Abstract
Liver regeneration after injury is normally mediated by proliferation of hepatocytes, although recent studies have suggested biliary epithelial cells (BECs) can differentiate into hepatocytes during severe liver injury when hepatocyte proliferation is impaired. We investigated the effect of hepatocyte‐specific β‐catenin deletion in recovery from severe liver injury and BEC‐to‐hepatocyte differentiation. To induce liver injury, we administered choline‐deficient, ethionine‐supplemented (CDE) diet to three different mouse models, the first being mice with deletion of β‐catenin in both BECs and hepatocytes (Albumin‐Cre; Ctnnb1flox/flox mice). In our second model, we performed hepatocyte lineage tracing by injecting Ctnnb1flox/flox; Rosa‐stopflox/flox‐EYFP mice with the adeno‐associated virus serotype 8 encoding Cre recombinase under the control of the thyroid binding globulin promoter, a virus that infects only hepatocytes. Finally, we performed BEC lineage tracing via Krt19‐CreERT; Rosa‐stopflox/flox‐tdTomato mice. To observe BEC‐to‐hepatocyte differentiation, mice were allowed to recover on normal diet following CDE diet–induced liver injury. Livers were collected from all mice and analyzed by quantitative real‐time polymerase chain reaction, western blotting, immunohistochemistry, and immunofluorescence. We show that mice with lack of β‐catenin in hepatocytes placed on the CDE diet develop severe liver injury with impaired hepatocyte proliferation, creating a stimulus for BECs to differentiate into hepatocytes. In particular, we use both hepatocyte and BEC lineage tracing to show that BECs differentiate into hepatocytes, which go on to repopulate the liver during long‐term recovery. Conclusion: β‐catenin is important for liver regeneration after CDE diet–induced liver injury, and BEC‐derived hepatocytes can permanently incorporate into the liver parenchyma to mediate liver regeneration.
Highlights
Liver regeneration after injury is normally mediated by proliferation of hepatocytes, recent studies have suggested biliary epithelial cells (BECs) can differentiate into hepatocytes during severe liver injury when hepatocyte proliferation is impaired
We show that mice with lack of β-catenin in hepatocytes placed on the CDE diet develop severe liver injury with impaired hepatocyte proliferation, creating a stimulus for BECs to differentiate into hepatocytes
Clusters of tdTomato-positive hepatocytes in Ctnnb1-small interfering RNA (siRNA)-injected mice would occasionally extend all the way to the central vein, resulting in localized re-expression of β-catenin target glutamine synthetase (GS) (Fig. 8B). These results demonstrate that BECs give rise to hepatocytes when hepatocyte proliferation is impaired, which was achieved in our model through the loss of β-catenin expression in hepatocytes in mice exposed to CDE diet–induced liver injury (Fig. 8C)
Summary
Liver regeneration after injury is normally mediated by proliferation of hepatocytes, recent studies have suggested biliary epithelial cells (BECs) can differentiate into hepatocytes during severe liver injury when hepatocyte proliferation is impaired. We show that mice with lack of β-catenin in hepatocytes placed on the CDE diet develop severe liver injury with impaired hepatocyte proliferation, creating a stimulus for BECs to differentiate into hepatocytes. The choline-deficient, ethionine-supplemented (CDE) diet is a well-known liver injury diet that induces proliferation of reactive BECs.[21,22] Recent lineage tracing studies have demonstrated limited contribution of BECs to hepatocytes in CDE diet– fed mice,(23-25) leading to the conclusion that BECs do not contribute significantly to the restoration of hepatocyte mass after chronic liver injury. The CDE diet does not block hepatocyte proliferation[25] and is unable to provide the correct milieu for BEC-to-hepatocyte transdifferentiation
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