RESUMEN
BCL6 is a potent transcriptional repressor that plays important roles in germinal center formation, T helper cell differentiation and lymphomagenesis and regulates expression of several chemokine genes in macrophages. In a further investigation of its role in macrophages, we show that BCL6 inactivation in primary bone marrow-derived macrophages leads to decreased polarization, motility and cell spreading accompanied by an increase in peripheral focal complexes, anchored F-actin bundles and cortical F-actin density. These changes were associated with excess RhoA activation. C3 transferase inhibition of RhoA activity reverted the adhesion structure phenotype, which was not affected by Rho kinase inhibitors, suggesting that other downstream effectors of Rho maintain this Bcl6(-/-) phenotype. Excess RhoA activation in BCL6-deficient macrophages is associated with a decrease in the p120RasGAP (RASA1)-mediated translocation of p190RhoGAP (GRLF1) to active RhoA at the plasma membrane and a reduction in cell surface expression of the CSF1R that has been reported to recruit RasGAP to the plasma membrane. Reconstitution of BCL6 expression in Bcl6(-/-) macrophages results in complete reversion of the morphological phenotype and a significant increase in cell surface CSF1R expression whereas overexpression of the CSF1R corrects the polarization and adhesion structure defects. These results demonstrate that BCL6 suppresses RhoA activity, largely through upregulation of surface CSF1R expression, to modulate cytoskeletal and adhesion structures and increase the motility of macrophages.
Asunto(s)
Proteínas de Unión al ADN/fisiología , Macrófagos/metabolismo , Proteína de Unión al GTP rhoA/fisiología , ADP Ribosa Transferasas/metabolismo , Actinas/metabolismo , Animales , Western Blotting , Células de la Médula Ósea/citología , Toxinas Botulínicas/metabolismo , Adhesión Celular , Membrana Celular/metabolismo , Movimiento Celular , Separación Celular , Células Cultivadas , Quimiocinas/metabolismo , Quimiotaxis , Citometría de Flujo , Inmunoprecipitación , Péptidos y Proteínas de Señalización Intracelular , Ratones , Ratones Transgénicos , Microscopía , Fenotipo , Proteínas Serina-Treonina Quinasas/metabolismo , Transporte de Proteínas , Proteínas Proto-Oncogénicas c-bcl-6 , Receptor de Factor Estimulante de Colonias de Macrófagos/biosíntesis , Retroviridae/genética , Factores de Tiempo , Transcripción Genética , Proteína Activadora de GTPasa p120/metabolismo , Quinasas Asociadas a rho , Proteína de Unión al GTP rhoA/metabolismoRESUMEN
BCL-6 is a transcription repressor frequently deregulated in non-Hodgkin's B cell lymphomas. Its activity is also critical to germinal center development and balanced Th1/Th2 differentiation. Previous studies have suggested that NF-kappaB activity is suppressed in germinal center and lymphoma B cells that express high levels of BCL-6, and yet the reason for this is unknown. We report in this study that BCL-6 can bind to three sequence motifs in the 5' regulatory region of NF-kappaB1 in vitro and in vivo, and repress NF-kappaB1 transcription both in reporter assays and in lymphoma B cell lines. BCL-6(-/-) mice further confirm the biological relevance of BCL-6-dependent regulation of NF-kappaB1 because BCL-6 inactivation caused notable increase in p105/p50 proteins in several cell types. Among these, BCL-6(-/-) macrophage cell lines displayed a hyperproliferation phenotype that can be reversed by NF-kappaB inhibitors, e.g., N-tosyl-l-phenylalanine chloromethyl ketone and SN50, a result that is consistent with increased nuclear kappaB-binding activity of p50 homodimer and p50/p65 heterodimer. Our results demonstrate that BCL-6 can negatively regulate NF-kappaB1 expression, thereby inhibiting NF-kappaB-mediated cellular functions.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , FN-kappa B/inmunología , Proteínas Proto-Oncogénicas/metabolismo , Factores de Transcripción/metabolismo , Transcripción Genética , Animales , Northern Blotting , Western Blotting , Línea Celular Tumoral , Proteínas de Unión al ADN/inmunología , Humanos , Inmunoprecipitación , Linfoma de Células B/metabolismo , Ratones , FN-kappa B/genética , Proteínas Proto-Oncogénicas/inmunología , Proteínas Proto-Oncogénicas c-bcl-6 , ARN Mensajero/análisis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Transcripción/inmunologíaRESUMEN
The transcription repressor BCL-6 is known to play critical roles in B-cell lymphomagenesis, germinal center formation, and balanced Th1/Th2 differentiation. In macrophages, although BCL-6 has also been shown to regulate the expression of several chemokine genes, its function in other aspects of macrophage biology has not been studied. In addition, the precise role of BCL-6 in cell proliferation is poorly understood in general. Here we report that BCL-6(-/-) macrophages hyperproliferate due to an accelerated G(1)/S transition accompanied by increased cyclin D2 and c-myc and decreased expression of p27. Crucial to this enhanced proliferation is spontaneous interleukin 6 (IL-6) production and signal transducer and activator of transcription 3 (STAT3) activation in BCL-6(-/-) macrophages. In colony-forming assays, BCL- 6(-/-) bone marrow progenitor cells form spontaneous macrophage colonies that can be inhibited by anti-IL-6 antibodies. Gene expression studies demonstrate that BCL-6 binds to several sequence motifs scattered in the IL-6 locus and can repress IL-6 transcription both in 293T cells and in macrophages. In conclusion, our results indicate that BCL-6 negatively regulates proliferation of the monocytic/macrophage lineage by suppressing an autocrine IL-6/STAT3-mediated gene expression program. Our work also suggests that BCL-6 prevents abnormal Th2 differentiation by suppressing basal level IL-6 production in antigen-presenting cells (APCs).