THE FUNCTIONAL EXPRESSION OF P47-PHOX AND P67-PHOX MAY CONTRIBUTE TO THE GENERATION OF SUPEROXIDE BY AN NADPH OXIDASE-LIKE SYSTEM IN HUMAN FIBROBLASTS

Recent evidence suggests that a number of non-phagocytic cell: types m ay contain a superoxide generating NADPH oxidase. Studies to date on c ultured human fibroblasts have primarily concerned the identification of cytochrome b(558) whilst expression of other NADPH oxidase componen ts have not been addressed. In this study we have investigated the exp ression of NADPH oxidase with particular reference to the cytosolic fa ctors p47-phox and p67-phox. Reverse transcriptase-polymerase chain re action (RT-PCR) showed that human fibroblasts express mRNA for p47-pho x, p67-phox and p22-phox. Expression of the gp91-phox transcript was n ot detected, indicating that human fibroblasts may possess an NADPH ox idase isoenzyme. Western blot analysis of human fibroblast cytosol, us ing an anti-p47-pkox antibody (JW-1), identified a 47 kDa protein. Cel l-free reconstitution assays showed that fibroblast cytosol could init iate superoxide generation when mixed with either human fibroblast mem branes (0.16 nmol superoxide/min/mu/g membrane protein), or resting hu man neutrophil membranes (0.20 nmol superoxide/min/mu g membrane prote in). These data indicate that the expression of p47-phox and p67-phox by human fibroblasts may contribute to the cells' generation of supero xide.