Enhanced expression of the alpha 7 beta 1 integrin reduces muscular dystrophy and restores viability in dystrophic mice.
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Muscle fibers attach to laminin in the basal lamina using two distinct mechanisms: the dystrophin glycoprotein complex and the alpha 7 beta 1 integrin. Defects in these linkage systems result in Duchenne muscular dystrophy (DMD), alpha 2 laminin congenital muscular dystrophy, sarcoglycan-related muscular dystrophy, and alpha 7 integrin congenital muscular dystrophy. Therefore, the molecular continuity between the extracellular matrix and cell cytoskeleton is essential for the structural and functional integrity of skeletal muscle. To test whether the alpha 7 beta 1 integrin can compensate for the absence of dystrophin, we expressed the rat alpha 7 chain in mdx/utr(-/-) mice that lack both dystrophin and utrophin. These mice develop a severe muscular dystrophy highly akin to that in DMD, and they also die prematurely. Using the muscle creatine kinase promoter, expression of the alpha 7BX2 integrin chain was increased 2.0-2.3-fold in mdx/utr(-/-) mice. Concomitant with the increase in the alpha 7 chain, its heterodimeric partner, beta 1D, was also increased in the transgenic animals. Transgenic expression of the alpha 7BX2 chain in the mdx/utr(-/-) mice extended their longevity by threefold, reduced kyphosis and the development of muscle disease, and maintained mobility and the structure of the neuromuscular junction. Thus, bolstering alpha 7 beta 1 integrin-mediated association of muscle cells with the extracellular matrix alleviates many of the symptoms of disease observed in mdx/utr(-/-) mice and compensates for the absence of the dystrophin- and utrophin-mediated linkage systems. This suggests that enhanced expression of the alpha 7 beta 1 integrin may provide a novel approach to treat DMD and other muscle diseases that arise due to defects in the dystrophin glycoprotein complex. A video that contrasts kyphosis, gait, joint contractures, and mobility in mdx/utr(-/-) and alpha 7BX2-mdx/utr(-/-) mice can be accessed at http://www.jcb.org/cgi/content/full/152/6/1207. (+info)
Interactions of the rapsyn RING-H2 domain with dystroglycan.
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Rapsyn, a peripheral membrane protein of skeletal muscle, is necessary for the formation of the highly organized structure of the vertebrate neuromuscular junction. For mice lacking rapsyn, there is a failure of postsynaptic specialization characterized by an absence of nicotinic acetylcholine receptors (nAChRs) and other integral and peripheral membrane proteins such as beta-dystroglycan and utrophin. Dystroglycan is necessary for the formation of the mature neuromuscular junction and has been shown to interact directly with rapsyn. Previous studies with rapsyn fragments and mutants, expressed in 293T cells along with nAChRs, establish that the rapsyn tetratricopeptide repeat (TPR) domain is involved in self-association and its coiled-coil domain is necessary for nAChR clustering. The function of the rapsyn RING-H2 domain, which is not necessary for rapsyn self-association or nAChR clustering, is unknown. To further characterize these domains, we have used a yeast two-hybrid assay to test for interactions at the plasma membrane between rapsyn domains and a nAChR beta-subunit fragment, the beta-dystroglycan cytoplasmic domain, or rapsyn domains. The rapsyn coiled-coil domain interacts with the nAChR beta-subunit cytoplasmic domain, but not with itself, other rapsyn domains, or beta-dystroglycan. The RING-H2 domain interacts only with the beta-dystroglycan cytoplasmic domain. Furthermore, when expressed in 293T cells, a rapsyn construct containing as few as two TPRs and the RING-H2 domain self-associates and clusters dystroglycan, but not nAChRs. These results emphasize the modular character of the rapsyn structural domains. (+info)
Muscle activity and muscle agrin regulate the organization of cytoskeletal proteins and attached acetylcholine receptor (AchR) aggregates in skeletal muscle fibers.
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In innervated skeletal muscle fibers, dystrophin and beta-dystroglycan form rib-like structures (costameres) that appear as predominantly transverse stripes over Z and M lines. Here, we show that the orientation of these stripes becomes longitudinal in denervated muscles and transverse again in denervated electrically stimulated muscles. Skeletal muscle fibers express nonneural (muscle) agrin whose function is not well understood. In this work, a single application of > or = 10 nM purified recombinant muscle agrin into denervated muscles preserved the transverse orientation of costameric proteins that is typical for innervated muscles, as did a single application of > or = 1 microM neural agrin. At lower concentration, neural agrin induced acetylcholine receptor aggregates, which colocalized with longitudinally oriented beta-dystroglycan, dystrophin, utrophin, syntrophin, rapsyn, and beta 2-laminin in denervated unstimulated fibers and with the same but transversely oriented proteins in innervated or denervated stimulated fibers. The results indicate that costameres are plastic structures whose organization depends on electrical muscle activity and/or muscle agrin. (+info)
iNOS expression in dystrophinopathies can be reduced by somatic gene transfer of dystrophin or utrophin.
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BACKGROUND: Nitric oxide (NO) is an inorganic gas produced by a family of NO synthase (NOS) proteins. The presence and the distribution of inducible-NOS (NOS II or iNOS), and NADPH-diaphorase (NADPH-d), a marker for NOS catalytic activity, were determined in muscle sections from control, DMD, and BMD patients. MATERIALS AND METHODS: NADPH-d reactivity, iNOS- and nNOS (NOS I)-immunolocalization were studied in muscles from mdx mice before and after somatic gene transfer of dystrophin or utrophin. RESULTS: In control patients, few fibers (<2%) demonstrated focal accumulation of iNOS in sarcolemma. In DMD patients, a strong iNOS immunoreactivity was observed in some necrotic muscle fibers as well as in some mononuclear cells, and regenerating muscle fibers had diffusely positive iNOS immunoreactivity. In DMD patients, NADPH-d reactivity was increased and mainly localized in regenerating muscle fibers. In mdx mice quadriceps, iNOS expression was mainly observed in regenerating muscle fibers, but not prior to 4 weeks postnatal, and was still present 8 weeks after birth. The expression of dystrophin and the overexpression of utrophin using adenovirus-mediated constructs reduced the number of iNOS-positive fibers in mdx quadriceps muscles. The correction of some pathology in mdx by dystrophin expression or utrophin overexpression was independent of the presence of nNOS. CONCLUSIONS: These results suggest that iNOS could play a role in the physiopathology of DMD and that the abnormal expression of iNOS could be corrected by gene therapy. (+info)
Dephosphorylation of beta2-syntrophin and Ca2+/mu-calpain-mediated cleavage of ICA512 upon stimulation of insulin secretion.
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Islet cell autoantigen (ICA) 512 is a receptor-tyrosine phosphatase-like protein associated with the secretory granules of neuroendocrine cells, including pancreatic beta-cells. Binding of its cytoplasmic tail to beta2-syntrophin suggests that ICA512 connects secretory granules to the utrophin complex and the actin cytoskeleton. Here we show that stimulation of insulin secretion from INS-1 cells triggers the biosynthesis of pro-ICA512 and the degradation of its mature form. Inhibition of calpain, which is activated upon stimulation of insulin secretion, prevents the Ca2+-dependent proteolysis of ICA512. In vitro mu-calpain cleaves ICA512 between a putative PEST domain and the beta2-syntrophin binding site, whereas binding of ICA512 to beta2-syntrophin protects the former from cleavage. beta2-syntrophin and its F-actin-binding protein utrophin are enriched in subcellular fractions containing secretory granules. ICA512 preferentially binds phospho-beta2-syntrophin and stimulation of insulin secretion induces the Ca2+-dependent, okadaic acid-sensitive dephosphorylation of beta2-syntrophin. Similarly to calpeptin, okadaic acid inhibits ICA512 proteolysis and insulin secretion. Thus, stimulation of insulin secretion might promote the mobilization of secretory granules by inducing the dissociation of ICA512 from beta2-syntrophin-utrophin complexes and the cleavage of the ICA512 cytoplasmic tail by mu-calpain. (+info)
Targeting transcription to the neuromuscular synapse.
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Concomitant with innervation, genes coding for components of the neuromuscular junction become exclusively expressed in subsynaptic nuclei. A six-base pair element, the N box, can confer synapse-specific transcription to the acetylcholine nicotinic receptor delta and epsilon subunit, utrophin, and acetylcholine esterase genes. N box-dependent synaptic expression is stimulated by the nerve-derived signal agrin and the trophic factor neuregulin, which triggers the MAPK and JNK signaling pathways, to ultimately allow activation by the N box binding Ets transcription factor GABP. (+info)
Increased expression of utrophin in a slow vs. a fast muscle involves posttranscriptional events.
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In addition to showing differences in the levels of contractile proteins and metabolic enzymes, fast and slow muscles also differ in their expression profile of structural and synaptic proteins. Because utrophin is a structural protein expressed at the neuromuscular junction, we hypothesize that its expression may be different between fast and slow muscles. Western blots showed that, compared with fast extensor digitorum longus (EDL) muscles, slow soleus muscles contain significantly more utrophin. Quantitative RT-PCR revealed that this difference is accompanied by a parallel increase in the expression of utrophin transcripts. Interestingly, the higher levels of utrophin and its mRNA appear to occur in extrasynaptic regions of muscle fibers as shown by immunofluorescence and in situ hybridization experiments. Furthermore, nuclear run-on assays showed that the rate of transcription of the utrophin gene was nearly identical between EDL and soleus muscles, indicating that increased mRNA stability accounts for the higher levels of utrophin in slow muscles. Direct plasmid injections of reporter gene constructs showed that cis-acting elements contained within the utrophin 3'-untranslated region (3'-UTR) confer greater stability to chimeric LacZ transcripts in soleus muscles. Finally, we observed a clear difference between EDL and soleus muscles in the abundance of RNA-binding proteins interacting with the utrophin 3'-UTR. Together, these findings highlight the contribution of posttranscriptional events in regulating the expression of utrophin in muscle. (+info)
Distinct regions in the 3' untranslated region are responsible for targeting and stabilizing utrophin transcripts in skeletal muscle cells.
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In this study, we have sought to determine whether utrophin transcripts are targeted to a distinct subcellular compartment in skeletal muscle cells, and have examined the role of the 3' untranslated region (UTR) in regulating the stability and localization of utrophin transcripts. Our results show that utrophin transcripts associate preferentially with cytoskeleton-bound polysomes via actin microfilaments. Because this association is not evident in myoblasts, our findings also indicate that the localization of utrophin transcripts with cytoskeleton-bound polysomes is under developmental influences. Transfection of LacZ reporter constructs containing the utrophin 3'UTR showed that this region is critical for targeting chimeric mRNAs to cytoskeleton-bound polysomes and controlling transcript stability. Deletion studies resulted in the identification of distinct regions within the 3'UTR responsible for targeting and stabilizing utrophin mRNAs. Together, these results illustrate the contribution of posttranscriptional events in the regulation of utrophin in skeletal muscle. Accordingly, these findings provide novel targets, in addition to transcriptional events, for which pharmacological interventions may be envisaged to ultimately increase the endogenous levels of utrophin in skeletal muscle fibers from Duchenne muscular dystrophy (DMD) patients. (+info)