RESUMEN
Transcription factor C/EBPalpha induces normal myeloid differentiation, inactivation of C/EBPalpha leads to a differentiation block in acute myeloid leukemias (AML), and overexpression of C/EBPalpha results in AML growth arrest and differentiation. Recent reports suggest that C/EBPalpha is activated or inactivated via protein-protein interactions. We previously reported that C/EBPalpha needs to inactivate the proto-oncogene c-Jun via leucine zipper domain interaction in order to induce granulocytic differentiation. We, therefore, hypothesized that c-Jun expression might be elevated in AML and subsequently inactivate C/EBPalpha. In fact, compared to normal bone marrow mononuclear cells, c-Jun expression is increased in AML patient samples (Affymetrix expression microarray analysis, n=166). c-Jun binds to C/EBPalpha via the leucine zipper domains and prevents C/EBPalpha from DNA binding. Inactivation of C/EBPalpha by c-Jun is necessary for c-Jun to induce proliferation because c-Jun-induced proliferation can be prevented by ectopic overexpression of C/EBPalpha. The dominant-negative 30-kDa C/EBPalpha protein, found in AML, fails to downregulate c-Jun mRNA expression in AML patient samples. Thus, our data suggest a model for AML in which c-Jun promotes proliferation and prevents differentiation by inhibiting C/EBPalpha DNA binding via leucine zipper domain interaction. It might depend on the expression levels of C/EBPalpha and c-Jun, if inhibition of C/EBPalpha by c-Jun or if inhibition of c-Jun by C/EBPalpha is more predominant: proliferation versus differentiation; AML versus normal myeloid development.
Asunto(s)
Proteína alfa Potenciadora de Unión a CCAAT/antagonistas & inhibidores , ADN/metabolismo , Leucemia Mieloide Aguda/enzimología , Proteínas Quinasas Activadas por Mitógenos/biosíntesis , Diferenciación Celular , División Celular , Regulación hacia Abajo , Citometría de Flujo , Humanos , Proteínas Quinasas JNK Activadas por Mitógenos , Leucina Zippers , Leucemia Mieloide Aguda/metabolismo , Proteínas Quinasas Activadas por Mitógenos/química , Modelos Moleculares , Análisis de Secuencia por Matrices de Oligonucleótidos , Fosforilación , Unión Proteica , Estructura Terciaria de Proteína , Proto-Oncogenes Mas , ARN Mensajero/metabolismo , Células Tumorales CultivadasRESUMEN
The transcription factor PU.1 plays a pivotal role in normal myeloid differentiation. PU.1(-/-) mice exhibit a complete block in myeloid differentiation. Heterozygous PU.1 mutations were reported in some patients with acute myeloid leukemia (AML), but not in AML with translocation t(8;21), which gives rise to the fusion gene AML1-ETO. Here we report a negative functional impact of AML1-ETO on the transcriptional activity of PU.1. AML1-ETO physically binds to PU.1 in t(8;21)(+) Kasumi-1 cells. AML1-ETO binds to the beta(3)beta(4) region in the DNA-binding domain of PU.1 and displaces the coactivator c-Jun from PU.1, thus down-regulating the transcriptional activity of PU.1. This physical interaction of AML1-ETO and PU.1 did not abolish the DNA-binding capacity of PU.1. AML1-ETO down-regulates the transactivation capacity of PU.1 in myeloid U937 cells, and the expression levels of PU.1 target genes in AML French-American-British (FAB) subtype M2 patients with t(8;21) were lower than in patients without t(8;21). Conditional expression of AML1-ETO causes proliferation in mouse bone marrow cells and inhibits antiproliferative function of PU.1. Overexpression of PU.1, however, differentiates AML1-ETO-expressing Kasumi-1 cells to the monocytic lineage. Thus, the function of PU.1 is down-regulated by AML1-ETO in t(8;21) myeloid leukemia, whereas overexpression of PU.1 restores normal differentiation.
Asunto(s)
Cromosomas Humanos Par 21 , Cromosomas Humanos Par 8 , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Proteínas de Fusión Oncogénica/fisiología , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/fisiología , Transactivadores/genética , Transactivadores/fisiología , Factores de Transcripción/fisiología , Translocación Genética , Animales , Sitios de Unión , Células de la Médula Ósea/citología , Diferenciación Celular , División Celular , Subunidad alfa 2 del Factor de Unión al Sitio Principal , Regulación hacia Abajo/efectos de los fármacos , Humanos , Leucemia Mieloide Aguda/etiología , Ratones , Proteínas de Fusión Oncogénica/metabolismo , Proteínas de Fusión Oncogénica/farmacología , Unión Proteica , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-jun , Proteína 1 Compañera de Translocación de RUNX1 , Transactivadores/metabolismo , Factores de Transcripción/metabolismo , Factores de Transcripción/farmacología , Transcripción Genética/efectos de los fármacos , Células Tumorales CultivadasRESUMEN
The transcription factor C/EBPalpha is crucial for the differentiation of granulocytes. Conditional expression of C/EBPalpha triggers neutrophilic differentiation, and C/EBPalpha can block 12-O-tetradecanoylphorbol-13-acetate-induced monocytic differentiation of bipotential myeloid cells. In C/EBPalpha knockout mice, no mature granulocytes are present. A dramatic increase of c-Jun mRNA in C/EBPalpha knockout mouse fetal liver was observed. c-Jun, a component of the AP-1 transcription factor complex and a coactivator of the transcription factor PU.1, is important for monocytic differentiation. Here we report that C/EBPalpha downregulates c-Jun expression to drive granulocytic differentiation. An ectopic increase of C/EBPalpha expression decreases the c-Jun mRNA level, and the human c-Jun promoter activity is downregulated eightfold in the presence of C/EBPalpha. C/EBPalpha and c-Jun interact through their leucine zipper domains, and this interaction prevents c-Jun from binding to DNA. This results in downregulation of c-Jun's capacity to autoregulate its own promoter through the proximal AP-1 site. Overexpression of c-Jun prevents C/EBPalpha-induced granulocytic differentiation. Thus, we propose a model in which C/EBPalpha needs to downregulate c-Jun expression and transactivation capacity for promoting granulocytic differentiation.
Asunto(s)
Proteína alfa Potenciadora de Unión a CCAAT/metabolismo , Diferenciación Celular/fisiología , Linaje de la Célula , Regulación hacia Abajo/fisiología , Regulación de la Expresión Génica , Granulocitos/fisiología , Proteínas Proto-Oncogénicas c-jun/metabolismo , Animales , Sitios de Unión , Proteína alfa Potenciadora de Unión a CCAAT/genética , Línea Celular , Genes Reporteros , Granulocitos/citología , Humanos , Leucina Zippers , Ratones , Ratones Noqueados , Regiones Promotoras Genéticas , Unión Proteica , Proteínas Proto-Oncogénicas c-jun/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Zinc/metabolismoRESUMEN
The transcription factor C/EBP alpha regulates early steps of normal granulocyte differentiation since mice with a disruption of the C/EBP alpha gene do not express detectable levels of the granulocyte colony-stimulating factor receptor and produce no neutrophils. We have recently shown that C/EBP alpha function is also impaired in acute myeloid leukemias. However, how the transcriptional activity of C/EBP alpha is regulated both in myelopoiesis and leukemogenesis is not fully understood. The current study demonstrates that activated Ras enhances the ability of C/EBP alpha to transactivate the granulocyte colony-stimulating factor receptor promoter and a minimal promoter containing only C/EBP DNA binding sites. Ras signaling activates C/EBP alpha via the transactivation domain because it enhances the transactivation function of a fusion protein containing a Gal4 DNA binding domain and the C/EBP alpha transactivation domain and does not change C/EBP alpha DNA binding. Ras acts on serine 248 of the C/EBP alpha transactivation domain, because it does not enhance the transactivation function of a C/EBP alpha serine 248 to alanine point mutant. Interestingly, serine 248 of C/EBP alpha is a protein kinase C (PKC) consensus site, and a PKC inhibitor blocks the activation of C/EB alpha by Ras. Ras signaling leads to phosphorylation of C/EBP alpha in vivo. Finally, mutation of serine 248 to alanine obviates the ability of C/EBP alpha to induce granulocytic differentiation. These data suggest a model where Ras signaling enhances the activity of C/EBP alpha to induce granulocytic differentiation by phosphorylation of serine 248.