Involvement of P2X4 receptor in P2X7 receptor-dependent cell death of mouse macrophages
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.
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These authors are contributed equally to this work, and share the first authorship.