Tumor necrosis factor-alpha reduces argininosuccinate synthase expression and nitric oxide production in aortic endothelial cells.
Endothelial dysfunction associated with elevated serum levels of TNF-alpha observed in diabetes, obesity, and congenital heart disease results, in part, from the impaired production of endothelial nitric oxide (NO). Cellular NO production depends absolutely on the availability of arginine, substrate of endothelial nitric oxide synthase (eNOS). In this report, evidence is provided demonstrating that treatment with TNF-alpha (10 ng/ml) suppresses not only eNOS expression but also the availability of arginine via the coordinate suppression of argininosuccinate synthase (AS) expression in aortic endothelial cells. Western blot and real-time RT-PCR demonstrated a significant and dose-dependent reduction of AS protein and mRNA when treated with TNF-alpha with a corresponding decrease in NO production. Reporter gene analysis demonstrated that TNF-alpha suppresses the AS proximal promoter, and EMSA analysis showed reduced binding to three essential Sp1 elements. Inhibitor studies suggested that the repression of AS expression by TNF-alpha may be mediated, in part, via the NF-kappaB signaling pathway. These findings demonstrate that TNF-alpha coordinately downregulates eNOS and AS expression, resulting in a severely impaired citrulline-NO cycle. The downregulation of AS by TNF-alpha is an added insult to endothelial function because of its important role in NO production and in endothelial viability.
Goodwin
Bonnie L
BL
Department of Molecular Medicine, University of South Florida College of Medicine, MDC Box 7, 12901 Bruce B. Downs Blvd., Tampa, FL 33612, USA.
Pendleton
Laura C
LC
Levy
Monique M
MM
Solomonson
Larry P
LP
Eichler
Duane C
DC
D000818:Animals; D001011:Aorta / Q000166:cytology / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D001120:Arginine / Q000378:metabolism*; D001124:Argininosuccinate Synthase / Q000096:biosynthesis* / Q000235:genetics; D002417:Cattle; D002467:Cell Nucleus / Q000378:metabolism; D002478:Cells, Cultured; D002956:Citrulline / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D015536:Down-Regulation; D042783:Endothelial Cells / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D004794:Enzyme Repression; D017930:Genes, Reporter; D008156:Luciferases; D016328:NF-kappa B / Q000378:metabolism; D009569:Nitric Oxide / Q000378:metabolism*; D052250:Nitric Oxide Synthase Type III / Q000096:biosynthesis* / Q000235:genetics; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D016329:Sp1 Transcription Factor / Q000378:metabolism; D051705:Sp3 Transcription Factor / Q000378:metabolism; D014158:Transcription, Genetic; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism* / Q000494:pharmacology
D016328:NF-kappa B; D012333:RNA, Messenger; D016329:Sp1 Transcription Factor; D014409:Tumor Necrosis Factor-alpha; D051705:Sp3 Transcription Factor; D002956:Citrulline; D009569:Nitric Oxide; D001120:Arginine; D008156:Luciferases; D052250:Nitric Oxide Synthase Type III; D001124:Argininosuccinate Synthase