Upregulation of Cox-2 Gene Transcription in Monkey Choroidal Retinal Cells by Hypoxia

Abstract

Abstract: : Purpose: Cyclooxygenase-2 (COX-2), an early response gene (ERG) is strongly implicated in pro-inflammatory (PI) signalling pathways associated with the onset of neovascularization in the ischemic retina. In this study we examined transcription factors (TFs)AP1-, hypoxia inducible factor-1-alpha (HIF-1α)-, NF-kB- and STAT1alpha-DNA binding in relation to COX-1 and COX-2 RNA message and protein levels in cultured monkey choroid-retinal cells. Methods: Cultured RF/6A cells were subjected to hypoxia (95% N2/5% O2) for 0, 0.5, 1, 3 and 4 hr. Cellular and nuclear extracts (CPXTs, NPXTs) and total RNA were prepared and gel shift assay was performed using CPXTs and NPXTs as TF sources. Relative COX-1 and COX-2 RNA message levels and COX-2 protein concentrations were determined by RT-PCR and Western immuno-dot blotting, respectively. Gel shift, RT-PCR and immunoblot signals were quantitated using a Typhoon 8600 Variable Mode Imager (Amersham-Pharmacia). DNA structural analyses identifying putative cis-acting DNA regulatory elements (-1200 bp and +100 bp of the COX-1 and COX-2 gene promoters) were also performed. The NF-kB inhibitor NVP-AGA048-NX-1 and sPLA₂ inhibitor CGP43182 were tested on hypoxia-induced NF-kB and COX-2 gene activation as well as tube formation in cultured RF/6A cells. Results: AP1-, HIF-1α, NF-kB and STAT1alpha-DNA binding in NPXTs were found increased by 0.4-, 4-, 3- and 0.9-fold, respectively after 1 hr hypoxia, suggesting that HIF-1α- and NF-kB activation are hypoxia-sensitive nuclear events. CPXT levels for AP1, HIF-1α, NF-kB and STAT1alpha TFs were slightly elevated after 3 hr hypoxia. COX-2 RNA message levels were elevated after 1 and 3 hr hypoxia to ∼5 and 4-fold, respectively vs. 0 time controls. NVP-AGA048-NX-1 and CGP43182 repressed both NF-kappaB p50/p65-DNA binding and COX-2 RNA message abundance and significantly reduced tubular morphogenesis induced by hypoxia. Conclusion: The COX-2 gene promoter contains nested targets for AP1-, HIF-1α-, NF-kB- and STAT1alpha-DNA binding. The mechanism by which the sPLA₂ inhibitor CGP43182 reduces NF-kB-DNA binding, COX-2 gene expression and tubular morphogenesis due to hypoxia needs further clarification. Decreased NF-kB-DNA binding and COX-2 RNA message abundance in the presence of NVP-AGA048-NX-1 suggests that NF-kB is critical to COX-2 gene activation in hypoxic RF/6A cells.

Keywords: 483 neovascularization • 554 retina • 605 transcription factors