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-induces a decrease in eNOS-promoter activity
1 The Center for Cardiovascular Science, Albany Medical College, Albany, NY, USA
2 Research Service, Upstate New York VA Healthcare Stratton Medical Center, Albany, NY, USA; The Center for Cardiovascular Science, Albany Medical College, Albany, NY, USA
* To whom correspondence should be addressed. E-mail: jmurd{at}msn.com.
We determined if TNF
-induces a decrease in activity of the promoter for the endothelial cell 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) following incubation with TNF
(50 ng/ml) for 0.5 hr or 4.0 hr. TNF caused a decrease in the Luc/Gal ratio in the PMEM transfected with the wild type-F1 and truncated-F2, -F4 and -F5 plasmids, but not with the 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 -[-186CACCC]- -176) were assessed using the electrophoretic mobility shift assay (EMSA). In response to TNF
treatment for 4.0 hr, nuclear protein binding to 32P-oligonucleotides was characterized as: (i) a significant increase in binding to -370CACCC, (ii) a significant decrease in binding to
-231GATA, and (iii) 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 indicates TNF causes a decrease in eNOS-promoter activity that may be mediated by GATA-4 and Sp3.
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