RESUMEN
Rho family GTPases activate intracellular kinase cascades to modulate transcription of multiple genes. Previous studies have examined the roles of the ubiquitously expressed Rho GTPase, Rac1, in regulation of gene expression in cell lines and implicated NF-kappaB, serum response factor, and kinase signaling pathways in this regulation. To understand the role of the closely related but hematopoiesis-specific Rho GTPase, Rac2, in regulation of gene transcription, we compared the gene expression profiles between wild-type and Rac2(-/-) bone marrow-derived mast cells. Our data demonstrate remarkable specificity in the regulation of gene expression by Rac2 versus Rac1. Microarray analysis demonstrated that expression of 38 known genes was significantly altered in Rac2(-/-) mast cells after cytokine stimulation compared with those in wild-type cells. Of these, the expression of the mouse mast cell protease 7 (MMCP-7) gene in wild-type cells was highly induced at the transcriptional level after stimulation with stem cell factor (SCF). In spite of compensatorily increased expression of Rac1 in Rac2-deficient cells, SCF-induced MMCP-7 transcription did not occur. Surprisingly, the loss of MMCP-7 induction was not due to decreased activation of NF-kappaB, a transcription factor postulated to lie downstream of Rac1 and known to play a critical role in hematopoietic cell differentiation and proliferation. However, the activities of c-Jun N-terminal kinases (JNKs) were markedly decreased in Rac2(-/-) mast cells. Our results suggest that cytokine-stimulated activation of MMCP-7 gene transcription is selectively regulated by a Rac2-dependent JNK signaling pathway in primary mast cells and imply a remarkable specificity in the regulation of transcriptional activity by these two highly related Rho GTPases.
Asunto(s)
Regulación Enzimológica de la Expresión Génica , Mastocitos/enzimología , Proteínas de Unión al GTP rac/metabolismo , Proteínas de Unión al GTP rac/fisiología , Proteínas de Unión al GTP rho/metabolismo , Secuencias de Aminoácidos , Animales , Northern Blotting , Western Blotting , Células de la Médula Ósea/metabolismo , Núcleo Celular/metabolismo , Dactinomicina/farmacología , Activación Enzimática , Immunoblotting , Proteínas Quinasas JNK Activadas por Mitógenos , Ratones , Ratones Endogámicos C57BL , Proteínas Quinasas Activadas por Mitógenos/metabolismo , FN-kappa B/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación , Plásmidos/metabolismo , Proteínas Proto-Oncogénicas c-kit/metabolismo , Serina Endopeptidasas/metabolismo , Transducción de Señal , Factor de Células Madre/metabolismo , Factores de Tiempo , Transcripción Genética , Triptasas , Regulación hacia Arriba , Proteína de Unión al GTP rac1/metabolismo , Proteína RCA2 de Unión a GTPRESUMEN
RhoH is an hematopoietic-specific, GTPase-deficient Rho GTPase that plays a role in T development. We investigated the mechanisms of RhoH function in TCR signaling. We found that the association between Lck and CD3ζ was impaired in RhoH-deficient T cells, due to defective translocation of both Lck and ZAP-70 to the immunological synapse. RhoH with Lck and ZAP-70 localizes in the detergent-soluble membrane fraction where the complex is associated with CD3ζ phosphorylation. To determine if impaired translocation of ZAP-70 was a major determinant of defective T cell development, Rhoh(-/-) bone marrow cells were transduced with a chimeric myristoylation-tagged ZAP-70. Myr-ZAP-70 transduced cells partially reversed the in vivo defects of RhoH-associated thymic development and TCR signaling. Together, our results suggest that RhoH regulates TCR signaling via recruitment of ZAP-70 and Lck to CD3ζ in the immunological synapse. Thus, we define a new function for a RhoH GTPase as an adaptor molecule in TCR signaling pathway.
Asunto(s)
Proteína Tirosina Quinasa p56(lck) Específica de Linfocito/metabolismo , Receptores de Antígenos de Linfocitos T/metabolismo , Transducción de Señal , Factores de Transcripción/metabolismo , Proteína Tirosina Quinasa ZAP-70/metabolismo , Proteínas de Unión al GTP rho/metabolismo , Animales , Complejo CD3/metabolismo , Femenino , Células HEK293 , Humanos , Immunoblotting , Espacio Intracelular/metabolismo , Células Jurkat , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito/genética , Masculino , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Fosforilación , Unión Proteica , Receptores de Antígenos de Linfocitos T/genética , Linfocitos T/metabolismo , Factores de Transcripción/genética , Proteína Tirosina Quinasa ZAP-70/genética , Proteínas de Unión al GTP rho/genéticaRESUMEN
RhoH is a hematopoietic-specific, GTPase-deficient member of the Rho GTPase family with unknown physiological function. Here we demonstrate that Rhoh-/- mice have impaired T cell receptor (TCR)-mediated thymocyte selection and maturation, resulting in T cell deficiency. RhoH deficiency resulted in defective CD3zeta phosphorylation, impaired translocation of the signaling molecule Zap70 to the immunological synapse and reduced activation of Zap70-mediated signaling in thymic and peripheral T cells. Proteomic analyses demonstrated that RhoH is a component of TCR signaling and is required for recruitment of Zap70 to the TCR through interaction with RhoH noncanonical immunoreceptor tyrosine-based activation motifs (ITAMs). In vivo reconstitution studies also demonstrated that RhoH function depends on phosphorylation of the RhoH ITAMs. These findings suggest that RhoH is a critical regulator of thymocyte development and TCR signaling by mediating recruitment and activation of Zap70.
Asunto(s)
Diferenciación Celular/inmunología , Receptores de Antígenos de Linfocitos T/fisiología , Transducción de Señal/inmunología , Timo/citología , Factores de Transcripción/fisiología , Proteína Tirosina Quinasa ZAP-70/metabolismo , Proteínas de Unión al GTP rho/fisiología , Animales , Línea Celular , Línea Celular Tumoral , Humanos , Células Jurkat , Linfopenia/genética , Linfopenia/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Timo/inmunología , Factores de Transcripción/deficiencia , Factores de Transcripción/genética , Proteínas de Unión al GTP rho/deficiencia , Proteínas de Unión al GTP rho/genéticaRESUMEN
Rho guanosine triphosphatases (GT-Pases) are recognized as critical mediators of signaling pathways regulating actin assembly, migration, proliferation, and survival in hematopoietic cells. Here, we have studied a recently identified hematopoietic-specific Rho GTPase, RhoH. Unlike most members of the Rho GTPase family, RhoH is GTPase deficient and does not cycle between GTP- and guanosine diphosphate (GDP)-bound forms, suggesting that regulation of RhoH expression may be critical in its activity. We found that RhoH is expressed in murine hematopoietic progenitor cells (HPCs) and fully differentiated myeloid and lymphoid lineages. In cytokine-stimulated HPCs, knockdown of RhoH expression via RNA interference stimulates proliferation, survival, and stromal cell-derived factor-1 alpha (SDF-1 alpha)-induced migration in vitro. Conversely, RhoH overexpression in these cells via retrovirus-mediated gene transfer is associated with impaired activation of Rac GTPases, reduced proliferation, increased apoptosis, and defective actin polymerization and chemotaxis. In vivo, HPCs with RhoH overexpression demonstrate defective hematopoietic reconstitution capability compared with control vector-transduced cells. Our results suggest that RhoH serves as a negative regulator of both growth and actin-based function of HPCs possibly via suppression of Rac-mediated signaling.
Asunto(s)
Movimiento Celular/fisiología , Proliferación Celular , Trasplante de Células Madre Hematopoyéticas , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/enzimología , Factores de Transcripción/fisiología , Proteínas de Unión al GTP rho/fisiología , Actinas/antagonistas & inhibidores , Actinas/metabolismo , Secuencia de Aminoácidos , Animales , Apoptosis/fisiología , Línea Celular , Supervivencia Celular/fisiología , Clonación Molecular , Regulación hacia Abajo/fisiología , Represión Enzimática/fisiología , Rechazo de Injerto/genética , Rechazo de Injerto/patología , Inhibidores de Crecimiento/biosíntesis , Inhibidores de Crecimiento/genética , Inhibidores de Crecimiento/fisiología , Células Madre Hematopoyéticas/patología , Masculino , Ratones , Datos de Secuencia Molecular , Procesamiento Proteico-Postraduccional/fisiología , Factores de Transcripción/antagonistas & inhibidores , Factores de Transcripción/biosíntesis , Factores de Transcripción/genética , Proteínas de Unión al GTP rac/antagonistas & inhibidores , Proteínas de Unión al GTP rac/biosíntesis , Proteínas de Unión al GTP rho/antagonistas & inhibidores , Proteínas de Unión al GTP rho/biosíntesis , Proteínas de Unión al GTP rho/genéticaRESUMEN
The Rho guanosine triphosphatases (GTPases) Rac1 and Rac2 are critical signaling regulators in mammalian cells. The deletion of both Rac1 and Rac2 murine alleles leads to a massive egress of hematopoietic stem/progenitor cells (HSC/Ps) into the blood from the marrow, whereas Rac1-/- but not Rac2-/- HSC/Ps fail to engraft in the bone marrow of irradiated recipient mice. In contrast, Rac2, but not Rac1, regulates superoxide production and directed migration in neutrophils, and in each cell type, the two GTPases play distinct roles in actin organization, cell survival, and proliferation. Thus, Rac1 and Rac2 regulate unique aspects of hematopoietic development and function.