The Stable Repression of Mesenchymal Program Is Required for Hepatocyte Identity: A Novel Role for Hepatocyte Nuclear Factor 4α

The concept that cellular terminal differentiation is stably maintained once development is complete has been questioned by numerous observations showing that differentiated epithelium may undergo an epithelial-to-mesenchymal transition (EMT) program. EMT and the reverse process, mesenchymal-to-epithelial transition (MET), are typical events of development, tissue repair, and tumor progression. In this study, we aimed to clarify the molecular mechanisms underlying these phenotypic conversions in hepatocytes. Hepatocyte nuclear factor 4 alpha (HNF4 alpha) was overexpressed in different hepatocyte cell lines and the resulting gene expression profile was determined by real-time quantitative polymerase chain reaction. HNF4 alpha recruitment on promoters of both mesenchymal and EMT regulator genes was determined by way of electrophoretic mobility shift assay and chromatin immunoprecipitation. The effect of HNF4 alpha depletion was assessed in silenced cells and in the context of the whole liver of HNF4 knockout animals. Our results identified key EMT regulators and mesenchymal genes as new targets of HNF4 alpha. HNF4 alpha, in cooperation with its target HNF1 alpha, directly inhibits transcription of the EMT master regulatory genes Snail, Slug, and HMGA2 and of several mesenchymal markers. HNF4 alpha-mediated repression of EMT genes induces MET in hepatomas, and its silencing triggers the mesenchymal program in differentiated hepatocytes both in cell culture and in the whole liver. Conclusion: The pivotal role of HNF4 alpha in the induction and maintenance of hepatocyte differentiation should also be ascribed to its capacity to continuously repress the mesenchymal program; thus, both HNF4 alpha activator and repressor functions are necessary for the identity of hepatocytes. (HEPATOLOGY 2011;53:2063-2074)