Hypoxia–Induced COX–2 and VEGF Gene Expression are Down–regulated by LY294002 and U0126 in Retinal Cells: Repression of HIF–1alpha and NF–B–DNA Binding

Abstract

Abstract: : Purpose: Up–regulation of cyclooxygenase–2 (COX–2) and vascular endothelial growth factor (VEGF) gene expression are associated with the initiation of retinal neovascularization (NV) in hypoxia triggered retinal cells. In this study we examined the effects of the MEK inhibitor U0126 and the IP3K inhibitor LY294004 on the oxygen sensitive transcription factor (TF) hypoxia inducible factor–1–alpha (HIF–1α)– and NF–kB–DNA binding in relation to COX–2 and VEGF RNA message levels in cultured monkey choroid–retinal cells exposed to hypoxia. Methods: Cultured RF/6A cells were subjected to hypoxia (90% N2/5% CO2/5%O2) for 0, 0.5, 1 and 3 hours. U0126 (Promega) and LY294002 (Calbiochem) were used at 0.5 µM and 5 µM respectively. Nuclear protein extracts (NPXTs) and total RNA were prepared and gel shift assay was performed using NPXTs as TF source. Relative COX–2 and VEGF RNA message levels were determined by RT–PCR. Results: Rapid HIF–1alpha and NF–kB activation were associated with an up–regulation of both COX–2 and VEGF gene activation during hypoxia. NF–kB–DNA binding and COX–2 gene activation were specifically suppressed by the MEK inhibitor U0126 while HIF–1alpha–DNA binding and VEGF expression was significantly repressed by the PI3K–specific inhibitor LY294002. Incubation of RF/6A cells with U0126+LY294002 prior to hypoxia treatment showed synergistic effects on the repression of COX–2 and VEGF gene expression. Conclusions: These data underscore the importance of HIF–1alpha– and NF–kB–DNA binding in orchestrating genetic programs that result in NV. Specific activation of HIF–1alpha and VEGF expression requires PI3K signaling and specific activation of NF–kB–DNA binding and COX–2 gene activation through MEK signalling suggests at least two converging kinase–signalling pathways for the induction of retinal NV in hypoxia triggered RF/6A choroid–retinal cells.

Keywords: oxidation/oxidative or free radical damage • hypoxia • gene/expression