RESUMEN
Sickle cell disease (SCD)-associated chronic hemolysis promotes oxidative stress, inflammation, and thrombosis leading to organ damage, including liver damage. Hemoglobin scavenger receptor CD163 plays a protective role in SCD by scavenging both hemoglobin-haptoglobin complexes and cell-free hemoglobin. A limited number of studies in the past have shown a positive correlation of CD163 expression with poor disease outcomes in patients with SCD. However, the role and regulation of CD163 in SCD-related hepatobiliary injury have not been fully elucidated yet. Here we show that chronic liver injury in SCD patients is associated with elevated levels of hepatic membrane-bound CD163. Hemolysis and increase in hepatic heme, hemoglobin, and iron levels elevate CD163 expression in the SCD mouse liver. Mechanistically we show that heme oxygenase-1 (HO-1) positively regulates membrane-bound CD163 expression independent of nuclear factor erythroid 2-related factor 2 (NRF2) signaling in SCD liver. We further demonstrate that the interaction between CD163 and HO-1 is not dependent on CD163-hemoglobin binding. These findings indicate that CD163 is a potential biomarker of SCD-associated hepatobiliary injury. Understanding the role of HO-1 in membrane-bound CD163 regulation may help identify novel therapeutic targets for hemolysis-induced chronic liver injury.
Asunto(s)
Anemia de Células Falciformes , Antígenos CD , Antígenos de Diferenciación Mielomonocítica , Biomarcadores , Hemo-Oxigenasa 1 , Hemoglobinas , Hemólisis , Receptores de Superficie Celular , Antígenos de Diferenciación Mielomonocítica/metabolismo , Antígenos de Diferenciación Mielomonocítica/genética , Anemia de Células Falciformes/metabolismo , Anemia de Células Falciformes/sangre , Anemia de Células Falciformes/complicaciones , Antígenos CD/metabolismo , Antígenos CD/genética , Animales , Receptores de Superficie Celular/metabolismo , Receptores de Superficie Celular/genética , Humanos , Biomarcadores/metabolismo , Biomarcadores/sangre , Hemo-Oxigenasa 1/metabolismo , Hemoglobinas/metabolismo , Ratones , Masculino , Hígado/metabolismo , Hígado/patología , Femenino , Ratones Endogámicos C57BL , Adulto , Factor 2 Relacionado con NF-E2/metabolismo , Hemo/metabolismo , Hepatopatías/metabolismo , Hepatopatías/patología , Transducción de Señal , Haptoglobinas/metabolismo , Proteínas de la MembranaRESUMEN
Acute lung injury, referred to as the acute chest syndrome, is a major cause of morbidity and mortality in patients with sickle cell disease (SCD), which often occurs in the setting of a vaso-occlusive painful crisis. P-selectin antibody therapy reduces hospitalization of patients with SCD by â¼50%, suggesting that an unknown P-selectin-independent mechanism promotes remaining vaso-occlusive events. In patients with SCD, intraerythrocytic polymerization of mutant hemoglobin promotes ischemia-reperfusion injury and hemolysis, which leads to the development of sterile inflammation. Using intravital microscopy in transgenic, humanized mice with SCD and in vitro studies with blood from patients with SCD, we reveal for the first time that the sterile inflammatory milieu in SCD promotes caspase-4/11-dependent activation of neutrophil-gasdermin D (GSDMD), which triggers P-selectin-independent shedding of neutrophil extracellular traps (NETs) in the liver. Remarkably, these NETs travel intravascularly from liver to lung, where they promote neutrophil-platelet aggregation and the development of acute lung injury. This study introduces a novel paradigm that liver-to-lung embolic translocation of NETs promotes pulmonary vascular vaso-occlusion and identifies a new GSDMD-mediated, P-selectin-independent mechanism of lung injury in SCD.
Asunto(s)
Lesión Pulmonar Aguda , Anemia de Células Falciformes , Trampas Extracelulares , Proteínas de Unión a Fosfato , Proteínas Citotóxicas Formadoras de Poros , Daño por Reperfusión , Lesión Pulmonar Aguda/etiología , Animales , Hígado , Pulmón/irrigación sanguínea , Ratones , Ratones Transgénicos , Selectina-P , Proteínas de Unión a Fosfato/metabolismo , Proteínas Citotóxicas Formadoras de Poros/metabolismo , Daño por Reperfusión/complicacionesRESUMEN
Sickle cell disease (SCD) is caused by a homozygous mutation in the ß-globin gene, which leads to erythrocyte sickling, vasoocclusion, and intense hemolysis. P-selectin inhibition has been shown to prevent vasoocclusive events in patients with SCD; however, the chronic effect of P-selectin inhibition in SCD remains to be determined. Here, we used quantitative liver intravital microscopy in our recently generated P-selectin-deficient SCD mice to show that chronic P-selectin deficiency attenuates liver ischemia but fails to prevent hepatobiliary injury. Remarkably, we find that this failure in resolution of hepatobiliary injury in P-selectin-deficient SCD mice is associated with the increase in cellular senescence and reduced epithelial cell proliferation in the liver. These findings highlight the importance of investigating the long-term effects of chronic P-selectin inhibition therapy on liver pathophysiology in patients with SCD.
Asunto(s)
Anemia de Células Falciformes/patología , Isquemia/patología , Hígado/irrigación sanguínea , Selectina-P/deficiencia , Anemia de Células Falciformes/fisiopatología , Animales , Arteriopatías Oclusivas/etiología , Arteriopatías Oclusivas/patología , Senescencia Celular , Células Epiteliales/patología , Hemo-Oxigenasa 1/análisis , Hemólisis , Hígado/patología , Hígado/fisiopatología , Proteínas de la Membrana/análisis , Ratones , Ratones Noqueados , Modelos Animales , Selectina-P/genéticaRESUMEN
Sickle cell disease (SCD) is a monogenic disorder that affects 100,000 African Americans and millions of people worldwide. Intra-erythrocytic polymerization of sickle hemoglobin (HbS) promotes erythrocyte sickling, impaired rheology, ischemia and hemolysis, leading to the development of progressive liver injury in SCD. Liver resident macrophages and monocytes are known to enable the clearance of HbS, however, the role of liver sinusoidal endothelial cells (LSECs) in HbS clearance and liver injury in SCD remains unknown. Using real-time intravital (in vivo) imaging in the mice liver as well as flow cytometric analysis and confocal imaging of primary human LSECs, we show for the first time that liver injury in SCD is associated with accumulation of HbS and iron in the LSECs, leading to LSEC senescence. Hb uptake by LSECs was mediated by micropinocytosis. Hepatic monocytes were observed to attenuate LSECsenescence by accelerating HbS clearance in the liver of SCD mice, however, this protection was impaired in P-selectin-deficient SCD mice secondary to reduced monocyte recruitment in the liver. These findings are the first to suggest that LSECs contribute to HbS clearance and HbS induced LSEC-senescence promotes progressive liver injury in SCD mice. Our results provide a novel insight into the pathogenesis of hemolysis induced chronic liver injury in SCD caused by LSEC senescence. Identifying the regulators of LSEC mediated HbS clearance may lead to new therapies to prevent the progression of liver injury in SCD.
RESUMEN
Sickle cell disease (SCD) is an autosomal recessive genetic disorder that affects â¼100,000 Americans and millions of people worldwide. Erythrocyte sickling, vaso-occlusion, sterile inflammation, and hemolysis are the major pathophysiological pathways leading to liver injury in SCD. Although hepatic dysfunction affects up to 10%-40% of patients with SCD, therapeutic approaches to prevent liver injury in SCD are not known, and the molecular mechanisms promoting progressive liver injury in SCD remain poorly understood. Animal models have been beneficial in bridging the gap between preclinical and translational research in SCD. Recent advances in methodology have allowed the development of several humanized animal models to address various aspects of SCD-related liver diseases. This review provides an overview of current knowledge of the molecular mechanisms and potential therapeutic options of SCD-associated liver dysfunction using the Townes mouse model.
Asunto(s)
Anemia de Células Falciformes , Hepatopatías , Anemia de Células Falciformes/complicaciones , Anemia de Células Falciformes/tratamiento farmacológico , Anemia de Células Falciformes/genética , Animales , Modelos Animales de Enfermedad , Eritrocitos/metabolismo , Hemólisis , Humanos , Hepatopatías/genética , RatonesRESUMEN
Aging is the most significant risk factor for the majority of chronic diseases, including liver disease. The cellular, molecular, and pathophysiological mechanisms that promote age-induced hepatovascular changes are unknown due to our inability to visualize changes in liver pathophysiology in live mice over time. We performed quantitative liver intravital microscopy (qLIM) in live C57BL/6J mice to investigate the impact of aging on the hepatovascular system over a 24-mo period. qLIM revealed that age-related hepatic alterations include reduced liver sinusoidal blood flow, increased sinusoidal vessel diameter, and loss of small hepatic vessels. The ductular cell structure deteriorates with age, along with altered expression of hepatic junctional proteins. Furthermore, qLIM imaging revealed increased inflammation in the aged liver, which was linked to increased expression of proinflammatory macrophages, hepatic neutrophils, liver sinusoidal endothelial cells, senescent cells, and procoagulants. Finally, we detected elevated NF-κB pathway activity in aged livers. Overall, these findings emphasize the importance of inflammation in age-related hepatic vasculo-epithelial alterations and highlight the utility of qLIM in studying age-related effects in organ pathophysiology.
Asunto(s)
Células Endoteliales , Hígado , Animales , Inflamación/metabolismo , Microscopía Intravital , Hígado/metabolismo , Ratones , Ratones Endogámicos C57BLRESUMEN
Hepatocytes are highly polarized epithelia. Loss of hepatocyte polarity is associated with various liver diseases, including cholestasis. However, the molecular underpinnings of hepatocyte polarization remain poorly understood. Loss of ß-catenin at adherens junctions is compensated by γ-catenin and dual loss of both catenins in double knockouts (DKOs) in mice liver leads to progressive intrahepatic cholestasis. However, the clinical relevance of this observation, and further phenotypic characterization of the phenotype, is important. Herein, simultaneous loss of ß-catenin and γ-catenin was identified in a subset of liver samples from patients of progressive familial intrahepatic cholestasis and primary sclerosing cholangitis. Hepatocytes in DKO mice exhibited defects in apical-basolateral localization of polarity proteins, impaired bile canaliculi formation, and loss of microvilli. Loss of polarity in DKO livers manifested as epithelial-mesenchymal transition, increased hepatocyte proliferation, and suppression of hepatocyte differentiation, which was associated with up-regulation of transforming growth factor-ß signaling and repression of hepatocyte nuclear factor 4α expression and activity. In conclusion, concomitant loss of the two catenins in the liver may play a pathogenic role in subsets of cholangiopathies. The findings also support a previously unknown role of ß-catenin and γ-catenin in the maintenance of hepatocyte polarity. Improved understanding of the regulation of hepatocyte polarization processes by ß-catenin and γ-catenin may potentially benefit development of new therapies for cholestasis.
Asunto(s)
Colestasis Intrahepática/patología , Factor Nuclear 4 del Hepatocito/metabolismo , Transducción de Señal , Factor de Crecimiento Transformador beta/metabolismo , beta Catenina/metabolismo , gamma Catenina/metabolismo , Uniones Adherentes/metabolismo , Animales , Línea Celular Tumoral , Polaridad Celular , Factor Nuclear 4 del Hepatocito/genética , Hepatocitos/metabolismo , Humanos , Hígado/metabolismo , Ratones , Ratones Noqueados , Factor de Crecimiento Transformador beta/genética , beta Catenina/genética , gamma Catenina/economía , gamma Catenina/genéticaRESUMEN
BACKGROUND AND AIMS: The Wnt/ß-catenin signaling pathway has a well-described role in liver pathobiology. Its suppression was recently shown to decrease bile acid (BA) synthesis, thus preventing the development of cholestatic liver injury and fibrosis after bile duct ligation (BDL). APPROACH AND RESULTS: To generalize these observations, we suppressed ß-catenin in Mdr2 knockout (KO) mice, which develop sclerosing cholangitis due to regurgitation of BA from leaky ducts. When ß-catenin was knocked down (KD) in KO for 2 weeks, hepatic and biliary injury were exacerbated in comparison to KO given placebo, as shown by serum biochemistry, ductular reaction, inflammation, and fibrosis. Simultaneously, KO/KD livers displayed increased oxidative stress and senescence and an impaired regenerative response. Although the total liver BA levels were similar between KO/KD and KO, there was significant dysregulation of BA transporters and BA detoxification/synthesis enzymes in KO/KD compared with KO alone. Multiphoton intravital microscopy revealed a mixing of blood and bile in the sinusoids, and validated the presence of increased serum BA in KO/KD mice. Although hepatocyte junctions were intact, KO/KD livers had significant canalicular defects, which resulted from loss of hepatocyte polarity. Thus, in contrast to the protective effect of ß-catenin KD in BDL model, ß-catenin KD in Mdr2 KO aggravated rather than alleviated injury by interfering with expression of BA transporters, hepatocyte polarity, canalicular structure, and the regenerative response. CONCLUSIONS: The resulting imbalance between ongoing injury and restitution led to worsening of the Mdr2 KO phenotype, suggesting caution in targeting ß-catenin globally for all cholestatic conditions.
Asunto(s)
Colangitis Esclerosante/complicaciones , Colangitis Esclerosante/genética , Colestasis/etiología , Colestasis/metabolismo , Vía de Señalización Wnt , beta Catenina/metabolismo , Subfamilia B de Transportador de Casetes de Unión a ATP/genética , Animales , Ácidos y Sales Biliares/metabolismo , Conductos Biliares/patología , Colestasis/genética , Modelos Animales de Enfermedad , Hepatocitos/metabolismo , Hepatocitos/patología , Uniones Intercelulares/metabolismo , Uniones Intercelulares/patología , Ratones Noqueados , Miembro 4 de la Subfamilia B de Casete de Unión a ATPRESUMEN
BACKGROUND AND AIMS: Hepatic crisis is an emergent complication affecting patients with sickle cell disease (SCD); however, the molecular mechanism of sickle cell hepatobiliary injury remains poorly understood. Using the knock-in humanized mouse model of SCD and SCD patient blood, we sought to mechanistically characterize SCD-associated hepato-pathophysiology applying our recently developed quantitative liver intravital imaging, RNA sequence analysis, and biochemical approaches. APPROACH AND RESULTS: SCD mice manifested sinusoidal ischemia, progressive hepatomegaly, liver injury, hyperbilirubinemia, and increased ductular reaction under basal conditions. Nuclear factor kappa B (NF-κB) activation in the liver of SCD mice inhibited farnesoid X receptor (FXR) signaling and its downstream targets, leading to loss of canalicular bile transport and altered bile acid pool. Intravital imaging revealed impaired bile secretion into the bile canaliculi, which was secondary to loss of canalicular bile transport and bile acid metabolism, leading to intrahepatic bile accumulation in SCD mouse liver. Blocking NF-κB activation rescued FXR signaling and partially ameliorated liver injury and sinusoidal ischemia in SCD mice. CONCLUSIONS: These findings identify that NF-κB/FXR-dependent impaired bile secretion promotes intrahepatic bile accumulation, which contributes to hepatobiliary injury of SCD. Improved understanding of these processes could potentially benefit the development of therapies to treat sickle cell hepatic crisis.
Asunto(s)
Anemia de Células Falciformes/complicaciones , Bilis/metabolismo , Colestasis/etiología , Insuficiencia Hepática/etiología , Hígado/patología , Adolescente , Adulto , Anemia de Células Falciformes/sangre , Anemia de Células Falciformes/tratamiento farmacológico , Anemia de Células Falciformes/genética , Animales , Conductos Biliares Intrahepáticos/diagnóstico por imagen , Conductos Biliares Intrahepáticos/patología , Colestasis/patología , Colestasis/prevención & control , Modelos Animales de Enfermedad , Femenino , Técnicas de Sustitución del Gen , Hemoglobina Falciforme/genética , Insuficiencia Hepática/patología , Insuficiencia Hepática/prevención & control , Humanos , Microscopía Intravital , Hígado/diagnóstico por imagen , Masculino , Ratones , Persona de Mediana Edad , FN-kappa B/antagonistas & inhibidores , FN-kappa B/efectos de los fármacos , FN-kappa B/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Transducción de Señal/efectos de los fármacos , Adulto JovenRESUMEN
Rationale: Intraerythrocytic polymerization of Hb S promotes hemolysis and vasoocclusive events in the microvasculature of patients with sickle cell disease (SCD). Although platelet-neutrophil aggregate-dependent vasoocclusion is known to occur in the lung and contribute to acute chest syndrome, the etiological mechanisms that trigger acute chest syndrome are largely unknown.Objectives: To identify the innate immune mechanism that promotes platelet-neutrophil aggregate-dependent lung vasoocclusion and injury in SCD.Methods:In vivo imaging of the lung in transgenic humanized SCD mice and in vitro imaging of SCD patient blood flowing through a microfluidic system was performed. SCD mice were systemically challenged with nanogram quantities of LPS to trigger lung vasoocclusion.Measurements and Main Results: Platelet-inflammasome activation led to generation of IL-1ß and caspase-1-carrying platelet extracellular vesicles (EVs) that bind to neutrophils and promote platelet-neutrophil aggregation in lung arterioles of SCD mice in vivo and SCD human blood in microfluidics in vitro. The inflammasome activation, platelet EV generation, and platelet-neutrophil aggregation were enhanced by the presence of LPS at a nanogram dose in SCD but not control human blood. Inhibition of the inflammasome effector caspase-1 or IL-1ß pathway attenuated platelet EV generation, prevented platelet-neutrophil aggregation, and restored microvascular blood flow in lung arterioles of SCD mice in vivo and SCD human blood in microfluidics in vitro.Conclusions: These results are the first to identify that platelet-inflammasome-dependent shedding of IL-1ß and caspase-1-carrying platelet EVs promote lung vasoocclusion in SCD. The current findings also highlight the therapeutic potential of targeting the platelet-inflammasome-dependent innate immune pathway to prevent acute chest syndrome.
Asunto(s)
Anemia de Células Falciformes/complicaciones , Anemia de Células Falciformes/inmunología , Vesículas Extracelulares/inmunología , Inflamasomas/inmunología , Lesión Pulmonar/etiología , Lesión Pulmonar/fisiopatología , Agregación Plaquetaria/inmunología , Síndrome Torácico Agudo/etiología , Síndrome Torácico Agudo/fisiopatología , Anemia de Células Falciformes/fisiopatología , Animales , Humanos , Ratones , Ratones Transgénicos , Modelos Animales , Neutrófilos/inmunologíaRESUMEN
Exposure of mice to a diet containing 3,5-diethoxycarbonyl-1, 4-dihydrocollidine (DDC) induces porphyrin accumulation, cholestasis, immune response, and hepatobiliary damage mimicking hepatic porphyria and sclerosing cholangitis. Although ß-catenin signaling promotes hepatocyte proliferation, and macrophages are a source of Wnts, the role of macrophage-derived Wnts in modulating hepatobiliary injury/repair remains unresolved. We investigated the effect of macrophage-specific deletion of Wntless, a cargo protein critical for cellular Wnt secretion, by feeding macrophage-Wntless-knockout (Mac-KO) and wild-type littermates a DDC diet for 14 days. DDC exposure induced Wnt11 up-regulation in macrophages. Mac-KO mice on DDC showed increased serum alkaline phosphatase, aspartate aminotransferase, direct bilirubin, and histologic evidence of more cell death, inflammation, and ductular reaction. There was impaired hepatocyte proliferation evidenced by Ki-67 immunostaining, which was associated with decreased hepatocyte ß-catenin activation and cyclin-D1 in Mac-KO. Mac-KO also showed increased CD45, F4/80, and neutrophil infiltration after DDC diet, along with increased expression of several proinflammatory cytokines and chemokines. Gene expression analyses of bone marrow-derived macrophages from Mac-KO mice and F4/80+ macrophages isolated from DDC-fed Mac-KO livers showed proinflammatory M1 polarization. In conclusion, this study shows that a lack of macrophage Wnt secretion leads to more DDC-induced hepatic injury due to impaired hepatocyte proliferation and increased M1 macrophages, which promotes immune-mediated cell injury.
Asunto(s)
Colangitis Esclerosante/metabolismo , Colestasis/metabolismo , Dieta/efectos adversos , Hepatocitos/metabolismo , Macrófagos/metabolismo , Piridinas/toxicidad , Proteínas Wnt/biosíntesis , Animales , Colangitis Esclerosante/inducido químicamente , Colangitis Esclerosante/genética , Colangitis Esclerosante/patología , Colestasis/inducido químicamente , Colestasis/genética , Colestasis/patología , Hepatocitos/patología , Macrófagos/patología , Ratones , Ratones Noqueados , Regulación hacia Arriba/efectos de los fármacos , Proteínas Wnt/genéticaRESUMEN
BACKGROUND & AIMS: Liver fibrosis, hepatocellular necrosis, inflammation, and proliferation of liver progenitor cells are features of chronic liver injury. Mouse models have been used to study the end-stage pathophysiology of chronic liver injury. However, little is known about differences in the mechanisms of liver injury among different mouse models because of our inability to visualize the progression of liver injury in vivo in mice. We developed a method to visualize bile transport and blood-bile barrier (BBlB) integrity in live mice. METHODS: C57BL/6 mice were fed a choline-deficient, ethionine-supplemented (CDE) diet or a diet containing 0.1% 3,5-diethoxycarbonyl-1, 4-dihydrocollidine (DDC) for up to 4 weeks to induce chronic liver injury. We used quantitative liver intravital microscopy (qLIM) for real-time assessment of bile transport and BBlB integrity in the intact livers of the live mice fed the CDE, DDC, or chow (control) diets. Liver tissues were collected from mice and analyzed by histology, immunohistochemistry, real-time polymerase chain reaction, and immunoblots. RESULTS: Mice with liver injury induced by a CDE or a DDC diet had breaches in the BBlB and impaired bile secretion, observed by qLIM compared with control mice. Impaired bile secretion was associated with reduced expression of several tight-junction proteins (claudins 3, 5, and 7) and bile transporters (NTCP, OATP1, BSEP, ABCG5, and ABCG8). A prolonged (2-week) CDE, but not DDC, diet led to re-expression of tight junction proteins and bile transporters, concomitant with the reestablishment of BBlB integrity and bile secretion. CONCLUSIONS: We used qLIM to study chronic liver injury, induced by a choline-deficient or DDC diet, in mice. Progression of chronic liver injury was accompanied by loss of bile transporters and tight junction proteins.
Asunto(s)
Bilis/metabolismo , Enfermedad Hepática Crónica Inducida por Sustancias y Drogas/metabolismo , Hepatocitos/metabolismo , Hígado/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Uniones Estrechas/metabolismo , Animales , Transporte Biológico , Enfermedad Hepática Crónica Inducida por Sustancias y Drogas/sangre , Enfermedad Hepática Crónica Inducida por Sustancias y Drogas/etiología , Enfermedad Hepática Crónica Inducida por Sustancias y Drogas/patología , Deficiencia de Colina/complicaciones , Claudinas/metabolismo , Modelos Animales de Enfermedad , Etionina , Hepatocitos/patología , Cinética , Hígado/patología , Ratones Endogámicos C57BL , Permeabilidad , Piridinas , Uniones Estrechas/patologíaRESUMEN
Hepatoblastoma (HB) is the most common pediatric liver malignant tumor. Previously, we reported co-activation of ß-catenin and Yes-associated protein-1 (YAP1) in 80% of HB. Hepatic co-expression of active ß-catenin and YAP1 via sleeping beauty transposon/transposase and hydrodynamic tail vein injection led to HB development in mice. Here, we identify lipocalin 2 (Lcn2) as a target of ß-catenin and YAP1 in HB and show that serum Lcn2 values positively correlated with tumor burden. Lcn2 was strongly expressed in HB tumor cells in our mouse model. A tissue array of 62 HB cases showed highest LCN2 expression in embryonal and lowest in fetal, blastemal, and small cell undifferentiated forms of HB. Knockdown of LCN2 in HB cells had no effect on cell proliferation but reduced NF-κB reporter activity. Next, liver-specific Lcn2 knockout (KO) mice were generated. No difference in tumor burden was observed between Lcn2 KO mice and wild-type littermate controls after sleeping beauty transposon/transposase and hydrodynamic tail vein injection delivery of active YAP1 and ß-catenin, although Lcn2 KO mice with HB lacked any serum Lcn2 elevation, demonstrating that transformed hepatocytes are the source of serum Lcn2. More blastemal areas and inflammation were observed within HB in Lcn2 KO compared with wild-type tumors. In conclusion, Lcn2 expressed in hepatocytes appears to be dispensable for the pathogenesis of HB. However, transformed hepatocytes secrete serum Lcn2, making Lcn2 a valuable biomarker for HB.
Asunto(s)
Biomarcadores de Tumor/sangre , Hepatoblastoma/patología , Hepatocitos/patología , Lipocalina 2/sangre , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Proteínas de Ciclo Celular , Proliferación Celular , Hepatoblastoma/sangre , Hepatocitos/metabolismo , Humanos , Ratones , Ratones Noqueados , FN-kappa B/metabolismo , Fosfoproteínas/metabolismo , Transducción de Señal , Factores de Transcripción , Carga Tumoral , Proteínas Señalizadoras YAPRESUMEN
ß-Catenin, the downstream effector of the Wnt signaling, plays important roles in hepatic development, regeneration, and tumorigenesis. However, its role at hepatocyte adherens junctions (AJ) is relatively poorly understood, chiefly due to spontaneous compensation by γ-catenin. We simultaneously ablated ß- and γ-catenin expression in mouse liver by interbreeding ß-catenin-γ-catenin double-floxed mice and Alb-Cre transgenic mice. Double knockout mice show failure to thrive, impaired hepatocyte differentiation, cholemia, ductular reaction, progressive cholestasis, inflammation, fibrosis, and tumorigenesis, which was associated with deregulation of tight junctions (TJ) and bile acid transporters, leading to early morbidity and mortality, a phenotype reminiscent of progressive familial intrahepatic cholestasis (PFIC). To address the mechanism, we specifically and temporally eliminated both catenins from hepatocytes using adeno-associated virus 8 carrying Cre-recombinase under the thyroid-binding globulin promoter (AAV8-TBG-Cre). This led to a time-dependent breach of the blood-biliary barrier associated with sequential disruption of AJ and TJ verified by ultrastructural imaging and intravital microscopy, which revealed unique paracellular leaks around individual hepatocytes, allowing mixing of blood and bile and leakage of blood from one sinusoid to another. Molecular analysis identified sequential losses of E-cadherin, occludin, claudin-3, and claudin-5 due to enhanced proteasomal degradation, and of claudin-2, a ß-catenin transcriptional target, which was also validated in vitro. CONCLUSION: We report partially redundant function of catenins at AJ in regulating TJ and contributing to the blood-biliary barrier. Furthermore, concomitant hepatic loss of ß- and γ-catenin disrupts structural and functional integrity of AJ and TJ via transcriptional and posttranslational mechanisms. Mice with dual catenin loss develop progressive intrahepatic cholestasis, providing a unique model to study diseases such as PFIC. (Hepatology 2018;67:2320-2337).
Asunto(s)
Uniones Adherentes , Colestasis Intrahepática/etiología , Uniones Estrechas , beta Catenina/fisiología , gamma Catenina/fisiología , Animales , Femenino , Hepatocitos , Masculino , Ratones , Ratones Noqueados , beta Catenina/genética , gamma Catenina/genéticaRESUMEN
Evolutionarily conserved intercellular signaling pathways regulate embryonic development and adult tissue homeostasis in metazoans. The precise control of the state and amplitude of signaling pathways is achieved in part through the kinase- and phosphatase-mediated reversible phosphorylation of proteins. In this study, we performed a genome-wide in vivo RNAi screen for kinases and phosphatases that regulate the Wnt pathway under physiological conditions in the Drosophila wing disc. Our analyses have identified 54 high-confidence kinases and phosphatases capable of modulating the Wnt pathway, including 22 novel regulators. These candidates were also assayed for a role in the Notch pathway, and numerous phospho-regulators were identified. Additionally, each regulator of the Wnt pathway was evaluated in the wing disc for its ability to affect the mechanistically similar Hedgehog pathway. We identified 29 dual regulators that have the same effect on the Wnt and Hedgehog pathways. As proof of principle, we established that Cdc37 and Gilgamesh/CK1γ inhibit and promote signaling, respectively, by functioning at analogous levels of these pathways in both Drosophila and mammalian cells. The Wnt and Hedgehog pathways function in tandem in multiple developmental contexts, and the identification of several shared phospho-regulators serve as potential nodes of control under conditions of aberrant signaling and disease.
Asunto(s)
Proteínas de Drosophila/metabolismo , Proteínas Wnt/metabolismo , Animales , Quinasa de la Caseína I/genética , Quinasa de la Caseína I/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Drosophila , Proteínas de Drosophila/genética , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Receptores Notch/genética , Receptores Notch/metabolismo , Receptor Smoothened , Alas de Animales/metabolismo , Proteínas Wnt/genética , Proteína Wnt1/genética , Proteína Wnt1/metabolismoRESUMEN
Platelet-derived growth factor receptor α (PDGFRα), a tyrosine kinase receptor, is up-regulated in hepatic stellate cells (HSCs) during chronic liver injury. HSCs mediate hepatic fibrosis through their activation from a quiescent state partially in response to profibrotic growth factors. HSC activation entails enhanced expression of profibrotic genes, increase in proliferation, and increase in motility, which facilitates migration within the hepatic lobule. We show colocalization of PDGFRα in murine carbon tetrachloride, bile duct ligation, and 0.1% 3,5-diethoxycarbonyl-1,4-dihydrocollidine models of chronic liver injury, and investigate the role of PDGFRα on proliferation, profibrotic gene expression, and migration in primary human HSCs (HHSteCs) using the PDGFRα-specific inhibitory monoclonal antibody olaratumab. Although lacking any effects on HHSteC transdifferentiation assessed by gene expression of ACTA2, TGFB1, COL1A1, SYP1, and FN1, olaratumab specifically reduced HHSteC proliferation (AlamarBlue assay) and cell migration (transwell migration assays). Using phospho-specific antibodies, we show that olaratumab attenuates PDGFRα activation in response to PDGF-BB, and reduced phosphorylation of extracellular signal-regulated kinase 1 and 2, Elk-1, p38, Akt, focal adhesion kinase, mechanistic target of rapamycin, C10 regulator of kinase II, and C10 regulator of kinase-like, suggesting that PDGFRα contributes to mitogenesis and actin reorganization through diverse downstream effectors. Our findings support a distinct contribution of PDGFRα signaling to HSC proliferation and migration and provide evidence that inhibition of PDGFRα signaling could alter the pathogenesis of hepatic fibrosis.
Asunto(s)
Movimiento Celular , Células Estrelladas Hepáticas/citología , Células Estrelladas Hepáticas/metabolismo , Receptor alfa de Factor de Crecimiento Derivado de Plaquetas/metabolismo , Animales , Anticuerpos Monoclonales/farmacología , Comunicación Autocrina/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Movimiento Celular/genética , Proliferación Celular/efectos de los fármacos , Proliferación Celular/genética , Densitometría , Modelos Animales de Enfermedad , Regulación de la Expresión Génica/efectos de los fármacos , Células Estrelladas Hepáticas/efectos de los fármacos , Humanos , Ligandos , Cirrosis Hepática/genética , Cirrosis Hepática/metabolismo , Cirrosis Hepática/patología , Ratones Endogámicos C57BL , Modelos Biológicos , Fosforilación/efectos de los fármacos , Factor de Crecimiento Derivado de Plaquetas/farmacología , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genéticaRESUMEN
The thyromimetic agent GC-1 induces hepatocyte proliferation via Wnt/ß-catenin signaling and may promote regeneration in both acute and chronic liver insufficiencies. However, ß-catenin activation due to mutations in CTNNB1 is seen in a subset of hepatocellular carcinomas (HCC). Thus, it is critical to address any effect of GC-1 on HCC growth and development before its use can be advocated to stimulate regeneration in chronic liver diseases. In this study, we first examined the effect of GC-1 on ß-catenin-T cell factor 4 activity in HCC cell lines harboring wild-type or mutated-CTNNB1. Next, we assessed the effect of GC-1 on HCC in FVB mice generated by hydrodynamic tail vein injection of hMet-S45Y-ß-catenin, using the sleeping beauty transposon-transposase. Four weeks following injection, mice were fed 5 mg/kg GC-1 or basal diet for 10 or 21 days. GC-1 treatment showed no effect on ß-catenin-T cell factor 4 activity in HCC cells, irrespective of CTNNB1 mutations. Treatment with GC-1 for 10 or 21 days led to a significant reduction in tumor burden, associated with decreased tumor cell proliferation and dramatic decreases in phospho-(p-)Met (Y1234/1235), p-extracellular signal-related kinase, and p-STAT3 without affecting ß-catenin and its downstream targets. GC-1 exerts a notable antitumoral effect on hMet-S45Y-ß-catenin HCC by inactivating Met signaling. GC-1 does not promote ß-catenin activation in HCC. Thus, GC-1 may be safe for use in inducing regeneration during chronic hepatic insufficiency.
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Acetatos/farmacología , Carcinoma Hepatocelular/patología , Neoplasias Hepáticas/tratamiento farmacológico , Fenoles/farmacología , beta Catenina/metabolismo , Carcinoma Hepatocelular/tratamiento farmacológico , Carcinoma Hepatocelular/metabolismo , Proliferación Celular/efectos de los fármacos , Insuficiencia Hepática/patología , Humanos , Neoplasias Hepáticas/metabolismoRESUMEN
Generating cellular heterogeneity is crucial to the development of complex organs. Organ-fate selector genes and signalling pathways generate cellular diversity by subdividing and patterning naïve tissues to assign them regional identities. The Drosophila eye-antennal imaginal disc is a well-characterised system in which to study regional specification; it is first divided into antennal and eye fates and subsequently retinal differentiation occurs within only the eye field. During development, signalling pathways and selector genes compete with and mutually antagonise each other to subdivide the tissue. Wingless (Wg) signalling is the main inhibitor of retinal differentiation; it does so by promoting antennal/head-fate via selector factors and by antagonising Hedgehog (Hh), the principal differentiation-initiating signal. Wg signalling must be suppressed by JAK/STAT at the disc posterior in order to initiate retinal differentiation. Ras/MEK/MAPK signalling has also been implicated in initiating retinal differentiation but its mode of action is not known. We find that compromising Ras/MEK/MAPK signalling in the early larval disc results in expanded antennal/head cuticle at the expense of the compound eye. These phenotypes correspond both to perturbations in selector factor expression, and to de-repressed wg. Indeed, STAT activity is reduced due to decreased mobility of the ligand Unpaired (Upd) along with a corresponding loss in Dally-like protein (Dlp), a heparan sulphate proteoglycan (HSPG) that aids Upd diffusion. Strikingly, blocking HSPG biogenesis phenocopies compromised Ras/MEK/MAPK, while restoring HSPG expression rescues the adult phenotype significantly. This study identifies a novel mode by which the Ras/MEK/MAPK pathway regulates regional-fate specification via HSPGs during development.
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Proteoglicanos de Heparán Sulfato/química , MAP Quinasa Quinasa 1/metabolismo , Retina/embriología , Proteínas ras/metabolismo , Animales , Linaje de la Célula , Cruzamientos Genéticos , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/embriología , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Larva/metabolismo , Ligandos , Sistema de Señalización de MAP Quinasas , Fenotipo , Proteínas Quinasas/metabolismo , Interferencia de ARN , Transducción de Señal , Proteína Wnt1/metabolismoRESUMEN
γ-Catenin, an important component of desmosomes, may also participate in Wnt signaling. Herein, we dissect the role of γ-catenin in liver by generating conditional γ-catenin knockout (KO) mice and assessing their phenotype after bile duct ligation (BDL) and diethylnitrosamine-induced chemical carcinogenesis. At baseline, KO and wild-type littermates showed comparable serum biochemistry, liver histology, and global gene expression. ß-Catenin protein was modestly increased without any change in Wnt signaling. Desmosomes were maintained in KO, and despite no noticeable changes in gene expression, differential detergent fractionation revealed quantitative and qualitative changes in desmosomal cadherins, plaque proteins, and ß-catenin. Enhanced association of ß-catenin to desmoglein-2 and plakophilin-3 was observed in KO. When subjected to BDL, wild-type littermates showed specific changes in desmosomal protein expression. In KO, BDL deteriorated baseline compensatory changes, which manifested as enhanced injury and fibrosis. KO also showed enhanced tumorigenesis to diethylnitrosamine treatment because of Wnt activation, as also verified in vitro. γ-Catenin overexpression in hepatoma cells increased its binding to T-cell factor 4 at the expense of ß-catenin-T-cell factor 4 association, induced unique target genes, affected Wnt targets, and reduced cell proliferation and viability. Thus, γ-catenin loss in liver is basally well tolerated. However, after insults like BDL, these compensations at desmosomes fail, and KO show enhanced injury. Also, γ-catenin negatively regulates tumor growth by affecting Wnt signaling.
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Colestasis/metabolismo , Desmosomas/metabolismo , Neoplasias Hepáticas Experimentales/metabolismo , Hígado/metabolismo , gamma Catenina/fisiología , Animales , Conductos Biliares/cirugía , Transformación Celular Neoplásica/metabolismo , Transformación Celular Neoplásica/patología , Colestasis/patología , Dietilnitrosamina , Femenino , Regulación de la Expresión Génica/fisiología , Regulación Neoplásica de la Expresión Génica/fisiología , Neoplasias Hepáticas Experimentales/patología , Masculino , Ratones Noqueados , Proteínas de Neoplasias/metabolismo , Proteínas de Neoplasias/fisiología , Transducción de Señal/fisiología , Células Tumorales Cultivadas , Vía de Señalización Wnt/fisiología , beta Catenina/fisiología , gamma Catenina/deficiencia , gamma Catenina/genéticaRESUMEN
Wingless (Wg) proteins are secreted-lipid-modified glycoproteins involved in tissue patterning and cell-fate specification. Wg secretion is regulated by a specialized mechanism involving a repertoire of proteins including Wntless (Wls). Here, we show that the Bro1-domain-containing protein Myopic (Mop) is indispensable for endosomal trafficking of Wg and Wls. Reductions in Mop leads to trapping of Wg and Wls in the early endosomes. Overexpression of the endosomal sorting protein Hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) rescues the trafficking defect caused by the mop loss of function. The vertebrate homolog of Mop, Histidine domain-containing protein tyrosine phosphatase (HDPTP), was also found to have a conserved role in Wnt trafficking. Our study highlights the importance of early endosomal trafficking for Wg secretion, and identifies a novel role for Mop in Wg signaling.