Implication Of Smad2and Smad3in Transforming Growth Factor-β Induced Posterior Capsular Opacification Of Human Lens Epithelial Cells

PurposePosterior capsular opacification (PCO), which is the common complication of cataract surgery, usually lead to further vision loss. Transforming growth factor-β2(TGF-β2) is a potent inducer of PCO, a critical Smad-dependent event. This study was conducted to investigate the regulation of Smad2and Smad3in human lens epithelial cells growth, apoptosis, migration, accumulation of extracellular matrix(ECM), epithelial-mesenchymal transition (EMT), which was based on selective over-expression of either Smad2or Smad3by expression plasmid.MethodsWe have chosen pcDNA3to be the vector, and contsructed expression plasmid of Smad2and Smad3successfully. We selectively activated the TGF-β/Smad pathway in cell lines transfected with expression plasmids containing Smad2or Smad3. These cell lines were then analyzed to determine the individual contributions of Smad2and Smad3to TGF-β2treatment response in an in vitro culture of HLE B-3cells. The effects of Smad2and Smad3on cell viability were assessed by MTT and flow cytometry assay. A transwell assay was used to observe the role of Smad2and Smad3in the migration of HLE B-3cells. Western blotting, real-time PCR, and immunocytofluorescence staining were performed to detect the accumulation of ECM proteins and EMT in response to selective Smad2or Smad3activation. The contents of soluble collagen Ⅰ, and fibronectin in the culture medium supernatant were detected by ELISA.Results1. We have constructed expression plasmid of Smad2and Smad3successfully, the resulting constructs were confirmed by DNA sequencing, then amplified and purified for transfection.2. Selective Smad3activation via gene transfection enhanced TGF-β2-responsive growth inhibition and apoptosis. Analysis by Western blot, RT-PCR and ELISA demonstrated that the determinant factor in ECM secretion was Smad3signaling rather than Smad2signaling. 3. Transwell assay results showed that TGF-β2-induced cell migration was Smad2dependent and Smad3independent. 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 Smad2signaling. Immunocytofluorescence staining confirmed the role of Smad2in the accumulation of a-SMA.ConclusionsSmad2and Smad3are both necessary for the formation of PCO. Smad2plays an important role in EMT and cell migration, Smad3is involved in the cell apoptosis and accumulation of ECM. The discovery of additional TGF-β2/Smad signaling mechanisms may provide potential therapeutic targets to help combat PCO.