Promoter Activity and Regulation of the Corneal CYP4B1 Gene by Hypoxia

Abstract

Abstract: : Purpose: Hypoxic injury to the ocular surface provokes an inflammatory response that is mediated, at least in part, by corneal epithelial-derived 12-hydroxyeicosanoids. These eicosanoids which exhibit potent inflammatory and angiogenic properties are formed by a cytochrome P450 enzyme, presumably CYP4B1. We have isolated and cloned the promoter region of the corneal epithelial CYP4B1 and studied its transcriptional regulation by hypoxia. Methods: GenomeWalker libraries were constructed from rabbit corneal epithelial genomic DNA and used to isolate the promoter region with gene- and adaptor-specific primers. DNA fragments of different lengths were cloned into the luciferase reporter vector (pGL3-Basic) and used for promoter analysis in the rabbit epithelial cell line (RCE). Results: A 3.4-kb DNA fragment of the 5'-flanking region of the corneal CYP4B1 promoter was isolated and cloned. Analysis of the promoter sequence revealed the presence of DNA binding motifs for hypoxia-sensitive transcription factors including HIF-1, NFkB, AP-1, HSF-1, Egr-1 and Sp1. Incubation of RCE cells transfected with luciferase reporter vectors containing different lengths of the promoter fragment under hypoxic condition resulted in a marked transcriptional activation of CYP4B1 gene that was correlated primarily with the presence of the HIF-1 binding motif. Promoter activity was also increased in response to CYP inducers including 3-MC and clofibrate suggesting the involvement of XRE and ARNT (HIF-1ß) as well as PPAR in hypoxic activation of CYP4B1 gene. Conclusions: The findings of sequences in the promoter region of the corneal CYP4B1 gene that are recognized by transcription factors whose activity is regulated by hypoxia provide a molecular mechanistic explanation for the induction of CYP4B1 and, thereby, the production of inflammatory eicosanoids by the corneal epithelium in response to hypoxic injury.

Keywords: eicosanoids • inflammation • hypoxia