Coordinate activation of HIF-1 and NF-kappaB DNA binding and COX-2 and VEGF expression in retinal cells by hypoxia.

Abstract

PURPOSE:

Proinflammatory signaling mechanisms are implicated in the induction of retinal neovascularization (NV) during ischemic retinopathies. This study examined transcription factor (TF) AP-1, HIF-1, and NF-kappaB DNA-binding in relation to cyclooxygenase (COX)-2 and VEGF RNA and protein levels in hypoxia-triggered monkey choroidal retinal (RF/6A) endothelial cells. Effects of the carboxamide CGP43182 were tested on COX-2 and VEGF activation and prostaglandin (PG)E(2) release.

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.

• 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.)

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

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