Hydrogen peroxide decreases endothelial nitric oxide synthase promoter activity through the inhibition of AP-1 activity
Hydrogen peroxide decreases endothelial nitric oxide synthase promoter activity through the inhibition of AP-1 activity
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DOI：
10.1152/ajplung.90205.2008
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发表时间：
2008-08-01
影响因子：
4.9
通讯作者：
Black, Stephen M.
中科院分区：
文献类型：
--
作者：
Kumar, Sanjiv;Sun, Xutong;Black, Stephen M.
以前，我们已经报道，内皮型一氧化氮合酶（eNOS）启动子活性降低肺动脉内皮细胞（PAECs）在响应过氧化氢（H2 O2）。因此，本研究的目的是确定负责氧化剂介导的eNOS基因下调的顺式元件和转录因子。初始启动子实验中的PAECs处理过氧化氢揭示了一个显着减少的活性的启动子片段，含有840 bp的上游序列的人eNOS基因融合到荧光素酶报告。然而，仅含有640 bp的上游序列的启动子构建体对H2 O2挑战的反应显着减弱。由于840-bp启动子构建体具有640-bp构建体中缺乏的转录因子激活蛋白-1（AP-1）的推定结合位点，因此我们评估了H2 O2对AP-1结合序列突变后启动子活性的影响（840-bp构建体中-661的TGAGTCA变为TGAGTtg）。与640 bp的结果相似，AP-1突变启动子对H2 O2的反应显著减弱。EMSA显示AP-1的结合在H2 O2处理过程中减少。超位移分析表明AP-1复合物由c-Jun和FosB异源二聚体组成。此外，体外EMSA分析表明H2 O2暴露后c-Jun结合显著降低。使用染色质免疫沉淀分析，我们证明了减少AP-1的eNOS启动子在体内响应H2 O2的结合。这些数据表明，AP-1结合的减少可能通过c-Jun在H（2）O（2）介导的eNOS启动子活性降低中发挥作用。
Previously, we have reported that endothelial nitric oxide synthase (eNOS) promoter activity is decreased in pulmonary arterial endothelial cells (PAECs) in response to hydrogen peroxide (H2O2). Thus the objective of this study was to identify the cis-element(s) and transcription factor(s) responsible for oxidant-mediated downregulation of the eNOS gene. Initial promoter experiments in PAECs treated with H2O2 revealed a significant decrease in activity of a promoter fragment containing 840 bp of upstream sequence of the human eNOS gene fused to a luciferase reporter. However, a promoter construct containing only 640 bp of upstream sequence had a significantly attenuated response to H2O2 challenge. As the 840-bp promoter construct had a putative binding site for the transcription factor activator protein-1 (AP-1) that was lacking in the 640-bp construct, we evaluated the effect of H2O2 on promoter activity after mutation of the AP-1 binding sequence (TGAGTCA at-661 to TGAGTtg in the 840-bp construct). Similar to the results seen with the 640 bp, the AP-1 mutant promoter had a significantly attenuated response to H2O2. EMSA revealed decreased binding of AP-1 during H2O2 treatment. Supershift analysis indicated that the AP-1 complex consisted of a c-Jun and FosB heterodimer. Furthermore, in vitro EMSA analysis indicated the c-Jun binding was significantly decreased after H2O2 exposure. Using chromatin immunoprecipitation analysis, we demonstrated decreased binding of AP-1 to the eNOS promoter in vivo in response to H2O2. These data suggest a role of decreased AP-1 binding likely through c-Jun in the H(2)O(2)mediated decrease in eNOS promoter activity.