TNF-α induces a decrease in eNOS promoter activity
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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TNFα downregulates NOS3 expression and abundance by decreasing the stability of NOS3 mRNA, effectively shortening its half-life.
“was shown to downregulate NOS3 expression and abundance [123, 131-134] by decreasing the stability of NOS3 mRNA [135, 136], effectively shortening its half-life [137].”
From Regulation of obesity and insulin resistance by nitric oxide (2014) · cites this paper as [133]
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“In multiple studies, tumor necrosis factor-α (TNFα), which is increased in obesity and implicated in the etiology of insulin resistance [130], was shown to downregulate NOS3 expression and abundance [123,131–134] by decreasing the stability of NOS3 mRNA [135,136], effectively shortening its half-life [137].”
Background
- Authors
- Neumann, P., Gertzberg, N., & Johnson, A.
- Affiliations
- Albany Medical College (US)
- Albany Stratton VA Medical Center Albany (US)
- Venue
- American Journal of Physiology-Lung Cellular and Molecular Physiology
- Publisher
- American Physiological Society
- Published
- 2004-02
- Volume
- 286
- Issue
- 2
- Pages
- L452-L459
- ISSN
- 1040-0605 (ISSN-L)
- 1522-1504
- OpenAlex
- W2078718611
- Cited by
- 118
- References
- 41
- Citations by year
- 2026: 5 · 2025: 7 · 2024: 12 · 2023: 10 · 2022: 11 · 2021: 8 · 2020: 3 · 2019: 4 · 2018: 3 · 2017: 10 · 2016: 3 · 2015: 6 · 2014: 3 · 2013: 2 · 2012: 2
- FWCI
- 2.67
- Type
- journal article
- Language
- en
- Open access
- closed
- Locations
- American Journal of Physiology-Lung Cellular and Molecular Physiology (publishedVersion)
- PubMed (publishedVersion)
- Funders
- U.S. Department of Veterans Affairs
- National Heart, Lung, and Blood Institute (R01HL-59901, R01HL059901)
- Topics
- Health Sciences › Medicine › Physiology › Nitric Oxide and Endothelin Effects
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Angiogenesis and VEGF in Cancer
- Health Sciences › Medicine › Immunology and Allergy › Cell Adhesion Molecules Research
- Keywords
- TNF-α, endothelial nitric oxide synthase, promoter regulation, pulmonary microvascular endothelial cells, GATA4, Sp3, GATA binding sites, transcription factor binding, lung endothelium
- MeSH terms
- Animals, Antineoplastic Agents, Cattle, Cell Count, Cell Survival, Cells, Cultured, DNA-Binding Proteins, Coloring Agents, Endothelium, Vascular, Genetic Complementation Test, Humans, Lung, Nuclear Proteins, Promoter Regions, Genetic, Transcription Factors, Trypan Blue, Tumor Necrosis Factor-alpha, Gene Expression Regulation, Enzymologic, Nitric Oxide Synthase, GATA4 Transcription Factor, Sp3 Transcription Factor, Nitric Oxide Synthase Type III
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