Attenuating the EGFR-ERK-SOX9 axis promotes liver progenitor cell‐mediated liver regeneration in zebrafish

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 pro‐inflammatory 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 since such promotion generates more functional hepatocytes and concomitantly reduces inflammation and fibrosis. Here, using zebrafish models of LPC‐mediated liver regeneration, we present a proof‐of‐principle of such therapeutics by demonstrating a role for the EGFR signaling pathway in differentiation of LPCs into hepatocytes. We found that suppression of EGFR signaling promoted LPC‐to‐hepatocyte differentiation via the MEK‐ERK‐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. Conclusion: The EGFR‐ERK‐SOX9 axis suppresses LPC‐to‐hepatocyte differentiation during LPC‐mediated liver regeneration. We suggest EGFR inhibitors as a pro‐regenerative therapeutic drug for patients with advanced liver disease

PPARα activation promotes liver progenitor cell-mediated liver regeneration by suppressing YAP signaling in zebrafish

Abstract Despite the robust regenerative capacity of the liver, prolonged and severe liver damage impairs liver regeneration, leading to liver failure. Since the liver co-opts the differentiation of liver progenitor cells (LPCs) into hepatocytes to restore functional hepatocytes, augmenting LPC-mediated liver regeneration may be beneficial to patients with chronic liver diseases. However, the molecular mechanisms underlying LPC-to-hepatocyte differentiation have remained largely unknown. Using the zebrafish model of LPC-mediated liver regeneration, Tg(fabp10a:pt-β-catenin), we present that peroxisome proliferator-activated receptor-alpha (PPARα) activation augments LPC-to-hepatocyte differentiation. We found that treating Tg(fabp10a:pt-β-catenin) larvae with GW7647, a potent PPARα agonist, enhanced the expression of hepatocyte markers and simultaneously reduced the expression of biliary epithelial cell (BEC)/LPC markers in the regenerating livers, indicating enhanced LPC-to-hepatocyte differentiation. Mechanistically, PPARα activation augments the differentiation by suppressing YAP signaling. The differentiation phenotypes resulting from GW7647 treatment were rescued by expressing a constitutively active form of Yap1. Moreover, we found that suppression of YAP signaling was sufficient to promote LPC-to-hepatocyte differentiation. Treating Tg(fabp10a:pt-β-catenin) larvae with the TEAD inhibitor K-975, which suppresses YAP signaling, phenocopied the effect of GW7647 on LPC differentiation. Altogether, our findings provide insights into augmenting LPC-mediated liver regeneration as a regenerative therapy for chronic liver diseases

Liver progenitor cell-driven liver regeneration

Regenerative medicine: Understanding regeneration in liver disease A deeper understanding of the biology of liver progenitor cells (LPCs) could give clinicians more effective tools for reversing the effects of liver disease. LPCs are precursors of the liver cells known as hepatocytes, and naturally appear when the tissue is injured or diseased. Juhoon So, Donghun Shin and colleagues at the University of Pittsburgh, USA, have reviewed the current state of knowledge of LPC biology. They explore the mechanisms by which LPCs are generated and activated in response to trauma. These molecular pathways could offer useful drug targets.The authors also point out that excessive LPC activity can promote liver damage and tumorigenesis. As a consequence, any regenerative medicine approach based on LPCs must be coupled with effective strategies for promoting the efficient maturation of these cells into healthy hepatocytes

Wnt/β-catenin signaling cell-autonomously converts non-hepatic endodermal cells to a liver fate

Summary Wnt/β-catenin signaling plays multiple roles in liver development including hepatoblast proliferation and differentiation, hepatocyte differentiation, and liver zonation. A positive role for Wnt/β-catenin signaling in liver specification was recently identified in zebrafish; however, its underlying cellular mechanisms are unknown. Here, we present two cellular mechanisms by which Wnt/β-catenin signaling regulates liver specification. First, using lineage tracing we show that ectopic hepatoblasts, which form in the endoderm posterior to the liver upon activation of Wnt/β-catenin signaling, are derived from the direct conversion of non-hepatic endodermal cells, but not from the posterior migration of hepatoblasts. We found that endodermal cells at the 4–6th somite levels, which normally give rise to the intestinal bulb or intestine, gave rise to hepatoblasts in Wnt8a-overexpressing embryos, and that the distribution of traced endodermal cells in Wnt8a-overexpressing embryos was similar to that in controls. Second, by using an endoderm-restricted cell-transplantation technique and mosaic analysis with transgenic lines that cell-autonomously suppress or activate Wnt/β-catenin signaling upon heat-shock, we show that Wnt/β-catenin signaling acts cell-autonomously in endodermal cells to induce hepatic conversion. Altogether, these data demonstrate that Wnt/β-catenin signaling can induce the fate-change of non-hepatic endodermal cells into a liver fate in a cell-autonomous manner. These findings have potential application to hepatocyte differentiation protocols for the generation of mature hepatocytes from induced pluripotent stem cells, supplying a sufficient amount of hepatocytes for cell-based therapies to treat patients with severe liver diseases

Identification and Characterization of a Novel Small-Molecule Inhibitor of β-Catenin Signaling

Hepatocellular carcinoma (HCC), the third most common cause of cancer-related deaths worldwide, lacks effective medical therapy. Large subsets of HCC demonstrate Wnt/β-catenin activation, making this an attractive therapeutic target. We report strategy and characterization of a novel small-molecule inhibitor, ICG-001, known to affect Wnt signaling by disrupting β-catenin–CREB binding protein interactions. We queried the ZINC online database for structural similarity to ICG-001 and identified PMED-1 as the lead compound, with ≥70% similarity to ICG-001. PMED-1 significantly reduced β-catenin activity in hepatoblastoma and several HCC cells, as determined by TOPflash reporter assay, with an IC50 ranging from 4.87 to 32 μmol/L. Although no toxicity was observed in primary human hepatocytes, PMED-1 inhibited Wnt target expression in HCC cells, including those with CTNNB1 mutations, and impaired cell proliferation and viability. PMED-1 treatment decreased β-catenin–CREB binding protein interactions without affecting total β-catenin levels or activity of other common kinases. PMED-1 treatment of Tg(OTM:d2EGFP) zebrafish expressing GFP under the β-catenin/Tcf reporter led to a notable decrease in β-catenin activity. The PMED effect on β-catenin signaling lasted from 12 to 24 hours in vitro and 6 to 15 hours in vivo. Thus, using a rapid and cost-effective computational methodology, we have identified a novel and specific small-molecule inhibitor of Wnt signaling that may have implications for HCC treatment

EGFR signaling regulates biliary morphogenesis.

<p>(A) Whole-mount in situ hybridization image showing <i>egfra</i> expression in the liver at 72 hpf. (B) Confocal images of the liver showing the intrahepatic biliary structure, as revealed by <i>Tp1</i>:GFP expression. <i>Tg(Tp1</i>:<i>GFP)</i> embryos were treated with DMSO or 4 μM AG1478 from 48 to 96 hpf, and processed for whole-mount immunostaining with anti-GFP antibody. The length of BEC filopodia was quantified as shown in a graph. Brackets delineate the length of BEC filopodia. (C) Confocal images of the liver showing the expression of <i>Tp1</i>:GFP (green) and Abcb11 (red) for biliary structure and bile canaliculi, respectively. (D) Confocal images of the liver showing the location of BEC nuclei in the entire liver, as assessed by <i>Tp1</i>:H2B-mCherry expression. Dashed lines outline clusters with four or more BECs. Graph showing the percentage of BECs present as single cells, doublets, triplets, or in clusters of four or more cells. (E) Epifluorescence images showing PED-6 accumulation in the gallbladder in DMSO- or AG1478-treated larvae at 5 dpf. Graph showing the percentage of larvae exhibiting different levels of PED-6 accumulation in the gallbladder. Arrows point to the gallbladder. All dotted lines outline the liver. n indicates the number of larvae examined; asterisks indicate statistical significance (* p<0.0001). Error bars, ± SEM; scale bars, 25 μm.</p