A Role for Mitogen-activated Protein KinaseErk1/2 Activation and Non-selective Pore Formation in P2X7 Receptor-mediated Thymocyte Death

Abstract

Extracellular ATP (ATPe) binds to P2X7 receptors (P2X7R) expressed on the surface of cells of hematopoietic lineage, including murine thymocytes. Activation of P2X7R by ATPe results in the opening of cation-specific channels, and prolonged ATPe exposure leads to the formation of non-selective pores enabling transmembrane passage of solutes up to 900 Da. In the presence of ATPe, P2X7R-mediated thymocyte death is due primarily to necrosis/lysis and not apoptosis, as measured by the release of lactate dehydrogenase indicative of a loss of plasma membrane integrity. The present study is focused on the identification of P2X7R signaling mediators in ATP-induced thymocyte necrosis/lysis. Thus, extracellular signal-regulated protein kinase 1/2 (Erk1/2) phosphorylation was found to be required for cell lysis, and both events were independent of ATP-induced calcium influx. P2X7R-dependent thymocyte death involved the chronological activation of Src family tyrosine kinase(s), phosphatidylinositol 3-kinase, the mitogen-activated protein (MAP) kinase Erk1/2 module, and the proteasome. Although independent of this signaling cascade, non-selective pore formation may modulate ATP-mediated thymocyte death. These results therefore suggest a role for both activation of MAP kinase Erk1/2 and non-selective pore opening in P2X7R-induced thymocyte death. Extracellular ATP interacts with P2 purinergic receptors that are expressed on a wide spectrum of tissues. Two classes of P2 receptors have been identified, the G-protein-coupled seventransmembrane P2Y receptors and the P2X ligand-gated cation channels (1). Seven members of the P2X receptor family have been cloned, which share the same predicted structure with two transmembrane-spanning domains, an extracellular loop and intracellular N-and C-terminal tails. The P2X7 receptor (P2X7R) 1 differs from the other P2X receptors; in particular, its C-terminal domain is 200 amino acids longer and it does not heteropolymerize with other members of the P2X family (2). Brief exposure to millimolar concentrations of ATP in its fully dissociated tetra-anionic form, ATP 4Ϫ

Intracellular and Extracellular ATP Coordinately Regulate the Inverse Correlation between Osteoclast Survival and Bone Resorption

“…In addition, ␣-fodrin degradation is proposed to contribute to cell blebbing and other morphologic changes (40). Extracellular ATP stimulation is also reported to induce c-Src activation and tyrosine phosphorylation of intracellular proteins (41,42). However, we could not detect degradation in cytoskeletal proteins including ␣-fodrin, actin, c-Src, and Pyk2.…”

P2X7 Receptor-Dependent Cell Death Is Modulated during Murine T Cell Maturation and Mediated by Dual Signaling Pathways

“…In contrast, Auger et al (9) reported that ERK1/2 phosphorylation plays a role in P2X 7 receptor-mediated necrotic cell death, and these events are independent of pore formation. Thus, we investigated the contributions of cell shrinkage and the ERK1/2 pathway to P2X 7 receptor-mediated splenocyte death.…”

“…The P2X 7 receptor C-terminal region of C57BL/6 mice bears a Pro-451 to Leu mutation that impairs nonselective pore formation (30), but there are no differences between the two mouse strains in terms of P2X 7 receptor mRNA and protein expressions, and ATP-induced phosphorylation of ERK1/2 (9). Reduced sensitivity to ATP-induced pore formation, cell shrinkage, and cell death were observed in splenocytes from C57BL/6 mice compared with BALB/c mice (Fig.…”

“…In contrast, P2X 7 receptor activation also leads to ERK1/2 phosphorylation (7) and this signaling cascade is dependent on the N terminus (8). Because the inhibition of ERK phosphorylation suppressed P2X 7 receptor-mediated necrotic cell death, an N terminus can also mediate cell death signaling (9). Activation of the P2X 7 receptor induces apoptotic DNA fragmentation, accompanied by the proteolytic processing of multiple caspases and caspase substrates.…”

Characterisation of the R276A gain-of-function mutation in the ectodomain of murine P2X7

“…Other important functions where P2X7 has been implicated include killing of mycobacteria and Chlamydia residing inside macrophages [33][34][35], apoptosis of immune cells [14,36,37], cell fusion [38,39] and shedding of the CD62L homing receptor and other membrane proteins including CD23 and CD27 [14,40,41].…”