Involvement of P2X4 receptor in P2X7 receptor-dependent cell death of mouse macrophages

Biochemical and Biophysical Research Communications 419 (2012) 374–380

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Involvement of P2X4 receptor in P2X7 receptor-dependent cell death of mouse macrophages Ayumi Kawano a,1, Mitsutoshi Tsukimoto a,⇑,1, Taisei Noguchi a, Noriyuki Hotta a, Hitoshi Harada b, Takato Takenouchi c, Hiroshi Kitani c, Shuji Kojima a a b c

Department of Radiation Biosciences, Faculty of Pharmaceutical Sciences, Tokyo University of Science, 2641 Yamazaki, Noda-shi Chiba 278-8510, Japan Faculty of Pharmaceutical Sciences, Suzuka University of Medical Science, 3500-3 Minamitamagaki-cho, Suzuka-shi, Mie, Japan Animal Immune and Cell Biology Research Unit, Division of Animal Sciences, National Institute of Agrobiological Sciences, 1-2 Ohwashi, Tsukuba-shi, Ibaraki, Japan

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Article history: Received 24 January 2012 Available online 14 February 2012 Keywords: P2X7 receptor P2X4 receptor Cell death Extracellular ATP Macrophage

a b s t r a c t Interaction of P2X7 receptor with P2X4 receptor has recently been suggested, but it remains unclear whether P2X4 receptor is involved in P2X7 receptor-mediated events, such as cell death of macrophages induced by high concentrations of extracellular ATP. Here, we present evidence that P2X4 receptor does play a role in P2X7 receptor-dependent cell death. Treatment of mouse macrophage RAW264.7 cells with 1 mM ATP induced Ca2+ influx, non-selective large pore formation, activation of extracellular signal-regulated protein kinase (ERK) 1/2 and p38 mitogen-activated protein kinase (MAPK), and cell death via activation of P2X7 receptor. P2X4-knockdown cells, established by transfecting RAW264.7 cells with two short hairpin RNAs (shRNAs) targeting P2X4 receptor, showed a decrease of the initial peak of intracellular Ca2+ after treatment with ATP, though pore formation and the P2X7-mediated activation of ERK1/2 and p38 MAPK were not affected. Intriguingly, P2X4 knockdown resulted in significant suppression of cell death induced by ATP or P2X7 agonist BzATP. In conclusion, our results suggest that P2X4 receptor is involved in P2X7 receptor-mediated cell death, but not pore formation or MAPK signaling. Ó 2012 Elsevier Inc. All rights reserved.

1. Introduction Extracellular ATP is able to evoke physiological responses in a wide spectrum of tissues via binding to P2 receptors. P2 receptors have classified into two major groups; ligand-gated ion channel P2X receptors and metabotropic G protein-coupled P2Y receptors [1]. These receptors and their ligand (extracellular ATP) play important roles in cell signaling, modulation of cell growth, differentiation and induction of cell death [2,3]. P2X7 receptor is the seventh member of the P2X receptor subfamily, and is expressed in immune cells, such as monocytes/macrophages, T cells, microglia, mast cells, and dendritic cells [4–8]. Activation of P2X7 receptor is linked to a number of cellular events, including the opening of ion channels leading to a rapid influx into the cytosol of divalent cations (in particular, Ca2+) [9], the opening of a large non-selective pore allowing the Abbreviations: BAPTA-AM, 1,2-bis(2-aminophenoxy)ethane-N,N,N,N-tetraacetic acid acetoxymethyl ester; BzATP, 20 - & 30 -O-(4-benzoyl) benzoyl-ATP; [Ca2+]i, cytosolic Ca2+ concentration; EtBr, ethidium bromide; ERK, extracellular signalregulated kinase; LDH, lactate dehydrogenase; MAPK, mitogen-activated protein kinase; shRNA, short hairpin RNA. ⇑ Corresponding author. Fax: +81 (0)4 7121 3613. E-mail addresses: [email protected], [email protected] (M. Tsukimoto). 1 These authors are contributed equally to this work, and share the first authorship. 0006-291X/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2012.01.156

passage of hydrophilic molecules of up to 900 Da in size [10], membrane blebbing [11], interleukin-1b release [12,13], and apoptotic and/or necrotic cell death [6]. Although P2X7 receptor can mediate activation of caspase, treatment with caspase inhibitors does not inhibit P2X7 receptor-mediated cell death, showing that caspase activation is not an obligatory step in P2X7 receptor-mediated cell death [2]. The cytoplasmic C-terminal region of P2X7 receptor is essential for opening of large pores [10] and activation of the p38 MAPK pathway [14], which correlate with apoptotic cell death [6,15]. On the other hand, the importance of the N-terminal region for the phosphorylation of ERK 1/2 and necrotic cell death has been demonstrated using P2X7 receptor C- and N-terminal mutants [16,17]. These observations suggest that the P2X7 receptor initiates both apoptotic-like signaling and necrotic signaling through Ca2+ influx, pore formation, ERK1/2 activation, and p38 MAPK activation [5,6,14,17]. Molecules released by injured tissues, called damage-associated molecular pattern molecules (DAMPs), such as ATP, trigger inflammation [18]. High concentrations of ATP leaked from damaged tissues activate P2X7 receptor, and thereby induce cell death. It is suggested that activation of P2X7 receptor plays a role in termination of inflammation through cell death [7]. P2X4 receptor, which is highly permeable to calcium [19], is more homologous to P2X7 receptor (40%) than are the other

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P2X receptor subtypes at the amino acid sequence level. P2X4 receptor is markedly up-regulated by LPS due to activation of Toll-like receptors [20]. It is abundantly expressed in activated microglia [21,22] and is also expressed in macrophages [23]. P2X4 receptor appears to play a prominent role in nucleotide-induced apoptosis of human mesangial cells, because the apoptosis is delayed by a selective P2X1–4 antagonist, 20 ,30 -O-(2,4,6-trinitrophenyl)adenosine 5-triphosphate (TNP-ATP) [24]. However, it is not yet known whether P2X4 receptor is involved in P2X7 receptor-dependent cell death. Since the P2X7 subtype differs from other members of the family in that it has a very long cytoplasmic C-terminal tail, and a low affinity for ATP, it has been widely assumed that P2X7 does not form heteromeric assemblies with other members of the P2X family. However, recent evidence has indicated a structural interaction between P2X7 and P2X4 receptors. First, P2X receptor currents recorded from airway-ciliated cells are reported to show a combination of P2X7-like and P2X4-like properties [25]. Second, P2X7 and P2X4 receptors could be co-immunoprecipitated from mouse bone marrow-derived macrophages and also from cells in which they were heterologously co-expressed [26]. P2X7 and P2X4 receptors are necessary for biglycan-dependent regulation of IL-1b in mouse peritoneal macrophages [27]. Thus, there is increasing evidence pointing to a major role of P2X7 or P2X4 receptors in various cells, but it is still unknown whether P2X4 receptor is involved in P2X7 receptor-dependent events, such as Ca2+ influx, pore formation and cell death. The objective of the present study is to examine the role of P2X4 receptor in P2X7 receptor-mediated cell death of RAW264.7 macrophages, focusing on Ca2+ influx, pore formation and MAPK activation. We found that decreased P2X4 expression resulted in suppression of the initial ATP-induced Ca2+ influx and P2X7 receptor-mediated LDH release, but did not influence pore formation or MAPK activation. These results indicate that P2X4 receptor is involved in P2X7 receptor-dependent cell death. 2. Materials and methods 2.1. Cell culture Macrophage-like RAW264.7 cells were routinely maintained in D-MEM (Wako Pure Chemical, Osaka, Japan) supplemented with 10% heat-inactivated FBS (Biowest, Nuaille, France), 100 U/mL penicillin, 100 lg/mL streptomycin. Cells were pre-incubated for 4 h with 1 lg/mL LPS. The conditioned medium was replaced with RPMI1640-based buffer [6] before experiments. 2.2. Mobilization of intracellular calcium Cells were loaded with the Ca2+-sensitive fluorescent dye Fluo4AM (Invitogen, Carlsbad, CA) for 30 min at 37 °C, and washed twice with Ca2+-free buffer. Cells were then suspended in RPMI 1640-based buffer. The samples were analyzed using a fluorescence spectrometer (F-2500, Hitachi) with laser excitation at 495 nm and emission at 518 nm. 2.3. Analysis of pore formation Cells were re-suspended in RPMI 1640-based buffer at 1.0  106 cells/mL and incubated with various concentrations of ATP (Sigma, St. Louis, MO) and ethidium bromide for 37 °C. After incubation, the sample was analyzed using a flow cytometer (FACSCaliber cytometer, Becton, Dickinson and Co., Franklin Lakes, NJ) with laser excitation at 488 nm and examined using an FL-2 filter for ethidium fluorescence. The forward- and side-scatter signals

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from 10,000 particles were collected. Signals of cellular debris and aggregates were gated out. 2.4. Quantification of lactate dehydrogenase (LDH) release Release of LDH into cell culture supernatant was quantified with a Cytotoxicity Detection Kit (Roche Applied Science, Penzberg, Germany), according to the supplied instructions. The cells (1.0  106 cells/mL) were incubated in a 96-well plate at 37 °C for the indicated times with ATP in RPMI1640 based-buffer. At the end of incubation, supernatants were collected and the LDH content was measured. LDH release is expressed as a percentage of the total content determined by lysing an equal amount of cells with 1% Triton X-100. 2.5. Immunoblotting Equal amounts of cell lysates were dissolved in 2  sample buffer (50% glycerin, 2% SDS, 125 mM Tris, 10 mM DTT) and boiled for 10 min. Aliquots of samples containing 4 lg of protein were analyzed by 10% SDS–PAGE and transferred onto a PVDF membrane. Blots were blocked in TBST with 1% bovine serum albumin at 4 °C overnight and incubated for 1.5 h at room temperature with phospho-p44/p42 MAPK (Thr202/Tyr204) rabbit monoclonal antibody (1:2000) (Cell Signaling Technology, Inc., Beverly, MA), p44/ p42 MAP kinase antibody (1:1000) (Cell Signaling Technology), phospho-p38 MAPK (Thr180/Tyr182) rabbit monoclonal antibody (1:1000) (Cell Signaling Technology), p38a MAP kinase mouse monoclonal antibody (1:1000) (Cell Signaling Technology), antiP2X4 receptor antibody (1:300) (Alomone Labs, Jerusalem, Israel), or anti-P2X7 receptor antibody (1:200) (Alomone Labs), or with mouse anti-b-actin antibody (1:1000) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) to confirm equal loading. Blots were washed with TBST, incubated with goat horseradish peroxidase (HRP)-conjugated anti-rabbit IgG antibody (1:20,000) (Cell Signaling Technology) or goat HRP-conjugated anti-mouse IgG antibody (1:10,000) (Santa Cruz Biotechnology) for 1.5 h at room temperature, and washed again with TBST. Specific proteins were visualized by using ECL Western blotting detection reagents (GE Healthcare, Piscataway, NJ). 2.6. Short hairpin RNA (shRNA) plasmid stable transfection Stable transfection with shRNA was performed using the SureSilencing™ shRNA Plasmid Kit for Mouse P2X4 (SABiosciences, Frederick, MD). shRNA plasmid targeting P2X4 or the negative control shRNA plasmid was transfected by lipofection using FuGENE 6 Transfection Reagent (Roche Applied Science). The transfected cells were selected in hygromycin-containing (100 lg/ml) culture medium for 2 weeks. 2.7. Real-time RT-PCR Total RNA was isolated from RAW264.7 cells using a Fast Pure RNA kit (Takara Bio, Shiga, Japan). The first-strand cDNA was synthesized from total RNA with PrimeScript Reverse Transcriptase (Takara Bio). The cDNA was used as the template for real-time PCR analysis: reactions were performed in a Stratagene Mx3000PÒ quantitative PCR system (Agilent Technologies, La Jolla, CA). Specific primers were designed with PrimerQuest and synthesized by Sigma Genosys. The sequences of specific primers for murine P2X4 were 50 -TCA TCC TGG CTT ACG TCA TTG GGT-30 (sense) and 50 -CCA CAC CTT TGG CTT TGG CTT TGG TTG TCA-30 (antisense). Each sample was assayed in a 20 ll amplification reaction mixture, containing cDNA, primer mixture (5 lM each of sense and antisense primers), and 2x KAPA SYBRÒ FAST qPCR Master Mix (KAPA

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A Fluorescence of Fluo-4 ATP

3.2. Extracellular ATP induces cell death through activation of P2X7 receptor We assessed cell death in terms of the release of cytosolic LDH. Treatment with ATP induced LDH release in a dose-dependent manner (Fig. 2A), and the release of LDH occurred 3 h after treatment with ATP (Fig. 2B). Though P2 receptors other than P2X7 receptor can be activated by 0.1 mM ATP, activation of the P2X7 receptor requires more than 0.1 mM ATP and peak activation is seen at 1 mM ATP [28]. Since cell death was not induced by ATP at concentrations lower than 0.5 mM, the P2X7 receptor appears to be involved in ATP-induced cell death. We also tested the effects of 20 - & 30 -O-(4-benzoyl) benzoyl-ATP (BzATP) (a P2X1, P2X7 receptor agonist) (Sigma) and A438079. BzATP, which activate P2X7

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Increase of intracellular Ca2+ ([Ca2+]i) plays a significant role in P2X7-mediated cell death [5]. As shown in Fig. 1A, we examined the effect of ATP on the elevation of [Ca2+]i. When RAW264.7 cells were stimulated with ATP, they showed an initial peak of [Ca2+]i followed by a sustained phase. Pretreatment with A438079 (a P2X7 receptor antagonist) (Tocris Bioscience, Bristol, UK) suppressed the sustained phase, but not the initial peak of [Ca2+]i. These results indicate that the sustained phase of [Ca2+]i elevation is induced by activation of P2X7 receptor, but other P2 receptors are involved in the initial peak in [Ca2+]i. ATP-mediated cell death has been shown to correlate with nonselective large pore formation [5,6,15,17]. To investigate the activity of non-selective large pore formation, we examined the uptake of EtBr into cells by flow cytometry, excluding dead cells. We found that ATP induced uptake of EtBr into the cells in dose-dependent manner. Pretreatment with A438079 significantly inhibited the ATP-induced uptake of EtBr (Fig. 1B), suggesting that large pore formation is dependent on activation of P2X7 receptor in RAW264.7 cells. It is known that activation of ERK1/2 contributes to P2X7 receptor-mediated cell death [17] and that the p38 MAPK pathway is required in part for ATP-induced apoptosis [14]. We therefore investigated activation of ERK1/2 and p38 MAPK after treatment of RAW264.7 cells with ATP. High concentrations of ATP induced phosphorylation of ERK1/2 and p38 MAPK, and pretreatment with A438079 inhibited activation of both ERK1/2 and p38 MAPK (Fig. 1C), suggesting that activation of P2X7 receptor induces activation of both ERK1/2 and p38 MAPK in these cells.

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Values are given as the mean ± SE. Comparison between two values was performed by means of the unpaired Student’s t-test. Multiple groups were compared using ANOVA followed by pairwise comparisons with Bonferroni’s post hoc analysis. Significance was defined as P < 0.05. Calculations were done using the Instat version 3.0 statistical package (GraphPad Software, San Diego, CA).

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Biosystems, Woburn, MA). The amplification program consisted of 40 cycles (each cycle: 95 °C for 3 s, annealing at 60 °C for 30 s) after 95 °C for 3 min. Fluorescent products were detected at the last step of each cycle. The obtained values were within the linear range of a standard curve and were normalized with respect to GAPDH mRNA.

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p-ERK 1/2 ERK 1/2 p-p38 MAPK p38 MAPK Fig. 1. ATP-induced Ca2+ influx, pore formation, and phosphorylation of MAPKs via activation of P2X7 receptor. (A) RAW264.7 cells loaded with Fluo-4 (10 lM) were stimulated with 1 mM ATP in the presence (red line) or absence (black line) of A438079 (50 lM). The fluorescence was analyzed with a fluorescence spectrometer. We obtained qualitatively similar results in three independent experiments, and typical data are presented. (B) Cells were pre-incubated with A438079 (100 lM) for 15 min, and incubated with various concentrations of ATP and EtBr (25 lM) in RPMI1640-based buffer for 10 min. The change in fluorescence intensity of EtBr was analyzed by flow cytometry. Error bars indicate ± SE (n = 4). Significant differences between the indicated groups are indicated with ⁄⁄⁄(p < 0.001). (C) Cells were preincubated with A438079 (50 lM) for 15 min, and incubated for 15 min with 1 mM or 3 mM ATP. Phosphorylation of ERK1/2 and p38 MAPK was determined by immunoblotting. The results are typical of those obtained in three independent experiments.

receptor but not P2X4 receptor, induced the release of LDH dosedependently (Fig. 2C). Pretreatment with A438079 significantly inhibited ATP- or BzATP-induced LDH release (Fig. 2C and D). These results indicated that treatment with ATP induced cell death via activation of the P2X7 receptor. To investigate the involvement of

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Fig. 2. ATP or BzATP-induced cell death through activation of P2X7 receptor. (A) Cells were incubated with vehicle (control) or various concentrations of ATP (0.1–3 mM) for 6 h. (B) Cells were incubated with 1 mM ATP for the indicated times. (C) Cells were pre-incubated with 50 lM A438079 for 30 min, and incubated with vehicle or BzATP (100– 300 lM) for 6 h. (D) Cells were pre-incubated with 50 lM A438079, 10 lM U0126, 10 lM SB203580, or 10 lM SP600125 for 30 min, and incubated with vehicle or 1 mM ATP for 6 h. At the end of incubation, supernatants were collected and LDH content was measured. The release of LDH is expressed as a percentage of total content determined by lysing an equal number of cells with 1% Triton X-100. Error bars indicate ± SE (n = 4–6). Significant differences between control and ATP (or BzATP)-treated cells are indicated with ⁄⁄⁄(p < 0.001). Significant differences between ATP (or BzATP)-treated cells and inhibitor-treated cells are indicated with #(p < 0.01).

MAPKs in P2X7-mediated cell death, we next examined the effect of MAPK inhibitors on ATP-induced LDH release. Although SP600125 (JNK-signaling inhibitor) (Calbiochem, San Diego, CA) did not affect ATP-induced LDH release, U0126 (MEK1/2 inhibitor) (Calbiochem) and SB203580 (p38 MAPK-signaling inhibitor) (Calbiochem) inhibited the LDH release (Fig. 2D). Since U0126 suppresses activation of ERK1/2, which is involved in P2X7-mediated necrotic cell death [6,17] and SB203580 inhibits activation of p38 MAPK, which is involved in P2X7 receptor-mediated apoptotic cell death [14], it is confirmed that P2X7-dependent macrophage death is partially regulated by both ERK1/2 and p38 MAPK signaling. These results suggest that ERK1/2 and p38 MAPK signaling pathways are involved in P2X7-dependent cell death of RAW264.7 cells. 3.3. Effect of decreased P2X4 expression on Ca2+ influx To investigate whether P2X4 receptor is involved in P2X7dependent events, such as intracellular Ca2+ elevation, pore formation, ERK1/2 and p38 MAPK activation, and cell death, we silenced the expression of P2X4 receptor with different shRNAs (clone ID: red and yellow). In cells transfected with P2X4-shRNA, P2X4 receptor mRNA and protein expression levels were decreased to <50% of that in scramble shRNA-transfected cells (negative control) (Fig. 3A

and B). This transfection did not affect P2X7 receptor expression (Fig. 3B). One of the hallmarks of P2X4 receptor is its very high calcium permeability [16]. Although Ca2+ influx is common for both P2X7 and P2X4 receptors, the P2X4-dependent Ca2+ influx is acute and transient. On the other hand, P2X7-dependent Ca2+ influx is associated with the sustained phase of [Ca2+]i increase. As shown in Fig. 3C, P2X4-knockdown cells showed a decrease of the initial peak in [Ca2+]i induced by ATP, although the sustained phase of [Ca2+]i increase was unaffected. These results indicated that P2X4-knockdown decreased the expression of P2X4 receptor and the acute-phase Ca2+ influx in these cells. 3.4. Involvement of P2X4 receptor in P2X7-dependent cell death To investigate the involvement of P2X4 receptor in other downstream events of P2X7 receptor signaling, we examined P2X7dependent pore formation and phosphorylation of MAPKs in P2X4-knockdown cells. Transfection of cells with P2X4-shRNA did not affect EtBr uptake or the activation of ERK1/2 and p38 MAPK (Fig. 4A and B), suggesting that these events are not dependent on P2X4 receptor. Next, we examined the role of P2X4 receptor in P2X7 receptor-mediated cell death. Decreased P2X4 expression resulted in suppression of LDH release in response to ATP or BzATP (Fig. 4C). Pre-treatment with A438079 completely inhibited ATP

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Fig. 3. Knockdown of P2X4 receptor in RAW264.7 cells. Cells were transfected with shRNA targeting P2X4 (clone ID: red or yellow) or the negative control shRNA (scramble shRNA). (A) Expression levels of P2X4 mRNA in transfectants were determined by real-time RT-PCR. Optical density of P2X4 mRNA was normalized to the optical density of GAPDH mRNA. (B) Protein expressions of P2X4 and P2X7 receptors were determined by immunoblotting. P2X4 and P2X7 expression levels were normalized for b-actin expression levels. (A and B) Values are expressed as ratios to scramble shRNA-transfected cells, and are means ± SE of three independent experiments. A statistically significant difference between scramble shRNA-transfected cells and P2X4-KD cells is indicated by ⁄⁄⁄(P < 0.001), ⁄⁄(P < 0.01) or ⁄(P < 0.05). (C and D) Cell transfected with shRNA targeting P2X4 (red (C) or yellow (D) or negative control shRNA (gray line) were loaded with Fluo-4 and stimulated with 1 mM ATP, and the fluorescence was analyzed with a fluorescence spectrometer.

or BzATP-induced LDH release in P2X4-knockdown as well as scramble shRNA-transfected cells (Fig. 4C), suggesting that P2X4 receptor does play a role in P2X7 receptor-mediated cell death. 3.5. Role of elevation of intracellular Ca2+ on ATP-induced cell death To investigate the involvement of Ca2+ influx in P2X7-dependent cell death, we examined the effect of removal of extracellular Ca2+ or pretreatment with an intracellular Ca2+ chelator, 1,2-bis(2aminophenoxy)ethane-N,N,N,N-tetraacetic acid acetoxymethyl ester (BAPTA-AM) (Dojindo, Kumamoto, Japan) on ATP-induced LDH release. As shown in Fig. 4D, LDH release was suppressed when cells were stimulated in Ca2+-free buffer or pretreated with BAPTA-AM, indicating that elevation of intracellular Ca2+ is in-

volved in P2X7-dependent cell death. These results support the idea that P2X4-dependent initial Ca2+ influx plays a role in P2X7dependent cell death. High concentrations of ATP induce Ca2+ influx, pore formation, activation of ERK1/2 and p38MAPK, and cell death via activation of P2X7 receptor in macrophages. Our findings indicate that P2X4 receptor is involved in P2X7 receptor-mediated macrophage death, but not in nonselective pore formation or activation of ERK1/2 and p38 MAPK, which are known to contribute to P2X7-dependent cell death. Although activation of P2X4 receptor alone does not induce cell death, our results suggest that co-expression of P2X4 receptor with P2X7 receptor facilitates P2X7 receptor-dependent cell death in activated macrophages. Specifically, P2X4 receptor-dependent acute-phase Ca2+ influx at least might contribute to P2X7

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Fig. 4. Effects of decreased P2X4 expression on P2X7-dependent pore formation, activation of MAPK and cell death. (A) Transfectants were incubated with 1 mM ATP and EtBr in RPMI1640-based buffer for 10 min. The change in fluorescence intensity of EtBr was analyzed by flow cytometry. (B) Transfectants were incubated for 15 min with 1 mM ATP. Phosphorylation of ERK1/2 and p38 MAPK was determined by immunoblotting. (C) Transfectants were preincubated with A438079 for 30 min and incubated with vehicle, 1 mM ATP or 200 lM BzATP for 6 h. (D) RAW264.7 cells were incubated with vehicle (control) or 1 mM ATP for 6 h in RPMI1640 buffer, Ca2+ free-RPMI1640 buffer or RPMI1640 buffer containing BAPTA-AM (10 lM). (C and D) At the end of incubation, supernatants were collected and LDH content was measured. The release of LDH is expressed as a percentage of total content determined by lysing an equal number of cells with 1% Triton X-100. Error bars indicate ± SE (n = 4). Significant differences between the indicated groups are indicated with ⁄⁄⁄(p < 0.001). Significant differences between ATP (or BzATP)-treated cells and inhibitor-treated cells are indicated with #(p < 0.01).

receptor-dependent cell death. P2X4 receptor may also have further roles in P2X7 receptor-mediated signaling. This is a first demonstration that P2X4 receptor plays a significant role in P2X7 receptor-dependent cell death in macrophages. Since ATP-induced cell death of immune cells plays an important role in termination of immune response [7], our results indicate that co-expression of P2X4 receptor with P2X7 receptor in macrophages may be important for immune regulation. Acknowledgment Parts of this work were supported by Grant-in-Aid for Young Scientists (B) (to M.T.) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.

References [1] G. Burnstock, Introduction: P2 receptors, Curr. Top Med. Chem. 4 (2004) 793– 803. [2] F. Di Virgilio, P. Chiozzi, S. Falzoni, D. Ferrari, J.M. Sanz, V. Venketaraman, O.R. Baricordi, Cytolytic P2X purinoceptors, Cell Death Differ. 5 (1998) 191–199. [3] S.C. Chow, G.E. Kass, S. Orrenius, Purines and their roles in apoptosis, Neuropharmacology 36 (1997) 1149–1156. [4] G. Collo, S. Neidhart, E. Kawashima, M. Kosco-Vilbois, R.A. North, G. Buell, Tissue distribution of the P2X7 receptor, Neuropharmacology 36 (1997) 1277– 1283. [5] E. Adinolfi, C. Pizzirani, M. Idzko, E. Panther, J. Norgauer, F. Di Virgilio, D. Ferrari, P2X(7) receptor: death or life?, Purinergic Signal 1 (2005) 219–227 [6] M. Tsukimoto, M. Maehata, H. Harada, A. Ikari, K. Takagi, M. Degawa, P2X7 receptor-dependent cell death is modulated during murine T cell maturation and mediated by dual signaling pathways, J. Immunol. 177 (2006) 2842–2850.

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[7] M. Tsukimoto, H. Harada, M. Degawa, Role of purinoceptors in immunemediated disease (therapies targeting the P2X7 receptor), Drug Discovery Today: Therapeutic Strategies 1 (2007) 33–37. [8] T. Takenouchi, M. Nakai, Y. Iwamaru, S. Sugama, M. Tsukimoto, M. Fujita, J. Wei, A. Sekigawa, M. Sato, S. Kojima, H. Kitani, M. Hashimoto, The activation of P2X7 receptor impairs lysosomal functions and stimulates the release of autophagolysosomes in microglial cells, J. Immunol. 182 (2009) 2051–2062. [9] B.R. Bianchi, K.J. Lynch, E. Touma, W. Niforatos, E.C. Burgard, K.M. Alexander, H.S. Park, H. Yu, R. Metzger, E. Kowaluk, M.F. Jarvis, T. van Biesen, Pharmacological characterization of recombinant human and rat P2X receptor subtypes, Eur. J. Pharmacol. 376 (1999) 127–138. [10] A. Surprenant, F. Rassendren, E. Kawashima, R.A. North, G. Buell, The cytolytic P2Z receptor for extracellular ATP identified as a P2X receptor (P2X7), Science 272 (1996) 735–738. [11] C. Virginio, A. MacKenzie, R.A. North, A. Surprenant, Kinetics of cell lysis, dye uptake and permeability changes in cells expressing the rat P2X7 receptor, J. Physiol. 519 (Pt. 2) (1999) 335–346. [12] D. Perregaux, C.A. Gabel, Interleukin-1 beta maturation and release in response to ATP and nigericin. Evidence that potassium depletion mediated by these agents is a necessary and common feature of their activity, J. Biol. Chem. 269 (1994) 15195–15203. [13] P. Pelegrin, A. Surprenant, Pannexin-1 mediates large pore formation and interleukin-1beta release by the ATP-gated P2X7 receptor, EMBO J. 25 (2006) 5071–5082. [14] T. Noguchi, K. Ishii, H. Fukutomi, I. Naguro, A. Matsuzawa, K. Takeda, H. Ichijo, Requirement of reactive oxygen species-dependent activation of ASK1-p38 MAPK pathway for extracellular ATP-induced apoptosis in macrophage, J. Biol. Chem. 283 (2008) 7657–7665. [15] M. Tsukimoto, H. Harada, A. Ikari, K. Takagi, Involvement of chloride in apoptotic cell death induced by activation of ATP-sensitive P2X7 purinoceptor, J. Biol. Chem. 280 (2005) 2653–2658. [16] J. Amstrup, I. Novak, P2X7 receptor activates extracellular signal-regulated kinases ERK1 and ERK2 independently of Ca2+ influx, Biochem. J. 374 (2003) 51–61. [17] R. Auger, I. Motta, K. Benihoud, D.M. Ojcius, J.M. Kanellopoulos, A role for mitogen-activated protein kinase (Erk1/2) activation and non-selective pore

[18]

[19] [20]

[21]

[22]

[23]

[24]

[25]

[26]

[27]

[28]

formation in P2X7 receptor-mediated thymocyte death, J. Biol. Chem. 280 (2005) 28142–28151. A. Rubartelli, M.T. Lotze, Inside, outside, upside down: damage-associated molecular-pattern molecules (DAMPs) and redox, Trends Immunol. 28 (2007) 429–436. T.M. Egan, B.S. Khakh, Contribution of calcium ions to P2X channel responses, J. Neurosci. 24 (2004) 3413–3420. R. Raouf, A.J. Chabot-Dore, A.R. Ase, D. Blais, P. Seguela, Differential regulation of microglial P2X4 and P2X7 ATP receptors following LPS-induced activation, Neuropharmacology 53 (2007) 496–504. M. Tsuda, Y. Shigemoto-Mogami, S. Koizumi, A. Mizokoshi, S. Kohsaka, M.W. Salter, K. Inoue, P2X4 receptors induced in spinal microglia gate tactile allodynia after nerve injury, Nature 424 (2003) 778–783. F. Cavaliere, F. Florenzano, S. Amadio, F.R. Fusco, M.T. Viscomi, N. D’Ambrosi, F. Vacca, G. Sancesario, G. Bernardi, M. Molinari, C. Volonte, Up-regulation of P2X2, P2X4 receptor and ischemic cell death: prevention by P2 antagonists, Neuroscience 120 (2003) 85–98. J.W. Bowler, R.J. Bailey, R.A. North, A. Surprenant, P2X4, P2Y1 and P2Y2 receptors on rat alveolar macrophages, Br. J. Pharmacol. 140 (2003) 567–575. A. Solini, E. Santini, D. Chimenti, P. Chiozzi, F. Pratesi, S. Cuccato, S. Falzoni, R. Lupi, E. Ferrannini, G. Pugliese, F. Di Virgilio, Multiple P2X receptors are involved in the modulation of apoptosis in human mesangial cells: evidence for a role of P2X4, Am. J. Physiol. Renal. Physiol. 292 (2007) F1537–F1547. W. Ma, A. Korngreen, S. Weil, E.B. Cohen, A. Priel, L. Kuzin, S.D. Silberberg, Pore properties and pharmacological features of the P2X receptor channel in airway ciliated cells, J. Physiol. 571 (2006) 503–517. O.S. Qureshi, A. Paramasivam, J.C. Yu, R.D. Murrell-Lagnado, Regulation of P2X4 receptors by lysosomal targeting, glycan protection and exocytosis, J. Cell Sci. 120 (2007) 3838–3849. A. Babelova, K. Moreth, W. Tsalastra-Greul, J. Zeng-Brouwers, O. Eickelberg, M.F. Young, P. Bruckner, J. Pfeilschifter, R.M. Schaefer, H.J. Grone, L. Schaefer, Biglycan, a danger signal that activates the NLRP3 inflammasome via toll-like and P2X receptors, J. Biol. Chem. 284 (2009) 24035–24048. R.A. North, Molecular physiology of P2X receptors, Physiol. Rev. 82 (2002) 1013–1067.