RESUMEN
Follistatin is an endogenous glycoprotein that promotes growth and repair of skeletal muscle by sequestering inhibitory ligands of the transforming growth factor-ß superfamily and may therefore have therapeutic potential for neuromuscular diseases. Here, we sought to determine the suitability of a newly engineered follistatin fusion protein (FST288-Fc) to promote localized, rather than systemic, growth of skeletal muscle by capitalizing on the intrinsic heparin-binding ability of the follistatin-288 isoform. As determined by surface plasmon resonance and cell-based assays, FST288-Fc binds to activin A, activin B, myostatin (growth differentiation factor GDF8), and GDF11 with high affinity and neutralizes their activity in vitro. Intramuscular administration of FST288-Fc in mice induced robust, dose-dependent growth of the targeted muscle but not of surrounding or contralateral muscles, in contrast to the systemic effects of a locally administered fusion protein incorporating activin receptor type IIB (ActRIIB-Fc). Furthermore, systemic administration of FST288-Fc in mice did not alter muscle mass or body composition as determined by NMR, which again contrasts with the pronounced systemic activity of ActRIIB-Fc when administered by the same route. Subsequent analysis revealed that FST288-Fc in the circulation undergoes rapid proteolysis, thereby restricting its activity to individual muscles targeted by intramuscular administration. These results indicate that FST288-Fc can produce localized growth of skeletal muscle in a targeted manner with reduced potential for undesirable systemic effects. Thus, FST288-Fc and similar agents may be beneficial in the treatment of disorders with muscle atrophy that is focal, asymmetric, or otherwise heterogeneous.
Asunto(s)
Folistatina/administración & dosificación , Inmunoglobulina G/administración & dosificación , Músculo Esquelético/efectos de los fármacos , Músculo Esquelético/crecimiento & desarrollo , Proteínas Recombinantes de Fusión/administración & dosificación , Secuencia de Aminoácidos , Animales , Relación Dosis-Respuesta a Droga , Folistatina/genética , Folistatina/metabolismo , Humanos , Inmunoglobulina G/genética , Inmunoglobulina G/metabolismo , Inyecciones Intramusculares , Masculino , Ratones , Ratones Endogámicos C57BL , Músculo Esquelético/metabolismo , Estructura Secundaria de Proteína , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
Erythropoietin (EPO) stimulates proliferation of early-stage erythrocyte precursors and is widely used for the treatment of chronic anemia. However, several types of EPO-resistant anemia are characterized by defects in late-stage erythropoiesis, which is EPO independent. Here we investigated regulation of erythropoiesis using a ligand-trapping fusion protein (ACE-536) containing the extracellular domain of human activin receptor type IIB (ActRIIB) modified to reduce activin binding. ACE-536, or its mouse version RAP-536, produced rapid and robust increases in erythrocyte numbers in multiple species under basal conditions and reduced or prevented anemia in murine models. Unlike EPO, RAP-536 promoted maturation of late-stage erythroid precursors in vivo. Cotreatment with ACE-536 and EPO produced a synergistic erythropoietic response. ACE-536 bound growth differentiation factor-11 (GDF11) and potently inhibited GDF11-mediated Smad2/3 signaling. GDF11 inhibited erythroid maturation in mice in vivo and ex vivo. Expression of GDF11 and ActRIIB in erythroid precursors decreased progressively with maturation, suggesting an inhibitory role for GDF11 in late-stage erythroid differentiation. RAP-536 treatment also reduced Smad2/3 activation, anemia, erythroid hyperplasia and ineffective erythropoiesis in a mouse model of myelodysplastic syndromes (MDS). These findings implicate transforming growth factor-ß (TGF-ß) superfamily signaling in erythroid maturation and identify ACE-536 as a new potential treatment for anemia, including that caused by ineffective erythropoiesis.
Asunto(s)
Receptores de Activinas Tipo II , Anemia/sangre , Proteínas Morfogenéticas Óseas/efectos de los fármacos , Células Precursoras Eritroides/efectos de los fármacos , Eritropoyesis/efectos de los fármacos , Factores de Diferenciación de Crecimiento/efectos de los fármacos , Hematínicos/farmacología , Síndromes Mielodisplásicos/sangre , Proteínas Recombinantes de Fusión/farmacología , Animales , Proteínas Morfogenéticas Óseas/antagonistas & inhibidores , Modelos Animales de Enfermedad , Quimioterapia Combinada , Recuento de Eritrocitos , Eritropoyetina/farmacología , Factores de Diferenciación de Crecimiento/antagonistas & inhibidores , Haplorrinos , Humanos , Ligandos , Ratones , Ratas , Recuento de Reticulocitos , Transducción de Señal/efectos de los fármacos , Proteína Smad2/efectos de los fármacos , Proteína smad3/efectos de los fármacosRESUMEN
Androgen deprivation, a consequence of hypogonadism, certain cancer treatments, or normal aging in men, leads to loss of muscle mass, increased adiposity, and osteoporosis. In the present study, using a soluble chimeric form of activin receptor type IIB (ActRIIB) we sought to offset the adverse effects of androgen deprivation on muscle, adipose tissue, and bone. Castrated (ORX) or sham-operated (SHAM) mice received either TBS [vehicle-treated (VEH)] or systemic administration of ActRIIB-mFc, a soluble fusion protein comprised of a form of the extracellular domain of ActRIIB fused to a murine IgG2aFc subunit. In vivo body composition imaging demonstrated that ActRIIB-mFc treatment results in increased lean tissue mass of 23% in SHAM mice [19.02 +/- 0.42 g (VEH) versus 23.43 +/- 0.35 g (ActRIIB-mFc), P < 0.00001] and 26% in ORX mice [15.59 +/- 0.26 g (VEH) versus 19.78 +/- 0.26 g (ActRIIB-mFc), P < 0.00001]. Treatment also caused a decrease in adiposity of 30% in SHAM mice [5.03 +/- 0.48 g (VEH) versus 3.53 +/- 0.19 g (ActRIIB-mFc), NS] and 36% in ORX mice [7.12 +/- 0.53 g (VEH) versus 4.57 +/- 0.28 g (ActRIIB-mFc), P < 0.001]. These changes were also accompanied by altered serum levels of leptin, adiponectin, and insulin, as well as by prevention of steatosis (fatty liver) in ActRIIB-mFc-treated ORX mice. Finally, ActRIIB-mFc prevented loss of bone mass in ORX mice as assessed by whole body dual x-ray absorptiometry and micro-computed tomography of proximal tibias. The data demonstrate that treatment with ActRIIB-mFc restored muscle mass, adiposity, and bone quality to normal levels in a mouse model of androgen deprivation, thereby alleviating multiple adverse consequences of such therapy.
Asunto(s)
Receptores de Activinas Tipo II/farmacología , Antagonistas de Andrógenos/farmacología , Composición Corporal/efectos de los fármacos , Densidad Ósea/efectos de los fármacos , Receptores de Activinas Tipo II/genética , Tejido Adiposo/efectos de los fármacos , Tejido Adiposo/metabolismo , Análisis de Varianza , Animales , Peso Corporal/efectos de los fármacos , Línea Celular , Humanos , Fragmentos Fc de Inmunoglobulinas/genética , Inmunoglobulina G/genética , Leptina/sangre , Masculino , Ratones , Ratones Endogámicos C57BL , Músculo Esquelético/efectos de los fármacos , Músculo Esquelético/metabolismo , Obesidad/sangre , Obesidad/prevención & control , Orquiectomía , Distribución Aleatoria , Proteínas Recombinantes de Fusión/farmacología , SolubilidadRESUMEN
The single transmembrane domain serine/threonine kinase activin receptor type IIB (ActRIIB) has been proposed to bind key regulators of skeletal muscle mass development, including the ligands GDF-8 (myostatin) and GDF-11 (BMP-11). Here we provide a detailed kinetic characterization of ActRIIB binding to several low and high affinity ligands using a soluble activin receptor type IIB-Fc chimera (ActRIIB.Fc). We show that both GDF-8 and GDF-11 bind the extracellular domain of ActRIIB with affinities comparable with those of activin A, a known high affinity ActRIIB ligand, whereas BMP-2 and BMP-7 affinities for ActRIIB are at least 100-fold lower. Using site-directed mutagenesis, we demonstrate that ActRIIB binds GDF-11 and activin A in different ways such as, for example, substitutions in ActRIIB Leu(79) effectively abolish ActRIIB binding to activin A yet not to GDF-11. Native ActRIIB has four isoforms that differ in the length of the C-terminal portion of their extracellular domains. We demonstrate that the C terminus of the ActRIIB extracellular domain is crucial for maintaining biological activity of the ActRIIB.Fc receptor chimera. In addition, we show that glycosylation of ActRIIB is not required for binding to activin A or GDF-11. Together, our findings reveal binding specificity and activity determinants of the ActRIIB receptor that combine to effect specificity in the activation of distinct signaling pathways.
Asunto(s)
Receptores de Activinas Tipo II/metabolismo , Receptores de Activinas Tipo II/química , Receptores de Activinas Tipo II/genética , Animales , Sitios de Unión , Células COS , Chlorocebus aethiops , ADN Complementario/genética , Genes Reporteros , Humanos , Ligandos , Mutagénesis , Proteínas Mutantes Quiméricas/química , Proteínas Mutantes Quiméricas/metabolismo , Miostatina/química , Miostatina/metabolismo , Plásmidos/genética , Activadores Plasminogénicos/química , Activadores Plasminogénicos/metabolismo , Unión Proteica , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
Activin A belongs to the TGF-beta superfamily and plays an important role in bone metabolism. It was reported that a soluble form of extracellular domain of the activin receptor type IIA (ActRIIA) fused to the Fc domain of murine IgG, an activin antagonist, has an anabolic effect on bone in intact and ovariectomized mice. The present study was designed to examine the skeletal effect of human ActRIIA-IgG1-Fc (ACE-011) in non-human primates. Young adult female Cynomolgus monkeys were given a biweekly subcutaneous injection of either 10mg/kg ACE-011 or vehicle (VEH) for 3months. Treatment effects were evaluated by histomorphometric analysis of the distal femur, femoral midshaft, femoral neck and 12th thoracic vertebrae, by muCT analysis of femoral neck and by biomarkers of bone turnover. Compared to VEH, at the distal femur ACE-011-treated monkeys had significantly increased cancellous bone volume (+93%), bone formation rate per bone surface (+166%) and osteoblast surface (+196%) indicating an anabolic action. Monkeys treated with ACE-011 also had decreased osteoclast surface and number. No differences were observed in parameters of cortical bone at the midshaft of the femur. Similar to distal femur, ACE-011-treated monkeys had significantly greater cancellous bone volume, bone formation rate and osteoblast surface at the femoral neck relative to VEH. A significant increase in bone formation rate and osteoblast surface with a decrease in osteoclast surface was observed in thoracic vertebrae. muCT analysis of femoral neck indicated more plate-like structure in ACE-011-treated monkeys. Monkeys treated with ACE-011 had no effect on serum bone-specific alkaline phosphatase and CTX at the end of the study. These observations demonstrate that ACE-011 is a dual anabolic-antiresorptive compound, improving cancellous bone volume by promoting bone formation and inhibiting bone resorption in non-human primates. Thus, soluble ActRIIA fusion protein may be useful in the prevention and/or treatment of osteoporosis and other diseases involving accelerated bone loss.
Asunto(s)
Activinas/metabolismo , Densidad Ósea/fisiología , Fémur/metabolismo , Proteínas Recombinantes de Fusión/administración & dosificación , Vértebras Torácicas/metabolismo , Animales , Densidad Ósea/efectos de los fármacos , Recuento de Células , Colágeno Tipo I/sangre , Ensayo de Inmunoadsorción Enzimática , Femenino , Fémur/efectos de los fármacos , Macaca fascicularis , Osteoclastos/efectos de los fármacos , Osteoclastos/metabolismo , Osteogénesis/efectos de los fármacos , Osteogénesis/fisiología , Distribución Aleatoria , Proteínas Recombinantes de Fusión/metabolismo , Estadísticas no Paramétricas , Vértebras Torácicas/efectos de los fármacosRESUMEN
A recent study suggests that activin inhibits bone matrix mineralization, whereas treatment of mice with a soluble form of the activin type IIA receptor markedly increases bone mass and strength. To further extend these observations, we determined the skeletal effects of inhibiting activin signaling through the ActRIIA receptor in a large animal model with a hormonal profile and bone metabolism similar to humans. Ten female cynomolgus monkeys (Macaca fascicularis) were divided into two weight-matched groups and treated biweekly, for 3 months, with either a subcutaneous injection 10 mg/kg of a soluble form of the ActRIIA receptor fused with the Fc portion of human IgG(1) (ACE-011) or vehicle (VEH). Bone mineral density (BMD), micro-architecture, compressive mechanical properties, and ash fraction were assessed at the end of the treatment period. BMD was significantly higher in ACE-011 treated individuals compared to VEH: +13% (p=0.003) in the 5th lumbar vertebral body and +15% (p=0.05) in the distal femur. In addition, trabecular volumetric bone density at the distal femur was 72% (p=0.0004) higher than the VEH-treated group. Monkeys treated with ACE-011 also had a significantly higher L5 vertebral body trabecular bone volume (p=0.002) and compressive mechanical properties. Ash fraction of L4 trabecular bone cores did not differ between groups. These results demonstrate that treatment with a soluble form of ActRIIA (ACE-011) enhances bone mass and bone strength in cynomolgus monkeys, and provide strong rationale for exploring the use of ACE-011 to prevent and/or treat skeletal fragility.
Asunto(s)
Receptores de Activinas Tipo II/farmacología , Densidad Ósea/efectos de los fármacos , Huesos/efectos de los fármacos , Huesos/metabolismo , Macaca fascicularis/metabolismo , Animales , Conservadores de la Densidad Ósea/farmacología , Femenino , HumanosRESUMEN
Myostatin is a secreted protein that normally functions as a negative regulator of muscle growth. Agents capable of blocking the myostatin signaling pathway could have important applications for treating human muscle degenerative diseases as well as for enhancing livestock production. Here we describe a potent myostatin inhibitor, a soluble form of the activin type IIB receptor (ACVR2B), which can cause dramatic increases in muscle mass (up to 60% in 2 weeks) when injected into wild-type mice. Furthermore, we show that the effect of the soluble receptor is attenuated but not eliminated in Mstn(-/-) mice, suggesting that at least one other ligand in addition to myostatin normally functions to limit muscle growth. Finally, we provide genetic evidence that these ligands signal through both activin type II receptors, ACVR2 and ACVR2B, to regulate muscle growth in vivo.
Asunto(s)
Receptores de Activinas Tipo II/metabolismo , Músculo Esquelético/crecimiento & desarrollo , Transducción de Señal/fisiología , Factor de Crecimiento Transformador beta/antagonistas & inhibidores , Receptores de Activinas Tipo II/genética , Receptores de Activinas Tipo II/farmacología , Animales , Ligandos , Ratones , Ratones Noqueados , Músculo Esquelético/efectos de los fármacos , Miostatina , Tamaño de los ÓrganosRESUMEN
The double-stranded (ds) RNA-activated protein kinase (PKR) plays an important role in control of viral infections and cell growth. We have studied the role of PKR in viral infection in mice that are defective in the PKR signaling pathway. Transgenic mice were derived that constitutively express a trans-dominant-negative kinase-defective mutant PKR under control of the beta-actin promoter. The trans-dominant-negative PKR mutant expressing transgenic mice do not have a detectable phenotype, similar to observations with PKR knock-out mice. The requirement for PKR in viral pathogenesis was studied by intracerebral infection of mice with a mouse-adapted poliovirus. Histopathological analysis revealed diffuse encephalomyelitis with severe inflammatory lesions throughout the central nervous system (CNS) in infected wild-type mice. In contrast, histopathological evaluation of virus-injected trans-dominant-negative PKR transgenic mice as well as PKR knock-out mice yielded no signs of tissue damage associated with inflammatory host responses. However, the virus did replicate in both models of PKR-deficient mice at a level equal to that observed in wild-type infected mice. Although the results indicate a clear difference in susceptibility to poliovirus-induced encephalitis, this difference manifests clinically as a slight delay in fatal neuropathy in trans-dominant-negative PKR transgenic and PKR knock-out animals. Our observations support the finding that viral-induced PKR activation may play a significant role in pathogenesis by mediating the host response to viral CNS infection. They support PKR to be an effective target to control tissue damage due to deleterious host responses to viral infection.
Asunto(s)
Encefalitis Viral/fisiopatología , Poliomielitis/fisiopatología , Poliovirus/patogenicidad , ARN Bicatenario/metabolismo , eIF-2 Quinasa/metabolismo , Animales , Sistema Nervioso Central/patología , Sistema Nervioso Central/virología , Encefalitis Viral/metabolismo , Encefalitis Viral/patología , Encefalitis Viral/virología , Factor 2 Eucariótico de Iniciación , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Células 3T3 NIH , Poliomielitis/metabolismo , Poliomielitis/patología , Poliomielitis/virología , ARN Bicatenario/genética , Transducción de SeñalRESUMEN
Myostatin is a transforming growth factor beta family member that acts as a negative regulator of skeletal muscle growth. Myostatin circulates in the blood of adult mice in a noncovalently held complex with other proteins, including its propeptide, which maintain the C-terminal dimer in a latent, inactive state. This latent form of myostatin can be activated in vitro by treatment with acid; however, the mechanisms by which latent myostatin is activated in vivo are unknown. Here, we show that members of the bone morphogenetic protein-1/tolloid (BMP-1/TLD) family of metalloproteinases can cleave the myostatin propeptide in this complex and can thereby activate latent myostatin. Furthermore, we show that a mutant form of the propeptide resistant to cleavage by BMP-1/TLD proteinases can cause significant increases in muscle mass when injected into adult mice. These findings raise the possibility that members of the BMP-1/TLD family may be involved in activating latent myostatin in vivo and that molecules capable of inhibiting these proteinases may be effective agents for increasing muscle mass for both human therapeutic and agricultural applications.
Asunto(s)
Proteínas Morfogenéticas Óseas/metabolismo , Metaloendopeptidasas/metabolismo , Metaloproteasas/metabolismo , Músculo Esquelético/crecimiento & desarrollo , Proteínas/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Animales , Proteína Morfogenética Ósea 1 , Células CHO , Cricetinae , Femenino , Genes Reporteros , Luciferasas/genética , Luciferasas/metabolismo , Ratones , Ratones Endogámicos BALB C , Miostatina , Precursores de Proteínas/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Metaloproteinasas Similares a TolloidRESUMEN
A human therapeutic that specifically modulates skeletal muscle growth would potentially provide a benefit for a variety of conditions including sarcopenia, cachexia, and muscular dystrophy. Myostatin, a member of the TGF-beta family of growth factors, is a known negative regulator of muscle mass, as mice lacking the myostatin gene have increased muscle mass. Thus, an inhibitor of myostatin may be useful therapeutically as an anabolic agent for muscle. However, since myostatin is expressed in both developing and adult muscles, it is not clear whether it regulates muscle mass during development or in adults. In order to test the hypothesis that myostatin regulates muscle mass in adults, we generated an inhibitory antibody to myostatin and administered it to adult mice. Here we show that mice treated pharmacologically with an antibody to myostatin have increased skeletal muscle mass and increased grip strength. These data show for the first time that myostatin acts postnatally as a negative regulator of skeletal muscle growth and suggest that myostatin inhibitors could provide a therapeutic benefit in diseases for which muscle mass is limiting.
Asunto(s)
Músculo Esquelético/crecimiento & desarrollo , Factor de Crecimiento Transformador beta/antagonistas & inhibidores , Factor de Crecimiento Transformador beta/metabolismo , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/administración & dosificación , Anticuerpos Monoclonales/inmunología , Peso Corporal , Células CHO , Cricetinae , Medios de Cultivo Condicionados , Femenino , Fuerza de la Mano , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Fibras Musculares Esqueléticas/citología , Músculo Esquelético/anatomía & histología , Músculo Esquelético/fisiología , Miostatina , Unión Proteica , Factor de Crecimiento Transformador beta/genética , Factor de Crecimiento Transformador beta/inmunologíaRESUMEN
Myostatin, also known as growth and differentiation factor 8, is a member of the transforming growth factor beta superfamily that negatively regulates skeletal muscle mass (1). Recent experiments have shown that myostatin activity is detected in serum by a reporter gene assay only after activation by acid, suggesting that native myostatin circulates as a latent complex (2). We have used a monoclonal myostatin antibody, JA16, to isolate the native myostatin complex from normal mouse and human serum. Analysis by mass spectrometry and Western blot shows that circulating myostatin is bound to at least two major proteins, the myostatin propeptide and the follistatin-related gene (FLRG). The myostatin propeptide is known to bind and inhibit myostatin in vitro (3). Here we show that this interaction is relevant in vivo, with a majority (>70%) of myostatin in serum bound to its propeptide. Studies with recombinant V5-His-tagged FLRG protein confirm a direct interaction between mature myostatin and FLRG. Functional studies show that FLRG inhibits myostatin activity in a reporter gene assay. These experiments suggest that the myostatin propeptide and FLRG are major negative regulators of myostatin in vivo.
Asunto(s)
Proteínas Relacionadas con la Folistatina/fisiología , Factor de Crecimiento Transformador beta/fisiología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Western Blotting , Cartilla de ADN , Proteínas Relacionadas con la Folistatina/genética , Proteínas Relacionadas con la Folistatina/metabolismo , Genes Reporteros , Humanos , Espectrometría de Masas , Ratones , Datos de Secuencia Molecular , Miostatina , Unión Proteica , Factor de Crecimiento Transformador beta/antagonistas & inhibidores , Factor de Crecimiento Transformador beta/química , Factor de Crecimiento Transformador beta/genéticaRESUMEN
Mice and cattle with genetic deficiencies in myostatin exhibit dramatic increases in skeletal muscle mass, suggesting that myostatin normally suppresses muscle growth. Whether this increased muscling results from prenatal or postnatal lack of myostatin activity is unknown. Here we show that myostatin circulates in the blood of adult mice in a latent form that can be activated by acid treatment. Systemic overexpression of myostatin in adult mice was found to induce profound muscle and fat loss analogous to that seen in human cachexia syndromes. These data indicate that myostatin acts systemically in adult animals and may be a useful pharmacologic target in clinical settings such as cachexia, where muscle growth is desired.