Phosphoproteomic Study of Human Tubular Epithelial Cell in Response to Transforming Growth Factor-Beta-1-Induced Epithelial-to-Mesenchymal Transition
Phosphoproteomic Study of Human Tubular Epithelial Cell in Response to Transforming Growth Factor-Beta-1-Induced Epithelial-to-Mesenchymal Transition
复制标题
人肾小管上皮细胞对转化生长因子-Beta-1 诱导的上皮间质转化反应的磷酸化蛋白质组学研究
DOI：
10.1159/000253865
复制
发表时间：
2010-01-01
影响因子：
4.2
通讯作者：
Chen, Nan
中科院分区：
文献类型：
--
作者：
Chen, Yong-Xi;Li, Ya;Chen, Nan
背景：转化生长因子-β(转化生长因子-β)诱导的上皮向间充质转化(EMT)在慢性肾脏疾病(CKD)的肾脏纤维化和进展中起重要作用。蛋白质的磷酸化是转化生长因子-β信号转导的关键。我们应用等压相对和绝对定量标记(ITRAQ)技术来分析肾小管上皮细胞对转化生长因子-β诱导的EMT反应中的磷蛋白，以进一步研究分子事件。方法：用转化生长因子-β1诱导HK-2细胞发生EMT。将细胞分为对照组(未经转化生长因子-β1处理)和转化生长因子-β1处理组。提取两组细胞中的磷蛋白，用iTRAQ试剂进行差示标记，经2D-Nano-Hplc-MS/MS处理后，进行蛋白质印迹验证iTRAQ分析。通过在线数据库进行生物信息学分析。结果：经iTRAQ-2D-Nano-HPLC-MS/MS分析，共鉴定出38个差异表达的磷蛋白，其中上调蛋白19个，下调蛋白19个。Western印迹证实磷酸化的moesin和HSP90α表达上调。生物信息学分析表明，大部分蛋白质定位于胞核和内质网腔。这些磷蛋白可分为17个分子功能分类。核酸结合蛋白、细胞骨架蛋白和伴侣蛋白是主要的分子功能类别。建立了一个生物网络来分析上调蛋白之间的相互作用。结论：我们证实了转化生长因子-β1介导的肾小管上皮细胞中EMT的转录后调控。Moesin和HSP90α的磷酸化可能在转化生长因子-β诱导的EMT中起作用。版权所有(C)2009年S.Karger AG，巴塞尔
Background: Transforming growth factor-beta (TGF-beta)-induced epithelial-to-mesenchymal transition (EMT) plays an important role in renal fibrosis and progression of chronic kidney disease (CKD). Phosphorylation of proteins is essential to TGF-beta signaling. We applied isobaric tags for relative and absolute quantification (iTRAQ) technology to profile the phosphoproteins in tubular epithelial cells in response to TGF-beta-induced EMT in order to further study molecular events. Methods: HK-2 cells were treated with TGF-beta 1 to induce EMT. The cells were divided into a control group (without TGF-beta 1 treatment) and a TGF-beta 1-treated group. Phosphoproteins from two groups were extracted and differentially labeled with iTRAQ reagents and processed by 2D-nano-HPLC-MS/MS. Validating of iTRAQ analysis was performed by western blot. Bioinformatic analysis was performed by on-line databases. Results: By iTRAQ-2D-nano-HPLC-MS/MS, 38 differentially expressed phosphoproteins were identified which included 19 up-regulated phosphoproteins and 19 down-regulated phosphoproteins. Western blot confirmed up-regulation of phosphorylated moesin and HSP90 alpha. Bioinformatic analysis suggested that the majority of proteins were located in the nucleus and endoplasmic reticulum lumen. The phosphoproteins were categorized into 17 molecular function classifications. Nucleic acid binding protein, cytoskeletal protein and chaperone were the major categories of molecular function. A biological network was built to analyze interaction between up-regulated proteins. Conclusion: We demonstrate a TGF-beta 1-mediated post-transcriptional regulation of EMT in tubular epithelial cells. Phosphorylation of moesin and HSP90 alpha might play a role in TGF-beta-induced EMT. Copyright (c) 2009 S. Karger AG, Basel