RESUMO
BACKGROUND: Biliary atresia (BA) is a congenital, progressive, fibro-obliterative disease of the extrahepatic biliary tree and the most common cause of end-stage liver disease in children. BA is characterized by extensive intrahepatic proliferating ductular reactions that may contribute to biliary fibrosis. Lineage tracing during experimental cholestasis indicates that cells within ductular reactions derive from PROM1-expressing hepatic progenitor cells. Given the role of Notch signaling in normal biliary development, we hypothesize that activated Notch signaling promotes the formation of ductular reactions in BA. METHODS: Liver samples collected from BA infants at Kasai portoenterostomy and age-matched controls, as well as from wild-type and Prom1 knockout mice with 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC)-induced experimental cholestasis were analyzed histologically using immunofluorescence and by quantitative polymerase chain reaction. RESULTS: Increased expression of genes encoding Notch ligand JAG1 and its receptor NOTCH2 was observed in BA livers compared with control by quantitative polymerase chain reaction analyses. Livers of DDC-treated mice, which exhibit cytokeratin-19-positive ductular reactions typical of BA livers, demonstrated significant increases in the expression level of the gene encoding Notch2, as well as downstream Notch target gene Hes1 compared with control. Prom1 knockout mice exhibit diminished ductular reactions and decreased levels of Jag1 and Hes1 compared with littermate controls. CONCLUSIONS: Human BA and cholestasis induced by DDC are associated with Notch signaling activation. Null mutation of Prom1 is associated with decreased ductular reactions and decreased Notch signaling activation during DDC treatment. These data are consistent with Notch signaling promoting ductular reactions of Prom1 expressing progenitor cells in BA.
Assuntos
Atresia Biliar/metabolismo , Atresia Biliar/patologia , Receptor Notch2/metabolismo , Transdução de Sinais , Antígeno AC133/genética , Antígeno AC133/metabolismo , Animais , Biomarcadores/metabolismo , Estudos de Casos e Controles , Humanos , Imuno-Histoquímica , Proteína Jagged-1/metabolismo , Camundongos , Camundongos Knockout , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Transcrição HES-1/metabolismo , Regulação para CimaRESUMO
Cancers are distributed unevenly across the body, but the importance of cell intrinsic factors such as stem cell function in determining organ cancer risk is unknown. Therefore, we used Cre-recombination of conditional lineage tracing, oncogene, and tumor suppressor alleles to define populations of stem and non-stem cells in mouse organs and test their life-long susceptibility to tumorigenesis. We show that tumor incidence is determined by the life-long generative capacity of mutated cells. This relationship held true in the presence of multiple genotypes and regardless of developmental stage, strongly supporting the notion that stem cells dictate organ cancer risk. Using the liver as a model system, we further show that damage-induced activation of stem cell function markedly increases cancer risk. Therefore, we propose that a combination of stem cell mutagenesis and extrinsic factors that enhance the proliferation of these cell populations, creates a "perfect storm" that ultimately determines organ cancer risk. VIDEO ABSTRACT.
Assuntos
Carcinogênese/genética , Carcinogênese/patologia , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Oncogenes , Células-Tronco , Alelos , Animais , Genes Supressores de Tumor , Humanos , Integrases , Camundongos , Modelos Biológicos , Mutagênese , Recombinação Genética , Risco , Células-Tronco/metabolismo , Células-Tronco/patologiaRESUMO
UNLABELLED: Biliary atresia (BA), the most common cause of end-stage liver disease and the leading indication for pediatric liver transplantation, is associated with intrahepatic ductular reactions within regions of rapidly expanding periportal biliary fibrosis. Whereas the extent of such biliary fibrosis is a negative predictor of long-term transplant-free survival, the cellular phenotypes involved in the fibrosis are not well established. Using a rhesus rotavirus-induced mouse model of BA, we demonstrate significant expansion of a cell population expressing the putative stem/progenitor cell marker, PROMININ-1 (PROM1), adjacent to ductular reactions within regions of periportal fibrosis. PROM1positive (pos) cells express Collagen-1α1. Subsets of PROM1pos cells coexpress progenitor cell marker CD49f, epithelial marker E-CADHERIN, biliary marker CYTOKERATIN-19, and mesenchymal markers VIMENTIN and alpha-SMOOTH MUSCLE ACTIN (αSMA). Expansion of the PROM1pos cell population is associated with activation of Fibroblast Growth Factor (FGF) and Transforming Growth Factor-beta (TGFß) signaling. In vitro cotreatment of PROM1-expressing Mat1a-/- hepatic progenitor cells with recombinant human FGF10 and TGFß1 promotes morphologic transformation toward a myofibroblastic cell phenotype with increased expression of myofibroblastic genes Collagen-1α1, Fibronectin, and α-Sma. Infants with BA demonstrate similar expansion of periportal PROM1pos cells with activated Mothers Against Decapentaplegic Homolog 3 (SMAD3) signaling in association with increased hepatic expression of FGF10, FGFR1, and FGFR2 as well as mesenchymal genes SLUG and SNAIL. Infants with perinatal subtype of BA have higher tissue levels of PROM1 expression than those with embryonic subtype. CONCLUSION: Expansion of collagen-producing PROM1pos cells within regions of periportal fibrosis is associated with activated FGF and TGFß pathways in both experimental and human BA. PROM1pos cells may therefore play an important role in the biliary fibrosis of BA.
Assuntos
Antígenos CD/biossíntese , Atresia Biliar/metabolismo , Glicoproteínas/biossíntese , Cirrose Hepática/metabolismo , Antígeno AC133 , Animais , Atresia Biliar/complicações , Modelos Animais de Doenças , Feminino , Fatores de Crescimento de Fibroblastos/metabolismo , Humanos , Cirrose Hepática/complicações , Cirrose Hepática/patologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Peptídeos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Infecções por Rotavirus/complicações , Fator de Crescimento Transformador beta/metabolismo , beta Catenina/metabolismoRESUMO
BACKGROUND & AIMS: Fibroblast Growth Factors (FGFs) promote the proliferation and survival of hepatic progenitor cells (HPCs) via AKT-dependent ß-catenin activation. Moreover, the emergence of hepatocytes expressing the HPC marker A6 during 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC)-induced liver injury is mediated partly by FGF and ß-catenin signaling. Herein, we investigate the role of FGF signaling and AKT-mediated ß-catenin activation in acute DDC liver injury. METHODS: Transgenic mice were fed DDC chow for 14days concurrent with either Fgf10 over-expression or inhibition of FGF signaling via expression of soluble dominant-negative FGF Receptor (R)-2IIIb. RESULTS: After 14days of DDC treatment, there was an increase in periportal cells expressing FGFR1, FGFR2, and AKT-activated phospho-Serine 552 (pSer552) ß-Catenin in association with up-regulation of genes encoding the FGFR2IIIb ligands, Fgf7, Fgf10, and Fgf22. In response to Fgf10 over-expression, there was an increase in the number of pSer552-ß-Catenin((positive)+ive) periportal cells as well as cells co-positive for A6 and hepatocyte marker, Hepatocyte Nuclear Factor-4α (HNF4α). A similar expansion of A6(+ive) cells was observed after Fgf10 over-expression with regular chow and after partial hepatectomy during ethanol toxicity. Inhibition of FGF signaling increased the periportal A6(+ive)HNF4α(+ive) cell population while reducing centrolobular A6(+ive) HNF4α(+ive) cells. AKT inhibition with Wortmannin attenuated FGF10-mediated A6(+ive)HNF4α(+ive) cell expansion. In vitro analyses using FGF10 treated HepG2 cells demonstrated AKT-mediated ß-Catenin activation but not enhanced cell migration. CONCLUSIONS: During acute DDC treatment, FGF signaling promotes the expansion of A6-expressing liver cells partly via AKT-dependent activation of ß-Catenin expansion of A6(+ive) periportal cells and possibly by reprogramming of centrolobular hepatocytes.
Assuntos
Fatores de Crescimento de Fibroblastos/metabolismo , Hepatócitos/citologia , Hepatócitos/metabolismo , Proteínas dos Microfilamentos/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , beta Catenina/metabolismo , Animais , Biomarcadores/metabolismo , Proliferação de Células , Doença Hepática Induzida por Substâncias e Drogas/genética , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Doença Hepática Induzida por Substâncias e Drogas/patologia , Fator 10 de Crescimento de Fibroblastos/genética , Fator 10 de Crescimento de Fibroblastos/metabolismo , Fatores de Crescimento de Fibroblastos/genética , Células Hep G2 , Fator 4 Nuclear de Hepatócito/genética , Fator 4 Nuclear de Hepatócito/metabolismo , Hepatócitos/efeitos dos fármacos , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Inibidores de Fosfoinositídeo-3 Quinase , Proteínas Tirosina Quinases/metabolismo , Piridinas/toxicidade , Receptores de Fatores de Crescimento de Fibroblastos/genética , Receptores de Fatores de Crescimento de Fibroblastos/metabolismo , Transdução de Sinais/efeitos dos fármacos , Células-Tronco/citologia , Células-Tronco/metabolismo , Regulação para Cima/efeitos dos fármacosRESUMO
UNLABELLED: Fibroblast Growth Factor (FGF)-10 promotes the proliferation and survival of murine hepatoblasts during early stages of hepatogenesis through a Wnt-ß-catenin dependent pathway. To determine the mechanism by which this occurs, we expanded primary culture of hepatoblasts enriched for progenitor markers CD133 and CD49f from embryonic day (E) 12.5 fetal liver and an established tumor initiating stem cell line from Mat1a(-/-) livers in media conditioned with recombinant (r) FGF10 or rFGF7. FGF Receptor (R) activation resulted in the downstream activation of MAPK, PI3K-AKT, and ß-catenin pathways, as well as cellular proliferation. Additionally, increased levels of nuclear ß-catenin phosphorylated at Serine-552 in cultured primary hepatoblasts, Mat1a(-/-) cells, and also in ex vivo embryonic liver explants indicate AKT-dependent activation of ß-catenin downstream of FGFR activation; conversely, the addition of AKT inhibitor Ly294002 completely abrogated ß-catenin activation. FGFR activation-induced cell proliferation and survival were also inhibited by the compound ICG-001, a small molecule inhibitor of ß-catenin-CREB Binding Protein (CBP) in hepatoblasts, further indicating a CBP-dependent regulatory mechanism of ß-catenin activity. CONCLUSION: FGF signaling regulates the proliferation and survival of embryonic and transformed progenitor cells in part through AKT-mediated activation of ß-catenin and downstream interaction with the transcriptional co-activator CBP.
Assuntos
Proteína de Ligação a CREB/metabolismo , Neoplasias Hepáticas/patologia , Fígado/citologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores de Fatores de Crescimento de Fibroblastos/metabolismo , Transdução de Sinais , beta Catenina/metabolismo , Animais , Ciclo Celular , Proliferação de Células , Sobrevivência Celular , Células-Tronco Embrionárias/citologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Humanos , Fígado/patologia , Metionina Adenosiltransferase/deficiência , Camundongos , Camundongos Endogâmicos C57BL , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologiaRESUMO
BACKGROUND: Understanding the pathways regulating mesenchymal progenitor cell fate during hepatogenesis may provide insight into postnatal liver injury or liver bioengineering. While ß-Catenin has been implicated in the proliferation of fetal hepatic epithelial progenitor cells, its role in mesenchymal precursors during hepatogenesis has not been established. MATERIALS AND METHODS: We used a murine model of conditional deletion of ß-Catenin in mesenchyme using the Dermo1 locus (ß-Catenin(Dermo1)) to characterize the role of ß-Catenin in liver mesenchyme during hepatogenesis. RESULTS: Lineage tracing using a LacZ reporter indicates that both hepatic stellate cells and pericytes derive from mesenchymal Dermo1 expressing precursor cells. Compared to control littermate livers, ß-Catenin(Dermo1) embryonic livers are smaller and filled with dilated sinusoids. While the fraction of mesenchymally-derived cells in ß-Catenin(Dermo1) embryos is unchanged compared to littermate controls, there is an increase in the expression of the mesenchymal markers, DESMIN, α-SMA, and extracellular deposition of COLLAGEN type I, particularly concentrated around dilated sinusoids. Analysis of the endothelial cell compartment in ß-Catenin(Dermo1)/Flk1(lacZ) embryos revealed a marked reorganization of the intrahepatic vasculature. Analysis of various markers for the endodermally-derived hepatoblast population revealed marked alterations in the spatial expression pattern of pan-cytokeratin but not E-cadherin, or albumin. ß-Catenin(Dermo1) phenocopies mesenchymal deletion of Pitx2, a known regulator of hepatic mesenchymal differentiation both during both organogenesis and postnatal injury. CONCLUSIONS: Our data implicate mesenchymal ß-Catenin signaling pathway in the differentiation of liver mesenchymal progenitor cells during organogenesis, possibly via Pitx2. Hepatic mesenchymal ß-Catenin signaling, in turn, modulates the development of both endothelium and endodermally-derived hepatoblasts, presumably via other downstream paracrine pathways.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Hepatócitos/fisiologia , Células-Tronco Mesenquimais/citologia , beta Catenina/farmacologia , Animais , Embrião de Mamíferos , Desenvolvimento Embrionário/efeitos dos fármacos , Hepatócitos/citologia , Hepatócitos/efeitos dos fármacos , Imuno-Histoquímica , Óperon Lac/efeitos dos fármacos , Células-Tronco Mesenquimais/efeitos dos fármacos , Camundongos , Camundongos Knockout , beta Catenina/deficiência , beta Catenina/toxicidade , beta-Galactosidase/metabolismoRESUMO
Perchlorate is a water soluble anion that is readily accumulated in vegetation. It inhibits uptake of iodide into thyroid gland tissue, thereby reducing production of thyroid hormones. Potential raccoon food items including berries, fish, and vegetation collected at a contaminated site contained quantifiable concentrations of perchlorate as determined by ion chromatography. Therefore, we monitored resident raccoons for exposure to perchlorate by examining plasma perchlorate and thyroid hormone concentrations. Resulting analytical data failed to demonstrate perchlorate exposure among raccoons that likely consumed food items collected along perchlorate-contaminated water bodies. There were no correlations between triiodothyronine or thyroxine and thyroid stimulating hormone concentrations, but triiodothyronine concentrations in raccoon plasma were significantly higher in 2000 than in 2001 (p = 0.0081). These data suggest that natural attenuation and remedial efforts initiated in January of 2001 may have reduced perchlorate exposure among raccoons inhabiting this site from 2000 to 2001. Temporal, spatial, and analytical factors limited our ability to quantify exposure among raccoons, however, our data do not indicate that raccoons currently inhabiting this site are at risk for significant exposure to perchlorate and subsequent effects.