RESUMO
Skeletal muscle atrophy is a severe condition of muscle mass loss. Muscle atrophy is caused by a down-regulation of protein synthesis and by an increase of protein breakdown due to the ubiquitin-proteasome system and autophagy activation. Up-regulation of specific genes, such as the muscle-specific E3 ubiquitin ligase MAFbx, by FoxO transcription factors is essential to initiate muscle protein ubiquitination and degradation during atrophy. HDAC6 is a particular HDAC, which is functionally related to the ubiquitin proteasome system via its ubiquitin binding domain. We show that HDAC6 is up-regulated during muscle atrophy. HDAC6 activation is dependent on the transcription factor FoxO3a, and the inactivation of HDAC6 in mice protects against muscle wasting. HDAC6 is able to interact with MAFbx, a key ubiquitin ligase involved in muscle atrophy. Our findings demonstrate the implication of HDAC6 in skeletal muscle wasting and identify HDAC6 as a new downstream target of FoxO3a in stress response. This work provides new insights in skeletal muscle atrophy development and opens interesting perspectives on HDAC6 as a valuable marker of muscle atrophy and a potential target for pharmacological treatments.
Assuntos
Fatores de Transcrição Forkhead/metabolismo , Regulação da Expressão Gênica , Histona Desacetilases/metabolismo , Músculo Esquelético/patologia , Atrofia Muscular/patologia , Animais , Western Blotting , Células Cultivadas , Imunoprecipitação da Cromatina , Ensaio de Desvio de Mobilidade Eletroforética , Proteína Forkhead Box O3 , Fatores de Transcrição Forkhead/genética , Desacetilase 6 de Histona , Histona Desacetilases/química , Histona Desacetilases/genética , Humanos , Imunoprecipitação , Integrases/metabolismo , Camundongos , Camundongos Knockout , Denervação Muscular , Músculo Esquelético/metabolismo , Atrofia Muscular/genética , Atrofia Muscular/metabolismo , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Human T lymphotropic virus type 1 (HTLV-1) is the etiologic agent of adult T-cell leukemia/lymphoma (ATLL). ATLL is a severe malignancy with no effective treatment. HTLV-1 regulatory proteins Tax and HTLV-1 basic leucine zipper factor (HBZ) play a major role in ATLL development, by interfering with cellular functions such as CD4(+) T-cell survival. In this study, we observed that the expression of Bfl-1, an antiapoptotic protein of the Bcl-2 family, is restricted to HTLV-1-infected T-cell lines and to T-cells expressing both Tax and HBZ proteins. We showed that Tax-induced bfl-1 transcription through the canonical NF-κB pathway. Moreover, we demonstrated that Tax cooperated with c-Jun or JunD, but not JunB, transcription factors of the AP-1 family to stimulate bfl-1 gene activation. By contrast, HBZ inhibited c-Jun-induced bfl-1 gene activation, whereas it increased JunD-induced bfl-1 gene activation. We identified one NF-κB, targeted by RelA, c-Rel, RelB, p105/p50, and p100/p52, and two AP-1, targeted by both c-Jun and JunD, binding sites in the bfl-1 promoter of T-cells expressing both Tax and HBZ. Analyzing the potential role of antiapoptotic Bcl-2 proteins in HTLV-1-infected T-cell survival, we demonstrated that these cells are differentially sensitive to silencing of Bfl-1, Bcl-x(L), and Bcl-2. Indeed, both Bfl-1 and Bcl-x(L) knockdowns decreased the survival of HTLV-1-infected T-cell lines, although no cell death was observed after Bcl-2 knockdown. Furthermore, we demonstrated that Bfl-1 knockdown sensitizes HTLV-1-infected T-cells to ABT-737 or etoposide treatment. Our results directly implicate Bfl-1 and Bcl-x(L) in HTLV-1-infected T-cell survival and suggest that both Bfl-1 and Bcl-x(L) represent potential therapeutic targets for ATLL treatment.
Assuntos
Linfócitos T CD4-Positivos/metabolismo , Produtos do Gene tax/metabolismo , Vírus Linfotrópico T Tipo 1 Humano/metabolismo , Leucemia-Linfoma de Células T do Adulto/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Adulto , Antineoplásicos Fitogênicos/farmacologia , Fatores de Transcrição de Zíper de Leucina Básica/genética , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Compostos de Bifenilo/farmacologia , Linfócitos T CD4-Positivos/patologia , Linfócitos T CD4-Positivos/virologia , Sobrevivência Celular , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Etoposídeo/farmacologia , Feminino , Técnicas de Silenciamento de Genes , Produtos do Gene tax/genética , Genes jun/genética , Células HeLa , Vírus Linfotrópico T Tipo 1 Humano/genética , Humanos , Leucemia-Linfoma de Células T do Adulto/dietoterapia , Leucemia-Linfoma de Células T do Adulto/genética , Leucemia-Linfoma de Células T do Adulto/patologia , Ligases/genética , Ligases/metabolismo , Masculino , Antígenos de Histocompatibilidade Menor , Subunidade p50 de NF-kappa B/genética , Subunidade p50 de NF-kappa B/metabolismo , Nitrofenóis/farmacologia , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Piperazinas/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-jun/genética , Proteínas Proto-Oncogênicas c-jun/metabolismo , Proteínas Proto-Oncogênicas c-rel , Proteínas dos Retroviridae , Sulfonamidas/farmacologia , Fator de Transcrição RelB/genética , Fator de Transcrição RelB/metabolismo , Transcrição Gênica/efeitos dos fármacos , Transcrição Gênica/genética , Proteínas Virais/genética , Proteínas Virais/metabolismo , Proteína bcl-X/genética , Proteína bcl-X/metabolismoRESUMO
The absence of dystrophin in Duchenne muscular dystrophy disrupts the dystrophin-associated glycoprotein complex resulting in skeletal muscle fiber fragility and atrophy, associated with fibrosis as well as microtubule and neuromuscular junction disorganization. The specific, non-conventional cytoplasmic histone deacetylase 6 (HDAC6) was recently shown to regulate acetylcholine receptor distribution and muscle atrophy. Here, we report that administration of the HDAC6 selective inhibitor tubastatin A to the Duchenne muscular dystrophy, mdx mouse model increases muscle strength, improves microtubule, neuromuscular junction, and dystrophin-associated glycoprotein complex organization, and reduces muscle atrophy and fibrosis. Interestingly, we found that the beneficial effects of HDAC6 inhibition involve the downregulation of transforming growth factor beta signaling. By increasing Smad3 acetylation in the cytoplasm, HDAC6 inhibition reduces Smad2/3 phosphorylation, nuclear translocation, and transcriptional activity. These findings provide in vivo evidence that Smad3 is a new target of HDAC6 and implicate HDAC6 as a potential therapeutic target in Duchenne muscular dystrophy.
Assuntos
Distrofina , Distrofia Muscular de Duchenne , Camundongos , Animais , Distrofina/genética , Distrofina/metabolismo , Camundongos Endogâmicos mdx , Desacetilase 6 de Histona/genética , Desacetilase 6 de Histona/metabolismo , Distrofia Muscular de Duchenne/tratamento farmacológico , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/metabolismo , Acetilação , Fator de Crescimento Transformador beta/metabolismo , Músculo Esquelético/metabolismo , Inibidores de Histona Desacetilases/farmacologia , Inibidores de Histona Desacetilases/metabolismo , Fibrose , Fenótipo , Atrofia Muscular/patologia , Glicoproteínas/metabolismoRESUMO
BACKGROUND: The protein kinase mechanistic target of rapamycin (mTOR) controls cellular growth and metabolism. Although balanced mTOR signalling is required for proper muscle homeostasis, partial mTOR inhibition by rapamycin has beneficial effects on various muscle disorders and age-related pathologies. Besides, more potent mTOR inhibitors targeting mTOR catalytic activity have been developed and are in clinical trials. However, the physiological impact of loss of mTOR catalytic activity in skeletal muscle is currently unknown. METHODS: We have generated the mTORmKOKI mouse model in which conditional loss of mTOR is concomitant with expression of kinase inactive mTOR in skeletal muscle. We performed a comparative phenotypic and biochemical analysis of mTORmKOKI mutant animals with muscle-specific mTOR knockout (mTORmKO) littermates. RESULTS: In striking contrast with mTORmKO littermates, mTORmKOKI mice developed an early onset rapidly progressive myopathy causing juvenile lethality. More than 50% mTORmKOKI mice died before 8 weeks of age, and none survived more than 12 weeks, while mTORmKO mice died around 7 months of age. The growth rate of mTORmKOKI mice declined beyond 1 week of age, and the animals showed profound alterations in body composition at 4 weeks of age. At this age, their body weight was 64% that of mTORmKO mice (P < 0.001) due to significant reduction in lean and fat mass. The mass of isolated muscles from mTORmKOKI mice was remarkably decreased by 38-56% (P < 0.001) as compared with that from mTORmKO mice. Histopathological analysis further revealed exacerbated dystrophic features and metabolic alterations in both slow/oxidative and fast/glycolytic muscles from mTORmKOKI mice. We show that the severity of the mTORmKOKI as compared with the mild mTORmKO phenotype is due to more robust suppression of muscle mTORC1 signalling leading to stronger alterations in protein synthesis, oxidative metabolism, and autophagy. This was accompanied with stronger feedback activation of PKB/Akt and dramatic down-regulation of glycogen phosphorylase expression (0.16-fold in tibialis anterior muscle, P < 0.01), thus causing features of glycogen storage disease type V. CONCLUSIONS: Our study demonstrates a critical role for muscle mTOR catalytic activity in the regulation of whole-body growth and homeostasis. We suggest that skeletal muscle targeting with mTOR catalytic inhibitors may have detrimental effects. The mTORmKOKI mutant mouse provides an animal model for the pathophysiological understanding of muscle mTOR activity inhibition as well as for mechanistic investigation of the influence of skeletal muscle perturbations on whole-body homeostasis.
Assuntos
Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Doenças Musculares/genética , Serina-Treonina Quinases TOR/genética , Animais , Modelos Animais de Doenças , Homeostase , Humanos , Masculino , Camundongos Transgênicos , Doenças Musculares/metabolismoRESUMO
New sources of red blood cells (RBCs) would improve the transfusion capacity of blood centers. Our objective was to generate cells for transfusion by inducing a massive proliferation of hematopoietic stem and progenitor cells, followed by terminal erythroid differentiation. We describe here a procedure for amplifying hematopoietic stem cells (HSCs) from human cord blood (CB) by the sequential application of specific combinations of growth factors in a serum-free culture medium. The procedure allowed the ex vivo expansion of CD34+ progenitor and stem cells into a pure erythroid precursor population. When injected into nonobese diabetic, severe combined immunodeficient (NOD/SCID) mice, the erythroid cells were capable of proliferation and terminal differentiation into mature enucleated RBCs. The approach may eventually be useful in clinical transfusion applications.
Assuntos
Eritrócitos/citologia , Eritrócitos/fisiologia , Animais , Antígenos CD34/biossíntese , Diferenciação Celular , Divisão Celular , Separação Celular , Células Cultivadas , Meios de Cultura Livres de Soro/farmacologia , Sangue Fetal/citologia , Citometria de Fluxo , Células-Tronco Hematopoéticas/citologia , Hemoglobinas/metabolismo , Humanos , Cinética , Camundongos , Camundongos SCID , Perfusão , Células-Tronco/metabolismo , Fatores de TempoRESUMO
Acetylcholine receptor (AChR) expression in innervated muscle is limited to the synaptic region. Neuron-induced electrical activity participates in this compartmentalization by promoting the repression of AChR expression in the extrasynaptic regions. Here, we show that the corepressor CtBP1 (C-terminal binding protein 1) is present on the myogenin promoter together with repressive histone marks. shRNA-mediated downregulation of CtBP1 expression is sufficient to derepress myogenin and AChR expression in innervated muscle. Upon denervation, CtBP1 is displaced from the myogenin promoter and relocates to the cytoplasm, while repressive histone marks are replaced by activating ones concomitantly to the activation of myogenin expression. We also observed that upon denervation the p21-activated kinase 1 (PAK1) expression is upregulated, suggesting that phosphorylation by PAK1 may be involved in the relocation of CtBP1. Indeed, preventing CtBP1 Ser158 phosphorylation induces CtBP1 accumulation in the nuclei and abrogates the activation of myogenin and AChR expression. Altogether, these findings reveal a molecular mechanism to account for the coordinated control of chromatin modifications and muscle gene expression by presynaptic neurons via a PAK1/CtBP1 pathway.
Assuntos
Oxirredutases do Álcool/metabolismo , Proteínas de Ligação a DNA/metabolismo , Denervação Muscular , Músculo Esquelético/inervação , Miogenina/biossíntese , Quinases Ativadas por p21/metabolismo , Transporte Ativo do Núcleo Celular/genética , Oxirredutases do Álcool/genética , Animais , Núcleo Celular/metabolismo , Cromatina/genética , Cromatina/metabolismo , Citoplasma/metabolismo , Proteínas de Ligação a DNA/genética , Histonas/genética , Histonas/metabolismo , Camundongos , Músculo Esquelético/metabolismo , Miogenina/genética , Fosforilação , Regiões Promotoras Genéticas/genética , Interferência de RNA , RNA Interferente Pequeno , Receptores Colinérgicos/genética , Receptores Colinérgicos/metabolismo , Ativação Transcricional/genética , Regulação para Cima , Quinases Ativadas por p21/biossíntese , Quinases Ativadas por p21/genéticaRESUMO
Mammalian target of rapamycin (mTOR) is a key regulator of cell growth that associates with raptor and rictor to form the mTOR complex 1 (mTORC1) and mTORC2, respectively. Raptor is required for oxidative muscle integrity, whereas rictor is dispensable. In this study, we show that muscle-specific inactivation of mTOR leads to severe myopathy, resulting in premature death. mTOR-deficient muscles display metabolic changes similar to those observed in muscles lacking raptor, including impaired oxidative metabolism, altered mitochondrial regulation, and glycogen accumulation associated with protein kinase B/Akt hyperactivation. In addition, mTOR-deficient muscles exhibit increased basal glucose uptake, whereas whole body glucose homeostasis is essentially maintained. Importantly, loss of mTOR exacerbates the myopathic features in both slow oxidative and fast glycolytic muscles. Moreover, mTOR but not raptor and rictor deficiency leads to reduced muscle dystrophin content. We provide evidence that mTOR controls dystrophin transcription in a cell-autonomous, rapamycin-resistant, and kinase-independent manner. Collectively, our results demonstrate that mTOR acts mainly via mTORC1, whereas regulation of dystrophin is raptor and rictor independent.