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1.
Hepatology ; 62(5): 1497-510, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26173433

RESUMO

UNLABELLED: The Yes-associated protein (YAP)/Hippo pathway has been implicated in tissue development, regeneration, and tumorigenesis. However, its role in cholangiocarcinoma (CC) is not established. We show that YAP activation is a common feature in CC patient biopsies and human CC cell lines. Using microarray expression profiling of CC cells with overexpressed or down-regulated YAP, we show that YAP regulates genes involved in proliferation, apoptosis, and angiogenesis. YAP activity promotes CC growth in vitro and in vivo by functionally interacting with TEAD transcription factors (TEADs). YAP activity together with TEADs prevents apoptosis induced by cytotoxic drugs, whereas YAP knockdown sensitizes CC cells to drug-induced apoptosis. We further show that the proangiogenic microfibrillar-associated protein 5 (MFAP5) is a direct transcriptional target of YAP/TEAD in CC cells and that secreted MFAP5 promotes tube formation of human microvascular endothelial cells. High YAP activity in human CC xenografts and clinical samples correlates with increased MFAP5 expression and CD31(+) vasculature. CONCLUSIONS: These findings establish YAP as a key regulator of proliferation and antiapoptotic mechanisms in CC and provide first evidence that YAP promotes angiogenesis by regulating the expression of secreted proangiogenic proteins.


Assuntos
Neoplasias dos Ductos Biliares/patologia , Ductos Biliares Intra-Hepáticos , Colangiocarcinoma/patologia , Proteínas de Ligação a DNA/fisiologia , Resistencia a Medicamentos Antineoplásicos , Neovascularização Patológica/etiologia , Proteínas Nucleares/fisiologia , Fatores de Transcrição/fisiologia , Animais , Apoptose , Neoplasias dos Ductos Biliares/irrigação sanguínea , Neoplasias dos Ductos Biliares/tratamento farmacológico , Proteínas de Ciclo Celular , Proliferação de Células , Colangiocarcinoma/irrigação sanguínea , Colangiocarcinoma/tratamento farmacológico , Proteínas Contráteis/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Glicoproteínas/genética , Humanos , Peptídeos e Proteínas de Sinalização Intercelular , Camundongos , Oncogenes , Fatores de Transcrição de Domínio TEA
2.
Dev Biol ; 390(2): 181-90, 2014 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-24680895

RESUMO

Lgr4 and Lgr5 are known markers of adult and embryonic tissue stem cells in various organs. However, whether Lgr4 and Lgr5 are important for embryonic development remains unclear. To study their functions during intestinal crypt, skin and kidney development we now generated mice lacking either Lgr4 (Lgr4KO), Lgr5 (Lgr5KO) or both receptors (Lgr4/5dKO). E16.5 Lgr4KO mice displayed complete loss of Lgr5+/Olfm4+intestinal stem cells, compromised Wnt signaling and impaired proliferation and differentiation of gut epithelium. Similarly, E16.5 Lgr4KO mice showed reduced basal cell proliferation and hair follicle numbers in the developing skin, as well as dilated kidney tubules and ectatic Bowman׳s spaces. Although Lgr4KO and Lgr5KO mice both died perinatally, Lgr5 deletion did not compromise embryonic development of gut, kidney or skin. Concomitant deletion of Lgr4 and Lgr5 did not prevent perinatal lethality, in contrast to a previous report that suggested rescue of Lgr5 KO perinatal lethality by a hypomorphic Lgr4 mutant. While the double deletion did not further promote the phenotypes observed in Lgr4KO intestines, impaired kidney cell proliferation, reduced epidermal thickness, loss of Lgr5+follicular epithelium and impaired hair follicle development were only observed in Lgr4/5dKO mice. This supports complementary functions of both receptors. Our findings clearly establish the importance of Lgr4 and Lgr5 during embryonic gut, skin and kidney development, with a dominant role of Lgr4.


Assuntos
Intestinos/embriologia , Rim/embriologia , Receptores Acoplados a Proteínas G/fisiologia , Pele/embriologia , Via de Sinalização Wnt/fisiologia , Animais , Southern Blotting , Primers do DNA/genética , Componentes do Gene , Genótipo , Proteínas de Fluorescência Verde , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Reação em Cadeia da Polimerase , Receptores Acoplados a Proteínas G/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células-Tronco/fisiologia , Via de Sinalização Wnt/genética
3.
Cell Stem Cell ; 31(4): 554-569.e17, 2024 Apr 04.
Artigo em Inglês | MEDLINE | ID: mdl-38579685

RESUMO

The YAP/Hippo pathway is an organ growth and size regulation rheostat safeguarding multiple tissue stem cell compartments. LATS kinases phosphorylate and thereby inactivate YAP, thus representing a potential direct drug target for promoting tissue regeneration. Here, we report the identification and characterization of the selective small-molecule LATS kinase inhibitor NIBR-LTSi. NIBR-LTSi activates YAP signaling, shows good oral bioavailability, and expands organoids derived from several mouse and human tissues. In tissue stem cells, NIBR-LTSi promotes proliferation, maintains stemness, and blocks differentiation in vitro and in vivo. NIBR-LTSi accelerates liver regeneration following extended hepatectomy in mice. However, increased proliferation and cell dedifferentiation in multiple organs prevent prolonged systemic LATS inhibition, thus limiting potential therapeutic benefit. Together, we report a selective LATS kinase inhibitor agonizing YAP signaling and promoting tissue regeneration in vitro and in vivo, enabling future research on the regenerative potential of the YAP/Hippo pathway.


Assuntos
Inibidores de Proteínas Quinases , Proteínas Serina-Treonina Quinases , Proteínas de Sinalização YAP , Animais , Humanos , Camundongos , Proliferação de Células , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/metabolismo , Células-Tronco/metabolismo , Fatores de Transcrição/metabolismo , Proteínas de Sinalização YAP/agonistas , Proteínas de Sinalização YAP/efeitos dos fármacos , Proteínas de Sinalização YAP/metabolismo , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia
4.
Nat Genet ; 55(4): 651-664, 2023 04.
Artigo em Inglês | MEDLINE | ID: mdl-36914834

RESUMO

Following severe liver injury, when hepatocyte-mediated regeneration is impaired, biliary epithelial cells (BECs) can transdifferentiate into functional hepatocytes. However, the subset of BECs with such facultative tissue stem cell potential, as well as the mechanisms enabling transdifferentiation, remains elusive. Here we identify a transitional liver progenitor cell (TLPC), which originates from BECs and differentiates into hepatocytes during regeneration from severe liver injury. By applying a dual genetic lineage tracing approach, we specifically labeled TLPCs and found that they are bipotent, as they either differentiate into hepatocytes or re-adopt BEC fate. Mechanistically, Notch and Wnt/ß-catenin signaling orchestrate BEC-to-TLPC and TLPC-to-hepatocyte conversions, respectively. Together, our study provides functional and mechanistic insights into transdifferentiation-assisted liver regeneration.


Assuntos
Regeneração Hepática , Fígado , Proliferação de Células/genética , Hepatócitos , Células Epiteliais , Células-Tronco , Diferenciação Celular/genética
5.
Cell Stem Cell ; 28(10): 1822-1837.e10, 2021 10 07.
Artigo em Inglês | MEDLINE | ID: mdl-34129813

RESUMO

AXIN2 and LGR5 mark intestinal stem cells (ISCs) that require WNT/ß-Catenin signaling for constant homeostatic proliferation. In contrast, AXIN2/LGR5+ pericentral hepatocytes show low proliferation rates despite a WNT/ß-Catenin activity gradient required for metabolic liver zonation. The mechanisms restricting proliferation in AXIN2+ hepatocytes and metabolic gene expression in AXIN2+ ISCs remained elusive. We now show that restricted chromatin accessibility in ISCs prevents the expression of ß-Catenin-regulated metabolic enzymes, whereas fine-tuning of WNT/ß-Catenin activity by ZNRF3 and RNF43 restricts proliferation in chromatin-permissive AXIN2+ hepatocytes, while preserving metabolic function. ZNRF3 deletion promotes hepatocyte proliferation, which in turn becomes limited by RNF43 upregulation. Concomitant deletion of RNF43 in ZNRF3 mutant mice results in metabolic reprogramming of periportal hepatocytes and induces clonal expansion in a subset of hepatocytes, ultimately promoting liver tumors. Together, ZNRF3 and RNF43 cooperate to safeguard liver homeostasis by spatially and temporally restricting WNT/ß-Catenin activity, balancing metabolic function and hepatocyte proliferation.


Assuntos
Fígado , Ubiquitina-Proteína Ligases/genética , Animais , Proliferação de Células , Hepatócitos/metabolismo , Fígado/crescimento & desenvolvimento , Fígado/metabolismo , Camundongos , Células-Tronco/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Via de Sinalização Wnt , beta Catenina/metabolismo
6.
Cell Stem Cell ; 26(1): 97-107.e6, 2020 01 02.
Artigo em Inglês | MEDLINE | ID: mdl-31866224

RESUMO

The existence of specialized liver stem cell populations, including AXIN2+ pericentral hepatocytes, that safeguard homeostasis and repair has been controversial. Here, using AXIN2 lineage tracing in BAC-transgenic mice, we confirm the regenerative potential of intestinal stem cells (ISCs) but find limited roles for pericentral hepatocytes in liver parenchyma homeostasis. Liver regrowth following partial hepatectomy is enabled by proliferation of hepatocytes throughout the liver, rather than by a pericentral population. Periportal hepatocyte injury triggers local repair as well as auxiliary proliferation in all liver zones. DTA-mediated ablation of AXIN2+ pericentral hepatocytes transiently disrupts this zone, which is reestablished by conversion of pericentral vein-juxtaposed glutamine synthetase (GS)- hepatocytes into GS+ hepatocytes and by compensatory proliferation of hepatocytes across liver zones. These findings show hepatocytes throughout the liver can upregulate AXIN2 and LGR5 after injury and contribute to liver regeneration on demand, without zonal dominance by a putative pericentral stem cell population.


Assuntos
Hepatócitos , Fígado , Animais , Proteína Axina , Homeostase , Regeneração Hepática , Camundongos , Células-Tronco
7.
Cell Stem Cell ; 25(1): 39-53.e10, 2019 07 03.
Artigo em Inglês | MEDLINE | ID: mdl-31080135

RESUMO

Biliary epithelial cells (BECs) form bile ducts in the liver and are facultative liver stem cells that establish a ductular reaction (DR) to support liver regeneration following injury. Liver damage induces periportal LGR5+ putative liver stem cells that can form BEC-like organoids, suggesting that RSPO-LGR4/5-mediated WNT/ß-catenin activity is important for a DR. We addressed the roles of this and other signaling pathways in a DR by performing a focused CRISPR-based loss-of-function screen in BEC-like organoids, followed by in vivo validation and single-cell RNA sequencing. We found that BECs lack and do not require LGR4/5-mediated WNT/ß-catenin signaling during a DR, whereas YAP and mTORC1 signaling are required for this process. Upregulation of AXIN2 and LGR5 is required in hepatocytes to enable their regenerative capacity in response to injury. Together, these data highlight heterogeneity within the BEC pool, delineate signaling pathways involved in a DR, and clarify the identity and roles of injury-induced periportal LGR5+ cells.


Assuntos
Lesão Pulmonar Aguda/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Ductos Biliares/patologia , Proteínas de Ciclo Celular/metabolismo , Células Epiteliais/fisiologia , Células-Tronco Pluripotentes Induzidas/fisiologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Proteína Axina/genética , Proteína Axina/metabolismo , Proteínas de Ciclo Celular/genética , Células Cultivadas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Modelos Animais de Doenças , Humanos , Regeneração Hepática , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Piridinas/toxicidade , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Trombospondinas/genética , Trombospondinas/metabolismo , Via de Sinalização Wnt , Proteínas de Sinalização YAP
9.
SLAS Discov ; 22(5): 571-582, 2017 06.
Artigo em Inglês | MEDLINE | ID: mdl-28345372

RESUMO

Oral and intestinal mucositis is a debilitating side effect of radiation treatment. A mouse model of radiation-induced mucositis leads to weight loss and tissue damage, reflecting the human ailment as it responds to keratinocyte growth factor (KGF), the standard-of-care treatment. Cultured intestinal crypt organoids allowed the development of an assay monitoring the effect of treatments of intestinal epithelium to radiation-induced damage. This in vitro assay resembles the mouse model as KGF and roof plate-specific spondin-1 (RSPO1) enhanced crypt organoid recovery following radiation. Screening identified compounds that increased the survival of organoids postradiation. Testing of these compounds revealed that the organoids changed their responses over time. Unbiased transcriptome analysis was performed on crypt organoid cultures at various time points in culture to investigate this adaptive behavior. A number of genes and pathways were found to be modulated over time, providing a rationale for the altered sensitivity of the organoid cultures. This report describes an in vitro assay that reflects aspects of human disease. The assay was used to identify bioactive compounds, which served as probes to interrogate the biology of crypt organoids over prolonged culture. The pathways that are changing over time may offer potential targets for treatment of mucositis.


Assuntos
Ensaios de Seleção de Medicamentos Antitumorais/métodos , Intestinos/efeitos dos fármacos , Organoides/efeitos dos fármacos , Animais , Técnicas de Cultura de Células/métodos , Fator 7 de Crescimento de Fibroblastos/metabolismo , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Organoides/metabolismo , Trombospondinas/metabolismo , Transcriptoma/fisiologia
10.
Acad Radiol ; 23(11): 1446-1453, 2016 11.
Artigo em Inglês | MEDLINE | ID: mdl-27595407

RESUMO

RATIONALE AND OBJECTIVES: The study aimed to validate magnetic resonance imaging (MRI)-based liver volumetry as a quantitative measure of hepatic regeneration in mice subjected to partial hepatectomy, in view of routine in vivo pharmacologic studies characterizing compounds aiming to accelerate liver regeneration. MATERIALS AND METHODS: Partial hepatectomy was performed in male B6 mice (n = 47). Images were acquired in 14.5 minutes from anesthetized and spontaneously respiring animals, without any gating and without administration of contrast material. Some of the mice (n = 6) were treated with 1, 4-bis [2-(3, 5-dichloropyridyloxy)] benzene (TCPOBOP), a synthetic agonist of mouse constitutive androstane receptor, or with the corresponding vehicle (n = 6). Postmortem analyses included total liver weight and histologic Ki67 expression. RESULTS: A highly significant correlation (R = 0.98, P = 1.5 × 10-14) was obtained between the MRI-derived liver volumes and the postmortem liver weights in hepatectomized, untreated mice. MRI reliably monitored enhanced murine liver regrowth following treatment with TCPOBOP, as confirmed by comparative hepatocyte proliferation (Ki67 expression) and liver weight analysis (R = 0.96, P = 2 × 10-6). CONCLUSIONS: MRI-based monitoring of liver regrowth in mice without the requirement of euthanizing animals at several time points has been established. In comparison to terminal procedures, the number of hepatectomized mice needed to derive a liver (re)growth curve was reduced by a factor of 6. The feasibility of using this imaging approach in pharmacologic studies in the context of liver regeneration has been demonstrated.


Assuntos
Hepatectomia/métodos , Regeneração Hepática , Fígado/diagnóstico por imagem , Imageamento por Ressonância Magnética , Animais , Proliferação de Células , Receptor Constitutivo de Androstano , Meios de Contraste , Modelos Animais de Doenças , Hepatócitos/fisiologia , Fígado/patologia , Regeneração Hepática/efeitos dos fármacos , Masculino , Camundongos , Tamanho do Órgão , Piridinas/administração & dosagem , Receptores Citoplasmáticos e Nucleares/agonistas
11.
Nat Cell Biol ; 18(5): 467-79, 2016 05.
Artigo em Inglês | MEDLINE | ID: mdl-27088858

RESUMO

LGR4/5 receptors and their cognate RSPO ligands potentiate Wnt/ß-catenin signalling and promote proliferation and tissue homeostasis in epithelial stem cell compartments. In the liver, metabolic zonation requires a Wnt/ß-catenin signalling gradient, but the instructive mechanism controlling its spatiotemporal regulation is not known. We have now identified the RSPO-LGR4/5-ZNRF3/RNF43 module as a master regulator of Wnt/ß-catenin-mediated metabolic liver zonation. Liver-specific LGR4/5 loss of function (LOF) or RSPO blockade disrupted hepatic Wnt/ß-catenin signalling and zonation. Conversely, pathway activation in ZNRF3/RNF43 LOF mice or with recombinant RSPO1 protein expanded the hepatic Wnt/ß-catenin signalling gradient in a reversible and LGR4/5-dependent manner. Recombinant RSPO1 protein increased liver size and improved liver regeneration, whereas LGR4/5 LOF caused the opposite effects, resulting in hypoplastic livers. Furthermore, we show that LGR4(+) hepatocytes throughout the lobule contribute to liver homeostasis without zonal dominance. Taken together, our results indicate that the RSPO-LGR4/5-ZNRF3/RNF43 module controls metabolic liver zonation and is a hepatic growth/size rheostat during development, homeostasis and regeneration.


Assuntos
Fígado/citologia , Receptores Acoplados a Proteínas G/metabolismo , Trombospondinas/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Animais Recém-Nascidos , Linhagem da Célula , Proliferação de Células , Citocromo P-450 CYP2E1/metabolismo , Deleção de Genes , Hepatócitos/citologia , Hepatócitos/metabolismo , Homeostase , Antígeno Ki-67/metabolismo , Fígado/crescimento & desenvolvimento , Fígado/metabolismo , Regeneração Hepática , Tamanho do Órgão , Transdução de Sinais , beta-Galactosidase/metabolismo
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