title: Impaired NADPH oxidase activity in Rac2-deficient murine neutrophils does not result from defective translocation of p47phox and p67phox and can be rescued by exogenous arachidonic acid.
abstract: Rac2 is a hematopoietic-specific Rho-GTPase that plays a stimulus-specific role in regulating reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activation and other functional responses in neutrophils. In this study, rac2-/- neutrophils were shown to have significantly decreased NADPH oxidase activity and actin remodeling in response to exogenous arachidonic acid (AA), as previously observed for phorbol 12-myristate 13-acetate (PMA) or formyl-Met-Leu-Phe (fMLP) as agonists. PMA-, fMLP-, or AA-induced translocation of p47phox and p67phox to the plasma membrane was not impaired in rac2-/- neutrophils. Combined stimulation of rac2-/- neutrophils with exogenous AA and PMA had a synergistic effect on NADPH oxidase activity, and superoxide production increased to a level that was at least as high as wild-type cells and had no effect on fMLP-elicited enzyme activity. Membrane translocation of p47phox and p67phox as well as Rac1 activation was not increased further by combined PMA and AA stimulation. Inhibitor studies were consistent with important roles for phorbol ester-activated protein kinase C (PKC) isoforms and an atypical isoform, PKCzeta, in superoxide production by wild-type and rac2-/- neutrophils stimulated with AA and PMA. In addition, PMA-stimulated release of AA and cytoplasmic phospholipase A2 expression in rac2-/- neutrophils were similar to wild-type, suggesting that deficient AA production by PMA-stimulated rac2-/- neutrophils does not explain the effect of exogenous AA on oxidase activity. Although not required for translocation of p47phox and p67phox, Rac2 is necessary for optimal activity of the assembled oxidase complex, an effect that can be replaced by exogenous AA, which may act directly or via an exogenous AA-induced mediator.
authors.affiliation: Herman B. Wells Center for Pediatric Research, Department of Pediatrics (Hematology/Oncology), James Whitcomb Riley Hospital for Children, Indiana University Medical Center, Indianapolis, IN, USA.
authors.forename: Chaekyun
authors.initials: C
authors.lastname: Kim
authors.forename: Mary C
authors.initials: MC
authors.lastname: Dinauer
mesh_terms: D000818:Animals; D016718:Arachidonic Acid / Q000276:immunology / Q000494:pharmacology*; D002273:Carcinogens / Q000494:pharmacology; D002462:Cell Membrane / Q000201:enzymology / Q000276:immunology; D002478:Cells, Cultured; D004789:Enzyme Activation / Q000187:drug effects / Q000276:immunology; D051379:Mice; D018345:Mice, Knockout; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D019255:NADPH Oxidases / Q000276:immunology / Q000378:metabolism*; D009479:Neuropeptides / Q000276:immunology / Q000378:metabolism; D009504:Neutrophils / Q000166:cytology / Q000201:enzymology* / Q000276:immunology; D010741:Phospholipases A / Q000276:immunology / Q000378:metabolism; D054467:Phospholipases A2; D010750:Phosphoproteins / Q000276:immunology / Q000378:metabolism*; D051744:Protein Kinase C-epsilon / Q000276:immunology / Q000378:metabolism; D021381:Protein Transport / Q000187:drug effects / Q000276:immunology; D013481:Superoxides / Q000276:immunology / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D020744:rac GTP-Binding Proteins / Q000172:deficiency / Q000276:immunology / Q000378:metabolism*; D020830:rac1 GTP-Binding Protein; D000097766:RAC2 GTP-Binding Protein
chemical_list: D016718:Arachidonic Acid; D002273:Carcinogens; D009240:N-Formylmethionine Leucyl-Phenylalanine; D019255:NADPH Oxidases; D009479:Neuropeptides; D010741:Phospholipases A; D054467:Phospholipases A2; D010750:Phosphoproteins; D051744:Protein Kinase C-epsilon; D013481:Superoxides; D013755:Tetradecanoylphorbol Acetate; D020744:rac GTP-Binding Proteins; D020830:rac1 GTP-Binding Protein; D000097766:RAC2 GTP-Binding Protein; C484602:Rac1 protein, mouse; C079789:neutrophil cytosol factor 67K; C060505:neutrophil cytosolic factor 1