Proteotoxic Stress Desensitizes TGF-beta Signaling Through Receptor Downregulation in Retinal Pigment Epithelial Cells.
Proteotoxic Stress Desensitizes TGF-beta Signaling Through Receptor Downregulation in Retinal Pigment Epithelial Cells.
复制标题
蛋白毒性应激通过视网膜色素上皮细胞中受体下调使 TGF-β 信号变得不敏感
DOI：
10.2174/1566524017666170619113435
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发表时间：
2017
影响因子：
2.5
通讯作者：
Liu Y
中科院分区：
文献类型：
--
作者：
Tan X;Chen C;Zhu Y;Deng J;Qiu X;Huang S;Shang F;Cheng B;Liu Y
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诱因，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沟通尚不清楚。 目的是研究蛋白毒性应激对视网膜色素上皮中 TGFβ 信号传导的调节作用。 ARPE-19 细胞和原代人视网膜色素上皮 (RPE) 细胞用蛋白酶体抑制剂 MG132 和 TGFβ 处理。通过CCK-8测定分析细胞增殖。通过实时聚合酶链反应（PCR）、蛋白质印迹和免疫荧光分析间充质标志物α-SMA、纤连蛋白和波形蛋白的水平。通过划痕实验分析细胞迁移。通过蛋白质印迹分析 p-Smad2、总 Smad2、p-细胞外信号调节激酶 1/2 (ERK1/2)、总 ERK1/2、p-焦点粘附激酶 (FAK) 和总 FAK 的水平。分别通过实时PCR和蛋白质印迹测量TGFβ受体-II (TGFβR-II)的mRNA和蛋白水平。 MG132 诱导的蛋白毒性应激导致细胞增殖减少。 MG132 显着抑制 TGFβ 诱导的 α-SMA、纤连蛋白和波形蛋白的上调，以及 TGFβ 诱导的细胞迁移。 Smad2、ERK1/2 和 FAK 的磷酸化水平也被 MG132 抑制。此外，MG132 治疗后 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。
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 real-time 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.