Abstract
Purpose :
Posterior Capsular Opacification (PCO) or secondary cataract is a common postoperative complication following cataract surgery that can result in blindness. Previous reports have shown that TGFβ-induced Epithelial to Mesenchymal Transition (EMT) is involved in PCO. However, the signaling events that cause observable cytoskeletal changes during TGFβ-induced PCO remains poorly understood. Here, we have examined the distribution of fascin, an actin bundling protein, during TGFb-induced EMT in lens epithelial cells and also relate it to known regulators of EMT, Matrix Metalloproteinase 9 (MMP9) and β-catenin.
Methods :
Wistar rats, 17-19 days old, or wild-type and MMP9 knock-out mice (MMP9KO) at 1.5-2 months old were utilized to obtain lens epithelial cell explants. Explants were cultured in medium M199 without serum and with or without TGFβ2, and TGFβ2 in combination with the β-catenin inhibitor (PNU-74654), that inhibits the interaction between β-catenin and T-cell factor (TCF) thereby inhibiting expression of downstream genes regulated by wnt/β-catenin signaling pathway, for 48 hrs. Western blot and Immunofluorescence analysis was carried for the treated and untreated rat lens explants.
Results :
An increase in the expression of fascin was observed in rat lens epithelial explants upon stimulation with TGFβ as determined by western blot and Immunofluorescence analysis. Furthermore, fascin was organized into filaments that co-localized with F-actin. Inhibition of β-catenin led to a decrease in the expression of TGFb-induced fascin and F-actin. Importantly, the lack of MMP9 in MMP9 KO mouse explants resulted in a complete abrogation in TGFb-induced fascin expression and actin polymerization. Finally, inhibition of β-catenin also led to a decrease in TGFb-induced expression of active MMP-9 and E-cadherin degradation.
Conclusions :
A lack of induction in fascin along with decrease in the stress-fiber formation upon MMP9 knock down demonstrates the requirement of MMP9 in mediating TGFb-induced fascin during EMT. The fact that inhibition of β-catenin also led to suppression in TGFb-induced fascin suggests that it is linked with MMP9 in inducing stress fiber formation in lens epithelial explants during EMT.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.