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TNF- induces a decrease in eNOS promoter activity

²Research Service, Upstate New York Veterans Affairs Healthcare Stratton Medical Center, and ¹Center for Cardiovascular Science, Albany Medical College, Albany, New York 12208

Submitted 5 November 2002 ; accepted in final form 8 October 2003

We determined whether TNF- 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- (50 ng/ml) for 0.5 or 4 h. TNF- 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 ^-370CACCC, ^-231GATA, and ^-186CACCC may regulate the effect of TNF- on the eNOS promoter. DNA-binding activity of ³²P-labeled oligonucleotide probes that span the GATA-binding site (^-239-[^-231GATA]-^-219) and the two different CACCC-binding regions (^-379-[^-370CACCC]-^-358 and ^-196-[^-186 CACCC]-^-176) were assessed using EMSA. In response to TNF- treatment for 4 h, nuclear protein binding to ³²P oligonucleotides was characterized as: 1) a significant increase in binding to ^-370CACCC, 2) a significant decrease in binding to ^-231GATA, and 3) no change in ^-186CACCC binding. EMSA supershift analysis indicated that the transcription factor protein GATA-4 bound to the ^-231GATA site, and Sp3 bound to the ^-370CACCC site. Our data indicate TNF causes a decrease in eNOS promoter activity that may be mediated by GATA-4 and Sp3.

decoy; edema; messenger ribonucleic acid; permeability; transcription; tumor necrosis factor-; endothelial nitric oxide synthase

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