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

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 real-time 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.

Elucidating the mechanism of action of alpha-1-antitrypsin using retinal pigment epithelium cells exposed to high glucose. Potential use in diabetic retinopathy

“…Different types of stress, like proteotoxic stress, or high glucose produce EMT in RPE cells [71,72]. In our work, we tested different pathways involved in this process that modulate expression of proteins that are vital to this process, like Akt/mTOR, and the Wnt canonical pathway.…”

Polarity and epithelial-mesenchymal transition of retinal pigment epithelial cells in proliferative vitreoretinopathy

“…Moreover, the ERK MAPK pathway plays a role in TGF-β-induced EMT and cooperates with other signaling pathways in the regulation of EMT in RPE cells. Recent studies ( Chen et al, 2014b ; Tan et al, 2017 ; Xiao et al, 2014 ) have shown that blocking the ERK1/2 pathway inhibits the phosphorylation of SMAD2 and the Jagged/Notch pathway. Inhibition of the Jagged/Notch signaling pathway can alleviate TGF-β2-induced EMT by regulating the expression of Snail, Slug and Zeb1 ( Fig.…”

The Association of CEP135 rs4865047 and NPY2R rs1902491 Single Nucleotide Polymorphisms (SNPs) with Rapid Progression of Proliferative Diabetic Retinopathy in Patients with Type 1 Diabetes Mellitus

“…CEP135 interacts with SMAD family member 9 (SMAD9), which is involved in transforming growth factor-β (TGF-β) signaling. During the development of proliferative diabetic retinopathy, TGF-β is significantly upregulated in the aqueous humor and vitreous body [19]. SNP rs4865047 is also 50.5 kb downstream of the exocyst complex component 1 gene, EXOC1 , and the exocyst complex is required for the insulin-stimulated transport of the glucose transporter type 4 (Glut4) to the plasma membrane [14].…”