RESUMEN
MiR-150 is a microRNA (miRNA) specifically expressed in mature lymphocytes, but not their progenitors. A top predicted target of miR-150 is c-Myb, a transcription factor controlling multiple steps of lymphocyte development. Combining loss- and gain-of-function gene targeting approaches for miR-150 with conditional and partial ablation of c-Myb, we show that miR-150 indeed controls c-Myb expression in vivo in a dose-dependent manner over a narrow range of miRNA and c-Myb concentrations and that this dramatically affects lymphocyte development and response. Our results identify a key transcription factor as a critical target of a stage-specifically expressed miRNA in lymphocytes and suggest that this and perhaps other miRNAs have evolved to control the expression of just a few critical target proteins in particular cellular contexts.
Asunto(s)
Linfocitos B/fisiología , Diferenciación Celular/fisiología , Regulación de la Expresión Génica , MicroARNs/metabolismo , Proteínas Proto-Oncogénicas c-myb/metabolismo , Regiones no Traducidas 3' , Animales , Linfocitos B/citología , Muerte Celular , Células Cultivadas , Marcación de Gen , Genes Reporteros , Humanos , Sistema Inmunológico/fisiología , Ratones , Ratones Noqueados , MicroARNs/genética , Proteínas Proto-Oncogénicas c-myb/genética , Linfocitos T/fisiologíaRESUMEN
The genomic region encoding the miR-17-92 microRNA (miRNA) cluster is often amplified in lymphoma and other cancers, and cancer cells carrying this amplification have higher expression of miRNA in this cluster. Retroviral expression of miR-17-92 accelerates c-Myc-induced lymphoma development, but precisely how higher expression of miR-17-92 promotes lymphomagenesis remains unclear. Here we generated mice with higher expression of miR-17-92 in lymphocytes. These mice developed lymphoproliferative disease and autoimmunity and died prematurely. Lymphocytes from these mice showed more proliferation and less activation-induced cell death. The miR-17-92 miRNA suppressed expression of the tumor suppressor PTEN and the proapoptotic protein Bim. This mechanism probably contributed to the lymphoproliferative disease and autoimmunity of miR-17-92-transgenic mice and contributes to lymphoma development in patients with amplifications of the miR-17-92 coding region.
Asunto(s)
Enfermedades Autoinmunes/genética , Linfocitos/inmunología , Trastornos Linfoproliferativos/genética , Trastornos Linfoproliferativos/inmunología , MicroARNs/biosíntesis , MicroARNs/genética , Animales , Enfermedades Autoinmunes/patología , Muerte Celular/genética , Muerte Celular/inmunología , Proliferación Celular , Células Cultivadas , Amplificación de Genes , Regulación Neoplásica de la Expresión Génica/inmunología , Humanos , Células Jurkat , Activación de Linfocitos/genética , Activación de Linfocitos/inmunología , Linfocitos/metabolismo , Linfoma/genética , Linfoma/inmunología , Trastornos Linfoproliferativos/patología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , MicroARNs/fisiologíaRESUMEN
Genome-wide association studies (GWASs) have identified a genetic variant of moderate effect size at 6p21.1 associated with erythrocyte traits in humans. We show that this variant affects an erythroid-specific enhancer of CCND3. A Ccnd3 knockout mouse phenocopies these erythroid phenotypes, with a dramatic increase in erythrocyte size and a concomitant decrease in erythrocyte number. By examining human and mouse primary erythroid cells, we demonstrate that the CCND3 gene product cyclin D3 regulates the number of cell divisions that erythroid precursors undergo during terminal differentiation, thereby controlling erythrocyte size and number. We illustrate how cell type-specific specialization can occur for general cell cycle components-a finding resulting from the biological follow-up of unbiased human genetic studies.
Asunto(s)
Ciclo Celular/fisiología , Diferenciación Celular , Ciclina D3/metabolismo , Eritrocitos/citología , Eritrocitos/metabolismo , Animales , Recuento de Células , Tamaño de la Célula , Células Cultivadas , Ciclina D3/genética , Eritropoyesis/fisiología , Regulación de la Expresión Génica , Técnicas de Silenciamiento del Gen , Humanos , Células K562 , Ratones , Ratones NoqueadosRESUMEN
Reactive oxygen species (ROS) such as hydrogen peroxide (H2O2) govern cellular homeostasis by inducing signaling. H2O2 modulates the activity of phosphatases and many other signaling molecules through oxidation of critical cysteine residues, which led to the notion that initiation of ROS signaling is broad and nonspecific, and thus fundamentally distinct from other signaling pathways. Here, we report that H2O2 signaling bears hallmarks of a regular signal transduction cascade. It is controlled by hierarchical signaling events resulting in a focused response as the results place the mitochondrial respiratory chain upstream of tyrosine-protein kinase Lyn, Lyn upstream of tyrosine-protein kinase SYK (Syk), and Syk upstream of numerous targets involved in signaling, transcription, translation, metabolism, and cell cycle regulation. The active mediators of H2O2 signaling colocalize as H2O2 induces mitochondria-associated Lyn and Syk phosphorylation, and a pool of Lyn and Syk reside in the mitochondrial intermembrane space. Finally, the same intermediaries control the signaling response in tissues and species responsive to H2O2 as the respiratory chain, Lyn, and Syk were similarly required for H2O2 signaling in mouse B cells, fibroblasts, and chicken DT40 B cells. Consistent with a broad role, the Syk pathway is coexpressed across tissues, is of early metazoan origin, and displays evidence of evolutionary constraint in the human. These results suggest that H2O2 signaling is under control of a signal transduction pathway that links the respiratory chain to the mitochondrial intermembrane space-localized, ubiquitous, and ancient Syk pathway in hematopoietic and nonhematopoietic cells.
Asunto(s)
Transporte de Electrón , Peróxido de Hidrógeno/metabolismo , Membranas Mitocondriales/metabolismo , Transducción de Señal , Animales , Células Cultivadas , Pollos , Activación Enzimática , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Ratones , Fosforilación , Proteínas Tirosina Quinasas/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Quinasa Syk , Tirosina/metabolismoRESUMEN
Global nuclear condensation, culminating in enucleation during terminal erythropoiesis, is poorly understood. Proteomic examination of extruded erythroid nuclei from fetal liver revealed a striking depletion of most nuclear proteins, suggesting that nuclear protein export had occurred. Expression of the nuclear export protein, Exportin 7 (Xpo7), is highly erythroid-specific, induced during erythropoiesis, and abundant in very late erythroblasts. Knockdown of Xpo7 in primary mouse fetal liver erythroblasts resulted in severe inhibition of chromatin condensation and enucleation but otherwise had little effect on erythroid differentiation, including hemoglobin accumulation. Nuclei in Xpo7-knockdown cells were larger and less dense than normal and accumulated most nuclear proteins as measured by mass spectrometry. Strikingly,many DNA binding proteins such as histones H2A and H3 were found to have migrated into the cytoplasm of normal late erythroblasts prior to and during enucleation, but not in Xpo7-knockdown cells. Thus, terminal erythroid maturation involves migration of histones into the cytoplasm via a process likely facilitated by Xpo7.
Asunto(s)
Eritroblastos/citología , Eritroblastos/metabolismo , Histonas/sangre , Carioferinas/sangre , Proteína de Unión al GTP ran/sangre , Animales , Núcleo Celular/metabolismo , Citosol/metabolismo , Eritropoyesis/genética , Eritropoyesis/fisiología , Técnicas de Silenciamiento del Gen , Carioferinas/antagonistas & inhibidores , Carioferinas/genética , Ratones , Ratones Endogámicos C57BL , Proteínas Nucleares/sangre , Proteína de Unión al GTP ran/antagonistas & inhibidores , Proteína de Unión al GTP ran/genéticaRESUMEN
MicroRNA-155 (miR-155) regulates antibody responses and subsequent B-cell effector functions to exogenous antigens. However, the role of miR-155 in systemic autoimmunity is not known. Using the death receptor deficient (Fas(lpr)) lupus-prone mouse, we show here that ablation of miR-155 reduced autoantibody responses accompanied by a decrease in serum IgG but not IgM anti-dsDNA antibodies and a reduction of kidney inflammation. MiR-155 deletion in Fas(lpr) B cells restored the reduced SH2 domain-containing inositol 5'-phosphatase 1 to normal levels. In addition, coaggregation of the Fc γ receptor IIB with the B-cell receptor in miR-155(-/-)-Fas(lpr) B cells resulted in decreased ERK activation, proliferation, and production of switched antibodies compared with miR-155 sufficient Fas(lpr) B cells. Thus, by controlling the levels of SH2 domain-containing inositol 5'-phosphatase 1, miR-155 in part maintains an activation threshold that allows B cells to respond to antigens.
Asunto(s)
Autoanticuerpos/inmunología , Lupus Eritematoso Sistémico/prevención & control , MicroARNs/genética , MicroARNs/inmunología , Animales , Autoanticuerpos/biosíntesis , Northern Blotting , Western Blotting , Ensayo de Inmunoadsorción Enzimática , Técnica del Anticuerpo Fluorescente , Eliminación de Gen , Técnicas Histológicas , Inmunohistoquímica , Lupus Eritematoso Sistémico/inmunología , Ratones , Ratones Noqueados , UrinálisisRESUMEN
The effects of adiponectin on hepatic glucose and lipid metabolism at transcriptional level are largely unknown. We profiled hepatic gene expression in adiponectin knockout (KO) and wild-type (WT) mice by RNA sequencing. Compared with WT mice, adiponectin KO mice fed a chow diet exhibited decreased mRNA expression of rate-limiting enzymes in several important glucose and lipid metabolic pathways, including glycolysis, tricarboxylic acid cycle, fatty-acid activation and synthesis, triglyceride synthesis, and cholesterol synthesis. In addition, binding of the transcription factor Hnf4a to DNAs encoding several key metabolic enzymes was reduced in KO mice, suggesting that adiponectin might regulate hepatic gene expression via Hnf4a. Phenotypically, adiponectin KO mice possessed smaller epididymal fat pads and showed reduced body weight compared with WT mice. When fed a high-fat diet, adiponectin KO mice showed significantly reduced lipid accumulation in the liver. These lipogenic defects are consistent with the down-regulation of lipogenic genes in the KO mice.
Asunto(s)
Adiponectina/metabolismo , Regulación de la Expresión Génica/genética , Glucosa/metabolismo , Metabolismo de los Lípidos/genética , Hígado/metabolismo , Adiponectina/genética , Tejido Adiposo/patología , Análisis de Varianza , Animales , Secuencia de Bases , Peso Corporal/genética , Inmunoprecipitación de Cromatina , ADN/metabolismo , ADN Complementario/genética , Factor Nuclear 4 del Hepatocito/metabolismo , Ratones , Ratones Noqueados , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Análisis de Secuencia de ADN , Triglicéridos/metabolismoRESUMEN
Pyogenic Arthritis, Pyoderma Gangrenosum, and Acne Syndrome (PAPA syndrome) is an autoinflammatory disease caused by aberrant production of the proinflammatory cytokine interleukin-1. Mutations in the gene encoding proline serine threonine phosphatase-interacting protein-1 (PSTPIP1) have been linked to PAPA syndrome. PSTPIP1 is an adaptor protein that interacts with PYRIN, the protein encoded by the Mediterranean Fever (MEFV) gene whose mutations cause Familial Mediterranean Fever (FMF). However, the pathophysiological function of PSTPIP1 remains to be elucidated. We have generated mouse strains that either are PSTPIP1 deficient or ectopically express mutant PSTPIP1. Results from analyzing these mice suggested that PSTPIP1 is not an essential regulator of the Nlrp3, Aim2, or Nlrc4 inflammasomes. Although common features of human PAPA syndrome such as pyogenic arthritis and skin inflammation were not recapitulated in the mouse model, ectopic expression of the mutant but not the wild type PSTPIP1 in mice lead to partial embryonic lethality, growth retardation, and elevated level of circulating proinflammatory cytokines.
Asunto(s)
Acné Vulgar/genética , Proteínas Adaptadoras Transductoras de Señales/genética , Artritis Infecciosa/genética , Proteínas del Citoesqueleto/genética , Mutación , Piodermia Gangrenosa/genética , Alelos , Animales , Enfermedades Autoinmunes/metabolismo , Caspasa 1/metabolismo , Citocinas/metabolismo , Regulación de la Expresión Génica , Humanos , Inmunidad Innata , Inflamación , Interleucina-1beta/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Fenotipo , Transducción de Señal , Síndrome , Trementina/farmacologíaRESUMEN
Igα serine 191 and 197 and threonine 203, which are located in proximity of the Igα ITAM, dampen Igα ITAM tyrosine phosphorylation. In this study, we show that mice with targeted mutations of Igα S191, 197, and T203 displayed elevated serum IgG2c and IgG2b concentrations and had elevated numbers of IgG2c- and IgG2b-secreting cells in the bone marrow. BCR-induced Igα tyrosine phosphorylation was slightly increased in splenic B cells. Our results suggest that Igα serine/threonines limit formation of IgG2c- and IgG2b-secreting bone marrow plasma cells, possibly by fine-tuning Igα tyrosine-mediated BCR signaling.
Asunto(s)
Células de la Médula Ósea/citología , Mutación/inmunología , Células Plasmáticas/citología , Receptores de Antígenos de Linfocitos B/química , Receptores de Antígenos de Linfocitos B/inmunología , Secuencia de Aminoácidos , Animales , Células de la Médula Ósea/inmunología , Células de la Médula Ósea/metabolismo , Diferenciación Celular , Separación Celular , Citoplasma/química , Citoplasma/inmunología , Citoplasma/metabolismo , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Immunoblotting , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Mutación/genética , Fosforilación , Células Plasmáticas/inmunología , Células Plasmáticas/metabolismo , Receptores de Antígenos de Linfocitos B/genética , Serina/química , Serina/inmunología , Transducción de Señal/genética , Transducción de Señal/inmunología , Treonina/química , Treonina/inmunología , Tirosina/metabolismoRESUMEN
Brown adipose tissue (BAT) is specialized to burn lipids for heat generation as a natural defense against cold and obesity. Previous studies established microRNAs (miRNAs) as essential regulators of brown adipocyte differentiation, but whether miRNAs are required for the feature maintenance of mature brown adipocytes remains unknown. To address this question, we ablated Dgcr8, a key regulator of the miRNA biogenesis pathway, in mature brown as well as in white adipocytes. Adipose tissue-specific Dgcr8 knockout mice displayed enlarged but pale interscapular brown fat with decreased expression of genes characteristic of brown fat and were intolerant to cold exposure. Primary brown adipocyte cultures in vitro confirmed that miRNAs are required for marker gene expression in mature brown adipocytes. We also demonstrated that miRNAs are essential for the browning of subcutaneous white adipocytes in vitro and in vivo. Using this animal model, we performed miRNA expression profiling analysis and identified a set of BAT-specific miRNAs that are upregulated during brown adipocyte differentiation and enriched in brown fat compared with other organs. We identified miR-182 and miR-203 as new regulators of brown adipocyte development. Taken together, our study demonstrates an essential role of miRNAs in the maintenance as well as in the differentiation of brown adipocytes.
Asunto(s)
Adipocitos Marrones/metabolismo , Tejido Adiposo Pardo/metabolismo , Diferenciación Celular/genética , MicroARNs/fisiología , ARN Mensajero/análisis , Adipocitos Blancos/metabolismo , Animales , Células Cultivadas , Técnicas de Inactivación de Genes , Ratones , Ratones Noqueados , Proteínas de Unión al ARN/genéticaRESUMEN
In addition to the tyrosines of the Igalpha and beta immunoreceptor tyrosine-based activation motifs (ITAMs), the evolutionarily conserved Igalpha non-ITAM tyrosine 204 becomes phosphorylated upon antigen recognition by the B cell receptor (BCR). Here we demonstrate that splenic B cells from mice with a targeted mutation of Igalpha Y204 exhibited an isolated defect in T cell-independent B cell activation, proliferation, and antibody response upon BCR engagement, yet normal BCR capping, antigen internalization, antigen presentation, and T cell-dependent antibody production. Mutant B cells, present in normal numbers, exhibited unimpaired BCR-induced spleen tyrosine kinase (Syk) phosphorylation but reduced B cell linker protein (BLNK) phosphorylation, calcium flux, and nuclear factor kappaB (NFkappaB), c-jun N-terminal kinase (JNK), and extracellular signal-regulated kinase (ERK) activation. These results suggest that Igalpha non-ITAM tyrosine 204 promotes a distinct cellular response, namely T-independent B cell proliferation and differentiation via phosphorylation of the adaptor BLNK.