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.
Anderson
Hope D I
HD
Diabetes Center and Department of Medicine, University of California at San Francisco, San Francisco, California 94143-0540, USA.
Rahmutula
Dolkun
D
Gardner
David G
DG
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