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

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Abstract

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.

Highlights

► Extracellular ATP induces P2X7 receptor-dependent cell death of macrophages. ► P2X4 receptor is co-expressed with P2X7 receptor in macrophages. ► Knockdown of P2X4 receptor attenuates P2X7 receptor-dependent cell death. ► P2X4 receptor plays a role in P2X7 receptor-dependent cell death.

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], [5], [6], [7], [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 passage of hydrophilic molecules of up to 900 Da in size [10], membrane blebbing [11], interleukin-1β 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 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, 2′,3′-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-1β 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.

Section snippets

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 μg/mL streptomycin. Cells were pre-incubated for 4 h with 1 μg/mL LPS. The conditioned medium was replaced with RPMI1640-based buffer [6] before experiments.

Mobilization of intracellular calcium

Cells were loaded with the Ca2+-sensitive fluorescent dye Fluo-4AM (Invitogen, Carlsbad, CA) for 30 min at 37 °C, and washed twice with Ca2+-free

Activation of P2X7 receptor induces intracellular Ca2+ mobilization, pore formation, and MAPK activation

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

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 (28)

  • F. Cavaliere et al.

    Up-regulation of P2X2, P2X4 receptor and ischemic cell death: prevention by P2 antagonists

    Neuroscience

    (2003)
  • A. Babelova et al.

    Biglycan, a danger signal that activates the NLRP3 inflammasome via toll-like and P2X receptors

    J. Biol. Chem.

    (2009)
  • G. Burnstock

    Introduction: P2 receptors

    Curr. Top Med. Chem.

    (2004)
  • F. Di Virgilio et al.

    Cytolytic P2X purinoceptors

    Cell Death Differ.

    (1998)
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    These authors are contributed equally to this work, and share the first authorship.

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