ENHANCEMENT OF SUPEROXIDE PRODUCTION AND PROTECTION AGAINST HEAT-SHOCK BY HSP27 IN FIBROBLASTS

Extracellular superoxide production in the Chinese hamster CCL39 fibro blast cell line was detected by direct counting of luminol-enhanced ch emiluminescence in the presence of horseradish peroxidase (HRP). This superoxide production, which is directly related to cell density, can be inhibited by diphenylene iodonium, a specific inhibitor of NADPH-ox idase. The O-2(-) generation is transiently inhibited following exposu re of the cells to elevated temperatures. To study the possible relati onship between HSP27 and its modulation of NADPH-oxidase activity we m easured the generation of O-2(-) activity in CCL39 cells containing ei ther the gene coding for human HSP27 or a mutant form of this gene lea ding to HSP27 unable to be phosphorylated. Cells with the enhanced exp ression of normal human HSP27 as well as the non-phosphorylatable prot ein exhibited a 20-fold higher superoxide production than control CCL3 9 cells. Furthermore, we demonstrate that accumulation of normal HSP27 appears to play a role in prevention of NADPH-oxidase inhibition by h eat. (C) 1996 Academic Press, Inc.