Hsu Yung-Ho, Chen Jin-Jer, Chang Nen-Chung, Chen Cheng-Hsien, Liu Ju-Chi, Chen Tso-Hsiao, Jeng Cherng-Jye, Chao Hung-Hsing, Cheng Tzu-Hurng
Department of Medicine, Taipei Medical University-Wan Fang Hospital, Taipei, Taiwan, ROC.
J Vasc Res. 2004 Jan-Feb;41(1):64-74. doi: 10.1159/000076247. Epub 2004 Jan 16.
Circulating angiotensin II (Ang II) increases vascular endothelin-1 (ET-1) tissue levels, which in turn mediate a major part of Ang II-stimulated vascular growth and hypertension in vivo. Ang II also stimulates the generation of reactive oxygen species (ROS) within vascular endothelial cells. However, whether ROS are involved in Ang II-induced ET-1 gene expression, and the related intracellular mechanisms occurring within vascular endothelial cells remain unclear.
Cultured endothelial cells were stimulated with Ang II, and the thus elicited ET-1 gene expression was examined by Northern blotting and a promoter activity assay. Antioxidant pretreatment of endothelial cells was performed prior to Ang II-induced extracellular signal-regulated kinase (ERK) phosphorylation in order to elucidate the redox-sensitive pathway for ET-1 gene expression.
The ET-1 gene was induced with Ang II, which was inhibited with Ang II type 1 receptor antagonist (irbesartan). Ang II-enhanced intracellular ROS levels were inhibited by irbesartan and several antioxidants, and antioxidants also suppressed Ang II-induced ET-1 gene expression. Further, Ang II-activated ERK phosphorylation was also significantly inhibited by certain antioxidants. An ERK inhibitor, U0126, inhibited Ang II-induced ET-1 expression completely. Cotransfection of the dominant negative mutant of Ras, Raf and MEK1 (ERK kinase) attenuated the Ang II-enhanced ET-1 promoter activity, suggesting that the Ras/Raf/ERK pathway is required for Ang II-induced ET-1 gene expression. Ang II-induced activator protein-1 (AP-1) reporter activities were inhibited by antioxidants. Moreover, mutational analysis of the ET-1 gene promoter showed that the AP-1 binding site was an important CIS element in Ang II-induced ET-1 gene expression.
Our data suggest that ROS are involved in Ang II-induced ET-1 gene expression within endothelial cells. The redox-sensitive ERK-mediated AP-1 transcriptional pathway plays an important role in Ang II-induced ET-1 gene expression.
循环血管紧张素II（Ang II）可提高血管内皮素-1（ET-1）的组织水平，而这反过来又介导了体内Ang II刺激的血管生长和高血压的主要部分。Ang II还刺激血管内皮细胞内活性氧（ROS）的生成。然而，ROS是否参与Ang II诱导的ET-1基因表达以及血管内皮细胞内发生的相关细胞内机制仍不清楚。
用Ang II刺激培养的内皮细胞，并通过Northern印迹法和启动子活性测定来检测由此引发的ET-1基因表达。在内皮细胞用抗氧化剂预处理后再进行Ang II诱导的细胞外信号调节激酶（ERK）磷酸化，以阐明ET-1基因表达的氧化还原敏感途径。
ET-1基因被Ang II诱导，而这被1型Ang II受体拮抗剂（厄贝沙坦）抑制。厄贝沙坦和几种抗氧化剂可抑制Ang II增强的细胞内ROS水平，并且抗氧化剂也抑制Ang II诱导的ET-1基因表达。此外，某些抗氧化剂也显著抑制了Ang II激活的ERK磷酸化。一种ERK抑制剂U0126完全抑制了Ang II诱导的ET-1表达。Ras、Raf和MEK1（ERK激酶）的显性负突变体的共转染减弱了Ang II增强的ET-1启动子活性，表明Ras/Raf/ERK途径是Ang II诱导的ET-1基因表达所必需的。抗氧化剂抑制了Ang II诱导的激活蛋白-1（AP-1）报告基因活性。此外，ET-1基因启动子的突变分析表明，AP-1结合位点是Ang II诱导的ET-1基因表达中的一个重要顺式元件。
我们的数据表明，ROS参与内皮细胞中Ang II诱导的ET-1基因表达。氧化还原敏感的ERK介导的AP-1转录途径在Ang II诱导的ET-1基因表达中起重要作用。
