RESUMEN
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.