RESUMEN
Among many factors of controlling stem cell differentiation, the key transcription factor upregulation via physical force is a good strategy on the lineage-specific differentiation of stem cells. The study aimed to compare growth and myogenic potentials between the parental cells (PCs) and the 1-day-old C2C12 spheroid-derived cells (SDCs) in two-dimensional (2D) and three-dimensional (3D) culture conditions through examination of the cell proliferation and the expression of myogenic genes. The data showed that 1-day-old spheroids had more intense expression of MyoD gene with respect to the PCs. The proliferation of the SDCs is significantly higher than the PCs in a time-dependent manner. The SDCs had also significantly higher myogenic potential than the PCs in 2D and 3D culture conditions. The results suggest that MyoD gene upregulation through cell-cell contacts is the good approach for preparation of seed cells in muscle tissue engineering.
Asunto(s)
Técnicas de Cultivo de Célula , Células Musculares/metabolismo , Desarrollo de Músculos/genética , Proteína MioD/genética , Mioblastos/metabolismo , Esferoides Celulares/metabolismo , Actinina/genética , Actinina/metabolismo , Animales , Diferenciación Celular , Línea Celular , Proliferación Celular/efectos de los fármacos , Colágeno/química , Colágeno/farmacología , Regulación de la Expresión Génica , Ratones , Células Musculares/citología , Células Musculares/efectos de los fármacos , Desarrollo de Músculos/efectos de los fármacos , Proteína MioD/antagonistas & inhibidores , Proteína MioD/metabolismo , Mioblastos/citología , Mioblastos/efectos de los fármacos , Miogenina/genética , Miogenina/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Esferoides Celulares/citología , Esferoides Celulares/efectos de los fármacos , Ingeniería de Tejidos/métodosRESUMEN
Satellite cells are skeletal muscle stem cells that provide myonuclei for postnatal muscle growth, maintenance, and repair/regeneration in adults. Normally, satellite cells are mitotically quiescent, but they are activated in response to muscle injury, in which case they proliferate extensively and exhibit up-regulated expression of the transcription factor MyoD, a master regulator of myogenesis. MyoD forms a heterodimer with E proteins through their basic helix-loop-helix domain, binds to E boxes in the genome and thereby activates transcription at muscle-specific promoters. The central role of MyoD in muscle differentiation has increased interest in finding potential MyoD regulators. Here we identified transducin-like enhancer of split (TLE3), one of the Groucho/TLE family members, as a regulator of MyoD function during myogenesis. TLE3 was expressed in activated and proliferative satellite cells in which increased TLE3 levels suppressed myogenic differentiation, and, conversely, reduced TLE3 levels promoted myogenesis with a concomitant increase in proliferation. We found that, via its glutamine- and serine/proline-rich domains, TLE3 interferes with MyoD function by disrupting the association between the basic helix-loop-helix domain of MyoD and E proteins. Our findings indicate that TLE3 participates in skeletal muscle homeostasis by dampening satellite cell differentiation via repression of MyoD transcriptional activity.
Asunto(s)
Proteínas Co-Represoras/metabolismo , Regulación del Desarrollo de la Expresión Génica , Desarrollo de Músculos , Fibras Musculares Esqueléticas/metabolismo , Proteína MioD/antagonistas & inhibidores , Mioblastos/metabolismo , Células Satélite del Músculo Esquelético/metabolismo , Factor de Transcripción Activador 3/química , Factor de Transcripción Activador 3/genética , Factor de Transcripción Activador 3/metabolismo , Animales , Proliferación Celular , Células Cultivadas , Proteínas Co-Represoras/antagonistas & inhibidores , Proteínas Co-Represoras/química , Proteínas Co-Represoras/genética , Eliminación de Gen , Secuencias Hélice-Asa-Hélice , Masculino , Ratones , Ratones Endogámicos C57BL , Fibras Musculares Esqueléticas/citología , Proteína MioD/química , Proteína MioD/genética , Proteína MioD/metabolismo , Mioblastos/citología , Fragmentos de Péptidos/antagonistas & inhibidores , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Dominios y Motivos de Interacción de Proteínas , Multimerización de Proteína , Interferencia de ARN , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Células Satélite del Músculo Esquelético/citologíaRESUMEN
BACKGROUND/AIMS: Vascular muscularity is a key event in vessel remodeling during pulmonary artery hypertension (PAH). Endothelial-mesenchymal transdifferentiation (EndMT) has been increasingly reported to play a role in disease occurrence. Galectin-3, a carbohydrate-binding protein regulates cell proliferation, differentiation, migration and neovascularization. However, whether galectin-3 controls endothelial cell transdifferentiation during the development of PAH is unknown. METHODS: Rats were exposed to normoxic or hypoxic conditions (fraction of inspired O2 0.10) for 21 d to establish PAH models. Hemodynamic changes were evaluated through surgery of the right jugular vein and ultrasound biomicroscopy inviVue. And vessel pathological alterations were detected by H&E staining. Galectin-3 (Gal-3)-induced pulmonary artery endothelium cell (PAEC) dynamic alterations were measured by MTT assays, Cell immunofluorescence, Flow cytometry, Real-time PCR and Western blot. RESULTS: Our study demonstrated that Gal-3 was expressed in hypoxic pulmonary vascular adventitia and intima. The increased Gal-3 expression was responsible for hypoxic vessel remodeling and PAH development in vivo. Gal-3 was found to inhibit cell proliferation and apoptosis in cultured endothelial cells. Meanwhile endothelial cell morphology was altered and exhibited smooth muscle-like cell features as demonstrated by the expression of α-SMA after Gal-3 treatment. Gal-3 activated Jagged1/Notch1 pathways and induced MyoD and SRF. When MyoD or SRF were silenced with siRNAs, Gal-3-initiated transdifferentiation in endothelial cells was blocked as indicated by a lack of α-SMA. CONCLUSION: These results suggest that Gal-3 induces PAECs to acquire an α-SMA phenotype via a transdifferentiation process which depends on the activation of Jagged1/Notch1 pathways that mediate MyoD and SRF expression.
Asunto(s)
Transdiferenciación Celular , Galectina 3/metabolismo , Remodelación Vascular , Animales , Proteínas de Ciclo Celular/metabolismo , Supervivencia Celular/efectos de los fármacos , Transdiferenciación Celular/efectos de los fármacos , Células Endoteliales/citología , Células Endoteliales/metabolismo , Galectina 3/antagonistas & inhibidores , Galectina 3/genética , Humanos , Hipertensión Pulmonar/metabolismo , Hipertensión Pulmonar/patología , Pulmón/metabolismo , Masculino , Proteína MioD/antagonistas & inhibidores , Proteína MioD/genética , Proteína MioD/metabolismo , Arteria Pulmonar/citología , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Ratas , Ratas Wistar , Receptor Notch1/metabolismo , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/farmacología , Factor de Respuesta Sérica/antagonistas & inhibidores , Factor de Respuesta Sérica/genética , Factor de Respuesta Sérica/metabolismo , Remodelación Vascular/efectos de los fármacosRESUMEN
Chromosome 3q alterations occur frequently in many types of tumours. In a genetic screen for loci present in rhabdomyosarcomas, we identified an isochromosome 3q [i(3q)], which inhibits muscle differentiation when transferred into myoblasts. The i(3q) inhibits MyoD function, resulting in a non-differentiating phenotype. Furthermore, the i(3q) induces a 'cut' phenotype, abnormal centrosome amplification, aneuploidy and loss of G1 arrest following gamma-irradiation. Testing candidate genes within this region reveals that forced expression of ataxia-telangiectasia and rad3-related (ATR) results in a phenocopy of the i(3q). Thus, genetic alteration of ATR leads to loss of differentiation as well as cell-cycle abnormalities.
Asunto(s)
Aneuploidia , Proteínas de Ciclo Celular/genética , Fase G1/efectos de la radiación , Familia de Multigenes , Proteína MioD/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas , Proteínas de la Ataxia Telangiectasia Mutada , División Celular , Cromosomas Humanos Par 3 , Humanos , Isocromosomas , Músculos/citología , Proteína MioD/fisiología , Rabdomiosarcoma/genética , Rabdomiosarcoma/patología , Células Tumorales CultivadasRESUMEN
Postnatal growth and regeneration of skeletal muscle requires a population of resident myogenic precursors named satellite cells. The transcription factor Pax7 is critical for satellite cell biogenesis and survival and has been also implicated in satellite cell self-renewal; however, the underlying molecular mechanisms remain unclear. Previously, we showed that Pax7 overexpression in adult primary myoblasts down-regulates MyoD and prevents myogenin induction, inhibiting myogenesis. We show that Pax7 prevents muscle differentiation independently of its transcriptional activity, affecting MyoD function. Conversely, myogenin directly affects Pax7 expression and may be critical for Pax7 down-regulation in differentiating cells. Our results provide evidence for a cross-inhibitory interaction between Pax7 and members of the muscle regulatory factor family. This could represent an additional mechanism for the control of satellite cell fate decisions resulting in proliferation, differentiation, and self-renewal, necessary for skeletal muscle maintenance and repair.
Asunto(s)
Diferenciación Celular/fisiología , Proteína MioD/fisiología , Factor de Transcripción PAX7/fisiología , Células Satélite del Músculo Esquelético/citología , Animales , Línea Celular , Proliferación Celular , Regulación de la Expresión Génica , Ratones , Desarrollo de Músculos/fisiología , Proteína MioD/antagonistas & inhibidores , Miogenina/fisiología , Factor de Transcripción PAX7/antagonistas & inhibidores , Factor de Transcripción PAX7/química , Mapeo de Interacción de Proteínas , Estructura Terciaria de ProteínaRESUMEN
Previously, we found that MRFs (myogenic regulatory factors) regulated the expression of PGC-1alpha (peroxisome-proliferator-activated receptor gamma co-activator 1alpha) by targeting a short region, from nt -49 to +2 adjacent to the transcription initiation site, that contained two E-boxes. However, only the E2-box had significant affinity for MRFs, and the E1-box was predicted to be the target of Bhlhe40 (basic helix-loop-helix family, member e40, also known as Stra13, Bhlhb2, DEC1 and Sharp2), a transcriptional repressor implicated in the regulation of several physiological processes. In the present study, by using EMSA (electrophoresis mobility-shift assay), we confirmed that Bhlhe40 targeted the E1-box and formed a complex with the basic helix-loop-helix transcription factor MyoD (myogenic differentiation factor D) on the PGC-1alpha core promoter. We demonstrate that Bhlhe40 binds to the promoters of PGC-1alpha and myogenic genes in vivo and that Bhlhe40 represses the MyoD-mediated transactivation of these promoters. Furthermore, we found that this repression could be relieved by P/CAF (p300/CBP-associated factor) in a dose-dependent manner, but not by CBP [CREB (cAMP-response-element-binding protein)-binding protein]. Bhlhe40 interacted with P/CAF and this interaction disrupted the interaction between P/CAF and MyoD. These results suggest that Bhlhe40 functions as a repressor of MyoD by binding to adjacent E-boxes and sequestering P/CAF from MyoD.
Asunto(s)
Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/fisiología , Proteína MioD/antagonistas & inhibidores , Proteína MioD/genética , Supresión Genética , Activación Transcripcional/fisiología , Factores de Transcripción p300-CBP/fisiología , Animales , Línea Celular , Ratones , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma , Transactivadores/antagonistas & inhibidores , Transactivadores/biosíntesis , Transactivadores/genética , Factores de TranscripciónRESUMEN
Cancer cells with mesenchymal attributes potentially display chemoresistance. Cancer stem cells (CSCs), which are intrinsically resistant to most chemotherapy agents, exhibit considerable phenotypic heterogeneity in their epithelial versus mesenchymal states. However, the drug response of CSCs in the epithelial and mesenchymal states has not been completely investigated. In this study, we found that epithelial-type (E-cadherinhigh/CD133high) CSCs displayed a higher sphere formation ability and chemoresistance than mesenchymal-type (E-cadherinlowCD133high) CSCs. Gene expression profiling of the CSC and non-CSC subpopulations with distinct epithelial-to-mesenchymal transition (EMT) states showed that MyoD family inhibitor domain-containing (MDFIC) was selectively upregulated in epithelial-type CSCs. Knockdown of MDFIC sensitized epithelial-type CSCs to chemotherapy agents. Ectopic expression of MDFIC increased the chemoresistance of mesenchymal-type CSCs. In a tissue microarray, high MDFIC expression was associated with poor prognosis of non-small cell lung cancer (NSCLC) patients. A mechanistic study showed that the MDFIC p32 isoform, which is located in the cytoplasm, interacted with the destruction complex, Axin/GSK-3/ß-catenin. This interaction stabilized ß-catenin by inhibiting ß-catenin phosphorylation at S33/37 and increased the nuclear translocation and transcriptional activity of ß-catenin. Knockdown of ß-catenin decreased MDFIC-enhanced chemoresistance. These results suggested that the upregulation of MDFIC enhanced the chemoresistance of epithelial-type CSCs by elevating ß-catenin activity. Thus, targeting MDFIC-regulated ß-catenin signaling of epithelial-type CSCs may be a potential strategy to overcome chemoresistance in NSCLC.
Asunto(s)
Resistencia a Antineoplásicos/genética , Proteína MioD/antagonistas & inhibidores , Células Madre Neoplásicas/metabolismo , beta Catenina/metabolismo , Transición Epitelial-Mesenquimal , HumanosRESUMEN
The muscle-specific intermediate filament protein, desmin, is one of the earliest myogenic markers whose functional role during myogenic commitment and differentiation is unknown. Sequence comparison of the presently isolated and fully characterized mouse desmin cDNA clones revealed a single domain of polypeptide similarity between desmin and the basic and helix-loop-helix region of members of the myoD family myogenic regulators. This further substantiated the need to search for the function of desmin. Constructs designed to express anti-sense desmin RNA were used to obtain stably transfected C2C12 myoblast cell lines. Several lines were obtained where expression of the anti-sense desmin RNA inhibited the expression of desmin RNA and protein down to basal levels. As a consequence, the differentiation of these myoblasts was blocked; complete inhibition of myoblast fusion and myotube formation was observed. Rescue of the normal phenotype was achieved either by spontaneous revertants, or by overexpression of the desmin sense RNA in the defective cell lines. In several of the cell lines obtained, inhibition of desmin expression was followed by differential inhibition of the myogenic regulators myoD and/or myogenin, depending on the stage and extent of desmin inhibition in these cells. These data suggested that myogenesis is modulated by at least more than one pathway and desmin, which so far was believed to be merely an architectural protein, seems to play a key role in this process.
Asunto(s)
Fusión Celular , Desmina/biosíntesis , Expresión Génica/efectos de los fármacos , Músculos/fisiología , Proteína MioD/biosíntesis , Miogenina/biosíntesis , ARN sin Sentido/farmacología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Línea Celular , Secuencia de Consenso , ADN Complementario/metabolismo , Desmina/antagonistas & inhibidores , Secuencias Hélice-Asa-Hélice , Ratones , Datos de Secuencia Molecular , Músculos/citología , Músculos/metabolismo , Proteína MioD/antagonistas & inhibidores , Proteína MioD/metabolismo , Miogenina/antagonistas & inhibidores , Miogenina/metabolismo , ARN Mensajero/biosíntesis , Homología de Secuencia de Aminoácido , TransfecciónRESUMEN
Although the myogenic regulator MyoD is expressed in proliferating myoblasts, differentiation of these cells is limited to the G0 phase of the cell cycle. Forced expression of cyclin D1, but not cyclins A, B, or E, inhibited the ability of MyoD to transactivate muscle-specific genes and correlated with phosphorylation of MyoD. Transfection of myoblasts with cyclin-dependent kinase (Cdk) inhibitors p21 and p16 augmented muscle-specific gene expression in cells maintained in high concentrations of serum, suggesting that an active cyclin-Cdk complex suppresses MyoD function in proliferating cells.
Asunto(s)
Quinasas Ciclina-Dependientes/metabolismo , Ciclinas/fisiología , Músculo Esquelético/citología , Proteína MioD/antagonistas & inhibidores , Proteínas Oncogénicas/fisiología , Animales , Proteínas Portadoras/biosíntesis , Proteínas Portadoras/fisiología , Ciclo Celular , Diferenciación Celular , Línea Celular , Ciclina D1 , Inhibidor p16 de la Quinasa Dependiente de Ciclina , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Ciclinas/biosíntesis , Activación Enzimática , Ratones , Músculo Esquelético/metabolismo , Proteína MioD/metabolismo , Fosforilación , Activación Transcripcional , TransfecciónRESUMEN
The mechanism by which activation of the Hedgehog (Hh) pathway modulates differentiation and promotes oncogenesis in specific tissues is poorly understood. We therefore, analysed rhabdomyosarcomas from mice that were haploinsufficient for the Hh-binding protein, Hip1, or for the Hh receptor, Patched 1 (Ptch1). Transfection of the Hh-regulated transcription factor Gli1, which is expressed in a subset of mouse and human rhabdomyosarcomas, suppressed differentiation of myogenic rhabdomyosarcoma lines generated from Hip1+/- and Ptch1+/- mice. The closely related factor, Gli2, had similar effects. Gli1 and Gli2 inhibited myogenesis by repressing the capacity of MyoD to activate transcription. Deletion analysis of Gli1 indicated that multiple domains of Gli1 are required for efficient inhibition of MyoD. Gli1 reduced the ability of MyoD to heterodimerize with E12 and bind DNA, providing one mechanism whereby the Gli proteins modulate the activity of MyoD. This novel activity of Gli proteins provides new insights into how Hh signaling modulates terminal differentiation through inhibition of tissue-specific factors such as MyoD. This mechanism may contribute to the broad role of Hh signaling and the Gli proteins in differentiation decisions and cancer formation.
Asunto(s)
Diferenciación Celular , Factores de Transcripción de Tipo Kruppel/fisiología , Proteína MioD/antagonistas & inhibidores , Mioblastos/citología , Proteínas Oncogénicas/fisiología , Oncogenes/fisiología , Transactivadores/fisiología , Animales , Diferenciación Celular/genética , Línea Celular Tumoral , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Dimerización , Regulación hacia Abajo , Proteínas Hedgehog/metabolismo , Humanos , Factores de Transcripción de Tipo Kruppel/genética , Ratones , Ratones Mutantes , Mutación , Proteína MioD/metabolismo , Mioblastos/metabolismo , Proteínas Oncogénicas/genética , Oncogenes/genética , Receptores Patched , Receptor Patched-1 , Estructura Terciaria de Proteína , Receptores de Superficie Celular/genética , Eliminación de Secuencia , Factores de Transcripción TCF/metabolismo , Transactivadores/genética , Proteína 1 Similar al Factor de Transcripción 7 , Activación Transcripcional , Proteína con Dedos de Zinc GLI1 , Proteína Gli2 con Dedos de ZincRESUMEN
Muscle satellite cells have long been considered a distinct myogenic lineage responsible for postnatal growth, repair and maintenance of skeletal muscle. Recent studies have demonstrated that they are multi-potential. Osterix (Osx), a novel zinc-finger-containing transcription factor of the sp family, is required for osteoblast differentiation and bone formation. It was hypothesized that Osx overexpression would enhance osteoblast differentiation of muscle satellite cells in vitro. Recombinant adenovirus-mediated Osx gene (Ad-Osx) was constructed and used to transfect muscle satellite cells. Osx overexpression inhibited myogenesis, as demonstrated by suppression of MyoD and myogenin mRNA levels and reduced myotube formation. Muscle satellite cells transduced with Ad-Osx exhibited apparent osteoblast differentiation as determined by the expression of related osteoblastic genes, increased activity of alkaline phosphatase and the formation of mineralized nodules. These results confirmed the ability of Osx to enhance osteoblast differentiation of muscle satellite cells in vitro, and the competence of muscle satellite cells as promising seed cells for bone tissue engineering.
Asunto(s)
Osteoblastos/citología , Osteogénesis/genética , Células Satélite del Músculo Esquelético/citología , Ingeniería de Tejidos/métodos , Factores de Transcripción/biosíntesis , Animales , Diferenciación Celular , Células Cultivadas , Colágeno Tipo I/biosíntesis , Expresión Génica , Sialoproteína de Unión a Integrina , Células Madre Multipotentes/citología , Células Madre Multipotentes/metabolismo , Desarrollo de Músculos , Proteína MioD/antagonistas & inhibidores , Miogenina/antagonistas & inhibidores , Osteoblastos/metabolismo , Osteocalcina/biosíntesis , Osteopontina/biosíntesis , ARN Mensajero/análisis , Ratas , Ratas Sprague-Dawley , Proteínas Recombinantes , Células Satélite del Músculo Esquelético/metabolismo , Sialoglicoproteínas/biosíntesis , Factores de Transcripción/genética , Factores de Transcripción/fisiología , Transfección , Regulación hacia ArribaRESUMEN
MyoD upstream noncoding RNA (MUNC) initiates in the distal regulatory region (DRR) enhancer of MYOD and is formally classified as an enhancer RNA (DRReRNA). MUNC is required for optimal myogenic differentiation, induces specific myogenic transcripts in trans (MYOD, MYOGENIN, and MYH3), and has a functional human homolog. The vast majority of eRNAs are believed to act in cis primarily on their neighboring genes (1, 2), making it likely that MUNC action is dependent on the induction of MYOD RNA. Surprisingly, MUNC overexpression in MYOD-/- C2C12 cells induces many myogenic transcripts in the complete absence of MyoD protein. Genomewide analysis showed that, while many genes are regulated by MUNC in a MyoD-dependent manner, there is a set of genes that are regulated by MUNC, both upward and downward, independently of MyoD. MUNC and MyoD even appear to act antagonistically on certain transcripts. Deletion mutagenesis showed that there are at least two independent functional sites on the MUNC long noncoding RNA (lncRNA), with exon 1 more active than exon 2 and with very little activity from the intron. Thus, although MUNC is an eRNA of MYOD, it is also a trans-acting lncRNA whose sequence, structure, and cooperating factors, which include but are not limited to MyoD, determine the regulation of many myogenic genes.
Asunto(s)
Desarrollo de Músculos/genética , Proteína MioD/genética , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo , Animales , Línea Celular , Elementos de Facilitación Genéticos , Regulación del Desarrollo de la Expresión Génica , Técnicas de Inactivación de Genes , Humanos , Ratones , Modelos Biológicos , Músculo Esquelético/crecimiento & desarrollo , Músculo Esquelético/metabolismo , Proteína MioD/antagonistas & inhibidores , Proteína MioD/metabolismo , Miogenina/biosíntesis , Miogenina/genética , Cadenas Pesadas de Miosina/biosíntesis , Cadenas Pesadas de Miosina/genética , ARN Largo no Codificante/químicaRESUMEN
One obvious phenotype of tumor cells is the lack of terminal differentiation. We previously classified rhabdomyosarcoma cell lines as having either a recessive or a dominant nondifferentiating phenotype. To study the genetic basis of the dominant nondifferentiating phenotype, we utilized microcell fusion to transfer chromosomes from rhabdomyosarcoma cells into C2C12 myoblasts. Transfer of a derivative chromosome 14 inhibits differentiation. The derivative chromosome 14 contains a DNA amplification. MDM2 is amplified and overexpressed in these nondifferentiating hybrids and in the parental rhabdomyosarcoma. Forced expression of MDM2 inhibits MyoD-dependent transcription. Expression of antisense MDM2 restores MyoD-dependent transcriptional activity. We conclude that amplification and overexpression of MDM2 inhibit MyoD function, resulting in a dominant nondifferentiating phenotype.
Asunto(s)
Amplificación de Genes , Células Híbridas/patología , Proteínas Musculares/fisiología , Músculos/citología , Proteína MioD/antagonistas & inhibidores , Proteínas de Neoplasias/fisiología , Proteínas Nucleares , Proteínas Proto-Oncogénicas/fisiología , Rabdomiosarcoma/genética , Animales , Ciclo Celular , Diferenciación Celular , Fusión Celular , Cromosomas Humanos Par 14/genética , Epistasis Genética , Regulación de la Expresión Génica , Humanos , Ratones , Proteínas Musculares/genética , Proteína MioD/fisiología , Proteínas de Neoplasias/genética , Fenotipo , Reacción en Cadena de la Polimerasa , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas c-mdm2 , Rabdomiosarcoma/patología , Transcripción Genética , Células Tumorales CultivadasRESUMEN
p202 is a primarily nuclear, interferon-inducible murine protein that is encoded by the Ifi 202 gene. Overexpression of p202 in transfected cells retards cell proliferation. p202 modulates the pattern of gene expression by inhibiting the activity of various transcription factors including NF-kappaB, c-Fos, c-Jun, E2F-1, and p53. Here we report that p202 was constitutively expressed in mouse skeletal muscle and that the levels of 202 RNA and p202 greatly increased during the differentiation of cultured C2C12 myoblasts to myotubes. When overexpressed in transfected myoblasts, p202 inhibited the expression of one muscle protein (MyoD) without affecting the expression of a second one (myogenin). Thus, the decrease in the level of MyoD (but not of myogenin) during muscle differentiation may be the consequence of the increase in p202 level. Overexpressed p202 also inhibited the transcriptional activity of both MyoD and myogenin. This inhibition was correlated with an interaction of p202 with both proteins, as well as the inhibition by p202 of the sequence-specific binding of both proteins to DNA. This inhibition of the expression of MyoD and of the transcriptional activity of MyoD and myogenin may account for the inhibition of the induction of myoblast differentiation by premature overexpression of p202.
Asunto(s)
Proteínas Portadoras/metabolismo , Péptidos y Proteínas de Señalización Intracelular , Músculo Esquelético/citología , Proteína MioD/antagonistas & inhibidores , Proteínas Nucleares/metabolismo , Fosfoproteínas/metabolismo , Animales , Proteínas Portadoras/biosíntesis , Bovinos , Diferenciación Celular , Línea Celular , Proteínas Cromosómicas no Histona , Proteínas de Unión al ADN , Expresión Génica , Caballos , Ratones , Ratones Endogámicos BALB C , Músculo Esquelético/metabolismo , Proteína MioD/biosíntesis , Miogenina/metabolismo , Proteínas Nucleares/biosíntesis , Fosfoproteínas/biosíntesis , ARN/metabolismo , Distribución Tisular , Transfección , Proteína 1 de Unión al Supresor Tumoral P53RESUMEN
MyoD and Myf5 belong to the family of basic helix-loop-helix transcription factors that are key operators in skeletal muscle differentiation. MyoD and Myf5 genes are selectively activated during development in a time and region-specific manner and in response to different stimuli. However, molecules that specifically regulate the expression of these two genes and the pathways involved remain to be determined. We have recently shown that the serum response factor (SRF), a transcription factor involved in activation of both mitogenic response and muscle differentiation, is required for MyoD gene expression. We have investigated here whether SRF is also involved in the control of Myf5 gene expression, and the potential role of upstream regulators of SRF activity, the Rho family G-proteins including Rho, Rac, and CDC42, in the regulation of MyoD and Myf5. We show that inactivation of SRF does not alter Myf5 gene expression, whereas it causes a rapid extinction of MyoD gene expression. Furthermore, we show that RhoA, but not Rac or CDC42, is also required for the expression of MyoD. Indeed, blocking the activity of G-proteins using the general inhibitor lovastatin, or more specific antagonists of Rho proteins such as C3-transferase or dominant negative RhoA protein, resulted in a dramatic decrease of MyoD protein levels and promoter activity without any effects on Myf5 expression. We further show that RhoA-dependent transcriptional activation required functional SRF in C2 muscle cells. These data illustrate that MyoD and Myf5 are regulated by different upstream activation pathways in which MyoD expression is specifically modulated by a RhoA/SRF signaling cascade. In addition, our results establish the first link between RhoA protein activity and the expression of a key muscle regulator.
Asunto(s)
Toxinas Botulínicas , Proteínas de Unión al ADN/fisiología , GTP Fosfohidrolasas/fisiología , Proteínas de Unión al GTP/fisiología , Proteína MioD/biosíntesis , Proteínas Nucleares/fisiología , Células 3T3 , ADP Ribosa Transferasas/fisiología , Animales , Línea Celular , Proteínas de Unión al ADN/antagonistas & inhibidores , GTP Fosfohidrolasas/antagonistas & inhibidores , Proteínas de Unión al GTP/antagonistas & inhibidores , Regulación de la Expresión Génica , Genes Dominantes , Ratones , Proteínas Musculares/genética , Músculo Esquelético/citología , Músculo Esquelético/metabolismo , Músculo Esquelético/fisiología , Proteína MioD/antagonistas & inhibidores , Factor 5 Regulador Miogénico , Proteínas Nucleares/antagonistas & inhibidores , Regiones Promotoras Genéticas/fisiología , Ratas , Proteínas Represoras/fisiología , Factor de Respuesta Sérica , Transactivadores/genética , Proteína de Unión al GTP rhoARESUMEN
I-mfa (inhibitor of the MyoD family) is a transcription modulator that binds to and suppresses the transcriptional activity of MyoD family members. I-mfa transcripts are expressed in sclerotome, suggesting a role of I-mfa in skeletogenesis. The aim of this study was to examine the expression and regulation of I-mfa in osteoblasts. We found that I-mfa is expressed at a low level in an osteoblast-like cell line, MC3T3E1, and a pluripotent differentiation modulator, 1,25-dihydroxyvitamin D(3), specifically enhanced I-mfa mRNA expression. This effect was completely blocked by the presence of an RNA polymerase inhibitor, but not by a protein synthesis inhibitor, suggesting that 1,25-dihydroxyvitamin D(3) upregulates transcription of the I-mfa gene without requirement for new protein synthesis. Western blot analysis indicated that 1,25-dihydroxyvitamin D(3) increased the I-mfa protein levels severalfold in MC3T3E1 cells. I-mfa expression was also observed in primary mouse calvaria cells and ROS17/2.8 cells and 1,25-dihydroxyvitamin D(3) enhanced I-mfa expression in these cells. These data indicate that I-mfa is a novel transcriptional regulator gene expressed in osteoblasts and that its level is under the control of 1,25-dihydroxyvitamin D(3).
Asunto(s)
Colecalciferol/farmacología , Factores Reguladores Miogénicos/biosíntesis , Osteoblastos/efectos de los fármacos , Animales , Células Cultivadas , Cicloheximida/farmacología , Diclororribofuranosil Benzoimidazol , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/farmacología , Ratones , Proteína MioD/antagonistas & inhibidores , Factores Reguladores Miogénicos/genética , Osteoblastos/metabolismo , Inhibidores de la Síntesis de la Proteína/farmacología , ARN Mensajero/biosíntesis , Cráneo/efectos de los fármacos , Cráneo/metabolismo , Regulación hacia ArribaRESUMEN
It has been reported that muscles of myoD-/- mice present a lower potential to regenerate, but there are no reports on the effect of acute interference with myoD expression limited in space and time to only a particular regenerating muscle. Here we relied on antisense inhibition of this factor. Four different oligos were tested. The suppression of regeneration indices (the expression of desmin, the formation of myotubes and the initiation of endplates) was the most pronounced, with the oligomer targeting a region encompassing the translation start site of myoD. A mixed backbone phosphorothioate-phosphate diester oligo (200 microl at 20 microM) was still detectable in the muscles 1 h after its administration and reverse transcription-polymerase chain reaction (RT-PCR) analysis showed that the level of the targeted 5' end of the myoD mRNA was selectively decreased. The level of myoD protein was also lowered. Four hours after the antisense treatment, when the oligos were no longer detectable, the myoD mRNA level was restored and 24 h later it exceeded controls together with that of myf-5 and myogenin. After 4 weeks, the antisense-treated soleus muscles were similar to the control-treated and the untreated regenerated soleus with respect to fiber types and motor endplates, however, they contained smaller fibers which reflected the asynchronity of regeneration. This shows that successfully targeted simple antisense oligonucleotides can be used as selective tools for inhibition of individual factors in studying the process of muscle regeneration.
Asunto(s)
Proteínas Musculares/genética , Músculo Esquelético/fisiología , Proteína MioD/antagonistas & inhibidores , Miogenina/genética , Oligodesoxirribonucleótidos Antisentido/farmacología , Regeneración/genética , Proteínas Represoras , Transactivadores , Animales , Secuencia de Bases , Bromodesoxiuridina/metabolismo , Proteínas de Unión al ADN/genética , Desmina/metabolismo , Expresión Génica , Proteína 1 Inhibidora de la Diferenciación , Proteína 2 Inhibidora de la Diferenciación , Masculino , Músculo Esquelético/efectos de los fármacos , Proteína MioD/genética , Proteína MioD/fisiología , Factor 5 Regulador Miogénico , Cadenas Pesadas de Miosina/metabolismo , Oligodesoxirribonucleótidos Antisentido/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Ratas Wistar , Regeneración/efectos de los fármacos , Factores de Transcripción/genéticaRESUMEN
The calcium-activated cysteine protease m-calpain plays a pivotal role during the earlier stages of myogenesis, particularly during fusion. The enzyme is a heterodimer, encoded by the genes capn2, for the large subunit, and capn4, for the small subunit. To study the regulation of m-calpain, the DNA sequence upstream of capn2 was analyzed for promoter elements, revealing the existence of five consensus-binding sites (E-box) for several myogenic regulatory factors and one binding site for myocyte enhancer factor-2 (MEF-2). Transient transfections with reporter gene constructs containing the E-box revealed that MyoD presents a high level of transactivation of reporter constructs containing this region, in particular the sequences including the MEF-2/E4-box. In addition, over-expression of various myogenic factors demonstrated that MyoD and myogenin with much less efficiency, can up-regulate capn2, both singly and synergistically, while Myf5 has no effect on synthesis of the protease. Experiments with antisense oligonucleotides directed against each myogenic factor revealed that MyoD plays a specific and pivotal role during capn2 regulation, and cannot be replaced wholly by myogenin and Myf5.
Asunto(s)
Calpaína/genética , Proteínas de Unión al ADN/fisiología , Regulación de la Expresión Génica , Desarrollo de Músculos/fisiología , Proteína MioD/genética , Mioblastos/fisiología , Miogenina/genética , Regiones Promotoras Genéticas , Transactivadores , Factores de Transcripción/fisiología , Activación Transcripcional/genética , Animales , Secuencia de Bases , Sitios de Unión , Western Blotting , Células COS , Calpaína/metabolismo , Células Cultivadas , Chlorocebus aethiops , Cartilla de ADN/química , Fibroblastos/fisiología , Humanos , Técnicas para Inmunoenzimas , Luciferasas/metabolismo , Factores de Transcripción MEF2 , Ratones , Datos de Secuencia Molecular , Proteínas Musculares/antagonistas & inhibidores , Proteínas Musculares/genética , Proteínas Musculares/farmacología , Proteína MioD/antagonistas & inhibidores , Proteína MioD/farmacología , Factor 5 Regulador Miogénico , Factores Reguladores Miogénicos , Miogenina/antagonistas & inhibidores , Miogenina/farmacología , Oligonucleótidos Antisentido/farmacología , Proteínas Recombinantes , Factores de Transcripción/metabolismo , Transfección , Regulación hacia ArribaRESUMEN
GH and IGF-I control over 80% of postnatal growth. We recently established that TNFalpha impairs the ability of IGF-I to increase protein synthesis and promote expression of myogenin in myoblasts. Here we extend these results by showing that ceramide, a second messenger in both TNFalpha and IL-1beta receptor signaling pathways, is a key downstream sphingosine-based lipid that leads to IGF-I resistance. A cell-permeable ceramide analog, C2-ceramide, inhibits IGF-I-induced protein synthesis by 65% and blocks the ability of IGF-I to increase expression of two key myogenic factors, myogenin and MyoD. Identical results were obtained with both TNFalpha and IL-1beta (1 ng/ml). Consistent with these data, neutral sphingomyelinase (N-SMase), an enzyme that catalyzes formation of ceramide from sphingomyelin, blocks IGF-I-induced protein synthesis and expression of both myogenin and MyoD. The possibility that cytokine-induced ceramide production is required for disruption of IGF-I biologic activity was confirmed by treating C2C12 myoblasts with inhibitors of all three ceramide-generating pathways. A N-SMase inhibitor, glutathione, as well as an acidic sphingomyelinase (A-SMase) inhibitor, D609, reverse the cytokine inhibition of IGF-I-induced protein synthesis by 80% and 45%, respectively. Likewise, an inhibitor of de novo ceramide synthesis, FB1, causes a 50% inhibition. Similarly, all three inhibitors significantly impair the ability of both TNFalpha and IL-1beta to suppress IGF-I-driven expression of myogenin. These experiments establish that ceramide, derived both from sphingomyelin and de novo synthesis, is a key intermediate by which proinflammatory cytokines impair the ability of IGF-I to promote protein synthesis and expression of critical muscle-specific transcription factors.
Asunto(s)
Ceramidas/fisiología , Citocinas/fisiología , Mediadores de Inflamación/fisiología , Factor I del Crecimiento Similar a la Insulina/farmacología , Proteínas Musculares/biosíntesis , Músculo Esquelético/metabolismo , Mioblastos/metabolismo , Animales , Línea Celular , Ceramidas/antagonistas & inhibidores , Sinergismo Farmacológico , Humanos , Proteínas Sustrato del Receptor de Insulina , Interleucina-1/farmacología , Ratones , Proteínas Musculares/antagonistas & inhibidores , Proteína MioD/antagonistas & inhibidores , Miogenina/antagonistas & inhibidores , Fosfoproteínas/metabolismo , Fosforilación , Proteínas Recombinantes/farmacología , Esfingomielina Fosfodiesterasa/farmacología , Factor de Necrosis Tumoral alfa/farmacología , Tirosina/metabolismoRESUMEN
Thyroid hormone stimulates myoblast differentiation, through an inhibition of AP-1 activity occurring at the onset of differentiation. In this study we found that the T3 nuclear receptor c-ErbAalpha1 (T3Ralpha1) is involved in a mechanism preserving the duration of myoblast proliferation. Independently of the hormone presence, T3Ralpha1 represses avian MyoD transcriptional activity. Using several mutants of T3Ralpha1, we found that the hinge region plays a crucial role in the inhibition of MyoD activity. In particular, mutations of two small basic sequences included in alpha helices abrogate the T3Ralpha1/MyoD functional interaction. Similarly, the T3 receptor also represses myogenin transcriptional activity. Therefore, despite stimulating avian myoblast differentiation by a T3-dependent pathway not involving myogenic factors, T3Ralpha1 contributes to maintain an optimal myoblast proliferation period by inhibiting MyoD and myogenin activity.