RESUMEN
It has been shown that p210(BCR-ABL) significantly impairs CXCR4 signaling. We report here that the migratory response to SDF-1 was profoundly altered in blast crisis, whereas chronic-phase CD34(+) cells migrated normally to this chemokine. This migratory defect was associated with a low CXCR4 membrane expression. In vitro STI-571 treatment of CD34(+) cells from patients in blast crisis markedly increased the CXCR4 transcript and CXCR4 membrane expression. Because p210(BCR-ABL) frequently increases with disease progression, we determined the effects of high and low p210(BCR-ABL) expression on CXCR4 protein in the granulocyte macrophage colony-stimulating factor-dependent human cell line MO7e. p210(BCR-ABL) expression distinctly alters CXCR4 protein through two different mechanisms depending on its expression level. At low expression, a signaling defect was detected with no modification of CXCR4 expression. However, higher p210(BCR-ABL) expression induced a marked down-regulation of CXCR4 that is related to its decreased transcription. The effect of p210(BCR-ABL) required its tyrosine kinase activity. Collectively, these data indicate that p210(BCR-ABL) could affect CXCR4 by more than one mechanism and suggest that down-regulation of CXCR4 may have important implications in chronic myelogenous leukemia pathogenesis.
Asunto(s)
Quimiocinas CXC/antagonistas & inhibidores , Proteínas de Fusión bcr-abl/fisiología , Receptores CXCR4/fisiología , Animales , Antígenos CD34/biosíntesis , Benzamidas , Crisis Blástica , Línea Celular , Quimiocina CXCL12 , Quimiocinas CXC/fisiología , Regulación hacia Abajo , Proteínas de Fusión bcr-abl/biosíntesis , Proteínas de Fusión bcr-abl/genética , Proteínas de Fusión bcr-abl/metabolismo , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , Células Madre Hematopoyéticas/fisiología , Humanos , Mesilato de Imatinib , Leucemia Mielógena Crónica BCR-ABL Positiva/genética , Leucemia Mielógena Crónica BCR-ABL Positiva/metabolismo , Leucemia Mielógena Crónica BCR-ABL Positiva/patología , Ratones , Células 3T3 NIH , Piperazinas/farmacología , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/metabolismo , Células Madre Pluripotentes/fisiología , Pirimidinas/farmacología , Receptores CXCR4/antagonistas & inhibidores , Receptores CXCR4/biosíntesis , Transducción de Señal , Transcripción GenéticaRESUMEN
Regulators of G-protein signaling (RGS) constitute a family of proteins involved in the negative regulation of signaling through heterotrimeric G protein-coupled receptors (GPCRs). Several RGS proteins have been implicated in the down-regulation of chemokine signaling in hematopoietic cells. The chemokine stromal-cell-derived factor 1 (SDF-1) activates migration of hematopoietic progenitors cells but fails to activate mature megakaryocytes despite high levels of CXC chemokine receptor 4 (CXCR4) receptor expression in these cells. This prompted us to analyze RGS expression and function during megakaryocyte differentiation. We found that RGS16 and RGS18 mRNA expression was up-regulated during this process. Overexpressing RGS16 mRNA in the megakaryocytic MO7e cell line inhibited SDF-1-induced migration, mitogen-activated protein kinase (MAPK) and protein kinase B (AKT) activation, whereas RGS18 overexpression had no effect on CXCR4 signaling. Knocking down RGS16 mRNA via lentiviral-mediated RNA interference increased CXCR4 signaling in MO7e cells and in primary megakaryocytes. Thus, our data reveal that RGS16 is a negative regulator of CXCR4 signaling in megakaryocytes. We postulate that RGS16 regulation is a mechanism that controls megakaryocyte maturation by regulating signals from the microenvironment.
Asunto(s)
Quimiocinas CXC/metabolismo , Megacariocitos/metabolismo , Proteínas/metabolismo , Proteínas RGS/metabolismo , Receptores CXCR4/metabolismo , Transducción de Señal , Plaquetas/citología , Plaquetas/metabolismo , Diferenciación Celular , Línea Celular , Quimiocina CXCL12 , Quimiocinas CXC/genética , Quimiotaxis , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Humanos , Megacariocitos/citología , Conformación de Ácido Nucleico , Proteínas/química , Proteínas/genética , Proteínas RGS/química , Proteínas RGS/genética , Regulación hacia ArribaRESUMEN
CXCR4, the stromal cell-derived factor-1 receptor, plays an important role in the migration of hematopoietic progenitor/stem cells. The surface and cytoplasmic expression of CXCR4 on human hematopoietic CD34(+) cells was investigated. We show that its surface expression is low, whereas a large part of CXCR4 protein is sequestered intracellularly. Using confocal microscopy, we demonstrated that CXCR4 is colocalized with EEA-1, Rab5, Rab4, and Rab11, which are localized in early and recycling endosomes. No significant colocalization of CXCR4 with lysosomal markers CD63 and Lamp-1 was detected. Using antibody feeding experiments, we report a role for CXCR4 constitutive endocytosis in subcellular localization in stably transduced UT7-CXCR4-GFP and CD34(+) cells. Agonist-independent endocytosis of CXCR4 occurs through clathrin-coated vesicles. These data implicate a constitutive endocytosis in the regulation of CXCR4 membrane expression and suggest that constitutive endocytosis may be involved in the regulation of trafficking the human hematopoietic progenitor/stem cells to and in the bone marrow microenvironment.