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

Background: Proteotoxic stress and transforming growth factor (TGF beta)-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 TGF beta signaling in retinal pigment epithelium. Methods: ARPE-19 cells and primary human retinal pigment epithelial (RPE) cells were treated with proteasome inhibitor MG132 and TGF beta. Cell proliferation was analyzed by CCK-8 assay. The levels of mesenchymal markers alpha-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 TGF beta receptor-II (TGF beta R-II) were measured by real-time PCR and western blot, respectively. Results: MG132-induced proteotoxic stress resulted in reduced cell proliferation. MG132 significantly suppressed TGF beta-induced upregulation of alpha-SMA, fibronectin, and vimentin, as well as TGF beta-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 TGF beta R-II decreased upon MG132 treatment. Conclusion: Proteotoxic stress suppressed TGF beta-induced EMT through downregulation of TGF beta R-II and subsequent blockade of Smad2, ERK1/2, and FAK activation.