Coordinate activation of HIF-1 and NF-kappaB DNA binding and COX-2 and VEGF expression in retinal cells by hypoxia.
OBJECTIVE
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.
D000818:Animals; D002460:Cell Line; D002722:Chlorobenzenes / Q000494:pharmacology; D051546:Cyclooxygenase 2; D004268:DNA-Binding Proteins / Q000378:metabolism*; D015232:Dinoprostone / Q000378:metabolism; D024202:Electrophoretic Mobility Shift Assay; D016228:Endothelial Growth Factors / Q000096:biosynthesis / Q000235:genetics*; D004730:Endothelium, Vascular / Q000378:metabolism; D015870:Gene Expression / Q000187:drug effects; D000860:Hypoxia / Q000378:metabolism*; D051793:Hypoxia-Inducible Factor 1; D036341:Intercellular Signaling Peptides and Proteins / Q000096:biosynthesis / Q000235:genetics*; D007527:Isoenzymes / Q000096:biosynthesis / Q000235:genetics*; D008222:Lymphokines / Q000096:biosynthesis / Q000235:genetics*; D008251:Macaca; D008858:Microscopy, Phase-Contrast; D016328:NF-kappa B / Q000378:metabolism*; D009687:Nuclear Proteins / Q000378:metabolism*; D011451:Prostaglandin-Endoperoxide Synthases / Q000096:biosynthesis / Q000235:genetics*; D012333:RNA, Messenger / Q000096:biosynthesis; D012160:Retina / Q000166:cytology / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D018808:Transcription Factor AP-1 / Q000378:metabolism; D014157:Transcription Factors; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors
D002722:Chlorobenzenes; D004268:DNA-Binding Proteins; D016228:Endothelial Growth Factors; D051793:Hypoxia-Inducible Factor 1; D036341:Intercellular Signaling Peptides and Proteins; D007527:Isoenzymes; D008222:Lymphokines; D016328:NF-kappa B; D009687:Nuclear Proteins; D012333:RNA, Messenger; D018808:Transcription Factor AP-1; D014157:Transcription Factors; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors; C084106:CGP 43182; D051546:Cyclooxygenase 2; D011451:Prostaglandin-Endoperoxide Synthases; D015232:Dinoprostone