RESUMO
Cellular reprogramming-the ability to interconvert distinct cell types with defined factors-is transforming the field of regenerative medicine. However, this phenomenon has rarely been observed in vivo without exogenous factors. Here, we report that activation of Notch, a signaling pathway that mediates lineage segregation during liver development, is sufficient to reprogram hepatocytes into biliary epithelial cells (BECs). Moreover, using lineage tracing, we show that hepatocytes undergo widespread hepatocyte-to-BEC reprogramming following injuries that provoke a biliary response, a process requiring Notch. These results provide direct evidence that mammalian regeneration prompts extensive and dramatic changes in cellular identity under injury conditions.
Assuntos
Células Epiteliais/citologia , Hepatócitos/citologia , Regeneração Hepática/fisiologia , Animais , Linhagem da Célula , Células Epiteliais/metabolismo , Hepatócitos/metabolismo , Camundongos , Receptores Notch/metabolismo , Transdução de Sinais , Células-Tronco/citologiaRESUMO
BACKGROUND & AIMS: The Notch signaling pathway is activated in leukemia and solid tumors (such as lung cancer), but little is known about its role in liver cancer. METHODS: The intracellular domain of Notch was conditionally expressed in hepatoblasts and their progeny (hepatocytes and cholangiocytes) in mice. This was achieved through Cre expression under the control of an albumin and α-fetoprotein (AFP) enhancer and promoter (AFP-Notch intracellular domain [NICD]). We used comparative functional genomics to integrate transcriptome data from AFP-NICD mice and human hepatocellular carcinoma (HCC) samples (n = 683). A Notch gene signature was generated using the nearest template prediction method. RESULTS: AFP-NICD mice developed HCC with 100% penetrance when they were 12 months old. Activation of Notch signaling correlated with activation of 3 promoters of insulin-like growth factor 2; these processes appeared to contribute to hepatocarcinogenesis. Comparative functional genomic analysis identified a signature of Notch activation in 30% of HCC samples from patients. These samples had altered expression in Notch pathway genes and activation of insulin-like growth factor signaling, despite a low frequency of mutations in regions of NOTCH1 associated with cancer. Blocking Notch signaling in liver cancer cells with the Notch activation signature using γ-secretase inhibitors or by expressing a dominant negative form of mastermind-like 1 reduced their proliferation in vitro. CONCLUSIONS: Notch signaling is activated in human HCC samples and promotes formation of liver tumors in mice. The Notch signature is a biomarker of response to Notch inhibition in vitro.
Assuntos
Carcinoma Hepatocelular/fisiopatologia , Regulação Neoplásica da Expressão Gênica/fisiologia , Neoplasias Hepáticas/fisiopatologia , Receptores Notch/fisiologia , Transdução de Sinais/fisiologia , Animais , Carcinoma Hepatocelular/patologia , Proliferação de Células , Sobrevivência Celular/fisiologia , Células Cultivadas , Modelos Animais de Doenças , Perfilação da Expressão Gênica , Humanos , Técnicas In Vitro , Fator de Crescimento Insulin-Like II/fisiologia , Neoplasias Hepáticas/patologia , Camundongos , Camundongos Endogâmicos , Mutação/genética , Receptores Notch/genéticaRESUMO
UNLABELLED: Whether or not cholangiocytes or their hepatic progenitors undergo an epithelial-to-mesenchymal transition (EMT) to become matrix-producing myofibroblasts during biliary fibrosis is a significant ongoing controversy. To assess whether EMT is active during biliary fibrosis, we used Alfp-Cre × Rosa26-YFP mice, in which the epithelial cells of the liver (hepatocytes, cholangiocytes, and their bipotential progenitors) are heritably labeled at high efficiency with yellow fluorescent protein (YFP). Primary cholangiocytes isolated from our reporter strain were able to undergo EMT in vitro when treated with transforming growth factor-ß1 alone or in combination with tumor necrosis factor-α, as indicated by adoption of fibroblastoid morphology, intracellular relocalization of E-cadherin, and expression of α-smooth muscle actin (α-SMA). To determine whether EMT occurs in vivo, we induced liver fibrosis in Alfp-Cre × Rosa26-YFP mice using the bile duct ligation (BDL) (2, 4, and 8 weeks), carbon tetrachloride (CCl(4) ) (3 weeks), and 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC; 2 and 3 weeks) models. In no case did we find evidence of colocalization of YFP with the mesenchymal markers S100A4, vimentin, α-SMA, or procollagen 1α2, although these proteins were abundant in the peribiliary regions. CONCLUSION: Hepatocytes and cholangiocytes do not undergo EMT in murine models of hepatic fibrosis.
Assuntos
Ductos Biliares/citologia , Linhagem da Célula , Transdiferenciação Celular , Células Epiteliais/citologia , Cirrose Hepática/patologia , Mesoderma/citologia , Animais , Células Cultivadas , Modelos Animais de Doenças , CamundongosRESUMO
BACKGROUND & AIMS: A number of diseases are characterized by defective formation of the intrahepatic bile ducts. In the embryo, hepatoblasts differentiate to cholangiocytes, which give rise to the bile ducts. Here, we investigated duct development in mouse liver and characterized the role of the SRY-related HMG box transcription factor 9 (SOX9). METHODS: We identified SOX9 as a new biliary marker and used it in immunostaining experiments to characterize bile duct morphogenesis. The expression of growth factors was determined by in situ hybridization and immunostaining, and their role was studied on cultured hepatoblasts. SOX9 function was investigated by phenotyping mice with a liver-specific inactivation of Sox9. RESULTS: Biliary tubulogenesis started with formation of asymmetrical ductal structures, lined on the portal side by cholangiocytes and on the parenchymal side by hepatoblasts. When the ducts grew from the hilum to the periphery, the hepatoblasts lining the asymmetrical structures differentiated to cholangiocytes, thereby allowing formation of symmetrical ducts lined only by cholangiocytes. We also provide evidence that transforming growth factor-beta promotes differentiation of the hepatoblasts lining the asymmetrical structures. In the absence of SOX9, the maturation of asymmetrical structures into symmetrical ducts was delayed. This was associated with abnormal expression of CCAAT/Enhancer Binding Protein alpha and Homolog of Hairy/Enhancer of Split-1, as well as of the transforming growth factor-beta receptor type II, which are regulators of biliary development. CONCLUSIONS: Our results suggest that biliary development proceeds according to a new mode of tubulogenesis characterized by transient asymmetry and whose timing is controlled by SOX9.
Assuntos
Ductos Biliares Intra-Hepáticos/embriologia , Regulação da Expressão Gênica no Desenvolvimento , Fatores de Transcrição SOX9/genética , Transdução de Sinais/genética , Animais , Ductos Biliares Intra-Hepáticos/crescimento & desenvolvimento , Diferenciação Celular , Fator de Crescimento Epidérmico/genética , Fator de Crescimento Epidérmico/metabolismo , Feminino , Hibridização In Situ , Fígado/embriologia , Fígado/crescimento & desenvolvimento , Camundongos , Camundongos Knockout , Modelos Animais , Morfogênese/genética , Gravidez , Probabilidade , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Transcrição SOX9/metabolismo , Sensibilidade e Especificidade , Transdução de Sinais/fisiologiaRESUMO
Focal adhesion kinase (FAK) is a non-receptor protein tyrosine kinase implicated in cell cycle progression and cell migration. Overexpression of FAK in a variety of tumors has suggested that FAK is a promising target for therapeutic intervention. In this study, we took advantage of a modified polyethylenimine (M-PEI) with high transfection efficiency for tumor cells and tissues, and targeted FAK function through both in vitro and in vivo approaches. The results demonstrated that both plasmid-encoded FAK small interfering RNA (siRNA) and overexpression of FAK-related non-kinase (FRNK, FAK dominant negative) dramatically inhibited in vitro B16F10 cell proliferation and invasion. We used two transplantable mouse tumor models of primary and metastatic melanoma to evaluate the therapeutic potential of PEI-complexed plasmids targeting FAK function. The results revealed that intratumoral delivery of PEI-complexed plasmids targeting FAK significantly suppressed primary tumor growth as well as metastasis of B16F10 cells into lung and lymph nodes. Both approaches prolonged the survival of the tumor-bearing mice. Taken together, these results indicate that intratumoral delivery of plasmid DNA targeting FAK function, using M-PEI as a gene carrier, represents a promising avenue for melanoma therapy.
Assuntos
Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Melanoma/enzimologia , Melanoma/terapia , Plasmídeos/genética , Polietilenoimina , Animais , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , DNA/genética , Regulação para Baixo , Proteína-Tirosina Quinases de Adesão Focal/genética , Vetores Genéticos/genética , Melanoma/genética , Melanoma/patologia , Camundongos , Camundongos Endogâmicos C57BL , Metástase Neoplásica , Transplante de Neoplasias , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/metabolismo , Interferência de RNA , RNA Mensageiro/genética , TransfecçãoRESUMO
The liver is thought to utilize facultative stem cells, also known as "oval cells" or "atypical ductal cells" (ADCs), for regeneration following various types of injury. However, this notion has been based largely on in vitro studies and transplantation models; where lineage tracing has been used, results have been conflicting and effect sizes have been small. Here, we used genetic and nucleoside analog-based tools to mark and track the origin and contribution of various cell populations to liver regeneration in vivo following several ADC-inducing insults. We report that, contrary to prevailing stem-cell-based models of regeneration, virtually all new hepatocytes come from preexisting hepatocytes.
Assuntos
Diferenciação Celular , Hepatócitos/citologia , Células-Tronco/citologia , Adulto , Animais , Sistema Biliar/citologia , Proliferação de Células , Dependovirus/metabolismo , Células Epiteliais/citologia , Hepatócitos/metabolismo , Humanos , Masculino , Camundongos Endogâmicos C57BLRESUMO
GATA transcription factors and their Friend of Gata (FOG) cofactors control the development of diverse tissues. GATA4 and GATA6 are essential for the expansion of the embryonic liver bud, but their expression patterns and functions in the adult liver are unclear. We characterized the expression of GATA and FOG factors in whole mouse liver and purified hepatocytes. GATA4, GATA6, and FOG1 are the most prominently expressed family members in whole liver and hepatocytes. GATA4 chromatin immunoprecipitation followed by high throughput sequencing (ChIP-seq) identified 4409 occupied sites, associated with genes enriched in ontologies related to liver function, including lipid and glucose metabolism. However, hepatocyte-specific excision of Gata4 had little impact on gross liver architecture and function, even under conditions of regenerative stress, and, despite the large number of GATA4 occupied genes, resulted in relatively few changes in gene expression. To address possible redundancy between GATA4 and GATA6, both factors were conditionally excised. Surprisingly, combined Gata4,6 loss did not exacerbate the phenotype resulting from Gata4 loss alone. This points to the presence of an unusually robust transcriptional network in adult hepatocytes that ensures the maintenance of liver function.
Assuntos
Fatores de Transcrição GATA/metabolismo , Fígado/metabolismo , Animais , Fatores de Transcrição GATA/deficiência , Fatores de Transcrição GATA/genética , Deleção de Genes , Regulação da Expressão Gênica no Desenvolvimento , Redes Reguladoras de Genes , Genômica , Hepatócitos/metabolismo , Fígado/citologia , Fígado/crescimento & desenvolvimento , Camundongos , Especificidade de Órgãos , TranscriptomaRESUMO
The liver is derived from the ventral foregut endoderm. After hepatic specification, liver progenitor cells delaminate from the endoderm and invade the septum transversum mesenchyme to form the liver bud. In addition to proliferation and expansion, liver progenitor cells differentiate into two epithelial cell types, each arranged into unique structures with distinctive function. Growth, morphogenesis, and differentiation during liver development are regulated by a variety of factors that are expressed in a spatially and temporally specific manner. A comprehensive understanding of the regulatory mechanisms underlying the liver development has influenced the diagnosis of liver diseases and further progress will be critical for future advances in therapy. This review highlights some of the best understood steps of liver development, summarizing progress in our understanding of the molecular mechanisms that underlie differentiation, morphogenesis, and functional integration of the liver.
Assuntos
Ductos Biliares/crescimento & desenvolvimento , Diferenciação Celular/genética , Endoderma/crescimento & desenvolvimento , Fígado/crescimento & desenvolvimento , Linhagem da Célula , Endoderma/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Hepatócitos/citologia , Hepatócitos/metabolismo , Humanos , Mesoderma/crescimento & desenvolvimento , Mesoderma/metabolismo , MorfogêneseRESUMO
The mammalian biliary system, consisting of the intrahepatic and extrahepatic bile ducts, is responsible for transporting bile from the liver to the intestine. Bile duct dysfunction, as is seen in some congenital biliary diseases such as Alagille syndrome and biliary atresia, can lead to the accumulation of bile in the liver, preventing the excretion of detoxification products and ultimately leading to liver damage. Bile duct formation requires coordinated cell-cell interactions, resulting in the regulation of cell differentiation and morphogenesis. Multiple signaling molecules and transcription factors have been identified as important regulators of bile duct development. This review summarizes recent progress in the field. Insights gained from studies of the molecular mechanisms of bile duct development have the potential to reveal novel mechanisms of differentiation and morphogenesis in addition to potential targets for therapy of bile duct disorders.
Assuntos
Ductos Biliares Extra-Hepáticos/embriologia , Ductos Biliares Extra-Hepáticos/crescimento & desenvolvimento , Ductos Biliares Intra-Hepáticos/embriologia , Ductos Biliares Intra-Hepáticos/crescimento & desenvolvimento , Animais , Doenças Biliares/metabolismo , Doenças Biliares/patologia , Diferenciação Celular , Regulação da Expressão Gênica no Desenvolvimento , Hepatócitos/citologia , Humanos , MicroRNAs/fisiologia , Morfogênese , Receptores Notch/fisiologia , Transdução de Sinais , Fator de Crescimento Transformador beta/fisiologia , Proteínas Wnt/fisiologia , beta Catenina/fisiologiaRESUMO
In the mammalian liver, bile is transported to the intestine through an intricate network of bile ducts. Notch signaling is required for normal duct formation, but its mode of action has been unclear. Here, we show in mice that bile ducts arise through a novel mechanism of tubulogenesis involving sequential radial differentiation. Notch signaling is activated in a subset of liver progenitor cells fated to become ductal cells, and pathway activation is necessary for biliary fate. Notch signals are also required for bile duct morphogenesis, and activation of Notch signaling in the hepatic lobule promotes ectopic biliary differentiation and tubule formation in a dose-dependent manner. Remarkably, activation of Notch signaling in postnatal hepatocytes causes them to adopt a biliary fate through a process of reprogramming that recapitulates normal bile duct development. These results reconcile previous conflicting reports about the role of Notch during liver development and suggest that Notch acts by coordinating biliary differentiation and morphogenesis.