Arterial flow reduces oxidative stress via an antioxidant response element and Oct-1 binding site within the NADPH oxidase 4 promoter in endothelial cells
Arterial flow reduces oxidative stress via an antioxidant response element and Oct-1 binding site within the NADPH oxidase 4 promoter in endothelial cells
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DOI：
10.1007/s00395-011-0170-3
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发表时间：
2011-06-01
影响因子：
9.5
通讯作者：
Morawietz, Henning
中科院分区：
文献类型：
--
作者：
Goettsch, Claudia;Goettsch, Winfried;Morawietz, Henning
血管壁中氧化应激的主要来源是尼古丁腺嘌呤二核苷酸磷酸（NADPH）氧化酶（Nox）复合物。内皮主要表达含Nox 4的复合物;然而，内皮细胞中剪切应力调节Nox 4的机制尚不清楚。这项研究表明，长期应用动脉层流剪切应力使用锥板粘度计减少内皮超氧阴离子的形成和Nox 4的表达。在原代培养的人内皮细胞中，我们用5 ′-RACE PCR技术鉴定了Nox 4 mRNA的一个47 bp的5 ′-非翻译区。人Nox 4启动子的克隆和功能分析揭示了在-1，490和-1，310 bp之间的范围内负责流量依赖性下调。在-1，376 bp处的重叠抗氧化反应元件（ARE）样和Oct-1结合位点的突变消除了剪切应力依赖性Nox 4下调。与这些观察结果一致，电泳迁移率变动试验（EMSA）证明含有ARE样/Oct-1结合位点的Nox 4寡核苷酸的剪切应力依赖性结合增强，这可以被针对转录因子核因子红细胞2相关因子2（Nrf 2）的特异性抗体抑制。和八聚体转录因子1（Oct-1）。此外，切应力引起的Nrf 2和Oct-1从细胞质的易位到细胞核。通过短发夹RNA（shRNA）敲低Nrf 2使Nox 4表达增加两倍，表明Nrf 2和Nox 4之间存在直接的串扰。总之，ARE样/Oct-1结合位点被认为是剪切应力依赖性下调Nox 4的关键。这一新的机制可能与内皮细胞超氧阴离子形成的流量依赖性下调有关。
The main sources of oxidative stress in the vessel wall are nicotine adenine dinucleotide phosphate (NADPH) oxidase (Nox) complexes. The endothelium mainly expresses the Nox4-containing complex; however, the mechanism by which shear stress in endothelial cells regulates Nox4 is not well understood. This study demonstrates that long-term application of arterial laminar shear stress using a cone-and-plate viscometer reduces endothelial superoxide anion formation and Nox4 expression. In primary human endothelial cells, we identified a 47 bp 5'-untranslated region of Nox4 mRNA by 5'-rapid amplification of cDNA ends (5'-RACE) PCR. Cloning and functional analysis of human Nox4 promoter revealed a range between -1,490 and -1,310 bp responsible for flow-dependent downregulation. Mutation of an overlapping antioxidative response element (ARE)-like and Oct-1 binding site at -1,376 bp eliminated shear stress-dependent Nox4 downregulation. Consistent with these observations, electrophoretic mobility shift assays (EMSA) demonstrated an enhanced shear stress-dependent binding of Nox4 oligonucleotide containing the ARE-like/Oct-1 binding site, which could be inhibited by specific antibodies against the transcription factors nuclear factor erythroid 2-related factor 2 (Nrf2) and octamer transcription factor 1 (Oct-1). Furthermore, shear stress caused the translocation of Nrf2 and Oct-1 from the cytoplasm to the nucleus. Knockdown of Nrf2 by short hairpin RNA (shRNA) increased Nox4 expression twofold, indicating a direct cross-talk between Nrf2 and Nox4. In conclusion, an ARE-like/Oct-1 binding site was noticed to be essential for shear stress-dependent downregulation of Nox4. This novel mechanism may be involved in the flow-dependent downregulation of endothelial superoxide anion formation.