Okumura Naoki, Fujii Keita, Kagami Takato, Makiko Nakahara, Kitahara Miu, Kinoshita Shigeru, Koizumi Noriko
Department of Biomedical Engineering, Faculty of Life and Medical Sciences, Doshisha University, Kyotanabe, Japan.
Department of Frontier Medical Science and Technology for Ophthalmology, Kyoto Prefectural University of Medicine, Kyoto, Japan.
Invest Ophthalmol Vis Sci. 2016 Dec 1;57(15):6843-6851. doi: 10.1167/iovs.16-20123.
Rho kinase (ROCK) pathways control fundamental cell functions, making ROCK an important therapeutic target in several pathophysiologic conditions. The purpose of this study was to investigate whether inhibition of ROCK can suppress apoptosis of the corneal endothelium and to determine the role of ROCK signaling in regulating apoptosis.
The effects of inhibitors of ROCK or myosin light chain (MLC) were evaluated in cultured monkey corneal endothelial cells (MCECs) irradiated with ultraviolet (UV) (100 J/m2) to induce apoptosis. Annexin V and TUNEL staining and Western blot for apoptosis-related proteins and focal adhesion complexes were then performed. RhoA activation was further evaluated by pull-down assays. ROCK inhibitor and caspase inhibitor effects on apoptosis were also evaluated in MCECs treated with ethylene glycol tetraacetic acid (EGTA) to induce MLC phosphorylation.
ROCK or MLC inhibition suppressed the caspase-3 cleavage and Annexin V and TUNEL expression typically seen during UV-mediated apoptosis of MCECs. The apoptotic stimulus activated RhoA and then induced phosphorylation of MLC via ROCK activation. EGTA-mediated phosphorylation of MLC was sufficient to induce the loss of cell contact with the substrate and subsequent apoptosis. Western blot showed that ROCK inhibition upregulated the expression of the focal adhesion complex in adhered cells, following UV stress.
Apoptotic stimuli activated Rho/ROCK/MLC phosphorylation in the corneal endothelium, and subsequent actomyosin contraction induced apoptosis by loss of cell adhesion. ROCK inhibition suppressed MLC phosphorylation and subsequent cell death, and it counteracted the loss of cell adhesion by activating the focal adhesion complex.
Rho激酶（ROCK）通路控制着细胞的基本功能，使ROCK成为多种病理生理状况下重要的治疗靶点。本研究旨在探讨抑制ROCK是否能抑制角膜内皮细胞凋亡，并确定ROCK信号在调节细胞凋亡中的作用。
在用紫外线（UV）（100 J/m2）照射以诱导凋亡的培养猴角膜内皮细胞（MCECs）中，评估ROCK或肌球蛋白轻链（MLC）抑制剂的作用。然后进行膜联蛋白V和TUNEL染色以及凋亡相关蛋白和粘着斑复合物的蛋白质印迹分析。通过下拉试验进一步评估RhoA的激活情况。在用乙二醇四乙酸（EGTA）处理以诱导MLC磷酸化的MCECs中，也评估了ROCK抑制剂和半胱天冬酶抑制剂对凋亡的影响。
ROCK或MLC抑制可抑制MCECs在UV介导的凋亡过程中通常出现的半胱天冬酶-3裂解以及膜联蛋白V和TUNEL表达。凋亡刺激激活RhoA，然后通过ROCK激活诱导MLC磷酸化。EGTA介导的MLC磷酸化足以诱导细胞与底物失去接触并随后发生凋亡。蛋白质印迹分析表明，在UV应激后，ROCK抑制上调了贴壁细胞中粘着斑复合物的表达。
凋亡刺激激活角膜内皮中的Rho/ROCK/MLC磷酸化，随后的肌动球蛋白收缩通过细胞粘附丧失诱导细胞凋亡。ROCK抑制可抑制MLC磷酸化及随后的细胞死亡，并通过激活粘着斑复合物抵消细胞粘附丧失。
