RESUMEN
The development of red blood cells from hematopoietic progenitors requires the interplay of specific extracellular factors and transcriptional regulators. Here we have identified an erythroid progenitor that is critically dependent on bFGF and requires expression of AMV v-Myb for sustained proliferation in vitro, indicating that bFGF and Myb proteins cooperate in these cells. In the presence of bFGF such v-Myb cells are completely blocked in their ability to differentiate and exhibit an exceptionally high proliferative potential and long lifespan in vitro. Interestingly, in the absence of bFGF cells effectively differentiate into mature erythrocytes, irrespective of constitutive and elevated levels of v-Myb. We also demonstrate that these cells express high levels of FGF receptor type 1 (FGFR1) and that phospholipase C(gamma) (PLC(gamma)) is one of the important molecules in FGF receptor signaling. Our studies suggest that bFGF, in cooperation with Myb proteins, represents an important factor for determining erythroid lineage choice. These findings unravel a so far unidentified link between extracellular signaling and Myb in hematopoietic cells.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Proteínas Adaptadoras del Transporte Vesicular , Células Precursoras Eritroides/citología , Factor 2 de Crecimiento de Fibroblastos/farmacología , Proteínas Oncogénicas v-myb/metabolismo , Animales , Western Blotting , Diferenciación Celular/efectos de los fármacos , División Celular/efectos de los fármacos , Tamaño de la Célula/efectos de los fármacos , Células Cultivadas , Senescencia Celular/efectos de los fármacos , Embrión de Pollo , Eritrocitos/citología , Eritrocitos/efectos de los fármacos , Eritrocitos/metabolismo , Células Precursoras Eritroides/efectos de los fármacos , Células Precursoras Eritroides/metabolismo , Fibroblastos , Citometría de Flujo , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Hemoglobinas/metabolismo , Isoenzimas/metabolismo , Mitógenos/farmacología , Fosfolipasa C gamma , Proteínas/fisiología , Proteínas Proto-Oncogénicas c-myb/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas Tirosina Quinasas Receptoras/metabolismo , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos , Receptores de Factores de Crecimiento de Fibroblastos/metabolismo , Proteínas Adaptadoras de la Señalización Shc , Transducción de Señal/efectos de los fármacos , Fosfolipasas de Tipo C/metabolismoRESUMEN
GATA-1 and c-Myb transcription factors represent key regulators of red blood cell development. GATA-1 is upregulated and c-myb proto-oncogene expression is downregulated when red cell progenitors differentiate into erythrocytes. Here we have employed a culture system, that faithfully recapitulates red blood cell differentiation in vitro, to follow the kinetics of GATA-1 and c-myb expression. We show that c-myb proto-oncogene expression is high in progenitors and effectively downregulated at the time when nuclear GATA-1 accumulates and cells differentiate into erythrocytes. Additionally, we identified two GATA-1 binding sites within the c-myb promoter and demonstrate that GATA-1 protein binds to these sites in vitro. Furthermore, GATA-1 represses c-myb expression through one of the GATA-1 binding sites in transient transfection experiments and this requires FOG-1. Thus, our study provides evidence for a direct molecular link between GATA-1 activity and c-myb proto-oncogene expression during terminal red cell differentiation.
Asunto(s)
Proteínas de Unión al ADN/fisiología , Células Precursoras Eritroides/metabolismo , Eritropoyesis/fisiología , Proteínas Proto-Oncogénicas c-myb/fisiología , Proteínas Represoras/fisiología , Factores de Transcripción/fisiología , Animales , Secuencia de Bases , Sitios de Unión , Proteínas Portadoras/fisiología , Embrión de Pollo , Pollos , Factores de Unión al ADN Específico de las Células Eritroides , Fibroblastos , Factor de Transcripción GATA1 , Regulación del Desarrollo de la Expresión Génica , Genes myb , Humanos , Cinética , Datos de Secuencia Molecular , Proteínas Nucleares/fisiología , Regiones Promotoras Genéticas/genética , Unión Proteica , Proto-Oncogenes Mas , Proteínas Proto-Oncogénicas c-myb/genética , Proteínas Recombinantes de Fusión/fisiología , Factor de Células Madre/farmacología , TransfecciónRESUMEN
Dendritic cells (DC) represent professional antigen-presenting cells that develop from hematopoietic progenitors through successive steps of differentiation. Employing DNA microarray technology, we analysed the specific changes in gene expression that occur when human progenitor cells differentiate into DC. CD34 progenitor cells were first amplified in vitro with stem cell factor (SCF), Flt3 ligand (FL), thrombopoietin and IL-6/soluble IL-6 receptor fusion protein, and cells were then induced to differentiate into DC with IL-4 and GM-CSF. DC maturation was induced by TNFalpha. Progenitor cells and DC were subjected to transcriptional profiling by DNA microarrays that represent 13000 human genes. Our analysis revealed specific changes in the expression of a large number of cell surface antigens including molecules involved in antigen uptake and processing, cell migration and antigen presentation. Genes encoding such molecules were upregulated during DC differentiation as were genes encoding cytokines, cytokine receptors, chemokines and chemokine receptors. Stem cell genes and genes related to the multilineage differentiation potential and proliferative state of progenitor cells were downregulated. Our analysis also provides information on the expression profiles of transcriptional regulators such as the NF-kappaB/rel and STAT transcription factors. Interestingly, NF-kappaB/rel factors were found to be expressed in both progenitor cells and DC at similar levels and were induced by TNFalpha. In contrast, expression of STAT factors increased during DC differentiation and their expression was virtually unaffected by TNFalpha.
Asunto(s)
Diferenciación Celular/genética , Células Dendríticas/metabolismo , Perfilación de la Expresión Génica , Antígenos CD/análisis , Antígenos CD34/análisis , Diferenciación Celular/efectos de los fármacos , División Celular/efectos de los fármacos , División Celular/genética , Células Cultivadas , Quimiocinas/genética , Citocinas/genética , Proteínas de Unión al ADN/genética , Células Dendríticas/citología , Células Dendríticas/efectos de los fármacos , Citometría de Flujo , Factor Estimulante de Colonias de Granulocitos y Macrófagos/farmacología , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/efectos de los fármacos , Células Madre Hematopoyéticas/inmunología , Humanos , Interleucina-4/farmacología , FN-kappa B/genética , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Receptores de Quimiocina/genética , Receptores de Citocinas/genética , Transactivadores/genética , Transcripción Genética , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
Dendritic cells (DC) represent key regulators of the immune system, yet their development from hemopoietic precursors is poorly defined. In this study, we describe an in vitro system for amplification of a Flt3(+)CD11b(+) progenitor from mouse bone marrow with specific cytokines. Such progenitor cells develop into both CD11b(+) and CD11b(-) DC, and CD8alpha(+) and CD8alpha(-) DC in vivo. Furthermore, with GM-CSF, these progenitors synchronously differentiated into fully functional DC in vitro. This two-step culture system yields homogeneous populations of Flt3(+)CD11b(+) progenitor cells in high numbers and allows monitoring the consecutive steps of DC development in vitro under well-defined conditions. We used phenotypic and functional markers and transcriptional profiling by DNA microarrays to study the Flt3(+)CD11b(+) progenitor and differentiated DC. We report here on an extensive analysis of the surface Ag expression of Flt3(+)CD11b(+) progenitor cells and relate that to surface Ag expression of hemopoietic stem cells. Flt3(+)CD11b(+) progenitors studied exhibit a broad overlap of surface Ags with stem cells and express several stem cell Ags such as Flt3, IL-6R, c-kit/SCF receptor, and CD93/AA4.1, CD133/AC133, and CD49f/integrin alpha(6). Thus, Flt3(+)CD11b(+) progenitors express several stem cell surface Ags and develop into both CD11b(+) and CD11b(-) DC, and CD8alpha(+) and CD8alpha(-) DC in vivo, and thus into both of the main conventional DC subtypes.