RESUMO
Lysosomal diseases are a class of genetic disorders predominantly caused by loss of lysosomal hydrolases, leading to lysosomal and cellular dysfunction. Enzyme replacement therapy (ERT), where recombinant enzyme is given intravenously, internalized by cells, and trafficked to the lysosome, has been applied to treat several lysosomal diseases. However, current ERT regimens do not correct disease phenotypes in all affected organs because the biodistribution of enzyme uptake does not match that of the affected cells that require the enzyme. We present here targeted ERT, an approach that utilizes antibody-enzyme fusion proteins to target the enzyme to specific cell types. The antibody moiety recognizes transmembrane proteins involved in lysosomal trafficking and that are also preferentially expressed in those cells most affected in disease. Using Pompe disease (PD) as an example, we show that targeted ERT is superior to ERT in treating the skeletal muscle phenotypes of PD mice both as a protein replacement therapeutic and as a gene therapy.
Assuntos
Doença de Depósito de Glicogênio Tipo II , Doenças por Armazenamento dos Lisossomos , Animais , Terapia de Reposição de Enzimas , Doença de Depósito de Glicogênio Tipo II/tratamento farmacológico , Doença de Depósito de Glicogênio Tipo II/genética , Hidrolases/metabolismo , Doenças por Armazenamento dos Lisossomos/tratamento farmacológico , Doenças por Armazenamento dos Lisossomos/genética , Lisossomos/metabolismo , Camundongos , Distribuição Tecidual , alfa-Glucosidases/genéticaRESUMO
Signaling mediated by the mechanistic target of rapamycin (mTOR) is believed to play a critical and positive role in adipogenesis, based on pharmacological evidence and genetic manipulation of mTOR regulators and targets. However, there is no direct genetic evidence for an autonomous role of mTOR itself in preadipocyte differentiation. To seek such evidence, we employed a conditional knockdown approach to deplete mTOR in preadipocytes. Surprisingly, while knockdown of S6K1, a target of mTOR, impairs 3T3-L1 preadipocyte differentiation, reduction of mTOR levels leads to increased differentiation. This enhanced adipogenesis requires the remaining mTOR activity, as mTOR inhibitors abolish differentiation in the mTOR knockdown cells. We also found that mTOR knockdown elevates the levels of CCAAT/enhancer-binding protein α (C/EBPα) and peroxisome proliferator-activated receptor γ (PPARγ). Furthermore, partial reduction of mTOR levels alleviates inhibition of Akt by mTORC1 via IRS1, while at the same time maintaining its positive input through mTORC1 into the adipogenic program. The greater sensitivity of the IRS1-Akt pathway to mTOR levels provides a mechanism that explains the net outcome of enhanced adipogenesis through PPARγ upon mTOR knockdown. Our observations reveal an unexpected role of mTOR in suppressing adipogenesis and suggest that mTOR governs the homeostasis of the adipogenic process by modulating multiple signaling pathways.
Assuntos
Adipogenia/efeitos dos fármacos , Homeostase/efeitos dos fármacos , Sirolimo/farmacologia , Células 3T3-L1 , Animais , Diferenciação Celular/efeitos dos fármacos , Camundongos , Proteínas Quinases S6 Ribossômicas 90-kDa/metabolismo , Relação Estrutura-AtividadeRESUMO
Genomic imprinting relies on establishing and maintaining the parental-specific methylation of DNA elements that control the differential expression of maternal and paternal alleles. Although the essential DNA methyltransferases have been discovered, proteins that regulate the sequence-specific establishment and maintenance of allelic methylation have not been identified. One candidate regulator of methylation, the zinc-finger protein CTCF, binds to the imprinting control region (ICR) of the genes Igf2 (encoding insulin-like growth factor 2) and H19 (fetal liver mRNA; refs. 1,2). The unmethylated maternal ICR is a chromatin boundary that prevents distant enhancers from activating Igf2 (refs. 3-6). In vitro experiments have suggested that CTCF mediates boundary activity of the maternal ICR, and that methylation of the paternal ICR abolishes this activity by preventing CTCF binding. Using mice with point mutations in all four CTCF sites in the ICR, we show that maternally transmitted mutant ICRs in neonatal mice acquire a substantial but heterogeneous degree of methylation. Mutant ICRs in oocytes and blastocysts are not methylated, however, indicating that binding of CTCF is not required to establish the unmethylated ICR during oogenesis. We also show that the mutant ICR lacks enhancer-blocking activity, as the expression of Igf2 is activated on mutant maternal chromosomes. Conversely, maternal H19 expression is reduced, suggesting a positive role for CTCF in the transcription of that gene. This study constitutes the first in vivo demonstration of the multiple functions of CTCF in an ICR.
Assuntos
Metilação de DNA , Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica , Fator de Crescimento Insulin-Like II/genética , RNA não Traduzido/genética , Proteínas Repressoras , Fatores de Transcrição/metabolismo , Alelos , Sequência de Aminoácidos , Animais , Animais Recém-Nascidos , Sítios de Ligação , Blastocisto/metabolismo , Fator de Ligação a CCCTC , Linhagem Celular , DNA/genética , DNA/metabolismo , Proteínas de Ligação a DNA/genética , Feminino , Impressão Genômica , Masculino , Camundongos , Dados de Sequência Molecular , Mutação , Oócitos/metabolismo , Ligação Proteica , RNA Longo não Codificante , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Sulfitos/metabolismo , Fatores de Transcrição/genéticaRESUMO
Methylation of CpGs is generally thought to repress transcription without significant influence from the sequence surrounding the methylated dinucleotides. Using the mouse Igf2/H19 imprinting control region (ICR), Igf2r differentially methylated region 2 (DMR2) and bacterial sequences, we addressed how methylation-dependent repression (MDR) from a distance varies with CpG number, density and surrounding sequence. In stably transfected F9 cells, the methylated ICR repressed expression from a CpG-free reporter plasmid more than 1000-fold compared with its unmethylated control. A segment of pBluescript, with a CpG number equal to the ICR's but with a higher density, repressed expression only 70-fold when methylated. A bacteriophage lambda fragment and the Igf2r DMR2 showed minimal MDR activity, despite having CpG numbers and densities similar to or greater than the ICR. By rearranging or deleting CpGs, we identified CpGs associated with three CTCF sites in the ICR that are necessary and sufficient for sequence-specific MDR. In contrast to F9 cells, the methylated ICR and pBS fragments exhibited only 3-fold reporter repression in Hela cells and none in Cos7. Our results show that the strength of MDR from a distance can vary a 1000-fold between different cell types and depends on the sequence surrounding the methylated CpGs, but does not necessarily increase with CpG number or density.
Assuntos
Ilhas de CpG , Metilação de DNA , Impressão Genômica , Fator de Crescimento Insulin-Like II/genética , RNA não Traduzido/genética , Animais , Sequência de Bases , Fator de Ligação a CCCTC , Linhagem Celular , Proteínas de Ligação a DNA/metabolismo , Inativação Gênica , Humanos , Camundongos , Dados de Sequência Molecular , Regiões Promotoras Genéticas , RNA Longo não Codificante , Proteínas Repressoras/metabolismoRESUMO
CTCF is a multipurpose transcription factor with activation, repression, and insulator activity. It also participates in regulating chromatin architecture by maintaining open chromatin and mediating long-range chromosomal interactions. Participation by CTCF in such diverse processes suggests that it has multiple functional domains that regulate transcription and modify chromatin structure. Using transient and integrated reporters, we identified a 107-amino-acid domain in CTCF's N-terminal region that is capable of transcriptional activation and chromatin decondensation. This domain demonstrated moderate transactivation when targeted to a promoter proximal position but showed little activity from more distal positions and on a natural promoter. By contrast, the activation domain dramatically decondensed the compact chromatin structure of a large transgene array, in a manner similar to the potent activation domain in VP16. In addition, the activation domain is subject to conjugation by SUMO, which reduced its transcriptional and chromatin opening activity. Moreover, mimicking full sumoylation by fusing Sumo-1 or -3 to the activation domain eliminated its transcriptional activity, but only Sumo-3 fusion prevented chromatin opening. We suggest that the activation domain's limited transactivation, but strong chromatin decondensation allows CTCF to establish and maintain open chromatin without necessarily activating transcription. Sumoylation may contribute to CTCF's enhancer blocking or repression functions by reducing transactivation and chromatin opening.
Assuntos
Montagem e Desmontagem da Cromatina , Proteínas Repressoras/fisiologia , Sumoilação/fisiologia , Ativação Transcricional , Animais , Fator de Ligação a CCCTC , Linhagem Celular , Cromatina , Humanos , Camundongos , Estrutura Terciária de Proteína , Proteínas Repressoras/genética , TransfecçãoRESUMO
Rapamycin-sensitive signaling is required for skeletal muscle differentiation and remodeling. In cultured myoblasts, the mammalian target of rapamycin (mTOR) has been reported to regulate differentiation at different stages through distinct mechanisms, including one that is independent of mTOR kinase activity. However, the kinase-independent function of mTOR remains controversial, and no in vivo studies have examined those mTOR myogenic mechanisms previously identified in vitro. In this study, we find that rapamycin impairs injury-induced muscle regeneration. To validate the role of mTOR with genetic evidence and to probe the mechanism of mTOR function, we have generated and characterized transgenic mice expressing two mutants of mTOR under the control of human skeletal actin (HSA) promoter: rapamycin-resistant (RR) and RR/kinase-inactive (RR/KI). Our results show that muscle regeneration in rapamycin-administered mice is restored by RR-mTOR expression. In the RR/KI-mTOR mice, nascent myofiber formation during the early phase of regeneration proceeds in the presence of rapamycin, but growth of the regenerating myofibers is blocked by rapamycin. Igf2 mRNA levels increase drastically during early regeneration, which is sensitive to rapamycin in wild-type muscles but partially resistant to rapamycin in both RR- and RR/KI-mTOR muscles, consistent with mTOR regulation of Igf2 expression in a kinase-independent manner. Furthermore, systemic ablation of S6K1, a target of mTOR kinase, results in impaired muscle growth but normal nascent myofiber formation during regeneration. Therefore, mTOR regulates muscle regeneration through kinase-independent and kinase-dependent mechanisms at the stages of nascent myofiber formation and myofiber growth, respectively.
Assuntos
Proteínas de Transporte/metabolismo , Músculo Esquelético/fisiologia , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Fosfotransferases/metabolismo , Regeneração/fisiologia , Animais , Proteínas de Transporte/genética , Crescimento/efeitos dos fármacos , Humanos , Fator de Crescimento Insulin-Like II/genética , Fator de Crescimento Insulin-Like II/metabolismo , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Fibras Musculares Esqueléticas/fisiologia , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/crescimento & desenvolvimento , Mutação , Fosfotransferases (Aceptor do Grupo Álcool)/genética , RNA Mensageiro/metabolismo , Regeneração/efeitos dos fármacos , Proteínas Quinases S6 Ribossômicas 90-kDa/deficiência , Proteínas Quinases S6 Ribossômicas 90-kDa/metabolismo , Sirolimo/farmacologia , Serina-Treonina Quinases TORRESUMO
Insulin-like growth factors (IGFs) are essential for skeletal muscle development, regeneration, and hypertrophy. Although autocrine actions of IGF-II are known to initiate myoblast differentiation, the regulatory elements and upstream signaling pathways for myogenic expression of IGF-II remain elusive. Here, we report the regulation of IGF-II transcription by mTOR, as well as by amino acid sufficiency, through the IGF-II promoter 3 and a downstream enhancer during C2C12 myoblast differentiation. Furthermore, we present evidence that IGF production, and not IGF signaling, is the primary target for mTOR's function in the initiation of differentiation. Moreover, myogenic signaling by mTOR is independent of its kinase activity and mediated by the PI3K-Akt pathway. Our findings represent the first identification of a signaling pathway that regulates IGF-II expression in myogenesis and implicate the mTOR-IGF axis as a molecular link between nutritional levels and skeletal muscle development.
Assuntos
Aminoácidos/metabolismo , Regulação da Expressão Gênica , Fator de Crescimento Insulin-Like II/metabolismo , Desenvolvimento Muscular/fisiologia , Músculo Esquelético/crescimento & desenvolvimento , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases , Transcrição Gênica , Animais , Células Cultivadas , Elementos Facilitadores Genéticos , Fator de Crescimento Insulin-Like II/genética , Camundongos , Músculo Esquelético/citologia , Músculo Esquelético/metabolismo , Mioblastos/citologia , Mioblastos/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-akt , RNA Mensageiro/metabolismo , Transdução de Sinais/fisiologia , Sirolimo/metabolismo , Serina-Treonina Quinases TORRESUMO
Fibrodysplasia ossificans progressiva (FOP) is a rare genetic disorder characterized by episodically exuberant heterotopic ossification (HO), whereby skeletal muscle is abnormally converted into misplaced, but histologically normal bone. This HO leads to progressive immobility with catastrophic consequences, including death by asphyxiation. FOP results from mutations in the intracellular domain of the type I BMP (bone morphogenetic protein) receptor ACVR1; the most common mutation alters arginine 206 to histidine (ACVR1(R206H)) and has been thought to drive inappropriate bone formation as a result of receptor hyperactivity. We unexpectedly found that this mutation rendered ACVR1 responsive to the activin family of ligands, which generally antagonize BMP signaling through ACVR1 but cannot normally induce bone formation. To test the implications of this finding in vivo, we engineered mice to carry the Acvr1(R206H) mutation. Because mice that constitutively express Acvr1[R206H] die perinatally, we generated a genetically humanized conditional-on knock-in model for this mutation. When Acvr1[R206H] expression was induced, mice developed HO resembling that of FOP; HO could also be triggered by activin A administration in this mouse model of FOP but not in wild-type controls. Finally, HO was blocked by broad-acting BMP blockers, as well as by a fully human antibody specific to activin A. Our results suggest that ACVR1(R206H) causes FOP by gaining responsiveness to the normally antagonistic ligand activin A, demonstrating that this ligand is necessary and sufficient for driving HO in a genetically accurate model of FOP; hence, our human antibody to activin A represents a potential therapeutic approach for FOP.