Tan X, Chen C, Zhu Y, Deng J, Qiu X, Huang S, Shang F, Cheng B, Liu Y
State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, Guangdong. China.
State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, 54 South Xianlie Road, Guangzhou, 510060. China.
Curr Mol Med. 2017;17(3):189-199. doi: 10.2174/1566524017666170619113435.
Proteotoxic stress and transforming growth factor (TGFβ)- induced epithelial-mesenchymal transition (EMT) are two main contributors of intraocular fibrotic disorders, including proliferative vitreoretinopathy (PVR) and proliferative diabetic retinopathy (PDR). However, how these two factors communicate with each other is not well-characterized.
The aim was to investigate the regulatory role of proteotoxic stress on TGFβ signaling in retinal pigment epithelium.
ARPE-19 cells and primary human retinal pigment epithelial (RPE) cells were treated with proteasome inhibitor MG132 and TGFβ. Cell proliferation was analyzed by CCK-8 assay. The levels of mesenchymal markers α-SMA, fibronectin, and vimentin were analyzed by real-time polymerase chain reaction (PCR), western blot, and immunofluorescence. Cell migration was analyzed by scratch wound assay. The levels of p-Smad2, total Smad2, p-extracellular signal-regulated kinase 1/2 (ERK1/2), total ERK1/2, p-focal adhesion kinase (FAK), and total FAK were analyzed by western blot. The mRNA and protein levels of TGFβ receptor-II (TGFβR-II) were measured by realtime PCR and western blot, respectively.
MG132-induced proteotoxic stress resulted in reduced cell proliferation. MG132 significantly suppressed TGFβ-induced upregulation of α-SMA, fibronectin, and vimentin, as well as TGFβ-induced cell migration. The phosphorylation levels of Smad2, ERK1/2, and FAK were also suppressed by MG132. Additionally, the mRNA level and protein level of TGFβR-II decreased upon MG132 treatment.
Proteotoxic stress suppressed TGFβ-induced EMT through downregulation of TGFβR-II and subsequent blockade of Smad2, ERK1/2, and FAK activation.
蛋白毒性应激和转化生长因子（TGFβ）诱导的上皮-间质转化（EMT）是眼内纤维化疾病的两个主要促成因素，包括增殖性玻璃体视网膜病变（PVR）和增殖性糖尿病视网膜病变（PDR）。然而，这两个因素如何相互作用尚未得到充分表征。
旨在研究蛋白毒性应激对视网膜色素上皮细胞中TGFβ信号传导的调节作用。
用蛋白酶体抑制剂MG132和TGFβ处理ARPE-19细胞和原代人视网膜色素上皮（RPE）细胞。通过CCK-8测定法分析细胞增殖。通过实时聚合酶链反应（PCR）、蛋白质免疫印迹和免疫荧光分析间充质标志物α-平滑肌肌动蛋白（α-SMA）、纤连蛋白和波形蛋白的水平。通过划痕试验分析细胞迁移。通过蛋白质免疫印迹分析磷酸化Smad2、总Smad2、磷酸化细胞外信号调节激酶1/2（ERK1/2）、总ERK1/2、磷酸化粘着斑激酶（FAK）和总FAK的水平。分别通过实时PCR和蛋白质免疫印迹测量TGFβ受体II（TGFβR-II）的mRNA和蛋白质水平。
MG132诱导的蛋白毒性应激导致细胞增殖减少。MG132显著抑制TGFβ诱导的α-SMA、纤连蛋白和波形蛋白上调，以及TGFβ诱导的细胞迁移。MG132还抑制了Smad2、ERK1/2和FAK的磷酸化水平。此外，MG132处理后TGFβR-II的mRNA水平和蛋白质水平降低。
蛋白毒性应激通过下调TGFβR-II并随后阻断Smad2、ERK1/2和FAK激活来抑制TGFβ诱导的EMT。
