Hua Li1, Xiaoyong Yuan, Jun Li, Xin Tang. 1. Clinical College of Ophthalmology, Tianjin Eye Hospital, Tianjin Medical University , Tianjin , China.
Abstract
PURPOSE: Transforming growth factor-β2 (TGF-β2) is a potent inducer of posterior capsular opacification (PCO), a critical Smad-dependent event. This study was conducted to investigate the contributions of Smad2 and Smad3 to PCO development based on selective over-expression of either Smad2 or Smad3. METHODS: We selectively activated the TGF-β/Smad pathway in cell lines transfected with expression plasmids containing Smad2 or Smad3. These cell lines were then analyzed to determine the individual contributions of Smad2 and Smad3 to TGF-β2 treatment response in an in vitro culture of HLE B-3 cells. The effects of Smad2 and Smad3 on cell viability were assessed by MTT and flow cytometry assay. A transwell assay was used to observe the role of Smad2 and Smad3 in the migration of HLE B-3 cells. Western blotting, real-time PCR, and immunocytofluorescence staining were performed to detect the accumulation of ECM proteins and EMT in response to selective Smad2 or Smad3 activation. The presence of soluble collagen I, and fibronectin in the culture medium supernatant were detected by ELISA. RESULTS: Selective Smad3 activation via gene transfection enhanced TGF-β2-responsive growth inhibition and apoptosis. Transwell assay results showed that TGF-β2-induced cell migration was Smad2 dependent and Smad3 independent. Analysis by Western blot, RT-PCR and ELISA demonstrated that the determinant factor in ECM secretion was Smad3 signaling rather than Smad2 signaling. Western blot and RT-PCR showed that the loss of E-Cadherin and acquisition of α-SMA, the hallmark of epithelial-mesenchymal transition (EMT), were both reliant on Smad2 signaling. Immunocytofluorescence staining confirmed the role of Smad2 in the accumulation of α-SMA. CONCLUSIONS: Smad2 and Smad3 are both necessary for the formation of PCO. The discovery of additional TGF-β2/Smad signaling mechanisms may provide potential therapeutic targets to help combat PCO.
PURPOSE: Transforming growth factor-β2 (TGF-β2) is a potent inducer of posterior capsular opacification (PCO), a critical Smad-dependent event. This study was conducted to investigate the contributions of Smad2 and Smad3 to PCO development based on selective over-expression of either Smad2 or Smad3. METHODS: We selectively activated the TGF-β/Smad pathway in cell lines transfected with expression plasmids containing Smad2 or Smad3. These cell lines were then analyzed to determine the individual contributions of Smad2 and Smad3 to TGF-β2 treatment response in an in vitro culture of HLE B-3 cells. The effects of Smad2 and Smad3 on cell viability were assessed by MTT and flow cytometry assay. A transwell assay was used to observe the role of Smad2 and Smad3 in the migration of HLE B-3 cells. Western blotting, real-time PCR, and immunocytofluorescence staining were performed to detect the accumulation of ECM proteins and EMT in response to selective Smad2 or Smad3 activation. The presence of soluble collagen I, and fibronectin in the culture medium supernatant were detected by ELISA. RESULTS: Selective Smad3 activation via gene transfection enhanced TGF-β2-responsive growth inhibition and apoptosis. Transwell assay results showed that TGF-β2-induced cell migration was Smad2 dependent and Smad3 independent. Analysis by Western blot, RT-PCR and ELISA demonstrated that the determinant factor in ECM secretion was Smad3 signaling rather than Smad2 signaling. Western blot and RT-PCR showed that the loss of E-Cadherin and acquisition of α-SMA, the hallmark of epithelial-mesenchymal transition (EMT), were both reliant on Smad2 signaling. Immunocytofluorescence staining confirmed the role of Smad2 in the accumulation of α-SMA. CONCLUSIONS:Smad2 and Smad3 are both necessary for the formation of PCO. The discovery of additional TGF-β2/Smad signaling mechanisms may provide potential therapeutic targets to help combat PCO.
Authors: Mahbubul H Shihan; Mallika Kanwar; Yan Wang; Erin E Jackson; Adam P Faranda; Melinda K Duncan Journal: Matrix Biol Date: 2020-03-12 Impact factor: 11.583
Authors: Leonid M Zukin; Michelle G Pedler; Sergio Groman-Lupa; Mina Pantcheva; David A Ammar; J Mark Petrash Journal: Invest Ophthalmol Vis Sci Date: 2018-07-02 Impact factor: 4.799
Authors: Mahbubul H Shihan; Samuel G Novo; Yan Wang; Dean Sheppard; Amha Atakilit; Thomas D Arnold; Nicole M Rossi; Adam P Faranda; Melinda K Duncan Journal: JCI Insight Date: 2021-11-08