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

Rodolphe Auger,Iris Motta,Karim Benihoud,Jean M Kanellopoulos,David M Ojcius

doi:10.1074/jbc.m501290200

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.

Highlights

Extracellular ATP interacts with P2 purinergic receptors that are expressed on a wide spectrum of tissues
We show that stimulation of P2X7 receptors (P2X7R) in thymocytes induces the activation of the three mitogenactivated protein (MAP) kinase modules, extracellular-signal regulated kinases 1/2 (Erk1/2), c-Jun N-terminal kinases 1/2 (JNK1/2), and p38 kinases
To further elucidate the basis for cell lysis, we took advantage of two assays that discriminate between the two types of cell death, namely, the release of high mobility group box 1 (HMGB1), a chromatin-associated nuclear protein that is liberated into the cytosol of necrotic but not apoptotic cells [40]; and the cleavage of poly(ADP-ribose) polymerase (PARP), a nuclear protein that is a specific substrate of caspase-3 and is an indicator of apoptotic cell death

Summary

Introduction

Extracellular ATP interacts with P2 purinergic receptors that are expressed on a wide spectrum of tissues. Prolonged triggering of P2X7R results in the formation of non-selective membrane pores permeable to molecules of molecular mass up to 900 Da. Formation of the non-selective pore is dependent on the cytoplasmic C-terminal domain of P2X7R [3,4,5]. Formation of the non-selective pore is dependent on the cytoplasmic C-terminal domain of P2X7R [3,4,5] It is still unclear whether it results from dilatation of the P2X7R-induced cation channel or from interaction of P2X7R with a distinct channel protein, which allows the entry of larger molecules (reviewed in Ref. 6). The physiological significance of cell death mediated by the ligation of P2X7R by ATP remains to be determined. A potential role for ATP in programmed cell death underlying positive and negative selection of immature thymocytes during thymus development has been suggested by Chvatchko et al [27]. The potential role of ATP in programmed cell death in the thymus still remains elusive

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Conclusion