P2Y Nucleotide Receptors Research Articles

The P2Y2 receptor mediates terminal adipocyte differentiation and insulin resistance: Evidence for a dual G-protein coupling mode

Several P2Y nucleotide receptors have been shown to be involved in the early stage of adipocyte differentiation in vitro and insulin resistance in obese mice; however, the exact receptor subtype(s) and its underlying molecular mechanism in relevant human cells are unclear. Here, using human primary visceral preadipocytes as a model, we found that during preadipocyte-to-mature adipocyte differentiation, the P2Y2 nucleotide receptor (P2Y2R) was the most upregulated subtype among the eight known P2Y receptors and the only one further dramatically upregulated after inflammatory TNFα treatment. Functional studies indicated that the P2Y2R induced intracellular Ca2+, ERK1/2, and JNK signaling but not the p38 pathway. In addition, stimulation of the P2Y2R suppressed basal and insulin-induced phosphorylation of AKT, accompanied by decreased GLUT4 membrane translocation and glucose uptake in mature adipocytes, suggesting a role of P2Y2R in insulin resistance. Mechanistically, we found that activation of P2Y2R did not increase lipolysis but suppressed PIP3 generation. Interestingly, activation of P2Y2R triggered Gi-protein coupling, and pertussis toxin pretreatment largely inhibited P2Y2R-mediated ERK1/2 signaling and cAMP suppression. Further, treatment of the cells with AR-C 118925XX, a selective P2Y2R antagonist, significantly inhibited adipogenesis, and P2Y2R knockout decreased mouse body weight gain with smaller eWAT mass infiltrated with fewer macrophages as compared to wild-type mice in response to a Western diet. Thus, we revealed that terminal adipocyte differentiation and inflammation selectively upregulate P2Y2R expression and that P2Y2R mediates insulin resistance by suppressing the AKT signaling pathway, highlighting P2Y2R as a potential new drug target to combat obesity and type-2 diabetes.

Knockdown of P2Y4 ameliorates sepsis-induced acute kidney injury in mice via inhibiting the activation of the NF-κB/MMP8 axis.

Sepsis-induced acute kidney injury (S-AKI) has emerged as a frequent and life-threatening complication in critically ill patients, which is characterized by a systematic inflammatory response and a rapid decline in kidney function. P2Y4, a member of G protein–coupled P2Y nucleotide receptor family, has been reported to serve as a crucial player in inflammatory responses during the development of neurocognitive disorder and myocardial infarction. Nonetheless, the biological role of P2Y4 in S-AKI remains largely unclear. This study aimed to decipher the biological role of P2Y4 in S-AKI and illuminate the potential mechanisms. In this study, S-AKI models were successfully established in mice via cecal ligation and puncture. Results showed that the kidney tissues from S-AKI mouse models exhibited a higher P2Y4 expression level than from the sham-operated group. Knockdown of P2Y4 was found to remarkably alleviate kidney damage and reduce inflammatory response in mice of S-AKI models. Moreover, P2Y4 ablation inhibited the activation of the NF-κB/MMP-8 signaling axis. Additionally, mechanistic studies revealed that rescuing MMP-8 reversed the alleviating effects of P2Y4 knockdown against renal cell damage. Collectively, our findings indicate that P2Y4 knockdown ameliorated S-AKI in mice via inhibiting the activation of the NF-κB/MMP-8 axis and that P2Y4 may represent a novel therapeutic target for S-AKI patients.

UTP Regulates the Cardioprotective Action of Transplanted Stem Cells Derived From Mouse Cardiac Adipose Tissue

Adipose tissue is a source of stem cells with a high potential of differentiation for cell-based regenerative therapies. We previously identified mouse P2Y2, an ATP and UTP nucleotide receptor, as a regulator of adipogenic and endothelial differentiation of cardiac adipose-derived stem cells (cADSC). We investigated here the potential involvement of P2Y2 receptor in the cardioprotective action of undifferentiated cADSC transplantation in mouse ischemic heart. Transplantation of cADSC was realized in the periphery of the infarcted zone of ischemic heart, 3 days after left anterior descending artery ligation. A strong reduction of collagen stained area was observed 14 days after cADSC injection, compared to PBS injection. Interestingly, loss of P2Y2 expression totally inhibits the ability of transplanted cADSC to reduce cardiac fibrosis. A detailed gene ontology enrichment analysis was realized by comparing RNA-sequencing data obtained for UTP-treated wild type cASDC and UTP-treated P2Y2-null cASDC. We identified UTP target genes linked to extracellular matrix organization such as matrix metalloproteinases and various collagen types, UTP target genes related to macrophage chemotaxis and differentiation into pro-fibrotic foam cells, and a significant number of UTP target genes linked to angiogenesis regulation. More particularly, we showed that UTP regulated the secretion of CCL5, CXCL5, and CCL12 chemokines and serum amyloid apolipoprotein 3, in the supernatants of UTP-treated cADSC. Interestingly, CCL5 is reported as a key factor in post-infarction heart failure and in the reparative and angiogenic action of transplanted ADSC on ischemic tissue. We investigated then if a UTP-pretreatment of cADSC amplifies their effect on cardiac revascularization in mouse ischemic heart. Transplantation of cADSC was able to increase peri-infarct capillary density, 14 days after their injection. This beneficial effect on cardiac revascularization was enhanced by a UTP-pretreatment of cADSC before their transplantation, and not observed using P2Y2-null cADSC. Our data support that the efficacy of transplanted cADSC can be regulated by the release of inflammatory mediators such as extracellular nucleotides in the ischemic site. The present study highlights the P2Y2 receptor as a regulator of cADSC cardioprotective action, and as a potential target for the therapeutic use of undifferentiated cADSC in post-ischemic cardiac ischemia.

UTP is a regulator of in vitro and in vivo angiogenic properties of cardiac adipose-derived stem cells.

The ability of cardiac adipose-derived stem cells (cADSC) to differentiate into multiple cell types has opened new perspectives in cardiac cell-based regenerative therapies. P2Y nucleotide receptors have already been described as regulators of adipogenic differentiation of cADSC and bone marrow-derived stem cells. In this study, we defined UTP as a regulator of cADSC endothelial differentiation. A daily UTP stimulation of cADSC during endothelial predifferentiation increased their capacity to form an endothelial network in matrigel. Additionally, pro-angiogenic UTP target genes such as epiregulin and hyaluronan synthase-1 were identified in predifferentiated cADSC by RNA sequencing experiments. Their regulation by UTP was confirmed by qPCR and ELISA experiments. We then evaluated the capacity of UTP-treated predifferentiated cADSC to increase post-ischemic revascularization in mice subjected to left anterior descending artery ligation. Predifferentiated cADSC treated or not with UTP were injected in the periphery of the infarcted zone, 3days after ligation. We observed a significant increase of capillary density 14 and 30days after UTP-treated predifferentiated cADSC injection, correlated with a reduction of cardiac fibrosis. This revascularization increase was not observed after injection of UTP-treated cADSC deficient for UTP and ATP nucleotide receptor P2Y2. The present study highlights the P2Y2 receptor as a regulator of cADSC endothelial differentiation and as a potential target for the therapeutic use of cADSC in post-ischemic heart revascularization.

P2Y2 receptor antagonism resolves sialadenitis and improves salivary flow in a Sjögren’s syndrome mouse model

ObjectiveSjögren’s syndrome (SS) is a chronic autoimmune exocrinopathy characterized by lymphocytic infiltration of the salivary and lacrimal glands and decreased saliva and tear production. Previous studies indicate that the G protein-coupled P2Y2 nucleotide receptor (P2Y2R) is upregulated in numerous models of salivary gland inflammation (i.e., sialadenitis), where it has been implicated as a key mediator of chronic inflammation. Here, we evaluate both systemic and localized P2Y2R antagonism as a means to resolve sialadenitis in the NOD.H-2h4,IFNγ−/−,CD28−/− (NOD.H-2h4 DKO) mouse model of SS. DesignFemale 4.5 month old NOD.H-2h4 DKO mice received daily intraperitoneal injections for 10 days of the selective P2Y2R antagonist, AR-C118925, or vehicle-only control. Single-dose localized intraglandular antagonist delivery into the Wharton’s duct was also evaluated. Carbachol-induced saliva was measured and then submandibular glands (SMGs) were isolated and either fixed and paraffin-embedded for H&E staining, homogenized for RNA isolation or dissociated for flow cytometry analysis. ResultsIntraperitoneal injection, but not localized intraglandular administration, of AR-C118925 significantly enhanced carbachol-induced salivation and reduced lymphocytic foci and immune cell markers in SMGs of 5 month old NOD.H-2h4 DKO mice, compared to vehicle-injected control mice. We found that B cells represent the primary immune cell population in inflamed SMGs of NOD.H-2h4 DKO mice that express elevated levels of P2Y2R compared to C57BL/6 control mice. We further demonstrate a role for P2Y2Rs in mediating B cell migration and the release of IgM. ConclusionOur findings suggest that the P2Y2R represents a novel therapeutic target for the treatment of Sjögren’s syndrome.

Nucleotides-Induced Changes in the Mechanical Properties of Living Endothelial Cells and Astrocytes, Analyzed by Atomic Force Microscopy.

Endothelial cells and astrocytes preferentially express metabotropic P2Y nucleotide receptors, which are involved in the maintenance of vascular and neural function. Among these, P2Y1 and P2Y2 receptors appear as main actors, since their stimulation induces intracellular calcium mobilization and activates signaling cascades linked to cytoskeletal reorganization. In the present work, we have analyzed, by means of atomic force microscopy (AFM) in force spectroscopy mode, the mechanical response of human umbilical vein endothelial cells (HUVEC) and astrocytes upon 2MeSADP and UTP stimulation. This approach allows for simultaneous measurement of variations in factors such as Young’s modulus, maximum adhesion force and rupture event formation, which reflect the potential changes in both the stiffness and adhesiveness of the plasma membrane. The largest effect was observed in both endothelial cells and astrocytes after P2Y2 receptor stimulation with UTP. Such exposure to UTP doubled the Young’s modulus and reduced both the adhesion force and the number of rupture events. In astrocytes, 2MeSADP stimulation also had a remarkable effect on AFM parameters. Additional studies performed with the selective P2Y1 and P2Y13 receptor antagonists revealed that the 2MeSADP-induced mechanical changes were mediated by the P2Y13 receptor, although they were negatively modulated by P2Y1 receptor stimulation. Hence, our results demonstrate that AFM can be a very useful tool to evaluate functional native nucleotide receptors in living cells.

P2Y2 receptors mediate nucleotide-induced EGFR phosphorylation and stimulate proliferation and tumorigenesis of head and neck squamous cell carcinoma cell lines.

To assess functional expression of the P2Y2 nucleotide receptor (P2Y2R) in head and neck squamous cell carcinoma (HNSCC) cell lines and define its role in nucleotide-induced epidermal growth factor receptor (EGFR) transactivation. The use of anti-EGFR therapeutics to treat HNSCC is hindered by intrinsic and acquired drug resistance. Defining novel pathways that modulate EGFR signaling could identify additional targets to treat HNSCC. In human HNSCC cell lines CAL27 and FaDu and the mouse oral cancer cell line MOC2, P2Y2R contributions to extracellular nucleotide-induced changes in intracellular free Ca2+ concentration and EGFR and extracellular signal-regulated kinase (ERK1/2) phosphorylation were determined using the ratiometric Ca2+ indicator fura-2 and immunoblot analysis, respectively. Genetic knockout of P2Y2Rs using CRISPR technology or pharmacological inhibition with P2Y2R-selective antagonist AR-C118925 defined P2Y2R contributions to in vivo tumor growth. P2Y2R agonists UTP and ATP increased intracellular Ca2+ levels and ERK1/2 and EGFR phosphorylation in CAL27 and FaDu cells, responses that were inhibited by AR-C118925 or P2Y2R knockout. P2Y2R-mediated EGFR phosphorylation was also attenuated by inhibition of the adamalysin family of metalloproteases or Src family kinases. P2Y2R knockout reduced UTP-induced CAL27 cell proliferation in vitro and significantly reduced CAL27 and FaDu tumor xenograft volume in vivo. In a syngeneic mouse model of oral cancer, AR-C118925 administration reduced MOC2 tumor volume. P2Y2Rs mediate HNSCC cell responses to extracellular nucleotides and genetic or pharmacological blockade of P2Y2R signaling attenuates tumor cell proliferation and tumorigenesis, suggesting that the P2Y2R represents a novel therapeutic target in HNSCC.

The P2Y2 Nucleotide Receptor Mediates Tissue Factor Expression in Human Monocytes

Recently we reported that in human coronary artery endothelial cells, activation of the P2Y2 receptor induces up‐regulation of tissue factor (TF), a key initiator of the coagulation cascade. However, it has been shown by others that monocyte TF is more important than endothelial TF in terms of provoking pro‐thrombotic state. Thus, we aimed to study whether human monocytes express P2Y2 receptor and its role in control of TF expression. RT‐PCR and receptor activity assays revealed that among the eight P2Y nucleotide receptors, the P2Y2 subtype was selectively and functionally expressed in human monocytic THP‐1 cells. Consistent with this, stimulation of the THP‐1 cells by ATP or UTP at micromolar concentration dramatically increased TF protein expression measured by Western blotting, which was abolished by AR‐C118925XX, a selective P2Y2 receptor antagonist, suggesting a key role for P2Y2 receptor. In addition, UTP or ATP treatment induced a rapid accumulation of TF mRNA, which was maximal at 2h and preceded with an increased TF pre‐mRNA, indicating enhanced TF gene transcription. However, we also observed a significant increase in TF mRNA stability after UTP and ATP treatment. Furthermore, activation of the monocyte P2Y2 receptor by a low dose of UTP or ATP (1μM) significantly activated the MAPK pathways including ERK1/2, JNK, and p38, along with their downstream transcription factors including c‐Jun, c‐Fos and ATF‐2, whereas blocking the ERK1/2, JNK or p38 pathways respectively, all significantly suppressed P2Y2 receptor‐mediated TF expression. These findings demonstrate for the first time that the P2Y2 receptor mediates TF expression in human monocytes through new mechanisms involving ERK1/2, JNK and p38, and that both transcriptional and posttranscriptional mechanisms contribute to a pro‐thrombotic status in monocytes through the P2Y2‐TF axis, highlighting monocyte P2Y2 receptor may be a new drug target for the prevention and/or treatment of relevant thrombotic diseases.Support or Funding InformationSupported by NIH funding 1R01HL125279‐01A1 (JS)

Cross-Talk in Nucleotide Signaling in Glioma C6 Cells.

The chapter is focused on the mechanism of action of metabotropic P2Y nucleotide receptors: P2Y1, P2Y2, P2Y12, P2Y14 and the ionotropic P2X7 receptor in glioma C6 cells. P2Y1 and P2Y12 both respond to ADP, but while P2Y1 links to PLC and elevates cytosolic Ca2+ concentration, P2Y12 negatively couples to adenylate cyclase, maintaining cAMP at low level. In glioma C6, these two P2Y receptors modulate activities of ERK1/2 and PI3K/Akt signaling and the effects depend on physiological conditions of the cells. During prolonged serum deprivation, cell growth is arrested, the expression of the P2Y1 receptor strongly decreases and P2Y12 becomes a major player responsible for ADP-evoked signal transduction. The P2Y12 receptor activates ERK1/2 kinase phosphorylation (a known cell proliferation regulator) and stimulates Akt activity, contributing to glioma invasiveness. In contrast, P2Y1 has an inhibitory effect on Akt pathway signaling. Furthermore, the P2X7 receptor, often responsible for apoptotic fate, is not involved in Ca2+elevation in C6 cells. The shift in nucleotide receptor expression from P2Y1 to P2Y12 during serum withdrawal, the cross talk between both receptors and the lack of P2X7 activity shows the precise self-regulating mechanism, enhancing survival and preserving the neoplastic features of C6 cells.

P2Y2 Nucleotide Receptor Is a Regulator of the Formation of Cardiac Adipose Tissue and Its Fat-Associated Lymphoid Clusters.

The formation of pericardial adipose tissue (PAT) and its regulatory function in cardiac inflammation are not well understood. We investigated the potential role of the ubiquitous ATP/UTP nucleotide receptor P2Y2 in the PAT by using P2Y2-null mice. We observed that P2Y2-null mice displayed a lower mass of PAT and a reduced density of its fat-associated lymphoid clusters (FALCs) and, more particularly, B cells. Loss of P2Y2 receptor in pericardial preadipocytes decreased their adipogenic differentiation and maturation abilities in vitro. Gene profiling identified P2Y2 target genes in PAT linked to immunomodulation. These data led to the identification of an increase of M2c anti-inflammatory macrophages correlated with increased apoptosis of B lymphocytes in P2Y2-null pericardial fat. In addition, follicular helper T cells, which contribute to B cell expansion in germinal centers, were dramatically decreased. The effect of P2Y2 loss was also investigated after ischemia-mediated expansion of FALCs in a model of myocardial infarct. Loss of P2Y2 led to reduced expansion of B and neutrophil populations in these clusters, whereas density of M2c anti-inflammatory macrophages was increased. Our study defines the P2Y2 nucleotide receptor as a regulator of the formation and inflammatory status of pericardial fat. The P2Y2 receptor could represent a therapeutic target in the regulation of PAT function before and during cardiac ischemia.

Purinergic Signaling in Mast Cell Degranulation and Asthma.

Mast cells are responsible for the majority of allergic conditions. It was originally thought that almost all allergic events were mediated directly only via the high-affinity immunoglobulin E receptors. However, recent evidence showed that many other receptors, such as G protein-coupled receptors and ligand-gated ion channels, are also directly involved in mast cell degranulation, the release of inflammatory mediators such as histamine, serine proteases, leukotrienes, heparin, and serotonin. These mediators are responsible for the symptoms in allergic conditions such as allergic asthma. In recent years, it has been realized that purinergic signaling, induced via the activation of G protein-coupled adenosine receptors and P2Y nucleotide receptors, as well as by ATP-gated P2X receptors, plays a significant role in mast cell degranulation. Both adenosine and ATP can induce degranulation and bronchoconstriction on their own and synergistically with allergens. All three classes of receptors, adenosine, P2X and P2Y are involved in tracheal mucus secretion. This review will summarize the currently available knowledge on the role of purinergic signaling in mast cell degranulation and its most relevant disease, asthma.

Mouse P2Y4 Nucleotide Receptor Is a Negative Regulator of Cardiac Adipose-Derived Stem Cell Differentiation and Cardiac Fat Formation.

Cardiac adipose tissue-derived stem cells (cASCs) have the ability to differentiate into multiple cell lineages giving them a high potential for use in regenerative medicine. Cardiac fat tissue still raises many unsolved questions related to its formation and features. P2Y nucleotide receptors have already been described as regulators of differentiation of bone-marrow derived stem cells, but remain poorly investigated in cASCs. We defined, in this study, the P2Y4 nucleotide receptor as a negative regulator of cardiac fat formation and cASC adipogenic differentiation. Higher expression of P2Y4 receptor in cardiac fat tissue was observed compared to other adipose tissues. P2Y4-null mice displayed a higher mass of cardiac adipose tissue specifically. We therefore examined the role of P2Y4 receptor in cASC adipogenic differentiation. An inhibitory effect of uridine 5'-triphosphate (UTP), ligand of P2Y4, was observed on the maturation state of differentiated cASCs, and on the expression of adipogenesis-linked genes and adiponectin, a cardioprotective adipokine. Higher adiponectin secretion by P2Y4-null adipocytes could be linked with cardioprotection previously observed in the heart of P2Y4-null ischemic mice. We realized here left anterior descending artery ligation on simple and double-knockout mice for P2Y4 and adiponectin. No cardioprotective effect of P2Y4 loss was observed in the absence of adiponectin secretion. In addition, P2Y4 loss was correlated with higher expression of UCP-1 (uncoupling protein-1) and CD137, two markers of brown/beige cardiac adipocytes. Our data highlight the P2Y4 receptor as an inhibitor of cardiac fat formation and cASC adipogenic differentiation, and as a potential therapeutic target in the regulation of cardioprotective function of cardiac fat.

The P2Y2 nucleotide receptor is an inhibitor of vascular calcification

Background and aimsMutations in the 5'-nucleotidase ecto (NT5E) gene that encodes CD73, a nucleotidase that converts AMP to adenosine, are linked to arterial calcification. However, the role of purinergic receptor signaling in the pathology of intimal calcification is not well understood. In this study, we examined whether extracellular nucleotides acting via P2Y2 receptor (P2Y2R) modulate arterial intimal calcification, a condition highly correlated with cardiovascular morbidity. MethodsApolipoprotein E, P2Y2R double knockout mice (ApoE−/−P2Y2R−/−) were used to determine the effect of P2Y2R deficiency on vascular calcification in vivo. Vascular smooth muscle cells (VSMC) isolated from P2Y2R−/− mice grown in high phosphate medium were used to assess the role of P2Y2R in the conversion of VSMC into osteoblasts. Luciferase-reporter assays were used to assess the effect of P2Y2R on the transcriptional activity of Runx2. ResultsP2Y2R deficiency in ApoE−/− mice caused extensive intimal calcification despite a significant reduction in atherosclerosis and macrophage plaque content. The ectoenzyme apyrase that degrades nucleoside di- and triphosphates accelerated high phosphate-induced calcium deposition in cultured VSMC. Expression of P2Y2R inhibits calcification in vitro inhibited the osteoblastic trans-differentiation of VSMC. Mechanistically, expression of P2Y2R inhibited Runx2 transcriptional activation of an osteocalcin promoter driven luciferase reporter gene. ConclusionsThis study reveals a role for vascular P2Y2R as an inhibitor of arterial intimal calcification and provides a new mechanistic insight into the regulation of the osteoblastic trans-differentiation of SMC through P2Y2R-mediated Runx2 antagonism. Given that calcification of atherosclerotic lesions is a significant clinical problem, activating P2Y2R may be an effective therapeutic approach for treatment or prevention of vascular calcification.

Upregulation of P2Y2 nucleotide receptor in human hepatocellular carcinoma cells.

Objective To examine if hypoxia inducible factor-1α (HIF-1α) can induce the upregulation of the purinergic receptor P2Y2 (P2Y2) and thereby promote the viability of human hepatocellular carcinoma (HCC) cells under hypoxic conditions.Methods Archival HCC tumour specimens and corresponding non-cancerous tissues were examined immunohistochemically for P2Y2 protein. A series of in vitro experiments were undertaken using HCC cell lines to determine the effect of hypoxia on HIF-1α and P2Y2 levels, the effect of HIF-1α upregulation on P2Y2 levels, and the effect of P2Y2 upregulation on cell viability under hypoxic conditions.Results Human HCC specimens were positive for P2Y2. Hypoxia and upregulated HIF-1α both upregulated the P2Y2 levels in HCC cell lines. P2Y2 upregulation using plasmid transfection resulted in enhanced cell viability under hypoxia. Treatment of HepG2 cells with the selective P2Y2 antagonist MRS2312 downregulated P2Y2 and reduced cell viability in five HCC cell lines. P2Y2 knockdown reduced HepG2 cell viability under hypoxia.Conclusions These present results suggest that HCC cells upregulate P2Y2 levels during hypoxia, which in turn promotes their growth. P2Y2 could be a potential therapeutic target for treating HCC.

Deletion of P2Y2 receptor reveals a role for lymphotoxin-α in fatty streak formation

BackgroundLymphotoxin alpha (LTα) is expressed in human atherosclerotic lesions and genetic variations in the LTα pathway have been linked to myocardial infarction. Activation of the P2Y2 nucleotide receptor (P2Y2R) regulates the production of LTα. in vitro. We aimed to uncover a potential pathway linking purinergic receptor to LTα-mediated inflammatory processes pivotal to the early stages of atherosclerosis in apolipoprotein E (ApoE−/−) deficient mice. Methods and resultsEn face immunostaining revealed that P2Y2R and VCAM-1 are preferentially expressed in the atherosclerosis prone site of the mouse aortic sinus. Deletion of the P2Y2R gene suppresses VCAM-1 expression. Compared with ApoE−/− mice, ApoE−/− mice lacking the P2Y2R gene (ApoE−/−/P2Y2R−/−) did not develop fatty streak lesions when fed a standard chow diet for 15weeks. Systemic and CD4+ T cell production of the pro-inflammatory cytokine lymphotoxin-alpha (LTα) were specifically inhibited in ApoE−/−/P2Y2R−/−mice. Anti-LTα preventive treatment was initiated in ApoE−/−mice with intraperitoneal administration of recombinant human tumor necrosis factor receptor 1 fusion protein (TNFR1-Fc) on 5 consecutive days before the disease onset. Remarkably, none of the TNFR1:Fc-treated ApoE−/−mice exhibited atherosclerotic lesions at any developmental stage. SignificanceApoE−/− mice deficient in P2Y2R exhibit low endothelial cell VCAM-1 levels, decreased production of LTα and delayed onset of atherosclerosis. These data suggest that targeting this nucleotide receptor could be an effective therapeutic approach in atherosclerosis.

Caveolin-1 Regulates the P2Y2 Receptor Signaling in Human 1321N1 Astrocytoma Cells

Damage to the CNS can cause a differential spatio-temporal release of multiple factors, such as nucleotides, ATP and UTP. The latter interact with neuronal and glial nucleotide receptors. The P2Y2 nucleotide receptor (P2Y2R) has gained prominence as a modulator of gliotic responses after CNS injury. Still, the molecular mechanisms underlying these responses in glia are not fully understood. Membrane-raft microdomains, such as caveolae, and their constituent caveolins, modulate receptor signaling in astrocytes; yet, their role in P2Y2R signaling has not been adequately explored. Hence, this study evaluated the role of caveolin-1 (Cav-1) in modulating P2Y2R subcellular distribution and signaling in human 1321N1 astrocytoma cells. Recombinant hP2Y2R expressed in 1321N1 cells and Cav-1 were found to co-fractionate in light-density membrane-raft fractions, co-localize via confocal microscopy, and co-immunoprecipitate. Raft localization was dependent on ATP stimulation and Cav-1 expression. This hP2Y2R/Cav-1 distribution and interaction was confirmed with various cell model systems differing in the expression of both P2Y2R and Cav-1, and shRNA knockdown of Cav-1 expression. Furthermore, shRNA knockdown of Cav-1 expression decreased nucleotide-induced increases in the intracellular Ca(2+) concentration in 1321N1 and C6 glioma cells without altering TRAP-6 and carbachol Ca(2+) responses. In addition, Cav-1 shRNA knockdown also decreased AKT phosphorylation and altered the kinetics of ERK1/2 activation in 1321N1 cells. Our findings strongly suggest that P2Y2R interaction with Cav-1 in membrane-raft caveolae of 1321N1 cells modulates receptor coupling to its downstream signaling machinery. Thus, P2Y2R/Cav-1 interactions represent a novel target for controlling P2Y2R function after CNS injury.

Abstract 346: Manipulating P2Y2 Receptor Biased Signaling to Limit Pro-thrombotic Gene Expression in Human Coronary Artery Endothelial Cells

Recently we reported that in human coronary artery endothelial cells (HCAEC), the P2Y2 nucleotide receptor (P2Y2R) regulates the pro-thrombotic gene tissue factor (TF) expression through positive and negative signaling mechanisms. Here, we report the identification of a P2Y2R ligand JZS0312 which selectively activates the negative, but not the positive pathways, leading to decreased TF expression. Unlike the endogenous agonist UTP, JZS0312 alone had no effect on TF mRNA transcription, but it unexpectedly inhibited UTP-induced TF expression. Mechanistic study showed that JZS0312 induced intracellular Ca 2+ mobilization in HCAEC which exhibited the same efficacy as UTP and was inhibited by AR-C118925, a P2Y2R-selective antagonist. However, when evaluated in their activation of the MAPK pathways, JZS0312 only activated the ERK1/2, but not JNK and p38, whereas UTP activated all the three, suggesting JZS0312 as a biased ligand for the P2Y2R. This notion was supported by the fact that JZS0312-induced ERK1/2 activation was inhibited by AR-C1189258 and it had no effect on ERK1/2 in P2Y2R-null cells. In addition, JZS0312 selectively activated the TF gene repressor Fra-1, but not the positive AP-1 subunits c-Jun and ATF-2, whereas UTP activated all of them. Furthermore, luciferase activity assay indicates that JZS0312 suppressed TF gene promoter activity via ERK1/2 activation, while UTP increased overall TF promoter activity. These findings reveal that JZS0312 acts as a biased agonist in human endothelial P2Y2R, in which it selectively activates the ERK1/2-Fra1 pathway, leading to suppression of TF gene transcription. Thus, JZS0312 represents the first identified biased ligand in the P2Y receptor family, opening new avenue for potential drug discovery in relevant inflammatory and thrombotic diseases.

Abstract 159: Empowering Cardiac Progenitor Cell-mediated Repair of Injured Myocardium by Overexpressing P2Y2 Nucleotide Receptor

Heart failure is a leading cause of death in the US due to the limited capability of adult mammalian heart to regenerate following injury. Autologous stem cell therapy holds promise for regeneration of injured myocardium after myocardial infarction. However, stem cells derived from diseased organs exhibit impaired proliferative and migratory capabilities and increased susceptibility to cell death. Empowering stem cells from diverse origins, including cardiac progenitor cells (CPCs), with pro-survival genes has been attempted. Despite the well-established roles of purinergic signaling mediated by extracellular nucleotides in regulating diverse cellular responses in cardiovascular diseases, it has not been well-defined in CPCs. Our preliminary data show, for the first time, that the majority of P2 purinergic receptors are expressed and exhibit functional responses to ATP and UTP in mouse and human CPCs. Since previous findings have shown that the G protein-coupled P2Y 2 receptor (P2Y 2 R) induces cardioprotective responses in animal models as well as human cardiomyocytes and regulates a wide range of signaling pathways that are crucial to tissue repair in various experimental models and in stem cells from diverse origins, we aim to determine whether P2Y 2 R plays similar roles in CPCs. Our preliminary data show that the P2Y 2 R agonists ATP and UTP enhance human CPC (hCPC) proliferation, migration and survival. Interestingly, hCPCs that exhibit relatively slower growth kinetics and higher levels of senescence markers show a dramatic decrease in P2Y 2 R expression as compared to fast-growing hCPCs consistent with our hypothesis that overexpressing P2Y 2 R participates in rejuvenating hCPCs and improving their growth capabilities. This hypothesis will be tested in vivo by determining whether P2Y 2 R overexpression in hCPCs improves their reparative potential for injured mouse myocardium. We also introduce the novel hypothesis that P2Y 2 R-induced regenerative responses in hCPCs involve the activation of Hippo signaling that is known to be regulated by different GPCRs, which links the extracellular nucleotides released during cardiac ischemia to extracellular matrix sensing and Hippo signaling that have been recently implicated in cardiac regeneration.

P2Y2 Receptor Control of Tissue Factor Transcription in Human Coronary Artery Endothelial Cells: New AP‐1 Site and Negative Regulator

Recently we reported that the P2Y2 receptor (P2Y2R) is the predominant nucleotide receptor expressed in human coronary artery endothelial cells (HCAEC), and that P2Y2R activation by ATP or UTP induces dramatic up‐regulation of tissue factor (TF), key initiator of the coagulation cascade. However, the molecular mechanism of this P2Y2R‐TF axis remains unclear. Here we report a role of a newly identified AP‐1 consensus sequence along with its new binding components in P2Y2R regulation of TF transcription. We identified with bioinformatics tools that a novel AP‐1 site at ‐1363 bp of human TF promoter region is highly conserved across multiple species. Activation of P2Y2R increased TF promoter activity and mRNA expression in HCAEC. Truncation, deletion and mutation of this new distal AP‐1 site all significantly supressed TF promoter activity in response to P2Y2R activation. EMSA and ChIP assays further confirmed that upon P2Y2R activation, c‐Jun, ATF‐2 and Fra‐1, but not the typical c‐Fos, bound to the new AP‐1 site. In addition, loss‐of‐function studies using siRNAs confirmed a positive transactivation role of c‐Jun and ATF‐2, but unexpectedly revealed a strong negative role of Fra‐1 in P2Y2R‐induced TF up‐regulation. Furthermore, we found that P2Y2R activation promoted ERK1/2 phosphorylation, leading to Fra‐1 activation while JNK activated c‐Jun and ATF‐2. These findings reveal the basis for P2Y nucleotide receptor control of endothelial TF expression and indicate that targeting the P2Y2R‐Fra‐1‐TF pathway may be an attractive new strategy in control of vascular thrombogenicity and/or inflammation associated with endothelial dysfunction.

P2Y2 receptor activation by nucleotides released from highly metastatic breast cancer cells increases tumor growth and invasion via crosstalk with endothelial cells.

IntroductionExtracellular nucleotides are released and detectable in a high concentration within the tumor microenvironment. G protein-coupled P2Y2 nucleotide receptor (P2Y2R) is activated equipotently by adenosine triphosphate (ATP) and uridine 5′-triphosphate (UTP), which mediate proinflammatory responses such as cell migration and proliferation. However, the role of P2Y2R in the process of cancer metastasis remains unclear. This study aimed to determine the role of P2Y2R in the proliferation, migration and invasion of highly metastatic MDA-MB-231 breast cancer cells through crosstalk with endothelial cells (ECs).MethodsATP release and P2Y2R activity between high metastatic breast cancer cell MDA-MB-231 and low metastatic breast cancer cell MCF-7 were compared. Then, the role of P2Y2R on tumor growth and invasion via crosstalk with ECs was examined in vitro, using MDA-MB-231 cells and ECs transfected with control- or P2Y2R-siRNA, and in vivo, using an animal model injected with control-shRNA- or P2Y2R-shRNA-transfected MDA-MB-231 cells.ResultsWe found that this highly metastatic breast cancer cell line released higher levels of ATP and showed a higher P2Y2R activity in comparison to a low metastatic breast cancer cell line, MCF-7. In MDA-MB-231 cells, P2Y2R activation by ATP or UTP increased proliferation at 24 or 72 hours, which was abolished by P2Y2R knock-down. In addition, the adhesion of MDA-MB-231 cells to ECs and cell migration were both significantly increased by ATP or UTP through the expression of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) in MDA-MB-231 or ECs but not in cells where P2Y2R was knocked down. Furthermore, ATP- or UTP-mediated activation of P2Y2R induced MDA-MB-231 invasion through ECs, increased matrix metalloproteinase-9 (MMP-9) activity and vascular endothelial growth factor (VEGF) production in MDA-MB-231 and induced the phosphorylation of vascular endothelial (VE)-cadherin in ECs. Tumor growth and metastasis to other tissues were dramatically reduced, and body weight was increased in mice injected with P2Y2R-shRNA-transfected MDA-MB-231 cells compared to mice injected with control shRNA-transfected MDA-MB-231 cells.ConclusionThis study suggests that P2Y2R may play an important role in cancer metastasis via modulation of the crosstalk between cancer cells and ECs.Electronic supplementary materialThe online version of this article (doi:10.1186/bcr3694) contains supplementary material, which is available to authorized users.