RESUMEN
BACKGROUND: Inflammatory Bowel Diseases (IBD) are a major public health issue with unclear aetiology. Changes in the composition and functionality of the intestinal microbiota are associated with these pathologies, including the depletion of strict anaerobes such as Feacalibacterium prausnitzii. Less evidence is observed for depletion in other anaerobes, among which bifidobacteria. This study characterized the taxonomic and functional diversity of bifidobacteria isolated from the human intestinal microbiota in active and non-active IBD patients by a culturomics approach and evaluated if these bifidobacteria might be used as probiotics for gut health. RESULTS: A total of 341 bifidobacteria were isolated from the intestinal microbiota of IBD patients (52 Crohn's disease and 26 ulcerative colitis patients), with a high proportion of Bifidobacterium dentium strains (28% of isolated bifidobacteria). In ulcerative colitis, the major species identified was B. dentium (39% of isolated bifidobacteria), in active and non-active ulcerative colitis. In Crohn's disease, B. adolescentis was the major species isolated from non-active patients (40%), while similar amounts of B. dentium and B. adolescentis were found in active Crohn's disease patients. The relative abundance of B. dentium was increased with age, both in Crohn's disease and ulcerative colitis and active and non-active IBD patients. Antibacterial capacities of bifidobacteria isolated from non-active ulcerative colitis against Escherichia coli LF82 and Salmonella enterica ATCC 14028 were observed more often compared to strains isolated from active ulcerative colitis. Finally, B. longum were retained as strains with the highest probiotic potential as they were the major strains presenting exopolysaccharide synthesis, antibacterial activity, and anti-inflammatory capacities. Antimicrobial activity and EPS synthesis were further correlated to the presence of antimicrobial and EPS gene clusters by in silico analysis. CONCLUSIONS: Different bifidobacterial taxonomic profiles were identified in the microbiota of IBD patients. The most abundant species were B. dentium, mainly associated to the microbiota of ulcerative colitis patients and B. adolescentis, in the intestinal microbiota of Crohn's disease patients. Additionally, the relative abundance of B. dentium significantly increased with age. Furthermore, this study evidenced that bifidobacteria with probiotic potential (antipathogenic activity, exopolysaccharide production and anti-inflammatory activity), especially B. longum strains, can be isolated from the intestinal microbiota of both active and non-active Crohn's disease and ulcerative colitis patients.
Asunto(s)
Bifidobacterium , Microbioma Gastrointestinal , Probióticos , Humanos , Bifidobacterium/aislamiento & purificación , Bifidobacterium/clasificación , Bifidobacterium/genética , Adulto , Femenino , Masculino , Persona de Mediana Edad , Enfermedades Inflamatorias del Intestino/microbiología , Adulto Joven , Anciano , Colitis Ulcerosa/microbiología , Enfermedad de Crohn/microbiología , Filogenia , Heces/microbiología , ARN Ribosómico 16S/genética , Fenotipo , Adolescente , Antibacterianos/farmacologíaRESUMEN
Novel molecules that directly target the neonatal Fc receptor (FcRn) and/or Fc gamma receptors (FcγRs) are emerging as promising treatments for immunoglobulin G (IgG)-dependent autoimmune pathologies. Mutated Fc regions and monoclonal antibodies that target FcRn are currently in clinical development and hold promise for reducing the levels of circulating IgG. Additionally, engineered structures containing multimeric Fc regions allow the dual targeting of FcRn and FcγRs; however, their tolerance needs to first be validated in phase I clinical studies. Here, for the first time, we have developed a modified monomeric recombinant Fc optimized for binding to all FcRns and FcγRs without the drawback of possible tolerance associated with FcγR cross-linking. A rational approach using Fc engineering allowed the selection of LFBD192, an Fc with a combination of six mutations that exhibits improved binding to human FcRn and FcγR as well as mouse FcRn and FcγRIV. The potency of LFBD192 was compared with that of intravenous immunoglobulin (IVIg), an FcRn blocker (Fc-MST-HN), and a trimeric Fc that blocks FcRn and/or immune complex-mediated cell activation through FcγR without triggering an immune reaction in several in vitro tests and validated in three mouse models of autoimmune disease.
Asunto(s)
Antirreumáticos/farmacología , Artritis Experimental/prevención & control , Autoinmunidad/efectos de los fármacos , Fragmentos Fc de Inmunoglobulinas/farmacología , Receptores Fc/antagonistas & inhibidores , Receptores de IgG/antagonistas & inhibidores , Animales , Antirreumáticos/metabolismo , Artritis Experimental/genética , Artritis Experimental/inmunología , Artritis Experimental/metabolismo , Unión Competitiva , Complemento C5a/metabolismo , Femenino , Antígenos de Histocompatibilidad Clase I/genética , Antígenos de Histocompatibilidad Clase I/inmunología , Antígenos de Histocompatibilidad Clase I/metabolismo , Humanos , Fragmentos Fc de Inmunoglobulinas/genética , Fragmentos Fc de Inmunoglobulinas/inmunología , Fragmentos Fc de Inmunoglobulinas/metabolismo , Interleucina-2/metabolismo , Células Jurkat , Cinética , Ratones Endogámicos C57BL , Ratones Transgénicos , Mutación , Fagocitosis/efectos de los fármacos , Agregación Plaquetaria/efectos de los fármacos , Unión Proteica , Ingeniería de Proteínas , Receptores Fc/genética , Receptores Fc/inmunología , Receptores Fc/metabolismo , Receptores de IgG/genética , Receptores de IgG/inmunología , Receptores de IgG/metabolismo , Vías Secretoras , Transducción de Señal , Células THP-1RESUMEN
The long serum t 1/2 of IgGs is ensured by their interaction with the neonatal Fc receptor (FcRn), which salvages IgG from intracellular degradation. Fc glycosylation is thought not to influence FcRn binding and IgG longevity in vivo. In this article, we demonstrate that hypersialylation of asparagine 297 (N297) enhances IgG serum persistence. This polarized glycosylation is achieved using a novel Fc mutation, a glutamate residue deletion at position 294 (Del) that endows IgGs with an up to 9-fold increase in serum lifespan. The strongest impact was observed when the Del was combined with Fc mutations improving FcRn binding (Del-FcRn+). Enzymatic desialylation of a Del-FcRn+ mutant or its production in a cell line unable to hypersialylate reduced the in vivo serum t 1/2 of the desialylated mutants to that of native FcRn+ mutants. Consequently, our study proves that sialylation of the N297 sugar moiety has a direct impact on human IgG serum persistence.
Asunto(s)
Anticuerpos/sangre , Anticuerpos/uso terapéutico , Fragmentos Fc de Inmunoglobulinas/sangre , Fragmentos Fc de Inmunoglobulinas/química , Inmunoglobulina G/sangre , Inmunoglobulina G/uso terapéutico , Animales , Anticuerpos/química , Células HEK293 , Semivida , Humanos , Inmunoglobulina G/química , Ratones , Ratones NoqueadosRESUMEN
Despite the reasonably long half-life of immunoglogulin G (IgGs), market pressure for higher patient convenience while conserving efficacy continues to drive IgG half-life improvement. IgG half-life is dependent on the neonatal Fc receptor (FcRn), which among other functions, protects IgG from catabolism. FcRn binds the Fc domain of IgG at an acidic pH ensuring that endocytosed IgG will not be degraded in lysosomal compartments and will then be released into the bloodstream. Consistent with this mechanism of action, several Fc-engineered IgG with increased FcRn affinity and conserved pH dependency were designed and resulted in longer half-life in vivo in human FcRn-transgenic mice (hFcRn), cynomolgus monkeys, and recently in healthy humans. These IgG variants were usually obtained by in silico approaches or directed mutagenesis in the FcRn-binding site. Using random mutagenesis, combined with a pH-dependent phage display selection process, we isolated IgG variants with improved FcRn-binding, which exhibited longer in vivo half-life in hFcRn mice. Interestingly, many mutations enhancing Fc/FcRn interaction were located at a distance from the FcRn-binding site validating our random molecular approach. Directed mutagenesis was then applied to generate new variants to further characterize our IgG variants and the effect of the mutations selected. Since these mutations are distributed over the whole Fc sequence, binding to other Fc effectors, such as complement C1q and FcγRs, was dramatically modified, even by mutations distant from these effectors' binding sites. Hence, we obtained numerous IgG variants with increased FcRn-binding and different binding patterns to other Fc effectors, including variants without any effector function, providing distinct "fit-for-purpose" Fc molecules. We therefore provide evidence that half-life and effector functions should be optimized simultaneously as mutations can have unexpected effects on all Fc receptors that are critical for IgG therapeutic efficacy.
RESUMEN
While glyco-engineered monoclonal antibodies (mAbs) with improved antibody-dependent cell-mediated cytotoxicity (ADCC) are reaching the market, extensive efforts have also been made to improve their pharmacokinetic properties to generate biologically superior molecules. Most therapeutic mAbs are human or humanized IgG molecules whose half-life is dependent on the neonatal Fc receptor FcRn. FcRn reduces IgG catabolism by binding to the Fc domain of endocytosed IgG in acidic lysosomal compartments, allowing them to be recycled into the blood. Fc-engineered mAbs with increased FcRn affinity resulted in longer in vivo half-life in animal models, but also in healthy humans. These Fc-engineered mAbs were obtained by alanine scanning, directed mutagenesis or in silico approach of the FcRn binding site. In our approach, we applied a random mutagenesis technology (MutaGen™) to generate mutations evenly distributed over the whole Fc sequence of human IgG1. IgG variants with improved FcRn-binding were then isolated from these Fc-libraries using a pH-dependent phage display selection process. Two successive rounds of mutagenesis and selection were performed to identify several mutations that dramatically improve FcRn binding. Notably, many of these mutations were unpredictable by rational design as they were located distantly from the FcRn binding site, validating our random molecular approach. When produced on the EMABling(®) platform allowing effector function increase, our IgG variants retained both higher ADCC and higher FcRn binding. Moreover, these IgG variants exhibited longer half-life in human FcRn transgenic mice. These results clearly demonstrate that glyco-engineering to improve cytotoxicity and protein-engineering to increase half-life can be combined to further optimize therapeutic mAbs.
Asunto(s)
Anticuerpos Monoclonales/farmacocinética , Antígenos de Histocompatibilidad Clase I/metabolismo , Inmunoglobulina G/metabolismo , Inmunoterapia/métodos , Ingeniería de Proteínas/métodos , Receptores Fc/metabolismo , Animales , Anticuerpos Monoclonales/genética , Citotoxicidad Celular Dependiente de Anticuerpos/genética , Técnicas de Visualización de Superficie Celular , Citotoxicidad Inmunológica/genética , Glicosilación , Semivida , Antígenos de Histocompatibilidad Clase I/genética , Humanos , Inmunoglobulina G/genética , Inmunoterapia/tendencias , Ratones , Ratones Transgénicos , Mutagénesis Sitio-Dirigida , Mutación/genética , Receptores Fc/genética , Receptores de IgG/antagonistas & inhibidores , Receptores de IgG/inmunología , Receptores de IgG/metabolismoRESUMEN
Blockade of the human epidermal growth factor receptor 3 (HER3) and of the downstream phosphatidylinositide 3-kinase (PI3K)/AKT pathway is a prerequisite for overcoming drug resistance and to develop novel treatments for cancers that are not eligible for the currently approved targeted therapies. To this end, we generated specific antibodies (Abs) against domain 1 (D1) and domain 3 (D3) of HER3 that recognize epitopes that do not overlap with the neuregulin-binding site. The fully human H4B-121 Ab and the mouse monoclonal Abs 16D3-C1 and 9F7-F11 inhibited tumor growth in nude mice xenografted with epidermoid, pancreatic, or triple-negative breast cancer cells. The combination of one anti-HER3 Ab and trastuzumab improved tumor growth inhibition in mice xenografted with HER2(low) cancer cell lines, for which trastuzumab alone shows no or moderate efficiency. Ab-induced disruption of tumor growth was associated with G1 cell cycle arrest, proliferation inhibition, and apoptosis of cancer cells. Anti-HER3 Abs blocked HER2/HER3 heterodimerization and HER3 phosphorylation at the cell membrane, leading to inhibition of phosphorylation of the downstream AKT targets murine double minute 2, X-linked inhibitor of apoptosis, and forkhead box O1. This study demonstrates that anti-HER3 D1 and D3 Abs could represent a new option for immunotherapy of pancreatic and triple-negative breast cancers.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Factores de Transcripción Forkhead/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Receptor ErbB-2/metabolismo , Receptor ErbB-3/metabolismo , Proteína Inhibidora de la Apoptosis Ligada a X/metabolismo , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/metabolismo , Anticuerpos Monoclonales Humanizados/farmacología , Especificidad de Anticuerpos , Apoptosis/efectos de los fármacos , Puntos de Control del Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Dimerización , Epítopos/química , Epítopos/inmunología , Femenino , Proteína Forkhead Box O1 , Humanos , Ratones , Datos de Secuencia Molecular , Neoplasias/metabolismo , Neoplasias/patología , Fosforilación/efectos de los fármacos , Unión Proteica , Receptor ErbB-2/química , Receptor ErbB-3/química , Receptor ErbB-3/inmunología , Trastuzumab , Carga Tumoral/efectos de los fármacosRESUMEN
As a growing number of therapeutic antibodies are developed, robust methods to efficiently improve the affinity and/or specificity of antibody candidates are needed. Here we describe our powerful platform that combines scFv affinity maturation and IgG high-throughput screening. After creating diversity with our random mutagenesis technology (MutaGen™), the scFv libraries are fully cleaned using a fusion system introducing the beta-lactamase gene to select in-frame and stop codon free variants on the basis of ampicillin resistance. The high-quality scFv libraries thereby constructed are then selected on the target in vitro using phage display technology. Contrary to standard procedures, instead of producing a limited number of affinity matured scFv as IgG molecules, we developed a cloning system to directly transfer the entire pool of selected scFv into an IgG expression vector permitting rapid IgG small-scale production (96 wells) in mammalian cells. Our integrated process allows us to generate high-quality scFv libraries and test numerous IgG variants, increasing the chances to select the best therapeutic antibody candidate.
Asunto(s)
Afinidad de Anticuerpos/inmunología , Ensayos Analíticos de Alto Rendimiento/métodos , Inmunoglobulina G/aislamiento & purificación , Biblioteca de Péptidos , Anticuerpos de Cadena Única/biosíntesis , Ampicilina/farmacología , Afinidad de Anticuerpos/efectos de los fármacos , Línea Celular , Clonación Molecular , Vectores Genéticos/genética , Humanos , Inmunoglobulina G/biosíntesis , Inmunoglobulina G/genética , Sistemas de Lectura Abierta/genéticaRESUMEN
Following infection of the central nervous system (CNS), the immune system is faced with the challenge of eliminating the pathogen without causing significant damage to neurons, which have limited capacities of renewal. In particular, it was thought that neurons were protected from direct attack by cytotoxic T lymphocytes (CTL) because they do not express major histocompatibility class I (MHC I) molecules, at least at steady state. To date, most of our current knowledge on the specifics of neuron-CTL interaction is based on studies artificially inducing MHC I expression on neurons, loading them with exogenous peptide and applying CTL clones or lines often differentiated in culture. Thus, much remains to be uncovered regarding the modalities of the interaction between infected neurons and antiviral CD8 T cells in the course of a natural disease. Here, we used the model of neuroinflammation caused by neurotropic Borna disease virus (BDV), in which virus-specific CTL have been demonstrated as the main immune effectors triggering disease. We tested the pathogenic properties of brain-isolated CD8 T cells against pure neuronal cultures infected with BDV. We observed that BDV infection of cortical neurons triggered a significant up regulation of MHC I molecules, rendering them susceptible to recognition by antiviral CTL, freshly isolated from the brains of acutely infected rats. Using real-time imaging, we analyzed the spatio-temporal relationships between neurons and CTL. Brain-isolated CTL exhibited a reduced mobility and established stable contacts with BDV-infected neurons, in an antigen- and MHC-dependent manner. This interaction induced rapid morphological changes of the neurons, without immediate killing or impairment of electrical activity. Early signs of neuronal apoptosis were detected only hours after this initial contact. Thus, our results show that infected neurons can be recognized efficiently by brain-isolated antiviral CD8 T cells and uncover the unusual modalities of CTL-induced neuronal damage.
Asunto(s)
Enfermedad de Borna/inmunología , Virus de la Enfermedad de Borna/inmunología , Neuronas/inmunología , Linfocitos T Citotóxicos/inmunología , Animales , Antígenos de Histocompatibilidad Clase I/biosíntesis , Neuronas/patología , Neuronas/virología , Ratas , Ratas Endogámicas LewRESUMEN
The neurotropic virus Borna disease virus (BDV) persists in the central nervous systems of a wide variety of vertebrates and causes behavioral disorders. BDV represents an intriguing example of a virus whose persistence in neurons leads to altered brain function in the absence of overt cytolysis and inflammation. The bases of BDV-induced behavioral impairment remain largely unknown. To better characterize the neuronal response to BDV infection, we compared the proteomes of primary cultures of cortical neurons with and without BDV infection. We used two-dimensional liquid chromatography fractionation, followed by protein identification by nanoliquid chromatography-tandem mass spectrometry. This analysis revealed distinct changes in proteins implicated in neurotransmission, neurogenesis, cytoskeleton dynamics, and the regulation of gene expression and chromatin remodeling. We also demonstrated the selective interference of BDV with processes related to the adaptative response of neurons, i.e., defects in proteins regulating synaptic function, global rigidification of the cytoskeleton network, and altered expression of transcriptional and translational repressors. Thus, this work provides a global view of the neuronal changes induced by BDV infection together with new clues to understand the mechanisms underlying the selective interference with neuronal plasticity and remodeling that characterizes BDV persistence.
Asunto(s)
Virus de la Enfermedad de Borna/fisiología , Neuronas/metabolismo , Secuencia de Aminoácidos , Animales , Células Cultivadas , Cromosomas/genética , Citoesqueleto/metabolismo , Bases de Datos de Proteínas , Regulación de la Expresión Génica , Histonas/metabolismo , Espectrometría de Masas , Proteómica , ARN Mensajero/genética , Ratas , Ratas Sprague-DawleyRESUMEN
Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase, initially discovered as part of the NPM-ALK fusion protein, resulting from the t(2;5) translocation that is frequently associated with anaplastic large-cell lymphomas. The native ALK protein is normally expressed in the developing and, at a weaker level, adult nervous system. We recently demonstrated that the oncogenic, constitutively kinase-activated NPM-ALK protein was antiapoptotic when expressed in Jurkat lymphoblastic cells treated with cytotoxic drugs. In contrast, we now show that Jurkat cells overexpressing the wild-type ALK receptor are more sensitive to doxorubicin-induced apoptosis than parental cells. Moreover, the ALK protein is cleaved during apoptosis in a caspase-dependent manner. Mutation of aspartic residues to asparagine allowed us to map the caspase cleavage site in the juxtamembrane region of ALK. In order to assess the role of ALK in neural cell-derived tissue, we transiently expressed ALK in the 13.S.1.24 rat neuroblast immortalized cell line. ALK expression led to apoptotic cell death of the neuroblasts. ALK ligation by specific activating antibodies decreased ALK-facilitated apoptosis in both lymphoid and neuronal cell lines. Moreover, ALK transfection reduced the survival of primary cultures of cortical neurons. Thus, ALK has a proapoptotic activity in the absence of ligand, whereas it is antiapoptotic in the presence of its ligand and when the kinase is intrinsically activated. These properties place ALK in the growing family of dependence receptors.
Asunto(s)
Apoptosis , Caspasas/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Receptores de Superficie Celular/metabolismo , Quinasa de Linfoma Anaplásico , Animales , Anticuerpos/inmunología , Apoptosis/efectos de los fármacos , Ácido Aspártico/genética , Caspasa 3 , Línea Celular Tumoral , Membrana Celular/metabolismo , Corteza Cerebral/citología , Corteza Cerebral/enzimología , Doxorrubicina/farmacología , Activación Enzimática , Expresión Génica , Humanos , Células Jurkat , Ratones , Mutación/genética , Neuronas/citología , Neuronas/enzimología , Procesamiento Proteico-Postraduccional , Ratas , Ratas Sprague-Dawley , Proteínas Tirosina Quinasas Receptoras , TransfecciónRESUMEN
Infection by Borna disease virus (BDV) enables the study of the molecular mechanisms whereby a virus can persist in the central nervous system and lead to altered brain function in the absence of overt cytolysis and inflammation. This neurotropic virus infects a wide variety of vertebrates and causes behavioral diseases. The basis of BDV-induced behavioral impairment remains largely unknown. Here, we investigated whether BDV infection of neurons affected synaptic activity, by studying the rate of synaptic vesicle (SV) recycling, a good indicator of synaptic activity. Vesicular cycling was visualized in cultured hippocampal neurons synapses, using an assay based on the uptake of an antibody directed against the luminal domain of synaptotagmin I. BDV infection did not affect elementary presynaptic functioning, such as spontaneous or depolarization-induced vesicular cycling. In contrast, infection of neurons with BDV specifically blocked the enhancement of SV recycling that is observed in response to stimuli-induced synaptic potentiation, suggesting defects in long-term potentiation. Studies of signaling pathways involved in synaptic potentiation revealed that this blockade was due to a reduction of the phosphorylation by protein kinase C (PKC) of proteins that regulate SV recycling, such as myristoylated alanine-rich C kinase substrate (MARCKS) and Munc18-1/nSec1. Moreover, BDV interference with PKC-dependent phosphorylation was identified downstream of PKC activation. We also provide evidence suggesting that the BDV phosphoprotein interferes with PKC-dependent phosphorylation. Altogether, our results reveal a new mechanism by which a virus can cause synaptic dysfunction and contribute to neurobehavioral disorders.
Asunto(s)
Enfermedad de Borna/fisiopatología , Enfermedad de Borna/virología , Virus de la Enfermedad de Borna/fisiología , Terminales Presinápticos/fisiología , Proteína Quinasa C/metabolismo , Transducción de Señal/fisiología , Animales , Células Cultivadas , Activación Enzimática , Datos de Secuencia Molecular , Neuronas/fisiología , Ratas , Sinapsis/fisiología , Vesículas Sinápticas/fisiologíaRESUMEN
We previously observed that cadherin-11, a type II cadherin, is expressed in growing motor and sensory axons in the mouse embryo. Here, we assessed its functional involvement in the regulation of axon elongation and fasciculation by evaluating the activity of a specific cadherin-11 homophilic ligand, cad11-Fc (cadherin-11 extracellular region fused to Fc fragment of IgG), on the length and organization of motor axons outgrowing from embryonic ventral spinal cord explants. Cad11-Fc substrate enhanced axon growth and prevented interactions occurring between growing axons, providing evidences for a role of cadherin-11 in the control of growth cone progression. Comparison of cadherin-11 with N-cadherin, a type I cadherin concomitantly expressed by motor axons, revealed similarities in their functional properties, including the ability to reorganize the actin cytoskeleton through interactions with catenins, but differences in their axon growth-promoting activity, arguing for subtle differences in their contributions to peripheral nerve elongation.
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Axones/fisiología , Cadherinas/fisiología , Aumento de la Célula , Neuronas Motoras/fisiología , Animales , Línea Celular , Células Cultivadas , Femenino , Humanos , Ratones , Neuronas Motoras/citología , EmbarazoRESUMEN
The physiological role of the prion protein is largely unknown. Here, clustering of prion at the surface of GT1-7 cells was observed upon anti-prion antibody treatments. This clustering was associated with a rapid and transient phosphorylation of the mitogen activated protein kinases (MAPKs) extracellular receptor kinases 1 and 2 (ERK1/2), and also of the microtubule-destabilizing protein stathmin at serine 16. The specificity of this antibody-mediated activation was ascertained by its inhibition by prion small interfering RNA. The phosphorylation of ERK1/2 but not that of stathmin was abolished by the MAPK/ERK kinase 1 inhibitor U0126, whereas both signaling pathways were blocked by the specific inhibitor of the epidermal growth factor receptor AG1478, suggesting the likely recruitment of this receptor upon prion clustering.
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Proteínas de Microtúbulos/metabolismo , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Neuronas/fisiología , Fosfoproteínas/metabolismo , Proteínas PrPC/fisiología , Animales , Anticuerpos Monoclonales/metabolismo , Western Blotting , Butadienos/farmacología , Células Cultivadas , Inhibidores Enzimáticos/farmacología , Técnica del Anticuerpo Fluorescente Indirecta , Ratones , Microscopía Confocal , Proteína Quinasa 1 Activada por Mitógenos/efectos de los fármacos , Proteína Quinasa 3 Activada por Mitógenos/efectos de los fármacos , Nitrilos/farmacología , Fosforilación/efectos de los fármacos , Quinazolinas , ARN Interferente Pequeño/antagonistas & inhibidores , Estatmina , Tirfostinos/farmacologíaRESUMEN
N-cadherin is expressed throughout skeletal myogenesis and has been proposed to be involved in the differentiation program of myogenic precursors. Here, we further characterize the N-cadherin involvement and its mechanism of action at the onset of differentiation, through controlled N-cadherin activation by plating isolated C2 myoblasts on surfaces coated with a chimeric Ncad-Fc homophilic ligand (N-cadherin ectodomain fused to the immunoglobulin G Fc fragment). We show that N-cadherin activation substitutes for the cell density in myogenic differentiation by promoting myogenin and troponin T expression. In addition, N-cadherin adhesion participates to the associated cell cycle arrest through the nuclear accumulation of cyclin-dependent kinase inhibitors p21 and p27. Mouse primary myoblast cultures exhibited similar responses to N-cadherin as C2 cells. RNA interference knockdowns of the N-cadherin-associated cytoplasmic proteins p120 and beta-catenin produced opposite effects on the differentiation pathway. p120 silencing resulted in a decreased myogenic differentiation, associated with a reduction in cadherin-catenin content, which may explain its action on myogenic differentiation. beta-Catenin silencing led to a stimulatory effect on myogenin expression, without any effect on cell cycle. Our results demonstrate that N-cadherin adhesion may account for cell-cell contact-dependent cell cycle arrest and differentiation of myogenic cells, involving regulation through p120 and beta-catenins.
Asunto(s)
Cadherinas/metabolismo , Proteínas del Citoesqueleto/metabolismo , Células Musculares/metabolismo , Transactivadores/metabolismo , Proteína Activadora de GTPasa p120/metabolismo , Animales , Western Blotting , Bromodesoxiuridina/farmacología , Cadherinas/química , Adhesión Celular , Comunicación Celular , Ciclo Celular , Proteínas de Ciclo Celular/metabolismo , Diferenciación Celular , División Celular , Línea Celular , Linaje de la Célula , Células Cultivadas , Inhibidor p27 de las Quinasas Dependientes de la Ciclina , ADN/metabolismo , Silenciador del Gen , Fragmentos de Inmunoglobulinas/química , Inmunoglobulina G/química , Ligandos , Ratones , Unión Proteica , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Factores de Tiempo , Transfección , Troponina T/química , Proteínas Supresoras de Tumor/metabolismo , beta CateninaRESUMEN
Juxtacrine cell interactions associated to cadherin-mediated cell-cell adhesion play a major role in the organization and homeostasis of tissues. Here, we review the intracellular molecules and regulations controlling the formation of cell-cell contacts initiated by homophilic interactions of cadherin ectodomain. These regulations involve proteins associated to cadherin cytoplasmic tail, named catenins, their association to the actin cytoskeleton and the stability of these complexes at the cell membrane. The underlying molecular mechanisms, which participate in the formation of dynamic cell-cell contacts, are intensively investigated.
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Cadherinas/fisiología , Adhesión Celular/fisiología , Actinas/fisiología , Animales , Cadherinas/química , Comunicación Celular/fisiología , Membrana Celular/fisiología , Citoesqueleto/fisiología , Homeostasis , HumanosRESUMEN
The normal cellular prion protein is a small sialoglycoprotein highly expressed in neurons, the physiological function of which is largely unknown. Due to extensive N-glycosylations with a wide range of oligosaccharides, the prion protein displays a complex glycosylation pattern that could be of relevance for its function. The cellular prion protein patterns in adult mouse and rat brain, and in neuronal cell lines, appeared highly heterogeneous, as distinct levels and glycoforms of cellular prion protein were revealed by immunoblotting of corresponding samples. Amongst neuronal cell lines, mouse N2a neuroblastoma cells expressed low levels of endogenous prion protein. Mouse hypothalamic GT1-7 cells and rat pheochromocytoma PC-12 cells expressed highly glycosylated forms of cellular prion protein that were found neither in adult mouse and rat brain, nor in mouse brain during development. In contrast, rat B104 neuroblastoma cells abundantly expressed N-glycosylated cellular prion protein forms similar to those observed in mouse and rat brain. In all these cell lines, the prion protein was normally exported to and expressed at the outer cell membrane. Our results suggest that B104 cells may represent an appropriate cell model to investigate the physiological role of cellular prion protein in further detail as they highly express the normal 'brain-like' cellular prion protein glycoforms. In addition, we observed that the various prion glycoforms in B104 cells were tightly regulated both as a function of cell density and during neuronal differentiation, implying a potential role of cellular prion protein in cell-cell interactions and differentiation.