RESUMEN
The molecular events that regulate engraftment and mobilization of hematopoietic stem cells and progenitors (HSC/Ps) are still incompletely defined. We have examined the role of the Rho GTPases Rac1 and Rac2 in HSC engraftment and mobilization. Rac1, but not the hematopoietic-specific Rac2, is required for the engraftment phase of hematopoietic reconstitution, because Rac1(-/-) HSCs did not rescue in vivo hematopoiesis after transplantation, but deletion of Rac1 after engraftment did not impair steady-state hematopoiesis. Rac1(-/-) HSC/Ps showed impaired spatial localization to the endosteum but near-normal homing to the medullary cavity in vivo. Interaction with the bone marrow microenvironment in vitro was markedly altered. Whereas post-engraftment deletion of Rac1 alone did not impair hematopoiesis, deficiency of both Rac1 and Rac2 led to massive mobilization of HSCs from the marrow associated with ineffective hematopoiesis and intense selection for Rac-expressing HSCs. This mobilization was reversible by re-expression of Rac1. In addition, a rationally designed, reversible small-molecule inhibitor of Rac activation led to transient mobilization of engraftable HSC/Ps. Rac proteins thus differentially regulate engraftment and mobilization phenotypes, suggesting that these biological processes and steady-state hematopoiesis are biochemically separable and that Rac proteins may be important molecular targets for stem cell modification.
Asunto(s)
Movimiento Celular/fisiología , Proliferación Celular , Hematopoyesis/fisiología , Movilización de Célula Madre Hematopoyética/métodos , Células Madre Hematopoyéticas/fisiología , Neuropéptidos/metabolismo , Transducción de Señal/fisiología , Proteínas de Unión al GTP rac/metabolismo , Aminoquinolinas , Animales , Trasplante de Médula Ósea/fisiología , Citometría de Flujo , Eliminación de Gen , Factor Estimulante de Colonias de Granulocitos , Hematopoyesis/genética , Células Madre Hematopoyéticas/enzimología , Ratones , Neuropéptidos/genética , Pirimidinas , Proteínas de Unión al GTP rac/genética , Proteína de Unión al GTP rac1RESUMEN
A region of interaction between the near N-terminal of cardiac troponin I (cTnI) and the C-lobe of troponin C (cTnC), where troponin T (cTnT) binds, appears to be critical in regulation of myofilament Ca(2+)-activation. We probed whether functional consequences of modulation of this interface influence the function of tropomyosin (Tm) in thin filament activation. We modified the C-lobe of cTnC directly by addition of the Ca(2+)-sensitizer, EMD 57033, and indirectly by replacing native cTnI with cTnI-containing Glu residues at Ser-43 and Ser-45 (cTnI-S43E/S45E) in myofilaments from hearts of non-transgenic (NTG) and transgenic (TG) mice expressing a point mutation on alpha-Tm (E180G) linked to familial hypertrophic cardiomyopathy. Introduction of cTnI-S43E/S45E induced a significantly greater reduction in tension in TG myofilaments compared to NTG controls. Furthermore, the effect of EMD 57033 to restore Ca(2+)-sensitivity was higher in TG compared to NTG fiber bundles containing cTnI-S43E/S45E and compared to TG or NTG fiber bundles containing native TnI. Our results indicate that alterations in regions of interaction among the N-terminal of cTnI, the C-lobe of cTnC, and the C-terminus of cTnT are important in the regulation of myofilament activity. Although levels of phosphorylation at protein kinase C-dependent sites were the same in TG and NTG myofilaments, our data indicate that the effects of phosphorylation were more depressive in TG hearts.
Asunto(s)
Cardiomiopatía Hipertrófica Familiar/genética , Mutación , Miocardio/metabolismo , Transducción de Señal , Tropomiosina/genética , Troponina C/química , Animales , Western Blotting , Calcio/metabolismo , Relación Dosis-Respuesta a Droga , Electroforesis en Gel de Poliacrilamida , Ácido Glutámico/química , Ratones , Ratones Transgénicos , Fibras Musculares Esqueléticas/metabolismo , Fosforilación , Unión Proteica , Proteína Quinasa C/metabolismo , Estructura Terciaria de Proteína , Quinolinas/farmacología , Serina/química , Tiadiazinas/farmacología , Factores de TiempoRESUMEN
Familial hypertrophic cardiomyopathy, a disease caused by mutations in cardiac contractile proteins, is characterized by left and/or right ventricular hypertrophy, myocyte disarray, fibrosis, and cardiac arrhythmias that may lead to premature sudden death. Five distinct point mutations within alpha-tropomyosin are associated with the development of familial hypertrophic cardiomyopathy. Two of these mutations are found within a troponin T binding site, located at amino acids 175 and 180. In this study, we analyze a transgenic mouse model for one of the mutations that occur at codon 180: a substitution of a glutamic acid for a glycine. These mice develop severe cardiac hypertrophy, substantial interstitial fibrosis, and have an increased heart weight/ body weight ratio. Results show that calcium-handling proteins associated with the sarcoplasmic reticulum exhibit decreased expression. These alterations in gene expression, coupled with the structurally-altered tropomyosin, may contribute to the demonstrated decreased physiological performance exhibited by these transgenic mice. A DNA hybridization microarray analysis of the transgenic vs. control ventricular RNAs shows that 50 transcripts are differentially expressed by more than 100% during the onset of the hypertrophic process, many of which are associated with the extracellular matrix. This study demonstrates that mutations within tropomyosin can be severely disruptive of sarcomeric function, triggering a hypertrophic response coupled with a cascade of alterations in gene expression.
Asunto(s)
Cardiomiopatía Hipertrófica Familiar/genética , Mutación Puntual , Tropomiosina/genética , Secuencia de Aminoácidos , Animales , Sitios de Unión , Peso Corporal , Cardiomiopatía Hipertrófica Familiar/fisiopatología , Cardiotónicos/farmacología , Codón , Relación Dosis-Respuesta a Droga , Expresión Génica , Ácido Glutámico/metabolismo , Corazón/efectos de los fármacos , Isoproterenol/farmacología , Ratones , Ratones Transgénicos , Tamaño de los Órganos , ARN/análisis , Tropomiosina/química , Troponina/químicaRESUMEN
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.