RESUMEN
The proliferation of all nontransformed adherent cells is dependent upon the development of mechanical tension within the cell; however, little is known about the mechanisms by which signals regulated by mechanical tension are integrated with those regulated by growth factors. We show here that Skp2, a component of a ubiquitin ligase complex that mediates the degradation of several proteins that inhibit proliferation, is upregulated when increased mechanical tension develops in intact smooth muscle and that its upregulation is critical for the smooth muscle proliferative response to increased mechanical tension. Notably, whereas growth factors regulate Skp2 at the level of protein stability, we found that mechanical tension regulates Skp2 at the transcriptional level. Importantly, we demonstrate that the calcium-regulated transcription factor NFATc1 is a critical mediator of the effect of increased mechanical tension on Skp2 transcription. These findings identify Skp2 as a node at which signals from mechanical tension and growth factors are integrated to regulate proliferation, and they define calcium-NFAT-Skp2 signaling as a critical pathway in the mechanoregulation of proliferation.
Asunto(s)
Mecanotransducción Celular , Animales , Secuencia de Bases , Sitios de Unión , Núcleo Celular/metabolismo , Proliferación Celular , Separación Celular , Secuencia de Consenso , Humanos , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Ratones , Datos de Secuencia Molecular , Miocitos del Músculo Liso/citología , Miocitos del Músculo Liso/metabolismo , Factores de Transcripción NFATC/metabolismo , Fosforilación , Regiones Promotoras Genéticas/genética , Transporte de Proteínas , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas Quinasas Asociadas a Fase-S/genética , Proteínas Quinasas Asociadas a Fase-S/metabolismo , Solubilidad , Estrés Mecánico , Técnicas de Cultivo de Tejidos , Transcripción GenéticaRESUMEN
The t(8;21)(q22;q22) translocation, present in 10-15% of acute myeloid leukemia (AML) cases results in the production of the AML1/ETO fusion protein. Expression of AML1/ETO in patients or mouse models is not sufficient to induce AML. Despite convincing evidence that AML1/ETO is directly involved in the pathogenesis of AML, the underlying mechanism is not well understood. Genetic and biochemical experiments suggest that AML1/ETO is a dominant inhibitor of the core binding factor (CBF) transcription complex that includes AML1 (RUNX1), the N-terminal fusion partner in the t(8;21). We generated and recently characterized a novel strain of transgenic mice in which the AML1/ETO cDNA was inserted into the Ly-6A gene that encodes Sca1, a well-characterized marker of murine hematopoietic stem cells. Unexpectedly, transgene expression assessed by flow cytometry was significantly lower than predicted in lymphocytes from these mice. We have confirmed this finding at the mRNA level and suggest that this phenotype is a consequence of dominant inhibition of transgene expression by AML1/ETO. The dominant negative characteristics of AML1/ETO may be important for AML pathogenesis and may provide a molecular target for therapeutic intervention.
Asunto(s)
Subunidad alfa 2 del Factor de Unión al Sitio Principal/fisiología , Leucemia Mieloide/etiología , Proteínas de Fusión Oncogénica/fisiología , Enfermedad Aguda , Animales , Ataxina-1 , Ataxinas , Subunidad alfa 2 del Factor de Unión al Sitio Principal/genética , Subunidades alfa del Factor de Unión al Sitio Principal/genética , Subunidades alfa del Factor de Unión al Sitio Principal/fisiología , ADN de Neoplasias/genética , Regulación Leucémica de la Expresión Génica , Genes Dominantes , Genes Relacionados con las Neoplasias , Hematopoyesis/genética , Humanos , Leucemia Mieloide/genética , Ratones , Ratones Transgénicos , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/fisiología , Proteínas Nucleares/genética , Proteínas Nucleares/fisiología , Proteínas de Fusión Oncogénica/genética , Proteína 1 Compañera de Translocación de RUNX1 , Translocación GenéticaRESUMEN
The t(8;21)(q22;q22) translocation, present in 10-15% of acute myeloid leukemia (AML) cases, generates the AML1/ETO fusion protein. To study the role of AML1/ETO in the pathogenesis of AML, we used the Ly6A locus that encodes the well characterized hematopoietic stem cell marker, Sca1, to target expression of AML1/ETO to the hematopoietic stem cell compartment in mice. Whereas germ-line expression of AML1/ETO from the AML1 promoter results in embryonic lethality, heterozygous Sca1(+/AML1-ETO ires EGFP) (abbreviated Sca(+/AE)) mutant mice are born in Mendelian ratios with no apparent abnormalities in growth or fertility. Hematopoietic cells from Sca(+/AE) mice have markedly extended survival in vitro and increasing myeloid clonogenic progenitor output over time. Sca(+/AE) mice develop a spontaneous myeloproliferative disorder with a latency of 6 months and a penetrance of 82% at 14 months. These results reinforce the notion that the phenotype of murine transgenic models of human leukemia is critically dependent on the cellular compartment targeted by the transgene. This model should provide a useful platform to analyze the effect of AML1/ETO on hematopoiesis and its potential cooperation with other mutations in the pathogenesis of leukemia.