Tumor necrosis factor-alpha 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.
Diabetes Center and Department of Medicine, University of California at San Francisco, San Francisco, California 94143-0540, USA.
Hope D I
HD
Anderson
Dolkun
D
Rahmutula
David G
DG
Gardner
D000818:Animals; D001011:Aorta / Q000166:cytology*; D015153:Blotting, Western; D002417:Cattle; D002467:Cell Nucleus / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D042783:Endothelial Cells / Q000166:cytology; D004730:Endothelium, Vascular / Q000166:cytology*; D005799:Genes, Dominant; D017930:Genes, Reporter; D006801:Humans; D051550:I-kappa B Kinase; D020672:I-kappa B Proteins / Q000378:metabolism; D008156:Luciferases / Q000378:metabolism; D009154:Mutation; D000072000:NF-KappaB Inhibitor alpha; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors* / Q000235:genetics*; D052250:Nitric Oxide Synthase Type III; D010766:Phosphorylation; D010957:Plasmids / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D012323:RNA Processing, Post-Transcriptional; D012333:RNA, Messenger / Q000378:metabolism; D016329:Sp1 Transcription Factor / Q000378:metabolism; D051705:Sp3 Transcription Factor; D013997:Time Factors; D014157:Transcription Factors / Q000378:metabolism; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism / Q000502:physiology*
D004268:DNA-Binding Proteins; D020672:I-kappa B Proteins; C000606204:NFKBIA protein, human; D012333:RNA, Messenger; C497121:SP3 protein, human; D016329:Sp1 Transcription Factor; D014157:Transcription Factors; D014409:Tumor Necrosis Factor-alpha; D000072000:NF-KappaB Inhibitor alpha; D051705:Sp3 Transcription Factor; D008156:Luciferases; C496314:NOS3 protein, human; D019001:Nitric Oxide Synthase; D052250:Nitric Oxide Synthase Type III; D017346:Protein Serine-Threonine Kinases; C496559:CHUK protein, human; D051550:I-kappa B Kinase; C496560:IKBKB protein, human; C496565:IKBKE protein, human