Enhancement of superoxide production and protection against heat shock by HSP27 in fibroblasts

Extracellular superoxide production in the Chinese hamster CCL39 fibroblast cell line was detected by direct counting of luminol-enhanced chemiluminescence 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-oxidase. The O-2(-) generation is transiently inhibited following exposure of the cells to elevated temperatures. To study the possible relationship between HSP27 and its modulation of NADPH-oxidase activity we measured the generation of O-2(-) activity in CCL39 cells containing either the gene coding for human HSP27 or a mutant form of this gene leading to HSP27 unable to be phosphorylated. Cells with the enhanced expression of normal human HSP27 as well as the non-phosphorylatable protein exhibited a 20-fold higher superoxide production than control CCL39 cells. Furthermore, we demonstrate that accumulation of normal HSP27 appears to play a role in prevention of NADPH-oxidase inhibition by heat. (C) 1996 Academic Press, Inc.