Isolation and Characterization of thr Promoter of the Corneal Epithelial CYP4B1 Gene: Molecular Regulation of the Synthesis of Inflammatory Eicosanoids

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 a corneal epithelial CYP4B1 cDNA and demonstrated that it is induced by hypoxia in vitro and in vivo. To further understand the molecular regulation that underlies the synthesis of these potent inflammatory eicosanoids, we isolated and cloned the CYP4B1 promoter. Methods: Genomic DNA was isolated from the rabbit corneal epithelium by standard methods. DNA was digested with different restriction enzymes. Each batch of digested genomic DNA was ligated to a Universal GenomeWalker Adaptor, which are referred to as GenomeWalker libraries. The libraries were used as templates in primary and nested PCR amplifications to isolate the promoter region with gene- and adaptor-specific primers. Amplified DNA fragments were sequenced and analyzed by computer software for the presence of known cis-acting elements. DNA fragments of different lengths were cloned into the luciferase reporter vector (pGL3-Basic) and further used for promoter analysis in the rabbit epithelial cell line (RCE). Results: A 3-kb DNA fragment of the 5'-flanking region of the CYP4B1 promoter was isolated and cloned. Analysis of the promoter sequence revealed the presence cis-acting elements including early growth response (EGR)-1, NFkB, SP-1, HIF and Ets. Transient transfection of luciferase reporter vectors containing different lengths of the promoter fragment in RCE cells revealed hypoxia-induced transcription. Conclusions: The findings of sequences on 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 in response to hypoxic injury. CR:None. Support: NIH grant EY06513.

Keywords: 428 hypoxia • 417 gene/expression • 392 eicosanoids