Angiotensin II stimulates DNA synthesis of rat pancreatic stellate cells by activating ERK through EGF receptor transactivation

Although angiotensin II (Ang II) is known to participate in pancreatic fibrosis, little is known as to the mechanism by which Ang II promotes pancreatic fibrosis. To elucidate the mechanism, we examined the action of Ang II on the proliferation of rat pancreatic stellate cells (PSCs) that play central roles in pancreatic fibrosis. Immunocytochemistry and Western blotting demonstrated that both Ang II type 1 and type 2 receptors were expressed in PSCs. [3H]Thymidine incorporation assay revealed that Ang II enhanced DNA synthesis in PSCs, which was blocked by Ang II type 1 receptor antagonist losartan. Western blotting using anti-phospho-epidermal growth factor (EGF) receptor and anti-phospho-extracellular signal regulated kinase (ERK) antibodies showed that Ang II-activated EGF receptor and ERK. Both EGF receptor kinase inhibitor AG1478 and MEK1 inhibitor PD98059 attenuated ERK activation and DNA synthesis enhanced by Ang II. These results indicate that Ang II stimulates PSC proliferation through EGF receptor transactivation-ERK activation pathway.     

Contribution of two ionotropic purinergic receptors to ATP responses in submandibular gland ductal cells

The effect of extracellular ATP on salivary gland function was compared in wild-type (WT) and P2X(7) knockout (KO) mice. The increase in the intracellular concentration of calcium ([Ca(2+)](i)) in response to carbachol was similar in submandibular ductal cells of WT and KO mice. ATP and its analog, benzoyl-ATP, induced a sustained increase in the [Ca(2+)](i) in WT animals. In KO mice, ATP slightly and transiently increased the [Ca(2+)](i) and benzoyl-ATP had no effect. The response to ATP of WT but not KO mice was blocked by KN-62, Coomassie blue and magnesium. The small response of ATP observed in KO mice was completely blocked in the absence of extracellular calcium, unchanged by U73122 and potentiated by ivermectin indicating the probable involvement of a P2X(4) receptor. A RT-PCR and a Western blot confirmed the presence of these receptors in ducts of both WT and KO mice. ATP increased the permeability of the cells to ethidium bromide and stimulated a phospholipase A(2) activity in WT but not KO mice. Mice submandibular gland cells secreted IL-1beta but this secretion was not modified by ATP and was similar in both groups of animals. The volume of saliva provoked by pilocarpine and the concentration of proteins, sodium and chloride in this saliva was similar in both groups of animals. The concentration of potassium was higher in KO mice. We can conclude that the major purinergic receptors expressed in mice submandibular ductal cells are P2X(7) receptors but that P2X(4) receptors are also involved in some ATP effects.     

Angiotensin II type 1 receptor interaction is an important regulator for the development of pancreatic fibrosis in mice

The renin-angiotensin system (RAS) plays important roles in various pathophysiological processes. However, the role of the RAS in pancreatic fibrosis has not been established. We investigated the role of angiotensin II (ANG II)-ANG II type 1 (AT(1)) receptor pathway in the development of pancreatic fibrosis with AT(1a) receptor-deficient [AT(1a)(-/-)] mice. To induce pancreatic fibrosis, AT(1a)(-/-) and wild-type (WT) mice were submitted to three episodes of acute pancreatitis induced by six intraperitoneal injections of 50 microg/kg body wt cerulein at hourly intervals, per week, for four consecutive weeks. Pancreatic fibrosis was assessed by histology and hydroxyproline content. Pancreatic stellate cell (PSC) activation and the localization of AT(1) receptors were assessed by Western blot analysis for alpha-smooth muscle actin and immunostaining. Transforming growth factor-beta(1) (TGF-beta(1)) mRNA expression in the pancreas was assessed by RT-PCR. Six intraperitoneal injections of cerulein induced acute pancreatitis in both AT(1a)(-/-) and WT mice. There were no significant differences between two groups with regard to serum amylase and histological changes. Pancreatic fibrosis induced by repeated episodes of acute pancreatitis was significantly attenuated in AT(1a)(-/-) mice compared with that in WT mice. This finding was accompanied by a reduction of activated PSCs. Dual-immunofluorescence staining in WT mice revealed that activated PSCs express AT(1) receptors. The level of TGF-beta(1) mRNA was lower in AT(1a)(-/-) mice than in WT mice. Our results demonstrate that the ANG II-AT(1) receptor pathway is not essential for the local pancreatic injury in acute pancreatitis but plays an important role in the development of pancreatic fibrosis through PSC activation and proliferation.     

The P2X7 nucleotide receptor mediates skeletal mechanotransduction

The P2X7 nucleotide receptor (P2X7R) is an ATP-gated ion channel expressed in many cell types including osteoblasts and osteocytes. Mice with a null mutation of P2X7R have osteopenia in load bearing bones, suggesting that the P2X7R may be involved in the skeletal response to mechanical loading. We found the skeletal sensitivity to mechanical loading was reduced by up to 73% in P2X7R null (knock-out (KO)) mice. Release of ATP in the primary calvarial osteoblasts occurred within 1 min of onset of fluid shear stress (FSS). After 30 min of FSS, P2X7R-mediated pore formation was observed in wild type (WT) cells but not in KO cells. FSS increased prostaglandin (PG) E2 release in WT cells but did not alter PGE2 release in KO cells. Studies using MC3T3-E1 osteoblasts and MLO-Y4 osteocytes confirmed that PGE2 release was suppressed by P2X7R blockade, whereas the P2X7R agonist BzATP enhanced PGE2 release. We conclude that ATP signaling through P2X7R is necessary for mechanically induced release of prostaglandins by bone cells and subsequent osteogenesis.     

Angiotensin II promotes the proliferation of activated pancreatic stellate cells by Smad7 induction through a protein kinase C pathway

Activated pancreatic stellate cells (PSCs) play major roles in promoting pancreatic fibrosis. We previously reported that angiotensin II (Ang II) enhances activated PSC proliferation through EGF receptor transactivation. In the present study, we elucidated a novel intracellular mechanism by which Ang II stimulates cellular proliferation. TGF-beta1 inhibits activated PSC proliferation via a Smad3 and Smad4-dependent pathway in an autocrine manner. We demonstrated that Ang II inhibited TGF-beta1-induced nuclear accumulation of Smad3 and Smad4. Furthermore, Ang II rapidly induced inhibitory Smad7 mRNA expression. Adenovirus-mediated Smad7 overexpression inhibited TGF-beta1-induced nuclear accumulation of Smad3 and Smad4, and potentiated activated PSC proliferation. PKC inhibitor Go6983 blocked the induction of Smad7 mRNA expression by Ang II. In addition, 12-O-tetradecanoyl-phorbol 13-acetate, a PKC activator, increased Smad7 mRNA expression. These results suggest that Ang II enhances activated PSC proliferation by blocking autocrine TGF-beta1-mediated growth inhibition by inducing Smad7 expression via a PKC-dependent pathway.     

Deletion of the P2X7 nucleotide receptor reveals its regulatory roles in bone formation and resorption

The P2X7 nucleotide receptor is an ATP-gated ion channel expressed widely in cells of hematopoietic origin. Our purpose was to explore the involvement of the P2X7 receptor in bone development and remodeling by characterizing the phenotype of mice genetically modified to disrupt the P2X7 receptor [knockout (KO)]. Femoral length did not differ between KO and wild-type (WT) littermates at 2 or 9 months of age, indicating that the P2X7 receptor does not regulate longitudinal bone growth. However, KO mice displayed significant reduction in total and cortical bone content and periosteal circumference in femurs, and reduced periosteal bone formation and increased trabecular bone resorption in tibias. Patch clamp recording confirmed expression of functional P2X7 receptors in osteoclasts from WT but not KO mice. Osteoclasts were present in vivo and formed in cultures of bone marrow from KO mice, indicating that this receptor is not essential for fusion of osteoclast precursors. Functional P2X7 receptors were also found in osteoblasts from WT but not KO mice, suggesting a direct role in bone formation. P2X7 receptor KO mice demonstrate a unique skeletal phenotype that involves deficient periosteal bone formation together with excessive trabecular bone resorption. Thus, the P2X7 receptor represents a novel therapeutic target for the management of skeletal disorders such as osteoporosis.     

A novel role for P2X7 receptor signalling in the survival of mouse embryonic stem cells

The growth of a pluripotent embryonic stem (ES) cell population is dependent on cell survival, proliferation and self-renewal. The nucleotide ATP represents an important extracellular signalling molecule that regulates the survival of differentiated cells, however, its role is largely undefined in embryonic stem cells. Here we report a role for ATP-gated P2X7 receptors in ES cell survival. The functional expression of P2X7 receptors in undifferentiated mouse ES cells is demonstrated using a selective P2X7 antagonist and small interfering RNA knockdown of these receptors. Our data illustrate a key role for the P2X7 receptor as an essential pro-survival signal required for optimal ES cell colony growth in the presence of leukemia inhibitor factor (LIF). However, chronic exposure to exogenous ATP leads to rapid P2X7-dependent cell death via necrosis. Together, these data demonstrate a novel role for P2X7 receptors in regulation of ES cell behaviour where they can mediate either a pro-survival or pro-death signal depending on the mode of activation.     

Peroxisome proliferator-activated receptor gamma overexpression inhibits pro-fibrogenic activities of immortalised rat pancreatic stellate cells

Pancreatic stellate cells (PSCs) play a key role in the development of pancreatic fibrosis, a constant feature of chronic pancreatitis and pancreatic cancer. In response to pro-fibrogenic mediators, PSCs undergo an activation process that involves proliferation, enhanced production of extracellular matrix proteins and a phenotypic transition towards myofibroblasts. Ligands of the peroxisome proliferator-activated receptor gamma (PPARgamma), such as thiazolidinediones, are potent inhibitors of stellate cell activation and fibrogenesis in pancreas and liver. The effects of PPARgamma ligands, however, are at least in part mediated through PPARgamma-independent pathways. Here, we have chosen a different approach to study regulatory functions of PPARgamma in PSCs. Using immortalised rat PSCs, we have established a model of tetracycline (tet)-regulated PPARgamma overexpression. Induction of PPARgamma expression strongly inhibited proliferation and enhanced the rate of apoptotic cell death. Furthermore, PPARgamma-overexpressing cells synthesised less collagen than controls. To monitor effects of PPARgamma on PSC gene expression, we employed Affymetrix microarray technology. Using stringent selection criteria, we identified 21 up- and 19 down-regulated genes in PPARgamma-overexpressing cells. Most of the corresponding gene products are either involved in lipid metabolism, play a role in signal transduction, or are secreted molecules that regulate cell growth and differentiation. In conclusion, our data suggest an active role of PPARgamma in the induction of a quiescent PSC phenotype. PPARgamma-regulated genes in PSCs may serve as novel targets for the development of antifibrotic therapies.     

Regulation by CRAMP of the responses of murine peritoneal macrophages to extracellular ATP

Peritoneal macrophages were isolated from wild type (WT) mice and from mice invalidated for the P2X₇ receptor (KO) which had been pretreated with thioglycolate. In cells from WT mice, 1 mM ATP increased the intracellular concentration of calcium ([Ca²⁺]_i), the uptake of ethidium bromide, the production of reactive oxygen species (ROS), the secretion of IL-1β, the release of oleic acid and of lactate dehydrogenase; it decreased the intracellular concentration of potassium ([K⁺]_i). In KO mice, ATP transiently increased the [Ca²⁺]_i confirming that the P2X₇ receptor is a major receptor of peritoneal macrophages. WKYMVm, an agonist of receptors for formylated peptides (FPR) also increased the [Ca²⁺]_i in murine macrophages. The slight increase of the [Ca²⁺]_i was strongly potentiated by ivermectin confirming the expression of functional P2X₄ receptors by murine peritoneal macrophages. CRAMP, the unique antimicrobial peptide derived from cathelin in mouse inhibited all the responses coupled to P2X₇ receptors in macrophages from WT mice. Agonists for FPR had no effect on the increase of the [Ca²⁺]_i in response to ATP. CRAMP had no effect on the increase of the [Ca²⁺]_i evoked by a combination of ATP and ivermectin in macrophages from P2X₇-KO mice.In summary CRAMP inhibits the responses secondary to the activation of the murine P2X₇ receptors expressed by peritoneal macrophages. This inhibition is not mediated by FPR receptors and is specific since CRAMP has no effect on the response coupled to P2X₄ receptors. It can thus be concluded that the interaction between P2X₇ receptors and cathelin-derived antimicrobial peptides is species-specific, in some cases (man) positive in others (mouse) negative.     

Ivermectin-dependent release of IL-1beta in response to ATP by peritoneal macrophages from P2X<Subscript>7</Subscript>-KO mice

The response to ATP of peritoneal macrophages from wild-type (WT) and P2X₇-invalidated (KO) mice was tested. Low concentrations (1–100 μM) of ATP transiently increased the intracellular concentration of calcium ([Ca²⁺]_i) in cells from both mice. The inhibition of the polyphosphoinositide-specific phospholipase C with U73122 inhibited this response especially in WT mice suggesting that the responses coupled to P2Y receptors were potentiated by the expression of P2X₇ receptors. One millimolar ATP provoked a sustained increase in the [Ca²⁺]_i only in WT mice. The response to 10 μM ATP was potentiated and prolonged by ivermectin in both mice. One millimolar ATP increased the influx of extracellular calcium, decreased the intracellular concentration of potassium ([K⁺]_i) and stimulated the secretion of interleukin-1β (IL-1β) only in cells from WT mice. Ten micromolar ATP in combination with 3 μM ivermectin reproduced these responses both in WT and KO mice. The secretion of IL-1β was also increased by nigericin in WT mice and the secretory effect of a combination of ivermectin with ATP in KO mice was suppressed in a medium containing a high concentration of potassium. In WT mice, 150 μM BzATP stimulated the uptake of YOPRO-1. Incubation of macrophages from WT and KO mice with 10 μM ATP resulted in a small increase of YOPRO-1 uptake, which was potentiated by addition of 3 μM ivermectin. The uptake of this dye was unaffected by pannexin-1 blockers. In conclusion, prolonged stimulation of P2X₄ receptors by a combination of low concentrations of ATP plus ivermectin produced a sustained activation of the non-selective cation channel coupled to this receptor. The ensuing variations of the [K⁺]_i triggered the secretion of IL-1β. Pore formation was also triggered by activation of P2X₄ receptors. Higher concentrations of ATP elicited similar responses after binding to P2X₇ receptors. The expression of the P2X₇ receptors was also coupled to a better response to P2Y receptors.     

Distinct roles of Smad2-, Smad3-, and ERK-dependent pathways in transforming growth factor-beta1 regulation of pancreatic stellate cellular functions

Pancreatic stellate cells (PSCs) play a major role in promoting pancreatic fibrosis. Transforming growth factor-beta(1) (TGF-beta(1)) regulates PSC activation and proliferation in an autocrine manner. The intracellular signaling pathways of the regulation were examined in this study. Immunoprecipitation and immunocytochemistry revealed that Smad2, Smad3, and Smad4 were functionally expressed in PSCs. Adenovirus-mediated expression of Smad2, Smad3, or dominant-negative Smad2/3 did not alter TGF-beta(1) mRNA expression level or the amount of autocrine TGF-beta(1) peptide. However, expression of dominant-negative Smad2/3 inhibited PSC activation and enhanced their proliferation. Co-expression of Smad2 with dominant-negative Smad2/3 restored PSC activation inhibited by dominant-negative Smad2/3 expression without changing their proliferation. By contrast, co-expression of Smad3 with dominant-negative Smad2/3 attenuated PSC proliferation enhanced by dominant-negative Smad2/3 expression without altering their activation. Exogenous TGF-beta(1) increased TGFbeta(1) mRNA expression in PSCs. However, PD98059, a specific inhibitor of mitogen-activated protein kinase kinase (MEK1), inhibited ERK activation by TGF-beta(1), and consequently attenuated TGF-beta(1) enhancement of its own mRNA expression in PSCs. We propose that TGF-beta(1) differentially regulates PSC activation, proliferation, and TGF-beta(1) mRNA expression through Smad2-, Smad3-, and ERK-dependent pathways, respectively.     

Adenosine‐5′‐triphosphate suppresses proliferation and migration capacity of human endometrial stem cells

Extracellular ATP through the activation of the P2X and P2Y purinergic receptors affects the migration, proliferation and differentiation of many types of cells, including stem cells. High plasticity, low immunogenicity and immunomodulation ability of mesenchymal stem cells derived from human endometrium (eMSCs) allow them to be considered a prominent tool for regenerative medicine. Here, we examined the role of ATP in the proliferation and migration of human eMSCs. Using a wound healing assay, we showed that ATP‐induced activation of purinergic receptors suppressed the migration ability of eMSCs. We found the expression of one of the ATP receptors, the P2X₇ receptor in eMSCs. In spite of this, cell activation with specific P2X₇ receptor agonist, BzATP did not significantly affect the cell migration. The allosteric P2X₇ receptor inhibitor, AZ10606120 also did not prevent ATP‐induced inhibition of cell migration, confirming that inhibition occurs without P2X₇ receptor involvement. Flow cytometry analysis showed that high concentrations of ATP did not have a cytotoxic effect on eMSCs. At the same time, ATP induced the cell cycle arrest, suppressed the proliferative and migration capacity of eMSCs and therefore could affect the regenerative potential of these cells.     

CCK1 and CCK2 Receptors Are Expressed on Pancreatic Stellate Cells and Induce Collagen Production

The gastrointestinal hormone cholecystokinin (CCK) can induce acute pancreatitis in rodents through its action on acinar cells. Treatment with CCK, in combination with other agents, represents the most commonly used model to induce experimental chronic pancreatitis. Pancreatic stellate cells (PSC) are responsible for pancreatic fibrosis and therefore play a predominant role in the genesis of chronic pancreatitis. However, it is not known whether PSC express CCK receptors. Using real time PCR techniques, we demonstrate that CCK1 and CCK2 receptors are expressed on rat PSC. Interestingly both CCK and gastrin significantly induced type I collagen synthesis. Moreover, both inhibit proliferation. These effects are comparable with TGF-β-stimulated PSC. Furthermore, the natural agonists CCK and gastrin induce activation of pro-fibrogenic pathways Akt, ERK, and Src. Using specific CCK1 and CCK2 receptor (CCK2R) inhibitors, we found that Akt activation is mainly mediated by CCK2R. Akt activation by CCK and gastrin could be inhibited by the PI3K inhibitor wortmannin. Activation of ERK and the downstream target Elk-1 could be inhibited by the MEK inhibitor U0126. These data suggest that CCK and gastrin have direct activating effects on PSC, are able to induce collagen synthesis in these cells, and therefore appear to be important regulators of pancreatic fibrogenesis. Furthermore, similar to TGF-β, both CCK and gastrin inhibit proliferation in PSC.     

Characterization of ATP‐induced cell death in the GL261 mouse glioma

Gliomas have one of the worst prognosis among cancers. Their resistance to cell death induced by endogenous neurotoxic agents, such as extracellular ATP, seems to play an important role in their pathobiology since alterations in the degradation rate of extracellular ATP drastically affects glioma growth in rats. In the present work we characterized the mechanisms of cell death induced by extracellular ATP in a murine glioma cell line, GL261. ATP and BzATP, a P2X7 agonist, induced cell death at concentrations that are described to activate the P2X7 receptor in mouse. oATP, an antagonist of P2X7, blocked the ATP‐induced cell death. Agonists of purinergic receptors expressed in GL261 such as adenosine, ADP, UTP did not cause any cell death, even at mM concentrations. A sub‐population of cells more sensitive to ATP expressed more P2X7 when compared to a less sensitive subpopulation. Accordingly, RNA interference of the P2X7 receptor drastically reduced ATP‐induced cell death, suggesting that this receptor is necessary for this effect. The mechanism of ATP‐induced cell death is predominantly necrotic, since cells presented shrinkage accompanied by membrane permeabilization, but not apoptotic, since no phosphatidylserine externalization or caspase activity was observed. These data show the importance of P2X7 in ATP‐induced cell death and shed light on the importance of ATP‐induced cell death in glioma development. J. Cell. Biochem. 109: 983–991, 2010. © 2010 Wiley‐Liss, Inc.     

Knock Out of S1P3 Receptor Signaling Attenuates Inflammation and Fibrosis in Bleomycin-Induced Lung Injury Mice Model

Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid metabolite involved in many critical cellular processes, including proliferation, migration, and angiogenesis, through interaction with a family of five G protein–coupled receptors (S1P1–5). Some reports have implicated S1P as an important inflammatory mediator of the pathogenesis of airway inflammation, but the role of S1P3 in the pathogenesis of lung diseases is not completely understood. We used S1P3-deficient (knockout (KO)) mice to clarify the role of S1P3 receptor signaling in the pathogenesis of pulmonary inflammation and fibrosis using a bleomycin-induced model of lung injury. On the seventh day after bleomycin administration, S1P3 KO mice exhibited significantly less body weight loss and pulmonary inflammation than wild-type (WT) mice. On the 28th day, there was less pulmonary fibrosis in S1P3 KO mice than in WT mice. S1P3 KO mice demonstrated a 56% reduction in total cell count in bronchoalveolar lavage fluid (BALF) collected on the seventh day compared with WT mice; however, the differential white blood cell profiles were similar. BALF analysis on the seventh day showed that connective tissue growth factor (CTGF) levels were significantly decreased in S1P3 KO mice compared with WT mice, although no differences were observed in monocyte chemotactic protein-1 (MCP-1) or transforming growth factor β1 (TGF-β1) levels. Finally, S1P levels in BALF collected on the 7th day after treatment were not significantly different between WT and S1P3 KO mice. Our results indicate that S1P3 receptor signaling plays an important role in pulmonary inflammation and fibrosis and that this signaling occurs via CTGF expression. This suggests that this pathway might be a therapeutic target for pulmonary fibrosis.     

P2X1 receptors mediate sympathetic postjunctional Ca2+ transients in mesenteric small arteries

Brief, spatially localized Ca(2+) transients occur in the smooth muscle adjacent to perivascular nerves of small arteries during neurogenic contractions. We named these "junctional Ca(2+) transients" (jCaTs) and postulated that they arose from Ca(2+) entering smooth muscle cells through P2X(1) receptors activated by neurally released ATP. Nevertheless, the lack of potent, subtype-selective P2X-receptor antagonists made determining the exact molecular identity of the channels difficult. Here we used small, pressurized mesenteric arteries from P2X(1)-receptor-deficient mice (KO) to test the hypothesis that jCaTs arise from Ca(2+) entering the smooth muscle cell via P2X(1) receptors. In wild-type (WT) arteries, confocal microscopy of fluo-4 fluorescence during electrical field stimulation (EFS) of perivascular sympathetic nerves revealed jCaTs in the smooth muscle cells adjacent to the perivascular nerves, similar to those reported previously in rat arteries, and alpha-latrotoxin (2.5 nM) markedly increased the frequency of "spontaneous" jCaTs. In the KO arteries, however, neither EFS nor alpha-latrotoxin elicited any jCaTs. A potent P2X-receptor agonist, alpha,beta-methylene ATP (10.0 microM), elicited strong contractions and increased intracellular Ca(2+) concentration in WT arteries but elicited neither in KO arteries. A biphasic vasoconstriction in response to EFS was observed in WT arteries. In KO arteries, however, the initial rapid, transient component of the biphasic vasoconstriction was absent. The data support the hypothesis that jCaTs represent Ca(2+) that enters the smooth muscle cells through P2X(1) receptors activated by neurally released ATP and that this Ca(2+) is involved in the initial rapid component of the sympathetic neurogenic contraction.     

P2X7 ionotropic receptor is functionally expressed in rabbit articular chondrocytes and mediates extracellular ATP cytotoxicity

Extracellular ATP regulates various cellular functions by engaging multiple subtypes of P2 purinergic receptors. In many cell types, the ionotropic P2X7 receptor mediates pathological events such as inflammation and cell death. However, the importance of this receptor in chondrocytes remains largely unexplored. Here, we report the functional identification of P2X7 receptor in articular chondrocytes and investigate the involvement of P2X7 receptors in ATP-induced cytotoxicity. Chondrocytes were isolated from rabbit articular cartilage, and P2X7 receptor currents were examined using the whole-cell patch-clamp technique. ATP-induced cytotoxicity was evaluated by measuring caspase-3/7 activity, lactate dehydrogenase (LDH) leakage, and prostagrandin E₂ (PGE₂) release using microscopic and fluorimetric/colorimetric evaluation. Extracellular ATP readily evoked a cationic current without obvious desensitization. This ATP-activated current was dose related, but required millimolar concentrations. A more potent P2X7 receptor agonist, BzATP, also activated this current but at 100-fold lower concentrations. ATP-induced currents were largely abolished by selective P2X7 antagonists, suggesting a predominant role for the P2X7 receptor. RT-PCR confirmed the presence of P2X7 in chondrocytes. Heterologous expression of a rabbit P2X7 clone successfully reproduced the ATP-induced current. Exposure of chondrocytes to ATP increased caspase-3/7 activities, an effect that was totally abrogated by P2X7 receptor antagonists. Extracellular ATP also enhanced LDH release, which was partially attenuated by the P2X7 inhibitor. The P2X7 receptor-mediated elevation in apoptotic caspase signaling was accompanied by increased PGE₂ release and was attenuated by inhibition of either phospholipase A₂ or cyclooxygenase-2. This study provides direct evidence for the presence of functional P2X7 receptors in articular chondrocytes. Our results suggest that the P2X7 receptor is a potential therapeutic target in chondrocyte death associated with cartilage injury and disorders including osteoarthritis.     

P2X7 receptor-dependent cell death is modulated during murine T cell maturation and mediated by dual signaling pathways

Extracellular ATP causes apoptosis and/or necrosis of the hemopoietic lineage through the activation of P2X7 receptors. In this study, we investigated P2X7 receptor-mediated cell death during murine T cell maturation. The expression level and activity of P2X7 receptors, as measured by induction of cell death and pore formation, were higher in splenocytes than thymocytes. Flow cytometric analysis revealed that cell shrinkage was induced by activation of the P2X7 receptor in murine lymphocytes and the responding cells were T cells. Splenic T cells were more responsive than their thymic counterpart. These observations indicate that the system of P2X7 receptor-mediated cell death in T cells could be modulated during T cell maturation. Furthermore, decreased extracellular Cl- suppressed ATP-induced cell shrinkage in splenocytes without inhibiting ERK1/2 phosphorylation, which is reported to mediate necrotic cell death. Treatment with U0126 (a MEK inhibitor) suppressed ATP-induced ERK1/2 phosphorylation without inhibiting cell shrinkage. Moreover, decreased extracellular Cl- and treatment with U0126 suppressed ATP-induced cell death. These observations indicate that the activation of P2X7 receptor leads to T cell death by two independent pathways, one of which is cell shrinkage dependent and the other of which involves the phosphorylation of ERK1/2. In conclusion, we demonstrate increasing P2X7 receptor activity during T cell maturation and the existence of two essential pathways in P2X7 receptor-mediated T cell death. Our findings suggest that ATP-induced cell death of peripheral T lymphocytes is important in P2X7 receptor-regulated immune responses.