RESUMEN
Utrophin is the autosomal homolog of dystrophin, the product of the Duchenne's muscular dystrophy (DMD) locus. Utrophin is of therapeutic interest since its over-expression can compensate dystrophin's absence. Utrophin is enriched at neuromuscular junctions due to heregulin-mediated utrophin-A promoter activation. We demonstrate that heregulin activated MSK1/2 and phosphorylated histone H3 at serine 10 in cultured C2C12 muscle cells, in an ERK-dependent manner. MSK1/2 inhibition suppressed heregulin-mediated utrophin-A activation. MSK1 over-expression potentiated heregulin-mediated utrophin-A activation and chromatin remodeling at the utrophin-A promoter. These results identify MSK1/2 as key effectors modulating utrophin-A expression as well as identify novel targets for DMD therapy.
Asunto(s)
Epigénesis Genética/efectos de los fármacos , Neurregulina-1/farmacología , Regiones Promotoras Genéticas/genética , Utrofina/genética , Animales , Células Cultivadas , Ensamble y Desensamble de Cromatina/efectos de los fármacos , Activación Enzimática/efectos de los fármacos , Histonas/metabolismo , Ratones , Modelos Genéticos , Células Musculares/efectos de los fármacos , Células Musculares/enzimología , Fosforilación/efectos de los fármacos , Proteínas Quinasas S6 Ribosómicas 90-kDa/metabolismo , Utrofina/metabolismoRESUMEN
Mutations in myostatin (GDF8) cause marked increases in muscle mass, suggesting that this transforming growth factor-beta (TGF-beta) superfamily member negatively regulates muscle growth. Myostatin blockade therefore offers a strategy for reversing muscle wasting in Duchenne's muscular dystrophy (DMD) without resorting to genetic manipulation. Here, we demonstrate that pharmacological blockade using a myostatin propeptide stabilized by fusion to IgG-Fc improved pathophysiology of the mdx mouse model of DMD. Functional benefits evidenced by specific force improvement, exceeded those reported previously using myostatin antibody-mediated blockade. More importantly, use of a propeptide blockade strategy obviates possibilities of anti-idiotypic responses that could potentially limit the effectiveness of antibody-mediated myostatin blockade strategies over time. This study provides a novel pharmacological strategy for treatment of diseases associated with muscle wasting such as DMD and since it uses an endogenous inhibitor of myostatin should help circumvent technical hurdles and toxicity associated with conventional gene or cell based therapies.
Asunto(s)
Distrofia Muscular Animal/terapia , Precursores de Proteínas/administración & dosificación , Factor de Crecimiento Transformador beta/antagonistas & inhibidores , Animales , Anticuerpos Monoclonales , Fragmentos Fc de Inmunoglobulinas/química , Inmunoglobulina G/química , Inyecciones Intraperitoneales , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos mdx , Músculo Esquelético/química , Músculo Esquelético/patología , Distrofia Muscular Animal/patología , Distrofia Muscular Animal/fisiopatología , Miostatina , Precursores de Proteínas/química , Precursores de Proteínas/inmunología , ARN Mensajero/análisis , Proteínas Recombinantes de Fusión , Factor de Crecimiento Transformador beta/inmunología , Utrofina/genéticaRESUMEN
Duchenne's muscular dystrophy (DMD) is a fatal neuromuscular disease caused by absence of dystrophin. Utrophin is a chromosome 6-encoded dystrophin-related protein (DRP), sharing functional motifs with dystrophin. Utrophin's ability to compensate for dystrophin during development and when transgenically overexpressed has provided an important impetus for identifying activators of utrophin expression. The utrophin promoter A is transcriptionally regulated in part by heregulin-mediated, extracellular signal-related kinase-dependent activation of the GABP(alpha/beta) transcription factor complex. Therefore, this pathway offers a potential mechanism to modulate utrophin expression in muscle. We tested the ability of heregulin to improve the dystrophic phenotype in the mdx mouse model of DMD. Intraperitoneal injections of a small peptide encoding the epidermal growth factor-like region of heregulin ectodomain for 3 months in vivo resulted in up-regulation of utrophin, a marked improvement in the mechanical properties of muscle as evidenced by resistance to eccentric contraction mediated damage, and a reduction of muscle pathology. The amelioration of dystrophic phenotype by heregulin-mediated utrophin up-regulation offers a pharmacological therapeutic modality and obviates many of the toxicity and delivery issues associated with viral vector-based gene therapy for DMD.
Asunto(s)
Distrofia Muscular Animal/genética , Neurregulina-1/uso terapéutico , Utrofina/genética , Animales , Inyecciones Intraperitoneales , Masculino , Ratones , Ratones Endogámicos mdx , Distrofia Muscular Animal/patología , Neurregulina-1/administración & dosificación , Neurregulina-1/genética , Fenotipo , Utrofina/deficienciaRESUMEN
Duchenne muscular dystrophy (DMD) is the most common X-linked neuromuscular disorder. The devastating nature of DMD has led to an intense effort toward finding a cure for this disease, dating back to the time when Duchenne first initiated clinical trials using faradic stimulation for DMD patients. Unfortunately despite the passage of some 150 years the disease remains incurable, and its medical management is largely supportive. However, the discovery of the DMD gene about 20 years ago has allowed a change in the focus of therapeutic strategy dramatically toward delivery of the missing gene/protein. Indeed, some degree of success has been achieved in preclinical animal studies using such strategies, and gene therapy trials are currently underway in humans. Pharmacological approaches for DMD are also being developed since they can circumvent some of the technical problems associated with gene and cell based therapy. This review explores developments in therapeutic approaches for DMD.
Asunto(s)
Distrofia Muscular de Duchenne/terapia , Proteínas del Citoesqueleto/genética , Proteínas del Citoesqueleto/metabolismo , Modelos Animales de Enfermedad , Diseño de Fármacos , Distrofina/genética , Distrofina/metabolismo , Regulación de la Expresión Génica/genética , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Distrofias Musculares/clasificación , Distrofias Musculares/genética , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/metabolismo , Neurregulina-1/metabolismo , Regiones Promotoras Genéticas/genética , UtrofinaRESUMEN
Mice and cattle with mutations in the myostatin (GDF8) gene show a marked increase in body weight and muscle mass, indicating that this new member of the TGF-beta superfamily is a negative regulator of skeletal muscle growth. Inhibition of the myostatin gene product is predicted to increase muscle mass and improve the disease phenotype in a variety of primary and secondary myopathies. We tested the ability of inhibition of myostatin in vivo to ameliorate the dystrophic phenotype in the mdx mouse model of Duchenne muscular dystrophy (DMD). Blockade of endogenous myostatin by using intraperitoneal injections of blocking antibodies for three months resulted in an increase in body weight, muscle mass, muscle size and absolute muscle strength in mdx mouse muscle along with a significant decrease in muscle degeneration and concentrations of serum creatine kinase. The functional improvement of dystrophic muscle by myostatin blockade provides a novel, pharmacological strategy for treatment of diseases associated with muscle wasting such as DMD, and circumvents the major problems associated with conventional gene therapy in these disorders.
Asunto(s)
Músculo Esquelético/fisiología , Distrofia Muscular Animal/fisiopatología , Distrofia Muscular de Duchenne/fisiopatología , Factor de Crecimiento Transformador beta/antagonistas & inhibidores , Animales , Anticuerpos/inmunología , Anticuerpos/farmacología , Anticuerpos/uso terapéutico , Peso Corporal/efectos de los fármacos , Creatina Quinasa/sangre , Masculino , Ratones , Ratones Endogámicos mdx , Fibras Musculares Esqueléticas/efectos de los fármacos , Fibras Musculares Esqueléticas/patología , Fibras Musculares Esqueléticas/fisiología , Músculo Esquelético/efectos de los fármacos , Músculo Esquelético/patología , Distrofia Muscular Animal/tratamiento farmacológico , Distrofia Muscular Animal/patología , Distrofia Muscular de Duchenne/tratamiento farmacológico , Distrofia Muscular de Duchenne/patología , Miostatina , Tamaño de los Órganos/efectos de los fármacos , Factor de Crecimiento Transformador beta/inmunología , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
Duchenne muscular dystrophy (DMD) is a fatal neuromuscular disease caused by the absence of dystrophin. Utrophin is the autosomal homolog of dystrophin and capable of compensating for the absence of dystrophin, when overexpressed. In skeletal muscle, utrophin plays an important role in the formation of neuromuscular junctions. This selective enrichment occurs, in part by transcriptional regulation of the utrophin gene at the sub-synaptic nuclei in muscle. Utrophin's complex transcriptional regulation is not yet fully understood, however, GABP alpha / beta has recently been shown to bind the N box and activate the utrophin promoter in response to heregulin. In this study, we show that the transcription factor Sp1 binds and activates the utrophin promoter in Drosophila S2 cells as well as define a Sp1 response element. We demonstrate that heregulin treatment of cultured muscle cells activates the ERK pathway and phosphorylates serine residue(s) in the consensus ERK recognition site of Sp1. Finally, Sp1 is shown to functionally cooperate with GABP alpha / beta and cause a 58-fold increase of de novo utrophin promoter transcription. Taken together, these findings help define mechanisms used for transcriptional regulation of utrophin expression as well as identify new targets for achieving potentially therapeutic upregulation of utrophin in DMD.