The role of c-met in liver biology

c-met 在肝脏生物学中的作用

• 批准号: 7965490
• 负责人: Snorri Thorgeirsson
• 金额: $ 13.57万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7965490
• 关键词: Ablation; Actins; Address; Adult; Affect; Alleles; Antibodies; Apoptosis; Apoptotic; Area; Basement membrane; Bile Acids; Bile fluid; Biliary; Biochemical; Biological; Biology; Cell Lineage; Cell Proliferation; Cell physiology; Cells; Cessation of life; Cholestasis; Cholesterol Homeostasis; Chromatin; Chromosome Condensation; Chronic; Collagen; Confocal Microscopy; Cytoskeleton; DNA biosynthesis; Data; Defect; Deposition; Development; Diet; Drug or chemical Tissue Distribution; Duct (organ) structure; Employee Strikes; Epidermal Growth Factor Receptor; Epithelial; Epithelial Cells; Exhibits; Failure; Fibrosis; Frequencies; Gelatin Zymography; Gelatinase B; Gene Targeting; Genes; Genotype; Goals; Growth; Growth Factor; Hepatic; Hepatic Mass; Hepatic Tissue; Hepatobiliary; Hepatocyte; Hepatocyte Growth Factor; Immunohistochemistry; In Situ; Infiltration; Injury; Knockout Mice; Kupffer Cells; Liver; Liver Regeneration; Liver diseases; MMP9 gene; Maintenance; Mediating; Membrane; Metabolism; Microtubule Stabilization; Mitotic Checkpoint; Modeling; Molecular; Mus; Mutant Strains Mice; Outcome; Partial Hepatectomy; Pattern; Phase; Phenotype; Phosphorylation; Physiology; Play; Process; Production; Proliferating; Protein Tyrosine Kinase; Proteins; Regulation; Resources; Role; Route; Signal Pathway; Signal Transduction; Sorting - Cell Movement; Staining method; Stains; Stem cells; Suspension substance; Suspensions; System; Tissue Inhibitor of Metalloproteinase-1; Tissues; Transgenes; Trees; Work; adult stem cell; aurora B kinase; bile duct; cell motility; cell type; cytokine; in utero; liver cell proliferation; macrophage; meetings; migration; monocyte; mouse model; novel; oval cell; postnatal; precursor cell; receptor; recombinase; research study; response; self-renewal; stellate cell; stem cell biology; stem cell division; tumorigenesis

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 by utilizing mice harboring c-met floxed alleles (Ctrl) and Alb-Cre or Mx1-Cre transgenes. 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. Inactivation of c-Met was achieved in both epithelial cell types (AlbCreMet model) and in all liver cells (MxCreMet model) allowing us to address the significance of the cross-talk between epithelial and nonparenchymal cell compartments. The phenotype of MxcreMet mice was very similar albeit more severe than that of AlbcreMet mice as determined by biochemical and morphological data. In the absence of c-Met signaling, both hepatocyte- and stem cell-mediated liver regeneration were severely impaired. There was no compensatory increase in liver mass due to the decrease in hepatocyte proliferation and increase in apoptotic death. At the molecular level, Met mutant mice were unable to activate the major downstream signaling pathways involved in cell proliferation, motility regulation and apoptosis protection, such as Erk1/2, Stat3, and Akt. The c-Met deficient livers displayed a classical bile duct proliferation restricted by a more severe periportal fibrosis reflecting a failure of oval cell outgrowth and/or redirection of their differentiation towards BEC lineage. Analysis of the frequency and tissue distribution using cell specific antibodies against BEC and oval cells (A6 and EpCam) revealed a striking decrease in the number of A6+/EpCam+ cells and confirmed almost complete lack of their migration inside the parenchyma. More significantly, we found a marked reduction in the number of A6+/EpCam-/HNF4+ cells, consistent with a reduced ability to differentiate into hepatocytes in the absence of c-Met signaling. This was accompanied by a progressive decline in the rate of ductular proliferation in c-Met deficient livers as estimated by Ki67 immunohistochemistry. Furthermore, experiments in culture showed that single cell suspensions of EpCam+/Lineage- cells isolated from c-Met KO mice consistently displayed a reduced frequency of spheroid-forming cells when seeded in BD MatrigelTm enriched with basement membrane matrix proteins and growth factors, suggesting that self-renewal of hepatic stem cells is affected by c-Met loss. Consistent with the important role of HGF/c-Met in protection against fibrosis, both models of c-Met conditional knockout mice developed a more extensive periportal fibrosis as compared to Ctrl mice despite the comparable protein levels of the known fibrogenic factors, such αSMA, TGFβ1, and TIMP-1. Nevertheless, the tissue distribution of collagen-producing stellate cells was dramatically different, with the majority being located strictly within the areas of periportal fibrosis. This was paralleled by defective recruitment of the tissue-infiltrating monocytes/macrophages and reduced MMP9 activity. Gelatin zymography of FACS-sorted F4/80+ Kupffer cells identified macrophages as the main cell type producing MMP-9. In Ctrl livers, a combination of A6 confocal microscopy either with in situ zymography or co-staining with F4/80 demonstrated increased MMP-9 activity along the routes of expanding oval cells, implying that fibrolytic activity of MMP-9 is contributing to migration of oval cells. Since these phenomena occurred regardless of the genotype of nonparenchymal liver cells, these data argue for a key functional role of the epithelial component in creating a microenvironment which may affect fate determination and terminal differentiation of hepatic precursor cells. c-Met gene ablation led to pleiotropic phenotypes due to c-Met involvement in the control of multiple cellular functions. Our study provides morphological and biochemical evidence that in the absence of c-Met signaling, considerable damage occurred in both epithelial compartments. After DDC treatment, AlbCreMet and MxCreMet mice exhibited impaired bile secretion with significant functional and molecular alterations as judged by hepatic expression of the major genes involved in bile acid production, metabolism and transport. In addition, we found defects in actin cytoskeleton organization and relocation of Bgp1 into the submembranous compartment which may affect sorting and maintenance of transporters in the canalicular membrane. The combined mechanisms of the excretory disfunction in c-Met deficient hepatocytes could contribute to an increased bile acid production and a defective hepatobiliary transport capacity thereby rendering c-Met mice more susceptible to toxic injury. The role of c-Met in cellular and molecular physiology of bile secretion and cholesterol homeostasis warrants further study. An understanding of the mechanisms involved in hepatobiliary transport systems has major applications in treating liver diseases such as cholestasis. In summary, our studies show that expression of c-Met is a major determinant of adult hepatic stem cell biology. Lack of c-Met affected (i) stem cell renewal, (ii) proliferation potential, (iii) capacity to migrate, (iv) pattern of differentiation, and (v) dynamic interaction with microenvironment. The latter may provide an explanation for the failure of stem cell mediated liver regeneration in cirrhotic livers and have important implications for fibrotic liver disease. Our previous work has established that HGF plays a pivotal role in regulating the onset of S phase and DNA replication following partial hepatectomy (PH). We used c-Met conditional knockout mice (AlbCreMet), in which the c-met gene is inactivated in postnatal hepatocytes by Alb-Cre recombinase to directly address the net biological outcome of c-Met on liver regeneration. Thecurrent work has begun to elucidate the molecular mechanisms behind c-Met signaling in regulation of G2/M progression during liver regeneration and highlights several novel and important issues: (i) c-Met signaling makes a unique contribution to hepatocyte proliferation by sustaining a long-term Erk1/2 phosphorylation which is not compensated either by other tyrosine kinases (e.g. EGFR) or other cytokines related to liver regeneration; (ii) c-Met through hyperactivation of the MAPK/Erk1/2 cascade regulates the mitotic checkpoints via stimulation of cdc2, Plk1, and Aurora B kinase; and (iii) the G2/M block caused by the lack of c-Met signaling in hepatocytes is associated with anomalous dynamics of chromosome condensation and related defects in chromatin-induced microtubule stabilization and spi [summary truncated at 7800 characters]

椭圆形细胞（肝干细胞后代）起源于位于实质和胆汁树之间界面的末端胆管管内的干细胞，并有可能分化为肝细胞和胆汁上皮细胞（BEC）。 为了应对严重的肝细胞损伤，椭圆形细胞形成了一种增殖的分支导管系统，该系统在实质内移动，最终将其区分为肝细胞谱系。多种分子因子和细胞类型有助于成年干细胞激活的过程。我们和其他人已经确定，肝组织的椭圆形细胞浸润与提供HGF的星状细胞密切相结合，并促进细胞周围的胶原蛋白沉积，从而产生了支撑膨胀导管细胞的生长的微环境。我们研究的目的是通过利用具有C-Met Floxed等位基因（CTRL）和Alb-CRE或MX1-CRE转基因的小鼠来确定C-MET在干细胞激活不同阶段的作用。为了激活椭圆形细胞，我们使用了含有含量卟啉剂3、5-二乙基骨1,4-二氢核苷（DDC）的饮食诱导的慢性肝损伤模型，该模型是由我们先前的工作引入现场的。在上皮细胞类型（Albcremet模型）和所有肝细胞（MXCREMET模型）中，C-MET的灭活均已灭活，使我们能够解决上皮细胞和非旁皮细胞室之间的串扰的重要性。 MXCREMET小鼠的表型非常相似，尽管比生化和形态学数据确定的Albcremet小鼠的表型非常严重。在没有C-MET信号的情况下，肝细胞和干细胞介导的肝脏再生均严重受损。由于肝细胞增殖的减少和凋亡死亡的增加，肝脏质量没有补偿性增加。在分子水平上，MET突变小鼠无法激活与细胞增殖，运动性调节和凋亡保护相关的主要下游信号通路，例如ERK1/2，STAT3和AKT。 C-MET缺乏的肝脏显示出经典的胆管增殖，受到更严重的周围纤维化的限制，反映了椭圆形细胞出生的失败和/或将其分化向BEC谱系的失败。使用针对BEC和椭圆形细胞（A6和EPCAM）的细胞特异性抗体对频率和组织分布的分析表明，A6+/EPCAM+细胞的数量显着减少，并且几乎完全缺乏实质内部的迁移。 更重要的是，我们发现A6+/EPCAM-/HNF4+细胞的数量显着减少，这与在没有C-MET信号传导的情况下降低了分化为肝细胞的能力。这伴随着ki67免疫组织化学估计的C-MET缺乏肝脏中的导管增殖率的逐渐下降。此外，在培养中的实验表明，从C-MET KO小鼠中分离的Epcam+/谱系细胞的单细胞悬浮液始终显示出在BD Matrigeltm中富集在BD Matrigeltm中的球体形成细胞的频率降低。与HGF/C-MET在防御纤维化中的重要作用一致，与CTRL小鼠相比，C-MET有条件敲除小鼠的C-MET模型都在更广泛的外围纤维化方面发展了更广泛的蛋白质，尽管已知的蛋白质水平是已知的纤维源性因子，例如SMA，TGF＆＃946; 1，and Timp-1。然而，产生胶原蛋白的星状细胞的组织分布截然不同，大多数严格位于外围纤维化区域。这与组织浸润的单核细胞/巨噬细胞和MMP9活性降低相似。 FACS分类的F4/80+库普弗细胞的明胶Zymography鉴定出巨噬细胞是产生MMP-9的主要细胞类型。在CTRL肝脏中，A6共聚焦显微镜与原位Zymography或与F4/80共同染色的组合表明，MMP-9的活性沿扩展的椭圆形细胞的途径增加，这表明MMP-9的纤维化活性对椭圆形细胞的迁移有助于卵子细胞的迁移。由于这些现象发生在非实质性肝细胞的基因型中，因此这些数据表明上皮成分在创建微环境中的关键功能作用，这可能会影响肝前体细胞的命运确定和终末分化。 C-Met基因消融导致C-MET参与控制多个细胞功能引起的多效性表型。我们的研究提供了形态学和生化证据，表明在没有C-MET信号传导的情况下，两个上皮室都发生了相当大的损害。 DDC处理后，Albcremet和MXCremet小鼠表现出受损的胆汁分泌物，具有显着的功能性和分子改变，这是由肝脏的主要基因涉及胆汁酸产生，代谢和运输的肝表达所判断的。此外，我们发现肌动蛋白细胞骨架组织中的缺陷以及将BGP1搬迁到膜室中，这可能会影响运输膜中转运蛋白的分类和维护。 C-MET缺乏肝细胞中排泄功能的结合机制可能导致胆汁酸的产生增加，并且有缺陷的肝胆管运输能力，从而使C-MET小鼠更容易受到毒性损伤。 C-MET在胆汁分泌和胆固醇稳态的细胞和分子生理学中的作用值得进一步研究。对肝胆运输系统涉及的机制的理解在治疗诸如胆汁淤积之类的肝脏疾病方面有主要应用。总而言之，我们的研究表明，C-MET的表达是成年肝干细胞生物学的主要决定因素。缺乏受影响的C-MET（I）干细胞更新，（II）增殖潜力，（iii）迁移的能力，（iv）分化模式以及（v）与微环境的动态相互作用。后者可以为肝硬化肝脏中干细胞介导的肝脏再生的失败提供解释，并对纤维化肝病具有重要意义。我们以前的工作已经确定，HGF在调节部分肝切除术（PH）后调节S相和DNA复制的发作中起关键作用。我们使用了条件敲除小鼠（Albcremet），其中C-MET基因通过Alb-Cre重组酶在产后肝细胞中灭活，以直接解决肝脏再生中C-MET的净生物学结果。 Thecurrent work has begun to elucidate the molecular mechanisms behind c-Met signaling in regulation of G2/M progression during liver regeneration and highlights several novel and important issues: (i) c-Met signaling makes a unique contribution to hepatocyte proliferation by sustaining a long-term Erk1/2 phosphorylation which is not compensated either by other tyrosine kinases (e.g. EGFR) or other cytokines与肝脏再生有关； （ii）通过MAPK/ERK1/2级联反激活C-MET通过刺激CDC2，PLK1和Aurora B激酶来调节有丝分裂检查点； （iii）由肝细胞中缺乏C-MET信号传导引起的G2/M块与染色质诱导的微管稳定和SPI的染色体凝结和相关缺陷的异常动态有关