RESUMEN
In ß-thalassaemia, anaemia results from ineffective erythropoiesis characterized by inhibition of late-stage erythroid differentiation. We earlier used luspatercept and RAP-536 protein traps for certain Smad2/3-pathway ligands to implicate Smad2/3-pathway overactivation in dysregulated erythroid differentiation associated with murine ß-thalassaemia and myelodysplasia. Importantly, luspatercept alleviates anaemia and has been shown to reduce transfusion burden in patients with ß-thalassaemia or myelodysplasia. Here, we investigated the molecular mechanisms underlying luspatercept action and pSmad2/3-mediated inhibition of erythroid differentiation. In murine erythroleukemic (MEL) cells in vitro, ligand-mediated overactivation of the Smad2/3 pathway reduced nuclear levels of GATA-1 (GATA-binding factor-1) and its transcriptional activator TIF1γ (transcription intermediary factor 1γ), increased levels of reactive oxygen species, reduced cell viability and haemoglobin levels, and inhibited erythroid differentiation. Co-treatment with luspatercept in MEL cells partially or completely restored each of these. In ß-thalassaemic mice, RAP-536 up-regulated Gata1 and its target gene signature in erythroid precursors determined by transcriptional profiling and gene set enrichment analysis, restored nuclear levels of GATA-1 in erythroid precursors, and nuclear distribution of TIF1γ in erythroblasts. Bone marrow cells from ß-thalassaemic mice treated with luspatercept also exhibited restored nuclear availability of GATA-1 ex vivo. Our results implicate GATA-1, and likely TIF1γ, as key mediators of luspatercept/RAP-536 action in alleviating ineffective erythropoiesis.
Asunto(s)
Receptores de Activinas Tipo II/farmacología , Diferenciación Celular , Células Eritroides/patología , Factor de Transcripción GATA1/metabolismo , Fragmentos Fc de Inmunoglobulinas/farmacología , Proteínas Recombinantes de Fusión/farmacología , Transducción de Señal , Proteína Smad2/metabolismo , Proteína smad3/metabolismo , Talasemia beta/patología , Anemia/complicaciones , Anemia/tratamiento farmacológico , Animales , Células de la Médula Ósea/efectos de los fármacos , Células de la Médula Ósea/metabolismo , Diferenciación Celular/efectos de los fármacos , Línea Celular Tumoral , Núcleo Celular/efectos de los fármacos , Núcleo Celular/metabolismo , Supervivencia Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Eritroblastos , Células Eritroides/efectos de los fármacos , Hemoglobinas/metabolismo , Leucemia Eritroblástica Aguda/patología , Ligandos , Ratones Endogámicos C57BL , Fosforilación/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo , Factores de Transcripción/metabolismo , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/genética , Talasemia beta/complicaciones , Talasemia beta/genéticaRESUMEN
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
INTRODUCTION: Difficulty in modeling congenital contractures (deformities of muscle-tendon unit development that include shortened muscles and lengthened tendons) has limited research of new treatments. METHODS: Early immobilization of the ankle in prepuberal mice was used to produce deformities similar to congenital contractures. Stretch treatment, electrostimulation, and local intramuscular injection of a follistatin analog (FST-288) were assessed as therapeutic interventions for these deformities. RESULTS: Ankle immobilization at full plantarflexion and 90 ° created tendon lengthening and muscle shortening in the tibialis anterior and soleus. Stretch treatment produced minimal evidence for longitudinal muscle growth and electrostimulation provided no additional benefit. Stretch treatment with FST-288 produced greater longitudinal muscle growth and less tendon lengthening, constituting the best treatment response. DISCUSSION: Ankle immobilization recapitulates key morphologic features of congenital contracture, and these features can be mitigated by a combination of stretch and pharmacological approaches that may be useful in patients. Muscle Nerve 58: 718-725, 2018.
Asunto(s)
Traumatismos del Tobillo/etiología , Traumatismos del Tobillo/patología , Inmovilización/efectos adversos , Músculo Esquelético/fisiopatología , Evaluación de Resultado en la Atención de Salud/métodos , Animales , Traumatismos del Tobillo/terapia , Fenómenos Biomecánicos , Modelos Animales de Enfermedad , Terapia por Estimulación Eléctrica , Femenino , Folistatina/uso terapéutico , Masculino , Ratones , Contracción Muscular , Sarcómeros/patología , Férulas (Fijadores) , Estadísticas no Paramétricas , Tendones , Factores de TiempoRESUMEN
INTRODUCTION: Osteogenesis imperfecta (OI) is characterized by skeletal fragility and muscle weakness. In this study we investigated the effects of soluble activin type IIB receptor (sActRIIB-mFc) on muscle mass and function in 2 distinct mouse models of OI: osteogenesis imperfecta murine (oim) and +/G610C. METHODS: Wild-type (WT), +/G610C, and oim/oim mice were treated from 2 to 4 months of age with Tris-buffered saline (vehicle) or sActRIIB-mFc and their hindlimb muscles evaluated for mass, morphology, and contractile function. RESULTS: sActRIIB-mFc-treated WT, +/G610C, and oim/oim mice had increased hindlimb muscle weights and myofiber cross-sectional area compared with vehicle-treated counterparts. sActRIIB-mFc-treated oim/oim mice also exhibited increased contractile function relative to vehicle-treated counterparts. DISCUSSION: Blocking endogenous ActRIIB was effective at increasing muscle size in mouse models of OI, and increasing contractile function in oim/oim mice. ActRIIB inhibitors may provide a potential mutation-specific therapeutic option for compromised muscle function in OI. Muscle Nerve 57: 294-304, 2018.
Asunto(s)
Receptores de Activinas Tipo II/genética , Músculo Esquelético/fisiopatología , Osteogénesis Imperfecta/genética , Osteogénesis Imperfecta/fisiopatología , Anatomía Transversal , Animales , Citrato (si)-Sintasa/metabolismo , Colágeno Tipo I/genética , Femenino , Miembro Posterior/fisiopatología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Actividad Motora , Contracción Muscular , Fibras Musculares Esqueléticas/patología , Fuerza Muscular , Mutación , Tamaño de los Órganos , Osteogénesis Imperfecta/patologíaRESUMEN
Nemaline myopathies (NMs) are a group of congenital muscle diseases caused by mutations in at least 10 genes and associated with a range of clinical symptoms. NM is defined on muscle biopsy by the presence of cytoplasmic rod-like structures (nemaline rods) composed of cytoskeletal material. Myofiber smallness is also found in many cases of NM and may represent a cause of weakness that can be counteracted by treatment. We have used i.p. injection of activin type IIB receptor (ActRIIB)-mFc (an inhibitor of myostatin signaling) to promote hypertrophy and increase strength in our prior murine work; we therefore tested whether ActRIIB-mFc could improve weakness in NM mice through myofiber hypertrophy. We report a study of ActRIIB-mFc treatment in the Acta1 H40Y mouse model of NM. Treatment of Acta1 H40Y mice produced significant increases in body mass, muscle mass, quadriceps myofiber size, and survival, but other measurements of strength (forelimb grip strength, ex vivo measurements of contractile function) did not improve. Our studies also identified that the complications of urethral obstruction are associated with mortality in male hemizygote Acta1 H40Y mice. The incidence of urethral obstruction and histologic evidence of chronic obstruction (inflammation) were significantly lower in Acta1 H40Y mice that had been treated with ActRIIB-mFc. ActRIIB-mFc treatment produces a mild benefit to the disease phenotype in Acta1 H40Y mice.
Asunto(s)
Receptores de Activinas Tipo II/antagonistas & inhibidores , Miofibrillas/efectos de los fármacos , Miopatías Nemalínicas/patología , Animales , Western Blotting , Modelos Animales de Enfermedad , Masculino , Ratones , Ratones Mutantes , Músculo Esquelético/efectos de los fármacos , Músculo Esquelético/patología , Miofibrillas/patologíaRESUMEN
RATIONALE: Transforming growth factor-ß (TGF-ß) ligands signal via type I and type II serine-threonine kinase receptors to regulate broad transcriptional programs. Excessive TGF-ß-mediated signaling is implicated in the pathogenesis of pulmonary arterial hypertension, based in part on the ability of broad inhibition of activin-like kinase (ALK) receptors 4/5/7 recognizing TGF-ß, activin, growth and differentiation factor, and nodal ligands to attenuate experimental pulmonary hypertension (PH). These broad inhibition strategies do not delineate the specific contribution of TGF-ß versus a multitude of other ligands, and their translation is limited by cardiovascular and systemic toxicity. OBJECTIVES: We tested the impact of a soluble TGF-ß type II receptor extracellular domain expressed as an immunoglobulin-Fc fusion protein (TGFBRII-Fc), serving as a selective TGF-ß1/3 ligand trap, in several experimental PH models. METHODS: Signaling studies used cultured human pulmonary artery smooth muscle cells. PH was studied in monocrotaline-treated Sprague-Dawley rats, SU5416/hypoxia-treated Sprague-Dawley rats, and SU5416/hypoxia-treated C57BL/6 mice. PH, cardiac function, vascular remodeling, and valve structure were assessed by ultrasound, invasive hemodynamic measurements, and histomorphometry. MEASUREMENTS AND MAIN RESULTS: TGFBRII-Fc is an inhibitor of TGF-ß1 and TGF-ß3, but not TGF-ß2, signaling. In vivo treatment with TGFBRII-Fc attenuated Smad2 phosphorylation, normalized expression of plasminogen activator inhibitor-1, and mitigated PH and pulmonary vascular remodeling in monocrotaline-treated rats, SU5416/hypoxia-treated rats, and SU5416/hypoxia-treated mice. Administration of TGFBRII-Fc to monocrotaline-treated or SU5416/hypoxia-treated rats with established PH improved right ventricular systolic pressures, right ventricular function, and survival. No cardiac structural or valvular abnormalities were observed after treatment with TGFBRII-Fc. CONCLUSIONS: Our findings are consistent with a pathogenetic role of TGF-ß1/3, demonstrating the efficacy and tolerability of selective TGF-ß ligand blockade for improving hemodynamics, remodeling, and survival in multiple experimental PH models.
Asunto(s)
Hipertensión Pulmonar/tratamiento farmacológico , Factor de Crecimiento Transformador beta/antagonistas & inhibidores , Animales , Modelos Animales de Enfermedad , Corazón/fisiopatología , Hemodinámica/fisiología , Hipertensión Pulmonar/fisiopatología , Fragmentos Fc de Inmunoglobulinas/metabolismo , Ligandos , Masculino , Ratones Endogámicos C57BL , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Ratas , Ratas Sprague-Dawley , Receptor Tipo II de Factor de Crecimiento Transformador beta , Receptores de Factores de Crecimiento Transformadores beta/genética , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Proteínas Recombinantes , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Factor de Crecimiento Transformador beta/fisiología , Remodelación Vascular/fisiologíaRESUMEN
In ß-thalassemia, unequal production of α- and ß-globin chains in erythroid precursors causes apoptosis and inhibition of late-stage erythroid differentiation, leading to anemia, ineffective erythropoiesis (IE), and dysregulated iron homeostasis. Here we used a murine model of ß-thalassemia intermedia (Hbb(th1/th1) mice) to investigate effects of a modified activin receptor type IIB (ActRIIB) ligand trap (RAP-536) that inhibits Smad2/3 signaling. In Hbb(th1/th1) mice, treatment with RAP-536 reduced overactivation of Smad2/3 in splenic erythroid precursors. In addition, treatment of Hbb(th1/th1) mice with RAP-536 reduced α-globin aggregates in peripheral red cells, decreased the elevated reactive oxygen species present in erythroid precursors and peripheral red cells, and alleviated anemia by promoting differentiation of late-stage erythroid precursors and reducing hemolysis. Notably, RAP-536 treatment mitigated disease complications of IE, including iron overload, splenomegaly, and bone pathology, while reducing erythropoietin levels, improving erythrocyte morphology, and extending erythrocyte life span. These results implicate signaling by the transforming growth factor-ß superfamily in late-stage erythropoiesis and reveal potential of a modified ActRIIB ligand trap as a novel therapeutic agent for thalassemia syndrome and other red cell disorders characterized by IE.
Asunto(s)
Receptores de Activinas Tipo II/genética , Eritropoyesis/efectos de los fármacos , Proteínas Recombinantes de Fusión/farmacología , Transducción de Señal/efectos de los fármacos , Talasemia beta/tratamiento farmacológico , Receptores de Activinas Tipo II/metabolismo , Anemia/sangre , Anemia/genética , Anemia/prevención & control , Animales , Western Blotting , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/genética , Eritrocitos/efectos de los fármacos , Eritrocitos/metabolismo , Células Precursoras Eritroides/efectos de los fármacos , Células Precursoras Eritroides/metabolismo , Eritropoyesis/genética , Hemólisis/efectos de los fármacos , Hemólisis/genética , Humanos , Fragmentos Fc de Inmunoglobulinas/genética , Fragmentos Fc de Inmunoglobulinas/metabolismo , Inmunoglobulina G/genética , Inmunoglobulina G/metabolismo , Sobrecarga de Hierro/metabolismo , Sobrecarga de Hierro/prevención & control , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mutación , Especies Reactivas de Oxígeno/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Transducción de Señal/genética , Proteína Smad2/metabolismo , Proteína smad3/metabolismo , Globinas beta/genética , Globinas beta/metabolismo , Talasemia beta/sangre , Talasemia beta/genéticaRESUMEN
Diseases such as osteoporosis are associated with reduced bone mass. Therapies to prevent bone loss exist, but there are few that stimulate bone formation and restore bone mass. Bone morphogenetic proteins (BMPs) are members of the TGFß superfamily, which act as pleiotropic regulators of skeletal organogenesis and bone homeostasis. Ablation of the BMPR1A receptor in osteoblasts increases bone mass, suggesting that inhibition of BMPR1A signaling may have therapeutic benefit. The aim of this study was to determine the skeletal effects of systemic administration of a soluble BMPR1A fusion protein (mBMPR1A-mFc) in vivo. mBMPR1A-mFc was shown to bind BMP2/4 specifically and with high affinity and prevent downstream signaling. mBMPR1A-mFc treatment of immature and mature mice increased bone mineral density, cortical thickness, trabecular bone volume, thickness and number, and decreased trabecular separation. The increase in bone mass was due to an early increase in osteoblast number and bone formation rate, mediated by a suppression of Dickkopf-1 expression. This was followed by a decrease in osteoclast number and eroded surface, which was associated with a decrease in receptor activator of NF-κB ligand (RANKL) production, an increase in osteoprotegerin expression, and a decrease in serum tartrate-resistant acid phosphatase (TRAP5b) concentration. mBMPR1A treatment also increased bone mass and strength in mice with bone loss due to estrogen deficiency. In conclusion, mBMPR1A-mFc stimulates osteoblastic bone formation and decreases bone resorption, which leads to an increase in bone mass, and offers a promising unique alternative for the treatment of bone-related disorders.
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Enfermedades Óseas Metabólicas/prevención & control , Receptores de Proteínas Morfogenéticas Óseas de Tipo 1/metabolismo , Huesos/efectos de los fármacos , Osteogénesis/efectos de los fármacos , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes de Fusión/farmacología , Transducción de Señal/efectos de los fármacos , Análisis de Varianza , Animales , Western Blotting , Densidad Ósea/efectos de los fármacos , Proteína Morfogenética Ósea 2/metabolismo , Proteína Morfogenética Ósea 4/metabolismo , Resorción Ósea/tratamiento farmacológico , Huesos/anatomía & histología , Huesos/fisiología , Cromatografía en Gel , Clonación Molecular , Densitometría , Electroforesis en Gel de Poliacrilamida , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Luciferasas , Ratones , Ratones Endogámicos C57BL , Osteoclastos/efectos de los fármacos , Osteoclastos/fisiología , Osteoprotegerina/metabolismo , Reacción en Cadena de la Polimerasa , Ligando RANK/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Proteínas Recombinantes de Fusión/administración & dosificación , Transducción de Señal/fisiologíaRESUMEN
Endoglin (CD105), a transmembrane protein of the transforming growth factor ß superfamily, plays a crucial role in angiogenesis. Mutations in endoglin result in the vascular defect known as hereditary hemorrhagic telangiectasia (HHT1). The soluble form of endoglin was suggested to contribute to the pathogenesis of preeclampsia. To obtain further insight into its function, we cloned, expressed, purified, and characterized the extracellular domain (ECD) of mouse and human endoglin fused to an immunoglobulin Fc domain. We found that mouse and human endoglin ECD-Fc bound directly, specifically, and with high affinity to bone morphogenetic proteins 9 and 10 (BMP9 and BMP10) in surface plasmon resonance (Biacore) and cell-based assays. We performed a function mapping analysis of the different domains of endoglin by examining their contributions to the selectivity and biological activity of the protein. The BMP9/BMP10 binding site was localized to the orphan domain of human endoglin composed of the amino acid sequence 26-359. We established that endoglin and type II receptors bind to overlapping sites on BMP9. In the in vivo chick chorioallantoic membrane assay, the mouse and the truncated human endoglin ECD-Fc both significantly reduced VEGF-induced vessel formation. Finally, murine endoglin ECD-Fc acted as an anti-angiogenic factor that decreased blood vessel sprouting in VEGF/FGF-induced angiogenesis in in vivo angioreactors and reduced the tumor burden in the colon-26 mouse tumor model. Together our findings indicate an important role of soluble endoglin ECD in the regulation of angiogenesis and highlight efficacy of endoglin-Fc as a potential anti-angiogenesis therapeutic agent.
Asunto(s)
Inhibidores de la Angiogénesis/farmacología , Antígenos CD/farmacología , Proteínas Morfogenéticas Óseas/metabolismo , Factor 2 de Diferenciación de Crecimiento/metabolismo , Factores de Diferenciación de Crecimiento/metabolismo , Péptidos y Proteínas de Señalización Intracelular/farmacología , Neoplasias/tratamiento farmacológico , Neovascularización Patológica/tratamiento farmacológico , Inhibidores de la Angiogénesis/genética , Inhibidores de la Angiogénesis/metabolismo , Animales , Antígenos CD/genética , Antígenos CD/metabolismo , Sitios de Unión , Receptores de Proteínas Morfogenéticas Óseas de Tipo II/genética , Receptores de Proteínas Morfogenéticas Óseas de Tipo II/metabolismo , Proteínas Morfogenéticas Óseas/genética , Línea Celular , Endoglina , Factor 2 de Diferenciación de Crecimiento/genética , Factores de Diferenciación de Crecimiento/genética , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Ratones , Neoplasias/genética , Neoplasias/metabolismo , Neovascularización Patológica/genética , Neovascularización Patológica/metabolismo , Unión Proteica , Estructura Terciaria de Proteína , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/metabolismoRESUMEN
Sotatercept is an activin receptor type IIA-Fc (ActRIIA-Fc) fusion protein that improves cardiopulmonary function in patients with pulmonary arterial hypertension (PAH) by selectively trapping activins and growth differentiation factors. However, the cellular and molecular mechanisms of ActRIIA-Fc action are incompletely understood. Here, we determined through genome-wide expression profiling that inflammatory and immune responses are prominently upregulated in the lungs of a Sugen-hypoxia rat model of severe angio-obliterative PAH, concordant with profiles observed in PAH patients. Therapeutic treatment with ActRIIA-Fc-but not with a vasodilator-strikingly reversed proinflammatory and proliferative gene expression profiles and normalized macrophage infiltration in diseased rodent lungs. Furthermore, ActRIIA-Fc normalized pulmonary macrophage infiltration and corrected cardiopulmonary structure and function in Bmpr2 haploinsufficient mice subjected to hypoxia, a model of heritable PAH. Three high-affinity ligands of ActRIIA-Fc each induced macrophage activation in vitro, and their combined immunoneutralization in PAH rats produced cardiopulmonary benefits comparable to those elicited by ActRIIA-Fc. Our results in complementary experimental and genetic models of PAH reveal therapeutic anti-inflammatory activities of ActRIIA-Fc that, together with its known anti-proliferative effects on vascular cell types, could underlie clinical activity of sotatercept as either monotherapy or add-on to current PAH therapies.
Asunto(s)
Hipertensión Pulmonar , Hipertensión Arterial Pulmonar , Animales , Modelos Animales de Enfermedad , Hipertensión Pulmonar Primaria Familiar , Humanos , Hipertensión Pulmonar/tratamiento farmacológico , Hipoxia/tratamiento farmacológico , Inflamación/tratamiento farmacológico , Ratones , Hipertensión Arterial Pulmonar/tratamiento farmacológico , Ratas , Proteínas Recombinantes de FusiónRESUMEN
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.
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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
Diseases that affect the regulation of bone turnover can lead to skeletal fragility and increased fracture risk. Members of the TGF-beta superfamily have been shown to be involved in the regulation of bone mass. Activin A, a TGF-beta signaling ligand, is present at high levels in bone and may play a role in the regulation of bone metabolism. Here we demonstrate that pharmacological blockade of ligand signaling through the high affinity receptor for activin, type II activin receptor (ActRIIA), by administration of the soluble extracellular domain of ActRIIA fused to a murine IgG2a-Fc, increases bone formation, bone mass, and bone strength in normal mice and in ovariectomized mice with established bone loss. These observations support the development of this pharmacological strategy for the treatment of diseases with skeletal fragility.
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Receptores de Activinas Tipo II/farmacología , Huesos/efectos de los fármacos , Osteogénesis/efectos de los fármacos , Receptores de Activinas Tipo II/administración & dosificación , Receptores de Activinas Tipo II/aislamiento & purificación , Animales , Fenómenos Biomecánicos , Resorción Ósea , Línea Celular , Femenino , Humanos , Inmunoglobulina G/administración & dosificación , Inmunoglobulina G/aislamiento & purificación , Inmunoglobulina G/farmacología , Vértebras Lumbares/efectos de los fármacos , Ratones , Tamaño de los Órganos/efectos de los fármacos , Ovariectomía , Proteínas Recombinantes de Fusión/administración & dosificación , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/farmacología , Solubilidad/efectos de los fármacosRESUMEN
Ligands of the transforming growth factor-ß (TGF-ß) superfamily are important targets for therapeutic intervention but present challenges because they signal combinatorially and exhibit overlapping activities in vivo. To obtain agents capable of sequestering multiple TGF-ß superfamily ligands with novel selectivity, we generated soluble, heterodimeric ligand traps by pairing the extracellular domain (ECD) of the native activin receptor type IIB (ActRIIB) alternately with the ECDs of native type I receptors activin receptor-like kinase 4 (ALK4), ALK7, or ALK3. Systematic analysis of these heterodimeric constructs by surface plasmon resonance, and comparison with their homodimeric counterparts, revealed that each type I receptor partner confers a distinct ligand-binding profile to the heterodimeric construct. Additional characterization in cell-based reporter gene assays confirmed that the heterodimeric constructs possessed different profiles of signaling inhibition in vitro, which translated into altered patterns of pharmacological activity when constructs were administered systemically to wild-type mice. Our results detail a versatile platform for the modular recombination of naturally occurring receptor domains, giving rise to inhibitory ligand traps that could aid in defining the physiological roles of TGF-ß ligand sets or be directed therapeutically to human diseases arising from dysregulated TGF-ß superfamily signaling.
Asunto(s)
Receptores de Activinas/metabolismo , Descubrimiento de Drogas/métodos , Ingeniería de Proteínas/métodos , Receptores de Activinas/química , Receptores de Activinas/genética , Animales , Sitios de Unión , Células CHO , Cricetinae , Cricetulus , Humanos , Ligandos , Ratones , Ratones Endogámicos C57BL , Unión Proteica , Multimerización de Proteína , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
Patients with neuromuscular disorders suffer from a lack of treatment options for skeletal muscle weakness and disease comorbidities. Here, we introduce as a potential therapeutic agent a heterodimeric ligand-trapping fusion protein, ActRIIB:ALK4-Fc, which comprises extracellular domains of activin-like kinase 4 (ALK4) and activin receptor type IIB (ActRIIB), a naturally occurring pair of type I and II receptors belonging to the TGF-ß superfamily. By surface plasmon resonance (SPR), ActRIIB:ALK4-Fc exhibited a ligand binding profile distinctly different from that of its homodimeric variant ActRIIB-Fc, sequestering ActRIIB ligands known to inhibit muscle growth but not trapping the vascular regulatory ligand bone morphogenetic protein 9 (BMP9). ActRIIB:ALK4-Fc and ActRIIB-Fc administered to mice exerted differential effects - concordant with SPR results - on vessel outgrowth in a retinal explant assay. ActRIIB:ALK4-Fc induced a systemic increase in muscle mass and function in wild-type mice and in murine models of Duchenne muscular dystrophy (DMD), amyotrophic lateral sclerosis (ALS), and disuse atrophy. Importantly, ActRIIB:ALK4-Fc improved neuromuscular junction abnormalities in murine models of DMD and presymptomatic ALS and alleviated acute muscle fibrosis in a DMD model. Furthermore, in combination therapy ActRIIB:ALK4-Fc increased the efficacy of antisense oligonucleotide M12-PMO on dystrophin expression and skeletal muscle endurance in an aged DMD model. ActRIIB:ALK4-Fc shows promise as a therapeutic agent, alone or in combination with dystrophin rescue therapy, to alleviate muscle weakness and comorbidities of neuromuscular disorders.
Asunto(s)
Receptores de Activinas Tipo II/farmacología , Receptores de Activinas Tipo I/farmacología , Esclerosis Amiotrófica Lateral/tratamiento farmacológico , Fragmentos Fc de Inmunoglobulinas/farmacología , Músculo Esquelético/metabolismo , Trastornos Musculares Atróficos/tratamiento farmacológico , Distrofia Muscular de Duchenne/tratamiento farmacológico , Proteínas Recombinantes de Fusión/farmacología , Receptores de Activinas Tipo I/genética , Receptores de Activinas Tipo II/genética , Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/metabolismo , Esclerosis Amiotrófica Lateral/patología , Animales , Células CHO , Cricetulus , Modelos Animales de Enfermedad , Humanos , Fragmentos Fc de Inmunoglobulinas/genética , Masculino , Ratones , Ratones Transgénicos , Músculo Esquelético/patología , Trastornos Musculares Atróficos/genética , Trastornos Musculares Atróficos/metabolismo , Trastornos Musculares Atróficos/patología , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/metabolismo , Distrofia Muscular de Duchenne/patología , Proteínas Recombinantes de Fusión/genéticaRESUMEN
The azoxymethane model of colorectal cancer (CRC) was used to gain insights into the genetic heterogeneity of nonfamilial CRC. We observed significant differences in susceptibility parameters across 40 mouse inbred strains, with 6 new and 18 of 24 previously identified mouse CRC modifier alleles detected using genome-wide association analysis. Tumor incidence varied in F1 as well as intercrosses and backcrosses between resistant and susceptible strains. Analysis of inheritance patterns indicates that resistance to CRC development is inherited as a dominant characteristic genome-wide, and that susceptibility appears to occur in individuals lacking a large-effect, or sufficient numbers of small-effect, polygenic resistance alleles. Our results suggest a new polygenic model for inheritance of nonfamilial CRC, and that genetic studies in humans aimed at identifying individuals with elevated susceptibility should be pursued through the lens of absence of dominant resistance alleles rather than for the presence of susceptibility alleles.
Asunto(s)
Neoplasias Colorrectales/genética , Predisposición Genética a la Enfermedad , Estudio de Asociación del Genoma Completo , Herencia Multifactorial/genética , Alelos , Animales , Azoximetano/toxicidad , Neoplasias Colorrectales/inducido químicamente , Neoplasias Colorrectales/patología , Modelos Animales de Enfermedad , Resistencia a Antineoplásicos , Heterogeneidad Genética , Herencia , Humanos , Ratones , Ratones Endogámicos/genética , Modelos GenéticosRESUMEN
Human genetics, biomarker, and animal studies implicate loss of function in bone morphogenetic protein (BMP) signaling and maladaptive transforming growth factor-ß (TGFß) signaling as drivers of pulmonary arterial hypertension (PAH). Although sharing common receptors and effectors with BMP/TGFß, the function of activin and growth and differentiation factor (GDF) ligands in PAH are less well defined. Increased expression of GDF8, GDF11, and activin A was detected in lung lesions from humans with PAH and experimental rodent models of pulmonary hypertension (PH). ACTRIIA-Fc, a potent GDF8/11 and activin ligand trap, was used to test the roles of these ligands in animal and cellular models of PH. By blocking GDF8/11- and activin-mediated SMAD2/3 activation in vascular cells, ACTRIIA-Fc attenuated proliferation of pulmonary arterial smooth muscle cells and pulmonary microvascular endothelial cells. In several experimental models of PH, prophylactic administration of ACTRIIA-Fc markedly improved hemodynamics, right ventricular (RV) hypertrophy, RV function, and arteriolar remodeling. When administered after the establishment of hemodynamically severe PH in a vasculoproliferative model, ACTRIIA-Fc was more effective than vasodilator in attenuating PH and arteriolar remodeling. Potent antiremodeling effects of ACTRIIA-Fc were associated with inhibition of SMAD2/3 activation and downstream transcriptional activity, inhibition of proliferation, and enhancement of apoptosis in the vascular wall. ACTRIIA-Fc reveals an unexpectedly prominent role of GDF8, GDF11, and activin as drivers of pulmonary vascular disease and represents a therapeutic strategy for restoring the balance between SMAD1/5/9 and SMAD2/3 signaling in PAH.
Asunto(s)
Hipertensión Pulmonar , Activinas , Animales , Diferenciación Celular , Células Endoteliales , Hipertensión Pulmonar/tratamiento farmacológico , Transducción de SeñalRESUMEN
Osteogenesis imperfecta (OI) is a heritable connective tissue disorder primarily due to mutations in the type I collagen genes (COL1A1 and COL1A2), leading to compromised biomechanical integrity in type I collagen-containing tissues such as bone. Bone is inherently mechanosensitive and thus responds and adapts to external stimuli, such as muscle mass and contractile strength, to alter its mass and shape. Myostatin, a member of the TGF-ß superfamily, signals through activin receptor type IIB to negatively regulate muscle fiber growth. Because of the positive impact of myostatin deficiency on bone mass, we utilized a soluble activin receptor type IIB-mFc (sActRIIB-mFc) fusion protein in two molecularly distinct OI mouse models (G610C and oim) and evaluated their bone properties. Wild-type (WT), +/G610C, and oim/oim mice were treated from 2 to 4 months of age with either vehicle (Tris-buffered saline) or sActRIIB-mFc (10 mg/kg). Femurs of sActRIIB-mFc-treated mice exhibited increased trabecular bone volume regardless of genotype, whereas the cortical bone microarchitecture and biomechanical strength were only improved in WT and +/G610C mice. Dynamic histomorphometric analyses suggest the improved cortical bone geometry and biomechanical integrity reflect an anabolic effect due to increased mineral apposition and bone formation rates, whereas static histomorphometric analyses supported sActRIIB-mFc treatment also having an anti-catabolic impact with decreased osteoclast number per bone surface on trabecular bone regardless of sex and genotype. Together, our data suggest that sActRIIB-mFc may provide a new therapeutic direction to improve both bone and muscle properties in OI. © 2018 American Society for Bone and Mineral Research.
Asunto(s)
Receptores de Activinas Tipo II/uso terapéutico , Huesos/patología , Osteogénesis Imperfecta/tratamiento farmacológico , Osteogénesis Imperfecta/patología , Receptores de Activinas Tipo II/farmacología , Animales , Biomarcadores/sangre , Fenómenos Biomecánicos , Huesos/fisiopatología , Modelos Animales de Enfermedad , Femenino , Fémur/patología , Masculino , Ratones Endogámicos C57BL , Osteoblastos/metabolismo , Osteoblastos/patología , Osteoclastos/metabolismo , Osteocitos/metabolismo , Osteogénesis Imperfecta/sangre , Osteogénesis Imperfecta/fisiopatología , Fragmentos de Péptidos/sangre , Procolágeno/sangre , Proteínas Recombinantes de Fusión/farmacología , Proteínas Recombinantes de Fusión/uso terapéutico , Solubilidad , Torsión MecánicaRESUMEN
Epiregulin, an epidermal growth factor family member, acts as a local signal mediator and shows dual biological activity, stimulating the proliferation of fibroblasts, hepatocytes, smooth muscle cells, and keratinocytes while inhibiting the growth of several tumor-derived epithelial cell lines. The epiregulin gene (Ereg) is located on mouse chromosome 5 adjacent to three other epidermal growth factor family members, epigen, amphiregulin, and betacellulin. Gene targeting was used to insert a lacZ reporter into the mouse Ereg locus and to ablate its function. Although epiregulin is broadly expressed and regulated both spatially and temporally, Ereg null mice show no overt developmental defects, reproductive abnormalities, or altered liver regeneration. Additionally, in contrast to previous hypotheses, Ereg deficiency does not alter intestinal cancer susceptibility, as assayed in the ApcMin model, despite showing robust expression in developing tumors. However, Ereg null mice are highly susceptible to cancer-predisposing intestinal damage caused by oral administration of dextran sulfate sodium.
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Factor de Crecimiento Epidérmico/genética , Factor de Crecimiento Epidérmico/metabolismo , Mucosa Intestinal/patología , Neoplasias Intestinales/metabolismo , Animales , Peso Corporal , Línea Celular , Colon/anatomía & histología , Colon/patología , Sulfato de Dextran/administración & dosificación , Sulfato de Dextran/farmacología , Modelos Animales de Enfermedad , Epirregulina , Células Epiteliales/citología , Células Epiteliales/metabolismo , Marcación de Gen , Genes Reporteros , Mucosa Intestinal/anatomía & histología , Mucosa Intestinal/efectos de los fármacos , Neoplasias Intestinales/patología , Regeneración Hepática/fisiología , Masculino , Ratones , Ratones Noqueados , Distribución TisularRESUMEN
Treatment of metastatic renal cell carcinoma (mRCC) with agents that block signaling through vascular endothelial growth factor receptor 2 (VEGFR2) induces disease regression or stabilization in some patients; however, these responses tend to be short-lived. Therefore, development of combination therapies that can extend the efficacy of VEGFR antagonists in mRCC remains a priority.We studied murine xenograft models of RCC that become refractory to treatment with the VEGFR tyrosine kinase inhibitor (TKI) sunitinib. Dalantercept is a novel antagonist of Activin receptor-like kinase 1 (ALK1)/Bone morphogenetic protein (BMP) 9 signaling. Dalantercept inhibited growth in the murine A498 xenograft model which correlated with hyperdilation of the tumor vasculature and an increase in tumor hypoxia. When combined with sunitinib, dalantercept induced tumor necrosis and prevented tumor regrowth and revascularization typically seen with sunitinib monotherapy in two RCC models. Combination therapy led to significant downregulation of angiogenic genes as well as downregulation of endothelial specific gene expression particularly of the Notch signaling pathway. We demonstrate that simultaneous targeting of molecules that control distinct phases of angiogenesis, such as ALK1 and VEGFR, is a valid strategy for treatment of mRCC. At the molecular level, combination therapy leads to downregulation of Notch signaling.
Asunto(s)
Receptores de Activinas Tipo II/antagonistas & inhibidores , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Carcinoma de Células Renales/tratamiento farmacológico , Neoplasias Renales/tratamiento farmacológico , Neovascularización Patológica/prevención & control , Receptores de Factores de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Receptores de Activinas Tipo II/administración & dosificación , Receptores de Activinas Tipo II/genética , Receptores de Activinas Tipo II/metabolismo , Animales , Axitinib , Carcinoma de Células Renales/irrigación sanguínea , Carcinoma de Células Renales/genética , Línea Celular Tumoral , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Imidazoles/administración & dosificación , Fragmentos Fc de Inmunoglobulinas/administración & dosificación , Indazoles/administración & dosificación , Indoles/administración & dosificación , Neoplasias Renales/irrigación sanguínea , Neoplasias Renales/genética , Ratones Desnudos , Neovascularización Patológica/genética , Neovascularización Patológica/metabolismo , Inhibidores de Proteínas Quinasas/administración & dosificación , Pirroles/administración & dosificación , Receptores de Factores de Crecimiento Endotelial Vascular/genética , Receptores de Factores de Crecimiento Endotelial Vascular/metabolismo , Proteínas Recombinantes de Fusión/administración & dosificación , Sunitinib , Carga Tumoral/efectos de los fármacos , Carga Tumoral/genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The azoxymethane (AOM) model has been widely used to investigate the pathology and genetics of colorectal cancer in rodents. However, there has been wide variation in treatment regimes, making it difficult to compare across studies. Consequently, standardizing AOM treatment and identifying sources of experimental variation would allow better comparisons across studies. In order to establish an optimal dosing regime for detecting experiment-dependent differences in tumorigenesis, we performed a dose curve analysis using AKR/J, SWR/J, and A/J mouse strains previously reported to vary widely in susceptibility to AOM. Although intraperitoneal or subcutaneous administration, but not in utero exposure, resulted in similar levels of tumor induction, significant dose- and strain-dependent effects of AOM were observed. No sex-dependent differences were observed. Increasing the number of treatments uncovered a significant strain-dependent effect on tumor promotion, independent of susceptibility to tumor initiation. Similarly, we used C57BL/6J and DBA/2J intercrosses to demonstrate that small diet modifications can significantly alter AOM-induced tumorigenesis in a background-dependent manner. These results provide experimental support for a standardized AOM treatment and for the importance of controlling both genetic and non-genetic factors when using this model.