Mastyugin Vladimir, Mezentsev Alexandre, Zhang Wen-Xiang, Ashkar Silvia, Dunn Michael W, Laniado-Schwartzman Michal
Department of Pharmacology, New York Medical College, Valhalla, New York 10595, USA.
J Cell Biochem. 2004 Apr 15;91(6):1218-38. doi: 10.1002/jcb.20018.
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 known cis-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, NFkappaB, 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 NFkappaB probe. The nuclear binding activity of AP-1 and NFkappaB 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.
眼表的缺氧损伤会引发炎症反应，部分是由角膜上皮衍生的12-羟基二十碳四烯酸介导的。最近的研究表明，一种被鉴定为CYP4B1的细胞色素P450（CYP）单加氧酶参与了这些具有强大炎症和血管生成特性的类花生酸的产生。我们已分离并克隆了角膜上皮CYP4B1全长cDNA，并证明CYP4B1 mRNA在体外和体内均可被缺氧诱导。为了进一步了解在缺氧损伤时这些强效炎症类花生酸合成背后的分子调控机制，我们分离并克隆了CYP4B1启动子区域。以从兔角膜上皮基因组DNA构建的GenomeWalker文库为模板，用基因特异性引物和接头特异性引物进行初次和巢式PCR扩增。分离出CYP4B1启动子5'侧翼区域的一个3.41 kb DNA片段，进行克隆、测序，并通过计算机软件分析是否存在已知的顺式作用元件。对启动子序列的分析揭示了已知可响应缺氧激活基因转录的因子（包括HIF-1、NFκB和AP-1）的共有DNA结合序列的存在。含有不同长度CYP4B1启动子片段的荧光素酶报告基因（pGL3-Basic）载体的瞬时转染证明了在兔角膜上皮（RCE）细胞中缺氧诱导的转录。电泳迁移率变动分析（EMSA）显示，在暴露于缺氧的细胞的核提取物中，来自CYP4B1启动子的标记HIF-1探针的核结合活性显著诱导。这种结合活性是由于与HIF-1的过量未标记探针竞争但不与未标记的NFκB探针竞争而与HIF-1寡核苷酸探针的序列特异性结合所致。来自CYP4B1启动子的AP-1和NFκB探针的核结合活性也因缺氧而增强，这表明这些转录因子有助于CYP4B1表达的缺氧诱导。本研究结果为CYP4B1的诱导以及因此在缺氧损伤时炎症类花生酸的产生提供了首个分子机制解释。需要进一步研究以全面评估该基因在炎症过程中的分子调控。
