Laboratory of Experimental Carcinogenesis, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892-4262, USA.
Hepatology. 2012 Apr;55(4):1215-26. doi: 10.1002/hep.24796.
Hepatocyte growth factor (HGF)/c-Met supports a pleiotrophic signal transduction pathway that controls stem cell homeostasis. Here, we directly addressed the role of c-Met in stem-cell-mediated liver regeneration by utilizing mice harboring c-met floxed alleles and Alb-Cre or Mx1-Cre transgenes. To activate oval cells, the hepatic stem cell (HSC) progeny, we used a model of liver injury induced by diet containing the porphyrinogenic agent, 3,5-diethocarbonyl-1,4-dihydrocollidine (DDC). Deletion of c-met in oval cells was confirmed in both models by polymerase chain reaction analysis of fluorescence-activated cell-sorted epithelial cell adhesion molecule (EpCam)-positive cells. Loss of c-Met receptor decreased the sphere-forming capacity of oval cells in vitro as well as reduced oval cell pool, impaired migration, and decreased hepatocytic differentiation in vivo, as demonstrated by double immunofluorescence using oval- (A6 and EpCam) and hepatocyte-specific (i.e. hepatocyte nuclear factor 4-alpha) antibodies. Furthermore, lack of c-Met had a profound effect on tissue remodeling and overall composition of HSC niche, which was associated with greatly reduced matrix metalloproteinase (MMP)9 activity and decreased expression of stromal-cell-derived factor 1. Using a combination of double immunofluorescence of cell-type-specific markers with MMP9 and gelatin zymography on the isolated cell populations, we identified macrophages as a major source of MMP9 in DDC-treated livers. The Mx1-Cre-driven c-met deletion caused the greatest phenotypic impact on HSCs response, as compared to the selective inactivation in the epithelial cell lineages achieved in c-Met(fl/fl); Alb-Cre(+/-) mice. However, in both models, genetic loss of c-met triggered a similar cascade of events, leading to the failure of HSC mobilization and death of the mice.
These results establish a direct contribution of c-Met in the regulation of HSC response and support a unique role for HGF/c-Met as an essential growth-factor-signaling pathway for regeneration of diseased liver.
肝细胞生长因子（HGF）/c-Met 支持一种多效信号转导途径，该途径控制干细胞的体内平衡。在这里，我们通过利用携带 c-met 基因 floxed 等位基因和 Alb-Cre 或 Mx1-Cre 转基因的小鼠，直接研究了 c-Met 在干细胞介导的肝再生中的作用。为了激活肝前体细胞（HSC）的祖细胞卵圆细胞，我们使用了一种含有卟啉原剂 3,5-二乙氧羰基-1,4-二氢吡啶（DDC）的饮食诱导的肝损伤模型。通过聚合酶链反应分析荧光激活细胞分选的上皮细胞黏附分子（EpCam）阳性细胞，证实了在这两种模型中卵圆细胞中 c-met 的缺失。c-Met 受体的缺失降低了卵圆细胞的球体形成能力，减少了卵圆细胞池，损害了体内迁移，并减少了肝祖细胞的分化，这一点通过使用卵圆细胞（A6 和 EpCam）和肝细胞特异性（即肝细胞核因子 4-α）抗体的双免疫荧光来证明。此外，缺乏 c-Met 对 HSC 龛位的组织重塑和整体组成有深远影响，这与基质金属蛋白酶（MMP）9 活性的大大降低和基质细胞衍生因子 1 的表达减少有关。通过细胞类型特异性标记物与 MMP9 的双免疫荧光和分离细胞群的明胶酶谱的组合，我们确定巨噬细胞是 DDC 处理的肝脏中 MMP9 的主要来源。与 c-Met(fl/fl); Alb-Cre(+/-)小鼠中上皮细胞谱系的选择性失活相比，Mx1-Cre 驱动的 c-met 缺失对 HSCs 反应产生了最大的表型影响。然而，在这两种模型中，c-met 的遗传缺失都触发了类似的级联事件，导致 HSC 动员失败和小鼠死亡。
这些结果确立了 c-Met 在调节 HSC 反应中的直接作用，并支持 HGF/c-Met 作为疾病肝脏再生的必需生长因子信号通路的独特作用。
