RESUMEN
The continuity of life depends on mechanisms in the germline that ensure the integrity of the genome. The DNA damage response/checkpoint kinases ATM and ATR are essential signaling factors in the germline. However, it remains unknown how a downstream transducer, Checkpoint Kinase 1 (CHEK1 or CHK1), mediates signaling in the male germline. Here, we show that CHEK1 has distinct functions in both the mitotic and meiotic phases of the male germline in mice. In the mitotic phase, CHEK1 is required for the resumption of prospermatogonia proliferation after birth and the maintenance of spermatogonia. In the meiotic phase, we uncovered two functions for CHEK1: one is the stage-specific attenuation of DNA damage signaling on autosomes, and the other is coordination of meiotic stage progression. On autosomes, the loss of CHEK1 delays the removal of DNA damage signaling that manifests as phosphorylation of histone variant H2AX at serine 139 (γH2AX). Importantly, CHEK1 does not have a direct function in meiotic sex chromosome inactivation (MSCI), an essential event in male meiosis, in which ATR is a key regulator. Thus, the functions of ATR and CHEK1 are uncoupled in MSCI, in contrast to their roles in DNA damage signaling in somatic cells. Our study reveals stage-speciï¬c functions for CHEK1 that ensure the integrity of the male germline.
Asunto(s)
Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1)/metabolismo , Daño del ADN , Meiosis , Transducción de Señal , Espermatogonias/metabolismo , Animales , Proteínas de la Ataxia Telangiectasia Mutada/genética , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1)/genética , Masculino , Ratones , Ratones Noqueados , Espermatogonias/citologíaRESUMEN
Fanconi anemia (FA) is characterized by a progressive bone marrow failure and an increased incidence of cancer. FA patients have high susceptibility to immune-related complications such as infection and posttransplant graft-versus-host disease. In this study, we investigated the effect of FA deficiency in B cell function using the Fancc mouse model. Fancc(-/-) B cells show a specific defect in IgG2a switch and impaired Ab-secreting cell (ASC) differentiation. Global transcriptome analysis of naive B cells by mRNA sequencing demonstrates that FA deficiency deregulates a network of genes involved in immune function. Significantly, many genes implicated in Wnt signaling were aberrantly expressed in Fancc(-/-) B cells. Consistently, Fancc(-/-) B cells accumulate high levels of ß-catenin under both resting and stimulated conditions, suggesting hyperactive Wnt signaling. Using an in vivo Wnt GFP reporter assay, we verified the upregulation of Wnt signaling as a potential mechanism responsible for the impaired Fancc(-/-) B cell differentiation. Furthermore, we showed that Wnt signaling inhibits ASC differentiation possibly through repression of Blimp1 and that Fancc(-/-) B cells are hypersensitive to Wnt activation during ASC differentiation. Our findings identify Wnt signaling as a physiological regulator of ASC differentiation and establish a role for the Wnt pathway in normal B cell function and FA immune deficiency.
Asunto(s)
Células Productoras de Anticuerpos/citología , Células Productoras de Anticuerpos/metabolismo , Diferenciación Celular/genética , Diferenciación Celular/inmunología , Proteína del Grupo de Complementación C de la Anemia de Fanconi/deficiencia , Vía de Señalización Wnt , Animales , Células Productoras de Anticuerpos/inmunología , Linfocitos B/citología , Linfocitos B/inmunología , Linfocitos B/metabolismo , Análisis por Conglomerados , Perfilación de la Expresión Génica , Cambio de Clase de Inmunoglobulina/genética , Cambio de Clase de Inmunoglobulina/inmunología , Inmunoglobulina G/genética , Inmunoglobulina G/inmunología , Ratones , Ratones Noqueados , Transcriptoma , Proteínas Wnt/metabolismoRESUMEN
Fanconi anemia (FA) is a recessive X-linked and autosomal genetic disease associated with bone marrow failure and increased cancer, as well as severe germline defects such as hypogonadism and germ cell depletion. Although deficiencies in FA factors are commonly associated with germ cell defects, it remains unknown whether the FA pathway is involved in unique epigenetic events in germ cells. In this study, we generated Fancb mutant mice, the first mouse model of X-linked FA, and identified a novel function of the FA pathway in epigenetic regulation during mammalian gametogenesis. Fancb mutant mice were infertile and exhibited primordial germ cell (PGC) defects during embryogenesis. Further, Fancb mutation resulted in the reduction of undifferentiated spermatogonia in spermatogenesis, suggesting that FANCB regulates the maintenance of undifferentiated spermatogonia. Additionally, based on functional studies, we dissected the pathway in which FANCB functions during meiosis. The localization of FANCB on sex chromosomes is dependent on MDC1, a binding partner of H2AX phosphorylated at serine 139 (γH2AX), which initiates chromosome-wide silencing. Also, FANCB is required for FANCD2 localization during meiosis, suggesting that the role of FANCB in the activation of the FA pathway is common to both meiosis and somatic DNA damage responses. H3K9me2, a silent epigenetic mark, was decreased on sex chromosomes, whereas H3K9me3 was increased on sex chromosomes in Fancb mutant spermatocytes. Taken together, these results indicate that FANCB functions at critical stages of germ cell development and reveal a novel function of the FA pathway in the regulation of H3K9 methylation in the germline.
Asunto(s)
Proteínas del Grupo de Complementación de la Anemia de Fanconi/genética , Genes Esenciales , Células Germinativas/metabolismo , Histonas/metabolismo , Meiosis/genética , Cromosomas Sexuales , Proteínas Adaptadoras Transductoras de Señales , Animales , Proteínas de Ciclo Celular , Diferenciación Celular/genética , Epigénesis Genética , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/genética , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/metabolismo , Proteínas del Grupo de Complementación de la Anemia de Fanconi/metabolismo , Femenino , Fertilidad/genética , Regulación de la Expresión Génica , Sitios Genéticos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Masculino , Metilación , Ratones , Ratones Noqueados , Modelos Biológicos , Mutación , Transporte de Proteínas , Recombinación Genética , Espermatogonias/citología , Espermatogonias/metabolismoRESUMEN
Fanconi anemia (FA) is an inherited bone marrow (BM) failure syndrome, presumably resulting from defects in hematopoietic stem cells (HSCs). Normal HSCs depend more on glycolysis than on oxidative phosphorylation (OXPHOS) for energy production. Here, we show that FA HSCs are more sensitive to the respiration inhibitor NaN3 treatment than to glycolytic inhibitor 2-deoxy-d-glucose (2-DG), indicating more dependence on OXPHOS. FA HSCs undergo glycolysis-to-OXPHOS switch in response to oxidative stress through a p53-dependent mechanism. Metabolic stresses induce upregulation of p53 metabolic targets in FA HSCs. Inactivation of p53 in FA HSCs prevents glycolysis-to-OXPHOS switch. Furthermore, p53-deficient FA HSCs are more sensitive to 2-DG-mediated metabolic stress. Finally, oxidative stress-induced glycolysis-to-OXPHOS switch is mediated by synthesis of cytochrome c oxidase 2 (SCO2). These findings demonstrate p53-mediated OXPHOS function as a compensatory alteration in FA HSCs to ensure a functional but mildly impaired energy metabolism and suggest a cautious approach to manipulating p53 signaling in FA.
Asunto(s)
Proteínas Portadoras/biosíntesis , Anemia de Fanconi/metabolismo , Células Madre Hematopoyéticas/metabolismo , Proteínas Mitocondriales/biosíntesis , Proteína p53 Supresora de Tumor/genética , Apoptosis/efectos de los fármacos , Proteínas Portadoras/genética , Desoxiglucosa/administración & dosificación , Metabolismo Energético , Anemia de Fanconi/genética , Anemia de Fanconi/patología , Glucólisis/efectos de los fármacos , Células Madre Hematopoyéticas/patología , Humanos , Proteínas Mitocondriales/genética , Chaperonas Moleculares , Fosforilación Oxidativa/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Azida Sódica/administración & dosificación , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
Functional maintenance of hematopoietic stem cells (HSCs) is constantly challenged by stresses like DNA damage and oxidative stress. Here we show that the Fanconi anemia protein Fancd2 and stress transcriptional factor Foxo3a cooperate to prevent HSC exhaustion in mice. Deletion of both Fancd2 and Foxo3a led to an initial expansion followed by a progressive decline of bone marrow stem and progenitor cells. Limiting dilution transplantation and competitive repopulating experiments demonstrated a dramatic reduction of competitive repopulating units and progressive decline in hematopoietic repopulating ability of double-knockout (dKO) HSCs. Analysis of the transcriptome of dKO HSCs revealed perturbation of multiple pathways implicated in HSC exhaustion. Fancd2 deficiency strongly promoted cytoplasmic localization of Foxo3a in HSCs, and re-expression of Fancd2 completely restored nuclear Foxo3a localization. By co-expressing a constitutively active CA-FOXO3a and WT or a nonubiquitinated Fancd2 in dKO bone marrow stem/progenitor cells, we demonstrated that Fancd2 was required for nuclear retention of CA-FOXO3a and for maintaining hematopoietic repopulation of the HSCs. Collectively, these results implicate a functional interaction between the Fanconi anemia DNA repair and FOXO3a pathways in HSC maintenance.
Asunto(s)
Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/metabolismo , Factores de Transcripción Forkhead/metabolismo , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , Animales , Ciclo Celular/fisiología , Daño del ADN/fisiología , Reparación del ADN/fisiología , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/genética , Fémur/metabolismo , Proteína Forkhead Box O3 , Factores de Transcripción Forkhead/genética , Humanos , Ratones , Ratones Noqueados , Estrés Oxidativo/fisiología , Tibia/metabolismoRESUMEN
Fanconi anemia (FA) is a genetic disorder associated with bone marrow (BM) failure and leukemia. Recent studies demonstrate variable immune defects in FA. However, the cause for FA immunodeficiency is unknown. Here we report that deletion of Fanca or Fancd2 dysregulates the suppressive activity of regulatory T cells (Tregs), shown functionally as exacerbation of graft-vs-host disease (GVHD) in mice. Recipient mice of Fanca(-/-) or Fancd2(-/-) BM chimeras exhibited severe acute GVHD after allogeneic BM transplantation (BMT). T cells from Fanca(-/-) or Fancd2(-/-) mice induced higher GVHD lethality than those from wild-type (WT) littermates. FA Tregs possessed lower proliferative suppression potential compared with WT Tregs, as demonstrated by in vitro proliferation assay and BMT. Analysis of CD25(+)Foxp3(+) Tregs indicated that loss of Fanca or Fancd2 dysregulated Foxp3 target gene expression. Additionally, CD25(+)Foxp3(+) Tregs of Fanca(-/-) or Fancd2(-/-) mice were less efficient in suppressing the production of GVHD-associated inflammatory cytokines. Consistently, aberrant NF-κB activity was observed in infiltrated T cells from FA GVHD mice. Conditional deletion of p65 in FA Tregs decreased GVHD mortality. Our study uncovers an essential role for FA proteins in maintaining Treg homeostasis, possibly explaining, at least in part, the immune deficiency reported in some FA patients.
Asunto(s)
Proteína del Grupo de Complementación A de la Anemia de Fanconi/deficiencia , Proteína del Grupo de Complementación A de la Anemia de Fanconi/inmunología , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/deficiencia , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/inmunología , Linfocitos T Reguladores/inmunología , Animales , Trasplante de Médula Ósea , Citocinas/biosíntesis , Anemia de Fanconi/genética , Anemia de Fanconi/inmunología , Anemia de Fanconi/metabolismo , Proteína del Grupo de Complementación A de la Anemia de Fanconi/genética , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/genética , Factores de Transcripción Forkhead/metabolismo , Expresión Génica , Enfermedad Injerto contra Huésped/inmunología , Enfermedad Injerto contra Huésped/patología , Humanos , Tolerancia Inmunológica , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Linfocitos T Reguladores/metabolismo , Quimera por TrasplanteRESUMEN
Fanconi anemia (FA) patients develop bone marrow (BM) failure or leukemia. One standard care for these devastating complications is hematopoietic stem cell transplantation. We identified a group of mesenchymal stromal cells (MSCs)-derived metabolites, glycerophospholipids, and their endogenous inhibitor, 5-(tetradecyloxy)-2-furoic acid (TOFA), as regulators of donor hematopoietic stem and progenitor cells. We provided two pieces of evidence that TOFA could improve hematopoiesis-supporting function of FA MSCs: (a) limiting-dilution cobblestone area-forming cell assay revealed that TOFA significantly increased cobblestone colonies in Fanca-/- or Fancd2-/- cocultures compared to untreated cocultures. (b) Competitive repopulating assay using output cells collected from cocultures showed that TOFA greatly alleviated the abnormal expansion of the donor myeloid (CD45.2+Gr1+Mac1+) compartment in both peripheral blood and BM of recipient mice transplanted with cells from Fanca-/- or Fancd2-/- cocultures. Furthermore, mechanistic studies identified Tlr4 signaling as the responsible pathway mediating the effect of glycerophospholipids. Thus, targeting glycerophospholipid biosynthesis in FA MSCs could be a therapeutic strategy to improve hematopoiesis and stem cell transplantation.
Asunto(s)
Diferenciación Celular/fisiología , Proteína del Grupo de Complementación A de la Anemia de Fanconi/deficiencia , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/deficiencia , Glicerofosfolípidos/biosíntesis , Células Madre Hematopoyéticas/metabolismo , Receptor Toll-Like 4/metabolismo , Animales , Células Cultivadas , Técnicas de Cocultivo , Anemia de Fanconi/genética , Proteína del Grupo de Complementación A de la Anemia de Fanconi/genética , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/genética , Células Madre Mesenquimatosas/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Transducción de Señal/fisiologíaRESUMEN
20-kDa FANCA-associated protein (FAAP20) is a recently identified protein that associates with the Fanconi anemia (FA) core complex component, FANCA. FAAP20 contains a conserved ubiquitin-binding zinc-finger domain and plays critical roles in the FA-BRCA pathway of DNA repair and genome maintenance. The function of FAAP20 in animals has not been explored. Here, we report that deletion of Faap20 in mice led to a mild FA-like phenotype with defects in the reproductive and hematopoietic systems. Specifically, hematopoietic stem and progenitor cells (HSPCs) from Faap20(-) (/) (-) mice showed defects in long-term multilineage reconstitution in lethally irradiated recipient mice, with milder phenotype as compared to HSPCs from Fanca(-) (/) (-) or Fancc(-) (/) (-) mice. Faap20(-) (/) (-) mice are susceptible to mitomycin C (MMC)-induced pancytopenia. That is, acute MMC stress induced a significant progenitor loss especially the erythroid progenitors and megakaryocyte-erythrocyte progenitors in Faap20(-) (/) (-) mice. Furthermore, Faap20(-) (/) (-) HSPCs displayed aberrant cell cycle pattern during chronic MMC treatment. Finally, using Faap20(-) (/) (-) Fanca(-) (/) (-) double-knockout mice, we demonstrated a possible dominant effect of FANCA in the interaction between FAAP20 and FANCA. This novel Faap20 mouse model may be valuable in studying the regulation of the FA pathway during bone marrow failure progress in FA patients.
Asunto(s)
Aminoacil-ARNt Sintetasas/metabolismo , Anemia de Fanconi/genética , Anemia de Fanconi/metabolismo , Células Madre Hematopoyéticas/metabolismo , Aminoacil-ARNt Sintetasas/genética , Animales , Diferenciación Celular , Daño del ADN , Anemia de Fanconi/mortalidad , Humanos , Ratones , Ratones Endogámicos C57BL , Células Madre , Análisis de SupervivenciaAsunto(s)
Proliferación Celular/fisiología , Elementos Transponibles de ADN/genética , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/metabolismo , Células Germinativas/metabolismo , Animales , Apoptosis/fisiología , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/genética , Ratones , Ratones TransgénicosRESUMEN
Hematopoietic stem cells (HSCs) can either self-renew or differentiate into various types of cells of the blood lineage. Signaling pathways that regulate this choice of self-renewal versus differentiation are currently under extensive investigation. In this study, we report that deregulation of Notch signaling skews HSC differentiation in mouse models of Fanconi anemia (FA), a genetic disorder associated with bone marrow failure and progression to leukemia and other cancers. In mice expressing a transgenic Notch reporter, deletion of the Fanca or Fancc gene enhances Notch signaling in multipotential progenitors (MPPs), which is correlated with decreased phenotypic long-term HSCs and increased formation of MPP1 progenitors. Furthermore, we found an inverse correlation between Notch signaling and self-renewal capacity in FA hematopoietic stem and progenitor cells. Significantly, FA deficiency in MPPs deregulates a complex network of genes in the Notch and canonical NF-κB pathways. Genetic ablation or pharmacologic inhibition of NF-κB reduces Notch signaling in FA MPPs to near wild type level, and blocking either NF-κB or Notch signaling partially restores FA HSC quiescence and self-renewal capacity. These results suggest a functional crosstalk between Notch signaling and NF-κB pathway in regulation of HSC differentiation.
Asunto(s)
Diferenciación Celular/fisiología , Anemia de Fanconi/metabolismo , Células Madre Hematopoyéticas/metabolismo , FN-kappa B/metabolismo , Receptores Notch/metabolismo , Transducción de Señal/fisiología , Animales , Modelos Animales de Enfermedad , Citometría de Flujo , Immunoblotting , Inflamación/metabolismo , Ratones , Ratones Noqueados , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptor Cross-Talk/fisiologíaRESUMEN
Tumor necrosis factor-alpha (TNF-α) is a major pro-inflammatory cytokine involved in systemic inflammation and the acute phase reaction. Dysregulation of TNF production has been implicated in a variety of human diseases including Fanconi anemia (FA). FA is a genomic instability syndrome characterized by progressive bone marrow failure and cancer susceptibility. The patients with FA are often found overproducing TNF-α, which may directly affect hematopoietic stem cell (HSC) function by impairing HSC survival, homing and proliferation, or indirectly change the bone marrow microenvironment critical for HSC homeostasis and function, therefore contributing to disease progression in FA. In this brief review, we discuss the link between TNF-α signaling and FA pathway with emphasis on the implication of inflammation in the pathophysiology and abnormal hematopoiesis in FA.
Asunto(s)
Médula Ósea/patología , Proteínas del Grupo de Complementación de la Anemia de Fanconi/genética , Anemia de Fanconi/genética , Células Madre Hematopoyéticas/patología , Transducción de Señal/genética , Factor de Necrosis Tumoral alfa/genética , Apoptosis , Médula Ósea/inmunología , Proliferación Celular , Supervivencia Celular , Transformación Celular Neoplásica/inmunología , Transformación Celular Neoplásica/patología , Anemia de Fanconi/inmunología , Anemia de Fanconi/patología , Proteínas del Grupo de Complementación de la Anemia de Fanconi/inmunología , Regulación de la Expresión Génica , Inestabilidad Genómica , Células Madre Hematopoyéticas/inmunología , Humanos , Inflamación/genética , Inflamación/inmunología , Inflamación/patología , Transducción de Señal/inmunología , Factor de Necrosis Tumoral alfa/inmunologíaRESUMEN
Salidroside is a phenylpropanoid glycoside isolated from the medicinal plant Rhodiola rosea, which has potent antioxidant properties. Here we show that salidroside prevented the loss of hematopoietic stem cells (HSCs) in mice under oxidative stress. Quiescent HSCs were recruited into cell cycling on in vivo challenge with oxidative stress, which was blocked by salidroside. Surprisingly, salidroside does not prevent the production of reactive oxygen species but reduces hydrogen peroxide-induced DNA-strand breaks in bone marrow cells enriched for HSCs. We tested whether salidroside enhances oxidative DNA damage repair in mice deficient for 5 DNA repair pathways known to be involved in oxidative DNA damage repair; we found that salidroside activated poly(ADP-ribose)polymerase-1 (PARP-1), a component of the base excision repair pathway, in mouse bone marrow HSCs as well as primary fibroblasts and human lymphoblasts. PARP-1 activation by salidroside protects quiescent HSCs from oxidative stress-induced cycling in native animals and self-renewal defect in transplanted recipients, which was abrogated by genetic ablation or pharmacologic inhibition of PARP-1. Together, these findings suggest that activation of PARP-1 by salidroside could affect the homeostasis and function of HSCs and contribute to the antioxidant effects of salidroside.
Asunto(s)
Antioxidantes/farmacología , Reparación del ADN/efectos de los fármacos , Glucósidos/farmacología , Células Madre Hematopoyéticas/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Fenoles/farmacología , Poli(ADP-Ribosa) Polimerasas/metabolismo , Animales , Ciclo Celular/efectos de los fármacos , ADN/efectos de los fármacos , Roturas del ADN de Doble Cadena , Evaluación Preclínica de Medicamentos , Proteína del Grupo de Complementación A de la Anemia de Fanconi/deficiencia , Proteína del Grupo de Complementación C de la Anemia de Fanconi/deficiencia , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , Peróxido de Hidrógeno/toxicidad , Ratones , Ratones Endogámicos C57BL , Oxidantes/toxicidad , Plantas Medicinales/química , Poli(ADP-Ribosa) Polimerasa-1 , Especies Reactivas de Oxígeno , Rhodiola/químicaRESUMEN
Fanconi anemia (FA) nuclear core complex is a multiprotein complex required for the functional integrity of the FA-BRCA pathway regulating DNA repair. This pathway is inactivated in FA, a devastating genetic disease, which leads to hematologic defects and cancer in patients. Here we report the isolation and characterization of a novel 20-kDa FANCA-associated protein (FAAP20). We show that FAAP20 is an integral component of the FA nuclear core complex. We identify a region on FANCA that physically interacts with FAAP20, and show that FANCA regulates stability of this protein. FAAP20 contains a conserved ubiquitin-binding zinc-finger domain (UBZ), and binds K-63-linked ubiquitin chains in vitro. The FAAP20-UBZ domain is not required for interaction with FANCA, but is required for DNA-damage-induced chromatin loading of FANCA and the functional integrity of the FA pathway. These findings reveal critical roles for FAAP20 in the FA-BRCA pathway of DNA damage repair and genome maintenance.
Asunto(s)
Reparación del ADN , Proteínas del Grupo de Complementación de la Anemia de Fanconi/metabolismo , Anemia de Fanconi/genética , Anemia de Fanconi/metabolismo , Transducción de Señal , Ubiquitina/metabolismo , Células Cultivadas , Cromatina/metabolismo , Daño del ADN , Proteína del Grupo de Complementación A de la Anemia de Fanconi/química , Proteína del Grupo de Complementación A de la Anemia de Fanconi/metabolismo , Proteínas del Grupo de Complementación de la Anemia de Fanconi/química , Proteínas del Grupo de Complementación de la Anemia de Fanconi/genética , Humanos , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Estabilidad Proteica , Dedos de ZincRESUMEN
Oxidative stress has been implicated in the pathogenesis of many human diseases including Fanconi anemia (FA), a genetic disorder associated with BM failure and cancer. Here we show that major antioxidant defense genes are down-regulated in FA patients, and that gene down-regulation is selectively associated with increased oxidative DNA damage in the promoters of the antioxidant defense genes. Assessment of promoter activity and DNA damage repair kinetics shows that increased initial damage, rather than a reduced repair rate, contributes to the augmented oxidative DNA damage. Mechanistically, FA proteins act in concert with the chromatin-remodeling factor BRG1 to protect the promoters of antioxidant defense genes from oxidative damage. Specifically, BRG1 binds to the promoters of the antioxidant defense genes at steady state. On challenge with oxidative stress, FA proteins are recruited to promoter DNA, which correlates with significant increase in the binding of BRG1 within promoter regions. In addition, oxidative stress-induced FANCD2 ubiquitination is required for the formation of a FA-BRG1-promoter complex. Taken together, these data identify a role for the FA pathway in cellular antioxidant defense.
Asunto(s)
Proteínas del Grupo de Complementación de la Anemia de Fanconi/fisiología , Anemia de Fanconi/metabolismo , Regulación de la Expresión Génica , Estrés Oxidativo/genética , Regiones Promotoras Genéticas/genética , 8-Hidroxi-2'-Desoxicoguanosina , Antioxidantes , Células de la Médula Ósea/metabolismo , Células Cultivadas/efectos de los fármacos , Daño del ADN , ADN Helicasas/metabolismo , Reparación del ADN , Desoxiguanosina/análogos & derivados , Desoxiguanosina/análisis , Regulación hacia Abajo , Anemia de Fanconi/genética , Anemia de Fanconi/patología , Proteínas del Grupo de Complementación de la Anemia de Fanconi/deficiencia , Proteínas del Grupo de Complementación de la Anemia de Fanconi/genética , Humanos , Peróxido de Hidrógeno/toxicidad , Complejos Multiproteicos , Proteínas Nucleares/metabolismo , Oxidación-Reducción , Unión Proteica , Procesamiento Proteico-Postraduccional , Factores de Transcripción/metabolismo , UbiquitinaciónAsunto(s)
Anemia de Fanconi/terapia , Proteínas Proto-Oncogénicas c-kit/antagonistas & inhibidores , Animales , Modelos Animales de Enfermedad , Anemia de Fanconi/genética , Anemia de Fanconi/patología , Proteína del Grupo de Complementación A de la Anemia de Fanconi/deficiencia , Proteína del Grupo de Complementación A de la Anemia de Fanconi/genética , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/deficiencia , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/genética , Supervivencia de Injerto , Trasplante de Células Madre Hematopoyéticas , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Acondicionamiento Pretrasplante/métodosRESUMEN
Patients with Fanconi anemia (FA) have a high risk of developing acute myeloid leukemia (AML). In this study, we attempted to identify cell-surface markers for leukemia-initiating cells in FA-AML patients. We found that the IL-3 receptor-α (IL-3Rα) is a promising candidate as an leukemia-initiating cell-specific antigen for FA-AML. Whereas IL-3Rα expression is undetectable on normal CD34(+)CD38(-) HSCs, it is overexpressed on CD34(+)CD38(-) cells from FA patients with AML. We examined the leukemia-initiating cell activity of IL-3Rα-positive FA-AML cells in a "humanized" FA xenotransplant model in which we separated AML cells into IL-3Rα-positive and IL-3Rα-negative CD34 fractions and transplanted them into irradiated recipient mice. In all 3 FA-AML samples, only IL-3Rα-positive cells showed significant levels of engraftment and developed leukemia in the recipient mice. The FA CD34(+)IL-3Rα(+) blasts isolated from leukemic mice exhibited hypersensitivity to IL-3 deprivation and JAK2-STAT5 overactivation after IL-3 treatment. Finally, treatment of FA CD34(+)IL-3Rα(+) blasts with an IL-3Rα-neutralizing antibody inhibited IL-3-mediated proliferation and STAT5 activation. These results demonstrate that IL-3Rα is a cell-surface marker present on FA-AML leukemia-initiating cells and may be a valuable therapeutic target.
Asunto(s)
ADP-Ribosil Ciclasa 1/inmunología , Antígenos CD34/inmunología , Anemia de Fanconi/complicaciones , Subunidad alfa del Receptor de Interleucina-3/genética , Leucemia Mieloide Aguda/inmunología , Células Madre/inmunología , Regulación hacia Arriba , Animales , Anemia de Fanconi/inmunología , Humanos , Subunidad alfa del Receptor de Interleucina-3/antagonistas & inhibidores , Subunidad alfa del Receptor de Interleucina-3/inmunología , Janus Quinasa 2/inmunología , Leucemia Mieloide Aguda/etiología , Ratones , Ratones Endogámicos NOD , Factor de Transcripción STAT5/inmunología , Trasplante HeterólogoRESUMEN
mTOR integrates signals from nutrients and growth factors to control protein synthesis, cell growth, and survival. Although mTOR has been established as a therapeutic target in hematologic malignancies, its physiological role in regulating hematopoiesis remains unclear. Here we show that conditional gene targeting of mTOR causes bone marrow failure and defects in multi-lineage hematopoiesis including myelopoiesis, erythropoiesis, thrombopoiesis, and lymphopoiesis. mTOR deficiency results in loss of quiescence of hematopoietic stem cells, leading to a transient increase but long-term exhaustion and defective engraftment of hematopoietic stem cells in lethally irradiated recipient mice. Furthermore, ablation of mTOR causes increased apoptosis in lineage-committed blood cells but not hematopoietic stem cells, indicating a differentiation stage-specific function. These results demonstrate that mTOR is essential for hematopoietic stem cell engraftment and multi-lineage hematopoiesis.
Asunto(s)
Marcación de Gen/métodos , Hematopoyesis/fisiología , Trasplante de Células Madre Hematopoyéticas/métodos , Serina-Treonina Quinasas TOR/fisiología , Animales , Supervivencia Celular/fisiología , Células Cultivadas , Ratones , Ratones Noqueados , Ratones SCIDRESUMEN
The molecular pathway by which Fanconi anemia (FA) proteins function in oxidative stress response has not been defined. Here we report the functional interaction of the FA protein Fanconi anemia complementation group D2 (FANCD2) and the forkhead transcription factor forkhead box O 3a (FOXO3a). FOXO3a colocalized with FANCD2 foci in response to oxidative stress. The FANCD2-FOXO3a complex was not detected in cells deficient for the FA core complex component FANCA but could be restored in corrected cells. Consistent with this, a nonmonoubiquitinated FANCD2 mutant failed to bind FOXO3a. Although both mitomycin C and ionizing radiation induced FANCD2 monoubiquitination, neither could induce the association of FANCD2 and FOXO3a. Overexpression of FOXO3a reduced abnormal accumulation of reactive oxygen species, enhanced cellular resistance to oxidative stress, and increased antioxidant gene expression in corrected but not mutant FA-D2 cells. The novel oxidative stress response pathway identified in this study, in which FANCD2 and FOXO3a converge, probably contributes to cellular antioxidant defense.
Asunto(s)
Antioxidantes/metabolismo , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/metabolismo , Factores de Transcripción Forkhead/metabolismo , Regulación de la Expresión Génica , Estrés Oxidativo , Especies Reactivas de Oxígeno/metabolismo , Línea Celular , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/genética , Proteína Forkhead Box O3 , Factores de Transcripción Forkhead/genética , Humanos , Mitomicina/farmacología , Mutación , Inhibidores de la Síntesis del Ácido Nucleico/farmacología , Unión Proteica/efectos de los fármacos , Unión Proteica/genética , Unión Proteica/efectos de la radiación , Radiación Ionizante , Ubiquitinación/efectos de los fármacos , Ubiquitinación/genética , Ubiquitinación/efectos de la radiaciónRESUMEN
Eight of the Fanconi anemia (FA) proteins form a core complex that activates the FA pathway. Some core complex components also form subcomplexes for yet-to-be-elucidated functions. Here, we have analyzed the interaction between a cytoplasmic FA subcomplex and the leukemic nucleophosmin (NPMc). Exogenous NPMc was degraded rapidly in FA acute myeloid leukemia bone marrow cells. Knockdown of FANCA or FANCC in leukemic OCI/AML3 cells induced rapid degradation of endogenous NPMc. NPMc degradation was mediated by the ubiquitin-proteasome pathway involving the IBR-type RING-finger E3 ubiquitin ligase IBRDC2, and genetic correction of FA-A or FA-C lymphoblasts prevented NPMc ubiquitination. Moreover, cytoplasmic FANCA and FANCC formed a cytoplasmic complex and interacted with NPMc. Using a patient-derived FANCC mutant and a nuclearized FANCC, we demonstrated that the cytoplasmic FANCA-FANCC complex was essential for NPMc stability. Finally, depletion of FANCA and FANCC in NPMc-positive leukemic cells significantly increased inflammation and chemoresistance through NF-κB activation. Our findings not only reveal the molecular mechanism involving cytoplasmic retention of NPMc but also suggest cytoplasmic function of FANCA and FANCC in NPMc-related leukemogenesis.
Asunto(s)
Citoplasma/metabolismo , Proteína del Grupo de Complementación A de la Anemia de Fanconi/metabolismo , Proteína del Grupo de Complementación C de la Anemia de Fanconi/metabolismo , Leucemia/metabolismo , Secuencia de Bases , Línea Celular Tumoral , Citoplasma/química , Citoplasma/genética , Anemia de Fanconi/genética , Anemia de Fanconi/metabolismo , Proteína del Grupo de Complementación A de la Anemia de Fanconi/genética , Proteína del Grupo de Complementación C de la Anemia de Fanconi/genética , Humanos , Leucemia/genética , Datos de Secuencia Molecular , Proteínas Nucleares , Nucleofosmina , Unión Proteica , Estabilidad Proteica , Transporte de Proteínas , UbiquitinaciónRESUMEN
An elevated level of nucleophosmin (NPM) is often found in actively proliferative cells including human tumors. To identify the regulatory role for NPM phosphorylation in proliferation and cell cycle control, a series of mutants targeting the consensus cyclin-dependent kinase (CDK) phosphorylation sites was created to mimic or abrogate either single-site or multi-site phosphorylation. Simultaneous inactivation of two CDK phosphorylation sites at Ser10 and Ser70 (NPM-AA) induced G(2)/M cell cycle arrest, phosphorylation of Cdk1 at Tyr15 (Cdc2(Tyr15)) and increased cytoplasmic accumulation of Cdc25C. Strikingly, stress-induced Cdk1(Tyr15) and Cdc25C sequestration was suppressed by expression of a phosphomimetic NPM mutant created on the same CDK sites (S10E/S70E, NPM-EE). Further analysis revealed that phosphorylation of NPM at both Ser10 and Ser70 was required for proper interaction between Cdk1 and Cdc25C. Moreover, NPM-EE directly bound to Cdc25C and prevented phosphorylation of Cdc25C at Ser216 during mitosis. Finally, NPM-EE overrided stress-induced G(2)/M arrest and increased leukemia blasts in a NOD/SCID xenograft model. Thus, these findings reveal a novel function of NPM on regulation of cell cycle progression, in which phosphorylation of NPM controls cell cycle progression at G(2)/M transition through modulation of Cdk1 and Cdc25C activities.