title: Implication of Smad2 and Smad3 in transforming growth factor-β-induced posterior capsular opacification of human lens epithelial cells.
abstract: OBJECTIVE
  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.affiliation: Clinical College of Ophthalmology, Tianjin Eye Hospital, Tianjin Medical University , Tianjin , China.
authors.forename: Hua
authors.initials: H
authors.lastname: Li
authors.forename: Xiaoyong
authors.initials: X
authors.lastname: Yuan
authors.forename: Jun
authors.initials: J
authors.lastname: Li
authors.forename: Xin
authors.initials: X
authors.lastname: Tang
mesh_terms: D015153:Blotting, Western; D058442:Capsule Opacification / Q000139:chemically induced / Q000378:metabolism* / Q000473:pathology; D002460:Cell Line; D002465:Cell Movement / Q000187:drug effects; D002470:Cell Survival; D024042:Collagen Type I / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D004847:Epithelial Cells / Q000187:drug effects* / Q000378:metabolism; D058750:Epithelial-Mesenchymal Transition / Q000187:drug effects; D005353:Fibronectins / Q000378:metabolism; D005434:Flow Cytometry; D019084:Fluorescent Antibody Technique, Indirect; D057809:HEK293 Cells; D006801:Humans; D007908:Lens, Crystalline / Q000166:cytology; D010957:Plasmids / Q000235:genetics; D057852:Posterior Capsule of the Lens / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D060888:Real-Time Polymerase Chain Reaction; D015398:Signal Transduction; D051899:Smad2 Protein / Q000502:physiology*; D051900:Smad3 Protein / Q000502:physiology*; D014162:Transfection; D053781:Transforming Growth Factor beta2 / Q000494:pharmacology*
chemical_list: D024042:Collagen Type I; D005353:Fibronectins; C497782:SMAD2 protein, human; C497790:SMAD3 protein, human; D051899:Smad2 Protein; D051900:Smad3 Protein; D053781:Transforming Growth Factor beta2
keywords: Human lens epithelial cells; Smad2; Smad3; posterior capsular opacification; transforming growth factor-β2