RESUMEN
Facioscapulohumeral muscular dystrophy (FSHD) is a common muscle disease whose molecular pathogenesis remains largely unknown. Over-expression of FSHD region gene 1 (FRG1) in mice, frogs, and worms perturbs muscle development and causes FSHD-like phenotypes. FRG1 has been implicated in splicing, and we asked how splicing might be involved in FSHD by conducting a genome-wide analysis in FRG1 mice. We find that splicing perturbations parallel the responses of different muscles to FRG1 over-expression and disease progression. Interestingly, binding sites for the Rbfox family of splicing factors are over-represented in a subset of FRG1-affected splicing events. Rbfox1 knockdown, over-expression, and RNA-IP confirm that these are direct Rbfox1 targets. We find that FRG1 is associated to the Rbfox1 RNA and decreases its stability. Consistent with this, Rbfox1 expression is down-regulated in mice and cells over-expressing FRG1 as well as in FSHD patients. Among the genes affected is Calpain 3, which is mutated in limb girdle muscular dystrophy, a disease phenotypically similar to FSHD. In FRG1 mice and FSHD patients, the Calpain 3 isoform lacking exon 6 (Capn3 E6-) is increased. Finally, Rbfox1 knockdown and over-expression of Capn3 E6- inhibit muscle differentiation. Collectively, our results suggest that a component of FSHD pathogenesis may arise by over-expression of FRG1, reducing Rbfox1 levels and leading to aberrant expression of an altered Calpain 3 protein through dysregulated splicing.
Asunto(s)
Calpaína , Proteínas Musculares , Distrofia Muscular Facioescapulohumeral , Proteínas , Proteínas de Unión al ARN/genética , Empalme Alternativo/genética , Animales , Calpaína/genética , Calpaína/metabolismo , Células Cultivadas , Modelos Animales de Enfermedad , Exones , Regulación del Desarrollo de la Expresión Génica , Células HEK293 , Humanos , Ratones , Proteínas de Microfilamentos , Desarrollo de Músculos/genética , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Distrofia Muscular Facioescapulohumeral/genética , Distrofia Muscular Facioescapulohumeral/metabolismo , Distrofia Muscular Facioescapulohumeral/patología , Mioblastos/citología , Mioblastos/metabolismo , Proteínas/genética , Proteínas/metabolismo , Factores de Empalme de ARN , Proteínas de Unión al ARN/metabolismoRESUMEN
Treatment of dominantly inherited muscle disorders remains a difficult task considering the need to eliminate the pathogenic gene product in a body-wide fashion. We show here that it is possible to reverse dominant muscle disease in a mouse model of facioscapulohumeral muscular dystrophy (FSHD). FSHD is a common form of muscular dystrophy associated with a complex cascade of epigenetic events following reduction in copy number of D4Z4 macrosatellite repeats located on chromosome 4q35. Several 4q35 genes have been examined for their role in disease, including FRG1. Overexpression of FRG1 causes features related to FSHD in transgenic mice and the FRG1 mouse is currently the only available mouse model of FSHD. Here we show that systemic delivery of RNA interference expression cassettes in the FRG1 mouse, after the onset of disease, led to a dose-dependent long-term FRG1 knockdown without signs of toxicity. Histological features including centrally nucleated fibers, fiber size reduction, fibrosis, adipocyte accumulation, and inflammation were all significantly improved. FRG1 mRNA knockdown resulted in a dramatic restoration of muscle function. Through RNA interference (RNAi) expression cassette redesign, our method is amenable to targeting any pathogenic gene offering a viable option for long-term, body-wide treatment of dominant muscle disease in humans.
Asunto(s)
Dependovirus/genética , Vectores Genéticos , Distrofia Muscular Facioescapulohumeral/terapia , ARN Interferente Pequeño/administración & dosificación , Animales , Modelos Animales de Enfermedad , Regulación hacia Abajo , Femenino , Regulación de la Expresión Génica , Silenciador del Gen , Vectores Genéticos/administración & dosificación , Vectores Genéticos/efectos adversos , Humanos , Inyecciones Intravenosas , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Proteínas de Microfilamentos , Músculo Esquelético/metabolismo , Músculo Esquelético/patología , Distrofia Muscular Facioescapulohumeral/genética , Distrofia Muscular Facioescapulohumeral/patología , Proteínas Nucleares/genética , Fenotipo , Proteínas de Unión al ARN , Factores de Tiempo , Transducción GenéticaRESUMEN
Pancreatic cancer is an aggressive malignancy resistant to most conventional and experimental therapies, including conditionally replicative adenoviruses (CRAds). The incorporation of immunostimulatory genes such as interleukin-12 (IL-12) in these viruses may overcome some of their limitations, but evaluation of such vectors requires suitable preclinical models. We describe a CRAd in which replication is dependent on hypoxia-inducible factor (HIF) activity and alterations of the pRB pathway in cancer cells. Transgenes (luciferase or IL-12) were incorporated into E3 region of the virus using a selective 6.7K/gp19K deletion. A novel permissive model of pancreatic cancer developed in immunocompetent Syrian hamsters was used for in vivo analysis. We show that, in contrast with nonreplicating adenoviruses (NR-Ad), active viral production and enhanced transgene expression took place in vivo. A single intratumor inoculation of the CRAd expressing IL-12 (Ad-DHscIL12) achieved a potent antitumor effect, whereas higher doses of replication-competent adenoviruses carrying luciferase did not. Compared to a standard NR-Ad expressing IL-12, Ad-DHscIL12 was less toxic in hamsters, with more selective tumor expression and shorter systemic exposure to the cytokine. We conclude that the expression of IL-12 in the context of a hypoxia-inducible oncolytic adenovirus is effective against pancreatic cancer in a relevant animal model.
Asunto(s)
Adenoviridae/genética , Interleucina-12/genética , Viroterapia Oncolítica , Neoplasias Pancreáticas/terapia , Adenoviridae/fisiología , Animales , Cricetinae , Neoplasias Hepáticas Experimentales/secundario , Mesocricetus , Neoplasias Pancreáticas/patología , Transgenes , Replicación ViralRESUMEN
Androgens are primarily involved in muscle growth, whilst disease-driven muscle wasting is frequently associated with hypogonadism. The Leydig cells of the testes also produce the peptide-hormone Insulin-like peptide 3 (INSL3). INSL3 displays anabolic activity on bone, a target tissue of androgens, and its plasma concentrations are diminished in male hypogonadism. Here we tested the role of INSL3 on muscle mass regulation, in physiological and pathological conditions. Studies on C2C12 cell line showed that INSL3, acting on his specific receptor RXFP2, promotes skeletal muscle protein synthesis through the Akt/mTOR/S6 pathway. Next, studies on Rxfp2 -/- mice showed that INSL3 is required to prevent excessive muscle loss after denervation. Mechanistically, denervated Rxfp2 -/- mice lacked the compensatory activation of the Akt/mTOR/S6 pathway and showed an abnormal ubiquitin-proteasome system activation. Lack of INSL3 activity resulted also in reduced contractile force. These findings underlie a role of INSL3/RXFP2 in protein turnover, contributing to muscle wasting in male hypogonadism.
RESUMEN
The preclinical evaluation of toxicity and antitumor effect of conditionally replicative (oncolytic) adenoviruses is hampered by the inability of human adenoviruses to replicate efficiently in murine cells. The Syrian golden hamster (Mesocricetus auratus) has been suggested as a permissive animal for adenoviral replication, and cancer cell lines derived from various hamster tumors are available. We provide evidence that wild-type adenovirus type 5 is able to infect and replicate in the pancreatic cancer cell lines HaP-T1 and H2T both in vitro and in vivo. Determination of cytopathic effect, viral spread, progeny production, and the expression of late viral proteins indicates that the complete viral cycle of adenovirus takes place, albeit less efficiently than in highly permissive human cancer cell lines A549 and HuH7. Intrahepatic inoculation of HaP-T1 and H2T cells gave rise to tumors in the liver of hamsters that resemble metastases of pancreatic cancer. The growth of HaP-T1-induced nodules was faster compared with those derived from H2T, but both caused progressive liver infiltration and peritoneal dissemination. When adenovirus was inoculated in these lesions, productive replication took place and newly formed infective virions could be recovered 4 days after administration. In conclusion, the Syrian hamster models described here offer the opportunity to evaluate the effect of oncolytic adenoviruses in an immunocompetent animal and may be a valuable tool in the preclinical evaluation of these agents.
Asunto(s)
Adenoviridae/fisiología , Neoplasias Pancreáticas/virología , Replicación Viral/fisiología , Animales , Línea Celular , Cricetinae , Femenino , Humanos , Mesocricetus , Ratones , Ratones Desnudos , Neoplasias Experimentales/inmunología , Neoplasias Experimentales/virología , Neoplasias Pancreáticas/inmunologíaRESUMEN
Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal dominant myopathy with a strong epigenetic component. It is associated with deletion of a macrosatellite repeat leading to over-expression of the nearby genes. Among them, we focused on FSHD region gene 1 (FRG1) since its over-expression in mice, Xenopus laevis and Caenorhabditis elegans, leads to muscular dystrophy-like defects, suggesting that FRG1 plays a relevant role in muscle biology. Here we show that, when over-expressed, FRG1 binds and interferes with the activity of the histone methyltransferase Suv4-20h1 both in mammals and Drosophila. Accordingly, FRG1 over-expression or Suv4-20h1 knockdown inhibits myogenesis. Moreover, Suv4-20h KO mice develop muscular dystrophy signs. Finally, we identify the FRG1/Suv4-20h1 target Eid3 as a novel myogenic inhibitor that contributes to the muscle differentiation defects. Our study suggests a novel role of FRG1 as epigenetic regulator of muscle differentiation and indicates that Suv4-20h1 has a gene-specific function in myogenesis.
Asunto(s)
N-Metiltransferasa de Histona-Lisina/metabolismo , Desarrollo de Músculos , Proteínas Nucleares/metabolismo , Proteínas/metabolismo , Animales , Proteínas Portadoras/metabolismo , Diferenciación Celular , Núcleo Celular/metabolismo , Drosophila melanogaster/metabolismo , Evolución Molecular , Técnicas de Silenciamiento del Gen , Células HEK293 , Humanos , Ratones , Ratones Noqueados , Proteínas de Microfilamentos , Células Musculares/metabolismo , Células Musculares/patología , Distrofia Muscular Animal/patología , Especificidad de Órganos , Fenotipo , Unión Proteica , Proteínas de Unión al ARNRESUMEN
Cell size is determined by the balance between protein synthesis and degradation. This equilibrium is affected by hormones, nutrients, energy levels, mechanical stress and cytokines. Mutations that inactivate myostatin lead to excessive muscle growth in animals and humans, but the signals and pathways responsible for this hypertrophy remain largely unknown. Here we show that bone morphogenetic protein (BMP) signaling, acting through Smad1, Smad5 and Smad8 (Smad1/5/8), is the fundamental hypertrophic signal in mice. Inhibition of BMP signaling causes muscle atrophy, abolishes the hypertrophic phenotype of myostatin-deficient mice and strongly exacerbates the effects of denervation and fasting. BMP-Smad1/5/8 signaling negatively regulates a gene (Fbxo30) that encodes a ubiquitin ligase required for muscle loss, which we named muscle ubiquitin ligase of the SCF complex in atrophy-1 (MUSA1). Collectively, these data identify a critical role for the BMP pathway in adult muscle maintenance, growth and atrophy.
Asunto(s)
Proteínas Morfogenéticas Óseas/metabolismo , Músculo Esquelético/metabolismo , Atrofia Muscular/genética , Proteína Smad4/metabolismo , Animales , Línea Celular , Activación Enzimática , Perfilación de la Expresión Génica , Células HEK293 , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Miostatina/genética , Interferencia de ARN , ARN Interferente Pequeño , Transducción de Señal , Proteína Smad1/genética , Proteína Smad1/metabolismo , Proteína Smad4/genética , Proteína Smad5/genética , Proteína Smad5/metabolismo , Proteína Smad8/genética , Proteína Smad8/metabolismo , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
Replication-competent (oncolytic) adenoviruses (OAV) can be adapted as vectors for the delivery of therapeutic genes, with the aim of extending the antitumor effect beyond direct cytolysis. Transgene expression using these vectors is usually intense but short-lived, and repeated administrations are hampered by the rapid appearance of neutralizing antibodies (NAbs). We have studied the performance of monocytes as cell carriers to improve transgene expression in cancer models established in athymic mice and immunocompetent Syrian hamsters. Human and hamster monocytic cell lines (MonoMac6 and HM-1, respectively) were loaded with replication-competent adenovirus-expressing luciferase. Intravenous administration of these cells caused a modest increase in transgene expression in tumor xenografts, but this effect was virtually lost in hamsters. In contrast, intratumoral administration of HM-1 cells allowed repeated cycles of expression and achieved partial protection from NAbs in preimmunized hamsters bearing pancreatic tumors. To explore the therapeutic potential of this approach, HM-1 cells were loaded with a hypoxia-inducible OAV expressing the immunostimulatory cytokine interleukin-12 (IL-12). Three cycles of treatment achieved a significant antitumor effect in the hamster model, and transgene expression was detected following each administration, in contrast with the rapid neutralization of the free virus. We propose monocytes as carriers for multiple intratumoral administrations of armed OAVs.
Asunto(s)
Adenoviridae/genética , Monocitos/virología , Neoplasias Experimentales/terapia , Viroterapia Oncolítica/métodos , Adenoviridae/patogenicidad , Animales , Cricetinae , Expresión Génica , Humanos , Interleucina-12/genética , Interleucina-12/metabolismo , Mesocricetus , Ratones , Ratones Desnudos , Neoplasias Experimentales/genética , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/terapia , Transgenes , Trasplantes/virología , Replicación Viral/genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
RNA interference with viral vectors that express short hairpin RNAs (shRNAs) has emerged as a powerful tool for functional genomics and therapeutic purposes. However, little is known about shRNA in vivo processing, accumulation, functional kinetics, and side effects related to shRNA saturation of the cellular gene silencing machinery. Therefore, we constructed first-generation recombinant adenoviruses encoding different shRNAs against murine ATP-binding cassette multidrug resistance protein 2 (Abcc2), which is involved in liver transport of bilirubin to bile, and analyzed Abcc2 silencing kinetics. C57/BL6 mice injected with these viruses showed significant impairment of Abcc2 function for up to 3 weeks, as reflected by increased serum bilirubin levels. The lack of Abcc2 function correlated with a specific reduction of Abcc2 mRNA and with high levels of processed shRNAs targeting Abcc2. Inhibition was lost at longer times postinfection, correlating with a decrease in the accumulation of processed shRNAs. This finding suggests that a minimal amount of processed shRNAs is required for efficient silencing in vivo. This system was also used to evaluate the effect of shRNA expression on the saturation of silencing factors. Saturation of the cellular silencing processing machinery alters the accumulation and functionality of endogenous microRNAs (miRNAs) and pre-miRNAs. However, expression of functional exogenous shRNAs did not change the levels of endogenous miRNAs or their precursors. In summary, this work shows that adenoviral vectors can deliver sufficient shRNAs to mediate inhibition of gene expression without saturating the silencing machinery.
Asunto(s)
Subfamilia B de Transportador de Casetes de Unión a ATP/metabolismo , Silenciador del Gen , MicroARNs/genética , Interferencia de ARN , Subfamilia B de Transportador de Casetes de Unión a ATP/genética , Adenoviridae , Animales , Secuencia de Bases , Bilirrubina/sangre , Línea Celular , Componentes del Gen , Vectores Genéticos/genética , Humanos , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Proteína 2 Asociada a Resistencia a Múltiples Medicamentos , Oligonucleótidos , Miembro 4 de la Subfamilia B de Casete de Unión a ATPRESUMEN
Recombinant adenovirus administration gives rise to transgene-independent effects caused by the ability of the vector to activate innate immunity mechanisms. We show that recombinant adenoviruses encoding reporter genes trigger IFN-alpha and IFN-beta transcription from both plasmacytoid and myeloid mouse dendritic cells. Interestingly, IFN-beta and IFN-alpha5 are the predominant transcribed type I IFN genes both in vitro and in vivo. In human peripheral blood leukocytes type I IFNs are induced by adenoviral vectors, with a preponderance of IFN-beta together with IFN-alpha1 and IFN-alpha5 subtypes. Accordingly, functional type I IFN is readily detected in serum samples from human cancer patients who have been treated intratumorally with a recombinant adenovirus encoding thymidine kinase. Despite inducing functional IFN-alpha release in both mice and humans, gene transfer by recombinant adenoviruses is not interfered with by type I IFNs either in vitro or in vivo. Moreover, IFN-alpha does not impair replication of wild-type adenovirus. As a consequence, cancer gene therapy strategies with defective or replicative-competent adenoviruses are not expected to be hampered by the effect of the type I IFNs induced by the vector itself. However, type I IFN might modulate antitumor and antiadenoviral immune responses and thus influence the outcome of gene immunotherapy.