RESUMO
Idiopathic Inflammatory Myopathies (IIMs) are a heterogeneous group of autoimmune diseases affecting skeletal muscle tissue homeostasis. They are characterized by muscle weakness and inflammatory infiltration with tissue damage. Amongst the cells in the muscle inflammatory infiltration, dendritic cells (DCs) are potent antigen-presenting and key components in autoimmunity exhibiting an increased activation in inflamed tissues. Since, the IIMs are characterized by the focal necrosis/regeneration and muscle atrophy, we hypothesized that DCs may play a role in these processes. Due to the absence of a reliable in vivo model for IIMs, we first performed co-culture experiments with immature DCs (iDC) or LPS-activated DCs (actDC) and proliferating myoblasts or differentiating myotubes. We demonstrated that both iDC or actDCs tightly interact with myoblasts and myotubes, increased myoblast proliferation and migration, but inhibited myotube differentiation. We also observed that actDCs increased HLA-ABC, HLA-DR, VLA-5, and VLA-6 expression and induced cytokine secretion on myoblasts. In an in vivo regeneration model, the co-injection of human myoblasts and DCs enhanced human myoblast migration, whereas the absolute number of human myofibres was unchanged. In conclusion, we suggest that in the early stages of myositis, DCs may play a crucial role in inducing muscle-damage through cell-cell contact and inflammatory cytokine secretion, leading to muscle regeneration impairment.
Assuntos
Diferenciação Celular , Proliferação de Células , Células Dendríticas/metabolismo , Mioblastos Esqueléticos/metabolismo , Adulto , Antígenos de Diferenciação/biossíntese , Células Dendríticas/citologia , Feminino , Humanos , Recém-Nascido , Lipopolissacarídeos/farmacologia , Masculino , Pessoa de Meia-Idade , Mioblastos Esqueléticos/citologiaRESUMO
Skeletal myogenesis comprises myoblast replication and differentiation into striated multinucleated myotubes. Agents that interfere with myoblast replication are important tools for the understanding of myogenesis. Recently, we showed that cholesterol depletion by methyl-ß-cyclodextrin (MCD) enhances the differentiation step in chick-cultured myogenic cells, involving the activation of the Wnt/ß-catenin signaling pathway. However, the effects of cholesterol depletion on myoblast replication have not been carefully studied. Here we show that MCD treatment increases cell proliferation in primary chick myogenic cell cultures. Treatment of myogenic cells with the anti-mitotic reagent cytosine arabinoside, immediately following cholesterol depletion, blocks the MCD-induced effects on proliferation. Cholesterol depletion induced an increase in the number of desmin-positive mononucleated cells, and an increase in desmin expression. MCD induces an increase in the expression of the cell cycle regulator p53 and the master switch gene MyoD1. Treatment with BIO, a specific inhibitor of GSK3ß, induced effects similar to MCD on cell proliferation; while treatment with Dkk1, a specific inhibitor of the Wnt/ß-catenin pathway, neutralized the effects of MCD. These findings indicate that rapid changes in the cholesterol content in cell membranes of myoblasts can induce cell proliferation, possibly by the activation of the Wnt/ß-catenin signaling pathway.
Assuntos
Colesterol/deficiência , Desmina/metabolismo , Mioblastos/citologia , Mioblastos/metabolismo , beta-Ciclodextrinas/farmacologia , Animais , Bromodesoxiuridina/metabolismo , Contagem de Células , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Embrião de Galinha , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Desenvolvimento Muscular/efeitos dos fármacos , Proteína MyoD/metabolismo , Mioblastos/efeitos dos fármacos , Especificidade de Órgãos , Transdução de Sinais/efeitos dos fármacos , Proteína Supressora de Tumor p53/metabolismo , Proteínas Wnt/metabolismoRESUMO
Cholesterol is one of the major lipids of plasma membranes. Recently, we have shown that cholesterol depletion by methyl-beta-cyclodextrin (M beta CD) induces the activation of the Wnt/beta-catenin pathway and enhances myogenic differentiation. Here, we show that M beta CD-conditioned media accelerates myogenesis in a similar way as M beta CD does, suggesting that the effects induced by M beta CD could be caused by soluble factors present in the culture medium. Soluble Wnt-3 protein is significantly enhanced in M beta CD-conditioned medium. Wnt-3a-enriched media induces myogenesis as much as M beta CD does, whereas Wnt-5a-enriched media inhibits. We suggest that Wnt-3a is involved in the myogenic induction observed after cholesterol depletion.
Assuntos
Diferenciação Celular , Colesterol/deficiência , Células Musculares/citologia , Desenvolvimento Muscular , Proteínas Wnt/metabolismo , Animais , Caderinas/metabolismo , Diferenciação Celular/efeitos dos fármacos , Fusão Celular , Células Cultivadas , Embrião de Galinha , Meios de Cultivo Condicionados/farmacologia , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Glicoproteínas/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Peso Molecular , Células Musculares/efeitos dos fármacos , Desenvolvimento Muscular/efeitos dos fármacos , Ratos , Solubilidade/efeitos dos fármacos , Proteína Wnt3 , Proteína Wnt3A , beta-Ciclodextrinas/farmacologiaRESUMO
Myogenic differentiation is a multistep process that begins with the commitment of mononucleated precursors that withdraw from cell cycle. These myoblasts elongate while aligning to each other, guided by the recognition between their membranes. This step is followed by cell fusion and the formation of long and striated multinucleated myotubes. We have recently shown that cholesterol depletion by methyl-beta-cyclodextrin (MbetaCD) induces myogenic differentiation by enhancing myoblast recognition and fusion. Here, we further studied the signaling pathways responsible for early steps of myogenesis. As it is known that Wnt plays a role in muscle differentiation, we used the chemical MbetaCD to deplete membrane cholesterol and investigate the involvement of the Wnt/beta-catenin pathway during myogenesis. We show that cholesterol depletion promoted a significant increase in expression of beta-catenin, its nuclear translocation and activation of the Wnt pathway. Moreover, we show that the activation of the Wnt pathway after cholesterol depletion can be inhibited by the soluble protein Frzb-1. Our data suggest that membrane cholesterol is involved in Wnt/beta-catenin signaling in the early steps of myogenic differentiation.
Assuntos
Colesterol/metabolismo , Fibras Musculares Esqueléticas/fisiologia , Proteínas Wnt/metabolismo , beta Catenina/metabolismo , Animais , Diferenciação Celular , Membrana Celular/metabolismo , Núcleo Celular/metabolismo , Núcleo Celular/ultraestrutura , Embrião de Galinha/metabolismo , Receptores Frizzled/metabolismo , Humanos , Modelos Biológicos , Fibras Musculares Esqueléticas/citologia , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/metabolismo , Transfecção , Proteínas Wnt/antagonistas & inibidores , beta Catenina/antagonistas & inibidores , beta-Ciclodextrinas/farmacologiaRESUMO
The muscle-specific intermediate filament protein desmin is expressed in mononucleated myoblasts and in differentiated myotubes. Desmin has been shown to associate with the sarcolemma in specific structures, such as neuromuscular junctions and the dystrophin-associated protein complex. Since these are specialized membrane regions, the study of a possible association between desmin and liquid-ordered membrane microdomains is of particular interest. We have carried out an analysis of the association between desmin and the muscle-specific protein caveolin-3, a major component of caveolar microdomains. Our results demonstrate that (1) desmin precisely co-localizes with caveolin-3 in myoblasts and multinucleated myotubes, (2) caveolin-3 is up-regulated during in vitro chick muscle development, (3) desmin is detectable in caveolae-enriched membrane fractions prepared from skeletal muscle, and (4) caveolin-3 co-immunoprecipitates with desmin. We have thus shown, for the first time, an association between the intermediate filament protein desmin and caveolin-3 in myogenic cells.
Assuntos
Caveolina 3/metabolismo , Desmina/metabolismo , Células Musculares/metabolismo , Desenvolvimento Muscular/fisiologia , Animais , Cavéolas/química , Cavéolas/metabolismo , Caveolina 3/análise , Diferenciação Celular/fisiologia , Células Cultivadas , Embrião de Galinha , Desmina/análise , Imunoprecipitação , Proteínas de Membrana/análise , Proteínas de Membrana/metabolismo , Microscopia de Fluorescência , Células Musculares/química , Fibras Musculares Esqueléticas/química , Fibras Musculares Esqueléticas/metabolismo , Mioblastos Esqueléticos/química , Mioblastos Esqueléticos/metabolismo , Ligação ProteicaRESUMO
Eukaryotic cells have highly organized, interconnected intracellular compartments. The nuclear surface and cytoplasmic cytoskeletal filaments represent compartments involved in such an association. Intermediate filaments are the major cytoskeletal elements in this association. Desmin is a muscle-specific structural protein and one of the earliest known muscle-specific genes to be expressed during cardiac and skeletal muscle development. Desmin filaments have been shown to be associated with the nuclear surface in the myogenic cell line C2C12. Previous studies have revealed that mice lacking desmin develop imperfect muscle, exhibiting the loss of nuclear shape and positioning. In the present work, we have analyzed the association between desmin filaments and the outer nuclear surface in nuclei isolated from pectoral skeletal muscle of chick embryos and in primary chick myogenic cell cultures by using immunofluorescence microscopy, negative staining, immunogold, and transmission electron microscopy. We show that desmin filaments remain firmly attached to the outer nuclear surface after the isolation of nuclei. Furthermore, positive localization of desmin persists after gentle washing of the nuclei with high ionic strength solutions. These data suggest that desmin intermediate filaments are stably and firmly connected to the outer nuclear surface in skeletal muscles cells in vivo and in vitro.
Assuntos
Núcleo Celular/metabolismo , Desmina/metabolismo , Filamentos Intermediários/ultraestrutura , Mioblastos/citologia , Animais , Fracionamento Celular , Núcleo Celular/ultraestrutura , Células Cultivadas , Embrião de Galinha , Desmina/ultraestrutura , Filamentos Intermediários/metabolismo , Microscopia Eletrônica de Transmissão , Microscopia de Fluorescência , Mioblastos/metabolismo , Mioblastos/ultraestrutura , Músculos Peitorais/citologiaRESUMO
The formation of a skeletal muscle fiber begins with the withdrawal of committed mononucleated precursors from the cell cycle. These myoblasts elongate while aligning with each other, guided by recognition between their membranes. This step is followed by cell fusion and the formation of long striated multinucleated myotubes. We used methyl-beta-cyclodextrin (MCD) in primary cultured chick skeletal muscle cells to deplete membrane cholesterol and investigate its role during myogenesis. MCD promoted a significant increase in the expression of troponin T, enhanced myoblast fusion, and induced the formation of large multinucleated myotubes with nuclei being clustered centrally and not aligned at the cell periphery. MCD myotubes were striated, as indicated by sarcomeric alpha-actinin staining, and microtubule and desmin filament distribution was not altered. Pre-fusion MCD-treated myoblasts formed large aggregates, with cadherin and beta-catenin being accumulated in cell adhesion contacts. We also found that the membrane microdomain marker GM1 was not present as clusters in the membrane of MCD-treated myoblasts. Our data demonstrate that cholesterol is involved in the early steps of skeletal muscle differentiation.