RESUMEN
Nemo-like kinase (NLK) is a member of the mitogen-activated protein kinase family of kinases and shares a highly conserved kinase domain with other mitogen-activated protein kinase family members. The activation of NLK contributes to the pathogenesis of Diamond-Blackfan anemia (DBA), reducing c-myb expression and mechanistic target of rapamycin activity, and is therefore a potential therapeutic target. Unlike other anemias, the hematopoietic effects of DBA are largely restricted to the erythroid lineage. Mutations in ribosomal genes induce ribosomal insufficiency and reduced protein translation, dramatically impacting early erythropoiesis in the bone marrow of patients with DBA. We sought to identify compounds that suppress NLK and increases erythropoiesis in ribosomal insufficiency. We report that the active component of ginseng, ginsenoside Rb1, suppresses NLK expression and improves erythropoiesis in in vitro models of DBA. Ginsenoside Rb1-mediated suppression of NLK occurs through the upregulation of miR-208, which binds to the 3'-UTR of NLK mRNA and targets it for degradation. We also compare ginsenoside Rb1-mediated upregulation of miR-208 with metformin-mediated upregulation of miR-26. We conclude that targeting NLK expression through miRNA binding of the unique 3'-UTR is a viable alternative to the challenges of developing small-molecule inhibitors to target the highly conserved kinase domain of this specific kinase.
Asunto(s)
Anemia de Diamond-Blackfan/patología , Eritropoyesis/efectos de los fármacos , Ginsenósidos/farmacología , Panax/química , Proteínas Serina-Treonina Quinasas/efectos de los fármacos , Regiones no Traducidas 3' , Animales , HumanosRESUMEN
Diamond-Blackfan anaemia (DBA) is a rare congenital disease causing severe anaemia and progressive bone marrow failure. The majority of patients carry mutations in ribosomal proteins, which leads to depletion of erythroid progenitors in the bone marrow. As many as 40% of all DBA patients receive glucocorticoids to alleviate their anaemia. However, despite their use in DBA treatment for more than half a century, the therapeutic mechanisms of glucocorticoids remain largely unknown. Therefore we sought to study disease specific effects of glucocorticoid treatment using a ribosomal protein s19 (Rps19) deficient mouse model of DBA. This study determines for the first time that a mouse model of DBA can respond to glucocorticoid treatment, similar to DBA patients. Our results demonstrate that glucocorticoid treatment reduces apoptosis, rescues erythroid progenitor depletion and premature differentiation of erythroid cells. Furthermore, glucocorticoids prevent Trp53 activation in Rps19-deficient cells- in a disease-specific manner. Dissecting the therapeutic mechanisms behind glucocorticoid treatment of DBA provides indispensible insight into DBA pathogenesis. Identifying mechanisms important for DBA treatment also enables development of more disease-specific treatments of DBA.
Asunto(s)
Anemia de Diamond-Blackfan/tratamiento farmacológico , Eritropoyesis/efectos de los fármacos , Prednisolona/uso terapéutico , Proteínas Ribosómicas/deficiencia , Proteína p53 Supresora de Tumor/fisiología , Adolescente , Anemia de Diamond-Blackfan/sangre , Animales , Apoptosis/efectos de los fármacos , Células Cultivadas , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/biosíntesis , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Dexametasona/farmacología , Modelos Animales de Enfermedad , Evaluación Preclínica de Medicamentos , Células Precursoras Eritroides/efectos de los fármacos , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Prednisolona/farmacología , Quimera por Radiación , Proteínas Ribosómicas/genética , Factores de Transcripción/biosíntesis , Factores de Transcripción/genética , Regulación hacia Arriba/efectos de los fármacos , Proteína X Asociada a bcl-2/biosíntesis , Proteína X Asociada a bcl-2/genéticaRESUMEN
We have developed a coculture system that establishes DLK(+) fetal hepatic progenitors as the authentic supportive cells for expansion of hematopoietic stem (HSCs) and progenitor cells. In 1-week cultures supplemented with serum and supportive cytokines, both cocultured DLK(+) fetal hepatic progenitors and their conditioned medium supported rapid expansion of hematopoietic progenitors and a small increase in HSC numbers. In 2- and 3-week cultures DLK(+) cells, but not their conditioned medium, continuously and significantly (>20-fold) expanded both hematopoietic stem and progenitor cells. Physical contact between HSCs and DLK(+) cells was crucial to maintaining this long-term expansion. Similar HSC expansion (approximately sevenfold) was achieved in cocultures using a serum-free, low cytokine- containing medium. In contrast, DLK(-) cells are incapable of expanding hematopoietic cells, demonstrating that hepatic progenitors are the principle supportive cells for HSC expansion in the fetal liver.
Asunto(s)
Técnicas de Cultivo de Célula/métodos , Proliferación Celular , Células Madre Hematopoyéticas/citología , Células Madre/citología , Animales , Proteínas de Unión al Calcio , Adhesión Celular/efectos de los fármacos , Células Cultivadas , Técnicas de Cocultivo , Medios de Cultivo Condicionados/farmacología , Medio de Cultivo Libre de Suero/farmacología , Citocinas/farmacología , Feto , Citometría de Flujo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células Madre Hematopoyéticas/metabolismo , Inmunohistoquímica , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Hígado/citología , Hígado/embriología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Células Madre/efectos de los fármacos , Células Madre/metabolismo , Factores de TiempoRESUMEN
Diamond-Blackfan anemia (DBA) is a congenital erythroid hypoplasia caused by a functional haploinsufficiency of genes encoding for ribosomal proteins. Recently, a case study reported a patient who became transfusion-independent in response to treatment with the amino acid L-leucine. Therefore, we have validated the therapeutic effect of L-leucine using our recently generated mouse model for RPS19-deficient DBA. Administration of L-leucine significantly improved the anemia in Rps19-deficient mice (19% improvement in hemoglobin concentration; 18% increase in the number of erythrocytes), increased the bone marrow cellularity, and alleviated stress hematopoiesis. Furthermore, the therapeutic response to L-leucine appeared specific for Rps19-deficient hematopoiesis and was associated with down-regulation of p53 activity. Our study supports the rationale for clinical trials of L-leucine as a therapeutic agent for DBA.
Asunto(s)
Anemia de Diamond-Blackfan/dietoterapia , Suplementos Dietéticos , Modelos Animales de Enfermedad , Hematínicos/uso terapéutico , Hematopoyesis , Leucina/uso terapéutico , Regulación hacia Arriba , Anemia de Diamond-Blackfan/sangre , Anemia de Diamond-Blackfan/metabolismo , Anemia de Diamond-Blackfan/patología , Animales , Células de la Médula Ósea/metabolismo , Células de la Médula Ósea/patología , Regulación hacia Abajo , Recuento de Eritrocitos , Técnicas de Silenciamiento del Gen , Células Madre Hematopoyéticas/metabolismo , Células Madre Hematopoyéticas/patología , Hemoglobinas/análisis , Ratones , Ratones Transgénicos , Terapia Molecular Dirigida , Interferencia de ARN , ARN Mensajero/metabolismo , ARN Interferente Pequeño , Proteínas Ribosómicas/antagonistas & inhibidores , Proteínas Ribosómicas/genética , Proteínas Ribosómicas/metabolismo , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
Diamond-Blackfan anemia (DBA) is a congenital erythroid hypoplastic anemia, characterized by macrocytic anemia, reticulocytopenia, and severely reduced numbers of erythroid precursors in the bone marrow. For more than fifty years, glucocorticoids have remained the main option for pharmacological treatment of DBA. While continuous glucocorticoid administration increases hemoglobin levels in a majority of DBA patients, it also causes severe side effects. There is therefore a great need for more specific and effective treatments to boost or replace the use of glucocorticoids. Over the years, many alternative therapies have been tried out, but most of them have shown to be ineffective. Here we review previous and current attempts to develop such alternative therapies for DBA. We further discuss how emerging knowledge regarding the pathological mechanism in DBA and the therapeutic mechanism of glucocorticoids treatment may reveal novel drug targets for DBA treatment.
Asunto(s)
Anemia de Diamond-Blackfan/terapia , Anemia de Diamond-Blackfan/genética , Ensayos Clínicos como Asunto , Terapia Genética , Glucocorticoides/administración & dosificación , Glucocorticoides/efectos adversos , Humanos , MutaciónRESUMEN
With the aim of finding small molecules that stimulate erythropoiesis earlier than erythropoietin and that enhance erythroid colony-forming unit (CFU-E) production, we studied the mechanism by which glucocorticoids increase CFU-E formation. Using erythroid burst-forming unit (BFU-E) and CFU-E progenitors purified by a new technique, we demonstrate that glucocorticoids stimulate the earliest (BFU-E) progenitors to undergo limited self-renewal, which increases formation of CFU-E cells > 20-fold. Interestingly, glucocorticoids induce expression of genes in BFU-E cells that contain promoter regions highly enriched for hypoxia-induced factor 1α (HIF1α) binding sites. This suggests activation of HIF1α may enhance or replace the effect of glucocorticoids on BFU-E self-renewal. Indeed, HIF1α activation by a prolyl hydroxylase inhibitor (PHI) synergizes with glucocorticoids and enhances production of CFU-Es 170-fold. Because PHIs are able to increase erythroblast production at very low concentrations of glucocorticoids, PHI-induced stimulation of BFU-E progenitors thus represents a conceptually new therapeutic window for treating erythropoietin-resistant anemia.