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1.
Dev Dyn ; 251(3): 444-458, 2022 03.
Artículo en Inglés | MEDLINE | ID: mdl-34374463

RESUMEN

BACKGROUND: Proper guidance of neuronal axons to their targets is required to assemble neural circuits during the development of the nervous system. However, the mechanism by which the guidance of axonal growth cones is regulated by specific intermediaries activated by receptor signaling pathways to mediate cytoskeleton dynamics is unclear. Vav protein members have been proposed to mediate this process, prompting us to investigate their role in the limb selection of the axon trajectory of spinal lateral motor column (LMC) neurons. RESULTS: We found Vav2 and Vav3 expression in LMC neurons when motor axons grew into the limb. Vav2, but not Vav3, loss-of-function perturbed LMC pathfinding, while Vav2 gain-of-function exhibited the opposite effects, demonstrating that Vav2 plays an important role in motor axon growth. Vav2 knockdown also attenuated the redirectional phenotype of LMC axons induced by Dcc, but not EphA4, in vivo and lateral LMC neurite growth preference to Netrin-1 in vitro. This study showed that Vav2 knockdown and ectopic nonphosphorylable Vav2 mutant expression abolished the Src-induced stronger growth preference of lateral LMC neurites to Netrin-1, suggesting that Vav2 is downstream of Src in this context. CONCLUSIONS: Vav2 is essential for Netrin-1-regulated LMC motor axon pathfinding through Src interaction.


Asunto(s)
Orientación del Axón , Conos de Crecimiento , Netrina-1 , Proteínas Proto-Oncogénicas c-vav , Animales , Orientación del Axón/fisiología , Axones/fisiología , Conos de Crecimiento/fisiología , Neuronas Motoras/fisiología , Netrina-1/fisiología , Proteínas Proto-Oncogénicas c-vav/fisiología
2.
Sci Rep ; 10(1): 7024, 2020 04 27.
Artículo en Inglés | MEDLINE | ID: mdl-32341385

RESUMEN

Vav1 regulates Rac activation as a hematopoietic-specific Rho/Rac-family guanine nucleotide exchange factor. Rac is a subfamily of Rho GTPases that regulates the bone-resorbing capacity of osteoclasts (OCs). In this study, we show that hematopoietic-specific Rac2 and Vav1 play opposing roles by enhancing or attenuating OC differentiation, respectively. This was demonstrated by higher and lower bone density in the femurs from Rac2-deficient (Rac2-/-) and Vav1-deficient (Vav1-/-) mice, respectively, compared to the wild-type (WT) mice. Accordingly, Rac2-/- cells displayed low numbers of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells (41%) compared to WT cells, whereas, Vav1-/- cells showed high TRAP-positive cell numbers (150%), and the double-knockout Rac2-/-Vav1-/- mice nullified the effects on OC numbers achieved by the individual knockouts. These reciprocal roles of Rac2 and Vav1 in OC differentiation were confirmed by reduced and increased levels of OC-specific markers, such as TRAP, calcitonin receptor, cathepsin K, and DC-STAMP in the Rac2-/- and Vav1-/- OCs, respectively. Our findings of decrease and increase in actin ring formation and αvß3 integrin-mediated adhesion in Rac2-/- and Vav1-/- mice, respectively, suggest that Vav1 and its downstream GTPase, Rac2, may counteract to fine-tune OC differentiation and bone resorption.


Asunto(s)
Diferenciación Celular/fisiología , Hematopoyesis , Osteoclastos/citología , Proteínas Proto-Oncogénicas c-vav/fisiología , Proteínas de Unión al GTP rac/fisiología , Animales , Ratones Noqueados , Osteoclastos/metabolismo , Proteína RCA2 de Unión a GTP
3.
BMB Rep ; 52(11): 659-664, 2019 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-31072447

RESUMEN

Vav1 is a Rho/Rac guanine nucleotide exchange factor primarily expressed in hematopoietic cells. In this study, we investigated the potential role of Vav1 in osteoclast (OC) differentiation by comparing the ability of bone marrow mononuclear cells (BMMCs) obtained from Vav1-deficient (Vav1-/-) and wild-type (WT) mice to differentiate into mature OCs upon stimulation with macrophage colony stimulating factor and receptor activator of nuclear kappa B ligand in vitro. Our results suggested that Vav1 deficiency promoted the differentiation of BMMCs into OCs, as indicated by the increased expression of tartrate-resistant acid phosphatase, cathepsin K, and calcitonin receptor. Therefore, Vav1 may play a negative role in OC differentiation. This hypothesis was supported by the observation of more OCs in the femurs of Vav1-/- mice than in WT mice. Furthermore, the bone status of Vav1-/- mice was analyzed in situ and the femurs of Vav1-/- mice appeared abnormal, with poor bone density and fewer number of trabeculae. In addition, Vav1-deficient OCs showed stronger adhesion to vitronectin, an αvß3 integrin ligand important in bone resorption. Thus, Vav1 may inhibit OC differentiation and protect against bone resorption. [BMB Reports 2019; 52(11): 659-664].


Asunto(s)
Osteoclastos/metabolismo , Proteínas Proto-Oncogénicas c-vav/metabolismo , Animales , Células de la Médula Ósea/metabolismo , Resorción Ósea/metabolismo , Huesos/metabolismo , Diferenciación Celular/fisiología , Integrina alfaVbeta3/metabolismo , Factor Estimulante de Colonias de Macrófagos/metabolismo , Factor Estimulante de Colonias de Macrófagos/farmacología , Masculino , Glicoproteínas de Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Osteoclastos/citología , Proteínas Proto-Oncogénicas c-vav/genética , Proteínas Proto-Oncogénicas c-vav/fisiología , Ligando RANK/metabolismo , Ligando RANK/fisiología , Receptor Activador del Factor Nuclear kappa-B/metabolismo , Fosfatasa Ácida Tartratorresistente
4.
Arterioscler Thromb Vasc Biol ; 39(2): 137-149, 2019 02.
Artículo en Inglés | MEDLINE | ID: mdl-30580573

RESUMEN

Objective- During atherosclerosis, LDLs (low-density lipoproteins) accumulate in the arteries, where they become modified, aggregated, and retained. Such deposits of aggregated LDL (agLDL) can be recognized by macrophages, which attempt to digest and clear them. AgLDL catabolism promotes internalization of cholesterol and foam cell formation, which leads to the progression of atherosclerosis. Therapeutic blockade of this process may delay disease progression. When macrophages interact with agLDL in vitro, they form a novel extracellular, hydrolytic compartment-the lysosomal synapse (LS)-aided by local actin polymerization to digest agLDL. Here, we investigated the specific regulators involved in actin polymerization during the formation of the LS. Approach and Results- We demonstrate in vivo that atherosclerotic plaque macrophages contacting agLDL deposits polymerize actin and form a compartment strikingly similar to those made in vitro. Live cell imaging revealed that macrophage cortical F-actin depolymerization is required for actin polymerization to support the formation of the LS. This depolymerization is cofilin-1 dependent. Using siRNA-mediated silencing, pharmacological inhibition, genetic knockout, and stable overexpression, we elucidate key roles for Cdc42 Rho GTPase and GEF (guanine nucleotide exchange factor) Vav in promoting actin polymerization during the formation of the LS and exclude a role for Rac1. Conclusions- These results highlight critical roles for dynamic macrophage F-actin rearrangement and polymerization via cofilin-1, Vav, and Cdc42 in LS formation, catabolism of agLDL, and foam cell formation. These proteins might represent therapeutic targets to treat atherosclerotic disease.


Asunto(s)
Actinas/química , Lipoproteínas LDL/metabolismo , Macrófagos/metabolismo , Agregado de Proteínas , Proteínas Proto-Oncogénicas c-vav/fisiología , Proteína de Unión al GTP cdc42/fisiología , Animales , Lipoproteínas LDL/química , Ratones , Polimerizacion , Células RAW 264.7
5.
PLoS Comput Biol ; 14(8): e1006321, 2018 08.
Artículo en Inglés | MEDLINE | ID: mdl-30071020

RESUMEN

Cellular morphology and associated morphodynamics are widely used for qualitative and quantitative assessments of cell state. Here we implement a framework to profile cellular morphodynamics based on an adaptive decomposition of local cell boundary motion into instantaneous frequency spectra defined by the Hilbert-Huang transform (HHT). Our approach revealed that spontaneously migrating cells with approximately homogeneous molecular makeup show remarkably consistent instantaneous frequency distributions, though they have markedly heterogeneous mobility. Distinctions in cell edge motion between these cells are captured predominantly by differences in the magnitude of the frequencies. We found that acute photo-inhibition of Vav2 guanine exchange factor, an activator of the Rho family of signaling proteins coordinating cell motility, produces significant shifts in the frequency distribution, but does not affect frequency magnitude. We therefore concluded that the frequency spectrum encodes the wiring of the molecular circuitry that regulates cell boundary movements, whereas the magnitude captures the activation level of the circuitry. We also used HHT spectra as multi-scale spatiotemporal features in statistical region merging to identify subcellular regions of distinct motion behavior. In line with our conclusion that different HHT spectra relate to different signaling regimes, we found that subcellular regions with different morphodynamics indeed exhibit distinct Rac1 activities. This algorithm thus can serve as an accurate and sensitive classifier of cellular morphodynamics to pinpoint spatial and temporal boundaries between signaling regimes.


Asunto(s)
Movimiento Celular/fisiología , Forma de la Célula/fisiología , Análisis Espacio-Temporal , Algoritmos , Animales , Células COS , Chlorocebus aethiops , Análisis de Datos , Factores de Intercambio de Guanina Nucleótido , Movimiento (Física) , Proteínas Proto-Oncogénicas c-vav/fisiología , Transducción de Señal , Análisis Espacial
6.
J Cell Biol ; 217(8): 2813-2830, 2018 08 06.
Artículo en Inglés | MEDLINE | ID: mdl-29858212

RESUMEN

Through multiple cell-cell and cell-matrix interactions, epithelial and endothelial sheets form tight barriers. Modulators of the cytoskeleton contribute to barrier stability and act as rheostats of vascular permeability. In this study, we sought to identify cytoskeletal regulators that underlie barrier diversity across vessels. To achieve this, we correlated functional and structural barrier features to gene expression of endothelial cells (ECs) derived from different vascular beds. Within a subset of identified candidates, we found that the guanosine nucleotide exchange factor Vav3 was exclusively expressed by microvascular ECs and was closely associated with a high-resistance barrier phenotype. Ectopic expression of Vav3 in large artery and brain ECs significantly enhanced barrier resistance and cortical rearrangement of the actin cytoskeleton. Mechanistically, we found that the barrier effect of Vav3 is dependent on its Dbl homology domain and downstream activation of Rap1. Importantly, inactivation of Vav3 in vivo resulted in increased vascular leakage, highlighting its function as a key regulator of barrier stability.


Asunto(s)
Citoesqueleto/metabolismo , Proteínas Proto-Oncogénicas c-vav/fisiología , Animales , Células Endoteliales/citología , Células Endoteliales/metabolismo , Humanos , Ratones Endogámicos C57BL , Microvasos/citología , Microvasos/metabolismo , Proteínas Proto-Oncogénicas c-vav/genética , Proteínas Proto-Oncogénicas c-vav/metabolismo
7.
Eur J Clin Invest ; 48 Suppl 2: e12939, 2018 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-29682742

RESUMEN

Rac-GTPases and their Rac-GEF activators play important roles in the recruitment and host defence functions of neutrophils. These proteins control the activation of adhesion molecules and the cytoskeletal dynamics that enable the adhesion, migration and tissue recruitment of neutrophils. They also regulate the effector functions that allow neutrophils to kill bacterial and fungal pathogens, and to clear debris. This review focuses on the roles of Rac-GTPases and Rac-GEFs in neutrophil adhesion, migration and recruitment.


Asunto(s)
Neutrófilos/fisiología , Factores de Intercambio de Guanina Nucleótido Rho/fisiología , Proteína de Unión al GTP rac1/fisiología , Adhesión Celular/fisiología , Movimiento Celular/fisiología , Factores de Intercambio de Guanina Nucleótido/fisiología , Humanos , Infiltración Neutrófila/fisiología , Neutrófilos/enzimología , Proteínas Proto-Oncogénicas c-vav/fisiología , Transducción de Señal/fisiología , Proteína 1 de Invasión e Inducción de Metástasis del Linfoma-T/fisiología , Proteínas de Unión al GTP rac/fisiología
8.
Biochem Pharmacol ; 132: 9-17, 2017 05 15.
Artículo en Inglés | MEDLINE | ID: mdl-28202288

RESUMEN

Glucose-stimulated insulin secretion [GSIS] from the islet ß-cell involves a well-orchestrated interplay between metabolic and cationic events. It is well established that intracellular generation of adenine and guanine nucleotide triphosphates [e.g., ATP and GTP] represents one of the requisite signaling steps in GSIS. The small molecular mass GTP-binding proteins [G-proteins; e.g., Rac1 and Cdc42] have been shown to regulate islet ß-cell function including actin cytoskeletal remodeling and fusion of insulin granules with the plasma membrane for GSIS to occur. In this context, several regulatory factors for these G-proteins have been identified in the pancreatic ß-cell; these include guanine nucleotide exchange factors [GEFs] and guanine nucleotide dissociation inhibitors [GDI]. Recent pharmacological and molecular biological evidence identified Tiam1 and Vav2 as GEFs for Rac1 in promoting physiological insulin secretion. Paradoxically, emerging evidence in multiple cell types, including the islet ß-cell, suggests key roles for Rac1 in the onset of cellular dysfunction under conditions of metabolic stress and diabetes. Furthermore, functional inactivation of either Tiam1 or Vav2 appears to attenuate sustained activation of Rac1 and its downstream signaling events [activation of stress kinases] under conditions of metabolic stress. Together, these findings suggest both "friendly" and "non-friendly" roles for Tiam1/Vav2-Rac1 signaling axis in islet ß-cell in health and diabetes. Our current understanding of the field and the knowledge gaps that exist in this area of islet biology are heighted herein. Furthermore, potential caveats in the specificity and selectivity of pharmacological inhibitors that are available currently are discussed in this Commentary.


Asunto(s)
Factores de Intercambio de Guanina Nucleótido/fisiología , Islotes Pancreáticos/fisiopatología , Proteínas Proto-Oncogénicas c-vav/fisiología , Proteína de Unión al GTP rac1/fisiología , Animales , Humanos , Insulina/metabolismo , Secreción de Insulina , Islotes Pancreáticos/metabolismo , Transducción de Señal , Proteína 1 de Invasión e Inducción de Metástasis del Linfoma-T
9.
Genetics ; 202(3): 1153-66, 2016 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-26801183

RESUMEN

Sleep is evolutionarily conserved and required for organism homeostasis and survival. Despite this importance, the molecular and cellular mechanisms underlying sleep are not well understood. Caenorhabditis elegans exhibits sleep-like behavioral quiescence and thus provides a valuable, simple model system for the study of cellular and molecular regulators of this process. In C. elegans, epidermal growth factor receptor (EGFR) signaling is required in the neurosecretory neuron ALA to promote sleep-like behavioral quiescence after cellular stress. We describe a novel role for VAV-1, a conserved guanine nucleotide exchange factor (GEF) for Rho-family GTPases, in regulation of sleep-like behavioral quiescence. VAV-1, in a GEF-dependent manner, acts in ALA to suppress locomotion and feeding during sleep-like behavioral quiescence in response to cellular stress. Additionally, VAV-1 activity is required for EGF-induced sleep-like quiescence and normal levels of EGFR and secretory dense core vesicles in ALA. Importantly, the role of VAV-1 in promoting cellular stress-induced behavioral quiescence is vital for organism health because VAV-1 is required for normal survival after cellular stress.


Asunto(s)
Proteínas de Caenorhabditis elegans/fisiología , Caenorhabditis elegans/fisiología , Interneuronas/fisiología , Locomoción/fisiología , Proteínas Proto-Oncogénicas c-vav/fisiología , Transducción de Señal , Animales , Animales Modificados Genéticamente , Conducta Animal/fisiología , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Factor de Crecimiento Epidérmico/fisiología , Receptores ErbB/fisiología , Conducta Alimentaria/fisiología , Mutagénesis Sitio-Dirigida , Proteínas Proto-Oncogénicas c-vav/genética , Estrés Fisiológico
10.
J Proteome Res ; 14(7): 2963-75, 2015 Jul 02.
Artículo en Inglés | MEDLINE | ID: mdl-26043137

RESUMEN

Vav1, a Rac/Rho guanine nucleotide exchange factor and a critical component of the T-cell receptor (TCR) signaling cascade is tyrosine phosphorylated rapidly in response to T-cell activation. Vav1 has established roles in proliferation, cytokine secretion, Ca(2+) responses, and actin cytoskeleton regulation; however, its function in the regulation of phosphorylation of TCR components, including the ζ chain, the CD3 δ, ε, γ chains, and the associated kinases Lck and ZAP-70, is not well established. To obtain a more comprehensive picture of the role of Vav1 in receptor proximal signaling, we performed a wide-scale characterization of Vav1-dependent tyrosine phosphorylation events using quantitative phosphoproteomic analysis of Vav1-deficient T cells across a time course of TCR stimulation. Importantly, this study revealed a new function for Vav1 in the negative feedback regulation of the phosphorylation of immunoreceptor tyrosine-based activation motifs within the ζ chains, CD3 δ, ε, γ chains, as well as activation sites on the critical T cell tyrosine kinases Itk, Lck, and ZAP-70. Our study also uncovered a previously unappreciated role for Vav1 in crosstalk between the CD28 and TCR signaling pathways.


Asunto(s)
Antígenos CD28/metabolismo , Activación de Linfocitos/fisiología , Proteínas Proto-Oncogénicas c-vav/fisiología , Receptor Cross-Talk , Receptores de Antígenos de Linfocitos T/fisiología , Linfocitos T/inmunología , Humanos , Células Jurkat , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación , Proteínas Proto-Oncogénicas c-vav/metabolismo , Transducción de Señal , Proteína Tirosina Quinasa ZAP-70/metabolismo
11.
Oncogene ; 33(31): 4021-35, 2014 Jul 31.
Artículo en Inglés | MEDLINE | ID: mdl-24037532

RESUMEN

The aberrant activity of Ras homologous (Rho) family small GTPases (20 human members) has been implicated in cancer and other human diseases. However, in contrast to the direct mutational activation of Ras found in cancer and developmental disorders, Rho GTPases are activated most commonly in disease by indirect mechanisms. One prevalent mechanism involves aberrant Rho activation via the deregulated expression and/or activity of Rho family guanine nucleotide exchange factors (RhoGEFs). RhoGEFs promote formation of the active GTP-bound state of Rho GTPases. The largest family of RhoGEFs is comprised of the Dbl family RhoGEFs with 70 human members. The multitude of RhoGEFs that activate a single Rho GTPase reflects the very specific role of each RhoGEF in controlling distinct signaling mechanisms involved in Rho activation. In this review, we summarize the role of Dbl RhoGEFs in development and disease, with a focus on Ect2 (epithelial cell transforming squence 2), Tiam1 (T-cell lymphoma invasion and metastasis 1), Vav and P-Rex1/2 (PtdIns(3,4,5)P3 (phosphatidylinositol (3,4,5)-triphosphate)-dependent Rac exchanger).


Asunto(s)
Neoplasias/metabolismo , Factores de Intercambio de Guanina Nucleótido Rho/metabolismo , Proteínas de Unión al GTP rho/metabolismo , Animales , División Celular , Anomalías Congénitas/metabolismo , Regulación Neoplásica de la Expresión Génica , Enfermedades Genéticas Congénitas/metabolismo , Humanos , Proteínas Proto-Oncogénicas c-vav/fisiología
12.
J Cell Biol ; 203(6): 1003-19, 2013 Dec 23.
Artículo en Inglés | MEDLINE | ID: mdl-24368807

RESUMEN

Lymphocyte recruitment is regulated by signaling modules based on the activity of Rho and Rap small guanosine triphosphatases that control integrin activation by chemokines. We show that Janus kinase (JAK) protein tyrosine kinases control chemokine-induced LFA-1- and VLA-4-mediated adhesion as well as human T lymphocyte homing to secondary lymphoid organs. JAK2 and JAK3 isoforms, but not JAK1, mediate CXCL12-induced LFA-1 triggering to a high affinity state. Signal transduction analysis showed that chemokine-induced activation of the Rho module of LFA-1 affinity triggering is dependent on JAK activity, with VAV1 mediating Rho activation by JAKs in a Gαi-independent manner. Furthermore, activation of Rap1A by chemokines is also dependent on JAK2 and JAK3 activity. Importantly, activation of Rap1A by JAKs is mediated by RhoA and PLD1, thus establishing Rap1A as a downstream effector of the Rho module. Thus, JAK tyrosine kinases control integrin activation and dependent lymphocyte trafficking by bridging chemokine receptors to the concurrent and hierarchical activation of the Rho and Rap modules of integrin activation.


Asunto(s)
Integrinas/fisiología , Quinasas Janus/fisiología , Linfocitos T/fisiología , Proteínas de Unión al GTP rap/metabolismo , Proteínas de Unión al GTP rho/metabolismo , Adhesión Celular , Quimiocina CXCL12/metabolismo , Humanos , Integrina alfa4beta1/metabolismo , Integrina alfa4beta1/fisiología , Integrinas/metabolismo , Antígeno-1 Asociado a Función de Linfocito/metabolismo , Antígeno-1 Asociado a Función de Linfocito/fisiología , Fosfolipasa D/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Proteínas Proto-Oncogénicas c-vav/metabolismo , Proteínas Proto-Oncogénicas c-vav/fisiología , Transducción de Señal , Linfocitos T/metabolismo , Proteínas de Unión al GTP rap/fisiología , Proteínas de Unión al GTP rho/fisiología
13.
Transl Res ; 162(3): 181-90, 2013 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-23856093

RESUMEN

Ovarian cancer is the most lethal gynecological malignancy in the western world. Although patients with early-stage ovarian cancer generally have a good prognosis, approximately 20%-30% of patients will die of the disease, and 5-year recurrence rates are 25%-45%, highlighting the need for improved detection and treatment. We investigated the role of VAV1, a protein with guanine nucleotide exchange factor activity, which is associated with survival in patients with early-stage ovarian cancer (International of Obstetrics and Gynecology [FIGO] stages I and II). We analyzed 88 samples from patients with primary epithelial ovarian cancer, which were divided into FIGO stages I and II (n = 46), and III and IV (n = 42). Prognostic analysis revealed that upregulated VAV1 expression correlated significantly with poor prognosis in patients with early-stage epithelial ovarian cancer (P ≤ 0.05), but not with other clinicopathologic features. Stable overexpression of VAV1 in human high-grade serous ovarian cancer SKOV3 cells induced morphologic changes indicative of loss of intercellular adhesions and organized actin stress fibers. Western blotting and real-time reverse transcriptase-polymerase chain reaction demonstrated that these cells had downregulated E-cadherin protein and messenger RNA levels, respectively. This downregulation is associated with epithelial-mesenchymal transition (EMT) and invasive cancer. Furthermore, VAV1 overexpression in both SKOV3 and human ovarian surface epithelial cells demonstrated that its upregulation of an E-cadherin transcriptional repressor, Snail and Slug, was not confined to ovarian cancer cells. Conversely, knockdown of VAV1 by RNA interference reduced Snail and Slug. Our findings suggest that VAV1 may play a role in the EMT of ovarian cancer, and may serve as a potential therapeutic target.


Asunto(s)
Cadherinas/metabolismo , Transición Epitelial-Mesenquimal , Neoplasias Glandulares y Epiteliales/metabolismo , Neoplasias Ováricas/metabolismo , Proteínas Proto-Oncogénicas c-vav/fisiología , Factores de Transcripción/metabolismo , Carcinoma Epitelial de Ovario , Regulación hacia Abajo , Femenino , Humanos , Persona de Mediana Edad , Neoplasias Glandulares y Epiteliales/patología , Neoplasias Ováricas/patología , Factores de Transcripción de la Familia Snail
14.
Hepatology ; 58(2): 642-53, 2013 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-23468064

RESUMEN

UNLABELLED: Hepatocellular carcinoma (HCC) is characterized by active angiogenesis and metastasis, which account for rapid recurrence and poor survival. There is frequent down-regulation of miR-195 expression in HCC tissues. In this study, the role of miR-195 in HCC angiogenesis and metastasis was investigated with in vitro capillary tube formation and transwell assays, in vivo orthotopic xenograft mouse models, and human HCC specimens. Reduction of miR-195 in HCC tissues was significantly associated with increased angiogenesis, metastasis, and worse recurrence-free survival. Both gain-of-function and loss-of-function studies of in vitro models revealed that miR-195 not only suppressed the ability of HCC cells to promote the migration and capillary tube formation of endothelial cells but also directly repressed the abilities of HCC cells to migrate and invade extracellular matrix gel. Based on mouse models, we found that the induced expression of miR-195 dramatically reduced microvessel densities in xenograft tumors and repressed both intrahepatic and pulmonary metastasis. Subsequent investigations disclosed that miR-195 directly inhibited the expression of the proangiogenic factor vascular endothelial growth factor (VEGF) and the prometastatic factors VAV2 and CDC42. Knockdown of these target molecules of miR-195 phenocopied the effects of miR-195 restoration, whereas overexpression of these targets antagonized the function of miR-195. Furthermore, we revealed that miR-195 down-regulation resulted in enhanced VEGF levels in the tumor microenvironment, which subsequently activated VEGF receptor 2 signaling in endothelial cells and thereby promoted angiogenesis. Additionally, miR-195 down-regulation led to increases in VAV2 and CDC42 expression, which stimulated VAV2/Rac1/CDC42 signaling and lamellipodia formation and thereby facilitated the metastasis of HCC cells. CONCLUSION: miR-195 deregulation contributes to angiogenesis and metastasis in HCC. The restoration of miR-195 expression may be a promising strategy for HCC therapy.


Asunto(s)
Carcinoma Hepatocelular/fisiopatología , Neoplasias Hepáticas/fisiopatología , MicroARNs/fisiología , Metástasis de la Neoplasia/fisiopatología , Neovascularización Patológica/fisiopatología , Proteínas Proto-Oncogénicas c-vav/antagonistas & inhibidores , Factor A de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Proteína de Unión al GTP cdc42/antagonistas & inhibidores , Animales , Línea Celular Tumoral , Regulación hacia Abajo/fisiología , Xenoinjertos , Humanos , Técnicas In Vitro , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Proteínas Proto-Oncogénicas c-vav/fisiología , Transducción de Señal/fisiología , Factor A de Crecimiento Endotelial Vascular/fisiología , Proteína de Unión al GTP cdc42/fisiología , Proteína de Unión al GTP rac1/fisiología
16.
J Biol Chem ; 288(8): 5463-74, 2013 Feb 22.
Artículo en Inglés | MEDLINE | ID: mdl-23281476

RESUMEN

Elevated androgen receptor (AR) activity in castration-resistant prostate cancer may occur through increased levels of AR co-activator proteins. Vav3, a guanine nucleotide exchange factor, is up-regulated following progression to castration resistance in preclinical models and is overexpressed in a significant number of human prostate cancers. Vav3 is a novel co-activator of the AR. We sought to identify Vav3 binding partners in an effort to understand the molecular mechanisms underlying Vav3 enhancement of AR activity and to identify new therapeutic targets. The cell division cycle 37 homolog (Cdc37), a protein kinase-specific co-chaperone for Hsp90, was identified as a Vav3 interacting protein by yeast two-hybrid screening. Vav3-Cdc37 interaction was confirmed by GST pulldown and, for native proteins, by co-immunoprecipitation experiments in prostate cancer cells. Cdc37 potentiated Vav3 co-activation of AR transcriptional activity and Vav3 enhancement of AR N-terminal-C-terminal interaction, which is essential for optimal receptor transcriptional activity. Cdc37 increased prostate cancer cell proliferation selectively in Vav3-expressing cells. Cdc37 did not affect Vav3 nucleotide exchange activity, Vav3 protein levels, or subcellular localization. Disruption of Vav3-Cdc37 interaction inhibited Vav3 enhancement of AR transcriptional activity and AR N-C interaction. Diminished Vav3-Cdc37 interaction also caused decreased prostate cancer cell proliferation selectively in Vav3-expressing cells. Taken together, we identified a novel Vav3 interacting protein that enhances Vav3 co-activation of AR and prostate cancer cell proliferation. Vav3-Cdc37 interaction may provide a new therapeutic target in prostate cancer.


Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Chaperoninas/metabolismo , Regulación Neoplásica de la Expresión Génica , Neoplasias de la Próstata/metabolismo , Proteínas Proto-Oncogénicas c-vav/fisiología , Animales , Células COS , Línea Celular Tumoral , Proliferación Celular , Chlorocebus aethiops , Glutatión Transferasa/metabolismo , Factores de Intercambio de Guanina Nucleótido/metabolismo , Células HEK293 , Humanos , Masculino , Chaperonas Moleculares/metabolismo , Unión Proteica , Proteínas Proto-Oncogénicas c-vav/metabolismo , Técnicas del Sistema de Dos Híbridos
18.
Immunity ; 36(6): 974-85, 2012 Jun 29.
Artículo en Inglés | MEDLINE | ID: mdl-22683124

RESUMEN

The adaptor SAP, mutated in X-linked lymphoproliferative disease, has critical roles in multiple immune cell types. Among these, SAP is essential for the ability of natural killer (NK) cells to eliminate abnormal hematopoietic cells. Herein, we elucidated the molecular and cellular bases of this activity. SAP enhanced NK cell responsiveness by a dual molecular mechanism. It coupled SLAM family receptors to the kinase Fyn, which triggered the exchange factor Vav-1 and augmented NK cell activation. SAP also prevented the inhibitory function of SLAM family receptors. This effect was Fyn independent and correlated with uncoupling of SLAM family receptors from the lipid phosphatase SHIP-1. Both mechanisms cooperated to enable conjugate formation with target cells and to stimulate cytotoxicity and cytokine secretion by NK cells. These data showed that SAP secures NK cell activation by a dichotomous molecular mechanism, which is required for conjugate formation. These findings may have implications for the role of SAP in other immune cell types.


Asunto(s)
Antígenos CD/inmunología , Péptidos y Proteínas de Señalización Intracelular/fisiología , Células Asesinas Activadas por Linfocinas/inmunología , Activación de Linfocitos/inmunología , Monoéster Fosfórico Hidrolasas/fisiología , Proteínas Proto-Oncogénicas c-fyn/fisiología , Proteínas Proto-Oncogénicas c-vav/fisiología , Receptores de Superficie Celular/inmunología , Animales , Antígenos CD/metabolismo , Sitios de Unión , Células CHO , Señalización del Calcio/efectos de los fármacos , Adhesión Celular , Línea Celular Tumoral , Cricetinae , Cricetulus , Citotoxicidad Inmunológica , Ácido Egtácico/análogos & derivados , Ácido Egtácico/farmacología , Inositol Polifosfato 5-Fosfatasas , Péptidos y Proteínas de Señalización Intracelular/química , Péptidos y Proteínas de Señalización Intracelular/genética , Células Asesinas Activadas por Linfocinas/enzimología , Linfoma de Células T/patología , Melanoma Experimental/patología , Ratones , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatasas , Fosfolipasa C gamma/fisiología , Estructura Terciaria de Proteína , Receptores de Superficie Celular/metabolismo , Proteína Asociada a la Molécula de Señalización de la Activación Linfocitaria , Miembro 1 de la Familia de Moléculas Señalizadoras de la Activación Linfocitaria
19.
Blood ; 120(4): 800-11, 2012 Jul 26.
Artículo en Inglés | MEDLINE | ID: mdl-22692505

RESUMEN

Despite the introduction of tyrosine kinase inhibitor therapy, the prognosis for p190-BCR-ABL(+) acute lymphoblastic leukemia remains poor. In the present study, we present the cellular and molecular roles of the Rho GTPase guanine nucleotide exchange factor Vav in lymphoid leukemogenesis and explore the roles of Vav proteins in BCR-ABL-dependent signaling. We show that genetic deficiency of the guanine nucleotide exchange factor Vav3 delays leukemogenesis by p190-BCR-ABL and phenocopies the effect of Rac2 deficiency, a downstream effector of Vav3. Compensatory up-regulation of expression and activation of Vav3 in Vav1/Vav2-deficient B-cell progenitors increases the transformation ability of p190-BCR-ABL. Vav3 deficiency induces apoptosis of murine and human leukemic lymphoid progenitors, decreases the activation of Rho GTPase family members and p21-activated kinase, and is associated with increased Bad phosphorylation and up-regulation of Bax, Bak, and Bik. Finally, Vav3 activation only partly depends on ABL TK activity, and Vav3 deficiency collaborates with tyrosine kinase inhibitors to inhibit CrkL activation and impair leukemogenesis in vitro and in vivo. We conclude that Vav3 represents a novel specific molecular leukemic effector for multitarget therapy in p190-BCR-ABL-expressing acute lymphoblastic leukemia.


Asunto(s)
Linfocitos B/patología , Transformación Celular Neoplásica/patología , Proteínas de Fusión bcr-abl/metabolismo , Células Progenitoras Linfoides/patología , Leucemia-Linfoma Linfoblástico de Células Precursoras/patología , Proteínas Proto-Oncogénicas c-vav/fisiología , Animales , Linfocitos B/metabolismo , Transformación Celular Neoplásica/metabolismo , Células Cultivadas , Femenino , Sangre Fetal/citología , Sangre Fetal/metabolismo , Humanos , Células Progenitoras Linfoides/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Fosforilación , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Tasa de Supervivencia , Ensayo de Tumor de Célula Madre , Proteínas de Unión al GTP rac/fisiología , Proteína RCA2 de Unión a GTP
20.
Sci Signal ; 5(225): ra38, 2012 May 22.
Artículo en Inglés | MEDLINE | ID: mdl-22623753

RESUMEN

Interferon-ß (IFN-ß) is widely used to treat multiple sclerosis (MS), and its efficacy was demonstrated in the setting of experimental autoimmune encephalomyelitis (EAE), an animal model of MS; however, IFN-ß is not effective in treating all cases of MS. Here, we demonstrate that signaling by IFNAR (the shared receptor for IFN-α and IFN-ß) on macrophages inhibits activation of Rac1 and the generation of reactive oxygen species (ROS) through suppressor of cytokine signaling 1 (SOCS1). The inhibition of Rac1 activation and ROS generation suppressed the activity of the Nod-like receptor (NLR) family, pyrin domain-containing 3 (NLRP3) inflammasome, which resulted in attenuated EAE pathogenicity. We further found that two subsets of EAE could be defined on the basis of their dependency on the NLRP3 inflammasome and that IFN-ß was not an effective therapy when EAE was induced in an NLRP3 inflammasome-independent fashion. Thus, our study demonstrates a previously uncharacterized signaling pathway that is involved in the suppression of EAE by IFN-ß and characterizes NLRP3-independent EAE, which cannot be treated with IFN-ß.


Asunto(s)
Proteínas Portadoras/fisiología , Encefalomielitis Autoinmune Experimental/tratamiento farmacológico , Inflamasomas/fisiología , Interferón beta/uso terapéutico , Receptor de Interferón alfa y beta/fisiología , Animales , Proteínas Reguladoras de la Apoptosis , Proteínas Adaptadoras de Señalización CARD , Linfocitos T CD4-Positivos/trasplante , Proteínas Portadoras/genética , Caspasa 1/fisiología , Proteínas del Citoesqueleto/deficiencia , Modelos Animales de Enfermedad , Encefalomielitis Autoinmune Experimental/inmunología , Encefalomielitis Autoinmune Experimental/fisiopatología , Activación Enzimática , Regulación de la Expresión Génica/efectos de los fármacos , Interferon beta-1b , Interleucina-1beta/biosíntesis , Interleucina-1beta/genética , Macrófagos/fisiología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Proteína con Dominio Pirina 3 de la Familia NLR , Neuropéptidos/antagonistas & inhibidores , Neuropéptidos/fisiología , Proteínas Proto-Oncogénicas c-vav/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-vav/fisiología , Especies Reactivas de Oxígeno/metabolismo , Receptor de Interferón alfa y beta/deficiencia , Proteínas Recombinantes/uso terapéutico , Proteína 1 Supresora de la Señalización de Citocinas , Proteínas Supresoras de la Señalización de Citocinas/antagonistas & inhibidores , Proteínas Supresoras de la Señalización de Citocinas/fisiología , Proteínas de Unión al GTP rac/antagonistas & inhibidores , Proteínas de Unión al GTP rac/fisiología , Proteína de Unión al GTP rac1
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