RESUMEN
OBJECTIVE: Stem cell-based tissue repair and regeneration require the regulation of cell migration and adhesion. As a regulator of cell polarization, Cdc42 (cell division control protein 42) plays a basic role at the initial stage of cell migration and adhesion. This study explores the effect of Cdc42 on the polarized migration and adhesion of hDPSCs (human dental pulp stem cells). DESIGN: HDPSCs were isolated from extracted third molars and transfected with siRNA targeted against Cdc42. Scratch wound assays and transwell assays were performed to detect the migration of human dental pulp stem cells. Polarization assays were applied to explore the polarized movement of Golgi bodies and nuclei. Western blot was used to examine the expression of related proteins. RESULTS: The expression of Cdc42 was knocked down by siRNA transfection, which inhibited the migration of hDPSCs in both the scratch wound assays and transwell assays. Meanwhile, the proportion of polarized hDPSCs during migration was also decreased, and the adhesion ability of hDPSCs was downregulated. Western blot demonstrated that these effects were dependent on FAK (focal adhesion kinase), ß-catenin and GSK3ß (Glycogen synthase kinase-3ß). CONCLUSION: Our study demonstrates that Cdc42 plays an essential role during the polarized movement and adhesion of hDPSCs.
Asunto(s)
Adhesión Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Pulpa Dental/citología , Proteína de Unión al GTP cdc42/farmacología , Adolescente , Western Blotting , Células Cultivadas , Citometría de Flujo , Aparato de Golgi/efectos de los fármacos , Humanos , Microscopía Confocal , Tercer Molar , Reacción en Cadena en Tiempo Real de la Polimerasa , Transfección , Adulto JovenRESUMEN
OBJECTIVES: The morphogenetic events that occur during angiogenic sprouting involve several members of the Rho family of GTPases, including Cdc42. However, the precise roles of Cdc42 in angiogenic sprouting have been difficult to elucidate owing to the lack of models to study these events in vitro. Here, we aim to identify the roles of Cdc42 in branching morphogenesis in angiogenesis. METHODS: Using a 3D biomimetic model of angiogenesis in vitro, where endothelial cells were seeded inside a cylindrical channel within collagen gel and sprouted from the channel in response to a defined biochemical gradient of angiogenic factors, we inhibited Cdc42 activity with a small molecule inhibitor ML141 and examined the effects of Cdc42 on the morphogenetic processes of angiogenic sprouting. RESULTS: We find that partial inhibition of Cdc42 had minimal effects on directional migration of endothelial cells, but led to fewer branching events without affecting the length of these branches. We also observed that antagonizing Cdc42 reduced collective migration in favor of single cell migration. Additionally, Cdc42 also regulated the initiation of filopodial extensions in endothelial tip cells. CONCLUSIONS: Our findings suggest that Cdc42 can affect multiple morphogenetic processes during angiogenic sprouting and ultimately impact the architecture of the vasculature.
Asunto(s)
Morfogénesis , Neovascularización Fisiológica , Proteína de Unión al GTP cdc42/fisiología , Biomimética , Movimiento Celular , Células Endoteliales/citología , Células Endoteliales/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana , Humanos , Modelos Cardiovasculares , Seudópodos , Pirazoles/farmacología , Sulfonamidas/farmacología , Proteína de Unión al GTP cdc42/antagonistas & inhibidores , Proteína de Unión al GTP cdc42/farmacologíaRESUMEN
The decline in hematopoietic function seen during aging involves a progressive reduction in the immune response and an increased incidence of myeloid malignancy, and has been linked to aging of hematopoietic stem cells (HSCs). The molecular mechanisms underlying HSC aging remain unclear. Here we demonstrate that elevated activity of the small RhoGTPase Cdc42 in aged HSCs is causally linked to HSC aging and correlates with a loss of polarity in aged HSCs. Pharmacological inhibition of Cdc42 activity functionally rejuvenates aged HSCs, increases the percentage of polarized cells in an aged HSC population, and restores the level and spatial distribution of histone H4 lysine 16 acetylation to a status similar to that seen in young HSCs. Our data therefore suggest a mechanistic role for Cdc42 activity in HSC biology and epigenetic regulation, and identify Cdc42 activity as a pharmacological target for ameliorating stem cell aging.
Asunto(s)
Senescencia Celular , Células Madre Hematopoyéticas/fisiología , Histonas/metabolismo , Tubulina (Proteína)/metabolismo , Proteína de Unión al GTP cdc42/metabolismo , Acetilación , Envejecimiento Prematuro/genética , Animales , Polaridad Celular/genética , Células Cultivadas , Proteínas Activadoras de GTPasa/genética , Hematopoyesis/genética , Células Madre Hematopoyéticas/efectos de los fármacos , Ratones , Ratones Noqueados , Células Mieloides/fisiología , Transporte de Proteínas/genética , Rejuvenecimiento , Proteína de Unión al GTP cdc42/farmacologíaRESUMEN
Cytoplasmic Ca2+ elevation and changes in Rho GTPase activity are both known to mediate axon guidance by extracellular factors, but the causal relationship between these two events has been unclear. Here we show that direct elevation of cytoplasmic Ca2+ by extracellular application of a low concentration of ryanodine, which activated Ca2+ release from intracellular stores, upregulated Cdc42/Rac, but downregulated RhoA, in cultured cerebellar granule cells and human embryonic kidney 293T cells. Chemoattractive turning of the growth cone triggered by a gradient of ryanodine was blocked by overexpression of mutant forms of Cdc42 but not of RhoA in Xenopus spinal cord neurons. Furthermore, Ca2+-induced GTPase activity correlated with activation of protein kinase C and required a basal activity of Ca2+/calmodulin-dependent protein kinase II. Thus, Rho GTPases may mediate axon guidance by linking upstream Ca2+ signals triggered by guidance factors to downstream cytoskeletal rearrangements.
Asunto(s)
Calcio/metabolismo , Conos de Crecimiento/fisiología , Neuronas/citología , Proteínas de Unión al GTP rho/metabolismo , Animales , Proteínas Bacterianas/farmacología , Toxinas Bacterianas/farmacología , Western Blotting/métodos , Factor Neurotrófico Derivado del Encéfalo/farmacología , Línea Celular , Quelantes/farmacología , Diagnóstico por Imagen/métodos , Relación Dosis-Respuesta a Droga , Interacciones Farmacológicas , Ácido Egtácico/análogos & derivados , Ácido Egtácico/farmacología , Embrión no Mamífero , Activación Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Proteínas Fluorescentes Verdes/farmacología , Conos de Crecimiento/efectos de los fármacos , Humanos , Factores de Crecimiento Nervioso/farmacología , Netrina-1 , Neuronas/fisiología , Rianodina/farmacología , Médula Espinal/citología , Tapsigargina/farmacología , Factores de Tiempo , Transfección/métodos , Proteínas Supresoras de Tumor/farmacología , Xenopus , Proteína de Unión al GTP cdc42/farmacologíaRESUMEN
The central nervous system (CNS) fails to regenerate after injury. A glial scar forms at the injury site, contributing to regenerative failure partly resulting from the chondroitin sulfate proteoglycans (CSPGs) in the glial scar. The family of Rho GTPases, which includes Cdc42, Rac1, and RhoA, is involved in growth cone dynamics. Although the response of neural cells to the inactivation of Rho when contacting myelin-related substrates, or CSPG, has been investigated, Rac1's and Cdc42's abilities to modulate CSPG-dependent inhibition have yet to be explored. In this study, a stripe assay was utilized to examine the effects of modulating all three Rho GTPases on neurite extension across inhibitory CSPG lanes. Alternating laminin (LN) and CSPG lanes were created and NG108-15 cells and E9 chick dorsal root ganglia (DRGs) were cultured on the lanes. By using the protein delivery agent Chariot, the neuronal response to exposure of constitutively active (CA) and dominant negative (DN) mutants of the Rho GTPases, along with the bacterial toxin C3, was determined by quantifying the percentage ratio of neurites crossing the CSPG lanes. CA-Cdc42, CA-Rac1, and C3 transferase significantly increased the number of neurites crossing into the CSPG lanes compared with the negative controls for both the NG108-15 cells and the E9 chick DRGs. We also show that these mutant proteins require the delivery vehicle, Chariot, to enter the neurons and affect neurite extension. Therefore, activation of Cdc42 and Rac, as well as inhibition of Rho, helps overcome the CSPG-dependent inhibition of neurite extension.
Asunto(s)
Proteoglicanos Tipo Condroitín Sulfato/metabolismo , Cicatriz/enzimología , Gliosis/enzimología , Regeneración Nerviosa/fisiología , Neuritas/enzimología , Proteínas de Unión al GTP rho/metabolismo , ADP Ribosa Transferasas/farmacología , Animales , Toxinas Botulínicas/farmacología , Línea Celular Tumoral , Sistema Nervioso Central/citología , Sistema Nervioso Central/enzimología , Sistema Nervioso Central/crecimiento & desarrollo , Embrión de Pollo , Cicatriz/fisiopatología , Cicatriz/prevención & control , Ganglios Espinales/citología , Ganglios Espinales/efectos de los fármacos , Ganglios Espinales/metabolismo , Gliosis/fisiopatología , Gliosis/prevención & control , Ratones , Neuritas/efectos de los fármacos , Neuritas/ultraestructura , Neuronas Aferentes/citología , Neuronas Aferentes/efectos de los fármacos , Neuronas Aferentes/metabolismo , Ratas , Proteína de Unión al GTP cdc42/genética , Proteína de Unión al GTP cdc42/metabolismo , Proteína de Unión al GTP cdc42/farmacología , Proteína de Unión al GTP rac1/genética , Proteína de Unión al GTP rac1/metabolismo , Proteína de Unión al GTP rac1/farmacología , Proteínas de Unión al GTP rho/genética , Proteínas de Unión al GTP rho/farmacología , Proteína de Unión al GTP rhoA/antagonistas & inhibidores , Proteína de Unión al GTP rhoA/metabolismoRESUMEN
Type I phosphatidylinositol 4-phosphate 5-kinase (PIP5K) catalyzes the formation of the phospholipid, phosphatidylinositol 4,5-bisphosphate (PIP(2)), which is implicated in many cellular processes. The Rho GTPases, RhoA and Rac1, have been shown previously to activate PIP5K and to bind PIP5K. Three type I PIP5K isoforms (Ialpha,Ibeta, and Igamma) have been identified; however, it is unclear whether these isoforms are differentially or even sequentially regulated by Rho GTPases. Here we show that RhoA and Rac1, as well as Cdc42, but not the Ras-like GTPases, RalA and Rap1A, markedly stimulate PIP(2) synthesis by all three PIP5K isoforms expressed in human embryonic kidney 293 cells, both in vitro and in vivo. RhoA-stimulated PIP(2) synthesis by the PIP5K isoforms was mediated by the RhoA effector, Rho-kinase. Stimulation of PIP5K isoforms by Rac1 and Cdc42 was apparently independent of and additive with RhoA- and Rho-kinase, as shown by studies with C3 transferase and Rho-kinase mutants. RhoA, and to a lesser extent Rac1, but not Cdc42, interacted in a nucleotide-independent form with all three PIP5K isoforms. Binding of PIP5K isoforms to GTP-bound, but not GDP-bound, RhoA could be displaced by Rho-kinase, suggesting a direct and constitutive PIP5K-Rho GTPase binding, which, however, does not trigger PIP5K activation. In summary, our findings indicate that synthesis of PIP(2) by the three PIP5K isoforms is controlled by RhoA, acting via Rho-kinase, as well as Rac1 and Cdc42, implicating that regulation of PIP(2) synthesis has a central position in signaling by these three Rho GTPases.
Asunto(s)
Isoenzimas/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Proteína de Unión al GTP cdc42/farmacología , Proteína de Unión al GTP rac1/farmacología , Proteína de Unión al GTP rhoA/farmacología , Animales , Línea Celular , Embrión de Mamíferos , Activación Enzimática/efectos de los fármacos , Expresión Génica , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Guanosina Difosfato/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intracelular , Riñón , Ratones , Mutagénesis , Fosfatidilinositol 4,5-Difosfato/biosíntesis , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Transfección , Proteína de Unión al GTP cdc42/genética , Proteína de Unión al GTP cdc42/metabolismo , Proteína de Unión al GTP rac1/genética , Proteína de Unión al GTP rac1/metabolismo , Quinasas Asociadas a rho , Proteína de Unión al GTP rhoA/genética , Proteína de Unión al GTP rhoA/metabolismoRESUMEN
The morphology of antigen-presenting dendritic cells (DC) is characterized by the possession of numerous long arborizing processes known as dendrites. The formation of these processes by DC, both in the periphery and in lymphoid organs, is believed to contribute to the remarkable efficiency with which they take up, process and present antigen to T cells. However, the process of dendrite formation and the signaling pathways that lead to the formation of these dendrites remain obscure. In this study we describe an in vitro model in which human immature DC form long processes similar to those formed in vivo. The formation of these processes involves initial attachment of a cell protrusion to the extracellular matrix substrate, and subsequent movement of the cell body away from the adhesion site, leaving behind a long slender dendrite. Dendrite formation, but not their maintenance, was found to be dependent on the activity of Rho GTPases. More specifically, Cdc42 and Rac1 were both required for the migration step of process formation, promoting cell spreading and extension. In contrast, Rho, and its downstream effector p160ROCK, regulated the release of adhesions to the substratum, and associated cellular contraction. Consequently, inhibition of Rho/p160ROCK leads to the formation of longer dendrites. DC therefore coordinate adhesion and protrusion to perform a specialized process of cellular morphogenesis, which differentiates these cells from all other cells of the immune system and may contribute to their distinctive function.
Asunto(s)
Células Dendríticas/fisiología , Proteína de Unión al GTP cdc42/metabolismo , Proteína de Unión al GTP rac1/metabolismo , Proteína de Unión al GTP rhoA/metabolismo , Adhesión Celular , Movimiento Celular/efectos de los fármacos , Movimiento Celular/fisiología , Células Cultivadas , Dendritas/efectos de los fármacos , Dendritas/fisiología , Células Dendríticas/citología , Células Dendríticas/efectos de los fármacos , Fibronectinas/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intracelular , Proteínas Serina-Treonina Quinasas/metabolismo , Proteína de Unión al GTP cdc42/farmacología , Proteína de Unión al GTP rac1/farmacología , Quinasas Asociadas a rho , Proteína de Unión al GTP rhoA/farmacologíaRESUMEN
Activation of Rho, Rac and Cdc42 GTPases by an Escherichia coli cytotoxin (CNF1) has been reported to induce a phagocytic-like activity by epithelial cells in terms of a ruffle-driven capture and ingestion of large material. More recently, it has been reported that treatment with CNF1 induces superoxide anion release by these cells following a phagocytic stimulus. We herein show that in epithelial cells both transfection with the dominant form of Rac (RacV12) and treatment with the Rac-activating epidermal growth factor (EGF) may increase the secretion of superoxide anions on challenge with latex beads. Moreover, exposure to CNF1 induces a significant augmentation of acidic vesicles where the internalized particles were detectable. Our results indicate that (i) Rac is a pivotal GTPase for inducing in epithelial cells superoxide anion generation and (ii) the internalized material travels trough acidic compartments in CNF1-treated epithelial cells. Altogether this suggests a novel role for epithelial cells that, following Rac activation, might share with professional phagocytes the task of eliminating unwanted pathogens.
Asunto(s)
Toxinas Bacterianas/farmacología , Citotoxinas/farmacología , Células Epiteliales/fisiología , Proteínas de Escherichia coli , Fagocitos/fisiología , Factor Rho/farmacología , Proteínas de Unión al GTP rac/farmacología , Células Epiteliales/efectos de los fármacos , Escherichia coli , Humanos , Transfección , Proteína de Unión al GTP cdc42/farmacología , Proteínas de Unión al GTP rac/biosíntesisRESUMEN
Although evidence exists that activation of the Rho family GTPase Cdc42 affects axonal development, its specific roles within a growth cone are not well delineated. To evaluate the model that Cdc42 activation regulates growth cone navigation by promoting filopodial activity, we adopted a live analysis strategy that uses transgenic Drosophila lines in which neurons coexpressed constitutively active Cdc42 (Cdc42(V12)) and membrane-targeted green fluorescent protein. We found that growth cones that displayed pathfinding defects exhibited little change in their filopodial activity, whereas others without pathfinding defects exhibited an similar50% increase in their filopodial activity. Moreover, effector loop mutations that were added to the constitutively active Cdc42 (Cdc42(V12C40) and Cdc42(V12A37)) exerted little influence over filopodial activity caused by Cdc42 activation but suppressed the pathfinding defects of the growth cones. Together, these data suggest that Cdc42 controls filopodial activity in axonal growth cones independently of its effects on their pathfinding.
Asunto(s)
Conos de Crecimiento/metabolismo , Seudópodos/metabolismo , Proteína de Unión al GTP cdc42/metabolismo , Sustitución de Aminoácidos , Animales , Animales Modificados Genéticamente , Axones/efectos de los fármacos , Axones/metabolismo , Axones/ultraestructura , Células Cultivadas , Drosophila , Embrión no Mamífero/inervación , Expresión Génica , Proteínas Fluorescentes Verdes , Conos de Crecimiento/efectos de los fármacos , Inmunohistoquímica , Técnicas In Vitro , Proteínas Luminiscentes/biosíntesis , Proteínas Luminiscentes/genética , Microscopía por Video , Neuronas Motoras/efectos de los fármacos , Neuronas Motoras/metabolismo , Neuronas Motoras/ultraestructura , Mutagénesis Sitio-Dirigida , Neuronas Aferentes/efectos de los fármacos , Neuronas Aferentes/metabolismo , Neuronas Aferentes/ultraestructura , Seudópodos/efectos de los fármacos , Seudópodos/ultraestructura , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Transgenes , Proteína de Unión al GTP cdc42/genética , Proteína de Unión al GTP cdc42/farmacologíaRESUMEN
Patients with the immunodeficiency disorder Wiskott-Aldrich syndrome (WAS) have lymphocytes with aberrant microvilli, and their T cells, macrophages, and dendritic cells are impaired in cytoskeletal-dependent processes. WAS is caused by a defective or a missing WAS protein (WASP). Signal mediators interleukin-4 (IL-4) and CD40 are important for actin-dependent morphology changes in B cells. A possible function of WASP and its interacting partners, Cdc42 and Rac1, was investigated for these changes. It was found that active Cdc42 and Rac1 induced filopodia and lamellipodia, respectively, in activated B cells. Evidence is given that IL-4 has a specific role in the regulated cycling of Cdc42 because IL-4 partially and transiently depleted active Cdc42 from detergent extract of activated B cells. WASP-deficient B lymphocytes were impaired in IL-4-- and CD40-dependent induction of polarized and spread cells. Microvilli were expressed on WASP-deficient B cells, but they appeared shorter and less dense in cell contacts than in wild-type cells. In conclusion, evidence is provided for the involvement of Cdc42, Rac1, and WASP in the cytoskeletal regulation of B lymphocytes. Aberrations in WASP-deficient B lymphocytes, described here, provide further evidence that WAS is a cytoskeletal disease of hematopoietic cells. (Blood. 2001;98:1086-1094)
Asunto(s)
Linfocitos B/efectos de los fármacos , Citoesqueleto/efectos de los fármacos , Proteínas/farmacología , Proteína de Unión al GTP cdc42/farmacología , Proteína de Unión al GTP rac1/farmacología , Animales , Linfocitos B/citología , Linfocitos B/ultraestructura , Antígenos CD40/farmacología , Adhesión Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Polaridad Celular/efectos de los fármacos , Interleucina-4/farmacología , Ratones , Ratones Noqueados , Microvellosidades/efectos de los fármacos , Proteínas del Tejido Nervioso/metabolismo , Proteínas/fisiología , Transducción de Señal/efectos de los fármacos , Síndrome de Wiskott-Aldrich/patología , Proteína del Síndrome de Wiskott-Aldrich , Proteína Neuronal del Síndrome de Wiskott-Aldrich , Proteína de Unión al GTP cdc42/efectos de los fármacos , Proteína de Unión al GTP cdc42/fisiología , Proteína de Unión al GTP rac1/fisiología , Proteínas de Unión al GTP rho/farmacología , Proteínas de Unión al GTP rho/fisiologíaRESUMEN
The cellular mechanisms that configure the cytoskeleton during migration of dendritic cells (DCs) are poorly understood. Immature DCs assemble specialized adhesion structures known as podosomes at their leading edge; these are associated with the localized recruitment of the Wiskott-Aldrich Syndrome protein (WASp) and the actin organizing actin-related protein 2/3 complex. In immature DCs lacking WASp, podosomes are absent, residual dysmorphic lamellipodia and filopodia are nonpolarized, and migration is severely compromised. Microinjection studies indicate that podosome assembly and polarization require concerted action of Cdc42, Rac, and Rho, thereby providing a link between sequential protrusive and adhesive activity. Formation of podosomes is restricted to cells with an immature phenotype, indicating a specific role for these structures during the early migratory phase. (Blood. 2001;98:1142-1149)
Asunto(s)
Proteínas del Citoesqueleto , Citoesqueleto/efectos de los fármacos , Células Dendríticas/citología , Células Dendríticas/ultraestructura , Proteínas/farmacología , Proteínas de Unión al GTP rho/farmacología , Proteína 2 Relacionada con la Actina , Proteína 3 Relacionada con la Actina , Actinas/efectos de los fármacos , Actinas/metabolismo , Actinas/fisiología , Diferenciación Celular/fisiología , Movimiento Celular/efectos de los fármacos , Citoesqueleto/enzimología , Células Dendríticas/enzimología , Humanos , Proteínas/fisiología , Proteína del Síndrome de Wiskott-Aldrich , Proteína de Unión al GTP cdc42/farmacología , Proteína de Unión al GTP cdc42/fisiología , Proteínas de Unión al GTP rac/farmacología , Proteínas de Unión al GTP rac/fisiología , Proteínas de Unión al GTP rho/fisiologíaRESUMEN
Superior cervical ganglion (SCG) sympathetic neurons die by apoptosis when deprived of nerve growth factor (NGF). It has been shown previously that the induction of apoptosis in these neurons at NGF withdrawal requires both the activity of the small GTP-binding protein Cdc42 and the activation of the c-Jun N-terminal kinase (JNK) pathway. The mixed lineage kinase 3 (MLK3) belongs to a family of mitogen-activated protein (MAP) kinase kinase kinases. MLK3 contains a Cdc42/Rac interactive-binding (CRIB) domain and activates both the JNK and the p38 MAP kinase pathways. In this study the role of MLK3 in the induction of apoptosis in sympathetic neurons has been investigated. Overexpression of an active MLK3 induces activation of the JNK pathway and apoptosis in SCG neurons. In addition, overexpression of kinase dead mutants of MLK3 blocks apoptosis as well as c-Jun phosphorylation induced by NGF deprivation. More importantly, MLK3 activity seems to increase by 5 hr after NGF withdrawal in both differentiated PC12 cells and SCG neurons. We also show that MLK3 lies downstream of Cdc42 in the neuronal death pathway. Regulation of MLK3 in neurons seems to be dependent on MLK3 activity and possibly on an additional cellular component, but not on its binding to Cdc42. These results suggest that MLK3, or a closely related kinase, is a physiological element of NGF withdrawal-induced activation of the Cdc42-c-Jun pathway and neuronal death. MLK3 therefore could be an interesting therapeutic target in a number of neurodegenerative diseases involving neuronal apoptosis.
Asunto(s)
Apoptosis/fisiología , Quinasas Quinasa Quinasa PAM/metabolismo , Neuronas/enzimología , Secuencias de Aminoácidos/fisiología , Animales , Apoptosis/efectos de los fármacos , Línea Celular , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Activación Enzimática/efectos de los fármacos , Activación Enzimática/genética , Expresión Génica , Genes Dominantes , Humanos , Proteínas Quinasas JNK Activadas por Mitógenos , Quinasas Quinasa Quinasa PAM/genética , Quinasas Quinasa Quinasa PAM/farmacología , Microinyecciones , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Mutagénesis Sitio-Dirigida , Factor de Crecimiento Nervioso/farmacología , Neuronas/citología , Neuronas/efectos de los fármacos , Fosforilación/efectos de los fármacos , Proteínas Proto-Oncogénicas c-bcl-2/biosíntesis , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-bcl-2/farmacología , Ratas , Ratas Sprague-Dawley , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Ganglio Cervical Superior , Proteína de Unión al GTP cdc42/metabolismo , Proteína de Unión al GTP cdc42/farmacología , Proteina Quinasa Quinasa Quinasa 11 Activada por MitógenoRESUMEN
It is well established that Rho-GTPases regulate vesicle fusion and fission events at the plasma membrane through their modulatory role on the cortical actin cytoskeleton. In contrast, their effects on intracellular transport processes and actin pools are less clear. It was recently shown that cdc42 associates with the Golgi apparatus in an ARF-dependent manner, similarly to coat proteins involved in vesicle formation and to several actin-binding proteins. We report here that mutants of cdc42 inhibited the exit of basolateral proteins from the trans-Golgi network (TGN), while stimulating the exit of an apical marker, in two different transport assays. This regulation may result from modulation of the actin cytoskeleton, as GTPase-deficient cdc42 depleted a perinuclear actin pool that rapidly exchanges with exogenous fluorescent actin.
Asunto(s)
Proteína de Unión al GTP cdc42/metabolismo , Red trans-Golgi/metabolismo , Citoesqueleto de Actina/efectos de los fármacos , Citoesqueleto de Actina/metabolismo , Actinas/química , Actinas/metabolismo , Animales , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Línea Celular , Permeabilidad de la Membrana Celular , Vesículas Citoplasmáticas/metabolismo , Citoesqueleto/metabolismo , Perros , Colorantes Fluorescentes/química , Proteínas Luminiscentes/genética , Mutagénesis Sitio-Dirigida , Moléculas de Adhesión de Célula Nerviosa/genética , Transporte de Proteínas/efectos de los fármacos , Transporte de Proteínas/fisiología , Receptor de Factor de Crecimiento Nervioso , Receptores de Factor de Crecimiento Nervioso/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Tiazoles/farmacología , Tiazolidinas , Proteína de Unión al GTP cdc42/genética , Proteína de Unión al GTP cdc42/farmacologíaRESUMEN
We previously demonstrated that Rac1 increased cyclin D1 promoter activity in an extracellular signal-regulated kinase (ERK)-independent, antioxidant-sensitive manner. Here, we examined the regulation of cyclin D1 expression by Cdc42 and RhoA. Overexpression of active Cdc42, but not of RhoA, induced transcription from the cyclin D1 promoter. Furthermore, dominant negative Cdc42, but not RhoA, attenuated platelet-derived growth factor-mediated activation of the cyclin D1 promoter. Overexpression of active Cdc42 increased cyclin D1 protein abundance in COS cells. Cdc42-induced cyclin D1 promoter activation was independent of ERK as evidenced by insensitivity to PD-98059, an inhibitor of mitogen-activated protein kinase/ERK kinase (MEK). Furthermore, Cdc42 was neither sufficient nor required for activation of ERK. Similar to Rac1-induced cyclin D1 expression, pretreatment with the antioxidants catalase and ebselen inhibited Cdc42-mediated transcription from the cyclin D1 promoter. Finally, like Rac1, active Cdc42 induced transactivation of the cyclin D1 promoter cAMP response element binding protein/activating transcription factor-2 binding site. Together, these data suggest that in airway smooth muscle cells, Cdc42 and Rac1 share a common signaling pathway to cyclin D1 promoter activation.
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Ciclina D1/metabolismo , Regulación de la Expresión Génica/fisiología , Músculo Liso/metabolismo , Proteína de Unión al GTP cdc42/metabolismo , Proteína de Unión al GTP rhoA/metabolismo , Animales , Antioxidantes/farmacología , Sitios de Unión/fisiología , Células COS , Bovinos , Células Cultivadas , Ciclina D1/genética , Inhibidores Enzimáticos/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Proteína Quinasa 1 Activada por Mitógenos/genética , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Quinasas de Proteína Quinasa Activadas por Mitógenos/antagonistas & inhibidores , Músculo Liso/citología , Mutación , Factor de Crecimiento Derivado de Plaquetas/farmacología , Regiones Promotoras Genéticas/efectos de los fármacos , Tráquea/citología , Tráquea/metabolismo , Transcripción Genética/efectos de los fármacos , Transfección , Proteína de Unión al GTP cdc42/genética , Proteína de Unión al GTP cdc42/farmacología , Proteína de Unión al GTP rac1/genética , Proteína de Unión al GTP rac1/metabolismo , Proteína de Unión al GTP rhoA/genéticaRESUMEN
In studies aimed at further characterizing the cellular immunodeficiency of the Wiskott-Aldrich syndrome (WAS), we found that T lymphocytes from WAS patients display abnormal chemotaxis in response to the T-cell chemoattractant stromal cell-derived factor (SDF)-1. The Wiskott- Aldrich syndrome protein (WASP), together with the Rho family GTPase Cdc42, control stimulus-induced actin cytoskeleton rearrangements that are involved in cell motility. Because WASP is an effector of Cdc42, we further studied how Cdc42 and WASP are involved in SDF-1-induced chemotaxis of T lymphocytes. We provide here direct evidence that SDF-1 activates Cdc42. We then specifically investigated the role of the interaction between Cdc42 and WASP in SDF-1-responsive cells. This was achieved by abrogating this interaction with a recombinant polypeptide (TAT-CRIB), comprising the Cdc42/Rac interactive binding (CRIB) domain of WASP and a human immunodeficiency virus-TAT peptide that renders the fusion protein cell-permeant. This TAT-CRIB protein was shown to bind specifically to Cdc42-GTP and to inhibit the chemotactic response of a T-cell line to SDF-1. Altogether, these data demonstrate that Cdc42-WASP interaction is critical for SDF-1-induced chemotaxis of T cells.
Asunto(s)
Quimiotaxis de Leucocito/efectos de los fármacos , Proteínas/metabolismo , Proteína de Unión al GTP cdc42/farmacología , Actinas/antagonistas & inhibidores , Actinas/metabolismo , Sitios de Unión , Línea Celular , Quimiocina CXCL12 , Quimiocinas CXC/farmacología , Interacciones Farmacológicas , Humanos , Unión Proteica , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas/fisiología , Linfocitos T/citología , Síndrome de Wiskott-Aldrich/sangre , Síndrome de Wiskott-Aldrich/etiología , Síndrome de Wiskott-Aldrich/metabolismo , Proteína del Síndrome de Wiskott-Aldrich , Proteína de Unión al GTP cdc42/efectos de los fármacos , Proteína de Unión al GTP cdc42/metabolismo , Quinasas p21 ActivadasRESUMEN
Dendritic spines mediate most excitatory transmission in the mammalian CNS and have been traditionally considered stable structures. Following the suggestion that spines may 'twitch', it has been recently shown that spines are capable of rapid morphological rearrangements. Because of the role of the small GTPases from the Rho family in controlling neuronal morphogenesis, we investigated the effects of several members of this biochemical signaling pathway in the maintenance of the morphology of extant dendritic spines by combining biolistic transfection of pyramidal neurons in cultured cortical and hippocampal slices with two-photon microscopy. We find a variety of effects on the density and morphology of dendritic spines by expressing either constitutively active or dominant negative forms of several small GTPases of the Rho family, by blocking the entire pathway with Clostridium difficile toxin B or by blocking Rho with C3 transferase. We propose a model where Rac promotes spine formation, while Rho prevents it. We conclude that the small GTPases provide antagonistic control mechanisms of spine maintenance in pyramidal neurons.
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Proteínas Bacterianas , Dendritas/fisiología , Dendritas/ultraestructura , Proteínas de Unión al GTP rho/fisiología , Animales , Toxinas Bacterianas/farmacología , Técnicas de Cultivo , Dendritas/efectos de los fármacos , Proteínas Fluorescentes Verdes , Indicadores y Reactivos/farmacología , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/farmacología , Ratones , Ratones Endogámicos C57BL , Transducción de Señal/fisiología , Transfección , Proteína de Unión al GTP cdc42/farmacología , Proteínas de Unión al GTP rac/farmacología , Proteínas de Unión al GTP rac/fisiología , Proteínas de Unión al GTP rho/farmacologíaRESUMEN
BACKGROUND: ERM (ezrin, radixin, and moesin) proteins function as membrane-cytoskeletal linkers, and are known to be localized at filopodia and microvilli-like structures. We have shown that Rho-associated kinase (Rho-kinase)/ROKalpha/ROCK II phosphorylates moesin at Thr-558 at the lower stream of Rho, and the phosphorylation is crucial to the formation of microvilli-like structures (Oshiro, N., Fukata, Y. & Kaibuchi, K. (1998) Phosphorylation of moesin by Rho-associated kinase (Rho-kinase) plays a crucial role in the formation of microvilli-like structures. J. Biol. Chem. 273, 34663- 34666). However, the role of ERM proteins in the formation of filopodia is less well characterized. RESULTS: Here we examined the phosphorylation state of ERM during filopodia formation induced by Cdc42 using the antibody recognizing ERM proteins phosphorylated at COOH (C)-terminal threonine. When NIH 3T3 cells were transfected with constitutively active Cdc42 (Cdc42V12), filopodia formation was induced and phosphorylation of ERM at C-terminal threonine was observed at the tip of filopodia, while the phosphorylation levels of ERM were lower and phosphorylated ERM was distributed throughout the cytoplasm in the control cells. We also showed that Myotonic dystrophy kinase-related Cdc42-binding kinase (MRCK) which has been identified as an effector of Cdc42, phosphorylated moesin at C-terminal threonine in a cell-free system. Coexpression of the dominant negative form of MRCK inhibited both the formation of filopodia and accumulation of C-terminal threonine-phosphorylated ERM proteins at filopodia induced by Cdc42V12. CONCLUSION: The formation of filopodia induced by Cdc42 is accompanied by phosphorylation of ERM proteins, and MRCK is a candidate for the kinase that phosphorylates ERM proteins at filopodia.
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Proteínas Sanguíneas/metabolismo , Proteínas del Citoesqueleto/metabolismo , Proteínas de Escherichia coli , Proteínas de la Membrana/metabolismo , Proteínas de Microfilamentos/metabolismo , Fosfoproteínas/metabolismo , Seudópodos/fisiología , Proteína de Unión al GTP cdc42/farmacología , Células 3T3/efectos de los fármacos , Células 3T3/metabolismo , Animales , Western Blotting , Proteínas de Unión a Calmodulina/biosíntesis , Células Cultivadas , Cartilla de ADN/química , Electroforesis en Gel de Poliacrilamida , Técnica del Anticuerpo Fluorescente , Genes Dominantes/genética , Genes Dominantes/fisiología , Glutatión Transferasa/biosíntesis , Glicoproteínas Hemaglutininas del Virus de la Influenza/biosíntesis , Péptidos y Proteínas de Señalización Intracelular , Ratones , Fragmentos de Péptidos/biosíntesis , Fosforilación , Plásmidos/biosíntesis , Reacción en Cadena de la Polimerasa , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Recombinantes , Proteínas Represoras/biosíntesis , Transducción de Señal , Treonina/química , Transfección , Quinasas Asociadas a rhoRESUMEN
To assess the role of cdc42 during neurite development, cmyc-tagged constitutively active (CA) and dominant negative (DN) cdc42 were expressed in dissociated primary chick spinal cord neurons using adenoviral-mediated gene transfer. Three days after infection, >85% of the neurons in infected cultures expressed cdc42 proteins, as detected by indirect immunofluorescence against cmyc. Growth cones of infected neurons displayed 1.83- (CAcdc42) and 1.93-fold (DNcdc42) higher cmyc immunofluorescence per square micrometer than uninfected controls. CAcdc42 expression stimulated growth cones, almost doubling growth cone size and number of filopodia, and increased neurite growth rates by 65-89%. In neurons plated onto fibronectin, the percent of growth cones with both filopodia and lamellipodia increased from 71 to 92%. Total Texas Red-phalloidin staining in these growth cones doubled, and the percent of growth cones with F-actin localized to peripheral regions increased from 52% in controls to 78% after CAcdc42 expression. Expression of DNcdc42 did not significantly alter growth cone morphology or neurite growth rates. Addition of soluble laminin to spinal cord neurons resulted in the identical phenotype as CAcdc42 expression, including changes in growth cone morphology, F-actin localization, and neurite growth rates. Significantly, expression of DNcdc42 blocked the effects of laminin on growth cones. These results show that cdc42 promotes neurite outgrowth and filopodial and lamellipodial formation in growth cones and suggests that cdc42 and laminin share a common signaling pathway during neurite development. Addition of laminin to CAcdc42-expressing neurons is inhibitory to growth cones, indicating that laminin also may activate some other pathways.
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Conos de Crecimiento/fisiología , Neuritas/fisiología , Seudópodos/fisiología , Proteína de Unión al GTP cdc42/fisiología , Actinas/metabolismo , Animales , Células Cultivadas , Embrión de Pollo , Regulación hacia Abajo , Genes Dominantes , Conos de Crecimiento/efectos de los fármacos , Laminina/antagonistas & inhibidores , Laminina/farmacología , Mutación/fisiología , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Neuronas/fisiología , Médula Espinal/citología , Médula Espinal/embriología , Distribución Tisular/fisiología , Proteína de Unión al GTP cdc42/genética , Proteína de Unión al GTP cdc42/metabolismo , Proteína de Unión al GTP cdc42/farmacologíaRESUMEN
The role of hippocalcin as a novel mediator in the PKC-independent Ca2+ -induced phospholipase D (PLD) activation pathway was investigated. Hippocalcin was expressed in the Sf9 insect cell expression system because the myristoylation of this protein is essential for its function. PLD and Cdc42 proteins were prepared from a rat brain cell membrane and cytosol, respectively. The recombinant hippocalcin was expressed in the Sf9 cell using expression vector pVL1393. The hippocalcin expressed was purified as a single band on PAGE following the hydrophobic phenyl HPLC and TSKgel G3000SW gel filtration HPLC. The molecular size of the rat brain hippocalcin expressed in this system was estimated to be 22 kDa. Myristoylated hippocalcin migrated faster than the non-myristoylated form on SDS-PAGE. Less than 10% of the total hippocalcin expressed was myristoylated in this baculovirus expression system. PLD was extracted from rat brain membranes and chromatographically enriched 70-fold. From the rat brain cytosol, Cdc42 was purified to near homogeneity. While hippocalcin alone did not activate PLD, it increased PLD activity activated with Cdc42 1.8-fold in the presence of calcium (300 nM free calcium). In the absence of calcium in the reaction mixture, the effect of hippocalcin to facilitate Cdc42-activated PLD activity was abolished. This result suggests that hippocalcin might be one of the regulatory proteins in the PKC-independent Ca2+ -mediated PLD activation pathway in conjunction with the Cdc42 protein.