Yes-associated protein promotes the proliferation and differentiation of liver progenitor cells during liver fibrosis.

Liver fibrosis is closely related to the proliferation and differentiation of liver progenitor cells (LPCs). Yes-associated protein (YAP) is a key effector molecule of the Hippo signaling pathway and plays an important role in regulating cell proliferation and liver homeostasis. However, its role in LPCs proliferation and differentiation during liver fibrosis are not well understood. Using immunohistochemistry, immunofluorescence staining, quantitative PCR and Western blotting, we discovered that LPCs expansion and enhanced YAP expression in LPCs in either choline-deficient, ethionine-supplemented (CDE) diet or 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) diet-induced fibrotic mice, as well as in patients with liver fibrosis. By injecting adeno-associated virus vectors under the transcriptional control of Lgr5 promoter, we found that targeted knockdown of YAP in LPCs attenuated the CDE/DDC diet-induced ductular reaction and liver fibrosis. Using EdU incorporation and Cell Counting Kit-8 assays, we demonstrated that YAP can modulate LPCs proliferation. Importantly, spleen transplantation of YAP-overexpressing LPCs improved their ability to differentiate into hepatocytes and alleviated carbon tetrachloride-induced liver fibrosis. Collectively, our findings indicate that LPCs expansion and differentiation during liver fibrosis could be modulated by YAP, further suggesting the possibility of manipulating YAP expression in LPCs as a potential treatment for chronic liver diseases.

YAP affects the efficacy of liver progenitor cells transplantation in CCl4-induced acute liver injury.

The liver is a highly regenerative organ. During acute liver injury, the remaining hepatocytes rapidly proliferate to restore liver parenchyma and liver function. However, hepatocytes-driven regeneration is compromised in severe liver injury; instead, liver progenitor cells (LPCs) proliferate and differentiate into hepatocytes or cholangiocytes to restore mass and function of liver. The Hippo signaling pathway is of vital importance in liver regeneration, and Yes-associated protein (YAP) is the key component of the Hippo pathway. The therapeutic role of YAP has been well studied in hepatocytes-driven liver regeneration. However, the role of LPCs transplantation in acute liver injury has not been defined. Here, we investigated the therapeutic effect of splenic-transplantation of LPCs in CCl4-induced acute liver injury and explored the role of YAP during the procedure. LPCs isolated from choline-deficient, ethionine-supplemented diet (CDE) model were infected with GFP-YAP cDNA lentiviral vector, GFP-YAP shRNA lentiviral vector, and GFP lentiviral vector as control, respectively. At 48 h after CCl4 injection, PBS (control group), GFP lentiviral vector-infected LPCs (GFP-LPC group), GFP-YAP cDNA lentiviral vector-infected LPCs (YAP-LPC group) and GFP-YAP shRNA lentiviral vector-infected LPCs (sh-YAP-LPC group) were injected into spleens in CCl4-treated mice. Histological and serological analyses were performed to evaluate pathology and liver function. The effect of LPCs on the proliferation of hepatocytes and inflammation was investigated. We demonstrated that intra-splenic transplantation of LPCs alleviates CCl4-induced acute liver injury and YAP signaling acts a key role during the procedure. Further studies suggested that LPCs alleviate acute liver injury by promoting pre-existing hepatocytes proliferation rather than differentiating into hepatocytes. Furthermore, intra-splenic transplantation of LPCs attenuates inflammation, which facilitates tissue repair in acute liver injury. In conclusion, LPCs transplantation is a potential treatment for acute liver injury and YAP is a prospective therapeutic target in acute liver injury.

Loss of YB-1 alleviates liver fibrosis by suppressing epithelial-mesenchymal transition in hepatic progenitor cells.

Previously, we reported that the nuclear translocation of Y-box binding protein 1 (YB-1) is induced by transforming growth factor-ß (TGF-ß) and promotes hepatic progenitor cells (HPCs) expansion. Here, we explored the mechanisms underlying YB-1 translocation and the impact of YB-1 on the epithelial-mesenchymal transition (EMT) in HPCs. YB-1flox/floxcre+/- (YB-1f/fcre+/-) mice and YB-1f/fcre-/- mice were fed with a 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) or a choline-deficient, ethionine-supplemented (CDE) diet. Liver injury and fibrosis were assessed by performing hematoxylin and eosin (HE) and Masson staining. The expression of collagen and EMT-related markers (E-cadherin, N-cadherin, and Snail) was detected by reverse transcription-polymerase chain reaction (RT-PCR), western blotting, and immunofluorescence analyses. Protein kinase B (AKT) expression in HPCs was silenced via RNA interference. Nuclear YB-1 expression in HPCs was detected via western blotting and immunofluorescence analyses. HPC proliferation was detected by immunofluorescence. Our results indicate that YB-1 transcriptionally regulated the biological behavior of HPCs. HPC-specific YB-1 knockout alleviated liver fibrosis in mice fed with DDC or CDE diet. YB-1 nuclear translocation promoted matrix metallopeptidase 9 transcription. YB-1 depletion in HPCs significantly dampened the EMT and inhibited AKT phosphorylation in vitro and in vivo. AKT knockdown compromised TGF-ß-induced YB-1 nuclear translocation, thereby inhibiting the EMT and HPC proliferation. EMT and AKT were highly activated in HPCs in cirrhotic livers. Collectively, our findings indicate that the loss of YB-1 suppressed EMT in HPCs and alleviated liver fibrosis in mice, and that AKT was essential for TGF-ß-induced YB-1 nuclear translocation and HPC proliferation.

Specific knockdown of Y-box binding protein 1 in hepatic progenitor cells inhibits proliferation and alleviates liver fibrosis.

The proliferation of hepatic progenitor cells (HPCs) contributes to liver regeneration and fibrogenesis during chronic liver injury; however, the mechanism modulating HPC proliferation remains unknown. Y-box binding protein-1 (YB-1) is a transcription factor that regulates the transcription of several genes and is highly expressed in liver injury. We explored the role of YB-1 in HPC proliferation and liver fibrosis. We detected increased expansion of HPCs and elevated levels of YB-1 in HPCs from patients with hepatitis B virus-related fibrosis and choline-deficient ethionine-supplemented or 5-diethoxycarbonyl-1,4-dihydrocollidine diet-induced mice compared with those in control groups. HPC-specific deletion of YB-1 using YB-1flox/flox; Foxl1-Cre+/- mice led to reduced HPC expansion and less collagen deposition in the liver tissues compared with that in Cre-/- mice. In cultured primary HPCs, YB-1 knockdown inhibited HPC proliferation. Further experiments indicated YB-1 negatively regulated p53 expression, and silencing of p53 blocked YB-1 knockdown-mediated inhibition of HPC proliferation. Collectively, YB-1 negatively regulates HPC proliferation and alleviates liver fibrosis by p53.

TGF-ß/YB-1/Atg7 axis promotes the proliferation of hepatic progenitor cells and liver fibrogenesis.

Hepatic fibrosis is characterized by excessive extracellular matrix deposition and ductular reactions, manifested as the expansion of hepatic progenitor cells (HPCs). We previously reported that the Y-box binding protein 1 (YB-1) in HPCs is involved in chronic liver injury. In this study, we constructed YB-1f/f Foxl1-Cre mice and investigated the role of YB-1 in HPC expansion in murine choline-deficient, ethionine-supplemented (CDE), and 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) models. Liver injury and fibrosis were measured using hematoxylin and eosin (HE), Masson, and Sirius Red staining. HPC proliferation was detected using EdU and immunofluorescence (IF). Autophagic flow was measured by mCherry-GFP-LC3B staining and transmission electron microscopy (TEM). YB-1 expression was measured by immunofluorescence and western blotting. CUT & Tag analysis, chromatin immunoprecipitation, and RT-PCR were performed to explore the regulation of autophagy-related protein 7 (Atg7) transcription by YB-1. Our results indicated that liver injury was accompanied by high expression of YB-1, proliferative HPCs, and activated autophagy in the CDE and DDC models. YB-1f/f Cre+/- mice displayed less liver injury and fibrosis than YB-1f/f Cre-/- mice in the CDE and DDC models. YB-1 promoted proliferation and autophagy of HPCs in vitro and in vivo. Transforming growth factor-ß (TGF-ß) induced YB-1 nuclear translocation and facilitated the proliferation and autophagy of HPCs. YB-1 nuclear translocation promoted the transcription of Atg7, which is essential for TGF-ß/YB-1 mediated HPCs expansion in vitro and in vivo. In summary, YB-1 nuclear translocation induced by TGF-ß in HPCs promotes the proliferation and autophagy of HPCs and Atg7 participates in YB-1-mediated HPC-expansion and liver fibrosis.