An Arg307 to Gln polymorphism within the ATP-binding site causes loss of function of the human P2X7 receptor

The P2X(7) receptor is a ligand-gated channel that is highly expressed on mononuclear cells of the immune system and that mediates ATP-induced apoptosis. Wide variations in the function of the P2X receptor have been observed, explained in part by (7)loss-of-function polymorphisms that change Glu(496) to Ala (E496A) and Ile(568) to Asn (I568N). In this study, a third polymorphism, which substitutes an uncharged glutamine for the highly positively charged Arg(307) (R307Q), has been found in heterozygous dosage in 12 of 420 subjects studied. P2X(7) function was measured by ATP-induced fluxes of Rb(+), Ba(2+), and ethidium(+) into peripheral blood monocytes or various lymphocyte subsets and was either absent or markedly decreased. Transfection experiments showed that P2X(7) carrying the R307Q mutation lacked either channel or pore function despite robust protein synthesis and surface expression of the receptor. The monoclonal antibody (clone L4) that binds to the extracellular domain of wild type P2X(7) and blocks P2X(7) function failed to bind to the R307Q mutant receptor. Differentiation of monocytes to macrophages up-regulated P2X(7) function in cells heterozygous for the R307Q to a value 10-40% of that for wild type macrophages. However, macrophages from a subject who was double heterozygous for R307Q/I568N remained totally non-functional for P2X(7), and lymphocytes from the same subject also lacked ATP-stimulated phospholipase D activity. These data identify a third loss-of-function polymorphism affecting the human P2X(7) receptor, and since the affected Arg(307) is homologous to those amino acids essential for ATP binding to P2X(1) and P2X(2), it is likely that this polymorphism abolishes the binding of ATP to the extracellular domain of P2X(7).     

A Thr357 to Ser polymorphism in homozygous and compound heterozygous subjects causes absent or reduced P2X7 function and impairs ATP-induced mycobacterial killing by macrophages

The P2X(7) receptor is a ligand-gated cation channel that is highly expressed on mononuclear leukocytes and that mediates ATP-induced apoptosis and killing of intracellular pathogens. There is a wide variation in P2X(7) receptor function between subjects, explained in part by four loss-of-function polymorphisms (R307Q, E496A, I568N, and a 5'-intronic splice site polymorphism), as well as rare mutations. In this study, we report the allele frequencies of 11 non-synonymous P2X(7) polymorphisms and describe a fifth loss-of-function polymorphism in the gene (1096C --> G), which changes Thr(357) to Ser (T357S) with an allele frequency of 0.08 in the Caucasian population. P2X(7) function was measured by ATP-induced ethidium(+) influx into peripheral blood lymphocytes and monocytes and, when compared with wild-type subjects, was reduced to 10-65% in heterozygotes, 1-18% in homozygotes, and 0-10% in compound heterozygotes carrying T357S and a second loss-of-function polymorphism. Overexpression of the T357S mutant P2X(7) in either HEK-293 cells or Xenopus oocytes gave P2X(7) function of approximately 50% that of wild-type constructs. Differentiation of monocytes to macrophages, which also up-regulates P2X(7), restored P2X(7) function to near normal in cells heterozygous for T357S and to a value 50-65% of wild-type in cells homozygous for T357S or compound heterozygous for T357S/E496A. However, macrophages from subjects that are compound heterozygous for either T357S/R307Q or T357S/stop codon had near-to-absent P2X(7) function. These functional deficits induced by T357S were paralleled by impaired ATP-induced apoptosis and mycobacteria killing in macrophages from these subjects. Lymphocytes, monocytes, and macrophages from subjects homozygous for T357S or compound heterozygous for T357S and a second loss-of-function allele have reduced or absent P2X(7) receptor function.     

A Glu-496 to Ala polymorphism leads to loss of function of the human P2X7 receptor

The P2X(7) receptor is a ligand-gated cation-selective channel that mediates ATP-induced apoptosis of cells of the immune system. We and others have shown that P2X(7) is nonfunctional both in lymphocytes and monocytes from some subjects. To study a possible genetic basis we sequenced DNA coding for the carboxyl-terminal tail of P2X(7). In 9 of 45 normal subjects a heterozygous nucleotide substitution (1513A-->C) was found, whereas 1 subject carried the homozygous substitution that codes for glutamic acid to alanine at amino acid position 496. Surface expression of P2X(7) on lymphocytes was not affected by this E496A polymorphism, demonstrated both by confocal microscopy and immunofluorescent staining. Monocytes and lymphocytes from the E496A homozygote subject expressed nonfunctional receptor, whereas heterozygotes showed P2X(7) function that was half that of germline P2X(7). Results of transfection experiments showed that the mutant P2X(7) receptor was nonfunctional when expressed at low receptor density but regained function at a high receptor density. This density dependence of mutant P2X(7) function was also seen on differentiation of fresh monocytes to macrophages with interferon-gamma, which up-regulated mutant P2X(7) and partially restored its function. P2X(7)-mediated apoptosis of lymphocytes was impaired in homozygous mutant P2X(7) compared with germline (8.6 versus 35.2%). The data suggest that the glutamic acid at position 496 is required for optimal assembly of the P2X(7) receptor.     

A His-155 to Tyr polymorphism confers gain-of-function to the human P2X7 receptor of human leukemic lymphocytes

The P2X(7)R is an ATP-gated cation channel expressed in hemopoietic cells that participates in both cell proliferation and apoptosis. Expression and function of the P2X(7)R have been associated with the clinical course of patients affected by chronic lymphocytic leukemia (CLL). Functional variants causing loss-of-function of the P2X(7)R have been identified, namely, polymorphisms 1513A>C (E496A), 1729T>A (I568N), and 946G>A (R307Q). Here we investigated other nonsynonymous polymorphisms located either in the extracellular portion of the receptor, such as the 489C>T (H155Y) variant, or in the long cytoplasmic tail of the receptor, such as the 1068G>A (A348T), 1096C>G (T357S), and 1405A>G (Q460R) variants. P2X(7)R function was monitored by measuring ATP-induced Ca(2+) influx in PBL of patients affected by CLL and in recombinant human embryonic kidney (HEK) 293 cells stably transfected with each single P2X(7) allelic variant. Ca(2+) influx was markedly reduced in association with the 1513C allele, whereas variants located in the same intracellular domain, such as the 1068A, 1096G, or 1405G variants, were associated with a minor functional decrease. Significant Ca(2+) flux increase was observed in lymphocytes from CLL patients bearing the 489C/T and 489T/T genotypes in association with the 1513A/A genotype. Functional analysis in recombinant HEK293 cells expressing P2X(7)R confirmed an increased ATP-dependent activation of the P2X(7) 489T mutant with respect to the wild type receptor, as assessed by both by [Ca(2+)](i) influx and ethidium uptake experiments. These data identify the 489C>T as a gain-of-function polymorphism of the P2X(7)R.     

Glu496 to Ala polymorphism in the P2X7 receptor impairs ATP-induced IL-1 beta release from human monocytes

Priming of monocytes with LPS produces large quantities of intracellular, biologically inactive IL-1beta that can be processed and released by subsequent activation of the P2X7 receptor by extracellular ATP. We examined whether a loss-of-function polymorphism of the human P2X7 receptor (Glu496Ala) impairs this process. Both ATP-induced ethidium+ uptake and ATP-induced shedding of L-selectin (CD62L) were nearly absent in monocytes from four subjects homozygous for Glu496Ala confirming that this polymorphism impairs P2X7 function. The level of ATP-induced IL-1beta released in 2 h from LPS-activated whole blood from homozygous subjects was 50% of that from wild-type samples. A more marked defect in IL-1beta release was observed from LPS-activated monocytes of homozygous subjects which was only 22% of that released from wild-type monocytes after a 30-min incubation with ATP. However, after a 60-min incubation with ATP, the amount of IL-1beta released from homozygous monocytes was 70% of that released from wild-type monocytes. Incubation of monocytes of either genotype with nigericin resulted in a similar release of IL-1beta. Western blotting demonstrated that ATP induced the release of mature 17-kDa IL-1beta from monocytes, and confirmed that this process was impaired in homozygous monocytes. Finally, ATP-induced 86Rb+ efflux was 9-fold lower from homozygous monocytes than from wild-type monocytes. The results indicate that ATP-induced release of IL-1beta is slower in monocytes from subjects homozygous for the Glu496Ala polymorphism in the P2X7 receptor and that this reduced rate of IL-1beta release is associated with a lower ATP-induced K+ efflux.     

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.     

Residues 155 and 348 contribute to the determination of P2X7 receptor function via distinct mechanisms revealed by single-nucleotide polymorphisms

P2X(7) receptors are important in mediating the physiological functions of extracellular ATP, and altered receptor expression and function have a causative role in the disease pathogenesis. Here, we investigated the mechanisms determining the P2X(7) receptor function by following two human single-nucleotide polymorphism (SNP) mutations that replace His-155 and Ala-348 in the human (h) P2X(7) receptor with the corresponding residues, Tyr-155 and Thr-348, in the rat (r) P2X(7) receptor. H155Y and A348T mutations in the hP2X(7) receptor increased ATP-induced currents, whereas the reciprocal mutations, Y155H and T348A, in the rP2X(7) receptor caused the opposite effects. Such a functional switch is a compelling indication that these residues are critical for P2X(7) receptor function. Additional mutations of His-155 and Ala-348 in the hP2X(7) receptor to residues with diverse side chains revealed a different dependence on the side chain properties, supporting the specificity of these two residues. Substitutions of the residues surrounding His-155 and Ala-348 in the hP2X(7) receptor with the equivalent ones in the rP2X(7) receptor also affected ATP-induced currents but were not fully reminiscent of the H155Y and A348T effects. Immunofluorescence imaging and biotin labeling assays showed that H155Y in the hP2X(7) receptor increased and Y155H in the rP2X(7) receptor decreased cell-surface expression. Such contrasting effects were not obvious with the reciprocal mutations of residue 348. Taken together, our results suggest that residues at positions 155 and 348 contribute to P2X(7) receptor function via determining the surface expression and the single-channel function, respectively. Such interpretations are consistent with the locations of the residues in the structural model of the hP2X(7) receptor.     

Extracellular ATP increases cation fluxes in human erythrocytes by activation of the P2X7 receptor

Canine erythrocytes are known to undergo a reversible increase in cation permeability when incubated with extracellular ATP. We have examined the expression and function of P2X receptors on human erythrocytes using confocal microscopy and a panel of anti-P2X(1-7) antibodies and have measured monovalent cation fluxes in the presence of various nucleotide agonists. Human erythrocytes expressed P2X7 receptors on all cells examined from eight of eight subjects, as well as P2X2 at a far lower staining intensity in six of eight subjects. ATP stimulated the efflux of 86Rb+ (K+) from human erythrocytes in a dose-dependent fashion with an EC50 of approximately 95 microM. Other nucleotides also induced an efflux of 86Rb+ from erythrocytes with an order of agonist potency of 2'- and 3'-O(4-benzoylbenzoyl) ATP (BzATP) > ATP > 2-methylthio-ATP (2MeSATP) > adenosine 5'-O-(3-thiotriphosphate) (ATPgammaS), whereas ADP or UTP had no effect. ATP-induced efflux of 86Rb+ from erythrocytes was inhibited by extracellular Na+ and oxidized ATP, as well as by KN-62, an antagonist specific for the human P2X7 receptor. When erythrocytes were incubated in isotonic KCl medium, the addition of ATP stimulated an 86Rb+ influx approximately equal in magnitude to ATP-stimulated 86Rb+ efflux from the same cells. BzATP also stimulated the influx of 22Na+ into erythrocytes incubated in isotonic NaCl medium. Both ATP-induced efflux and influx of 86Rb+ and 22Na+ were impaired in erythrocytes from subjects who had inherited loss-of-function polymorphisms in the P2X7 receptor. These results suggest that the reversible permeabilization of erythrocytes by extracellular ATP is mediated by the P2X7 receptor.     

N-Glycans mutations rule oligomeric assembly and functional expression of P2X3 receptor for extracellular ATP

N-Glycosylation affects the function of ion channels at the level of multisubunit assembly, protein trafficking, ligand binding and channel opening. Like the majority of membrane proteins, ionotropic P2X receptors for extracellular ATP are glycosylated in their extracellular moiety. Here, we used site-directed mutagenesis to the four predicted N-glycosylation sites of P2X(3) receptor (Asn(139), Asn(170), Asn(194) and Asn(290)) and performed comparative analysis of the role of N-glycans on protein stability, plasma membrane delivery, trimer formation and inward currents. We have found that in transiently transfected HEK293 cells, Asn(170) is apparently the most important site for receptor stability, since its mutation causes a primary loss in protein content and indirect failure in membrane expression, oligomeric association and inward current responses. Even stronger effects are obtained when mutating Thr(172) in the same glycosylation consensus. Asn(194) and Asn(290) are the most dispensable, since even their simultaneous mutation does not affect any tested receptor feature. All double mutants containing Asn(170) mutation or the Asn(139)/Asn(290) double mutant are instead almost unable to assemble into a functional trimeric structure. The main emerging finding is that the inability to assemble into trimers might account for the impaired function in P2X(3) mutants where residue Asn(170) is replaced. These results improve our knowledge about the role of N-glycosylation in proper folding and oligomeric association of P2X(3) receptor.     

Expression of P2X(7) purinoceptors on human lymphocytes and monocytes: evidence for nonfunctional P2X(7) receptors

Lymphocytes from normal subjects and patients with B-chroniclymphocytic leukemia (B-CLL) show functional responses to extracellular ATP characteristic of the P2X₇ receptor (previously termedP2Z). These responses include opening of a cation-selectivechannel/pore that allows entry of the fluorescent dye ethidium andactivation of a membrane metalloprotease that sheds the adhesionmolecule L-selectin. The surface expression of P2X₇receptors was measured in normal leucocytes, platelets, and B-CLLlymphocytes and correlated with their functional responses. Monocytesshowed four- to fivefold greater expression of P2X₇ than B,T, and NK lymphocytes, whereas P2X₇ expression onneutrophils and platelets was weak. All cell types demonstratedabundant intracellular expression of this receptor. All 12 subjectswith B-CLL expressed lymphocyte P2X₇ at about the samelevel as B lymphocytes from normal subjects. P2X₇ function, measured by ATP-induced uptake of ethidium, correlated closely withsurface expression of this receptor in normal and B-CLL lymphocytes andmonocytes (n = 47, r = 0.70; P< 0.0001). However, in three patients the ATP-induced uptake ofethidium into the malignant B lymphocytes was low or absent. The lackof P2X₇ function in these B lymphocytes was confirmed bythe failure of ATP to induce Ba²⁺ uptake into theirlymphocytes. This lack of function of the P2X₇ receptorresulted in a failure of ATP-induced shedding of L-selectin, anadhesion molecule that directs the recirculation of lymphocytes fromblood into the lymph node.

     

Co-Expression of Wild-Type P2X7R with Gln460Arg Variant Alters Receptor Function

The P2X7 receptor is a member of the P2X family of ligand-gated ion channels. A single-nucleotide polymorphism leading to a glutamine (Gln) by arginine (Arg) substitution at codon 460 of the purinergic P2X7 receptor (P2X7R) has been associated with mood disorders. No change in function (loss or gain) has been described for this SNP so far. Here we show that although the P2X7R-Gln460Arg variant per se is not compromised in its function, co-expression of wild-type P2X7R with P2X7R-Gln460Arg impairs receptor function with respect to calcium influx, channel currents and intracellular signaling in vitro. Moreover, co-immunoprecipitation and FRET studies show that the P2X7R-Gln460Arg variant physically interacts with P2X7R-WT. Specific silencing of either the normal or polymorphic variant rescues the heterozygous loss of function phenotype and restores normal function. The described loss of function due to co-expression, unique for mutations in the P2RX7 gene so far, explains the mechanism by which the P2X7R-Gln460Arg variant affects the normal function of the channel and may represent a mechanism of action for other mutations.     

Specific detection of non-functional human P2X(7) receptors in HEK293 cells and B-lymphocytes

P2X(7) receptor/channels mediate ATP-induced apoptosis in a range of cells including lymphocytes. HEK293 cells were transfected with wild-type human P2X(7) receptor or site-directed mutant constructs (K193A, K311A and E496A) known to be non-functional from measurements of barium/ethidium influx in the presence of ATP or 2',3'-O-(4-benzoylbenzoyl)-ATP. An antibody was designed against an epitope from a loop adjacent to the extracellular ATP site. The epitope was unavailable in cells expressing normal functional surface receptors. Non-functional surface receptors as well as intracellular receptors selectively bound the antibody. So did B-lymphocytes from chronic lymphocytic leukemia patients expressing non-functional (E496A) mutant receptor.     

A 5' intronic splice site polymorphism leads to a null allele of the P2X7 gene in 1-2% of the Caucasian population

The P2X(7) gene is important for the innate immune response but known polymorphisms do not explain all subjects with loss of P2X(7) function. A splice site mutation (g-->t) was found at position +1 of the first intron of the P2X(7) gene in 7 of 336 Caucasians and 1 of 39 subjects of Indian ethnicity. All eight subjects were heterozygous for the uncommon 1513A-->C polymorphism of the P2X(7) gene. RT-PCR and sequencing showed the splice site mutation was on the 1513C allele in the Caucasians and on the 1513A allele in the Indian subject. The splice site mutation is an inherited polymorphism and gives rise to a P2X(7) null allele in 1-2% of the Caucasian population.     

P2X(4) receptors mediate ATP-induced calcium influx in human vascular endothelial cells

ATP induces Ca(2+) influx across the cell membrane and activates release from intracellular Ca(2+) pools in vascular endothelial cells (ECs). Ca(2+) signaling leads to the modification of a variety of EC functions, including the production of vasoactive substances such as nitric oxide and prostacyclin. However, the molecular mechanisms for ATP-induced Ca(2+) influx in ECs have not been thoroughly clarified. Here we demonstrate evidence that a P2X(4) receptor for an ATP-gated cation channel is predominantly expressed in human ECs and is involved in the ATP-induced Ca(2+) influx. Northern blot analysis distinctly showed the expression of P2X(4) mRNA in human ECs cultured from the umbilical vein, aorta, pulmonary artery, and skin microvessels. Competitive PCR revealed that P2X(4) mRNA expression was much higher in ECs than was the expression of other subtypes, including P2X(1), P2X(3), P2X(5), and P2X(7). Treatment of ECs with antisense oligonucleotides designed to target the P2X(4) receptor decreased the P2X(4) mRNA and protein levels to approximately 25% of control levels and markedly prevented the ATP-induced Ca(2+) influx.     

Cutting edge: a natural P451L mutation in the cytoplasmic domain impairs the function of the mouse P2X7 receptor

The P2X7 receptor (P2X(7)R) is an ATP-gated channel that mediates apoptosis of cells of the immune system. The capacity of P2X(7)R to form large pores depends on its large cytoplasmic tail, which harbors a putative TNFR-related death domain. Previous transfection studies indicated that mouse P2X(7)R forms pores much less efficiently than its counterparts from humans and rats. In this study, we demonstrate that an allelic mutation (P451L) in the predicted death domain of P2X(7)R confers a drastically reduced sensitivity to ATP-induced pore formation in cells from some commonly used strains of mice, i.e., C57BL/6 and DBA/2. In contrast, most other strains of mice, including strains derived from wild mice, carry P451 at this position as do rats and humans. The effects of the P451L mutation resemble those of the E496A mutation in human P2X(7)R. These P2X(7)R mutants may provide useful tools to decipher the molecular mechanisms leading to pore formation.     

TGF-β1 prevents up-regulation of the P2X7 receptor by IFN-γ and LPS in leukemic THP-1 monocytes

The P2X7 receptor is an extracellular ATP-gated cation channel critical in inflammation and immunity, and can be up-regulated by IFN-γ and LPS. This study aimed to examine the effect of TGF-β1 on the up-regulation of P2X7 function and expression in leukemic THP-1 monocytes differentiated with IFN-γ and LPS. Cell-surface molecules including P2X7 were examined by immunofluorescence staining. Total P2X7 protein and mRNA was assessed by immunoblotting and RT-PCR respectively. P2X7 function was evaluated by ATP-induced cation dye uptake measurements. Cell-surface P2X7 was present on THP-1 cells differentiated for 3 days with IFN-γ and LPS but not on undifferentiated THP-1 cells. ATP induced ethidium⁺ uptake into differentiated but not undifferentiated THP-1 cells, and the P2X7 antagonist, KN-62, impaired ATP-induced ethidium⁺ uptake. Co-incubation of cells with TGF-β1 plus IFN-γ and LPS prevented the up-regulation of P2X7 expression and ATP-induced ethidium⁺ uptake in a concentration-dependent fashion with a maximum effect at 5 ng/ml and with an IC₅₀ of ~ 0.4 ng/ml. Moreover, ATP-induced YO-PRO-1²⁺ uptake and IL-1β release were abrogated in cells co-incubated with TGF-β1. TGF-β1 also abrogated the amount of total P2X7 protein and mRNA induced by IFN-γ and LPS. Finally, TGF-β1 prevented the up-regulation of cell-surface CD86, but not CD14 and MHC class II, by IFN-γ and LPS. These results indicate that TGF-β1 prevents the up-regulation of P2X7 function and expression by IFN-γ and LPS in THP-1 monocytes. This suggests that TGF-β1 may limit P2X7-mediated processes in inflammation and immunity.     

Epistasis with HLA DR3 implicates the P2X7 receptor in the pathogenesis of primary Sj?gren's syndrome

Introduction

The aim of this study was to examine the association between functional polymorphisms in the pro-inflammatory P2X7 receptor and the Ro/La autoantibody response in primary Sjögren''s syndrome (pSS).

Methods

Twelve functional P2RX7 polymorphisms were genotyped in 114 pSS patients fulfilling the Revised American-European Consensus Criteria for pSS, and 136 controls. Genotyping of the A1405G (rs2230912) polymorphism was performed on a replication cohort consisting of 281 pSS patients and 534 controls. P2X7 receptor function in lymphocytes and monocytes was assessed by measurement of ATP-induced ethidium+ uptake. Serum IL-18 levels were determined by ELISA.

Results

The minor allele of P2RX7 A1405G is a tag for a common haplotype associated with gain in receptor function, as assessed by ATP-induced ethidium+ uptake. A positive association between 1405G and anti-Ro±La seropositive pSS patients was observed in Cohort 1. Although not replicated in Cohort 2, there was a consistent, significant, negative epistatic interaction effect with HLA-DR3 in seropositive pSS patients from both cohorts, thereby implicating this gain of function variant in the pathogenesis of pSS. Serum IL-18 was elevated in seropositive pSS patients, but was not influenced by P2RX7 A1405G.

Conclusions

The P2RX7 1405G gain-of-function haplotype may be a risk factor for seropositive pSS in a subset of subjects who do not carry HLA risk alleles, but has no effect in subjects who do (epistasis). Potential mechanisms relate to autoantigen exposure and inflammatory cytokine expression. The observed elevation of IL-18 levels is consistent with P2X7 receptor activation in seropositive pSS patients. Collectively these findings implicate P2X7 receptor function in the pathogenesis of pSS.     

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.     

Regulation of P2X7-dependent inflammatory functions by P2X4 receptor in mouse macrophages

Activation of the P2X7 receptor of macrophages plays an important role in inflammation. We recently reported that co-expression of P2X4 receptor with P2X7 receptor facilitates P2X7 receptor-mediated cell death via Ca(2+) influx. However, it remained unclear whether P2X4 receptor is involved in P2X7 receptor-mediated inflammatory responses, such as cytokine production. Here, we present evidence that P2X4 receptor modulates P2X7 receptor-dependent inflammatory functions. Treatment of mouse macrophage RAW264.7 cells with 1mM ATP induced high mobility group box 1 (HMGB1) release and IL-1β production via activation of P2X7 receptor. Knockdown of P2X4 receptor or removal of extracellular Ca(2+) suppressed ATP-induced release of both HMGB1 and IL-1β. On the other hand, knockdown of P2X4 receptor or removal of extracellular Ca(2+) enhanced P2X7-dependent LC3-II expression (an index of autophagy), suggesting that P2X4 receptor suppresses P2X7-mediated autophagy. Since LC3-II expression was inhibited by pretreatment with antioxidant and NADPH oxidase inhibitor, we examined P2X7-mediated production of reactive oxygen species (ROS). We found that activation of P2X7 receptor-mediated production of ROS was significantly facilitated in P2X4-knockdown cells, suggesting that co-expression of P2X4 receptor with P2X7 receptor may suppress anti-inflammatory function-related autophagy via suppression of ROS production. We conclude that co-expression of P2X4 receptor with P2X7 receptor enhances P2X7-mediated inflammation through both facilitation of release of cytokines and suppression of autophagy.