RESUMEN
Skeletal muscle stem cells, also called Satellite Cells (SCs), are actively maintained in quiescence but can activate quickly upon extrinsic stimuli. However, the mechanisms of how quiescent SCs (QSCs) activate swiftly remain elusive. Here, using a whole mouse perfusion fixation approach to obtain bona fide QSCs, we identify massive proteomic changes during the quiescence-to-activation transition in pathways such as chromatin maintenance, metabolism, transcription, and translation. Discordant correlation of transcriptomic and proteomic changes reveals potential translational regulation upon SC activation. Importantly, we show Cytoplasmic Polyadenylation Element Binding protein 1 (CPEB1), post-transcriptionally affects protein translation during SC activation by binding to the 3' UTRs of different transcripts. We demonstrate phosphorylation-dependent CPEB1 promoted Myod1 protein synthesis by binding to the cytoplasmic polyadenylation elements (CPEs) within its 3' UTRs to regulate SC activation and muscle regeneration. Our study characterizes CPEB1 as a key regulator to reprogram the translational landscape directing SC activation and subsequent proliferation.
Asunto(s)
Músculo Esquelético/lesiones , Biosíntesis de Proteínas/genética , Regeneración/genética , Células Satélite del Músculo Esquelético/fisiología , Factores de Transcripción/metabolismo , Factores de Escisión y Poliadenilación de ARNm/metabolismo , Regiones no Traducidas 3'/genética , Animales , Línea Celular , Células Cultivadas , Modelos Animales de Enfermedad , Regulación de la Expresión Génica , Células HEK293 , Humanos , Masculino , Ratones , Ratones Transgénicos , Músculo Esquelético/citología , Proteína MioD/biosíntesis , Proteómica , RNA-SeqRESUMEN
Purpose: Myoblast determination protein 1 (MYOD) is a critical myogenic regulatory factor in muscle development, differentiation, myofiber repair, and regeneration. As the extraocular muscles significantly remodel their myofibers throughout life compared with limb skeletal muscles, we hypothesized that the absence of MYOD would result in their abnormal structure and function. To assess structural and functional changes in the extraocular muscles in MyoD-/- mice, fiber size and number and optokinetic nystagmus reflex (OKN) responses were examined. Methods: OKN was measured in MyoD-/- mice and littermate wild-type controls at 3, 6, and 12 months. The extraocular muscles were examined histologically for changes in mean myofiber cross-sectional area, total myofiber number, and nuclei immunostained for PAX7 and PITX2, markers of myogenic precursor cells. Results: The MyoD-/- mice developed nystagmus, with both jerk and pendular waveforms, in the absence and in the presence of moving visual stimulation. At 12 months, there were significant losses in mean myofiber cross-sectional area and in total number of orbital layer fibers in all rectus muscles, as well as in global layer fibers in the superior and inferior rectus muscles. Haploinsufficient mice showed abnormal OKN responses. PITX2-positive cell entry into myofibers of the MyoD-/- mice was significantly reduced. Conclusions: This study is the first demonstration of the development of nystagmus in the constitutive absence of expression of the muscle-specific transcription factor MYOD. We hypothesize that myofiber loss over time may alter anterograde and/or retrograde communication between the motor nerves and extraocular muscles that are critical for maintaining normalcy of extraocular muscle function.
Asunto(s)
Regulación de la Expresión Génica , Proteína MioD/genética , Nistagmo Patológico/genética , Músculos Oculomotores/metabolismo , Animales , Modelos Animales de Enfermedad , Estudios de Seguimiento , Ratones , Proteína MioD/biosíntesis , Nistagmo Patológico/diagnóstico , Nistagmo Patológico/metabolismo , Músculos Oculomotores/diagnóstico por imagenRESUMEN
Transdifferentiation of human non-muscle cells directly into myogenic cells by forced expression of MyoD represents one route to obtain highly desirable human myogenic cells. However, functional properties of the tissue constructs derived from these transdifferentiated cells have been rarely studied. Here, we report that three-dimensional (3D) tissue constructs engineered with iMyoD-hTERT-NHDFs, normal human dermal fibroblasts transduced with genes encoding human telomerase reverse transcriptase and doxycycline-inducible MyoD, generate detectable contractile forces in response to electrical stimuli upon MyoD expression. Withdrawal of doxycycline in the middle of 3D culture results in 3.05 and 2.28 times increases in twitch and tetanic forces, respectively, suggesting that temporally-controlled MyoD expression benefits functional myogenic differentiation of transdifferentiated myoblast-like cells. Treatment with CHIR99021, a Wnt activator, and DAPT, a Notch inhibitor, leads to further enhanced contractile forces. The ability of these abundant and potentially patient-specific and disease-specific cells to develop into functional skeletal muscle constructs makes them highly valuable for many applications, such as disease modeling.
Asunto(s)
Transdiferenciación Celular , Fibroblastos/metabolismo , Desarrollo de Músculos , Músculo Esquelético/metabolismo , Proteína MioD/biosíntesis , Telomerasa/sangre , Fibroblastos/citología , Células HEK293 , Humanos , Músculo Esquelético/citología , Proteína MioD/genética , Telomerasa/genéticaRESUMEN
Activating foreign genes in bovine skeletal muscle is necessary in the study of the role of related genes in skeletal muscle development and the effects on skeletal muscle formation, especially in the study of transgenic cattle. At this time, a skeletal muscle-specific promoter should be selected to initiate a functional foreign gene. Here, calpain3 (CAPN3) was found to be highly expressed in skeletal muscle and skeletal muscle cells by real-time PCR. Next, 5' deletion analysis of the bovine CAPN3 promoter was performed and showed that Q5(-495/+40) region was the core promoter of the bovine CAPN3. A key regulatory site (-465/-453) in CAPN3 core promoter was associated with the transcription factor, MyoD, which is a skeletal muscle-specific transcription factor. Furthermore, the mRNA and protein expression levels of MyoD and CAPN3 were positively correlated during skeletal muscle cell differentiation. The overexpression of MyoD enhanced the activity of the bovine CAPN3 core promoter. The core promoter Q5(-495/+40) could drive the exogenous gene EGFP and the fat-specific expression gene PPARγ in skeletal muscle cells. In summary, our study obtained a bovine skeletal muscle-specific promoter and provided a basis for studying the role of functional genes in the growth and development of skeletal muscle. It also provides a basis for studying the transcriptional regulation mechanism of CAPN3.
Asunto(s)
Calpaína/genética , Proteínas Fluorescentes Verdes/metabolismo , Músculo Esquelético/fisiología , Proteína MioD/genética , Animales , Calpaína/metabolismo , Bovinos , Diferenciación Celular/fisiología , Células Cultivadas , Regulación de la Expresión Génica , Proteínas Fluorescentes Verdes/genética , Desarrollo de Músculos/fisiología , Músculo Esquelético/citología , Músculo Esquelético/metabolismo , Proteína MioD/biosíntesis , Proteína MioD/metabolismo , PPAR gamma/genética , PPAR gamma/metabolismo , Regiones Promotoras Genéticas , Activación TranscripcionalRESUMEN
Proliferative vitreoretinopathy (PVR) is a complication of rhegmatogenous retinal detachment and ocular trauma. The disease is characterized by development of membranes that may apply traction to the retina and cause redetachment. Membrane contractions are attributed to myofibroblasts arising from retinal pigment epithelial cells, glia and fibroblasts. The progenitors of myofibrobasts in the lens are Myo/Nog cells that express the skeletal muscle transcription factor MyoD and bone morphogenetic protein inhibitor Noggin. The retina and choroid also contain Myo/Nog cells that respond to stress. We examined preretinal PVR membranes from three ocular trauma patients with retinal detachment for Myo/Nog cells and their expression of muscle proteins. Myo/Nog cells were identified by co-localization of antibodies to the G8 antigen and Noggin. Greater than 80% of all cells in sections from two of three patients expressed both G8 and Noggin. Myo/Nog cells lacked pigment. Alpha smooth muscle actin (α-SMA) and striated myosin II heavy chain were present in the majority of Myo/Nog cells in these two patients. Differentiation of Myo/Nog cells was paralleled by low levels of MyoD. Membrane sections from the third patient consisted mostly of connective tissue with very few cells. A small subpopulation in these sections expressed both G8 and Noggin, and muscle proteins were detected in only a minority of G8-positive (+) cells. In all three patients, greater than 99% of cells with MyoD, α-SMA and striated muscle myosin co-expressed G8. These findings suggest that contractile myofibroblasts in PVR membranes may be derived from differentiating Myo/Nog cells.
Asunto(s)
Proteínas Musculares/biosíntesis , Proteína MioD/biosíntesis , Retina/patología , Vitreorretinopatía Proliferativa/metabolismo , Animales , Humanos , Retina/metabolismo , Vitreorretinopatía Proliferativa/diagnósticoRESUMEN
MYOD is a master regulator of the skeletal myogenic program. But what regulates expression of Myod? More than 20 years ago, studies established that Myod expression is largely controlled by just two enhancer regions located within a region 24 kb upstream of the transcription start site in mammals, which regulate Myod expression in the embryo, fetus and adult. Despite this apparently simple arrangement, Myod regulation is complex, with different combinations of transcription factors acting on these enhancers in different muscle progenitor cells and phases of differentiation. A range of epigenetic modifications in the Myod upstream region also play a part in activating and repressing Myod expression during development and regeneration. Here the evidence for this binding at Myod control regions is summarized, giving an overview of our current understanding of Myod expression regulation in mammals.
Asunto(s)
Diferenciación Celular , Elementos de Facilitación Genéticos , Regulación de la Expresión Génica , Proteína MioD/biosíntesis , Mioblastos/metabolismo , Transcripción Genética , Animales , Historia del Siglo XX , Historia del Siglo XXI , Humanos , Proteína MioD/genética , Proteína MioD/historiaRESUMEN
OBJECTIVE: To reveal the potential role of the basic helix-loop-helix myogenic transcription regulator MyoD in the regulation of castration-resistant prostate cancer. METHODS: Expression level of MyoD was assessed in prostate cancer tissues using quantitative reverse transcription polymerase chain reaction and immunohistochemistry and in experimentally induced castration-resistant LNCaP/R cells using quantitative reverse transcription polymerase chain reaction and immunoblotting. Effect of MyoD knockdown on LNCaP/R cell progression was determined by assessing cell proliferation, apoptosis, and colony formation rate. The effect of MyoD knockdown on the oxidative stress state in PC3 cells was determined by assessing antioxidant response gene expression and glutathione synthetase-to-glutathione ratio. Finally, the functional link between the nuclear factor erythroid-derived 2-related factor 1 (NRF1) and the regulation of antioxidant response element-driven transcription by MyoD was studied at both molecular and functional levels. RESULTS: MyoD expression was significantly upregulated in hormone-refractory prostate cancer tissues and in experimentally induced castration-resistant LNCaP/R cells, and MyoD knockdown effectively impaired LNCaP/R cell proliferation and promoted apoptosis under androgen-depleted condition. Moreover, MyoD enhanced the glutathione production and protected against oxidative stress by positively regulating a cluster of antioxidant genes known to be the downstream targets of NRF1. Mechanistically, MyoD could augment the antioxidant response element-driven transcription in an NRF1-dependent manner, and the stimulatory effect of MyoD on the antioxidant response was substantially compromised in the presence of NRF1 small interfering RNA treatment. CONCLUSION: We have identified an unexpected collaboration between MyoD and NRF1 under androgen-depleted condition, which may serve as an important adaptive mechanism during the pathogenesis of castration-resistant prostate cancer.
Asunto(s)
Proteína MioD/fisiología , Neoplasias de la Próstata Resistentes a la Castración/etiología , Antioxidantes , Proliferación Celular , Humanos , Masculino , Proteína MioD/biosíntesis , Neoplasias de la Próstata Resistentes a la Castración/metabolismo , Neoplasias de la Próstata Resistentes a la Castración/patología , Células Tumorales CultivadasRESUMEN
The enhancer regions of the myogenic master regulator MyoD give rise to at least two enhancer RNAs. Core enhancer eRNA (CEeRNA) regulates transcription of the adjacent MyoD gene, whereas DRReRNA affects expression of Myogenin in trans. We found that DRReRNA is recruited at the Myogenin locus, where it colocalizes with Myogenin nascent transcripts. DRReRNA associates with the cohesin complex, and this association correlates with its transactivating properties. Despite being expressed in undifferentiated cells, cohesin is not loaded on Myogenin until the cells start expressing DRReRNA, which is then required for cohesin chromatin recruitment and maintenance. Functionally, depletion of either cohesin or DRReRNA reduces chromatin accessibility, prevents Myogenin activation, and hinders muscle cell differentiation. Thus, DRReRNA ensures spatially appropriate cohesin loading in trans to regulate gene expression.
Asunto(s)
Proteínas de Ciclo Celular/biosíntesis , Proteínas Cromosómicas no Histona/biosíntesis , Elementos de Facilitación Genéticos , Músculo Esquelético/metabolismo , Miogenina/biosíntesis , ARN no Traducido/metabolismo , Transcripción Genética , Animales , Proteínas de Ciclo Celular/genética , Diferenciación Celular , Cromatina/genética , Cromatina/metabolismo , Proteínas Cromosómicas no Histona/genética , Células HEK293 , Humanos , Ratones , Músculo Esquelético/citología , Proteína MioD/biosíntesis , Proteína MioD/genética , Miogenina/genética , ARN no Traducido/genética , CohesinasRESUMEN
Many genes related to muscle growth modulate myoblast proliferation and differentiation and promote muscle hypertrophy. MyoD is a myogenic determinant that contributes to myoblast determination, and insulin-like growth factor 1 (IGF-I) interacts with MyoD to regulate muscle hypertrophy and muscle mass. In this study, we aimed to assess DNA methylation and mRNA expression patterns of MyoD and IGF-I during different developmental stages of Japanese flounder, and to examine the relationship between MyoD and IGF-I gene. DNA and RNA were extracted from muscles, and DNA methylation of MyoD and IGF-I promoter and exons was detected by bisulfite sequencing. The relative expression of MyoD and IGF-I was measured by quantitative polymerase chain reaction. IGF-I was measured by radioimmunoassay. Interestingly, the lowest expression of MyoD and IGF-I emerged at larva stage, and the mRNA expression was negatively associated with methylation. We hypothesized that many skeletal muscle were required to complete metamorphosis; thus, the expression levels of MyoD and IGF-I genes increased from larva stage and then decreased. The relative expression levels of MyoD and IGF-I exhibited similar patterns, suggesting that MyoD and IGF-I regulated muscle growth through combined effects. Changes in the concentrations of IGF-I hormone were similar to those of IGF-I gene expression. Our results the mechanism through which MyoD and IGF-I regulate muscle development and demonstrated that MyoD interacted with IGF-I to regulate muscle growth during different developmental stages.
Asunto(s)
Metilación de ADN/fisiología , Proteínas de Peces/biosíntesis , Lenguado/embriología , Factor I del Crecimiento Similar a la Insulina/biosíntesis , Músculo Esquelético/embriología , Proteína MioD/biosíntesis , Animales , Proteínas de Peces/genética , Lenguado/genética , Factor I del Crecimiento Similar a la Insulina/genética , Proteína MioD/genéticaRESUMEN
The metabolic perturbation caused by calorie restriction enhances muscle repair by playing a critical role in regulating satellite cell availability and activity in the muscles of young and old mice. To clarify the underlying mechanisms we asked whether myoblast replication and differentiation are affected by metformin, a calorie restriction-mimicking drug. C2C12, a mouse myoblast cell line, readily differentiate in vitro and fuse to form myotubes. However, when incubated with metformin, C2C12 slow their replication and do not differentiate. Interestingly, lower doses of metformin promote myogenic differentiation. We observe that metformin treatment modulates the expression of cyclins and cyclin inhibitors thereby inducing a cell cycle perturbation that causes a delay in the G2/M transition. The effect of metformin treatment is reversible since after drug withdrawal, myoblasts can re-enter the cell cycle and/or differentiate, depending on culture conditions. Myoblasts cultured under metformin treatment fail to up-regulate MyoD and p21cip1, a key step in cell cycle exit and terminal differentiation. Although the details of the molecular mechanisms underlying the effect of the drug on myoblasts still need to be clarified, we propose that metformin negatively affects myogenic differentiation by inhibiting irreversible exit from the cell cycle through reduction of MyoD and p21cip1 levels.
Asunto(s)
Ciclo Celular/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Metformina/farmacología , Desarrollo de Músculos/efectos de los fármacos , Animales , Restricción Calórica , Línea Celular , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/biosíntesis , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Humanos , Ratones , Proteína MioD/biosíntesis , Mioblastos/efectos de los fármacosRESUMEN
Quercetin is a natural product that is sold as a supplement in health food stores. While there are reported benefits for this flavonoid as a dietary supplement due to antioxidant properties, the full scope of its biological interactions has not been fully addressed. To learn more about the mechanisms of action related to quercetin, we exposed zebrafish (Danio rerio) embryos to 1 and 10µg/L quercetin for 96h starting at 3h post fertilization. Quercetin up to 10µg/L did not induce significant mortality in developing fish, but did increase prevalence of an upward-curved dorsal plane in hatched larvae. To determine whether this developmental defect was potentially related to mitochondrial bioenergetics during development, we measured oxygen consumption rate in whole embryos following a 24-hour exposure to quercetin. Basal mitochondrial and ATP-linked respiration were decreased at 1 and 10µg/L quercetin, and maximal respiration was decreased at 10µg/L quercetin, suggesting that quercetin impairs mitochondrial bioenergetics. This is proposed to be related to the deformities observed during development. Due to the fact that ATP production was affected by quercetin, larval behaviors related to locomotion were investigated, as well as transcriptional responses of six myogenesis transcripts. Quercetin at 10µg/L significantly reduced the swimming velocity of zebrafish larvae. The expression levels of both myostatin A (mstna) and myogenic differentiation (myoD) were also altered by quercetin. Mstna, an inhibitory factor for myogenesis, was significantly increased at 1µg/L quercetin exposure, while myoD, a stimulatory factor for myogenesis, was significantly increased at 10µg/L quercetin exposure. There were no changes in transcripts related to apoptosis (bcl2, bax, casp3, casp7), but we did observe a decrease in mRNA levels for catalase (cat) in fish exposed to each dose, supporting an oxidative stress response. Our data support the hypothesis that quercetin may affect locomotion and induce deformities in zebrafish larvae by diminishing ATP production and by altering the expression of transcripts related to muscle formation and activity.
Asunto(s)
Suplementos Dietéticos/toxicidad , Metabolismo Energético/efectos de los fármacos , Locomoción/efectos de los fármacos , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Quercetina/toxicidad , Anomalías Inducidas por Medicamentos/patología , Adenosina Trifosfato/biosíntesis , Animales , Conducta Animal/efectos de los fármacos , Femenino , Larva , Masculino , Desarrollo de Músculos/efectos de los fármacos , Desarrollo de Músculos/genética , Proteína MioD/biosíntesis , Proteína MioD/genética , Estrés Oxidativo/efectos de los fármacos , Consumo de Oxígeno/efectos de los fármacos , Natación , Transcripción Genética/efectos de los fármacos , Contaminantes Químicos del Agua , Pez CebraRESUMEN
Muscle regeneration is an important process for skeletal muscle growth and recovery. Repair of muscle damage is exquisitely programmed by cellular mechanisms inherent in myogenic stem cells, also known as muscle satellite cells. We demonstrated previously the involvement of homeobox transcription factors, SIX1, SIX4 and SIX5, in the coordinated proliferation and differentiation of isolated satellite cells in vitro. However, their roles in adult muscle regeneration in vivo remain elusive. To investigate SIX4 and SIX5 functions during muscle regeneration, we introduced knockout alleles of Six4 and Six5 into an animal model of Duchenne Muscular Dystrophy (DMD), mdx (Dmd(mdx) /Y) mice, characterized by frequent degeneration-regeneration cycles in muscles. A lower number of small myofibers, higher number of thick ones and lower serum creatine kinase and lactate dehydrogenase activities were noted in 50-week-old Six4(+/-) 5(+/-) Dmd(mdx) /Y mice than Dmd(mdx) /Y mice, indicating improvement of dystrophic phenotypes of Dmd(mdx) /Y mice. Higher proportions of cells positive for MYOD1 and MYOG (markers of regenerating myonuclei) and SIX1 (a marker of regenerating myoblasts and newly regenerated myofibers) in 12-week-old Six4(+/-) 5(+/-) Dmd(mdx) /Y mice suggested enhanced regeneration, compared with Dmd(mdx) /Y mice. Although grip strength was comparable in Six4(+/-) 5(+/-) Dmd(mdx) /Y and Dmd(mdx) /Y mice, treadmill exercise did not induce muscle weakness in Six4(+/-) 5(+/-) Dmd(mdx) /Y mice, suggesting higher regeneration capacity. In addition, Six4(+/-) 5(+/-) Dmd(mdx) /Y mice showed 33.8% extension of life span. The results indicated that low Six4 and Six5 gene dosage improved dystrophic phenotypes of Dmd(mdx) /Y mice by enhancing muscle regeneration, and suggested that SIX4 and SIX5 are potentially useful de novo targets in therapeutic applications against muscle disorders, including DMD.
Asunto(s)
Dosificación de Gen/fisiología , Proteínas de Homeodominio/metabolismo , Longevidad/fisiología , Músculo Esquelético/fisiología , Regeneración/fisiología , Transactivadores/metabolismo , Animales , Proteínas de Homeodominio/genética , Ratones , Ratones Endogámicos mdx , Ratones Noqueados , Proteína MioD/biosíntesis , Proteína MioD/genética , Miogenina/biosíntesis , Miogenina/genética , Transactivadores/genéticaRESUMEN
Muscle weakness is considered the pivotal sign of amyotrophic lateral sclerosis (ALS). Knowledge about the skeletal muscle degeneration/regeneration process and the myogenic potential is limited in ALS patients. Therefore, we investigate these processes in a time course perspective by analysing skeletal muscle biopsies from ALS patients collected before and after a 12-week period of normal daily activities and compare these with healthy age-matched control tissue. We do this by evaluating mRNA and protein (immunohistochemical) markers of regeneration, neurodegeneration, myogenesis, cell cycle regulation, and inflammation. Our results show morphological changes indicative of active denervation and reinnervation and an increase in small atrophic fibres. We demonstrate differences between ALS and controls in pathways controlling skeletal muscle homeostasis, cytoskeletal and regenerative markers, neurodegenerative factors, myogenic factors, cell cycle determinants, and inflammatory markers. Our results on Pax7 and MyoD protein expression suggest that proliferation and differentiation of skeletal muscle stem cells are affected in ALS patients, and the myogenic processes cannot overcome the denervation-induced wasting.
Asunto(s)
Esclerosis Amiotrófica Lateral/fisiopatología , Inflamación/genética , Desarrollo de Músculos/genética , Proteína MioD/biosíntesis , Factor de Transcripción PAX7/biosíntesis , Anciano , Biopsia , Proteínas de Ciclo Celular/biosíntesis , Proteínas de Ciclo Celular/genética , Diferenciación Celular/genética , Regulación del Desarrollo de la Expresión Génica , Voluntarios Sanos , Humanos , Inflamación/patología , Inflamación/fisiopatología , MicroARNs/biosíntesis , MicroARNs/genética , Persona de Mediana Edad , Músculo Esquelético/inervación , Músculo Esquelético/metabolismo , Músculo Esquelético/fisiopatología , Proteína MioD/genética , Factor de Transcripción PAX7/genética , Regeneración/genética , Células Madre/metabolismoRESUMEN
In Duchenne muscular dystrophy (DMD), the search for new biomarkers to follow the evolution of the disease is of fundamental importance in the light of the evolving gene and pharmacological therapies. In addition to the lack of dystrophin, secondary events including changes in calcium levels, inflammation and fibrosis greatly contribute to DMD progression and the molecules involved in these events may represent potential biomarkers. In this study, we performed a comparative evaluation of the progression of dystrophy within muscles that are differently affected by dystrophy (diaphragm; DIA and quadriceps; QDR) or spared (intrinsic laryngeal muscles) using the mdx mice model of DMD. We assessed muscle levels of calsequestrin (calcium-related protein), tumour necrosis factor (TNF-α; pro-inflammatory cytokine), tumour growth factor (TGF-ß; pro-fibrotic factor) and MyoD (muscle proliferation) vs. histopathology at early (1 and 4 months of age) and late (9 months of age) stages of dystrophy. Fibrosis was the primary feature in the DIA of mdx mice (9 months: 32% fibrosis), which was greater than in the QDR (9 months: 0.6% fibrosis). Muscle regeneration was the primary feature in the QDR (9 months: 90% of centrally nucleated fibres areas vs. 33% in the DIA). The QDR expressed higher levels of calsequestrin than the DIA. Laryngeal muscles showed normal levels of TNF-α, TGF-ß and MyoD. A positive correlation between histopathology and cytokine levels was observed only in the diaphragm, suggesting that TNF-α and TGF-ß serve as markers of dystrophy primarily for the diaphragm.
Asunto(s)
Biomarcadores/análisis , Diafragma/metabolismo , Músculos Laríngeos/metabolismo , Distrofia Muscular de Duchenne/metabolismo , Músculo Cuádriceps/metabolismo , Animales , Western Blotting , Calsecuestrina/análisis , Calsecuestrina/biosíntesis , Diafragma/patología , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Femenino , Técnica del Anticuerpo Fluorescente , Músculos Laríngeos/patología , Masculino , Ratones , Ratones Endogámicos mdx , Proteína MioD/análisis , Proteína MioD/biosíntesis , Músculo Cuádriceps/patología , Factor de Crecimiento Transformador beta/análisis , Factor de Crecimiento Transformador beta/biosíntesis , Factor de Necrosis Tumoral alfa/análisis , Factor de Necrosis Tumoral alfa/biosíntesisRESUMEN
The transcription factor Pax7 regulates skeletal muscle stem cell (satellite cells) specification and maintenance through various mechanisms, including repressing the activity of the muscle regulatory factor MyoD. Hence, Pax7-to-MyoD protein ratios can determine maintenance of the committed-undifferentiated state or activation of the differentiation program. Pax7 expression decreases sharply in differentiating myoblasts but is maintained in cells (re)acquiring quiescence, yet the mechanisms regulating Pax7 levels based on differentiation status are not well understood. Here we show that Pax7 levels are directly regulated by the ubiquitin-ligase Nedd4. Our results indicate that Nedd4 is expressed in quiescent and activated satellite cells, that Nedd4 and Pax7 physically interact during early muscle differentiation-correlating with Pax7 ubiquitination and decline-and that Nedd4 loss of function prevented this effect. Furthermore, even transient nuclear accumulation of Nedd4 induced a drop in Pax7 levels and precocious muscle differentiation. Consequently, we propose that Nedd4 functions as a novel Pax7 regulator, which activity is temporally and spatially controlled to modulate the Pax7 protein levels and therefore satellite cell fate.
Asunto(s)
Diferenciación Celular/genética , Complejos de Clasificación Endosomal Requeridos para el Transporte/biosíntesis , Desarrollo de Músculos , Factor de Transcripción PAX7/biosíntesis , Células Satélite del Músculo Esquelético/metabolismo , Ubiquitina-Proteína Ligasas/biosíntesis , Animales , Proliferación Celular/genética , Complejos de Clasificación Endosomal Requeridos para el Transporte/genética , Regulación del Desarrollo de la Expresión Génica , Humanos , Ratones , Músculo Esquelético/crecimiento & desarrollo , Músculo Esquelético/metabolismo , Proteína MioD/biosíntesis , Ubiquitina-Proteína Ligasas Nedd4 , Factor de Transcripción PAX7/genética , Complejo de la Endopetidasa Proteasomal/genética , Células Satélite del Músculo Esquelético/citología , Ubiquitina-Proteína Ligasas/genética , UbiquitinaciónRESUMEN
There is increasing evidence that WT1 protein expression is found not only at nuclear, but also at cytoplasmic, level in several developing and neoplastic tissues. In order to better understand the possible role of WT1 protein in human skeletal myogenesis and oncogenesis of rhabdomyosarcoma, we assessed immunohistochemically its comparative expression in a large series of human developing, adult and neoplastic skeletal muscle tissues. The present study shows that WT1 protein is developmentally expressed in the cytoplasm of human myoblasts from the 6 weeks of gestational age. This expression was maintained in the myotubes of developing muscles of the trunk, head, neck, and extremities, while it was down-regulated in fetal skeletal fibers from 20 weeks of gestational age as well as in adult normal skeletal muscle. Notably, WT1 immunostaining disappeared from rhabdomyomas, whereas it was strongly and diffusely re-expressed in all cases (27/27) of embryonal and alveolar rhabdomyosarcoma. The comparative evaluation of the immunohistochemical findings revealed that WT1 cytoplasmic expression in rhabdomyosarcoma may represent an ontogenetic reversal, and this nuclear transcription factor can also be considered an oncofetal protein which can be exploitable as an additional, highly sensitive immunomarker, together with desmin, myogenin and MyoD1, of this tumor. Moreover, our observations support the rationale for the use of WT1 protein-based target therapy in high risk rhabdomyosarcomas in children and adolescents.
Asunto(s)
Feto/embriología , Regulación del Desarrollo de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Neoplasias de los Músculos/metabolismo , Mioblastos Esqueléticos/metabolismo , Rabdomiosarcoma/metabolismo , Proteínas WT1/biosíntesis , Adolescente , Adulto , Niño , Preescolar , Femenino , Feto/patología , Humanos , Lactante , Masculino , Neoplasias de los Músculos/patología , Proteína MioD/biosíntesis , Mioblastos Esqueléticos/patología , Rabdomiosarcoma/patologíaRESUMEN
Wnt proteins are secreted cytokines and several Wnts are expressed in the developing somites and surrounding tissues. Without proper Wnt stimulation, the organization of the dermomyotome and myotome can become defective. These Wnt signals received by somitic cells can lead to activation of Pax3/Pax7 and myogenic regulatory factors (MRFs), especially Myf5 and MyoD. However, it is currently unknown whether Wnts activate Myf5 and MyoD through direct targeting of their cis-regulatory elements or via indirect pathways. To clarify this issue, in the present study, we tested the regulation of MyoD cis-regulatory elements by Wnt3a secreted from human embryonic kidney (HEK)-293T cells. We found that Wnt3a activated the MyoD proximal 6.0k promoter (P6P) only marginally, but highly enhanced the activity of the composite P6P plus distal enhancer (DE) reporter through canonical and non-canonical pathways. Further screening of the intervening fragments between the DE and the P6P identified a strong Wnt-response element (WRE) in the upstream -8 to -9k region (L fragment) that acted independently of the DE, but was dependent on the P6P. Deletion of a Pax3/Pax7-targeted site in the L fragment significantly reduced its response to Wnt3a, implying that Wnt3a activates the L fragment partially through Pax3/Pax7 action. Binding of ß-catenin and Pax7 to their target sites in the DE and the L fragment respectively was also demonstrated by ChIP. These observations demonstrated the first time that Wnt3a can directly activate MyoD expression through targeting cis-elements in the DE and the L fragment.
Asunto(s)
Regulación de la Expresión Génica/fisiología , Proteína MioD/biosíntesis , Elementos de Respuesta/fisiología , Vía de Señalización Wnt/fisiología , Proteína Wnt3A/metabolismo , Línea Celular , Humanos , Proteína MioD/genética , Factor 5 Regulador Miogénico/genética , Factor 5 Regulador Miogénico/metabolismo , Factor de Transcripción PAX3 , Factor de Transcripción PAX7/genética , Factor de Transcripción PAX7/metabolismo , Factores de Transcripción Paired Box/genética , Factores de Transcripción Paired Box/metabolismo , Proteína Wnt3A/genéticaRESUMEN
Setdb1, an H3-K9 specific histone methyltransferase, is associated with transcriptional silencing of euchromatic genes through chromatin modification. Functions of Setdb1 during development have been extensively studied in embryonic and mesenchymal stem cells as well as neurogenic progenitor cells. But the role of Sedtdb1 in myogenic differentiation remains unknown. In this study, we report that Setdb1 is required for myogenic potential of C2C12 myoblast cells through maintaining the expressions of MyoD and muscle-specific genes. We find that reduced Setdb1 expression in C2C12 myoblast cells severely delayed differentiation of C2C12 myoblast cells, whereas exogenous Setdb1 expression had little effect on. Gene expression profiling analysis using oligonucleotide micro-array and RNA-Seq technologies demonstrated that depletion of Setdb1 results in downregulation of MyoD as well as the components of muscle fiber in proliferating C2C12 cells. In addition, exogenous expression of MyoD reversed transcriptional repression of MyoD promoter-driven lucif-erase reporter by Setdb1 shRNA and rescued myogenic differentiation of C2C12 myoblast cells depleted of endogenous Setdb1. Taken together, these results provide new insights into how levels of key myogenic regulators are maintained prior to induction of differentiation.
Asunto(s)
Proteína MioD/biosíntesis , Mioblastos/citología , Proteína Metiltransferasas/genética , Proteína Metiltransferasas/metabolismo , Animales , Diferenciación Celular/fisiología , Línea Celular , N-Metiltransferasa de Histona-Lisina , Humanos , Ratones , Ratones Endogámicos C3H , Mioblastos/metabolismo , Transcriptoma , TransfecciónRESUMEN
Many studies have reported that CCN family protein 2 (also known as connective tissue growth factor) induces fibrotic response in skeletal muscle, thus emphasizing the pathological role of CCN2 in muscle tissues. However, the physiological role of CCN2 in myogenesis is still unknown. This study clarified the CCN2 functions during myogenesis. Recombinant CCN2 (rCCN2) promoted proliferation and MyoD production in C2C12 cells and primary myoblasts, but inhibited myogenin production. In accordance with these findings, the gene expression levels of myosin heavy chain, which is a marker of terminally differentiated myoblasts and desmin, which is the main intermediate filament protein of muscle cells, were decreased by rCCN2 treatment. In vivo analyses with Ccn2-deficient skeletal muscle revealed decreased proliferating cell nuclear antigen (PCNA)/MyoD double positive cells and muscle hypoplasia. Consistent with this finding, myogenic marker genes and myotube formation were repressed in Ccn2-deficient myoblasts. The protein production of CCN2 was increased in C2C12 myoblasts treated with tumor necrosis factor-α, which is a pro-inflammatory cytokine, suggesting its role in muscle regeneration after inflammation. These findings indicate that CCN2 promotes proliferation and early differentiation but inhibits the terminal differentiation of myoblasts, thus suggesting that CCN2 plays a physiological role in myogenesis.
Asunto(s)
Diferenciación Celular/genética , Factor de Crecimiento del Tejido Conjuntivo/metabolismo , Desarrollo de Músculos/genética , Factor de Crecimiento del Tejido Conjuntivo/genética , Regulación del Desarrollo de la Expresión Génica , Humanos , Músculo Esquelético/crecimiento & desarrollo , Músculo Esquelético/metabolismo , Proteína MioD/biosíntesis , Proteína MioD/metabolismo , Mioblastos/metabolismoRESUMEN
Heat stress could promote skeletal muscle regeneration. But, in the regeneration process, effects of heat stress on myogenic cells and the regulating factors is unknown. Therefore, Influences of heat stress soon after injury on distribution of the myogenic cells and chronological changes in expression of MyoD and myogenin were examined. The first peak of MyoD expression was temporally correlated with the time when proliferating satellite cells began to appear, and the rapid decline of the MyoD expression from the first peak, with the appearance time of myoblasts, respectively in both the non-Heat and Heat groups. The first peak of myogenin expression was temporally correlated with the time when multinuclear cells began to form in the both groups. Due to the heat stress, proliferation and differentiation of myogenic cells and chronological changes in these factors were accelerated one day earlier than in the non-Heat group. As MyoD and myogenin are regulating factor of proliferation and differentiation, heat stress soon after the muscle injury could accelerate the proliferation and differentiation of myogenic cells and the expression of their regulating factors MyoD and myogenin.