Tumor necrosis factor- reduces argininosuccinate synthase expression and nitric oxide production in aortic endothelial cells

Goodwin BL, Pendleton LC, Levy MM, Solomonson LP, Eichler DC. Tumor necrosis factorreduces argininosuccinate synthase expression and nitric oxide production in aortic endothelial cells. Am J Physiol Heart Circ Physiol 293: H1115–H1121, 2007. First published May 11, 2007; doi:10.1152/ajpheart.01100.2006.—Endothelial dysfunction associated with elevated serum levels of TNFobserved 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(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 TNFwith a corresponding decrease in NO production. Reporter gene analysis demonstrated that TNFsuppresses 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 TNFmay be mediated, in part, via the NFB signaling pathway. These findings demonstrate that TNFcoordinately downregulates eNOS and AS expression, resulting in a severely impaired citrulline-NO cycle. The downregulation of AS by TNFis an added insult to endothelial function because of its important role in NO production and in endothelial viability.