Okumura Naoki, Nakamura Takahiro, Kay EunDuck P, Nakahara Makiko, Kinoshita Shigeru, Koizumi Noriko
Department of Biomedical Engineering, Faculty of Life and Medical Sciences, Doshisha University, Kyotanabe, Japan Department of Ophthalmology, Kyoto Prefectural University of Medicine, Kyoto, Japan.
Research Center for Tissue Engineering and Inflammation, Faculty of Life and Medical Sciences, Doshisha University, Kyotanabe, Japan.
Invest Ophthalmol Vis Sci. 2014 Oct 2;55(10):6861-9. doi: 10.1167/iovs.14-14091.
To evaluate the effect of Roof plate-specific spondin 1 (R-spondin1) on the proliferation of corneal endothelial cells (CECs) and to determine whether the Wnt/β-catenin pathway is involved in the activities of R-spondin1.
The proliferation of rabbit CECs (RCECs) and human CECs (HCECs) was measured by 5-bromo-2'-deoxyuridine (BrdU) incorporation into DNA. The effect of R-spondin1 on CEC density was evaluated in ex vivo organ-cultured rabbit and human corneal tissues. The cell density of HCECs cultured with R-spondin1 was also evaluated in vitro. The subcellular localization of function-associated markers of CECs (zona occludens 1 [ZO-1] and Na+/K+-ATPase) was determined by immunohistocytochemistry. The expression of cell cycle proteins and localization of β-catenin were determined by immunoblotting.
The in vitro proliferation of RCECs and HCECs increased by 1.2- to 1.3-fold in response to R-spondin1. The CEC densities of rabbit and human corneal tissues were increased significantly by R-spondin1 treatment. Na+/K+-ATPase and ZO-1 were well preserved on the plasma membranes. When HCECs were maintained in the presence of R-spondin1 for up to 90 days, the maximum cell density was observed at approximately 50 days, and the cell density was maintained for up to 90 days. R-spondin1 facilitated the nuclear import of β-catenin in RCECs within 30 minutes, which subsequently upregulated cyclin D and downregulated p27, leading to G1/S progression by hyperphosphorylation of the retinoblastoma protein.
The unique effects of R-spondin1 on the proliferation of CECs, regardless of species, indicate that R-spondin1 may play a key role in maintaining corneal endothelium homeostasis through the Wnt/β-catenin pathway.
评估顶板特异性促腱蛋白1（R-spondin1）对角膜内皮细胞（CECs）增殖的影响，并确定Wnt/β-连环蛋白信号通路是否参与R-spondin1的作用机制。
通过检测5-溴-2'-脱氧尿苷（BrdU）掺入DNA的情况来测定兔角膜内皮细胞（RCECs）和人角膜内皮细胞（HCECs）的增殖。在离体器官培养的兔和人角膜组织中评估R-spondin1对CEC密度的影响。同时也在体外评估R-spondin1对培养的HCECs细胞密度的影响。通过免疫细胞化学法确定CECs功能相关标志物（紧密连接蛋白1[ZO-1]和钠钾ATP酶）的亚细胞定位。通过免疫印迹法检测细胞周期蛋白的表达和β-连环蛋白的定位。
R-spondin1可使RCECs和HCECs的体外增殖增加1.2至1.3倍。R-spondin1处理可显著提高兔和人角膜组织中的CEC密度。钠钾ATP酶和ZO-1在质膜上保存良好。当HCECs在R-spondin1存在的情况下培养长达90天时，在大约50天时观察到最大细胞密度，并且细胞密度维持长达90天。R-spondin1在30分钟内促进β-连环蛋白向RCECs细胞核内转运，随后上调细胞周期蛋白D并下调p27，通过视网膜母细胞瘤蛋白的过度磷酸化导致G1/S期进展。
R-spondin1对CECs增殖具有独特作用，无论物种如何，这表明R-spondin1可能通过Wnt/β-连环蛋白信号通路在维持角膜内皮细胞稳态中发挥关键作用。
