Genetic Labeling Does Not Detect Epithelial-to-Mesenchymal Transition of Cholangiocytes in Liver Fibrosis in Mice

Abstract

Chronic injury changes the fate of certain cellular populations, inducing epithelial cells to generate fibroblasts by epithelial-to-mesenchymal transition (EMT) and mesenchymal cells to generate epithelial cells by mesenchymal-to-epithelial transition (MET). Although contribution of EMT/MET to embryogenesis, renal fibrosis, and lung fibrosis is well documented, role of EMT/MET in liver fibrosis is unclear. We determined whether cytokeratin-19 positive (K19(+)) cholangiocytes give rise to myofibroblasts (EMT) and/or whether glial fibrillary acidic protein positive (GFAP(+)) hepatic stellate cells (HSCs) can express epithelial markers (MET) in response to experimental liver injury. EMT was studied with Cre-loxP system to map cell fate of K19(+) cholangiocytes in K19(YFP) or fibroblast-specific protein-1 (FSP-1)(YFP) mice, generated by crossing tamoxifen-inducible K19(CreERT) mice or FSP-1(Cre) mice with Rosa26(f/f-YFP) mice. MET of GFAP(+) HSCs was studied in GFAP(GFP) mice. Mice were subjected to bile duct ligation or CCl(4)-liver injury, and livers were analyzed for expression of mesodermal and epithelial markers. On Cre-loxP recombination, >40% of genetically labeled K19(+) cholangiocytes expressed yellow fluorescent protein (YFP). All mice developed liver fibrosis. However, specific immunostaining of K19(YFP) cholangiocytes showed no expression of EMT markers alpha-smooth muscle actin, desmin, or FSP-1. Moreover, cells genetically labeled by FSP-1(YFP) expression did not coexpress cholangiocyte markers K19 or E-cadherin. Genetically labeled GFAP(GFP) HSCs did not express epithelial or liver progenitor markers in response to liver injury. EMT of cholangiocytes identified by genetic labeling does not contribute to hepatic fibrosis in mice. Likewise, GFAP(Cre)-labeled HSCs showed no coexpression of epithelial markers, providing no evidence for MET in HSCs in response to fibrogenic liver injury.