Adenosine A2A Receptor Stimulation Research Articles

Functional Interaction between Adenosine A2A and mGlu5 Receptors Mediates STEP Phosphatase Activation and Promotes STEP/mGlu5R Binding in Mouse Hippocampus and Neuroblastoma Cell Line.

(1) Background: Recently, we found that adenosine A2A receptor (A2AR) stimulation results in an increase in STEP phosphatase activity. In order to delve into the mechanism through which A2AR stimulation induced STEP activation, we investigated the involvement of mGlu5R since it is well documented that A2AR and mGlu5R physically and functionally interact in several brain areas. (2) Methods: In a neuroblastoma cell line (SH-SY5Y) and in mouse hippocampal slices, we evaluated the enzymatic activity of STEP by using a para-nitrophenyl phosphate colorimetric assay. A co-immunoprecipitation assay and a Western blot analysis were used to evaluate STEP/mGlu5R binding. (3) Results: We found that the A2AR-dependent activation of STEP was mediated by the mGlu5R. Indeed, the A2AR agonist CGS 21680 significantly increased STEP activity, and this effect was prevented not only by the A2AR antagonist ZM 241385, as expected, but also by the mGlu5R antagonist MPEP. In addition, we found that mGlu5R agonist DHPG-induced STEP activation was reversed not only by the mGlu5R antagonist MPEP but also by ZM 241385. Finally, via co-immunoprecipitation experiments, we found that mGlu5R and STEP physically interact when both receptors are activated (4) Conclusions: These results demonstrated a close functional interaction between mGlu5 and A2A receptors in the modulation of STEP activity.

Adenosine A2A receptor activation reduces chondrocyte senescence.

Osteoarthritis (OA) pathogenesis is associated with reduced chondrocyte homeostasis and increased levels of cartilage cellular senescence. Chondrosenescence is the development of cartilage senescence that increases with aging joints and disrupts chondrocyte homeostasis and is associated with OA. Adenosine A2A receptor (A2AR) activation in cartilage via intra-articular injection of liposomal A2AR agonist, liposomal-CGS21680, leads to cartilage regeneration in vivo and chondrocyte homeostasis. A2AR knockout mice develop early OA isolated chondrocytes demonstrate upregulated expression of cellular senescence and aging-associated genes. Based on these observations, we hypothesized that A2AR activation would ameliorate cartilage senescence. We found that A2AR stimulation of chondrocytes reduced beta-galactosidase staining and regulated levels and cell localization of common senescence mediators p21 and p16 in vitro in the human TC28a2 chondrocyte cell line. In vivo analysis similarly showed A2AR activation reduced nuclear p21 and p16 in obesity-induced OA mice injected with liposomal-CGS21680 and increased nuclear p21 and p16 in A2AR knockout mouse chondrocytes compared to wild-type mice. A2AR agonism also increased activity of the chondrocyte Sirt1/AMPK energy-sensing pathway by enhancing nuclear Sirt1 localization and upregulating T172-phosphorylated (active) AMPK protein levels. Lastly, A2AR activation in TC28a2 and primary human chondrocytes reduced wild-type p53 and concomitantly increased p53 alternative splicing leading to increase in an anti-senescent p53 variant, Δ133p53α. The results reported here indicate that A2AR signaling promotes chondrocyte homeostasis in vitro and reduces OA cartilage development in vivo by reducing chondrocyte senescence.

Expression and Impact of Adenosine A3 Receptors on Calcium Homeostasis in Human Right Atrium.

Increased adenosine A2A receptor (A2AR) expression and activation underlies a higher incidence of spontaneous calcium release in atrial fibrillation (AF). Adenosine A3 receptors (A3R) could counteract excessive A2AR activation, but their functional role in the atrium remains elusive, and we therefore aimed to address the impact of A3Rs on intracellular calcium homeostasis. For this purpose, we analyzed right atrial samples or myocytes from 53 patients without AF, using quantitative PCR, patch-clamp technique, immunofluorescent labeling or confocal calcium imaging. A3R mRNA accounted for 9% and A2AR mRNA for 32%. At baseline, A3R inhibition increased the transient inward current (ITI) frequency from 0.28 to 0.81 events/min (p < 0.05). Simultaneous stimulation of A2ARs and A3Rs increased the calcium spark frequency seven-fold (p < 0.001) and the ITI frequency from 0.14 to 0.64 events/min (p < 0.05). Subsequent A3R inhibition caused a strong additional increase in the ITI frequency (to 2.04 events/min; p < 0.01) and increased phosphorylation at s2808 1.7-fold (p < 0.001). These pharmacological treatments had no significant effects on L-type calcium current density or sarcoplasmic reticulum calcium load. In conclusion, A3Rs are expressed and blunt spontaneous calcium release at baseline and upon A2AR-stimulation in human atrial myocytes, pointing to A3R activation as a means to attenuate physiological and pathological elevations of spontaneous calcium release events.

Myelin Defects in Niemann-Pick Type C Disease: Mechanisms and Possible Therapeutic Perspectives.

Niemann–Pick type C (NPC) disease is a wide-spectrum clinical condition classified as a neurovisceral disorder affecting mainly the liver and the brain. It is caused by mutations in one of two genes, NPC1 and NPC2, coding for proteins located in the lysosomes. NPC proteins are deputed to transport cholesterol within lysosomes or between late endosome/lysosome systems and other cellular compartments, such as the endoplasmic reticulum and plasma membrane. The first trait of NPC is the accumulation of unesterified cholesterol and other lipids, like sphingosine and glycosphingolipids, in the late endosomal and lysosomal compartments, which causes the blockade of autophagic flux and the impairment of mitochondrial functions. In the brain, the main consequences of NPC are cerebellar neurodegeneration, neuroinflammation, and myelin defects. This review will focus on myelin defects and the pivotal importance of cholesterol for myelination and will offer an overview of the molecular targets and the pharmacological strategies so far proposed, or an object of clinical trials for NPC. Finally, it will summarize recent data on a new and promising pharmacological perspective involving A2A adenosine receptor stimulation in genetic and pharmacological NPC dysmyelination models.

PDRN, a natural bioactive compound, blunts inflammation and positively reprograms healing genes in an “in vitro” model of oral mucositis

Oral mucositis is a side effect hard to treat following high dose chemotherapy or radiotherapy. Adenosine A2A receptor stimulation blocks NF-κB and boosts the Wnt/β-catenin signaling, thus blunting inflammation and triggering growth factor codifying genes. Polydeoxyribonucleotide (PDRN) is a registered drug that activates the A2A receptor. Therefore, the aim of this study was to evaluate PDRN effects in an “in vitro” model of oral mucositis induced by prompting an inflammatory phenotype in human gingival fibroblasts (GF) and human oral mucosal epithelial cells (EC). GF and EC were stimulated with LPS (2 μg/ml) alone or in combination with i) PDRN (100 μg/ml); ii) PDRN plus ZM241385 (1 μM) as an A2AR antagonist; iii) CGS21680 (1 μM) as an A2AR agonist. LPS boosted NF-κB, TNF-α and IL-6 expression, decreased IL-10 levels and downregulated both Wnt/β-catenin, VEGF and EGF expression. PDRN reverted the LPS-induced phenotype as well as CGS21680. Co-incubation with ZM241385 abolished PDRN effects, thus confirming A2A receptor involvement in PDRN mechanism of action. These results suggest that PDRN efficacy may be due to a “dual mode” of action: NF-κB inhibition and Wnt/β-catenin signaling activation. However, these interesting findings need to be confirmed by animal and clinical studies.

Adenosine A2A receptor signaling promotes FoxO associated autophagy in chondrocytes

Autophagy, a homeostatic pathway upregulated during cellular stress, is decreased in osteoarthritic chondrocytes and this reduction in autophagy is thought to contribute to the development and progression of osteoarthritis (OA). The adenosine A2A receptor (A2AR) is a potent anti-inflammatory receptor and deficiency of this receptor leads to the development of OA in mice. Moreover, treatment using liposomally conjugated adenosine or a specific A2AR agonist improved joint scores significantly in both rats with post-traumatic OA (PTOA) and mice subjected to a high fat diet obesity induced OA. Importantly, A2AR ligation is beneficial for mitochondrial health and metabolism in vitro in primary and the TC28a2 human cell line. An additional set of metabolic, stress-responsive, and homeostatic mediators include the Forkhead box O transcription factors (FoxOs). Data has shown that mouse FoxO knockouts develop early OA with reduced cartilage autophagy, indicating that FoxO-induced homeostasis is important for articular cartilage. Given the apparent similarities between A2AR and FoxO signaling, we tested the hypothesis that A2AR stimulation improves cartilage function through activation of the FoxO proteins leading to increased autophagy in chondrocytes. We analyzed the signaling pathway in the human TC28a2 cell line and corroborated these findings in vivo in a metabolically relevant obesity-induced OA mouse model. We found that A2AR stimulation increases activation and nuclear localization of FoxO1 and FoxO3, promotes an increase in autophagic flux, improves metabolic function in chondrocytes, and reduces markers of apoptosis in vitro and reduced apoptosis by TUNEL assay in vivo. A2AR ligation additionally enhances in vivo activation of FoxO1 and FoxO3 with evidence of enhanced autophagic flux upon injection of the liposome-associated A2AR agonist in a mouse obesity-induced OA model. These findings offer further evidence that A2AR may be an excellent target for promoting chondrocyte and cartilage homeostasis.

Adenosine A2a Receptor Stimulation Attenuates Ischemia-Reperfusion Injury and Improves Survival in A Porcine Model of DCD Liver Transplantation.

Orthotopic liver transplantation (OLT) using allografts from donation after circulatory death (DCD) is potentially associated with compromised clinical outcomes due to ischemia-reperfusion injury (IRI)-induced organ damage and graft-related complications. The aim of this study was to provide in vivo data on the effects of adenosine A2a receptor stimulation in a clinically relevant large animal model of DCD liver transplantation. Cardiac arrest was induced in German Landrace pigs (n = 10; 20–25 kg). After 30 min of warm ischemia, the donor liver was retrieved following a cold flush with 3 L of histidine-tryptophan-ketoglutarate-HTK solution. Animals of the treatment group (n = 5/group) received a standard dose of the selective adenosine receptor agonist CGS 21680 added to the cold flush. All grafts were stored for 4.5 h at 4 °C in HTK-solution before OLT. Hepatocellular injury, apoptosis, protein kinase A-PKA activity, graft microcirculation, liver function, and animal survival were assessed. Compared to untreated livers, adenosine A2a receptor stimulation resulted in improved tissue microcirculation (103% ± 5% vs. 38% ± 4% compared to baseline; p < 0.05), accelerated functional recovery of the graft (indocyanine green-plasma disappearance rate (ICG-PDR) of 75% ± 18% vs. 40% ± 30% after 3 h), increased PKA activity ratio (56% ± 3% vs. 32% ± 3%; p < 0.001 after 1 h), and consequently reduced tissue necrosis and apoptosis. The potent protective effects were clinically manifested in significantly improved survival in the treatment group after 72 h (100% vs. 40%; p = 0.04). The ex vivo administration of adenosine A2a receptor agonist during the back-table flush mitigates IRI-mediated tissue damage and improves functional graft recovery and survival in a large animal model of DCD liver transplantation.

Abstract 4517: Targeting Notch1 via adenosine A2A receptor to modulate tumor immunity

Abstract One of the hallmarks of cancer is the ability to evade the host immune system and this is achieved through different mechanisms. Several studies have showed that the accumulation of adenosine in the tumor microenvironment suppresses T-cell functions, thus helping the tumor to evade the immune system. Finding a way to counteract adenosine-mediated immune suppression might greatly enhance the host endogenous immune response against cancer and the efficacy of adoptive immunotherapies. To achieve this, we need to understand how adenosine regulates T-cell functions. In a collaborative project, our group recently showed that stimulation of adenosine A2A receptor (A2AR) reduces T-cell receptor (TCR)-signaling and consequently Notch1 activation and expression in CD8+ T-cells, which are critical to cancer immunity. This suggests that A2AR stimulation suppresses CD8+ T-cell function through inhibition of TCR-induced Notch1, which is required for the activation and function of CD8+ T-cells. Based on these observations, we hypothesized that while stimulation of A2AR suppresses CD8+ T-cells, inhibition of A2AR should protect CD8+ T-cells from the adenosine-mediated immune suppression. Notch1 is likely to be critical in this process since it was shown that ectopic expression of Notch1 intracellular domain prevents the adenosine-mediated immune suppression in CD8+ T-cells. Therefore, we aim to investigate the effect of A2AR inhibition on TCR activation and Notch1 to evaluate the A2AR-Notch axis as a novel immunotherapeutic target. Our data show that pharmacological inhibition of A2AR with a selective antagonist induces tumor cell death and increases the number of CD8+ T-cells in tumor-derived spheroids from a mouse triple-negative breast cancer (TNBC) model. The effect of A2AR inhibition appears to be immune-mediated since the same agent did not induce cell death in tumor-derived spheroids from immunocompromised mice. Along the same lines, we show that, inhibition of A2AR restored Notch1 activation and proliferation in primary murine CD8+ T-cells, but did not affect Notch1 and proliferation in TNBC cell lines. This effect is likely to be strictly dependent on Notch since A2AR inhibition failed to rescue CD8+ T-cell proliferation from the suppressive effect of gamma-secretase inhibition. It is not yet known how A2AR regulates Notch1, however, our latest preliminary results suggest that A2AR stimulation might promote the endosomal degradation of Notch1, whereas, A2AR inhibition might switch Notch1 fate from endosomal degradation to activation. Further investigation is needed to establish how A2AR regulates Notch1 and whether A2AR inhibition can protect CD8+ T-cell function in vivo in the tumor microenvironment. Overall our current data provides a rationale for the evaluation of A2AR antagonists as a Notch-modulating immunotherapy. Citation Format: Giulia Monticone, Fokhrul M. Hossain, Deniz A. Ucar, Samarpan Majumder, Claudia Sorrentino, Paulo C. Rodriguez, Rosa A. Sierra, Antonio Pannuti, Stephen Hatfield, Barbara A. Osborne, Lisa M. Minter, Silvana Morello, Lucio Miele. Targeting Notch1 via adenosine A2A receptor to modulate tumor immunity [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4517.

Adenosine A2A receptor (A2AR) stimulation enhances mitochondrial metabolism and mitigates reactive oxygen species-mediated mitochondrial injury.

In OA chondrocytes, there is diminished mitochondrial production of ATP and diminished extracellular adenosine resulting in diminished adenosine A2A receptor (A2AR) stimulation and altered chondrocyte homeostasis which contributes to the pathogenesis of OA. We tested the hypothesis that A2AR stimulation maintains or enhances mitochondrial function in chondrocytes. The effect of A2AR signaling on mitochondrial health and function was determined in primary murine chondrocytes, a human chondrocytic cell line (T/C-28a2), primary human chondrocytes, and a murine model of OA by transmission electron microscopy analysis, mitochondrial stress testing, confocal live imaging for mitochondrial inner membrane polarity, and immunohistochemistry. In primary murine chondrocytes from A2AR-/- null mice, which develop spontaneous OA by 16 weeks, there is mitochondrial swelling, dysfunction, and reduced mitochondrial content with increased reactive oxygen species (ROS) burden and diminished mitophagy, as compared to chondrocytes from WT animals. IL-1-stimulated T/C-28a2 cells treated with an A2AR agonist had reduced ROS burden with increased mitochondrial dynamic stability and function, findings which were recapitulated in primary human chondrocytes. In an obesity-induced OA mouse model, there was a marked increase in mitochondrial oxidized material which was markedly improved after intraarticular injections of liposomal A2AR agonist. These results are consistent with the hypothesis that A2AR ligation is mitoprotective in OA.

Adenosine A2A receptor stimulation restores cell functions and differentiation in Niemann-Pick type C-like oligodendrocytes

Niemann Pick type C (NPC) disease is a rare neurovisceral disorder. Mutations in npc1 gene induce an intracellular accumulation of unesterified cholesterol in the endosomal/lysosomal system causing cell death. We recently showed that stimulation of adenosine A2A receptors (A2AR) restores cholesterol accumulation in late endosomes/lysosomes in human NPC fibroblasts and neural cell lines transiently transfected with NPC1 siRNA, suggesting that these receptors might be targeted to contrast the disease. Since NPC1 disease is characterized by dysmyelination and maturational arrest of oligodendrocyte progenitors (OPs), in this study, we investigated whether A2AR stimulation could promote oligodendrocyte differentiation and myelin formation, thus overcoming these important neurological abnormalities. We developed a NPC1 pharmacological model, in which primary cultures of OPs are exposed to a cholesterol transport inhibitor to induce a NPC1-like phenotype characterized by several typical features such as (i) cholesterol accumulation, (ii) altered mitochondrial morphology and membrane potential, (iii) defect of autophagy and (iv) maturation arrest. The A2AR agonist CGS21680 normalized all NPC1-like features. The ability of CGS21680 of rescuing OP from maturational arrest and promoting their differentiation to mature OL, suggests that A2AR stimulation might be exploited to correct dysmyelination in NPC1, further supporting their therapeutic potential in the disease.

Adenosine A2A Receptor Stimulation Inhibits TCR-Induced Notch1 Activation in CD8+T-Cells.

Notch receptors signaling is required for optimal T-cell activation and function. T-cell receptor (TCR) engagement can activate Notch receptors in T-cells in a ligand-independent fashion. In this study, we examined the role of adenosine A2A receptor (A2AR) signaling pathway in regulating the activity of Notch1 induced by TCR stimulation in CD8+T-cells. A selective A2AR agonist decreased Notch1 protein expression and Notch1 cleavage, and reduced transcripts of Notch1-target genes Hes1 and Myc in activated CD8+T-cells. Inhibition of TCR-induced Notch1 expression by an A2AR agonist was accompanied by increased cAMP concentration and mimicked by forskolin. This effect was associated with reduced IFN-γ and granzyme B production. The effect of an A2AR agonist was abrogated by a selective A2AR antagonist and absent in CD8+T-cells harvested from A2AR−/− mice. Stimulation of A2AR reduced Notch1 receptor levels by inhibiting upstream TCR signals, including ZAP70 phosphorylation, in turn impairing the generation of the active Notch1 intracellular domain (N1ICD). Direct activation of PKC with PMA and ionomycin bypassed A2AR-induced Notch1 inhibition. Overexpression of N1ICD in CD8+T-cells prevented the suppressive effects of an A2AR agonist on proliferation and cytokine release during activation. Our results identify the A2AR signaling pathway as an important regulator of TCR-induced Notch1 receptor activation in CD8+T-cells, and Notch as an important target of the immune suppressive effects of A2AR. We propose a mechanism whereby A2AR impairs CD8 T-cells function through inhibition of Notch1 receptor activation.

A2a adenosine receptor agonist improves endoplasmic reticulum stress in MIN6 cell line through protein kinase A/ protein kinase B/ Cyclic adenosine monophosphate response element-binding protein/ and Growth Arrest And DNA-Damage-Inducible 34/ eukaryotic Initiation Factor 2α pathways.

Endoplasmic reticulum (ER) stress is one of the main molecular events underlying pancreatic beta cell (PBC) failure, apoptosis, and a decrease in insulin secretion. Recent studies have highlighted the fundamental role of A2a adenosine receptor (A2aR) in potentiation of insulin secretion and proliferation of PBCs. However, possible protective effects of A2aR signaling against ER stress have not been elucidated yet. Thus, in the present study, we aimed to investigate the effects of A2aR activation in MIN6 beta cells undergoing tunicamycin (TM)-mediated ER stress. A2aR expression and activity were evaluated using real-time polymerase chain reaction and measurement of the cyclic adenosine monophosphate (cAMP), protein kinase A (PKA), phospho-protein kinase B or Akt (p-Akt)/Akt, and phospho-Cyclic adenosine monophosphate response element-binding protein/CREB levels in response to a specific agonist (CGS 21680). Survival and proliferation in TM and CGS 21680 cotreated cells were evaluated using 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), annexin V-fluorescein isothiocyanate (FITC)/propidium iodide staining, colony formation, and 5-bromo-2'-deoxyuridine (Brdu) assays. In addition, the effects of A2aR stimulation on insulin secretion were evaluated using the enzyme-linked immunosorbent assay. B-cell lymphoma 2 (Bcl-2), phospho-eukaryotic Initiation Factor 2α (p-eIF2α)/eIF2α, growth arrest and DNA-damage-inducible 34 (GADD34), X-box binding protein 1 (XBP-1), spliced X-box binding protein 1 (XBP-1s), immunoglobulin heavy-chain-binding protein (BIP), and CCAAT-enhancer-binding protein homologous protein (CHOP) levels were evaluated using western blotting. Our results showed a decrease in A2aR expression and p-Akt/Akt and p-CREB/CREB levels in TM-pretreated cells. We also mentioned that CGS 21680 effectively increased cell survival, proliferation, and insulin secretion in TM-treated cells. The antiapoptotic effects were possibly mediated through Bcl-2 upregulation. Our western blotting results indicated that A2aR effectively downregulated p-eIF2α/eIF2α, XBP-1, XBP-1s, BIP, and CHOP levels, whereas GADD34 was upregulated. Altogether, the present study revealed that A2aR signaling through PKA/Akt/CREB mediators alleviated TM cytotoxicity effects in MIN6 beta cells. Thus, the stimulation of this receptor was seen as a new approach to control ER stress in the PBC cells.

Adenosine A2A receptor (A2AR) stimulation modulates expression of semaphorins 4D and 3A, regulators of bone homeostasis.

The axonal guidance proteins semaphorin (Sema)4D and Sema3A play important roles in communication between osteoclasts and osteoblasts. As stimulation of adenosine A2A receptors (A2AR) regulates both osteoclast and osteoblast function, we asked whether A2AR regulates both osteoclast and osteoblast expression of Semas. In vivo bone formation and Sema3A/PlexinA1/Neuropilin-1, Sema4D/PlexinB1 protein expression were studied in a murine model of wear particle-induced osteolysis. Osteoclast/osteoblast differentiation were studied in vitro as the number of tartrate-resistant acid phosphatase+/Alizarin Red+ cells after challenge with CGS21680 (A2AR agonist, 1 µM) or ZM241385 (A2AR antagonist, 1 µM), with or without Sema4D or Sema3A (10 ng/ml). Sema3A/PlexinA1/Neuropilin-1, Sema4D/PlexinB1, and receptor activator of NF-κB ligand/osteoprotegerin (RANKL/OPG) expression was studied by RT-PCR and Western blot. β-Catenin activation and cytoskeleton changes were studied by fluorescence microscopy and Western blot. In mice with wear particles implanted over the calvaria, CGS21680 treatment increased bone formation in vivo, reduced Sema4D, and increased Sema3A expression compared with mice with wear particle-induced osteolysis treated with vehicle alone. During osteoclast differentiation, CGS21680 abrogated RANKL-induced Sema4D mRNA expression (1.3 ± 0.3- vs. 2.5 ± 0.1-fold change, P < 0.001, n = 4). PlexinA1, but not Neuropilin-1, mRNA was enhanced by CGS21680 treatment. CGS21680 enhanced Sema3A mRNA expression during osteoblast differentiation (8.7 ± 0.2-fold increase, P < 0.001, n = 4); PlexinB1 mRNA was increased 2-fold during osteoblast differentiation and was not altered by CGS21680. Similar changes were observed at the protein level. CGS21680 decreased RANKL, increased OPG, and increased total/nuclear β-catenin expression in osteoblasts. Sema4D increased Ras homolog gene family, member A phosphorylation and focal adhesion kinase activation in osteoclast precursors, and CGS21680 abrogated these effects. In summary, A2AR activation diminishes secretion of Sema4D by osteoclasts, inhibits Sema4D-mediated osteoclast activation, and enhances secretion of Sema3A by osteoblasts, increasing osteoblast differentiation and diminishing inflammatory osteolysis.-Mediero, A., Wilder, T., Shah, L., Cronstein, B. N. Adenosine A2A receptor (A2AR) stimulation modulates expression of semaphorins 4D and 3A, regulators of bone homeostasis.

GSK3\u03b2 negatively regulates TRAX, a scaffold protein implicated in mental disorders, for NHEJ-mediated DNA repair in neurons

Translin-associated protein X (TRAX) is a scaffold protein with various functions and has been associated with mental illnesses, including schizophrenia. We have previously demonstrated that TRAX interacts with a Gsα protein-coupled receptor, the A2A adenosine receptor (A2AR), and mediates the function of this receptor in neuritogenesis. In addition, stimulation of the A2AR markedly ameliorates DNA damage evoked by elevated oxidative stress in neurons derived from induced pluripotent stem cells (iPSCs). Here, we report that glycogen synthase kinase 3 beta (GSK3β) and disrupted-in-schizophrenia 1 (DISC1) are two novel interacting proteins of TRAX. We present evidence to suggest that the stimulation of A2AR markedly facilitated DNA repair through the TRAX/DISC1/GSK3β complex in a rat neuronal cell line (PC12), primary mouse neurons, and human medium spiny neurons derived from iPSCs. A2AR stimulation led to the inhibition of GSK3β, thus dissociating the TRAX/DISC1/GSK3β complex and facilitating the non-homologous end-joining pathway (NHEJ) by enhancing the activation of a DNA-dependent protein kinase via phosphorylation at Thr2609. Similarly, pharmacological inhibition of GSK3β by SB216763 also facilitated the TRAX-mediated repair of oxidative DNA damage. Collectively, GSK3β binds with TRAX and negatively affects its ability to facilitate NHEJ repair. The suppression of GSK3β by A2AR activation or a GSK3β inhibitor releases TRAX for the repair of oxidative DNA damage. Our findings shed new light on the molecular mechanisms underlying diseases associated with DNA damage and provides a novel target (i.e., the TRAX/DISC1/GSK3β complex) for future therapeutic development for mental disorders.

Adenosine A2a Receptor Blockade Diminishes Wnt/β-Catenin Signaling in a Murine Model of Bleomycin-Induced Dermal Fibrosis

Adenosine A2a receptor (A2aR) stimulation promotes the synthesis of collagens I and III, and we have recently demonstrated that there is crosstalk between the A2aR and WNT/β-catenin signaling pathway. In invitro studies, A2aR signaling for collagen III expression was mediated by WNT/β-catenin signaling in human dermal fibroblasts; we further verified whether the crosstalk between A2aR and Wnt/β-catenin signaling was involved in diffuse dermal fibrosis invivo. Wnt-signaling reporter mice (Tcf/Lef:H2B-GFP) were challenged with bleomycin and treated with the selective A2aR antagonist istradefylline (KW6002) or vehicle. Dermal fibrosis was quantitated and nuclear translocation of β-catenin in fibroblasts was assessed by double-staining for Green fluorescent protein or dephosphorylated β-catenin or β-catenin phosphorylated at Ser552, and vimentin. KW6002 significantly reduced skin thickness, skinfold thickness, breaking tension, dermal hydroxyproline content, myofibroblast accumulation, and collagen alignment in bleomycin-induced dermal fibrosis. Also, there was increased expression of Tcf/Lef:H2B-GFP reporter in bleomycin-induced dermal fibrosis, an effect that was diminished by treatment with KW6002. Moreover, KW6002 significantly inhibited nuclear translocation of Tcf/Lef:H2B-GFP reporter, as well as dephosphorylated β-catenin and β-catenin phosphorylated at Ser552. Our work supports the hypothesis that pharmacologic blockade of A2aR inhibits the WNT/β-catenin signaling pathway, contributing to its capacity to inhibit dermal fibrosis in diseases such as scleroderma.

CD39 limits P2X7 receptor inflammatory signaling and attenuates sepsis-induced liver injury

The severity of sepsis can be linked to excessive inflammatory responses resulting in hepatic injury. P2X7 receptor activation by extracellular ATP (eATP) exacerbates inflammation by augmenting cytokine production; while CD39 (ENTPD1) scavenges eATP to generate adenosine, thereby limiting P2X7 activation and resulting in A2A receptor stimulation. We aim to determine how the functional interaction of P2X7 receptor and CD39 control the macrophage response, and consequently impact on sepsis and liver injury. Sepsis was induced by cecal ligation and puncture in C57BL/6 wild-type (WT) and CD39-/- mice. Several in vitro assays were performed using peritoneal or bone marrow derived macrophages to determine CD39 ectonucleotidase activity and its role in sepsis-induced liver injury. CD39 expression in macrophages limits ATP-P2X7 receptor pro-inflammatory signaling. P2X7 receptor paradoxically boosts CD39 activity. Inhibition and/or deletion of P2X7 receptor in LPS-primed macrophages attenuates cytokine production and inflammatory signaling as well as preventing ATP-induced increases in CD39 activity. Septic CD39-/- mice exhibit higher levels of inflammatory cytokines and show more pronounced liver injury than WT mice. Pharmacological P2X7 blockade largely prevents tissue damage, cell apoptosis, cytokine production, and the activation of inflammatory signaling pathways in the liver from septic WT, while only attenuating these outcomes in CD39-/- mice. Furthermore, the combination of P2X7 blockade with adenosine A2A receptor stimulation completely inhibits cytokine production, the activation of inflammatory signaling pathways, and protects septic CD39-/- mice against liver injury. CD39 attenuates sepsis-associated liver injury by scavenging eATP and ultimately generating adenosine. We propose boosting of CD39 would suppress P2X7 responses and trigger adenosinergic signaling to limit systemic inflammation and restore liver homeostasis during the acute phase of sepsis. Lay summary: CD39 expression in macrophages limits P2X7-mediated pro-inflammatory responses, scavenging extracellular ATP and ultimately generating adenosine. CD39 genetic deletion exacerbates sepsis-induced experimental liver injury. Combinations of a P2X7 antagonist and adenosine A2A receptor agonist are hepatoprotective during the acute phase of abdominal sepsis.

Adenosine A2a receptor stimulation blocks development of nonalcoholic steatohepatitis in mice by multilevel inhibition of signals that cause immunolipotoxicity

Lipotoxicity and immunoinflammation are associated with the evolution of steatosis toward nonalcoholic steatohepatitis (NASH). This study reports the ability of adenosine A2a receptor (A2aR) activation to inhibit NASH development by modulating the responses of CD4+ T-helper (Th) cells to avoid an immuno-mediated potentiation of lipotoxicity. The effect of the A2aR agonist CGS21680 on immunoinflammatory signals, CD4+Th cell infiltration and immunolipotoxicity was analyzed in steatotic C57BL/6 mice fed with a methionine-choline-deficient (MCD) diet and in mouse hepatocytes exposed to palmitic acid (PA). CGS21680 inhibited NASH development in steatotic mice and decreased cytokines and chemokines involved in Th cell recruitment or polarization (namely CXCL10, CCL2, tumor necrosis factor alfa [TNFα], tumor growth factor [TGFβ], and IL-12). CGS21680 also reduced the expansion of Th17, Th22, and Th1 cells and increased the immunosuppressive activity of T regulatory cells. In PA-treated mice hepatocytes, CGS21680 inhibited the production of CXCL10, TNFα, TGFβ, IL-12, and CCL2; CGS21680 also prevented JNK-dependent lipotoxicity and its intensification by IL-17 or IL-17 plus IL-22 through Akt/PI3-kinase stimulation and inhibition of the negative regulator of PI3-kinase, (phosphatase and tensin homologue deleted from chromosome 10 (PTEN), which is upregulated by IL-17. In MCD livers, CGS21680 reduced JNK activation and PTEN expression and increased Akt phosphorylation. In conclusion, A2aR stimulation inhibited NASH development by reducing Th17cell expansion and inhibiting the exacerbation of the IL-17-induced JNK-dependent lipotoxicity. These data promote the implementation of further studies to evaluate the potential clinical application of A2aR agonists that, by being able to function as both cytoprotective and immunomodulatory agents, could efficiently antagonizethe multi-faced pathogenesis of NASH.

Adenosine A2A receptor promotes collagen type III synthesis via β-catenin activation in human dermal fibroblasts.

Adenosine A2A receptor stimulation promotes the synthesis of collagen type I and type III (Col1 and Col3), mediators of fibrosis and scarring. The A2A receptor modulates collagen balance via cAMP/PKA/p38-MAPK/Akt pathways. Wnt signalling is important in fibrosis and the cAMP and Wnt pathways converge. Because the A2A receptor is Gs-linked and increases cAMP, we determined whether A2A receptors and Wnt signalling interact. Total β-catenin, de-phosphorylated β-catenin (canonical activation, de-phospho β-catenin) and phosphorylated β-catenin at Ser552 (non-canonical activation, p-Ser552 β-catenin) levels were determined in primary human dermal fibroblasts, cytosol and nucleus, by western blot analysis and fluorescence microscopy, before and after stimulation by A2A receptor-selective agonist CGS21680, with/without A2A receptor-selective antagonist (SCH56261) pretreatment. β-Catenin was knocked down by transfection with scrambled-siRNA or specific-siRNA, and Col1 and Col3 levels determined by western blots. CGS21680 stimulation rapidly (15min) increased cellular β-catenin levels. Both de-phospho β-catenin and p-Ser552 β-catenin levels were also increased. CGS21680 stimulated the translocation of total de-phospho and p-Ser552 β-catenin to the nucleus. A2A receptor-stimulation increased Col1 synthesis similarly in β-catenin knockeddown and scrambled cells. However, β-catenin knockdown abolished the increase in Col3 synthesis induced in A2A receptor-stimulated fibroblasts. A2A receptor stimulation promotes Col3 synthesis via the activation of canonical and non-canonical β-catenin, consistent with a role for A2A receptors in dermal fibrosis and scarring.

TRAX mediates the protective role of the A2A adenosine receptor in detecting the DNA damage induced by oxidative stress

The translin‐associated protein X (TRAX) was originally identified as an interacting protein of Translin, a DNA/RNA binding protein that controls mRNA transport, translation, DNA repair, and DNA recombination. It interacts specifically with an activator (i.e., C1D) of DNA‐dependent protein kinase (DNA‐PK) upon γ‐irradiation and thus is implicated in repair of double‐strand breaks (DSBs). We recently reported that TRAX interacts with phosphorylated ATM (Ser1981) and contributes to the ATM‐mediated DNA repair in response to genotoxic stresses. The A2A adenosine receptor (A2AR) is a G‐protein‐coupled receptor that binds to adenosine and exists in many brain areas. Using a yeast two‐hybrid approach, we found that TRAX is an interacting protein of the cytoplasmic C terminus of A2AR, and mediates the neuritogenesis process via KIF2A. Given that stimulation of A2AR by two A2AR‐selective agonists markedly ameliorates the DSBs evoked by elevated oxidative stress in human iPSCs‐derived neurons, the role of TRAX in mediating the protective role of A2AR is of great interest. In the present study, we present evidence to suggest that TRAX is required for the A2AR‐mediated facilitation of DNA repair in a neuronal cell line (PC12) and primary neurons. Stimulation of A2AR appears to enhance the non‐homologous end joining (NHEJ) repair activity by increasing the activation and phosphorylation of DNA‐PK at Thr2609. Collectively, TRAX contributes to the activation of DNA‐PK and the detection of DSBs evoked by oxidative stress.

Activation of the adenosine A2A receptor exacerbates experimental autoimmune neuritis in Lewis rats in association with enhanced humoral immunity

Accumulated evidence demonstrated that Adenosine A2A receptor (A2AR) is involved in the inflammatory diseases. In the present study, we showed that a selective A2AR agonist, CGS21680, exacerbated experimental autoimmune neuritis in Lewis rats induced with bovine peripheral myelin. The exacerbation was accompanied with reduced CD4+Foxp3+ T cells, increased CD4+CXCR5+ T cells, B cells, dendritic cells and antigen-specific autoantibodies, which is possibly due to the inhibition of IL-2 induced by CGS21680. Combined with previous studies, our data indicate that the effects of A2AR stimulation in vivo are variable in different diseases. Caution should be taken in the use of A2AR agonists.