and B- and T-lymphocyte attenuator (BTLA) genes in dendritic cells (DCs) to develop a tolerogenic phenotype in DCs and investigate their capabilities for induction of immunosuppression. MethodsPlasmid vectors were designed harboring A20, BTLA, and A20 + BTLA genes and were transfected to HEK 293T cells to produce lentiviruses. DCs were transduced by the gene carrying viruses and evaluated for the surface expression of MHCII, CD40, and CD86 molecules by flow-cytometry. The mRNA expression of A20, BTLA, and CCR7 were determined. Mixed-lymphocyte reaction was conducted to evaluate the T cell stimulation potency and ELISA was used to measure the production of IL-10, TGF-β, and TNF-α. The potential of DCs for migration to lymph nodes and Treg induction were assessed by in vivo experiments. ResultsTransduction of DCs resulted in significantly decreased surface expression of CD40 and CD86 co-stimulators and upregulated A20, BTLA, and CCR7 mRNA expression. The IL-10 and TGF-β levels were enhanced significantly in the supernatant of LPS-treated DCs transduced with A20 + BTLA-containing virus group relative to the DCs transduced with pCDH vectors. DCs transduced with A20 + BTLA harboring vectors had higher migratory potential to mouse lymph nodes and caused the development of higher numbers of Treg cells compared with the DCs transduced with pCDH vectors. ConclusionsSimultaneous overexpression of A20 and BTLA genes in DCs caused development of tolerogenic DCs with a promoted potential in induction of Treg cells, accompanied by remarkable stability after inflammatory stimulation. All these offer a promising potential of such DCs in treating autoimmune and inflammatory disorders.

A20/TNFAIP3 Increases ENOS Expression in an ERK5/KLF2-Dependent Manner to Support Endothelial Cell Health in the Face of Inflammation.

Rationale: Decreased expression and activity of endothelial nitric oxide synthase (eNOS) in response to inflammatory and metabolic insults is the hallmark of endothelial cell (EC) dysfunction that preludes the development of atherosclerosis and hypertension. We previously reported the atheroprotective properties of the ubiquitin-editing and anti-inflammatory protein A20, also known as TNFAIP3, in part through interrupting nuclear factor-kappa B (NF-κB) and interferon signaling in EC and protecting these cells from apoptosis. However, A20's effect on eNOS expression and function remains unknown. In this study, we evaluated the impact of A20 overexpression or knockdown on eNOS expression in EC, at baseline and after tumor necrosis factor (TNF) treatment, used to mimic inflammation.Methods and Results: A20 overexpression in human coronary artery EC (HCAEC) significantly increased basal eNOS mRNA (qPCR) and protein (western blot) levels and prevented their downregulation by TNF. Conversely, siRNA-induced A20 knockdown decreased eNOS mRNA levels, identifying A20 as a physiologic regulator of eNOS expression. By reporter assays, using deletion and point mutants of the human eNOS promoter, and knockdown of eNOS transcriptional regulators, we demonstrated that A20-mediated increase of eNOS was transcriptional and relied on increased expression of the transcription factor Krüppel-like factor (KLF2), and upstream of KLF2, on activation of extracellular signal-regulated kinase 5 (ERK5). Accordingly, ERK5 knockdown or inhibition significantly abrogated A20's ability to increase KLF2 and eNOS expression. In addition, A20 overexpression in HCAEC increased eNOS phosphorylation at Ser-1177, which is key for the function of this enzyme.Conclusions: This is the first report demonstrating that overexpression of A20 in EC increases eNOS transcription in an ERK5/KLF2-dependent manner and promotes eNOS activating phosphorylation. This effect withstands eNOS downregulation by TNF, preventing EC dysfunction in the face of inflammation. This novel function of A20 further qualifies its therapeutic promise to prevent/treat atherosclerosis.

Correction to: Targeted Cardiac Overexpression of A20 Improves Left Ventricular Performance and Reduces Compensatory Hypertrophy After Myocardial Infarction.

Correction to: Targeted Cardiac Overexpression of A20 Improves Left Ventricular Performance and Reduces Compensatory Hypertrophy After Myocardial Infarction.

Pharmacological inhibition of GSK3 promotes TNFα-induced GM-CSF via up-regulation of ERK signaling in nasopharyngeal carcinoma (NPC)

Granulocyte-macrophage colony stimulating factor (GM-CSF) functions to drive nasopharyngeal cancer (NPC) metastasis via recruitment and activation of macrophages. However, the source and the regulation of GM-CSF in tumor microenvironment of NPC are not fully understood. In this study, we found that TNFα induced GM-CSF production in NPC CNE1, CNE2, and 5-8F cells in time- and dose-dependent manners. GM-CSF production was tolerant, because the pre-treatment of NPC cells with TNFα down-regulated the GM-CSF production induced by TNFα re-treatment. TNFα activated glycogen synthase kinase-3 (GSK-3), which is an enzyme to regulate glycogen synthesis, and also is a critical downstream element of the PI3K/Akt to regulate cell survival. GSK3 inhibitors up-regulated TNFα-induced GM-CSF, and reversed GM-CSF tolerance induced by TNFα pre-treatment, suggesting that GSK3 activation down-regulated GM-CSF production. GM-CSF down-regulation was not related to ubiquitin-editing enzyme A20. The over-expression of A20 did not regulate GM-CSF production induced by TNFα. However, GSK3 inhibitors up-regulated ERK activation, which contributed to the production of GM-CSF induced by TNFα, suggesting that GSK3 negatively regulated TNFα-induced GM-CSF via down-regulation of ERK signaling. Taking together, these results suggested that GSK3 pathway may be a target for the regulation of TNFα-induced GM-CSF in the tumor microenvironment.

The ubiquitin-editing enzyme A20 regulates synapse remodeling and efficacy

Ubiquitination and its reverse process, deubiquitination, play essential roles in neural development, function, and plasticity. A20, a ubiquitin editing enzyme that can remove K63-polyubiquitin chains from substrates and attach K48-polyubiquitin chains to them, is a critical component in the NF-κB signaling pathway in the immune system. This dual ubiquitin enzyme is also present in mammalian brains, but its potential role in neurons and synapses is unknown. We show that A20 in pyramidal neurons potently regulates dendritic arborization, spine morphogenesis, and synaptic transmission through an NF-κB-dependent mechanism. In cultured hippocampal neurons, overexpression of A20 reduced dendritic complexity and spine size and density, whereas A20 knockdown increased spine size and density, as well as clustering of the postsynaptic scaffold PSD-95 and glutamate receptor subunit GluA1. A20 effects in vitro were recapitulated in vivo where increasing or decreasing A20 expression in mouse brains reduced and enhanced spine density, respectively. Functionally, A20 knockdown significantly increased the amplitude, but not frequency of miniature excitatory postsynaptic currents, suggesting a role in postsynaptic efficacy. A20 negatively regulated NF-κB activation in neurons and A20 mutants deficient in either the deubiquitinase or the ubiquitin ligase activity failed to suppress NF-κB activation or reduce spine morphogenesis. Finally, selective inhibition of NF-κB abolished A20 knockdown-elicited spine formation, suggesting that A20 exerts its modulation on synapses through NF-κB signaling. Together, our study reveals a previously unknown role for A20, the only known ubiquitin editing enzyme with both deubiquitinase and ubiquitin ligase activity, in dendritic arborization, spine remodeling, and synaptic plasticity.

Regulation of dendritic cell function by A20 through high glucose-induced Akt2 signaling

A20 is a negative regulator of nuclear factor (NF)-κB-dependent inflammatory reaction in response to different stimuli by immune cells including dendritic cells (DCs), the most potent antigen-presenting cells involved in both the innate and adaptive immune response. Dendritic cells use glucose as carbon source to synthesize fatty acid and generate energy. Glucose enhances cell apoptosis mediated through PI3K/Akt, ERK1/2, and Bax/Bcl-2 pathways. The protein kinase Akt2/PKBβ is expressed in DCs and a regulator of Ca2+ influx, Na+/H+ exchanger activity, and migration of DCs. This study explored whether regulation of high glucose-induced DC function through Akt2 signaling is influenced by overexpression of A20. To this end, A20 protein expression was determined by western blotting and immunoprecipitation, secretion of inflammatory cytokines by ELISA, and expression of apoptotic markers by flow cytometry. As a result, treatment of mice with 10% high glucose enriched water increased secretion of insulin/IGF1 and reduced A20 protein level, the effects were blunted in Akt2−/− mice. Incubation of DCs with high glucose significantly decreased A20 protein expression in both control and Akt1-silenced DCs, but not in Akt2−/− DCs. Importantly, treatment of DCs with high glucose increased ceramide synthesis, caspase 8 activity, and annexin V binding in control DCs, the effects were abolished in Akt2−/− DCs or by A20 overexpression. In conclusion, regulation of A20 sensitive DC function by high glucose is mediated through insulin/IGF-1/Akt2 signaling.