Han Jung Woo, Lyu Jungmook, Park Yeo Jin, Jang Sun Young, Park Tae Kwann
Department of Ophthalmology, Soonchunhyang University, College of Medicine, Soonchunhyang University Bucheon Hospital, Bucheon, Korea.
Myung-Gok Eye Research Institute, Department of Medical Science, Konyang University, Daejeon, Korea.
Invest Ophthalmol Vis Sci. 2015 Dec;56(13):8314-24. doi: 10.1167/iovs.15-18359.
Laser photocoagulation of retinal pigment epithelium (RPE) is used to stimulate the regenerative processes of the RPE. However, the molecular mechanisms that control RPE proliferation and the epithelial-mesenchymal transition (EMT) during regeneration remain poorly understood. We investigated the role of Wnt/β-catenin signaling in the regeneration of mouse RPE after laser photocoagulation.
C57BL/6J mice were photocoagulated unilaterally. To determine the β-catenin-dependent Wnt signal transduction in the photocoagulated RPE, the expression levels of Wnts, β-catenin, and their target genes were analyzed using real time-PCR and Western blotting. Retinal pigment epithelium proliferation and EMT were determined by 5-ethynyl-2'-deoxyuridine (EdU) incorporation assay and by profiling expression of EMT markers, respectively, in eyes injected intravitreally with a Wnt/β-catenin signaling antagonist, Dkk-1, after laser photocoagulation.
Expression of several of the 19 Wnt genes was significantly increased in laser-treated RPE. The expression levels of β-catenin signaling target genes cyclin D1, Otx2, and Mitf were higher in laser-treated RPE than in control RPE. The number of EdU-positive cells in the laser-treated area was significantly lower in the Dkk-1-injected group than in the laser-treated group or laser-treated + vehicle-injected group. There were more Otx2- and Mitf-positive cells after laser photocoagulation and markedly fewer in the Dkk-1-injected group. Otx2- and Mitf-positive cells were colocalized with EdU-positive cells. The EMT markers vimentin and α-smooth muscle actin (α-SMA) were upregulated in the laser-treated area and significantly downregulated in the Dkk-1-injected group.
Laser photocoagulation activates a Wnt/β-catenin signal transduction pathway, promoting both RPE proliferation and EMT. Wnt/β-catenin signaling also upregulates the expression of Otx2 and Mitf, key transcription factors in RPE formation. Our results demonstrate an important role for Wnt/β-catenin signaling in RPE proliferation and EMT, and suggest that manipulating Wnt/β-catenin signaling may have therapeutic potential for RPE regeneration.
视网膜色素上皮（RPE）激光光凝用于刺激RPE的再生过程。然而，在再生过程中控制RPE增殖和上皮-间质转化（EMT）的分子机制仍知之甚少。我们研究了Wnt/β-连环蛋白信号通路在激光光凝后小鼠RPE再生中的作用。
对C57BL/6J小鼠进行单侧光凝。为了确定光凝RPE中β-连环蛋白依赖性Wnt信号转导，使用实时PCR和蛋白质印迹分析Wnts、β-连环蛋白及其靶基因的表达水平。在激光光凝后，通过5-乙炔基-2'-脱氧尿苷（EdU）掺入试验以及分别分析EMT标志物的表达谱，来确定视网膜色素上皮增殖和EMT情况，其中EMT标志物的表达谱分析是在玻璃体内注射Wnt/β-连环蛋白信号拮抗剂Dkk-1的眼中进行的。
19种Wnt基因中的几种在激光处理的RPE中表达显著增加。β-连环蛋白信号靶基因细胞周期蛋白D1、Otx2和Mitf在激光处理的RPE中的表达水平高于对照RPE。在注射Dkk-1的组中，激光处理区域内EdU阳性细胞的数量显著低于激光处理组或激光处理+注射溶剂组。激光光凝后Otx2和Mitf阳性细胞较多，而在注射Dkk-1的组中明显较少。Otx2和Mitf阳性细胞与EdU阳性细胞共定位。EMT标志物波形蛋白和α-平滑肌肌动蛋白（α-SMA）在激光处理区域上调，而在注射Dkk-1的组中显著下调。
激光光凝激活Wnt/β-连环蛋白信号转导通路，促进RPE增殖和EMT。Wnt/β-连环蛋白信号通路还上调RPE形成中的关键转录因子Otx2和Mitf的表达。我们的结果证明Wnt/β-连环蛋白信号通路在RPE增殖和EMT中起重要作用，并表明操纵Wnt/β-连环蛋白信号通路可能对RPE再生具有治疗潜力。
