Competitive binding of CREB and ATF2 to cAMP/ATF responsive element regulates eNOS gene expression in endothelial cells
Competitive binding of CREB and ATF2 to cAMP/ATF responsive element regulates eNOS gene expression in endothelial cells
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DOI：
10.1161/01.atv.0000215179.76144.39
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发表时间：
2006-05-01
影响因子：
8.7
通讯作者：
Kurabayashi, M
中科院分区：
文献类型：
--
作者：
Niwano, K;Arai, M;Kurabayashi, M
目的-内皮型一氧化氮合酶（eNOS）的表达是血管稳态的关键决定因素.我们研究的影响贝前列素钠（BPS），前列环素的稳定类似物，eNOS基因表达的存在下，炎症细胞因子白细胞介素（IL）-1 β在培养的内皮cells.Method和Results -暴露的人和牛内皮细胞IL-1 β降低eNOS的表达。使用磷酸化特异性抗体的蛋白质印迹分析显示IL-1 β刺激p38 MAP激酶和磷酸化ATF 2。BPS通过激活蛋白激酶A（PKA）/cAMP反应元件结合蛋白（CREB）抑制这些作用。使用位点特异性突变构建体的转染试验表明，位于人eNOS启动子内-733和-603的CRE/ATF元件对于完全IL-1 β反应是必需的。BPS通过PKA途径减轻IL-1 β介导的eNOS启动子活性降低和eNOS基因表达的降低。电泳凝胶迁移率变动分析表明，IL-1 β增加了磷酸化ATF 2与CRE/ATF的结合。在治疗与BPS，磷酸化CREB主要结合CRE/ATF.Conclusions -这些结果表明，IL-1 β和BPS拮抗调节eNOS的表达，通过激活p38和PKA，分别。此外，结合CREB和ATF 2的能力暗示CRE/ATF序列是eNOS基因转录调控中多种信号传导途径的潜在靶点。
Objective - Expression of endothelial nitric oxide synthase ( eNOS) is a critical determinant for vascular homeostasis. We examined the effects of Beraprost sodium (BPS), a stable analogue of prostacyclin, on the eNOS gene expression in the presence of inflammatory cytokine interleukin (IL)-1 beta in cultured endothelial cells.Method and Results - Exposure of human and bovine endothelial cells to IL-1 beta decreased eNOS expression. Western blot analysis using phospho-specific antibodies showed that IL-1 beta stimulated p38 MAP kinase and phosphorylated ATF2. BPS inhibited these effects via protein kinase A (PKA)/cAMP- responsive element binding protein ( CREB) activation. Transfection assays using site-specific mutation constructs showed that CRE/ATF elements located at - 733 and - 603 within the human eNOS promoter are necessary for full IL-1 beta responsiveness. BPS attenuated the IL-1 beta-mediated decrease in eNOS promoter activity and the expression of eNOS gene through PKA pathway. Electrophoretic gel mobility shift assays showed that IL-1 beta increased the binding of phosphorylated ATF2 to CRE/ATF. On treatment with BPS, phosphorylated CREB predominantly bound to CRE/ATF.Conclusions - These results indicate that IL-1 beta and BPS antagonistically regulates the eNOS expression through the activation of p38 and PKA, respectively. Furthermore, the ability to bind both CREB and ATF2 implicates the CRE/ATF sequence as a potential target for multiple signaling pathways in the regulation of the eNOS gene transcription.