背景:视网膜色素上皮(RPE)细胞的上皮-间质转化(EMT)与包括年龄相关性黄斑变性(AMD)在内的各种视网膜病变的发病机理有关。氧化应激是招致RPE细胞变性的主要身分,与AMD的病因学相干。
目的:碘酸钠(NaIO3)产生细胞内活性氧(ROS),由于选择性诱导视网膜变性而被广泛用于建立AMD模型。进行这项研究以阐明多个NaIO3刺激的信号通路对RPE细胞中EMT的影响。
方法:分析了NaIO3处理的人ARPE-19细胞和小鼠眼睛的RPE细胞中的EMT特征。研究了多种氧化应激诱导的调节剂,以及用Ca2螯合剂预处理的效果,细胞外信号相关激酶(ERK)抑制剂,或表皮生长因子受体(EGFR)抑制剂对NaIO3诱导的EMT进行了测定。解剖了ERK抑制剂后处理对NaIO3诱导的信号通路调节的功效,并通过使用组织学横截面和谱域光学相干断层扫描评估了其在视网膜厚度和形态中的作用。
结果:我们发现NaIO3在小鼠眼睛的ARPE-19细胞和RPE细胞中诱导EMT。细胞内ROS,Ca2+,内质网(ER)应激标记,磷酸化ERK,和磷酸EGFR在NaIO3刺激的细胞中增加。我们的结果表明,用Ca2+螯合剂预处理,ERK抑制剂,或EGFR抑制剂降低NaIO3诱导的EMT,有趣的是,ERK的抑制表现出最显著的效果。此外,用特定的ERK抑制剂FR180204治疗后,降低细胞内ROS和Ca2+水平,下调的磷酸-EGFR和ER应激标志物,减毒的RPE细胞的EMT,并恢复了NaIO3诱导的视网膜结构紊乱。
结论:ERK是协调RPE细胞中EMT程序的多个NaIO3诱导的信号通路的关键调节因子。抑制ERK可能是治疗AMD的潜在治疗策略。
BACKGROUND: Epithelial-mesenchymal transition (EMT) of retinal pigment epithelial (RPE) cells is related to the pathogenesis of various retinopathies including age-related macular degeneration (AMD). Oxidative stress is the major factor that induces degeneration of RPE cells associated with the etiology of AMD.
OBJECTIVE: Sodium iodate (NaIO3) generates intracellular reactive oxygen species (ROS) and is widely used to establish a model of AMD due to the selective induction of retinal degeneration. This study was performed to clarify the effects of multiple NaIO3-stimulated signaling pathways on EMT in RPE cells.
METHODS: The EMT characteristics in NaIO3-treated human ARPE-19 cells and RPE cells of the mouse eyes were analyzed. Multiple oxidative stress-induced modulators were investigated and the effects of pre-treatment with Ca2+ chelator, extracellular signal-related kinase (ERK) inhibitor, or epidermal growth factor receptor (EGFR) inhibitor on NaIO3-induced EMT were determined. The efficacy of post-treatment with ERK inhibitor on the regulation of NaIO3-induced signaling pathways was dissected and its role in retinal thickness and morphology was evaluated by using histological cross-sections and spectral domain optical coherence tomography.
RESULTS: We found that NaIO3 induced EMT in ARPE-19 cells and in RPE cells of the mouse eyes. The intracellular ROS, Ca2+, endoplasmic reticulum (ER) stress marker, phospho-ERK, and phospho-EGFR were increased in NaIO3-stimulated cells. Our results showed that pre-treatment with Ca2+ chelator, ERK inhibitor, or EGFR inhibitor decreased NaIO3-induced EMT, interestingly, the inhibition of ERK displayed the most prominent effect. Furthermore, post-treatment with FR180204, a specific ERK inhibitor, reduced intracellular ROS and Ca2+ levels, downregulated phospho-EGFR and ER stress marker, attenuated EMT of RPE cells, and prevented structural disorder of the retina induced by NaIO3.
CONCLUSIONS: ERK is a crucial regulator of multiple NaIO3-induced signaling pathways that coordinate EMT program in RPE cells. Inhibition of ERK may be a potential therapeutic strategy for the treatment of AMD.