Extracellular ATP Causes ROCK I-dependent Bleb Formation in P2X₇-transfected HEK293 Cells

Abstract

The P2X7 ATP receptor mediates the cytotoxic effect of extracellular ATP. P2X7-dependent cell death is heralded by dramatic plasma membrane bleb formation. Membrane blebbing is a complex phenomenon involving as yet poorly characterized intracellular pathways. We have investigated the effect of extracellular ATP on HEK293 cells transfected with the cytotoxic/pore-forming P2X7 receptor. Addition of ATP to P2X7-transfected, but not to wt P2X7-less, HEK293 cells caused massive membrane blebbing within 1-2 min. UTP, a nucleotide incapable of activating P2X7, had no early effects on cell shape and bleb formation. Bleb formation triggered by ATP was reversible and required extracellular Ca2+ and an intact cytoskeleton. Furthermore, it was completely prevented by preincubation with the P2X blocker oxidized ATP. It was recently observed that the ROCK protein is a key determinant of bleb formation. Preincubation of HEK293-P2X7 cells with the ROCK blocker Y-27632 completely prevented P2X7-dependent blebbing. Although ATP triggered cleavage of the ROCK I isoform in P2X7-transfected HEK293 cells, the wide range caspase inhibitor z-VAD-fluoromethylketone had no effect. These observations suggest that P2X7-dependent plasma membrane blebbing depends on the activation of the serine/threonine kinase ROCK I.

Autocrine signaling via release of ATP and activation of P2X7 receptor influences motile activity of human lung cancer cells

“…This suggests that P2X7-dependent signaling contributes to restricting lamellipodia broadening. It is known that activation of P2X7 receptor causes activation of Rho kinase [32,33]. Therefore, blockade of P2X7 receptor would suppress activation of the Rho pathway and result in aberrant activation of Rac1.…”

Rho kinase in the regulation of cell death and survival

“…However, caspase 3-independent cleavage of ROCK1 was observed in extracellular ATP-induced and P2X7 ATP receptor-mediated apoptosis [91] and in cancer cells subjected to combined BGC9331 (a thymidylate synthase inhibitor) and SN-38 (a topoisomerase I inhibitor) treatment [17]. The nature of the proteases involved and the cleavage site for caspase 3-independent cleavage of ROCK1 remain to be identified [17,91]. On the other hand, during cytotoxic lymphocyte granule-induced cell death, human ROCK2 can be cleaved by the proapoptotic protease granzyme B at IGLD1131 site, and this site is not present in ROCK1 [118].…”

“…In addition, ROCK1 cleavage by caspase 3 can be inhibited by caspase inhibitors in a variety of apoptotic cells [16,23,70,86,104,119,127,139,140,155]. However, caspase 3-independent cleavage of ROCK1 was observed in extracellular ATP-induced and P2X7 ATP receptor-mediated apoptosis [91] and in cancer cells subjected to combined BGC9331 (a thymidylate synthase inhibitor) and SN-38 (a topoisomerase I inhibitor) treatment [17]. The nature of the proteases involved and the cleavage site for caspase 3-independent cleavage of ROCK1 remain to be identified [17,91].…”

P2X7: a growth-promoting receptor—implications for cancer

“…Although P2X 7 has been reported to cause caspase, MAP kinase or Rho/ROCK protein activation [20,21,22], ion fluxes are still thought to be the most important signal transduction device associated to P2X 7 . Uncontrolled Na + and Ca 2+ entry is the main trigger of the well-known P2X 7 -dependent cytotoxicity, while K + efflux is thought to be the main mechanism by which P2X 7 drives pro-IL-1β processing.…”