RESUMEN
T cells form immunological synapses with professional antigen-presenting cells (APCs) resulting in T cell activation and the acquisition of peptide antigen-MHC (pMHC) complexes from the plasma membrane of the APC. They thus become APCs themselves. We investigate the functional outcome of T-T cell antigen presentation by CD4 T cells and find that the antigen-presenting T cells (Tpres) predominantly differentiate into regulatory T cells (Treg), whereas T cells that have been stimulated by Tpres cells predominantly differentiate into Th17 pro-inflammatory cells. Using mice deficient in pMHC uptake by T cells, we show that T-T antigen presentation is important for the development of experimental autoimmune encephalitis and Th17 cell differentiation in vivo. By varying the professional APC:T cell ratio, we can modulate Treg versus Th17 differentiation in vitro and in vivo, suggesting that T-T antigen presentation underlies proinflammatory responses in conditions of antigen scarcity.
Asunto(s)
Presentación de Antígeno/inmunología , Antígenos/metabolismo , Polaridad Celular/inmunología , Células Th17/inmunología , Animales , Antígenos CD28/metabolismo , Diferenciación Celular/inmunología , Membrana Celular/metabolismo , Células Dendríticas/inmunología , Modelos Animales de Enfermedad , Encefalomielitis Autoinmune Experimental/inmunología , Encefalomielitis Autoinmune Experimental/patología , Regulación de la Expresión Génica , Genoma , Antígenos de Histocompatibilidad Clase II/inmunología , Ratones Endogámicos C57BL , Linfocitos T Reguladores/inmunología , Transcripción Genética , Trogocitosis , Proteínas de Unión al GTP rho/deficiencia , Proteínas de Unión al GTP rho/metabolismoRESUMEN
BACKGROUND & AIMS: It is not clear how regulation of T-cell function is altered during development of inflammatory bowel diseases (IBD). We studied the mechanisms by which geranylgeranyltransferase-mediated prenylation controls T-cell localization to the intestine and chronic inflammation. METHODS: We generated mice with T-cell-specific disruption of the geranylgeranyltransferase type I, beta subunit gene (Pggt1b), called Pggt1bΔCD4 mice, or the ras homolog family member A gene (Rhoa), called RhoaΔCD4 mice. We also studied mice with knockout of CDC42 or RAC1 and wild-type mice (controls). Intestinal tissues were analyzed by histology, multiphoton and confocal microscopy, and real-time polymerase chain reaction. Activation of CDC42, RAC1, and RHOA were measured with G-LISA, cell fractionation, and immunoblots. T cells and lamina propria mononuclear cells from mice were analyzed by flow cytometry or transferred to Rag1-/- mice. Mice were given injections of antibodies against integrin alpha4beta7 or gavaged with the RORC antagonist GSK805. We obtained peripheral blood and intestinal tissue samples from patients with and without IBD and analyzed them by flow cytometry. RESULTS: Pggt1bΔCD4 mice developed spontaneous colitis, characterized by thickening of the intestinal wall, edema, fibrosis, accumulation of T cells in the colon, and increased expression of inflammatory cytokines. Compared with control CD4+ T cells, PGGT1B-deficient CD4+ T cells expressed significantly higher levels of integrin alpha4beta7, which regulates their localization to the intestine. Inflammation induced by transfer of PGGT1B-deficient CD4+ T cells to Rag1-/- mice was blocked by injection of an antibody against integrin alpha4beta7. Lamina propria of Pggt1bΔCD4 mice had increased numbers of CD4+ T cells that expressed RORC and higher levels of cytokines produced by T-helper 17 cells (granulocyte-macrophage colony-stimulating factor, interleukin [IL]17A, IL17F, IL22, and tumor necrosis factor [TNF]). The RORC inverse agonist GSK805, but not antibodies against IL17A or IL17F, prevented colitis in Pggt1bΔCD4 mice. PGGT1B-deficient CD4+ T cells had decreased activation of RHOA. RhoAΔCD4 mice had a similar phenotype to Pggt1bΔCD4 mice, including development of colitis, increased numbers of CD4+ T cells in colon, increased expression of integrin alpha4beta7 by CD4+ T cells, and increased levels of IL17A and other inflammatory cytokines in lamina propria. T cells isolated from intestinal tissues from patients with IBD had significantly lower levels of PGGT1B than tissues from individuals without IBD. CONCLUSION: Loss of PGGT1B from T cells in mice impairs RHOA function, increasing CD4+ T-cell expression of integrin alpha4beta7 and localization to colon, resulting in increased expression of inflammatory cytokines and colitis. T cells isolated from gut tissues from patients with IBD have lower levels of PGGT1B than tissues from patients without IBD.
Asunto(s)
Transferasas Alquil y Aril/deficiencia , Quimiotaxis de Leucocito , Colitis/enzimología , Colon/enzimología , Integrinas/metabolismo , Linfocitos T/enzimología , Proteínas de Unión al GTP rho/metabolismo , Inmunidad Adaptativa , Transferasas Alquil y Aril/genética , Animales , Estudios de Casos y Controles , Células Cultivadas , Colitis/genética , Colitis/inmunología , Colitis/patología , Colon/inmunología , Colon/patología , Citocinas/metabolismo , Modelos Animales de Enfermedad , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Inmunidad Innata , Mediadores de Inflamación/metabolismo , Activación de Linfocitos , Ratones Noqueados , Neuropéptidos/genética , Neuropéptidos/metabolismo , Transducción de Señal , Linfocitos T/inmunología , Linfocitos T/patología , 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 , Proteínas de Unión al GTP rho/deficiencia , Proteínas de Unión al GTP rho/genética , Proteína de Unión al GTP rhoARESUMEN
Typical Rho GTPases, such as Rac1, Cdc42, and RhoA, act as molecular switches regulating various aspects of platelet cytoskeleton reorganization. The loss of these enzymes results in reduced platelet functionality. Atypical Rho GTPases of the RhoBTB subfamily are characterized by divergent domain architecture. One family member, RhoBTB3, is expressed in platelets, but its function is unclear. In the present study we examined the role of RhoBTB3 in platelet function using a knockout mouse model. We found the platelet count, size, numbers of both alpha and dense granules, and surface receptor profile in these mice were comparable to wild-type mice. Deletion of Rhobtb3 had no effect on aggregation and dense granule secretion in response to a range of agonists including thrombin, collagen, and adenosine diphosphate (ADP). By contrast, alpha-granule secretion increased in mice lacking RhoBTB3 in response to thrombin, collagen related peptide (CRP) and U46619/ADP. Integrin activation and spreading on fibrinogen and collagen under static conditions were also unimpaired; however, we observed reduced platelet accrual on collagen under flow conditions. These defects did not translate into alterations in tail bleeding time. We conclude that genetic deletion of Rhobtb3 leads to subtle alterations in alpha-granule secretion and adhesion to collagen without significant effects on hemostasis in vivo.
Asunto(s)
Plaquetas/metabolismo , Colágeno/farmacología , Gránulos Citoplasmáticos/metabolismo , Adhesividad Plaquetaria , Reología , Proteínas de Unión al GTP rho/deficiencia , Animales , Plaquetas/efectos de los fármacos , Plaquetas/ultraestructura , Línea Celular , Gránulos Citoplasmáticos/efectos de los fármacos , Humanos , Megacariocitos/efectos de los fármacos , Megacariocitos/metabolismo , Ratones Endogámicos C57BL , Ratones Noqueados , Adhesividad Plaquetaria/efectos de los fármacos , Agregación Plaquetaria/efectos de los fármacos , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas de Unión al GTP rho/genética , Proteínas de Unión al GTP rho/metabolismoRESUMEN
Growth cones are complex, motile structures at the tip of an outgrowing neurite. They often exhibit a high density of filopodia (thin actin bundles), which complicates the unbiased quantification of their morphologies by software. Contemporary image processing methods require extensive tuning of segmentation parameters, require significant manual curation, and are often not sufficiently adaptable to capture morphology changes associated with switches in regulatory signals. To overcome these limitations, we developed Growth Cone Analyzer (GCA). GCA is designed to quantify growth cone morphodynamics from time-lapse sequences imaged both in vitro and in vivo, but is sufficiently generic that it may be applied to nonneuronal cellular structures. We demonstrate the adaptability of GCA through the analysis of growth cone morphological variation and its relation to motility in both an unperturbed system and in the context of modified Rho GTPase signaling. We find that perturbations inducing similar changes in neurite length exhibit underappreciated phenotypic nuance at the scale of the growth cone.
Asunto(s)
Conos de Crecimiento/ultraestructura , Procesamiento de Imagen Asistido por Computador/estadística & datos numéricos , Imagen Molecular/normas , Neuronas/ultraestructura , Programas Informáticos , Imagen de Lapso de Tiempo/normas , Proteínas de Unión al GTP rho/genética , Animales , Línea Celular Tumoral , Movimiento Celular , Forma de la Célula/genética , Regulación de la Expresión Génica , Heterogeneidad Genética , Conos de Crecimiento/metabolismo , Factores de Intercambio de Guanina Nucleótido/deficiencia , Factores de Intercambio de Guanina Nucleótido/genética , Ratones , Imagen Molecular/métodos , Neuronas/metabolismo , Neuropéptidos/deficiencia , Neuropéptidos/genética , Fosfoproteínas/deficiencia , Fosfoproteínas/genética , Proteínas Serina-Treonina Quinasas/deficiencia , Proteínas Serina-Treonina Quinasas/genética , Seudópodos/metabolismo , Seudópodos/ultraestructura , Factores de Intercambio de Guanina Nucleótido Rho/deficiencia , Factores de Intercambio de Guanina Nucleótido Rho/genética , Transducción de Señal , Imagen de Lapso de Tiempo/métodos , Proteína de Unión al GTP cdc42/deficiencia , Proteína de Unión al GTP cdc42/genética , Proteína de Unión al GTP rac1/deficiencia , Proteína de Unión al GTP rac1/genética , Proteínas de Unión al GTP rho/deficiencia , Proteína de Unión al GTP rhoARESUMEN
RhoA, a member of Rho GTPases family, is known to play an important role in remodeling actin cytoskeleton. During the development of the peripheral nervous system (PNS), Schwann cells undergo proliferation, migration, and radial sorting and finally wrap the related axons compactly to form myelin sheath. All these processes involve actin cytoskeletal remodeling. However, the role of RhoA on Schwann cell during development is still unclear. To address this question, we first used a lentiviral vector-mediated short hairpin (sh) RNA targeting RhoA to knock down the expression of RhoA in the cultured Schwann cells in vitro. Effects of RhoA on Schwann cell proliferation and migration were examined by BrdU assay and transwell assay, respectively. Results of the present study indicated that downregulated RhoA expression in cultured Schwann cells significantly slacked the cells' capabilities of migration and proliferation. Then, we investigated the role of RhoA in the developing rat sciatic nerves. Immunohistology and Western blotting showed that RhoA was mainly expressed in Schwann cells in the sciatic nerves and was peaked at 2 weeks postnatal then kept in low level up to 8 weeks. In the subjected rats whose sciatic nerves were microinjected with lentiviral vectors at postnatal 3 days, we found that the lentiviruses mainly transfected Schwann cells, and the RhoA expression in the transfected Schwann cells was significantly knocked down. Four weeks after lentivirus microinjection, immunohistology and transmission electron microscopy illustrated that RhoA knockdown resulted in hypomyelination and significant decrease of the thickness of myelin in the transfected area. Overall data of current study suggested that RhoA plays a critical role in Schwann cell biology and is essential for myelination in developing peripheral nerve.
Asunto(s)
Movimiento Celular/fisiología , Proliferación Celular/fisiología , Vaina de Mielina/fisiología , Interferencia de ARN/fisiología , Células de Schwann/fisiología , Proteínas de Unión al GTP rho/deficiencia , Animales , Animales Recién Nacidos , Células Cultivadas , Técnicas de Silenciamiento del Gen/métodos , Marcación de Gen/métodos , Lentivirus/genética , Ratas , Ratas Sprague-Dawley , Proteínas de Unión al GTP rho/genéticaRESUMEN
Retinal degenerative diseases can have many possible causes and are currently difficult to treat. As an alternative to therapies that require genetic manipulation or the implantation of electronic devices, photopharmacology has emerged as a viable approach to restore visual responses. Here, we present a new photopharmacological strategy that relies on a photoswitchable excitatory amino acid, ATA. This freely diffusible molecule selectively activates AMPA receptors in a light-dependent fashion. It primarily acts on amacrine and retinal ganglion cells, although a minor effect on bipolar cells has been observed. As such, it complements previous pharmacological approaches based on photochromic channel blockers and increases the potential of photopharmacology in vision restoration.
Asunto(s)
Ceguera/tratamiento farmacológico , Luz , Receptores AMPA/metabolismo , Receptores de Ácido Kaínico/metabolismo , Células Ganglionares de la Retina/fisiología , Potenciales de Acción/efectos de los fármacos , Potenciales de Acción/genética , Animales , Animales Recién Nacidos , Ceguera/genética , Ceguera/patología , Canales Catiónicos Regulados por Nucleótidos Cíclicos/deficiencia , Canales Catiónicos Regulados por Nucleótidos Cíclicos/genética , Modelos Animales de Enfermedad , GABAérgicos/farmacología , Células HEK293 , Hipocampo/citología , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neuronas/efectos de los fármacos , Neuronas/fisiología , Ácidos Fosfínicos/farmacología , Picrotoxina/análogos & derivados , Picrotoxina/farmacología , Piridinas/farmacología , Receptores de Ácido Kaínico/genética , Células Ganglionares de la Retina/efectos de los fármacos , Opsinas de Bastones/deficiencia , Opsinas de Bastones/genética , Sesterterpenos , Proteínas de Unión al GTP rho/deficiencia , Proteínas de Unión al GTP rho/genética , Receptor de Ácido Kaínico GluK2RESUMEN
Wnt ligands regulate heart morphogenesis but the underlying mechanisms remain unclear. Two Formin-related proteins, DAAM1 and 2, were previously found to bind the Wnt effector Disheveled. Here, since DAAM1 and 2 nucleate actin and mediate Wnt-induced cytoskeletal changes, a floxed-allele of Daam1 was used to disrupt its function specifically in the myocardium and investigate Wnt-associated pathways. Homozygous Daam1 conditional knockout (CKO) mice were viable but had misshapen hearts and poor cardiac function. The defects in Daam1 CKO mice were observed by mid-gestation and were associated with a loss of protrusions from cardiomyocytes invading the outflow tract. Further, these mice exhibited noncompaction cardiomyopathy (NCM) and deranged cardiomyocyte polarity. Interestingly, Daam1 CKO mice that were also homozygous for an insertion disrupting Daam2 (DKO) had stronger NCM, severely reduced cardiac function, disrupted sarcomere structure, and increased myocardial proliferation, suggesting that DAAM1 and DAAM2 have redundant functions. While RhoA was unaffected in the hearts of Daam1/2 DKO mice, AKT activity was lower than in controls, raising the issue of whether DAAM1/2 are only mediating Wnt signaling. Daam1-floxed mice were thus bred to Wnt5a null mice to identify genetic interactions. The hearts of Daam1 CKO mice that were also heterozygous for the null allele of Wnt5a had stronger NCM and more severe loss of cardiac function than Daam1 CKO mice, consistent with DAAM1 and Wnt5a acting in a common pathway. However, deleting Daam1 further disrupted Wnt5a homozygous-null hearts, suggesting that DAAM1 also has Wnt5a-independent roles in cardiac development.
Asunto(s)
Proteínas de Microfilamentos/metabolismo , Miocardio/metabolismo , Sarcómeros/metabolismo , Proteínas de Unión al GTP rho/metabolismo , Animales , Adhesión Celular , Proliferación Celular , Citoesqueleto/metabolismo , Embrión de Mamíferos/metabolismo , Eliminación de Gen , Regulación del Desarrollo de la Expresión Génica , Glucógeno Sintasa Quinasa 3/metabolismo , Pruebas de Función Cardíaca , Ventrículos Cardíacos/patología , Ventrículos Cardíacos/fisiopatología , Heterocigoto , Ratones Noqueados , Proteínas de Microfilamentos/deficiencia , Proteínas de Microfilamentos/genética , Morfogénesis , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/patología , Especificidad de Órganos , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal , Proteínas Wnt , Proteína Wnt-5a , Proteínas de Unión al GTP rho/deficiencia , Proteínas de Unión al GTP rho/genética , Proteína de Unión al GTP rhoA/metabolismoRESUMEN
Stroma mediated wound healing signals may share similarities with the ones produced by tumor's microenvironment and their modulation may impact tumor response to the various anti-cancer treatments including radiation therapy. Therefore we conducted this study, to assess the crosstalk between stromal and carcinoma cells in response to radiotherapy by genetic modulation of the stroma and irradiation. We found that fibroblasts irrespective of their RhoB status do not modulate intrinsic radiosensitivity of TC-1 but produce diffusible factors able to modify tumor cell fate. Then we found that Wt and RhoB deficient fibroblasts stimulated TC-1 migration through distinct mechanisms which are TGF-ß1 and MMP-mediated respectively. Lastly, we found that simultaneous irradiation of fibroblasts and TC-1 abrogated the pro-migratory phenotype by repression of TGF-ß and MMP secretion. This last result is highly relevant to the clinical situation and suggests that conversely to, the current view; irradiated stroma would not enhance carcinoma migration and could be manipulated to promote anti-tumor immune response.
Asunto(s)
Carcinoma/radioterapia , Movimiento Celular/efectos de la radiación , Fibroblastos/efectos de la radiación , Neoplasias Pulmonares/radioterapia , Proteínas de Unión al GTP rho/genética , Animales , Carcinoma/metabolismo , Carcinoma/patología , Diferenciación Celular/efectos de la radiación , Línea Celular Tumoral , Fibroblastos/metabolismo , Fibroblastos/patología , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Ratones , Comunicación Paracrina/efectos de la radiación , Radiación , Tolerancia a Radiación , Factor de Crecimiento Transformador beta1/metabolismo , Vimentina/metabolismo , Cicatrización de Heridas/efectos de la radiación , Proteínas de Unión al GTP rho/deficienciaRESUMEN
Epithelial ovarian cancer (EOC) is the second leading cause of death from gynecological malignancies worldwide. Enhancer of zeste homology 2 (EZH2), participating in gene expression silencing by trimethylating histone 3 lysine 27 (H3K27me3), is often up-regulated in EOC. ARHI, an imprinted tumor-suppressor gene, is markedly down-regulated or even undetectable in the majority of EOC. To explore the correlation between EZH2 and ARHI expression in EOC as well as the possible mechanism of EZH2-ARH1 interaction. We used immunohistochemical staining to evaluate the expression of EZH2 and ARHI in EOC and normal ovarian tissue specimens; western blotting, shRNA, and chromatin immunoprecipitation were used to study the expression correlation of EZH2 and ARHI in EOC and normal ovarian epithelial cells and to further explore the mechanism of EZH2 regulation of ARHI expression. Cell viability assay was used to evaluate the influence of these two genes on cell survival. (1) The expression of EZH2 inversely correlated with ARHI expression levels and predicted shorter overall survival in EOC patients; (2) EZH2 promoted repression of ARHI by catalyzing trimethylation on H3K27; (3) ARHI was synergistically silenced by DNA methylation and histone modification; and (4) DZNep, an inhibitor of EZH2, significantly reduced survival rate of EOC cells by restoring ARHI expression. EZH2-â³induced H3K27me3 is associated with epigenetic repression of the ARHI tumor-suppressor gene in EOC. Suppression of EZH2 by DZNep, as a way of restoring the expression of ARHI, could be a potential treatment modality to EOC.
Asunto(s)
Represión Epigenética , Histonas/metabolismo , Lisina/metabolismo , Neoplasias Glandulares y Epiteliales/genética , Neoplasias Ováricas/genética , Complejo Represivo Polycomb 2/metabolismo , Proteínas Supresoras de Tumor/genética , Proteínas de Unión al GTP rho/genética , Adenosina/análogos & derivados , Adenosina/farmacología , Carcinogénesis/efectos de los fármacos , Carcinogénesis/genética , Carcinoma Epitelial de Ovario , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Proliferación Celular/genética , Proteína Potenciadora del Homólogo Zeste 2 , Represión Epigenética/efectos de los fármacos , Femenino , Técnicas de Silenciamiento del Gen , Histonas/química , Humanos , Metilación/efectos de los fármacos , Neoplasias Glandulares y Epiteliales/patología , Neoplasias Ováricas/patología , Complejo Represivo Polycomb 2/deficiencia , Complejo Represivo Polycomb 2/genética , ARN Interferente Pequeño/genética , Análisis de Supervivencia , Proteínas Supresoras de Tumor/deficiencia , Proteínas de Unión al GTP rho/deficienciaRESUMEN
The subventricular zone represents an important reservoir of progenitor cells in the adult brain. Cells from the subventricular zone migrate along the rostral migratory stream and reach the olfactory bulb, where they originate different types of interneurons. In this work, we have analyzed the role of the small GTPase RhoE/Rnd3 in subventricular zone cell development using mice-lacking RhoE expression. Our results show that RhoE null mice display a remarkable postnatal broadening of the subventricular zone and caudal rostral migratory stream. This broadening was caused by an increase in progenitor proliferation, observed in the second postnatal week but not before, and by an altered migration of the cells, which appeared in disorganized cell arrangements that impaired the appropriate contact between cells in the rostral migratory stream. In addition, the thickness of the granule cell layer in the olfactory bulb was reduced, although the density of granule cells did not differ between wild-type and RhoE null mice. Finally, the lack of RhoE expression affected the olfactory glomeruli inducing a severe reduction of calbindin-expressing interneurons in the periglomerular layer. This was already evident in the newborns and even more pronounced 15 days later when RhoE null mice displayed 89% less cells than control mice. Our results indicate that RhoE has pleiotropic functions on subventricular cells because of its role in proliferation and tangential migration, affecting mainly the development of calbindin-expressing cells in the olfactory bulb.
Asunto(s)
Calbindinas/biosíntesis , Ventrículos Laterales/metabolismo , Neuronas/metabolismo , Bulbo Olfatorio/metabolismo , Proteínas de Unión al GTP rho/deficiencia , Proteínas de Unión al GTP rho/metabolismo , Animales , Animales Recién Nacidos , Encéfalo/citología , Encéfalo/crecimiento & desarrollo , Encéfalo/metabolismo , Encéfalo/fisiología , Calbindinas/metabolismo , Diferenciación Celular/fisiología , Movimiento Celular/fisiología , Ventrículos Laterales/citología , Ventrículos Laterales/crecimiento & desarrollo , Ratones , Neuronas/citología , Bulbo Olfatorio/citología , Zona Incerta/citología , Zona Incerta/crecimiento & desarrollo , Zona Incerta/metabolismoRESUMEN
RATIONALE: Rnd3, a small Rho GTPase, is involved in the regulation of cell actin cytoskeleton dynamics, cell migration, and proliferation. The biological function of Rnd3 in the heart remains unexplored. OBJECTIVE: To define the functional role of the Rnd3 gene in the animal heart and investigate the associated molecular mechanism. METHODS AND RESULTS: By loss-of-function approaches, we discovered that Rnd3 is involved in calcium regulation in cardiomyocytes. Rnd3-null mice died at the embryonic stage with fetal arrhythmias. The deletion of Rnd3 resulted in severe Ca(2+) leakage through destabilized ryanodine receptor type 2 Ca(2+) release channels. We further found that downregulation of Rnd3 attenuated ß2-adrenergic receptor lysosomal targeting and ubiquitination, which in turn resulted in the elevation of ß2-adrenergic receptor protein levels leading to the hyperactivation of protein kinase A (PKA) signaling. The PKA activation destabilized ryanodine receptor type 2 channels. This irregular spontaneous Ca(2+) release can be curtailed by PKA inhibitor treatment. Increases in the PKA activity along with elevated cAMP levels were detected in Rnd3-null embryos, in neonatal rat cardiomyocytes, and noncardiac cell lines with Rnd3 knockdown, suggesting a general mechanism for Rnd3-mediated PKA signaling activation. ß2-Adrenergic receptor blocker treatment reduced arrhythmia and improved cardiac function. CONCLUSIONS: Rnd3 is a novel factor involved in intracellular Ca(2+) homeostasis regulation in the heart. Deficiency of the protein induces ryanodine receptor type 2 dysfunction by a mechanism that attenuates Rnd3-mediated ß2-adrenergic receptor ubiquitination, which leads to the activation of PKA signaling. Increased PKA signaling in turn promotes ryanodine receptor type 2 hyperphosphorylation, which contributes to arrhythmogenesis and heart failure.
Asunto(s)
Calcio/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/biosíntesis , Eliminación de Gen , Miocitos Cardíacos/metabolismo , Proteínas de Unión al GTP rho/deficiencia , Proteínas de Unión al GTP rho/genética , Animales , Animales Recién Nacidos , Células Cultivadas , Femenino , Corazón/fisiología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratas , Transducción de Señal/fisiología , Regulación hacia Arriba/fisiologíaRESUMEN
Rho proteins are a large family of GTPases involved in the control of actin cytoskeleton dynamics, proliferation and survival. Rnd1, Rnd2 and RhoE/Rnd3 form a subfamily of Rho proteins characterized by being constitutively active. The role of these proteins has been studied during the last years in several systems; however, little is known about their expression and functions in the reproductive organs. In this work we analysed the localization and the effect of RhoE deficiency in the testes using mice lacking RhoE expression (RhoE gt/gt), and our research shows some unexpected and relevant results. First, we have observed that RhoE is only expressed in Leydig cells within the testicular parenchyma and it is absent of seminiferous tubules. In addition, RhoE is expressed in the excurrent ducts of the testis, including the ductuli efferentes, epididymis and ductus deferens. Moreover, the testes of postnatal 15-day-old RhoE null mice are smaller, both in absolute values and in relation to the body weight. Furthermore, the dimensions of their seminiferous tubules are also reduced compared with wild-types. In order to study the role of RhoE in the adult, we analysed heterozygous animals as RhoE null mice die early postnatally. Our results show that the testes of adult RhoE heterozygous mice are also smaller than those of the wild-types, with a 17% decrease in the ratio testis weight/body weight. In addition, their seminiferous tubules have reduced tubular diameter (12%) and a thinner epithelial wall (33%) that appears disorganized and with a swollen lumen. Finally, and probably as a consequence of those alterations, the sperm concentration of heterozygous animals was found to be lower than in the wild-types. These results indicate that accurate levels of RhoE in the testes are necessary for a correct development and function of male gonads, and suggest novel and unexpected roles of Rnd GTPases in the reproductive physiology.
Asunto(s)
Epidídimo/metabolismo , Testículo/metabolismo , Conducto Deferente/metabolismo , Proteínas de Unión al GTP rho/metabolismo , Animales , Masculino , Ratones , Testículo/anatomía & histología , Proteínas de Unión al GTP rho/deficiencia , Proteínas de Unión al GTP rho/fisiologíaRESUMEN
PURPOSE: Epithelial cell migration is required for regeneration of tissues and can be defective in a number of ocular surface diseases. This study aimed to determine the expression pattern of Rho family small G-proteins in human corneal epithelial cells to test their requirement in directional cell migration. METHODS: Rho family small G-protein expression was assessed by reverse transcription-polymerase chain reaction. Dominant-inhibitory constructs encoding Rho proteins or Rho protein targeting small interfering RNA were transfected into human corneal epithelial large T antigen cells, and wound closure rate were evaluated by scratch wounding assay, and a complementary non-traumatic cell migration assay. Immunofluorescence staining was performed to study cell polarization and to assess Cdc42 downstream effector. RESULTS: Cdc42, Chp, Rac1, RhoA, TC10 and TCL were expressed in human corneal epithelial cells. Among them, Cdc42 and TCL were found to significantly affect cell migration in monolayer scratch assays. These results were confirmed through the use of validated siRNAs directed to Cdc42 and TCL. Scramble siRNA transfected cells had high percentage of polarized cells than Cdc42 or TCL siRNA transfected cells at the wound edge. We showed that the Cdc42-specific effector p21-activated kinase 4 localized predominantly to cell-cell junctions in cell monolayers, but failed to translocate to the leading edge in Cdc42 siRNA transfected cells after monolayer wounding. CONCLUSION: Rho proteins expressed in cultured human corneal epithelial cells, and Cdc42, TCL facilitate two-dimensional cell migration in-vitro. Although silencing of Cdc42 and TCL did not noticeably affect the appearance of cell adhesions at the leading edge, the slower migration of these cells indicates both GTP-binding proteins play important roles in promoting cell movement of human corneal epithelial cells.
Asunto(s)
Movimiento Celular , Polaridad Celular , Epitelio Corneal/citología , Proteínas de Unión al GTP rho/metabolismo , Silenciador del Gen , Humanos , Transporte de Proteínas , ARN Interferente Pequeño/genética , Proteína de Unión al GTP cdc42/deficiencia , Proteína de Unión al GTP cdc42/genética , Proteína de Unión al GTP cdc42/metabolismo , Quinasas p21 Activadas/metabolismo , Proteínas de Unión al GTP rho/deficiencia , Proteínas de Unión al GTP rho/genéticaRESUMEN
Cortical interneurons arise from the ganglionic eminences in the ventral telencephalon and migrate tangentially to the cortex. Although RhoA and Cdc42, members of the Rho family of small GTPases, have been implicated in regulating neuronal migration, their respective roles in the tangential migration of cortical interneurons remain unknown. Here we show that loss of RhoA and Cdc42 in the ventricular zone (VZ) of the medial ganglionic eminence (MGE) using Olig2-Cre mice causes moderate or severe defects in the migration of cortical interneurons, respectively. Furthermore, RhoA- or Cdc42-deleted MGE cells exhibit impaired migration in vitro. To determine whether RhoA and Cdc42 directly regulate the motility of cortical interneurons during migration, we deleted RhoA and Cdc42 in the subventricular zone (SVZ), where more fate-restricted progenitors are located within the ganglionic eminences, using Dlx5/6-Cre-ires-EGFP (Dlx5/6-CIE) mice. Deletion of either gene within the SVZ does not cause any obvious defects in cortical interneuron migration, indicating that cell motility is not dependent upon RhoA or Cdc42. These findings provide genetic evidence that RhoA and Cdc42 are required in progenitors of the MGE in the VZ, but not the SVZ, for proper cortical interneuron migration.
Asunto(s)
Corteza Cerebral/embriología , Corteza Cerebral/metabolismo , Interneuronas/metabolismo , Células-Madre Neurales/metabolismo , Proteína de Unión al GTP cdc42/metabolismo , Proteínas de Unión al GTP rho/metabolismo , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/deficiencia , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Muerte Celular , Diferenciación Celular , Movimiento Celular , Proliferación Celular , Corteza Cerebral/citología , Femenino , Eminencia Media/citología , Eminencia Media/embriología , Eminencia Media/metabolismo , Ratones , Ratones Noqueados , Ratones Transgénicos , Red Nerviosa/citología , Red Nerviosa/embriología , Red Nerviosa/metabolismo , Proteínas del Tejido Nervioso/deficiencia , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Neurogénesis , Factor de Transcripción 2 de los Oligodendrocitos , Embarazo , Proteína de Unión al GTP cdc42/deficiencia , Proteína de Unión al GTP cdc42/genética , Proteínas de Unión al GTP rho/deficiencia , Proteínas de Unión al GTP rho/genética , Proteína de Unión al GTP rhoARESUMEN
RhoH is a new member of the atypical G proteins exclusively expressed in hematopoietic lineage cells. It has been shown to act as an adaptor for ZAP70, Syk, Lck and Csk kinases in signal transduction, and is required for positive selection of thymocytes as well as activation of peripheral T cells and mast cells. In the present study, we showed that RhoH is required not only for positive selection but also for negative selection of thymocytes. Regarding development of unconventional T cell subsets, development of NKT and regulatory T cells was also inhibited, whereas development of TCRαß CD8αα intestinal intraepithelial lymphocytes (IEL) was not affected by the absence of RhoH. TCR-dependent in vitro activation of TCRαß CD8αα IEL required RhoH, suggesting that overall development of IEL does not critically depend on TCR signaling but more on cytokine-dependent expansion and survival in the periphery. Our current results indicate differential requirements for RhoH in the development of TCRαß CD8αα IELs compared to other subsets of T cells including agonist selected T cells.
Asunto(s)
Antígenos CD8/metabolismo , Diferenciación Celular , Mucosa Intestinal/inmunología , Receptores de Antígenos de Linfocitos T alfa-beta/metabolismo , Subgrupos de Linfocitos T/citología , Subgrupos de Linfocitos T/metabolismo , Factores de Transcripción/metabolismo , Proteínas de Unión al GTP rho/metabolismo , Animales , Diferenciación Celular/genética , Diferenciación Celular/inmunología , Femenino , Interferón gamma/biosíntesis , Células Progenitoras Linfoides/citología , Células Progenitoras Linfoides/metabolismo , Masculino , Ratones , Ratones Noqueados , Ratones Transgénicos , Células T Asesinas Naturales/metabolismo , Bazo/citología , Bazo/inmunología , Subgrupos de Linfocitos T/inmunología , Linfocitos T Reguladores/metabolismo , Timo/citología , Timo/inmunología , Factores de Transcripción/deficiencia , Factores de Transcripción/genética , Proteínas de Unión al GTP rho/deficiencia , Proteínas de Unión al GTP rho/genéticaRESUMEN
Retinitis pigmentosa (RP) relates to a group of hereditary neurodegenerative diseases of the retina. On the cellular level, RP results in the primary death of rod photoreceptors, caused by rod-specific mutations, followed by a secondary degeneration of genetically normal cones. Different mechanisms may influence the spread of cell death from one photoreceptor type to the other. As one of these mechanisms a gap junction-mediated bystander effect was proposed, i.e., toxic molecules generated in dying rods and propagating through gap junctions induce the death of healthy cone photoreceptors. We investigated whether disruption of rod-cone coupling can prevent secondary cone death and reduce the spread of degeneration. We tested this hypothesis in two different mouse models for retinal degeneration (rhodopsin knockout and rd1) by crossbreeding them with connexin36-deficient mice as connexin36 represents the gap junction protein on the cone side and lack thereof most likely disrupts rod-cone coupling. Using immunohistochemistry, we compared the progress of cone degeneration between connexin36-deficient mouse mutants and their connexin36-expressing littermates at different ages and assessed the accompanied morphological changes during the onset (rhodopsin knockout) and later stages of secondary cone death (rd1 mutants). Connexin36-deficient mouse mutants showed the same time course of cone degeneration and the same morphological changes in second order neurons as their connexin36-expressing littermates. Thus, our results indicate that disruption of connexin36-mediated rod-cone coupling does not stop, delay or spatially restrict secondary cone degeneration and suggest that the gap junction-mediated bystander effect does not contribute to the progression of RP.
Asunto(s)
Conexinas/metabolismo , Uniones Comunicantes/metabolismo , Células Fotorreceptoras Retinianas Conos/metabolismo , Retinitis Pigmentosa/metabolismo , Animales , Muerte Celular , Conexinas/genética , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Eliminación de Gen , Regulación de la Expresión Génica , Ratones , Ratones Noqueados , Células Fotorreceptoras Retinianas Conos/patología , Degeneración Retiniana/genética , Degeneración Retiniana/metabolismo , Retinitis Pigmentosa/genética , Proteínas de Unión al GTP rho/deficiencia , Proteína delta-6 de Union ComunicanteRESUMEN
The fibrous sheath (FS) is a flagellar cytoskeletal structure unique to sperm that surrounds the outer dense fibers and axoneme. Its primary components are A-kinase anchoring proteins (AKAPs) 3 and 4, which suggests that the FS affects flagellar beating via the scaffolding of signaling pathways necessary for motility. Sperm proteins ROPN1 and ROPN1L bind AKAP3. To determine the role of ROPN1 and ROPN1L in sperm function, we created mice deficient in ROPN1 (RKO), mice deficient in ROPN1L (RLKO), and double knockout mice (DKO). All three strains of mice had normal testicular morphology and spermatogenesis. Only the DKOs had obvious defects in sperm morphology (thinning and shredding of the principal piece), which was accompanied by a reduction in AKAP3 levels. RLKO mice had slightly reduced sperm motility and increased levels of ROPN1. RKO mice had moderately impaired motility and increased levels of ROPN1L. DKO sperm were immotile. We have previously determined that RKO male mice are subfertile, and DKO males are infertile. Together these data indicate that ROPN1L and ROPN1 compensate for each other in the absence of the opposing protein, possibly to maintain AKAP3 incorporation in the FS. Sperm from mice lacking ROPN1L exhibited reductions in both cAMP-dependent protein kinase (PKA) phosphorylation of a 270-kDa protein (perhaps FSCB), and in capacitation-induced tyrosine phosphorylation. Sperm from mice lacking ROPN1 had reduced levels of FSCB and increased tyrosine phosphorylation of noncapacitated sperm. These data demonstrate that mutations in ROPN1 and ROPN1L can cause defects in FS integrity, sperm motility, and PKA-dependent signaling processes, leading to male infertility.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/deficiencia , Axonema/fisiología , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Proteínas de la Membrana/deficiencia , Motilidad Espermática/fisiología , Cola del Espermatozoide/fisiología , Proteínas de Unión al GTP rho/deficiencia , Proteínas de Anclaje a la Quinasa A/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/fisiología , Animales , Infertilidad Masculina/metabolismo , Infertilidad Masculina/fisiopatología , Masculino , Proteínas de la Membrana/genética , Proteínas de la Membrana/fisiología , Ratones , Ratones Noqueados , Modelos Animales , Fosforilación/fisiología , Transducción de Señal/fisiología , Capacitación Espermática/fisiología , Tirosina/metabolismo , Proteínas de Unión al GTP rho/genética , Proteínas de Unión al GTP rho/fisiologíaRESUMEN
Epidermodysplasia verruciformis (EV) is a rare genetic disorder characterized by increased susceptibility to specific human papillomaviruses, the betapapillomaviruses. These EV-HPVs cause warts and increase the risk of skin carcinomas in otherwise healthy individuals. Inactivating mutations in epidermodysplasia verruciformis 1 (EVER1) or EVER2 have been identified in most, but not all, patients with autosomal recessive EV. We found that 2 young adult siblings presenting with T cell deficiency and various infectious diseases, including persistent EV-HPV infections, were homozygous for a mutation creating a stop codon in the ras homolog gene family member H (RHOH) gene. RHOH encodes an atypical Rho GTPase expressed predominantly in hematopoietic cells. Patients' circulating T cells contained predominantly effector memory T cells, which displayed impaired TCR signaling. Additionally, very few circulating T cells expressed the ß7 integrin subunit, which homes T cells to specific tissues. Similarly, Rhoh-null mice exhibited a severe overall T cell defect and abnormally small numbers of circulating ß7-positive cells. Expression of the WT, but not of the mutated RHOH, allele in Rhoh-/- hematopoietic stem cells corrected the T cell lymphopenia in mice after bone marrow transplantation. We conclude that RHOH deficiency leads to T cell defects and persistent EV-HPV infections, suggesting that T cells play a role in the pathogenesis of chronic EV-HPV infections.
Asunto(s)
Epidermodisplasia Verruciforme/genética , Linfocitos T/patología , Factores de Transcripción/deficiencia , Proteínas de Unión al GTP rho/deficiencia , Adulto , Animales , Secuencia de Bases , Betapapillomavirus , Estudios de Casos y Controles , Codón sin Sentido , Consanguinidad , Susceptibilidad a Enfermedades , Epidermodisplasia Verruciforme/inmunología , Epidermodisplasia Verruciforme/patología , Epidermodisplasia Verruciforme/virología , Estudio de Asociación del Genoma Completo , Genotipo , Humanos , Integrinas/metabolismo , Recuento de Linfocitos , Ratones , Ratones Noqueados , Linaje , Polimorfismo de Nucleótido Simple , Receptores de Antígenos de Linfocitos T/metabolismo , Análisis de Secuencia de ADN , Transducción de Señal , Factores de Transcripción/genética , Proteínas de Unión al GTP rho/genéticaRESUMEN
Rho family GTPases are molecular switches best known for their pivotal role in dynamic regulation of the actin cytoskeleton. The prototypic members of this family are Cdc42, Rac1, and RhoA; these GTPases contribute to the breakdown of glomerular filtration and the resultant proteinuria, but their functions in normal podocyte physiology remain poorly understood. Here, mice lacking Cdc42 in podocytes developed congenital nephropathy and died as a result of renal failure within 2 weeks after birth. In contrast, mice lacking Rac1 or RhoA in podocytes were overtly normal and lived to adulthood. Kidneys from Cdc42-mutant mice exhibited protein-filled microcysts with hallmarks of collapsing glomerulopathy, as well as extensive effacement of podocyte foot processes with abnormal junctional complexes. Furthermore, we observed aberrant expression of several podocyte markers and cell polarity proteins in the absence of Cdc42, indicating a disruption of the slit diaphragm. Kidneys from Rac1- and RhoA-mutant mice, however, had normal glomerular morphology and intact foot processes. A nephrin clustering assay suggested that Cdc42 deficiency, but not Rac1 or RhoA deficiency, impairs the polymerization of actin at sites of nephrin aggregates. Taken together, these data highlight the physiological importance of Cdc42, but not Rac1 or RhoA, in establishing podocyte architecture and glomerular function.
Asunto(s)
Enfermedades Renales/congénito , Enfermedades Renales/etiología , Podocitos/metabolismo , Proteína de Unión al GTP cdc42/deficiencia , Animales , Modelos Animales de Enfermedad , Femenino , Barrera de Filtración Glomerular/metabolismo , Enfermedades Renales/metabolismo , Proteínas de la Membrana/metabolismo , Ratones , Ratones Noqueados , Ratones Transgénicos , Podocitos/patología , Embarazo , Proteína de Unión al GTP cdc42/genética , Proteína de Unión al GTP cdc42/metabolismo , Proteína de Unión al GTP rac1/deficiencia , Proteína de Unión al GTP rac1/genética , Proteína de Unión al GTP rac1/metabolismo , Proteínas de Unión al GTP rho/deficiencia , Proteínas de Unión al GTP rho/genética , Proteínas de Unión al GTP rho/metabolismo , Proteína de Unión al GTP rhoARESUMEN
RhoA is a member of the Rho family small GTPases that are implicated in various cell functions including proliferation and survival. However, the physiological role of RhoA in vivo remains largely unknown. Here, we deleted RhoA in the B cell and hematopoietic stem cell (HSC) populations in RhoA(flox/flox) mice with CD19 and Mx promoter-driven Cre expression, respectively. Deletion of RhoA by CD19(Cre/+) significantly blocked B cell development in spleen, leading to a marked reduction in the number of transitional, marginal zone, and follicular B cells. Surprisingly, neither B cell proliferation in response to either LPS or B cell receptor (BCR) engagement nor B cell survival rate in vivo was affected by RhoA deletion. Furthermore, RhoA(-/-) B cells, like control cells, were rescued from apoptosis by BCR crosslinking in vitro. In contrast, RhoA deficiency led to a defect in B cell activating factor (BAFF)-mediated B cell survival that was associated with a dampened expression of BAFF receptor and a loss of BAFF-mediated Akt activation. Finally, HSC deletion of RhoA by Mx-Cre severely reduced proB/preB and immature B cell populations in bone marrow while common lymphoid progenitors were increased, indicating that RhoA is also required for B cell progenitor/precursor differentiation. Taken together, our results uncover an important role for RhoA at multiple stages of B cell development.