title: MEK-ERK Activity Regulates the Proliferative Activity of Fetal Hepatoblasts Through Accumulation of p16/19(cdkn2a).
abstract: Hepatoblasts are somatic progenitor cells in the fetal liver, which retain a high proliferative capacity and differentiate into both hepatocytes and cholangiocytes in vivo. Although efficient expansion of hepatoblasts in vitro has been difficult without genetic modification, we have previously demonstrated that the interaction with mesenchymal cells is important for expansion of hepatoblasts in vitro. In this study, we show cell signaling pathways regulating the long-term proliferative ability of hepatoblasts. Individual primary hepatoblasts derived from mouse fetal livers formed large colonies when cocultured with mesenchymal feeder cells; however, secondary colony formation was unsuccessful, indicating that in vitro culture could induce short-term, but not long-term, proliferation. When the MEK inhibitor, PD0325901, was added to these cultures, hepatoblasts formed large colonies containing many Ki-67-positive cells. Expression of p16/19(cdkn2a), a cyclin-dependent kinase inhibitor, was induced after 3-6 days culture of hepatoblasts, whereas PD0325901 significantly suppressed this expression. Consistent with these observations, fetal hepatoblasts derived from p16/19(cdkn2a) knockout mice showed long-term proliferation without PD0325901, suggesting that MEK activity induced cell cycle arrest through accumulation of p16/19(cdkn2a). In transplantation assays, we could demonstrate that in vitro expanded hepatoblasts could proliferate and differentiate into hepatocytic and cholangiocytic cells in injured livers. It should also be noted that ERK in primary hepatoblasts was not highly activated during fetal liver development. Collectively, all these findings suggest that the MEK/ERK-independent pathway in the fetal liver is involved in hepatoblast proliferation to avoid accumulation of cyclin-dependent kinase inhibitor.
authors.affiliation: 1 Department of Molecular Life Sciences, Tokai University School of Medicine , Isehara, Kanagawa, Japan .|2 Division of Stem Cell Therapy, Center for Stem Cell and Regenerative Medicine, The Institute of Medical Science, The University of Tokyo , Minato-ku, Tokyo, Japan .
authors.forename: Akihide
authors.initials: A
authors.lastname: Kamiya
authors.affiliation: 2 Division of Stem Cell Therapy, Center for Stem Cell and Regenerative Medicine, The Institute of Medical Science, The University of Tokyo , Minato-ku, Tokyo, Japan .
authors.forename: Keiichi
authors.initials: K
authors.lastname: Ito
authors.affiliation: 2 Division of Stem Cell Therapy, Center for Stem Cell and Regenerative Medicine, The Institute of Medical Science, The University of Tokyo , Minato-ku, Tokyo, Japan .
authors.forename: Ayaka
authors.initials: A
authors.lastname: Yanagida
authors.affiliation: 1 Department of Molecular Life Sciences, Tokai University School of Medicine , Isehara, Kanagawa, Japan .
authors.forename: Hiromi
authors.initials: H
authors.lastname: Chikada
authors.affiliation: 3 Department of Cellular and Molecular Medicine, Graduate School of Medicine, Chiba University , Chiba, Japan .
authors.forename: Atsushi
authors.initials: A
authors.lastname: Iwama
authors.affiliation: 2 Division of Stem Cell Therapy, Center for Stem Cell and Regenerative Medicine, The Institute of Medical Science, The University of Tokyo , Minato-ku, Tokyo, Japan .|4 Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine , Stanford, California.
authors.forename: Hiromitsu
authors.initials: H
authors.lastname: Nakauchi
mesh_terms: D000818:Animals; D002454:Cell Differentiation / Q000502:physiology; D049109:Cell Proliferation / Q000502:physiology; D002478:Cells, Cultured; D019941:Cyclin-Dependent Kinase Inhibitor p16 / Q000378:metabolism*; D061252:Feeder Cells / Q000378:metabolism; D005333:Fetus / Q000166:cytology*; D018507:Gene Expression Regulation, Developmental / Q000502:physiology*; D022781:Hepatocytes / Q000166:cytology*; D008099:Liver / Q000166:cytology; D020935:MAP Kinase Signaling System / Q000502:physiology*; D059630:Mesenchymal Stem Cells / Q000378:metabolism; D051379:Mice; D008822:Mice, Transgenic
chemical_list: C417743:Cdkn2a protein, mouse; D019941:Cyclin-Dependent Kinase Inhibitor p16