title: Expression of Smad7 inhibits fibrogenic responses of keratocytes to transforming growth factor β2.
abstract: BACKGROUND
  Transforming growth factor β (TGFβ) is one of the most important growth factors in the development of fibrosis and scarring on cornea. Smad7, an inhibitory Smad, can inhibit TGFβ signal transduction. In recent years, effects of lentiviral-mediated Smad7 on inhibition of fibrosis on some organs have been studied, while little is known about the effects on cornea. This study aimed to determine the effects of lentiviral-mediated Smad7 gene expression on keratocyte proliferation and fibrosis induced by TGF β2 in vitro.
  
  
  METHODS
  Keratocytes were cultured from corneal tissue isolated from Sprague-Dawley (SD) rats and transfected with Smad7 expressing lentiviral vector (Lv-Smad7) or non-functioning control vector (Lv-blank). Following the exposure to TGFβ2, keratocytes were processed for immunoblotting to assess the phosphorylation of Smad2 as down-stream event of TGFβ/Smad signaling. Expression of fibrotic markers α-smooth muscle actin (α-SMA), type III collagen (collagen III) were measured by Western blotting and quantitative real time polymerase chain reaction (PCR). Overall cell proliferation was determined by 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and the expression of cell cycle-related marker Ki67 at both mRNA and protein levels.
  
  
  RESULTS
  The Smad7 gene transfer suppressed TGFβ/Smad signaling in keratocytes by down-regulating phosphorylation of Smad2. Markers of cell proliferation and fibrosis including Ki67, α-SMA, collagen III were inhibited by introduction of Smad 7 into TGFβ exposed keratocytes. Consequently, the rate of cell proliferation was attenuated.
  
  
  CONCLUSIONS
  Smad7 gene transfer inhibited fibrogenic responses of keratocytes to TGFβ2.
authors.affiliation: Department of Ophthalmology, Eye & ENT Hospital of Fudan University, Shanghai 200031, China.
authors.forename: Ti
authors.initials: T
authors.lastname: Wang
authors.forename: Xing-tao
authors.initials: XT
authors.lastname: Zhou
authors.forename: Yan
authors.initials: Y
authors.lastname: Yu
authors.forename: Jin-hui
authors.initials: JH
authors.lastname: Dai
authors.forename: Xiao-mei
authors.initials: XM
authors.lastname: Qu
authors.forename: Qi-hua
authors.initials: QH
authors.lastname: LE
authors.forename: Ren-yuan
authors.initials: RY
authors.lastname: Chu
mesh_terms: D000199:Actins / Q000235:genetics / Q000378:metabolism; D000818:Animals; D015153:Blotting, Western; D049109:Cell Proliferation; D002478:Cells, Cultured; D024061:Collagen Type III / Q000235:genetics / Q000378:metabolism; D060527:Corneal Keratocytes / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism*; D005822:Genetic Vectors / Q000235:genetics; D019394:Ki-67 Antigen / Q000235:genetics / Q000378:metabolism; D016086:Lentivirus / Q000235:genetics; D051381:Rats; D017207:Rats, Sprague-Dawley; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction / Q000187:drug effects / Q000235:genetics; D051906:Smad7 Protein / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D053781:Transforming Growth Factor beta2 / Q000494:pharmacology*
chemical_list: D000199:Actins; D024061:Collagen Type III; D019394:Ki-67 Antigen; D051906:Smad7 Protein; D053781:Transforming Growth Factor beta2; C498862:Acta2 protein, rat