Abstract
Purpose:
The proliferation, migration and epithelial-mesenchymal transition (EMT) of retinal pigment epithelial (RPE) cells play key roles in proliferative vitreoretinopathy (PVR), a catastrophic complication secondary to retinal detachment surgery which can lead to profound visual loss. Previous studies have shown that the durability of the ubiquitin proteasome pathway is a potential contributor to cell proliferation, migration and EMT. This study was conducted to investigate suppression of PVR development through the usage of proteasome inhibitor MG132.
Methods:
Human retinal pigment epithelial ARPE-19 cells were treated with proteasome inhibitor MG132 (2.5, 5, 10, 20, or 50μM) for 24h, 48h or 72h. Cell proliferation was determined using the CCK-8 reagent. Cell cycle and cell apoptosis were analyzed through propidium iodide (PI) and Annexin V-PI staining using cell flow cytometry. Cell migration was tested by cell scratch assay. Also, ARPE-19 cells were treated with transforming growth factor-β (TGF-β) alone or plus MG132 for 24h, 48h or 72h. Cell morphology was observed with phase-contrast microscope. The expression of EMT markers was determined by RT-PCR, western blot and immunofluorescence.
Results:
Compared with control group, cell proliferation was greatly suppressed by MG132 at 24h, 48h and 72h. Cell cycle was delayed and apoptosis rate was increased after MG132 treatment. Cell scratch assay indicated that the number of APRE cells migrated into the wounded area was lower in MG132 treated group. TGF-β treatment caused cells converted to a fibroblast-like shape, and upregulated the expression of EMT markers (α-SMA, fibronectin, N-cadherin, vimentin and Collagen IV). Inhibition of proteasome reversed TGF-β-induced cell change to a mesenchymal phenotype, and downregulated the expression of EMT markers.
Conclusions:
These data suggest that proteasome inhibition decreases the proliferation, migration and EMT of APRE-19 cells. These findings indicate that proteasome inhibitor MG132 may be an attractive candidate for blocking development of PVR.
Keywords: 655 proliferative vitreoretinopathy •
654 proliferation •
512 EMT (epithelial mesenchymal transition)