RESUMEN
Epigenetic changes are regarded as emerging major players for hematopoietic stem cell (HSC) biology. Although some histone deacetylase (HDAC) inhibitors, such as valproic acid (VA), induce differentiation and apoptosis in a variety of leukemic cells in vitro, they produce a favorable effect on the expansion of normal HSCs. In this study, we have identified the VA target HDAC3 as a negative regulator of umbilical cord blood HSC expansion. We demonstrate that knockdown of the transcript dramatically improves CD34+ cell expansion, which correlates with a higher potential to generate colony-forming units in functional assays. We show that this effect is mediated at the level of primitive hematopoietic cells and that it is not due to negative effects on specific cell commitment or alterations in the cell cycle. HDAC3 inhibition does not block commitment to the monocytic lineage and the maturation of monocyte precursors, which are the main inhibited pathways in the presence of VA. Therefore, our results identify HDAC3 as a promising target for therapies aiming to expand HSCs.
Asunto(s)
Proliferación Celular , Células Madre Hematopoyéticas/enzimología , Histona Desacetilasas/metabolismo , Antígenos CD34/metabolismo , Diferenciación Celular/efectos de los fármacos , División Celular/efectos de los fármacos , Linaje de la Célula , Proliferación Celular/efectos de los fármacos , Sangre Fetal/citología , Citometría de Flujo , Técnicas de Silenciamiento del Gen , Células HEK293 , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/efectos de los fármacos , Inhibidores de Histona Desacetilasas/farmacología , Histona Desacetilasas/genética , Humanos , Lentivirus/genética , Monocitos/citología , Monocitos/efectos de los fármacos , Monocitos/enzimología , ARN Interferente Pequeño/genética , Ácido Valproico/farmacologíaRESUMEN
Retinoic acid and Wnt/ß-catenin signals play important roles during neuronal differentiation but less is known about noncanonical Wnt signals in this context. We examined retinoic acid and Wnt signaling in two human embryonal carcinoma cell lines, NTERA-2 (clone D1), which undergoes neuronal differentiation in response to retinoic acid, and 2102Ep, which does not. Retinoic acid treatment inhibited ß-catenin/Tcf activity in NTERA-2 cells but not in 2102Ep cells. Inhibition occurred downstream of ß-catenin but did not involve competition between retinoic acid receptors and ß-catenin for binding to p300 or Tcf-4. Ectopic expression of FZD1 partially restored inhibition in 2102Ep cells, suggesting the involvement of Wnt ligands. Retinoic acid treatment of NTERA-2 cells induced the expression of Wnt-4 and Wnt-11, both of which were able to inhibit ß-catenin/Tcf activity. Wnt-4 and Wnt-11 were found at cell borders in islands of cells that expressed OCT4 and GFAP and were predominantly negative for Nestin, PAX6, and GATA6. Gene silencing of Wnt-4, but not Wnt-11, reduced retinoic acid downregulation of OCT4 and Nanog and upregulation of PAX6, ASCL1, HOXC5, and NEUROD1, suggesting that Wnt-4 promotes early neuronal differentiation. Gene expression analysis of NTERA-2 cells stably overexpressing Wnt-11 suggested that Wnt-11 potentiates retinoic acid induction of early neurogenesis. Consistent with this, overexpression of Wnt-11 maintained a population of proliferating progenitor cells in cultures treated with retinoic acid for several weeks. These observations highlight the distinct roles of two noncanonical Wnts during the early stages of retinoic acid-induced neuronal differentiation.
Asunto(s)
Células Madre Embrionarias/fisiología , Células-Madre Neurales/fisiología , Neurogénesis/fisiología , Neuronas/citología , Tretinoina/fisiología , Proteínas Wnt/fisiología , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/antagonistas & inhibidores , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Línea Celular Tumoral , Células Madre Embrionarias/citología , Células Madre Embrionarias/efectos de los fármacos , Receptores Frizzled/metabolismo , Humanos , Células-Madre Neurales/citología , Células-Madre Neurales/efectos de los fármacos , Neurogénesis/efectos de los fármacos , Neurogénesis/genética , Transducción de Señal , Transfección , Tretinoina/farmacología , Regulación hacia Arriba , Proteínas Wnt/genética , Proteína Wnt4 , beta Catenina/antagonistas & inhibidores , beta Catenina/metabolismoRESUMEN
It is widely accepted that the neurological alterations in Down syndrome (DS) are principally due to modifications in developmental processes. Accordingly, a large part of the research on DS in recent years has focused on chromosome 21 genes that influence brain development. MNB/DYRK1A is one of the genes on human chromosome 21 that has raised most interest, due to its relationship with the brain functions that are altered in DS. Although a number of interesting experimental mouse models for DS are being developed, we still know little about the expression of Mnb/Dyrk1A during mouse brain development. Here, we report that Mnb/Dyrk1A displays a rather dynamic spatio-temporal expression pattern during mouse central nervous system development. Our data indicate that Mnb/Dyrk1A is specifically expressed in four sequential developmental phases: transient expression in preneurogenic progenitors, cell cycle-regulated expression in neurogenic progenitors, transient expression in recently born neurones, and persistent expression in late differentiating neurones. Our results also suggest that the subcellular localization of MNB/DYRK1A, including its translocation to the nucleus, is finely regulated. Thus, the MNB/DYRK1A protein kinase could be a key element in the molecular machinery that couples sequential events in neuronal development. This rich repertoire of potential functions in the developing central nervous system is suitable to be linked to the neurological alterations in DS through the use of mouse experimental models.