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TNF-alpha induces a decrease in eNOS promoter activity.

Paul Neumann·Nancy Gertzberg·Arnold Johnson
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Abstract

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.

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AnimalsAntineoplastic AgentsCattleCell CountCell SurvivalCells, CulturedColoring AgentsDNA-Binding ProteinsEndothelium, VascularGATA4 Transcription FactorGene Expression Regulation, EnzymologicGenetic Complementation TestHumansLungNitric Oxide SynthaseNitric Oxide Synthase Type IIINuclear ProteinsPromoter Regions, GeneticSp3 Transcription FactorTranscription FactorsTrypan BlueTumor Necrosis Factor-alphaJournal ArticleResearch Support, Non-U.S. Gov'tResearch Support, U.S. Gov't, Non-P.H.S.Research Support, U.S. Gov't, P.H.S.

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Paul Neumann, Nancy Gertzberg, Arnold Johnson. 2003-10-10. TNF-alpha induces a decrease in eNOS promoter activity.. https://doi.org/10.1152/ajplung.00378.2002

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