RESUMO
Collagen Q (COLQ) is a specific collagen that anchors acetylcholinesterase (AChE) in the synaptic cleft of the neuromuscular junction. So far, no mutation has been identified in the ACHE human gene but over 50 different mutations in the COLQ gene are causative for a congenital myasthenic syndrome (CMS) with AChE deficiency. Mice deficient for COLQ mimic most of the functional deficit observed in CMS patients. At the molecular level, a striking consequence of the absence of COLQ is an increase in the levels of acetylcholine receptor (AChR) mRNAs and proteins in vivo and in vitro in murine skeletal muscle cells. Here, we decipher the mechanisms that drive AChR mRNA upregulation in cultured muscle cells deficient for COLQ. We show that the levels of AChR ß-subunit mRNAs are post-transcriptionally regulated by an increase in their stability. We demonstrate that this process results from an activation of p38 MAPK and the cytoplasmic translocation of the nuclear RNA-binding protein human antigen R (HuR) that interacts with the AU-rich element located within AChR ß-subunit transcripts. This HuR/AChR transcript interaction induces AChR ß-subunit mRNA stabilization and occurs at a specific stage of myogenic differentiation. In addition, pharmacological drugs that modulate p38 activity cause parallel modifications of HuR protein and AChR ß-subunit levels. Thus, our study provides new insights into the signaling pathways that are regulated by ColQ-deficiency and highlights for the first time a role for HuR and p38 in mRNA stability in a model of congenital myasthenic syndrome.
RESUMO
Myotonic dystrophy type 1 (DM1) is a debilitating multisystemic disorder caused by a triplet repeat expansion in the 3' untranslated region of dystrophia myotonica protein kinase mRNAs. Mutant mRNAs accumulate in the nucleus of affected cells and misregulate RNA-binding proteins, thereby promoting characteristic missplicing events. However, little is known about the signaling pathways that may be affected in DM1. Here, we investigated the status of activated protein kinase (AMPK) signaling in DM1 skeletal muscle and found that the AMPK pathway is markedly repressed in a DM1 mouse model (human skeletal actin-long repeat, HSALR) and patient-derived DM1 myoblasts. Chronic pharmacological activation of AMPK signaling in DM1 mice with 5-aminoimidazole-4-carboxamide-1-ß-D-ribofuranoside (AICAR) has multiple beneficial effects on the DM1 phenotype. Indeed, a 6-week AICAR treatment of DM1 mice promoted expression of a slower, more oxidative phenotype, improved muscle histology and corrected several events associated with RNA toxicity. Importantly, AICAR also had a dose-dependent positive effect on the spliceopathy in patient-derived DM1 myoblasts. In separate experiments, we also show that chronic treatment of DM1 mice with resveratrol as well as voluntary wheel running also rescued missplicing events in muscle. Collectively, our findings demonstrate the therapeutic potential of chronic AMPK stimulation both physiologically and pharmacologically for DM1 patients.
Assuntos
Aminoimidazol Carboxamida/análogos & derivados , Distrofia Miotônica/tratamento farmacológico , Proteínas Quinases/genética , Proteínas de Ligação a RNA/genética , Ribonucleotídeos/administração & dosagem , Quinases Proteína-Quinases Ativadas por AMP , Aminoimidazol Carboxamida/administração & dosagem , Animais , Modelos Animais de Doenças , Humanos , Camundongos , Atividade Motora/efeitos dos fármacos , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/fisiopatologia , Mioblastos/efeitos dos fármacos , Distrofia Miotônica/genética , Distrofia Miotônica/fisiopatologia , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/genética , Resveratrol/administração & dosagem , Expansão das Repetições de Trinucleotídeos/genéticaRESUMO
Myotonic Dystrophy type 1 (DM1) is caused by an expansion of CUG repeats in DMPK mRNAs. This mutation affects alternative splicing through misregulation of RNA-binding proteins. Amongst pre-mRNAs that are mis-spliced, several code for proteins involved in calcium homeostasis suggesting that calcium-handling and signaling are perturbed in DM1. Here, we analyzed expression of such proteins in DM1 mouse muscle. We found that the levels of several sarcoplasmic reticulum proteins (SERCA1, sarcolipin and calsequestrin) are altered, likely contributing to an imbalance in calcium homeostasis. We also observed that calcineurin (CnA) signaling is hyperactivated in DM1 muscle. Indeed, CnA expression and phosphatase activity are both markedly increased in DM1 muscle. Coherent with this, we found that activators of the CnA pathway (MLP, FHL1) are also elevated. Consequently, NFATc1 expression is increased in DM1 muscle and becomes relocalized to myonuclei, together with an up-regulation of its transcriptional targets (RCAN1.4 and myoglobin). Accordingly, DM1 mouse muscles display an increase in oxidative metabolism and fiber hypertrophy. To determine the functional consequences of this CnA hyperactivation, we administered cyclosporine A, an inhibitor of CnA, to DM1 mice. Muscles of treated DM1 mice showed an increase in CUGBP1 levels, and an exacerbation of key alternative splicing events associated with DM1. Finally, inhibition of CnA in cultured human DM1 myoblasts also resulted in a splicing exacerbation of the insulin receptor. Together, these findings show for the first time that calcium-CnA signaling is hyperactivated in DM1 muscle and that such hyperactivation represents a beneficial compensatory adaptation to the disease.
Assuntos
Calcineurina/metabolismo , Distrofia Miotônica/genética , Miotonina Proteína Quinase/genética , Processamento Alternativo , Animais , Antígenos CD , Calcineurina/genética , Cálcio/metabolismo , Sinalização do Cálcio , Técnicas de Cultura de Células , Modelos Animais de Doenças , Fibroblastos/metabolismo , Homeostase , Humanos , Camundongos , Camundongos Transgênicos , Músculo Esquelético/metabolismo , Mioblastos/metabolismo , Distrofia Miotônica/metabolismo , Miotonina Proteína Quinase/metabolismo , Fatores de Transcrição NFATC , Splicing de RNA , RNA Mensageiro/genética , Proteínas de Ligação a RNA , Receptor de Insulina , Retículo Sarcoplasmático/genética , Retículo Sarcoplasmático/metabolismo , Transdução de Sinais , Regulação para CimaRESUMO
Myotonic dystrophy (DM1) is caused by an expansion of CUG repeats (CUG(exp)) in the DMPK mRNA 3'UTR. CUG(exp)-containing mRNAs become toxic to cells by misregulating RNA-binding proteins. Here we investigated the consequence of this RNA toxicity on the cellular stress response. We report that cell stress efficiently triggers formation of stress granules (SGs) in proliferating, quiescent, and differentiated muscle cells, as shown by the appearance of distinct cytoplasmic TIA-1- and DDX3-containing foci. We show that Staufen1 is also dynamically recruited into these granules. Moreover, we discovered that DM1 myoblasts fail to properly form SGs in response to arsenite. This blockage was not observed in DM1 fibroblasts, demonstrating a cell type-specific defect. DM1 myoblasts display increased expression and sequestration of toxic CUG(exp) mRNAs compared with fibroblasts. Of importance, down-regulation of Staufen1 in DM1 myoblasts rescues SG formation. Together our data show that Staufen1 participates in the inhibition of SG formation in DM1 myoblasts. These results reveal that DM1 muscle cells fail to properly respond to stress, thereby likely contributing to the complex pathogenesis of DM1.
Assuntos
Grânulos Citoplasmáticos/metabolismo , Proteínas do Citoesqueleto/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Distrofia Miotônica/metabolismo , Proteínas de Ligação a RNA/metabolismo , Processamento Alternativo , Animais , Técnicas de Cultura de Células , Diferenciação Celular/fisiologia , Linhagem Celular , Células Cultivadas , Citoplasma/metabolismo , Grânulos Citoplasmáticos/patologia , Regulação para Baixo , Humanos , Camundongos , Fibras Musculares Esqueléticas/patologia , Mioblastos/metabolismo , Mioblastos/patologia , Distrofia Miotônica/genética , RNA Mensageiro/metabolismo , Estresse Fisiológico/genéticaRESUMO
Acetylcholine receptors (AChRs) are heteromeric membrane proteins essential for neurotransmission at the neuromuscular junction. Previous work showed that muscle denervation increases expression of AChR mRNAs due to transcriptional activation of AChR subunit genes. However, it remains possible that post-transcriptional mechanisms are also involved in controlling the levels of AChR mRNAs following denervation. We examined whether post-transcriptional events indeed regulate AChR ß-subunit mRNAs in response to denervation. First, in vitro stability assays revealed that the half-life of AChR ß-subunit mRNAs was increased in the presence of denervated muscle protein extracts. A bioinformatics analysis revealed the existence of a conserved AU-rich element (ARE) in the 3'-untranslated region (UTR) of AChR ß-subunit mRNA. Furthermore, denervation of mouse muscle injected with a luciferase reporter construct containing the AChR ß-subunit 3'UTR, caused an increase in luciferase activity. By contrast, mutation of this ARE prevented this increase. We also observed that denervation increased expression of the RNA-binding protein human antigen R (HuR) and induced its translocation to the cytoplasm. Importantly, HuR binds to endogenous AChR ß-subunit transcripts in cultured myotubes and in vivo, and this binding is increased in denervated versus innervated muscles. Finally, p38 MAPK, a pathway known to activate HuR, was induced following denervation as a result of MKK3/6 activation and a decrease in MKP-1 expression, thereby leading to an increase in the stability of AChR ß-subunit transcripts. Together, these results demonstrate the important contribution of post-transcriptional events in regulating AChR ß-subunit mRNAs and point toward a central role for HuR in mediating synaptic gene expression. SIGNIFICANCE STATEMENT: Muscle denervation is a convenient model to examine expression of genes encoding proteins of the neuromuscular junction, especially acetylcholine receptors (AChRs). Despite the accepted model of AChR regulation, which implicates transcriptional mechanisms, it remains plausible that such events cannot fully account for changes in AChR expression following denervation. We show that denervation increases expression of the RNA-binding protein HuR, which in turn, causes an increase in the stability of AChR ß-subunit mRNAs in denervated muscle. Our findings demonstrate for the first time the contribution of post-transcriptional events in controlling AChR expression in skeletal muscle, and points toward a central role for HuR in mediating synaptic development while also paving the way for developing RNA-based therapeutics for neuromuscular diseases.
Assuntos
Proteínas ELAV/metabolismo , Músculo Esquelético/inervação , Músculo Esquelético/metabolismo , Receptores Colinérgicos/metabolismo , Animais , Células Cultivadas , Proteínas ELAV/genética , Proteína Semelhante a ELAV 1 , Feminino , Membro Posterior/inervação , Camundongos , Denervação Muscular , Fibras Musculares Esqueléticas/citologia , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/citologia , Junção Neuromuscular/fisiologia , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Receptores Colinérgicos/genéticaRESUMO
Recent work has shown that Staufen1 plays key roles in skeletal muscle, yet little is known about its pattern of expression during embryonic and postnatal development. Here we first show that Staufen1 levels are abundant in mouse embryonic muscles and that its expression decreases thereafter, reaching low levels in mature muscles. A similar pattern of expression is seen as cultured myoblasts differentiate into myotubes. Muscle degeneration/regeneration experiments revealed that Staufen1 increases after cardiotoxin injection before returning to the low levels seen in mature muscles. We next prevented the decrease in Staufen1 during differentiation by generating stable C2C12 muscle cell lines overexpressing Staufen1. Cells overexpressing Staufen1 differentiated poorly, as evidenced by reductions in the differentiation and fusion indices and decreases in MyoD, myogenin, MEF2A, and MEF2C, independently of Staufen-mediated mRNA decay. However, levels of c-myc, a factor known to inhibit differentiation, were increased in C2C12 cells overexpressing Staufen1 through enhanced translation. By contrast, the knockdown of Staufen1 decreased c-myc levels in myoblasts. Collectively our results show that Staufen1 is highly expressed during early stages of differentiation/development and that it can impair differentiation by regulating c-myc, thereby highlighting the multifunctional role of Staufen1 in skeletal muscle cells.
Assuntos
Diferenciação Celular/genética , Desenvolvimento Muscular , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas de Ligação a RNA/genética , Animais , Regulação da Expressão Gênica no Desenvolvimento , Fatores de Transcrição MEF2/biossíntese , Camundongos , Músculos/metabolismo , Proteína MyoD/biossíntese , Proteínas Proto-Oncogênicas c-myc/biossíntese , Proteínas de Ligação a RNA/biossíntese , Regeneração/genéticaRESUMO
Several reports have previously highlighted the potential role of miR-206 in the post-transcriptional downregulation of utrophin A in cultured cells. Along those lines, we recently identified K-homology splicing regulator protein (KSRP) as an important negative regulator in the post-transcriptional control of utrophin A in skeletal muscle. We sought to determine whether these two pathways act together to downregulate utrophin A expression in skeletal muscle. Surprisingly, we discovered that miR-206 overexpression in cultured cells and dystrophic muscle fibers causes upregulation of endogenous utrophin A levels. We further show that this upregulation of utrophin A results from the binding of miR-206 to conserved sites located in the 3'-UTR (untranslated region) of KSRP, thus causing the subsequent inhibition of KSRP expression. This miR-206-mediated decrease in KSRP levels leads, in turn, to an increase in the expression of utrophin A due to a reduction in the activity of this destabilizing RNA-binding protein. Our work shows that miR-206 can oscillate between direct repression of utrophin A expression via its 3'-UTR and activation of its expression through decreased availability of KSRP and interactions with AU-rich elements located within the 3'-UTR of utrophin A. Our study thus reveals that two apparent negative post-transcriptional pathways can act distinctively as molecular switches causing repression or activation of utrophin A expression.
Assuntos
Regulação da Expressão Gênica , MicroRNAs/metabolismo , Músculo Esquelético/metabolismo , Proteínas de Ligação a RNA/metabolismo , Transativadores/metabolismo , Utrofina/metabolismo , Regiões 3' não Traduzidas , Animais , Diferenciação Celular , Linhagem Celular , Masculino , Camundongos , Camundongos Endogâmicos mdx , Músculo Esquelético/citologia , Proteínas de Ligação a RNA/genética , Transativadores/genética , Regulação para Cima , Utrofina/genéticaRESUMO
Several therapeutic approaches are currently being developed for Duchenne muscular dystrophy (DMD) including upregulating the levels of endogenous utrophin A in dystrophic fibers. Here, we examined the role of post-transcriptional mechanisms in controlling utrophin A expression in skeletal muscle. We show that activation of p38 leads to an increase in utrophin A independently of a transcriptional induction. Rather, p38 controls the levels of utrophin A mRNA by extending the half-life of transcripts via AU-rich elements (AREs). This mechanism critically depends on a decrease in the functional availability of KSRP, an RNA-binding protein known to promote decay of ARE-containing transcripts. In vitro and in vivo binding studies revealed that KSRP interacts with specific AREs located within the utrophin A 3' UTR. Electroporation experiments to knockdown KSRP led to an increase in utrophin A in wild-type and mdx mouse muscles. In pre-clinical studies, treatment of mdx mice with heparin, an activator of p38, causes a pronounced increase in utrophin A in diaphragm muscle fibers. Together, these studies identify a pathway that culminates in the post-transcriptional regulation of utrophin A through increases in mRNA stability. Furthermore, our results constitute proof-of-principle showing that pharmacological activation of p38 may prove beneficial as a novel therapeutic approach for DMD.
Assuntos
Elementos Ricos em Adenilato e Uridilato , Músculo Esquelético/metabolismo , Estabilidade de RNA , Proteínas de Ligação a RNA/metabolismo , Transdução de Sinais , Transativadores/metabolismo , Utrofina/genética , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Regiões 3' não Traduzidas , Animais , Ativação Enzimática , Regulação da Expressão Gênica/efeitos dos fármacos , Heparina/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos mdx , Distrofia Muscular Animal , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/metabolismo , Ligação Proteica , Processamento Pós-Transcricional do RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Utrofina/metabolismoRESUMO
The RBP (RNA-binding protein) and Hu/ELAV family member HuD regulates mRNA metabolism of genes directly or indirectly involved in neuronal differentiation, learning and memory, and several neurological diseases. Given the important functions of HuD in a variety of processes, we set out to determine the mechanisms that promote HuD mRNA expression in neurons using a mouse model. Through several complementary approaches, we determined that the abundance of HuD mRNA is predominantly under transcriptional control in developing neurons. Bioinformatic and 5'RACE (rapid amplification of cDNA ends) analyses of the 5' genomic flanking region identified eight conserved HuD leader exons (E1s), two of which are novel. Expression of all E1 variants was determined in mouse embryonic (E14.5) and adult brains. Sequential deletion of the 5' regulatory region upstream of the predominantly expressed E1c variant revealed a well conserved 400 bp DNA region that contains five E-boxes and is capable of directing HuD expression specifically in neurons. Using EMSA (electrophoretic mobility shift assay), ChIP (chromatin immunoprecipitation), and 5' regulatory region deletion and mutation analysis, we found that two of these E-boxes are targets of Neurogenin 2 (Ngn2) and that this mechanism is important for HuD mRNA induction. Together, our findings reveal that transcriptional regulation of HuD involves the use of alternate leader exons and Ngn2 mediates neuron-specific mRNA expression. To our knowledge, this is the first study to identify molecular events that positively regulate HuD mRNA expression.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Proteínas ELAV/genética , Éxons/genética , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismo , RNA Mensageiro/genética , Animais , Encéfalo/citologia , Encéfalo/embriologia , Encéfalo/metabolismo , Bovinos , Linhagem Celular Transformada , Nucléolo Celular/genética , Nucléolo Celular/metabolismo , Imunoprecipitação da Cromatina , Biologia Computacional , Dactinomicina/farmacologia , Elementos E-Box/efeitos dos fármacos , Elementos E-Box/genética , Proteínas ELAV/metabolismo , Proteína Semelhante a ELAV 4 , Ensaio de Desvio de Mobilidade Eletroforética , Embrião de Mamíferos , Feminino , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Regulação da Expressão Gênica no Desenvolvimento/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Mutagênese/fisiologia , Proteínas do Tecido Nervoso/genética , Neurônios/efeitos dos fármacos , Neurônios/ultraestrutura , Inibidores da Síntese de Proteínas/farmacologia , Ratos , Alinhamento de SequênciaRESUMO
In myotonic dystrophy type 1 (DM1), dystrophia myotonica protein kinase messenger ribonucleic acids (RNAs; mRNAs) with expanded CUG repeats (CUG(exp)) aggregate in the nucleus and become toxic to cells by sequestering and/or misregulating RNA-binding proteins, resulting in aberrant alternative splicing. In this paper, we find that the RNA-binding protein Staufen1 is markedly and specifically increased in skeletal muscle from DM1 mouse models and patients. We show that Staufen1 interacts with mutant CUG(exp) mRNAs and promotes their nuclear export and translation. This effect is critically dependent on the third double-stranded RNA-binding domain of Staufen1 and shuttling of Staufen1 into the nucleus via its nuclear localization signal. Moreover, we uncover a new role of Staufen1 in splicing regulation. Overexpression of Staufen1 rescues alternative splicing of two key pre-mRNAs known to be aberrantly spliced in DM1, suggesting its increased expression represents an adaptive response to the pathology. Altogether, our results unravel a novel function for Staufen1 in splicing regulation and indicate that it may positively modulate the complex DM1 phenotype, thereby revealing its potential as a therapeutic target.
Assuntos
Proteínas do Citoesqueleto/genética , Músculo Esquelético/metabolismo , Distrofia Miotônica/genética , Precursores de RNA/metabolismo , Splicing de RNA , Proteínas de Ligação a RNA/genética , Animais , Proteínas do Citoesqueleto/metabolismo , Feminino , Imunofluorescência , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Distrofia Miotônica/metabolismo , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Transfecção , Expansão das Repetições de TrinucleotídeosRESUMO
Brain-derived neurotrophic factor (BDNF) is required for efficient skeletal-muscle regeneration and perturbing its expression causes abnormalities in the proliferation and differentiation of skeletal muscle cells. In this study, we investigated the mechanism of BDNF suppression that occurs during myogenic differentiation. BDNF is expressed at the mRNA level as two isoforms that differ in the length of their 3'UTRs as a result of alternative cleavage and polyadenylation. Sequence analysis revealed the presence of three miR-206 target sites in the long BDNF 3'UTR (BDNF-L), whereas only one site was found in the short mRNA BDNF 3'UTR (BDNF-S). miR-206 is known to regulate the differentiation of C2C12 myoblasts and its expression is induced during the transition from myoblasts to myotubes. We thus examined whether miR-206-mediated suppression is responsible for the expression pattern of BDNF during myogenic differentiation. BDNF-L was suppressed to a greater extent than BDNF-S during differentiation of C2C12 myoblasts. Transfection of a miR-206 precursor decreased activity of reporters representative of the BDNF-L 3'UTR, but not BDNF-S 3'UTR, and repressed endogenous BDNF mRNA levels. This suppression was found to be dependent on the presence of multiple miR-206 target sites in the BDNF-L 3'UTR. Conversely, suppression of miR-206 levels resulted in de-repression of BDNF 3'UTR reporter activity and increased endogenous BDNF-L mRNA levels. A receptor for BDNF, p75(NTR) , was also suppressed during differentiation and in response to miR-206, but this appeared to not be entirely mediated via a miR-206 target site its 3'UTR. Based on these observations, BDNF represents a novel target through which miR-206 controls the initiation and maintenance of the differentiated state of muscle cells. These results further suggest that miR-206 might play a role in regulating retrograde signaling of BDNF at the neuromuscular junction.
Assuntos
Fator Neurotrófico Derivado do Encéfalo/metabolismo , Diferenciação Celular/fisiologia , Regulação da Expressão Gênica/fisiologia , MicroRNAs/fisiologia , Desenvolvimento Muscular/fisiologia , Regiões 3' não Traduzidas/fisiologia , Animais , Fator Neurotrófico Derivado do Encéfalo/genética , Diferenciação Celular/genética , Linhagem Celular Transformada , Inibidores Enzimáticos/farmacologia , Ensaio de Imunoadsorção Enzimática/métodos , Camundongos , Mutação/genética , Mioblastos/fisiologia , Isoformas de RNA/metabolismo , RNA Mensageiro/metabolismo , Receptor de Fator de Crescimento Neural/genética , Receptor de Fator de Crescimento Neural/metabolismo , TransfecçãoRESUMO
A therapeutic strategy to treat Duchenne muscular dystrophy (DMD) involves identifying compounds that can elevate utrophin A expression in muscle fibers of affected patients. The dystrophin homologue utrophin A can functionally substitute for dystrophin when its levels are enhanced in the mdx mouse model of DMD. Utrophin A expression in skeletal muscle is regulated by mechanisms that promote the slow myofiber program. Since activation of peroxisome proliferator-activated receptor (PPAR) beta/delta promotes the slow oxidative phenotype in skeletal muscle, we initiated studies to determine whether pharmacological activation of PPARbeta/delta provides functional benefits to the mdx mouse. GW501516, a PPARbeta/delta agonist, was found to stimulate utrophin A mRNA levels in C2C12 muscle cells through an element in the utrophin A promoter. Expression of PPARbeta/delta was greater in skeletal muscles of mdx versus wild-type mice. We treated 5-7-week-old mdx mice with GW501516 for 4 weeks. This treatment increased the percentage of muscle fibers expressing slower myosin heavy chain isoforms and stimulated utrophin A mRNA levels leading to its increased expression at the sarcolemma. Expression of alpha1-syntrophin and beta-dystroglycan was restored to the sarcolemma. Improvement of mdx sarcolemmal integrity was evidenced by decreased intracellular IgM staining and decreased in vivo Evans blue dye (EBD) uptake. GW501516 treatment also conferred protection against eccentric contraction (ECC)-induced damage of mdx skeletal muscles, as shown by a decreased contraction-induced force drop and reduction of dye uptake during ECC. These results demonstrate that pharmacological activation of PPARbeta/delta might provide functional benefits to DMD patients through enhancement of utrophin A expression.
Assuntos
Expressão Gênica/efeitos dos fármacos , Fibras Musculares Esqueléticas/metabolismo , Distrofia Muscular de Duchenne/tratamento farmacológico , PPAR alfa/metabolismo , PPAR beta/metabolismo , Tiazóis/administração & dosagem , Utrofina/genética , Animais , Linhagem Celular , Modelos Animais de Doenças , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos mdx , Fibras Musculares Esqueléticas/efeitos dos fármacos , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/metabolismo , PPAR alfa/agonistas , PPAR alfa/genética , PPAR beta/agonistas , PPAR beta/genética , Sarcolema/efeitos dos fármacos , Sarcolema/metabolismo , Utrofina/metabolismoRESUMO
In wild-type mice, a single injection of streptozotocin (STZ, 200 mg/kg body wt) caused within 4 days severe hyperglycemia, hypoinsulinemia, significant glucose intolerance, loss of body weight, and the disappearance of pancreatic beta-cells. However, in ATP-sensitive K(+) channel (K(ATP) channel)-deficient mice (Kir6.2(-/-) mice), STZ had none of these effects. Exposing isolated pancreatic islets to STZ caused severe damage in wild-type but not in Kir6.2(-/-) islets. Following a single injection, plasma STZ levels were slightly less in Kir6.2(-/-) mice than in wild-type mice. Despite the difference in plasma STZ, wild-type and Kir6.2(-/-) liver accumulated the same amount of STZ, whereas Kir6.2(-/-) pancreas accumulated 4.1-fold less STZ than wild-type pancreas. Kir6.2(-/-) isolated pancreatic islets also transported less glucose than wild-type ones. Quantification of glucose transporter 2 (GLUT2) protein content by Western blot using an antibody with an epitope in the extracellular loop showed no significant difference in GLUT2 content between wild-type and Kir6.2(-/-) pancreatic islets. However, visualization by immunofluorescence with the same antibody gave rise to 32% less fluorescence in Kir6.2(-/-) pancreatic islets. The fluorescence intensity using another antibody, with an epitope in the COOH terminus, was 5.6 times less in Kir6.2(-/-) than in wild-type pancreatic islets. We conclude that 1) Kir6.2(-/-) mice are STZ resistant because of a decrease in STZ transport by GLUT2 in pancreatic beta-cells and 2) the decreased transport is due to a downregulation of GLUT2 activity involving an effect at the COOH terminus.
Assuntos
Antibióticos Antineoplásicos/farmacologia , Transportador de Glucose Tipo 2/metabolismo , Células Secretoras de Insulina/metabolismo , Canais KATP/fisiologia , Estreptozocina/farmacologia , Animais , Antibióticos Antineoplásicos/sangue , Antibióticos Antineoplásicos/metabolismo , Glicemia/metabolismo , Western Blotting , Citosol/metabolismo , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/patologia , Diabetes Mellitus Experimental/fisiopatologia , Resistência a Medicamentos , Transportador de Glucose Tipo 2/genética , Técnicas In Vitro , Insulina/sangue , Ilhotas Pancreáticas/efeitos dos fármacos , Ilhotas Pancreáticas/metabolismo , Canais KATP/deficiência , Canais KATP/genética , Fígado/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia de Fluorescência , Pâncreas/metabolismo , Canais de Potássio Corretores do Fluxo de Internalização/genética , Canais de Potássio Corretores do Fluxo de Internalização/fisiologia , Estreptozocina/sangue , Estreptozocina/metabolismoRESUMO
We examined the role of post-transcriptional mechanisms in controlling utrophin A mRNA expression in slow versus fast skeletal muscles. First, we determined that the half-life of utrophin A mRNA is significantly shorter in the presence of proteins isolated from fast muscles. Direct plasmid injection experiments using reporter constructs containing the full-length or truncated variants of the utrophin 3'UTR into slow soleus and fast extensor digitorum longus muscles revealed that a region of 265 nucleotides is sufficient to confer lower levels of reporter mRNA in fast muscles. Further analysis of this region uncovered a conserved AU-rich element (ARE) that suppresses expression of reporter mRNAs in cultured muscle cells. Moreover, stability of reporter mRNAs fused to the utrophin full-length 3'UTR was lower in the presence of fast muscle protein extracts. This destabilization effect seen in vivo was lost upon deletion of the conserved ARE. Finally, we observed that calcineurin signaling affects utrophin A mRNA stability through the conserved ARE. These results indicate that ARE-mediated mRNA decay is a key mechanism that regulates expression of utrophin A mRNA in slow muscle fibers. This is the first demonstration of ARE-mediated mRNA decay regulating the expression of a gene associated with the slow myogenic program.
Assuntos
Regiões 3' não Traduzidas/química , Calcineurina/metabolismo , Fibras Musculares de Contração Rápida/metabolismo , Fibras Musculares de Contração Lenta/metabolismo , Estabilidade de RNA , Sequências Reguladoras de Ácido Ribonucleico , Utrofina/genética , Adenosina/análise , Animais , Sequência de Bases , Linhagem Celular , Sequência Conservada , Regulação da Expressão Gênica , Genes Reporter , Camundongos , Camundongos Endogâmicos C57BL , Músculo Esquelético/metabolismo , RNA Mensageiro/metabolismo , Transdução de Sinais , Uridina/análise , Utrofina/metabolismoAssuntos
Sistemas de Apoio a Decisões Clínicas/organização & administração , Necessidades e Demandas de Serviços de Saúde/organização & administração , Recepcionistas de Consultório Médico/organização & administração , Informática em Enfermagem/organização & administração , Registros de Enfermagem , Humanos , Objetivos Organizacionais , Quebeque , Sociedades de Enfermagem/organização & administraçãoRESUMO
Over the last few years, several laboratories have focused their attention on elucidating the molecular events that control the expression and localization of acetylcholinesterase (AChE) in neurons and skeletal muscle cells. In this context, results from a number of studies have clearly shown the important contribution of transcriptional events in regulating AChE expression. Specifically, these studies have highlighted the roles of several cis- and trans-acting factors that control transcription of the AChE gene in these excitable cells. However, it has also become apparent that changes in the transcriptional activity of the AChE gene cannot fully account for the alterations seen in the overall abundance of AChE transcripts in neurons and muscle cells placed under a variety of experimental conditions. This indicates, therefore, that post-transcriptional mechanisms also play a significant role in controlling AChE mRNA expression. With this in mind, we have recently begun to address this issue in greater detail. Here, we provide a summary of our most recent findings dealing with the post-transcriptional regulation of AChE. Together, our studies have shown so far the important contribution of an AU-rich element located in the 3'UTR of AChE transcripts and of the stabilizing RNA-binding proteins of the ELAV-like family in regulating AChE expression in differentiating neuronal and muscle cells.
Assuntos
Acetilcolinesterase/metabolismo , Proteínas ELAV/metabolismo , Regulação Enzimológica da Expressão Gênica/genética , Músculo Esquelético/enzimologia , Neurônios/enzimologia , Acetilcolinesterase/genética , Animais , Diferenciação Celular , Proteínas ELAV/química , Humanos , Músculo Esquelético/citologia , Ratos , Transcrição GênicaRESUMO
We examined whether calcineurin-NFAT (nuclear factors of activated T cells) signaling plays a role in specifically directing the expression of utrophin in the synaptic compartment of muscle fibers. Immunofluorescence experiments revealed the accumulation of components of the calcineurin-NFAT signaling cascade within the postsynaptic membrane domain of the neuromuscular junction. RT-PCR analysis using synaptic vs. extrasynaptic regions of muscle fibers confirmed these findings by showing an accumulation of calcineurin transcripts within the synaptic compartment. We also examined the effect of calcineurin on utrophin gene expression. Pharmacological inhibition of calcineurin in mice with either cyclosporin A or FK506 resulted in a marked decrease in utrophin A expression at synaptic sites, whereas constitutive activation of calcineurin had the opposite effect. Mutation of the previously identified NFAT binding site in the utrophin A promoter region, followed by direct gene transfer studies in mouse muscle, led to an inhibition in the synaptic expression of a lacZ reporter gene construct. Transfection assays performed with cultured myogenic cells indicated that calcineurin acted additively with GA binding protein (GABP) to transactivate utrophin A gene expression. Because both GABP- and calcineurin-mediated pathways are targeted by peroxisome proliferator-activated receptor-gamma coactivator-1alpha (PGC-1alpha), we examined whether this coactivator contributes to utrophin gene expression. In vitro and in vivo transfection experiments showed that PGC-1alpha alone induces transcription from the utrophin A promoter. Interestingly, this induction is largely potentiated by coexpression of PGC-1alpha with GABP. Together, these studies indicate that the synaptic expression of utrophin is also driven by calcineurin-NFAT signaling and occurs in conjunction with signaling events that involve GABP and PGC-1alpha.
Assuntos
Calcineurina/fisiologia , Proteínas de Ligação a DNA/fisiologia , Regulação da Expressão Gênica/fisiologia , Junção Neuromuscular/metabolismo , Proteínas Nucleares/fisiologia , Transativadores/fisiologia , Fatores de Transcrição/fisiologia , Utrofina/biossíntese , Animais , Ciclosporina/farmacologia , Fator de Transcrição de Proteínas de Ligação GA , Imunossupressores/farmacologia , Camundongos , Músculo Esquelético/metabolismo , Fatores de Transcrição NFATC , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo , Transdução de Sinais , Tacrolimo/farmacologiaRESUMO
During myogenic differentiation, acetylcholinesterase (AChE) transcript levels are known to increase dramatically. Although this increase can be attributed in part to increased transcriptional activity, posttranscriptional mechanisms have also been implicated in the high levels of AChE mRNA in myotubes. In this study, we observed that transfection of a luciferase reporter construct containing the full-length AChE 3'-untranslated region (UTR) resulted in significantly higher (5-fold) luciferase activity in differentiated myotubes versus myoblasts. RNA-electrophoretic mobility shift assays (REMSAs) performed with a full-length AChE 3'-UTR probe and the AU-rich element revealed that the intensity of RNA-binding protein complexes increased as myogenic differentiation proceeded. Using several complementary approaches including supershift REMSA, mRNA-binding protein pull-down assays, and immunoprecipitation followed by reverse transcription-PCR, we found that the mRNA-stabilizing protein HuR interacts directly with AChE transcripts. Stable overexpression of HuR in C2C12 cells increased the expression of endogenous AChE transcripts as well as that of the luciferase reporter construct containing the AChE 3'-UTR. In vitro stability assays performed with protein extracts from these cells versus controls resulted in a slower rate of AChE mRNA decay. The down-regulation of HuR expression mediated through small interfering RNA further confirmed the role of HuR in the regulation of AChE mRNA levels. Taken together, these studies demonstrate that HuR interacts with the AChE 3'-UTR to regulate posttranscriptionally the expression of AChE mRNA during myogenic differentiation.
Assuntos
Acetilcolinesterase/genética , Antígenos de Superfície/metabolismo , Regulação Enzimológica da Expressão Gênica/fisiologia , Mioblastos Esqueléticos/citologia , Mioblastos Esqueléticos/fisiologia , Proteínas de Ligação a RNA/metabolismo , Regiões 3' não Traduzidas/fisiologia , Animais , Diferenciação Celular/fisiologia , Células Cultivadas , Proteínas ELAV , Proteína Semelhante a ELAV 1 , Camundongos , Estabilidade de RNA/fisiologia , RNA Mensageiro/metabolismoRESUMO
Staufen is an RNA-binding protein, first identified for its role in oogenesis and CNS development in Drosophila. Two mammalian homologs of Staufen have been identified and shown to bind double-stranded RNA and tubulin, and to function in the somatodendritic transport of mRNA in neurons. Here, we examined whether Staufen proteins are expressed in skeletal muscle in relation to the neuromuscular junction. Immunofluorescence experiments revealed that Staufen1 (Stau1) and Staufen2 (Stau2) accumulate preferentially within the postsynaptic sarcoplasm of muscle fibers as well as at newly formed ectopic synapses. Western blot analyses showed that the levels of Stau1 and Stau2 are greater in slow muscles than in fast-twitch muscles. Muscle denervation induced a significant increase in the expression of Stau1 and Stau2 in the extrasynaptic compartment of both fast and slow muscles. Consistent with these observations, we also demonstrated that expression of Stau1 and Stau2 is increased during myogenic differentiation and that treatment of myotubes with agrin and neuregulin induces a further increase in the expression of both Staufen proteins. We propose that Stau1 and Stau2 are key components of the postsynaptic apparatus in muscle, and that they contribute to the maturation and plasticity of the neuromuscular junction.
Assuntos
Proteínas do Tecido Nervoso/biossíntese , Junção Neuromuscular/metabolismo , Proteínas de Ligação a RNA/biossíntese , Agrina/farmacologia , Animais , Western Blotting , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/fisiologia , Linhagem Celular , Camundongos , Camundongos Endogâmicos C57BL , Denervação Muscular , Fibras Musculares de Contração Rápida/química , Fibras Musculares de Contração Rápida/metabolismo , Fibras Musculares Esqueléticas/citologia , Fibras Musculares Esqueléticas/efeitos dos fármacos , Fibras Musculares Esqueléticas/metabolismo , Fibras Musculares de Contração Lenta/química , Fibras Musculares de Contração Lenta/metabolismo , Músculo Esquelético/química , Músculo Esquelético/citologia , Músculo Esquelético/metabolismo , Mioblastos/citologia , Mioblastos/metabolismo , Proteínas do Tecido Nervoso/análise , Neurregulinas/farmacologia , Proteínas de Ligação a RNA/análiseRESUMO
Expression of acetylcholinesterase (AChE) is greatly enhanced during neuronal differentiation, but the nature of the molecular mechanisms remains to be fully defined. In this study, we observed that nerve growth factor treatment of PC12 cells leads to a progressive increase in the expression of AChE transcripts, reaching approximately 3.5-fold by 72 h. Given that the AChE 3'-untranslated region (UTR) contains an AU-rich element, we focused on the potential role of the RNA-binding protein HuD in mediating the increase in AChE mRNA seen in differentiating neurons. Using PC12 cells engineered to stably express HuD or an antisense to HuD, our studies indicate that HuD can regulate the abundance of AChE transcripts in neuronal cells. Furthermore, transfection of a reporter construct containing the AChE 3'-UTR showed that this 3'-UTR can increase expression of the reporter gene product in cells expressing HuD but not in cells expressing the antisense. RNA gel shifts and Northwestern blots revealed an increase in the binding of several protein complexes in differentiated neurons. Immunoprecipitation experiments demonstrated that HuD can bind directly AChE transcripts. These results show the importance of post-transcriptional mechanisms in regulating AChE expression in differentiating neurons and implicate HuD as a key trans-acting factor in these events.