The Ubiquitin Conjugating Enzyme, UbcH7, Affects Both Migration and the Epithelial to Mesenchymal Transition in Lens Epithelial Cells:A Promising Drug Target for Secondary Cataracts.
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Deubiquitinating enzyme
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Epithelial-mesenchymal transition (EMT) of human lens epithelial cells (LECs) contributes to posterior capsule opacification (PCO). C-terminal binding protein 2 (CtBP2) has been reported to be essential in EMT and embryonic development. However, the function of CtBP2 in EMT of LECs is unknown. The goal of this study was to investigate the role of CtBP2 through Notch signaling in transforming growth factor β2 (TGFβ2)-induced EMT in LECs.The human LEC line SRA01/04 was cultured in the presence of TGFβ2 for different periods of time or with γ-Secretase Inhibitor IX (DAPT), a specific inhibitor of Notch receptor cleavage, for 24 h, utilizing plasmid-based method. The levels of protein expression of CtBP2, EMT markers, and Notch signaling molecules were measured by Western bolts.Treatment of SRA01/04 cells with TGFβ2 induced typical molecular changes of EMT and increased the expression of CtBP2 in a time-dependent manner. Similarly, the expressions of Jagged1 and Notch1 were increased after TGFβ2 treatment. Knockdown of CtBP2 by specific siRNA inhibited TGFβ2-induced changes of Connexin 43 (CX43), α-smooth muscle actin (α-SMA), Notch1, and Notch intracellular domain (NICD). In addition, treatment of LECs with ectopic expression of CtBP2 changed the expressions of CX43, α-SMA, Notch1, and NICD, but blockade of Notch pathway with DAPT inhibited CtBP2-induced changes of α-SMA and CX43.Our data suggest that CtBP2 plays a critical role in TGFβ2-induced EMT via the Jagged/Notch signaling pathway in human LECs and may contribute to the development of PCO.
Ectopic expression
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Purpose.: Cataract surgery involves removal of lens tissue, but is associated with a high complication rate due to regrowth of residual lens epithelial cells to produce posterior capsule opacification (PCO) and diminished visual acuity. As inhibitors of aldose reductase (AR) have been shown to suppress markers of PCO, our studies were designed to identify a role for AR in the pathogenesis of PCO. Methods.: Sorbinil-mediated AR inhibition was determined by measuring sorbitol accumulation. Cell migration was measured using both transwell and scratch assays. Proteins in the SMAD signaling pathway were measured by Western blotting. The interactions of AR and SMADs were demonstrated by co-immunoprecipitation (Co-IP) and proximity ligation assay (PLA). Epithelial-to-mesenchymal transition (EMT) expression was measured by Western blot and quantitative PCR (q-PCR). Matrix metalloproteinase (MMP)-2 and MMP-9 activities were measured in conditioned medium by zymography. Results.: We observed that either Sorbinil-mediated AR inhibition or siRNA-mediated AR gene knockdown prevented migration of lens epithelial cells following exposure to TGF-β2. AR inhibition or AR knockdown reduced SMAD and MMP activation triggered by TGF-β2. In addition, we demonstrated AR inhibition or AR knockdown decreased TGF-β2–induced expression of EMT markers. Co-IP studies and PLA were used to demonstrate that AR and SMAD2 interact either directly or in close concert with additional factor(s) in a nonenzymatic manner. Conclusions.: This study demonstrates that AR participates in the response of lens epithelial cells to TGF-β2. Our studies raise the possibility that AR inhibition may be effective in preventing development of PCO by disrupting the TGF-β2/SMAD pathway.
Sorbinil
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