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-induced MCP-1 gene expression in endothelial cells: role of Rac1 and NADPH oxidase
1Discovery Research, AtheroGenics Incorporated, Alpharetta 30004; and 2Division of Cardiology, Emory University School of Medicine, Atlanta, Georgia 30322
Submitted 24 July 2003 ; accepted in final form 16 October 2003
Reactive oxygen species (ROS) play an important but not yet fully defined role in the expression of inflammatory genes such as monocyte chemoattractant protein (MCP)-1. We used complementary molecular and biochemical approaches to explore the roles of specific ROS and their molecular linkage to inflammatory signaling in endothelial cells. Adenovirus-mediated expression of superoxide dismutase and catalase inhibited TNF-
-induced MCP-1 gene expression, suggesting important roles of superoxide (
) and H2O2 in MCP-1 gene activation. In addition, the iron chelator 1,2-dimethyl-3-hydroxypyridin-4-one and the hydroxyl radical scavengers dimethylthiourea and dimethyl sulfoxide inhibited TNF--induced MCP-1 expression, suggesting important roles of iron and hydroxyl radicals in inflammatory signal activation. In contrast, scavenging of peroxynitrite with 5,10,15,20-tetrakis-(4-sulfonatophenyl)prophyrinato iron (III) chloride had no effect on TNF--induced MCP-1 expression. Inhibition of NADPH oxidase, the major oxidase responsible for generation, with diphenylene iodonium suppressed TNF--induced MCP-1 mRNA accumulation. Rac1 is an upstream signaling molecule for the activation of NADPH oxidase and generation. Expression of dominant negative N17Rac1 by adenovirus suppressed TNF--induced MCP-1 mRNA levels and MCP-1 protein secretion. Expression of N17Rac1 inhibited TNF--induced MCP-1 and NF-
B transcriptional activity. These data suggest that ROS such as superoxide and H2O2 derived from Rac1-activated NADPH oxidase mediate TNF--induced MCP-1 expression in endothelial cells.
monocyte chemoattractant protein-1; tumor necrosis factor-; reactive oxygen species; endothelial cells
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