Laminar shear stress modulates phosphorylation and localization of RNA polymerase II on the endothelial nitric oxide synthase gene.
Laminar shear stress modulates phosphorylation and localization of RNA polymerase II on the endothelial nitric oxide synthase gene.
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DOI：
10.1161/atvbaha.109.199554
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发表时间：
2010-03
期刊：
影响因子：
--
通讯作者：
Searles CD
中科院分区：
文献类型：
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作者：
Moore JP;Weber M;Searles CD
在暴露于单向层流剪切应力 (LSS) 的内皮细胞中，eNOS 转录、mRNA 稳定性和蛋白质水平均得到增强。我们之前已经证明这些变化与 eNOS mRNA 3' 聚腺苷酸化增加有关。在这里，我们研究了 LSS 对 RNA 聚合酶 (Pol) II 磷酸化和定位的影响，该酶主要负责协调转录和转录后加工。使用蛋白质印迹和染色质免疫沉淀 (ChIP) 分析，评估暴露于 LSS 的牛主动脉内皮细胞 (BAEC) 中 eNOS 基因的 Pol II 磷酸化和定位。 LSS 后，总 Pol II（磷酸化和非磷酸化）水平增加了 65%。这与 Pol II 磷酸丝氨酸 2 的增加有关，但未磷酸化或磷酸丝氨酸 5 亚型的水平没有变化。定量 ChIP 分析表明，LSS 增强了 Pol II 磷酸丝氨酸 2 与 eNOS 基因 3' 末端的结合，特别是编码 3'UTR 的外显子 26。用 DRB 处理细胞减弱了 LSS 诱导的 Pol II 磷酸化、eNOS 3' 聚腺苷酸化和 eNOS 表达。这些数据表明，LSS 通过调节 Pol II 的磷酸化和定位来增强 eNOS mRNA 3' 多腺苷酸化。
In endothelial cells exposed to unidirectional, laminar shear stress (LSS), eNOS transcription, mRNA stability, and protein levels are enhanced. We have previously demonstrated that these changes are associated with increased 3′ polyadenylation of eNOS mRNA. Here, we investigated the effect of LSS on the phosphorylation and localization RNA Polymerase (Pol) II, the enzyme primarily responsible for coordinating transcription and posttranscriptional processing. Using Western and chromatin immunoprecipitation (ChIP) analyses, Pol II phosphorylation and localization on the eNOS gene were assessed in bovine aortic endothelial cells (BAECs) exposed to LSS. Total Pol II (phosphorylated and unphosphorylated) levels were increased 65% in response to LSS. This was associated with an increase in Pol II phospho-serine 2, but no change in levels of the unphosphorylated or phospho-serine 5 isoforms. Quantitative ChIP analysis showed that LSS enhanced binding of Pol II phospho-serine 2 to the 3′ end of the eNOS gene, particularly exon 26, which encodes the 3′UTR. Treatment of cells with DRB attenuated LSS-induced Pol II phosphorylation, eNOS 3′ polyadenylation, and eNOS expression. These data suggest that LSS enhances eNOS mRNA 3′ polyadenylation by modulating phosphorylation and localization of Pol II.