title: 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.
authors.affiliation: Department of Biomecular Chemistry, University of Wisconsin Medical School, 1300 University Avenue, Madison, WI 53706-0450, USA.
authors.forename: Zachary A
authors.initials: ZA
authors.lastname: Pfeiffer
authors.forename: Mini
authors.initials: M
authors.lastname: Aga
authors.forename: Usha
authors.initials: U
authors.lastname: Prabhu
authors.forename: Jyoti J
authors.initials: JJ
authors.lastname: Watters
authors.forename: David J
authors.initials: DJ
authors.lastname: Hall
authors.forename: Paul J
authors.initials: PJ
authors.lastname: Bertics
mesh_terms: D008841:Actin Cytoskeleton / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000494:pharmacology; D000577:Amides / Q000494:pharmacology; D000818:Animals; D002462:Cell Membrane / Q000378:metabolism*; D015638:Cytochalasin D / Q000494:pharmacology; D003599:Cytoskeleton / Q000378:metabolism; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D047908:Intracellular Signaling Peptides and Proteins; D008264:Macrophages / Q000166:cytology / Q000187:drug effects / Q000201:enzymology*; D051379:Mice; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D019384:Nucleic Acid Synthesis Inhibitors / Q000494:pharmacology; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D010975:Platelet Aggregation Inhibitors / Q000494:pharmacology; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D011725:Pyridines / Q000494:pharmacology; D018048:Receptors, Purinergic P2 / Q000378:metabolism*; D058486:Receptors, Purinergic P2X7; D048051:p38 Mitogen-Activated Protein Kinases; D054460:rho-Associated Kinases
chemical_list: D000255:Adenosine Triphosphate; D000577:Amides; D015638:Cytochalasin D; D004791:Enzyme Inhibitors; D047908:Intracellular Signaling Peptides and Proteins; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D019384:Nucleic Acid Synthesis Inhibitors; D019869:Phosphatidylinositol 3-Kinases; D010975:Platelet Aggregation Inhibitors; D017346:Protein Serine-Threonine Kinases; D011725:Pyridines; D018048:Receptors, Purinergic P2; D058486:Receptors, Purinergic P2X7; D048051:p38 Mitogen-Activated Protein Kinases; D054460:rho-Associated Kinases; C546469:P2rx7 protein, mouse; C108830:Y 27632; C033901:3'-O-(4-benzoyl)benzoyladenosine 5'-triphosphate