RESUMEN
The bones that comprise the axial skeleton have distinct morphological features characteristic of their positions along the anterior/posterior axis. We previously described a novel TGF-beta family member, myostatin (encoded by the gene Mstn, formerly Gdf8), that has an essential role in regulating skeletal muscle mass. We also identified a gene related to Mstn by low-stringency screening. While the work described here was being completed, the cloning of this gene, designated Gdf11 (also called Bmp11), was also reported by other groups. Here we show that Gdf11, a new transforming growth factor beta(TGFbeta) superfamily member, has an important role in establishing this skeletal pattern. During early mouse embryogenesis, Gdf11 is expressed in the primitive streak and tail bud regions, which are sites where new mesodermal cells are generated. Homozygous mutant mice carrying a targeted deletion of Gdf11 exhibit anteriorly directed homeotic transformations throughout the axial skeleton and posterior displacement of the hindlimbs. The effect of the mutation is dose dependent, as Gdf11+/- mice have a milder phenotype than Gdf11-/- mice. Mutant embryos show alterations in patterns of Hox gene expression, suggesting that Gdf11 acts upstream of the Hox genes. Our findings suggest that Gdf11 is a secreted signal that acts globally to specify positional identity along the anterior/posterior axis.
Asunto(s)
Desarrollo Óseo/genética , Proteínas Morfogenéticas Óseas/genética , Proteínas Morfogenéticas Óseas/fisiología , Animales , Tipificación del Cuerpo/genética , Huesos/anomalías , Femenino , Regulación del Desarrollo de la Expresión Génica , Factores de Diferenciación de Crecimiento , Heterocigoto , Homocigoto , Humanos , Hibridación in Situ , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Fenotipo , EmbarazoRESUMEN
The growth factor myostatin (MSTN) negatively regulates skeletal muscle mass. Mstn gene deletion in mice causes increased muscle mass, reduced adiposity and resistance to genetic or diet-induced obesity (DIO). Pharmacologic MSTN inhibition in mice also causes increased muscle mass and resistance to DIO. To test whether MSTN inhibition causes weight loss in mice that are already obese, we inhibited MSTN in mice fed a high-fat diet (HFD). Mice were fed a diet containing 60% kcal from fat for 12 weeks followed by treatment with a soluble MSTN receptor derived from the activin receptor type IIB extracellular domain. During the next 12 weeks of soluble receptor treatment and HFD feeding, lean mass increased without a loss of adipose mass. Glucose metabolism was also similar between groups. Our results suggest that MSTN inhibition may be ineffective at inducing weight loss in obese patients.
Asunto(s)
Receptores de Activinas Tipo II/farmacología , Tejido Adiposo/efectos de los fármacos , Músculo Esquelético/efectos de los fármacos , Miostatina/antagonistas & inhibidores , Miostatina/metabolismo , Obesidad/tratamiento farmacológico , Tejido Adiposo/metabolismo , Animales , Dieta Alta en Grasa , Ingestión de Energía , Resistencia a la Insulina , Masculino , Ratones , Ratones Endogámicos C57BL , Músculo Esquelético/metabolismo , Miostatina/farmacología , Obesidad/etiología , Obesidad/metabolismoRESUMEN
The mechanisms by which tissue size is controlled are poorly understood. Over 30 years ago, Bullough proposed the existence of chalones, which act as tissue-specific negative growth regulators. The recent discovery of myostatin suggests that negative regulation of tissue growth may be an important mechanism for controlling skeletal muscle mass and raises the possibility that growth inhibitors may also be involved in regulating the size of other tissues.
Asunto(s)
Músculo Esquelético/anatomía & histología , Tamaño de los Órganos/fisiología , Factor de Crecimiento Transformador beta/fisiología , Animales , Proteínas Morfogenéticas Óseas/fisiología , Factores de Diferenciación de Crecimiento , Inhibidores de Crecimiento/fisiología , Ratones , MiostatinaRESUMEN
GDF-8, also known as myostatin, is a member of the transforming growth factor-beta (TGF-beta) superfamily of secreted growth and differentiation factors that is expressed in vertebrate skeletal muscle. Myostatin functions as a negative regulator of skeletal muscle growth and myostatin null mice show a doubling of muscle mass compared with normal mice. We examined femoral morphology of adult myostatin-deficient mice to assess the effects of muscle fiber hypertrophy and hyperplasia on bone shape and cross-sectional geometry. Femora of age- and weight-matched adult mice homozygous for the disrupted myostatin sequence were compared with those of wild-type controls (n = 8 per group). Results show that, as was the case in previous studies, myostatin null mice have hindlimb muscle masses that are approximately double those of controls. Myostatin-deficient mice exhibit third trochanters that are significantly larger than those of controls, whereas the femoral midshafts of the control and experimental mice do not differ significantly from one another in cortical area, bending moment of inertia, and polar moment of inertia. Our findings indicate that the increased muscle mass of myostatin-deficient mice primarily affects sites of muscle insertion, but does not induce additional cortical bone deposition in the diaphysis relative to controls. We therefore conclude that the expanded third trochanters of myostatin-deficient subjects result from tendon and Sharpey fiber expansion associated with muscle growth rather than cortical bone deposition in response to increased levels of mechanical stress.
Asunto(s)
Fémur/patología , Factor de Crecimiento Transformador beta/deficiencia , Animales , Fenómenos Biomecánicos , Fémur/crecimiento & desarrollo , Fémur/fisiopatología , Miembro Posterior , Hiperplasia , Hipertrofia , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Desarrollo de Músculos , Músculo Esquelético/crecimiento & desarrollo , Músculo Esquelético/patología , Miostatina , Factor de Crecimiento Transformador beta/genética , Factor de Crecimiento Transformador beta/fisiologíaRESUMEN
Two new mammalian members (growth/differentiation factor 3 (GDF-3) and GDF-9) of the transforming growth factor-beta superfamily were identified using degenerate oligonucleotides corresponding to conserved regions among known family members. By Northern analysis, GDF-3 transcripts were detected primarily in adult bone marrow, spleen, thymus, and adipose tissue. In contrast, GDF-9 transcripts were detected only in the ovary. Based on their cDNA sequences, the predicted GDF-3 and GDF-9 polypeptides each contain a potential signal sequence for secretion, a putative tetrabasic proteolytic processing site, and a COOH-terminal region that shows significant homology to the known members of the transforming growth factor-beta superfamily. In the COOH-terminal region, GDF-3 and GDF-9 are most homologous to Xenopus Vg-1 (57%) and human bone morphogenetic protein 4 (34%), respectively. Unlike all previously described members of this superfamily, both GDF-3 and GDF-9 lack the conserved cysteine residue that is believed to form the sole disulfide linkage between subunits in other family members. These findings raise new possibilities regarding subunit interactions among members of this superfamily.
Asunto(s)
Cisteína , Sustancias de Crecimiento/genética , Péptidos y Proteínas de Señalización Intercelular , Familia de Multigenes , Factor de Crecimiento Transformador beta/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Proteína Morfogenética Ósea 15 , ADN/genética , ADN/aislamiento & purificación , Femenino , Factor 3 de Diferenciación de Crecimiento , Factor 9 de Diferenciación de Crecimiento , Humanos , Masculino , Ratones , Datos de Secuencia Molecular , Oligodesoxirribonucleótidos , Especificidad de Órganos , Reacción en Cadena de la Polimerasa/métodos , ARN Mensajero/metabolismo , Homología de Secuencia de Aminoácido , XenopusRESUMEN
Myostatin is a transforming growth factor-beta family member that acts as a negative regulator of skeletal muscle mass. To identify possible myostatin inhibitors that may have applications for promoting muscle growth, we investigated the regulation of myostatin signaling. Myostatin protein purified from mammalian cells consisted of a noncovalently held complex of the N-terminal propeptide and a disulfide-linked dimer of C-terminal fragments. The purified C-terminal myostatin dimer was capable of binding the activin type II receptors, Act RIIB and, to a lesser extent, Act RIIA. Binding of myostatin to Act RIIB could be inhibited by the activin-binding protein follistatin and, at higher concentrations, by the myostatin propeptide. To determine the functional significance of these interactions in vivo, we generated transgenic mice expressing high levels of the propeptide, follistatin, or a dominant-negative form of Act RIIB by using a skeletal muscle-specific promoter. Independent transgenic mouse lines for each construct exhibited dramatic increases in muscle mass comparable to those seen in myostatin knockout mice. Our findings suggest that the propeptide, follistatin, or other molecules that block signaling through this pathway may be useful agents for enhancing muscle growth for both human therapeutic and agricultural applications.
Asunto(s)
Desarrollo de Músculos , Músculo Esquelético/crecimiento & desarrollo , Factor de Crecimiento Transformador beta/metabolismo , Receptores de Activinas Tipo II , Animales , Células CHO , Células COS , Cricetinae , Femenino , Masculino , Ratones , Ratones Transgénicos , Miostatina , Unión Proteica , Receptores de Factores de Crecimiento/genética , Receptores de Factores de Crecimiento/metabolismoRESUMEN
Myostatin (GDF-8) is a member of the transforming growth factor beta superfamily of secreted growth and differentiation factors that is essential for proper regulation of skeletal muscle mass in mice. Here we report the myostatin sequences of nine other vertebrate species and the identification of mutations in the coding sequence of bovine myostatin in two breeds of double-muscled cattle, Belgian Blue and Piedmontese, which are known to have an increase in muscle mass relative to conventional cattle. The Belgian Blue myostatin sequence contains an 11-nucleotide deletion in the third exon which causes a frameshift that eliminates virtually all of the mature, active region of the molecule. The Piedmontese myostatin sequence contains a missense mutation in exon 3, resulting in a substitution of tyrosine for an invariant cysteine in the mature region of the protein. The similarity in phenotypes of double-muscled cattle and myostatin null mice suggests that myostatin performs the same biological function in these two species and is a potentially useful target for genetic manipulation in other farm animals.
Asunto(s)
Mutación del Sistema de Lectura , Músculo Esquelético/anatomía & histología , Factor de Crecimiento Transformador beta/genética , Secuencia de Aminoácidos , Animales , Bovinos , Clonación Molecular , Ratones , Datos de Secuencia Molecular , Miostatina , Tamaño de los Órganos/genética , Alineación de Secuencia , Análisis de Secuencia , Especificidad de la EspecieRESUMEN
Myostatin (GDF-8), a member of the transforming growth factor-b superfamily of secreted growth and differentiation factors, is a negative regulator of skeletal muscle growth. We investigated the effects of increased muscle mass on bone morphology by examining bone mineral content and density in the humeri of myostatin-deficient mice. We compared the humeri of 11 mixed-gender, adult mice homozygous for the disrupted myostatin sequence with those from 11 mixed-gender, adult wild-type mice. Body mass, deltoid mass, and triceps mass were recorded from each animal and densitometric and geometric parameters were collected from the humerus using peripheral quantitative computed tomography (pQCT). Cross-sectional slices were scanned at four different positions along the humerus corresponding to 15%, 40%, 60%, and 85% of total humerus length. Results show that the myostatin- deficient mice weigh more than controls and have significantly larger triceps and deltoid muscles. The myostatin-deficient animals also have significantly (P < 0.05) higher trabecular area and trabecular bone mineral content (BMC) in the proximal humerus (15% length) and significantly (P < 0.01) higher cortical BMC, cortical area, and periosteal circumference in the region of the deltoid crest (40% length). The myostatin knockouts otherwise do not differ from controls in cortical BMC. Moreover, experimental and control mice do not differ significantly from one another in cortical bone mineral density (BMD) at any of the sites examined. These results suggest that the effects of increased muscle mass on the mouse humerus are localized to regions where muscles attach; furthermore, these effects include increased mineral content of both trabecular and cortical bone.
Asunto(s)
Densidad Ósea/fisiología , Huesos/metabolismo , Húmero/fisiología , Factor de Crecimiento Transformador beta/deficiencia , Factor de Crecimiento Transformador beta/fisiología , Animales , Peso Corporal , Desarrollo Óseo/genética , Desarrollo Óseo/fisiología , Femenino , Húmero/anatomía & histología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Minerales/metabolismo , Músculo Esquelético/anatomía & histología , Músculo Esquelético/crecimiento & desarrollo , Músculo Esquelético/fisiología , Miostatina , Tamaño de los Órganos , Factor de Crecimiento Transformador beta/genéticaRESUMEN
The transforming growth factor-beta (TGF-beta) superfamily encompasses a large group of growth and differentiation factors playing important roles in regulating embryonic development and in maintaining tissue homeostasis in adult animals. Using degenerate polymerase chain reaction, we have identified a new murine TGF-beta family member, growth/differentiation factor-8 (GDF-8), which is expressed specifically in developing and adult skeletal muscle. During early stages of embryogenesis, GDF-8 expression is restricted to the myotome compartment of developing somites. At later stages and in adult animals, GDF-8 is expressed in many different muscles throughout the body. To determine the biological function of GDF-8, we disrupted the GDF-8 gene by gene targeting in mice. GDF-8 null animals are significantly larger than wild-type animals and show a large and widespread increase in skeletal muscle mass. Individual muscles of mutant animals weigh 2-3 times more than those of wild-type animals, and the increase in mass appears to result from a combination of muscle cell hyperplasia and hypertrophy. These results suggest that GDF-8 functions specifically as a negative regulator of skeletal muscle growth.