The Pro-451 to Leu Polymorphism within the C-terminal Tail of P2X7 Receptor Impairs Cell Death but Not Phospholipase D Activation in Murine Thymocytes
2004; Elsevier BV; Volume: 279; Issue: 17 Linguagem: Inglês
10.1074/jbc.m313064200
ISSN1083-351X
AutoresHervé Le Stunff, Rodolphe Auger, Jean Kanellopoulos, Marie‐Noëlle Raymond,
Tópico(s)Calcium signaling and nucleotide metabolism
ResumoThe P2X family of ATP receptors (P2XR) are ligandgated channels that have been proposed to regulate cell death of immature thymocytes. However, the nature of the P2XR subtype involved has been controversial until recently. In agreement with previous studies, we found that extracellular ATP (ATPe) induces a caspase-dependent apoptosis of BALB/c thymocytes, as observed by DNA fragmentation. Additionally, ATPe induces a predominant caspase-independent thymocytes lysis characterized by plasma membrane disruption. Both responses to ATPe can be induced by a potent P2X7R agonist, benzoylbenzoyl-ATP, whereas P2X7R antagonists, oxidized ATP and pyridoxalphosphate-6-azophenyl-2′,4′-disulfonic acid, inhibited the effect of ATPe. These results are further supported by observations where disruption of the P2X7R gene (P2X7R–/– mice) completely abolishes thymocytes death induced by ATPe. Interestingly, the natural P451L mutation in the C-terminal tail of P2X7R present in C57BL/6 mice, which impairs ATPe-dependent pore formation in T lymphocytes, significantly reduces thymocytes death triggered by ATPe. Furthermore, we found that P2X7R from BW5147 thymoma cells also harbors this point mutation, accounting for their insensitivity to ATPe-induced cell death. Concentrations of ATPe effective in inducing cell death also increase phosphatidylcholine-hydrolyzing phospholipase D (PC-PLD) activity in BALB/c thymocytes through the stimulation of P2X7R. However, in contrast to ATPe-induced cell death, PC-PLD activation is totally Ca2+-dependent. Moreover, the stimulation of PC-PLD by ATPe is not affected by the P451L mutation present in C57BL/6 thymocytes and BW5147 cells, suggesting that cell death and PC-PLD activity are regulated through distinct domains of the P2X7R. Finally, the inhibition of ATPe-induced PC-PLD stimulation does not affect thymocytes death. Altogether, these data suggest that P2X7R-induced thymocytes death is independent of the stimulation of PC-PLD activity. The P2X family of ATP receptors (P2XR) are ligandgated channels that have been proposed to regulate cell death of immature thymocytes. However, the nature of the P2XR subtype involved has been controversial until recently. In agreement with previous studies, we found that extracellular ATP (ATPe) induces a caspase-dependent apoptosis of BALB/c thymocytes, as observed by DNA fragmentation. Additionally, ATPe induces a predominant caspase-independent thymocytes lysis characterized by plasma membrane disruption. Both responses to ATPe can be induced by a potent P2X7R agonist, benzoylbenzoyl-ATP, whereas P2X7R antagonists, oxidized ATP and pyridoxalphosphate-6-azophenyl-2′,4′-disulfonic acid, inhibited the effect of ATPe. These results are further supported by observations where disruption of the P2X7R gene (P2X7R–/– mice) completely abolishes thymocytes death induced by ATPe. Interestingly, the natural P451L mutation in the C-terminal tail of P2X7R present in C57BL/6 mice, which impairs ATPe-dependent pore formation in T lymphocytes, significantly reduces thymocytes death triggered by ATPe. Furthermore, we found that P2X7R from BW5147 thymoma cells also harbors this point mutation, accounting for their insensitivity to ATPe-induced cell death. Concentrations of ATPe effective in inducing cell death also increase phosphatidylcholine-hydrolyzing phospholipase D (PC-PLD) activity in BALB/c thymocytes through the stimulation of P2X7R. However, in contrast to ATPe-induced cell death, PC-PLD activation is totally Ca2+-dependent. Moreover, the stimulation of PC-PLD by ATPe is not affected by the P451L mutation present in C57BL/6 thymocytes and BW5147 cells, suggesting that cell death and PC-PLD activity are regulated through distinct domains of the P2X7R. Finally, the inhibition of ATPe-induced PC-PLD stimulation does not affect thymocytes death. Altogether, these data suggest that P2X7R-induced thymocytes death is independent of the stimulation of PC-PLD activity. Extracellular ATP (ATPe) 1The abbreviations used are: ATPe, extracellular ATP; P2XR, purinergic 2X receptors; BzATP, 2′,3′-O-(benzoyl-4-benzoyl)-ATP; oATP, oxidized ATP; PPADS, pyridoxalphosphate-6-azophenyl-2′,4′-disulfonic acid; 2,3-DPG, 2,3-diphosphoglycerate; PC, phosphatidylcholine; PLD, phospholipase D; PC-PLD, phosphatidylcholine-hydrolyzing PLD; PBut, phosphatidylbutanol; PS, phosphatidylserine; TNFα, tumor necrosis factor α; TNF-DD, TNF receptor 1 death domain; LDH, lactate dehydrogenase; FBS, fetal bovine serum; TcR, T cell receptor. 1The abbreviations used are: ATPe, extracellular ATP; P2XR, purinergic 2X receptors; BzATP, 2′,3′-O-(benzoyl-4-benzoyl)-ATP; oATP, oxidized ATP; PPADS, pyridoxalphosphate-6-azophenyl-2′,4′-disulfonic acid; 2,3-DPG, 2,3-diphosphoglycerate; PC, phosphatidylcholine; PLD, phospholipase D; PC-PLD, phosphatidylcholine-hydrolyzing PLD; PBut, phosphatidylbutanol; PS, phosphatidylserine; TNFα, tumor necrosis factor α; TNF-DD, TNF receptor 1 death domain; LDH, lactate dehydrogenase; FBS, fetal bovine serum; TcR, T cell receptor. interacts with P2 purinergic receptors, which are widely distributed in different cell types. Two distinct subfamilies of P2 receptors have been described, namely the G-protein-coupled seven-transmembrane P2Y receptors and the ligand-gated ionotropic P2X receptors (P2XR). Among the P2XR, the P2X7R subclass is notably expressed in hemopoietic tissue (1North R.A. Physiol. Rev. 2002; 82: 1013-1067Crossref PubMed Scopus (2438) Google Scholar). Activation of the P2X7R by ATPe opens ion channels, which mediate fast permeability changes to monovalent and divalent cations (Na+, K+, and Ca2+). Upon prolonged activation, P2X7R induces the formation of nonselective membrane pores permeable to molecules up to 800 Da (2Di Virgilio F. Chiozzi P. Ferrari D. Falzoni S. Sanz J.M. Morelli A. Torboli M. Bolognesi G. Baricordi O.R. Blood. 2001; 97: 587-600Crossref PubMed Scopus (616) Google Scholar). At present, the mechanisms underlying the opening of the cation channel and its transition or coupling to a pore are still unknown. It has been proposed that P2X7R is able to undergo a progressive increase in size, possibly by recruitment of additional receptor subunits (2Di Virgilio F. Chiozzi P. Ferrari D. Falzoni S. Sanz J.M. Morelli A. Torboli M. Bolognesi G. Baricordi O.R. Blood. 2001; 97: 587-600Crossref PubMed Scopus (616) Google Scholar). However, Schilling et al. (3Schilling W.P. Wasylyna T. Dubyak G.R. Humphreys B.D. Sinkins W.G. Am. J. Physiol. 1999; 277: C766-C776Crossref PubMed Google Scholar) propose another attractive hypothesis, where P2X7R might interact with a distinct cytolytic pore allowing the entry of large molecules into the cell. Moreover, formation of the nonselective pore has been shown recently to be dissociated from large scale changes in P2X7R density (4Smart M.L. Panchal R.G. Bowser D.N. Williams D.A. Petrou S. Am. J. Physiol. 2002; 283: C77-C84Crossref PubMed Scopus (32) Google Scholar) but is dependent on the cytoplasmic C-terminal part of the P2X7R (5Gu B.J. Zhang W. 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The function of P2X7R in the latter is still controversial, because previous reports have proposed an additional role of P2X1R in thymocyte death (14Chvatchko Y. Valera S. Aubry J.P. Renno T. Buell G. Bonnefoy J.Y. Immunity. 1996; 5: 275-283Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar, 15Nagy P.V. Feher T. Morga S. Matko J. Immunol. Lett. 2000; 72: 23-30Crossref PubMed Scopus (26) Google Scholar). In addition, although caspases have been implicated in ATPe-triggered cell death in some cell types (8Ferrari D. Los M. Bauer M.K. Vandenabeele P. Wesselborg S. Schulze-Osthoff K. FEBS Lett. 1999; 447: 71-75Crossref PubMed Scopus (241) Google Scholar, 10Le Feuvre R.A. Brough D. Iwakura Y. Takeda K. Rothwell N.J. J. Biol. Chem. 2002; 277: 3210-3218Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar, 16Humphreys B.D. Rice J. Kertesy S.B. Dubyak G.R. J. Biol. 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Dubyak G.R. J. Biol. Chem. 1992; 267: 23664-23673Abstract Full Text PDF PubMed Google Scholar), lymphocytes (20Gargett C.E. Cornish E.J. Wiley J.S. Biochem. J. 1996; 313: 529-535Crossref PubMed Scopus (65) Google Scholar), astrocytes (21Sun S.H. Lin L.B. Hung A.C. Kuo J.S. J. Neurochem. 1999; 73: 334-343Crossref PubMed Scopus (64) Google Scholar), and PC12 pheochromocytoma (22Lee C.S. Bae Y.S. Lee S.D. Suh P.G. Ryu S.H. Neurosci. Lett. 2001; 313: 117-120Crossref PubMed Scopus (8) Google Scholar). In mammals, two types of PLDs have been described, those that are dependent of phosphatidylinositol 4,5-bisphosphate for activity and those that are activated by oleate. Among the first type of PLDs, PLD1 and PLD2 isoforms have been cloned and characterized (23Frohman M.A. Sung T.C. Morris A.J. Biochim. Biophys. Acta. 1999; 1439: 175-186Crossref PubMed Scopus (276) Google Scholar, 24Liscovitch M. Czarny M. Fiucci G. Tang X. Biochem. J. 2000; 345: 401-415Crossref PubMed Scopus (456) Google Scholar). The mechanisms of regulation of PLDs are highly dependent on the cell-type studied (25Exton J.H. Rev. Physiol. Biochem. Pharmacol. 2002; 144: 1-94Crossref PubMed Google Scholar, 26Rizzo M. Romero G. Pharmacol. Ther. 2002; 94: 35-50Crossref PubMed Scopus (58) Google Scholar). PLDs are involved in a variety of physiological processes such as vesicular trafficking and secretion, superoxide generation, bacterial killing, proliferation, differentiation, and possibly apoptosis (25Exton J.H. Rev. Physiol. Biochem. Pharmacol. 2002; 144: 1-94Crossref PubMed Google Scholar, 27Nozawa Y. Biochim. Biophys. Acta. 2002; 1585: 77-86Crossref PubMed Scopus (65) Google Scholar). Indeed, PLD activity increases when apoptosis is induced by tumor necrosis factor α (TNFα) or anti-Fas/Apo1 monoclonal antibody in HL60 cells and in murine B cell lymphoma A20 cells (28Kang J.H. Shin I. Han J.S. Exp. Mol. Med. 1998; 30: 21-27Crossref PubMed Scopus (25) Google Scholar). Oleate-stimulated PLD activity is also associated with apoptosis induced by actinomycin D, TNFα, or hydrogen peroxide (H2O2) in Jurkat T cells (29Nakashima S. Nozawa Y. Chem. Phys. Lipids. 1999; 98: 153-164Crossref PubMed Scopus (64) Google Scholar). However, PLD activity has also been shown to be involved in anti-apoptotic responses. Thus, PLD2 plays a suppressive role in H2O2-induced apoptosis (30Lee S.D. Lee B.D. Han J.M. Kim J.H. Kim Y. Suh P.G. Ryu S.H. J. Neurochem. 2000; 75: 1053-1059Crossref PubMed Scopus (51) Google Scholar) and in hypoxia-induced cell death (31Yamakawa H. Banno Y. Nakashima S. Sawada M. Yamada J. Yoshimura S. Nishimura Y. Nozawa Y. Sakai N. Neuroreport. 2000; 11: 3647-3650Crossref PubMed Scopus (26) Google Scholar).Taking into account the divergent roles of PLD in apoptosis, we examined whether PLD activation via P2X7R is associated with ATPe-triggered cell death of thymocytes. During their development immature thymocytes undergo two selection processes. Positive selection insures that thymocytes can interact with self-major histocompatibility complex molecules via their receptor for antigen (TcR). Thymocytes die by neglect when they fail to undergo positive selection. Negative selection eliminates thymocytes bearing a TcR with too high an avidity for self-major histocompatibility complex molecules. Thus, a large proportion of thymocytes fails to develop and dies by apoptosis (32van Meerwijk J.P.M. Marguerat S. Lees R.K. Germain R.N. Fowlkes B.J. MacDonald H.R. J. Exp. Med. 1997; 185: 377-383Crossref PubMed Scopus (164) Google Scholar). Interestingly, in addition to antigen stimulation through the TcR and the glucocorticoid receptor, a third pathway involving ATPe has been proposed to regulate apoptosis of immature thymocytes (14Chvatchko Y. Valera S. Aubry J.P. Renno T. Buell G. Bonnefoy J.Y. Immunity. 1996; 5: 275-283Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar, 33Freedman B.D. Liu Q.H. Gaulton G. Kotlikoff M.I. Hescheler J. Fleischmann B.K. Eur. J. Immunol. 1999; 29: 1635-1646Crossref PubMed Scopus (29) Google Scholar). It has been suggested that intrathymic elimination of self-reactive thymocytes is mediated by P2X1R (14Chvatchko Y. Valera S. Aubry J.P. Renno T. Buell G. Bonnefoy J.Y. Immunity. 1996; 5: 275-283Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar), whereas death by neglect may be due to one or more PPADS-sensitive P2X1R, -2R, and -7R (33Freedman B.D. Liu Q.H. Gaulton G. Kotlikoff M.I. Hescheler J. Fleischmann B.K. Eur. J. Immunol. 1999; 29: 1635-1646Crossref PubMed Scopus (29) Google Scholar). In dexamethasone-induced apoptosis, it was also shown that P2X1Rs were up-regulated and antagonists to these purinoreceptors prevented cell death (14Chvatchko Y. Valera S. Aubry J.P. Renno T. Buell G. Bonnefoy J.Y. Immunity. 1996; 5: 275-283Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). However, the role of P2X1R in negative selection has been challenged recently (34Koshiba M. Apasov S. Sverdlov V. Chen P. Erb L. Turner J.T. Weisman G.A. Sitkovsky M.V. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 831-836Crossref PubMed Scopus (77) Google Scholar).In the present work, we have examined the consequences of P2X7R stimulation by ATPe in thymocytes by taking advantage of the P2X7R knock-out mice (P2X7R–/–) (35Solle M. Labasi J. Perregaux D.G. Stam E. Petrushova N. Koller B.H. Griffiths R.J. Gabel C.A. J. Biol. Chem. 2001; 276: 125-132Abstract Full Text Full Text PDF PubMed Scopus (776) Google Scholar). We showed that ATPe stimulation of P2X7R triggers phosphatidylcholinehydrolyzing PLD (PC-PLD) activity and cell death of mouse thymocytes. We also found that, in contrast to cell death, PC-PLD activation is calcium-dependent and not modified by the P451L mutation present in the C-terminal cytoplasmic tail of P2X7R of some strains of mice. Altogether, our results strongly suggest that PC-PLD is not directly involved in the pathways leading to ATPe-induced cell death of thymocytes.EXPERIMENTAL PROCEDURESMaterials—Tissue culture media were from Invitrogen SARL. [3H]Myristic acid (49 Ci/mmol, 1.81 TBq/mmol; 1 mCi/ml, 37 MBq/ml) was purchased from PerkinElmer Life Sciences. ATP, UTP, 2′,3′-O-(benzoyl-4-benzoyl)-ATP (BzATP), oxidized ATP (oATP), pyridoxalphosphate-6-azophenyl-2′,4′-disulfonic acid (PPADS), 2,3-diphosphoglycerate (2,3-DPG), ionomycin and lactate dehydrogenase (LDH) detection kit were from Sigma Chemicals Co. All solvents were from Merck Eurolab. Annexin-V-fluos and the "In situ Cell Death Detection Kit, Fluorescein" were from Roche Applied Science, and U73122 was from Calbiochem.Thymocytes were prepared from thymus of 4- to 6-week-old male BALB/c, BDF1 (C57BL/6 x DBA/2 F1) or C57BL/6 mice (Charles River) by standard procedures. Their viability was always above 98%, as tested by trypan blue exclusion. Thymocytes (about 200–250 million/mouse) were suspended before the experiments in RPMI medium supplemented with 1 mg/ml lipid-free BSA. Fetal bovine serum (FBS) was not used, because it may contain components able to stimulate thymocytes. P2X7R–/– BDF1 mice were previously described (35Solle M. Labasi J. Perregaux D.G. Stam E. Petrushova N. Koller B.H. Griffiths R.J. Gabel C.A. J. Biol. Chem. 2001; 276: 125-132Abstract Full Text Full Text PDF PubMed Scopus (776) Google Scholar). BW5147 cells, a murine thymoma cell line derived from the AKR mouse strain, were grown in RPMI medium supplemented with 10% FBS and 1% antibiotics.Measurement of PC-PLD Activity—For determination of PC-PLD activity, we measured the accumulation of phosphatidylbutanol (PBut) (24Liscovitch M. Czarny M. Fiucci G. Tang X. Biochem. J. 2000; 345: 401-415Crossref PubMed Scopus (456) Google Scholar). Murine thymocytes (8.5 × 106) suspended in 2.5 ml of RPMI containing 1 mg/ml lipid-free BSA, were labeled with 12.5 μCi of [3H]myristic acid at 37 °C for 5 h. BW5147 cells (8.5 × 105) suspended in 1 ml of RPMI containing 1 mg/ml lipid-free BSA, were labeled with 6.4 μCi of [3H]myristic acid at 37 °C for 24 h. Analysis of [3H]myristic acid-labeled lipids, performed as previously described (36Auger R. Robin P. Camier B. Vial G. Rossignol B. Tenu J.P. Raymond M.N. J. Biol. Chem. 1999; 274: 28652-28659Abstract Full Text Full Text PDF PubMed Scopus (10) Google Scholar), revealed that PC represents 86 ± 4% and 70 ± 5% of the labeled phospholipids in BALB/c thymocytes and BW5147 cells, respectively. After labeling, the thymocytes and BW5147 cells were washed with RPMI and preincubated at 37 °C for 15 min, in the presence of 0.3% butanol-1 in 2 ml of RPMI supplemented with 1 mg/ml lipid-free BSA and 0.5 mm CaCl2 (CaCl2 final concentration: 0.925 mm). ATPe or other agents were then added for 45-min incubation. At the end of the experiment, cellular lipids were extracted and separated by thin layer chromatography as described previously (36Auger R. Robin P. Camier B. Vial G. Rossignol B. Tenu J.P. Raymond M.N. J. Biol. Chem. 1999; 274: 28652-28659Abstract Full Text Full Text PDF PubMed Scopus (10) Google Scholar). The amounts of [3H]PBut formed were expressed as a percentage of the amount of total radiolabeled lipids.Calculations of Concentrations of ATP4– Species—In the medium routinely used, Ca2+ and Mg2+ concentrations were 0.925 and 0.406 mm, respectively. The concentrations of ATP4–, ATP4–-Ca2+, ATP4–-Mg2+, free Ca2+ were calculated using the equilibrium values reported in Data for Biochemical Research (55O'Sullivan W.J. Dawson R.M.C. Elliott D.C. Elliott W.H. Jones K.M. Data for Biochemical Research. Oxford University Press, Oxford1969: 423-434Google Scholar).Measurement of LDH Release—Cell lysis was determined by measuring the release of lactate dehydrogenase (LDH). Murine thymocytes (5 × 106) and BW5147 cells (8 × 105) were incubated in 0.5 ml of RPMI supplemented with 1 mg/ml lipid-free BSA and 0.5 mm CaCl2 (CaCl2 final concentration: 0.925 mm). ATPe or other agonists were added, and at the end of the incubations, the cells were pelleted for 10 min at 200 × g and the LDH activity released from dead cells was measured in the media according to the manufacturer's instructions. Total LDH activity was measured by lysing thymocytes and BW5147 cells with 0.1% Triton X-100. The percentage of LDH released represents the fraction of LDH activity found in the supernatants with respect to the overall enzyme activity. Student's t test was used for statistical analysis; p < 0.05 was accepted as a significant difference.Flow Cytometric Analysis of Phosphatidylserine Externalization— The analysis of this phospholipid on the outer leaflet of apoptotic thymocyte-membranes was performed by using fluorescein isothiocyanate-labeled Annexin-V, which binds to phosphatidylserine (PS), and propidium iodide which allows the differentiation from lytic cells. Thymocytes (5 × 105) were incubated in 0.5 ml of RPMI supplemented with 1 mg/ml lipid-free BSA and 0.5 mm CaCl2 (CaCl2 final concentration: 0.925 mm) in the absence or the presence of ATPe. At the end of the incubation time, cells were pelleted for 10 min at 200 × g, washed with 1 mg/ml lipid-free BSA in PBS, and then incubated for 15 min at 37 °C with 0.5 μg/ml propidium iodide and annexin V diluted in a buffer containing 5 mm CaCl2, 140 mm NaCl, and 10 mm Hepes, pH 7.4. The cells were then immediately analyzed by flow cytometry. Values of externalized PS correspond to the percentage of cells found in the quadrant annexin V-positive/propidium iodide-negative cells.Flow Cytometric Analysis of DNA Fragmentation—DNA fragmentation of thymocytes was followed by flow cytometry using the sensitive TUNEL method. Murine thymocytes (4–5 × 106) or BW5147 cells (1.2 × 106) were incubated in 1 ml of RPMI supplemented with 1 mg/ml lipid-free BSA and 0.5 mm CaCl2 (CaCl2 final concentration: 0.925 mm) in the absence or the presence of ATPe. At the end of the incubation time, the cells were pelleted for 10 min at 200 × g, fixed with 1% paraformaldehyde, and permeabilized in 0.1% Triton X-100. Cells were then labeled with fluorescein dUTP at strand breaks by terminal deoxynucleotidyl transferase and analyzed by flow cytometry.RESULTSATPe Induces Thymocytes Lysis—It is well established that in lytic cells, plasma membrane disruption leads to the release of cytosolic proteins as lactate dehydrogenase (LDH). To check that LDH release discriminates lytic from apoptotic cells, we verified that dexamethasone, a well established apoptotic agent for thymocytes (37Distelhorst C.W. Cell Death Differ. 2002; 9: 6-19Crossref PubMed Scopus (266) Google Scholar), does not induce LDH release. Indeed, after 2 h of incubation, LDH released represents 5.0 ± 0.8% and 4.9 ± 0.7% of total LDH in untreated and 0.1 μm dexamethasone-treated BALB/c thymocytes, respectively. In contrast, 1 mm ATPe (74.5 μm ATP4–) leads to a time-dependent release of LDH, reaching a maximum of about 50% of total LDH after 5 h (Fig. 1A). Increasing the ATPe concentration from 0.1 to 3 mm enhanced LDH release by about 6-fold (Fig. 1B) with a maximal effect at 1 mm ATPe. Cell lysis measured through the index of LDH release is in agreement with the number of trypan blue-positive cells (data not shown). To determine whether LDH released originates from cells of the T cell lineage, macrophages, B lymphocytes, and dendritic cells were removed from thymocytes suspension by immunomagnetic depletion with monoclonal anti-mouse major histocompatibility complex class II antibodies. We found that ATPe-triggered LDH release in purified thymocytes is identical to that of the unfractionated preparation of thymocytes (data not shown).To examine whether cell lysis is due to a sustained stimulation with ATPe, we performed experiments in which the medium containing 1 mm ATPe was removed after a 30-min incubation and then replaced by fresh medium for a further 4.5 h. In those conditions, the amount of LDH released in response to ATPe was reduced by about 40% (Fig. 1C), indicating that prolonged purinergic stimulation is required for a cytolytic effect. ATPe-triggered cell lysis has been shown in some cases to be a caspase-mediated process (10Le Feuvre R.A. Brough D. Iwakura Y. Takeda K. Rothwell N.J. J. Biol. Chem. 2002; 277: 3210-3218Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar). However, a pan-caspase inhibitor, z-VAD (50 μm), was unable to inhibit LDH released after 2 h of incubation of BALB/c thymocytes with 1 mm ATPe (Fig. 1D), whereas it completely abrogates dexamethasone-induced apoptosis of thymocytes (data not shown and Ref. 37Distelhorst C.W. Cell Death Differ. 2002; 9: 6-19Crossref PubMed Scopus (266) Google Scholar). This result indicates that ATPe-triggered LDH release is a caspase-independent process in thymocytes.Involvement of P2X7R in ATPe-induced Thymocytes Lysis—To determine the identity of the purinergic receptors involved in ATPe-triggered cell lysis, we first examined the expression of P2XR mRNAs in murine thymocytes. C57BL/6 thymocytes were shown to express P2X1, P2X2, P2X6, and P2X7 purinergic receptors (33Freedman B.D. Liu Q.H. Gaulton G. Kotlikoff M.I. Hescheler J. Fleischmann B.K. Eur. J. Immunol. 1999; 29: 1635-1646Crossref PubMed Scopus (29) Google Scholar). We found that P2X1, P2X2, and P2X7 receptors are expressed in BALB/c thymocytes. 2R. Auger, unpublished data. We then tested the effects of different purinergic receptor agonists or antagonists. As indicated in Fig. 2A, 250 μm BzATP, a potent P2X7R agonist, triggers cell lysis, whereas 1 mm UTP, a P2Y agonist, has no effect, suggesting that P2X7R may be implicated in this phenomenon. Thus, we determined the ATPe-triggered cell lysis in the presence of two well known P2X7R antagonists, oATP and PPADS. oATP and PPADS used alone did not affect thymocyte survival significantly. However, they reduced LDH release induced by 1 mm ATPe, to 7 and 15%, respectively (Fig. 2A). In addition, ATPe does not trigger LDH release from P2X7R–/– thymocytes, whereas about 20% LDH release is observed in P2X7R+/+ thymocytes (Fig. 2B). Thus, ATPe-induced cell lysis appears to be controlled by P2X7R.Fig. 2Implication of P2X7R on ATPe-induced LDH release of murine thymocytes. A, effect of different purinergic receptor agonists or antagonists on LDH release. BALB/c thymocytes were treated with 1 mm ATPe, 0.25 mm BzATP, or 1 mm UTP for 3 h. Supernatants were collected for LDH content determination. When antagonists were tested, thymocytes were preincubated for 2 h with 0.3 mm oATP and for 15 min with 0.3 mm PPADS. The amounts of LDH released are expressed as a percentage of the amounts of total LDH. Data are means ± S.E. of three independent experiments. B, effect of ATPe on LDH release in thymocytes from P2X7R-deficient mice. Thymocytes from wild-type BDF1 (WT) mice or P2X7R–/– mice were stimulated with 1 mm ATPe for 2 h. Supernatants were collected for LDH content determination. Data are means ± S.E. of three independent experiments. C, effect of ATPe-triggered LDH release from C57BL/6 thymocytes and BW5147 cells. Cells were incubated with 1 mm ATPe for 5 h, and supernatants were collected for LDH content determination. Data are means ± S.E. of three independent experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT)ATPe-induced Thymocytes Lysis Is Affected by the P451L Mutation in the C-terminal Tail of P2X7R—Recently, mutation analyses have shown the importance of the C-terminal tail of P2X7R in pore formation in response to ATPe stimulation (5Gu B.J. Zhang W. Worthington R.A. Sluyter R. Dao-Ung P. Petrou S. Barden J.A. Wiley J.S. J. Biol. Chem. 2001; 276: 11135-11142Abstract Full Text Full Text PDF PubMed Scopus (276) Google Scholar, 6Adriouch S. Dox C. Welge V. Seman M. Koch-Nolte F. Haag F. J. Immunol. 2002; 169: 4108-4112Crossref PubMed Scopus (169) Google Scholar, 7Smart M.L. Gu B. Panchal R.G. Wiley J. Cromer B. Williams D.A. Petrou S. J. Biol. Chem. 2003; 278: 8853-8860Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar). To analyze the role of the C-terminal region of P2X7R in ATPe-induced cell lysis, we used thymocytes from C57BL/6 mice, which bear a natural mutation (P451L) in the C-terminal tail of P2X7R, associated to a drastic reduction of ATPe-dependent pore formation and PS externalization (6Adriouch S. Dox C. Welge V. Seman M. Koch-Nolte F. Haag F. J. Immunol. 2002; 169: 4108-4112Crossref PubMed Scopus (169) Google Scholar). Fig. 2C shows that ATPe-triggered lysis in C57BL/6 thymocytes is reduced by almost 45% compared with BALB/c thymocytes. We next used BW5147 thymoma cells, which have also impaired responses to ATPe. Indeed, upon ATPe stimulation, a functional Ca2+ channel is formed in these cells while there is no opening of the nonspecific pore (38Humphreys B.D. Virginio C. Surprenant A. Rice J. Dubyak G.R. Mol. Pharmacol. 1998; 54: 22-32Crossref PubMed Scopus (183) Google Scholar). Fig. 2C shows that BW5147 cells are marginall
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