Superoxide, H2O2, and iron are required for TNF-alpha-induced MCP-1 gene expression in endothelial cells: role of Rac1 and NADPH oxidase.
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-alpha-induced MCP-1 gene expression, suggesting important roles of superoxide (O(2)(-).) and H(2)O(2) 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-alpha-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-alpha-induced MCP-1 expression. Inhibition of NADPH oxidase, the major oxidase responsible for O(2)(-). generation, with diphenylene iodonium suppressed TNF-alpha-induced MCP-1 mRNA accumulation. Rac1 is an upstream signaling molecule for the activation of NADPH oxidase and O(2)(-). generation. Expression of dominant negative N17Rac1 by adenovirus suppressed TNF-alpha-induced MCP-1 mRNA levels and MCP-1 protein secretion. Expression of N17Rac1 inhibited TNF-alpha-induced MCP-1 and NF-kappaB transcriptional activity. These data suggest that ROS such as superoxide and H(2)O(2) derived from Rac1-activated NADPH oxidase mediate TNF-alpha-induced MCP-1 expression in endothelial cells.
Athero-Genics, Inc., 8995 Westside Parkway, Alpharetta, GA 30004, USA. xchen@atherogenics.com
Xi-Lin
XL
Chen
Qiang
Q
Zhang
Ruozhi
R
Zhao
Russell M
RM
Medford
D000970:Antineoplastic Agents / Q000494:pharmacology; D001011:Aorta / Q000166:cytology; D002374:Catalase / Q000378:metabolism; D002478:Cells, Cultured; D018932:Chemokine CCL2 / Q000235:genetics*; D000077543:Deferiprone; D004121:Dimethyl Sulfoxide / Q000494:pharmacology; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism*; D016166:Free Radical Scavengers / Q000494:pharmacology; D015870:Gene Expression / Q000187:drug effects; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism; D006878:Hydroxides / Q000378:metabolism; D007501:Iron / Q000378:metabolism*; D007502:Iron Chelating Agents / Q000494:pharmacology; D008665:Metalloporphyrins / Q000494:pharmacology; D019255:NADPH Oxidases / Q000378:metabolism*; D016328:NF-kappa B / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011728:Pyridones / Q000494:pharmacology; D012333:RNA, Messenger / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology; D013482:Superoxide Dismutase / Q000235:genetics; D013481:Superoxides / Q000378:metabolism; D013890:Thiourea / Q000031:analogs & derivatives* / Q000494:pharmacology; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology; D020830:rac1 GTP-Binding Protein / Q000235:genetics / Q000378:metabolism*
C466420:5,10,15,20-tetrakis(4-sulfonatophenyl)porphyrinato iron(III) chloride; D000970:Antineoplastic Agents; D018932:Chemokine CCL2; D016166:Free Radical Scavengers; D006878:Hydroxides; D007502:Iron Chelating Agents; D008665:Metalloporphyrins; D016328:NF-kappa B; D011728:Pyridones; D012333:RNA, Messenger; D014409:Tumor Necrosis Factor-alpha; D013481:Superoxides; D000077543:Deferiprone; C038983:1,3-dimethylthiourea; C031356:hydroxide ion; D006861:Hydrogen Peroxide; D007501:Iron; D002374:Catalase; D013482:Superoxide Dismutase; D019255:NADPH Oxidases; D020830:rac1 GTP-Binding Protein; D013890:Thiourea; D004121:Dimethyl Sulfoxide