Activator protein-1 mediates shear stress-induced prostaglandin D synthase gene expression endothelial cells
Activator protein-1 mediates shear stress-induced prostaglandin D synthase gene expression endothelial cells
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DOI：
10.1161/01.atv.0000159702.68591.0d
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发表时间：
2005-05-01
影响因子：
8.7
通讯作者：
Sasaguri, T
中科院分区：
文献类型：
--
作者：
Miyagi, M;Miwa, Y;Sasaguri, T
我们试图确定流体剪切应力诱导的血管内皮细胞中脂质运载蛋白型前列腺素D合成酶（L-PGDS）表达的分子机制。方法和结果我们通过加载层流剪切应力检测L-PGDS基因的启动子区。（20达因/厘米（2）），使用平行板流动室，在用含有人L-PGDS基因的5 '侧翼区的荧光素酶报告载体转染的内皮细胞上。缺失突变体分析表明，剪切应力响应元件位于-2607和-2523 bp之间的区域。一个突变引入到假定的结合位点激活蛋白-1（AP-1）在这个区域内消除了对剪切应力的反应。在电泳迁移率变动分析中，剪切应力刺激核蛋白结合到AP-1结合位点，这是supershifted由c-Fos和c-Jun的抗体。剪切应力升高c-Jun磷酸化水平的时间依赖性的方式，类似于L-PGDS基因的表达。SP 600125是一种c-Jun N-末端激酶抑制剂，可降低切应力诱导的c-Jun磷酸化、AP-1的DNA结合和L-PGDS表达。此外，使用放线菌素D的mRNA追逐实验表明，剪切应力不稳定L-PGDS mRNA. Conclusions剪切应力诱导L-PGDS的表达通过转录激活通过AP-1结合位点。
Objective-We attempted to determine the molecular mechanism of fluid shear stress-induced lipocalin-type prostaglandin D synthase (L-PGDS) expression in vascular endothelial cells.Methods and Results-We examined the promoter region of the L-PGDS gene by loading laminar shear stress (20 dyne/cm(2)), using a parallel-plate flow chamber, on endothelial cells transfected with luciferase reporter vectors containing the 5'-flanking regions of the human L-PGDS gene. A deletion mutant analysis revealed that a shear stress-responsive element resided in the region between -2607 and -2523 bp. A mutation introduced into the putative binding site for activator protein-1 (AP-1) within this region eliminated the response to shear stress. In an electrophoretic mobility shift assay, shear stress stimulated nuclear protein binding to the AP-1 binding site, which was supershifted by antibodies to c-Fos and c-Jun. Shear stress elevated the c-Jun phosphorylation level in a time-dependent manner, similar to that of L-PGDS gene expression. SP600125, a c-Jun N-terminal kinase inhibitor, decreased the c-Jun phosphorylation, DNA binding of AP-1, and L-PGDS expression induced by shear stress. Additionally, an mRNA chase experiment using actinomycin D demonstrated that shear stress did not stabilize L-PGDS mRNA.Conclusions-Shear stress induces L-PGDS expression by transcriptional activation through the AP-1 binding site.