Hydrogen peroxide decreases endothelial nitric oxide synthase promoter activity through the inhibition of Sp1 activity.
Hydrogen peroxide decreases endothelial nitric oxide synthase promoter activity through the inhibition of Sp1 activity.
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DOI：
10.1089/dna.2008.0775
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发表时间：
2009-03
影响因子：
3.1
通讯作者：
Black SM
中科院分区：
文献类型：
--
作者：
Kumar S;Sun X;Wiseman DA;Tian J;Umapathy NS;Verin AD;Black SM
我们先前已经证明，内皮细胞中的内皮型一氧化氮合酶(ENOS)启动子活性因过氧化氢(H_2O_2)的加入而降低，这至少部分涉及对AP-1活性的抑制。因此，本研究的目的是确定是否有其他顺式元件(S)和转录因子(S)参与氧化剂介导的eNOS下调。我们的初步实验表明，尽管过氧化氢处理增加了绵羊肺动脉内皮细胞(OPAECs)中enos mRNA的水平，但含有840bp上游序列的enos启动子的启动子活性显著降低。然而，缺少AP-1元件的截短启动子结构(650bp)也被H_2O_2抑制。将650bp的人eNOS启动子载体导入人肺内皮细胞，也观察到类似的作用。我们还发现，尽管细胞暴露在聚乙二醇过氧化氢酶阻止了对eNOS启动子活性的抑制，但羟基自由基清除剂去铁胺肉豆蔻酸盐却没有。我们也不能确定暴露在过氧化氢中的细胞中的羟基自由基水平的增加。暴露于PAECs可引起活性锌水平显著升高，以响应过氧化氢。由于eNOS启动子有一个与Sp1结合的顺式元件，我们评估了Sp1在H_2O_2应答中的作用。正如先前报道的那样，Sp1共识的突变导致eNOS启动子活性的完全丧失，证实了Sp1在调节基础eNOS启动子活性中的关键作用。此外，我们发现，使用电泳迁移率和超移动分析，H_2O_2减少了Sp1的结合。最后，利用染色质免疫沉淀分析，我们发现在体内，过氧化氢处理后，Sp1与eNOS启动子的结合显著减少。综上所述，这些数据表明，Sp1活性的抑制可能是通过蛋白质中锌的丢失，在H_2O_2诱导的eNOS启动子活性的抑制中起作用。
We have previously shown that endothelial nitric oxide synthase (eNOS) promoter activity is decreased in endothelial cells in response to the addition of hydrogen peroxide (H2O2), and this involves, at least in part, the inhibition of AP-1 activity. Thus, the objective of this study was to determine if other cis-element(s) and transcription factor(s) are involved in the oxidant-mediated downregulation of eNOS. Our initial experiments indicated that although H2O2 treatment increased eNOS mRNA levels in ovine pulmonary arterial endothelial cells (OPAECs), there was a significant decrease in the promoter activity of an eNOS promoter construct containing 840 bp of upstream sequence. However, a truncated promoter construct that lacked the AP-1 element (650 bp) was also inhibited by H2O2. A similar effect was observed when the 650 bp human eNOS promoter construct was transfected into human PAECs. We also found that although exposure of the cells to PEG-catalase prevented the inhibitory effect on eNOS promoter activity, the hydroxyl radical scavenger, deferoxamine myslate, did not. Nor could we identify an increase in hydroxyl radical levels in cells exposed to H2O2. Exposure of PAECs caused a significant increase in labile zinc levels in response to H2O2. As the eNOS promoter has a cis-element for Sp1 binding, we evaluated the role of Sp1 in response to H2O2. As previously reported, mutation of the Sp1 consensus lead to the complete loss of eNOS promoter activity, confirming the key role of Sp1 in regulating basal eNOS promoter activity. In addition, we found, using electrophoretic mobility and supershift assays, that H2O2 decreased Sp1 binding. Finally, using chromatin immunoprecipitation analysis, we found a significant decrease in Sp1 binding to the eNOS promoter in vivo in response to treatment with H2O2. Together, these data suggest that the inhibition of Sp1 activity, possibly through loss of zinc in the protein, plays a role in the H2O2-induced inhibition of eNOS promoter activity.
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影响因子：
4
作者：
Black, Stephen M.;Fineman, Jeffrey R.
通讯作者：
Fineman, Jeffrey R.
DOI：
10.1073/pnas.89.16.7345
发表时间：
1992-08-15
影响因子：
11.1
作者：
DESJARLAIS, JR;BERG, JM
通讯作者：
BERG, JM
影响因子：
20.1
作者：
Drummond, GR;Cai, H;Harrison, DG
通讯作者：
Harrison, DG
影响因子：
56.9
作者：
BRIGGS, MR;KADONAGA, JT;TJIAN, R
通讯作者：
TJIAN, R
影响因子：
3.6
作者：
Black, SM;Johengen, MJ;Soifer, SJ
通讯作者：
Soifer, SJ