P2Y Nucleotide Receptors Research Articles

Abstract 5543: The P2Y 2 Nucleotide Receptor Contributes To Intimal Lesion Growth And Regulates Lymphotoxin-α Secretion

Inflammatory processes play a crucial role in atherosclerotic lesion growth. A number of pro-inflammatory molecules have been implicated in the pathogenesis of atherosclerosis, including lymphotoxin-α (LTA). Our research has shown that activation of a G protein-coupled P2Y 2 nucleotide receptor (P2Y 2 R) expressed in vascular cells mediates inflammatory responses. To examine the role of the P2Y 2 R in lesion growth, we developed P2Y 2 R over-expressing transgenic rats. Collar-induced injury to the carotid artery of P2Y 2 R transgenic rats caused a dramatic increase in intimal lesion growth and significant macrophage accumulation, which nearly blocked the arterial lumen. Immunohistochemical staining showed that LTA and galectin-2 (gal-2) were abundantly expressed in smooth muscle cells (SMC) and macrophages in carotid lesions of P2Y 2 R transgenic rats. We also identified galectin-2 as a P2Y 2 R binding partner using the yeast two-hybrid system and a co-immunoprecipitation assay. P2Y 2 R agonist, UTP, stimulated gal-2 mRNA expression in rat carotid SMC. Transient transfection of SMC with a genomic fragment including the rat galectin (LGALS2) promoter incorporated into a luciferase (pGL-3) reporter vector showed that UTP markedly increased LGALS2 promoter activity in a dose-dependent manner. Moreover, UTP induced LTA secretion in cultured aortic SMC from wild type, but not P2Y 2 R −/− mice. Adenoviral expression of the full length P2Y 2 R in SMC from P2Y 2 R −/− mice fully restored UTP-induced LTA secretion. However, expression of a mutant P2Y 2 R that does not bind filamin A, an actin-binding protein that interacts with the P2Y 2 R, only partially restored UTP-induced LTA secretion in P2Y 2 R −/− SMC. Gal-2 siRNA partially inhibited LTA release into medium of cultured SMC expressing the P2Y 2 R. In contrast, gal-2 siRNA abolished UTP-induced LTA secretion in SMC isolated from P2Y 2 R −/− mice expressing the filamin A binding mutant P2Y 2 R. These results indicate that P2Y 2 Rs regulate gal-2-dependent LTA secretion in SMC via a filamin A-dependent mechanism that likely contributes to vascular inflammation.

Proinflammatory cytokines tumor necrosis factor-α and interferon-γ alter tight junction structure and function in the rat parotid gland Par-C10 cell line

Sjögren's syndrome (SS) is an autoimmune disorder characterized by inflammation and dysfunction of salivary glands, resulting in impaired secretory function. The production of the proinflammatory cytokines tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) is elevated in exocrine glands of patients with SS, although little is known about the effects of these cytokines on salivary epithelial cell functions necessary for saliva secretion, including tight junction (TJ) integrity and the establishment of transepithelial ion gradients. The present study demonstrates that chronic exposure of polarized rat parotid gland (Par-C10) epithelial cell monolayers to TNF-alpha and IFN-gamma decreases transepithelial resistance (TER) and anion secretion, as measured by changes in short-circuit current (I(sc)) induced by carbachol, a muscarinic cholinergic receptor agonist, or UTP, a P2Y(2) nucleotide receptor agonist. In contrast, TNF-alpha and IFN-gamma had no effect on agonist-induced increases in the intracellular calcium concentration [Ca(2+)](i) in Par-C10 cells. Furthermore, treatment of Par-C10 cell monolayers with TNF-alpha and IFN-gamma increased paracellular permeability to normally impermeant proteins, altered cell and TJ morphology, and downregulated the expression of the TJ protein, claudin-1, but not other TJ proteins expressed in Par-C10 cells. The decreases in TER, agonist-induced transepithelial anion secretion, and claudin-1 expression caused by TNF-alpha, but not IFN-gamma, were reversible by incubation of Par-C10 cell monolayers with cytokine-free medium for 24 h, indicating that IFN-gamma causes irreversible inhibition of cellular activities associated with fluid secretion in salivary glands. Our results suggest that cytokine production is an important contributor to secretory dysfunction in SS by disrupting TJ integrity of salivary epithelium.

Identification of hydrolytically stable and selective P2Y 1 receptor agonists

P2Y nucleotide receptors (P2YRs) are attractive pharmaceutical targets. Most P2YR agonists proposed as drugs consist of a nucleotide scaffold, but their use is limited due to their chemical and enzymatic instabilities. To identify drug candidates, we developed non-hydrolyzable P2YR agonists. We synthesized ATP-β,γ-CH 2 analogues 2– 4, and evaluated their chemical and metabolic stabilities and activities at P2Y 1,2,4,6 receptors. Analogues 2– 4 exhibited t 1/2 values of 14.5–65 h in gastric juice pH. They were completely resistant to alkaline phosphatase for 30 min at 37 °C and slowly hydrolyzed in human blood serum ( t 1/2 12.7–71.9 h). In comparison to ATP, analogues 2– 4 were barely hydrolyzed by nucleoside triphosphate diphosphohydrolases, NTPDase1,2,3,8 (<8% hydrolysis), and nucleotide pyrophosphatases, NPP1,3 (≤10% hydrolysis). Analogues 2 and 4B were selective agonists of the P2Y 1R with EC 50s of 0.08 and 17.2 μM, respectively. These features make analogues 2 and 4B potential therapeutic agents for health disorders involving the P2Y 1R.

P3-371: Interleukin-1β enhances nucleotide-induced α-secretase–dependent amyloid precursor protein processing in rat primary cortical neurons via upregulation of the P2Y 2 receptor

The P2Y2 nucleotide receptor (P2Y2R) stimulates α-secretase-dependent cleavage of the amyloid precursor protein (APP) in human 1321N1 astrocytoma cells, causing extracellular release of the neuroprotective protein sAPPα. In this study, we analyzed functional upregulation of the P2Y2R by cytokines and the P2Y2 receptor-mediated production of sAPPα in rat primary cortical neurons (rPCNs). In rPCNs, P2Y2R mRNA and receptor activity were virtually absent in unstimulated cells, whereas overnight treatment with the inflammatory cytokine interleukin 1β (IL-1β) upregulated both P2Y2R mRNA expression and receptor activity. The upregulation of P2Y2R mRNA was abrogated by pre-incubation with an IkappaBα phosphorylation inhibitor Bay 11, which suggests that P2Y2R mRNA transcript levels are regulated through NF–κB signaling. Furthermore, the P2Y2R agonist UTP enhanced the release of sAPPα in rPCNs treated with IL-1β or transfected with P2Y2R mRNA. UTP-induced release of sAPPα from rPCNs was completely inhibited by pretreatment of the cells with the metalloproteinase inhibitor TAPI-2 or the phosphatidylinositol 3-kinase (PI3K) inhibitor LY2904, and was partially inhibited by the MEK inhibitor U0126 and unaffected by the protein kinase C (PKC) inhibitor GF109203. The data indicate that P2Y2R-mediated release of sAPPα from neurons is dependent on ADAM10/17 and PI3K activity, partially dependent on ERK1/2 activity, and does not require activation of PKC. These results suggest that under inflammatory conditions, IL-1β evokes the functional upregulation of the P2Y2R in neurons to promote ADAM10/17-dependent APP processing through P2Y2R-mediated activation of PI3K. Ongoing studies with the TgCRND8 mouse model of Alzheimer's Disease and P2Y2 knockout mice are evaluating these findings in vivo.

Selective loss of P2Y2 nucleotide receptor immunoreactivity is associated with Alzheimer’s disease neuropathology

The uridine nucleotide-activated P2Y2, P2Y4 and P2Y6 receptors are widely expressed in the brain and are involved in many CNS processes, including those which malfunction in Alzheimer's disease (AD). However, the status of these receptors in the AD neocortex, as well as their putative roles in the pathogenesis of neuritic plaques and neurofibrillary tangles, remain unclear. In this study, we used immunoblotting to measure P2Y2, P2Y4 and P2Y6 receptors in two regions of the postmortem neocortex of neuropathologically assessed AD patients and aged controls. P2Y2 immunoreactivity was found to be selectively reduced in the AD parietal cortex, while P2Y4 and P2Y6 levels were unchanged. In contrast, all three receptors were preserved in the occipital cortex, which is known to be minimally affected by AD neuropathology. Furthermore, reductions in parietal P2Y2 immunoreactivity correlated both with neuropathologic scores and markers of synapse loss. These results provide a basis for considering P2Y2 receptor changes as a neurochemical substrate of AD, and point towards uridine nucleotide-activated P2Y receptors as novel targets for disease-modifying AD pharmacotherapeutic strategies.

Binding of the P2Y 2 Nucleotide Receptor to Filamin A Regulates Migration of Vascular Smooth Muscle Cells

The functional expression of the G protein-coupled P2Y(2) nucleotide receptor (P2Y(2)R) has been associated with proliferation and migration of vascular smooth muscle cells (SMCs), two processes involved in atherosclerosis and restenosis. Activation of the P2Y(2)R causes dynamic reorganization of the actin cytoskeleton, which transmits biochemical signals and forces necessary for cell locomotion, suggesting that P2Y(2)Rs may be linked to the actin cytoskeleton. Here, we identified filamin A (FLNa) as a P2Y(2)R-interacting protein using a yeast 2-hybrid system screen with the C-terminal region of the P2Y(2)R as bait. The FLNa binding site in the P2Y(2)R is localized between amino acids 322 and 333. Deletion of this region led to selective loss of FLNa binding to the P2Y(2)R and abolished Tyr phosphorylation of FLNa induced by the P2Y(2)R agonist UTP. Using both time-lapse microscopy and the Transwell cell migration assay, we showed that UTP significantly increased SMC spreading on collagen I (6.8 fold; P < or = 0.01) and migration (3.6 fold; P < or = 0.01) of aortic SMCs isolated from wild-type mice, as compared with unstimulated SMCs. UTP-induced spreading and migration of aortic SMCs did not occur with cells isolated from P2Y(2)R knockout mice. Expression of the full-length P2Y(2)R in SMCs isolated from P2Y(2)R knockout mice restored both UTP-induced spreading and migration. In contrast, UTP-induced spreading and migration did not occur in SMCs isolated from P2Y(2)R knockout mice transfected with a mutant P2Y(2)R that does not bind FLNa. Furthermore, ex vivo studies showed that both ATP and UTP (10 micromol/L) promoted migration of SMCs out of aortic explants isolated from wild-type but not P2Y(2)R knockout mice. Thus, this study demonstrates that P2Y(2)R/FLNa interaction selectively regulates spreading and migration of vascular SMCs.

Involvement of P2Y Receptors in TGFβ‐Induced EMT of MDCK Cells

Epithelial‐to‐mesenchymal transition (EMT) contributes to renal tubulointerstitial fibrosis, a common pathway leading to end‐stage renal disease. EMT can be induced in certain renal cells by transforming growth factor beta (TGFβ) and results in the phenotypic conversion of tubular epithelia to fibroblasts, reduction of E‐Cadherin expression and induction of α‐smooth muscle actin (αSMA). Since nucleotides can be released during renal injury, we hypothesized that P2Y (nucleotide) receptors contribute to EMT of renal epithelial cells. To test this hypothesis, we used a well‐characterized renal epithelial cell line, Madin Darby Kidney Cells (MDCK), and examined the role of P2Y receptors in TGFβ‐induced EMT. Quantative PCR experiments (used to identify P2Y receptor expression) revealed P2Y2 &gt; P2Y11 &gt; P2Y1 = P2Y14 &gt; P2Y6 = P2Y13 &gt; P2Y12 = P2Y4 as the relative order of abundance. TGFβ treatment reduced the expression of P2Y13 mRNA, elevated P2Y11 and P2Y14 mRNA levels without substantial changes in mRNA expression of P2Y1 and P2Y2. The P2Y agonist ATPγS inhibited protein expression of the EMT marker αSMA in response to TGFβ without influencing the E‐Cadherin decrease or morphological changes. These results indicate that TGFβ‐promoted EMT differentially regulates expression of P2Y receptor subtypes of MDCK cells and that P2Y receptor activation potentially blunts features of the EMT phenotype. Supported by grants from NIH.

The glucose moiety of uridine 5′–diphosphoglucose is structurally permissive in activation of the human P2Y14 receptor

The P2Y14 receptor is a signaling protein in the eight member P2Y nucleotide receptor family that is activated by UDP‐glucose (UDPG) and other uridine nucleotides. The P2Y14 receptor plays a role in the neuroimmune system, with expression in T cells, dendritic cells, and hematopoietic stem cells, and also is expressed in other tissues. We have systematically explored the structure activity relationships of UDPG analogues at this receptor. With the exception of 2‐ and 4‐thio modifications, most modifications of the nucleobase or ribose moieties diminished activity. UDPG also activates the P2Y2 receptor, but its 2‐thio analogue (MRS2690) was selective for the P2Y14 receptor and activated this receptor with 7‐fold greater potency than UDPG. Molecules in which the glucose moiety was replaced with a simple β‐methyloxy group retained agonist activity at the P2Y14 receptor. Replacement of the ribose moiety with a rigid methanocarba group, either in the North or South conformation, abolished agonist activity. Many sugar moieties, such as mannose and fructose, may be substituted for the glucose with retention of activity. Thus, the glucose moiety is the most structurally permissive region of the ligand for derivatization.

Abstract 732: The P2Y 2 Receptor Selectively Binds to Filamin A to Regulate Spreading and Migration of Vascular Smooth Muscle Cells

The functional expression of the G protein-coupled P2Y 2 nucleotide receptor has been associated with the development of intimal lesions. Activation of this receptor also stimulates actin cytoskeleton reorganization and migration of vascular smooth muscle cells (SMCs). Since cell migration has been linked to the dynamic reorganization of the actin cytoskeleton, which transmits biochemical signals and forces necessary for cell locomotion, the aim of the present study was to identify cytoskeletal proteins that bind to the P2Y 2 receptor and potentially regulate SMC migration. Using the yeast two-hybrid system, we isolated filamin A, a filamentous actin-cross linking protein that interacts with the C-terminal domain of the P2Y 2 receptor and we used deletion mapping to identify amino acid deletion in the P2Y 2 receptor that led to selective loss of filamin A binding. Ex-vivo treatment of aortic explants with the P2Y 2 receptor agonist UTP (10 μmol/L) significantly promoted migration of SMCs in wild type but not in P2Y 2 receptor −/− mice. Likewise, using a Transwell migration assay we showed that UTP increased migration of SMCs in wild type (3.5 folds, P&lt;0.01 vs. untreated cells) but not P2Y 2 receptor −/− mice (P&lt;0.4 vs. untreated cells). Adenoviral infection of the full length P2Y 2 receptor restored UTP-mediated cell migration in P2Y 2 receptor −/− mice whereas infection of a mutant P2Y 2 receptor that does not bind filamin A did not. UTP-induced migration was preceded by a rapid phosphorylation of filamin A that was not observed either in P2Y 2 receptor −/− SMCs or in P2Y 2 receptor −/− SMCs infected with the mutant P2Y 2 receptor that does not bind filamin A. Treatment of SMCs from wild type mice with UTP (10 μmol/L) caused a 4-fold increase in spreading to collagen I as compared to unstimulated cells. The UTP-mediated increase of SMC spreading was not found in P2Y 2 receptor −/− SMCs but was restored by adenoviral infection of the full length P2Y 2 receptor cDNA into these cells. However, adenoviral delivery of a mutant receptor −/− which does not bind to filamin A did not restore UTP-mediated spreading of P2Y 2 receptor SMCs to collagen. This study demonstrates that P2Y 2 -dependent modulation of the actin cytoskeleton selectively regulates spreading and migration of SMC.

P2Y2 nucleotide receptors inhibit trauma‐induced death of astrocytic cells

Nucleotides as well as other neurotransmitters are known to be released to the extracellular space upon injury. To determine whether nucleotides acting on P2Y(2) nucleotide receptors promote protective or degenerative events after trauma in astrocytic cells, a well-established model of in vitro brain trauma was applied to 1321N1 cells expressing recombinant P2Y(2) nucleotide receptors (P2Y(2)R-1321N1). Cellular death was examined by measuring DNA fragmentation and caspase activation. Fragmented DNA was observed 48 h post-injury in 1321N1 cells, while P2Y(2) nucleotide receptor expressing cells did not show DNA fragmentation. A laddering pattern of fragmented DNA following injury was observed upon inhibition of P2Y(2) nucleotide receptors with suramin. Time-dependent increases of cleaved caspase-9, a mitochondrial-associated caspase, correlated with injury-induced cellular death. A decreased bax/bcl-2 gene expression ratio was observed in P2Y(2)R-1321N1 cells after traumatic injury, while untransfected 1321N1 cells showed a significant time-dependent increase of the bax/bcl-2 gene expression ratio. Activation of protein kinases was assessed to determine the signaling pathways involved in cell death and survival responses following traumatic injury. In P2Y(2)R-1321N1 and 1321N1 cells p38 phosphorylation was stimulated in a time-dependent manner but the phosphatidylinositol 3-kinase-dependent activation of extracellular signal-regulated kinase 1/2 and protein kinase B (PKB)/Akt was only observed in P2Y(2)R-1321N1 cells after injury. The stress-activated protein kinase/c-Jun NH2-terminal kinase (SAPK/JNK) signaling pathway was not activated by traumatic injury in either astrocytic cell line. Inhibition of p38 kinase signaling pathway by treatment with PD1693, a MKK3/6 inhibitor, abolished the expression of cleaved caspase-9, the increase in the bax/bcl-2 gene expression ratio, as well as the fragmentation of DNA that followed injury of 1321N1 cells. Taken together, our results demonstrate a novel role for P2Y(2) nucleotide receptors and extracellular nucleotides in mediating survival responses to glial cells undergoing cellular death induced by trauma.

P2Y nucleotide receptor signaling through MAPK/ERK is regulated by extracellular matrix: Involvement of β3 integrins

Extracellular matrix influences cell behavior through receptors such as integrins and through transmission of mechanical forces. Nucleotides are released in response to mechanical stimuli and bind to P2 nucleotide receptors. As chondrocytes are subjected to frequent mechanical stimulation within a rich extracellular matrix, they are an excellent model for studying integration of signals induced by matrix and nucleotides. We investigated signaling of G protein-coupled P2Y receptors to MAPK/ERK and how this is influenced by matrix. Rat articular chondrocytes expressed transcripts for P2Y1, P2Y2, P2Y4, and P2Y6 receptors and responded to extracellular nucleotides by transient elevation of cytosolic calcium and MAPK/ERK phosphorylation. ERK1/2 activation was suppressed by the protein kinase C (PKC) inhibitors bisindolylmaleimide I and rottlerin, and by the phospholipase D inhibitor 1-butanol. Thus, nucleotides stimulate P2Y receptors to activate ERK1/2 through a mechanism dependent on PKC and phospholipase D. We next examined the involvement of integrins. Both an RGD-containing pentapeptide and a beta3 integrin blocking antibody, but not a beta1 integrin blocking antibody, abolished nucleotide-induced ERK1/2 phosphorylation. Moreover, chondrocytes adhering to fibronectin (which binds to beta1 and beta3 containing integrins in an RGD-dependent manner) displayed prolonged ERK1/2 signaling compared to cells grown on type I or II collagen (which bind to beta1-containing integrins in an RGD-independent manner). In conclusion, P2Y receptor signaling through ERK1/2 is gated selectively by matrix proteins. Thus, nucleotides released in response to mechanical stimulation will have differing effects on cell function due to changes in the composition of the extracellular matrix during development and disease.

Gα(q/11)‐coupled P2Y2nucleotide receptor inhibits human keratinocyte spreading and migration

Reepithelialization is a critical step in wound healing. It is initiated by keratinocyte migration at the wound edges. After wounding, extracellular nucleotides are released by keratinocytes and other skin cells. Here, we report that activation of P2Y2 nucleotide receptor by ATP/UTP inhibits keratinocyte cell spreading and induces lamellipodium withdrawal. Kymography analysis demonstrates that these effects correlate with a durable decrease of lamellipodium dynamics. P2Y2 receptor activation also induces a dramatic dismantling of the actin network, the loss of alpha3 integrin expression at the cell periphery, and the dissolution of focal contacts as indicated by the alteration of alpha(v) integrins and focal contact protein distribution. In addition, activation of P2Y2R prevents growth factor-induced phosphorylation of Erk(1,2) and Akt/PkB. The use of a specific pharmacological inhibitor (YM-254890), the depletion of G alpha(q/11) by siRNA, or the expression of a constitutively active G alpha(q/11) mutant (Q209L) show that activation of G alpha(q/11) is responsible for these ATP/UTP-induced effects. Finally, we report that ATP delays growth factor-induced wound healing of keratinocyte monolayers. Collectively, these findings provide evidence for a unique and important role for extracellular nucleotides as efficient autocrine/paracrine regulators of keratinocyte shape and migration during wound healing.

P2Y2 nucleotide receptor activation up-regulates vascular cell adhesion molecular-1 expression and enhances lymphocyte adherence to a human submandibular gland cell line

Sjögren's syndrome (SS) is a chronic inflammatory autoimmune disease that causes salivary and lacrimal gland tissue destruction resulting in impaired secretory function. Although lymphocytic infiltration of salivary epithelium is associated with SS, the mechanisms involved have not been adequately elucidated. Our previous studies have shown that the G protein-coupled P2Y2 nucleotide receptor (P2Y2R) is up-regulated in response to damage or stress of salivary gland epithelium, and in salivary glands of the NOD.B10 mouse model of SS-like autoimmune exocrinopathy. Additionally, we have shown that P2Y2R activation up-regulates vascular cell adhesion molecule-1 (VCAM-1) expression in endothelial cells leading to the binding of monocytes. The present study demonstrates that activation of the P2Y2R in dispersed cell aggregates from rat submandibular gland (SMG) and in human submandibular gland ductal cells (HSG) up-regulates the expression of VCAM-1. Furthermore, P2Y2R activation mediated the up-regulation of VCAM-1 expression in HSG cells leading to increased adherence of lymphocytic cells. Inhibitors of EGFR phosphorylation and metalloprotease activity abolished P2Y2R-mediated VCAM-1 expression and decreased lymphocyte binding to HSG cells. Moreover, silencing of EGFR expression abolished UTP-induced VCAM-1 up-regulation in HSG cells. These results suggest that P2Y2R activation in salivary gland cells increases the EGFR-dependent expression of VCAM-1 and the binding of lymphocytes, a pathway relevant to inflammation associated with SS.

The P2Y2 nucleotide receptor requires interaction with αv integrins to access and activate G12

The P2Y2 nucleotide receptor (P2Y2R) interacts with alpha v integrins to activate G(o) and induce chemotaxis in human 1321N1 astrocytoma cells. In this study, it was determined that the P2Y2R also requires interaction with alpha v integrins to activate G12 and associated signaling pathways that control chemotaxis in 1321N1 cells. Mutation of the Arg-Gly-Asp (RGD) integrin-binding sequence in the first extracellular loop of the human P2Y2R to Arg-Gly-Glu (RGE), which prevents integrin interaction, did not inhibit G(q) or ERK1/2 signaling by the P2Y2R agonist UTP but completely inhibited activation of G12 and G12-mediated events, including Rho activation, cofilin and myosin light chain-2 phosphorylation, stress fiber formation and chemotaxis towards UTP. The involvement of G12 in all these events was verified by using a dominant negative G alpha12 construct. G12 activation by the P2Y2R also was inhibited by anti-alpha v beta5 integrin antibodies and alpha v integrin antisense oligonucleotides, suggesting that alpha v integrin activity and expression are required for the P2Y2R to activate G12. Co-immunoprecipitation experiments confirmed that G alpha12 protein associates with the wild-type P2Y2R and with alpha v integrins but not with the RGE mutant P2Y2R or with alpha3 integrins. Collectively, these results suggest that alpha v integrin complexes provide the P2Y2R with access to G12, thereby allowing activation of this heterotrimeric G protein that controls actin cytoskeletal rearrangements required for chemotaxis.

Expression and functional characterization of P2Y1 and P2Y12 nucleotide receptors in long-term serum-deprived glioma C6 cells

We characterized the expression and functional properties of the ADP-sensitive P2Y(1) and P2Y(12) nucleotide receptors in glioma C6 cells cultured in medium devoid of serum for up to 96 h. During this long-term serum starvation, cell morphology changed from fibroblast-like flat to round, the adhesion pattern changed, cell-cycle arrest was induced, extracellular signal-regulated kinase (ERK1/2) phosphorylation was reduced, Akt phosphorylation was enhanced, and expression of the P2Y(12) receptor relative to P2Y(1) was increased. These processes did not reflect differentiation into astrocytes or oligodendrocytes, as expression of glial fibrillary acidic protein and NG2 proteoglycan (standard markers of glial cell differentiation) was not increased during the serum deprivation. Transfer of the cells into fresh medium containing 10% fetal bovine serum reversed the changes. This demonstrates that serum starvation caused only temporary growth arrest of the glioma C6 cells, which were ready for rapid division as soon as the environment became more favorable. In cells starved for 72 and 96 h, expression of the P2Y(1) receptor was low, and the P2Y(12) receptor was the major player, responsible for ADP-evoked signal transduction. The P2Y(12) receptor activated ERK1/2 kinase phosphorylation (a known cell proliferation regulator) and stimulated Akt activity. These effects were reduced by AR-C69931MX, a specific antagonist of the P2Y(12) receptor. On the other hand, Akt phosphorylation increased in parallel with the low expression of the P2Y(1) receptor, indicating the inhibitory role of P2Y(1) in Akt pathway signaling. The shift in nucleotide receptor expression from P2Y(1) to P2Y(12) would appear to be a new and important self-regulating mechanism that promotes cell growth rather than differentiation and is a defense mechanism against effects of serum deprivation.

Identification and Functional Characterization of the Hepatic Stellate Cell CD38 Cell Surface Molecule

The activation of hepatic stellate cells (HSCs) is a critical event in hepatic fibrosis, because these cells are the main producers of extracellular matrix proteins in the liver and contribute to the modulation of inflammatory responses via the secretion of several cytokines and the expression of adhesion molecules. The goal of the present study was to characterize cell surface proteins that regulate HSC activation. To this end, a panel of monoclonal antibodies (mAbs) was generated. mAb 14.27 recognized a protein of 45 kd that was highly expressed on HSCs. Affinity purification of this protein followed by sequencing revealed that protein to be CD38. We subsequently demonstrated that CD38 was constitutively expressed by HSCs and that its expression increased after in vitro and in vivo activation. mAb 14.27 induced an increase in cytosolic Ca2+ levels in HSCs, showing that it functions as an agonistic antibody. Moreover, the effects mediated by the CD38 mAb included induction of the proinflammatory cytokine interleukin-6 and up-regulation of the adhesion molecules intercellular adhesion molecule-1, vascular cell adhesion molecule-1, and neural cell adhesion molecule. Collectively, our data suggest that CD38 can act as a regulator of HSC activation and effector functions.

Expression Changes of P2Y2 and P2Y6 Nucleotide Receptors During C6 Glioma Cell Differentiation

Nucleotide receptors, which are activated by ATP, UTP and their metabolites, can induce a diversity of physiological responses in most tissues. Studies provide evidence of nucleotide signaling roles in the nervous system. Nevertheless, little is known about nucleotide receptors function during glial differentiation. Using C6 glioma cells, we studied the expression of four P2Y receptor subtypes: P2Y1, P2Y2, P2Y6, and P2Y12, during cellular differentiation. A nucleotide receptors time course was prepared using real-time RT-PCR and immunoblotting. In addition, sucrose density gradient fractionation with Na2CO3 was executed for P2Y2 and P2Y6. Laser scanning confocal microscopy was used to determine P2Y receptors expression. Time course analysis results indicate that P2Y2 receptors are down-regulated opposed to P2Y6 which shows a slightly up-regulation. P2Y2 and P2Y6 detected between fractions 3–5, which are characterized to be caveolar microdomains fractions. Indirect immunofluorescence analysis of P2Y2 and caveolin-1 in undifferentiated cells shows co-localization of them; a result confirmed using C6 glioma cells transfected with a cav1-EGFP chimera. Nucleotide receptors expression may be correlated to astrogliosis process observed during physiological and pathophysiological responses in nervous system. Supported by NIH GM08102, NIH SCORE GM08224, and NIH RISE GM61838.

Possible Role of P2Y2 Nucleotide Receptor in the Environment Generated After Injury

Spinal cord injury (SCI) increases inhibitory factors that may restrict neurite outgrowth after trauma. Release of inhibitory molecules and proliferation of reactive astrocytes at the injury site produce a nonpermissive environment for axonal regeneration. The mechanism that triggers astrogliosis is unknown, but the release of nucleotides has been linked to this hypertrophic state. Our goal is to investigate the spatio-temporal profile of P2Y2 after SCI. Molecular biology, immunohistochemical (IHC) studies, and behavioral assays were used to evaluate the expression and role of these receptors in rats injured at the T-10 level using the NYU impactor device. P2Y2 gene temporal profile using standardized RT-PCR showed a two-fold increase after 4–7 days post-injury (DPI) and returned to basal levels by 14 DPI. IHC studies demonstrated a low level of P2Y2 receptor in sham samples. After SCI, P2Y2 receptor expression in GFAP positive cells increased. Intrathecal infusion of suramin and PPADS antagonists resulted in some locomotor recovery. Our results suggest that P2Y2 receptors may play a role in the establishment of the restrictive environment for axonal regeneration after SCI. Supported by NIH-MRISP 2R2MH48190-14, NIH-SNRP NS39405, MBRS-SCORE S06-GM008224, MBRS-RISE GM-68138, PR-EPSCOR EPS-9874782.

P2Y2 receptors induced cell surface redistribution of αv integrin is required for activation of ERK 1/2 in U937 cells

Nucleotides released from cells due to stress, injury or inflammation, induce mitogenic effects in monocytes via activation of P2Y(2) nucleotide receptors (P2Y(2)Rs). Here we show that P2Y(2) nucleotide receptors in U937 monocytic cells regulate the activation of extracellular signal-regulated kinases 1 and 2 (ERK 1/2) by inducing the clustering of alpha(v) integrins. The activation of phosphatidylinositol 3-kinase by P2Y(2)R ligands was required for alpha(v) clustering, suggesting a means whereby two different classes of receptors communicate to induce mitogenic responses in monocytic cells. P2Y(2)R-induced alpha(v) clustering was also associated with a flattened phenotype of the U937 cells, consistent with the role of the P2Y(2)R in regulating early events in cell migration.

ATP Release Guides Neutrophil Chemotaxis via P2Y2 and A3 Receptors

Cells must amplify external signals to orient and migrate in chemotactic gradient fields. We find that human neutrophils release adenosine triphosphate (ATP) from the leading edge of the cell surface to amplify chemotactic signals and direct cell orientation by feedback through P2Y2 nucleotide receptors. Neutrophils rapidly hydrolyze released ATP to adenosine that then acts via A3-type adenosine receptors, which are recruited to the leading edge, to promote cell migration. Thus, ATP release and autocrine feedback through P2Y2 and A3 receptors provide signal amplification, controlling gradient sensing and migration of neutrophils.