Proteotoxic Stress Desensitizes TGF-beta Signaling Through Receptor Downregulation in Retinal Pigment Epithelial Cells

Author(s): X. Tan, C. Chen, Y. Zhu, J. Deng, X. Qiu, S. Huang, F. Shang, B. Cheng*, Y. Liu*.

Journal Name:Current Molecular Medicine

Volume 17 , Issue 3 , 2017

Abstract:

Background: 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.

Objective: The aim was to investigate the regulatory role of proteotoxic stress on TGFβ signaling in retinal pigment epithelium.

Methods: 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.

Results: 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.

Conclusion: Proteotoxic stress suppressed TGFβ-induced EMT through downregulation of TGFβR-II and subsequent blockade of Smad2, ERK1/2, and FAK activation.

Keywords: Proteotoxic stress, retinal pigment epithelium, transforming growth factor, epithelial-mesenchymal transition, proliferative vitreoretinopathy, diabetic retinopathy.

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Article Details

VOLUME: 17
ISSUE: 3
Year: 2017
Page: [189 - 199]
Pages: 11
DOI: 10.2174/1566524017666170619113435