Hypoxia induces high-mobility-group protein I(Y) and transcription of the cyclooxygenase-2 gene in human vascular endothelium
Hypoxia induces high-mobility-group protein I(Y) and transcription of the cyclooxygenase-2 gene in human vascular endothelium
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DOI：
10.1161/01.res.83.3.295
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发表时间：
1998-08-10
影响因子：
20.1
通讯作者：
Schmedtje, JF
中科院分区：
文献类型：
--
作者：
Ji, YS;Xu, Q;Schmedtje, JF
环加氧酶催化血管内皮前列腺素合成的限速步骤。缺氧时，诱导型环氧合酶-2 （COX-2）基因通过核因子(NF)- κ B p65转录因子表达增加，这是缺氧时COX-2转录完全诱导的必要条件，但不是充分条件。在发现细胞质NF-kappa B p65和I kappa B α（一种结合NF-kappa B p65前体的抑制蛋白）水平不因缺氧而改变后，我们假设其他因素可能在调节COX-2启动子中起作用，如高流动性组（HMG） I(Y)家族蛋白，其具有多个A.T挂钩并与NF-kappa B介导的转激活相关。利用NF-kappa B-3’元件探针进行电泳迁移迁移试验时，将从人脐静脉内皮细胞（HUVECs）中获得的核蛋白添加HMG I(Y)。这些数据表明，HMG I(Y)蛋白与NF-kappa B p65相互作用，诱导COX-2启动子活性。我们还发现，在缺氧HUVEC核蛋白或添加HMG I(Y)的常氧核蛋白孵育后，TATA-box DNA显示出增加的电泳转移，表明DNA结合。用含有连接HMG I(Y) cDNA的COX-2启动子的表达载体转染HUVECs，在缺氧条件下对COX-2启动子活性有正反馈。我们通过核径流试验证实COX-2受缺氧的转录调节。缺氧增加HMG I(Y) mRNA的稳态细胞水平是一个早期事件，与HMG I(Y)蛋白的增加相对应。在缺氧huvec中，HMG I(Y)的过表达与COX-2蛋白的增加呈剂量-反应关系，此外，通过荧光素酶报告基因表达测量，正（和反义）HMG I(Y)过表达引起COX-2启动子活性的刺激（或抑制）。这些发现的生理学意义通过缺氧条件下HUVECs环氧化酶依赖性释放前列腺素E-2得到证实。我们得出结论，缺氧增加HMG I(Y)蛋白的表达，同时促进COX-2启动子的反激活。因此，HMG I(Y)家族蛋白可能作为缺氧诱导的增强体的一部分，有助于促进COX-2的转录。
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)-kappa B p65 transcription factor, which is necessary but not sufficient to fully induce COX-2 transcription in response to hypoxia. After finding that cytoplasmic NF-kappa B p65 and I kappa B alpha (an inhibitory protein that binds NF-kappa B 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-kappa B-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-kappa B-3' element probe. These data suggested that HMG I(Y) proteins interact with NF-kappa B 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 E-2 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.