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.