Expression of Smad7 inhibits fibrogenic responses of keratocytes to transforming growth factor β2

Background Transforming growth factor β (TGFβ) is one of the most important growth factors in the development offibrosis and scarring on cornea. Smad7, an inhibitory Smad, can inhibit TGFβ signal transduction. In recent years, effectsof lentiviral-mediated Smad7 on inhibition of fibrosis on some organs have been studied, while little is known about theeffects on cornea. This study aimed to determine the effects of lentiviral-mediated Smad7 gene expression on keratocyteproliferation and fibrosis induced by TGF β2 in vitro.Methods Keratocytes were cultured from corneal tissue isolated from Sprague-Dawley (SD) rats and transfected withSmad7 expressing lentiviral vector (Lv-Smad7) or non-functioning control vector (Lv-blank). Following the exposure toTGFβ2, keratocytes were processed for immunoblotting to assess the phosphorylation of Smad2 as down-stream eventof 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 proliferationwas determined by 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and the expression of cellcycle-related marker Ki67 at both mRNA and protein levels.Results The Smad7 gene transfer suppressed TGFβ/Smad signaling in keratocytes by down-regulatingphosphorylation of Smad2. Markers of cell proliferation and fibrosis including Ki67, α-SMA, collagen Ⅲ were inhibited byintroduction of Smad 7 into TGFβ exposed keratocytes. Consequently, the rate of cell proliferation was attenuated.Conclusion Smad7 gene transfer inhibited fibrogenic responses of keratocytes to TGFβ2.