Abstract
Purpose :
Posterior Capsular Opacification (PCO) or secondary cataract is a common postoperative complication following cataract surgery that results in blindness. Previous reports show that TGFβ-induced Epithelial to Mesenchymal (myofibroblast) Transition (EMT) is involved in PCO. However, the signaling events that lead to EMT during TGFβ-induced PCO are not fully understood. Here, we have examined the role of interaction between β-catenin and Smad3 in regulation of EMT in TGFβ stimulated LECs. We hypothesize that the interaction between β-catenin and Smad3 is indispensable during TGFβ-induced EMT in lens.
Methods :
Wistar rats 17-19 days old were sacrificed by asphyxiation and their eyes were removed. Lens epithelial explants were prepared by dissecting out the posterior lens fiber cell mass, leaving the anterior epithelium intact on the capsule. Explants were cultured in serum free medium with or without TGF-β2, and TGF-β2 in combination with the β-catenin/CBP interaction inhibitor, ICG-001, or the Smad3 inhibitor, SIS3, for 48 hrs. Western blot and immunofluorescence analysis was carried out and data was statistically analyzed using Graphpad Prism.
Results :
Western blot and Immunofluorescence analyses revealed a decrease in expression of the known EMT marker, αSMA, along with an absence in stress fiber formation upon inhibition of TGFβ-induced Smad3 activation by SIS3. In addition, inhibition of Smad3 activation abrogated TGFβ-induced expression of fascin, an actin bundling protein that facilitates stress fiber formation. Inhibition of β-catenin/CBP-dependent signaling, by ICG-001 as well as inhibition of Smad3 activation suppressed nuclear translocation of myocardin-related transcription factor (MRTFa), the major transcription factor that regulates cytoskeletal remodeling during EMT.
Conclusions :
The fact that inhibition of TGFβ-induced Smad3 activation resulted in a decrease in the expression of αSMA and fascin along with decrease in stress-fiber formation indicates that Smad3 is important for transformation of LECs into myofibroblasts. The absence of nuclear MRTFa upon inhibition of either β-catenin or Smad3 shows the requirement of both β-catenin and Smad3 in translocation of MRTFa to the nucleus.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.