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1.
Int J Biol Macromol ; 137: 1221-1231, 2019 Sep 15.
Artículo en Inglés | MEDLINE | ID: mdl-31279058

RESUMEN

Osteosarcoma (OS) is the most common primary malignancy of bone and is characterized by a high malignant and metastatic potential. Microarray-based differentially expressed gene screening determined RAC2 as the candidate gene related to OS. Highly expressed RAC2 and activated Wnt signaling pathway were determined in OS tissues using reverse transcription quantitative polymerase chain reaction (RT-qPCR) and Western blot analysis. The OS cells were transfected with siRNA-RAC2 or treated with BIO (activator of Wnt pathway), whereby the effects of siRNA-RAC2 on cell proliferation, invasion, cycle and apoptosis were analyzed by CCK-8, Transwell assay and flow cytometry. The mRNA and protein levels of RAC2 and the Wnt signaling pathway-, proliferation- and apoptosis-related genes in OS cells were determined by RT-qPCR and Western blot assay. Importantly, siRNA-mediated RAC2 silencing inhibited the activation of the Wnt signaling pathway in OS. siRNA-RAC2 inhibited the proliferation and invasion, while impeded OS cell cycle progression and facilitated cell apoptosis. However, activation of Wnt signaling pathway reversed the effects of siRNA-RAC2. Finally, orthotopic xenograft OS mouse model confirmed the in vivo anti-tumor effects by silencing RAC2. Taken together, RAC2 gene silencing could suppress OS progression. The mechanism was obtained by inhibiting the activation of the Wnt signaling pathway.


Asunto(s)
Neoplasias Óseas/patología , Progresión de la Enfermedad , Osteosarcoma/patología , ARN Interferente Pequeño/genética , Vía de Señalización Wnt/genética , Proteínas de Unión al GTP rac/deficiencia , Proteínas de Unión al GTP rac/genética , Animales , Apoptosis/genética , Ciclo Celular/genética , Línea Celular Tumoral , Proliferación Celular/genética , Regulación hacia Abajo/genética , Humanos , Masculino , Ratones , Invasividad Neoplásica/genética , Proteína RCA2 de Unión a GTP
2.
Hippocampus ; 29(7): 569-578, 2019 07.
Artículo en Inglés | MEDLINE | ID: mdl-30387892

RESUMEN

The hippocampus is generally considered as a brain center for learning and memory. We have recently established an electroporation-mediated gene transfer method to investigate the development of neonatal dentate granule cells in vivo. Using this new technique, we introduced knockdown vectors against Rac1 small GTPase into precursors for dentate granule cells at postnatal day 0. After 21 days, Rac1-deficient cells were frequently mispositioned between the granule cell layer (GCL) and hilus. About 60% of these mislocalized cells expressed a dentate granule cell marker, Prox1. Both the dendritic spine density and the ratio of mature spine were reduced when Rac1 was silenced. Notably, the deficient cells have immature thin processes during migrating in the early neonatal period. Knockdown of another Rac isoform, Rac3, also resulted in mislocalization of neonatally born dentate granule cells. In addition, knockdown of Cdc42, another Rho family protein, also caused mislocalization of the cell, although the effects were moderate compared to Rac1 and 3. Despite the ectopic localization, Rac3- or Cdc42-disrupted mispositioned cells expressed Prox1. These results indicate that Rho signaling pathways differentially regulate the proper localization and differentiation of dentate granule cells.


Asunto(s)
Giro Dentado/enzimología , Giro Dentado/crecimiento & desarrollo , Neuropéptidos/metabolismo , Proteína de Unión al GTP cdc42/metabolismo , Proteínas de Unión al GTP rac/metabolismo , Proteína de Unión al GTP rac1/metabolismo , Animales , Animales Recién Nacidos , Diferenciación Celular , Movimiento Celular , Giro Dentado/citología , Técnicas de Silenciamiento del Gen , Técnicas de Transferencia de Gen , Proteínas de Homeodominio/metabolismo , Ratones , Ratones Endogámicos ICR , Neurogénesis , Neuropéptidos/deficiencia , Neuropéptidos/genética , Interferencia de ARN , Transducción de Señal , Proteínas Supresoras de Tumor/metabolismo , Proteína de Unión al GTP cdc42/deficiencia , Proteína de Unión al GTP cdc42/genética , Proteínas de Unión al GTP rac/deficiencia , Proteínas de Unión al GTP rac/genética , Proteína de Unión al GTP rac1/deficiencia , Proteína de Unión al GTP rac1/genética
3.
J Pharmacol Exp Ther ; 367(1): 9-19, 2018 10.
Artículo en Inglés | MEDLINE | ID: mdl-30021868

RESUMEN

Histamine induces chemotaxis of mast cells through the H4 receptor. However, little is known about the precise intracellular signaling pathway that mediates this process. In this study, we identified small GTPases Rac1 and Rac2 as intracellular binding partners of the H4 receptor and characterized their roles in H4 receptor signaling. We showed that histamine induced Rac GTPase activation via the H4 receptor. A Rac inhibitor NSC23766 attenuated chemotaxis of mast cells toward histamine, as well as histamine-induced calcium mobilization and extracellular signal-regulated kinase (ERK) activation. Histamine-induced migration of mast cells was also sensitive to PD98059, an inhibitor of the mitogen-activated protein kinase kinase, indicating that the Rac-ERK pathway was involved in chemotaxis through the H4 receptor. Inhibition of phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) by LY294002 suppressed the histamine-induced chemotaxis and activation of Rac GTPases, suggesting that PI3K regulates chemotaxis upstream of Rac activation. Specific knockdown of Rac1 and Rac2 by short-hairpin RNA revealed that both Rac GTPases are necessary for histamine-induced migration. Downregulation of Rac1 and Rac2 led to attenuated response in calcium mobilization and ERK activation, respectively. These observations suggested that Rac1 and Rac2 have distinct and essential roles in intracellular signaling downstream of H4 receptor-PI3K in histamine-induced chemotaxis of mast cells.


Asunto(s)
Quimiotaxis , Mastocitos/citología , Receptores Histamínicos H4/metabolismo , Proteínas de Unión al GTP rac/metabolismo , Proteína de Unión al GTP rac1/metabolismo , Animales , Calcio/metabolismo , Activación Enzimática , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Femenino , Técnicas de Silenciamiento del Gen , Histamina/metabolismo , Ratones , Ratones Endogámicos C57BL , Fosfatidilinositol 3-Quinasas/metabolismo , ARN Interferente Pequeño/genética , Transducción de Señal , Proteínas de Unión al GTP rac/deficiencia , Proteínas de Unión al GTP rac/genética , Proteína de Unión al GTP rac1/deficiencia , Proteína de Unión al GTP rac1/genética , Proteína RCA2 de Unión a GTP
4.
J Cell Biol ; 216(12): 4331-4349, 2017 12 04.
Artículo en Inglés | MEDLINE | ID: mdl-29061650

RESUMEN

The initial step of metastasis is the local invasion of tumor cells into the surrounding tissue. Invadopodia are actin-based protrusions that mediate the matrix degradation necessary for invasion and metastasis of tumor cells. We demonstrate that Rac3 GTPase is critical for integrating the adhesion of invadopodia to the extracellular matrix (ECM) with their ability to degrade the ECM in breast tumor cells. We identify two pathways at invadopodia important for integrin activation and delivery of matrix metalloproteinases: through the upstream recruiter CIB1 as well as the downstream effector GIT1. Rac3 activity, at and surrounding invadopodia, is controlled by Vav2 and ßPIX. These guanine nucleotide exchange factors regulate the spatiotemporal dynamics of Rac3 activity, impacting GIT1 localization. Moreover, the GTPase-activating function of GIT1 toward the vesicular trafficking regulator Arf6 GTPase is required for matrix degradation. Importantly, Rac3 regulates the ability of tumor cells to metastasize in vivo. The Rac3-dependent mechanisms we show in this study are critical for balancing proteolytic activity and adhesive activity to achieve a maximally invasive phenotype.


Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/genética , Neoplasias de la Mama/genética , Proteínas de Ciclo Celular/genética , Regulación Neoplásica de la Expresión Génica , Integrina beta1/genética , Neoplasias Mamarias Animales/genética , Proteínas de Unión al GTP rac/genética , Factor 6 de Ribosilación del ADP , Factores de Ribosilacion-ADP/genética , Factores de Ribosilacion-ADP/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Proteínas de Unión al Calcio/genética , Proteínas de Unión al Calcio/metabolismo , Adhesión Celular , Proteínas de Ciclo Celular/metabolismo , Línea Celular Tumoral , Matriz Extracelular/metabolismo , Matriz Extracelular/patología , Femenino , Células HEK293 , Humanos , Integrina beta1/metabolismo , Neoplasias Mamarias Animales/metabolismo , Neoplasias Mamarias Animales/patología , Ratones , Invasividad Neoplásica , Metástasis de la Neoplasia , Proteínas Proto-Oncogénicas c-vav/genética , Proteínas Proto-Oncogénicas c-vav/metabolismo , Ratas , Factores de Intercambio de Guanina Nucleótido Rho/genética , Factores de Intercambio de Guanina Nucleótido Rho/metabolismo , Transducción de Señal , Proteínas de Unión al GTP rac/deficiencia
5.
Biomed Pharmacother ; 94: 140-149, 2017 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-28759751

RESUMEN

Oxidative stress is a leading cause to liver injury. Rac2 is a Ras-associated guanosine triphosphatase, an important molecule modulating a large number of cells and involved in the regulation of reactive oxygen species (ROS). For the study described here, we supposed that Rac2 knockout protects mice against CCl4-induced acute liver injury. We found that Rac2 expressed highly in CCl4-induced liver tissues. CCl4-treated Rac2 knockout (Rac2-/-) mice had reduced CD24 levels and steatosis. In addition, CCl4-induced high expression of pro-inflammatory cytokines and chemokine were reversed by Rac2 deficiency compared to CCl4-treated wild type (WT) mice. We also found that fibrosis-related signals of MMP-9, MMP-2 and TGF-ß1 were also down-regulated in Rac2 knockout mice induced by CCl4. Significantly, oxidative stress induced by CCl4 was also suppressed owing to the lack of Rac2, evidenced by enhanced superoxide dismutase (SOD) activity, and reduced malondialdehyde (MDA) levels, superoxide radical, H2O2, xanthine oxidase (XO), xanthine dehydrogenase (XDH) and XO/XDH ratio. Moreover, c-Jun N-terminal protein kinase mitogen-activated protein kinases (JNK MAPK) was activated by CCl4, which was reversed in the liver of Rac2-/- mice through western blot and immunohistochemical analysis. In vitro, endotoxin (LPS) was treated to hepatocytes isolated from WT mice and Rac2-/- mice. The data further confirmed the role of Rac2 deficiency suppressed pro-inflammatory cytokines and chemokine, as well as fibrosis-related signals. Of note, production of ROS induced by LPS was reduced in Rac2-/- cells, accompanied with enhanced SOD1, SOD2 and reduced XO and phosphorylated-JNK expressions. Our results indicated that Rac2 played an essential role in acute liver injury induced by CCl4, providing the compelling information of the effects of Rac2 on liver injury, and revealing a novel regulatory mechanism for acute liver injury.


Asunto(s)
Enfermedad Hepática Inducida por Sustancias y Drogas/metabolismo , Enfermedad Hepática Inducida por Sustancias y Drogas/patología , Inflamación/sangre , Estrés Oxidativo , Proteínas de Unión al GTP rac/deficiencia , Animales , Tetracloruro de Carbono , Enfermedad Hepática Inducida por Sustancias y Drogas/sangre , Enfermedad Hepática Inducida por Sustancias y Drogas/complicaciones , Quimiocinas/sangre , Regulación hacia Abajo/efectos de los fármacos , Activación Enzimática/efectos de los fármacos , Técnicas de Inactivación de Genes , Inflamación/complicaciones , Inflamación/patología , Mediadores de Inflamación/metabolismo , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Lipopolisacáridos/farmacología , Hígado/patología , Masculino , Metaloproteinasa 3 de la Matriz/sangre , Metaloproteinasa 9 de la Matriz/sangre , Ratones Endogámicos C57BL , Estrés Oxidativo/efectos de los fármacos , Factor de Crecimiento Transformador beta1/sangre , Factor de Crecimiento Transformador beta1/metabolismo , Proteínas de Unión al GTP rac/metabolismo , Proteína RCA2 de Unión a GTP
6.
J Neurosci ; 37(32): 7682-7699, 2017 08 09.
Artículo en Inglés | MEDLINE | ID: mdl-28747385

RESUMEN

In the developing CNS, the midline barrier, which comprises guidance molecule-expressing midline glial somata and processes, plays a pivotal role in midline axon guidance. Accumulating evidence has revealed the molecular mechanisms by which the midline barrier ensures proper midline guidance for axons. In contrast, the mechanisms for establishing the midline barrier remain obscure. Here, we report that Rac-specific GTPase-activating protein (RacGAP) α-chimaerin is required for both axonal repulsion at and establishment of the midline barrier in the spinal cord. We generated cortex-specific and spinal-cord-specific α-chimaerin gene (Chn1) knock-out mice (Cx-Chn1KO and Sp-Chn1KO mice, respectively) and found that both showed aberrant corticospinal tract (CST) axon midline crossing in the spinal cord. Strikingly, Sp-Chn1KO mice had breaks (holes) in the ephrinB3(+) spinal midline barrier and EphA4(+) CST axons aberrantly crossed the midline through these holes. During normal embryonic development, EphA4(+) spinal cells are located in juxta-midline areas but are excluded from the midline. In contrast, in Chn1KO embryos, several EphA4(+) cells were aberrantly relocated into the midline and the midline barrier was broken around these cells. Similarly, the spinal cord midline of Epha4KO mice was invaded by juxta-midline EphA4 cells (i.e., Epha4 promoter-active cells) during the embryonic stage and holes were formed in the midline barrier. Juxta-midline EphA4 cells in the spinal cord expressed α-chimaerin. We propose that spinal α-chimaerin aids in establishing an intact spinal midline barrier by mediating juxta-midline EphA4(+) cell repulsion, thus preventing these cells from breaking into the ephrinB3(+) midline barrier.SIGNIFICANCE STATEMENT The midline barrier plays a critical role in midline axon guidance, which is fundamental to the formation of neural circuits that are responsible for proper left-right coordination of the body. Studies have revealed some of the mechanisms underlying how the midline barrier navigates axons. In contrast, the establishment of the midline barrier during embryonic development remains unclear. In this study, we determined that α-chimaerin is required for the formation of an intact midline barrier. Spinal-cord-specific α-chimaerin knock-out mice had spinal midline barriers with numerous breaks (holes), through which corticospinal axons aberrantly crossed the midline. We propose that α-chimaerin protects the midline barrier by mediating cell-repulsive signaling in juxta-midline cells, which prevents these cells from invading the midline.


Asunto(s)
Orientación del Axón/fisiología , Axones/metabolismo , Quimerina 1/metabolismo , Tractos Piramidales/metabolismo , Médula Espinal/metabolismo , Proteínas de Unión al GTP rac/deficiencia , Animales , Animales Recién Nacidos , Quimerina 1/genética , Ratones , Ratones Noqueados , Ratones Transgénicos , Tractos Piramidales/embriología , Tractos Piramidales/crecimiento & desarrollo , Médula Espinal/embriología , Médula Espinal/crecimiento & desarrollo , Proteínas de Unión al GTP rac/genética
7.
Arterioscler Thromb Vasc Biol ; 37(2): 328-340, 2017 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-27834690

RESUMEN

OBJECTIVE: The calcium composition of atherosclerotic plaque is thought to be associated with increased risk for cardiovascular events, but whether plaque calcium itself is predictive of worsening clinical outcomes remains highly controversial. Inflammation is likely a key mediator of vascular calcification, but immune signaling mechanisms that promote this process are minimally understood. APPROACH AND RESULTS: Here, we identify Rac2 as a major inflammatory regulator of signaling that directs plaque osteogenesis. In experimental atherogenesis, Rac2 prevented progressive calcification through its suppression of Rac1-dependent macrophage interleukin-1ß (IL-1ß) expression, which in turn is a key driver of vascular smooth muscle cell calcium deposition by its ability to promote osteogenic transcriptional programs. Calcified coronary arteries from patients revealed decreased Rac2 expression but increased IL-1ß expression, and high coronary calcium burden in patients with coronary artery disease was associated with significantly increased serum IL-1ß levels. Moreover, we found that elevated IL-1ß was an independent predictor of cardiovascular death in those subjects with high coronary calcium burden. CONCLUSIONS: Overall, these studies identify a novel Rac2-mediated regulation of macrophage IL-1ß expression, which has the potential to serve as a powerful biomarker and therapeutic target for atherosclerosis.


Asunto(s)
Enfermedades de la Aorta/enzimología , Aterosclerosis/enzimología , Enfermedad de la Arteria Coronaria/enzimología , Mediadores de Inflamación/metabolismo , Interleucina-1beta/metabolismo , Macrófagos/enzimología , Placa Aterosclerótica , Calcificación Vascular/enzimología , Proteínas de Unión al GTP rac/metabolismo , Animales , Aorta/enzimología , Aorta/patología , Enfermedades de la Aorta/genética , Enfermedades de la Aorta/patología , Enfermedades de la Aorta/prevención & control , Apolipoproteínas E/deficiencia , Apolipoproteínas E/genética , Aterosclerosis/genética , Aterosclerosis/patología , Aterosclerosis/prevención & control , Células Cultivadas , Enfermedad de la Arteria Coronaria/mortalidad , Enfermedad de la Arteria Coronaria/patología , Vasos Coronarios/enzimología , Vasos Coronarios/patología , Femenino , Predisposición Genética a la Enfermedad , Humanos , Proteína Antagonista del Receptor de Interleucina 1/farmacología , Macrófagos/patología , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Músculo Liso Vascular/enzimología , Músculo Liso Vascular/patología , Miocitos del Músculo Liso/enzimología , Miocitos del Músculo Liso/patología , Neuropéptidos/metabolismo , Fenotipo , Pronóstico , Transducción de Señal , Transfección , Regulación hacia Arriba , Calcificación Vascular/mortalidad , Calcificación Vascular/patología , Proteínas de Unión al GTP rac/deficiencia , Proteínas de Unión al GTP rac/genética , Proteína de Unión al GTP rac1/metabolismo , Proteína RCA2 de Unión a GTP
8.
Cereb Cortex ; 26(2): 873-890, 2016 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-26582364

RESUMEN

Rac GTPases regulate the development of cortical/hippocampal GABAergic interneurons by affecting the early development and migration of GABAergic precursors. We have addressed the function of Rac1 and Rac3 proteins during the late maturation of hippocampal interneurons. We observed specific phenotypic differences between conditional Rac1 and full Rac3 knockout mice. Rac1 deletion caused greater generalized hyperactivity and cognitive impairment compared with Rac3 deletion. This phenotype matched with a more evident functional impairment of the inhibitory circuits in Rac1 mutants, showing higher excitability and reduced spontaneous inhibitory currents in the CA hippocampal pyramidal neurons. Morphological analysis confirmed a differential modification of the inhibitory circuits: deletion of either Rac caused a similar reduction of parvalbumin-positive inhibitory terminals in the pyramidal layer. Intriguingly, cannabinoid receptor-1-positive terminals were strongly increased only in the CA1 of Rac1-depleted mice. This increase may underlie the stronger electrophysiological defects in this mutant. Accordingly, incubation with an antagonist for cannabinoid receptors partially rescued the reduction of spontaneous inhibitory currents in the pyramidal cells of Rac1 mutants. Our results show that Rac1 and Rac3 have independent roles in the formation of GABAergic circuits, as highlighted by the differential effects of their deletion on the late maturation of specific populations of interneurons.


Asunto(s)
Conducta Animal/fisiología , Neuronas GABAérgicas/fisiología , Hipocampo/citología , Red Nerviosa/metabolismo , Proteínas de Unión al GTP rac/deficiencia , Proteína de Unión al GTP rac1/deficiencia , Adaptación Ocular/genética , Animales , Condicionamiento Clásico/fisiología , Emociones/fisiología , Fármacos actuantes sobre Aminoácidos Excitadores/farmacología , Conducta Exploratoria/fisiología , Regulación de la Expresión Génica/genética , Glutamato Descarboxilasa/genética , Glutamato Descarboxilasa/metabolismo , Técnicas In Vitro , Potenciales Postsinápticos Inhibidores/efectos de los fármacos , Potenciales Postsinápticos Inhibidores/genética , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Proteínas del Tejido Nervioso/metabolismo , Células Piramidales/metabolismo , Sinapsinas/genética , Sinapsinas/metabolismo , Proteínas de Unión al GTP rac/genética , Proteína de Unión al GTP rac1/genética
9.
Blood ; 125(20): 3105-13, 2015 May 14.
Artículo en Inglés | MEDLINE | ID: mdl-25824687

RESUMEN

Hematopoietic stem cells (HSCs) are localized within specialized microenvironments throughout the BM. Nestin-expressing (Nestin(+)) mesenchymal stromal cells (MSCs) are important in the perivascular space. Rac is critical for MSC cell shape in vitro, whereas its function in MSCs in vivo remains poorly characterized. We hypothesized that deletion of Rac in the Nestin(+) cells would perturb the perivascular space, altering HSC localization and hematopoiesis. Nestin-Cre-directed excision of Rac1 in Rac3(-/-) mice reduces Nestin(+) cells in the marrow. We observed a 2.7-fold decrease in homing of labeled wild-type hematopoietic cells into Rac1(Δ/Δ)Rac3(-/-) mice compared with control mice. Rac1(Δ/Δ)Rac3(-/-) mice demonstrated a marked decrease in arterioles and an increase in the number and volume of venous sinusoids in the marrow that was associated with a reduction in the numbers of immunophenotypically and functionally-defined long-term HSCs in the marrow, a decrease in colony-forming cells and a reduction in circulating progenitors. Rac-deleted animals demonstrated a significant increase in trabecular bone. These data demonstrate that Rac GTPases play an important role in the integrity of perivascular space. Increased trabecular bone and sinusoidal space and decreased arteriolar volume in this model were associated with decreased HSC, underscoring the complexity of regulation of hematopoiesis in the perivascular space.


Asunto(s)
Médula Ósea/metabolismo , Médula Ósea/patología , Hematopoyesis/genética , Proteínas de Unión al GTP rac/genética , Animales , Apoptosis/genética , Vasos Sanguíneos , Huesos/metabolismo , Huesos/patología , Microambiente Celular , Quimiocina CXCL12/genética , Quimiocina CXCL12/metabolismo , Células Endoteliales/metabolismo , Células Madre Hematopoyéticas/metabolismo , Inmunofenotipificación , Ratones , Ratones Noqueados , Nestina/genética , Nestina/metabolismo , Osteoblastos/metabolismo , Factor de Células Madre/metabolismo , Proteínas de Unión al GTP rac/deficiencia
10.
Respir Res ; 15: 71, 2014 Jun 27.
Artículo en Inglés | MEDLINE | ID: mdl-24970330

RESUMEN

BACKGROUND: Pulmonary fibrotic diseases induce significant morbidity and mortality, for which there are limited therapeutic options available. Rac2, a ras-related guanosine triphosphatase expressed mainly in hematopoietic cells, is a crucial molecule regulating a diversity of mast cell, macrophage, and neutrophil functions. All these cell types have been implicated in the development of pulmonary fibrosis in a variety of animal models. For the studies described here we hypothesized that Rac2 deficiency protects mice from bleomycin-induced pulmonary fibrosis. METHODS: To determine the role of Rac2 in pulmonary fibrosis we used a bleomycin-induced mouse model. Anesthetized C57BL/6 wild type and rac2-/- mice were instilled intratracheally with bleomycin sulphate (1.25 U/Kg) or saline as control. Bronchoalveolar lavage (BAL) samples were collected at days 3 and 7 of treatment and analyzed for matrix metalloproteinases (MMPs). On day 21 after bleomycin treatment, we measured airway resistance and elastance in tracheotomized animals. Lung sections were stained for histological analysis, while homogenates were analyzed for hydroxyproline and total collagen content. RESULTS: BLM-treated rac2-/- mice had reduced MMP-9 levels in the BAL on day 3 and reduced neutrophilia and TNF and CCL3/MIP-1α levels in the BAL on day 7 compared to BLM-treated WT mice. We also showed that rac2-/- mice had significantly lower mortality (30%) than WT mice (70%) at day 21 of bleomycin treatment. Lung function was diminished in bleomycin-treated WT mice, while it was unaffected in bleomycin-treated rac2-/- mice. Histological analysis of inflammation and fibrosis as well as collagen and hydroxyproline content in the lungs did not show significant differences between BLM-treated rac2-/- and WT and mice that survived to day 21. CONCLUSION: Rac2 plays an important role in bleomycin-induced lung injury. It is an important signaling molecule leading to BLM-induced mortality and it also mediates the physiological changes seen in the airways after BLM-induced injury.


Asunto(s)
Bleomicina/toxicidad , Neumonía/inducido químicamente , Neumonía/metabolismo , Fibrosis Pulmonar/inducido químicamente , Fibrosis Pulmonar/metabolismo , Proteínas de Unión al GTP rac/deficiencia , Animales , Femenino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neumonía/mortalidad , Fibrosis Pulmonar/mortalidad , Proteína RCA2 de Unión a GTP
11.
PLoS One ; 8(4): e61629, 2013.
Artículo en Inglés | MEDLINE | ID: mdl-23613889

RESUMEN

Recent genetic-based studies have implicated a number of immune-related genes in the pathogenesis of inflammatory bowel disease (IBD). Our recent genetic studies showed that RAC2 is associated with human IBD; however, its role in disease pathogenesis is unclear. Given Rac2's importance in various fundamental immune cell processes, we investigated whether a defect in Rac2 may impair host immune responses in the intestine and promote disease in the context of an infection-based (Citrobacter rodentium) model of colitis. In response to infection, Rac2(-/-) mice showed i) worsened clinical symptoms (days 13-18), ii) increased crypt hyperplasia at days 11 and 22 (a time when crypt hyperplasia was largely resolved in wild-type mice; WT), and iii) marked mononuclear cell infiltration characterized by higher numbers of T (CD3(+)) cells (day 22), compared to WT-infected mice. Moreover, splenocytes harvested from infected Rac2(-/-) mice and stimulated in vitro with C. rodentium lysate produced considerably higher levels of interferon-γ and interleukin-17A. The augmented responses observed in Rac2(-/-) mice did not appear to stem from Rac2's role in NADPH oxidase-driven reactive oxygen species production as no differences in crypt hyperplasia, nor inflammation, were observed in infected NOX2(-/-) mice compared to WT. Collectively, our findings demonstrate that Rac2(-/-) mice develop more severe disease when subjected to a C. rodentium-induced model of infectious colitis, and suggest that impaired Rac2 function may promote the development of IBD in humans.


Asunto(s)
Citrobacter rodentium/patogenicidad , Colitis/etiología , Colitis/metabolismo , Infecciones por Enterobacteriaceae/complicaciones , Proteínas de Unión al GTP rac/deficiencia , Animales , Colitis/genética , Colitis/patología , Colon/microbiología , Masculino , Ratones , Ratones Noqueados , Proteínas de Unión al GTP rac/genética , Proteína RCA2 de Unión a GTP
12.
Cell Death Dis ; 4: e558, 2013 Mar 21.
Artículo en Inglés | MEDLINE | ID: mdl-23519127

RESUMEN

To investigate the importance of the Ras-homologous GTPase Rac1 for the hepatic response to genotoxic insults and liver aging, rac1 was deleted in liver of mice by Mx1-Cre-based recombination. Knockout of rac1 caused complex changes in basal as well as doxorubicin and ionizing radiation-induced mRNA expression of various genotoxic stress response-related genes, including hspa1b, rad51, wrn and xpc. Rac1 deletion protected the liver from acute toxicity following doxorubicin treatment. Moreover, the level of S139 phosphorylated histone H2AX (γH2AX), which is indicative of DNA damage, and mRNA expression of pro-inflammatory (IL-6) and pro-fibrotic (CTGF, TGFß, αSMA) factors were mitigated in rac1 knockout animals. By contrast, lack of rac1 promoted subacute hepatotoxicity, which was determined 3 weeks after injection of multiple low doses of doxorubicin by assaying the γH2AX level, mitotic index and pro-fibrotic gene expression. Regarding ionizing radiation, rac1 deficiency had no major effects on DNA damage induction or acute pro-inflammatory and pro-fibrotic stress responses. Mice lacking hepatic rac1 for extended period of time (15 months) revealed increased mRNA expression of fibrosis-related factors (CTGF, TGFß, collagen, MMP1) and fibrotic tissue remodeling. In addition, protein expression of the senescence marker p16 was enhanced in the absence of rac1. Taken together, the data provide evidence that Rac1 is required for doxorubicin-induced DNA damage induction. It is also involved in both the acute and delayed inflammatory and fibrotic stress response in the liver following doxorubicin, but not ionizing radiation, treatment and, furthermore, protects against endogenous liver aging.


Asunto(s)
Envejecimiento/genética , Doxorrubicina/toxicidad , Hígado/metabolismo , Mutágenos/toxicidad , Neuropéptidos/genética , Proteínas de Unión al GTP rac/genética , Actinas/genética , Actinas/metabolismo , Envejecimiento/efectos de los fármacos , Envejecimiento/efectos de la radiación , Animales , Apoptosis/efectos de los fármacos , Apoptosis/efectos de la radiación , Factor de Crecimiento del Tejido Conjuntivo/genética , Factor de Crecimiento del Tejido Conjuntivo/metabolismo , Daño del ADN , Femenino , Fibrosis , Rayos gamma , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/efectos de la radiación , Histonas/genética , Histonas/metabolismo , Hígado/efectos de los fármacos , Hígado/patología , Hígado/efectos de la radiación , Masculino , Ratones , Ratones Noqueados , Neuropéptidos/deficiencia , Estrés Oxidativo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/efectos de la radiación , Factor de Crecimiento Transformador beta/genética , Factor de Crecimiento Transformador beta/metabolismo , Proteínas de Unión al GTP rac/deficiencia , Proteína de Unión al GTP rac1
13.
Cardiovasc Res ; 97(1): 77-87, 2013 Jan 01.
Artículo en Inglés | MEDLINE | ID: mdl-23027656

RESUMEN

AIMS: Doxorubicin causes damage to the heart, often leading to irreversible cardiomyopathy, which is fatal. Reactive oxygen species (ROS) or oxidative stress is involved in cardiomyocyte death, contributing to doxorubicin-induced cardiotoxicity. This study investigated the role of Rac1, an important subunit of NADPH oxidase, in doxorubicin-induced cardiotoxicity and the underlying mechanisms. METHODS AND RESULTS: In a mouse model of acute doxorubicin-induced cardiotoxicity, cardiomyocyte-specific deletion of Rac1 inhibited NADPH oxidase activation and ROS production, prevented cardiac cell death, and improved myocardial function in Rac1 knockout mice. Therapeutic administration of the specific Rac1 inhibitor NSC23766 achieved similar cardio-protective effects in doxorubicin-stimulated mice. In rat cardiomyoblasts (H9c2 cells) and cultured neonatal mouse cardiomyocytes, Rac1 inhibition attenuated apoptosis as evidenced by decreases in caspase-3 activity and DNA fragmentation in response to doxorubicin, which correlated with a reduction in ROS production and down-regulation of p53 acetylation and histone H2AX phosphorylation. In contrast, overexpression of Rac1 enhanced apoptosis. Doxorubicin also inhibited the activity of classical histone deacetylases (HDAC), which was preserved by Rac1 inhibition and further decreased by Rac1 overexpression. Interestingly, scavenging ROS mitigated apoptosis but did not change HDAC activity and p53 acetylation stimulated by doxorubicin, suggesting both ROS-dependent and -independent pathways are involved in Rac1-mediated cardiotoxicity. Furthermore, the HDAC inhibitor trichostatin A enhanced apoptosis, p53 acetylation and H2AX phosphorylation in doxorubicin-treated cardiomyocytes. CONCLUSIONS: Rac1 signalling contributes to doxorubicin-induced cardiotoxicity through both a ROS-dependent mechanism and ROS-independent HDAC/p53 signalling in cardiomyocytes. Thus, inhibition of Rac1 may be a useful therapy for doxorubicin-induced cardiotoxicity.


Asunto(s)
Doxorrubicina/toxicidad , Cardiopatías/inducido químicamente , Miocitos Cardíacos/efectos de los fármacos , Neuropéptidos/metabolismo , Estrés Oxidativo/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal/efectos de los fármacos , Proteínas de Unión al GTP rac/metabolismo , Proteína de Unión al GTP rac1/metabolismo , Acetilación , Animales , Apoptosis , Caspasa 3/metabolismo , Línea Celular , Fragmentación del ADN , Modelos Animales de Enfermedad , Activación Enzimática , Inhibidores Enzimáticos/farmacología , Depuradores de Radicales Libres/farmacología , Cardiopatías/genética , Cardiopatías/metabolismo , Cardiopatías/patología , Cardiopatías/prevención & control , Inhibidores de Histona Desacetilasas/farmacología , Histona Desacetilasas/metabolismo , Histonas/metabolismo , Ratones , Ratones Noqueados , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/patología , NADPH Oxidasas/metabolismo , Neuropéptidos/antagonistas & inhibidores , Neuropéptidos/deficiencia , Neuropéptidos/genética , Fosforilación , Ratas , Transfección , Proteína p53 Supresora de Tumor/metabolismo , Proteínas de Unión al GTP rac/antagonistas & inhibidores , Proteínas de Unión al GTP rac/deficiencia , Proteínas de Unión al GTP rac/genética
14.
Am J Physiol Cell Physiol ; 304(1): C102-11, 2013 Jan 01.
Artículo en Inglés | MEDLINE | ID: mdl-23135700

RESUMEN

The epithelial Na(+) channel (ENaC) is a key transporter participating in the fine tuning of Na(+) reabsorption in the nephron. ENaC activity is acutely upregulated by epidermal growth factor (EGF), insulin, and insulin-like growth factor-1 (IGF-1). It was also proposed that reactive oxygen species (ROS) have a stimulatory effect on ENaC. Here we studied whether effects of EGF, insulin, and IGF-1 correlate with ROS production in the mouse cortical collecting duct (mpkCCD(c14)) cells. Western blotting confirmed the expression of the NADPH oxidase complex subunits in these cells. Treatment of mpkCCD(c14) cells with EGF, insulin, or IGF-1 evoked an increase in ROS production as measured by CM-H(2)DCF-DA fluorescence. ROS production caused by a xanthine-xanthine oxidase reaction also resulted in a significant elevation in short-circuit current through the mpkCCD(c14) monolayer. Transepithelial current measurements showed an acute increase of amiloride-sensitive current through the mpkCCD(c14) monolayer in response to EGF, insulin, or IGF-1. Pretreatment with the nonselective NADPH oxidase activity inhibitor apocynin blunted both ROS production and increase in ENaC-mediated current in response to these drugs. To further test whether NADPH oxidase subunits are involved in the effect of EGF, we used a stable M-1 cell line with a knockdown of Rac1, which is one of the key subunits of the NADPH oxidase complex, and measured amiloride-sensitive currents in response to EGF. In contrast to control cells, EGF had no effect in Rac1 knockdown cells. We hypothesize that EGF, insulin, and IGF-1 have a common stimulatory effect on ENaC mediated by ROS production.


Asunto(s)
Factor de Crecimiento Epidérmico/fisiología , Canales Epiteliales de Sodio/metabolismo , Factor I del Crecimiento Similar a la Insulina/fisiología , Insulina/fisiología , Túbulos Renales Colectores/metabolismo , Animales , Línea Celular Transformada , Canales Epiteliales de Sodio/fisiología , Hipoglucemiantes/química , Hipoglucemiantes/metabolismo , Túbulos Renales Colectores/citología , Túbulos Renales Colectores/fisiología , Ratones , Neuropéptidos/deficiencia , Neuropéptidos/genética , Estrés Oxidativo/fisiología , Especies Reactivas de Oxígeno/metabolismo , Proteínas de Unión al GTP rac/deficiencia , Proteínas de Unión al GTP rac/genética , Proteína de Unión al GTP rac1
15.
Arterioscler Thromb Vasc Biol ; 32(11): 2761-8, 2012 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-22995516

RESUMEN

OBJECTIVE: The platelet receptor for von Willebrand factor, the glycoprotein Ib-IX (GPIb-IX) complex, mediates platelet adhesion at sites of vascular injury and transmits signals leading to platelet activation. von Willebrand factor/GPIb-IX interaction sequentially activates the Src family kinase Lyn (SFK), phosphoinositide 3-kinase (PI3K), and Akt, leading to activation of integrin α(IIb)ß(3) and integrin-dependent stable platelet adhesion and aggregation. It remains unclear how Lyn activates the PI3K/Akt pathway after ligand binding to GPIb-IX. METHODS AND RESULTS: Using platelet-specific Rac1(-/-) mice and the Rac1 inhibitor NSC23766, we examined the role of Rac1 in GPIb-IX-dependent platelet activation. Rac1(-/-) mouse platelets and NSC23766-treated human platelets were defective in GPIb-dependent stable adhesion to von Willebrand factor under shear stress, integrin activation, thromboxane A(2) synthesis, and platelet aggregation. Interestingly, GPIb-induced activation of Rac1 and the guanine nucleotide exchange factor for Rac1, Vav, was abolished in both Lyn(-/-) and SFK inhibitor-treated platelets but was unaffected by the PI3K inhibitor LY294002, indicating that Lyn mediates activation of Vav and Rac1 independently of PI3K. Furthermore, GPIb-induced activation of Akt was abolished in Rac1-deficient platelets, suggesting that Rac1 is upstream of the PI3K/Akt pathway. CONCLUSIONS: A Lyn-Vav-Rac1-PI3K-Akt pathway mediates von Willebrand factor-induced activation of integrin α(IIb)ß(3) to promote GPIb-IX-dependent platelet activation.


Asunto(s)
Plaquetas/metabolismo , Neuropéptidos/metabolismo , Adhesividad Plaquetaria , Agregación Plaquetaria , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/metabolismo , Complejo GPIb-IX de Glicoproteína Plaquetaria/metabolismo , Transducción de Señal , Proteínas de Unión al GTP rac/metabolismo , Animales , Plaquetas/efectos de los fármacos , Activación Enzimática , Inhibidores Enzimáticos/farmacología , Humanos , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Noqueados , Neuropéptidos/antagonistas & inhibidores , Neuropéptidos/deficiencia , Neuropéptidos/genética , Fosfatidilinositol 3-Quinasa/metabolismo , Inhibidores de las Quinasa Fosfoinosítidos-3 , Fosforilación , Adhesividad Plaquetaria/efectos de los fármacos , Agregación Plaquetaria/efectos de los fármacos , Proteínas Proto-Oncogénicas c-vav/metabolismo , Transducción de Señal/efectos de los fármacos , Tromboxano A2/metabolismo , Factores de Tiempo , Proteínas de Unión al GTP rac/antagonistas & inhibidores , Proteínas de Unión al GTP rac/deficiencia , Proteínas de Unión al GTP rac/genética , Proteína de Unión al GTP rac1 , Familia-src Quinasas/antagonistas & inhibidores , Familia-src Quinasas/genética , Familia-src Quinasas/metabolismo , Factor de von Willebrand/metabolismo
16.
J Cell Sci ; 125(Pt 22): 5379-90, 2012 Nov 15.
Artículo en Inglés | MEDLINE | ID: mdl-22956547

RESUMEN

Crosstalk between keratinocytes and immune cells is crucial for the immunological barrier function of the skin, and aberrant crosstalk contributes to inflammatory skin diseases. Using mice with a keratinocyte-restricted deletion of the RAC1 gene we found that RAC1 in keratinocytes plays an important role in modulating the interferon (IFN) response in skin. These RAC1 mutant mice showed increased sensitivity in an irritant contact dermatitis model, abnormal keratinocyte differentiation, and increased expression of immune response genes including the IFN signal transducer STAT1. Loss of RAC1 in keratinocytes decreased actin polymerization in vivo and in vitro and caused Arp2/3-dependent expression of STAT1, increased interferon sensitivity and upregulation of aberrant keratinocyte differentiation markers. This can be inhibited by the AP-1 inhibitor tanshinone IIA. Loss of RAC1 makes keratinocytes hypersensitive to inflammatory stimuli both in vitro and in vivo, suggesting a major role for RAC1 in regulating the crosstalk between the epidermis and the immune system.


Asunto(s)
Complejo 2-3 Proteico Relacionado con la Actina/metabolismo , Queratinocitos/enzimología , Leucocitos/metabolismo , Neuropéptidos/metabolismo , Factor de Transcripción STAT1/metabolismo , Proteínas de Unión al GTP rac/metabolismo , Abietanos/farmacología , Citoesqueleto de Actina/efectos de los fármacos , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Animales , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/genética , Activación Enzimática/efectos de los fármacos , Epidermis/efectos de los fármacos , Epidermis/enzimología , Epidermis/patología , Epidermis/ultraestructura , Regulación de la Expresión Génica/efectos de los fármacos , Inflamación/patología , Interferón gamma/farmacología , Queratinocitos/efectos de los fármacos , Queratinocitos/patología , Queratinocitos/ultraestructura , Leucocitos/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neuropéptidos/deficiencia , Polimerizacion/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Piel/efectos de los fármacos , Piel/metabolismo , Piel/patología , Acetato de Tetradecanoilforbol/farmacología , Proteínas de Unión al GTP rac/deficiencia , Proteína de Unión al GTP rac1
17.
PLoS One ; 7(9): e44358, 2012.
Artículo en Inglés | MEDLINE | ID: mdl-22970203

RESUMEN

The Rho GTPases Rac1 and Cdc42 regulate a variety of cellular functions by signaling to different signal pathways. It is believed that the presence of a specific effector at the location of GTPase activation determines the route of downstream signaling. We previously reported about EGF-induced Ser-71 phosphorylation of Rac1/Cdc42. By using the phosphomimetic S71E-mutants of Rac1 and Cdc42 we investigated the impact of Ser-71 phosphorylation on binding to selected effector proteins. Binding of the constitutively active (Q61L) variants of Rac1 and Cdc42 to their specific interaction partners Sra-1 and N-WASP, respectively, as well as to their common effector protein PAK was abrogated when Ser-71 was exchanged to glutamate as phosphomimetic substitution. Interaction with their common effector proteins IQGAP1/2/3 or MRCK alpha was, however, hardly affected. This ambivalent behaviour was obvious in functional assays. In contrast to Rac1 Q61L, phosphomimetic Rac1 Q61L/S71E was not able to induce increased membrane ruffling. Instead, Rac1 Q61L/S71E allowed filopodia formation, which is in accordance with abrogation of the dominant Sra-1/Wave signalling pathway. In addition, in contrast to Rac1 transfected cells Rac1 S71E failed to activate PAK1/2. On the other hand, Rac1 Q61L/S71E was as effective in activation of NF-kappaB as Rac1 Q61L, illustrating positive signal transduction of phosphorylated Rac1. Together, these data suggest that phosphorylation of Rac1 and Cdc42 at serine-71 represents a reversible mechanism to shift specificity of GTPase/effector coupling, and to preferentially address selected downstream pathways.


Asunto(s)
Neuropéptidos/metabolismo , Fosfoserina/metabolismo , Transducción de Señal , Proteínas de Unión al GTP rac/metabolismo , Animales , Activación Enzimática , Células HEK293 , Humanos , Ratones , Proteínas Mutantes/metabolismo , FN-kappa B/metabolismo , Neuropéptidos/deficiencia , Fenotipo , Fosforilación , Unión Proteica , Seudópodos/metabolismo , Relación Estructura-Actividad , Proteína de Unión al GTP cdc42/metabolismo , Quinasas p21 Activadas/metabolismo , Proteínas de Unión al GTP rac/deficiencia , Proteína de Unión al GTP rac1
18.
Cereb Cortex ; 22(3): 680-92, 2012 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-21690261

RESUMEN

Cortical γ-aminobutyric acid (GABA)ergic interneurons are characterized by extraordinary neurochemical and functional diversity. Although recent studies have uncovered some of the molecular components underlying interneuron development, including the cellular and molecular mechanisms guiding their migration to the cortex, the intracellular components involved are still unknown. Rac1, a member of the Rac subfamily of Rho-GTPases, has been implicated in various cellular processes such as cell cycle dynamics, axonogenesis, and migration. In this study, we have addressed the specific role of Rac1 in interneuron progenitors originating in the medial ganglionic eminence, via Cre/loxP technology. We show that ablation of Rac1 from Nkx2.1-positive progenitors, results in a migratory impairment. As a consequence, only half of GABAergic interneurons are found in the postnatal cortex. The rest remain aggregated in the ventral telencephalon and show morphological defects in their growing processes in vitro. Ablation of Rac1 from postmitotic progenitors does not result in similar defects, thus underlying a novel cell autonomous and stage-specific requirement for Rac1 activity, within proliferating progenitors of cortical interneurons. Rac1 is necessary for their transition from G1 to S phase, at least in part by regulating cyclin D levels and retinoblastoma protein phosphorylation.


Asunto(s)
Puntos de Control del Ciclo Celular , Movimiento Celular , Corteza Cerebral/fisiología , Interneuronas/fisiología , Eminencia Media/fisiología , Células-Madre Neurales/fisiología , Neuropéptidos/fisiología , Proteínas de Unión al GTP rac/fisiología , Animales , Puntos de Control del Ciclo Celular/genética , Movimiento Celular/genética , Corteza Cerebral/citología , Corteza Cerebral/patología , Femenino , Fase G1/genética , Interneuronas/citología , Interneuronas/patología , Eminencia Media/citología , Eminencia Media/patología , Ratones , Ratones Noqueados , Células-Madre Neurales/citología , Células-Madre Neurales/patología , Neuropéptidos/deficiencia , Neuropéptidos/genética , Embarazo , Cultivo Primario de Células , Proteínas de Unión al GTP rac/deficiencia , Proteínas de Unión al GTP rac/genética , Proteína de Unión al GTP rac1
19.
Blood ; 118(19): 5235-45, 2011 Nov 10.
Artículo en Inglés | MEDLINE | ID: mdl-21940819

RESUMEN

The Rac family of small Rho GTPases coordinates diverse cellular functions in hematopoietic cells including adhesion, migration, cytoskeleton rearrangements, gene transcription, proliferation, and survival. The integrity of Rac signaling has also been found to critically regulate cellular functions in the initiation and maintenance of hematopoietic malignancies. Using an in vivo gene targeting approach, we demonstrate that Rac2, but not Rac1, is critical to the initiation of acute myeloid leukemia in a retroviral expression model of MLL-AF9 leukemogenesis. However, loss of either Rac1 or Rac2 is sufficient to impair survival and growth of the transformed MLL-AF9 leukemia. Rac2 is known to positively regulate expression of Bcl-2 family proteins toward a prosurvival balance. We demonstrate that disruption of downstream survival signaling through antiapoptotic Bcl-2 proteins is implicated in mediating the effects of Rac2 deficiency in MLL-AF9 leukemia. Indeed, overexpression of Bcl-xL is able to rescue the effects of Rac2 deficiency and MLL-AF9 cells are exquisitely sensitive to direct inhibition of Bcl-2 family proteins by the BH3-mimetic, ABT-737. Furthermore, concurrent exposure to NSC23766, a small-molecule inhibitor of Rac activation, increases the apoptotic effect of ABT-737, indicating the Rac/Bcl-2 survival pathway may be targeted synergistically.


Asunto(s)
Leucemia Bifenotípica Aguda/tratamiento farmacológico , Leucemia Bifenotípica Aguda/metabolismo , Neuropéptidos/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-bcl-2/antagonistas & inhibidores , Proteínas de Unión al GTP rac/antagonistas & inhibidores , Aminoquinolinas/farmacología , Animales , Compuestos de Bifenilo/farmacología , Línea Celular Tumoral , Expresión Génica , Técnicas de Silenciamiento del Gen , Humanos , Leucemia Bifenotípica Aguda/genética , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Ratones Noqueados , Ratones SCID , Ratones Transgénicos , Neuropéptidos/deficiencia , Neuropéptidos/genética , Nitrofenoles/farmacología , Piperazinas/farmacología , Pirimidinas/farmacología , Transducción de Señal , Sulfonamidas/farmacología , Trasplante Heterólogo , Proteína bcl-X/genética , Proteínas de Unión al GTP rac/deficiencia , Proteínas de Unión al GTP rac/genética , Proteína de Unión al GTP rac1 , Proteína RCA2 de Unión a GTP
20.
Dev Biol ; 360(1): 30-43, 2011 Dec 01.
Artículo en Inglés | MEDLINE | ID: mdl-21945075

RESUMEN

Morphogenesis and shape of the ocular lens depend on epithelial cell elongation and differentiation into fiber cells, followed by the symmetric and compact organization of fiber cells within an enclosed extracellular matrix-enriched elastic capsule. The cellular mechanisms orchestrating these different events however, remain obscure. We investigated the role of the Rac1 GTPase in these processes by targeted deletion of expression using the conditional gene knockout (cKO) approach. Rac1 cKO mice were derived from two different Cre (Le-Cre and MLR-10) transgenic mice in which lens-specific Cre expression starts at embryonic day 8.75 and 10.5, respectively, in both the lens epithelium and fiber cells. The Le-Cre/Rac1 cKO mice exhibited an early-onset (E12.5) and severe lens phenotype compared to the MLR-10/Rac1 cKO (E15.5) mice. While the Le-Cre/Rac1 cKO lenses displayed delayed primary fiber cell elongation, lenses from both Rac1 cKO strains were characterized by abnormal shape, impaired secondary fiber cell migration, sutural defects and thinning of the posterior capsule which often led to rupture. Lens fiber cell N-cadherin/ß-catenin/Rap1/Nectin-based cell-cell junction formation and WAVE-2/Abi-2/Nap1-regulated actin polymerization were impaired in the Rac1 deficient mice. Additionally, the Rac1 cKO lenses were characterized by a shortened epithelial sheet, reduced levels of extracellular matrix (ECM) proteins and increased apoptosis. Taken together, these data uncover the essential role of Rac1 GTPase activity in establishment and maintenance of lens shape, suture formation and capsule integrity, and in fiber cell migration, adhesion and survival, via regulation of actin cytoskeletal dynamics, cell adhesive interactions and ECM turnover.


Asunto(s)
Cristalino/embriología , Neuropéptidos/deficiencia , Proteínas de Unión al GTP rac/deficiencia , Actinas/metabolismo , Animales , Apoptosis/genética , Apoptosis/fisiología , Comunicación Celular/genética , Comunicación Celular/fisiología , Movimiento Celular/genética , Movimiento Celular/fisiología , Supervivencia Celular/genética , Supervivencia Celular/fisiología , Citoesqueleto/metabolismo , Células Epiteliales/patología , Células Epiteliales/fisiología , Femenino , Regulación del Desarrollo de la Expresión Génica , Cápsula del Cristalino/anomalías , Cápsula del Cristalino/citología , Cápsula del Cristalino/embriología , Cápsula del Cristalino/fisiología , Cristalino/anomalías , Cristalino/citología , Cristalino/fisiología , Ratones , Ratones Noqueados , Ratones Transgénicos , Neuropéptidos/genética , Neuropéptidos/fisiología , Fenotipo , Embarazo , Proteínas de Unión al GTP rac/genética , Proteínas de Unión al GTP rac/fisiología , Proteína de Unión al GTP rac1
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