TNF-alpha induces a decrease in eNOS promoter activity.
We determined whether TNF-alpha induces a decrease in activity of the promoter for the endothelial nitric oxide synthase (eNOS) gene in pulmonary microvessel endothelial monolayers (PMEM). eNOS promoter activity was assessed in PMEM transfected with plasmids coding the wild-type (F1: -1600 nt from transcription start site) and truncated (F2: -1189, F4: -779, F5: -494, F6: -166) human eNOS promoters linked to a luciferase reporter. PMEM lysates were analyzed for the luciferase/galactosidase ratio (Luc/Gal) after incubation with TNF-alpha (50 ng/ml) for 0.5 or 4 h. TNF-alpha caused a decrease in the Luc/Gal ratio in the PMEM transfected with wild-type F1 and truncated F2, F4, and F5 plasmids but not with truncated F6 plasmid. Truncated-promoter analysis indicated the response elements (-370)CACCC, (-231)GATA, and (-186)CACCC may regulate the effect of TNF-alpha on the eNOS promoter. DNA-binding activity of (32)P-labeled oligonucleotide probes that span the GATA-binding site ((-239)-[(-231)GATA]-(-219)) and the two different CACCC-binding regions ((-379)-[(-370)CACCC]-(-358) and (-196)-[(-186) CACCC]-(-176)) were assessed using EMSA. In response to TNF-alpha treatment for 4 h, nuclear protein binding to (32)P oligonucleotides was characterized as: 1) a significant increase in binding to (-370)CACCC, 2) a significant decrease in binding to (-231)GATA, and 3) no change in (-186)CACCC binding. EMSA supershift analysis indicated that the transcription factor protein GATA-4 bound to the (-231)GATA site, and Sp3 bound to the (-370)CACCC site. Our data indicate TNF causes a decrease in eNOS promoter activity that may be mediated by GATA-4 and Sp3.
Neumann
Paul
P
Center for Cardiovascular Science, Albany Medical College, Albany, NY 12208, USA.
Gertzberg
Nancy
N
Johnson
Arnold
A
D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology*; D002417:Cattle; D002452:Cell Count; D002470:Cell Survival; D002478:Cells, Cultured; D004396:Coloring Agents; D004268:DNA-Binding Proteins / Q000378:metabolism; D004730:Endothelium, Vascular / Q000166:cytology*; D050991:GATA4 Transcription Factor; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects / Q000502:physiology; D005816:Genetic Complementation Test; D006801:Humans; D008168:Lung / Q000098:blood supply*; D019001:Nitric Oxide Synthase / Q000235:genetics*; D052250:Nitric Oxide Synthase Type III; D009687:Nuclear Proteins / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000502:physiology; D051705:Sp3 Transcription Factor; D014157:Transcription Factors / Q000378:metabolism; D014343:Trypan Blue; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*
D000970:Antineoplastic Agents; D004396:Coloring Agents; D004268:DNA-Binding Proteins; D050991:GATA4 Transcription Factor; D009687:Nuclear Proteins; C497121:SP3 protein, human; D014157:Transcription Factors; D014409:Tumor Necrosis Factor-alpha; D051705:Sp3 Transcription Factor; C496314:NOS3 protein, human; D019001:Nitric Oxide Synthase; D052250:Nitric Oxide Synthase Type III; D014343:Trypan Blue