Abstract | PURPOSE: METHODS: RF/6A cells were subjected to hypoxia for 1 and 3 hours, at which times RNA and proteins were isolated. Potential AP-1, hypoxia-inducible factor (HIF)-1 and NF-kappaB DNA-binding sites were identified using DNA sequence search algorithms and were analyzed using gel-shift assay. COX-2 and VEGF RNA, protein, and PGE(2) levels were quantified by RT-PCR, Western analysis, and enzyme immunoassay, respectively. Tubular morphogenesis was analyzed with phase-contrast imaging microscopy. RESULTS: Nuclear AP-1, HIF-1 and NF-kappaB promoter DNA binding increased 1.5-, 4-, and 3-fold, respectively, after 1 hour of hypoxia. COX-2 RNA was elevated five- and fourfold after 1 and 3 hours of hypoxia, respectively. VEGF RNA and protein abundance lagged behind COX-2 induction but were each increased two- to threefold 3 hours after hypoxia. CGP43182 was found to inhibit NF-kappaB DNA binding, COX-2 and VEGF gene expression, PGE(2) release, and hypoxia-induced tubular morphogenesis. CONCLUSIONS: Maximum HIF-1 and NF-kappaB DNA binding immediately before COX-2 expression suggests that these TFs are important regulators of COX-2 induction in hypoxic RF/6A cells. IL-1beta emulated AP-1, HIF-1, and NF-kappaB DNA binding during hypoxia and may be a novel cytokine trigger for NV. CGP43182 appears to be an effective inhibitor of NV. VEGF expression appears to be regulated through dual interdependent mechanisms involving HIF-1 directly and indirectly through NF-kappaB-mediated COX-2 expression and PGE(2) production.
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Authors | Walter J Lukiw, Anna Ottlecz, George Lambrou, Monika Grueninger, Joelle Finley, Hilary W Thompson, Nicolas G Bazan |
Journal | Investigative ophthalmology & visual science
(Invest Ophthalmol Vis Sci)
Vol. 44
Issue 10
Pg. 4163-70
(Oct 2003)
ISSN: 0146-0404 [Print] United States |
PMID | 14507857
(Publication Type: Journal Article, Research Support, Non-U.S. Gov't, Research Support, U.S. Gov't, P.H.S.)
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Chemical References |
- Chlorobenzenes
- DNA-Binding Proteins
- Endothelial Growth Factors
- Hypoxia-Inducible Factor 1
- Intercellular Signaling Peptides and Proteins
- Isoenzymes
- Lymphokines
- NF-kappa B
- Nuclear Proteins
- RNA, Messenger
- Transcription Factor AP-1
- Transcription Factors
- Vascular Endothelial Growth Factor A
- Vascular Endothelial Growth Factors
- CGP 43182
- Cyclooxygenase 2
- Prostaglandin-Endoperoxide Synthases
- Dinoprostone
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Topics |
- Animals
- Cell Line
- Chlorobenzenes
(pharmacology)
- Cyclooxygenase 2
- DNA-Binding Proteins
(metabolism)
- Dinoprostone
(metabolism)
- Electrophoretic Mobility Shift Assay
- Endothelial Growth Factors
(biosynthesis, genetics)
- Endothelium, Vascular
(metabolism)
- Gene Expression
(drug effects)
- Hypoxia
(metabolism)
- Hypoxia-Inducible Factor 1
- Intercellular Signaling Peptides and Proteins
(biosynthesis, genetics)
- Isoenzymes
(biosynthesis, genetics)
- Lymphokines
(biosynthesis, genetics)
- Macaca
- Microscopy, Phase-Contrast
- NF-kappa B
(metabolism)
- Nuclear Proteins
(metabolism)
- Prostaglandin-Endoperoxide Synthases
(biosynthesis, genetics)
- RNA, Messenger
(biosynthesis)
- Retina
(cytology, metabolism)
- Reverse Transcriptase Polymerase Chain Reaction
- Transcription Factor AP-1
(metabolism)
- Transcription Factors
- Vascular Endothelial Growth Factor A
- Vascular Endothelial Growth Factors
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