Role of Formation of an ERK-FAK-Paxillin Complex in Migration of Human Corneal Epithelial Cells during Wound Closure In Vitro
Role of Formation of an ERK-FAK-Paxillin Complex in Migration of Human Corneal Epithelial Cells during Wound Closure In Vitro
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DOI：
10.1167/iovs.08-2534
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发表时间：
2009-12-01
影响因子：
4.4
通讯作者：
Nishida, Teruo
中科院分区：
文献类型：
--
作者：
Teranishi, Shinichiro;Kimura, Kazuhiro;Nishida, Teruo
目的.角膜上皮细胞的迁移是角膜创伤愈合过程中的重要环节。研究细胞外信号调节激酶（ERK）在创伤愈合过程中对细胞迁移的调节作用。在不存在或存在ERK信号传导抑制剂PD 98059的情况下，将划痕伤口引入人角膜上皮细胞中。通过免疫印迹和免疫荧光分析检测创伤闭合过程中ERK的磷酸化和定位。通过免疫沉淀和免疫印迹分析来评估粘着斑激酶（FAK）和桩蛋白的酪氨酸磷酸化，以及FAK与桩蛋白和ERK的结合。MEK 1的突变形式对细胞迁移和增殖的影响通过转染确定。PD 98059以浓度和时间依赖性方式抑制细胞迁移。创伤以对PD 98059敏感的方式增加ERK的磷酸化以及FAK和桩蛋白的酪氨酸磷酸化。此外，创伤诱导ERK-FAK-桩蛋白复合物的形成，这种效应以及创伤诱导的局灶性粘连、膜皱褶和F-肌动蛋白束的形成均被PD 98059抑制。磷酸化ERK定位于伤口边缘，并且在存在PD 98059的情况下未观察到这种定位。显性失活突变型MEK 1的表达在伤口闭合过程中抑制细胞迁移，而不影响细胞增殖。ERK通过调节FAK和桩蛋白的磷酸化以及随后的局灶性粘连的形成来调节体外伤口愈合过程中的细胞迁移。ERK-FAK-桩蛋白信号通路可能在体内角膜上皮创伤愈合中起重要作用。（Invest Ophthalmol维斯科学。2009; 50：5646-5652）DOI：10.1167/iovs.08-2534
PURPOSE. Migration of corneal epithelial cells is an important step in the corneal wound healing. The role of extracellular signal regulated kinase (ERK) for the regulation of cell migration during wound closure was examined.METHODS. Scratch wounds were introduced into human corneal epithelial cells in the absence or presence of PD98059, an ERK signaling inhibitor. The phosphorylation and localization of ERK during wound closure were examined by immunoblot and immunofluorescence analyses. The tyrosine phosphorylation of focal adhesion kinase (FAK) and paxillin, as well as the association of FAK with paxillin and ERK, were evaluated by immunoprecipitation and immunoblot analysis. The effect of a mutant form of MEK1 on cell migration and proliferation was determined by transfection.RESULTS. PD98059 inhibited cell migration in a concentration- and time-dependent manner. Wounding increased the phosphorylation of ERK as well as the tyrosine phosphorylation of FAK and paxillin in a manner sensitive to PD98059. Furthermore, wounding induced the formation of an ERK-FAK-paxillin complex and this effect as well as the wounding-induced formation of focal adhesions, membrane ruffles, and bundles of F-actin, were inhibited by PD98059. Phosphorylated ERK localized at the wound margin, and such localization was not observed in the presence of PD98059. Expression of dominant negative mutant form of MEK1 inhibited cell migration during wound closure without the effect of cell proliferation.CONCLUSIONS. ERK regulates cell migration during wound healing in vitro by modulating the phosphorylation of FAK and paxillin and the consequent formation of focal adhesions. An ERK-FAK-paxillin signaling pathway may play an important role in corneal epithelial wound healing in vivo. (Invest Ophthalmol Vis Sci. 2009; 50: 5646-5652) DOI: 10.1167/iovs.08-2534