Promoter activity and regulation of the corneal CYP4B1 gene by hypoxia

Journal of Cellular Biochemistry - Tập 91 Số 6 - Trang 1218-1238 - 2004
Vladimir Mastyugin1, Alexandre Mezentsev1, Wenxiang Zhang1, Silvia Ashkar1, Michael W. Dunn1, Michal L. Schwartzman2
1Department of Pharmacology, New York Medical College, Valhalla, New York.
2Department of Pharmacology, New York Medical College, Valhalla, NY 10595

Tóm tắt

Abstract

Hypoxic injury to the ocular surface provokes an inflammatory response that is mediated, in part, by corneal epithelial‐derived 12‐hydroxyeicosanoids. Recent studies indicate that a cytochrome P450 (CYP) monooxygenase, identified as CYP4B1, is involved in the production of these eicosanoids which exhibit potent inflammatory and angiogenic properties. We have isolated and cloned a corneal epithelial CYP4B1 full‐length cDNA and demonstrated that the CYP4B1 mRNA is induced by hypoxia in vitro and in vivo. To further understand the molecular regulation that underlies the synthesis of these potent inflammatory eicosanoids in response to hypoxic injury, we isolated and cloned the CYP4B1 promoter region. GenomeWalker libraries constructed from rabbit corneal epithelial genomic DNA were used as templates for primary and nested PCR amplifications with gene‐ and adaptor‐specific primers. A 3.41‐kb DNA fragment of the 5′‐flanking region of the CYP4B1 promoter was isolated, cloned, sequenced, and analyzed by computer software for the presence of knowncis‐acting elements. Analysis of the promoter sequence revealed the presence of consensus DNA binding sequences for factors known to activate gene transcription in response to hypoxia including HIF‐1, NFκB, and AP‐1. Transient transfection of luciferase reporter (pGL3‐Basic) vectors containing different lengths of the CYP4B1 promoter fragment demonstrated hypoxia‐induced transcription in rabbit corneal epithelial (RCE) cells. Electrophoretic mobility shift assay (EMSA) revealed a marked induction of nuclear binding activity for the labeled HIF‐1 probe from the CYP4B1 promoter in nuclear extracts of cells exposed to hypoxia. This binding activity was due to sequence‐specific binding to the HIF‐1 oligonucleotide probe as shown by competition with excess unlabeled probe for the HIF‐1 but not with unlabeled NFκB probe. The nuclear binding activity of AP‐1 and NFκB probes from the CYP4B1 promoter was also enhanced in response to hypoxia suggesting that these transcription factors contribute to the hypoxic induction of CYP4B1 expression. The results of this study provide the first molecular mechanistic explanation for the induction of CYP4B1 and, thereby, the production of inflammatory eicosanoids in response to hypoxic injury. Further studies are needed to fully evaluate the molecular regulation of this gene during inflammation. © 2004 Wiley‐Liss, Inc.

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