RESUMEN
Emerging evidence supports that mitochondrial dysfunction contributes to systemic lupus erythematosus (SLE) pathogenesis. Here we show that programmed mitochondrial removal, a hallmark of mammalian erythropoiesis, is defective in SLE. Specifically, we demonstrate that during human erythroid cell maturation, a hypoxia-inducible factor (HIF)-mediated metabolic switch is responsible for the activation of the ubiquitin-proteasome system (UPS), which precedes and is necessary for the autophagic removal of mitochondria. A defect in this pathway leads to accumulation of red blood cells (RBCs) carrying mitochondria (Mito+ RBCs) in SLE patients and in correlation with disease activity. Antibody-mediated internalization of Mito+ RBCs induces type I interferon (IFN) production through activation of cGAS in macrophages. Accordingly, SLE patients carrying both Mito+ RBCs and opsonizing antibodies display the highest levels of blood IFN-stimulated gene (ISG) signatures, a distinctive feature of SLE.
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Interferón Tipo I/metabolismo , Lupus Eritematoso Sistémico/metabolismo , Mitocondrias/metabolismo , Células Mieloides/metabolismo , Adolescente , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Niño , Preescolar , Eritroblastos/metabolismo , Eritroblastos/ultraestructura , Eritrocitos/metabolismo , Eritropoyesis , Humanos , Mitofagia , Complejo de la Endopetidasa Proteasomal/metabolismo , Ubiquitina/metabolismoRESUMEN
Erythropoiesis is an intricate process starting in hematopoietic stem cells and leading to the daily production of 200 billion red blood cells (RBCs). Enucleation is a greatly complex and rate-limiting step during terminal maturation of mammalian RBC production involving expulsion of the nucleus from the orthochromatic erythroblasts, resulting in the formation of reticulocytes. The dynamic enucleation process involves many factors ranging from cytoskeletal proteins to transcription factors to microRNAs. Lack of optimum terminal erythroid maturation and enucleation has been an impediment to optimum RBC production ex vivo. Major efforts in the past two decades have exposed some of the mechanisms that govern the enucleation process. This review focuses in detail on mechanisms implicated in enucleation and discusses the future perspectives of this fascinating process.
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Núcleo Celular , Eritroblastos/ultraestructura , Eritrocitos/ultraestructura , Eritropoyesis , Reticulocitos/ultraestructura , Animales , Aves/sangre , Calcio/fisiología , Cromatina/ultraestructura , Ensayo de Unidades Formadoras de Colonias , Biología Computacional , Citocinas/fisiología , Proteínas del Citoesqueleto/fisiología , Proteínas de Unión al ADN/fisiología , Eritroblastos/citología , Eritrocitos/citología , Péptidos y Proteínas de Señalización Intercelular/fisiología , Mamíferos/sangre , Ratones , MicroARNs/fisiología , Proteínas Proto-Oncogénicas/fisiología , Receptores de Hormona Tiroidea/fisiología , Proteínas Represoras/fisiología , Reticulocitos/citología , Factores de Transcripción/fisiología , Vesículas Transportadoras/fisiología , Saco Vitelino/citología , Proteínas de Unión al GTP rho/fisiologíaRESUMEN
DISEASE OVERVIEW: Ring sideroblasts (RS) are erythroid precursors with abnormal perinuclear mitochondrial iron accumulation. Two myeloid neoplasms defined by the presence of RS, include myelodysplastic syndromes with RS (MDS-RS) and MDS/myeloproliferative neoplasm with RS and thrombocytosis (MDS/MPN-RS-T). DIAGNOSIS: MDS-RS is a lower risk MDS, with single or multilineage dysplasia (MDS-RS-SLD/MLD), <5% bone marrow (BM) blasts, <1% peripheral blood blasts and ≥15% BM RS (≥5% in the presence of SF3B1 mutations). MDS/MPN-RS-T, now a formal entity in the MDS/MPN overlap syndromes, has diagnostic features of MDS-RS-SLD, along with a platelet count ≥450 × 109 /L and large atypical megakaryocytes. MUTATIONS AND KARYOTYPE: Mutations in SF3B1 are seen in ≥80% of patients with MDS-RS-SLD and MDS/MPN-RS-T, and strongly correlate with the presence of BM RS; MDS/MPN-RS-T patients also demonstrate JAK2V617F (50%), DNMT3A, TET2 and ASXL1 mutations. Cytogenetic abnormalities are uncommon in both. RISK STRATIFICATION: Most patients with MDS-RS-SLD are stratified into lower risk groups by the revised-IPSS. Disease outcome in MDS/MPN-RS-T is better than that of MDS-RS-SLD, but worse than that of essential thrombocythemia (MPN). Both diseases are associated with a low risk of leukemic transformation. TREATMENT: Anemia and iron overload are complications seen in both and are managed similar to lower risk MDS and MPN. Luspatercept, a first-in-class erythroid maturation agent is now approved for the management of anemia in patients with MDS-RS and MDS/MPN-RS-T. Aspirin therapy is reasonable in MDS/MPN-RS-T, especially in the presence of JAK2V617F, but the value of platelet-lowering drugs remains to be defined.
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Anemia Sideroblástica , Enfermedades Mielodisplásicas-Mieloproliferativas , Aloinjertos , Anemia Sideroblástica/diagnóstico , Anemia Sideroblástica/etiología , Anemia Sideroblástica/patología , Anemia Sideroblástica/terapia , Médula Ósea/patología , Linaje de la Célula , Células Clonales/patología , Terapia Combinada , Metilación de ADN/efectos de los fármacos , Manejo de la Enfermedad , Eritroblastos/ultraestructura , Ferritinas/análisis , Hematínicos/uso terapéutico , Trasplante de Células Madre Hematopoyéticas , Humanos , Quelantes del Hierro/uso terapéutico , Mitocondrias/química , Mutación , Enfermedades Mielodisplásicas-Mieloproliferativas/diagnóstico , Enfermedades Mielodisplásicas-Mieloproliferativas/genética , Enfermedades Mielodisplásicas-Mieloproliferativas/terapia , Fosfoproteínas/genética , Pronóstico , Factores de Empalme de ARN/genética , Medición de Riesgo , Trombocitosis/diagnóstico , Trombocitosis/terapiaRESUMEN
The final stages of mammalian erythropoiesis involve enucleation, membrane and proteome remodeling, and organelle clearance. Concomitantly, the erythroid membrane skeleton establishes a unique pseudohexagonal spectrin meshwork that is connected to the membrane through junctional complexes. The mechanism and signaling pathways involved in the coordination of these processes are unclear. The results of our study revealed an unexpected role of the membrane skeleton in the modulation of proteome remodeling and organelle clearance during the final stages of erythropoiesis. We found that diaphanous-related formin mDia2 is a master regulator of the integrity of the membrane skeleton through polymerization of actin protofilament in the junctional complex. The mDia2-deficient terminal erythroid cell contained a disorganized and rigid membrane skeleton that was ineffective in detaching the extruded nucleus. In addition, the disrupted skeleton failed to activate the endosomal sorting complex required for transport-III (ESCRT-III) complex, which led to a global defect in proteome remodeling, endolysosomal trafficking, and autophagic organelle clearance. Chmp5, a component of the ESCRT-III complex, is regulated by mDia2-dependent activation of the serum response factor and is essential for membrane remodeling and autophagosome-lysosome fusion. Mice with loss of Chmp5 in hematopoietic cells in vivo resembled the phenotypes in mDia2-knockout mice. Furthermore, overexpression of Chmp5 in mDia2-deficient hematopoietic stem and progenitor cells significantly restored terminal erythropoiesis in vivo. These findings reveal a formin-regulated signaling pathway that connects the membrane skeleton to proteome remodeling, enucleation, and organelle clearance during terminal erythropoiesis.
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Eritroblastos/metabolismo , Membrana Eritrocítica/metabolismo , Orgánulos/metabolismo , Proteoma/metabolismo , Animales , Autofagosomas/metabolismo , Secuencia de Bases , Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Endosomas/metabolismo , Eritroblastos/ultraestructura , Membrana Eritrocítica/ultraestructura , Eritropoyesis , Lisosomas/metabolismo , Fusión de Membrana , Ratones Endogámicos C57BL , Proteínas Asociadas a Microtúbulos/deficiencia , Proteínas Asociadas a Microtúbulos/metabolismo , NADPH Deshidrogenasa/deficiencia , NADPH Deshidrogenasa/metabolismo , Orgánulos/ultraestructura , Reticulocitos/metabolismo , Reticulocitos/ultraestructuraAsunto(s)
Eritroblastos/ultraestructura , Eritrocitos/ultraestructura , Cuerpos de Inclusión/química , Globinas alfa/análisis , Niño , Femenino , Hemoglobina Fetal/análisis , Hemoglobinopatías/sangre , Hemoglobinopatías/genética , Heterocigoto , Humanos , Esplenectomía , Adulto Joven , Talasemia beta/sangre , Talasemia beta/genética , Talasemia beta/cirugíaAsunto(s)
Anemia/patología , Médula Ósea/patología , Eritema Infeccioso/patología , Eritroblastos/ultraestructura , Mitosis , Complicaciones Posoperatorias/patología , Adulto , Anemia/etiología , Cromosomas Humanos/ultraestructura , Células Gigantes/ultraestructura , Humanos , Huésped Inmunocomprometido , Trasplante de Riñón , Masculino , Poliploidía , Complicaciones Posoperatorias/virologíaRESUMEN
The widely expressed bromodomain and extraterminal motif (BET) proteins bromodomain-containing protein 2 (BRD2), BRD3, and BRD4 are multifunctional transcriptional regulators that bind acetylated chromatin via their conserved tandem bromodomains. Small molecules that target BET bromodomains are being tested for various diseases but typically do not discern between BET family members. Genomic distributions and protein partners of BET proteins have been described, but the basis for differences in BET protein function within a given lineage remains unclear. By establishing a gene knockout-rescue system in a Brd2-null erythroblast cell line, here we compared a series of mutant and chimeric BET proteins for their ability to modulate cell growth, differentiation, and gene expression. We found that the BET N-terminal halves bearing the bromodomains convey marked differences in protein stability but do not account for specificity in BET protein function. Instead, when BET proteins were expressed at comparable levels, their specificity was largely determined by the C-terminal half. Remarkably, a chimeric BET protein comprising the N-terminal half of the structurally similar short BRD4 isoform (BRD4S) and the C-terminal half of BRD2 functioned similarly to intact BRD2. We traced part of the BRD2-specific activity to a previously uncharacterized short segment predicted to harbor a coiled-coil (CC) domain. Deleting the CC segment impaired BRD2's ability to restore growth and differentiation, and the CC region functioned in conjunction with the adjacent ET domain to impart BRD2-like activity onto BRD4S. In summary, our results identify distinct BET protein domains that regulate protein turnover and biological activities.
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Proteínas de Ciclo Celular/genética , Relación Estructura-Actividad , Factores de Transcripción/genética , Acetilación , Secuencias de Aminoácidos/genética , Proteínas de Ciclo Celular/ultraestructura , Diferenciación Celular/genética , Línea Celular , Proliferación Celular/genética , Cromatina/genética , Eritroblastos/química , Eritroblastos/metabolismo , Eritroblastos/ultraestructura , Regulación de la Expresión Génica/genética , Humanos , Dominios Proteicos/genética , Isoformas de Proteínas/genética , Bibliotecas de Moléculas Pequeñas/química , Factores de Transcripción/ultraestructuraRESUMEN
Sixteen patients with mild anemia and hemolysis were difficult to be classified into any known category based on laboratory examinations and light microscopy. To make a definite diagnosis and investigate the pathomechanism, ultrastructural study was performed on erythroid cells from 16 patients. Transmission electron microscopy demonstrated a series of alterations of cytoplasm, including cytoplasm sequestration, membranous transformation, and degeneration in erythroblasts and reticulocytes at different stages. The affected erythroblasts were usually complicated with chromatin condensation, karyorrhexis, nuclear membrane lysis, and megaloblastic changes. The reticulocytes with the cytoplasm alterations had a huge size from 10 um to 15 um in diameter. The membranous cytoplasm degeneration revealed a unique pathomechanism of dyserythropoiesis and ineffective erythropoiesis in 16 patients with anemia, and suggested a novel anemia category though more details remained to be investigated.
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Anemia/patología , Membrana Celular/ultraestructura , Eritroblastos/ultraestructura , Reticulocitos/ultraestructura , Adulto , Anciano , Médula Ósea/ultraestructura , Núcleo Celular/ultraestructura , Citoplasma/ultraestructura , Eritrocitos/ultraestructura , Femenino , Humanos , Masculino , Persona de Mediana EdadRESUMEN
The bone marrow is the primary site of erythropoiesis in healthy adult mammals. In the bone marrow, erythroid cells mature within specialized microenvironments termed erythroblastic islands (EBIs). EBIs are multi-cellular clusters comprised of a central macrophage surrounded by red blood cell (erythroid) progenitors. It has been proposed that the central macrophage functions as a "nurse-cell" providing iron, cytokines, and growth factors for the developing erythroid cells. The central macrophage also engulfs and destroys extruded erythroid nuclei. EBIs have recently been shown to play clinically important roles during human hematological disease. The molecular mechanisms regulating this hematopoietic niche are largely unknown. In this chapter, we detail protocols to study isolated EBIs using multiple microscopy platforms. Adhesion molecules regulate cell-cell interactions within the EBI and maintain the integrity of the niche. To improve our understanding of the molecular regulation of erythroid cells in EBIs, we have developed protocols for immuno-gold labeling of erythroid surface antigens to combine with scanning electron microscopy. These protocols have allowed imaging of EBIs at the nanometer scale, offering novel insights into the processes regulating red blood cell production.
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Médula Ósea/fisiología , Diferenciación Celular , Microambiente Celular , Eritroblastos/citología , Eritropoyesis , Animales , Ensayo de Unidades Formadoras de Colonias/métodos , Eritroblastos/metabolismo , Eritroblastos/ultraestructura , Técnica del Anticuerpo Fluorescente , Humanos , Macrófagos/citología , Macrófagos/metabolismo , Ratones , Microscopía Confocal , Nicho de Células MadreAsunto(s)
Citoplasma/ultraestructura , Errores Diagnósticos , Eritroblastos/ultraestructura , Leucemia Eritroblástica Aguda/diagnóstico , Mieloma Múltiple/diagnóstico , Síndromes Mielodisplásicos/sangre , Anciano , Protocolos de Quimioterapia Combinada Antineoplásica/efectos adversos , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Azacitidina/uso terapéutico , Ciclofosfamida/administración & dosificación , Ciclofosfamida/efectos adversos , Dexametasona/administración & dosificación , Dexametasona/efectos adversos , Resultado Fatal , Trasplante de Células Madre Hematopoyéticas , Humanos , Lenalidomida , Leucemia Eritroblástica Aguda/sangre , Leucemia Eritroblástica Aguda/patología , Masculino , Mieloma Múltiple/sangre , Síndromes Mielodisplásicos/inducido químicamente , Síndromes Mielodisplásicos/patología , Talidomida/análogos & derivados , Talidomida/uso terapéuticoRESUMEN
INTRODUCTION: Congenital dyserythropoietic anemias are rare blood disorders characterized by congenital anemia and a wide range of morphological and functional abnormalities of erythroid precursors. OBJECTIVES: To analyze the relative frequency of both light microscopic (LM) and electron microscopic (EM) morphological features of erythroblasts in a large group of patients with molecular proven congenital dyserythropoietic anemia type I (CDAI). METHODS: We retrospectively evaluated the LM and EM of bone marrow (BM) erythroblasts in 35 patients with CDAI. Thirty-four patients carried the CDAN1 Arg1042Trp founder mutation and one the p.Pro1130Leu mutation. BM slides of 24 patients were available for LM examination. EM studies were performed in all 35 patients. RESULTS: On LM, marked erythroid hyperplasia, binuclear erythroblasts, and various non-specific dyserythropoietic features were documented in every case; internuclear chromatin bridges were detected in 19 patients (79%). In all, EM of erythroblasts revealed a spongy appearance of heterochromatin, a widening of nuclear pores, and invagination of cytoplasm into the nuclear region. CONCLUSIONS: EM studies revealed high morphological frequency of specific ultrastructural changes in erythroblasts which facilitate prompt diagnosis of CDAI. Due to low specificity of BM LM findings, when BM EM is unavailable diagnostic approach should also include other inherited anemias.
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Anemia Diseritropoyética Congénita/diagnóstico , Médula Ósea/patología , Eritroblastos/patología , Anemia Diseritropoyética Congénita/sangre , Médula Ósea/ultraestructura , Eritroblastos/ultraestructura , Humanos , Microscopía , Microscopía ElectrónicaRESUMEN
Erythroblastic islands are multicellular clusters in which a central macrophage supports the development and maturation of red blood cell (erythroid) progenitors. These clusters play crucial roles in the pathogenesis observed in animal models of hematological disorders. The precise structure and function of erythroblastic islands is poorly understood. Here, we have combined scanning electron microscopy and immuno-gold labeling of surface proteins to develop a better understanding of the ultrastructure of these multicellular clusters. The erythroid-specific surface antigen Ter-119 and the transferrin receptor CD71 exhibited distinct patterns of protein sorting during erythroid cell maturation as detected by immuno-gold labeling. During electron microscopy analysis we observed two distinct classes of erythroblastic islands. The islands varied in size and morphology, and the number and type of erythroid cells interacting with the central macrophage. Assessment of femoral marrow isolated from a cavid rodent species (guinea pig, Cavis porcellus) and a marsupial carnivore species (fat-tailed dunnarts, Sminthopsis crassicaudata) showed that while the morphology of the central macrophage varied, two different types of erythroblastic islands were consistently identifiable. Our findings suggest that these two classes of erythroblastic islands are conserved in mammalian evolution and may play distinct roles in red blood cell production.
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Células de la Médula Ósea/ultraestructura , Médula Ósea/anatomía & histología , Eritroblastos/ultraestructura , Microscopía Electrónica de Rastreo , Animales , Antígenos CD/análisis , Antígenos de Grupos Sanguíneos/análisis , Cobayas , Marsupiales , Proteínas de la Membrana/análisis , Microscopía Inmunoelectrónica , Receptores de Transferrina/análisisRESUMEN
Clathrin-dependent endocytosis is an essential cellular process shared by all cell types. Despite this, precisely how endocytosis is regulated in a cell-type-specific manner and how this key pathway functions physiologically or pathophysiologically remain largely unknown. PICALM, which encodes the clathrin adaptor protein PICALM, was originally identified as a component of the CALM/AF10 leukemia oncogene. Here we show, by employing a series of conditional Picalm knockout mice, that PICALM critically regulates transferrin uptake in erythroid cells by functioning as a cell-type-specific regulator of transferrin receptor endocytosis. While transferrin receptor is essential for the development of all hematopoietic lineages, Picalm was dispensable for myeloid and B-lymphoid development. Furthermore, global Picalm inactivation in adult mice did not cause gross defects in mouse fitness, except for anemia and a coat color change. Freeze-etch electron microscopy of primary erythroblasts and live-cell imaging of murine embryonic fibroblasts revealed that Picalm function is required for efficient clathrin coat maturation. We showed that the PICALM PIP2 binding domain is necessary for transferrin receptor endocytosis in erythroblasts and absolutely essential for erythroid development from mouse hematopoietic stem/progenitor cells in an erythroid culture system. We further showed that Picalm deletion entirely abrogated the disease phenotype in a Jak2(V617F) knock-in murine model of polycythemia vera. Our findings provide new insights into the regulation of cell-type-specific transferrin receptor endocytosis in vivo. They also suggest a new strategy to block cellular uptake of transferrin-bound iron, with therapeutic potential for disorders characterized by inappropriate red blood cell production, such as polycythemia vera.
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Hematopoyesis/genética , Proteínas de Ensamble de Clatrina Monoméricas/genética , Policitemia Vera/genética , Anemia Hipocrómica/genética , Animales , Diferenciación Celular , Vesículas Cubiertas por Clatrina/metabolismo , Vesículas Cubiertas por Clatrina/ultraestructura , Modelos Animales de Enfermedad , Endocitosis , Eritroblastos/metabolismo , Eritroblastos/ultraestructura , Eritropoyesis/genética , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , Inmunofenotipificación , Linfopoyesis/genética , Ratones , Ratones Noqueados , Proteínas de Ensamble de Clatrina Monoméricas/química , Proteínas de Ensamble de Clatrina Monoméricas/deficiencia , Proteínas de Ensamble de Clatrina Monoméricas/metabolismo , Mielopoyesis/genética , Fenotipo , Fosfatidilinositol 4,5-Difosfato/metabolismo , Policitemia Vera/mortalidad , Dominios y Motivos de Interacción de Proteínas , Receptores de Transferrina/metabolismoRESUMEN
Here we demonstrate the feasibility of a novel approach to quantify DNA in cell nuclei. This approach is based on spectroscopy analysis of Raman light scattering, and avoids the problem of nonstoichiometric binding of dyes to DNA, as it directly measures the signal from DNA. Quantitative analysis of nuclear DNA contribution to Raman spectrum could be reliably performed using intensity of a phosphate mode at 1096 cm(-1) . When compared to the known DNA standards from cells of different animals, our results matched those values at error of 10%. We therefore suggest that this approach will be useful to expand the list of DNA standards, to properly adjust the duration of hydrolysis in Feulgen staining, to assay the applicability of fuchsines for DNA quantification, as well as to measure DNA content in cells with complex hydrolysis patterns, when Feulgen densitometry is inappropriate.
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Núcleo Celular/ultraestructura , ADN/análisis , Eritroblastos/ultraestructura , Linfocitos/ultraestructura , Espectrometría Raman , Animales , Pollos , Humanos , Fosfatos/química , Colorantes de Rosanilina , Salamandridae , Pez CebraRESUMEN
Definitive erythropoiesis takes place at erythroblastic islands, where erythroblasts proliferate and differentiate in association with central macrophages. At the final stage of erythropoiesis, pyrenocytes (nuclei surrounded by plasma membranes) are excluded from erythroblasts, expose phosphatidylserine (PtdSer), and are engulfed by the macrophages in a PtdSer-dependent manner. However, the molecular mechanism(s) involved in the engulfment of pyrenocytes are incompletely understood. Here, we constructed an in vitro assay system for the enucleation and engulfment of pyrenocytes using a methylcellulose-based culture. As reported previously, erythroblasts were bound to macrophages via interactions between integrin-α4ß1 on erythroblasts and Vcam1 on macrophages. After enucleation, the resulting pyrenocytes exhibited a reduced affinity for Vcam1 that correlated with the presence of inactive integrin-α4ß1 complexes. The pyrenocytes were then engulfed by the macrophages via a MerTK-protein S-dependent mechanism. Protein S appeared to function as a bridge between the pyrenocytes and macrophages by binding to PtdSer on the pyrenocytes and MerTK on the macrophages. Normally, NIH3T3 cells do not engulf pyrenocytes, but when they were transformed with MerTK, they efficiently engulfed pyrenocytes in the presence of protein S. These results suggest that macrophages use similar mechanisms to engulf both pyrenocytes and apoptotic cells.
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Eritroblastos/metabolismo , Eritrocitos/metabolismo , Macrófagos/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Tirosina Quinasas Receptoras/metabolismo , Animales , Apoptosis , Línea Celular , Núcleo Celular/metabolismo , Células Cultivadas , Eritroblastos/citología , Eritroblastos/ultraestructura , Membrana Eritrocítica/metabolismo , Eritrocitos/citología , Eritropoyesis , Células HEK293 , Humanos , Integrina alfa4beta1/genética , Integrina alfa4beta1/metabolismo , Macrófagos/citología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microscopía Confocal , Microscopía Electrónica de Transmisión , Células 3T3 NIH , Fagocitosis , Unión Proteica , Proteína S/metabolismo , Proteínas Proto-Oncogénicas/genética , Proteínas Tirosina Quinasas Receptoras/genética , Reticulocitos/citología , Reticulocitos/metabolismo , Molécula 1 de Adhesión Celular Vascular/genética , Molécula 1 de Adhesión Celular Vascular/metabolismo , Tirosina Quinasa c-MerRESUMEN
During hematopoiesis, lineage- and stage-specific transcription factors work in concert with chromatin modifiers to direct the differentiation of all blood cells. We explored the role of KRAB-containing zinc finger proteins (KRAB-ZFPs) and their cofactor KAP1 in this process. In mice, hematopoietic-restricted deletion of Kap1 resulted in severe hypoproliferative anemia. Kap1-deleted erythroblasts failed to induce mitophagy-associated genes and retained mitochondria. This was due to persistent expression of microRNAs (miRNAs) targeting mitophagy transcripts, itself secondary to a lack of repression by stage-specific KRAB-ZFPs. The KRAB/KAP1-miRNA regulatory cascade is evolutionarily conserved, as it also controls mitophagy during human erythropoiesis. Thus, a multilayered transcription regulatory system is present, in which protein- and RNA-based repressors are superimposed in combinatorial fashion to govern the timely triggering of an important differentiation event.
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Autofagia/genética , Eritroblastos/metabolismo , Eritropoyesis/genética , MicroARNs/metabolismo , Mitocondrias/fisiología , Proteínas Nucleares/metabolismo , Proteínas Represoras/metabolismo , Factores de Transcripción/metabolismo , Dedos de Zinc , Anemia/genética , Animales , Eritroblastos/ultraestructura , Femenino , Masculino , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , MicroARNs/genética , Mitocondrias/genética , Proteínas Mitocondriales/metabolismo , Proteínas Nucleares/genética , Proteínas Represoras/genética , Proteína 28 que Contiene Motivos TripartitoRESUMEN
Wholesale depletion of membrane organelles and extrusion of the nucleus are hallmarks of mammalian erythropoiesis. Using quantitative EM and fluorescence imaging we have investigated how autophagy contributes to organelle removal in an ex vivo model of human erythroid differentiation. We found that autophagy is induced at the polychromatic erythroid stage, and that autophagosomes remain abundant until enucleation. This stimulation of autophagy was concomitant with the transcriptional upregulation of many autophagy genes: of note, expression of all ATG8 mammalian paralog family members was stimulated, and increased expression of a subset of ATG4 family members (ATG4A and ATG4D) was also observed. Stable expression of dominant-negative ATG4 cysteine mutants (ATG4B (C74A) ; ATG4D (C144A) ) did not markedly delay or accelerate differentiation of human erythroid cells; however, quantitative EM demonstrated that autophagosomes are assembled less efficiently in ATG4B (C74A) -expressing progenitor cells, and that cells expressing either mutant accumulate enlarged amphisomes that cannot be degraded. The appearance of these hybrid autophagosome/endosome structures correlated with the contraction of the lysosomal compartment, suggesting that the actions of ATG4 family members (particularly ATG4B) are required for the control of autophagosome fusion with late, degradative compartments in differentiating human erythroblasts.
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Autofagia , Diferenciación Celular , Cisteína Endopeptidasas/metabolismo , Fagosomas/metabolismo , Homología de Secuencia de Aminoácido , Proteínas Relacionadas con la Autofagia , Compartimento Celular , Cisteína/genética , Eritroblastos/citología , Eritroblastos/metabolismo , Eritroblastos/ultraestructura , Células HEK293 , Células HeLa , Humanos , Membranas Intracelulares/metabolismo , Membranas Intracelulares/ultraestructura , Mitocondrias/metabolismo , Mitocondrias/ultraestructura , Proteínas Mutantes/metabolismo , Fagosomas/ultraestructura , Regulación hacia ArribaRESUMEN
Ultrastructural answer of bone marrow erythroid series and of red blood cells (RBCs) in Wistar rats to bee venom (BV) was analyzed by transmission and scanning electron microscopy, and corroborated with hematological data. A 5-day and a 30-day treatment with daily doses of 700 µg BV/kg and an acute-lethal treatment with a single dose of 62 mg BV/kg were performed. The 5-day treatment resulted in a reduced cellularity of the bone marrow, with necrosed proerythroblasts, polymorphous erythroblasts, and reticulocytes with cytoplasmic extensions, and a lower number of larger RBCs, with poikilocytosis (acanthocytosis) and anisocytosis, and reduced concentrations of hemoglobin. After the 30-day treatment, the bone marrow architecture was restored, but polymorphous erythroblasts and reticulocytes with thin extensions could still be observed, while the RBCs in higher number were smaller, many with abnormal shapes, especially acanthocytes. The acute treatment produced a partial depopulation of the bone marrow and ultrastructural changes of erythroblasts including abnormal mitochondrial cristae. The RBCs in lower number were bigger and crenated, with reduced concentrations of hemoglobin. Overall, BV was able to promote stress erythropoiesis in a time- and dose-related manner, mitochondrial cristae modification being a critical factor involved in the toxicity of the BV high doses.
Asunto(s)
Venenos de Abeja/farmacología , Células de la Médula Ósea/efectos de los fármacos , Eritrocitos/efectos de los fármacos , Eritropoyesis/efectos de los fármacos , Animales , Venenos de Abeja/administración & dosificación , Médula Ósea/fisiología , Médula Ósea/ultraestructura , Células de la Médula Ósea/ultraestructura , Eritroblastos/ultraestructura , Recuento de Eritrocitos , Eritrocitos/ultraestructura , Microscopía Electrónica de Transmisión de Rastreo , Ratas , Ratas WistarRESUMEN
PURPOSE: The mode of development of the human hyaloid vascular system (HVS) remains unclear. Early studies suggested that these blood vessels formed by vasculogenesis, while the current concept seems to favor angiogenesis as the mode of development. We examined embryonic and fetal human HVS using a variety of techniques to gain new insights into formation of this vasculature. METHODS: Embryonic and fetal human eyes from 5.5 to 12 weeks gestation (WG) were prepared for immunohistochemical analysis or for light and electron microscopy. Immunolabeling of sections with a panel of antibodies directed at growth factors, transcription factors, and hematopoietic stem cell markers was employed. RESULTS: Light microscopic examination revealed free blood islands (BI) in the embryonic vitreous cavity (5.5-7 WG). Giemsa stain revealed that BI were aggregates of mesenchymal cells and primitive nucleated erythroblasts. Free cells were also observed. Immunolabeling demonstrated that BI were composed of mesenchymal cells that expressed hemangioblast markers (CD31, CD34, C-kit, CXCR4, Runx1, and VEGFR2), erythroblasts that expressed embryonic hemoglobin (Hb-ε), and cells that expressed both. Few cells were proliferating as determined by lack of Ki67 antigen. As development progressed (12 WG), blood vessels became more mature structurally with pericyte investment and basement membrane formation. Concomitantly, Hb-ε and CXCR4 expression was down-regulated and von Willebrand factor expression was increased with the formation of Weibel-Palade bodies. CONCLUSIONS: Our results support the view that the human HVS, like the choriocapillaris, develops by hemo-vasculogenesis, the process by which vasculogenesis, erythropoiesis, and hematopoiesis occur simultaneously from common precursors, hemangioblasts.