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首页> 外文期刊>Journal of Cellular Physiology >Thrombin promotes actin stress fiber formation in RPE through Rho/ROCK-mediated MLC phosphorylation.
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Thrombin promotes actin stress fiber formation in RPE through Rho/ROCK-mediated MLC phosphorylation.

机译：凝血酶促进肌动蛋白纤维形成压力RPE无论是Rho - ROCK-mediated MLC磷酸化。

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摘要

The retinal pigment epithelium (RPE) forms the outer blood-retina barrier (BRB). Most retinal diseases involve BRB breakdown, whereupon thrombin contained in serum directly contacts the RPE. Thrombin is known to promote actin stress fiber formation, an important determinant in eye diseases involving the epithelial-mesenchymal transition (EMT) and migration of RPE cells, such as proliferative vitreoretinopathy. We analyzed thrombin effect on signaling pathways leading to myosin light chain (MLC) phosphorylation and actin stress fiber formation in primary cultures of rat RPE cells, in order to support a role for thrombin in RPE transdifferentiation. MLC phosphorylation was measured by Western blot; actin cytoskeleton was visualized using immunofluorescent phalloidin, and Rho GTPase activation was assessed by ELISA. We showed that thrombin/PAR-1 induces the time- and dose-dependent phosphorylation of MLC through the activation of Rho/ROCK and myosin light chain kinase (MLCK). ROCK increased phospho-MLC by phosphorylating MLC and by inhibiting MLC phosphatase. Thrombin effect was abolished by the ROCK inhibitor Y-27632, whereas MLCK inhibitor ML-7 and PLC-beta inhibitor U73122 attenuated MLC phosphorylation by approximately 50%, suggesting the activation of MLCK by PLC-beta-mediated calcium increase. Additionally, thrombin-induced MLC phosphorylation was blocked by the inhibitory PKCzeta pseudosubstrate, wortmannin, and LY294002, indicating IP(3)/PKCzeta involvement in the control of MLC phosphorylation. Moreover, we demonstrated that thrombin effect on MLC induces actin stress fiber formation, since this effect was prevented by inhibiting the pathways leading to MLC phosphorylation. We conclude that thrombin stimulation of MLC phosphorylation and actin stress fiber formation may be involved in thrombin-induced RPE cell transformation subsequent to BRB dysfunction.
机译：视网膜色素上皮(RPE)的形式外blood-retina屏障(马上回来)。疾病包括马上回来崩溃,于是凝血酶中含有血清直接接触RPE。纤维的形成,眼睛的重要决定因素疾病包括epithelial-mesenchymal过渡(EMT)和RPE细胞的迁移增生性玻璃体。对信号通路导致凝血酶的影响肌球蛋白轻链磷酸化和(多层陶瓷)在主文化中肌动蛋白纤维形成压力老鼠的RPE细胞,以支持的角色凝血酶在RPE分化转移。磷酸化是用免疫印迹;肌动蛋白细胞骨架是可视化使用immunofluorescent phalloidin,ρGTPase激活被ELISA评估。凝血酶/ PAR-1诱导时间,多层陶瓷的磷酸化存在剂量依赖的相关性激活ρ/岩石和肌球蛋白轻链激酶(MLCK)。磷酸化的多层陶瓷,通过抑制多层陶瓷磷酸酶。岩石抑制剂y - 27632,而MLCK抑制剂ML-7和PLC-beta抑制剂U73122减毒多层陶瓷磷酸化的大约50%,暗示由PLC-beta-mediated MLCK的激活钙增加。多层陶瓷磷酸化作用被抑制PKCzeta pseudosubstrate、渥曼青霉素LY294002,表明IP (3) / PKCzeta参与多层陶瓷磷酸化的控制。证明了对多层陶瓷诱发凝血酶的影响肌动蛋白纤维形成压力,因为这种效果预防是通过抑制途径多层陶瓷磷酸化。刺激的多层陶瓷磷酸化和肌动蛋白应力纤维的形成可能参与thrombin-induced RPE细胞转换随后马上回来功能障碍。

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