RESUMEN
ABSTRACT: Lysyl oxidase (LOX) is a facilitator of extracellular matrix cross-linking. Using newly developed megakaryocyte-specific LOX knockout mice, we show that LOX expressed in these scarce bone marrow cells affects bone volume and collagen architecture in a sex-dependent manner.
Asunto(s)
Megacariocitos , Ratones Noqueados , Proteína-Lisina 6-Oxidasa , Animales , Proteína-Lisina 6-Oxidasa/metabolismo , Proteína-Lisina 6-Oxidasa/genética , Megacariocitos/metabolismo , Megacariocitos/citología , Ratones , Masculino , Femenino , Huesos/metabolismo , Caracteres Sexuales , Colágeno/metabolismo , Eliminación de Gen , Factores Sexuales , Proteínas de la Matriz ExtracelularRESUMEN
Mechanisms through which mature megakaryocytes (Mks) and their progenitors sense the bone marrow extracellular matrix to promote lineage differentiation in health and disease are still partially understood. We found PIEZO1, a mechanosensitive cation channel, to be expressed in mouse and human Mks. Human mutations in PIEZO1 have been described to be associated with blood cell disorders. Yet, a role for PIEZO1 in megakaryopoiesis and proplatelet formation has never been investigated. Here, we show that activation of PIEZO1 increases the number of immature Mks in mice, while the number of mature Mks and Mk ploidy level are reduced. Piezo1/2 knockout mice show an increase in Mk size and platelet count, both at basal state and upon marrow regeneration. Similarly, in human samples, PIEZO1 is expressed during megakaryopoiesis. Its activation reduces Mk size, ploidy, maturation, and proplatelet extension. Resulting effects of PIEZO1 activation on Mks resemble the profile in Primary Myelofibrosis (PMF). Intriguingly, Mks derived from Jak2V617F PMF mice show significantly elevated PIEZO1 expression, compared to wild-type controls. Accordingly, Mks isolated from bone marrow aspirates of JAK2V617F PMF patients show increased PIEZO1 expression compared to Essential Thrombocythemia. Most importantly, PIEZO1 expression in bone marrow Mks is inversely correlated with patient platelet count. The ploidy, maturation, and proplatelet formation of Mks from JAK2V617F PMF patients are rescued upon PIEZO1 inhibition. Together, our data suggest that PIEZO1 places a brake on Mk maturation and platelet formation in physiology, and its upregulation in PMF Mks might contribute to aggravating some hallmarks of the disease.
Asunto(s)
Mielofibrosis Primaria , Trombocitemia Esencial , Humanos , Animales , Ratones , Megacariocitos/metabolismo , Mielofibrosis Primaria/genética , Médula Ósea , Trombopoyesis/genética , Trombocitemia Esencial/metabolismo , Plaquetas/metabolismo , Canales Iónicos/genética , Canales Iónicos/metabolismoRESUMEN
Excessive accumulation of extracellular matrix (ECM) is a hallmark of bone marrow (BM) milieu in primary myelofibrosis (PMF). Because cells have the ability to adhere to the surrounding ECM through integrin receptors, we examined the hypothesis that an abnormal ECM-integrin receptor axis contributes to BM megakaryocytosis in JAK2V617F+ PMF. Secretion of ECM protein fibronectin (FN) by BM stromal cells from PMF patients correlates with fibrosis and disease severity. Here, we show that Vav1-hJAK2V617F transgenic mice (JAK2V617F+) have high BM FN content associated with megakaryocytosis and fibrosis. Further, megakaryocytes from JAK2V617F+ mice have increased cell surface expression of the α5 subunit of the α5ß1 integrin, the major FN receptor in megakaryocytes, and augmented adhesion to FN compared with wild-type controls. Reducing adhesion to FN by an inhibitory antibody to the α5 subunit effectively reduces the percentage of CD41+ JAK2V617F+ megakaryocytes in vitro and in vivo. Corroborating our findings in mice, JAK2V617F+ megakaryocytes from patients showed elevated expression of α5 subunit, and a neutralizing antibody to α5 subunit reduced adhesion to FN and megakaryocyte number derived from CD34+ cells. Our findings reveal a previously unappreciated contribution of FN-α5ß1 integrin to megakaryocytosis in JAK2V617F+ PMF.
Asunto(s)
Integrina alfa5beta1/fisiología , Megacariocitos/patología , Mielofibrosis Primaria/patología , Animales , Médula Ósea/metabolismo , Adhesión Celular , Células Cultivadas , Matriz Extracelular/metabolismo , Femenino , Humanos , Integrina alfa5/biosíntesis , Integrina alfa5/genética , Integrina alfa5/inmunología , Integrina alfa5beta1/antagonistas & inhibidores , Janus Quinasa 2/genética , Masculino , Megacariocitos/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Mutación Missense , Mielofibrosis Primaria/genéticaRESUMEN
OBJECTIVE: The risk of thrombosis in myeloproliferative neoplasms, such as primary myelofibrosis varies depending on the type of key driving mutation (JAK2 [janus kinase 2], CALR [calreticulin], and MPL [myeloproliferative leukemia protein or thrombopoietin receptor]) and the accompanying mutations in other genes. In the current study, we sought to examine the propensity for thrombosis, as well as platelet activation properties in a mouse model of primary myelofibrosis induced by JAK2V617F (janus kinase 2 with valine to phenylalanine substitution on codon 617) mutation. Approach and Results: Vav1-hJAK2V617F transgenic mice show hallmarks of primary myelofibrosis, including significant megakaryocytosis and bone marrow fibrosis, with a moderate increase in red blood cells and platelet number. This mouse model was used to study responses to 2 models of vascular injury and to investigate platelet properties. Platelets derived from the mutated mice have reduced aggregation in response to collagen, reduced thrombus formation and thrombus size, as demonstrated using laser-induced or FeCl3-induced vascular injury models, and increased bleeding time. Strikingly, the mutated platelets had a significantly reduced number of dense granules, which could explain impaired ADP secretion upon platelet activation, and a diminished second wave of activation. CONCLUSIONS: Together, our study highlights for the first time the influence of a hyperactive JAK2 on platelet activation-induced ADP secretion and dense granule homeostasis, with consequent effects on platelet activation properties.
Asunto(s)
Coagulación Sanguínea , Plaquetas/enzimología , Traumatismos de las Arterias Carótidas/enzimología , Janus Quinasa 2/sangre , Megacariocitos/enzimología , Activación Plaquetaria , Mielofibrosis Primaria/enzimología , Trombosis/enzimología , Animales , Traumatismos de las Arterias Carótidas/sangre , Traumatismos de las Arterias Carótidas/genética , Modelos Animales de Enfermedad , Janus Quinasa 2/genética , Ratones Transgénicos , Mutación , Agregación Plaquetaria , Mielofibrosis Primaria/sangre , Mielofibrosis Primaria/genética , Trombopoyesis , Trombosis/sangre , Trombosis/genéticaRESUMEN
It has been suspected that cell-cycle progression might be functionally coupled with RNA processing. However, little is known about the role of the precise splicing control in cell-cycle progression. Here, we report that SON, a large Ser/Arg (SR)-related protein, is a splicing cofactor contributing to efficient splicing of cell-cycle regulators. Downregulation of SON leads to severe impairment of spindle pole separation, microtubule dynamics, and genome integrity. These molecular defects result from inadequate RNA splicing of a specific set of cell-cycle-related genes that possess weak splice sites. Furthermore, we show that SON facilitates the interaction of SR proteins with RNA polymerase II and other key spliceosome components, suggesting its function in efficient cotranscriptional RNA processing. These results reveal a mechanism for controlling cell-cycle progression through SON-dependent constitutive splicing at suboptimal splice sites, with strong implications for its role in cancer and other human diseases.
Asunto(s)
Ciclo Celular/genética , Proteínas de Unión al ADN/metabolismo , Genes cdc , Empalme del ARN , Empalmosomas/metabolismo , Segregación Cromosómica , Citocinesis , Proteínas de Unión al ADN/genética , Inestabilidad Genómica , Células HEK293 , Células HeLa , Humanos , Células K562 , Microtúbulos/metabolismo , Antígenos de Histocompatibilidad Menor , Interferencia de ARN , ARN Polimerasa II/metabolismo , Huso Acromático/metabolismo , Factores de Tiempo , TransfecciónRESUMEN
Individuals with CKD are particularly predisposed to thrombosis after vascular injury. Using mouse models, we recently described indoxyl sulfate, a tryptophan metabolite retained in CKD and an activator of tissue factor (TF) through aryl hydrocarbon receptor (AHR) signaling, as an inducer of thrombosis across the CKD spectrum. However, the translation of findings from animal models to humans is often challenging. Here, we investigated the uremic solute-AHR-TF thrombosis axis in two human cohorts, using a targeted metabolomics approach to probe a set of tryptophan products and high-throughput assays to measure AHR and TF activity. Analysis of baseline serum samples was performed from 473 participants with advanced CKD from the Dialysis Access Consortium Clopidogrel Prevention of Early AV Fistula Thrombosis trial. Participants with subsequent arteriovenous thrombosis had significantly higher levels of indoxyl sulfate and kynurenine, another uremic solute, and greater activity of AHR and TF, than those without thrombosis. Pattern recognition analysis using the components of the thrombosis axis facilitated clustering of the thrombotic and nonthrombotic groups. We further validated these findings using 377 baseline samples from participants in the Thrombolysis in Myocardial Infarction II trial, many of whom had CKD stage 2-3. Mechanistic probing revealed that kynurenine enhances thrombosis after vascular injury in an animal model and regulates thrombosis in an AHR-dependent manner. This human validation of the solute-AHR-TF axis supports further studies probing its utility in risk stratification of patients with CKD and exploring its role in other diseases with heightened risk of thrombosis.
Asunto(s)
Indicán/sangre , Quinurenina/sangre , Receptores de Hidrocarburo de Aril/sangre , Insuficiencia Renal Crónica/sangre , Tromboplastina/metabolismo , Trombosis/sangre , Lesiones del Sistema Vascular/sangre , Lesiones del Sistema Vascular/complicaciones , Adulto , Anciano , Ensayos Clínicos como Asunto , Femenino , Humanos , Masculino , Metabolómica , Persona de Mediana Edad , Reconocimiento de Normas Patrones Automatizadas , Insuficiencia Renal Crónica/complicaciones , Transducción de Señal , Trombosis/etiología , Uremia/sangre , Uremia/complicacionesRESUMEN
Lysyl oxidase (LOX) is overexpressed in various pathologies associated with thrombosis, such as arterial stenosis and myeloproliferative neoplasms (MPNs). LOX is elevated in the megakaryocytic lineage of mouse models of MPNs and in patients with MPNs. To gain insight into the role of LOX in thrombosis and platelet function without compounding the influences of other pathologies, transgenic mice expressing LOX in wild-type megakaryocytes and platelets (Pf4-Lox(tg/tg)) were generated. Pf4-Lox(tg/tg) mice had a normal number of platelets; however, time to vessel occlusion after endothelial injury was significantly shorter in Pf4-Lox(tg/tg) mice, indicating a higher propensity for thrombus formation in vivo. Exploring underlying mechanisms, we found that Pf4-Lox(tg/tg) platelets adhere better to collagen and have greater aggregation response to lower doses of collagen compared with controls. Platelet activation in response to the ligand for collagen receptor glycoprotein VI (cross-linked collagen-related peptide) was unaffected. However, the higher affinity of Pf4-Lox(tg/tg) platelets to the collagen sequence GFOGER implies that the collagen receptor integrin α2ß1 is affected by LOX. Taken together, our findings demonstrate that LOX enhances platelet activation and thrombosis.
Asunto(s)
Plaquetas/efectos de los fármacos , Colágeno/farmacología , Activación Plaquetaria/fisiología , Proteína-Lisina 6-Oxidasa/fisiología , Trombofilia/enzimología , Animales , Plaquetas/citología , Traumatismos de las Arterias Carótidas/complicaciones , Trombosis de las Arterias Carótidas/etiología , Integrina alfa2beta1/fisiología , Megacariocitos/enzimología , Ratones , Ratones Transgénicos , Fragmentos de Péptidos/farmacología , Adhesividad Plaquetaria/genética , Adhesividad Plaquetaria/fisiología , Agregación Plaquetaria/efectos de los fármacos , Agregación Plaquetaria/genética , Factor Plaquetario 4/genética , Regiones Promotoras Genéticas , Proteína-Lisina 6-Oxidasa/genética , Ratas , Trombofilia/genéticaRESUMEN
RUNX1 is an important transcription factor for hematopoiesis. There are multiple alternatively spliced isoforms of RUNX1. The best known isoforms are RUNX1a from use of exon 7A and RUNX1b and c from use of exon 7B. RUNX1a has unique functions due to its lack of C-terminal regions common to RUNX1b and c. Here, we report that the ortholog of human RUNX1a was only found in primates. Furthermore, we characterized 3 Runx1 isoforms generated by exon 6 alternative splicing. Runx1bEx6(-) (Runx1b without exon 6) and a unique mouse Runx1bEx6e showed higher colony-forming activity than the full-length Runx1b (Runx1bEx6(+)). They also facilitated the transactivation of Runx1bEx6(+). To gain insight into in vivo functions, we analyzed a knock-in (KI) mouse model that lacks isoforms Runx1b/cEx6(-) and Runx1bEx6e. KI mice had significantly fewer lineage-Sca1(+)c-Kit(+) cells, short-term hematopoietic stem cells (HSCs) and multipotent progenitors than controls. In vivo competitive repopulation assays demonstrated a sevenfold difference of functional HSCs between wild-type and KI mice. Together, our results show that Runx1 isoforms involving exon 6 support high self-renewal capacity in vitro, and their loss results in reduction of the HSC pool in vivo, which underscore the importance of fine-tuning RNA splicing in hematopoiesis.
Asunto(s)
Subunidad alfa 2 del Factor de Unión al Sitio Principal/fisiología , Hematopoyesis/genética , Animales , Secuencia de Bases , Células Cultivadas , Subunidad alfa 2 del Factor de Unión al Sitio Principal/genética , Exones , Regulación de la Expresión Génica , Células HEK293 , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Datos de Secuencia Molecular , Isoformas de Proteínas/genética , Isoformas de Proteínas/fisiología , Sitios de Empalme de ARN , Homología de SecuenciaRESUMEN
RUNX1 is a master transcription factor in hematopoiesis and mediates the specification and homeostasis of hematopoietic stem and progenitor cells (HSPCs). Disruptions in RUNX1 are well known to lead to hematologic disease. In this study, we sought to identify and characterize RUNX1 target genes in HSPCs by performing RUNX1 chromatin immunoprecipitation with high-throughput sequencing (ChIP-seq) using a murine HSPC line and complementing this data with our previously described gene expression profiling of primary wild-type and RUNX1-deficient HSPCs (Lineage(-)/cKit(+)/Sca1(+)). From this analysis, we identified and confirmed that Hmga2, a known oncogene, as a direct target of RUNX1. Hmga2 was strongly upregulated in RUNX1-deficient HSPCs, and the promoter of Hmga2 was responsive in a cell-type dependent manner upon coexpression of RUNX1. Conditional Runx1 knockout mice exhibit expansion of their HSPCs and myeloid progenitors as hallmark phenotypes. To further validate and establish that Hmga2 plays a role in inducing HSPC expansion, we generated mouse models of HMGA2 and RUNX1 deficiency. Although mice lacking both factors continued to display higher frequencies of HSPCs, the expansion of myeloid progenitors was effectively rescued. The data presented here establish Hmga2 as a transcriptional target of RUNX1 and a critical regulator of myeloid progenitor expansion.
Asunto(s)
Subunidad alfa 2 del Factor de Unión al Sitio Principal/metabolismo , Regulación de la Expresión Génica , Proteína HMGA2/metabolismo , Células Progenitoras Mieloides/citología , Animales , Sitios de Unión , Línea Celular , Hematopoyesis/fisiología , Células Madre Hematopoyéticas/metabolismo , Humanos , Células Jurkat , Células K562 , Ratones , Ratones Noqueados , Ratones Transgénicos , Células 3T3 NIH , Fenotipo , Factores de Transcripción/metabolismo , Regulación hacia ArribaRESUMEN
Throughout the plant and animal kingdoms specific cell types become polyploid, increasing their DNA content to attain a large cell size. In mammals, megakaryocytes (MKs) become polyploid before fragmenting into platelets. The mammalian trophoblast giant cells (TGCs) exploit their size to form a barrier between the maternal and embryonic tissues. The mechanism of polyploidization has been investigated extensively in Drosophila, in which a modified cell cycle--the endocycle, consisting solely of alternating S and gap phases--produces polyploid tissues. During S phase in the Drosophila endocycle, heterochromatin and specific euchromatic regions are underreplicated and reduced in copy number. Here we investigate the properties of polyploidization in murine MKs and TGCs. We induced differentiation of primary MKs and directly microdissected TGCs from embryonic day 9.5 implantation sites. The copy number across the genome was analyzed by array-based comparative genome hybridization. In striking contrast to Drosophila, the genome was uniformly and integrally duplicated in both MKs and TGCs. This was true even for heterochromatic regions analyzed by quantitative PCR. Underreplication of specific regions in polyploid cells is proposed to be due to a slower S phase, resulting from low expression of S-phase genes, causing failure to duplicate late replicating genomic intervals. We defined the transcriptome of TGCs and found robust expression of S-phase genes. Similarly, S-phase gene expression is not repressed in MKs, providing an explanation for the distinct endoreplication parameters compared with Drosophila. Consistent with TGCs endocycling rather than undergoing endomitosis, they have low expression of M-phase genes.
Asunto(s)
Células Gigantes/citología , Megacariocitos/citología , Poliploidía , Fase S/fisiología , Trofoblastos/citología , Animales , Diferenciación Celular/fisiología , Hibridación Genómica Comparativa , Variaciones en el Número de Copia de ADN , Cartilla de ADN/genética , Perfilación de la Expresión Génica , Regulación de la Expresión Génica/fisiología , Heterocromatina/metabolismo , Ratones , Microdisección , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
SON is a DNA- and RNA-binding protein localized in nuclear speckles. Although its function in RNA splicing for effective cell cycle progression and genome stability was recently unveiled, other mechanisms of SON functions remain unexplored. Here, we report that SON regulates GATA-2, a key transcription factor involved in hematopoietic stem cell maintenance and differentiation. SON is highly expressed in undifferentiated hematopoietic stem/progenitor cells and leukemic blasts. SON knockdown leads to significant depletion of GATA-2 protein with marginal down-regulation of GATA-2 mRNA. We show that miR-27a is up-regulated upon SON knockdown and targets the 3'-UTR of GATA-2 mRNA in hematopoietic cells. Up-regulation of miR-27a was due to activation of the promoter of the miR-23aâ¼27aâ¼24-2 cluster, suggesting that SON suppresses this promoter to lower the microRNAs from this cluster. Our data revealed a previously unidentified role of SON in microRNA production via regulating the transcription process, thereby modulating GATA-2 at the protein level during hematopoietic differentiation.
Asunto(s)
Proteínas de Unión al ADN/fisiología , Factor de Transcripción GATA2/metabolismo , Regulación de la Expresión Génica , MicroARNs/metabolismo , Regiones no Traducidas 3' , Animales , Diferenciación Celular , Proteínas de Unión al ADN/genética , Hematopoyesis , Humanos , Células K562 , Leucemia Mieloide Aguda/metabolismo , Ratones , Ratones Endogámicos C57BL , Antígenos de Histocompatibilidad Menor , Modelos Biológicos , Regiones Promotoras Genéticas , Empalme del ARN , ARN Mensajero/metabolismo , Células U937 , Regulación hacia ArribaRESUMEN
Megakaryocytes (MKs), the platelet precursors, are capable of accumulating DNA greater than a diploid content as part of their cell cycle. MKs have been recognized as mediating fibrosis in a subset of hematologic malignancies, including acute megakaryoblastic leukemia and a subset of myeloproliferative neoplasms. The mechanisms responsible for fibrosis remain only partially understood. Past studies highlighted the role of growth factors in such pathologies, and recently, the protein lysyl oxidase (LOX) has been implicated in proliferation of MKs, ploidy and deposition of fibers. LOX was initially characterized as a protein responsible for the intermolecular cross-linking of elastin and collagen, and in recent years it has been identified as regulator of various pathologies, such as cancer and inflammation. Here, we review recent advances in the understanding of the contribution of MKs to the progression of myelofibrosis, highlighting the newly identified role of LOX.
Asunto(s)
Megacariocitos/enzimología , Mielofibrosis Primaria/enzimología , Proteína-Lisina 6-Oxidasa/metabolismo , Animales , Médula Ósea/enzimología , Médula Ósea/patología , Humanos , Leucemia Megacarioblástica Aguda/enzimología , Leucemia Megacarioblástica Aguda/patología , Megacariocitos/patología , Modelos Biológicos , Trastornos Mieloproliferativos/enzimología , Trastornos Mieloproliferativos/patología , Mielofibrosis Primaria/patologíaAsunto(s)
Plaquetas/enzimología , Megacariocitos/enzimología , Adhesividad Plaquetaria/fisiología , Mielofibrosis Primaria/enzimología , Proteína-Lisina 6-Oxidasa/fisiología , Aminopropionitrilo/farmacología , Células Cultivadas , Colágeno Tipo I/química , Colágeno Tipo I/farmacología , Inducción Enzimática , Colágenos Asociados a Fibrillas/farmacología , Humanos , Adhesividad Plaquetaria/efectos de los fármacos , Mielofibrosis Primaria/sangre , Mielofibrosis Primaria/patología , Proteína-Lisina 6-Oxidasa/antagonistas & inhibidores , Proteína-Lisina 6-Oxidasa/biosíntesis , Regulación hacia ArribaRESUMEN
The t(8;21)(q22;q22) is common in adult acute myeloid leukemia (AML). The RUNX1-ETO fusion protein that is expressed by this translocation is poorly leukemogenic and requires additional mutations for transformation. Loss of sex chromosome (LOS) is frequently observed in t(8;21) AML. In the present study, to evaluate whether LOS cooperates with t(8;21) in leukemogenesis, we first used a retroviral transduction/transplantation model to express RUNX1-ETO in hematopoietic cells from XO mice. The low frequency of leukemia in these mice suggests that the potentially critical gene for suppression of t(8;21) leukemia in humans is not conserved on mouse sex chromosomes. The gene encoding the GM-CSF receptor α subunit (CSF2RA) is located on X and Y chromosomes in humans but on chromosome 19 in mice. GM-CSF promotes myeloid cell survival, proliferation, and differentiation. To determine whether GM-CSF signaling affects RUNX1-ETO leukemogenesis, hematopoietic stem/progenitor cells that lack GM-CSF signaling were used to express RUNX1-ETO and transplanted into lethally irradiated mice, and a high penetrance of AML was observed in recipients. Furthermore, GM-CSF reduced the replating ability of RUNX1-ETO-expressing cells. These results suggest a possible tumor-suppressor role of GM-CSF in RUNX1-ETO leukemia. Loss of the CSF2RA gene may be a critical mutation explaining the high incidence of LOS associated with the t(8;21)(q22;q22) translocation.
Asunto(s)
Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Factor Estimulante de Colonias de Granulocitos y Macrófagos/farmacología , Leucemia Mieloide Aguda/genética , Transducción de Señal/fisiología , Translocación Genética , Adulto , Animales , Células Cultivadas , Cromosomas Humanos Par 21/genética , Cromosomas Humanos Par 8/genética , Cromosomas de los Mamíferos/genética , Subunidad alfa 2 del Factor de Unión al Sitio Principal/genética , Proteínas de Unión al ADN/genética , Factor Estimulante de Colonias de Granulocitos y Macrófagos/genética , Factor Estimulante de Colonias de Granulocitos y Macrófagos/fisiología , Humanos , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Proteínas de Fusión Oncogénica/genética , Proteínas Proto-Oncogénicas/genética , Receptores de Factor Estimulante de Colonias de Granulocitos y Macrófagos/genética , Receptores de Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Receptores de Factor Estimulante de Colonias de Granulocitos y Macrófagos/fisiología , Cromosomas Sexuales/genética , Cromosomas Sexuales/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Factores de Transcripción/genéticaRESUMEN
Fusion protein AML1-ETO, resulting from t(8;21) translocation, is highly related to leukemia development. It has been reported that full-length AML1-ETO blocks AML1 function and requires additional mutagenic events to promote leukemia. We have previously shown that the expression of AE9a, a splice isoform of AML1-ETO, can rapidly cause leukemia in mice. To understand how AML1-ETO is involved in leukemia development, we took advantage of our AE9a leukemia model and sought to identify its interacting proteins from primary leukemic cells. Here, we report the discovery of a novel AE9a binding partner PRMT1 (protein arginine methyltransferase 1). PRMT1 not only interacts with but also weakly methylates arginine 142 of AE9a. Knockdown of PRMT1 affects expression of a specific group of AE9a-activated genes. We also show that AE9a recruits PRMT1 to promoters of AE9a-activated genes, resulting in enrichment of H4 arginine 3 methylation, H3 Lys9/14 acetylation, and transcription activation. More importantly, knockdown of PRMT1 suppresses the self-renewal capability of AE9a, suggesting a potential role of PRMT1 in regulating leukemia development.
Asunto(s)
Proliferación Celular , Subunidad alfa 2 del Factor de Unión al Sitio Principal/metabolismo , Proteínas de Fusión Oncogénica/metabolismo , Proteína-Arginina N-Metiltransferasas/metabolismo , Proteínas Represoras/metabolismo , Células Madre/fisiología , Activación Transcripcional , Animales , Células Cultivadas , Subunidad alfa 2 del Factor de Unión al Sitio Principal/fisiología , Perfilación de la Expresión Génica , Regulación Leucémica de la Expresión Génica , Células HEK293 , Humanos , Células K562 , Ratones , Análisis por Micromatrices , Proteínas de Fusión Oncogénica/fisiología , Unión Proteica/fisiología , Proteína 1 Compañera de Translocación de RUNX1 , Células Madre/metabolismo , Activación Transcripcional/genética , Regulación hacia Arriba/genética , Regulación hacia Arriba/fisiologíaRESUMEN
Mutations of RUNX1 are detected in patients with myelodysplastic syndrome (MDS). In particular, C-terminal truncation mutations lack a transcription regulatory domain and have increased DNA binding through the runt homology domain. The expression of the runt homology domain, RUNX1(41-214), in mouse hematopoietic cells induced progression to MDS and acute myeloid leukemia. Analysis of premyelodysplastic animals found expansion of c-Kit(+)Sca-1(+)Lin(-) cells and skewed differentiation to myeloid at the expense of the lymphoid lineage. These abnormalities correlate with the phenotype of Runx1-deficient animals, as expected given the reported dominant-negative role of C-terminal mutations over the full-length RUNX1. However, MDS is not observed in Runx1-deficient animals. Gene expression profiling found that RUNX1(41-214) c-Kit(+)Sca-1(+)Lin(-) cells have an overlapping yet distinct gene expression profile from Runx1-deficient animals. Moreover, an unexpected parallel was observed between the hematopoietic phenotype of RUNX1(41-214) and aged animals. Genes deregulated in RUNX1(41-214), but not in Runx1-deficient animals, were inversely correlated with the aging gene signature of HSCs, suggesting that disruption of the expression of genes related to normal aging by RUNX1 mutations contributes to development of MDS. The data presented here provide insights into the mechanisms of development of MDS in HSCs by C-terminal mutations of RUNX1.
Asunto(s)
Transformación Celular Neoplásica/genética , Subunidad alfa 2 del Factor de Unión al Sitio Principal/genética , Regulación de la Expresión Génica , Células Madre Hematopoyéticas/metabolismo , Síndromes Mielodisplásicos/genética , Síndromes Mielodisplásicos/metabolismo , Animales , Apoptosis/genética , Médula Ósea/patología , Ciclo Celular/genética , Línea Celular , Análisis por Conglomerados , Perfilación de la Expresión Génica , Hematopoyesis/genética , Trasplante de Células Madre Hematopoyéticas , Homeostasis/genética , Humanos , Leucemia Experimental , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Ratones , Transducción GenéticaRESUMEN
Chromosome translocation 8q22;21q22 [t(8;21)] is commonly associated with acute myeloid leukemia (AML), and the resulting AML1-ETO fusion proteins are involved in the pathogenesis of AML. To identify novel molecular and therapeutic targets, we performed combined gene expression microarray and promoter occupancy (ChIP-chip) profiling using Lin(-)/Sca1(-)/cKit(+) cells, the major leukemia cell population, from an AML mouse model induced by AML1-ETO9a (AE9a). Approximately 30% of the identified common targets of microarray and ChIP-chip assays overlap with the human t(8;21)-gene expression molecular signature. CD45, a protein tyrosine phosphatase and a negative regulator of cytokine/growth factor receptor and JAK/STAT signaling, is among those targets. Its expression is substantially down-regulated in leukemia cells. Consequently, JAK/STAT signaling is enhanced. Re-expression of CD45 suppresses JAK/STAT activation, delays leukemia development, and promotes apoptosis of t(8;21)-positive cells. This study demonstrates the benefit of combining gene expression and promoter occupancy profiling assays to identify molecular and potential therapeutic targets in human cancers and describes a previously unappreciated signaling pathway involving t(8;21) fusion proteins, CD45, and JAK/STAT, which could be a potential novel target for treating t(8;21) AML.
Asunto(s)
Cromosomas Humanos Par 21/genética , Cromosomas Humanos Par 8/genética , ADN de Neoplasias/metabolismo , Perfilación de la Expresión Génica , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/genética , Translocación Genética , Animales , Antineoplásicos/uso terapéutico , Línea Celular Tumoral , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/patología , Inmunoprecipitación de Cromatina , Activación Enzimática , Regulación Leucémica de la Expresión Génica , Redes Reguladoras de Genes/genética , Genes Relacionados con las Neoplasias/genética , Humanos , Quinasas Janus/metabolismo , Antígenos Comunes de Leucocito/metabolismo , Ratones , Análisis de Secuencia por Matrices de Oligonucleótidos , Regiones Promotoras Genéticas/genética , Reproducibilidad de los Resultados , Factores de Transcripción STAT/metabolismo , Transducción de Señal/genéticaRESUMEN
Ubiquitin specific protease 18 (USP18) serves as a potent inhibitor of Type I interferon (IFN) signaling. Previous studies have shown that Usp18 deficient (homozygous Usp18 gene knockout) mice exhibit hydrocephalus; however, the precise molecular mechanism underlying hydrocephalus development remains elusive. In this study, we demonstrate that mice lacking both type I IFN receptor subunit 1 (Ifnar1) and Usp18 (Ifnar1/Usp18 double knockout mice) are viable and do not display a hydrocephalus phenotype. Moreover, we observed that suppression of USP18 in ependymal cells treated with IFN significantly increased cell death, including pyroptosis, and decreased proliferation. These findings suggest that heightened sensitivity to type I IFN during brain development contributes to the onset of hydrocephalus. Furthermore, they imply that inhibition of IFN signaling may hold promise as a therapeutic strategy for hydrocephalus.
Asunto(s)
Hidrocefalia , Interferón Tipo I , Ratones Noqueados , Receptor de Interferón alfa y beta , Ubiquitina Tiolesterasa , Animales , Hidrocefalia/genética , Hidrocefalia/patología , Interferón Tipo I/metabolismo , Ratones , Ubiquitina Tiolesterasa/genética , Ubiquitina Tiolesterasa/metabolismo , Receptor de Interferón alfa y beta/genética , Receptor de Interferón alfa y beta/metabolismo , Ratones Endogámicos C57BL , Transducción de Señal/efectos de los fármacos , Encéfalo/metabolismo , Encéfalo/patología , Encéfalo/efectos de los fármacos , Epéndimo/metabolismo , Proliferación Celular/efectos de los fármacos , Piroptosis/efectos de los fármacos , Piroptosis/fisiologíaRESUMEN
Reactive oxygen species (ROS), generated as a result of various reactions, control an array of cellular processes. The role of ROS during megakaryocyte (MK) development has been a subject of interest and research. The bone marrow niche is a site of MK differentiation and maturation. In this environment, a gradient of oxygen tension, from normoxia to hypoxia results in different levels of ROS, impacting cellular physiology. This article provides an overview of major sources of ROS, their implication in different signaling pathways, and their effect on cellular physiology, with a focus on megakaryopoiesis. The importance of ROS-generating oxidases in MK biology and pathology, including myelofibrosis, is also described.
Asunto(s)
Hematopoyesis/fisiología , Megacariocitos/metabolismo , Oxidorreductasas/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Animales , Humanos , Megacariocitos/citologíaRESUMEN
Mature megakaryocytes, the platelet precursors, originate from hematopoietic stem cell progenitors, which, once committed to this lineage, undergo endomitosis leading to polyploidization. The process entails repeated rounds of DNA replication without cell division, yielding polyploid cells. Supporting the cell's developmental process and various cellular functions are integrin receptors, a conduit of communication between the extracellular environment and the cell actin cytoskeleton. Integrins are heterodimers of α and ß subunits, where different combinations of the known 18 α and 8 ß subunits confer specificity to the receptor. Integrin ligands range from extracellular matrices through soluble ligands, infectious agents, and counterreceptors, to cells. In this review, we describe the different integrins expressed on bone marrow megakaryocytes and their attributed roles in lineage development and cellular functions, including adhesion, spreading, proplatelet formation, and functional interaction with other cells. Pathologies associated with dysregulated megakaryocyte integrin expression are also reviewed.