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
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.