Notch‐dependent expression of epithelial–mesenchymal transition markers in cholangiocytes after liver transplantation

Abstract

Epithelial-mesenchymal transition (EMT) has been identified in chronic cholestatic liver diseases, which are characterized by biliary proliferation and fibrosis. Activation of Notch signaling mediates EMT in a variety of epithelial cell types. In the present study, we investigated the role of Notch signaling in the regulation of EMT marker expression in cholangiocytes after liver transplantation. Orthotopic liver transplantation was performed in Sprague-Dawley rats. Liver tissues and isolated cholangiocytes were collected 1 week after transplantation. The expression of mesenchymal and biliary epithelial markers was evaluated by immunohistochemistry, quantitative polymerase chain reaction (PCR) and western blotting in liver sections and isolated cholangiocytes. Quantitative real-time PCR and western blotting for Jagged1 and HES1 were utilized to evaluate the activation of Notch signaling. Proliferation and migration of cholangiocytes were assessed by 5-bromodeoxyuridine and transwell assays, respectively. Cholangiocyte proliferation, migration and expression of EMT markers were also evaluated following the inhibition of Notch signaling with N,(N-[3,5-difluorophenacetyl]-L-alanyl)-S-phenylglycine t-butylester (γ-secretase inhibitor) and a Jagged1-neutralizing antibody. Expression of EMT markers by cholangiocytes was observed in liver grafts and isolated cholangiocytes obtained 1 week after transplantation. Inhibition of Notch signaling prevented the expression of EMT markers in bile ducts of liver sections and isolated cholangiocytes. Cholangiocyte proliferative and migratory capacities were also suppressed by the inhibition of Notch signaling. Activation of Notch signaling promotes cholangiocyte proliferation and expression of EMT markers after liver transplantation.