Department of Ophthalmology, Yamaguchi University Graduate School of Medicine, Yamaguchi, Japan.
Invest Ophthalmol Vis Sci. 2019 Feb 1;60(2):528-537. doi: 10.1167/iovs.18-25678.
Epithelial-mesenchymal transition (EMT) in retinal pigment epithelial (RPE) cells is related to the pathogenesis of subretinal fibrosis such as that associated with macular degeneration. The role of myocardin-related transcription factor A (MRTF-A) in EMT of RPE cells and subretinal fibrosis was investigated.
The migratory activity of human RPE-1 cells in culture was evaluated using a scratch assay. The subcellular distribution of MRTF-A in RPE-1 cells, as well as the extent of subretinal fibrosis in a mouse model, were determined by immunofluorescence analysis. Expression of α-smooth muscle actin (α-SMA), collagen type I (COL1), connective tissue growth factor (CTGF), and paxillin was examined by immunoblot analysis or reverse transcription and quantitative polymerase chain reaction analysis, whereas that of pro-matrix metalloproteinase-2 (MMP-2) was assessed by gelatin zymography.
The MRTF-A signaling inhibitor CCG-1423 suppressed RPE-1 cell migration in a concentration-dependent manner. Transforming growth factor-beta (TGF-β2) induced MRTF-A translocation from the cytoplasm to the nucleus of RPE-1 cells, and this effect was attenuated by CCG-1423. TGF-β2 up-regulated the abundance of α-SMA, paxillin, and pro-MMP-2 proteins as well as the amounts of α-SMA, COL1, and CTGF mRNAs in a manner sensitive to inhibition by CCG-1423. Finally, intravitreal injection of CCG-1423 markedly attenuated the development of subretinal fibrosis induced by photocoagulation in vivo.
Our results implicate MRTF-A in EMT of RPE cells and in the development of subretinal fibrosis in vivo, suggesting that MRTF-A is a potential therapeutic target for retinal diseases characterized by subretinal fibrosis.
视网膜色素上皮（RPE）细胞的上皮-间充质转化（EMT）与黄斑变性等相关的视网膜下纤维化的发病机制有关。本研究旨在探讨心肌营养素相关转录因子 A（MRTF-A）在 RPE 细胞 EMT 和视网膜下纤维化中的作用。
采用划痕实验评估人 RPE-1 细胞在培养中的迁移活性。通过免疫荧光分析确定 MRTF-A 在 RPE-1 细胞中的亚细胞分布以及在小鼠模型中视网膜下纤维化的程度。通过免疫印迹分析或逆转录和定量聚合酶链反应分析检测α-平滑肌肌动蛋白（α-SMA）、I 型胶原（COL1）、结缔组织生长因子（CTGF）和桩蛋白的表达，通过明胶酶谱法评估前基质金属蛋白酶-2（MMP-2）的表达。
MRTF-A 信号抑制剂 CCG-1423 呈浓度依赖性地抑制 RPE-1 细胞迁移。转化生长因子-β2（TGF-β2）诱导 RPE-1 细胞的 MRTF-A 从细胞质向细胞核易位，CCG-1423 可减弱此作用。TGF-β2 以上调α-SMA、桩蛋白和前 MMP-2 蛋白的丰度以及 α-SMA、COL1 和 CTGF mRNA 的量的方式诱导 EMT，这种作用对 CCG-1423 敏感。最后，玻璃体内注射 CCG-1423 可显著减轻体内光凝诱导的视网膜下纤维化的发展。
我们的结果表明 MRTF-A 参与了 RPE 细胞的 EMT 以及体内视网膜下纤维化的发展，提示 MRTF-A 可能是一种具有潜在治疗价值的靶点，可用于治疗以视网膜下纤维化为特征的眼部疾病。
