RESUMEN
Genetically unstable tetraploid cells can promote tumorigenesis. Recent estimates suggest that â¼37% of human tumors have undergone a genome-doubling event during their development. This potentially oncogenic effect of tetraploidy is countered by a p53-dependent barrier to proliferation. However, the cellular defects and corresponding signaling pathways that trigger growth suppression in tetraploid cells are not known. Here, we combine RNAi screening and in vitro evolution approaches to demonstrate that cytokinesis failure activates the Hippo tumor suppressor pathway in cultured cells, as well as in naturally occurring tetraploid cells in vivo. Induction of the Hippo pathway is triggered in part by extra centrosomes, which alter small G protein signaling and activate LATS2 kinase. LATS2 in turn stabilizes p53 and inhibits the transcriptional regulators YAP and TAZ. These findings define an important tumor suppression mechanism and uncover adaptive mechanisms potentially available to nascent tumor cells that bypass this inhibitory regulation.
Asunto(s)
Citocinesis , Proteínas Serina-Treonina Quinasas/metabolismo , Transducción de Señal , Línea Celular Tumoral , Centrosoma/metabolismo , Células Epiteliales/metabolismo , Hepatocitos/metabolismo , Vía de Señalización Hippo , Humanos , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Neoplasias/metabolismo , Neoplasias/patología , Tetraploidía , Proteína p53 Supresora de Tumor/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Proteína de Unión al GTP rhoA/metabolismoRESUMEN
The Hippo-signaling pathway is an important regulator of cellular proliferation and organ size. However, little is known about the role of this cascade in the control of cell fate. Employing a combination of lineage tracing, clonal analysis, and organoid culture approaches, we demonstrate that Hippo pathway activity is essential for the maintenance of the differentiated hepatocyte state. Remarkably, acute inactivation of Hippo pathway signaling in vivo is sufficient to dedifferentiate, at very high efficiencies, adult hepatocytes into cells bearing progenitor characteristics. These hepatocyte-derived progenitor cells demonstrate self-renewal and engraftment capacity at the single-cell level. We also identify the NOTCH-signaling pathway as a functional important effector downstream of the Hippo transducer YAP. Our findings uncover a potent role for Hippo/YAP signaling in controlling liver cell fate and reveal an unprecedented level of phenotypic plasticity in mature hepatocytes, which has implications for the understanding and manipulation of liver regeneration.
Asunto(s)
Desdiferenciación Celular , Hígado/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Transducción de Señal , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Proteínas de Ciclo Celular , Hepatocitos/metabolismo , Vía de Señalización Hippo , Hígado/citología , Ratones , Fosfoproteínas/metabolismo , Receptores Notch/metabolismo , Células Madre/citología , Células Madre/metabolismo , Proteínas Señalizadoras YAPRESUMEN
Nonalcoholic fatty liver disease (NAFLD) is the most common chronic liver disease, in which prognosis is determined by liver fibrosis. A common variant in hydroxysteroid 17-beta dehydrogenase 13 (HSD17B13, rs72613567-A) is associated with a reduced risk of fibrosis in NAFLD, but the underlying mechanism(s) remains unclear. We investigated the effects of this variant in the human liver and in Hsd17b13 knockdown in mice by using a state-of-the-art metabolomics approach. We demonstrate that protection against liver fibrosis conferred by the HSD17B13 rs72613567-A variant in humans and by the Hsd17b13 knockdown in mice is associated with decreased pyrimidine catabolism at the level of dihydropyrimidine dehydrogenase. Furthermore, we show that hepatic pyrimidines are depleted in two distinct mouse models of NAFLD and that inhibition of pyrimidine catabolism by gimeracil phenocopies the HSD17B13-induced protection against liver fibrosis. Our data suggest pyrimidine catabolism as a therapeutic target against the development of liver fibrosis in NAFLD.
Asunto(s)
Enfermedad del Hígado Graso no Alcohólico , Animales , Humanos , Ratones , Hígado/metabolismo , Cirrosis Hepática/patología , Enfermedad del Hígado Graso no Alcohólico/patología , Pirimidinas/farmacología , Pirimidinas/metabolismoRESUMEN
Maintenance of a healthy pregnancy is reliant on a successful balance between the fetal and maternal immune systems. Although the maternal mechanisms responsible have been well studied, those used by the fetal immune system remain poorly understood. Using suspension mass cytometry and various imaging modalities, we report a complex immune system within the mid-gestation (17-23â weeks) human placental villi (PV). Consistent with recent reports in other fetal organs, T cells with memory phenotypes, although rare in abundance, were detected within the PV tissue and vasculature. Moreover, we determined that T cells isolated from PV samples may be more proliferative after T cell receptor stimulation than adult T cells at baseline. Collectively, we identified multiple subtypes of fetal immune cells within the PV and specifically highlight the enhanced proliferative capacity of fetal PV T cells.
Asunto(s)
Vellosidades Coriónicas/inmunología , Placenta/inmunología , Antígenos CD/metabolismo , Antígenos de Diferenciación Mielomonocítica/metabolismo , Linfocitos B/citología , Linfocitos B/inmunología , Linfocitos B/metabolismo , Antígeno B7-H1/genética , Antígeno B7-H1/metabolismo , Vellosidades Coriónicas/metabolismo , Femenino , Feto/inmunología , Feto/metabolismo , Citometría de Flujo , Antígenos HLA-DR/genética , Antígenos HLA-DR/metabolismo , Humanos , Células Asesinas Naturales/citología , Células Asesinas Naturales/inmunología , Células Asesinas Naturales/metabolismo , Antígenos Comunes de Leucocito/metabolismo , Activación de Linfocitos , Macrófagos/citología , Macrófagos/inmunología , Macrófagos/metabolismo , Células T de Memoria/citología , Células T de Memoria/inmunología , Células T de Memoria/metabolismo , Placenta/citología , Placenta/metabolismo , Embarazo , Segundo Trimestre del Embarazo , Receptores de Superficie Celular/metabolismo , Receptores de Quimiocina/genética , Receptores de Quimiocina/metabolismo , Análisis de la Célula Individual/métodos , Linfocitos T/citología , Linfocitos T/inmunología , Linfocitos T/metabolismoRESUMEN
BACKGROUND AND AIMS: The liver is remarkably regenerative and can completely recover even when 80% of its mass is surgically removed. Identification of secreted factors that regulate liver growth would help us understand how organ size and regeneration are controlled but also provide candidate targets to promote regeneration or impair cancer growth. APPROACH AND RESULTS: To enrich for secreted factors that regulate growth control, we induced massive liver overgrowth with either YAP or MYC . Differentially expressed secreted factors were identified in these livers using transcriptomic analysis. To rank candidates by functionality, we performed in vivo CRISPR screening using the Fah knockout model of tyrosinemia. We identified secreted phosphoprotein-2 (SPP2) as a secreted factor that negatively regulates regeneration. Spp2 -deficient mice showed increased survival after acetaminophen poisoning and reduced fibrosis after repeated carbon tetrachloride injections. We examined the impact of SPP2 on bone morphogenetic protein signaling in liver cells and found that SPP2 antagonized bone morphogenetic protein signaling in vitro and in vivo. We also identified cell-surface receptors that interact with SPP2 using a proximity biotinylation assay coupled with mass spectrometry. We showed that SPP2's interactions with integrin family members are in part responsible for some of the regeneration phenotypes. CONCLUSIONS: Using an in vivo CRISPR screening system, we identified SPP2 as a secreted factor that negatively regulates liver regeneration. This study provides ways to identify, validate, and characterize secreted factors in vivo.
Asunto(s)
Regeneración Hepática , Neoplasias , Ratones , Animales , Hígado/metabolismo , Hepatocitos/metabolismo , Transducción de SeñalRESUMEN
ANKS6 is a ciliary protein that localizes to the proximal compartment of the primary cilium, where it regulates signaling. Mutations in the ANKS6 gene cause multiorgan ciliopathies in humans, which include laterality defects of the visceral organs, renal cysts as part of nephronophthisis and congenital hepatic fibrosis (CHF) in the liver. Although CHF together with liver ductal plate malformations are common features of several human ciliopathy syndromes, including nephronophthisis-related ciliopathies, the mechanism by which mutations in ciliary genes lead to bile duct developmental abnormalities is not understood. Here, we generated a knockout mouse model of Anks6 and show that ANKS6 function is required for bile duct morphogenesis and cholangiocyte differentiation. The loss of Anks6 causes ciliary abnormalities, ductal plate remodeling defects and periportal fibrosis in the liver. Our expression studies and biochemical analyses show that biliary abnormalities in Anks6-deficient livers result from the dysregulation of YAP transcriptional activity in the bile duct-lining epithelial cells. Mechanistically, our studies suggest, that ANKS6 antagonizes Hippo signaling in the liver during bile duct development by binding to Hippo pathway effector proteins YAP1, TAZ and TEAD4 and promoting their transcriptional activity. Together, this study reveals a novel function for ANKS6 in regulating Hippo signaling during organogenesis and provides mechanistic insights into the regulatory network controlling bile duct differentiation and morphogenesis during liver development.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Portadoras/genética , Proteínas de Unión al ADN/genética , Hígado/crecimiento & desarrollo , Proteínas Musculares/genética , Factores de Transcripción/genética , Animales , Conductos Biliares/crecimiento & desarrollo , Conductos Biliares/metabolismo , Conductos Biliares/patología , Diferenciación Celular/genética , Ciliopatías/genética , Ciliopatías/metabolismo , Ciliopatías/patología , Humanos , Hígado/anomalías , Hígado/metabolismo , Hígado/patología , Ratones , Ratones Noqueados , Morfogénesis/genética , Transducción de Señal/genética , Factores de Transcripción de Dominio TEA , Proteínas Señalizadoras YAPRESUMEN
The Hippo pathway and its nuclear effector Yap regulate organ size and cancer formation. While many modulators of Hippo activity have been identified, little is known about the Yap target genes that mediate these growth effects. Here, we show that yap-/- mutant zebrafish exhibit defects in hepatic progenitor potential and liver growth due to impaired glucose transport and nucleotide biosynthesis. Transcriptomic and metabolomic analyses reveal that Yap regulates expression of glucose transporter glut1, causing decreased glucose uptake and use for nucleotide biosynthesis in yap-/- mutants, and impaired glucose tolerance in adults. Nucleotide supplementation improves Yap deficiency phenotypes, indicating functional importance of glucose-fueled nucleotide biosynthesis. Yap-regulated glut1 expression and glucose uptake are conserved in mammals, suggesting that stimulation of anabolic glucose metabolism is an evolutionarily conserved mechanism by which the Hippo pathway controls organ growth. Together, our results reveal a central role for Hippo signaling in glucose metabolic homeostasis.
Asunto(s)
Glucosa/metabolismo , Hígado/embriología , Nucleótidos/biosíntesis , Transducción de Señal/fisiología , Transactivadores/metabolismo , Proteínas de Pez Cebra/metabolismo , Pez Cebra/embriología , Animales , Glucosa/genética , Transportador de Glucosa de Tipo 1/genética , Transportador de Glucosa de Tipo 1/metabolismo , Ratones , Nucleótidos/genética , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Serina-Treonina Quinasa 3 , Transactivadores/genética , Proteínas Señalizadoras YAP , Pez Cebra/genética , Proteínas de Pez Cebra/genéticaRESUMEN
BACKGROUND & AIMS: Calcineurin is a ubiquitously expressed central Ca2+-responsive signaling molecule that mediates acute pancreatitis, but little is known about its effects. We compared the effects of calcineurin expression by hematopoietic cells vs pancreas in mouse models of pancreatitis and pancreatitis-associated lung inflammation. METHODS: We performed studies with mice with hematopoietic-specific or pancreas-specific deletion of protein phosphatase 3, regulatory subunit B, alpha isoform (PPP3R1, also called CNB1), in mice with deletion of CNB1 (Cnb1UBCâ³/â³) and in the corresponding controls for each deletion of CNB1. Acute pancreatitis was induced in mice by administration of caerulein or high-pressure infusion of radiocontrast into biliopancreatic ducts; some mice were also given intraductal infusions of an adeno-associated virus vector that expressed nuclear factor of activated T -cells (NFAT)-luciferase into pancreas. Pancreas, bone marrow, liver, kidney, heart, and lung were collected and analyzed by histopathology, immunohistochemistry, and immunoblots; levels of cytokines were measured in serum. Mouse and human primary pancreatic acinar cells were transfected with a vector that expressed NFAT-luciferase and incubated with an agent that blocks interaction of NFAT with calcineurin; cells were analyzed by immunofluorescence. Calcineurin-mediated neutrophil chemotaxis and reactive oxygen species production were measured in neutrophils from mice. RESULTS: Mice with hematopoietic-specific deletion of CNB1 developed the same level of local pancreatic inflammation as control mice after administration of caerulein or infusion of radiocontrast into biliopancreatic ducts. Cnb1UBCâ³/â³ mice or mice with pancreas-specific deletion of CNB1 developed less severe pancreatitis and reduced pancreatic inflammation after administration of caerulein or infusion of radiocontrast into biliopancreatic ducts compared with control mice. NFAT was activated in pancreas of Swiss Webster mice given caerulein or infusions of radiocontrast into biliopancreatic ducts. Blocking the interaction between calcineurin and NFAT did not reduce pancreatic acinar cell necrosis in response to caerulein or infusions of radiocontrast. Mice with hematopoietic-specific deletion of CNB1 (but not mice with pancreas-specific deletion of CNB1) had reduced infiltration of lung tissues by neutrophils. Neutrophil chemotaxis and production of reactive oxygen species were decreased after incubation with a calcineurin inhibitor. CONCLUSIONS: Hematopoietic and neutrophil expression of calcineurin promotes pancreatitis-associated lung inflammation, whereas pancreatic calcineurin promotes local pancreatic inflammation. The findings indicate that the protective effects of blocking or deleting calcineurin on pancreatitis are mediated by the source of its expression. This information should be used in the development of strategies to inhibit calcineurin for the prevention of pancreatitis and pancreatitis-associated lung inflammation.
Asunto(s)
Lesión Pulmonar Aguda/inmunología , Inhibidores de la Calcineurina/uso terapéutico , Calcineurina/metabolismo , Proteínas de Unión al Calcio/metabolismo , Proteínas Musculares/metabolismo , Pancreatitis/inmunología , Células Acinares/metabolismo , Lesión Pulmonar Aguda/sangre , Lesión Pulmonar Aguda/patología , Lesión Pulmonar Aguda/prevención & control , Animales , Células de la Médula Ósea/inmunología , Células de la Médula Ósea/metabolismo , Calcineurina/genética , Calcineurina/inmunología , Proteínas de Unión al Calcio/genética , Células Cultivadas , Ceruletida/administración & dosificación , Ceruletida/toxicidad , Citocinas/sangre , Citocinas/metabolismo , Modelos Animales de Enfermedad , Femenino , Humanos , Masculino , Ratones , Ratones Transgénicos , Proteínas Musculares/genética , Factores de Transcripción NFATC/antagonistas & inhibidores , Factores de Transcripción NFATC/metabolismo , Neutrófilos/inmunología , Neutrófilos/metabolismo , Páncreas/citología , Páncreas/inmunología , Páncreas/metabolismo , Pancreatitis/inducido químicamente , Pancreatitis/complicaciones , Pancreatitis/tratamiento farmacológico , Cultivo Primario de CélulasRESUMEN
BACKGROUND AND AIMS: Activated hepatocytes are hypothesized to be a major source of signals that drive cirrhosis, but the biochemical pathways that convert hepatocytes into such a state are unclear. We examined the role of the Hippo pathway transcriptional coactivators Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ) in hepatocytes to facilitate cell-cell interactions that stimulate liver inflammation and fibrosis. APPROACH AND RESULTS: Using a variety of genetic, metabolic, and liver injury models in mice, we manipulated Hippo signaling in hepatocytes and examined its effects in nonparenchymal cells to promote liver inflammation and fibrosis. YAP-expressing hepatocytes rapidly and potently activate the expression of proteins that promote fibrosis (collagen type I alpha 1 chain, tissue inhibitor of metalloproteinase 1, platelet-derived growth factor c, transforming growth factor ß2) and inflammation (tumor necrosis factor, interleukin 1ß). They stimulate expansion of myofibroblasts and immune cells, followed by aggressive liver fibrosis. In contrast, hepatocyte-specific YAP and YAP/TAZ knockouts exhibit limited myofibroblast expansion, less inflammation, and decreased fibrosis after CCl4 injury despite a similar degree of necrosis as controls. We identified cellular communication network factor 1 (CYR61) as a chemokine that is up-regulated by hepatocytes during liver injury but is expressed at significantly lower levels in mice with hepatocyte-specific deletion of YAP or TAZ. Gain-of-function and loss-of-function experiments with CYR61 in vivo point to it being a key chemokine controlling liver fibrosis and inflammation in the context of YAP/TAZ. There is a direct correlation between levels of YAP/TAZ and CYR61 in liver tissues of patients with high-grade nonalcoholic steatohepatitis. CONCLUSIONS: Liver injury in mice and humans increases levels of YAP/TAZ/CYR61 in hepatocytes, thus attracting macrophages to the liver to promote inflammation and fibrosis.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas de Ciclo Celular/metabolismo , Hepatocitos/metabolismo , Cirrosis Hepática/metabolismo , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Estrés Fisiológico , Transactivadores/metabolismo , Factores de Transcripción/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Proteínas de Ciclo Celular/genética , Cadena alfa 1 del Colágeno Tipo I , Proteína 61 Rica en Cisteína/genética , Proteína 61 Rica en Cisteína/metabolismo , Modelos Animales de Enfermedad , Mutación con Ganancia de Función , Humanos , Cirrosis Hepática/genética , Mutación con Pérdida de Función , Ratones , Enfermedad del Hígado Graso no Alcohólico/genética , Transactivadores/genética , Factores de Transcripción/genética , Proteínas Coactivadoras Transcripcionales con Motivo de Unión a PDZ , Proteínas Señalizadoras YAPRESUMEN
BACKGROUND & AIMS: Pancreatitis after endoscopic retrograde cholangiopancreatography (PEP) is thought to be provoked by pancreatic ductal hypertension, via unknown mechanisms. We investigated the effects of hydrostatic pressures on the development of pancreatitis in mice. METHODS: We performed studies with Swiss Webster mice, B6129 mice (controls), and B6129 mice with disruption of the protein phosphatase 3, catalytic subunit, ßisoform gene (Cnab-/- mice). Acute pancreatitis was induced in mice by retrograde biliopancreatic ductal or intraductal infusion of saline with a constant hydrostatic pressure while the proximal common bile duct was clamped -these mice were used as a model of PEP. Some mice were given pancreatic infusions of adeno-associated virus 6-nuclear factor of activated T-cells-luciferase to monitor calcineurin activity or the calcineurin inhibitor FK506. Blood samples and pancreas were collected at 6 and 24 hours and analyzed by enzyme-linked immunosorbent assay, histology, immunohistochemistry, or fluorescence microscopy. Ca2+ signaling and mitochondrial permeability were measured in pancreatic acinar cells isolated 15 minutes after PEP induction. Ca2+-activated phosphatase calcineurin within the pancreas was tracked in vivo over 24 hours. RESULTS: Intraductal pressures of up to 130 mm Hg were observed in the previously reported model of PEP; we found that application of hydrostatic pressures of 100 and 150 mm Hg for 10 minutes consistently induced pancreatitis. Pancreatic tissues had markers of inflammation (increased levels of interleukin [IL] 6, IL1B, and tumor necrosis factor), activation of signal transducer and activator of transcription 3, increased serum amylase and IL6, and loss of tight junction integrity. Transiently high pressures dysregulated Ca2+ processing (reduced Ca2+ oscillations and an increased peak plateau Ca2+ signal) and reduced the mitochondrial membrane potential. We observed activation of pancreatic calcineurin in the pancreas in mice. Cnab-/- mice, which lack the catalytic subunit of calcineurin, and mice given FK506 did not develop pressure-induced pancreatic inflammation, edema, or loss of tight junction integrity. CONCLUSIONS: Transient high ductal pressure produces pancreatic inflammation and loss of tight junction integrity in a mouse model of PEP. These processes require calcineurin signaling. Calcineurin inhibitors might be used to prevent acute pancreatitis that results from obstruction.
Asunto(s)
Ampolla Hepatopancreática/enzimología , Calcineurina/metabolismo , Señalización del Calcio , Mecanotransducción Celular , Pancreatitis/enzimología , Uniones Estrechas/enzimología , Ampolla Hepatopancreática/efectos de los fármacos , Ampolla Hepatopancreática/patología , Amilasas/sangre , Animales , Calcineurina/deficiencia , Calcineurina/genética , Inhibidores de la Calcineurina/farmacología , Señalización del Calcio/efectos de los fármacos , Colangiopancreatografia Retrógrada Endoscópica , Modelos Animales de Enfermedad , Femenino , Presión Hidrostática , Interleucina-1beta/metabolismo , Interleucina-6/sangre , Masculino , Mecanotransducción Celular/efectos de los fármacos , Potencial de la Membrana Mitocondrial , Ratones Noqueados , Mitocondrias/metabolismo , Pancreatitis/etiología , Pancreatitis/patología , Pancreatitis/prevención & control , Factor de Transcripción STAT3/metabolismo , Tacrolimus/farmacología , Uniones Estrechas/efectos de los fármacos , Uniones Estrechas/patología , Factores de Tiempo , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
The Hippo signaling pathway, also known as the Salvador-Warts-Hippo pathway, is a regulator of organ size. The pathway takes its name from the Drosophila protein kinase, Hippo (STK4/MST1 and STK3/MST2 in mammals), which, when inactivated, leads to considerable tissue overgrowth. In mammals, MST1 and MST2 negatively regulate the transcriptional co-activators yes-associated protein 1 and WW domain containing transcription regulator 1 (WWTR1/TAZ), which together regulate expression of genes that control proliferation, survival, and differentiation. Yes-associated protein 1 and TAZ activation have been associated with liver development, regeneration, and tumorigenesis. How their activity is dynamically regulated in these contexts is just beginning to be elucidated. We review the mechanisms of Hippo signaling in the liver and explore outstanding questions for future research.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Carcinoma Hepatocelular/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Neoplasias Hepáticas/metabolismo , Hígado/metabolismo , Fosfoproteínas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Factores de Transcripción/metabolismo , Aciltransferasas , Carcinogénesis , Factor de Crecimiento de Hepatocito , Vía de Señalización Hippo , Humanos , Hígado/patología , Regeneración Hepática , Tamaño de los Órganos , Proteínas Proto-Oncogénicas , Serina-Treonina Quinasa 3 , Transducción de Señal , Transactivadores , Proteínas Coactivadoras Transcripcionales con Motivo de Unión a PDZ , Proteínas Señalizadoras YAPRESUMEN
The Hippo pathway and its regulatory target, YAP, has recently emerged as an important biochemical signaling pathway that tightly governs epithelial tissue growth. Initially defined in Drosophilia, this pathway has shown remarkable conservation in vertebrate systems with many components of the Hippo/YAP pathway showing biochemical and functional conservation. The liver is particularly sensitive to changes in Hippo/YAP signaling with rapid increases in liver size becoming manifest on the order of days to weeks after perturbation. The first identified direct targets of Hippo/YAP signaling were pro-proliferative and anti-apoptotic gene programs, but recent work has now implicated this pathway in cell fate choice, stem cell maintenance/renewal, epithelial to mesenchymal transition, and oncogenesis. The mechanisms by which Hippo/YAP signaling is changed endogenously are beginning to come to light as well as how this pathway interacts with other signaling pathways, and important details for designing new therapeutic interventions. This review focuses on the known roles for Hippo/YAP signaling in the liver and promising avenues for future study.
Asunto(s)
Neoplasias Hepáticas/fisiopatología , Hígado/fisiología , Animales , Proteínas de Drosophila/fisiología , Humanos , Péptidos y Proteínas de Señalización Intracelular/fisiología , Regeneración Hepática/fisiología , Modelos Biológicos , Proteínas Nucleares/fisiología , Tamaño de los Órganos/fisiología , Proteínas Serina-Treonina Quinasas/fisiología , Transducción de Señal/fisiología , Transactivadores/fisiología , Proteínas Señalizadoras YAPRESUMEN
UNLABELLED: The purpose of the pretransplant assessment in lung transplantation is to determine a patient's need for transplant as well as their potential survival post-procedure. In 2005, the UNOS introduced the LAS, a calculation based on multiple physiologic measures to determine need and likelihood for survival. Measures include NYHA class and the 6-MWT. Some adult studies indicate a positive correlation with 6-MWT and waiting list survival. In pediatric/adolescent patients, there are minimal data regarding the predictive value of physiologic markers in either wait list survival or post-transplant outcome. A retrospective cohort study of 60 consecutive lung transplantations from 1990 to 2008 was performed at a pediatric tertiary care facility. Functional pretransplant assessments were abstracted from the medical record and compared with outcomes after transplantation. RESULTS: a 6-MWT of >1000 ft (305 m) prior to transplantation correlated with a shorter ICU stay (7 vs. 11 days, p = 0.046) and fewer days of mechanical ventilation (2 vs. 4, p = 0.04). A pretransplant 6-MWT greater than 750 ft (229 m) correlated with shorter overall hospitalization (37 vs. 20 days, p = 0.03). Measuring pretransplant 6-MWT tests for pediatric patients is valuable in predicting peri-operative outcomes after lung transplantation.
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Prueba de Esfuerzo , Enfermedades Pulmonares/diagnóstico , Trasplante de Pulmón/métodos , Caminata , Adolescente , Adulto , Índice de Masa Corporal , Niño , Preescolar , Femenino , Hospitalización , Humanos , Inmunosupresores/uso terapéutico , Lactante , Unidades de Cuidados Intensivos , Tiempo de Internación , Masculino , Complicaciones Posoperatorias/prevención & control , Periodo Posoperatorio , Valor Predictivo de las Pruebas , Pronóstico , Estudios Retrospectivos , Índice de Severidad de la Enfermedad , Resultado del Tratamiento , Adulto JovenRESUMEN
Obesity is increasing worldwide and leads to a multitude of metabolic diseases, including cardiovascular disease, type 2 diabetes, nonalcoholic fatty liver disease, and nonalcoholic steatohepatitis (NASH). Cysteine-rich angiogenic inducer 61 (CYR61) is associated with the progression of NASH, but it has been described to have anti- and proinflammatory properties. We sought to examine the role of liver CYR61 in NASH progression. CYR61 liver-specific knockout mice on a NASH diet showed improved glucose tolerance, decreased liver inflammation, and reduced fibrosis. CYR61 polarized infiltrating monocytes promoting a proinflammatory/profibrotic phenotype through an IRAK4/SYK/NF-κB signaling cascade. In vitro, CYR61 activated a profibrotic program, including PDGFa/PDGFb expression in macrophages, in an IRAK4/SYK/NF-κB-dependent manner. Furthermore, targeted-antibody blockade reduced CYR61-driven signaling in macrophages in vitro and in vivo, reducing fibrotic development. This study demonstrates that CYR61 is a key driver of liver inflammation and fibrosis in NASH.
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Diabetes Mellitus Tipo 2 , Enfermedad del Hígado Graso no Alcohólico , Ratones , Animales , Enfermedad del Hígado Graso no Alcohólico/patología , Quinasas Asociadas a Receptores de Interleucina-1/metabolismo , FN-kappa B/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Modelos Animales de Enfermedad , Hígado/metabolismo , Hepatocitos/metabolismo , Fibrosis , Macrófagos/metabolismo , Ratones Noqueados , Ratones Endogámicos C57BLRESUMEN
Nonalcoholic fatty liver disease (NAFLD) is the most common type of chronic liver disease in children. The mechanisms that drive NAFLD disease progression in this specific patient population remain poorly defined. In this study, we obtained liver biopsy samples from a multiethnic cohort of pediatric patients with NAFLD (n = 52, mean age = 13.6 years) and healthy liver controls (n = 5). We analyzed transcriptomic changes associated with NAFLD stages using high-throughput RNA sequencing. Unsupervised clustering as well as pairwise transcriptome comparison distinguished NAFLD from healthy livers. We identified perturbations in pathways including calcium and insulin/glucose signaling occurring early in NAFLD disease, before the presence of histopathologic evidence of advanced disease. Transcriptomic comparisons identified a 25-gene signature associated with the degree of liver fibrosis. We also identified expression of the insulin-like growth factor binding protein (IGFBP) gene family (1/2/3/7) as correlating with disease stages, and it has the potential to be used as a peripheral biomarker in NAFLD. Comparing our data set with publicly available adult and adolescent transcriptomic data, we identified similarities and differences in pathway enrichment and gene-expression profiles between adult and pediatric patients with NAFLD. Regulation of genes including interleukin-32, IGFBP1, IGFBP2, and IGFBP7 was consistently found in both NAFLD populations, whereas IGFBP3 was specific to pediatric NAFLD. Conclusion: This paper expands our knowledge on the molecular mechanisms underlying pediatric NAFLD. It identifies potential biomarkers and directs us toward new therapies in this population.
Asunto(s)
Enfermedad del Hígado Graso no Alcohólico , Adolescente , Adulto , Biomarcadores , Niño , Perfilación de la Expresión Génica , Humanos , Insulina/genética , Cirrosis Hepática/genética , Enfermedad del Hígado Graso no Alcohólico/genética , Transcriptoma/genéticaRESUMEN
Tumour cell invasiveness is crucial for cancer metastasis and is not yet understood. Here we describe two functional screens for proteins required for the invasion of fibrosarcoma cells that identified the molecular chaperone heat shock protein 90 (hsp90). The hsp90 alpha isoform, but not hsp90 beta, is expressed extracellularly where it interacts with the matrix metalloproteinase 2 (MMP2). Inhibition of extracellular hsp90 alpha decreases both MMP2 activity and invasiveness. This role for extracellular hsp90 alpha in MMP2 activation indicates that cell-impermeant anti-hsp90 drugs might decrease invasiveness without the concerns inherent in inhibiting intracellular hsp90.
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Membrana Celular/metabolismo , Matriz Extracelular/metabolismo , Fibrosarcoma/fisiopatología , Proteínas HSP90 de Choque Térmico/metabolismo , Metaloproteinasa 2 de la Matriz/metabolismo , Invasividad Neoplásica/fisiopatología , Membrana Basal/metabolismo , Sitios de Unión/fisiología , Línea Celular Tumoral , Inhibidores Enzimáticos/farmacología , Fibrosarcoma/metabolismo , Proteínas HSP90 de Choque Térmico/antagonistas & inhibidores , Humanos , Unión Proteica/fisiología , Mapeo de Interacción de Proteínas , Isoformas de Proteínas/antagonistas & inhibidores , Isoformas de Proteínas/metabolismo , Estructura Terciaria de Proteína/fisiología , ProteómicaRESUMEN
Malignant tumors exhibit high degrees of genomic heterogeneity at the cellular level, leading to the view that subpopulations of tumor cells drive growth and treatment resistance. To examine the degree to which tumors also exhibit metabolic heterogeneity at the level of individual cells, we employed multi-isotope imaging mass spectrometry (MIMS) to quantify utilization of stable isotopes of glucose and glutamine along with a label for cell division. Mouse models of melanoma and malignant peripheral nerve sheath tumors (MPNSTs) exhibited striking heterogeneity of substrate utilization, evident in both proliferating and non-proliferating cells. We identified a correlation between metabolic heterogeneity, proliferation, and therapeutic resistance. Heterogeneity in metabolic substrate usage as revealed by incorporation of glucose and glutamine tracers is thus a marker for tumor proliferation. Collectively, our data demonstrate that MIMS provides a powerful tool with which to dissect metabolic functions of individual cells within the native tumor environment.
RESUMEN
Hepatic stellate cells (HSCs) drive hepatic fibrosis. Therapies that inactivate HSCs have clinical potential as antifibrotic agents. We previously identified acid ceramidase (aCDase) as an antifibrotic target. We showed that tricyclic antidepressants (TCAs) reduce hepatic fibrosis by inhibiting aCDase and increasing the bioactive sphingolipid ceramide. We now demonstrate that targeting aCDase inhibits YAP/TAZ activity by potentiating its phosphorylation-mediated proteasomal degradation via the ubiquitin ligase adaptor protein ß-TrCP. In mouse models of fibrosis, pharmacologic inhibition of aCDase or genetic knockout of aCDase in HSCs reduces fibrosis, stromal stiffness, and YAP/TAZ activity. In patients with advanced fibrosis, aCDase expression in HSCs is increased. Consistently, a signature of the genes most down-regulated by ceramide identifies patients with advanced fibrosis who could benefit from aCDase targeting. The findings implicate ceramide as a critical regulator of YAP/TAZ signaling and HSC activation and highlight aCDase as a therapeutic target for the treatment of fibrosis.
Asunto(s)
Ceramidasa Ácida , Células Estrelladas Hepáticas , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Fibrosis , Células Estrelladas Hepáticas/metabolismo , Humanos , Ratones , Transducción de SeñalRESUMEN
The myosin family of motor proteins is implicated in mediating actin-based growth cone motility, but the roles of many myosins remain unclear. We previously implicated myosin 1c (M1c; formerly myosin I beta) in the retention of lamellipodia (Wang et al., 1996). Here we address the role of myosin II (MII) in chick dorsal root ganglion neuronal growth cone motility and the contribution of M1c and MII to retrograde F-actin flow using chromophore-assisted laser inactivation (CALI). CALI of MII reduced neurite outgrowth and growth cone area by 25%, suggesting a role for MII in lamellipodial expansion. Micro-CALI of MII caused a rapid reduction in local lamellipodial protrusion in growth cones with no effects on filopodial dynamics. This is opposite to micro-CALI of M1c, which caused an increase in lamellipodial protrusion. We used fiduciary beads (Forscher et al., 1992) to observe retrograde F-actin flow during the acute loss of M1c or MII. Micro-CALI of M1c reduced retrograde bead flow by 76%, whereas micro-CALI of MII or the MIIB isoform did not. Thus, M1c and MIIB serve opposite and nonredundant roles in regulating lamellipodial dynamics, and M1c activity is specifically required for retrograde F-actin flow.
Asunto(s)
Movimiento Celular/fisiología , Ganglios Espinales/embriología , Ganglios Espinales/metabolismo , Conos de Crecimiento/fisiología , Miosina Tipo I/metabolismo , Neuronas/metabolismo , Miosina Tipo IIB no Muscular/metabolismo , Actinas/química , Animales , Formación de Anticuerpos , Especificidad de Anticuerpos , Embrión de Pollo , Rayos Láser , Miosina Tipo I/inmunología , Neuritas/metabolismo , Miosina Tipo IIB no Muscular/inmunología , Fragmentos de Péptidos/inmunología , ConejosRESUMEN
One million patients with congenital heart disease (CHD) live in the United States. They have a lifelong risk of developing heart failure. Current concepts do not sufficiently address mechanisms of heart failure development specifically for these patients. Here, analysis of heart tissue from an infant with tetralogy of Fallot with pulmonary stenosis (ToF/PS) labeled with isotope-tagged thymidine demonstrated that cardiomyocyte cytokinesis failure is increased in this common form of CHD. We used single-cell transcriptional profiling to discover that the underlying mechanism of cytokinesis failure is repression of the cytokinesis gene ECT2, downstream of ß-adrenergic receptors (ß-ARs). Inactivation of the ß-AR genes and administration of the ß-blocker propranolol increased cardiomyocyte division in neonatal mice, which increased the number of cardiomyocytes (endowment) and conferred benefit after myocardial infarction in adults. Propranolol enabled the division of ToF/PS cardiomyocytes in vitro. These results suggest that ß-blockers could be evaluated for increasing cardiomyocyte division in patients with ToF/PS and other types of CHD.