title: Creation of a genetic system for analysis of the phagocyte respiratory burst: high-level reconstitution of the NADPH oxidase in a nonhematopoietic system.
abstract: The phagocyte nicotinamide adenine dinucleotide phosphate (reduced form) (NADPH) oxidase was functionally reconstituted in monkey kidney COS-7 cells by transfection of essential subunits, gp91(phox), p22(phox), p47(phox), and p67(phox). COS-7 cells express the essential small guanosine 5'-triphosphatase, Rac1. Transgenic COS-phox cells were capable of arachidonic acid-induced NADPH oxidase activity up to 80% of that of human neutrophils, and of phorbol myristate acetate (PMA)-induced activity up to 20% of that of neutrophils. Expression of all 4 phox components was required for enzyme activity, and enzyme activation was associated with membrane translocation of p47(phox), p67(phox), and Rac1. Expression of p47(phox) Ser303Ala/Ser304Ala or Ser379Ala phosphorylation-deficient mutants resulted in significantly impaired NAPDH oxidase activity, compared with expression of wild-type p47(phox) or the p47(phox) Ser303Glu/Ser304Glu phosphorylation mimic, suggesting that p47(phox) phosphorylation contributes to enzyme activity in the COS system, as is the case in neutrophils. Hence, COS-phox cells should be useful as a new whole-cell model that is both capable of high-level superoxide production and readily amenable to genetic manipulation for investigation of NADPH oxidase function. PMA-elicited superoxide production in COS-phox cells was regulated by activation of protein kinase C (PKC) and Rac. Although COS-7 cells differ from human neutrophils in PKC isoform expression, transient expression of major neutrophil isoforms in COS-phox cells did not increase PMA-induced superoxide production, suggesting that endogenous isoforms were not rate limiting. Val204 in p67(phox), previously shown to be required for NADPH oxidase activity under cell-free conditions, was found to be essential for superoxide production by intact COS-phox cells, on the basis of transfection studies using a p67(phox) (Val204Ala) mutant.
authors.affiliation: Herman B Wells Center for Pediatric Research, Department of Pediatrics, James Whitcomb Riley Hospital for Children, Indiana University Medical Center, Indianapolis 46202, USA.
authors.forename: Marianne O
authors.initials: MO
authors.lastname: Price
authors.forename: Linda C
authors.initials: LC
authors.lastname: McPhail
authors.forename: J David
authors.initials: JD
authors.lastname: Lambeth
authors.forename: Chang-Hoon
authors.initials: CH
authors.lastname: Han
authors.forename: Ulla G
authors.initials: UG
authors.lastname: Knaus
authors.forename: Mary C
authors.initials: MC
authors.lastname: Dinauer
mesh_terms: D000818:Animals; D019556:COS Cells; D006801:Humans; D019255:NADPH Oxidases / Q000235:genetics* / Q000378:metabolism / Q000502:physiology; D009504:Neutrophils / Q000201:enzymology / Q000378:metabolism; D030781:Organisms, Genetically Modified / Q000378:metabolism*; D010586:Phagocytes / Q000201:enzymology* / Q000378:metabolism; D010750:Phosphoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D011493:Protein Kinase C / Q000502:physiology; D021122:Protein Subunits; D016897:Respiratory Burst / Q000187:drug effects; D013481:Superoxides / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014162:Transfection; D020830:rac1 GTP-Binding Protein / Q000502:physiology
chemical_list: D010750:Phosphoproteins; D021122:Protein Subunits; C079789:neutrophil cytosol factor 67K; D013481:Superoxides; D019255:NADPH Oxidases; C060505:neutrophil cytosolic factor 1; D011493:Protein Kinase C; D020830:rac1 GTP-Binding Protein; D013755:Tetradecanoylphorbol Acetate