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The TGF-ß superfamily plays a crucial role in regulating biological processes of virtually every tissue and system in the body, including hemostasis and hematopoiesis. Elritercept (KER-050) is an investigational, modified activin receptor type IIA ligand trap designed to bind and inhibit activin A and other select TGF-ß superfamily ligands including activin B and growth differentiation factor (GDF)-8 and GDF-11. The objectives of this Phase 1 randomized, placebo-controlled study of elritercept were to evaluate safety, tolerability, pharmacokinetics, and pharmacodynamic markers of activin inhibition and hematopoiesis in healthy, post-menopausal women (N=48). This study was comprised of 2 parts: single ascending doses ranging from 0.05 to 4.5 mg/kg, and multiple (up to 2 doses) ascending doses of 0.75 mg/kg administered subcutaneously (SC) every 4 weeks. Elritercept was generally well-tolerated at all dose levels, with no dose limiting toxicities observed. There were no severe or serious adverse events nor clinically significant changes in safety laboratory measures. Serum concentrations increased in dose-proportional manner following single SC doses, with peak concentrations achieved in 4.5 to 6 days and a mean elimination half-life of 12 days. These parameters were comparable following multiple doses. Elritercept elicited rapid, sustained, and dose-dependent increases in reticulocytes, red blood cells, hemoglobin, and platelets without eliciting detrimental changes in white blood cells such as neutrophils and lymphocytes. The time course and duration of changes in these cell populations supported a differentiated pharmacologic profile that is consistent with stimulation of both early- and late-stage hematologic pathways. Australian New Zealand Clinical Trial Registry (ACTRN12619000318189).
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Degenerative myopathies typically display a decline in satellite cells coupled with a replacement of muscle fibers by fat and fibrosis. During this pathological remodeling, satellite cells are present at lower numbers and do not display a proper regenerative function. Whether a decline in satellite cells directly contributes to disease progression or is a secondary result is unknown. In order to dissect these processes, we used a genetic model to reduce the satellite cell population by ~70-80% which leads to a nearly complete loss of regenerative potential. We observe that while no overt tissue damage is observed following satellite cell depletion, muscle fibers atrophy accompanied by changes in the stem cell niche cellular composition. Treatment of these mice with an Activin receptor type-2B (AcvR2B) pathway blocker reverses muscle fiber atrophy as expected, but also restores regenerative potential of the remaining satellite cells. These findings demonstrate that in addition to controlling fiber size, the AcvR2B pathway acts to regulate the muscle stem cell niche providing a more favorable environment for muscle regeneration.
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Exercise training benefits many organ systems and offers protection against metabolic disorders such as obesity and diabetes. Using the recently identified isoform of PGC1-α (PGC1-α4) as a discovery tool, we report the identification of meteorin-like (Metrnl), a circulating factor that is induced in muscle after exercise and in adipose tissue upon cold exposure. Increasing circulating levels of Metrnl stimulates energy expenditure and improves glucose tolerance and the expression of genes associated with beige fat thermogenesis and anti-inflammatory cytokines. Metrnl stimulates an eosinophil-dependent increase in IL-4 expression and promotes alternative activation of adipose tissue macrophages, which are required for the increased expression of the thermogenic and anti-inflammatory gene programs in fat. Importantly, blocking Metrnl actions in vivo significantly attenuates chronic cold-exposure-induced alternative macrophage activation and thermogenic gene responses. Thus, Metrnl links host-adaptive responses to the regulation of energy homeostasis and tissue inflammation and has therapeutic potential for metabolic and inflammatory diseases.
Assuntos
Tecido Adiposo Marrom/metabolismo , Fatores de Crescimento Neural/metabolismo , Animais , Glucose/metabolismo , Interleucina-13/metabolismo , Interleucina-4/metabolismo , Fígado/metabolismo , Macrófagos/metabolismo , Camundongos , Camundongos Transgênicos , Músculo Esquelético/metabolismo , Fatores de Crescimento Neural/genética , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo , Termogênese , Fatores de Transcrição/genéticaRESUMO
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.
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Receptores de Activinas Tipo II , Anemia/sangue , Proteínas Morfogenéticas Ósseas/efeitos dos fármacos , Células Precursoras Eritroides/efeitos dos fármacos , Eritropoese/efeitos dos fármacos , Fatores de Diferenciação de Crescimento/efeitos dos fármacos , Hematínicos/farmacologia , Síndromes Mielodisplásicas/sangue , Proteínas Recombinantes de Fusão/farmacologia , Animais , Proteínas Morfogenéticas Ósseas/antagonistas & inibidores , Modelos Animais de Doenças , Quimioterapia Combinada , Contagem de Eritrócitos , Eritropoetina/farmacologia , Fatores de Diferenciação de Crescimento/antagonistas & inibidores , Haplorrinos , Humanos , Ligantes , Camundongos , Ratos , Contagem de Reticulócitos , Transdução de Sinais/efeitos dos fármacos , Proteína Smad2/efeitos dos fármacos , Proteína Smad3/efeitos dos fármacosRESUMO
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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Doenças Ósseas Metabólicas/prevenção & controle , Receptores de Proteínas Morfogenéticas Ósseas Tipo I/metabolismo , Osso e Ossos/efeitos dos fármacos , Osteogênese/efeitos dos fármacos , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes de Fusão/farmacologia , Transdução de Sinais/efeitos dos fármacos , Análise de Variância , Animais , Western Blotting , Densidade Óssea/efeitos dos fármacos , Proteína Morfogenética Óssea 2/metabolismo , Proteína Morfogenética Óssea 4/metabolismo , Reabsorção Óssea/tratamento farmacológico , Osso e Ossos/anatomia & histologia , Osso e Ossos/fisiologia , Cromatografia em Gel , Clonagem Molecular , Densitometria , Eletroforese em Gel de Poliacrilamida , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Luciferases , Camundongos , Camundongos Endogâmicos C57BL , Osteoclastos/efeitos dos fármacos , Osteoclastos/fisiologia , Osteoprotegerina/metabolismo , Reação em Cadeia da Polimerase , Ligante RANK/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Proteínas Recombinantes de Fusão/administração & dosagem , Transdução de Sinais/fisiologiaRESUMO
Activin receptor-like kinase 1 (ALK1), an endothelial cell-specific type I receptor of the TGF-ß superfamily, is an important regulator of normal blood vessel development as well as pathological tumor angiogenesis. As such, ALK1 is an important therapeutic target. Thus, several ALK1-directed agents are currently in clinical trials as anti-angiogenic cancer therapeutics. Given the biological and clinical importance of the ALK1 signaling pathway, we sought to elucidate the biophysical and structural basis underlying ALK1 signaling. The TGF-ß family ligands BMP9 and BMP10 as well as the three type II TGF-ß family receptors ActRIIA, ActRIIB, and BMPRII have been implicated in ALK1 signaling. Here, we provide a kinetic and thermodynamic analysis of BMP9 and BMP10 interactions with ALK1 and type II receptors. Our data show that BMP9 displays a significant discrimination in type II receptor binding, whereas BMP10 does not. We also report the crystal structure of a fully assembled ternary complex of BMP9 with the extracellular domains of ALK1 and ActRIIB. The structure reveals that the high specificity of ALK1 for BMP9/10 is determined by a novel orientation of ALK1 with respect to BMP9, which leads to a unique set of receptor-ligand interactions. In addition, the structure explains how BMP9 discriminates between low and high affinity type II receptors. Taken together, our findings provide structural and mechanistic insights into ALK1 signaling that could serve as a basis for novel anti-angiogenic therapies.
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Receptores de Activinas Tipo II/química , Proteínas Morfogenéticas Ósseas/química , Fatores de Diferenciação de Crescimento/química , Receptores de Activinas Tipo II/genética , Receptores de Activinas Tipo II/metabolismo , Inibidores da Angiogênese/química , Inibidores da Angiogênese/uso terapêutico , Proteínas Morfogenéticas Ósseas/genética , Proteínas Morfogenéticas Ósseas/metabolismo , Cristalografia por Raios X , Fator 2 de Diferenciação de Crescimento , Fatores de Diferenciação de Crescimento/genética , Fatores de Diferenciação de Crescimento/metabolismo , Células HEK293 , Humanos , Neovascularização Patológica/tratamento farmacológico , Neovascularização Patológica/genética , Neovascularização Patológica/metabolismo , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genéticaRESUMO
Obesity results from disproportionately high energy intake relative to energy expenditure. Many therapeutic strategies have focused on the intake side of the equation, including pharmaceutical targeting of appetite and digestion. An alternative approach is to increase energy expenditure through physical activity or adaptive thermogenesis. A pharmacological way to increase muscle mass and hence exercise capacity is through inhibition of the activin receptor type IIB (ActRIIB). Muscle mass and strength is regulated, at least in part, by growth factors that signal via ActRIIB. Administration of a soluble ActRIIB protein comprised of a form of the extracellular domain of ActRIIB fused to a human Fc (ActRIIB-Fc) results in a substantial muscle mass increase in normal mice. However, ActRIIB is also present on and mediates the action of growth factors in adipose tissue, although the function of this system is poorly understood. In the current study, we report the effect of ActRIIB-Fc to suppress diet-induced obesity and linked metabolic dysfunctions in mice fed a high-fat diet. ActRIIB-Fc induced a brown fat-like thermogenic gene program in epididymal white fat, as shown by robustly increased expression of the thermogenic genes uncoupling protein 1 and peroxisomal proliferator-activated receptor-γ coactivator 1α. Finally, we identified multiple ligands capable of reducing thermogenesis that represent likely target ligands for the ActRIIB-Fc effects on the white fat depots. These data demonstrate that novel therapeutic ActRIIB-Fc improves obesity and obesity-linked metabolic disease by both increasing skeletal muscle mass and by inducing a gene program of thermogenesis in the white adipose tissues.
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Receptores de Activinas Tipo II/metabolismo , Obesidade/metabolismo , Transativadores/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Animais , Ensaio de Imunoadsorção Enzimática/métodos , Perfilação da Expressão Gênica , Humanos , Imuno-Histoquímica/métodos , Ligantes , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Músculo Esquelético/citologia , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo , Receptores Ativados por Proliferador de Peroxissomo , Ressonância de Plasmônio de Superfície , Termogênese , Tomografia Computadorizada por Raios X/métodos , Fatores de TranscriçãoRESUMO
OBJECTIVE: The expression of bone morphogenetic proteins (BMPs) is enhanced in human atherosclerotic and calcific vascular lesions. Although genetic gain- and loss-of-function experiments in mice have supported a causal role of BMP signaling in atherosclerosis and vascular calcification, it remains uncertain whether BMP signaling might be targeted pharmacologically to ameliorate both of these processes. METHODS AND RESULTS: We tested the impact of pharmacological BMP inhibition on atherosclerosis and calcification in LDL receptor-deficient (LDLR-/-) mice. LDLR-/- mice fed a high-fat diet developed abundant vascular calcification within 20 weeks. Prolonged treatment of LDLR-/- mice with the small molecule BMP inhibitor LDN-193189 was well-tolerated and potently inhibited development of atheroma, as well as associated vascular inflammation, osteogenic activity, and calcification. Administration of recombinant BMP antagonist ALK3-Fc replicated the antiatherosclerotic and anti-inflammatory effects of LDN-193189. Treatment of human aortic endothelial cells with LDN-193189 or ALK3-Fc abrogated the production of reactive oxygen species induced by oxidized LDL, a known early event in atherogenesis. Unexpectedly, treatment of mice with LDN-193189 lowered LDL serum cholesterol by 35% and markedly decreased hepatosteatosis without inhibiting HMG-CoA reductase activity. Treatment with BMP2 increased, whereas LDN-193189 or ALK3-Fc inhibited apolipoprotein B100 secretion in HepG2 cells, suggesting that BMP signaling contributes to the regulation of cholesterol biosynthesis. CONCLUSION: These results definitively implicate BMP signaling in atherosclerosis and calcification, while uncovering a previously unidentified role for BMP signaling in LDL cholesterol metabolism. BMP inhibition may be helpful in the treatment of atherosclerosis and associated vascular calcification.
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Aterosclerose/prevenção & controle , Proteínas Morfogenéticas Ósseas/antagonistas & inibidores , Fármacos Cardiovasculares/farmacologia , Pirazóis/farmacologia , Pirimidinas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Calcificação Vascular/prevenção & controle , Animais , Anti-Inflamatórios/farmacologia , Antioxidantes/farmacologia , Aterosclerose/etiologia , Aterosclerose/genética , Aterosclerose/metabolismo , Aterosclerose/patologia , Receptores de Proteínas Morfogenéticas Ósseas Tipo I/metabolismo , Proteínas Morfogenéticas Ósseas/metabolismo , LDL-Colesterol/sangue , Dieta Hiperlipídica , Modelos Animais de Doenças , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Fígado Gorduroso/etiologia , Fígado Gorduroso/metabolismo , Fígado Gorduroso/prevenção & controle , Feminino , Células Hep G2 , Humanos , Lipoproteínas LDL/metabolismo , Fígado/efeitos dos fármacos , Fígado/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Espécies Reativas de Oxigênio/metabolismo , Receptores de LDL/deficiência , Receptores de LDL/genética , Proteínas Recombinantes de Fusão/metabolismo , Fatores de Tempo , Calcificação Vascular/etiologia , Calcificação Vascular/genética , Calcificação Vascular/metabolismo , Calcificação Vascular/patologiaRESUMO
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.
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Inibidores da Angiogênese/farmacologia , Antígenos CD/farmacologia , Proteínas Morfogenéticas Ósseas/metabolismo , Fator 2 de Diferenciação de Crescimento/metabolismo , Fatores de Diferenciação de Crescimento/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/farmacologia , Neoplasias/tratamento farmacológico , Neovascularização Patológica/tratamento farmacológico , Inibidores da Angiogênese/genética , Inibidores da Angiogênese/metabolismo , Animais , Antígenos CD/genética , Antígenos CD/metabolismo , Sítios de Ligação , Receptores de Proteínas Morfogenéticas Ósseas Tipo II/genética , Receptores de Proteínas Morfogenéticas Ósseas Tipo II/metabolismo , Proteínas Morfogenéticas Ósseas/genética , Linhagem Celular , Endoglina , Fator 2 de Diferenciação de Crescimento/genética , Fatores de Diferenciação de Crescimento/genética , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Camundongos , Neoplasias/genética , Neoplasias/metabolismo , Neovascularização Patológica/genética , Neovascularização Patológica/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismoRESUMO
INTRODUCTION: In this study we investigated the action of RAP-031, a soluble activin receptor type IIB (ActRIIB) comprised of a form of the ActRIIB extracellular domain linked to a murine Fc, and the NF-κB inhibitor, ursodeoxycholic acid (UDCA), on the whole body strength of mdx mice. METHODS: The whole body tension (WBT) method of assessing the forward pulling tension (FPT) exerted by dystrophic (mdx) mice was used. RESULTS: RAP-031 produced a 41% increase in body mass and a 42.5% increase in FPT without altering the FPT normalized for body mass (WBT). Coadministration of RAP-031 with UDCA produced increases in FPT that were associated with an increase in WBT. CONCLUSIONS: Myostatin inhibition increases muscle mass without altering the fundamental weakness characteristic of dystrophic muscle. Cotreatment with an NF-κB inhibitor potentiates the effects of myostatin inhibition in improving FPT in mdx mice.
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Receptores de Activinas Tipo II/fisiologia , Tono Muscular/fisiologia , Músculo Esquelético/fisiologia , Distrofia Muscular Animal/fisiopatologia , Receptores de Activinas Tipo II/farmacologia , Animais , Feminino , Masculino , Camundongos , Camundongos Endogâmicos mdx , Contração Muscular/efeitos dos fármacos , Contração Muscular/fisiologia , Tono Muscular/efeitos dos fármacos , Músculo Esquelético/efeitos dos fármacos , Distrofia Muscular Animal/genética , SolubilidadeRESUMO
The activin receptor type IIB (ActRIIB) is a transmembrane receptor for transforming growth factor-ß superfamily members, including myostatin, that are involved in the negative regulation of skeletal muscle mass. We tested the translational hypothesis that blocking ligand binding to ActRIIB for 12 weeks would stimulate skeletal muscle growth and improve muscle function in the mdx mouse. ActRIIB was targeted using a novel inhibitor comprised of the extracellular portion of the ActRIIB fused to the Fc portion of murine IgG (sActRIIB), at concentrations of 1.0 and 10.0 mg/kg(-1) body weight. After 12 weeks of treatment, the 10.0 mg/kg(-1) dose caused a 27% increase in body weight with a concomitant 33% increase in lean muscle mass. Absolute force production of the extensor digitorum longus muscle ex vivo was higher in mice after treatment with either dose of sActRIIB, and the specific force was significantly higher after the lower dose (1.0 mg/kg(-1)), indicating functional improvement in the muscle. Circulating creatine kinase levels were significantly lower in mice treated with sActRIIB, compared with control mice. These data show that targeting the ActRIIB improves skeletal muscle mass and functional strength in the mdx mouse model of DMD, providing a therapeutic rationale for use of this molecule in treating skeletal myopathies.
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Receptores de Activinas Tipo II/antagonistas & inibidores , Músculo Esquelético/patologia , Músculo Esquelético/fisiopatologia , Distrofia Muscular Animal/fisiopatologia , Recuperação de Função Fisiológica/fisiologia , Receptores de Activinas Tipo II/metabolismo , Animais , Fenômenos Biomecânicos , Peso Corporal , Creatina Quinase/sangue , Hidroxiprolina/metabolismo , Técnicas In Vitro , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos mdx , Contração Muscular/fisiologia , Distrofia Muscular Animal/sangue , Distrofia Muscular Animal/patologia , Tamanho do ÓrgãoRESUMO
Anemia of inflammation develops in settings of chronic inflammatory, infectious, or neoplastic disease. In this highly prevalent form of anemia, inflammatory cytokines, including IL-6, stimulate hepatic expression of hepcidin, which negatively regulates iron bioavailability by inactivating ferroportin. Hepcidin is transcriptionally regulated by IL-6 and bone morphogenetic protein (BMP) signaling. We hypothesized that inhibiting BMP signaling can reduce hepcidin expression and ameliorate hypoferremia and anemia associated with inflammation. In human hepatoma cells, IL-6-induced hepcidin expression, an effect that was inhibited by treatment with a BMP type I receptor inhibitor, LDN-193189, or BMP ligand antagonists noggin and ALK3-Fc. In zebrafish, the induction of hepcidin expression by transgenic expression of IL-6 was also reduced by LDN-193189. In mice, treatment with IL-6 or turpentine increased hepcidin expression and reduced serum iron, effects that were inhibited by LDN-193189 or ALK3-Fc. Chronic turpentine treatment led to microcytic anemia, which was prevented by concurrent administration of LDN-193189 or attenuated when LDN-193189 was administered after anemia was established. Our studies support the concept that BMP and IL-6 act together to regulate iron homeostasis and suggest that inhibition of BMP signaling may be an effective strategy for the treatment of anemia of inflammation.
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Anemia/etiologia , Anemia/prevenção & controle , Proteínas Morfogenéticas Ósseas/antagonistas & inibidores , Inflamação/complicações , Animais , Peptídeos Catiônicos Antimicrobianos/metabolismo , Receptores de Proteínas Morfogenéticas Ósseas Tipo I/antagonistas & inibidores , Proteínas de Transporte/farmacologia , Células-Tronco Hematopoéticas/efeitos dos fármacos , Células Hep G2 , Hepcidinas , Humanos , Interleucina-6/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Pirazóis/farmacologia , Pirimidinas/farmacologia , Proteínas Recombinantes/farmacologia , Transdução de Sinais/efeitos dos fármacos , Terebintina/toxicidade , Peixe-Zebra , Proteínas de Peixe-Zebra/metabolismoRESUMO
X-linked myotubular myopathy (XLMTM) is a congenital disorder caused by deficiency of the lipid phosphatase, myotubularin. Patients with XLMTM often have severe perinatal weakness that requires mechanical ventilation to prevent death from respiratory failure. Muscle biopsy specimens from patients with XLMTM exhibit small myofibers with central nuclei and central aggregations of organelles in many cells. It was postulated that therapeutically increasing muscle fiber size would cause symptomatic improvement in myotubularin deficiency. Recent studies have elucidated an important role for the activin-receptor type IIB (ActRIIB) in regulation of muscle growth and have demonstrated that ActRIIB inhibition results in significant muscle hypertrophy. To evaluate whether promoting muscle hypertrophy can attenuate symptoms resulting from myotubularin deficiency, the effect of ActRIIB-mFC treatment was determined in myotubularin-deficient (Mtm1δ4) mice. Compared with wild-type mice, untreated Mtm1δ4 mice have decreased body weight, skeletal muscle hypotrophy, and reduced survival. Treatment of Mtm1δ4 mice with ActRIIB-mFC produced a 17% extension of lifespan, with transient increases in weight, forelimb grip strength, and myofiber size. Pathologic analysis of Mtm1δ4 mice during treatment revealed that ActRIIB-mFC produced marked hypertrophy restricted to type 2b myofibers, which suggests that oxidative fibers in Mtm1δ4 animals are incapable of a hypertrophic response in this setting. These results support ActRIIB-mFC as an effective treatment for the weakness observed in myotubularin deficiency.
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Receptores de Activinas Tipo II/antagonistas & inibidores , Longevidade/fisiologia , Força Muscular/fisiologia , Proteínas Tirosina Fosfatases não Receptoras/deficiência , Receptores de Activinas Tipo II/metabolismo , Animais , Comportamento Animal/efeitos dos fármacos , Peso Corporal/efeitos dos fármacos , Membro Anterior/efeitos dos fármacos , Membro Anterior/fisiologia , Gravitação , Força da Mão/fisiologia , Longevidade/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Força Muscular/efeitos dos fármacos , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Músculo Esquelético/ultraestrutura , Miostatina/metabolismo , Proteínas Tirosina Fosfatases não Receptoras/metabolismo , Proteínas Recombinantes de Fusão/farmacologia , Análise de SobrevidaRESUMO
Cancers that grow in bone, such as myeloma and breast cancer metastases, cause devastating osteolytic bone destruction. These cancers hijack bone remodeling by stimulating osteoclastic bone resorption and suppressing bone formation. Currently, treatment is targeted primarily at blocking bone resorption, but this approach has achieved only limited success. Stimulating osteoblastic bone formation to promote repair is a novel alternative approach. We show that a soluble activin receptor type IIA fusion protein (ActRIIA.muFc) stimulates osteoblastogenesis (p < .01), promotes bone formation (p < .01) and increases bone mass in vivo (p < .001). We show that the development of osteolytic bone lesions in mice bearing murine myeloma cells is caused by both increased resorption (p < .05) and suppression of bone formation (p < .01). ActRIIA.muFc treatment stimulates osteoblastogenesis (p < .01), prevents myeloma-induced suppression of bone formation (p < .05), blocks the development of osteolytic bone lesions (p < .05), and increases survival (p < .05). We also show, in a murine model of breast cancer bone metastasis, that ActRIIA.muFc again prevents bone destruction (p < .001) and inhibits bone metastases (p < .05). These findings show that stimulating osteoblastic bone formation with ActRIIA.muFc blocks the formation of osteolytic bone lesions and bone metastases in models of myeloma and breast cancer and paves the way for new approaches to treating this debilitating aspect of cancer.
Assuntos
Ativinas/metabolismo , Neoplasias Ósseas/complicações , Reabsorção Óssea/etiologia , Reabsorção Óssea/prevenção & controle , Osteogênese , Transdução de Sinais , Animais , Neoplasias Ósseas/patologia , Neoplasias Ósseas/fisiopatologia , Neoplasias Ósseas/secundário , Reabsorção Óssea/patologia , Reabsorção Óssea/fisiopatologia , Calcificação Fisiológica/efeitos dos fármacos , Linhagem Celular Tumoral , Células HEK293 , Humanos , Camundongos , Mieloma Múltiplo/complicações , Mieloma Múltiplo/patologia , Mieloma Múltiplo/fisiopatologia , Transplante de Neoplasias , Tamanho do Órgão/efeitos dos fármacos , Osteoblastos/efeitos dos fármacos , Osteoblastos/patologia , Osteogênese/efeitos dos fármacos , Osteólise/sangue , Osteólise/complicações , Osteólise/fisiopatologia , Osteólise/prevenção & controle , Paraproteínas/metabolismo , Proteínas Recombinantes de Fusão/farmacologia , Transdução de Sinais/efeitos dos fármacos , Análise de Sobrevida , Carga Tumoral/efeitos dos fármacosRESUMO
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.
Assuntos
Receptores de Activinas Tipo II/farmacologia , Antagonistas de Androgênios/farmacologia , Composição Corporal/efeitos dos fármacos , Densidade Óssea/efeitos dos fármacos , Receptores de Activinas Tipo II/genética , Tecido Adiposo/efeitos dos fármacos , Tecido Adiposo/metabolismo , Análise de Variância , Animais , Peso Corporal/efeitos dos fármacos , Linhagem Celular , Humanos , Fragmentos Fc das Imunoglobulinas/genética , Imunoglobulina G/genética , Leptina/sangue , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/metabolismo , Obesidade/sangue , Obesidade/prevenção & controle , Orquiectomia , Distribuição Aleatória , Proteínas Recombinantes de Fusão/farmacologia , SolubilidadeRESUMO
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.
Assuntos
Receptores de Activinas Tipo II/metabolismo , Receptores de Activinas Tipo II/química , Receptores de Activinas Tipo II/genética , Animais , Sítios de Ligação , Células COS , Chlorocebus aethiops , DNA Complementar/genética , Genes Reporter , Humanos , Ligantes , Mutagênese , Proteínas Mutantes Quiméricas/química , Proteínas Mutantes Quiméricas/metabolismo , Miostatina/química , Miostatina/metabolismo , Plasmídeos/genética , Ativadores de Plasminogênio/química , Ativadores de Plasminogênio/metabolismo , Ligação Proteica , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismoRESUMO
Understanding the pathogenesis of cancer-related bone disease is crucial to the discovery of new therapies. Here we identify activin A, a TGF-beta family member, as a therapeutically amenable target exploited by multiple myeloma (MM) to alter its microenvironmental niche favoring osteolysis. Increased bone marrow plasma activin A levels were found in MM patients with osteolytic disease. MM cell engagement of marrow stromal cells enhanced activin A secretion via adhesion-mediated JNK activation. Activin A, in turn, inhibited osteoblast differentiation via SMAD2-dependent distal-less homeobox-5 down-regulation. Targeting activin A by a soluble decoy receptor reversed osteoblast inhibition, ameliorated MM bone disease, and inhibited tumor growth in an in vivo humanized MM model, setting the stage for testing in human clinical trials.
Assuntos
Ativinas/metabolismo , Mieloma Múltiplo/complicações , Osteólise/etiologia , Ativinas/antagonistas & inibidores , Animais , Células da Medula Óssea/metabolismo , Células da Medula Óssea/patologia , Diferenciação Celular , Linhagem Celular Tumoral , Regulação para Baixo , Ativação Enzimática , Proteínas de Homeodomínio/metabolismo , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Camundongos , Mieloma Múltiplo/enzimologia , Mieloma Múltiplo/patologia , Osteoblastos/patologia , Osteólise/patologia , Receptores de Superfície Celular/metabolismo , Proteína Smad2/metabolismo , Células Estromais/metabolismo , Células Estromais/patologiaRESUMO
Activin receptor-like kinase-1 (ALK1) is a type I, endothelial cell-specific member of the transforming growth factor-beta superfamily of receptors known to play an essential role in modulating angiogenesis and vessel maintenance. In the present study, we sought to examine the angiogenic and tumorigenic effects mediated upon the inhibition of ALK1 signaling using a soluble chimeric protein (ALK1-Fc). Of 29 transforming growth factor-beta-related ligands screened by surface plasmon resonance, only bone morphogenetic protein (BMP9) and BMP10 displayed high-affinity binding to ALK1-Fc. In cell-based assays, ALK1-Fc inhibited BMP9-mediated Id-1 expression in human umbilical vein endothelial cells and inhibited cord formation by these cells on a Matrigel substrate. In a chick chorioallantoic membrane assay, ALK1-Fc reduced vascular endothelial growth factor-, fibroblast growth factor-, and BMP10-mediated vessel formation. The growth of B16 melanoma explants was also inhibited significantly by ALK1-Fc in this assay. Finally, ALK1-Fc treatment reduced tumor burden in mice receiving orthotopic grafts of MCF7 mammary adenocarcinoma cells. These data show the efficacy of chimeric ALK1-Fc proteins in mitigating vessel formation and support the view that ALK1-Fc is a powerful antiangiogenic agent capable of blocking vascularization.
Assuntos
Receptores de Activinas Tipo II/metabolismo , Fragmentos Fc das Imunoglobulinas/metabolismo , Neoplasias/metabolismo , Neovascularização Patológica , Proteínas Recombinantes de Fusão/metabolismo , Animais , Proteínas Morfogenéticas Ósseas/metabolismo , Células CHO , Cricetinae , Cricetulus , Células Endoteliais/citologia , Endotélio Vascular/citologia , Fator 2 de Diferenciação de Crescimento/metabolismo , Humanos , Camundongos , Ressonância de Plasmônio de Superfície , Telangiectasia Hemorrágica Hereditária/metabolismoRESUMO
Members of the transforming growth factor beta (TGF-beta) family have been genetically linked to vascular formation during embryogenesis. However, contradictory studies about the role of TGF-beta and other family members with reported vascular functions, such as bone morphogenetic protein (BMP) 9, in physiological and pathological angiogenesis make the need for mechanistic studies apparent. We demonstrate, by genetic and pharmacological means, that the TGF-beta and BMP9 receptor activin receptor-like kinase (ALK) 1 represents a new therapeutic target for tumor angiogenesis. Diminution of ALK1 gene dosage or systemic treatment with the ALK1-Fc fusion protein RAP-041 retarded tumor growth and progression by inhibition of angiogenesis in a transgenic mouse model of multistep tumorigenesis. Furthermore, RAP-041 significantly impaired the in vitro and in vivo angiogenic response toward vascular endothelial growth factor A and basic fibroblast growth factor. In seeking the mechanism for the observed effects, we uncovered an unexpected signaling synergy between TGF-beta and BMP9, through which the combined action of the two factors augmented the endothelial cell response to angiogenic stimuli. We delineate a decisive role for signaling by TGF-beta family members in tumor angiogenesis and offer mechanistic insight for the forthcoming clinical development of drugs blocking ALK1 in oncology.
Assuntos
Receptores de Activinas Tipo II , Receptores de Ativinas Tipo I , Células Endoteliais/metabolismo , Fragmentos Fc das Imunoglobulinas/farmacologia , Neoplasias Experimentais , Neovascularização Patológica , Proteínas Recombinantes de Fusão/farmacologia , Receptores de Ativinas Tipo I/genética , Receptores de Ativinas Tipo I/metabolismo , Receptores de Ativinas Tipo I/farmacologia , Receptores de Activinas Tipo II/genética , Receptores de Activinas Tipo II/metabolismo , Receptores de Activinas Tipo II/farmacologia , Animais , Linhagem Celular , Células Endoteliais/patologia , Fator 2 de Crescimento de Fibroblastos/genética , Fator 2 de Crescimento de Fibroblastos/metabolismo , Dosagem de Genes/genética , Fator 2 de Diferenciação de Crescimento/genética , Fator 2 de Diferenciação de Crescimento/metabolismo , Fatores de Diferenciação de Crescimento/genética , Fatores de Diferenciação de Crescimento/metabolismo , Humanos , Fragmentos Fc das Imunoglobulinas/genética , Camundongos , Camundongos Transgênicos , Neoplasias Experimentais/tratamento farmacológico , Neoplasias Experimentais/genética , Neoplasias Experimentais/metabolismo , Neovascularização Patológica/tratamento farmacológico , Neovascularização Patológica/genética , Neovascularização Patológica/metabolismo , Neovascularização Patológica/patologia , Proteínas Recombinantes de Fusão/genética , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Fator de Crescimento Transformador beta , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
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.