RESUMO
The formation of multinucleated myofibers is essential for the growth of skeletal muscle. The nuclear factor of activated T cells (NFAT) promotes skeletal muscle growth. How NFAT responds to changes in extracellular cues to regulate skeletal muscle growth remains to be fully defined. In this study, we demonstrate that mice containing a skeletal muscle-specific deletion of the tyrosine phosphatase SHP-2 (muscle creatine kinase [MCK]-SHP-2 null) exhibited a reduction in both myofiber size and type I slow myofiber number. We found that interleukin-4, an NFAT-regulated cytokine known to stimulate myofiber growth, was reduced in its expression in skeletal muscles of MCK-SHP-2-null mice. When SHP-2 was deleted during the differentiation of primary myoblasts, NFAT transcriptional activity and myotube multinucleation were impaired. Finally, SHP-2 coupled myotube multinucleation to an integrin-dependent pathway and activated NFAT by stimulating c-Src. Thus, SHP-2 transduces extracellular matrix stimuli to intracellular signaling pathways to promote skeletal muscle growth.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Músculo Esquelético/crescimento & desenvolvimento , Fatores de Transcrição NFATC/metabolismo , Proteínas Tirosina Fosfatases/fisiologia , Transdução de Sinais , Animais , Animais Geneticamente Modificados , Diferenciação Celular , Creatina Quinase Forma MM/genética , Creatina Quinase Forma MM/metabolismo , Deleção de Genes , Regulação da Expressão Gênica , Genes src , Interleucina-4/genética , Interleucina-4/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Camundongos , Fibras Musculares Esqueléticas/citologia , Fibras Musculares Esqueléticas/fisiologia , Músculo Esquelético/citologia , Proteína Tirosina Fosfatase não Receptora Tipo 11 , Proteínas Tirosina Fosfatases/genéticaRESUMO
Mitogens activate cell signaling and gene expression cascades that culminate in expression of cyclin D1 during the G(0)-to-G(1) transition of the cell cycle. Using cell cycle arrest in response to oxidative stress, we have delineated a dynamic program of chromatin trafficking of c-Fos and Fra-1 required for cyclin D1 expression during cell cycle reentry. In serum-stimulated lung epithelial cells, c-Fos was expressed, recruited to chromatin, phosphorylated at extracellular signal-regulated kinase 1- and 2 (ERK1,2)-dependent sites, and degraded prior to prolonged recruitment of Fra-1 to chromatin. Immunostaining showed that expression of nuclear c-Fos and that of cyclin D1 are mutually exclusive, whereas nuclear Fra-1 and cyclin D1 are coexpressed as cells traverse G(1). Oxidative stress prolonged the accumulation of phospho-ERK1,2 and phospho-c-Fos on chromatin, inhibited entry of Fra-1 into the nucleus, and blocked cyclin D1 expression. After induction of the immediate-early gene response in the presence of oxidative stress, inhibition of ERK1,2 signaling promoted degradation of c-Fos, recruitment of Fra-1 to chromatin, and expression of cyclin D1. Our data indicate that termination of nuclear ERK1,2 signaling is required for an exchange of Fra-1 for c-Fos on chromatin and initiation of cyclin D1 expression at the G(0)-to-G(1) transition of the cell cycle.
Assuntos
Cromatina/metabolismo , Ciclina D1/biossíntese , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteínas Serina-Treonina Quinases , Proteínas Proto-Oncogênicas c-fos/metabolismo , Animais , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/fisiologia , Ciclina D1/genética , Inibidores Enzimáticos/farmacologia , Peróxido de Hidrogênio/farmacologia , Camundongos , Proteína Quinase 3 Ativada por Mitógeno , Molsidomina/análogos & derivados , Molsidomina/farmacologia , Oxidantes/farmacologia , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-aktRESUMO
One of the goals of the Critical Path Institute's Predictive Safety Testing Consortium (PSTC) is to promote best practices for evaluating novel markers of drug induced injury. This includes the use of sound statistical methods. For rat studies, these practices have centered around comparing the area under the receiver-operator characteristic curve for each novel injury biomarker to those for the standard markers. In addition, the PSTC has previously used the net reclassification index (NRI) and integrated discrimination index (IDI) to assess the increased certainty provided by each novel injury biomarker when added to the information already provided by the standard markers. Due to their relatively simple interpretations, NRI and IDI have generally been popular measures of predictive performance. However recent literature suggests that significance tests for NRI and IDI can have inflated false positive rates and thus, tests based on these metrics should not be relied upon. Instead, when parametric models are employed to assess the added predictive value of a new marker, following (Pepe, M. S., Kerr, K. F., Longton, G., and Wang, Z. (2013). Testing for improvement in prediction model performance. Stat. Med. 32, 1467-1482), the PSTC recommends that likelihood based methods be used for significance testing.
Assuntos
Biomarcadores/metabolismo , Avaliação Pré-Clínica de Medicamentos , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos/diagnóstico , Drogas em Investigação/efeitos adversos , Modelos Estatísticos , Testes de Toxicidade , Xenobióticos/toxicidade , Animais , Biomarcadores/sangue , Biomarcadores/urina , Avaliação Pré-Clínica de Medicamentos/tendências , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos/sangue , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos/metabolismo , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos/urina , Drogas em Investigação/classificação , Reações Falso-Positivas , Humanos , Doenças Musculares/induzido quimicamente , Doenças Musculares/diagnóstico , Doenças Musculares/metabolismo , Organizações sem Fins Lucrativos , Valor Preditivo dos Testes , Curva ROC , Insuficiência Renal/induzido quimicamente , Insuficiência Renal/diagnóstico , Insuficiência Renal/metabolismo , Testes de Toxicidade/tendências , Estados Unidos , Xenobióticos/classificaçãoRESUMO
BACKGROUND: The treatments currently approved for Duchenne muscular dystrophy (DMD), a progressive skeletal muscle wasting disease, address the needs of only a small proportion of patients resulting in an urgent need for therapies that benefit all patients regardless of the underlying mutation. Myostatin is a member of the transforming growth factor-ß (TGF-ß) family of ligands and is a negative regulator of skeletal muscle mass. Loss of myostatin has been shown to increase muscle mass and improve muscle function in both normal and dystrophic mice. Therefore, myostatin blockade via a specific antibody could ameliorate the muscle weakness in DMD patients by increasing skeletal muscle mass and function, thereby reducing patients' functional decline. METHODS: A murine anti-myostatin antibody, mRK35, and its humanized analog, domagrozumab, were developed and their ability to inhibit several TGB-ß ligands was measured using a cell-based Smad-activity reporter system. Normal and mdx mice were treated with mRK35 to examine the antibody's effect on body weight, lean mass, muscle weights, grip strength, ex vivo force production, and fiber size. The humanized analog (domagrozumab) was tested in non-human primates (NHPs) for changes in skeletal muscle mass and volume as well as target engagement via modulation of circulating myostatin. RESULTS: Both the murine and human antibodies are specific and potent inhibitors of myostatin and GDF11. mRK35 is able to increase body weight, lean mass, and muscle weights in normal mice. In mdx mice, mRK35 significantly increased body weight, muscle weights, grip strength, and ex vivo force production in the extensor digitorum longus (EDL) muscle. Further, tibialis anterior (TA) fiber size was significantly increased. NHPs treated with domagrozumab demonstrated a dose-dependent increase in lean mass and muscle volume and exhibited increased circulating levels of myostatin demonstrating target engagement. CONCLUSIONS: We demonstrated that the potent anti-myostatin antibody mRK35 and its clinical analog, domagrozumab, were able to induce muscle anabolic activity in both rodents, including the mdx mouse model of DMD, and non-human primates. A Phase 2, potentially registrational, clinical study with domagrozumab in DMD patients is currently underway.
Assuntos
Anticorpos/administração & dosagem , Contração Muscular , Força Muscular , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/fisiologia , Distrofia Muscular de Duchenne/tratamento farmacológico , Miostatina/imunologia , Animais , Células CHO , Cricetulus , Modelos Animais de Doenças , Humanos , Macaca fascicularis , Masculino , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos mdx , Distrofia Muscular de Duchenne/imunologia , Distrofia Muscular de Duchenne/fisiopatologia , Miostatina/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacosRESUMO
Myostatin is a highly conserved protein secreted primarily from skeletal muscle that can potently suppress muscle growth. This ability to regulate skeletal muscle mass has sparked intense interest in the development of anti-myostatin therapies for a wide array of muscle disorders including sarcopenia, cachexia and genetic neuromuscular diseases. While a number of studies have examined the circulating myostatin concentrations in healthy and sarcopenic populations, very little data are available from inherited muscle disease patients. Here, we have measured the myostatin concentration in serum from seven genetic neuromuscular disorder patient populations using immunoaffinity LC-MS/MS. Average serum concentrations of myostatin in all seven muscle disease patient groups were significantly less than those measured in healthy controls. Furthermore, circulating myostatin concentrations correlated with clinical measures of disease progression for five of the muscle disease patient populations. These findings greatly expand the understanding of myostatin in neuromuscular disease and suggest its potential utility as a biomarker of disease progression.
Assuntos
Miostatina/sangue , Doenças Neuromusculares/sangue , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Biomarcadores/sangue , Análise Química do Sangue , Criança , Pré-Escolar , Estudos de Coortes , Progressão da Doença , Feminino , Humanos , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Pessoa de Meia-Idade , Doenças Neuromusculares/genética , Adulto JovemRESUMO
NADPH oxidases produce reactive oxygen species (ROS) that serve as co-stimulatory signals for cell proliferation. In mouse lung epithelial cells that express Nox1, Nox2, Nox4, p22(phox), p47(phox), p67(phox), and Noxo1, overexpression of Nox1 delayed cell cycle withdrawal by maintaining AP-1-dependent expression of cyclin D1 in low serum conditions. In cycling cells, the effects of Nox1 were dose dependent: levels of Nox1 that induced 3- to 10-fold increases in ROS promoted phosphorylation of ERK1/2 and expression of cyclin D1, whereas expression of Nox1 with Noxo1 and Noxa1 (or expression of Nox4 alone) that induced substantial increases in intracellular ROS inhibited cyclin D1 and proliferation. Catalase reversed the effects of Nox1 on cyclin D1 and cell proliferation. Diphenylene iodonium, an inhibitor of NADPH oxidase activity, did not affect dosedependent responses of ERK1/2 or Akt to serum, but markedly inhibited the sequential expression of c-Fos and Fra-1 required for induction of cyclin D1 during cell cycle re-entry. These results indicate that Nox1 stimulates cell proliferation in actively cycling cells by reducing the requirement for growth factors to maintain expression of cyclin D1, whereas during cell cycle re-entry, NADPH oxidase activity is required for transcriptional activation of Fos family genes during the immediate early gene response.
Assuntos
Proliferação de Células , Ciclinas/metabolismo , Células Epiteliais/metabolismo , NADH NADPH Oxirredutases/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Proteínas Adaptadoras de Transporte Vesicular/genética , Animais , Ciclo Celular/efeitos dos fármacos , Linhagem Celular , Ciclina D , Ciclinas/genética , Inibidores Enzimáticos/farmacologia , Células Epiteliais/citologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Regulação da Expressão Gênica , Humanos , Peróxido de Hidrogênio/metabolismo , Peróxido de Hidrogênio/farmacologia , Pulmão/citologia , Camundongos , NADH NADPH Oxirredutases/antagonistas & inibidores , NADPH Oxidase 1 , NADPH Oxidases/genética , Oniocompostos/farmacologia , Oxirredução , Fosforilação , Regiões Promotoras Genéticas/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-fos/metabolismo , TransfecçãoRESUMO
Novel skeletal muscle (SKM) injury biomarkers that have recently been identified may outperform or add value to the conventional SKM injury biomarkers aspartate transaminase (AST) and creatine kinase (CK). The relative performance of these novel biomarkers of SKM injury including skeletal troponin I (sTnI), myosin light chain 3 (Myl3), CK M Isoform (Ckm), and fatty acid binding protein 3 (Fabp3) was assessed in 34 rat studies including both SKM toxicants and compounds with toxicities in tissues other than SKM. sTnI, Myl3, Ckm, and Fabp3 all outperformed CK or AST and/or added value for the diagnosis of drug-induced SKM injury (ie, myocyte degeneration/necrosis). In addition, when used in conjunction with CK and AST, sTnI, Myl3, CKm, and Fabp3 individually and collectively improved diagnostic sensitivity and specificity, as well as diagnostic certainty, for SKM injury and responded in a sensitive manner to low levels of SKM degeneration/necrosis in rats. These findings support the proposal that sTnI, Myl3, Ckm, and Fabp3 are suitable for voluntary use, in conjunction with CK and AST, in regulatory safety studies in rats to monitor drug-induced SKM injury and the potential translational use of these exploratory biomarkers in early clinical trials to ensure patient safety.
Assuntos
Biomarcadores/sangue , Músculo Esquelético/efeitos dos fármacos , Doenças Musculares/sangue , Doenças Musculares/induzido quimicamente , Animais , Creatina Quinase Forma MM/sangue , Relação Dose-Resposta a Droga , Proteína 3 Ligante de Ácido Graxo , Proteínas de Ligação a Ácido Graxo/sangue , Feminino , Masculino , Músculo Esquelético/enzimologia , Músculo Esquelético/metabolismo , Doenças Musculares/enzimologia , Doenças Musculares/metabolismo , Cadeias Leves de Miosina/sangue , Preparações Farmacêuticas/administração & dosagem , Preparações Farmacêuticas/química , Ratos Endogâmicos F344 , Ratos Sprague-Dawley , Ratos Wistar , Projetos de Pesquisa , Sensibilidade e Especificidade , Troponina I/sangueRESUMO
Reactive oxygen and nitrogen species inhibit or promote cell proliferation by modulating the cell signaling pathways that dictate decisions between cell survival, proliferation, and death. In the growth factor-dependent pathways that regulate mitogenesis, numerous positive and negative effectors of signaling are influenced by physiological fluctuations of oxidants, including receptor tyrosine kinases, small GTPases, mitogen-activated protein kinases, protein phosphatases, and transcription factors. The same mitogenic pathways that are sensitive to oxidant levels also directly regulate the expression of cyclin D1, a labile factor required for progression through the G1 phase on the cell cycle. Because the transition from G0 to G1 is the only phase of the cell cycle that is not regulated by cyclin-dependent kinases, but rather by redox-dependent signaling pathways, expression of cyclin D1 represents a primary regulatory node for the dose-dependent effects of oxidants on the induction of cell growth. We suggest that expression of cyclin D1 represents a useful marker for assessing the integration of proliferative and growth inhibitory effects of oxidants on the redox-dependent signaling events that control reentry into the cell cycle.
Assuntos
Ciclo Celular , Ciclina D1/metabolismo , Mitógenos/metabolismo , Espécies Reativas de Nitrogênio/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Animais , Humanos , Transdução de SinaisRESUMO
BACKGROUND: Identifying translatable, non-invasive biomarkers of muscular dystrophy that better reflect the disease pathology than those currently available would aid the development of new therapies, the monitoring of disease progression and the response to therapy. OBJECTIVE: The goal of this study was to evaluate a panel of serum protein biomarkers with the potential to specifically detect skeletal muscle injury. METHOD: Serum concentrations of skeletal troponin I (sTnI), myosin light chain 3 (Myl3), fatty acid binding protein 3 (FABP3) and muscle-type creatine kinase (CKM) proteins were measured in 74 Duchenne muscular dystrophy (DMD), 38 Becker muscular dystrophy (BMD) and 49 Limb-girdle muscular dystrophy type 2B (LGMD2B) patients and 32 healthy controls. RESULTS: All four proteins were significantly elevated in the serum of these three muscular dystrophy patient populations when compared to healthy controls, but, interestingly, displayed different profiles depending on the type of muscular dystrophy. Additionally, the effects of patient age, ambulatory status, cardiac function and treatment status on the serum concentrations of the proteins were investigated. Statistical analysis revealed correlations between the serum concentrations and certain clinical endpoints including forced vital capacity in DMD patients and the time to walk ten meters in LGMD2B patients. Serum concentrations of these proteins were also elevated in two preclinical models of muscular dystrophy, the mdx mouse and the golden-retriever muscular dystrophy dog. CONCLUSIONS: These proteins, therefore, are potential muscular dystrophy biomarkers for monitoring disease progression and therapeutic response in both preclinical and clinical studies.
RESUMO
Gain-of-function mutations in SHP-2/PTPN11 cause Noonan syndrome, a human developmental disorder. Noonan syndrome is characterized by proportionate short stature, facial dysmorphia, increased risk of leukemia, and congenital heart defects in approximately 50% of cases. Congenital heart abnormalities are common in Noonan syndrome, but the signaling pathway(s) linking gain-of-function SHP-2 mutants to heart disease is unclear. Diverse cell types coordinate cardiac morphogenesis, which is regulated by calcium (Ca2+) and the nuclear factor of activated T-cells (NFAT). It has been shown that the frequency of Ca2+ oscillations regulates NFAT activity. Here, we show that in fibroblasts, Ca2+ oscillations in response to FGF-2 require the phosphatase activity of SHP-2. Conversely, gain-of-function mutants of SHP-2 enhanced FGF-2-mediated Ca2+ oscillations in fibroblasts and spontaneous Ca2+ oscillations in cardiomyocytes. The enhanced frequency of cardiomyocyte Ca2+ oscillations induced by a gain-of-function SHP-2 mutant correlated with reduced nuclear translocation and transcriptional activity of NFAT. These data imply that gain-of-function SHP-2 mutants disrupt the Ca2+ oscillatory control of NFAT, suggesting a potential mechanism for congenital heart defects in Noonan syndrome.