RESUMO
BACKGROUND: Tyrosine kinase inhibitors (TKIs) are effective therapies with demonstrated antineoplastic activity. Nilotinib is a second-generation FDA-approved TKI designed to overcome Imatinib resistance and intolerance in patients with chronic myelogenous leukemia (CML). Interestingly, TKIs have also been shown to be an efficient treatment for several non-malignant disorders such fibrotic diseases, including those affecting skeletal muscles. METHODS: We investigated the role of Nilotinib on skeletal myogenesis using the well-established C2C12 myoblast cell line. We evaluated the impact of Nilotinib during the time course of skeletal myogenesis. We compared the effect of Nilotinib with the well-known p38 MAPK inhibitor SB203580. MEK1/2 UO126 and PI3K/AKT LY294002 inhibitors were used to identify the signaling pathways involved in Nilotinib-related effects on myoblast. Adult primary myoblasts were also used to corroborate the inhibition of myoblasts fusion and myotube-nuclei positioning by Nilotinib. RESULTS: We found that Nilotinib inhibited myogenic differentiation, reducing the number of myogenin-positive myoblasts and decreasing myogenin and MyoD expression. Furthermore, Nilotinib-mediated anti-myogenic effects impair myotube formation, myosin heavy chain expression, and compromise myotube-nuclei positioning. In addition, we found that p38 MAPK is a new off-target protein of Nilotinib, which causes inhibition of p38 phosphorylation in a similar manner as the well-characterized p38 inhibitor SB203580. Nilotinib induces the activation of ERK1/2 and AKT on myoblasts but not in myotubes. We also found that Nilotinib stimulates myoblast proliferation, a process dependent on ERK1/2 and AKT activation. CONCLUSIONS: Our findings suggest that Nilotinib may have important negative effects on muscle homeostasis, inhibiting myogenic differentiation but stimulating myoblasts proliferation. Additionally, we found that Nilotinib stimulates the activation of ERK1/2 and AKT. On the other hand, we suggest that p38 MAPK is a new off-target of Nilotinib. Thus, there is a necessity for future studies to investigate the long-term effects of TKIs on skeletal muscle homeostasis, along with potential detrimental effects in cell differentiation and proliferation in patients receiving TKI therapies.
Assuntos
Desenvolvimento Muscular/efeitos dos fármacos , Mioblastos Esqueléticos/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Pirimidinas/farmacologia , Animais , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/fisiologia , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/fisiologia , Células Cultivadas , Regulação da Expressão Gênica/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/fisiologia , Camundongos Endogâmicos C57BL , Desenvolvimento Muscular/fisiologia , Fibras Musculares Esqueléticas/citologia , Fibras Musculares Esqueléticas/efeitos dos fármacos , Fibras Musculares Esqueléticas/metabolismo , Mioblastos Esqueléticos/citologia , Mioblastos Esqueléticos/enzimologia , Miogenina/biossíntese , Miogenina/genética , Fosforilação/efeitos dos fármacos , Fosforilação/fisiologia , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteostase/efeitos dos fármacos , Proteostase/fisiologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Mensageiro/genética , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
In the present study, real time-polymerase chain reaction was applied to analyze the expression of IGF-I and MyoG genes in Hu sheep longissimus dorsi at different growth stages and their association with meat traits. Expression of the IGF-I gene in Hu sheep differed significantly between males and females at the two day-old (0.01 < P < 0.05), one-month old (0.01 < P < 0.05), and three month-old (P < 0.01) stages. IGF-I gene expression in male longissimus muscles was higher than that of females at all growth stages, except for the three month-old stage. There was no significant difference (P > 0.05) between males and females at any growth stage in expression of the MyoG gene. MyoG gene expression in male longissimus muscles tended to be higher than that of females at all growth stages, except for the six month-old stage. IGF-I gene expression was significantly and positively correlated with live weight (P < 0.01) and carcass weight (0.01< P < 0.05), and was non-significantly positively correlated with net meat weight (P > 0.05). In contrast, MyoG gene expression was non-significantly and positively correlated with live weight, carcass, and net meat weight (P > 0.05). Carcass traits showed highly significant positive correlations (P < 0.01). Furthermore, expressions of IGF-I and MyoG genes showed highly significant positive correlations (P < 0.01). We conclude that the expressions of IGF-I and MyoG genes are significantly and positively correlated with early muscle traits of Hu sheep.
Assuntos
Fator de Crescimento Insulin-Like I/biossíntese , Carne , Miogenina/biossíntese , Carneiro Doméstico/genética , Animais , Peso Corporal/genética , Regulação da Expressão Gênica no Desenvolvimento , Fator de Crescimento Insulin-Like I/genética , Músculo Esquelético/crescimento & desenvolvimento , Miogenina/genética , FenótipoRESUMO
To confirm the entire developmental process and transition point of embryonic Pekin duck pectoral muscle, and to investigate the association between pectoral muscle development and their regulating genes, anatomical and morphological analyses of embryonic Pekin duck skeletal muscles were performed, and the expression patterns of its regulating genes were investigated. The anatomical analysis revealed that body weight increased with age, while increases in pectoral muscle weight nearly ceased after the embryo was 20 days of hatching (E20). The developmental morphological characteristics of Pekin duck pectoral muscle at the embryonic stage showed that E20 was the transition point (from proliferation to fusion) of Pekin duck pectoral muscle. The expression patterns of MRF4, MyoG, and MSTN indicated that E19 or E20 was the fastest point of pectoral muscle development and the crucial transition for Pekin duck pectoral muscle development during the embryonic stage. Together, these findings imply that E20 is the crucial transition point (from proliferation to fusion) of Pekin duck pectoral muscle and that there is no muscle fiber hypertrophy after E20. Results of this study provide further understanding of the developmental process and transition point of Pekin duck pectoral muscle during the embryo stage.
Assuntos
Patos/embriologia , Perfilação da Expressão Gênica/veterinária , Músculos Peitorais/embriologia , Animais , Peso Corporal , Embrião não Mamífero/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/genética , Fatores de Regulação Miogênica/biossíntese , Fatores de Regulação Miogênica/genética , Miogenina/biossíntese , Miogenina/genética , Miostatina/biossíntese , Miostatina/genética , Músculos Peitorais/anatomia & histologia , Músculos Peitorais/crescimento & desenvolvimento , RNA Mensageiro/biossínteseRESUMO
Skeletal muscle growth is regulated by differential expression of myogenic regulatory factors (MRFs). We evaluated hyperplasia, hypertrophy and quantitative expression of MRFs MyoD and myogenin in 45, 90, 180, and 400 days post-hatching (dph) and adult pacu (Piaractus mesopotamicus) skeletal muscle. Transverse sections of white dorsal muscles were obtained to evaluate hypertrophy and hyperplasia. MyoD and myogenin gene expression was determined by reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR). Pacu skeletal muscle had similar morphology at all stages. The highest and the lowest frequencies of fiber diameters <20 µm were found at the 45 dph and adult stages, respectively. Their frequency was similar in the 90, 180, and 400 dph stages. The highest percentage of >50 µm diameter fibers were found in 180 and 400 dph, and adult fish. Hyperplasia was the main mechanism observed in pacu skeletal muscle growth at 45dph; this declined through 90, 180, and 400 dph and remained low in adult fish; the latter presented hypertrophy as the main mechanism responsible for skeletal muscle growth. The high frequencies of 20-50 µm diameter fibers at 90, 180, and 400 dph can be related to intense hypertrophy. The mRNA levels for MyoD and myogenin were similar in 45, 90, and 400 dph and adult fish, peaking at 180 dph. The high MyoD expression at 180 dph can be related to intense myoblast proliferation and hyperplasia, while high myogenin expression can be related to intense myoblast differentiation and fusion during hypertrophy. MyoD and myogenin expression patterns in adults can respectively be associated with myoblast proliferation and differentiation, which both contribute to hypertrophy. Differential MyoD and myogenin expression in pacu white muscle probably is associated with differences in growth patterns during the stages analyzed. In this study, the 180 dph pacu could represent an interesting phase to investigate suitable strategies in commercial fish production focusing on skeletal muscle growth improvement to raise healthy, fast-growing fish.
Assuntos
Peixes/fisiologia , Regulação da Expressão Gênica , Músculo Esquelético/fisiologia , Proteína MyoD/biossíntese , Miogenina/biossíntese , Animais , Peixes/genética , Peixes/crescimento & desenvolvimento , Perfilação da Expressão Gênica , Hipertrofia , Músculo Esquelético/crescimento & desenvolvimento , Proteína MyoD/genética , Miogenina/genética , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Skeletal muscle differentiation follows an organized sequence of events including commitment, cell cycle withdrawal, and cell fusion to form multinucleated myotubes. The role of adenosine 5'-triphosphate (ATP)-mediated signaling in differentiation of skeletal muscle myoblasts was evaluated in C(2)C(12) cells, a myoblast cell line. Cell differentiation was inhibited by P2X receptor blockers or by degradation of endogenous ATP with apyrase. However, pertussis toxin, known to block only a group of P2Y receptors, did not alter the differentiation process. Cells were heterogeneous in their expression of functional P2X receptors, evaluated by the uptake of fluorescent permeability tracers (Lucifer yellow and ethidium bromide), and by immunofluorescence of P2X(7) receptors. Moreover, xestospongin C, a selective and membrane-permeable inhibitor of IP(3) receptors, inhibited both myotube formation and myogenin expression. Based on these results, we suggest that the known increase in intracellular Ca(2+) concentration required for differentiation is due at least in part to Ca(2+) influx through P2X receptors and Ca(2+) release from intracellular stores. The possible involvement of P2X receptors and other pathways that might set the intracellular Ca(2+) at the level required for myoblast differentiation as well as the possible involvement of gap junction channels in the intercellular transfer of second messengers involved in coordinating myogenesis is proposed.
Assuntos
Trifosfato de Adenosina/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/crescimento & desenvolvimento , Receptores Purinérgicos P2/metabolismo , Sarcolema/metabolismo , Animais , Cálcio/metabolismo , Canais de Cálcio/metabolismo , Sinalização do Cálcio/efeitos dos fármacos , Sinalização do Cálcio/fisiologia , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/fisiologia , Linhagem Celular , Líquido Extracelular/metabolismo , Corantes Fluorescentes/metabolismo , Junções Comunicantes/metabolismo , Receptores de Inositol 1,4,5-Trifosfato , Líquido Intracelular/efeitos dos fármacos , Líquido Intracelular/metabolismo , Camundongos , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/metabolismo , Miogenina/biossíntese , Miogenina/efeitos dos fármacos , Antagonistas do Receptor Purinérgico P2 , Receptores Citoplasmáticos e Nucleares/antagonistas & inibidores , Receptores Citoplasmáticos e Nucleares/metabolismo , Receptores Purinérgicos P2X , Receptores Purinérgicos P2X7RESUMO
Decorin is a member of the family of the small leucine-rich proteoglycans. In addition to its function as an extracellular matrix organizer, it has the ability to activate the epidermal growth factor receptor, and it forms complexes with various isoforms of transforming growth factor beta (TGF-beta). Decorin is expressed during skeletal muscle differentiation and is up-regulated in dystrophic muscle. In this study we investigated the role of decorin in TGF-beta-dependent inhibition of myogenesis. To probe the function of decorin during myogenesis, C(2)C(12) myoblasts were stably transfected with a plasmid expressing antisense decorin mRNA. The resulting inhibition of decorin expression led to the expression of myogenin, a master transcription factor for muscle differentiation, under growth conditions and accelerated skeletal muscle differentiation as determined by the expression of creatine kinase. In contrast myogenin expression was inhibited by adenovirally induced decorin expression or by adding exogenous decorin. Reduced synthesis of decorin resulted in a 7-fold decreased sensitivity to TGF-beta-mediated inhibition of myogenin expression. In contrast, adenovirally induced decorin expression in wild type cells resulted in a 5-fold increased sensitivity to TGF-beta-mediated inhibition of myogenin expression. Transfection studies with the TGF-beta-dependent promoter of the plasminogen activator inhibitor-1 coupled with luciferase revealed that the transducing receptors for TGF-beta1 and TGF-beta2 were involved in the different responses of wild type and antisense decorin myoblasts. These results demonstrate that a reduction of decorin expression or of decorin availability results in a decreased responsiveness to TGF-beta. These findings strongly suggest a new role for decorin during skeletal muscle terminal differentiation by activating TGF-beta-dependent signaling pathways.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Músculo Esquelético/efeitos dos fármacos , Oligonucleotídeos Antissenso/farmacologia , Proteoglicanas/antagonistas & inibidores , Fator de Crescimento Transformador beta/metabolismo , Animais , Células Cultivadas , Decorina , Proteínas da Matriz Extracelular , Expressão Gênica/efeitos dos fármacos , Inativação Gênica , Camundongos , Músculo Esquelético/patologia , Miogenina/biossíntese , Oligonucleotídeos Antissenso/genética , Proteoglicanas/genética , Proteoglicanas/metabolismo , Transdução de Sinais/efeitos dos fármacos , TransfecçãoRESUMO
Expression of syndecan-1, a cell-surface heparan sulfate proteoglycan, is down-regulated during skeletal muscle differentiation (Larraín, J., Cizmeci-Smith, G., Troncoso, V., Stahl, R. C., Carey, D. J., and Brandan, E. (1997) J. Biol. Chem. 272, 18418-18424). We examined the role of syndecan-1 in basic fibroblast growth factor (bFGF)-dependent inhibition of myogenesis. C2C12 myoblasts were stably transfected with an expression plasmid containing the rat syndecan-1 coding region cDNA. Constitutive syndecan-1 expression resulted in a strongly diminished capacity of the transfected clones to differentiate and to express skeletal muscle-specific markers such as fusion, creatine kinase, and myosin. The expression of myogenin, a master transcription factor for muscle differentiation, was also reduced and delayed. Analysis of the induction of a myogenin promoter-driven reporter revealed that syndecan-1 expression resulted in a 6-7-fold increase in sensitivity to bFGF-dependent inhibition of myogenin expression. Transfecting the cells with a plasmid containing myogenin cDNA reversed the inhibition of myogenin transcriptional activation and myosin expression in syndecan-1-transfected cells; however, cell fusion was not observed. These results demonstrate that syndecan-1 expression enhances cell responsiveness to bFGF and inhibits myoblast fusion and suggest that muscle terminal differentiation is regulated by syndecan-1 expression.
Assuntos
Fator 2 de Crescimento de Fibroblastos/fisiologia , Glicoproteínas de Membrana/fisiologia , Músculo Esquelético/citologia , Músculo Esquelético/metabolismo , Proteoglicanas/fisiologia , Receptores Proteína Tirosina Quinases , Animais , Biomarcadores , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/fisiologia , Fusão Celular , Linhagem Celular , Cloranfenicol O-Acetiltransferase/biossíntese , Creatina Quinase/genética , Fator 2 de Crescimento de Fibroblastos/farmacologia , Genes Reporter , Biblioteca Genômica , Heparina/farmacologia , Cinética , Glicoproteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos BALB C , Músculo Esquelético/efeitos dos fármacos , Miogenina/biossíntese , Miogenina/genética , Cadeias Pesadas de Miosina/análise , Cadeias Pesadas de Miosina/genética , Proteoglicanas/genética , Ratos , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos , Receptores de Fatores de Crescimento de Fibroblastos/análise , Receptores de Fatores de Crescimento de Fibroblastos/genética , Proteínas Recombinantes de Fusão/biossíntese , Sindecana-1 , Sindecanas , Fatores de Tempo , TransfecçãoRESUMO
Syndecan-1 is an integral membrane proteoglycan involved in the interaction of cells with extracellular matrix proteins and growth factors. It is transiently expressed in several condensing mesenchymal tissues after epithelial induction. In this study we evaluated the expression of syndecan-1 during skeletal muscle differentiation. The expression of syndecan-1 as determined by Northern blot analyses and immunofluorescence microscopy is down-regulated during differentiation. The transcriptional activity of a syndecan-1 promoter construct is also down-regulated in differentiating muscle cells. The decrease in syndecan-1 gene expression is not dependent on the presence of E-boxes, binding sites for the MyoD family of transcription factors in the promoter region, or myogenin expression. Deletion of the region containing the E-boxes or treatment of differentiating cells with sodium butyrate, an inhibitor of myogenin expression, had no effect on syndecan-1 expression. Basic fibroblast growth factor and transforming growth factor type beta, which are inhibitors of myogenesis, had little effect on syndecan-1 expression. When added together, however, they induced syndecan-1 expression. Retinoic acid, an inducer of myogenesis, inhibited syndecan-1 expression and abolished the effect of the growth factors. These results indicate that syndecan-1 expression is down-regulated during myogenesis and that growth factors and retinoic acid modulate syndecan-1 expression by a mechanism that is independent of myogenin.