The nucleotide receptor P2X7 mediates actin reorganization and membrane blebbing in RAW 264.7 macrophages via p38 MAP kinase and Rho

Abstract

Extracellular nucleotides regulate macrophage function via P2X nucleotide receptors that form ligand-gated ion channels. In particular, P2X7 activation is characterized by pore formation, membrane blebbing, and cytokine release. P2X7 is also linked to mitogen-activated protein kinases (MAPK) and Rho-dependent pathways, which are known to affect cytoskeletal structure in other systems. As cytoskeletal function is critical for macrophage behavior, we have tested the importance of these pathways in actin filament reorganization during P2X7 stimulation in RAW 264.7 macrophages. We observed that the P2X7 agonists adenosine 5'-triphosphate (ATP) and 3'-O-(4-benzoylbenzoyl) ATP (BzATP) stimulated actin reorganization and concomitant membrane blebbing within 5 min. Disruption of actin filaments with cytochalasin D attenuated membrane blebbing but not P2X7-dependent pore formation or extracellular-regulated kinase (ERK)1/ERK2 and p38 activation, suggesting that these latter processes do not require intact actin filaments. However, we provide evidence that p38 MAPK and Rho activation but not ERK1/ERK2 activation is important for P2X7-mediated actin reorganization and membrane blebbing. First, activation of p38 and Rho was detected within 5 min of BzATP treatment, which is coincident with membrane blebbing. Second, the p38 inhibitors SB202190 and SB203580 reduced nucleotide-induced blebbing and actin reorganization, whereas the MAPK kinase-1/2 inhibitor U0126, which blocks ERK1/ERK2 activation, had no discernable effect. Third, the Rho-selective inhibitor C3 exoenzyme and the Rho effector kinase, Rho-associated coiled-coil kinase, inhibitor Y-27632, markedly attenuated BzATP-stimulated actin reorganization and membrane blebbing. These data support a model wherein p38- and Rho-dependent pathways are critical for P2X7-dependent actin reorganization and membrane blebbing, thereby facilitating P2X7 involvement in macrophage inflammatory responses.

P2X7 receptors regulate multiple types of membrane trafficking responses and non-classical secretion pathways

“…Disruption of actin filaments by cytochalasin D, which inhibits actin polymerization and depolymerization, diminished the P2X7R-induced blebbing without affecting the receptor's ionotropic function. Activation of Rho and p38 MAPKs occurred concurrently with P2X7R-induced membrane blebbing [46]. Likewise, pharmacological inhibitors of p38, Rho, and Rho kinases all reduced P2X7R-stimulated actin reorganization as well as the blebbing [44,46,47].…”

“…Studies with different cell types have indicated that P2X7R-stimulated membrane blebbing appears to involve several common signaling pathways [44,45]. ATP-induced membrane blebbing was found to be concurrent with actin reorganization in murine macrophages [46] and could be dissociated from the non-classical release of mature IL-1β that is also stimulated in parallel by P2X7R in such macrophages [47]. Disruption of actin filaments by cytochalasin D, which inhibits actin polymerization and depolymerization, diminished the P2X7R-induced blebbing without affecting the receptor's ionotropic function.…”

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.…”

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.…”