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

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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.

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