Wiesener M S, Turley H, Allen W E, Willam C, Eckardt K U, Talks K L, Wood S M, Gatter K C, Harris A L, Pugh C W, Ratcliffe P J, Maxwell P H
Institute of Molecular Medicine and the Department of Cellular Science, John Radcliffe Hospital, Oxford, UK.
Blood. 1998 Oct 1;92(7):2260-8.
Hypoxia results in adaptive changes in the transcription of a range of genes including erythropoietin. An important mediator is hypoxia-inducible factor-1 (HIF-1), a DNA binding complex shown to contain at least two basic helix-loop-helix PAS-domain (bHLH-PAS) proteins, HIF-1alpha and aryl hydrocarbon nuclear receptor translocator (ARNT). In response to hypoxia, HIF-1alpha is activated and accumulates rapidly in the cell. Endothelial PAS domain protein 1 (EPAS-1) is a recently identified bHLH-PAS protein with 48% identity to HIF-1alpha, raising the question of its role in responses to hypoxia. We developed specific antibodies and studied expression and regulation of EPAS-1 mRNA and protein across a range of human cell lines. EPAS-1 was widely expressed, and strongly induced by hypoxia at the level of protein but not mRNA. Comparison of the effect of a range of activating and inhibitory stimuli showed striking similarities in the EPAS-1 and HIF-1alpha responses. Although major differences were observed in the abundance of EPAS-1 and HIF-1alpha in different cell types, differences in the inducible response were subtle with EPAS-1 protein being slightly more evident in normoxic and mildly hypoxic cells. Functional studies in a mutant cell line (Ka13) expressing neither HIF-1alpha nor EPAS-1 confirmed that both proteins interact with hypoxically responsive targets, but suggest target specificity with greater EPAS-1 transactivation (relative to HIF-1alpha transactivation) of the VEGF promoter than the LDH-A promoter.
缺氧会导致一系列基因转录发生适应性变化，其中包括促红细胞生成素。一个重要的介导因子是缺氧诱导因子-1（HIF-1），这是一种DNA结合复合物，已证明其至少包含两种碱性螺旋-环-螺旋PAS结构域（bHLH-PAS）蛋白，即HIF-1α和芳烃核受体转运蛋白（ARNT）。在缺氧反应中，HIF-1α被激活并在细胞内迅速积累。内皮PAS结构域蛋白1（EPAS-1）是最近发现的一种bHLH-PAS蛋白，与HIF-1α有48%的同源性，这就引发了其在缺氧反应中作用的问题。我们制备了特异性抗体，并研究了EPAS-1 mRNA和蛋白在一系列人类细胞系中的表达及调控情况。EPAS-1广泛表达，在蛋白水平上受到缺氧的强烈诱导，但在mRNA水平上未受诱导。对一系列激活和抑制刺激效应的比较显示，EPAS-1和HIF-1α的反应存在显著相似性。尽管在不同细胞类型中观察到EPAS-1和HIF-1α丰度存在主要差异，但诱导反应的差异很细微，EPAS-1蛋白在常氧和轻度缺氧细胞中略显明显。在既不表达HIF-1α也不表达EPAS-1的突变细胞系（Ka13）中进行的功能研究证实，这两种蛋白都与缺氧反应靶点相互作用，但表明相对于乳酸脱氢酶A（LDH-A）启动子，EPAS-1对血管内皮生长因子（VEGF）启动子具有更高的反式激活作用（相对于HIF-1α反式激活作用），具有靶点特异性。
