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

Abstract Recent evidence suggests that a number of non-phagocytic cell types may contain a superoxide generating NADPH oxidase. Studies to date on cultured human fibroblasts have primarily concerned the identification of cytochrome b 558 , whilst expression of other NADPH oxidase components have not been addressed. In this study we have investigated the expression of NADPH oxidase with particular reference to the cytosolic factors p47- phox and p67- phox . Reverse transcriptase-polymerase chain reaction (RT-PCR) showed that human fibroblasts express mRNA for p47- phox , p67- phox and p22- phox . Expression of the gp91- phox transcript was not detected, indicating that human fibroblasts may possess an NADPH oxidase isoenzyme. Western blot analysis of human fibroblast cytosol, using an anti-p47- phox antibody (JW-1), identified a 47 kDa protein. Cell-free reconstitution assays showed that fibroblast cytosol could initiate superoxide generation when mixed with either human fibroblast membranes (0.16 nmol superoxide/min/μg membrane protein), or resting human neutrophil membranes (0.20 nmol superoxide/min/μg membrane protein). These data indicate that the expression of p47- phox and p67- phox by human fibroblasts may contribute to the cells' generation of superoxide.