Tumor Necrosis Factor-α Inhibits Endothelial Nitric-oxide Synthase Gene Promoter Activity in Bovine Aortic Endothelial Cells

Tumor necrosis factor-alpha (TNF-alpha) has been shown to reduce endothelial nitric-oxide synthase (eNOS) gene expression through post-transcriptional regulation of mRNA stability. The current study documented an independent effect of the cytokine on the eNOS gene promoter. TNF-alpha effected a time- and dose-dependent reduction in activity of a transiently transfected human -1197 eNOS-luciferase reporter. This reduction was inhibited by co-transfection of dominant negative IKKbeta as well as a nonphosphorylatable constitutively suppressive mutant of IkappaB implying involvement of the NFkappaB cascade in the inhibitory effect. The locus of the TNF-alpha-dependent inhibition was traced to two Sp1-binding sites positioned between -109 and -95 and -81 and -67 relative to the transcription start site. Electrophoretic mobility shift analysis and immunoperturbation studies showed evidence for Sp1 and Sp3 binding to each element. TNF-alpha treatment had no effect on the binding pattern to the downstream (-81 to -67) site but did suppress association of Sp1 and Sp3 to the upstream (-109 to -95) site. Collectively, these data indicate that TNF-alpha exerts transcriptional, as well as post-transcriptional, effects on eNOS gene expression and suggest a potential mechanism to account for the endothelial dysfunction that accompanies disorders such as diabetes mellitus and heart failure.