A20 Regulation Research Articles

Ethanol suppresses the pulmonary response to Streptococcus pneumoniae: Role of NOD2 and NF-kB signaling in alveolar macrophages exposed to muramyl dipeptide.

Abstract Alcohol use decreases host immune defense and increases susceptibility to pathogens such as Streptococcus pneumoniae. NOD2 is an intracellular pattern recognition receptor that binds muramyl dipeptide (MDP), a cell wall component of S. pneumoniae, and activates NF-kB signaling. To limit inflammation, NF-kB signaling is negatively regulated in part by TNFAIP3 (TNF Alpha Induced Protein 3), or A20. We hypothesize that ethanol impairs NOD2 signaling and A20 expression in alveolar macrophages, leading to excessive inflammation and reduced bacterial clearance after infection. For in vivo experiments, we gavaged mice once daily for 3 consecutive days with water or ethanol (blood alcohol concentration ~ 80 mg/dL at 30 min), and infected with 104 cfu S. pneumoniae. In parallel experiments, MH-S cells were treated with 25 mM ethanol and 10 μg/ml MDP. We found that ethanol treated animals, compared to vehicle treated, had impaired respiratory function (17% lowered respiration rate [p < 0.01] and 33% increased penh [p = 0.09]) and increased lung bacteria (log10 increase of 0.8 [p = 0.11]) at 24 hours. RNA sequencing of alveolar macrophages from vehicle- or ethanol-gavaged mice revealed that ethanol intake led to 25% less Nod2 and 60% less A20 (p < 0.05) in the absence of infection. These results were confirmed by qPCR in ethanol-treated MH-S cells (40% decrease in Nod2 and 50% reduction in A20 [p < 0.01]), along with 15% lower A20 protein levels, which was sustained after MDP stimulation (29% less A20 compared to vehicle-treated, stimulated cells). Future experiments aim to further investigate ethanol’s effect on A20 regulation of NF-kB signaling, and will provide insight into immune dysregulation seen in alcohol-consuming patients with pneumonia.

MiR-873a-5p Targets A20 to Facilitate Morphine Tolerance in Mice.

Long-term morphine administration leads to tolerance and a gradual reduction in analgesic potency. Noncoding microRNAs (miRNAs) modulate gene expression in a posttranscriptional manner, and their dysregulation causes various diseases. Emerging evidence suggests that miRNAs play a regulatory role in the development of morphine tolerance. In the present study, we hypothesized that miR-873a-5p is a key functional small RNA that participates in the development and maintenance of morphine tolerance through the regulation of A20 (tumor necrosis factor α-induced protein 3, TNFAIP3) in mice. We measured the percentage of maximum possible effect (MPE %) to evaluate the analgesic effect of morphine. The expression of miR-873a-5p and its target gene A20 were determined after the morphine-tolerant model was successfully established. Intrathecal injection with lentivirus to intervene in the expression of A20 and the miR-873a-5p antagomir was used to explore the role of miR-873a-5p in the development of morphine tolerance. Chronic morphine administration significantly increased the expression of miR-873a-5p, which was inversely correlated with decreased A20 expression in the spinal cord of morphine-tolerant mice. Downregulation of miR-873a-5p in the spinal cord attenuated and partly reversed the development of morphine tolerance accompanied by overexpression of A20. Similarly, A20 was upregulated by a recombinant lentivirus vector, which attenuated and reversed the pathology of morphine tolerance by inhibiting the activation of nuclear factor (NF)-κB. Collectively, our results indicated that miR-873a-5p targets A20 in the spinal cord to facilitate the development of morphine tolerance in mice. Downregulating the expression of miR-873a-5p may be a potential strategy to ameliorate morphine tolerance.

Regulators of A20 (TNFAIP3): new drug-able targets in inflammation.

Persistent activation of the transcription factor Nuclear factor-κB (NF-κB) is central to the pathogenesis of many inflammatory disorders, including those of the lung such as cystic fibrosis (CF), asthma, and chronic obstructive pulmonary disease (COPD). Despite recent advances in treatment, management of the inflammatory component of these diseases still remains suboptimal. A20 is an endogenous negative regulator of NF-κB signaling, which has been widely described in several autoimmune and inflammatory disorders and more recently in terms of chronic lung disorders. However, the underlying mechanism for the apparent lack of A20 in CF, COPD, and asthma has not been investigated. Transcriptional regulation of A20 is complex and requires coordination of different transcription factors. In this review we examine the existing body of research evidence on the regulation of A20, concentrating on pulmonary inflammation. Special focus is given to the repressor downstream regulatory element antagonist modulator (DREAM) and its nuclear and cytosolic action to regulate inflammation. We provide evidence that would suggest the A20-DREAM axis to be an important player in (airway) inflammatory responses and point to DREAM as a potential future therapeutic target for the modification of phenotypic changes in airway inflammatory disorders. A schematic summary describing the role of DREAM in inflammation with a focus on chronic lung diseases as well as the possible consequences of altered DREAM expression on immune responses is provided.

Tumor necrosis factor α-induced protein 3 (A20) is dysregulated in pediatric Crohn disease.

PurposeA significant feature of pediatric inflammatory bowel diseases (IBD), which include Crohn disease (CD), and ulcerative colitis (UC), is failure to suppress inflammation. The inability to regulate inflammation renders a major challenge toward establishing effective treatments in IBD. Nuclear factor kappa-light-chain-enhancer of activated B-cells-induced inflammation is inhibited by A20 through interactions with TAX1BP1 (Tax1-binding protein 1) and A20-binding inhibitor of NF-κβ activation (ABIN)-1 (A20 binding and inhibitor of NF-κβ) and upon phosphorylation by inhibitor of nuclear factor kappa-β kinase subunit beta (IKKβ), which stabilizes it. We hypothesized that dysregulation of A20 is an important factor in uncontrolled inflammation in pediatric IBD.Patients and methodsGene expression of A20, IKKβ, ABIN-1, TAX1BP1, A20 protein, cytokine levels, and A20 phosphorylation was analyzed in the terminal ileum (TI) of 39 patients (14 non-IBD, 15 CD, and 10 UC). A20 expression and protein in T-84 cells and ex vivo biopsies of patients were measured after treatment with Escherichia coli strains or tumor necrosis factor (TNF)-α.ResultsTNF-α levels and A20 expression were increased in the TI of CD patients. A20 protein levels and ABIN-1 expression were low, TAX1BP1 expression was high, and IKKβ was unchanged. A20 expression positively correlated with biopsy TNF-α levels and inflammatory markers in CD patients. A20 phosphorylation appeared lower in CD patients. A20 expression in TI biopsies from CD patients and T84 cells was triggered with E. coli, strain LF82, while A20 protein levels remained unchanged.ConclusionWe describe a potential mechanism related to failure of A20 to suppress inflammation in CD, characterized by high A20 expression and low A20 protein levels. The dysregulation of A20 is potentially due to alterations in ABIN-1, and infection with E. coli strain LF82 could affect the function and stability of A20. Our study signifies an important finding in A20 regulation in IBD, which prevents it from suppressing inflammation.

TNFα induces tolerant production of CXC chemokines in colorectal cancer HCT116 cells via A20 inhibition of ERK signaling

Ubiquitin editing enzyme A20 functions as a tumor suppressor in various cancer. However, the mechanism for A20 regulation of cancer progress is not fully understood. In this study, we found that in human colorectal cancer HCT116 cells, TNFα induced a tolerant production of CXC chemokines, including CXCL1, 2, and 8 in a dose and time dependent manner. TNFα pre-treatment of HCT116 cells down-regulated the chemokine production induced by TNFα re-treatment. TNFα induced the phosphorylation of MAPKs ERK, JNK, P38 and NF-κB P65, but only ERK inhibition decreased TNFα-induced chemokine production. Both RT-PCR and FACS results showed that TNFα treatment did not regulate the expression of TNF receptors. However, TNFα up-regulated the expression of A20 at both mRNA and protein levels significantly. TNFα pre-treatment inhibited the signal transduction of MAPKs induced by TNFα re-stimulation, and A20 over-expression decreased the signal transduction of ERK and P38. Meanwhile, A20 inhibition by RNA interference reversed chemokine down-regulation induced by TNFα re-stimulation after TNFα pre-treatment. Taken together, these results suggested that in human colorectal cancer cells, A20 may function to inhibit cancer progression via down-regulation of TNFα-induced chemokine production by suppression of ERK signaling.

The Effects of miR-136-5p-Mediated Regulation of A20 in Astrocytes from Cultured Spinal Cord Cultured Cells In Vitro

Background/Aims: This study focused on investigating the regulatory mechanism of miR-136-5p in mouse astrocytes stimulated with interleukin-17(IL-17). Methods: C57BL/6 mouse astrocytes were stimulated with IL-17 (100ng/ml) for various periods of time (0-48 hours) and at various doses (0-200 ng), and the expression levels of inflammatory cytokine and chemokine genes (IL-6, TNF-α, MCP-1, MCP-5 and MIP-2) were then detected by real-time PCR. The expression of the A20 gene was measured with real-time PCR in cells that were stimulated with IL-17 (50 ng/ml) for various periods of time (0-48 hours). C57BL/6 mouse astrocytes were transfected with Ctrl-anti-miR-136-5p or LNA -anti-miR-136-5p for 48 h. Thereafter, the cells were stimulated with or without IL-17 (50ng/ml) for 6 h. The level of A20 protein (TNFα-induced protein 3, TNFAIP3) was detected by Western blot analysis. Results: (1) Compared with the DMEM control group, within six hours, IL-17 stimulation significantly increased the expression levels of inflammatory cytokine and chemokine genes and clearly decreased the expression level of the A20 protein. (2) Without IL-17 stimulation, the expression level of the miR-136-5p gene was significantly decreased, whereas in the miR-136-5p-inhibition group, the A20 protein expression was elevated. IL-17 stimulation slightly decreased the expression of the A20 protein in the miR-136-5p-inhibition group, but it was still slightly higher than in the control group. Conclusion: This study demonstrated that miR-136-5p affected the expression of A20 in IL-17-stimulated astrocytes.

Aberrant TNFAIP3 Induces Mesenchymal Stem Cell Impairment Via the NF-Kb/SMAD7 Signaling Pathway in Immune Thrombocytopenia

IntroductionImmune thrombocytopenia (ITP) is an autoimmune disease that is mainly caused by antibody-mediated platelet destruction and a variable reduction of thrombopoiesis. Mesenchymal stem cells (MSCs) play important roles in the physiology and homeostasis of the hematopoietic system, including the support of megakaryocytic differentiation from CD34+ hematopoietic progenitor cells. Tumor necrosis factor alpha-induced protein 3(TNFAIP3, A20) plays a key role in the termination of NF-kB signaling, which is a central regulator of immunopathology. Human genetic studies have linked single-nucleotide polymorphisms of TNFAIP3 with susceptibility to multiple human diseases, including ITP. SMAD7 is another important factor that regulates megakaryocytic differentiation. All-trans retinoic acid (ATRA) could inhibit NF-kB signaling, and ATRA therapy could induce significant changes of Treg cells to induce a response in patients with chronic ITP.Our previous study confirmed the enhanced senescence and apoptosis of BM-MSCs in patients with ITP, and MSCs from ITP patients showed a lower capacity for inhibiting the proliferation of activated T-cells inducing Treg cells, though whether TNFAIP3 plays a role in this deficiency remains unclear.MethodsBM-MSCs and CD34+cells were isolated from ITP patients and healthy control subjects. Representative immunostaining for p-p65, A20, and SMAD7 were analyzed by staining ITP BM pathological sections. The expression of cytokines as well as A20, NF-kB, and phospho-SMAD7 were assessed by western blot analysis. Real-time transcription-polymerase chain reaction (RT-PCR) analysis was used to evaluate the expression of TNFAIP3, NF-kB and SMAD mRNA in MSC cells. Vectors carrying either IkB/IKKβ-specific shRNA, A20 cDNA/TNFAIP3-specific shRNA or SMAD7-specific shRNA were transduced into the MSCs from either healthy control subjects or ITP patients. The different MSCs were co-cultured with CD34+ cells separately. We compared the ability of MSCs to support megakaryocytopoiesis and thrombopoiesisby estimating the relative percentages of these cells using flow cytometryas well as CFU counts of megakaryocytes and proplatelets and platelets in culture.ResultsThe ability of ITP-MSCs to support megakaryocytic differentiation and thrombopoiesis of CD34+ cells was impaired. Highly significant correlations between A20 and phospho-p65, A20 and SMAD7, p-p65 and SMAD7 immunostaining intensities were revealed in the BM pathological sections from the ITP patients. There was a significant decrease in A20 expression in the MSCs from patients with ITP. Increased NF-kB/SMAD7 signaling was also observed in BM-MSCs from ITP patients. In the co-cultures with CD34+ cells, NF-kB was overexpressed in MSCs from healthy control subjects after transfection with IKKβ-specific shRNA, resulting in MSC deficiency and a reduction in megakaryocytic differentiation and thrombopoiesis. Knockdown of A20 expression using A20-specific shRNA in MSCs affected megakaryocytopoiesis and thrombopoiesis. However, in the ITP-MSCs, IkB knockdown rescued the inhibition of megakaryocytopoiesis and thrombopoiesis by decreasing the expression of NF-kB. Because the native expression of A20 was lower in ITP-MSCs, exogenous expression from A20-cDNA was amplified, which facilitated the differentiation of megakaryocytes. Knockdown of SMAD7 expression by siRNA rescued the impaired function of MSCs from patients with ITP. ATRA could address the abnormal A20/NF-kB/SMAD signaling pathway and thus improve the promotional activity of ITP-MSCs in megakaryocytopoiesis.ConclusionsWe explored the decreased expression of A20 and increased expression of NF-kB and SMAD7 in ITP-MSCs. The ability of ITP-MSCs to support megakaryocytic differentiation and thrombopoiesis of CD34+ cells was impaired. We demonstrated firstly that a pathological reduction of the A20 levels induced NF-kB/SMAD7 pathway activation, causing a deficiency in MSCs from patients with ITP. Therefore, the aberrant signaling of the A20/NF-kB/SMAD pathway may be a cause of the pathogenesis of ITP. ATRA could restore the abnormal signaling of the A20/NF-kB/SMAD pathway and thus improve the promotional activity of ITP-MSCs in megakaryocytopoiesis. This study indicates that the regulation of A20 could be involved in the therapeutic strategies against NF-kB signaling in ITP. DisclosuresNo relevant conflicts of interest to declare.

A20 suppresses canonical Smad-dependent fibroblast activation: novel function for an endogenous inflammatory modulator.

BackgroundThe ubiquitin-editing cytosolic enzyme A20, the major negative regulator of toll-like receptor (TLR)-mediated cellular inflammatory responses, has tight genetic linkage with systemic sclerosis (SSc). Because recent studies implicate endogenous ligand-driven TLR signaling in SSc pathogenesis, we sought to investigate the regulation, role and mechanism of action of A20 in skin fibroblasts.MethodA20 expression and the effects of forced A20 expression or siRNA-mediated A20 knockdown on fibrotic responses induced by transforming growth factor-ß (TGF-ß) were evaluated was evaluated in explanted human skin fibroblasts. Additionally, A20 regulation by TGF-ß, and by adiponectin, a pleiotropic adipokine with anti-fibrotic activity, was evaluated.ResultsIn normal fibroblasts, TGF-ß induced sustained downregulation of A20, and abrogated its TLR4-dependent induction. Forced expression of A20 aborted the stimulation of collagen gene expression and myofibroblast transformation induced by TGF-ß, and disrupted canonical Smad signaling and Smad-dependent transcriptional responses. Conversely, siRNA-mediated knockdown of A20 enhanced the amplitude of fibrotic responses elicited by TGF-ß. Adiponectin, previously shown to block TLR-dependent fibrotic responses, elicited rapid and sustained increase in A20 accumulation in fibroblasts.ConclusionThese results identify the ubiquitin-editing enzyme A20 as a novel endogenous mechanism for negative regulation of fibrotic response intensity. Systemic sclerosis-associated genetic variants of A20 that cause impaired A20 expression or function, combined with direct suppression of A20 by TGF-ß within the fibrotic milieu, might play a significant functional role in persistence of fibrotic responses, while pharmacological augmentation of A20 inhibitory pathway activity might represent a novel therapeutic strategy.Electronic supplementary materialThe online version of this article (doi:10.1186/s13075-016-1118-7) contains supplementary material, which is available to authorized users.

Glucocorticoid and TNF signaling converge at A20 (TNFAIP3) to repress airway smooth muscle cytokine expression.

Airway smooth muscle is a major target tissue for glucocorticoid (GC)-based asthma therapies, however, molecular mechanisms through which the GC receptor (GR) exerts therapeutic effects in this key airway cell type have not been fully elucidated. We previously identified the nuclear factor-κB (NF-κB) inhibitor, A20 (TNFAIP3), as a mediator of cytokine repression by glucocorticoids (GCs) in airway epithelial cells and defined cooperative regulation of anti-inflammatory genes by GR and NF-κB as a key mechanistic underpinning of airway epithelial GR function. Here, we expand on these findings to determine whether a similar mechanism is operational in human airway smooth muscle (HASM). Using HASM cells derived from normal and fatal asthma samples as an in vitro model, we demonstrate that GCs spare or augment TNF-mediated induction of A20 (TNFAIP3), TNIP1, and NFKBIA, all implicated in negative feedback control of NF-κB-driven inflammatory processes. We applied chromatin immunoprecipitation and reporter analysis to show that GR and NF-κB directly regulate A20 expression in HASM through cooperative induction of an intronic enhancer. Using overexpression, we show for the first time that A20 and its interacting partner, TNIP1, repress TNF signaling in HASM cells. Moreover, we applied small interfering RNA-based gene knockdown to demonstrate that A20 is required for maximal cytokine repression by GCs in HASM. Taken together, our data suggest that inductive regulation of A20 by GR and NF-κB contributes to cytokine repression in HASM.

Biphasic regulation of A20 gene expression during human cytomegalovirus infection.

BackgroundThe A20 ubiquitin-editing enzyme is a target of nuclear factor kappa B (NF-κB) and also plays a key role in regulating the NF-κB signaling pathway. NF-κB activity is increased during human cytomegalovirus (HCMV) infection and HCMV appears to be adapted to this change. To better understand the regulation of NF-κB signaling during HCMV infection, we investigated how A20 expression is controlled during HCMV infection.MethodsThe expression level of A20 in human fibroblast cells infected with HCMV or UV-inactivated virus (UV-HCMV) was measured by immunoblot analysis, cell staining, and quantitative real-time PCR. Changes of histone modifications on the A20 promoter were determined by chromatin immunoprecipitation assays. Lentiviral vectors were used to knockdown A20 in fibroblast cells.ResultsA20 expression was increased at early times after HCMV infection. This increase of the A20 protein level was promoted by viral gene expression under low viral load conditions. The viral IE1 protein, which is known to activate NF-κB, increased the A20 promoter activity through the upstream NF-κB sites in reporter assays, suggesting that IE1 is at least partly involved in A20 induction. Analysis of A20 expression with a high viral load demonstrated that the A20 regulation by HCMV was biphasic; both A20 protein and mRNA levels were increased at the early stage of infection, but decreased at the late stage. Under high viral load conditions, A20 upregulation was more profound with UV-HCMV than with HCMV, indicating a role of the viral gene product(s) in limiting A20 induction. Consistently, more histone modifications for euchromatin were found on the A20 promoter during UV-HCMV infection than with HCMV infection. A20 knockdown by shRNA reduced HCMV growth.ConclusionThese results suggest that the biphasic regulation of A20 expression may be important for productive HCMV infection.

Imiquimod induces apoptosis of squamous cell carcinoma (SCC) cells via regulation of A20.

Imiquimod, a nucleoside analogue of the imidazoquinoline family, is being used to treat various cutaneous cancers including squamous cell carcinoma (SCC). Imiquimod activates anti-tumor immunity via Toll-like receptor 7 (TLR7) in macrophage and other immune cells. Imiquimod can also affect tumor cells directly, regardless of its impact on immune system. In this study, we demonstrated that imiquimod induced apoptosis of SCC cells (SCC12) and A20 was involved in this process. When A20 was overexpressed, imiquimod-induced apoptosis was markedly inhibited. Conversely, knockdown of A20 potentiated imiquimod-induced apoptosis. Interestingly, A20 counteracted activation of c-Jun N-terminal kinase (JNK), suggesting that A20-regulated JNK activity was possible mechanism underlying imiquimod-induced apoptosis of SCC12 cells. Finally, imiquimod-induced apoptosis of SCC12 cells was taken place in a TLR7-independent manner. Our data provide new insight into the mechanism underlying imiquimod effect in cutaneous cancer treatment.

A20 expressing tumors and anticancer drug resistance.

Resistance to anticancer drugs is a major impediment to treating patients with cancer. The molecular mechanisms deciding whether a tumor cell commits to cell death or survives under chemotherapy are complex. Mounting evidence indicates a critical role of cell death and survival pathways in determining the response of human cancers to chemotherapy. Nuclear factor-kappaB (NF-kappaB) is a eukaryotic transcription factor on the crossroad of a cell's decision to live or die. Under physiological conditions, NF-kappaB is regulated by a complex network of endogenous pathway modulators. Tumor necrosis factor alpha induced protein 3 (tnfaip3), a gene encoding the A20 protein, is one of the cell's own inhibitory molecule, which regulates canonical NF-kappaB activation by interacting with upstream signaling pathway components. Interestingly, A20 is also itself a NF-kappaB dependent gene, that has been shown to also exert cell-type specific anti- or pro-apoptotic functions. Recent reports suggest that A20 expression is increased in a number of solid human tumors. This likely contributes to both carcinogenesis and response to chemotherapy. These data uncover the complexities of the mechanisms involved in A20's impact on tumor development and response to treatment, highlighting tumor and drug-type specific outcomes. While A20-targeted therapies may certainly add to the chemotherapeutic armamentarium, better understanding of A20 regulation, molecular targets and function(s) in every single tumor and in response to any given drug is required prior to any clinical implementation. Current renewed appreciation of the unique molecular signature of each tumor holds promise for personalized chemotherapeutic regimen hopefully comprising specific A20-targeting agents i.e., both inhibitors and enhancers.

Regulation of A20 and other OTU deubiquitinases by reversible oxidation

Protein ubiquitination is a highly versatile posttranslational modification that regulates as diverse processes as protein degradation and kinase activation. Deubiquitinases hydrolyse ubiquitin modifications from proteins and are hence key regulators of the ubiquitin system. Ovarian Tumour (OTU) deubiquitinases comprise a family of fourteen human enzymes, many of which regulate cellular signalling pathways. OTU deubiquitinases are cysteine proteases that cleave polyubiquitin (polyUb) chains in vitro and in cells, but little is currently known about their regulation. Here we show that OTU deubiquitinases are susceptible to reversible oxidation of the catalytic cysteine residue. High-resolution crystal structures of the catalytic domain of A20 in four different oxidation states reveal that the reversible form of A20 oxidation is a cysteine sulphenic acid intermediate, which is stabilised by the architecture of the catalytic centre. Using chemical tools to detect sulphenic acid intermediates, we show that many OTU deubiquitinases undergo reversible oxidation upon treatment with H2O2, revealing a new mechanism to regulate deubiquitinase activity.

Effect of pulsed electromagnetic field on inflammatory pathway markers in RAW 264.7 murine macrophages

In the treatment of bacterial infections, antibiotics have proven to be very effective, but the way in which antibiotics are dosed can create a lag time between the administration of the drug and its absorption at the site of insult. The time it takes an antibiotic to reach therapeutic levels can often be significantly increased if the vascular system is compromized. Bacteria can multiply pending the delivery of the drug, therefore, developing treatments that can inhibit the inflammatory response while waiting for antibiotics to take effect could help prevent medical conditions such as septic shock. The aim of this study was to examine the effect of a pulsed electromagnetic field on the production of inflammatory markers tumor necrosis factor (TNF), transcription factor nuclear factor kappa B (NFkB), and the expression of the A20 (tumor necrosis factor-alpha-induced protein 3), in an inflamed-cell model. Lipopolysaccharide-challenged cells were exposed to a pulsed electromagnetic field at various frequencies in order to determine which, if any, frequency would affect the TNF-NFkB-A20 inflammatory response pathway. Our study revealed that cells continuously exposed to a pulsed electromagnetic field at 5 Hz demonstrated significant changes in the downregulation of TNF-α and NFkB and also showed a trend in the down regulation of A20, as compared with controls. This treatment could be beneficial in modulating the immune response, in the presence of infection.

DSIF Restricts NF-κB Signaling by Coordinating Elongation with mRNA Processing of Negative Feedback Genes

NF-κB is central for immune response and cell survival, and its deregulation is linked to chronic inflammation and cancer through poorly defined mechanisms. IκBα and A20 are NF-κB target genes and negative feedback regulators. Upon their activation by NF-κB, DSIF is recruited, P-TEFb is released, and their elongating polymerase II (Pol II) C-terminal domain (CTD) remains hypophosphorylated. We show that upon DSIF knockdown, mRNA levels of a subset of NF-κB targets are not diminished; yet much less IκBα and A20 protein are synthesized, and NF-κB activation is abnormally prolonged. Further analysis of IκBα and A20 mRNA revealed that a significant portion is uncapped, unspliced, and retained in the nucleus. Interestingly, the Spt5 C-terminal repeat (CTR) domain involved in elongation stimulation through P-TEFb is dispensable for IκBα and A20 regulation. These findings assign a function for DSIF in cotranscriptional mRNA processing when elongating Pol II is hypophosphorylated and define DSIF as part of the negative feedback regulation of NF-κB.

A20 Regulation of Nuclear Factor–κB

Persistent activation of NF-κB is central to the pathogenesis of many inflammatory lung disorders, including cystic fibrosis, asthma, and chronic obstructive pulmonary disease. A20 is an endogenous negative regulator of NF-κB signaling, which has been widely described in autoimmune and inflammatory disorders, including diabetes and Crohn's disease, but which has received little attention in terms of chronic lung disorders. This review examines the existing body of research on A20 regulation of NF-κB signaling and details the mechanism and regulation of A20 action focusing, where possible, on pulmonary inflammation. A20 and its associated signaling molecules are highlighted as being of potential therapeutic interest for the treatment of inflammatory disorders, and a proposed model of A20 activity in inflammatory lung disease is provided.

Nuclear Factor-κB Regulates β-Cell Death

Apoptotic β-cell death is central to the pathogenesis of type 1 diabetes and may be important in islet graft rejection. Despite this, genetic control of β-cell apoptosis is only poorly understood. We report that inhibition of gene transcription sensitized β-cells to tumor necrosis factor (TNF)-α–induced apoptosis, indicating the presence of a regulated antiapoptotic response. Using oligonucleotide microarrays and real-time PCR, we identified TNFAIP3/A20 as the most highly regulated antiapoptotic gene expressed in cytokine-stimulated human and mouse islets. Cytokine induction of A20 mRNA in primary islets and insulinoma cells was rapid and observed within 1 h, consistent with A20 being an immediate early response gene in β-cells. Regulation of A20 was nuclear factor-κB (NF-κB)–dependent, two NF-κB sites within the A20 promoter were found to be necessary and sufficient for A20 expression in β-cells. Activation of NF-κB by TNF receptor–associated factor (TRAF) 2, TRAF6, NF-κB–inducing kinase, or protein kinase D, which transduce signals downstream of Toll-like receptors, TNF receptors, and free radicals, respectively, were all potent activators of the A20 promoter. Moreover, A20 expression was induced in transplanted islets in vivo. Finally, A20 expression was sufficient to protect β-cells from TNF-induced apoptosis. These data demonstrate that A20 is the cardinal antiapoptotic gene in β-cells. Further, A20 expression is NF-κB dependent, thus linking islet proinflammatory gene responses with protection from apoptosis.

ABIN-2 protects endothelial cells from death and has a role in the antiapoptotic effect of angiopoietin-1

AbstractA20 binding inhibitor of NF-κB activation-2, ABIN-2, is a newly identified intracellular protein that interacts with the zinc finger protein A20. ABIN-2 inhibits nuclear factor-κB (NF-κB) activity and is a possible effector of A20 regulation of NF-κB. Although A20 is a potent inhibitor of endothelial apoptosis, the effect of ABIN-2 on apoptosis is not known. ABIN-2 also interacts with the endothelial receptor Tie2. This receptor is essential for blood vessel formation and promotes endothelial survival. Here we examine the effects of ABIN-2 on endothelial cell apoptosis and its potential involvement in Tie2-mediated endothelial survival. ABIN-2 was found to inhibit endothelial apoptosis and rescue cells from death following growth factor deprivation. The inhibitors of phosphatidylinositol-3 kinase, wortmannin and LY294002, suppressed ABIN-2 inhibition of endothelial cell death. Deletion of the carboxy-terminus of ABIN-2 removed its ability to inhibit apoptosis. Expression of truncated ABIN-2 prevented the Tie2-activating ligand angiopoietin-1 from inhibiting endothelial cell death. (Blood. 2003;102:4407-4409)

Lymphoid expression and regulation of A20, an inhibitor of programmed cell death.

A20 is a cytokine-inducible primary response gene that encodes a protein that inhibits apoptosis. We cloned and characterized a murine A20 cDNA and mapped the gene to mouse chromosome 10, 3.5 centimorgans proximal to the c-myb locus. Using the murine cDNA, we conducted in situ hybridization studies to examine patterns of A20 expression in mouse embryos and postnatal tissues. Both temporally restricted and tissue-specific patterns of A20 expression were observed, with strikingly high levels in lymphoid organs, including the thymus, spleen, and gut-associated lymphoid tissue. Northern analysis using purified human lymphocyte populations demonstrated that A20 is constitutively expressed in both immature and mature thymocyte subpopulations, as well as in resting peripheral T cells. Activation leads to a down-regulation of A20 expression in both mature thymocytes and peripheral T cells. This pattern of expression and regulation differs markedly from that observed in prior studies with cell lines in which stimulation led to an induction of A20 expression. Additionally, this is the first demonstration of constitutive expression of A20 in primary cells. The data suggest a role for A20 in the function of the lymphoid system.