Attenuating the Epidermal Growth Factor Receptor-Extracellular Signal-Regulated Kinase-Sex-Determining Region Y-Box 9 Axis Promotes Liver Progenitor Cell-Mediated Liver Regeneration in Zebrafish.

BACKGROUND AND AIMS: The liver is a highly regenerative organ, but its regenerative capacity is compromised in severe liver injury settings. In chronic liver diseases, the number of liver progenitor cells (LPCs) correlates proportionally to disease severity, implying that their inefficient differentiation into hepatocytes exacerbates the disease. Moreover, LPCs secrete proinflammatory cytokines; thus, their prolonged presence worsens inflammation and induces fibrosis. Promoting LPC-to-hepatocyte differentiation in patients with advanced liver disease, for whom liver transplantation is currently the only therapeutic option, may be a feasible clinical approach because such promotion generates more functional hepatocytes and concomitantly reduces inflammation and fibrosis. APPROACH AND RESULTS: Here, using zebrafish models of LPC-mediated liver regeneration, we present a proof of principle of such therapeutics by demonstrating a role for the epidermal growth factor receptor (EGFR) signaling pathway in differentiation of LPCs into hepatocytes. We found that suppression of EGFR signaling promoted LPC-to-hepatocyte differentiation through the mitogen-activated ERK kinase (MEK)-extracellular signal-regulated kinase (ERK)-sex-determining region Y-box 9 (SOX9) cascade. Pharmacological inhibition of EGFR or MEK/ERK promoted LPC-to-hepatocyte differentiation as well as genetic suppression of the EGFR-ERK-SOX9 axis. Moreover, Sox9b overexpression in LPCs blocked their differentiation into hepatocytes. In the zebrafish liver injury model, both hepatocytes and biliary epithelial cells contributed to LPCs. EGFR inhibition promoted the differentiation of LPCs regardless of their origin. Notably, short-term treatment with EGFR inhibitors resulted in better liver recovery over the long term. CONCLUSIONS: The EGFR-ERK-SOX9 axis suppresses LPC-to-hepatocyte differentiation during LPC-mediated liver regeneration. We suggest EGFR inhibitors as a proregenerative therapeutic drug for patients with advanced liver disease.

Farnesoid X Receptor Activation Impairs Liver Progenitor Cell-Mediated Liver Regeneration via the PTEN-PI3K-AKT-mTOR Axis in Zebrafish.

BACKGROUND AND AIMS: Following mild liver injury, pre-existing hepatocytes replicate. However, if hepatocyte proliferation is compromised, such as in chronic liver diseases, biliary epithelial cells (BECs) contribute to hepatocytes through liver progenitor cells (LPCs), thereby restoring hepatic mass and function. Recently, augmenting innate BEC-driven liver regeneration has garnered attention as an alternative to liver transplantation, the only reliable treatment for patients with end-stage liver diseases. Despite this attention, the molecular basis of BEC-driven liver regeneration remains poorly understood. APPROACH AND RESULTS: By performing a chemical screen with the zebrafish hepatocyte ablation model, in which BECs robustly contribute to hepatocytes, we identified farnesoid X receptor (FXR) agonists as inhibitors of BEC-driven liver regeneration. Here we show that FXR activation blocks the process through the FXR-PTEN (phosphatase and tensin homolog)-PI3K (phosphoinositide 3-kinase)-AKT-mTOR (mammalian target of rapamycin) axis. We found that FXR activation blocked LPC-to-hepatocyte differentiation, but not BEC-to-LPC dedifferentiation. FXR activation also suppressed LPC proliferation and increased its death. These defects were rescued by suppressing PTEN activity with its chemical inhibitor and ptena/b mutants, indicating PTEN as a critical downstream mediator of FXR signaling in BEC-driven liver regeneration. Consistent with the role of PTEN in inhibiting the PI3K-AKT-mTOR pathway, FXR activation reduced the expression of pS6, a marker of mTORC1 activation, in LPCs of regenerating livers. Importantly, suppressing PI3K and mTORC1 activities with their chemical inhibitors blocked BEC-driven liver regeneration, as did FXR activation. CONCLUSIONS: FXR activation impairs BEC-driven liver regeneration by enhancing PTEN activity; the PI3K-AKT-mTOR pathway controls the regeneration process. Given the clinical trials and use of FXR agonists for multiple liver diseases due to their beneficial effects on steatosis and fibrosis, the detrimental effects of FXR activation on LPCs suggest a rather personalized use of the agonists in the clinic.

Wnt/ß-catenin signaling controls intrahepatic biliary network formation in zebrafish by regulating notch activity.

Malformations of the intrahepatic biliary structure cause cholestasis, a liver pathology that corresponds to poor bile flow, which leads to inflammation, fibrosis, and cirrhosis. Although the specification of biliary epithelial cells (BECs) that line the bile ducts is fairly well understood, the molecular mechanisms underlying intrahepatic biliary morphogenesis remain largely unknown. Wnt/ß-catenin signaling plays multiple roles in liver biology; however, its role in intrahepatic biliary morphogenesis remains unclear. Using pharmacological and genetic tools that allow one to manipulate Wnt/ß-catenin signaling, we show that in zebrafish both suppression and overactivation of Wnt/ß-catenin signaling impaired intrahepatic biliary morphogenesis. Hepatocytes, but not BECs, exhibited Wnt/ß-catenin activity; and the global suppression of Wnt/ß-catenin signaling reduced Notch activity in BECs. Hepatocyte-specific suppression of Wnt/ß-catenin signaling also reduced Notch activity in BECs, indicating a cell nonautonomous role for Wnt/ß-catenin signaling in regulating hepatic Notch activity. Reducing Notch activity to the same level as that observed in Wnt-suppressed livers also impaired biliary morphogenesis. Intriguingly, expression of the Notch ligand genes jag1b and jag2b in hepatocytes was reduced in Wnt-suppressed livers and enhanced in Wnt-overactivated livers, revealing their regulation by Wnt/ß-catenin signaling. Importantly, restoring Notch activity rescued the biliary defects observed in Wnt-suppressed livers. CONCLUSION: Wnt/ß-catenin signaling cell nonautonomously controls Notch activity in BECs by regulating the expression of Notch ligand genes in hepatocytes, thereby regulating biliary morphogenesis. (Hepatology 2018;67:2352-2366).

Glutathione antioxidant pathway activity and reserve determine toxicity and specificity of the biliary toxin biliatresone in zebrafish.

UNLABELLED: Biliatresone is an electrophilic isoflavone isolated from Dysphania species plants that has been causatively linked to naturally occurring outbreaks of a biliary atresia (BA)-like disease in livestock. Biliatresone has selective toxicity for extrahepatic cholangiocytes (EHCs) in zebrafish larvae. To better understand its mechanism of toxicity, we performed transcriptional profiling of liver cells isolated from zebrafish larvae at the earliest stage of biliatresone-mediated biliary injury, with subsequent comparison of biliary and hepatocyte gene expression profiles. Transcripts encoded by genes involved in redox stress response, particularly those involved in glutathione (GSH) metabolism, were among the most prominently up-regulated in both cholangiocytes and hepatocytes of biliatresone-treated larvae. Consistent with these findings, hepatic GSH was depleted at the onset of biliary injury, and in situ mapping of the hepatic GSH redox potential using a redox-sensitive green fluorescent protein biosensor showed that it was significantly more oxidized in EHCs both before and after treatment with biliatresone. Pharmacological and genetic manipulation of GSH redox homeostasis confirmed the importance of GSH in modulating biliatresone-induced injury given that GSH depletion sensitized both EHCs and the otherwise resistant intrahepatic cholangiocytes to the toxin, whereas replenishing GSH level by N-acetylcysteine administration or activation of nuclear factor erythroid 2-like 2 (Nrf2), a transcriptional regulator of GSH synthesis, inhibited EHC injury. CONCLUSION: These findings strongly support redox stress as a critical contributing factor in biliatresone-induced cholangiocyte injury, and suggest that variations in intrinsic stress responses underlie the susceptibility profile. Insufficient antioxidant capacity of EHCs may be critical to early pathogenesis of human BA. (Hepatology 2016;64:894-907).