RESUMEN
Shear stress generated by urinary fluid flow is an important regulator of renal function. Its dysregulation is observed in various chronic and acute kidney diseases. Previously, we demonstrated that primary cilium-dependent autophagy allows kidney epithelial cells to adapt their metabolism in response to fluid flow. Here, we show that nuclear YAP/TAZ negatively regulates autophagy flux in kidney epithelial cells subjected to fluid flow. This crosstalk is supported by a primary cilium-dependent activation of AMPK and SIRT1, independently of the Hippo pathway. We confirm the relevance of the YAP/TAZ-autophagy molecular dialog in vivo using a zebrafish model of kidney development and a unilateral ureteral obstruction mouse model. In addition, an in vitro assay simulating pathological accelerated flow observed at early stages of chronic kidney disease (CKD) activates YAP, leading to a primary cilium-dependent inhibition of autophagic flux. We confirm this YAP/autophagy relationship in renal biopsies from patients suffering from diabetic kidney disease (DKD), the leading cause of CKD. Our findings demonstrate the importance of YAP/TAZ and autophagy in the translation of fluid flow into cellular and physiological responses. Dysregulation of this pathway is associated with the early onset of CKD.
Asunto(s)
Insuficiencia Renal Crónica , Sirtuina 1 , Animales , Ratones , Humanos , Sirtuina 1/genética , Proteínas Quinasas Activadas por AMP , Pez Cebra , Autofagia/fisiología , Insuficiencia Renal Crónica/genética , Células Epiteliales/fisiología , RiñónRESUMEN
Cognitive decline and mood disorders increase in frequency with age. Many efforts are focused on the identification of molecules and pathways to treat these conditions. Here, we demonstrate that systemic administration of growth differentiation factor 11 (GDF11) in aged mice improves memory and alleviates senescence and depression-like symptoms in a neurogenesis-independent manner. Mechanistically, GDF11 acts directly on hippocampal neurons to enhance neuronal activity via stimulation of autophagy. Transcriptomic and biochemical analyses of these neurons reveal that GDF11 reduces the activity of mammalian target of rapamycin (mTOR), a master regulator of autophagy. Using a murine model of corticosterone-induced depression-like phenotype, we also show that GDF11 attenuates the depressive-like behavior of young mice. Analysis of sera from young adults with major depressive disorder (MDD) reveals reduced GDF11 levels. These findings identify mechanistic pathways related to GDF11 action in the brain and uncover an unknown role for GDF11 as an antidepressant candidate and biomarker.
Asunto(s)
Depresión , Trastorno Depresivo Mayor , Ratones , Animales , Depresión/tratamiento farmacológico , Trastorno Depresivo Mayor/tratamiento farmacológico , Factores de Diferenciación de Crecimiento/genética , Fenotipo , Autofagia/genética , Mamíferos/metabolismo , Proteínas Morfogenéticas Óseas/genéticaRESUMEN
Organs and cells must adapt to shear stress induced by biological fluids, but how fluid flow contributes to the execution of specific cell programs is poorly understood. Here we show that shear stress favours mitochondrial biogenesis and metabolic reprogramming to ensure energy production and cellular adaptation in kidney epithelial cells. Shear stress stimulates lipophagy, contributing to the production of fatty acids that provide mitochondrial substrates to generate ATP through ß-oxidation. This flow-induced process is dependent on the primary cilia located on the apical side of epithelial cells. The interplay between fluid flow and lipid metabolism was confirmed in vivo using a unilateral ureteral obstruction mouse model. Finally, primary cilium-dependent lipophagy and mitochondrial biogenesis are required to support energy-consuming cellular processes such as glucose reabsorption, gluconeogenesis and cytoskeletal remodelling. Our findings demonstrate how primary cilia and autophagy are involved in the translation of mechanical forces into metabolic adaptation.
Asunto(s)
Autofagia/fisiología , Cilios/metabolismo , Cilios/fisiología , Células Epiteliales/metabolismo , Células Epiteliales/fisiología , Riñón/metabolismo , Riñón/fisiología , Adenosina Trifosfato/metabolismo , Animales , Línea Celular , Gluconeogénesis/fisiología , Glucosa/metabolismo , Metabolismo de los Lípidos/fisiología , Ratones , Ratones Endogámicos C57BL , Mitocondrias/metabolismo , Estrés MecánicoRESUMEN
mTOR activation is essential and sufficient to cause polycystic kidneys in Tuberous Sclerosis Complex (TSC) and other genetic disorders. In disease models, a sharp increase of proliferation and cyst formation correlates with a dramatic loss of oriented cell division (OCD). We find that OCD distortion is intrinsically due to S6 kinase 1 (S6K1) activation. The concomitant loss of S6K1 in Tsc1-mutant mice restores OCD but does not decrease hyperproliferation, leading to non-cystic harmonious hyper growth of kidneys. Mass spectrometry-based phosphoproteomics for S6K1 substrates revealed Afadin, a known component of cell-cell junctions required to couple intercellular adhesions and cortical cues to spindle orientation. Afadin is directly phosphorylated by S6K1 and abnormally decorates the apical surface of Tsc1-mutant cells with E-cadherin and α-catenin. Our data reveal that S6K1 hyperactivity alters centrosome positioning in mitotic cells, affecting oriented cell division and promoting kidney cysts in conditions of mTOR hyperactivity.
Asunto(s)
División Celular , Cinesinas/metabolismo , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Miosinas/metabolismo , Enfermedades Renales Poliquísticas/patología , Proteínas Quinasas S6 Ribosómicas 90-kDa/metabolismo , Animales , Línea Celular , Cinesinas/genética , Ratones , Ratones Mutantes , Mutación , Miosinas/genética , Fosforilación , Enfermedades Renales Poliquísticas/genética , Enfermedades Renales Poliquísticas/metabolismo , Proteínas Quinasas S6 Ribosómicas 90-kDa/genética , Transducción de Señal , Esclerosis Tuberosa/genética , Esclerosis Tuberosa/metabolismo , Esclerosis Tuberosa/patología , Proteína 1 del Complejo de la Esclerosis Tuberosa/genética , Proteína 1 del Complejo de la Esclerosis Tuberosa/metabolismoRESUMEN
Aging is a negative regulator of general homeostasis, tissue function, and regeneration. Changes in organismal energy levels and physiology, through systemic manipulations such as calorie restriction and young blood infusion, can regenerate tissue activity and increase lifespan in aged mice. However, whether these two systemic manipulations could be linked has never been investigated. Here, we report that systemic GDF11 triggers a calorie restriction-like phenotype without affecting appetite or GDF15 levels in the blood, restores the insulin/IGF-1 signaling pathway, and stimulates adiponectin secretion from white adipose tissue by direct action on adipocytes, while repairing neurogenesis in the aged brain. These findings suggest that GDF11 has a pleiotropic effect on an organismal level and that it could be a linking mechanism of rejuvenation between heterochronic parabiosis and calorie restriction. As such, GDF11 could be considered as an important therapeutic candidate for age-related neurodegenerative and metabolic disorders.
Asunto(s)
Adiponectina/metabolismo , Proteínas Morfogenéticas Óseas/uso terapéutico , Restricción Calórica/métodos , Factores de Diferenciación de Crecimiento/uso terapéutico , Envejecimiento , Animales , Proteínas Morfogenéticas Óseas/farmacología , Factores de Diferenciación de Crecimiento/farmacología , Ratones , FenotipoRESUMEN
Age-related declines in cognitive fitness are associated with a reduction in autophagy, an intracellular lysosomal catabolic process that regulates protein homeostasis and organelle turnover. However, the functional significance of autophagy in regulating cognitive function and its decline during aging remains largely elusive. Here, we show that stimulating memory upregulates autophagy in the hippocampus. Using hippocampal injections of genetic and pharmacological modulators of autophagy, we find that inducing autophagy in hippocampal neurons is required to form novel memory by promoting activity-dependent structural and functional synaptic plasticity, including dendritic spine formation, neuronal facilitation, and long-term potentiation. We show that hippocampal autophagy activity is reduced during aging and that restoring its levels is sufficient to reverse age-related memory deficits. Moreover, we demonstrate that systemic administration of young plasma into aged mice rejuvenates memory in an autophagy-dependent manner, suggesting a prominent role for autophagy to favor the communication between systemic factors and neurons in fostering cognition. Among these youthful factors, we identify osteocalcin, a bone-derived molecule, as a direct hormonal inducer of hippocampal autophagy. Our results reveal that inducing autophagy in hippocampal neurons is a necessary mechanism to enhance the integration of novel stimulations of memory and to promote the influence of systemic factors on cognitive fitness. We also demonstrate the potential therapeutic benefits of modulating autophagy in the aged brain to counteract age-related cognitive impairments.
Asunto(s)
Envejecimiento/fisiología , Autofagia/fisiología , Hipocampo/fisiología , Trastornos de la Memoria , Memoria/fisiología , Animales , Autofagia/efectos de los fármacos , Autofagia/genética , Modelos Animales de Enfermedad , Masculino , Memoria/efectos de los fármacos , Trastornos de la Memoria/fisiopatología , Ratones , Ratones Endogámicos C57BLRESUMEN
Genome-wide association studies have struggled to identify functional genes and variants underlying complex phenotypes. We recruited a multi-ethnic cohort of healthy volunteers (n = 91) and used their tissue to generate induced pluripotent stem cells (iPSCs) and hepatocyte-like cells (HLCs) for genome-wide mapping of expression quantitative trait loci (eQTLs) and allele-specific expression (ASE). We identified many eQTL genes (eGenes) not observed in the comparably sized Genotype-Tissue Expression project's human liver cohort (n = 96). Focusing on blood lipid-associated loci, we performed massively parallel reporter assays to screen candidate functional variants and used genome-edited stem cells, CRISPR interference, and mouse modeling to establish rs2277862-CPNE1, rs10889356-DOCK7, rs10889356-ANGPTL3, and rs10872142-FRK as functional SNP-gene sets. We demonstrated HLC eGenes CPNE1, VKORC1, UBE2L3, and ANGPTL3 and HLC ASE gene ACAA2 to be lipid-functional genes in mouse models. These findings endorse an iPSC-based experimental framework to discover functional variants and genes contributing to complex human traits.
Asunto(s)
Sitios Genéticos , Variación Genética , Hepatocitos/citología , Células Madre Pluripotentes Inducidas/citología , Lípidos/sangre , Animales , Secuencia de Bases , Estudios de Cohortes , Perfilación de la Expresión Génica , Predisposición Genética a la Enfermedad , Estudio de Asociación del Genoma Completo , Hepatocitos/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Metabolismo de los Lípidos/genética , Hígado/metabolismo , Ratones , Especificidad de Órganos/genética , Polimorfismo de Nucleótido Simple/genética , Sitios de Carácter Cuantitativo/genéticaRESUMEN
Recent genome wide association studies have linked tribbles pseudokinase 1 (TRIB1) to the risk of coronary artery disease (CAD). Based on the observations that increased expression of TRIB1 reduces secretion of VLDL and is associated with lower plasma levels of LDL cholesterol and triglycerides, higher plasma levels of HDL cholesterol and reduced risk for myocardial infarction, we carried out a high throughput phenotypic screen based on quantitative RT-PCR assay to identify compounds that induce TRIB1 expression in human HepG2 hepatoma cells. In a screen of a collection of diversity-oriented synthesis (DOS)-derived compounds, we identified a series of benzofuran-based compounds that upregulate TRIB1 expression and phenocopy the effects of TRIB1 cDNA overexpression, as they inhibit triglyceride synthesis and apoB secretion in cells. In addition, the compounds downregulate expression of MTTP and APOC3, key components of the lipoprotein assembly pathway. However, CRISPR-Cas9 induced chromosomal disruption of the TRIB1 locus in HepG2 cells, while confirming its regulatory role in lipoprotein metabolism, demonstrated that the effects of benzofurans persist in TRIB1-null cells indicating that TRIB1 is sufficient but not necessary to transmit the effects of the drug. Remarkably, active benzofurans, as well as natural products capable of TRIB1 upregulation, also modulate hepatic cell cholesterol metabolism by elevating the expression of LDLR transcript and LDL receptor protein, while reducing the levels of PCSK9 transcript and secreted PCSK9 protein and stimulating LDL uptake. The effects of benzofurans are not masked by cholesterol depletion and are independent of the SREBP-2 regulatory circuit, indicating that these compounds represent a novel class of chemically tractable small-molecule modulators that shift cellular lipoprotein metabolism in HepG2 cells from lipogenesis to scavenging.
Asunto(s)
Regulación de la Expresión Génica/efectos de los fármacos , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Lipoproteínas/metabolismo , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Análisis por Conglomerados , Perfilación de la Expresión Génica , Células Hep G2 , Ensayos Analíticos de Alto Rendimiento , Humanos , Metabolismo de los Lípidos/efectos de los fármacos , Metabolismo de los Lípidos/genética , Lipoproteínas LDL/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Oncostatina M/farmacología , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Bibliotecas de Moléculas PequeñasRESUMEN
Transcription activator-like effector nucleases (TALENs) are a new class of engineered nucleases that are easier to design to cleave at desired sites in a genome than previous types of nucleases. We report here the use of TALENs to rapidly and efficiently generate mutant alleles of 15 genes in cultured somatic cells or human pluripotent stem cells, the latter for which we differentiated both the targeted lines and isogenic control lines into various metabolic cell types. We demonstrate cell-autonomous phenotypes directly linked to disease-dyslipidemia, insulin resistance, hypoglycemia, lipodystrophy, motor-neuron death, and hepatitis C infection. We found little evidence of TALEN off-target effects, but each clonal line nevertheless harbors a significant number of unique mutations. Given the speed and ease with which we were able to derive and characterize these cell lines, we anticipate TALEN-mediated genome editing of human cells becoming a mainstay for the investigation of human biology and disease.
Asunto(s)
Desoxirribonucleasas/genética , Células Madre/enzimología , Alelos , Genoma Humano/genética , Humanos , MutaciónRESUMEN
Recent genome-wide association studies (GWASs) have identified a locus on chromosome 1p13 strongly associated with both plasma low-density lipoprotein cholesterol (LDL-C) and myocardial infarction (MI) in humans. Here we show through a series of studies in human cohorts and human-derived hepatocytes that a common noncoding polymorphism at the 1p13 locus, rs12740374, creates a C/EBP (CCAAT/enhancer binding protein) transcription factor binding site and alters the hepatic expression of the SORT1 gene. With small interfering RNA (siRNA) knockdown and viral overexpression in mouse liver, we demonstrate that Sort1 alters plasma LDL-C and very low-density lipoprotein (VLDL) particle levels by modulating hepatic VLDL secretion. Thus, we provide functional evidence for a novel regulatory pathway for lipoprotein metabolism and suggest that modulation of this pathway may alter risk for MI in humans. We also demonstrate that common noncoding DNA variants identified by GWASs can directly contribute to clinical phenotypes.
Asunto(s)
Proteínas Adaptadoras del Transporte Vesicular/metabolismo , LDL-Colesterol/metabolismo , Cromosomas Humanos Par 1/genética , Predisposición Genética a la Enfermedad/genética , Polimorfismo de Nucleótido Simple/genética , Proteínas Adaptadoras del Transporte Vesicular/biosíntesis , Proteínas Adaptadoras del Transporte Vesicular/deficiencia , Proteínas Adaptadoras del Transporte Vesicular/genética , Animales , Secuencia de Bases , Sitios de Unión , Proteínas Potenciadoras de Unión a CCAAT/metabolismo , Células Cultivadas , LDL-Colesterol/sangre , Estudios de Cohortes , Enfermedad de la Arteria Coronaria/sangre , Enfermedad de la Arteria Coronaria/genética , Europa (Continente)/etnología , Regulación de la Expresión Génica , Técnicas de Silenciamiento del Gen , Estudio de Asociación del Genoma Completo , Haplotipos/genética , Hepatocitos/metabolismo , Humanos , Lípidos/sangre , Lipoproteínas VLDL/sangre , Lipoproteínas VLDL/metabolismo , Hígado/citología , Hígado/metabolismo , Ratones , Infarto del Miocardio/sangre , Infarto del Miocardio/genética , Fenotipo , Transcripción GenéticaRESUMEN
We demonstrate that deltaNp63alpha is an essential survival factor in head and neck squamous cell carcinoma (HNSCC) through its ability to suppress p73-dependent apoptosis. Inhibition of endogenous p63 expression by RNAi induces apoptosis selectively in HNSCC cells that overexpress deltaNp63alpha. Knockdown of p63 induces the proapoptotic bcl-2 family members Puma and Noxa, and both their induction and subsequent cell death are p53 independent but require transactivating isoforms of p73. Inhibition of p73-dependent transcription by deltaNp63alpha involves both direct promoter binding and physical interaction with p73. In HNSCC cells lacking endogenous deltaNp63alpha expression, bcl-2 is instead upregulated and can suppress p73-mediated death. Together, these data define a pathway whereby deltaNp63alpha promotes survival in squamous epithelial malignancy by repressing a p73-dependent proapoptotic transcriptional program.
Asunto(s)
Apoptosis , Carcinoma de Células Escamosas/metabolismo , Supervivencia Celular , Proteínas de Unión al ADN/fisiología , Genes Supresores de Tumor/fisiología , Neoplasias de Cabeza y Cuello/metabolismo , Proteínas Nucleares/fisiología , Fosfoproteínas/fisiología , Transactivadores/fisiología , Proteínas Reguladoras de la Apoptosis/metabolismo , Carcinoma de Células Escamosas/patología , Línea Celular Tumoral , Proteínas de Unión al ADN/genética , Genes bcl-2 , Genes p53 , Neoplasias de Cabeza y Cuello/patología , Humanos , Proteínas Nucleares/genética , Fosfoproteínas/antagonistas & inhibidores , Regiones Promotoras Genéticas , Unión Proteica , Isoformas de Proteínas/genética , Isoformas de Proteínas/fisiología , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Transducción de Señal , Transactivadores/antagonistas & inhibidores , Factores de Transcripción , Activación Transcripcional , Proteína Tumoral p73 , Proteínas Supresoras de TumorRESUMEN
To investigate the role of 3' end formation in yeast mRNA export, we replaced the mRNA cleavage and polyadenylation signal with a self-cleaving hammerhead ribozyme element. The resulting RNA is unadenylated and accumulates near its site of synthesis. Nonetheless, a significant fraction of this RNA reaches the cytoplasm. Nuclear accumulation was relieved by insertion of a stretch of DNA-encoded adenosine residues immediately upstream of the ribozyme element (a synthetic A tail). This indicates that a 3' stretch of adenosines can promote export, independently of cleavage and polyadenylation. We further show that a synthetic A tail-containing RNA is unaffected in 3' end formation mutant strains, in which a normally cleaved and polyadenylated RNA accumulates within nuclei. Our results support a model in which a polyA tail contributes to efficient mRNA progression away from the gene, most likely through the action of the yeast polyA-tail binding protein Pab1p.
Asunto(s)
ARN Catalítico/metabolismo , ARN de Hongos/metabolismo , ARN Mensajero/metabolismo , Secuencia de Bases , Núcleo Celular/metabolismo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Hibridación Fluorescente in Situ , Modelos Biológicos , Mutación , Poli A/química , Poli A/genética , Poli A/metabolismo , Proteínas de Unión a Poli(A)/genética , Proteínas de Unión a Poli(A)/metabolismo , ARN de Hongos/química , ARN de Hongos/genética , ARN Mensajero/química , ARN Mensajero/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Transcripción GenéticaRESUMEN
Nonsense-mediated decay (NMD) is a eukaryotic regulatory process that degrades mRNAs with premature termination codons (PTCs). Although NMD is a translation-dependent process, there is evidence from mammalian systems that PTC recognition and mRNA degradation takes place in association with nuclei. Consistent with this notion, degradation of mammalian PTC-containing mRNAs occurs when they are bound by the cap binding complex (CBC) during a "pioneer" round of translation. Moreover, there are reports indicating that a PTC can trigger other nuclear events such as alternative splicing, abnormal 3' end processing, and accumulation of pre-mRNA at transcription sites. To examine whether a PTC can elicit similar nuclear events in yeast, we used RNA export-defective mutants to sequester mRNAs within nuclei. The results indicate that nuclear PTC-containing yeast RNAs are NMD insensitive. We also observed by fluorescent in situ hybridization that there was no PTC effect on mRNA accumulated at the site of transcription. Finally, we show that yeast NMD occurs minimally if at all on CBC-bound transcripts, arguing against a CBC-mediated pioneer round of translation in yeast. The data taken together indicate that there are no direct consequences of a PTC within the yeast nucleus.