RESUMEN
BACKGROUND AND AIMS: Glycogen storage disease type Ib (GSD1b) is a rare metabolic and immune disorder caused by a deficiency in the glucose-6-phosphate transporter (G6PT) and characterized by impaired glucose homeostasis, myeloid dysfunction, and long-term risk of hepatocellular adenomas. Despite maximal therapy, based on a strict diet and on granulocyte colony-stimulating factor treatment, long-term severe complications still develop. Understanding the pathophysiology of GSD1b is a prerequisite to develop new therapeutic strategies and depends on the availability of animal models. The G6PT-KO mouse mimics the human disease but is very fragile and rarely survives weaning. We generated a conditional G6PT-deficient mouse as an alternative model for studying the long-term pathophysiology of the disease. We utilized this conditional mouse to develop an inducible G6PT-KO model to allow temporally regulated G6PT deletion by the administration of tamoxifen (TM). METHODS: We generated a conditional G6PT-deficient mouse utilizing the CRElox strategy. Histology, histochemistry, and phenotype analyses were performed at different times after TM-induced G6PT inactivation. Neutrophils and monocytes were isolated and analyzed for functional activity with standard techniques. RESULTS: The G6PT-inducible KO mice display the expected disturbances of G6P metabolism and myeloid dysfunctions of the human disorder, even though with a milder intensity. CONCLUSIONS: TM-induced inactivation of G6PT in these mice leads to a phenotype which mimics that of human GSD1b patients. The conditional mice we have generated represent an excellent tool to study the tissue-specific role of the G6PT gene and the mechanism of long-term complications in GSD1b.
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Antiportadores/deficiencia , Modelos Animales de Enfermedad , Glucosa/metabolismo , Enfermedad del Almacenamiento de Glucógeno Tipo I/genética , Homeostasis , Proteínas de Transporte de Monosacáridos/deficiencia , Animales , Antiportadores/genética , Enfermedad del Almacenamiento de Glucógeno Tipo I/etiología , Enfermedad del Almacenamiento de Glucógeno Tipo I/patología , Ratones , Ratones Noqueados , Proteínas de Transporte de Monosacáridos/genética , Neutropenia/etiología , Tamoxifeno/administración & dosificaciónRESUMEN
The proteins of the Dbl family are guanine nucleotide exchange factors (GEFs) of Rho GTPases and are known to be involved in cell growth regulation. Alterations of the normal function of these proteins lead to pathological processes such as developmental disorders, neoplastic transformation, and tumor metastasis. We have previously demonstrated that expression of Dbl oncogene in lens epithelial cells modulates genes encoding proteins involved in epithelial-mesenchymal-transition (EMT) and induces angiogenesis in the lens. Our present study was undertaken to investigate the role of Dbl oncogene in epithelial cells transformation, providing new insights into carcinoma progression.To assess how Dbl oncogene can modulate EMT, cell migration, morphogenesis, and expression of pro-apoptotic and angiogenic factors we utilized bi- and 3-dimensional cultures of MCF-10 A cells. We show that upon Dbl expression MCF-10 A cells undergo EMT. In addition, we found that Dbl overexpression sustains Cdc42 and Rac activation inducing morphological alterations, characterized by the presence of lamellipodia and conferring a high migratory capacity to the cells. Moreover, Dbl expressing MCF-10 A cells form altered 3D structures and can induce angiogenesis by producing proangiogenic factors such as CCL2. These results support a role for Dbl oncogene in epithelial cell differentiation and transformation and suggest the relevance of GEF deregulation in tumor onset and progression.
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Células Acinares/enzimología , Proteínas Angiogénicas/metabolismo , Neoplasias de la Mama/enzimología , Transformación Celular Neoplásica/metabolismo , Células Epiteliales/enzimología , Transición Epitelial-Mesenquimal , Factores de Intercambio de Guanina Nucleótido/metabolismo , Glándulas Mamarias Humanas/enzimología , Proteínas Proto-Oncogénicas/metabolismo , Células Acinares/metabolismo , Células Acinares/patología , Apoptosis , Neoplasias de la Mama/irrigación sanguínea , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Diferenciación Celular , Línea Celular , Movimiento Celular , Forma de la Célula , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/patología , Quimiocina CCL2/metabolismo , Células Epiteliales/metabolismo , Células Epiteliales/patología , Femenino , Factores de Intercambio de Guanina Nucleótido/genética , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Glándulas Mamarias Humanas/metabolismo , Glándulas Mamarias Humanas/patología , Neovascularización Fisiológica , Proteínas Proto-Oncogénicas/genética , Transducción de Señal , Transfección , Regulación hacia Arriba , Proteína de Unión al GTP cdc42/metabolismo , Proteínas de Unión al GTP rac/metabolismoRESUMEN
Glycogen storage disease type 1a (GSD-1a) is caused by a deficiency in glucose-6-phosphatase-α (G6Pase-α), and is characterized by impaired glucose homeostasis and a high risk of developing hepatocellular adenomas (HCAs). A globally G6Pase-α-deficient (G6pc(-/-)) mouse model that shows pathological features similar to those of humans with GSD-1a has been developed. These mice show a very severe phenotype of disturbed glucose homeostasis and rarely live beyond weaning. We generated liver-specific G6Pase-α-deficient (LSG6pc(-/-)) mice as an alternative animal model for studying the long-term pathophysiology of the liver and the potential treatment strategies, such as cell therapy. LSG6pc(-/-) mice were viable and exhibited normal glucose profiles in the fed state, but showed significantly lower blood glucose levels than their control littermates after 6 hours of fasting. LSG6pc(-/-) mice developed hepatomegaly with glycogen accumulation and hepatic steatosis, and progressive hepatic degeneration. Ninety percent of the mice analyzed developed amyloidosis by 12 months of age. Finally, 25% of the mice sacrificed at age 10-20 months showed the presence of multiple HCAs and in one case late development of hepatocellular carcinoma (HCC). In conclusion, LSG6pc(-/-) mice manifest hepatic symptoms similar to those of human GSD-1a and, therefore, represent a valid model to evaluate long-term liver pathogenesis of GSD-1a.
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Adenoma/patología , Carcinoma Hepatocelular/patología , Glucosafosfato Deshidrogenasa/genética , Neoplasias Hepáticas/patología , Adenoma/enzimología , Animales , Carcinoma Hepatocelular/enzimología , Neoplasias Hepáticas/enzimología , RatonesRESUMEN
The Rho guanine nucleotide exchange factor protoDbl is involved in different biochemical pathways affecting cell proliferation and migration. The N-terminal sequence of protoDbl contains negative regulatory elements that restrict the catalytic activity of the DH-PH module. Here, we report the identification of a new mouse protoDbl splice variant lacking exon 3. We found that the splice variant mRNA is expressed in the spleen and bone marrow lymphocytes, adrenal gland, gonads and brain. The protoDbl variant protein was detectable in the brain. The newly identified variant displays the disruption of the SEC14 domain, positioned on exons 2 and 3 in the protoDbl N-terminal region. We show here that an altered SEC14 sequence leads to enhanced Dbl translocation to the plasma membrane and to augmented transforming and exchange activity.
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Empalme Alternativo , Factores de Intercambio de Guanina Nucleótido/genética , Factores de Intercambio de Guanina Nucleótido/metabolismo , Proto-Oncogenes , Secuencia de Aminoácidos , Animales , Linfocitos B/metabolismo , Encéfalo/metabolismo , Células COS , Membrana Celular/metabolismo , Exones , Femenino , Humanos , Masculino , Ratones , Datos de Secuencia Molecular , Especificidad de Órganos , Proteínas de Transferencia de Fosfolípidos/metabolismo , Estructura Terciaria de Proteína , Proto-Oncogenes Mas , Proteínas Proto-Oncogénicas/genética , ARN Mensajero , Proteínas de Saccharomyces cerevisiae/metabolismo , Bazo/metabolismo , Proteína de Unión al GTP cdc42/metabolismoRESUMEN
Bradykinin drives normal lung fibroblasts into myofibroblasts, induces fibroblast proliferation and activates mitogen activated protein kinase pathways (MAPK) but its effects on bronchial fibroblasts from asthmatics (HBAFb) have not been yet studied. We studied bradykinin-induced fibroblast proliferation and differentiation and the related intracellular mechanisms in HBAFb compared to normal bronchial fibroblasts (HNBFb). Bradykinin-stimulated HBAFb and HNBFb were used to assess: bradykinin B2 receptor expression by Western blot analysis; cell proliferation by [(3)H] thymidine incorporation; α-smooth muscle actin (SMA) expression/polymerization by Western blot and immunofluorescence; epidermal growth factor (EGF) receptor, extracellular-regulated kinase (ERK) 1/2 and p38 MAPK activation by immunoprecipitation and Western blot, respectively. Constitutive bradykinin B2 receptor and α-SMA expression was higher in HBAFb as compared to HNBFb. Bradykinin increased bradykinin B2 receptor expression in HBAFb. Bradykinin, via bradykinin B2 receptor, significantly increased fibroblast proliferation at lower concentration (10(-11)M) and α-SMA expression/polymerization at higher concentration (10(-6)M) in both cells. Bradykinin increased ERK1/2 and p38 phosphorylation via bradykinin B2 receptor; EGF receptor inhibitor AG1478 and panmetalloproteinase inhibitor GM6001 blocked bradykinin-induced ERK1/2 activation but not p38 phosphorylation. Bradykinin, via bradykinin B2 receptor, induced EGF receptor phosphorylation that was suppressed by AG1478. In HBAFb AG1478, GM6001, the ERK1/2-inhibitor U0126 and the p38 inhibitor SB203580 suppressed bradykinin-induced cell proliferation, but only SB203580 reduced myofibroblast differentiation. These data indicate that bradykinin is actively involved in asthmatic bronchial fibroblast proliferation and differentiation, through MAPK pathways and EGF receptor transactivation, by which bradykinin may contribute to airway remodeling in asthma, opening new horizons for potential therapeutic implications in asthmatic patients.
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Asma/metabolismo , Bradiquinina/farmacología , Fibroblastos/efectos de los fármacos , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Miofibroblastos/efectos de los fármacos , Receptor de Bradiquinina B2/metabolismo , Actinas/metabolismo , Diferenciación Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Receptores ErbB/metabolismo , Fibroblastos/citología , Fibroblastos/metabolismo , Humanos , Miofibroblastos/citología , Miofibroblastos/metabolismoRESUMEN
The Rho guanine nucleotide exchange factor (GEF) Dbl binds to the N-terminal region of ezrin, a member of the ERM (ezrin, radixin, moesin) proteins known to function as linkers between the plasma membrane and the actin cytoskeleton. Here we have characterized the interaction between ezrin and Dbl. We show that binding of Dbl with ezrin involves positively charged amino acids within the region of the pleckstrin homology (PH) domain comprised between ß1 and ß2 sheets. In addition, we show that Dbl forms a complex with the tuberous sclerosis-1 (TSC-1) gene product hamartin and with ezrin. We demonstrate that hamartin and ezrin are both required for activation of Dbl. In fact, the knock-down of ezrin and hamartin, as well as the expression of a mutant hamartin, unable to bind ezrin, inhibit Dbl transforming and exchange activity. These results suggest that Dbl is regulated by hamartin through association with ezrin.
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Proteínas del Citoesqueleto/metabolismo , Factores de Intercambio de Guanina Nucleótido/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Animales , Células COS , Chlorocebus aethiops , Proteínas del Citoesqueleto/genética , Factores de Intercambio de Guanina Nucleótido/genética , Humanos , Ratones , Ratones Noqueados , Células 3T3 NIH , Unión Proteica , Estructura Secundaria de Proteína , Proteína 1 del Complejo de la Esclerosis Tuberosa , Proteínas Supresoras de Tumor/genéticaRESUMEN
BACKGROUND & AIMS: Several studies have shown that bone marrow-derived committed myelomonocytic cells can repopulate diseased livers by fusing with host hepatocytes and can restore normal liver function. These data suggest that myelomonocyte transplantation could be a promising approach for targeted and well-tolerated cell therapy aimed at liver regeneration. We sought to determine whether bone marrow-derived myelomonocytic cells could be effective for liver reconstitution in newborn mice knock-out for glucose-6-phosphatase-α. METHODS: Bone marrow-derived myelomonocytic cells obtained from adult wild type mice were transplanted in newborn knock-out mice. Tissues of control and treated mice were frozen for histochemical analysis, or paraffin-embedded and stained with hematoxylin and eosin for histological examination or analyzed by immunohistochemistry or fluorescent in situ hybridization. RESULTS: Histological sections of livers of treated knock-out mice revealed areas of regenerating tissue consisting of hepatocytes of normal appearance and partial recovery of normal architecture as early as 1 week after myelomonocytic cells transplant. FISH analysis with X and Y chromosome paints indicated fusion between infused cells and host hepatocytes. Glucose-6-phosphatase activity was detected in treated mice with improved profiles of liver functional parameters. CONCLUSIONS: Our data indicate that bone marrow-derived myelomonocytic cell transplant may represent an effective way to achieve liver reconstitution of highly degenerated livers in newborn animals.
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Enfermedad del Almacenamiento de Glucógeno Tipo I/patología , Enfermedad del Almacenamiento de Glucógeno Tipo I/terapia , Hígado/patología , Células Mieloides/trasplante , Animales , Animales Recién Nacidos , Trasplante de Médula Ósea , Femenino , Glucosa-6-Fosfatasa/genética , Glucosa-6-Fosfatasa/metabolismo , Enfermedad del Almacenamiento de Glucógeno Tipo I/genética , Enfermedad del Almacenamiento de Glucógeno Tipo I/fisiopatología , Hígado/fisiopatología , Regeneración Hepática , Masculino , Ratones , Ratones Congénicos , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , EmbarazoRESUMEN
Dbl is the prototype of a large family of GDP-GTP exchange factors for small GTPases of the Rho family. In vitro, Dbl is known to activate Rho, Rac, and Cdc42 and to induce a transformed phenotype in murine fibroblasts. We previously reported that Dbl-null mice are viable and fertile but display defective dendrite elongation of distinct subpopulations of cortical neurons, suggesting a role of Dbl in controlling dendritic growth. To gain deeper insights into the role of Dbl in development and disease, we attempted a knock-in approach to create an endogenous allele that encodes a missense-mutation-mediated loss of function in the DH domain. We generated, by gene targeting technology, a mutant mouse strain by inserting a mutagenized human proto-Dbl cDNA clone expressing only the Dbl N terminus regulatory sequence at the starting codon of murine exon 1. Animals were monitored over a 21-month period, and necropsy specimens were collected for histological examination and immunohistochemistry analysis. Dbl knock-in mice are viable and did not manifest either decreased reproductive performances or gross developmental phenotype but revealed a reduced lifespan compared to wild-type (w.t.) mice and showed, with aging, a B cell lymphoproliferation that often has features of a frank diffuse large B cell lymphoma. Moreover, Dbl knock-in male mice displayed an increased incidence of lung adenoma compared to w.t. mice. These data indicate that Dbl is a tumor susceptibility gene in mice and that loss of function of Dbl DH domain by genetic missense mutations is responsible for induction of diffuse large B cell lymphoma.
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Factores de Intercambio de Guanina Nucleótido/metabolismo , Linfoma de Células B/metabolismo , Linfoma de Células B Grandes Difuso/metabolismo , Animales , Western Blotting , Factores de Intercambio de Guanina Nucleótido/genética , Humanos , Inmunohistoquímica , Etiquetado Corte-Fin in Situ , Linfoma de Células B/genética , Linfoma de Células B Grandes Difuso/genética , Masculino , Ratones , Células 3T3 NIH , Proteínas de Unión al GTP rho/genética , Proteínas de Unión al GTP rho/metabolismoRESUMEN
Dendritic cells (DCs) are a heterogeneous group of professional antigen-presenting cells functioning as sentinels of the immune system and playing a key role in the initiation and amplification of innate and adaptive immune responses. DC development and functions are acquired during a complex differentiation and maturation process influenced by several factors present in the local milieu. A common feature at pathologic sites is represented by hypoxia, a condition of low pO(2), which creates a unique microenvironment affecting cell phenotype and behavior. Little is known about the impact of hypoxia on the generation of mature DCs (mDCs). In this study, we identified by gene expression profiling a significant cluster of genes coding for immune-related cell surface receptors strongly up-regulated by hypoxia in monocyte-derived mDCs and characterized one of such receptors, TREM-1, as a new hypoxia-inducible gene in mDCs. TREM-1 associated with DAP12 in hypoxic mDCs, and its engagement elicited DAP12-linked signaling, resulting in ERK-1, Akt, and IκBα phosphorylation and proinflammatory cytokine and chemokine secretion. Finally, we provided the first evidence that TREM-1 is expressed on mDCs infiltrating the inflamed hypoxic joints of children affected by juvenile idiopathic arthritis, representing a new in vivo marker of hypoxic mDCs endowed with proinflammatory properties.
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Diferenciación Celular/genética , Células Dendríticas/citología , Células Dendríticas/metabolismo , Perfilación de la Expresión Génica , Glicoproteínas de Membrana/metabolismo , Receptores Inmunológicos/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Artritis Juvenil/genética , Artritis Juvenil/patología , Biomarcadores/metabolismo , Diferenciación Celular/efectos de los fármacos , Hipoxia de la Célula/efectos de los fármacos , Hipoxia de la Célula/genética , Quimiocinas/metabolismo , Reactivos de Enlaces Cruzados/farmacología , Minería de Datos , Células Dendríticas/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Mediadores de Inflamación/metabolismo , Glicoproteínas de Membrana/genética , Proteínas de la Membrana/metabolismo , Monocitos/citología , Oxígeno/farmacología , Fenotipo , Receptores Inmunológicos/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Líquido Sinovial/efectos de los fármacos , Líquido Sinovial/metabolismo , Receptor Activador Expresado en Células Mieloides 1RESUMEN
BACKGROUND: The Dbl family of proteins represents a large group of proto-oncogenes involved in cell growth regulation. The numerous domains that are present in many Dbl family proteins suggest that they act to integrate multiple inputs in complicated signaling networks involving the Rho GTPases. Alterations of the normal function of these proteins lead to pathological processes such as developmental disorders and neoplastic transformation. We generated transgenic mice introducing the cDNA of Dbl oncogene linked to the metallothionein promoter into the germ line of FVB mice and found that onco-Dbl expression in mouse lenses affected proliferation, migration and differentiation of lens epithelial cells. RESULTS: We used high density oligonucleotide microarray to define the transcriptional profile induced by Dbl in the lenses of 2 days, 2 weeks, and 6 weeks old transgenic mice. We observed modulation of genes encoding proteins promoting epithelial-mesenchymal transition (EMT), such as down-regulation of epithelial cell markers and up-regulation of fibroblast markers. Genes encoding proteins involved in the positive regulation of apoptosis were markedly down regulated while anti-apoptotic genes were strongly up-regulated. Finally, several genes encoding proteins involved in the process of angiogenesis were up-regulated. These observations were validated by histological and immunohistochemical examination of the transgenic lenses where vascularization can be readily observed. CONCLUSION: Onco-Dbl expression in mouse lens correlated with modulation of genes involved in the regulation of EMT, apoptosis and vasculogenesis leading to disruption of the lens architecture, epithelial cell proliferation, and aberrant angiogenesis. We conclude that onco-Dbl has a potentially important, previously unreported, capacity to dramatically alter epithelial cell migration, replication, polarization and differentiation and to induce vascularization of an epithelial tissue.
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Epitelio/metabolismo , Regulación de la Expresión Génica , Factores de Intercambio de Guanina Nucleótido/genética , Factores de Intercambio de Guanina Nucleótido/fisiología , Cristalino/irrigación sanguínea , Cristalino/metabolismo , Mesodermo/metabolismo , Animales , Diferenciación Celular , Movimiento Celular , Proliferación Celular , Femenino , Masculino , Metalotioneína/química , Ratones , Ratones Transgénicos , Proteínas de Unión al GTP rho/metabolismoRESUMEN
Dendritic cells (DCs) are the most potent antigen-presenting cells and fine-tune the immune response. We have investigated hypoxia's effects on the differentiation and maturation of DCs from human monocytes in vitro, and have shown that it affects DC functions. Hypoxic immature DCs (H-iDCs) significantly fail to capture antigens through down-modulation of the RhoA/Ezrin-Radixin-Moesin pathway and the expression of CD206. Moreover, H-iDCs released higher levels of CXCL1, VEGF, CCL20, CXCL8, and CXCL10 but decreased levels of CCL2 and CCL18, which predict a different ability to recruit neutrophils rather than monocytes and create a proinflammatory and proangiogenic environment. By contrast, hypoxia has no effect on DC maturation. Hypoxic mature DCs display a mature phenotype and activate both allogeneic and specific T cells like normoxic mDCs. This study provides the first demonstration that hypoxia inhibits antigen uptake by DCs and profoundly changes the DC chemokine expression profile and may have a critical role in DC differentiation, adaptation, and activation in inflamed tissues.
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Antígenos/metabolismo , Diferenciación Celular/fisiología , Quimiocinas/metabolismo , Células Dendríticas/citología , Hipoxia/metabolismo , Movimiento Celular , Proliferación Celular , Células Cultivadas , Citocinas/metabolismo , Proteínas de Unión al ADN/metabolismo , Células Dendríticas/metabolismo , Regulación hacia Abajo , Citometría de Flujo , Humanos , Activación de Linfocitos , Macrófagos/citología , Macrófagos/inmunología , Macrófagos/metabolismo , Monocitos/citología , Monocitos/inmunología , Monocitos/metabolismo , Fenotipo , Fosforilación , Linfocitos T/inmunología , Linfocitos T/metabolismo , Factores de Transcripción/metabolismo , Proteínas de Unión al GTP rho/metabolismoRESUMEN
Hypoxia is a condition of low oxygen tension occurring in inflammatory tissues. Dendritic cells (DC) are professional antigen-presenting cells whose differentiation, migration, and activities are intrinsically linked to the microenvironment. DCs will home and migrate through pathologic tissues before reaching their final destination in the lymph node. We studied the differentiation of human monocytes into immature DCs (iDCs) in a hypoxic microenvironment. We generated iDC in vitro under normoxic (iDCs) or hypoxic (Hi-DCs) conditions and examined the hypoxia-responsive element in the promoter, gene expression, and biochemical KEGG pathways. Hi-DCs had an interesting phenotype represented by up-regulation of genes associated with cell movement/migration. In addition, the Hi-DC cytokine/receptor pathway showed a dichotomy between down-regulated chemokines and up-regulated chemokine receptor mRNA expression. We showed that CCR3, CX3CR1, and CCR2 are hypoxia-inducible genes and that CCL18, CCL23, CCL26, CCL24, and CCL14 are inhibited by hypoxia. A strong chemotactic response to CCR2 and CXCR4 agonists distinguished Hi-DCs from iDCs at a functional level. The hypoxic microenvironment promotes the differentiation of Hi-DCs, which differs from iDCs for gene expression profile and function. The most prominent characteristic of Hi-DCs is the expression of a mobility/migratory rather than inflammatory phenotype. We speculate that Hi-DCs have the tendency to leave the hypoxic tissue and follow the chemokine gradient toward normoxic areas where they can mature and contribute to the inflammatory process.
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Quimiocinas/genética , Células Dendríticas/citología , Células Dendríticas/metabolismo , Perfilación de la Expresión Génica , Receptores de Quimiocina/genética , Hipoxia de la Célula , Movimiento Celular , Regulación de la Expresión Génica , Humanos , Análisis de Secuencia por Matrices de Oligonucleótidos , Fenotipo , Reproducibilidad de los Resultados , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Rho family GTPases play important roles in the regulation of intracellular signals induced by activated heterotrimeric G proteins of the alpha(12/13) family. The alpha(12/13) subunits activate Rho GTPases through direct binding to a group of Rho guanine nucleotide exchange factors (GEFs) characterized by the presence of a G protein signaling-like (RGL) domain. The Rho GEF proto-Dbl, that does not contain a RGL domain, was also found to link Galpha(12/13) signals to Rho. We have explored the effects of activated Galpha(13) and Galpha(13)-associated G protein-coupled receptor (GPCR) agonists on proto-Dbl regulation. We show that activated Galpha(13), but not Galpha(12) or Galpha(q), induces translocation of proto-Dbl to the cell membrane with consequent enlargement of cell body and membrane ruffling. These effects were evident also when Galpha(13)-associated GPCR agonists were used on cells expressing proto-Dbl and were accompanied by the activation of Cdc42 and RhoA GTPases and further downstream effector JNK and p38 kinases. Moreover, we show that both activated Galpha(13) and GPCR agonists stimulate proto-Dbl interaction with ezrin to promote ezrin translocation to the plasma membrane. These results suggest a mechanism by which proto-Dbl and its effector pathways are regulated by Galpha(13)-mediated signals through association with ezrin.
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Subunidades alfa de la Proteína de Unión al GTP G12-G13/metabolismo , Factores de Intercambio de Guanina Nucleótido/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Transducción de Señal/fisiología , Animales , Transporte Biológico/efectos de los fármacos , Western Blotting , Bradiquinina/farmacología , Células COS , Chlorocebus aethiops , Proteínas del Citoesqueleto/metabolismo , Técnica del Anticuerpo Fluorescente , Subunidades alfa de la Proteína de Unión al GTP G12-G13/genética , Subunidades alfa de la Proteína de Unión al GTP G12-G13/fisiología , Factores de Intercambio de Guanina Nucleótido/genética , Factores de Intercambio de Guanina Nucleótido/fisiología , Inmunoprecipitación , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Lisofosfolípidos/farmacología , Ratones , Células 3T3 NIH , Unión Proteica/efectos de los fármacos , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/fisiología , ARN Interferente Pequeño/genética , Factores de Intercambio de Guanina Nucleótido Rho , Transducción de Señal/genética , Trombina/farmacología , Transfección , Proteína de Unión al GTP cdc42/genética , Proteína de Unión al GTP cdc42/metabolismo , Proteína de Unión al GTP cdc42/fisiología , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Proteína de Unión al GTP rhoA/genética , Proteína de Unión al GTP rhoA/metabolismo , Proteína de Unión al GTP rhoA/fisiologíaRESUMEN
Dystroglycan is an essential laminin binding cell adhesion molecule, which is also an adaptor for several SH2 domain-containing signaling molecules and as a scaffold for the ERK-MAP kinase cascade. Loss of dystroglycan function is implicated in muscular dystrophies and the aetiology of epithelial cancers. We have previously demonstrated a role for dystroglycan and ezrin in the formation of filopodia structures. Here we demonstrate the existence of a dystroglycan:ezrin:Dbl complex that is targeted to the membrane by dystroglycan where it drives local Cdc42 activation and the formation of filopodia. Deletion of an ezrin binding site in dystroglycan prevented the association with ezrin and Dbl and the formation of filopodia. Furthermore, expression of the dystroglycan cytoplasmic domain alone had a dominant-negative effect on filopodia formation and Cdc42 activation by sequestering ezrin and Dbl away from the membrane. Depletion of dystroglycan inhibited Cdc42-induced filopodia formation. For the first time we also demonstrate co-localization of Cdc42 and dystroglycan at the tips of dynamic filopodia.
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Proteínas del Citoesqueleto/metabolismo , Distroglicanos/metabolismo , Factores de Intercambio de Guanina Nucleótido/metabolismo , Seudópodos/metabolismo , Proteína de Unión al GTP cdc42/metabolismo , Actinas/metabolismo , Animales , Sitios de Unión , Células COS , Línea Celular , Chlorocebus aethiops , Proteínas del Citoesqueleto/genética , Distroglicanos/genética , Técnica del Anticuerpo Fluorescente , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Factores de Intercambio de Guanina Nucleótido/genética , Immunoblotting , Inmunoprecipitación , Ratones , Mutación , Unión Proteica , Interferencia de ARN , Ratas , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Células 3T3 Swiss , Proteína de Unión al GTP cdc42/genética , Proteínas de Unión al GTP rho/metabolismoRESUMEN
The Dbl oncogene is a guanine nucleotide exchange factor for Rho GTPases and its activity has been linked to the regulation of gene transcription. Dbl oncogene expression in NIH3T3 cells leads to changes in morphological and proliferative properties of these cells, inducing a highly transformed phenotype. To gain insights into Dbl oncogene-induced transformation we compared gene expression profiles between Dbl oncogene-transformed and parental NIH3T3 cells by cDNA microarray. We found that Dbl oncogene expression is associated with gene expression modulation involving upregulation of 51 genes and downregulation of 49 genes. Five of the overexpressed genes identified are known to exert antiproliferative functions. Our observations suggest that the expression of Dbl oncogene in NIH3T3 may lead to the induction of genes associated with cell cycle arrest, possibly through the activation of stress-induced kinases.
Asunto(s)
Expresión Génica/fisiología , Factores de Intercambio de Guanina Nucleótido/genética , Animales , Northern Blotting , Western Blotting , Ciclo Celular , Línea Celular Transformada , Células Cultivadas , Activación Enzimática , Fibroblastos/metabolismo , Perfilación de la Expresión Génica , Factores de Intercambio de Guanina Nucleótido/metabolismo , Ratones , Células 3T3 NIH , Análisis de Secuencia por Matrices de Oligonucleótidos , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
Proto-Dbl protein, a guanine nucleotide exchange factor (GEF) for Rho GTPases, is tightly regulated by a combination of mechanisms that involve intra- and intermolecular interaction and N- and C-terminal domain-dependent turnover of the protein. Moreover, the interaction of the PH domain of proto-Dbl with phosphoinositides regulates its subcellular localization and biological activity. Here we show that inhibition of the phosphatidylinositol 3-kinase (PI3K) by molecular and pharmacological inhibitors causes a strong inhibition of proto-Dbl-induced cell proliferation and transformation. Conversely, inhibition of PI3K results in the translocation of proto-Dbl to the plasma membrane, Rac activation and increased membrane ruffles and cell motility. Furthermore, we investigated the signaling molecules involved in proto-Dbl-induced cell transformation and motility and observed that inhibition of PI3K in proto-Dbl expressing cells induces an increase in p38 activity and a decrease in ERK phosphorylation. Our results suggest that proto-Dbl activates distinct downstream effectors to induce morphological changes and cell transformation.
Asunto(s)
Membrana Celular/ultraestructura , Factores de Intercambio de Guanina Nucleótido/metabolismo , Inhibidores de las Quinasa Fosfoinosítidos-3 , Proteínas Proto-Oncogénicas c-akt/metabolismo , Animales , Células COS , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Chlorocebus aethiops , Cromonas/farmacología , Inhibidores Enzimáticos/farmacología , Ratones , Microscopía Fluorescente , Morfolinas/farmacología , Células 3T3 NIH , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación , Proteínas Proto-Oncogénicas/metabolismo , Transducción de Señal , TransfecciónRESUMEN
The Rho family small GTPase Cdc42 is critical for diverse cellular functions including the regulation of actin organization, cell polarity, intracellular membrane trafficking, transcription, cell cycle progression and cell transformation. Like other members of the Rho family, Cdc42 cycles between the GTP-bound, active state, and the inactive, GDP-bound state under tight regulation, and it is believed that the GTP bound form of Cdc42 represents the active signaling module in eliciting effector activation and cellular responses. The constitutively active mutant, V12, derived from the analogous mutations found in oncogenic Ras that are GTPase-defective, and a "fast-cycling" self-activating mutant, F28, of Cdc42, have been widely in use to study the cellular effects of Cdc42. Here we report that the constitutively active V12 mutant of Cdc42, when stably expressed in cells, could behave in a dominant negative fashion in inhibiting cell proliferation while the F28 mutant was growth stimulatory. The V12 mutant failed to transform NIH3T3 cells while F28 potently stimulated anchorage-independent growth. The growth inhibitory effect of the V12 mutant correlated with activation of JNK2 and suppression of the cyclin D1 and NF-kappaB expressions that were instead upregulated by the F28 mutant. Furthermore, the V12 mutant could suppress, whereas the F28 mutant potentiated or had no effect on, a wide variety of oncogene-induced cell transformation, including that by the Dbl family GEFs Dbl, Vav and Lbc and the oncogenic Ras, ErbB-2, PDGF B or Raf. These results raise the possibility that over-saturation or constitutive activation of Cdc42 signal may negatively impact on cell proliferation and that both the activation and deactivation steps, or the complete GTPase cycle, of Cdc42 is required for proper function.
Asunto(s)
Línea Celular Transformada/citología , Inhibidores de Crecimiento/genética , Inhibidores de Crecimiento/metabolismo , Mutación , Proteína de Unión al GTP cdc42/genética , Proteína de Unión al GTP cdc42/metabolismo , Animales , Línea Celular Transformada/metabolismo , Inhibidores de Crecimiento/fisiología , Ratones , Células 3T3 NIH , Proteína Oncogénica p21(ras)/fisiología , Proteína de Unión al GTP cdc42/fisiologíaRESUMEN
Ezrin, a widespread protein involved in cell migration, morphogenesis and cell adhesion, belongs to a large family of proteins known as ERM (ezrin, radixin, moesin). These three closely related proteins are thought to function as linkers between plasma membrane and actin cytoskeleton and their function is regulated by the small GTP-binding protein Rho. It has been previously shown that the active form of radixin can bind in vitro to Dbl, a Rho-specific guanine nucleotide exchange factor, although an in vivo interaction has not yet been demonstrated. In this paper, we attempted to investigate whether ezrin can also associate with Dbl. We show here that Dbl protein can effectively bind both in vitro and in vivo to the N-terminal region (amino acids 1-531) of a constitutively active mutant of ezrin and with the full-length molecule. We found that this binding is mediated by the Dbl pleckstrin homology domain, responsible for the proper subcellular localization of the Dbl protein. Moreover, we show that Dbl induces localization to the plasma membrane of both the active deletion mutant and the full-length ezrin proteins. Finally, we show that the relocalization of ezrin is independent of Dbl GEF activity. These results indicate that Dbl could induce translocation of ezrin to the plasma membrane through a mechanism that does not require ezrin C-terminus phosphorylation by Rho-associated kinases.
Asunto(s)
Membrana Celular/metabolismo , Fosfoproteínas/metabolismo , Proteínas Oncogénicas de Retroviridae/metabolismo , Animales , Células COS , Proteínas del Citoesqueleto , Factores de Intercambio de Guanina Nucleótido/metabolismo , Ratones , Células 3T3 NIH , Fosforilación , Transporte de Proteínas/fisiologíaRESUMEN
Dbl is the prototype of a large family of GDP-GTP exchange factors for small GTPases of the Rho family. In vitro, Dbl is known to activate Rho and Cdc42 and to induce a transformed phenotype. Dbl is specifically expressed in brain and gonads, but its in vivo functions are largely unknown. To assess its role in neurogenesis and gametogenesis, targeted deletion of the murine Dbl gene was accomplished in embryonic stem cells. Dbl-null mice are viable and did not show either decreased reproductive performances or obvious neurological defects. Histological analysis of mutant testis showed normal morphology and unaltered proliferation and survival of spermatogonia. Dbl-null brains indicated a correct disposition of the major neural structures. Analysis of cortical stratification indicated that Dbl is not crucial for neuronal migration. However, in distinct populations of Dbl-null cortical pyramidal neurons, the length of dendrites was significantly reduced, suggesting a role for Dbl in dendrite elongation.
Asunto(s)
Dendritas/metabolismo , Dendritas/patología , Fertilidad , Proteínas Activadoras de GTPasa/metabolismo , Factores de Intercambio de Guanina Nucleótido/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Animales , Western Blotting , Encéfalo/crecimiento & desarrollo , Encéfalo/metabolismo , Encéfalo/patología , Movimiento Celular , Femenino , GTP Fosfohidrolasas/metabolismo , Proteínas Activadoras de GTPasa/deficiencia , Proteínas Activadoras de GTPasa/genética , Eliminación de Gen , Gónadas/embriología , Gónadas/metabolismo , Factores de Intercambio de Guanina Nucleótido/deficiencia , Factores de Intercambio de Guanina Nucleótido/genética , Masculino , Ratones , Ratones Noqueados , Ovario/citología , Ovario/metabolismo , Proteínas Proto-Oncogénicas/deficiencia , Proteínas Proto-Oncogénicas/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Espermatogénesis , Testículo/citología , Testículo/metabolismoRESUMEN
The pleckstrin homology (PH) domain of onco-Dbl, a guanine nucleotide exchange factor (GEF) for Cdc42 and RhoA GTPases, interacts with phosphoinositides (PIPs). This interaction modulates both the GEF activity and the targeting to the plasma membrane of onco-Dbl. Conversely, we have previously shown that in proto-Dbl an intramolecular interaction between the N-terminal domain and the PH domain imposes a negative regulation on both the DH and PH functions, suppressing its transforming activity. Here we have further investigated the mode of regulation of proto-Dbl by generating proto-Dbl mutants deleted of the last C-terminal 50 amino acids, which contain a PEST motif, and/or unable to bind to PIPs due to substitutions of the positively charged residues of the PH domain. The PH mutants of proto-Dbl retained a relative weak GEF activity toward Cdc42 and RhoA in vitro, but their RhoA activating potential was impaired in vivo. Further, these mutants lost both the plasma membrane targeting and the transforming activities, contrary to the PH mutants of onco-Dbl that retained the exchange activity both in vitro and in vivo and showed significant, but partially, reduced transforming activity. Deletion of the C-terminal sequences from onco-Dbl did not affect its function, whereas similar deletion of proto-Dbl led to an increase of transforming activity. Analysis of the half-life of the proto-Dbl mutants revealed that deletion of the C-terminal sequences increases the stability of the protein. Overall, the transformation potential of proto-Dbl mutants was associated with an augmented localization of the protein to the plasma membrane and a strong activation of Jun N-terminal kinase activity and transcription of cyclin D1. Together with previous observations, these data suggest that the biological activity of proto-Dbl is tightly regulated by a combination of mechanisms that involve intramolecular interaction, PH binding to PIPs, and the N- and C-terminal domain-dependent turnover of the protein.