RESUMO
The transcriptional regulation of skeletal muscle (SKM) development (myogenesis) has been documented for over 3 decades and served as a paradigm for tissue-specific cell type determination and differentiation. Myogenic stem cells (MuSC) in embryos and adult SKM are regulated by the transcription factors Pax3 and Pax7 for their stem cell characteristics, while their lineage determination and terminal differentiation are both dictated by the myogenic regulatory factors (MRF) that comprise Mrf4, Myf5, Myogenin, and MyoD. The myocyte enhancer factor Mef2c is activated by MRF during terminal differentiation and collaborates with them to promote myoblast fusion and differentiation. Recent studies have found critical regulation of these myogenic transcription factors at mRNA level, including subcellular localization, stability, and translational regulation. Therefore, the regulation of Pax3/7, MRFs and Mef2c mRNAs by RNA-binding factors and non-coding RNAs (ncRNA), including microRNAs and long non-coding RNAs (lncRNA), will be the focus of this review and the impact of this regulation on myogenesis will be further addressed. Interestingly, the stem cell characteristics of MuSC has been found to be critically regulated by ncRNAs, implying the involvement of ncRNAs in SKM homeostasis and regeneration. Current studies have further identified that some ncRNAs are implicated in the etiology of some SKM diseases and can serve as valuable tools/indicators for prediction of prognosis. The roles of ncRNAs in the MuSC biology and SKM disease etiology will also be discussed in this review.
Assuntos
Músculo Esquelético , Proteína MyoD , Proteína MyoD/genética , Músculo Esquelético/metabolismo , Regulação da Expressão Gênica , Fator de Transcrição PAX3/genética , Fator de Transcrição PAX3/metabolismo , Diferenciação Celular/genética , Desenvolvimento Muscular/genéticaRESUMO
Skeletal muscle (SKM) is the largest organ in mammalian body and it can repair damages by using the residential myogenic stem cells (MuSC), but this repairing capacity reduces with age and in some genetic muscular dystrophy. Under these circumstances, artificial amplification of autologous MuSC in vitro might be necessary to repair the damaged SKM. The amplification of MuSC is highly dependent on myogenic signals, such as sonic hedgehog (Shh), Wnt3a, and fibroblast growth factors, so formulating an optimum myogenic kit composed of specific myogenic signals might increase the proliferation and differentiation of MuSC efficiently. In this study, various myogenic signals have been tested on C2C12 myoblasts and primary MuSC, and a myogenic kit consists of insulin, lithium chloride, T3, and retinoic acid has been formulated, and we found it significantly increased the fusion index and MHC expression level of both C2C12 and MuSC myotubes. A novel bioreactor providing cyclic stretching (CS) and electrical stimulation (ES) has been fabricated to enhance the myogenic differentiation of both C2C12 and MuSC. We further found that coating the bioreactor substratum with collagen gave the best effect on proliferation and differentiation of MuSC. Furthermore, combining the collagen coating and physical stimuli (CS + ES) in the bioreactor can generate more proliferative primary MuSC cells. Our results have demonstrated that the combination of myogenic kit and bioreactor can provide environment for efficient MuSC proliferation and differentiation. These MuSC and mature myotubes amplified in the bioreactor might be useful for clinical grafting into damaged SKM in the future.
RESUMO
During embryogenesis, basic fibroblast growth factor (bFGF) is released from neural tube and myotome to promote myogenic fate in the somite, and is routinely used for the culture of adult skeletal muscle (SKM) stem cells (MuSC, called satellite cells). However, the mechanism employed by bFGF to promote SKM lineage and MuSC proliferation has not been analyzed in detail. Furthermore, the question of if the post-translational modification (PTM) of bFGF is important to its stemness-promoting effect has not been answered. In this study, GST-bFGF was expressed and purified from E.coli, which lacks the PTM system in eukaryotes. We found that both GST-bFGF and commercially available bFGF activated the Akt-Erk pathway and had strong cell proliferation effect on C2C12 myoblasts and MuSC. GST-bFGF reversibly compromised the myogenesis of C2C12 myoblasts and MuSC, and it increased the expression of Myf5, Pax3/7, and Cyclin D1 but strongly repressed that of MyoD, suggesting the maintenance of myogenic stemness amid repressed MyoD expression. The proliferation effect of GST-bFGF was conserved in C2C12 over-expressed with MyoD (C2C12-tTA-MyoD), implying its independence of the down-regulation of MyoD. In addition, the repressive effect of GST-bFGF on myogenic differentiation was almost totally rescued by the over-expression of MyoD. Together, these evidences suggest that (1) GST-bFGF and bFGF have similar effects on myogenic cell proliferation and differentiation, and (2) GST-bFGF can promote MuSC stemness and proliferation by differentially regulating MRFs and Pax3/7, (3) MyoD repression by GST-bFGF is reversible and independent of the proliferation effect, and (4) GST-bFGF can be a good substitute for bFGF in sustaining MuSC stemness and proliferation.
Assuntos
Proliferação de Células , Fator 2 de Crescimento de Fibroblastos , Desenvolvimento Muscular , Proteína MyoD , Mioblastos , Desenvolvimento Muscular/genética , Animais , Camundongos , Proteína MyoD/metabolismo , Proteína MyoD/genética , Fator 2 de Crescimento de Fibroblastos/metabolismo , Fator 2 de Crescimento de Fibroblastos/farmacologia , Fator 2 de Crescimento de Fibroblastos/genética , Mioblastos/metabolismo , Mioblastos/citologia , Linhagem Celular , Fator de Transcrição PAX7/metabolismo , Fator de Transcrição PAX7/genética , Fator de Transcrição PAX3/metabolismo , Fator de Transcrição PAX3/genética , Fator Regulador Miogênico 5/metabolismo , Fator Regulador Miogênico 5/genética , Ciclina D1/metabolismo , Ciclina D1/genética , Células Satélites de Músculo Esquelético/metabolismo , Células Satélites de Músculo Esquelético/citologia , Diferenciação Celular , Proteínas Proto-Oncogênicas c-akt/metabolismo , Músculo Esquelético/metabolismo , Músculo Esquelético/citologiaRESUMO
Mitochondria (MITO) and peroxisomes (PEXO) are the major organelles involved in the oxidative metabolism of cells, but detailed examination of their dynamics and functional adaptations during skeletal muscle (SKM) development (myogenesis) is still lacking. In this study, we found that during myogenesis, MITO DNA, ROS level, and redox ratio increased in myotubes, but the membrane potential (Δψm) and ATP content reduced, implying that the MITO efficiency might reduce during myogenesis. The PEXO number and density both increased during myogenesis, which probably resulted from the accumulation and increased biogenesis of PEXO. The expression of PEXO biogenesis factors was induced during myogenesis in vitro and in utero, and their promoters were also activated by MyoD. Knockdown of the biogenesis factors Pex3 repressed not only the PEXO density and functions but also the levels of MITO genes and functions, suggesting a close coupling between PEXO biogenesis and MITO functions. Surprisingly, Pex3 knockdown by the CRISPRi system repressed myogenic differentiation, indicating critical involvement of PEXO biogenesis in myogenesis. Taken together, these observations suggest that the dynamics and functions of both MITO and PEXO are coupled with each other and with the metabolic changes that occur during myogenesis, and these metabolic couplings are critical to myogenesis.
Assuntos
Fibras Musculares Esqueléticas , Peroxissomos , Peroxissomos/metabolismo , Diferenciação Celular/genética , Fibras Musculares Esqueléticas/metabolismo , Mitocôndrias/metabolismo , Desenvolvimento Muscular/genética , Músculo Esquelético/metabolismoRESUMO
M-cadherin is a skeletal muscle-specific transmembrane protein mediating the cell-cell adhesion of myoblasts during myogenesis. It is expressed in the proliferating satellite cells and highly induced by myogenic regulatory factors (MRFs) during terminal myogenic differentiation. Several conserved cis-elements, including 5 E-boxes, 2 GC boxes, and 1 conserved downstream element (CDE) were identified in the M-cadherin proximal promoter. We found that E-box-3 and -4 close to the transcription initiation site (TIS) mediated most of its transactivation by MyoD, the strongest myogenic MRF. Including of any one of the other E-boxes restored the full activation by MyoD, suggesting an essential collaboration between E-boxes. Stronger activation of M-cadherin promoter than that of muscle creatine kinase (MCK) by MyoD was observed regardless of culture conditions and the presence of E47. Furthermore, MyoD/E47 heterodimer and MyoD â¼ E47 fusion protein achieved similar levels of activation in differentiation medium (DM), suggesting high affinity of MyoD/E47 to E-boxes 3/4 under DM. We also found that GC boxes and CDE positively affected MyoD mediated activation. The CDE element was predicted to be the target of the chromatin-modifying factor Meis1/Pbx1 heterodimer. Knockdown of Pbx1 significantly reduced the expression level of M-cadherin, but increased that of N-cadherin. Using ChIP assay, we further found significant reduction in MyoD recruitment to M-cadherin promoter when CDE was deleted. Taken together, these observations suggest that the chromatin-modifying function of Pbx1/Meis1 is critical to M-cadherin promoter activation before MyoD is recruited to E-boxes to trigger transcription.
Assuntos
Caderinas/genética , Elementos E-Box/genética , Regulação da Expressão Gênica/genética , Desenvolvimento Muscular/genética , Regiões Promotoras Genéticas/genética , Animais , Sequência de Bases , Células Cultivadas , Sequência Conservada , Fibroblastos , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Camundongos , Proteína Meis1/fisiologia , Proteína MyoD/metabolismo , Mioblastos , Fator de Transcrição 1 de Leucemia de Células Pré-B/fisiologia , Interferência de RNA , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/farmacologia , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência do Ácido NucleicoRESUMO
The NS4A protein of dengue virus (DENV) has a cytosolic N terminus and four transmembrane domains. NS4A participates in RNA replication and the host antiviral response. However, the roles of amino acid residues within the N-terminus of NS4A during the life cycle of DENV are not clear. Here we explore the function of DENV NS4A by introducing a series of alanine substitutions into the N-terminus of NS4A in the context of a DENV infectious clone or subgenomic replicon. Nine of 17 NS4A mutants displayed a lethal phenotype due to the impairment of RNA replication. M2 and M14 displayed a more than 10â000-fold reduction in viral yields and moderate defects in viral replication by a replicon assay. Sequencing analyses of pseudorevertant viruses derived from M2 and M14 viruses revealed one consensus reversion mutation, A21V, within NS4A. The A21V mutation apparently rescued viral RNA replication in the M2 and M14 mutants although not to wild-type (WT) levels but resulted in 100- and 1000-fold lower titres than that of the WT, respectively. M2 Rev1 (M2+A21V) and M14 Rev1 (M14+A21V) mutants displayed phenotypes of smaller plaque size and WT-like assembly/secretion by a transpackaging assay. A defect in the virus-induced cytopathic effect (CPE) was observed in HEK-293 cells infected with either M2 Rev1 or M14 Rev1 mutant virus by MitoCapture staining, cell proliferation and lactate dehydrogenase release assays. In conclusion, the results revealed the essential roles of the N-terminal NS4A in both RNA replication and virus-induced CPE. Intramolecular interactions in the N-terminus of NS4A were implicated.
Assuntos
Efeito Citopatogênico Viral , Vírus da Dengue/metabolismo , Dengue/virologia , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/genética , Sequência de Aminoácidos , Substituição de Aminoácidos , Vírus da Dengue/genética , Vírus da Dengue/fisiologia , Células HEK293 , Humanos , Mutagênese , Domínios Proteicos , Proteínas não Estruturais Virais/metabolismo , Replicação ViralRESUMO
Skeletal myogenesis involves highly coordinated steps that integrate developmental cues at the chromatin of muscle progenitors. Here, we identify Myb-binding protein 1a (Mybbp1a) as a novel negative regulator of muscle-specific gene expression and myoblast differentiation. The mode of action of Mybbp1a was linked to promoter regulation as illustrated by its interaction with MyoD at the genomic regions of silent muscle-specific genes as well as its negative effect on MyoD-mediated transcriptional activity. We propose that Mybbp1a exerts its repressive role by inducing a less permissible chromatin structure following recruitment of negative epigenetic modifiers such as HDAC1/2 and Suv39h1. At the onset of differentiation, Mybbp1a undergoes a promoter disengagement that may be due to the differentiation-responsive, miR-546-mediated downregulation of Mybbp1a expression. Moreover, such alteration gave rise to promoter enrichment of activators and histone acetylation, an epigenetic status amenable to gene activation. Together, these findings unveil a hitherto unrecognized transcriptional co-repressor role of Mybbp1a in proliferating muscle progenitor cells, and highlight an epigenetic mechanism by which Mybbp1a and miR-546 interplay to control myoblast differentiation transition.
Assuntos
Proteínas de Transporte/metabolismo , Inativação Gênica , Desenvolvimento Muscular/genética , Proteínas Nucleares/metabolismo , Regiões Promotoras Genéticas , Animais , Proteínas de Transporte/genética , Células Cultivadas , Proteínas de Ligação a DNA , Regulação para Baixo , Expressão Gênica , Humanos , Camundongos , MicroRNAs/metabolismo , Músculo Esquelético/metabolismo , Proteína MyoD/genética , Proteína MyoD/metabolismo , Proteínas Nucleares/genética , Proteínas de Ligação a RNA , Fatores de TranscriçãoRESUMO
The objective of the current study was to investigate the effects of Ca(2+) levels on myofibril alignment during zebrafish embryogenesis. To investigate how altered cytoplasmic Ca(2+) levels affect myofibril alignment, we exposed zebrafish embryos to 2-aminothoxyldiphenyl borate (2-APB; an inositol 1,4,5-trisphosphate receptor inhibitor that reduces cytosolic Ca(2+) levels) and caffeine (a ryanodine receptor activator that enhances cytosolic Ca(2+) levels). The results demonstrated that the most evident changes in zebrafish embryos treated with 2-APB were shorter body length, curved trunk and malformed somite boundary. In contrast, such malformed phenotypes were evident neither in untreated controls nor in caffeine-treated embryos. Subtle morphological changes, including changes in muscle fibers, F-actin and ultrastructures were easily observed by staining with specific monoclonal antibodies (F59 and α-laminin), fluorescent probes (phalloidin) and by transmission electron microscopy. Our data suggested that: (1) the exposure to 2-APB and/or caffeine led to myofibril misalignment; (2) 2-APB-treated embryos displayed split and short myofibril phenotypes, whereas muscle fibers from caffeine-treated embryos were twisted and wavy; and (3) zebrafish embryos co-exposed to 2-APB and caffeine resulted in normal myofibril alignment. In conclusion, we proposed that cytosolic Ca(2+) is important for myogenesis, particularly for myofibril alignment.
Assuntos
Compostos de Boro/toxicidade , Cafeína/toxicidade , Cálcio/metabolismo , Citosol/efeitos dos fármacos , Desenvolvimento Muscular/efeitos dos fármacos , Miofibrilas/efeitos dos fármacos , Peixe-Zebra/embriologia , Animais , Citosol/metabolismo , Embrião não Mamífero/efeitos dos fármacos , Embrião não Mamífero/metabolismo , Embrião não Mamífero/ultraestrutura , Microscopia Eletrônica de Transmissão , Miofibrilas/ultraestruturaRESUMO
Homeobox genes encode transcription factors that regulate embryonic development programs including organogenesis, axis formation and limb development. Previously, we identified and cloned a mouse double homeobox gene, Duxbl, whose homeodomain exhibits the highest identity (67 %) to human DUX4, a candidate gene of facioscapulohumeral muscular dystrophy (FSHD). Duxbl proteins have been shown to be expressed in elongated myocytes and myotubes of trunk and limb muscles during embryogenesis. In this study, we found that Duxbl maintained low expression levels in various adult muscles. Duxbl proteins were induced to express in activated satellite cells and colocalized with MyoG, a myogenic differentiating marker. Furthermore, Duxbl proteins were not detected in quiescent satellite cells but detected in regenerated myocytes and colocalized with MyoD and MyoG following cardiotoxin-induced muscle injury. Ectopic Duxbl overexpressions in C2C12 myoblast cells promoted cell proliferation through mainly enhancing cyclin D1 and hyper-phosphorylated retinoblastoma protein but reducing p21 expression. However, Duxbl overexpression in C2C12 cells inhibited myogenic differentiation by decreasing MyoD downstream gene expressions, including M-cadherin, MyoG, p21 and cyclin D3 but not MyoD itself. Duxbl overexpressions also promoted cell proliferation but blocked MyoD-induced myogenic conversion in multipotent mesenchymal C3H10T1/2 cells. In addition, results of a luciferase reporter assay suggest that Duxbl negatively regulated MyoG promoter activity through the proximal two E boxes. In conclusion, these results indicate that Duxbl may play a crucial role in myogenesis and postnatal muscle regeneration by activating and proliferating satellite and myoblast cells.
Assuntos
Diferenciação Celular , Proteínas de Homeodomínio/genética , Proteína MyoD/genética , Mioblastos/citologia , Mioblastos/metabolismo , Fatores de Transcrição/genética , Ativação Transcricional/genética , Envelhecimento/metabolismo , Animais , Proteínas de Ciclo Celular/metabolismo , Diferenciação Celular/genética , Proliferação de Células , Imunofluorescência , Proteínas de Homeodomínio/metabolismo , Humanos , Camundongos , Desenvolvimento Muscular , Proteína MyoD/metabolismo , Miogenina/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Regeneração , Células Satélites de Músculo Esquelético/metabolismo , Fatores de Transcrição/metabolismoRESUMO
A multifunctional bioreactor was fabricated in this study to investigate the facilitation efficiency of electrical and mechanical stimulations on myogenic differentiation. This bioreactor consisted of a highly stretchable conductive membrane prepared by depositing polypyrrole (PPy) on a flexible polydimethylsiloxane (PDMS) film. The tensile deformation of the PPy/PDMS membrane can be tuned by adjusting the channel depth. In addition, PPy/PDMS maintained its electrical conductivity under continuous cyclic stretching in the strain range of 6.5%-13% for 24 h. This device can be used to individually or simultaneously perform cyclic stretching and electrical stimulation. The results of single stimulation showed that either cyclic stretching or electrical stimulation upregulated myogenic gene expression and promoted myotube formation, where electrical stimulation improved better than cyclic stretching. However, only cyclic stretching can align C2C12 cells perpendicular to the stretching direction, and electrical stimulation did not affect cell morphology. Myosin heavy chain (MHC) immunostaining demonstrated that oriented cells under cyclic stretching resulted in parallel myotubes. The combination of these two stimuli exhibited synergetic effects on both myogenic gene regulation and myotube formation, and the incorporated electrical field did not affect the orientation effect of the cyclic stretching. These results suggested that these two treatments likely influenced cells through different pathways. Overall, the simultaneous application of cyclic stretching and electrical stimulation preserved both stimuli's advantages, so myo-differentiation can be highly improved to obtain abundant parallel myotubes, suggesting that our developed multifunctional bioreactor should benefit muscle tissue engineering applications.
RESUMO
M- and N-cadherin are members of the Ca(2+)-dependent cell-cell adhesion molecule family. M-cadherin is expressed predominantly in developing skeletal muscles and has been implicated in terminal myogenic differentiation, particularly in myoblast fusion. N-cadherin-mediated cell-cell adhesion also plays an important role in skeletal myogenesis. In the present study, we found that both genes were differentially expressed in C2C12 and Sol8 myoblasts during myogenic differentiation and that the expression of M-cadherin was preferentially enhanced in slow-twitch muscle. Interestingly, most MRFs (myogenic regulatory factors) significantly activated the promoter of M-cadherin, but not that of N-cadherin. In line with this, overexpression of MyoD in C3H10T1/2 fibroblasts strongly induced endogenous M-cadherin expression. Promoter analysis in silico and in vitro identified an E-box (from -2 to +4) abutting the transcription initiation site within the M-cadherin promoter that is bound and differentially activated by different MRFs. The activation of the M-cadherin promoter by MRFs was also modulated by Bhlhe40 (basic helix-loop-helix family member e40). Finally, chromatin immunoprecipitation proved that MyoD as well as myogenin binds to the M-cadherin promoter in vivo. Taken together, these observations identify a molecular mechanism by which MRFs regulate M-cadherin expression directly to ensure the terminal differentiation of myoblasts.
Assuntos
Caderinas/genética , Fatores de Regulação Miogênica/fisiologia , Regiões Promotoras Genéticas/genética , Animais , Linhagem Celular , Imunoprecipitação da Cromatina , Elementos E-Box/genética , Ensaio de Desvio de Mobilidade Eletroforética , Fatores de Transcrição MEF2 , Camundongos , Proteína MyoD/genética , Proteína MyoD/metabolismo , Proteína MyoD/fisiologia , Fatores de Regulação Miogênica/genética , Fatores de Regulação Miogênica/metabolismo , Miogenina/genética , Miogenina/metabolismo , Miogenina/fisiologia , Ligação Proteica , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Induced pluripotent stem (iPS) cells are reprogrammed from somatic cells through ectopic expression of stem cell-specific transcription factors, including Oct4, Nanog, Sox2, Lin28, Klf4, and c-Myc. Although iPS cells are similar to embryonic stem (ES) cells in their pluripotency, their inherited defects, such as insertion mutagenesis, employment of oncogenes, and low efficiency, associated with the reprogramming procedure have hindered their clinical application. A study has shown that valproic acid (VPA) treatment can significantly enhance the reprogramming efficiency and avoid the usage of oncogenes. To understand how VPA can enhance pluripotency, we stably transfected an Oct4 promoter driven luciferase reporter (Oct4-1.9k-Luc) into P19 embryonic carcinoma (EC) cells and C2C12 myoblasts and examined their response to VPA. We found that VPA could both activate Oct4 promoter and rescue its inhibition by retinoic acid (RA). In C2C12 myoblasts, VPA treatment also enhanced endogenous Oct4 expression but repressed that of MyoD. Furthermore, both RARalpha over-expression and mutation of a proximal hormone response element (HRE) blocked the activation effect of VPA on Oct4 promoter, implying that VPA may exert its activation effect through factors targeting this HRE. Taken together, these observations identify a molecular mechanism by which VPA directly regulate Oct4 expression to ensure the acquirement and maintenance of pluripotency.
Assuntos
Músculos/metabolismo , Fator 3 de Transcrição de Octâmero/genética , Regiões Promotoras Genéticas , Ácido Valproico/farmacologia , Animais , Sequência de Bases , Linhagem Celular , Primers do DNA , Imuno-Histoquímica , Fator 4 Semelhante a Kruppel , Camundongos , Músculos/citologia , Reação em Cadeia da Polimerase/métodosRESUMO
The level of di-(2-ethylhexyl) phthalate (DEHP) is elevated in chronic kidney disease patients undergoing dialysis. However, statins are unable to reduce the cardiovascular events in chronic dialysis patients. In this study, we investigated the effects of DEHP on statin-conferred pleiotropic effects and the underlying molecular mechanism in peritoneal dialysis (PD) patients and endothelial cells (ECs). In PD patients with serum DEHP level ≥0.0687 µg/mL, statin treatment was not associated with lower risk of cardiovascular disease. In ECs, exposure to DEHP abrogated the simvastatin-induced NO bioavailability and EC-related functions. Additionally, DEHP abolished the anti-inflammatory effect of simvastatin on the tumor necrosis factor α-induced upregulation of adhesion molecules and monocyte adhesion to ECs. Mechanistically, DEHP blunted the activation of transient receptor potential vanilloid type 1 (TRPV1), which is required for NO production by simvastatin in ECs. Notably, DEHP increased the activity and expression of protein phosphatase 2B (PP2B), a negative regulator of TRPV1 activity. The effect of DEHP on PP2B activation was mediated by the activation of the NADPH oxidase/reactive oxygen species (NOX-ROS) pathway. Inhibition of PP2B activity by pharmacological antagonists prevented the inhibitory effects of DEHP on simvastatin-induced Ca2+ influx, NO bioavailability, and EC migration, proliferation, tube formation, and anti-inflammatory action. Collectively, DEHP activates the NOX-ROS-PP2B pathway, which in turns inhibits TRPV1/Ca2+-dependent signaling and abrogates the statin-conferred pleiotropic protection in ECs.
Assuntos
Dietilexilftalato , Inibidores de Hidroximetilglutaril-CoA Redutases , Insuficiência Renal Crônica , Dietilexilftalato/toxicidade , Células Endoteliais , Humanos , Ácidos Ftálicos , Diálise Renal , Insuficiência Renal Crônica/terapiaRESUMO
Di (2-ethylhexyl) phthalate (DEHP) is a plasticizer frequently leached out from polyvinyl chloride (PVC) products and is quickly metabolized to its monoester equivalent mono(2-ethylhexyl) phthalate (MEHP) once enters organisms. Exposure to DEHP/MEHP through food chain intake has been shown to modified metabolism but its effect on the development of metabolic myopathy of skeletal muscle (SKM) has not been revealed so far. Here, we found that MEHP repressed myogenic terminal differentiation of proliferating myoblasts (PMB) and confluent myoblasts (CMB) but had weak effect on this process once it had been initiated. The transition of mitochondria (MITO) morphology from high efficient filamentary network to low efficient vesicles was triggered by MEHP, implying its negative effects on MITO functions. The impaired MITO functions was further demonstrated by reduced MITO DNA (mtDNA) level and SDH enzyme activity as well as highly increased reactive oxygen species (ROS) in cells after MEHP treatment. The expression of metabolic genes, including PDK4, CPT1b, UCP2, and HO1, was highly increased by MEHP and the promoters of PDK4 and CPT1b were also activated by MEHP. Additionally, the stability of some subunits in the oxidative phosphorylation system (OXPHOS) complexes was found to be reduced by MEHP, implying defective oxidative metabolism in MITO and which was confirmed by repressed palmitic acid oxidation in MEHP-treated cells. Besides, MEHP also blocked insulin-induced glucose uptake. Taken together, our results suggest that MEHP is inhibitory to myogenesis and is harmful to MITO functions in SKM, so its exposure should be avoided or limited.
Assuntos
Dietilexilftalato/análogos & derivados , Mitocôndrias/efeitos dos fármacos , Músculo Esquelético/efeitos dos fármacos , Mioblastos/efeitos dos fármacos , Plastificantes/toxicidade , Animais , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Dietilexilftalato/metabolismo , Dietilexilftalato/toxicidade , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Camundongos , Mitocôndrias/patologia , Miopatias Mitocondriais/induzido quimicamente , Miopatias Mitocondriais/patologia , Desenvolvimento Muscular/efeitos dos fármacos , Músculo Esquelético/citologia , Músculo Esquelético/patologia , Mioblastos/citologia , Mioblastos/patologia , Oxirredução/efeitos dos fármacos , Fosforilação Oxidativa/efeitos dos fármacos , Plastificantes/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Testes de Toxicidade AgudaRESUMO
Mono(2-ethylhexyl)phthalate (MEHP) promotes adipogenesis via PPARγ. PPARγ agonists, e.g., rosiglitazone (RSG), enhance adipocyte browning. However, scientific evidence regarding MEHP as a browning chemical is lacking. This study combined 3T3-L1 adipocytes and C57BL/6J mice to examine the potential roles of MEHP in browning. MEHP and the browning agent RSG caused similar energy metabolism in adipocytes. Both MEHP and RSG caused transcriptional changes involved in browning-associated thermogenesis, energy homeostasis, inflammatory response, and glucose uptake. MEHP-treated adipocytes exhibited brown adipocyte-like characteristics, i.e., increased mitochondrial proton leak, triiodothyronine-induced Bmp8b expression, decreased inflammation, and smaller lipid droplets. Increased PDK4 and PEPCK1 in MEHP/RSG-treated adipocytes could block glucose utilization for mitochondrial respiration. Mitochondrial/peroxisomal biogenesis and fatty acid ß-oxidation in MEHP-treated adipocytes were enhanced. Candidate genes in promoting browning of MEHP-treated adipocytes were highlighted. In di(2-ethylhexyl)phthalate (DEHP)-treated mice, transcriptional changes in white adipose tissue (WAT) were associated with adipocyte differentiation, lipid synthesis, carbohydrate uptake, and WAT/brown adipose tissue (BAT) quantity. PPARγ and NR4A1 were predicted as the top two upstream regulators in orchestrating transcriptional changes. DEHP-treated mice exhibited actively expressed browning marker genes (i.e., Pparg, Adrb1, Adrb3, Ppargc1a, and Ucp1) in WAT, increased blood FGF21 levels, and higher amounts of BAT, supporting the browning-like effects in vivo.
Assuntos
Adipócitos Marrons/efeitos dos fármacos , Dietilexilftalato/análogos & derivados , Células 3T3-L1 , Adipócitos Marrons/metabolismo , Animais , Dietilexilftalato/toxicidade , Metabolismo Energético/efeitos dos fármacos , Expressão Gênica/efeitos dos fármacos , Glucose/metabolismo , Técnicas In Vitro , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/metabolismo , Fosfoenolpiruvato Carboxiquinase (ATP)/metabolismo , Piruvato Desidrogenase Quinase de Transferência de AcetilRESUMO
Oct4 and Nanog are two embryonic stem (ES) cell-specific transcription factors that play critical roles in the maintenance of ES cell pluripotency. In this study, we investigated the effects of Oct4 and Nanog expression on the differentiation state of myogenic cells, which is sustained by a strong positive feedback loop. Oct4 and Nanog, either independently or simultaneously, were overexpressed in C2C12 myoblasts, and the expression of myogenic lineage-specific genes and terminal differentiation was observed by RT-PCR. Overexpression of Oct4 in C2C12 cultures repressed, while exogenous Nanog did not significantly alter C2C12 terminal differentiation. The expression of Pax7 was reduced in all Oct4-overexpressing myoblasts, and we identified a major Oct4-binding site in the Pax7 promoter. Simultaneous expression of Oct4 and Nanog in myoblasts inhibited the formation of myotubes, concomitant with a reduction in the endogenous levels of hallmark myogenic markers. Furthermore, overexpression of Oct4 and Nanog induced the expression of their endogenous counterparts along with the expression of Sox2. Using mammalian two-hybrid assays, we confirmed that Oct4 functions as a transcriptional repressor whereas Nanog functions as a transcriptional activator during muscle terminal differentiation. Importantly, in nonobese diabetic (NOD) severe combined immunodeficiency (SCID) mice, the pluripotency of C2C12 cells was conferred by overexpression of Oct4 and Nanog. These results suggest that Oct4 in cooperation with Nanog strongly suppresses the myogenic differentiation program and promotes pluripotency in myoblasts.
Assuntos
Diferenciação Celular , Proteínas de Homeodomínio/metabolismo , Desenvolvimento Muscular , Fibras Musculares Esqueléticas/metabolismo , Mioblastos Esqueléticos/metabolismo , Fator 3 de Transcrição de Octâmero/metabolismo , Células-Tronco Pluripotentes/metabolismo , Animais , Sequência de Bases , Sítios de Ligação , Diferenciação Celular/genética , Linhagem Celular , Linhagem da Célula , Retroalimentação Fisiológica , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/genética , Fatores de Transcrição MEF2 , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Dados de Sequência Molecular , Desenvolvimento Muscular/genética , Fatores de Regulação Miogênica/genética , Fatores de Regulação Miogênica/metabolismo , Proteína Homeobox Nanog , Fator 3 de Transcrição de Octâmero/genética , Fator de Transcrição PAX7/genética , Fator de Transcrição PAX7/metabolismo , Regiões Promotoras Genéticas , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismo , Teratoma/genética , Teratoma/metabolismo , Ativação Transcricional , Regulação para CimaRESUMO
In the past decade, the number of islands fixed link with mainland has been increasing rapidly. The construction of such projects improves the accessibility of islands, which results in a rapid increase of tourists and economic income. However, the rapid change of land use and increases of tourists will make the islands more vulnerable. It is urgent to formulate ecosystem management strategy for island ecosystems based on the scientifical prediction of the island ecological vulnerability and accurate identification of sensitive areas. Island ecological vulnerability assessment model was used to evaluate the ecological vulnerability of Zhujiajian Island. Taking 2015 as the starting date, we simulated the changes of tourists and land use, as well as the changes of island ecological vulnerability in the next 20 years. Then, the management strategy was formulated based on the vulnerability assessment and sensitive analysis. The results showed that the ecosystem of Zhujiajian Island was in good condition now, with limited area at moderate and severe vulnerable status. With the rapid increases of tourists and island development intensity, the ecological vulnerability of the island tended to more vulnerability with the increases of severe vulnerability and shrink of low vulnerability. According to the vulnerability assessment and sensitivity analysis, the Zhujiajian Island could be divided into prohibited development zones, restricted development zones and conditional development zones with different ecosystem management strategies.
Assuntos
Ecologia , Ecossistema , China , Conservação dos Recursos Naturais , Ilhas , Modelos TeóricosRESUMO
PGC-1α is a key regulator of oxidative metabolism facilitating the expression of genes critical for the function and biogenesis of the two key oxidative organelles, mitochondria and peroxisomes, in skeletal muscle (SKM) and other organs. Our recent studies have found that the transcription factor Bhlhe40 negatively regulates PGC-1α gene expression and its coactivational activity, therefore, this factor should have profound influence on the biogenesis and metabolic activity of mitochondria and peroxisomes. Here we found that both the number and activity of peroxisomes were increased upon knockdown of Bhlhe40 expression but were repressed by its over-expression. Mitochondrial efficiency was significantly reduced by Bhlhe40 knockdown, resulting in the burst of ROS. Over-expression of a constitutively active PGC-1α-interactive domain (named as VBH135) of Bhlhe40 mimicked the effects of its knockdown on peroxisomes but simultaneously reduced ROS level. Furthermore, the efficiency, but not the number, of mitochondria was also increased by VBH135, suggesting differential regulation of peroxisomes and mitochondria by Bhlhe40. Unsaturated fatty acid oxidation, insulin response, and oxidative respiration were highly enhanced in Bhlhe40 knockdown or VBH135 over-expressed cells, suggesting the importance of Bhlhe40 in the regulation of unsaturated fatty acid and glucose oxidative metabolism. Expression profiling of genes important for either organelle also supports differential regulation of peroxisomes and mitochondria by Bhlhe40. These observations have established the important role of Bhlhe40 in SKM oxidative metabolism as the critical regulator of peroxisome and mitochondrion biogenesis and functions, and thus should provide a novel route for developing drugs targeting SKM metabolic diseases.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Proteínas de Homeodomínio/genética , Mitocôndrias/genética , Mitocôndrias/metabolismo , Desenvolvimento Muscular/genética , Mioblastos/metabolismo , Peroxissomos/genética , Peroxissomos/metabolismo , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/antagonistas & inibidores , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Biomarcadores , Catalase/metabolismo , Ácidos Graxos/metabolismo , Expressão Gênica , Técnicas de Silenciamento de Genes , Glucose/metabolismo , Proteínas de Homeodomínio/antagonistas & inibidores , Proteínas de Homeodomínio/metabolismo , Humanos , Imuno-Histoquímica , Camundongos , Oxirredução , Consumo de Oxigênio , RNA Interferente Pequeno/genética , Ratos , Espécies Reativas de Oxigênio/metabolismoRESUMO
The objective of this study was to identify genes regulated by thyroid hormone (T(3)) and associated with tumor invasion. The gene encoding furin, as previously identified by cDNA microarray, is known to be up-regulated by T(3) treatment, and stimulated furin production occurs in thyroidectomized rats after administration of T(3). Presently, by using serial deletion of the promoter and EMSAs, the T(3) response element on the furin promoter was localized to the -6317/-6302 region. T(3)-mediated furin up-regulation was cooperative with TGF-beta because T(3) induction increased after Smad3/4 addition. Furthermore, the invasiveness of HepG2-thyroid hormone receptor (TR) cells was significantly increased by T(3) treatment, perhaps due to furin processing of matrix metalloproteinase-2 and -9. In addition, furin up-regulation either by stable overexpression or T(3) and/or TGF-beta induction was evident in severe-combined immune-deficient mice inoculated with HepG2-TRalpha1 cells. The HepG2-furin mice displayed a higher metastasis index and tumor size than HepG2-neo mice. Notably, the increased liver and lung tumor number or size in the hyperthyroid severe-combined immune-deficient mice as well as TGF-beta mice was attributed specifically to furin overexpression in the HepG2-TRalpha1 cells. Furthermore, this study demonstrated that furin overexpression in some types of hepatocellular carcinomas is TR dependent and might play a crucial role in the development of hepatocellular carcinoma. Thus, T(3) regulates furin gene expression via a novel mechanism or in cooperation with TGF-beta to enhance tumor metastasis in vitro and in vivo.
Assuntos
Carcinoma Hepatocelular/patologia , Movimento Celular/efeitos dos fármacos , Furina/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Neoplasias Hepáticas/patologia , Hormônios Tireóideos/farmacologia , Animais , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Adesão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Células Cultivadas , Chlorocebus aethiops , Furina/metabolismo , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Masculino , Camundongos , Camundongos SCID , Modelos Biológicos , Invasividade Neoplásica , Ratos , Ratos Sprague-Dawley , Receptores dos Hormônios Tireóideos/fisiologia , Fator de Crescimento Transformador beta/farmacologiaRESUMO
Peroxisomal proliferator-activated receptor-gamma coactivator-1alpha (PGC-1alpha), a transcriptional coactivator, is selectively expressed in slow-twitch fibers in skeletal muscle. Ectopic expression of the PGC-1alpha gene in either a cell or an animal has been shown to promote fast to slow fiber-type switch. The expression of PGC-1alpha in muscle is regulated by myocyte enhancer factor 2 and Forkhead in rhabdomyosarcoma, two transcription factors implicated in terminal muscle differentiation. In this study we found that PGC-1alpha expression was activated during terminal muscle differentiation in both C2C12 and Sol8 myoblasts. Using retrovirus-mediated MyoD overexpression in C3H10T1/2 cells, we also demonstrated that MyoD, the master regulator of terminal differentiation, could activate PGC-1alpha expression in vivo. Our transient transfection results also show that myogenic basic helix-loop-helix (bHLH) proteins, especially MyoD, can activate PGC-1alpha expression by targeting its promoter. Myogenic bHLH protein target sites on PGC-1alpha promoter were localized to a short region (-49 to approximately +2) adjacent to the transcription start site, which contains two putative E boxes. Mutation of either site significantly reduced MyoD-mediated transactivation in the cells, suggesting that both sites are required for myogenic bHLH protein-mediated activation. However, only one site, the E2 box, was directly bound by glutathione-S-transferase-MyoD protein in EMSAs. Our results indicate that myogenic bHLH proteins not only are involved in lineage determination and terminal differentiation, but also are directly implicated in activation of the key fiber-type and metabolic switch gene, PGC-1alpha.