Expression of the P2X7 Receptor Increases the Ca2+ Content of the Endoplasmic Reticulum, Activates NFATc1, and Protects from Apoptosis

Abstract

The P2X 7 receptor is known for the cytotoxic activity because of its ability to cause opening of non-selective pores in the plasma membrane and activate apoptotic caspases. A key factor of P2X 7 -dependent cytotoxicity is the massive intracellular Ca 2؉ increase triggered by its activation. Here we show that P2X 7 transfection increased the ability of the endoplasmic reticulum to accumulate, store, and release Ca 2؉ . This caused a larger agonist-stimulated increase in cytosol and mitochondrial Ca 2؉ in P2X 7 transfectants than in mock transfected cells. P2X 7 transfectants survived and even proliferated in serum-free conditions and were resistant to apoptosis triggered by ceramide, staurosporin, or intracellular Zn 2؉ chelation. Finally, the nuclear factor of activated T cells complex 1 (NFATc1) was strongly activated in the P2X 7 transfectants. These observations support our previous finding that the P2X 7 receptor under tonic conditions of stimulation, i.e. those observed in response to basal ATP release, has an anti-apoptotic or even growth promoting rather than cytotoxic activity.Cell responses to extracellular ATP are mediated by P2 receptors: ionotropic P2X and metabotropic P2Y (1). The P2X 7 receptor (P2X 7 R) 4 subtype stands out in the P2X subfamily for its ability to trigger a host of physiologic or pathologic responses: plasma membrane blebbing (2, 3), rapid release of interleukin-1␤ via microvesicle shedding (2, 4), cell fusion (5), lymphoid cell proliferation (6), cell death (7,8), and bone formation/ resorption (9). This receptor is characterized by low affinity for ATP and by two states of permeability (10, 11). At high micromolar ATP concentrations, P2X 7 behaves as a cation-selective channel, whereas a prolonged exposure to nearly millimolar concentrations triggers the transition to a nonselective pore that admits hydrophilic solutes of molecular mass up to 900 Da (12-14). A common feature of both conductance states is a strong elevation of free cytoplasmic calcium levels ([Ca 2ϩ ] i ), a response critical for the biological role of this receptor.Recent evidence from our group shows that basal levels of ATP, naturally present in the extracellular milieu, cause a tonic activation of the P2X 7 R, which in turn triggers [Ca 2ϩ ] i increase and Ca 2ϩ entry into the mitochondria. The increased intramitochondrial Ca 2ϩ concentration ([Ca 2ϩ ] mt ) then enhances mitochondrial potential, increases ATP synthesis, and promotes survival and proliferation in the absence of serum (15). Increased mitochondrial potential and ability to grow under serum-free conditions are hallmark of tumor transformation (16). Furthermore, overexpression of the P2X 7 R is associated with several cancers or growth disturbances such as chronic lymphocytic leukemia (17), prostate (18)

Ivermectin-dependent release of IL-1beta in response to ATP by peritoneal macrophages from P2X7-KO mice

“…Considering that the activation of P2X 7 receptor inhibits the PPI-PLC signaling pathway [21] it might look paradoxical that the response coupled to P2Y receptors was larger in cells expressing the P2X 7 receptor. The group of Di Virgilio has very recently reported that transfection of HEK293 cells with P2X 7 receptors increased their capacity to accumulate calcium in the endoplasmic reticulum [22]. As a consequence, the agonists mobilizing this intracellular pool of calcium (carbachol or bradykinin in their hands) provoked a larger increase in the cytosolic concentration of calcium in the cells expressing the P2X 7 receptor than in cells which did not express it.…”

Expression of P2X7 Receptor IncreasesIn VivoTumor Growth

“…The trophic signaling cascade activated by P2X7 involves subtle changes in intracellular Ca 2þ homeostasis, stimulation of mitochondrial metabolism, activation of NFATc1, and suppression of apoptosis (8,9). NFATc1, a key transcription factor in lymphocyte division and cancer cell growth, exerts a central role in P2X7-mediated proliferation as it is strongly upregulated in HEK293 cells expressing different P2X7 isoforms (9,12), and its blockade by selective inhibitors, such as cyclosporine or VIVIT, obliterates P2X7-dependent cell growth (9).…”

“…NFATc1, a key transcription factor in lymphocyte division and cancer cell growth, exerts a central role in P2X7-mediated proliferation as it is strongly upregulated in HEK293 cells expressing different P2X7 isoforms (9,12), and its blockade by selective inhibitors, such as cyclosporine or VIVIT, obliterates P2X7-dependent cell growth (9). P2X7 expression not only stimulates growth but also enhances invasiveness of HEK293 cells, as shown in vitro, in Matrigel infiltration experiments (12), and in vivo, in a zebrafish model of metastasis (30).…”

“…Characterization of B16, CT26-mP2X7, and ACNshRNA clones Expression of P2X7 by B16, CT26, and ACNshRNA clones was verified by semiquantitative PCR and Western blot analysis as previously described (9). For Western blot analysis, primary antibodies (anti-P2X7 and anti-actin A2668; Sigma-Aldrich) were used at 1:200 dilution and incubated for 16 hours at 4 C (anti-P2X7) or 1 hour at room temperature (anti-actin).…”

“…ATP is the key energy currency as well as a ubiquitous extracellular messenger (6). Depending on its dose and the purinergic P2 receptor subtype engaged, ATP can trigger many different cell responses, including cell death or proliferation (7)(8)(9)(10). Among the receptors engaged by extracellular ATP (P2 receptors), P2X7 is the one most consistently expressed (or even overexpressed) by tumor cells (see ref.…”