ABSTRACT: PURPOSE: Continuous culture of limbal epithelial stem cells (LSCs) slows down proliferation, which inevitably results in differentiation. Transforming growth factor-beta (TGF?)-assisted epithelial-mesenchymal transition (EMT) is often found in the late stage of LSC culture. Thus, EMT is proposed to be part of the mechanism responsible for the loss of LSCs in culture. To explore the regulation mechanism of EMT, we investigated the early stage culture for factor(s) that may potentially prevent EMT. METHODS: LSCs from the corneal limbus region of rabbits were isolated and expanded to confluence in culture (P0), and then serial passage of these LSCs (P1 to P3) was performed. EMT in LSCs was induced with TGF?1, and the corresponding EMT signaling was confirmed with Smad2/3 phosphorylation. The expression of mesenchymal markers, including alpha-smooth muscle actin (?-SMA) and vimentin, was determined with western blot analysis. Proteins extracted from different passaged cells were also subjected to western blot analysis of TGF? signaling components, including TGF?1, TGF? receptor I/II, and Smad2/3 as well as Smad7, the main negative regulator of TGF? signaling. The mitogenic response was measured with the bromodeoxyuridine (BrdU) labeling index and real-time PCR using primers for Ki67. N-(N-[3,5-difluorophenacetyl]-l-alanyl)-S-phenylglycine t-butyl ester (DAPT), a gamma-secretase inhibitor, and Jagged-1 Notch ligand were used to block and activate Notch signaling, respectively, and their efficacy was evaluated by determining the expression of Hes1, a Notch signaling target. RESULTS: Mesenchymal marker induction and growth arrest were found in the TGF?1-treated P1 cells, and the changes were less significant in the TGF?1-treated P0 cells. Western blot analysis confirmed that the expressed levels of TGF? signaling components, including TGF?1, TGF? receptor I/II, and Smad2/3, were relatively stable with passages. In contrast, the expression of Hes1 and Smad7 markedly decreased after the first passage, and with each passage, the levels diminished even further. Hes1 and Smad7 were expressed only in the limbal epithelium and not in the corneal epithelium. DAPT effectively blocked the expression of Hes1. DAPT also dose-dependently suppressed Smad7 expression in P0 cells, which was associated with the susceptibility of P0 cells to TGF?1-induced Smad2/3 phosphorylation, EMT formation, and growth arrest. Reciprocally, Jagged-1 upregulated Smad7 expression in LSCs against TGF? signaling. CONCLUSIONS: These findings indicate that Smad7 plays a crucial role in antagonizing EMT induced by TGF? signaling and support our proposition that Smad7 is a Notch signaling target in LSCs, and may mediate the Notch function in preventing the occurrence of EMT.
