Loss of β-catenin in cholangiocytes promotes hepatocyte reprogramming and vascular remodeling during murine cholestasis.

BACKGROUND & AIMS: The Wnt/β-catenin signaling pathway is critical for liver homeostasis. We have previously shown that hepatocyte β-catenin plays a pleiotropic role in cholestatic injury. However, the role of cholangiocyte β-catenin signaling during cholestasis remains unclear. METHODS: Inducible-Osteopontin (OPN)-Cre-β-catenin-floxed C57BL/6 mice were used in two cholestasis models. Mdr2 knockout (KO)-β-catenin-floxed:OPN-Cre mice were administered tamoxifen to delete β-catenin from cholangiocytes. Wild-type and cholangiocyte β-catenin KO mice were also administered a 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) diet to induce cholestasis. Serum was collected to evaluate liver enzymes. qRT-PCR and immunohistochemistry/immunofluorescence assays were performed on whole livers to assess injury, vascular remodeling, and hepatocyte reprogramming. Livers were isolated for transmission electron microscopy. Isolated cholangiocytes were analyzed by RNA-seq. Cholangiocytes were treated with β-catenin siRNA and lipopolysaccharide in vitro to determine changes in angiogenic factors and NF-κB activation. Conditioned media from cholangiocytes were used to evaluate endothelial cell proliferation in vitro. RESULTS: Mice lacking cholangiocyte β-catenin showed similar levels of hepatobiliary injury compared to controls. We observed more hepatocytes expressing cholangiocyte markers and ductular cells expressing β-catenin in β-catenin KO animals, indicating enhanced hepatocyte reprogramming. Interestingly, cholangiocyte β-catenin KO also had fibrotic hepatic arteries and increased angiogenesis versus controls. Histology and transmission electron microscopy revealed increased basement membrane formation and loss of fenestrations in the sinusoids of β-catenin KO animals. RNA-seq of isolated β-catenin KO cholangiocytes revealed increased expression of angiogenesis pathways that was associated with NF-κB activation. In vitro studies silencing β-catenin in cholangiocytes induced Vegf and Pdgfb expression. Lipopolysaccharide stimulation increased NF-κB nuclear localization in β-catenin-silenced cholangiocytes. Stimulated media from these cells promoted endothelial cell proliferation, recapitulating the angiogenic phenotype found in vivo. CONCLUSIONS: β-catenin signaling in cholangiocytes is a novel mediator of cell-cell communication, and its loss induces a pro-angiogenic phenotype and supports hepatocyte reprogramming during cholestasis, both of which may prevent accelerated liver injury.