Ji Y S, Xu Q, Schmedtje J F
Sealy Center for Molecular Cardiology, Department of Medicine, The University of Texas Medical Branch, Galveston, USA.
Circ Res. 1998 Aug 10;83(3):295-304. doi: 10.1161/01.res.83.3.295.
Cyclooxygenases catalyze a rate-limiting step in the synthesis of vascular endothelial prostaglandins. Expression of the inducible cyclooxygenase-2 (COX-2) gene is increased by hypoxia in human vascular endothelial cells via the nuclear factor (NF)-kappaB p65 transcription factor, which is necessary but not sufficient to fully induce COX-2 transcription in response to hypoxia. After finding that cytoplasmic NF-kappaB p65 and IkappaBalpha (an inhibitory protein that binds NF-kappaB p65 precursors) levels are not changed by hypoxia, we hypothesized that other factors might play a role in regulating the COX-2 promoter, like the high-mobility-group (HMG) I(Y) family of proteins, which features multiple A.T hooks and is associated with NF-kappaB-mediated transactivation. Nuclear protein obtained from human umbilical vein endothelial cells (HUVECs) was supplemented with HMG I(Y) during electrophoretic mobility shift assays using an NF-kappaB-3' element probe. These data suggested that HMG I(Y) proteins interact with NF-kappaB p65 to induce COX-2 promoter activity. We also found that TATA-box DNA demonstrated increased electrophoretic shifting indicative of DNA binding after incubation with either hypoxic HUVEC nuclear protein or normoxic nuclear protein supplemented with HMG I(Y). Transfection of HUVECs with an expression vector containing the COX-2 promoter ligated to HMG I(Y) cDNA demonstrated positive feedback on COX-2 promoter activity in hypoxia. We confirmed that COX-2 is transcriptionally regulated by hypoxia using a nuclear runoff assay. Hypoxia increased steady-state cellular levels of HMG I(Y) mRNA as an early event, corresponding with increases in HMG I(Y) protein. Overexpression of HMG I(Y) was associated in a dose-response relationship with increasing prevalence of the COX-2 protein in hypoxic HUVECs. Furthermore, sense (and antisense) HMG I(Y) overexpression caused stimulation (or inhibition) of COX-2 promoter activity as measured by luciferase reporter gene expression. The physiological significance of these findings was demonstrated by cyclooxygenase-dependent release of prostaglandin E2 by HUVECs in hypoxia. We concluded that hypoxia increases expression of HMG I(Y) proteins while facilitating transactivation of the COX-2 promoter. The HMG I(Y) family of proteins may therefore function as part of a hypoxia-induced enhanceosome that helps to promote transcription of COX-2.
环氧化酶催化血管内皮前列腺素合成中的限速步骤。在人血管内皮细胞中，缺氧通过核因子（NF）-κB p65转录因子增加诱导型环氧化酶-2（COX-2）基因的表达，该转录因子对于响应缺氧充分诱导COX-2转录是必要的，但并不充分。在发现缺氧不会改变细胞质中NF-κB p65和IκBα（一种结合NF-κB p65前体的抑制性蛋白）的水平后，我们推测其他因子可能在调节COX-2启动子中发挥作用，比如高迁移率族（HMG）I（Y）蛋白家族，其具有多个A.T钩并与NF-κB介导的反式激活相关。在使用NF-κB-3'元件探针进行电泳迁移率变动分析期间，从人脐静脉内皮细胞（HUVECs）获得的核蛋白中添加了HMG I（Y）。这些数据表明HMG I（Y）蛋白与NF-κB p65相互作用以诱导COX-2启动子活性。我们还发现，与缺氧的HUVEC核蛋白或添加了HMG I（Y）的常氧核蛋白孵育后，TATA框DNA显示出电泳迁移增加，表明存在DNA结合。用含有与HMG I（Y）cDNA连接的COX-2启动子的表达载体转染HUVECs，证明了缺氧时对COX-2启动子活性的正反馈。我们使用核转录分析证实了COX-2受缺氧转录调控。缺氧作为早期事件增加了HMG I（Y）mRNA的稳态细胞水平，这与HMG I（Y）蛋白的增加相对应。HMG I（Y）的过表达与缺氧的HUVECs中COX-2蛋白患病率增加呈剂量反应关系。此外，如通过荧光素酶报告基因表达所测，正义（和反义）HMG I（Y）过表达导致COX-2启动子活性的刺激（或抑制）。缺氧时HUVECs依赖环氧化酶释放前列腺素E2证明了这些发现的生理学意义。我们得出结论，缺氧增加HMG I（Y）蛋白的表达，同时促进COX-2启动子的反式激活。因此，HMG I（Y）蛋白家族可能作为缺氧诱导增强体的一部分发挥作用，有助于促进COX-2的转录。
