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, gp91phox, p22phox, p47phox, and p67phox. 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 p47phox, p67phox, and Rac1. Expression of p47phox Ser303Ala/Ser304Ala or Ser379Ala phosphorylation-deficient mutants resulted in significantly impaired NAPDH oxidase activity, compared with expression of wild-type p47phox or the p47phox Ser303Glu/Ser304Glu phosphorylation mimic, suggesting that p47phoxphosphorylation 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 p67phox, 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 p67phox(Val204Ala) mutant.

Reconstitution of Chemotactic Peptide-Induced Nicotinamide Adenine Dinucleotide Phosphate (Reduced) Oxidase Activation in Transgenic COS-phox Cells

“…PKC␦ is a novel PKC isoform and is activated in a Ca 2ϩ -insensitive and diacyl glycerol-dependent manner. PKC␦ and PKC␤II are the two most abundant PKC isoforms in neutrophils, whereas COS-7 cells express only a low level of PKC␦ (17). Like several other PKC isoforms, PKC␦ responds to PMA stimulation with binding to p47 phox (36) and translocation to the cytoskeletal fraction (29).…”

“…COS-phox cells respond to PMA and arachidonic acid with potent O 2 . production (17), and our initial reconstitution of fMLP-induced NADPH oxidase activity was achieved without p40 phox (Fig. 2).…”

“…compared with neutrophils. More recently, one of our laboratories generated a stable COS-7 line expressing gp91 phox , p22 phox , p67 phox , and p47 phox (the COS-phox cell line) (17). Stimulation of these cells with PMA and arachidonic acid led to potent production of O 2 .…”

“…The transgenic COS-phox cells were generated as described previously (17). The stable cell line was maintained at 37°C with 5% CO 2 in DMEM supplemented with 10% heat-inactivated FBS, 2 mM L-glutamine, 100 IU/ml penicillin, and 50 g/ml streptomycin.…”

“…The COS-phox cell line was generated by stable expression of gp91 phox , p22 phox , p67 phox , and p47 phox in the monkey epithelial cell line COS-7 (17). Expression of these components enables the epithelial cells to respond to arachidonic acid and PMA with potent O 2 .…”