RESUMO
Phenotypic drug discovery (PDD) continues to fuel the research and development pipelines with first-in-class therapeutic modalities, but success rates critically depend on the quality of the underlying model system. Here, we employed a stem cell-based approach for the target-agnostic, yet pathway-centric discovery of small-molecule cytokine signaling activators to act as morphogens during development and regeneration. Unbiased screening identified triazolo[1,5-c]quinazolines as a new-in-class in vitro and in vivo active amplifier of the bone morphogenetic protein (BMP) pathway. Cellular BMP outputs were stimulated via enhanced and sustained availability of BMP-Smad proteins, strictly dependent on a minimal BMP input. Holistic target deconvolution unveiled a unique mechanism of dual targeting of casein kinase 1 and phosphatidyl inositol 3-kinase isoforms as key effectors for efficient amplification of osteogenic BMP signaling. This work underscores the asset of PDD to discover unrecognized polypharmacology signatures, in this case significantly expanding the chemical and druggable space of BMP modulators.
Assuntos
Proteínas Morfogenéticas Ósseas , Quinazolinas , Triazóis , Proteína Morfogenética Óssea 2/metabolismo , Proteínas Morfogenéticas Ósseas/efeitos dos fármacos , Proteínas Morfogenéticas Ósseas/metabolismo , Diferenciação Celular , Osteogênese , Quinazolinas/farmacologia , Proteínas Smad/metabolismo , Triazóis/farmacologiaRESUMO
Currently, no effective therapies exist for fibrodysplasia ossificans progressiva (FOP), a rare congenital syndrome in which heterotopic bone is formed in soft tissues owing to dysregulated activity of the bone morphogenetic protein (BMP) receptor kinase ALK2 (also known as ACVR1). From a screen of known biologically active compounds, we identified saracatinib as a potent ALK2 kinase inhibitor. In enzymatic and cell-based assays, saracatinib preferentially inhibited ALK2, compared with other receptors of the BMP/TGF-ß signaling pathway, and induced dorsalization in zebrafish embryos consistent with BMP antagonism. We further tested the efficacy of saracatinib using an inducible ACVR1Q207D-transgenic mouse line, which provides a model of heterotopic ossification (HO), as well as an inducible ACVR1R206H-knockin mouse, which serves as a genetically and physiologically faithful FOP model. In both models, saracatinib was well tolerated and potently inhibited the development of HO, even when administered transiently following soft tissue injury. Together, these data suggest that saracatinib is an efficacious clinical candidate for repositioning in FOP treatment, offering an accelerated path to clinical proof-of-efficacy studies and potentially significant benefits to individuals with this devastating condition.
Assuntos
Receptores de Ativinas Tipo I/genética , Benzodioxóis/farmacologia , Proteínas Morfogenéticas Ósseas/efeitos dos fármacos , Músculos/efeitos dos fármacos , Miosite Ossificante/genética , Quinazolinas/farmacologia , Receptores de Ativinas Tipo I/antagonistas & inibidores , Animais , Benzodioxóis/uso terapêutico , Proteínas Morfogenéticas Ósseas/metabolismo , Avaliação Pré-Clínica de Medicamentos , Técnicas de Introdução de Genes , Camundongos , Camundongos Transgênicos , Músculos/metabolismo , Miosite Ossificante/metabolismo , Miosite Ossificante/patologia , Ossificação Heterotópica/genética , Ossificação Heterotópica/metabolismo , Ossificação Heterotópica/patologia , Quinazolinas/uso terapêutico , Peixe-ZebraRESUMO
GDF11 has been reported to play a critical role in rejuvenating hypertrophy heart, skeletal muscle, and blood vessel regeneration in aged mice. Whether GDF11 can regulate autophagy in cardiomyocytes remains largely unknown. Thus, the purpose of the present study was to investigate the effects of GDF11 on cardiomyocyte autophagy induced by hypoxia, in addition to the underlying mechanisms. By using the MTT assay, Flow cytometry assay, LIVE/DEAD® Viability/Cytotoxicity Kit Stains and TUNEL assay, we found that exogenous GDF11 decreased apoptosis caused by prolonged hypoxia in cardiomyocytes. The expression of GDF11 was decreased obviously both in the cardiac tissue of myocardial infarction mice and the hypoxia treated cardiomyocytes. Protein levels of cleaved caspase-3, p-AMPK, SQSTM1, LC3B-I/II and GDF11 were detected by western blot. Autophagosomes and autolysosomes were identified by confocal laser microscopy after transfecting with the mRFP-eGFP-LC3 plasmids. Antibody against GDF11 (anti-GDF11) was used to inhibit the function of GDF11. At the molecular level, exogenous GDF11 increased AMPK function and enhanced autophagy activity. Anti-GDF11 inhibited autophagy and aggravated hypoxia-induced apoptosis in cardiomyocytes. Thus, GDF11 might be a potential target for myocardial infarction therapy.
Assuntos
Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Proteínas Morfogenéticas Ósseas/genética , Hipóxia Celular/efeitos dos fármacos , Fatores de Diferenciação de Crescimento/genética , Miócitos Cardíacos/efeitos dos fármacos , Animais , Anticorpos Bloqueadores/farmacologia , Proteínas Morfogenéticas Ósseas/antagonistas & inibidores , Proteínas Morfogenéticas Ósseas/efeitos dos fármacos , Eletrocardiografia/efeitos dos fármacos , Fatores de Diferenciação de Crescimento/antagonistas & inibidores , Fatores de Diferenciação de Crescimento/efeitos dos fármacos , Lisossomos/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fagossomos/efeitos dos fármacos , Ratos , Ratos Sprague-DawleyRESUMO
OBJECTIVE: Obesity has increased to pandemic levels and enhanced understanding of adipose regulation is required for new treatment strategies. Although bone morphogenetic proteins (BMPs) influence adipogenesis, the effect of BMP antagonists such as Noggin is largely unknown. The aim of the study was to define the role of Noggin, an extracellular BMP inhibitor, in adipogenesis. METHODS: We generated adipose-derived progenitor cells and a mouse model with adipocyte-specific Noggin deletion using the AdiponectinCre transgenic mouse, and determined the adipose phenotype of Noggin-deficiency. RESULTS: Our studies showed that Noggin is expressed in progenitor cells but declines in adipocytes, possibly allowing for lipid accumulation. Correspondingly, adipocyte-specific Noggin deletion in vivo promoted age-related obesity in both genders with no change in food intake. Although the loss of Noggin caused white adipose tissue hypertrophy, and whitening and impaired function in brown adipose tissue in both genders, there were clear gender differences with the females being most affected. The females had suppressed expression of brown adipose markers and thermogenic genes including peroxisome proliferator activated receptor gamma coactivator 1 alpha (PGC1alpha) and uncoupling protein 1 (UCP1) as well as genes associated with adipogenesis and lipid metabolism. The males, on the other hand, had early changes in a few BAT markers and thermogenic genes, but the main changes were in the genes associated with adipogenesis and lipid metabolism. Further characterization revealed that both genders had reductions in VO2, VCO2, and RER, whereas females also had reduced heat production. Noggin was also reduced in diet-induced obesity in inbred mice consistent with the obesity phenotype of the Noggin-deficient mice. CONCLUSIONS: BMP signaling regulates female and male adipogenesis through different metabolic pathways. Modulation of adipose tissue metabolism by select BMP antagonists may be a strategy for long-term regulation of age-related weight gain and obesity.
Assuntos
Adipócitos/metabolismo , Adipogenia/fisiologia , Proteínas Morfogenéticas Ósseas/metabolismo , Proteínas de Transporte/metabolismo , Obesidade/metabolismo , Adipócitos/patologia , Adipogenia/genética , Tecido Adiposo/metabolismo , Tecido Adiposo Marrom/metabolismo , Tecido Adiposo Branco/metabolismo , Animais , Proteínas Morfogenéticas Ósseas/efeitos dos fármacos , Proteínas de Transporte/genética , Modelos Animais de Doenças , Ingestão de Alimentos , Feminino , Deleção de Genes , Regulação da Expressão Gênica , Estudos de Associação Genética , Produto da Acumulação Lipídica , Metabolismo dos Lipídeos , Masculino , Camundongos , Camundongos Obesos , Camundongos Transgênicos , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/genética , Transdução de Sinais/genética , Termogênese/genética , Termogênese/fisiologia , Transcriptoma , Proteína Desacopladora 1/genéticaRESUMO
Craniosynostosis is defined as congenital premature fusion of one or more cranial sutures. While the genetic basis for about 30% of cases is known, the causative genes for the diverse presentations of the remainder of cases are unknown. The recently discovered cranial suture stem cell population affords an opportunity to identify early signaling pathways that contribute to craniosynostosis. We previously demonstrated that enhanced BMP signaling in neural crest cells (caA3 mutants) leads to premature cranial suture fusion resulting in midline craniosynostosis. Since enhanced mTOR signaling in neural crest cells leads to craniofacial bone lesions, we investigated the extent to which mTOR signaling is involved in the pathogenesis of BMP-mediated craniosynostosis by affecting the suture stem cell population. Our results demonstrate a loss of suture stem cells in the caA3 mutant mice by the newborn stage. We have found increased activation of mTOR signaling in caA3 mutant mice during embryonic stages, but not at the newborn stage. Our study demonstrated that inhibition of mTOR signaling via rapamycin in a time specific manner partially rescued the loss of the suture stem cell population. This study provides insight into how enhanced BMP signaling regulates suture stem cells via mTOR activation.
Assuntos
Craniossinostoses/genética , Sirolimo/farmacologia , Serina-Treonina Quinases TOR/efeitos dos fármacos , Animais , Proteínas Morfogenéticas Ósseas/efeitos dos fármacos , Proteínas Morfogenéticas Ósseas/fisiologia , Suturas Cranianas/embriologia , Craniossinostoses/tratamento farmacológico , Modelos Animais de Doenças , Camundongos , Camundongos Endogâmicos C57BL , Crista Neural/metabolismo , Fenótipo , Transdução de Sinais/efeitos dos fármacos , Sirolimo/metabolismo , Crânio/embriologiaRESUMO
Psoralen and angelicin are two effective compounds isolated from psoraleae, a traditional Chinese medicine. They have a wide range of applications for bone disease treatment and immune modulation. In this study, we explored their new applications for the treatment of periodontal diseases. This study aimed to investigate the effects of psoralen and angelicin on Porphyromonas gingivalis growth and P. gingivalis-derived lipopolysaccharide (Pg-LPS)-induced inflammation, and further to evaluate their effects on osteogenesis. Finally, the effects of angelicin on a mouse model of periodontitis were also investigated. The results showed that psoralen and angelicin had beneficial dose-dependent effects regarding the inhibition of planktonic P. gingivalis and biofilms of P. gingivalis. There were no significant differences in the viability of monocyte-like THP-1 cells and human periodontal ligament cells (hPDLCs) treated with either psoralen or angelicin compared to the untreated control cells. Psoralen and angelicin also markedly decreased the mRNA expression and release of inflammatory cytokines (interleukin [IL]-1ß and IL-8) by THP-1 cells in a dose-dependent manner. They significantly enhanced the alkaline phosphatase (ALP) activity of hPDLCs and up-regulated the expression of osteogenic proteins (runt-related transcription factor 2 [RUNX2], distal-less homeobox 5 [DLX5], and osteopontin [OPN]). Angelicin significantly attenuated alveolar bone loss and inflammation response in the mice with periodontitis. In conclusion, our data demonstrated that psoralen and angelicin could inhibit the growth of planktonic P. gingivalis and P. gingivalis biofilm. It is also the first report on the anti-inflammatory effect of psoralen and angelicin against Pg-LPS. They also had an osteogenesis-potentiating effect on hPDLCs. The in vivo study also indicated the effect of angelicin regarding protection against periodontitis. Our study highlighted the potential ability of psoralen and angelicin to act as novel natural agents to prevent and treat periodontitis.
Assuntos
Antibacterianos/farmacologia , Anti-Inflamatórios/farmacologia , Ficusina/farmacologia , Furocumarinas/farmacologia , Osteogênese/efeitos dos fármacos , Periodontite/tratamento farmacológico , Periodontite/prevenção & controle , Fosfatase Alcalina/metabolismo , Perda do Osso Alveolar/diagnóstico por imagem , Perda do Osso Alveolar/patologia , Perda do Osso Alveolar/prevenção & controle , Animais , Biofilmes/efeitos dos fármacos , Proteínas Morfogenéticas Ósseas/efeitos dos fármacos , Proteínas Morfogenéticas Ósseas/genética , Sobrevivência Celular/efeitos dos fármacos , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Citocinas/metabolismo , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Combinação de Medicamentos , Ficusina/química , Furocumarinas/química , Expressão Gênica/efeitos dos fármacos , Proteínas de Homeodomínio/metabolismo , Humanos , Inflamação , Interleucina-1beta/metabolismo , Interleucina-8/metabolismo , Lipopolissacarídeos/efeitos adversos , Masculino , Medicina Tradicional Chinesa , Camundongos , Camundongos Endogâmicos C57BL , Testes de Sensibilidade Microbiana , Osteopontina/metabolismo , Doenças Periodontais/tratamento farmacológico , Ligamento Periodontal , Periodontite/diagnóstico por imagem , Periodontite/patologia , Porphyromonas gingivalis/efeitos dos fármacos , Porphyromonas gingivalis/crescimento & desenvolvimento , RNA Mensageiro/metabolismo , Células THP-1 , Fatores de Transcrição/metabolismo , Regulação para CimaRESUMO
OBJECTIVE: To evaluate the dose-response relationship of exercise loading in the cartilage-subchondral bone (SB) unit in surgically-induced post-traumatic osteoarthritis (PTOA) of the knee. DESIGN: Destabilized medial meniscus (DMM) surgery was performed on the right knee of 12-week-old male Wistar rats, and sham surgery was performed on the contralateral knee. Four weeks after the surgery, the animals were subjected to moderate (12 m/min) or intense (21 m/min) treadmill exercises for 30 min/day, 5 days/week for 4 weeks. PTOA development in articular cartilage and SB was examined using histological and immunohistochemical analyses, micro-computed tomography (micro-CT) analysis, and biomechanical testing at 8 weeks after surgery. Gremlin-1 was injected to determine the role of bone morphogenetic protein (BMP) signaling on PTOA development following moderate exercise. RESULTS: Moderate exercise increased BMP-2, BMP-4, BMP-6, BMP receptor 2, pSmad-5, and inhibitor of DNA binding protein-1 expression in the superficial zone chondrocytes and suppressed cartilage degeneration, osteophyte growth, SB damage, and osteoclast-mediated SB resorption. However, intense exercise had little effect on BMP expression and even caused progression of these osteoarthritis (OA) changes. Gremlin-1 injection following moderate exercise caused progression of the PTOA development down to the level of the non-exercise DMM-operated knee. CONCLUSIONS: Exercise regulated cartilage-SB PTOA development in DMM-operated knees in a dose-dependent manner. Our findings shed light on the important role of BMP expression in superficial zone chondrocytes in attenuation of PTOA development following physiological exercise loading. Further studies to support a mechanism by which BMPs would be beneficial in preventing PTOA progression are warranted.
Assuntos
Proteínas Morfogenéticas Ósseas/metabolismo , Cartilagem Articular/metabolismo , Condrócitos/metabolismo , Articulação do Joelho/metabolismo , Osteoartrite do Joelho/metabolismo , Condicionamento Físico Animal , Suporte de Carga , Animais , Proteína Morfogenética Óssea 2/efeitos dos fármacos , Proteína Morfogenética Óssea 2/metabolismo , Proteína Morfogenética Óssea 4/efeitos dos fármacos , Proteína Morfogenética Óssea 4/metabolismo , Proteína Morfogenética Óssea 6/efeitos dos fármacos , Proteína Morfogenética Óssea 6/metabolismo , Receptores de Proteínas Morfogenéticas Ósseas Tipo II/efeitos dos fármacos , Receptores de Proteínas Morfogenéticas Ósseas Tipo II/metabolismo , Proteínas Morfogenéticas Ósseas/efeitos dos fármacos , Cartilagem Articular/efeitos dos fármacos , Condrócitos/efeitos dos fármacos , Citocinas , Proteína 1 Inibidora de Diferenciação , Articulação do Joelho/efeitos dos fármacos , Masculino , Osteoartrite do Joelho/etiologia , Proteínas/farmacologia , Ratos , Ratos Wistar , Proteína Smad5/efeitos dos fármacos , Proteína Smad5/metabolismo , Lesões do Menisco Tibial/complicações , Lesões do Menisco Tibial/metabolismoRESUMO
Lack of bone formation-related health problems are a major problem for the aging population in the modern world. As a part of the ongoing trend of developing natural substances that attenuate osteoporotic bone loss conditions, dioxinodehydroeckol (DHE) from edible brown alga Ecklonia cava was tested for its effects on osteoblastogenic differentiation in MC3T3-E1 pre-osteoblasts. DHE was observed to successfully enhance osteoblast differentiation, as indicated by elevated cell proliferation, alkaline phosphatase activity, intracellular cell mineralization, along with raised levels of osteoblastogenesis indicators at the concentration of 20 µM. Results suggested a possible intervening of DHE on the bone morphogenetic protein (BMP) signaling pathway, according to elevated protein levels of BMP-2, collagen-I, and Smads. In addition, the presence of DHE was also able to raise the phosphorylated extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) levels which are also activated by the BMP signaling pathway. In conclusion, DHE is suggested to be a potential bioactive compound against bone loss that could enhance osteoblastogenesis with a suggested BMP pathway interaction.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Dioxinas/farmacologia , Osteoblastos/efeitos dos fármacos , Proteínas Smad/biossíntese , Proteínas Smad/genética , Células 3T3 , Fosfatase Alcalina/metabolismo , Animais , Proteína Morfogenética Óssea 2/biossíntese , Proteína Morfogenética Óssea 2/genética , Proteínas Morfogenéticas Ósseas/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Colágeno Tipo I/biossíntese , Colágeno Tipo I/genética , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Camundongos , Osteoporose/prevenção & controle , Phaeophyceae/química , RNA/biossíntese , RNA/genética , Transdução de Sinais/efeitos dos fármacosRESUMO
Bone differs from other tissues in its capacity to self-repair after a fracture. The low bone mass and structural deterioration of bone associated with osteoporosis increases the risk of fragility fracture compared with healthy individuals. The intention of this article is to review the complex process of fracture repair and essential requirements for a successful fracture healing response summarized as the "diamond concept" in terms of aging and osteoporosis. The current preclinical and clinical evidence for a beneficial or harmful influence of anti-osteoporosis medications such as bisphosphonates, parathyroid hormone (PTH), strontium ranelate and antibodies of Wnt-inhibiting signaling proteins on bone healing is presented and discussed. Literature suggests that there are no detrimental consequences of such therapeutics on fracture repair processes. Following a fragility fracture, it seems that early start of preventive anti-osteoporotic treatment right after surgery does not delay the union of the fracture, except perhaps in the case of very rigidly fixed fracture requiring direct bone healing. There is some promising experimental and clinical evidence for possible enhancement of the bone repair process via administration of systemic agents. Further well designed studies in humans are necessary to accumulate more evidence on the positive effects and to translate this knowledge into valid therapeutic applications.
Assuntos
Osso e Ossos/metabolismo , Difosfonatos/uso terapêutico , Osteoporose/prevenção & controle , Osteoporose/fisiopatologia , Fraturas por Osteoporose/prevenção & controle , Densidade Óssea/efeitos dos fármacos , Conservadores da Densidade Óssea/uso terapêutico , Proteínas Morfogenéticas Ósseas/efeitos dos fármacos , Osso e Ossos/efeitos dos fármacos , Calo Ósseo , Consolidação da Fratura/efeitos dos fármacos , Humanos , Fraturas por Osteoporose/metabolismo , Fraturas por Osteoporose/patologia , Hormônio Paratireóideo/metabolismoRESUMO
Development of anti-angiogenic therapy including the vascular endothelial growth factor (VEGF) antibodies and VEGF-tyrosine kinase receptors has been a major landmark in cancer therapy leading improvement in survival in several cancers. While anti-angiogenic therapy is effective in some settings, resistance often develops owing to evasive, alternative pathways. Novel targets for anti-angiogenic therapy are urgently required to provide treatment alternatives in patients whose tumors are unresponsive to approved anti-angiogenic agents; one such pathway is the bone morphogenetic proteins (BMP 9 and BMP 10) that activate the type I activin receptor-like kinase-1 (ALK1), which has been implicated in the development of functional vasculature. Dalantercept (ACE-041) is a novel anti-angiogenic agent, which is a soluble form of ALK1, and acts as a ligand trap for BMP 9 and BMP 10, inhibiting their interaction with ALK1, which further disrupts the process of vascular development. This review will discuss the preclinical and clinical development of dalantercept as a novel anti-angiogenic therapy in treating a variety of cancers and its distinct safety profile compared to other anti-VEGF agents. We will also discuss the ongoing and completed studies of dalantercept, including combination studies with other VEGF-directed therapies.
Assuntos
Receptores de Activinas Tipo II/antagonistas & inibidores , Receptores de Activinas Tipo II/farmacologia , Inibidores da Angiogênese/farmacologia , Proteínas Morfogenéticas Ósseas/metabolismo , Fragmentos Fc das Imunoglobulinas/farmacologia , Neoplasias/tratamento farmacológico , Proteínas Recombinantes de Fusão/farmacologia , Antineoplásicos/farmacologia , Proteínas Morfogenéticas Ósseas/efeitos dos fármacos , Ensaios Clínicos como Assunto , Fator 2 de Diferenciação de Crescimento , Fatores de Diferenciação de Crescimento/metabolismo , Humanos , Neoplasias/mortalidade , Transdução de Sinais/efeitos dos fármacos , Resultado do Tratamento , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidoresRESUMO
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.
Assuntos
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
Fatty acid metabolism plays an important role in brain development and function. Mutations in acyl-CoA synthetase long-chain family member 4 (ACSL4), which converts long-chain fatty acids to acyl-CoAs, result in nonsyndromic X-linked mental retardation. ACSL4 is highly expressed in the hippocampus, a structure critical for learning and memory. However, the underlying mechanism by which mutations of ACSL4 lead to mental retardation remains poorly understood. We report here that dAcsl, the Drosophila ortholog of ACSL4 and ACSL3, inhibits synaptic growth by attenuating BMP signaling, a major growth-promoting pathway at neuromuscular junction (NMJ) synapses. Specifically, dAcsl mutants exhibited NMJ overgrowth that was suppressed by reducing the doses of the BMP pathway components, accompanied by increased levels of activated BMP receptor Thickveins (Tkv) and phosphorylated mothers against decapentaplegic (Mad), the effector of the BMP signaling at NMJ terminals. In addition, Rab11, a small GTPase involved in endosomal recycling, was mislocalized in dAcsl mutant NMJs, and the membrane association of Rab11 was reduced in dAcsl mutant brains. Consistently, the BMP receptor Tkv accumulated in early endosomes but reduced in recycling endosomes in dAcsl mutant NMJs. dAcsl was also required for the recycling of photoreceptor rhodopsin in the eyes, implying a general role for dAcsl in regulating endocytic recycling of membrane receptors. Importantly, expression of human ACSL4 rescued the endocytic trafficking and NMJ phenotypes of dAcsl mutants. Together, our results reveal a novel mechanism whereby dAcsl facilitates Rab11-dependent receptor recycling and provide insights into the pathogenesis of ACSL4-related mental retardation.
Assuntos
Proteínas Morfogenéticas Ósseas/fisiologia , Coenzima A Ligases/farmacologia , Sinapses/efeitos dos fármacos , Vesículas Transportadoras/efeitos dos fármacos , Animais , Western Blotting , Proteínas Morfogenéticas Ósseas/efeitos dos fármacos , Drosophila , Proteínas de Drosophila/metabolismo , Processamento de Imagem Assistida por Computador , Imuno-Histoquímica , Microscopia Eletrônica , Músculos/metabolismo , Mutação/genética , Mutação/fisiologia , Junção Neuromuscular/efeitos dos fármacos , Células Fotorreceptoras de Invertebrados/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Receptores de Superfície Celular/metabolismo , Receptores Pré-Sinápticos/efeitos dos fármacos , Rodopsina/metabolismo , Transdução de Sinais/efeitos dos fármacos , Frações Subcelulares/metabolismo , Proteínas rab de Ligação ao GTP/metabolismoRESUMO
OBJECTIVE: Serum sclerostin levels have been reported to be inversely associated with serum 25OHD levels, but the effect of vitamin D and calcium supplementation on serum sclerostin levels is unknown. This study was carried out to determine whether vitamin D and calcium supplementation altered serum sclerostin levels in healthy older adults. DESIGN: We measured serum sclerostin levels at baseline and after 2 years in 279 men and women who participated in a placebo-controlled vitamin D (700âIU/day) and calcium (500âmg/day) intervention trial carried out in men and women aged ≥65 years. METHOD: Serum sclerostin levels were measured using the MesoScale Discovery chemiluminescence assay. RESULTS: In the men, sclerostin levels increased over 2 years by 4.11±1.81âng/l (13.1%) in the vitamin D plus calcium-supplemented group and decreased by 3.16±1.78âng/l (10.9%) in the placebo group (P=0.005 for difference in change). Adjustments for the season of measurement, baseline physical activity levels, baseline serum sclerostin levels, and total body bone mineral content did not substantially alter the changes. In the women, there was no significant group difference in change in serum sclerostin levels either before or after the above-mentioned adjustments. In both the sexes, vitamin D and calcium supplementation significantly increased serum ionized calcium levels and decreased parathyroid hormone levels. CONCLUSION: Men and women appear to have different serum sclerostin responses to vitamin D and calcium supplementation. The reason for this difference remains to be determined.
Assuntos
Proteínas Morfogenéticas Ósseas/efeitos dos fármacos , Cálcio/farmacologia , Suplementos Nutricionais , Marcadores Genéticos/efeitos dos fármacos , Vitamina D/farmacologia , Vitaminas/farmacologia , Proteínas Adaptadoras de Transdução de Sinal , Idoso , Cálcio/uso terapêutico , Feminino , Humanos , Estudos Longitudinais , Masculino , Osteoporose/prevenção & controle , Osteoporose Pós-Menopausa/prevenção & controle , Fatores Sexuais , Resultado do Tratamento , Vitamina D/uso terapêutico , Vitaminas/uso terapêuticoRESUMO
BACKGROUND: A number of medications are approved for treatment of osteoporosis. As mode of action usually is anti-catabolic/anti-resorptive or anabolic, it is of interest to know whether these drugs affect not only normal bone remodeling, but also fracture healing. OBJECTIVE: The purpose of this paper is to give a short overview of the potential effect of various anti-osteoporotic medication on fracture healing. METHODS: A narrative literature review was performed to describe the current knowledge. RESULTS: Anti-catabolic/anti-resorptive drugs: for bisphosphonates, the most common class of drugs in this group, experimental studies have shown a larger and stronger callus and delayed remodeling but no evidence of delayed healing. A human monoclonal antibody to RANKL is another anti-catabolic drug, with the only report to date showing enhanced healing in an animal model. Strontium ranelate is a drug where both anti-catabolic and a weak anabolic effect have been proposed, with experimental data ranging from no effect to significant increase in both callus volume and strength. Anabolic drugs: PTH has demonstrated accelerated healing of various experimental fractures and of distal radius and pelvic fractures in humans. While the exact mechanism is not fully understood, PTH results in increased recruitment and differentiation of chondrocytes and enhancement of endochondral ossification. A monoclonal antibody to block sclerostin is another potential anabolic pathway, where animal data have shown increase in bone mass and strength. The potential effect on fracture healing is yet to be studied. CONCLUSION: There are still large gaps in the understanding of the potential effect of anti-osteoporotic drugs on fracture healing, although based on present knowledge a recent or present fracture should not be considered as a contraindication to such treatment.
Assuntos
Conservadores da Densidade Óssea/uso terapêutico , Consolidação da Fratura/efeitos dos fármacos , Fraturas por Osteoporose/prevenção & controle , Animais , Densidade Óssea/efeitos dos fármacos , Proteínas Morfogenéticas Ósseas/efeitos dos fármacos , Remodelação Óssea , Osso e Ossos/metabolismo , Calo Ósseo , Difosfonatos/uso terapêutico , Humanos , Compostos Organometálicos , Fraturas por Osteoporose/fisiopatologia , Hormônio Paratireóideo/fisiologia , Ligante RANK , Tiofenos , Cicatrização/efeitos dos fármacosRESUMO
BACKGROUND: Although enamel matrix derivative (EMD) has demonstrated the ability to promote angiogenesis and osteogenesis both in vitro and in vivo, the specific elements within the EMD compound responsible for these effects remain unknown. METHODS: Nine different protein pools from a commercially produced EMD were collected based on molecular weight. Six of these pools, along with the complete EMD unfractionated compound and positive and negative controls, were tested for their ability to induce bone formation in a calvarial induction assay. Immunocytochemistry of phosphorylated SMAD1/5/8 (phospho-SMAD), osterix, and vascular endothelial growth factor A (VEGF-A) was carried out at selected time points. Finally, proteomic analysis was completed to determine the specific protein-peptide content of the various osteoinductive pools. RESULTS: One of the lower-molecular-weight pools tested, pool 7, showed bone induction responses significantly greater than those of the other pools and the complete EMD compound and was concentration dependent. Dynamic bone formation rate analysis demonstrated that pool 7 was optimally active at the 5- to 10-µg concentration. It was demonstrated that EMD and pool 7 induced phospho-SMAD, osterix, and VEGF-A, which is indicative of increased bone morphogenetic protein (BMP) signaling. Proteomic composition analysis demonstrated that pool 7 had the highest concentration of the biologically active amelogenin-leucine-rich amelogenin peptide and ameloblastin 17-kDa peptides. CONCLUSIONS: These studies demonstrate that the low-molecular-weight protein pools (7 to 17 kDa) within EMD have greater osteoinductive potential than the commercially available complete EMD compound and that the mechanism of action, in part, is through increased BMP signaling and increased osterix and VEGF-A. With this information, selected components of EMD can now be formulated for optimal osteo- and angio-genesis.
Assuntos
Proteínas do Esmalte Dentário/análise , Amelogenina/análise , Animais , Proteínas Morfogenéticas Ósseas/efeitos dos fármacos , Cromatografia em Gel , Cromatografia Líquida de Alta Pressão , Proteínas do Esmalte Dentário/fisiologia , Relação Dose-Resposta a Droga , Eletroforese em Gel de Poliacrilamida , Espectrometria de Massas , Camundongos , Modelos Animais , Peso Molecular , Osteogênese/efeitos dos fármacos , Osso Parietal/efeitos dos fármacos , Periósteo/efeitos dos fármacos , Proteoma/análise , Proteína Smad1/análise , Proteína Smad1/farmacologia , Proteína Smad5/análise , Proteína Smad5/farmacologia , Proteína Smad8/análise , Proteína Smad8/farmacologia , Fator de Transcrição Sp7 , Fatores de Transcrição/análise , Fatores de Transcrição/farmacologia , Fator A de Crescimento do Endotélio Vascular/análise , Fator A de Crescimento do Endotélio Vascular/farmacologiaRESUMO
INTRODUCTION: During root formation, Smad-4 plays a key role during the epithelial-mesenchymal interactions and the Hertwig's epithelial root sheath (HERS) apical proliferation. The root formation and eruption of rat molars is impeded by alendronate treatment due to the inhibition of bone resortion by this drug. The present study aimed to examine the structures affected in the developing root and immunodetect the presence of Smad-4 in rats treated with alendronate. METHODS: Newborn Wistar rats were daily injected 2.5 mg/kg alendronate (ALN) during 9, 12 and 30 days. The controls (CON) were injected with saline. The maxillae were fixed and embedded in paraffin or Spurr resin. Paraffin sections were incubated in Smad-4 antibody that was labelled with DAB. The ultrathin sections were examined in a transmission electron microscope. RESULTS: In ALN, a short portion of root dentine was formed; the epithelial diaphragm (ED) and the dental follicle (DF) were disorganized by the contact of bone trabeculae. The (CON) molar roots developed normally. Smad-4 labelling was detected in the cytoplasm of fibroblasts and cementoblasts adjacent to the cementum in CON; in ALN group, few ED cells presented weak immunolabelling. Ultrastructurally, the ED and DF appeared disrupted due to the presence of thin bone trabeculae between its cells. It resulted in the lack of apical proliferation of HERS and, consequently, arrest of root formation. CONCLUSION: The immunodetection of Smad-4 in the DF cells of ALN specimens indicates that the signalling for the differentiation of these cells into cementum-forming fibroblasts and cementoblasts occurs, despite the impairment of root elongation.
Assuntos
Alendronato/efeitos adversos , Reabsorção Óssea/etiologia , Cementogênese/efeitos dos fármacos , Cemento Dentário/efeitos dos fármacos , Dente Molar/efeitos dos fármacos , Proteína Smad4/análise , Raiz Dentária/efeitos dos fármacos , Animais , Proteínas Morfogenéticas Ósseas/efeitos dos fármacos , Proteínas Morfogenéticas Ósseas/metabolismo , Diferenciação Celular/efeitos dos fármacos , Imuno-Histoquímica , Marcação In Situ das Extremidades Cortadas , Microscopia Eletrônica , Dente Molar/citologia , Dente Molar/crescimento & desenvolvimento , Ratos , Ratos Wistar , Transdução de Sinais/efeitos dos fármacos , Proteína Smad4/imunologia , Raiz Dentária/citologia , Raiz Dentária/crescimento & desenvolvimento , Fator de Crescimento Transformador beta1/efeitos dos fármacos , Fator de Crescimento Transformador beta1/metabolismoRESUMO
UNLABELLED: We determined whether suppression of sclerostin levels by estrogen treatment was mediated by anti-resorptive effect. Raloxifene, but not bisphosphonates, suppressed circulating sclerostin concentration, suggesting that sclerostin may mediate the action of estrogen on bone metabolism, independently of their anti-resorptive effects. INTRODUCTION: Circulating sclerostin concentrations are higher in postmenopausal than in premenopausal women, and estrogen treatment suppresses sclerostin levels in both men and women. We determined whether anti-resorptives may suppress the circulating sclerostin levels. METHODS: We conducted a retrospective observational study. Eighty postmenopausal women were treated with raloxifene for 19.4 ± 7.7 months (n = 16), bisphosphonates for 19.2 ± 6.7 months (n = 32), or were untreated (n = 32) for 17.1 ± 4.6 months. Plasma sclerostin concentrations were measured before and after treatment. RESULTS: Plasma sclerostin levels after treatment were significantly lower in the raloxifene than in the control group (55.8 ± 23.4 pmol/l vs. 92.1 ± 50.4 pmol/l, p = 0.046), but were similar between the bisphosphonate and control groups. Relative to baseline, raloxifene treatment markedly reduced plasma sclerostin concentration (-40.7 ± 22.8%, p < 0.001), with respect to both control (-7.5 ± 29.1%) and bisphosphonate (-3.1 ± 35.2%) groups. Changes in bone-specific alkaline phosphatase and osteocalcin levels showed reverse associations with sclerostin concentration changes in the raloxifene (γ = -0.505, p = 0.017) and control (γ = -0.410, p = 0.020) groups. CONCLUSIONS: Raloxifene, but not bisphosphonates, significantly suppressed circulating sclerostin concentration, suggesting that sclerostin may mediate the action of estrogen on bone metabolism, independently of their anti-resorptive effects.
Assuntos
Conservadores da Densidade Óssea/farmacologia , Proteínas Morfogenéticas Ósseas/efeitos dos fármacos , Difosfonatos/farmacologia , Marcadores Genéticos/efeitos dos fármacos , Osteoporose Pós-Menopausa/sangue , Cloridrato de Raloxifeno/farmacologia , Proteínas Adaptadoras de Transdução de Sinal , Idoso , Fosfatase Alcalina/sangue , Biomarcadores/sangue , Densidade Óssea/efeitos dos fármacos , Conservadores da Densidade Óssea/administração & dosagem , Conservadores da Densidade Óssea/uso terapêutico , Proteínas Morfogenéticas Ósseas/sangue , Difosfonatos/administração & dosagem , Difosfonatos/uso terapêutico , Esquema de Medicação , Feminino , Humanos , Pessoa de Meia-Idade , Osteocalcina/sangue , Osteoporose Pós-Menopausa/tratamento farmacológico , Osteoporose Pós-Menopausa/fisiopatologia , Cloridrato de Raloxifeno/administração & dosagem , Cloridrato de Raloxifeno/uso terapêutico , Estudos Retrospectivos , Moduladores Seletivos de Receptor Estrogênico/administração & dosagem , Moduladores Seletivos de Receptor Estrogênico/farmacologia , Moduladores Seletivos de Receptor Estrogênico/uso terapêuticoRESUMO
UNLABELLED: Sclerostin is expressed by osteocytes and inhibits bone formation by osteoblasts. In this study, serum sclerostin was positively correlated with either lumbar spinal bone mineral density or T-score. Furthermore, serum sclerostin was increased after 6 months treatment with risedronate, whereas remained unchanged after 6 months teriparatide treatment. INTRODUCTION: The primary aim of this study was the evaluation of serum sclerostin levels in postmenopausal women and their association with bone mineral density (BMD) and bone turnover markers. The secondary aim was the evaluation of treatment with either teriparatide (TPTD) or risedronate (RIS) on serum sclerostin levels in women with postmenopausal osteoporosis. METHODS: Women with postmenopausal osteoporosis, assigned to receive either TPTD (TPTD group, n = 13) or RIS (RIS group, n = 36) for 6 months, and non-osteoporotic early postmenopausal women (NOEP group, n = 13) were recruited. Main outcome measure was serum sclerostin levels. RESULTS: Serum sclerostin was higher in the NOEP group at baseline compared with either TPTD group (p = 0.007) or RIS group (p = 0.049). Sclerostin was positively correlated with both lumbar spinal (LS) BMD (r = 0.353; p = 0.005) and T-score (r = 0.501; p < 0.001) and negatively correlated with intact parathyroid hormone (r = -0.343; p = 0.024) at baseline. Multiple regression analysis showed that either LS BMD (Beta = 0.653; p = 0.018) or T-score (Beta = 0.711; p = 0.005) were independent predictors of serum sclerostin levels. No significant correlation was observed between serum sclerostin and bone turnover markers or estradiol at baseline. Sclerostin was significantly increased 6 months post-treatment in RIS group (p = 0.002), whereas remained statistically unaffected in the TPTD group. CONCLUSIONS: Serum sclerostin is decreased in women with postmenopausal osteoporosis compared with non-osteoporotic early postmenopausal women and is positively correlated to either LS BMD or LS T-score. Furthermore, serum sclerostin was increased after 6 months treatment with RIS, whereas remained essentially unchanged after 6 months TPTD treatment.
Assuntos
Conservadores da Densidade Óssea/farmacologia , Densidade Óssea/fisiologia , Proteínas Morfogenéticas Ósseas/sangue , Vértebras Lombares/fisiopatologia , Osteoporose Pós-Menopausa/sangue , Proteínas Adaptadoras de Transdução de Sinal , Idoso , Idoso de 80 Anos ou mais , Biomarcadores/sangue , Densidade Óssea/efeitos dos fármacos , Conservadores da Densidade Óssea/uso terapêutico , Proteínas Morfogenéticas Ósseas/efeitos dos fármacos , Remodelação Óssea/efeitos dos fármacos , Remodelação Óssea/fisiologia , Ácido Etidrônico/análogos & derivados , Ácido Etidrônico/farmacologia , Ácido Etidrônico/uso terapêutico , Feminino , Marcadores Genéticos/efeitos dos fármacos , Humanos , Vértebras Lombares/efeitos dos fármacos , Pessoa de Meia-Idade , Osteoporose Pós-Menopausa/tratamento farmacológico , Pós-Menopausa/sangue , Estudos Retrospectivos , Ácido Risedrônico , Teriparatida/farmacologia , Teriparatida/uso terapêuticoRESUMO
OBJECTIVE: We recently reported that lowering of macrophage free intracellular iron increases expression of cholesterol efflux transporters ABCA1 and ABCG1 by reducing generation of reactive oxygen species. In this study, we explored whether reducing macrophage intracellular iron levels via pharmacological suppression of hepcidin can increase macrophage-specific expression of cholesterol efflux transporters and reduce atherosclerosis. METHODS AND RESULTS: To suppress hepcidin, increase expression of the iron exporter ferroportin, and reduce macrophage intracellular iron, we used a small molecule inhibitor of bone morphogenetic protein (BMP) signaling, LDN 193189 (LDN). LDN (10 mg/kg IP b.i.d.) was administered to mice, and its effects on atherosclerosis, intracellular iron, oxidative stress, lipid efflux, and foam cell formation were measured in plaques and peritoneal macrophages. Long-term LDN administration to apolipoprotein E-/- mice increased ABCA1 immunoreactivity within intraplaque macrophages by 3.7-fold (n=8; P=0.03), reduced Oil Red O-positive lipid area by 50% (n=8; P=0.02), and decreased total plaque area by 43% (n=8; P=0.001). LDN suppressed liver hepcidin transcription and increased macrophage ferroportin, lowering intracellular iron and hydrogen peroxide production. LDN treatment increased macrophage ABCA1 and ABCG1 expression, significantly raised cholesterol efflux to ApoA-1, and decreased foam cell formation. All preceding LDN-induced effects on cholesterol efflux were reversed by exogenous hepcidin administration, suggesting modulation of intracellular iron levels within macrophages as the mechanism by which LDN triggers these effects. CONCLUSIONS: These data suggest that pharmacological manipulation of iron homeostasis may be a promising target to increase macrophage reverse cholesterol transport and limit atherosclerosis.
Assuntos
Peptídeos Catiônicos Antimicrobianos/antagonistas & inibidores , Aterosclerose/prevenção & controle , Diferenciação Celular/efeitos dos fármacos , Colesterol/metabolismo , Células Espumosas/patologia , Macrófagos/metabolismo , Pirazóis/farmacologia , Pirimidinas/farmacologia , Transportador 1 de Cassete de Ligação de ATP , Membro 1 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Apolipoproteínas E/deficiência , Apolipoproteínas E/genética , Aterosclerose/metabolismo , Aterosclerose/fisiopatologia , Proteínas Morfogenéticas Ósseas/antagonistas & inibidores , Proteínas Morfogenéticas Ósseas/efeitos dos fármacos , Modelos Animais de Doenças , Hepcidinas , Ferro/metabolismo , Lipoproteínas/metabolismo , Macrófagos/efeitos dos fármacos , Macrófagos/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Transdução de Sinais/efeitos dos fármacosRESUMO
BACKGROUND AND PURPOSE: Sclerostin is produced by osteocytes and is an inhibitor of bone formation. Thus, inhibition of sclerostin by a monoclonal antibody increases bone formation and improves fracture repair. Sclerostin expression is upregulated in unloaded bone and is downregulated by loading. We wanted to determine whether an anti-sclerostin antibody would stimulate metaphyseal healing in unloaded bone in a rat model. METHODS: 10-week-old male rats (n = 48) were divided into 4 groups, with 12 in each. In 24 rats, the right hind limb was unloaded by paralyzing the calf and thigh muscles with an injection of botulinum toxin A (Botox). 3 days later, all the animals had a steel screw inserted into the right proximal tibia. Starting 3 days after screw insertion, either anti-sclerostin antibody (Scl-Ab) or saline was given twice weekly. The other 24 rats did not receive Botox injections and they were treated with Scl-Ab or saline to serve as normal-loaded controls. Screw pull-out force was measured 4 weeks after insertion, as an indicator of the regenerative response of bone to trauma. RESULTS: Unloading reduced the pull-out force. Scl-Ab treatment increased the pull-out force, with or without unloading. The response to the antibody was similar in both groups, and no statistically significant relationship was found between unloading and antibody treatment. The cancellous bone at a distance from the screw showed changes in bone volume fraction that followed the same pattern as the pull-out force. INTERPRETATION: Scl-Ab increases bone formation and screw fixation to a similar degree in loaded and unloaded bone.