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OBJECTIVE: There are currently no specific biomarkers to identify patients with abdominal aortic aneurysms (AAAs). Circulating exosomes contain microRNAs (miRNA) that are potential biomarkers for the presence of disease. This study aimed to characterize the exosomal miRNA expression profile of patients with AAAs in order to identify novel biomarkers of disease. METHODS: Patients undergoing duplex ultrasound (US) or computed tomography (CT) for screening or surveillance of an AAA were screened to participate in the study. Cases with AAA were defined as having a max aortic diameter >3 cm. Circulating plasma exosomes were isolated using Cushioned-Density Gradient Ultracentrifugation and total RNA was extracted. Next Generation Sequencing was performed on the Illumina HiSeq4000 SE50. Differential miRNA expression analysis was performed using DESeq2 software with a Benjamini-Hochberg correction. MicroRNA expression profiles were validated by Quantitative Real-Time PCR. RESULTS: A total of 109 patients were screened to participate in the study. Eleven patients with AAA and 15 non-aneurysmal controls met study criteria and were enrolled. Ultrasound measured aortic diameter was significantly larger in the AAA group (mean maximum diameter 4.3 vs 2.0 cm, P = 6.45x10-6). More AAA patients had coronary artery disease (5/11 vs 1/15, P = 0.05) as compared to controls, but the groups did not differ significantly in the rates of peripheral arterial disease and chronic obstructive pulmonary disease. A total of 40 miRNAs were differentially expressed (P<0.05). Of these, 18 miRNAs were downregulated and 22 were upregulated in the AAA group compared to controls. After false discovery rate (FDR) adjustment, only miR-122-5p was expressed at significantly different levels in the AAA group compared to controls (fold change = 5.03 controls vs AAA; raw P = 1.8x10-5; FDR P = 0.02). CONCLUSION: Plasma exosomes from AAA patients have significantly reduced levels of miRNA-122-5p compared to controls. This is a novel exosome-associated miRNA that warrants further investigation to determine its use as a diagnostic biomarker and potential implications in AAA pathogenesis.
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Aneurisma de la Aorta Abdominal , Exosomas , MicroARNs , Humanos , Exosomas/metabolismo , MicroARNs/metabolismo , Aneurisma de la Aorta Abdominal/diagnóstico por imagen , Aneurisma de la Aorta Abdominal/genética , Aneurisma de la Aorta Abdominal/metabolismo , Biomarcadores , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
Cancers display significant heterogeneity with respect to tissue of origin, driver mutations, and other features of the surrounding tissue. It is likely that individual tumors engage common patterns of the immune system-here "archetypes"-creating prototypical non-destructive tumor immune microenvironments (TMEs) and modulating tumor-targeting. To discover the dominant immune system archetypes, the University of California, San Francisco (UCSF) Immunoprofiler Initiative (IPI) processed 364 individual tumors across 12 cancer types using standardized protocols. Computational clustering of flow cytometry and transcriptomic data obtained from cell sub-compartments uncovered dominant patterns of immune composition across cancers. These archetypes were profound insofar as they also differentiated tumors based upon unique immune and tumor gene-expression patterns. They also partitioned well-established classifications of tumor biology. The IPI resource provides a template for understanding cancer immunity as a collection of dominant patterns of immune organization and provides a rational path forward to learn how to modulate these to improve therapy.
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Censos , Neoplasias/genética , Neoplasias/inmunología , Transcriptoma/genética , Microambiente Tumoral/inmunología , Biomarcadores de Tumor , Análisis por Conglomerados , Estudios de Cohortes , Biología Computacional/métodos , Citometría de Flujo/métodos , Regulación Neoplásica de la Expresión Génica , Humanos , Neoplasias/clasificación , Neoplasias/patología , RNA-Seq/métodos , San Francisco , UniversidadesRESUMEN
Rationale: The 17q12-21.1 locus is one of the most highly replicated genetic associations with asthma. Individuals of African descent have lower linkage disequilibrium in this region, which could facilitate identifying causal variants.Objectives: To identify functional variants at 17q12-21.1 associated with early-onset asthma among African American individuals.Methods: We evaluated African American participants from SAPPHIRE (Study of Asthma Phenotypes and Pharmacogenomic Interactions by Race-Ethnicity) (n = 1,940), SAGE II (Study of African Americans, Asthma, Genes and Environment) (n = 885), and GCPD-A (Study of the Genetic Causes of Complex Pediatric Disorders-Asthma) (n = 2,805). Associations with asthma onset at ages under 5 years were meta-analyzed across cohorts. The lead signal was reevaluated considering haplotypes informed by genetic ancestry (i.e., African vs. European). Both an expression-quantitative trait locus analysis and a phenome-wide association study were performed on the lead variant.Measurements and Main Results: The meta-analyzed results from SAPPHIRE, SAGE II, and the GCPD-A identified rs11078928 as the top association for early-onset asthma. A haplotype analysis suggested that the asthma association partitioned most closely with the rs11078928 genotype. Genetic ancestry did not appear to influence the effect of this variant. In the expression-quantitative trait locus analysis, rs11078928 was related to alternative splicing of GSDMB (gasdermin-B) transcripts. The phenome-wide association study of rs11078928 suggested that this variant was predominantly associated with asthma and asthma-associated symptoms.Conclusions: A splice-acceptor polymorphism appears to be a causal variant for asthma at the 17q12-21.1 locus. This variant appears to have the same magnitude of effect in individuals of African and European descent.
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Negro o Afroamericano/genética , Cromosomas Humanos Par 17 , Estudios de Asociación Genética , Predisposición Genética a la Enfermedad/genética , Población Blanca/genética , Adolescente , Adulto , Edad de Inicio , Asma/genética , Niño , Preescolar , Mapeo Cromosómico , Femenino , Variación Genética , Humanos , Lactante , Recién Nacido , Desequilibrio de Ligamiento , Masculino , Persona de Mediana Edad , Polimorfismo de Nucleótido Simple , Sitios de Carácter Cuantitativo , Estados Unidos , Adulto JovenRESUMEN
BACKGROUND: Mitochondria support critical cellular functions, such as energy production through oxidative phosphorylation, regulation of reactive oxygen species, apoptosis, and calcium homeostasis. OBJECTIVE: Given the heightened level of cellular activity in patients with asthma, we sought to determine whether mitochondrial DNA (mtDNA) copy number measured in peripheral blood differed between individuals with and without asthma. METHODS: Whole genome sequence data was generated as part of the Trans-Omics for Precision Medicine (TOPMed) Program on participants from the Study of Asthma Phenotypes and Pharmacogenomic Interactions by Race-ethnicity (SAPPHIRE) and the Study of African Americans, Asthma, Genes, & Environment II (SAGE II). We restricted our analysis to individuals who self-identified as African American (3,651 asthma cases and 1,344 controls). Mitochondrial copy number was estimated using the sequencing read depth ratio for the mitochondrial and nuclear genomes. Respiratory complex expression was assessed using RNA-sequencing. RESULTS: Average mitochondrial copy number was significantly higher among individuals with asthma when compared with controls (SAPPHIRE: 218.60 vs. 200.47, P<0.001; SAGE II: 235.99 vs. 223.07, P<0.001). Asthma status was significantly associated with mitochondrial copy number after accounting for potential explanatory variables, such as participant age, sex, leukocyte counts, and mitochondrial haplogroup. Despite the consistent relationship between asthma status and mitochondrial copy number, the latter was not associated with time-to-exacerbation or patient-reported asthma control. Mitochondrial respiratory complex gene expression was disproportionately lower in individuals with asthma when compared with individuals without asthma and other protein-encoding genes. CONCLUSIONS: We observed a robust association between asthma and higher mitochondrial copy number. Asthma having an effect on mitochondria function was also supported by lower respiratory complex gene expression in this group.
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Asma/genética , Negro o Afroamericano/genética , Variaciones en el Número de Copia de ADN , ADN Mitocondrial/genética , Adulto , Asma/etnología , Secuencia de Bases , Estudios de Cohortes , ADN Mitocondrial/sangre , Proteínas del Complejo de Cadena de Transporte de Electrón/genética , Femenino , Citometría de Flujo , Humanos , Leucocitos/ultraestructura , Modelos Logísticos , Masculino , Persona de Mediana Edad , Modelos de Riesgos Proporcionales , ARN/genética , Sensibilidad y Especificidad , Investigación Biomédica Traslacional , Secuenciación Completa del Genoma , Adulto JovenRESUMEN
BACKGROUND: The frontonasal ectodermal zone (FEZ) is a signaling center that regulates patterned development of the upper jaw, and Sonic hedgehog (SHH) mediates FEZ activity. Induction of SHH expression in the FEZ results from SHH-dependent signals from the brain and neural crest cells. Given the role of miRNAs in modulating gene expression, we investigated the extent to which miRNAs regulate SHH expression and FEZ signaling. RESULTS: In the FEZ, the miR-199 family appears to be regulated by SHH-dependent signals from the brain; expression of this family increased from HH18 to HH22, and upon activation of SHH signaling in the brain. However, the miR-199 family is more broadly expressed in the mesenchyme of the frontonasal process and adjacent neuroepithelium. Downregulating the miR-199 genes expanded SHH expression in the FEZ, resulting in wider faces, while upregulating miR-199 genes resulted in decreased SHH expression and narrow faces. Hypoxia inducible factor 1 alpha (HIF1A) and mitogen-activated protein kinase kinase kinase 4 (MAP3K4) appear to be potential targets of miR-199b. Reduction of MAP3K4 altered beak development but increased apoptosis, while reducing HIF1A reduced expression of SHH in the FEZ and produced malformations independent of apoptosis. CONCLUSIONS: Our results demonstrate that this miRNA family appears to participate in regulating SHH expression in the FEZ; however, specific molecular mechanisms remain unknown.
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Proteínas Aviares/biosíntesis , Pollos , Huesos Faciales/embriología , Regulación del Desarrollo de la Expresión Génica , Proteínas Hedgehog/biosíntesis , MicroARNs/biosíntesis , Transducción de Señal , Animales , Tipificación del Cuerpo , Embrión de Pollo , Ectodermo/embriologíaRESUMEN
Given the increasing interest in their use as disease biomarkers, the establishment of reproducible, accurate, sensitive, and specific platforms for microRNA (miRNA) quantification in biofluids is of high priority. We compare four platforms for these characteristics: small RNA sequencing (RNA-seq), FirePlex, EdgeSeq, and nCounter. For a pool of synthetic miRNAs, coefficients of variation for technical replicates are lower for EdgeSeq (6.9%) and RNA-seq (8.2%) than for FirePlex (22.4%); nCounter replicates are not performed. Receiver operating characteristic analysis for distinguishing present versus absent miRNAs shows small RNA-seq (area under curve 0.99) is superior to EdgeSeq (0.97), nCounter (0.94), and FirePlex (0.81). Expected differences in expression of placenta-associated miRNAs in plasma from pregnant and non-pregnant women are observed with RNA-seq and EdgeSeq, but not FirePlex or nCounter. These results indicate that differences in performance among miRNA profiling platforms impact ability to detect biological differences among samples and thus their relative utility for research and clinical use.
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Perfilación de la Expresión Génica/métodos , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , MicroARNs/sangre , MicroARNs/genética , Placenta/metabolismo , Análisis de Secuencia de ARN/métodos , Adulto , Femenino , Humanos , Masculino , Persona de Mediana Edad , Embarazo , Curva ROC , Reproducibilidad de los Resultados , Adulto JovenRESUMEN
BACKGROUND: Although inhaled corticosteroid (ICS) medication is considered the cornerstone treatment for patients with persistent asthma, few ICS pharmacogenomic studies have involved nonwhite populations. OBJECTIVE: We sought to identify genetic predictors of ICS response in multiple population groups with asthma. METHODS: The discovery group comprised African American participants from the Study of Asthma Phenotypes and Pharmacogenomic Interactions by Race-Ethnicity (SAPPHIRE) who underwent 6 weeks of monitored ICS therapy (n = 244). A genome-wide scan was performed to identify single nucleotide polymorphism (SNP) variants jointly associated (ie, the combined effect of the SNP and SNP × ICS treatment interaction) with changes in asthma control. Top associations were validated by assessing the joint association with asthma exacerbations in 3 additional groups: African Americans (n = 803 and n = 563) and Latinos (n = 1461). RNA sequencing data from 408 asthmatic patients and 405 control subjects were used to examine whether genotype was associated with gene expression. RESULTS: One variant, rs3827907, was significantly associated with ICS-mediated changes in asthma control in the discovery set (P = 7.79 × 10-8) and was jointly associated with asthma exacerbations in 3 validation cohorts (P = .023, P = .029, and P = .041). RNA sequencing analysis found the rs3827907 C-allele to be associated with lower RNASE2 expression (P = 6.10 × 10-4). RNASE2 encodes eosinophil-derived neurotoxin, and the rs3827907 C-allele appeared to particularly influence ICS treatment response in the presence of eosinophilic inflammation (ie, high pretreatment eosinophil-derived neurotoxin levels or blood eosinophil counts). CONCLUSION: We identified a variant, rs3827907, that appears to influence response to ICS treatment in multiple population groups and likely mediates its effect through eosinophils.
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Corticoesteroides/uso terapéutico , Asma/tratamiento farmacológico , Negro o Afroamericano , Neurotoxina Derivada del Eosinófilo/genética , Eosinófilos/inmunología , Genotipo , Hispánicos o Latinos , Adolescente , Adulto , Asma/epidemiología , Asma/genética , Niño , Estudios de Cohortes , Progresión de la Enfermedad , Estudio de Asociación del Genoma Completo , Humanos , Recuento de Leucocitos , Masculino , Inhaladores de Dosis Medida , Persona de Mediana Edad , Variantes Farmacogenómicas , Fenotipo , Polimorfismo de Nucleótido Simple , Resultado del Tratamiento , Estados Unidos/epidemiología , Adulto JovenRESUMEN
Extracellular microRNAs (miRNAs) and other small RNAs are implicated in cellular communication and may be useful as disease biomarkers. We systematically compared small RNAs in 12 human biofluid types using RNA sequencing (RNA-seq). miRNAs and tRNA-derived RNAs (tDRs) accounted for the majority of mapped reads in all biofluids, but the ratio of miRNA to tDR reads varied from 72 in plasma to 0.004 in bile. miRNA levels were highly correlated across all biofluids, but levels of some miRNAs differed markedly between biofluids. tDR populations differed extensively between biofluids. Y RNA fragments were seen in all biofluids and accounted for >10% of reads in blood plasma, serum, and cerebrospinal fluid (CSF). Reads mapping exclusively to Piwi-interacting RNAs (piRNAs) were very rare, except in seminal plasma. These results demonstrate extensive differences in small RNAs between human biofluids and provide a useful resource for investigating extracellular RNA biology and developing biomarkers.
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Líquidos Corporales/metabolismo , MicroARNs/genética , ARN de Transferencia/genética , Adulto , Aminoácidos/genética , Anticodón/genética , Femenino , Humanos , Masculino , MicroARNs/metabolismo , Persona de Mediana Edad , ARN Interferente Pequeño/genética , ARN de Transferencia/metabolismo , Análisis de Secuencia de ARNRESUMEN
RNA-seq is increasingly used for quantitative profiling of small RNAs (for example, microRNAs, piRNAs and snoRNAs) in diverse sample types, including isolated cells, tissues and cell-free biofluids. The accuracy and reproducibility of the currently used small RNA-seq library preparation methods have not been systematically tested. Here we report results obtained by a consortium of nine labs that independently sequenced reference, 'ground truth' samples of synthetic small RNAs and human plasma-derived RNA. We assessed three commercially available library preparation methods that use adapters of defined sequence and six methods using adapters with degenerate bases. Both protocol- and sequence-specific biases were identified, including biases that reduced the ability of small RNA-seq to accurately measure adenosine-to-inosine editing in microRNAs. We found that these biases were mitigated by library preparation methods that incorporate adapters with degenerate bases. MicroRNA relative quantification between samples using small RNA-seq was accurate and reproducible across laboratories and methods.
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MicroARNs/genética , Análisis de Secuencia de ARN/métodos , Adenosina/genética , Humanos , Inosina/genética , MicroARNs/sangre , MicroARNs/normas , Edición de ARN , Estándares de Referencia , Reproducibilidad de los ResultadosRESUMEN
Thymic dendritic cells (tDCs) play an important role in central tolerance by eliminating self-reactive thymocytes or differentiating them to regulatory T (Treg) cells. However, the molecular and cellular mechanisms underlying these functions are not completely understood. We found that mouse tDCs undergo maturation following cognate interaction with self-reactive CD4+ thymocytes and that this maturation is dependent on CD40 signaling. Ablation of CD40 expression in tDCs resulted in a significant reduction in the number of Treg cells in association with a significant reduction in the number of mature tDCs. In addition, CD40-deficient DCs failed to fully mature upon cognate interaction with CD4+ thymocytes in vitro and failed to differentiate them into Treg cells to a sufficient number. These findings suggest that tDCs mature and potentiate Treg cell development in feedback response to self-reactive CD4+ thymocytes.
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Antígenos CD40/inmunología , Células Dendríticas/citología , Autotolerancia/inmunología , Timocitos/citología , Timo/citología , Animales , Linfocitos T CD4-Positivos/citología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Antígenos CD40/metabolismo , Diferenciación Celular/inmunología , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Ratones , Transducción de Señal/inmunología , Linfocitos T Reguladores/citología , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/metabolismo , Timocitos/inmunología , Timocitos/metabolismo , Timo/inmunología , Timo/metabolismoRESUMEN
Thymic epithelial cells (TECs) support T cell development in the thymus. Cortical thymic epithelial cells (cTECs) facilitate positive selection of developing thymocytes whereas medullary thymic epithelial cells (mTECs) facilitate the deletion of self-reactive thymocytes in order to prevent autoimmunity. The mTEC compartment is highly dynamic with continuous maturation and turnover, but the genetic regulation of these processes remains poorly understood. MicroRNAs (miRNAs) are important regulators of TEC genetic programs since miRNA-deficient TECs are severely defective. However, the individual miRNAs important for TEC maintenance and function and their mechanisms of action remain unknown. Here, we demonstrate that miR-205 is highly and preferentially expressed in mTECs during both thymic ontogeny and in the postnatal thymus. This distinct expression is suggestive of functional importance for TEC biology. Genetic ablation of miR-205 in TECs, however, neither revealed a role for miR-205 in TEC function during homeostatic conditions nor during recovery from thymic stress conditions. Thus, despite its distinct expression, miR-205 on its own is largely dispensable for mTEC biology.
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Células Epiteliales/metabolismo , Regulación de la Expresión Génica/fisiología , MicroARNs/biosíntesis , Timo/metabolismo , Animales , Células Epiteliales/citología , Ratones , Ratones Transgénicos , MicroARNs/genética , Timocitos/citología , Timocitos/metabolismo , Timo/citologíaRESUMEN
Airway remodeling, caused by inflammation and fibrosis, is a major component of chronic obstructive pulmonary disease (COPD) and currently has no effective treatment. Transforming growth factor-ß (TGF-ß) has been widely implicated in the pathogenesis of airway remodeling in COPD. TGF-ß is expressed in a latent form that requires activation. The integrin αvß8 (encoded by the itgb8 gene) is a receptor for latent TGF-ß and is essential for its activation. Expression of integrin αvß8 is increased in airway fibroblasts in COPD and thus is an attractive therapeutic target for the treatment of airway remodeling in COPD. We demonstrate that an engineered optimized antibody to human αvß8 (B5) inhibited TGF-ß activation in transgenic mice expressing only human and not mouse ITGB8. The B5 engineered antibody blocked fibroinflammatory responses induced by tobacco smoke, cytokines, and allergens by inhibiting TGF-ß activation. To clarify the mechanism of action of B5, we used hydrodynamic, mutational, and electron microscopic methods to demonstrate that αvß8 predominantly adopts a constitutively active, extended-closed headpiece conformation. Epitope mapping and functional characterization of B5 revealed an allosteric mechanism of action due to locking-in of a low-affinity αvß8 conformation. Collectively, these data demonstrate a new model for integrin function and present a strategy to selectively target the TGF-ß pathway to treat fibroinflammatory airway diseases.
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Traqueítis/terapia , Factor de Crecimiento Transformador beta/metabolismo , Animales , Humanos , Ratones , Ratones TransgénicosRESUMEN
AIMS: Animal studies show that transforming growth factor-ß1 (TGF-ß1) is an important mediator of atrial fibrosis and atrial fibrillation (AF). This study investigated the role of TGF-ß1 in human AF and the mechanism of atrial-selective fibrosis. METHODS AND RESULTS: Atrial specimens from 17 open heart surgery patients and left atrial and ventricular specimens from 17 explanted hearts were collected to assess the relationship between TGF-ß1, AF, and differential atrial vs. ventricular TGF-ß1 levels. A transgenic mouse model overexpressing active TGF-ß1 was used to study the mechanisms underlying the resultant atrial-selective fibrosis. Higher right atrial total TGF-ß1 levels (2.58 ± 0.16-fold, P < 0.0001) and active TGF-ß1 (3.7 ± 0.7-fold, P = 0.013) were observed in those that developed post-operative AF. Although no ventricular differences were observed, 11 explanted heart failure hearts exhibited higher atrial TGF-ß1 levels than 6 non-failing hearts (2.30 ± 0.87 fold higher, P < 0.001). In the transgenic mouse, TGF-ß1 receptor-1 kinase blockade resulted in decreased atrial expression of fibrosis-related genes. By RNA microarray analyses in that model, 80 genes in the atria and only 2 genes in the ventricle were differentially expressed. Although these mice atria, but not the ventricles, exhibited increased expression of fibrosis-related genes and phosphorylation of Smad2, there were no differences in TGF-ß1 receptor levels or Smads in the atria compared with the ventricles. CONCLUSIONS: TGF-ß1 mediates selective atrial fibrosis in AF that occurs via TGF-ß Receptor 1/2 and the classical Smad pathway. The differential atrial vs. ventricular fibrotic response occurs at the level of TGF-ß1 receptor binding or phosphorylation.
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Fibrilación Atrial/etiología , Atrios Cardíacos/patología , Factor de Crecimiento Transformador beta1/fisiología , Animales , Fibrosis , Humanos , Ratones , Ratones Transgénicos , Visón , Receptores de Factores de Crecimiento Transformadores beta/análisis , Factor de Crecimiento Transformador beta1/análisisRESUMEN
The glucocorticoid receptor (GR) regulates adaptive transcriptional programs that alter metabolism in response to stress. Network properties that allow GR to tune gene expression to match specific physiologic demands are poorly understood. We analyzed the transcriptional consequences of GR activation in murine lungs deficient for KLF15, a transcriptional regulator of amino acid metabolism that is induced by glucocorticoids and fasting. Approximately 7% of glucocorticoid-regulated genes had altered expression in Klf15-knockdown (Klf15(-/-)) mice. KLF15 formed coherent and incoherent feed-forward circuits with GR that correlated with the expression dynamics of the glucocorticoid response. Coherent feed-forward gene regulation by GR and KLF15 was characterized by combinatorial activation of linked GR-KLF15 regulatory elements by both factors and increased GR occupancy, while expression of KLF15 reduced GR occupancy at the incoherent target, MT2A. Serum deprivation, which increased KLF15 expression in a GR-independent manner in vitro, enhanced glucocorticoid-mediated induction of feed-forward targets of GR and KLF15, such as the loci for the amino acid-metabolizing enzymes proline dehydrogenase and alpha-aminoadipic semialdehyde synthase. Our results establish feed-forward architecture as an organizational principle for the GR network and provide a novel mechanism through which GR integrates signals and regulates expression dynamics.
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Proteínas de Unión al ADN/genética , Receptores de Glucocorticoides/metabolismo , Factores de Transcripción/genética , Animales , Inmunoprecipitación de Cromatina , Proteínas de Unión al ADN/metabolismo , Dexametasona/farmacología , Femenino , Regulación de la Expresión Génica , Factores de Transcripción de Tipo Kruppel , Pulmón/efectos de los fármacos , Pulmón/fisiología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Prolina Oxidasa/metabolismo , Regiones Promotoras Genéticas , Receptores de Glucocorticoides/genética , Elementos de Respuesta , Transducción de Señal/genética , Factores de Transcripción/metabolismoRESUMEN
Activation induces extensive changes in the gene expression program of naive CD4(+) T cells, promoting their differentiation into helper T cells that coordinate immune responses. MicroRNAs (miRNAs) play a critical role in this process, and miRNA expression also changes dramatically during T cell differentiation. Quantitative analyses revealed that T cell activation induces global posttranscriptional miRNA down-regulation in vitro and in vivo. Argonaute (Ago) proteins, the core effector proteins of the miRNA-induced silencing complex (miRISC), were also posttranscriptionally down-regulated during T cell activation. Ago2 was inducibly ubiquitinated in activated T cells and its down-regulation was inhibited by the proteasome inhibitor MG132. Therefore, activation-induced miRNA down-regulation likely occurs at the level of miRISC turnover. Measurements of miRNA-processing intermediates uncovered an additional layer of activation-induced, miRNA-specific transcriptional regulation. Thus, transcriptional and posttranscriptional mechanisms cooperate to rapidly reprogram the miRNA repertoire in differentiating T cells. Altering Ago2 expression in T cells revealed that Ago proteins are limiting factors that determine miRNA abundance. Naive T cells with reduced Ago2 and miRNA expression differentiated more readily into cytokine-producing helper T cells, suggesting that activation-induced miRNA down-regulation promotes acquisition of helper T cell effector functions by relaxing the repression of genes that direct T cell differentiation.
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Proteínas Argonautas/metabolismo , MicroARNs/genética , MicroARNs/metabolismo , Linfocitos T/inmunología , Linfocitos T/metabolismo , Animales , Diferenciación Celular , Citocinas/biosíntesis , Regulación hacia Abajo , Humanos , Activación de Linfocitos , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Complejo de la Endopetidasa Proteasomal/metabolismo , Complejo Silenciador Inducido por ARN/metabolismo , Linfocitos T/citología , Linfocitos T Colaboradores-Inductores/citología , Linfocitos T Colaboradores-Inductores/inmunología , Linfocitos T Colaboradores-Inductores/metabolismo , UbiquitinaciónRESUMEN
RATIONALE: Changes in airway epithelial cell differentiation, driven in part by IL-13, are important in asthma. Micro-RNAs (miRNAs) regulate cell differentiation in many systems and could contribute to epithelial abnormalities in asthma. OBJECTIVES: To determine whether airway epithelial miRNA expression is altered in asthma and identify IL-13-regulated miRNAs. METHODS: We used miRNA microarrays to analyze bronchial epithelial brushings from 16 steroid-naive subjects with asthma before and after inhaled corticosteroids, 19 steroid-using subjects with asthma, and 12 healthy control subjects, and the effects of IL-13 and corticosteroids on cultured bronchial epithelial cells. We used quantitative polymerase chain reaction to confirm selected microarray results. MEASUREMENTS AND MAIN RESULTS: Most (12 of 16) steroid-naive subjects with asthma had a markedly abnormal pattern of bronchial epithelial miRNA expression by microarray analysis. Compared with control subjects, 217 miRNAs were differentially expressed in steroid-naive subjects with asthma and 200 in steroid-using subjects with asthma (false discovery rate < 0.05). Treatment with inhaled corticosteroids had modest effects on miRNA expression in steroid-naive asthma, inducing a statistically significant (false discovery rate < 0.05) change for only nine miRNAs. qPCR analysis confirmed differential expression of 22 miRNAs that were highly differentially expressed by microarrays. IL-13 stimulation recapitulated changes in many differentially expressed miRNAs, including four members of the miR-34/449 family, and these changes in miR-34/449 family members were resistant to corticosteroids. CONCLUSIONS: Dramatic alterations of airway epithelial cell miRNA levels are a common feature of asthma. These alterations are only modestly corrected by inhaled corticosteroids. IL-13 effects may account for some of these alterations, including repression of miR-34/449 family members that have established roles in airway epithelial cell differentiation. Clinical trial registered with www.clinicaltrials.gov (NCT 00595153).
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Asma/metabolismo , Bronquios/metabolismo , Células Epiteliales/metabolismo , MicroARNs/metabolismo , Administración por Inhalación , Adulto , Asma/tratamiento farmacológico , Asma/genética , Bronquios/efectos de los fármacos , Budesonida/administración & dosificación , Células Cultivadas , Relación Dosis-Respuesta a Droga , Células Epiteliales/efectos de los fármacos , Femenino , Glucocorticoides/administración & dosificación , Humanos , Interleucina-13/farmacología , Masculino , MicroARNs/genética , MicroARNs/fisiología , Análisis por Micromatrices , Reacción en Cadena de la PolimerasaRESUMEN
The mammalian target of rapamycin (mTOR) is a central regulator of cell growth and proliferation in response to growth factor and nutrient signaling. Consequently, this kinase is implicated in metabolic diseases including cancer and diabetes, so there is great interest in understanding the complete spectrum of mTOR-regulated networks. mTOR exists in two functionally distinct complexes, mTORC1 and mTORC2, and whereas the natural product rapamycin inhibits only a subset of mTORC1 functions, recently developed ATP-competitive mTOR inhibitors have revealed new roles for both complexes. A number of studies have highlighted mTORC1 as a regulator of lipid homeostasis. We show that the ATP-competitive inhibitor PP242, but not rapamycin, significantly down-regulates cholesterol biosynthesis genes in a 4E-BP1-dependent manner in NIH 3T3 cells, whereas S6 kinase 1 is the dominant regulator in hepatocellular carcinoma cells. To identify other rapamycin-resistant transcriptional outputs of mTOR, we compared the expression profiles of NIH 3T3 cells treated with rapamycin versus PP242. PP242 caused 1,666 genes to be differentially expressed whereas rapamycin affected only 88 genes. Our analysis provides a genomewide view of the transcriptional outputs of mTOR signaling that are insensitive to rapamycin.
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Colesterol/biosíntesis , Regulación de la Expresión Génica/efectos de los fármacos , Sirolimus/farmacología , Serina-Treonina Quinasas TOR/metabolismo , Transcripción Genética/efectos de los fármacos , Proteínas Adaptadoras Transductoras de Señales , Animales , Carcinoma Hepatocelular/enzimología , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patología , Proteínas Portadoras/metabolismo , Proteínas de Ciclo Celular , Línea Celular Tumoral , Factores Eucarióticos de Iniciación , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Neoplasias Hepáticas/enzimología , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patología , Diana Mecanicista del Complejo 1 de la Rapamicina , Ratones , Complejos Multiproteicos , Células 3T3 NIH , Fosfoproteínas/metabolismo , Proteínas/metabolismo , Proteínas Quinasas S6 Ribosómicas 70-kDa/metabolismo , Proteína 2 de Unión a Elementos Reguladores de Esteroles/metabolismoRESUMEN
The response of primary human endothelial (ECs) and vascular smooth muscle cells (VSMCs) to TiO2 nanotube arrays is studied through gene expression analysis. Microarrays revealed that nanotubes enhanced EC proliferation and motility, decreased VSMC proliferation, and decreased expression of molecules involved in inflammation and coagulation in both cell types. Networks generated from significantly affected genes suggest that cells may be sensing nanotopographical cues via pathways previously implicated in sensing shear stress.
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Células Endoteliales/citología , Genoma , Nanopartículas del Metal/química , Músculo Liso Vascular/citología , Nanotubos/química , Titanio/química , Adhesión Celular , Movimiento Celular , Proliferación Celular , Humanos , Inflamación , Fenotipo , Estrés MecánicoRESUMEN
Protein disulfide isomerases (PDIs) aid protein folding and assembly by catalyzing formation and shuffling of cysteine disulfide bonds in the endoplasmic reticulum (ER). Many members of the PDI family are expressed in mammals, but the roles of specific PDIs in vivo are poorly understood. A recent homology-based search for additional PDI family members identified anterior gradient homolog 2 (AGR2), a protein originally presumed to be secreted by intestinal epithelial cells. Here, we show that AGR2 is present within the ER of intestinal secretory epithelial cells and is essential for in vivo production of the intestinal mucin MUC2, a large, cysteine-rich glycoprotein that forms the protective mucus gel lining the intestine. A cysteine residue within the AGR2 thioredoxin-like domain forms mixed disulfide bonds with MUC2, indicating a direct role for AGR2 in mucin processing. Mice lacking AGR2 were viable but were highly susceptible to colitis, indicating a critical role for AGR2 in protection from disease. We conclude that AGR2 is a unique member of the PDI family, with a specialized and nonredundant role in intestinal mucus production.