P2Y4, P2Y6 and P2Y11 receptors: From the early days of cloning to their function.

The family of P2Y nucleotide receptors is composed of eight members differentiated by their pharmacology and their coupling to specific G-proteins and transduction mechanisms. The laboratory studying these nucleotide receptors at IRIBHM institute (Free University of Brussels) has participated actively in their cloning. We used classical cloning by homology strategies relying on polymerase chain reactions with degenerate primers or on DNA libraries screening with P2Y receptors-related primers or probes, respectively. We identified and characterised four of the eight human P2Y receptors cloned so far: P2Y4, P2Y6, P2Y11 and P2Y13 receptors. These human receptors displayed specific features in terms of pharmacology such as affinity for pyrimidine nucleotides for P2Y4 and P2Y6 receptors and differential G-protein coupling. Their specific and restricted tissue distribution compared to ubiquitous P2Y1 and P2Y2 receptors led us to study their physiological role in chosen cell systems or using mice deficient for these P2Y subtypes. These studies revealed over the years that the P2Y11 receptor was able to confer tolerogenic and tumorigenic properties to human dendritic cells and that P2Y4 and P2Y6 receptors were involved in mouse heart post-natal development and cardioprotection. P2Y receptors and their identified target genes could constitute therapeutic targets to regulate cardiac hypertrophy and regeneration. The multiple roles of P2Y receptors identified in the ischemic heart and cardiac adipose tissue could have multiple innovative clinical applications and present a major interest in the field of cardiovascular diseases. P2Y receptors can induce cardioprotection by the regulation of cardiac inflammation and the modulation of the volume and composition of cardiac adipose tissue. These findings might lead to the pre-clinical validation of P2Y receptors as new targets for the treatment of myocardial ischemia.

The purinergic receptor subtype P2Y2 mediates chemotaxis of neutrophils and fibroblasts in fibrotic lung disease.

Idiopathic pulmonary fibrosis (IPF) is a devastating disease with few available treatment options. Recently, the involvement of purinergic receptor subtypes in the pathogenesis of different lung diseases has been demonstrated. Here we investigated the role of the purinergic receptor subtype P2Y2 in the context of fibrotic lung diseases.The concentration of different nucleotides was measured in the broncho-alveolar lavage (BAL) fluid derived from IPF patients and animals with bleomycin-induced pulmonary fibrosis. In addition expression of P2Y2 receptors by different cell types was determined. To investigate the functional relevance of P2Y2 receptors for the pathogenesis of the disease the bleomycin model of pulmonary fibrosis was used. Finally, experiments were performed in pursuit of the involved mechanisms.Compared to healthy individuals or vehicle treated animals, extracellular nucleotide levels in the BAL fluid were increased in patients with IPF and in mice after bleomycin administration, paralleled by a functional up-regulation of P2Y2R expression. Both bleomycin-induced inflammation and fibrosis were reduced in P2Y2R-deficient compared to wild type animals. Mechanistic studies demonstrated that recruitment of neutrophils into the lungs, proliferation and migration of lung fibroblasts as well as IL6 production are key P2Y2R mediated processes.Our results clearly demonstrate the involvement of P2Y2R subtypes in the pathogenesis of fibrotic lung diseases in humans and mice and hence support the development of selective P2Y2R antagonists for the treatment of IPF.

Central Role of P2Y6 UDP Receptor in Arteriolar Myogenic Tone.

OBJECTIVE: Myogenic tone (MT) of resistance arteries ensures autoregulation of blood flow in organs and relies on the intrinsic property of smooth muscle to contract in response to stretch. Nucleotides released by mechanical strain on cells are responsible for pleiotropic vascular effects, including vasoconstriction. Here, we evaluated the contribution of extracellular nucleotides to MT. APPROACH AND RESULTS: We measured MT and the associated pathway in mouse mesenteric resistance arteries using arteriography for small arteries and molecular biology. Of the P2 receptors in mouse mesenteric resistance arteries, mRNA expression of P2X1 and P2Y6 was dominant. P2Y6 fully sustained UDP/UTP-induced contraction (abrogated in P2ry6(-/-) arteries). Preventing nucleotide hydrolysis with the ectonucleotidase inhibitor ARL67156 enhanced pressure-induced MT by 20%, whereas P2Y6 receptor blockade blunted MT in mouse mesenteric resistance arteries and human subcutaneous arteries. Despite normal hemodynamic parameters, P2ry6(-/-) mice were protected against MT elevation in myocardial infarction-induced heart failure. Although both P2Y6 and P2Y2 receptors contributed to calcium mobilization, P2Y6 activation was mandatory for RhoA-GTP binding, myosin light chain, P42-P44, and c-Jun N-terminal kinase phosphorylation in arterial smooth muscle cells. In accordance with the opening of a nucleotide conduit in pressurized arteries, MT was altered by hemichannel pharmacological inhibitors and impaired in Cx43(+/-) and P2rx7(-/-) mesenteric resistance arteries. CONCLUSIONS: Signaling through P2 nucleotide receptors contributes to MT. This mechanism encompasses the release of nucleotides coupled to specific autocrine/paracrine activation of the uracil nucleotide P2Y6 receptor and may contribute to impaired tissue perfusion in cardiovascular diseases.

Increased atherosclerosis in P2Y13/apolipoprotein E double-knockout mice: contribution of P2Y13 to reverse cholesterol transport.

AIMS: High-density lipoproteins (HDLs) protect against atherosclerosis mainly due to their function in hepatobiliary reverse cholesterol transport (RCT). This is a process whereby excess cholesterol from peripheral tissues is transported by HDL particles to the liver for further metabolism and biliary excretion. Hepatic uptake of HDL holoparticles involves the P2Y13 receptor, independently of the selective cholesteryl ester uptake mediated by scavenger receptor class B, type I (SR-BI). Accordingly, P2Y13-deficient mice (P2Y13 (-/-)) have impaired RCT. This study assessed whether P2Y13 deficiency would affect atherosclerotic development. METHODS AND RESULTS: P2Y13 (-/-) mice were crossbred with atherosclerosis-prone apoE(-/-) mice. When 15 weeks old, P2Y13 (-/-)/apoE(-/-) mice had more aortic sinus lesions than apoE(-/-) mice. Bone marrow transplantation showed that the absence of the P2Y13 receptor in blood cells did not lead to significantly greater atherosclerotic plaque size formation compared with control apoE(-/-) reconstituted animals. Conversely, the absence of the P2Y13 receptor, except in blood cells, resulted in lesion sizes similar to that in P2Y13 (-/-)/apoE(-/-) reconstituted mice, pointing to a role for non-haematopoietic-derived P2Y13. Unexpectedly, P2Y13 (-/-)/apoE(-/-) mice displayed a lower HDL-cholesterol level than apoE(-/-) mice, which might be due to greater SR-BI expression in the liver. However, P2Y13 deficiency in apoE(-/-) mice was translated into reduced biliary and faecal sterol excretion and impaired RCT from macrophage to faeces, suggesting that an alteration in hepatobiliary RCT could be solely responsible for the greater atherosclerosis observed. CONCLUSION: The P2Y13 receptor protects against atherosclerosis, primarily through its role in hepatobiliary RCT.

Enhancement of glucose uptake in mouse skeletal muscle cells and adipocytes by P2Y6 receptor agonists.

Glucose uptake by peripheral tissues such as skeletal muscles and adipocytes is important in the maintenance of glucose homeostasis. We previously demonstrated that P2Y6 receptor (P2Y6R) agonists protect pancreatic islet cells from apoptosis and stimulate glucose-dependent insulin release. Here, we investigated the effects of P2Y6R activation on glucose uptake in insulin target tissues. An agonist of the P2Y6R, P1-(5'-uridine)-P3-(5'-N4-methoxycytidine)-triphosphate (MRS2957), significantly increased the uptake of [3H]2-deoxyglucose in mouse C2C12 myotubes and 3T3-L1 adipocytes, and this stimulation was significantly decreased by a selective P2Y6R antagonist N,N″-1,4-butanediyl-bis[N'-(3-isothiocyanatophenyl)thiourea] (MRS2578). Pre-incubation with Compound C (an inhibitor of 5'-AMP-activated protein kinase, AMPK), or AMPK siRNA abolished the stimulatory effect of MRS2957 on glucose uptake. Also, MRS2957 (60 min incubation) increased recruitment of the facilitated glucose transporter-4 (GLUT4) to the cell membrane, which was blocked by MRS2578. Treatment of C2C12 myotubes with MRS2957 induced significant phosphorylation of AMPK, which increase GLUT4 expression through histone deacetylase (HDAC)5 signaling. Glucose uptake in primary mouse adipocytes from wild-type mice was stimulated upon P2Y6R activation by either MRS2957 or native agonist UDP, and the P2Y6R effect was antagonized by MRS2578. However, in adipocytes from P2Y6R-knockout mice P2Y6R agonists had no effect on glucose uptake, and there was no change in the glucose uptake by insulin. Our results indicate that the P2Y6R promotes glucose metabolism in peripheral tissues, which may be mediated through AMPK signaling.

Purinergic signalling and immune cells.

This review article provides a historical perspective on the role of purinergic signalling in the regulation of various subsets of immune cells from early discoveries to current understanding. It is now recognised that adenosine 5'-triphosphate (ATP) and other nucleotides are released from cells following stress or injury. They can act on virtually all subsets of immune cells through a spectrum of P2X ligand-gated ion channels and G protein-coupled P2Y receptors. Furthermore, ATP is rapidly degraded into adenosine by ectonucleotidases such as CD39 and CD73, and adenosine exerts additional regulatory effects through its own receptors. The resulting effect ranges from stimulation to tolerance depending on the amount and time courses of nucleotides released, and the balance between ATP and adenosine. This review identifies the various receptors involved in the different subsets of immune cells and their effects on the function of these cells.

Role of the P2Y13 receptor in the differentiation of bone marrow stromal cells into osteoblasts and adipocytes.

Accumulating evidence indicates that extracellular nucleotides, signaling through purinergic receptors, play a significant role in bone remodeling. Mesenchymal stem cells (MSCs) express functional P2Y receptors whose expression level is regulated during osteoblast or adipocyte differentiation. P2Y13 -deficient mice were previously shown to exhibit a decreased bone turnover associated with a reduction in the number of both osteoblasts and osteoclasts on the bone surfaces. We therefore examined whether P2Y13 R activation was involved in the osteogenic differentiation of MSC. Our study demonstrated that ADP stimulation of P2Y13 R(+/+) (but not P2Y13 R(-/-) ) adherent bone marrow stromal cells (BMSCs) increased significantly the formation of alkaline phosphatase-colony-forming units (CFU-ALP) as well as the expression of osteoblastic markers (osterix, alkaline phosphatase, and collagen I) involved in the maturation of preosteoblasts into osteoblasts. The number of CFU-ALP obtained from P2Y13 R(-/-) BMSC and the level of osteoblastic gene expression after osteogenic stimulation were strongly reduced compared to those obtained in wild-type cell cultures. In contrast, when P2Y13 R(-/-) BMSCs were incubated in an adipogenic medium, the number of adipocytes generated and the level of adipogenic gene expression (PPARγ2 and Adipsin) were higher than those obtained in P2Y13 R(+/+) MSC. Interestingly, we observed a significant increase of the number of bone marrow adipocytes in tibia of P2Y13 R(-/-) mice. In conclusion, our findings indicate that the P2Y13 R plays an important role in the balance of osteoblast and adipocyte terminal differentiation of bone marrow progenitors. Therefore, the P2Y13 receptor can be considered as a new pharmacological target for the treatment of bone diseases like osteoporosis. STEM Cells 2013;31:2747-2758.

The P2Y13 receptor regulates extracellular ATP metabolism and the osteogenic response to mechanical loading.

ATP release and subsequent activation of purinergic receptors has been suggested to be one of the key transduction pathways activated by mechanical stimulation of bone. The P2Y(13) receptor, recently found to be expressed by osteoblasts, has been suggested to provide a negative feedback pathway for ATP release in different cell types. Therefore, we hypothesized that the P2Y(13) receptor may contribute to the mediation of osteogenic responses to mechanical stimulation by regulating ATP metabolism by osteoblasts. To test this hypothesis, wild-type (WT) and P2Y(13) receptor knockout (P2Y(13)R-/-) mice were subject to non-invasive axial mechanical loading of the left tibiae to induce an osteogenic response. Micro-computed tomography analysis showed mechanical loading induced an osteogenic response in both strains of mice in terms of increased total bone volume and cortical bone volume, with the P2Y(13)R-/- mice having a significantly greater response. The extent of the increased osteogenic response was defined by dynamic histomorphometry data showing dramatically increased bone formation and mineral apposition rates in P2Y(13)R-/- mice compared with controls. In vitro, primary P2Y(13)R-/- osteoblasts had an accumulation of mechanically induced extracellular ATP and reduced levels of hydrolysis. In addition, P2Y(13)R-/- osteoblasts also had a reduction in their maximal alkaline phosphatase (ALP) activity, one of the main ecto-enzymes expressed by osteoblasts, which hydrolyzes extracellular ATP. In conclusion, deletion of the P2Y(13) receptor leads to an enhanced osteogenic response to mechanical loading in vivo, possibly because of the reduced extracellular ATP degradation by ALP. The augmented osteogenic response to mechanical stimulation, combined with suppressed bone remodeling activities and protection from OVX-induced bone loss after P2Y(13) receptor depletion as previously described, suggests a potential role for P2Y(13) receptor antagonist-based therapy, possibly in combination with mechanical loading, for the treatment of osteoporosis.

Protective role of P2Y2 receptor against lung infection induced by pneumonia virus of mice.

ATP released in the early inflammatory processes acts as a danger signal by binding to purinergic receptors expressed on immune cells. A major contribution of the P2Y(2) receptor of ATP/UTP to dendritic cell function and Th2 lymphocyte recruitment during asthmatic airway inflammation was previously reported. We investigated here the involvement of P2Y(2) receptor in lung inflammation initiated by pneumonia virus of mice infection. We demonstrated that P2Y(2) (-/-) mice display a severe increase in morbidity and mortality rate in response to the virus. Lower survival of P2Y(2) (-/-) mice was not significantly correlated with excessive inflammation despite the higher level of neutrophil recruiters in their bronchoalveolar fluids. Interestingly, we observed reduced ATP level and lower numbers of dendritic cells, CD4(+) T cells and CD8(+) T cells in P2Y(2) (-/-) compared to P2Y(2) (+/+) infected lungs. Lower level of IL-12 and higher level of IL-6 in bronchoalveolar fluid support an inhibition of Th1 response in P2Y(2) (-/-) infected mice. Quantification of DC recruiter expression revealed comparable IP-10 and MIP-3α levels but a reduced BRAK level in P2Y(2) (-/-) compared to P2Y(2) (+/+) bronchoalveolar fluids. The increased morbidity and mortality of P2Y(2) (-/-) mice could be the consequence of a lower viral clearance leading to a more persistent viral load correlated with the observed higher viral titer. The decreased viral clearance could result from the defective Th1 response to PVM with a lack of DC and T cell infiltration. In conclusion, P2Y(2) receptor, previously described as a target in cystic fibrosis therapy and as a mediator of Th2 response in asthma, may also regulate Th1 response protecting mice against lung viral infection.

Purinergic P2Y2 receptors promote neutrophil infiltration and hepatocyte death in mice with acute liver injury.

BACKGROUND & AIMS: During progression of liver disease, inflammation affects survival of hepatocytes. Endogenous release of adenosine triphosphate (ATP) in the liver activates purinergic P2 receptors (P2R), which regulate inflammatory responses, but little is known about the roles of these processes in the development of acute hepatitis. METHODS: We induced acute hepatitis in C57BL/6 mice by intravenous injection of concanavalin A and then analyzed liver concentrations of ATP and expression of P2R. We assessed P2Y(2)R(-/-) mice and C57BL/6 wild-type mice injected with suramin, a pharmacologic inhibitor of P2YR. Toxic liver failure was induced in mice by intraperitoneal injection of acetaminophen. Hepatocyte-specific functions of P2R signaling were analyzed in primary mouse hepatocytes. RESULTS: Induction of acute hepatitis in wild-type C57BL/6 mice released large amounts of ATP from livers and induced expression of P2Y(2)R. Liver damage and necrosis were greatly reduced in P2Y(2)R(-/-) mice and C57BL/6 mice given injections of suramin. Acetaminophen-induced liver damage was reduced in P2Y(2)R(-/-) mice. Analysis of liver-infiltrating immune cells during acute hepatitis revealed that expression of P2Y(2)R in bone marrow-derived cells was required for liver infiltration by neutrophils and subsequent liver damage. Hepatic expression of P2Y(2)R interfered with expression of genes that regulate cell survival, and promoted tumor necrosis factor-α-mediated cell death, in a cell-autonomous manner. CONCLUSIONS: Extracellular ATP and P2Y(2)R have cell-type specific, but synergistic functions during liver damage that regulate cellular immune responses and promote hepatocyte death. Reagents designed to target P2Y(2)R might be developed to treat inflammatory liver disease.

Reduced bone turnover in mice lacking the P2Y13 receptor of ADP.

Osteoporosis is a condition of excessive and uncoupled bone turnover, in which osteoclastic resorption exceeds osteoblastic bone formation, resulting in an overall net bone loss, bone fragility, and morbidity. Although numerous treatments have been developed to inhibit bone loss by blocking osteoclastic bone resorption, understanding of the mechanisms behind bone loss is incomplete. The purinergic signaling system is emerging to be a pivotal regulator of bone homeostasis, and extracellular ADP has previously been shown to be a powerful osteolytic agent in vitro. We report here that deletion of the P2Y(13) receptor, a G protein-coupled receptor for extracellular ADP, leads to a 40% reduction in trabecular bone mass, 50% reduction in osteoblast and osteoclast numbers in vivo, as well as activity in vitro, and an overall 50% reduction in the rate of bone remodeling in mice in vivo. Down-regulation of RhoA/ROCK I signaling and a reduced ratio of receptor activator of nuclear factor κB ligand/osteoprotegerin observed in osteoblasts from P2Y(13)R(-/-) mice might explain this bone phenotype. Furthermore, because one of the main causes of osteoporosis in older women is lack of estrogen, we examined the effect of ovariectomy of the P2Y(13)R(-/-) mice and found them to be protected from ovariectomy-induced bone loss by up to 65%. These data confirm a role of purinergic ADP signaling in the skeleton, whereby deletion of the P2Y(13) receptor leads to reduced bone turnover rates, which provide a protective advantage in conditions of accelerated bone turnover such as oestrogen deficiency-induced osteoporosis.

Purinergic receptor type 6 contributes to airway inflammation and remodeling in experimental allergic airway inflammation.

RATIONALE: Extracellular nucleotides have recently been identified as proinflammatory mediators involved in asthma pathogenesis by signaling via purinergic receptors, but the role of the purinergic receptor type 6 (P2Y6R) has not been previously investigated. OBJECTIVES: To investigate the role of P2Y6R in asthma pathogenesis. METHODS: Acute and chronic OVA model and also HDM model of allergic inflammation in C57Bl/6 mice treated with specific P2Y6R antagonist and P2Y6R(-/-) mice were evaluated for classical features of asthmatic inflammation. In addition, primary epithelial cell culture from human and epithelial cell lines from mouse and human were stimulated with P2Y6R agonist and treated with P2Y6R antagonist and assessed for IL-6, IL-8/CXCL8 and KC levels. Experiments with P2Y6R(-/-) and P2Y6R(+/+) chimera were performed to discriminate the role of P2Y6R activation in structural lung cells and in cells from hematopoietic system. MEASUREMENTS AND MAIN RESULTS: We observed that the intratracheal application of a P2Y6R antagonist (MRS2578) and P2Y6R deficiency inhibited cardinal features of asthma, such as bronchoalveolar lavage eosinophilia, airway remodeling, Th2 cytokine production, and bronchial hyperresponsiveness in the ovalbumin-alum model. MRS2578 was also effective in reducing airway inflammation in a model using house dust mite extracts to induce allergic lung inflammation. Experiments with bone marrow chimeras revealed the importance of the P2Y6R expression on lung structural cells in airway inflammation. In accordance with this finding, we found a strong up-regulation of P2Y6 expression on airway epithelial cells of animals with experimental asthma. Concerning the underlying mechanism, we observed that MRS2578 inhibited the release of IL-6 and IL-8/KC by lung epithelial cells in vivo, whereas intrapulmonary application of the P2Y6R agonist uridine-5'-diphosphate increased the bronchoalveolar levels of IL-6 and KC. In addition, selective activation of P2Y6 receptors induced the release of IL-6 and KC/IL-8 by murine and human lung epithelial cells in vitro. CONCLUSIONS: P2Y6R expression on airway epithelial cells is up-regulated during acute and chronic allergic airway inflammation, and selective blocking of P2Y6R or P2Y6R deficiency on the structural cells reduces cardinal features of experimental asthma. Thus, blocking pulmonary P2Y6R might be a target for the treatment of allergic airway inflammation.

Selective induction of endothelial P2Y6 nucleotide receptor promotes vascular inflammation.

During a systemic inflammatory response endothelial-expressed surface molecules have been strongly implicated in orchestrating immune responses. Previous studies have shown enhanced extracellular nucleotide release during acute inflammatory conditions. Therefore, we hypothesized that endothelial nucleotide receptors could play a role in vascular inflammation. To address this hypothesis, we performed screening experiments and exposed human microvascular endothelia to inflammatory stimuli, followed by measurements of P2Y or P2X transcriptional responses. These studies showed a selective induction of the P2Y(6) receptor (> 4-fold at 24 hours). Moreover, studies that used real-time reverse transcription-polymerase chain reaction, Western blot analysis, or immunofluorescence confirmed time- and dose-dependent induction of P2Y(6) with tumor necrosis factor α or Lipopolysaccharide (LPS) stimulation in vitro and in vivo. Studies that used MRS 2578 as P2Y(6) receptor antagonist showed attenuated nuclear factor κB reporter activity and proinflammatory gene expression in human microvascular endothelial cells in vitro. Moreover, pharmacologic or genetic in vivo studies showed attenuated inflammatory responses in P2Y(6)(-/-) mice or after P2Y(6) antagonist treatment during LPS-induced vascular inflammation. These studies show an important contribution of P2Y(6) signaling in enhancing vascular inflammation during systemic LPS challenge and implicate the P2Y(6) receptor as a therapeutic target during systemic inflammatory responses.

Role of the P2Y12 receptor in the modulation of murine dendritic cell function by ADP.

The effects of ADP on the biology of dendritic cells have been studied much less than those of ATP or adenosine. In this study, we showed that adenosine-5'-O-(2-thiodiphosphate) (ADPßS) induced intracellular Ca(2+) transients in murine dendritic cells (DCs). This effect was abolished by AR-C69931MX, a dual P2Y(12) and P2Y(13) receptor antagonist. RT-PCR experiments revealed the expression of both P2Y(12) and P2Y(13) mRNA in DCs. The Ca(2+) response to ADPßS was maintained in P2Y(13)-deficient DCs, whereas it was abolished completely in P2Y(12)(-/-) DCs. ADPßS stimulated FITC-dextran and OVA capture in murine DCs through macropinocytosis, and this effect was abolished in P2Y(12)(-/-) DCs. ADPßS had a similar effect on FITC-dextran uptake by human monocyte-derived DCs. OVA loading in the presence of ADPßS increased the capacity of DCs to stimulate OVA-specific T cells, whereas ADPßS had no effect on the ability of DCs to stimulate allogeneic T cells. Moreover, after immunization against OVA, the serum level of anti-OVA IgG1 was significantly lower in P2Y(12)(-/-) mice than that in wild-type controls. In conclusion, we have shown that the P2Y(12) receptor is expressed in murine DCs and that its activation increased Ag endocytosis by DCs with subsequent enhancement of specific T cell activation.

P2Y13 receptor is critical for reverse cholesterol transport.

UNLABELLED: A major atheroprotective functionality of high-density lipoproteins (HDLs) is to promote "reverse cholesterol transport" (RCT). In this process, HDLs mediate the efflux and transport of cholesterol from peripheral cells and its subsequent transport to the liver for further metabolism and biliary excretion. We have previously demonstrated in cultured hepatocytes that P2Y(13) (purinergic receptor P2Y, G protein-coupled, 13) activation is essential for HDL uptake but the potential of P2Y(13) as a target to promote RCT has not been documented. Here, we show that P2Y(13)-deficient mice exhibited a decrease in hepatic HDL cholesterol uptake, hepatic cholesterol content, and biliary cholesterol output, although their plasma HDL and other lipid levels were normal. These changes translated into a substantial decrease in the rate of macrophage-to-feces RCT. Therefore, hallmark features of RCT are impaired in P2Y(13)-deficient mice. Furthermore, cangrelor, a partial agonist of P2Y(13), stimulated hepatic HDL uptake and biliary lipid secretions in normal mice and in mice with a targeted deletion of scavenger receptor class B type I (SR-BI) in liver (hypomSR-BI-knockout(liver)) but had no effect in P2Y(13) knockout mice, which indicate that P2Y(13)-mediated HDL uptake pathway is independent of SR-BI-mediated HDL selective cholesteryl ester uptake. CONCLUSION: These results establish P2Y(13) as an attractive novel target for modulating RCT and support the emerging view that steady-state plasma HDL levels do not necessarily reflect the capacity of HDL to promote RCT.

ATP confers tumorigenic properties to dendritic cells by inducing amphiregulin secretion.

ATP, which has an important proinflammatory action as danger signal, induces the semimaturation of dendritic cells (DCs) that can be associated with immune tolerance. We identified epidermal growth factor receptor ligands as target genes of ATPγS, a slowly hydrolyzed ATP derivative, by a gene profiling approach in DCs. Amphiregulin was the most highly up-regulated gene in response to ATPγS. Human monocyte-derived DCs and mouse bone marrow-derived DCs released amphiregulin (AREG) after purinergic receptor activation, with a contribution of P2Y(11) and A(2B) receptor, respectively. Supernatants of LPS+ATPγS-stimulated DCs induced smooth muscle cell and Lewis Lung Carcinoma (LLC) cell growth in vitro. The coinjection of LPS+ATPγS-stimulated DCs or their supernatants with LLC cells increased tumor weight in mice compared with LPS-treated DCs. The preincubation of LPS+ATPγS-treated DC supernatants with an anti-AREG blocking antibody inhibited their positive effect on smooth muscle cell density and tumor growth. The present study demonstrates for the first time that DCs can be a source of AREG. ATP released from tumor cells might exert a tumorigenic action by stimulating the secretion of AREG from DCs. Antagonists of purinergic receptors expressed on DCs and anti-AREG blocking antibodies could have a therapeutic potential as antitumor agents.

P2Y nucleotide receptors: promise of therapeutic applications.

Extracellular nucleotides, such as ATP and UTP, have distinct signaling roles through a class of G-protein-coupled receptors, termed P2Y. The receptor ligands are typically charged molecules of low bioavailability and stability in vivo. Recent progress in the development of selective agonists and antagonists for P2Y receptors and study of knockout mice have led to new drug concepts based on these receptors. The rapidly accelerating progress in this field has already resulted in drug candidates for cystic fibrosis, dry eye disease and thrombosis. On the horizon are novel treatments for cardiovascular diseases, inflammatory diseases and neurodegeneration.

Purinergic receptor inhibition prevents the development of smoke-induced lung injury and emphysema.

Extracellular ATP acts as a "danger signal" and can induce inflammation by binding to purinergic receptors. Chronic obstructive pulmonary disease is one of the most common inflammatory diseases associated with cigarette smoke inhalation, but the underlying mechanisms are incompletely understood. In this study, we show that endogenous pulmonary ATP levels are increased in a mouse model of smoke-induced acute lung inflammation and emphysema. ATP neutralization or nonspecific P2R-blockade markedly reduced smoke-induced lung inflammation and emphysema. We detected an upregulation the purinergic receptors subtypes on neutrophils (e.g., P2Y2R), macrophages, and lung tissue from animals with smoke-induced lung inflammation. By using P2Y(2)R deficient ((-/-)) animals, we show that ATP induces the recruitment of blood neutrophils to the lungs via P2Y(2)R. Moreover, P2Y(2)R deficient animals had a reduced pulmonary inflammation following acute smoke-exposure. A series of experiments with P2Y(2)R(-/-) and wild type chimera animals revealed that P2Y(2)R expression on hematopoietic cell plays the pivotal role in the observed effect. We demonstrate, for the first time, that endogenous ATP contributes to smoke-induced lung inflammation and then development of emphysema via activation of the purinergic receptor subtypes, such as P2Y(2)R.

Lithium treatment arrests the development of neurofibrillary tangles in mutant tau transgenic mice with advanced neurofibrillary pathology.

Neurofibrillary tangles (NFTs) made of phosphorylated tau proteins are a key lesion of Alzheimer's disease and other neurodegenerative diseases, and previous studies have indicated that lithium can decrease tau phosphorylation in tau transgenic models. In this study, we have reassessed the effectiveness of treatment per os with lithium on the prevention, the arrest, or the reversal of NFT development in a tau transgenic line (Tg30tau) developing severe neurofibrillary pathology in the brain and the spinal cord. Wild-type and Tgtau30 mice were treated per os with lithium carbonate or with natrium carbonate by chronic chow feeding for 8 months starting at the age of 3 months (to test for a preventive effect on NFT formation) or by oral gavage for 1 month starting at the age of 9 months (after development of NFTs). In mice treated by oral gavage, a decrease of tau phosphorylation and of Sarkosyl-insoluble aggregated tau was observed in the brain and in the spinal cord. The density of NFTs identified by Gallyas staining in the hippocampus and in the spinal cord was also significantly reduced and was similar to that observed at the beginning of the lithium treatment. In these animals, the level of brain beta-catenin was increased probably as a result of its stabilization by glycogen synthase kinase-3beta inhibition. Despite this inhibitory effect of lithium on NFT development, the motor and working memory deficits were not significantly rescued in these aged animals. Chronic chow feeding with lithium did not alter the development of NFT. Nevertheless, this study indicates that even a relatively short-term per os treatment leading to high blood concentration of lithium is effective in arresting the formation of NFTs in the hippocampus and the spinal cord of a tau transgenic model.

NF546 [4,4'-(carbonylbis(imino-3,1-phenylene-carbonylimino-3,1-(4-methyl-phenylene)-carbonylimino))-bis(1,3-xylene-alpha,alpha'-diphosphonic acid) tetrasodium salt] is a non-nucleotide P2Y11 agonist and stimulates release of interleukin-8 from human monocyte-derived dendritic cells.

The G protein-coupled P2Y(11) receptor is involved in immune system modulation. In-depth physiological evaluation is hampered, however, by a lack of selective and potent ligands. By screening a library of sulfonic and phosphonic acid derivatives at P2Y(11) receptors recombinantly expressed in human 1321N1 astrocytoma cells (calcium and cAMP assays), the selective non-nucleotide P2Y(11) agonist NF546 [4,4'-(carbonylbis(imino-3,1-phenylene-carbonylimino-3,1-(4-methyl-phenylene)carbonylimino))-bis(1,3-xylene-alpha,alpha'-diphosphonic acid) tetrasodium salt] was identified. NF546 had a pEC(50) of 6.27 and is relatively selective for P2Y(11) over P2Y(1), P2Y(2), P2Y(4), P2Y(6), P2Y(12), P2X(1), P2X(2), and P2X(2)-X(3). Adenosine-5'-O-(3-thio)triphosphate (ATPgammaS), a nonhydrolyzable analog of the physiological P2Y(11) agonist ATP, and NF546 use a common binding site as suggested by molecular modeling studies and their competitive behavior toward the nanomolar potency antagonist NF340 [4,4'-(carbonylbis(imino-3,1-(4-methyl-phenylene)carbonylimino))bis(naphthalene-2,6-disulfonic acid) tetrasodium salt] in Schild analysis. The pA(2) of NF340 was 8.02 against ATPgammaS and 8.04 against NF546 (calcium assays). NF546 was further tested for P2Y(11)-mediated effects in monocyte-derived dendritic cells. Similarly to ATPgammaS, NF546 led to thrombospondin-1 secretion and inhibition of lipopolysaccharide-stimulated interleukin-12 release, whereas NF340 inhibited these effects. Further, for the first time, it was shown that ATPgammaS or NF546 stimulation promotes interleukin 8 (IL-8) release from dendritic cells, which could be inhibited by NF340. In conclusion, we have described the first selective, non-nucleotide agonist NF546 for P2Y(11) receptors in both recombinant and physiological expression systems and could show a P2Y(11)-stimulated IL-8 release, further supporting the immunomodulatory role of P2Y(11) receptors.