Loss of β-catenin in cholangiocytes promotes hepatocyte reprogramming and vascular remodeling during murine
Matthew Carson1,2,3, Jamie Fornsaglio4, Ridgeway Case Iv1,2,3
1Organ Pathobiology and Therapeutics Institute, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA.
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.
