内质网应激作为抑制人视网膜色素上皮细胞转分化的新靶点。
Endoplasmic reticulum stress as a novel target to inhibit transdifferentiation of human retinal pigment epithelial cells.
机构信息
Department of Ophthalmology, The Third Xiangya Hospital, Central South University, 410000 Changsha, Hunan, China.
Eye Center of Xiangya Hospital, Central South University, 410008 Changsha, Hunan, China.
出版信息
Front Biosci (Landmark Ed). 2022 Jan 24;27(2):38. doi: 10.31083/j.fbl2702038.
BACKGROUND
The epithelial to mesenchymal transition (EMT) of retinal pigment epithelial (RPE) cells is a critical event in the pathogenesis of proliferative vitreoretinopathy and neovascular age-related macular degeneration, which are the leading causes of severe vision loss. Endoplasmic reticulum (ER) stress has been implicated in the EMT of many cell types and various ocular diseases. However, the relationship between ER stress and EMT in RPE cells remains unknown. Therefore, in the study, we explored the impact of ER stress on EMT in RPE cells.
METHODS
Different concentrations of tunicamycin (TM) and thapsigargin (TG) were used to induce ER stress in human RPE cells. The expression of epithelial marker, mesenchymal markers and some of genes/proteins involved in TGF-β/Smad signaling were analized by qPCR, western blot or immunostaining at the condition with or without stimulation of TGF-β2 (10 ng/mL). Boyden chamber and scratch assay were used to evaluate the migration of RPE cells, while cell viability and apoptosis of RPE cells were measured by MTT and TUNEL assay, respectively.
RESULTS
Treatment of RPE cells with TM and TG (24 h) reduced the expression of α -SMA and FN, and increased the expression of Occludin in a dose dependent manner at protein level, which was highly associated with the expression of GRP78. Treatment with TGF-β2 significantly increased the expression of α-SMA and FN, and decreased the expression of Occludin both in protein and mRNA levels, which was significantly inhibited by a 4h pre-treatment with TM. In addition, the expression of TGF-βRII and Smad2/3, and mRNAs of TGF-βRII and Smad3 were also decreased by the TM treatment. TM-induced ER stress inhibited RPE cell migration, and high concentrations of TM and TG reduced cell viability and induced apoptosis of RPE cells.
CONCLUSIONS
Chemical induction of ER stress inhibited EMT and migration in RPE cells, possibly by inactivation of TGF-β signaling, suggesting that regulation of ER stress in RPE cells may be a new approach to prevent the development of intraocular fibrosis.
背景
视网膜色素上皮（RPE）细胞的上皮间质转化（EMT）是增生性玻璃体视网膜病变和新生血管性年龄相关性黄斑变性发病机制中的关键事件，这两种疾病是导致严重视力丧失的主要原因。内质网（ER）应激已被牵涉到多种细胞类型和各种眼部疾病的 EMT 中。然而，ER 应激与 RPE 细胞 EMT 之间的关系尚不清楚。因此，在这项研究中，我们探讨了 ER 应激对 RPE 细胞 EMT 的影响。
方法
用不同浓度的衣霉素（TM）和他普西醇（TG）诱导人 RPE 细胞发生 ER 应激。在有无 TGF-β2（10ng/ml）刺激的条件下，通过 qPCR、western blot 或免疫染色分析上皮标志物、间充质标志物以及 TGF-β/Smad 信号通路相关的一些基因/蛋白的表达。Boyden 室和划痕实验用于评估 RPE 细胞的迁移，而 MTT 和 TUNEL 实验分别用于测量 RPE 细胞的活力和凋亡。
结果
用 TM 和 TG（24 小时）处理 RPE 细胞，在蛋白水平上，呈剂量依赖性地降低 α-SMA 和 FN 的表达，增加 Occludin 的表达，这与 GRP78 的表达高度相关。用 TGF-β2 处理，在蛋白和 mRNA 水平上，均显著增加 α-SMA 和 FN 的表达，降低 Occludin 的表达，而 TM 的 4 小时预处理显著抑制了这种表达。此外，TM 处理还降低了 TGF-βRII 和 Smad2/3 的表达，以及 TGF-βRII 和 Smad3 的 mRNA 表达。TM 诱导的 ER 应激抑制了 RPE 细胞的迁移，且高浓度的 TM 和 TG 降低了 RPE 细胞的活力，并诱导了其凋亡。
结论
化学诱导的 ER 应激抑制了 RPE 细胞的 EMT 和迁移，可能是通过 TGF-β 信号失活实现的，这表明调节 RPE 细胞中的 ER 应激可能是防止眼内纤维化发展的新方法。
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