A20 deficiency causes spontaneous neuroinflammation in mice.

BACKGROUND: A20 (TNFAIP3) is a pleiotropic NFκB-dependent gene that terminates NFκB activation in response to inflammatory stimuli. The potent anti-inflammatory properties of A20 are well characterized in several organs. However, little is known about its role in the brain. In this study, we investigated the brain phenotype of A20 heterozygous (HT) and knockout (KO) mice. METHODS: The inflammatory status of A20 wild type (WT), HT and KO brain was determined by immunostaining, quantitative PCR, and Western blot analysis. Cytokines secretion was evaluated by ELISA. Quantitative results were statistically analyzed by ANOVA followed by a post-hoc test. RESULTS: Total loss of A20 caused remarkable reactive microgliosis and astrogliosis, as determined by F4/80 and GFAP immunostaining. Glial activation correlated with significantly higher mRNA and protein levels of the pro-inflammatory molecules TNF, IL-6, and MCP-1 in cerebral cortex and hippocampus of A20 KO, as compared to WT. Basal and TNF/LPS-induced cytokine production was significantly higher in A20 deficient mouse primary astrocytes and in a mouse microglia cell line. Brain endothelium of A20 KO mice demonstrated baseline activation as shown by increased vascular immunostaining for ICAM-1 and VCAM-1, and mRNA levels of E-selectin. In addition, total loss of A20 increased basal brain oxidative/nitrosative stress, as indicated by higher iNOS and NADPH oxidase subunit gp91phox levels, correlating with increased protein nitration, gauged by nitrotyrosine immunostaining. Notably, we also observed lower neurofilaments immunostaining in A20 KO brains, suggesting higher susceptibility to axonal injury. Importantly, A20 HT brains showed an intermediate phenotype, exhibiting considerable, albeit not statistically significant, increase in markers of basal inflammation when compared to WT. CONCLUSIONS: This is the first characterization of spontaneous neuroinflammation caused by total or partial loss of A20, suggesting its key role in maintenance of nervous tissue homeostasis, particularly control of inflammation. Remarkably, mere partial loss of A20 was sufficient to cause chronic, spontaneous low-grade cerebral inflammation, which could sensitize these animals to neurodegenerative diseases. These findings carry strong clinical relevance in that they question implication of identified A20 SNPs that lower A20 expression/function (phenocopying A20 HT mice) in the pathophysiology of neuroinflammatory diseases.

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.

A20--an omnipotent protein in the liver: prometheus myth resolved?

Contribution of NF-kappaB inhibitory and ubiquitin-editing A20 (tnfaip3) to the liver's protective response to injury, particularly to its anti-inflammatory armamentarium, is exemplified by the dramatic phenotype of A20 knockout mice that die prematurely of unfettered inflammation predominantly in the liver. A number of additional studies originating from our laboratory and others clearly demonstrate that A20 is part of the liver response to injury and resection. Upregulation of A20 in hepatocytes serves a broad hepatoprotective goal through combined anti-inflammatory, anti-apoptotic, anti-oxidant and pro-regenerative functions. The molecular basis for A20's hepatoprotective functions were partially resolved and include blockade of NF-kappaB activation in support of its anti-inflammatory function, inhibition of pro-caspase 8 cleavage in support of its anti-apoptotic function, increasing Peroxisome Proliferator Activated Receptor alpha (PPARalpha) expression in support of its anti-oxidant function, and decreasing Cyclin Dependent Kinase Inhibitor p21 while boosting IL-6/STAT3 proliferative signals as part of its pro-regenerative function. In experimental animal models, overexpression of A20 in the liver protects from radical acute fulminant toxic hepatitis, lethal hepatectomy, and severe liver ischemia reperfusion injury (IRI), and allows successful engraftment of marginal liver grafts. Conversely, partial loss of A20, as in A20 heterozygote mice, significantly impairs liver regeneration and damage, which confers high lethality to an otherwise safe procedure i.e., 2/3 partial hepatectomy. This is the ultimate proof of the physiologic role of A20 in liver regeneration and repair. In recent work, A20's functions in the liver have expanded to encompass regulation of lipid and glucose metabolism, unlocking a whole new set of metabolic diseases that could be affected by A20. In this chapter we review all available data regarding A20's physiologic role in the liver, and Reflect on the clinical implication of these findings with regard to A20-based therapies in the context of liver transplantation, resection of large liver tumors, liver fibrosis, and metabolic liver diseases.

Mechanism of purinergic activation of endothelial nitric oxide synthase in endothelial cells.

BACKGROUND: Decreased endothelial nitric oxide (NO) synthase (eNOS) activity and NO production are critical contributors to the endothelial dysfunction and vascular complications observed in many diseases, including diabetes mellitus. Extracellular nucleotides activate eNOS and increase NO generation; however, the mechanism of this observation is not fully clarified. METHODS AND RESULTS: To elucidate the signaling pathway(s) leading to nucleotide-mediated eNOS phosphorylation at Ser-1177, human umbilical vein endothelial cells were treated with several nucleotides, including ATP, UTP, and ADP, in the presence or absence of selective inhibitors. These experiments identified P2Y1, P2Y2, and possibly P2Y4 as the purinergic receptors involved in eNOS phosphorylation and demonstrated that this process was adenosine independent. Nucleotide-induced eNOS phosphorylation and activity were inhibited by BAPTA-AM (an intracellular free calcium chelator), rottlerin (a protein kinase Cdelta inhibitor), and protein kinase Cdelta siRNA. In contrast, blockade of AMP-activated protein kinase, calcium/calmodulin-dependent kinase II, calcium/calmodulin-dependent kinase kinase, serine/threonine protein kinase B, protein kinase A, extracellular signal-regulated kinase 1/2, and p38 mitogen-activated protein kinase did not affect nucleotide-mediated eNOS phosphorylation. CONCLUSIONS: The present study indicates that extracellular nucleotide-mediated eNOS phosphorylation is calcium and protein kinase Cdelta dependent. This newly identified signaling pathway opens new therapeutic avenues for the treatment of endothelial dysfunction.

Extracellular nucleotides and adenosine independently activate AMP-activated protein kinase in endothelial cells: involvement of P2 receptors and adenosine transporters.

AMP-activated protein kinase (AMPK) plays a key role in the regulation of energy homeostasis and is activated in response to cellular stress, including hypoxia/ischemia and hyperglycemia. The stress events are accompanied by rapid release of extracellular nucleotides from damaged tissues or activated endothelial cells (EC) and platelets. We demonstrate that extracellular nucleotides (ATP, ADP, and UTP, but not UDP) and adenosine independently induce phosphorylation and activation of AMPK in human umbilical vein EC (HUVEC) by the mechanism that is not linked to changes in AMP:ATP ratio. HUVEC express NTPDases, as well as 5'-nucleotidase; hence, nucleotides can be metabolized to adenosine. However, inhibition of 5'-nucleotidase had no effect on ATP/ADP/UTP-induced phospho- rylation of AMPK, indicating that AMPK activation occurred as a direct response to nucleotides. Nucleotide-evoked phosphorylation of AMPK in HUVEC was mediated by P2Y1, P2Y2, and/or P2Y4 receptors, whereas P2Y6, P2Y11, and P2X receptors were not involved. The nucleotide-induced phosphorylation of AMPK was affected by changes in the concentration of intracellular Ca2+ and by Ca2+/calmodulin-dependent kinase kinase (CaMKK), although most likely it was not dependent on LKB1 kinase. Adenosine-induced phosphorylation of AMPK was not mediated by P1 receptors but required adenosine uptake by equilibrative nucleoside transporters followed by its (intracellular) metabolism to AMP. Moreover, adenosine effect was Ca2+ and CaMKK independent, although probably associated with upstream LKB1. We hypothesize that P2 receptors and adenosine transporters could be novel targets for the pharmacological regulation of AMPK activity and its downstream effects on EC function.

The P2X7 Receptor in Inflammatory Diseases: Angel or Demon?

Under physiological conditions, adenosine triphosphate (ATP) is present at low levels in the extracellular milieu, being massively released by stressed or dying cells. Once outside the cells, ATP and related nucleotides/nucleoside generated by ectonucleotidases mediate a high evolutionary conserved signaling system: the purinergic signaling, which is involved in a variety of pathological conditions, including inflammatory diseases. Extracellular ATP has been considered an endogenous adjuvant that can initiate inflammation by acting as a danger signal through the activation of purinergic type 2 receptors-P2 receptors (P2Y G-protein coupled receptors and P2X ligand-gated ion channels). Among the P2 receptors, the P2X7 receptor is the most extensively studied from an immunological perspective, being involved in both innate and adaptive immune responses. P2X7 receptor activation induces large-scale ATP release via its intrinsic ability to form a membrane pore or in association with pannexin hemichannels, boosting purinergic signaling. ATP acting via P2X7 receptor is the second signal to the inflammasome activation, inducing both maturation and release of pro-inflammatory cytokines, such as IL-1ß and IL-18, and the production of reactive nitrogen and oxygen species. Furthermore, the P2X7 receptor is involved in caspases activation, as well as in apoptosis induction. During adaptive immune response, P2X7 receptor modulates the balance between the generation of T helper type 17 (Th17) and T regulatory (Treg) lymphocytes. Therefore, this receptor is involved in several inflammatory pathological conditions. In infectious diseases and cancer, P2X7 receptor can have different and contrasting effects, being an angel or a demon depending on its level of activation, cell studied, type of pathogen, and severity of infection. In neuroinflammatory and neurodegenerative diseases, P2X7 upregulation and function appears to contribute to disease progression. In this review, we deeply discuss P2X7 receptor dual function and its pharmacological modulation in the context of different pathologies, and we also highlight the P2X7 receptor as a potential target to treat inflammatory related diseases.

Mitochondrial energy metabolism is markedly impaired by D-2-hydroxyglutaric acid in rat tissues.

Tissue accumulation of high amounts of D-2-hydroxyglutaric acid (DGA) and l-2-hydroxyglutaric acid (LGA) is the biochemical hallmark of the inherited neurometabolic disorders D-2-hydroxyglutaric aciduria (DHGA) and l-2-hydroxyglutaric aciduria (LHGA), respectively. Patients affected by DHGA predominantly present neurological and cardiomuscular symptoms, while those with LHGA have mainly severe neurological symptoms. Lactic aciduria and/or lactic acidemia may also occur in both disorders, suggesting mitochondrial dysfunction. We have previously reported that cytochrome c oxidase (COX) activity is severely inhibited by DGA in rat cerebral cortex and human skeletal muscle. In the present study, we initially evaluated the role of DGA and LGA on the mitochondrial respiratory chain complex activities, as well as CO2 on production in cardiac and skeletal muscle from 30-day-old Wistar rats. DGA significantly inhibited COX and ATP synthase (F0F1-ATP synthase) activities, in contrast to the other activities of the respiratory chain enzymes which were not affected by DGA in both muscular tissues. In addition, CO2 production was also markedly reduced by DGA in rat skeletal and cardiac muscles. On the other hand, LGA did not interfere with any of the respiratory chain complex activities studied, neither with CO2 generation. We also measured mitochondrial respiratory parameters in rat brain mitochondrial preparations in the presence of DGA and LGA. Both metabolites significantly lowered the respiratory control ratio in the presence of glutamate/malate and succinate. Since the metabolites stimulated oxygen consumption in state IV and compromised ATP formation, it can be presumed that these organic acids might act as endogenous uncouplers of mitochondria respiration. Moreover, COX activity linked to TMPD-ascorbate was significantly reduced by DGA in the brain mitochondrial enriched fractions. Finally, DGA and LGA reduced cell viability of rat cerebral cortex slices, as determined by the MTT assay. In case our in vitro data also occur in vivo, it may be presumed that impairment of energy metabolism may contribute to the understanding of the clinical features mainly of patients affected by DHGA.