Oval cells (hepatic stem cell progeny) originate from stem cells which reside within the terminal bile ductules located at the interface between the parenchyma and the bile tree and have the potential to differentiate into hepatocytes and biliary epithelial cells (BEC). In response to severe hepatocyte injury, oval cells form a system of proliferating branching ducts that move inside the parenchyma where they finally differentiate towards hepatocytic lineage. Multiple molecular factors and cell types contribute to the process of adult stem cell activation. We and others have established that oval cell infiltration of hepatic tissue occurs in a close conjunction with accompanying stellate cells which provide HGF, and also promote pericellular collagen deposition, thus creating a microenvironment supporting growth of expanding ductular cells. The goal of our study was to establish the role for c-Met in different phases of stem cell activation and interaction of c-Met and EGFR signaling pathways by utilizing conditional knock-out mouse models for both receptors. To activate oval cells, we used a model of chronic liver injury induced by diet containing the porphyrinogenic agent 3, 5-diethocarbonyl-1,4-dihydrocollidine (DDC), which was introduced into the field by our previous work. Our previous work has established that HGF/c-Met signaling plays a pivotal role in regulating the onset of S phase following partial hepatectomy (PH). In this study, we used Metfl/fl;Alb-Cre+/- conditional knockout mice to determine the effects of c-Met dysfunction in hepatocytes on kinetics of liver regeneration. The priming events appeared to be intact in Metfl/fl;Alb-Cre+/- livers. Up-regulation of stress response (MAFK, IKBZ, SOCS3) and early growth response (c-Myc, c-Jun, c-Fos, DUSP1) genes as assessed by RT-qPCR and/or microarray profiling was unchanged. This was consistent with an early induction of MAPK/Erk and STAT3. However, after a successful completion of the first round of DNA replication, c-Met deficient hepatocytes were blocked in early/mid G2 phase as shown by staining with phosphorylated form of histone H3. Furthermore, loss of c-Met in hepatocytes diminished the subsequent G1/S progression and delayed liver recovery after partial hepatectomy. Upstream signaling pathways involved in the blockage of G2/M transition included lack of persistent Erk1/2 activation and inability to up-regulate the levels of Cdk1, Plk1, Aurora A and B, and Mad2 along with a defective histone 3 phosphorylation and lack of chromatin condensation. Continuous supplementation with EGF in vitro increased proliferation of Metfl/fl;Alb-Cre+/- primary hepatocytes and partially restored expression levels of mitotic cell cycle regulators albeit to a lesser degree as compared to control cultures. Conclusion/Significance. From these results we conclude that our results assign a novel non-redundant function for HGF/c-Met signaling in regulation of G2/M gene expression program via maintaining a persistent Erk1/2 activation throughout liver regeneration. The results from our study aimed at elucidating the role of c-Met and EGFR, the tyrosine kinase receptors for the most potent liver mitogens (HGF and EGF, respectively), in hepatic progenitor cell biology include: To investigate the role of c-Met and/or EGFR in self-renewal, we monitored the sphere-forming capacity of oval cells in vitro. HGF and EGF had a similar impact of the size and number of spheres generated by oval cells with intact Met and EGFR signaling. Combined treatment with HGF+EGF produced a further increase in sphere growth whereas knockdown of c-Met and EGFR signaling completely abolished the sphere-forming ability of oval cells. The HGF+EGFR treatment also significantly increased and prolonged phosphorylation of each receptor as well as activation of ERK, Gab1, and PLC-gamma downstream signaling suggesting that a cross-talk between c-Met and EGFR contributes to self-renewal of oval cells. Conversely, in the differentiation assays, HGF alone was more effective than EGF in inducing oval cell differentiation along hepatocytic and biliary epithelial cell lineage. This was consistent with a stronger and more persistent Stat3 and Akt phosphorylation following HGF treatment as compared to that caused by EGF or combination of EGF+HGF. From these results we conclude that c-Met and EGFR have similar potency in maintaining self-renewing proliferation of hepatic progenitor cells which can be further enhanced by simultaneous activation of both receptors. However, c-Met is a major driving force of oval cell differentiation towards both hepatic epithelial cell lineages through sustaining a long term Stat3 and AKT signaling.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Investigator-Initiated Intramural Research Projects (ZIA)
Project #
1ZIABC010638-07
Application #
8157376
Study Section
Project Start
Project End
Budget Start
Budget End
Support Year
7
Fiscal Year
2010
Total Cost
$133,650
Indirect Cost
Name
National Cancer Institute Division of Basic Sciences
Department
Type
DUNS #
City
State
Country
Zip Code
Clavijo-Cornejo, Denise; Enriquez-Cortina, Cristina; López-Reyes, Alberto et al. (2013) Biphasic regulation of the NADPH oxidase by HGF/c-Met signaling pathway in primary mouse hepatocytes. Biochimie 95:1177-84
Coulouarn, Cédric; Corlu, Anne; Glaise, Denise et al. (2012) Hepatocyte-stellate cell cross-talk in the liver engenders a permissive inflammatory microenvironment that drives progression in hepatocellular carcinoma. Cancer Res 72:2533-42
Ishikawa, Tsuyoshi; Factor, Valentina M; Marquardt, Jens U et al. (2012) Hepatocyte growth factor/c-met signaling is required for stem-cell-mediated liver regeneration in mice. Hepatology 55:1215-26
Marquardt, Jens U; Seo, Daekwan; Gómez-Quiroz, Luis E et al. (2012) Loss of c-Met accelerates development of liver fibrosis in response to CCl(4) exposure through deregulation of multiple molecular pathways. Biochim Biophys Acta 1822:942-51
Thorgeirsson, S S (2012) The central role of the c-Met pathway in rebuilding the liver. Gut 61:1105-6
Hwang, Chang-Il; Matoso, Andres; Corney, David C et al. (2011) Wild-type p53 controls cell motility and invasion by dual regulation of MET expression. Proc Natl Acad Sci U S A 108:14240-5
Factor, Valentina M; Seo, Daekwan; Ishikawa, Tsuyoshi et al. (2010) Loss of c-Met disrupts gene expression program required for G2/M progression during liver regeneration in mice. PLoS One 5:
Ishibe, Shuta; Karihaloo, Anil; Ma, Hong et al. (2009) Met and the epidermal growth factor receptor act cooperatively to regulate final nephron number and maintain collecting duct morphology. Development 136:337-45
Gomez-Quiroz, Luis E; Factor, Valentina M; Kaposi-Novak, Pal et al. (2008) Hepatocyte-specific c-Met deletion disrupts redox homeostasis and sensitizes to Fas-mediated apoptosis. J Biol Chem 283:14581-9
del Castillo, Gaelle; Factor, Valentina M; Fernandez, Margarita et al. (2008) Deletion of the Met tyrosine kinase in liver progenitor oval cells increases sensitivity to apoptosis in vitro. Am J Pathol 172:1238-47