Department of Ophthalmology, Faculty of Life Sciences, Kumamoto University, Kumamoto, Japan.
Invest Ophthalmol Vis Sci. 2012 Oct 11;53(11):7097-108. doi: 10.1167/iovs.12-9989.
We investigated the involvement of the RhoA/Rho kinase (ROCK) signal transduction pathway in dexamethasone (DEX)-induced changes in aqueous outflow.
Using trabecular meshwork (TM) and Schlemm's canal endothelial (SCE) cells, RhoA activation was evaluated with a pull-down assay and myosin light chain phosphorylation was evaluated by Western blot analysis. Outflow facility was measured in perfused porcine anterior segment organ cultures treated with DEX and/or Y-27632, a selective ROCK inhibitor. The barrier function of the cultured cells on a micropore filter was evaluated by measuring the transendothelial electrical resistance. Collagen, fibronectin, and integrin mRNA expression levels were evaluated by quantitative real-time RT-PCR.
Relative RhoA activities increased following stimulation with 100 nM DEX in TM and SCE cells. Perfusion with DEX decreased outflow facility by 31.9 ± 14.3% compared to controls at 24 hours, but not by 50 μM Y-27632 in addition to DEX. The transendothelial electrical resistance of the SCE cell monolayer was increased by 48.6 ± 6.4% and 5.3 ± 5.0% following DEX treatments without and with 10 μM Y-27632, respectively, compared to controls. In TM cells, the mRNA expressions of COL4A1 and fibronectin were increased significantly by DEX treatment, but combined treatment with Y-27632 and DEX significantly inhibited the increase in COL4A1 and fibronectin expression.
Activation of the Rho/ROCK pathway in SCE cells contributes to the mechanism of DEX-induced changes in aqueous outflow.
我们研究了 RhoA/ Rho 激酶（ROCK）信号转导通路在地塞米松（DEX）诱导房水流出改变中的作用。
使用小梁网（TM）和施莱姆氏管内皮（SCE）细胞，通过下拉测定评估 RhoA 激活，通过 Western blot 分析评估肌球蛋白轻链磷酸化。用 DEX 和/或 Y-27632（一种选择性 ROCK 抑制剂）处理灌注的猪前节器官培养物来测量流出物的便利性。通过测量跨内皮电阻来评估培养细胞在微孔滤器上的屏障功能。通过定量实时 RT-PCR 评估胶原、纤维连接蛋白和整合素 mRNA 的表达水平。
在 TM 和 SCE 细胞中，刺激 100 nM DEX 后相对 RhoA 活性增加。与对照组相比，DEX 灌注 24 小时后，流出物便利性降低了 31.9%±14.3%，但 DEX 加 50 μM Y-27632 则没有降低。与对照组相比，DEX 处理后 SCE 细胞单层的跨内皮电阻分别增加了 48.6%±6.4%和 5.3%±5.0%，而无 10 μM Y-27632 的 DEX 处理则分别增加了 5.3%±5.0%。在 TM 细胞中，DEX 处理显著增加 COL4A1 和纤维连接蛋白的 mRNA 表达，但 Y-27632 和 DEX 的联合处理显著抑制了 COL4A1 和纤维连接蛋白表达的增加。
SCE 细胞中 Rho/ROCK 通路的激活有助于解释 DEX 诱导的房水流出改变的机制。
