Comparative Genomic and Proteomic Analysis of Cytoskeletal Changes in Dexamethasone-Treated Trabecular Meshwork Cells
Comparative Genomic and Proteomic Analysis of Cytoskeletal Changes in Dexamethasone-Treated Trabecular Meshwork Cells
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DOI：
10.1074/mcp.m112.019745
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发表时间：
2013-01-01
影响因子：
7
通讯作者：
Peters, Donna M.
中科院分区：
文献类型：
--
作者：
Clark, Ross;Nosie, Amanda;Peters, Donna M.
肌动蛋白细胞骨架的改变，特别是交联型肌动蛋白网络(CLANS)的形成被认为是导致原发性开角型青光眼和激素性青光眼眼压升高的原因。为了更好地了解糖皮质激素的作用，我们使用鸟枪法分析了地塞米松(DEX)处理人小梁网络(HTM)细胞后细胞骨架和整合素信号通路的整体变化。用地塞米松和地塞米松共同孵育HTM细胞，获得RNA和细胞裂解产物。细胞裂解产物差速离心法浓缩低丰度细胞骨架和信号蛋白，蛋白水解物消化，二氧化钛柱富集磷酸肽后，用质谱仪(MS)测定蛋白质表达的变化。结果经Western blotts验证。用基因芯片和RT-PCR检测RNA水平的变化。总体而言，MS确定了318个细胞骨架相关蛋白。其中5种蛋白(PDLIM1、FGFR1OP、Leiomodin-1、ZO-2和LRP16A)仅在DEX处理的细胞中被检测到，但只有PDLIM1在RNA水平上有统计学意义的增加。其他在RNA和蛋白质水平上存在差异的蛋白质包括β3整合素、小窝蛋白-1、Borg2、raftlin1、PI-3激酶调节亚基α、转明胶和细丝B。免疫荧光显微镜显示，细丝B和PDLIM1在人眼小梁细胞中表达增强，但只有PDLIM1在家族中有明显的定位。MS分析表明，部分细胞骨架蛋白(Borg2、Leiomodin-1、LRP16A、raftlin1和CKAP4)含有磷酸化残基。这项研究表明，DEX在转录和翻译水平上影响细胞骨架蛋白的表达，并表明基因组和蛋白质组相结合的方法可以用于TM中蛋白质的快速分析。它还表明，地塞米松改变了参与家族形成的成分(PDLIM1和β3整合素)的表达，并为糖皮质激素对细胞骨架的影响提供了新的发现。分子与细胞蛋白质组学12：10.1074/mcp.M112.019745,194-206,2013年。
Changes in the actin cytoskeleton, especially the formation of cross-linked actin networks (CLANs) are thought to contribute to the increased intraocular pressure observed in primary open-angle and steroid-induced glaucoma. To better understand the effects of glucocorticoids, we employed a shotgun method to analyze global changes in the cytoskeleton and integrin signaling pathways following dexamethasone (DEX) treatment of human trabecular meshwork (HTM) cells. RNA and cell lysates were obtained from HTM cells incubated with or without DEX. Changes in protein expression were determined by mass spectrometry (MS) following differential centrifugation of cell lysates to enrich for low-abundance cytoskeletal and signaling proteins, proteolytic digestion, and a titanium dioxide column to enrich for phosphopeptides. Results were validated by Western blots. Changes in RNA levels were determined with gene arrays and RT-PCR. Overall, MS identified 318 cytoskeleton associated proteins. Five of these proteins (PDLIM1, FGFR1OP, leiomodin-1, ZO-2 and LRP16A) were only detected in DEX-treated cells by MS. However, only PDLIM1 showed a statistically significant increase at the RNA level. Other proteins with differences at both the RNA and protein levels included beta 3 integrin, caveolin-1, Borg2, raftlin1, PI-3 kinase regulatory subunit alpha, transgelin, and filamin B. By immunofluorescence microscopy filamin B and PDLIM1 showed enhanced expression in human trabecular meshwork cells, but only PDLIM1 demonstrated significant localization within CLANs. Finally, MS showed that some of the cytoskeleton proteins (Borg2, leiomodin-1, LRP16A, raftlin1 and CKAP4) contained phosphorylated residues. This study suggests that DEX affects the expression of cytoskeleton proteins at the transcriptional and translational level and shows that a combined genomic and proteomic approach can be used for rapid analysis of proteins in the TM. It also shows that DEX altered the expression of components (PDLIM1 and beta 3 integrins) involved in CLAN formation and provides new findings into the effects of glucocorticoids on the cytoskeleton. Molecular & Cellular Proteomics 12: 10.1074/mcp.M112.019745, 194-206, 2013.