Our most recent results in this project include: (1)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 Met(fl/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 Met(fl/fl);Alb-Cre(+/-) livers. Up-regulation of stress response (MAFK, IKBZ, SOCS3) and early growth response (c-Myc, c-Jun, c-Fos, DUSP1 and 6) 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 Met(fl/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.(2)Redox signaling is emerging as an essential mechanism in the regulation of biological activities of the cell. The HGF/c-Met signaling pathway has been implicated as a key regulator of the cellular redox homeostasis and oxidative stress. We previously demonstrated that genetic deletion of c-met in hepatocytes disrupts redox homeostasis by a mechanism involving NADPH oxidase. Here, we identified biphasic mechanism of NADPH oxidase regulation by HGF/c-Met signaling in mouse hepatocytes. HGF induced a biphasic mechanism of NADPH oxidase regulation. The first phase employed the rapid increase in production of ROS as signaling effectors to activate the Nrf2-mediated protective response resulting in up-regulation of the antioxidant proteins, such as NAD(P)H quinone oxidoreductase and gamma-glutamyl cysteine synthetase. The second phase operated under a prolonged HGF exposure, caused a suppression of the NADPH oxidase components, including NOX2, NOX4, p22 and p67, and was able to abrogate the TGFb-induced ROS production and improve cell viability. In addition, we discovered a direct interaction between p22 and c-Met possibly involved in the regulation of HGF-mediated survival.(3)EGFR and c-Met are involved in both activation and differentiation of hepatic progenitor cell (HPC) . However, the underlying mechanisms have not been elucidated. Here we have addressed the impact of EGFR and c-Met signaling on the differentiation of HPC into heptatocytic and biliary epithelial lineages using clonally derived progenitor cell lines from EGFRfl/fl and Met fl/fl conditional knockout mice. EGFRfl/fl and c-Met fl/fl mice were treated with DDC for 2 weeks to activate the HPC compartment. Early HPCs lines were established from FACS sorted EpCAM+/Lineage Cocktail- cells isolated from the bulk nonparenchymal cell fraction after selecting for a self renewal capacity in 3D culture. EGFR and c-Met were deleted by Adeno-Cre transfection in vitro to generate early clonal MetKO and EGFRKO cell lines (used within the first 10 passages). Hepatocytic (HC) differentiation was induced by a combined treatment with dexamethasone and HGF/EGF. For biliary epithelial cell (BEC) differentiation, cells were cultured in matrigel/collagen in the presence of HGF and EGF. Confocal microscopy and western blotting as well as chemical inhibitors of the major downstream effectors (Erk, Akt, Stat3) were used to evaluate the impact of EGFR and c-Met on the efficiency of differentiation and the involvement of the downstream signaling. EGFRfl/fl and c-Metfl/fl HPC lines expressed both HC and BEC markers, were capable of branching tubologenesis in 3D cultures, and successfully repopulated the diseased liver in MUP-upA/SCID transgenic mice consistent with their stem/progenitor cell origin. MetKO HPCs failed to undergo HC differentiation concomitant with a strong downregulation of Akt and Stat3. Inhibition of both Akt and Stat3 but not Erk1/2 in c-Metfl/fl cells similarly reduced the HC differentiation. In BEC differentiation assay, loss of c-Met reduced tubulogenesis but did not affect either branching or induction of BEC marker expression. Inhibition of Akt and Stat3 in c-Metfl/fl also significantly attenuated tubular formation. In contrast, in the absence of EGFR, HPCs were only capable of forming long tubular structures without branching upon HGF treatment and lost the expression of BEC markers. Furthermore, EGFRKO cells did not express Notch1, known to be required for biliary differentiation. In conclusion, c-Met and EGFR, two major growth factor receptors in the liver, have a distinct impact on HPC differentiation. Met-driven Akt and Stat3 signaling is critical for both HC differentiation and biliary tubulogenesis, whereas EGFR has a dominant role in driving liver progenitor cells towards BEC via Notch1 pathway. A strong regenerative potential of HPC suggest their therapeutic potential

National Institute of Health (NIH)
National Cancer Institute (NCI)
Investigator-Initiated Intramural Research Projects (ZIA)
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National Cancer Institute Division of Basic Sciences
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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