RESUMO
Gene delivery vectors derived from adeno-associated virus (AAV) have great potential as therapeutic agents. rAAV1 and rAAV6, efficiently target striated muscle, but the mechanisms that determine their tropism remain unclear. It is known that AAV6, but not AAV1, interacts with heparin-sulfate proteoglycans (HSPG). HSPGs are not primary receptors for AAV6, but heparin interactions may affect tissue tropism and transduction. To investigate these possibilities, we generated rAAV1 and rAAV6 capsids that do or do not bind heparin. We evaluated the transduction profile of these vectors in vivo across multiple routes of administration, and found that heparin-binding capability influences tissue transduction in striated muscle and neuronal tissues. Heparin-binding capsids transduce striated muscle more efficiently than non-binding capsids, via both intramuscular and intravenous injection. However, rAAV6 achieved greater muscle transduction than the heparin-binding rAAV1 variant, suggesting that there are additional factors that influence differences in transduction efficiency between AAV1 and AAV6. Interestingly, the opposite trend was found when vectors were delivered via intracranial injection. Non-binding vectors achieved robust and widespread gene expression, whereas transduction via heparin-binding serotypes was substantially reduced. These data indicate that heparin-binding capability is an important determinant of transduction that should be considered in the design of rAAV-mediated gene therapies.
Assuntos
Dependovirus/genética , Técnicas de Transferência de Genes , Heparina/metabolismo , Músculo Estriado/metabolismo , Animais , Capsídeo/metabolismo , Dependovirus/metabolismo , Terapia Genética , Proteoglicanas de Heparan Sulfato , Heparina/genética , Humanos , Camundongos , Transdução Genética , Tropismo/genéticaRESUMO
The dystrophin gene encodes several tissue-specific protein isoforms that are generated by alternative splicing and by transcription from at least three separate promoters. We have characterized the mutation in a new strain of mdx mice that results in aberrant splicing of both the 14 and 4.8 kilobase dystrophin mRNAs and disrupts expression of the muscle and brain 427K and nonmuscle 70K isoforms of dystrophin. In contrast, we have determined that expression of the 70K isoform is normal in the original mdx mutant. We have cloned the unique 5' exon of the murine 4.8 kb mRNA and have analysed the tissue distribution and aberrant splicing of this transcript in the mdx3Cv mutant. This new mdx mutant will provide an improved model system for functional studies of the dystrophin C-terminus in muscle and nonmuscle tissues.
Assuntos
Distrofina/genética , Regulação da Expressão Gênica , Camundongos Mutantes/genética , Proteínas Musculares/genética , Distrofia Muscular Animal/genética , Splicing de RNA , RNA Mensageiro/genética , Animais , Sequência de Bases , Clonagem Molecular , Análise Mutacional de DNA , Éxons , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Proteínas Musculares/biossíntese , Mutagênese , Especificidade de Órgãos , Fenótipo , Polimorfismo de Fragmento de RestriçãoRESUMO
Two lines of transgenic mdx mice have been generated that express a 71 kD non-muscle isoform of dystrophin (Dp71) in skeletal muscle. This isoform contains the cysteine-rich and C-terminal domains of dystrophin, but lacks the N-terminal actin-binding and central spectrin-like repeat domains. Dp71 was associated with the sarcolemma membrane, where it restored normal expression and localization of all members of the dystrophin-associated glycoprotein complex. However, the skeletal muscle pathology of the transgenic mdx mice remained severe. These results indicate that the dystrophin C terminus cannot function independently to prevent dystrophic symptoms and confirms predictions based on patient data that both the N and C-terminal domains are required for normal dystrophin function.
Assuntos
Distrofina/análogos & derivados , Distrofias Musculares/genética , Animais , Linhagem Celular , Modelos Animais de Doenças , Distrofina/genética , Distrofina/metabolismo , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Distrofias Musculares/prevenção & controleRESUMO
Progressive force loss in Duchenne muscular dystrophy is characterized by degeneration/regeneration cycles and fibrosis. Disease progression may involve structural remodeling of muscle tissue. An effect on molecular motorprotein function may also be possible. We used second harmonic generation imaging to reveal vastly altered subcellular sarcomere microarchitecture in intact single dystrophic mdx muscle cells (approximately 1 year old). Myofibril tilting, twisting, and local axis deviations explain at least up to 20% of force drop during unsynchronized contractile activation as judged from cosine angle sums of myofibril orientations within mdx fibers. In contrast, in vitro motility assays showed unaltered sliding velocities of single mdx fiber myosin extracts. Closer quantification of the microarchitecture revealed that dystrophic fibers had significantly more Y-shaped sarcomere irregularities ("verniers") than wild-type fibers (approximately 130/1000 microm(3) vs. approximately 36/1000 microm(3)). In transgenic mini-dystrophin-expressing fibers, ultrastructure was restored (approximately 38/1000 microm(3) counts). We suggest that in aged dystrophic toe muscle, progressive force loss is reflected by a vastly deranged micromorphology that prevents a coordinated and aligned contraction. Second harmonic generation imaging may soon be available in routine clinical diagnostics, and in this work we provide valuable imaging tools to track and quantify ultrastructural worsening in Duchenne muscular dystrophy, and to judge the beneficial effects of possible drug or gene therapies.
Assuntos
Proteínas Motores Moleculares/química , Proteínas Motores Moleculares/metabolismo , Músculo Esquelético/metabolismo , Distrofias Musculares/metabolismo , Envelhecimento , Algoritmos , Animais , Fenômenos Biomecânicos , Distrofina/metabolismo , Regulação da Expressão Gênica , Humanos , Imageamento Tridimensional , Camundongos , Camundongos Endogâmicos mdx , Camundongos Transgênicos , Microscopia , Imagem Molecular , Movimento , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/patologia , Músculo Esquelético/fisiologia , Distrofias Musculares/fisiopatologia , Sarcômeros/metabolismoRESUMO
Becker muscular dystrophy is an X-linked disease due to mutations of the dystrophin gene. We now show that neuronal-type nitric oxide synthase (nNOS), an identified enzyme in the dystrophin complex, is uniquely absent from skeletal muscle plasma membrane in many human Becker patients and in mouse models of dystrophinopathy. An NH2-terminal domain of nNOS directly interacts with alpha 1-syntrophin but not with other proteins in the dystrophin complex analyzed. However, nNOS does not associate with alpha 1-syntrophin on the sarcolemma in transgenic mdx mice expressing truncated dystrophin proteins. This suggests a ternary interaction of nNOS, alpha 1-syntrophin, and the central domain of dystrophin in vivo, a conclusion supported by developmental studies in muscle. These data indicate that proper assembly of the dystrophin complex is dependent upon the structure of the central rodlike domain and have implications for the design of dystrophin-containing vectors for gene therapy.
Assuntos
Distrofias Musculares/enzimologia , Óxido Nítrico Sintase/metabolismo , Animais , Biópsia , Proteínas de Ligação ao Cálcio , Proteínas do Citoesqueleto/metabolismo , Técnica Indireta de Fluorescência para Anticorpo , Humanos , Glicoproteínas de Membrana/metabolismo , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Mutantes , Proteínas Musculares/metabolismo , Músculos/metabolismo , Sarcoglicanas , Sarcolema/enzimologia , UtrofinaRESUMO
Genetic defects in a number of components of the dystrophin-glycoprotein complex (DGC) lead to distinct forms of muscular dystrophy. However, little is known about how alterations in the DGC are manifested in the pathophysiology present in dystrophic muscle tissue. One hypothesis is that the DGC protects the sarcolemma from contraction-induced damage. Using tracer molecules, we compared sarcolemmal integrity in animal models for muscular dystrophy and in muscular dystrophy patient samples. Evans blue, a low molecular weight diazo dye, does not cross into skeletal muscle fibers in normal mice. In contrast, mdx mice, a dystrophin-deficient animal model for Duchenne muscular dystrophy, showed significant Evans blue accumulation in skeletal muscle fibers. We also studied Evans blue dispersion in transgenic mice bearing different dystrophin mutations, and we demonstrated that cytoskeletal and sarcolemmal attachment of dystrophin might be a necessary requirement to prevent serious fiber damage. The extent of dye incorporation in transgenic mice correlated with the phenotypic severity of similar dystrophin mutations in humans. We furthermore assessed Evans blue incorporation in skeletal muscle of the dystrophia muscularis (dy/dy) mouse and its milder allelic variant, the dy2J/dy2J mouse, animal models for congenital muscular dystrophy. Surprisingly, these mice, which have defects in the laminin alpha2-chain, an extracellular ligand of the DGC, showed little Evans blue accumulation in their skeletal muscles. Taken together, these results suggest that the pathogenic mechanisms in congenital muscular dystrophy are different from those in Duchenne muscular dystrophy, although the primary defects originate in two components associated with the same protein complex.
Assuntos
Fibras Musculares Esqueléticas/patologia , Músculo Esquelético/patologia , Distrofias Musculares/patologia , Distrofia Muscular Animal/patologia , Sarcolema/patologia , Animais , Distrofina/biossíntese , Distrofina/deficiência , Distrofina/genética , Azul Evans , Humanos , Camundongos , Camundongos Endogâmicos mdx , Camundongos Mutantes , Camundongos Transgênicos , Mutagênese , Mutação , Miocárdio/patologia , Albumina Sérica/análiseRESUMO
Dystrophin is a multidomain protein that links the actin cytoskeleton to laminin in the extracellular matrix through the dystrophin associated protein (DAP) complex. The COOH-terminal domain of dystrophin binds to two components of the DAP complex, syntrophin and dystrobrevin. To understand the role of syntrophin and dystrobrevin, we previously generated a series of transgenic mouse lines expressing dystrophins with deletions throughout the COOH-terminal domain. Each of these mice had normal muscle function and displayed normal localization of syntrophin and dystrobrevin. Since syntrophin and dystrobrevin bind to each other as well as to dystrophin, we have now generated a transgenic mouse deleted for the entire dystrophin COOH-terminal domain. Unexpectedly, this truncated dystrophin supported normal muscle function and assembly of the DAP complex. These results demonstrate that syntrophin and dystrobrevin functionally associate with the DAP complex in the absence of a direct link to dystrophin. We also observed that the DAP complexes in these different transgenic mouse strains were not identical. Instead, the DAP complexes contained varying ratios of syntrophin and dystrobrevin isoforms. These results suggest that alternative splicing of the dystrophin gene, which naturally generates COOH-terminal deletions in dystrophin, may function to regulate the isoform composition of the DAP complex.
Assuntos
Proteínas Associadas à Distrofina , Distrofina , Distrofias Musculares/metabolismo , Actinas/metabolismo , Animais , Sítios de Ligação/fisiologia , Núcleo Celular/patologia , Proteínas do Citoesqueleto/análise , Proteínas do Citoesqueleto/metabolismo , Modelos Animais de Doenças , Distrofina/química , Distrofina/genética , Distrofina/metabolismo , Éxons , Proteínas de Membrana/análise , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos mdx , Camundongos Transgênicos , Microssomos/química , Microssomos/metabolismo , Contração Muscular/genética , Fibras Musculares Esqueléticas/química , Fibras Musculares Esqueléticas/metabolismo , Fibras Musculares Esqueléticas/patologia , Proteínas Musculares/análise , Proteínas Musculares/metabolismo , Distrofias Musculares/patologia , Neuropeptídeos/análise , Neuropeptídeos/metabolismo , Estrutura Terciária de Proteína , UtrofinaRESUMO
Dystrophin plays an important role in skeletal muscle by linking the cytoskeleton and the extracellular matrix. The amino terminus of dystrophin binds to actin and possibly other components of the subsarcolemmal cytoskeleton, while the carboxy terminus associates with a group of integral and peripheral membrane proteins and glycoproteins that are collectively known as the dystrophin-associated protein (DAP) complex. We have generated transgenic/mdx mice expressing "full-length" dystrophin constructs, but with consecutive deletions within the COOH-terminal domains. These mice have enabled analysis of the interaction between dystrophin and members of the DAP complex and the effects that perturbing these associations have on the dystrophic process. Deletions within the cysteine-rich region disrupt the interaction between dystrophin and the DAP complex, leading to a severe dystrophic pathology. These deletions remove the beta-dystroglycan-binding site, which leads to a parallel loss of both beta-dystroglycan and the sarcoglycan complex from the sarcolemma. In contrast, deletion of the alternatively spliced domain and the extreme COOH terminus has no apparent effect on the function of dystrophin when expressed at normal levels. The proteins resulting from these latter two deletions supported formation of a completely normal DAP complex, and their expression was associated with normal muscle morphology in mdx mice. These data indicate that the cysteine-rich domain is critical for functional activity, presumably by mediating a direct interaction with beta-dystroglycan. However, the remainder of the COOH terminus is not required for assembly of the DAP complex.
Assuntos
Distrofina/fisiologia , Músculos/ultraestrutura , Animais , Éxons , Técnica Indireta de Fluorescência para Anticorpo , Substâncias Macromoleculares , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Camundongos Transgênicos , Distrofia Muscular Animal/patologia , Deleção de Sequência , Relação Estrutura-AtividadeRESUMO
The functional significance of the actin-binding domain of dystrophin, the protein lacking in patients with Duchenne muscular dystrophy, has remained elusive. Patients with deletions of this domain (domain I) typically express low levels of the truncated protein. Whether the moderate to severe phenotypes associated with such deletions result from loss of an essential function, or from reduced levels of a functional protein, is unclear. To address this question, we have generated transgenic mice that express wild-type levels of a dystrophin deleted for the majority of the actin-binding domain. The transgene derived protein lacks amino acids 45-273, removing 2 of 3 in vitro identified actin interacting sites and part of hinge 1. Examination of the effect of this deletion in mice lacking wild-type dystrophin (mdx) suggests that a functional domain I is not essential for prevention of a dystrophic phenotype. However, in contrast to deletions in the central rod domain and to full-length dystrophin, both of which are functional at only 20% of wild-type levels, proteins with a deletion in domain I must be expressed at high levels to prevent a severe dystrophy. These results are also in contrast to the severe dystrophy resulting from truncation of the COOH-terminal domain that links dystrophin to the extracellular matrix. The mild phenotype observed in mice with domain I-deletions indicates that an intact actin-binding domain is not essential, although it does contribute to an important function of dystrophin. These studies also suggest the link between dystrophin and the subsarcolemmal cytoskeleton involves more than a simple attachment of domain I to actin filaments.
Assuntos
Distrofina/fisiologia , Distrofia Muscular Animal/patologia , Deleção de Sequência , Actinina/análise , Actinas/metabolismo , Animais , Diafragma/química , Diafragma/patologia , Diafragma/fisiopatologia , Distrofina/análise , Distrofina/genética , Distrofina/metabolismo , Expressão Gênica , Camundongos , Camundongos Endogâmicos mdx , Camundongos Transgênicos , Contração Muscular , Músculo Esquelético/química , Músculo Esquelético/patologia , Distrofia Muscular Animal/fisiopatologia , Fenótipo , Ligação Proteica , Sarcolema/química , Transgenes/genéticaRESUMO
The dystrophin-glycoprotein complex (DGC) is a multisubunit complex that spans the muscle plasma membrane and forms a link between the F-actin cytoskeleton and the extracellular matrix. The proteins of the DGC are structurally organized into distinct subcomplexes, and genetic mutations in many individual components are manifested as muscular dystrophy. We recently identified a unique tetraspan-like dystrophin-associated protein, which we have named sarcospan (SPN) for its multiple sarcolemma spanning domains (Crosbie, R.H., J. Heighway, D.P. Venzke, J.C. Lee, and K.P. Campbell. 1997. J. Biol. Chem. 272:31221-31224). To probe molecular associations of SPN within the DGC, we investigated SPN expression in normal muscle as a baseline for comparison to SPN's expression in animal models of muscular dystrophy. We show that, in addition to its sarcolemma localization, SPN is enriched at the myotendinous junction (MTJ) and neuromuscular junction (NMJ), where it is a component of both the dystrophin- and utrophin-glycoprotein complexes. We demonstrate that SPN is preferentially associated with the sarcoglycan (SG) subcomplex, and this interaction is critical for stable localization of SPN to the sarcolemma, NMJ, and MTJ. Our experiments indicate that assembly of the SG subcomplex is a prerequisite for targeting SPN to the sarcolemma. In addition, the SG- SPN subcomplex functions to stabilize alpha-dystroglycan to the muscle plasma membrane. Taken together, our data provide important information about assembly and function of the SG-SPN subcomplex.
Assuntos
Proteínas de Transporte/metabolismo , Proteínas do Citoesqueleto/fisiologia , Distrofina/fisiologia , Glicoproteínas de Membrana/fisiologia , Proteínas de Membrana/metabolismo , Distrofia Muscular Animal/metabolismo , Proteínas de Neoplasias , Sequência de Aminoácidos , Animais , Transporte Biológico , Células CHO , Cardiomiopatia Hipertrófica/genética , Cardiomiopatia Hipertrófica/metabolismo , Cardiomiopatia Hipertrófica/patologia , Cricetinae , Proteínas do Citoesqueleto/deficiência , Proteínas do Citoesqueleto/genética , Distroglicanas , Distrofina/deficiência , Distrofina/genética , Humanos , Concentração de Íons de Hidrogênio , Substâncias Macromoleculares , Masculino , Glicoproteínas de Membrana/deficiência , Glicoproteínas de Membrana/genética , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Proteínas de Membrana/fisiologia , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Modelos Moleculares , Dados de Sequência Molecular , Músculo Esquelético/metabolismo , Distrofia Muscular Animal/genética , Junção Neuromuscular/metabolismo , Coelhos , Sarcoglicanas , Sarcolema/metabolismo , Sarcolema/ultraestrutura , Alinhamento de Sequência , Especificidade da Espécie , Tendões/metabolismo , Transfecção , UtrofinaRESUMO
Complementary DNA clones were isolated that represent the 5' terminal 2.5 kilobases of the murine Duchenne muscular dystrophy (Dmd) messenger RNA (mRNA). Mouse Dmd mRNA was detectable in skeletal and cardiac muscle and at a level approximately 90 percent lower in brain. Dmd mRNA is also present, but at much lower than normal levels, in both the muscle and brain of three different strains of dystrophic mdx mice. The identification of Dmd mRNA in brain raises the possibility of a relation between human Duchenne muscular dystrophy (DMD) gene expression and the mental retardation found in some DMD males. These results also provide evidence that the mdx mutations are allelic variants of mouse Dmd gene mutations.
Assuntos
Encéfalo/metabolismo , Regulação da Expressão Gênica , Músculos/metabolismo , Distrofia Muscular Animal/genética , Animais , DNA/genética , DNA Recombinante , Deficiência Intelectual/genética , Camundongos , Camundongos Endogâmicos ICR , Camundongos Mutantes , Mutação , Hibridização de Ácido Nucleico , RNA Mensageiro/metabolismo , Ribonuclease Pancreático/metabolismoRESUMO
Skeletal muscle unloaded shortening has been indirectly determined in the past. Here, we present a novel high-speed optical tracking technique that allows recording of unloaded shortening in single intact, voltage-clamped mammalian skeletal muscle fibers with 2-ms time resolution. L-type Ca(2+) currents were simultaneously recorded. The time course of shortening was biexponential: a fast initial phase, tau(1), and a slower successive phase, tau(2,) with activation energies of 59 kJ/mol and 47 kJ/mol. Maximum unloaded shortening speed, v(u,max), was faster than that derived using other techniques, e.g., approximately 14.0 L(0) s(-1) at 30 degrees C. Our technique also allowed direct determination of shortening acceleration. We applied our technique to single fibers from C57 wild-type, dystrophic mdx, and minidystrophin-expressing mice to test whether unloaded shortening was affected in the pathophysiological mechanism of Duchenne muscular dystrophy. v(u,max) and a(u,max) values were not significantly different in the three strains, whereas tau(1) and tau(2) were increased in mdx fibers. The results were complemented by myosin heavy and light chain (MLC) determinations that showed the same myosin heavy chain IIA profiles in the interossei muscles from the different strains. In mdx muscle, MLC-1f was significantly increased and MLC-2f and MLC-3f somewhat reduced. Fast initial active shortening seems almost unaffected in mdx muscle.
Assuntos
Distrofina/metabolismo , Microscopia de Vídeo/métodos , Fibras Musculares Esqueléticas/citologia , Fibras Musculares Esqueléticas/fisiologia , Músculo Esquelético/citologia , Músculo Esquelético/fisiologia , Animais , Células Cultivadas , Distrofina/genética , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos mdx , Camundongos Transgênicos , Microscopia de Vídeo/instrumentação , Técnicas de Patch-Clamp , Processamento de Sinais Assistido por Computador/instrumentaçãoRESUMO
Progress in understanding the role of dystrophin raises promising hopes for a treatment for Duchenne muscular dystrophy. In addition, great improvements have been made in the ability to diagnose this disease using simple molecular methods.
Assuntos
Distrofina/deficiência , Distrofias Musculares/genética , Cálcio/metabolismo , Distrofina/genética , Distrofina/fisiologia , Feminino , Triagem de Portadores Genéticos , Terapia Genética , Humanos , Masculino , Músculos/patologia , Distrofias Musculares/diagnóstico , Distrofias Musculares/terapia , Fenótipo , Reação em Cadeia da Polimerase , Transplante de Células-TroncoRESUMO
A screen for proteins that interact with beta 2-syntrophin led to the isolation of MAST205 (microtubule-associated serine/threonine kinase-205 kD) and a newly identified homologue, SAST (syntrophin-associated serine/threonine kinase). Binding studies showed that beta 2-syntrophin and MAST205/SAST associated via a PDZ-PDZ domain interaction. MAST205 colocalized with beta 2-syntrophin and utrophin at neuromuscular junctions. SAST colocalized with syntrophin in cerebral vasculature, spermatic acrosomes and neuronal processes. SAST and syntrophin were highly associated with purified microtubules and microtubule-associated proteins, whereas utrophin and dystrophin were only partially associated with microtubules. Our data suggest that MAST205 and SAST link the dystrophin/utrophin network with microtubule filaments via the syntrophins.
Assuntos
Proteínas de Membrana/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Sequência de Aminoácidos , Animais , Encéfalo/enzimologia , Células Cultivadas , Proteínas Associadas à Distrofina , Hipocampo/enzimologia , Masculino , Proteínas de Membrana/química , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos mdx , Proteínas Associadas aos Microtúbulos/química , Proteínas Associadas aos Microtúbulos/genética , Dados de Sequência Molecular , Músculo Esquelético/enzimologia , Neurônios/metabolismo , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/genética , Células Piramidais/enzimologia , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Testículo/enzimologiaRESUMO
A new animal model for studying muscular dystrophy, a mutant form of the nematode Caenorhabditis elegans, brings the power of worm genetics to bear on the search for a cure for this disease; work on this worm has already led to the identification of a novel component that can suppress the mutant phenotype.
Assuntos
Caenorhabditis elegans/genética , Distrofina/genética , Distrofia Muscular de Duchenne/genética , Animais , Modelos Animais de Doenças , Distrofina/metabolismo , Humanos , Modelos Biológicos , Músculo Esquelético/fisiologia , Distrofia Muscular de Duchenne/fisiopatologia , MutaçãoRESUMO
Duchenne Muscular Dystrophy (DMD) originates from deleterious mutations in the dystrophin gene, with a complete loss of the protein product. Subsequently, the disease is manifested in severe striated muscle wasting and death in early adulthood. Dystrophin provides a structural base for the assembly of an integral membrane protein complex. As such, dystrophin deficiency leads to an altered mechanical integrity of the myofiber and a predisposition to contraction-induced damage. However, the development of myofiber degeneration prior to an observed mechanical defect has been documented in various dystrophic models. Although activation of a detrimental signal transduction pathway has been suggested as a probable cause, a specific cellular cascade has yet to be defined. Here, it is shown that murine models of DMD displayed a muscle-specific activation of JNK1. Independent activation of JNK1 resulted in defects in myotube viability and integrity in vitro, similar to a dystrophic phenotype. In addition, direct muscle injection of an adenoviral construct containing the JNK1 inhibitory protein, JIP1, dramatically attenuated the progression of dystrophic myofiber destruction. Taken together, these results suggest that a JNK1-mediated signal cascade is a conserved feature of dystrophic muscle and contributes to the progression of the disease pathogenesis.
Assuntos
Proteínas Quinases Ativadas por Mitógeno/metabolismo , Músculo Esquelético/patologia , Distrofia Muscular de Duchenne/enzimologia , Distrofia Muscular de Duchenne/patologia , Adenoviridae/genética , Animais , Células Cultivadas , Ativação Enzimática , Proteínas de Fluorescência Verde , Humanos , Indicadores e Reagentes/metabolismo , Proteínas Luminescentes/metabolismo , Sistema de Sinalização das MAP Quinases , Camundongos , Camundongos Endogâmicos mdx , Camundongos Transgênicos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno , Proteína Quinase 8 Ativada por Mitógeno , Proteínas Quinases Ativadas por Mitógeno/genética , Músculo Esquelético/enzimologia , Músculo Esquelético/metabolismo , Distrofia Muscular Animal/enzimologia , Distrofia Muscular Animal/genética , Distrofia Muscular Animal/patologia , Distrofia Muscular de Duchenne/genética , Miocárdio/enzimologia , Miocárdio/metabolismo , Miocárdio/patologia , Fosforilação , TransfecçãoRESUMO
The regulation of creatine kinase (CK) induction during muscle differentiation was analyzed with MM14 mouse myoblasts. These cells withdraw from the cell cycle and commit to terminal differentiation when fed with mitogen-depleted medium. Myoblasts contained trace amounts of an isozyme of brain CK (designated BB-CK), but differentiation was accompanied by the induction of two other isozymes of muscle and brain CKs (designated MM-CK and MB-CK). Increased CK activity was detectable within 6 h of mitogen removal, 3 h after the first cells committed to differentiation and 6 h before fusion began. By 48 h, MM-CK activity increased more than 400-fold, MB-CK activity increased more than 150-fold, and BB-CK activity increased more than 10-fold. Antibodies prepared against purified mouse MM-CK cross-reacted with muscle and brain CKs (designated M-CK and B-CK, respectively) from a variety of species and were used to demonstrate that the increase in enzymatic activity was paralleled by an increase in the protein itself. CK antibodies were also used to aid in identifying cDNA clones to M-CK. cDNA sequences which corresponded to protein-coding regions cross-hybridized with B-CK mRNA; however, a subclone containing the 3'-nontranslated region was unique and was used to quantitate M-CK mRNA levels during myoblast differentiation. M-CK mRNA was not detectable in myoblasts, but within 5 to 6 h of mitogen withdrawal (6 to 7 h before fusion begins) it accumulated to about 30 molecules per cell. By 24 h, myotubes contained approximately 1,100 molecules per nucleus of M-CK mRNA.
Assuntos
Creatina Quinase/biossíntese , Isoenzimas/biossíntese , Proteínas Musculares/biossíntese , Músculos/enzimologia , Animais , Diferenciação Celular , Fusão Celular , Células Cultivadas , Embrião de Galinha , Indução Enzimática , Humanos , Camundongos , Mitógenos/farmacologia , Músculos/citologia , RNA Mensageiro/análise , CoelhosRESUMO
The muscle-specific form of creatine kinase (MCK) is induced in differentiating myoblast cultures, and a dramatic increase in mRNA levels precedes and parallels the increase in MCK protein. To study this induction, the complete MCK gene was cloned and characterized. The transcription unit was shown to span 11 kilobases and to contain seven introns. The splice junctions were identified and shown to conform to the appropriate consensus sequences. Close homology with branchpoint consensuses was found upstream of the 3' splice sites in six of seven cases. Transcriptional regulation of the gene in differentiating myoblast cultures was demonstrated by nuclear run-on experiments; increases in transcription accounted for a major part of the increased mRNA levels. Regulated expression of a transfected MCK gene containing the entire transcription unit with 3.3 kilobases of 5'-flanking sequence was also demonstrated during differentiation of the MM14 mouse myoblast cell line. The MCK 5'-flanking region was sufficient to confer transcriptional regulation to a heterologous structural gene, since chloramphenicol acetyl transferase activity was induced during differentiation of cultures transfected with an MCK-chloramphenicol acetyl transferase fusion construct. Examination of the DNA sequence immediately upstream of the transcription start site revealed a 17-nucleotide element which occurred three times. Comparisons with other muscle-specific genes which are also transcriptionally regulated during myogenesis revealed upstream homologies in the alpha-actin and myosin heavy chain genes, but not in the myosin light-chain genes, with the regions containing these repeats. We suggest that coordinate control of a subset of muscle genes may occur via recognition of these common sequences.
Assuntos
Creatina Quinase/genética , Regulação da Expressão Gênica , Músculos/enzimologia , Transcrição Gênica , Transfecção , Animais , Sequência de Bases , Diferenciação Celular , Clonagem Molecular , Camundongos , Splicing de RNA , RNA Mensageiro/metabolismo , Sequências Repetitivas de Ácido NucleicoRESUMO
A lack of dystrophin results in muscle degeneration in Duchenne muscular dystrophy. Dystrophin-deficient human and mouse muscle cells have higher resting levels of intracellular free calcium ([Ca2+]i) and show a related increase in single-channel open probabilities of calcium leak channels. Elevated [Ca2+]i results in high levels of calcium-dependent proteolysis, which in turn increases calcium leak channel activity. This process could initiate muscle degeneration by further increasing [Ca2+]i and proteolysis in a positive feedback loop. Here, we tested the direct effect of restoration of dystrophin on [Ca2+]i and channel activity in primary myotubes from mdx mice made transgenic for full-length dystrophin. Transgenic mdx mice have been previously shown to have normal dystrophin localization and no muscle degeneration. Fura-2 calcium measurements and single-channel patch recordings showed that resting [Ca2+]i levels and open probabilities of calcium leak channels of transgenic mdx myotubes were similar to normal levels and significantly lower than mdx littermate controls (mdx) that lack dystrophin. Thus, restoration of normal calcium regulation in transgenic mdx mice may underlie the resulting absence of degeneration.
Assuntos
Cálcio/metabolismo , Distrofina/genética , Distrofina/metabolismo , Terapia Genética , Músculo Esquelético/metabolismo , Distrofia Muscular Animal/metabolismo , Distrofia Muscular Animal/terapia , Animais , Canais de Cálcio/metabolismo , Células Cultivadas , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos mdx , Camundongos Transgênicos , Distrofia Muscular Animal/genéticaRESUMO
Secondary lymphoid tissue chemokine (SLC) is a CC chemokine that is selective in its recruitment of naive T cells and dendritic cells (DCs). In the lymph node, SLC is believed to play an important role in the initiation of an immune response by colocalizing naive T cells with DC-presenting antigen. Here, we used SLC as a treatment for tumors established from the poorly immunogenic B16 melanoma. Intratumoral injections of SLC inhibited tumor growth in a CD8+, T cell-dependent manner. SLC elicited a substantial infiltration of DCs and T cells into the tumor, coincident with the antitumor response. We next used SLC gene-modified DCs as a treatment of established tumors. Intratumoral injections of SLC-expressing DCs resulted in tumor growth inhibition that was significantly better than either control DCs or SLC alone. Distal site immunization of tumor-bearing mice with SLC gene-modified DCs pulsed with tumor lysate elicited an antitumor response whereas control DCs did not. We also found that s.c. injection of lysate-pulsed DCs expressing SLC promoted the migration of T cells to the immunization site. This report demonstrates that SLC can both induce antitumor responses and enhance the antitumor immunity elicited by DCs.