Adenosine A2A Receptor Signaling Research Articles

Deletion of adenosine A2A receptors on CD8 T cells enhances their accumulation in tumors and tumor-draining lymph nodes, as well as their ability to control tumor outgrowth (40.6)

Abstract Previous work has demonstrated that ablation of adenosine A2A receptor (A2AR) expression in tumor-bearing mice enhances tumor control. However, it is not yet clear what cells and functions are influenced by A2AR signaling. Here we show that the ability of adoptively transferred OT-I CD8 T cells to control ovalbumin expressing B16 melanomas in A2aR+/+ recipients is enhanced if the A2AR gene is ablated in the OT-I cells. This demonstrates directly that the ability of CD8 T cells to control tumors is influenced by adenosine. To further understand the functions of CD8 T cells influenced by A2AR deletion, we examined the activation, proliferation, and survival of A2AR+/+ and A2AR-/- OT-I cells in tumors and tumor-draining lymph nodes (TDLN) after co-transfer. While activation and proliferation of both populations in TDLN was comparable, the accumulation of A2AR-/- cells in both TDLN and tumor was enhanced relative to A2AR+/+ cells. Preliminary data suggest that this is due to enhanced survival of A2AR-/- CD8 T cells in TDLN. This enhanced accumulation does not occur in the lymph nodes of mice immunized with bone marrow derived dendritic cells or Vaccinia virus, suggesting that it reflects alterations in the level of adenosine specifically in TDLN. We theorize that enhanced accumulation of A2AR-/- CD8 T cells is at least partially responsible for their increased ability to control tumors. Support: NIH R01 CA78400, F31 CA130117

Distinct Roles of PDE4 and PDE10A in the Regulation of cAMP/PKA Signaling in the Striatum

Phosphodiesterase (PDE) is a critical regulator of cAMP/protein kinase A (PKA) signaling in cells. Multiple PDEs with different substrate specificities and subcellular localization are expressed in neurons. Dopamine plays a central role in the regulation of motor and cognitive functions. The effect of dopamine is largely mediated through the cAMP/PKA signaling cascade, and therefore controlled by PDE activity. We used in vitro and in vivo biochemical techniques to dissect the roles of PDE4 and PDE10A in dopaminergic neurotransmission in mouse striatum by monitoring the ability of selective PDE inhibitors to regulate phosphorylation of presynaptic [e.g., tyrosine hydroxylase (TH)] and postsynaptic [e.g., dopamine- and cAMP-regulated phosphoprotein of M(r) 32 kDa (DARPP-32)] PKA substrates. The PDE4 inhibitor, rolipram, induced a large increase in TH Ser40 phosphorylation at dopaminergic terminals that was associated with a commensurate increase in dopamine synthesis and turnover in striatum in vivo. Rolipram induced a small increase in DARPP-32 Thr34 phosphorylation preferentially in striatopallidal neurons by activating adenosine A(2A) receptor signaling in striatum. In contrast, the PDE10A inhibitor, papaverine, had no effect on TH phosphorylation or dopamine turnover, but instead robustly increased DARPP-32 Thr34 and GluR1 Ser845 phosphorylation in striatal neurons. Inhibition of PDE10A by papaverine activated cAMP/PKA signaling in both striatonigral and striatopallidal neurons, resulting in potentiation of dopamine D(1) receptor signaling and inhibition of dopamine D(2) receptor signaling. These biochemical results are supported by immunohistochemical data demonstrating differential localization of PDE10A and PDE4 in striatum. These data underscore the importance of individual brain-enriched cyclic-nucleotide PDE isoforms as therapeutic targets for neuropsychiatric and neurodegenerative disorders affecting dopamine neurotransmission.

Altered intracellular and extracellular signaling leads to impaired T-cell functions in ADA-SCID patients

AbstractMutations in the adenosine deaminase (ADA) gene are responsible for a form of severe combined immunodeficiency (SCID) caused by the lymphotoxic accumulation of ADA substrates, adenosine and 2′-deoxy-adenosine. The molecular mechanisms underlying T-cell dysfunction in humans remain to be elucidated. Here, we show that CD4+ T cells from ADA-SCID patients have severely compromised TCR/CD28-driven proliferation and cytokine production, both at the transcriptional and protein levels. Such an impairment is associated with an intrinsically reduced ZAP-70 phosphorylation, Ca2+ flux, and ERK1/2 signaling and to defective transcriptional events linked to CREB and NF-κB. Moreover, exposure to 2′-deoxy-adenosine results in a stronger inhibition of T-cell activation, mediated by the aberrant A2A adenosine receptor signaling engagement and PKA hyperactivation, or in a direct apoptotic effect at higher doses. Conversely, in T cells isolated from patients after gene therapy with retrovirally transduced hematopoietic stem/progenitor cells, the biochemical events after TCR triggering occur properly, leading to restored effector functions and normal sensitivity to apoptosis. Overall, our findings provide a better understanding of the pathogenesis of the immune defects associated with an altered purine metabolism and confirm that ADA gene transfer is an efficacious treatment for ADA-SCID. The trials in this study are enrolled at www.ClinicalTrials.gov as #NCT00598481 and #NCT0059978.

Inhibition of T cell and natural killer cell function by adenosine and its contribution to immune evasion by tumor cells (Review)

The resistance of many human cancers to immune-based therapies, including adoptive immunotherapy and the administration of therapeutic cancer vaccines, has been attributed to tumor-associated immune suppression, due in part to immunosuppressive molecules located within the tumor microenvironment. Adenosine is a purine nucleoside found within the interstitial fluid of solid tumors at concentrations that are able to inhibit cell-mediated immune responses to tumor cells. It is well established that extracellular adenosine inhibits T lymphocyte activation and effector function, including T cell adhesion to tumor cells and cytotoxic activity, by signaling primarily through A2a and A3 adenosine receptors on the surface of T cells. Importantly, A2a adenosine receptor-deficient mice exhibit enhanced anti-tumor immune responses by CD8+ T cells, as well as a dramatic reduction in the growth of experimental tumors in comparison to wild-type controls. A2a adenosine receptor signaling has also been implicated in adenosine-mediated inhibition of cytokine production and cytotoxic activity by activated natural killer (NK) cells, although the process of NK cell granule exocytosis is apparently suppressed via a distinct and as yet uncharacterized adenosine receptor. In this report, we review the evidence that adenosine is a potent inhibitor of cellular immune responses and may therefore be a major barrier to the successful immunotherapy of human carcinomas. The signaling pathways through which adenosine exerts its inhibitory effects on cell-mediated immune responses are also discussed. The accumulated evidence suggests that the effectiveness of immune-based therapies for solid tumors may be enhanced by selective antagonism of the adenosine receptor subtypes through which adenosine inhibits the anti-tumor activity of T lymphocytes and NK cells.

Critical Role of Hypoxia and A2A Adenosine Receptors in Liver Tissue-Protecting Physiological Anti-Inflammatory Pathway

Whole body exposure of wild type control littermates and A2A adenosine receptor (A2AR) gene deleted mice to low oxygen containing inspired gas mixture allowed the investigation of the mechanism that controls inflammatory liver damage and protects the liver using a mouse model of T cell-mediated viral and autoimmune hepatitis. We tested the hypothesis that the inflammatory tissue damage-associated hypoxia and extracellular adenosine --> A2AR signaling plays an important role in the physiological anti-inflammatory mechanism that limits liver damage during fulminant hepatitis. After induction of T cell-mediated hepatitis, mice were kept in modular chambers either under normoxic (21% oxygen) or hypoxic (10% oxygen) conditions for 8 h. It was shown that the whole body exposure to hypoxic atmosphere caused tissue hypoxia in healthy animals as evidenced by a decrease in the arterial blood oxygen tension and increase of the plasma adenosine concentration (P < 0.05). This "hypoxic" treatment resulted in significantly reduced hepatocellular damage and attenuated levels of serum cytokines in mice with acute liver inflammation. The anti-inflammatory effects of hypoxia were not observed in the absence of A2AR in studies of A2AR gene-deficient mice or when A2AR have been pharmacologically antagonized with synthetic antagonist. The presented data demonstrate that total body hypoxia-triggered pathway provides protection in acute hepatitis and that hypoxia (upstream) and A2AR (downstream) function in the same immunosuppressive and liver tissue-protecting pathway.

Adenosine A2A receptor signaling regulation of cardiac NADPH oxidase activity

Cardiac tissues express constitutively an NADPH oxidase, which generates reactive oxygen species (ROS) and is involved in redox signaling. Myocardial metabolism generates abundant adenosine, which binds to its receptors and plays important roles in cardiac function. The adenosine A2A receptor (A2AR) has been found to be expressed in cardiac myocytes and coronary endothelial cells. However, the role of the A2AR in the regulation of cardiac ROS production remains unknown. We found that knockout of A2AR significantly decreased (39±8%) NADPH-dependent O2− production in mouse hearts compared to age (10 weeks)-matched wild-type controls. This was accompanied by a significant decrease in Nox2 (a catalytic subunit of NADPH oxidase) protein expression, and down-regulation of ERK1/2, p38MAPK, and JNK phosphorylation (all P<0.05). In wild-type mice, intraperitoneal injection of the selective A2AR antagonist SCH58261 (3–10 mg/kg body weight for 90 min) inhibited phosphorylation of p47phox (a regulatory subunit of Nox2), which was accompanied by a down-regulated cardiac ROS production (48±8%), and decreased JNK and ERK1/2 activation by 54±28% (all P<0.05). In conclusion, A2AR through MAPK signaling regulates p47phox phosphorylation and cardiac ROS production by NADPH oxidase. Modulation of A2AR activity may have potential therapeutic applications in controlling ROS production by NADPH oxidase in the heart.

Wound Healing Is Impaired in MyD88-Deficient Mice

Synergy between Toll-like receptor (TLR) and adenosine A2A receptor (A2AR) signaling switches macrophages from production of inflammatory cytokines such as tumor necrosis factor-alpha to production of the angiogenic growth factor vascular endothelial growth factor (VEGF). We show in this study that this switch critically requires signaling through MyD88, IRAK4, and TRAF6. Macrophages from mice lacking MyD88 (MyD88(-/-)) or IRAK4 (IRAK4(-/-)) lacked responsiveness to TLR agonists and did not respond to A2AR agonists by expressing VEGF. Suppression of TRAF6 expression with siRNA in RAW264.7 macrophages also blocked their response to TLR and A2AR agonists. Excisional skin wounds in MyD88(-/-) mice healed at a markedly slower rate than wounds in wild-type MyD88(+/+) mice, showing delayed contraction, decreased and delayed granulation tissue formation, and reduced new blood vessel density. Although macrophages accumulated to higher levels in MyD88(-/-) wounds than in controls, expression of VEGF and HIF1-alpha mRNAs was elevated in MyD88(+/+) wounds. CGS21680, an A2AR agonist, promoted repair in MyD88(+/+) wounds and stimulated angiogenesis but had no significant effect on healing of MyD88(-/-) wounds. These results suggest that the synergistic interaction between TLR and A(2A)R signaling observed in vitro that switches macrophages from an inflammatory to an angiogenic phenotype also plays a role in wound healing in vivo.

Genetic removal of the A2Aadenosine receptor enhances pulmonary inflammation, mucin production, and angiogenesis in adenosine deaminase-deficient mice

Adenosine is generated at sites of tissue injury where it serves to regulate inflammation and damage. Adenosine signaling has been implicated in the regulation of pulmonary inflammation and damage in diseases such as asthma and chronic obstructive pulmonary disease; however, the contribution of specific adenosine receptors to key immunoregulatory processes in these diseases is still unclear. Mice deficient in the purine catabolic enzyme adenosine deaminase (ADA) develop pulmonary inflammation and mucous metaplasia in association with adenosine elevations making them a useful model for assessing the contribution of specific adenosine receptors to adenosine-mediated pulmonary disease. Studies suggest that the A(2A) adenosine receptor (A(2A)R) functions to limit inflammation and promote tissue protection; however, the contribution of A(2A)R signaling has not been examined in the ADA-deficient model of adenosine-mediated lung inflammation. The purpose of the current study was to examine the contribution of A(2A)R signaling to the pulmonary phenotype seen in ADA-deficient mice. This was accomplished by generating ADA/A(2A)R double knockout mice. Genetic removal of the A(2A)R from ADA-deficient mice resulted in enhanced inflammation comprised largely of macrophages and neutrophils, mucin production in the bronchial airways, and angiogenesis, relative to that seen in the lungs of ADA-deficient mice with the A(2A)R. In addition, levels of the chemokines monocyte chemoattractant protein-1 and CXCL1 were elevated, whereas levels of cytokines such as TNF-alpha and IL-6 were not. There were no compensatory changes in the other adenosine receptors in the lungs of ADA/A(2A)R double knockout mice. These findings suggest that the A(2A)R plays a protective role in the ADA-deficient model of pulmonary inflammation.

Adenosine and anergy

T cells must integrate multiple environmental cues when deciding whether to mount an immunogenic or tolerogenic response. Since not all self-reactive T cells are eliminated during thymic development, mechanisms of peripheral tolerance such as T cell anergy contribute to preventing autoimmunity. Recent studies have implicated extracellular adenosine and the adenosine A2A receptor as playing an important role in inhibiting T cell effector function. Herein, we review the current literature regarding T cell anergy and the emerging literature implicating the A2A receptor as critical regulator of immune activation. Finally, we present evidence to suggest a possible role for adenosine A2A receptor signaling in T cell anergy.

Early and transient alteration of adenosine A2A receptor signaling in a mouse model of Huntington disease

Huntington Disease (HD) is characterized by choreic involuntary movements and striatal vulnerability. A2A receptors expressed on GABAergic striatal neurons have been suggested to play a pathogenetic role. Previous data demonstrated the presence of an aberrant alteration of A2A receptor-dependent adenylyl cyclase in an in vitro model of the disease (striatal cells expressing mutant huntingtin) and in peripheral circulating cells of HD patients. Here, we investigated whether this dysfunction is present in the R6/2 HD transgenic mouse model, by analyzing striatal A2A receptor-binding and adenylyl cyclase activity at different developmental stages in comparison with age-matched wild type animals. A transient increase in A2A receptor density (Bmax) and A2A receptor-dependent cAMP production at early presymptomatic ages (7-14 postnatal days) was found. Both alterations normalized to control values starting from postnatal day 21. In contrast, A2A receptor mRNA, as detected by real time PCR, dramatically decreased starting from PND21 until late symptomatic stages (12 weeks of age). The discrepancy between A2A receptor expression and density suggests compensatory mechanisms. These data, reproducing ex vivo the previous observations in vitro, support the hypothesis that an alteration of A2A receptor signaling is present in HD and might represent an interesting target for neuroprotective therapies.

Role of adenosine A1 receptors in the modulation of dopamine D1 and adenosine A2a receptor signaling in the neostriatum

Adenosine is known to modulate the function of neostriatal neurons. Adenosine acting on A2A receptors increases the phosphorylation of dopamine- and cAMP-regulated phosphoprotein of Mr 32 kDa (DARPP-32) at Thr34 (the cAMP-dependent protein kinase [PKA] site) in striatopallidal neurons, and opposes dopamine D2 receptor signaling. In contrast, the role of adenosine A1 receptors in the regulation of dopamine/DARPP-32 signaling is not clearly understood. Here, we investigated the effect of adenosine A1 receptors on D1, D2 and A2A receptor signaling using mouse neostriatal slices. An A1 receptor agonist, 2-chloro-N6-cyclopentyladenosine (100 nM), caused a transient increase, followed by a transient decrease, in DARPP-32 Thr34 phosphorylation. Our data support the following model for the actions of the A1 receptor agonist. The A1 receptor-induced early increase in Thr34 phosphorylation was mediated by presynaptic inhibition of dopamine release, and the subsequent removal of tonic inhibition by D2 receptors of A2A receptor/Golf/cAMP/PKA signaling. The A1 receptor-induced late decrease in Thr34 phosphorylation was mediated by a postsynaptic Gi mechanism, resulting in inhibition of D1 and A2A receptor-coupled Golf/cAMP/PKA signaling in direct and indirect pathway neurons, respectively. In conclusion, A1 receptors play a major modulatory role in dopamine and adenosine receptor signaling.

Modification of Cytokine Milieu by A2A Adenosine Receptor Signaling–Possible Application for Inflammatory Diseases

Pro‐inflammatory cytokine TNF‐alpha (TNF) production from in vitro lipopolysaccharide (LPS)‐stimulated human peripheral blood CD14+ cells (PB‐CD14) was inhibited by A2A adenosine receptor (AdoR) (A2AR) or ß2 adrenergic receptor (ADR) (ß2R) signaling in a concentration‐dependent manner. These inhibitory effects were presumably mediated by the increase in intracellular cAMP. Furthermore A2AR agonist and ß2R agonist synergistically inhibited the TNF production of LPS‐stimulated PB‐CD14 cells. These results suggest that the anti‐inflammatory effect of extracellular adenosine is, at least in part, due to the modification of the cytokine milieu via A2A signaling, and that the targeting of both A2AR and ß2R may have strong therapeutic potential for the inflammatory diseases.

Receptor heteromerization in adenosine A2A receptor signaling: relevance for striatal function and Parkinson's disease.

Recently evidence has been presented that adenosine A2A and dopamine D2 receptors form functional heteromeric receptor complexes as demonstrated in human neuroblastoma cells and mouse fibroblast Ltk- cells. These A2A/D2 heteromeric receptor complexes undergo coaggregation, cointernalization, and codesensitization on D2 or A2A receptor agonist treatments and especially after combined agonist treatment. It is hypothesized that the A2A/D2 receptor heteromer represents the molecular basis for the antagonistic A2A/D2 receptor interactions demonstrated at the biochemical and behavioral levels. Functional heteromeric complexes between A2A and metabotropic glutamate 5 receptors (mGluR5) have also recently been demonstrated in HEK-293 cells and rat striatal membrane preparations. The A2A/mGluR5 receptor heteromer may account for the synergism found after combined agonist treatments demonstrated in different in vitro and in vivo models. D2, A2A, and mGluR5 receptors are found together in the dendritic spines of the striatopallidal GABA neurons. Therefore, possible D2/A2A/mGluR5 multimeric receptor complexes and the receptor interactions within them may have a major role in controlling the dorsal and ventral striatopallidal GABA neurons involved in Parkinson's disease and in schizophrenia and drug addiction, respectively.

Sensitization of neuronal A2A adenosine receptors after persistent D2 dopamine receptor activation.

Acute activation of Galpha(i/o)-coupled D2 dopamine receptors inhibits A2A adenosine receptor stimulation of adenylate cyclase. This antagonistic interaction between D2 dopamine and A2A adenosine receptors has been well documented; however, the effects of persistent activation of D2 dopamine receptors on subsequent A2A adenosine receptor signaling have not been explored. The present study investigated the effects of short-term (3-h) and long-term (18-h) activation of D2L dopamine receptors on subsequent A2A adenosine receptor stimulation of adenylate cyclase in CAD-D2L and NS20Y-D2L neuroblastoma cells. Short- and long-term activation of D2L dopamine receptors markedly increased 5'-N-methylcarboxamidoadenosine (MECA)-stimulated cyclic AMP accumulation 1.4-fold and 1.7-fold, respectively. D2L receptor-induced sensitization of A2A-stimulated cyclic AMP accumulation was blocked by the D2 antagonist spiperone and pertussis toxin pretreatment. In addition, persistent activation of A2A adenosine receptors resulted in 50% desensitization of subsequent MECA-stimulated cyclic AMP accumulation; however, MECA-induced desensitization of A2A adenosine receptors did not prevent completely quinpirole-induced sensitization of adenylate cyclase. These studies revealed a novel mode of regulation between D2L dopamine and A2A adenosine receptors and suggest a cooperative interaction in the regulation of cyclic AMP signaling.

Metabotropic mGlu5 receptors regulate adenosine A2A receptor signaling.

Dopamine, by activating dopamine D1-type receptors, and adenosine, by activating adenosine A(2A) receptors, stimulate phosphorylation of DARPP-32 (dopamine- and cAMP-regulated phosphoprotein of M(r) 32,000) at Thr-34. In this study, we investigated the effect of metabotropic glutamate (mGlu) receptors on DARPP-32 phosphorylation at Thr-34 in neostriatal slices. A broad-spectrum mGlu receptor agonist, trans-ACPD, and a group I mGlu receptor agonist, DHPG, stimulated DARPP-32 phosphorylation at Thr-34. Studies with mGlu receptor antagonists revealed that the effects of trans-ACPD and DHPG were mediated through activation of mGlu5 receptors. The action of mGlu5 receptors required activation of adenosine A(2A) receptors by endogenous adenosine. Conversely, the action of adenosine A(2A) receptors required activation of mGlu5 receptors by endogenous glutamate. Coactivation of mGlu5 and adenosine A(2A) receptors by exogenous agonists synergistically increased DARPP-32 phosphorylation. mGlu5 receptors did not require activation of dopamine D1-type receptors by endogenous dopamine, nor did dopamine D1-type receptors require activation of mGlu5 receptors by endogenous glutamate. DHPG potentiated the effect of forskolin, but not that of 8-bromo-cAMP, and stimulated DARPP-32 phosphorylation in the presence of the phosphodiesterase inhibitor IBMX, suggesting that mGlu5 receptors stimulate the rate of cAMP formation coupled to adenosine A(2A) receptors. The action of mGlu5 receptors was attenuated by inhibitors of extracellular signal-regulated kinase, but not by inhibitors of phospholipase C, p38, casein kinase 1, or Cdk5. The results demonstrate that mGlu5 receptors potentiate adenosine A(2A)DARPP-32 signaling by stimulating the adenosine A(2A) receptor-mediated formation of cAMP in an extracellular signal-regulated kinase-dependent manner.