From the Department of Cellular and Molecular
Medicine, Faculty of Medicine, University of Ottawa, Ottawa, Ontario,
Canada K1H 8M5; ¶ Department of Biochemistry, Genetics Unit,
University of Oxford, South Parks Road, Oxford OX1 3QU, United Kingdom;
** Centre for Research in Neuroscience, Montreal General Hospital
Research Institute, Montreal, Quebec, Canada H3G 1A4; and
Biologie Cellulaire des Membranes, Institut
Jacques Monod, Université Denis Diderot, 75251 Paris Cédex 05, France
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ABSTRACT |
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Duchenne muscular dystrophy is a prevalent
X-linked neuromuscular disease for which there is currently no cure.
Recently, it was demonstrated in a transgenic mouse model that utrophin could functionally compensate for the lack of dystrophin and alleviate the muscle pathology (Tinsley, J. M., Potter, A. C., Phelps,
S. R., Fisher, R., Trickett, J. I., and Davies, K. E. (1996) Nature 384, 349-353). In this context, it thus
becomes essential to determine the cellular and molecular mechanisms
presiding over utrophin expression in attempts to overexpress the
endogenous gene product throughout skeletal muscle fibers. In a recent
study, we showed that the nerve exerts a profound influence on utrophin
gene expression and postulated that nerve-derived trophic factors
mediate the local transcriptional activation of the utrophin gene
within nuclei located in the postsynaptic sarcoplasm (Gramolini,
A. O., Dennis, C. L., Tinsley, J. M., Robertson, G. S., Davies, K. E, Cartaud, J., and Jasmin, B. J. (1997)
J. Biol. Chem. 272, 8117-8120). In the present study,
we have therefore focused on the effect of agrin on utrophin expression
in cultured C2 myotubes. In response to Torpedo-, muscle-,
or nerve-derived agrin, we observed a significant 2-fold increase in
utrophin mRNAs. By contrast, CGRP treatment failed to affect
expression of utrophin transcripts. Western blotting experiments also
revealed that the increase in utrophin mRNAs was accompanied by an
increase in the levels of utrophin. To determine whether these changes
were caused by parallel increases in the transcriptional activity of
the utrophin gene, we transfected muscle cells with a 1.3-kilobase pair
utrophin promoter-reporter (nlsLacZ) gene construct and treated them
with agrin for 24-48 h. Under these conditions, both muscle- and
nerve-derived agrin increased the activity of -galactosidase,
indicating that agrin treatment led, directly or indirectly, to the
transcriptional activation of the utrophin gene. Furthermore, this
increase in transcriptional activity in response to agrin resulted from
a greater number of myonuclei expressing the 1.3-kilobase pair utrophin promoter-nlsLacZ construct. Deletion of 800 base pairs 5
from this
fragment decreased the basal levels of nlsLacZ expression and abolished
the sensitivity of the utrophin promoter to exogenously applied agrin.
In addition, site-directed mutagenesis of an N-box motif contained
within this 800-base pair fragment demonstrated its essential
contribution in this regulatory mechanism. Finally, direct gene
transfer studies performed in vivo further revealed the
importance of this DNA element for the synapse-specific expression of
the utrophin gene along multinucleated muscle fibers. These data show
that both muscle and neural isoforms of agrin can regulate expression
of the utrophin gene and further indicate that agrin is not only
involved in the mechanisms leading to the formation of clusters
containing presynthesized synaptic molecules but that it can also
participate in the local regulation of genes encoding synaptic
proteins. Together, these observations are therefore relevant for our
basic understanding of the events involved in the assembly and
maintenance of the postsynaptic membrane domain of the neuromuscular
junction and for the potential use of utrophin as a therapeutic
strategy to counteract the effects of Duchenne muscular dystrophy.
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INTRODUCTION |
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Duchenne muscular dystrophy (DMD)1 is the most severe and prevalent neuromuscular disease affecting 1 in 3,500 male births (1). This disease is characterized by repeated cycles of muscle fiber degeneration and regeneration with an eventual failure to regenerate, thereby leading to a loss of muscle mass and function. The genetic defect underlying DMD, located on the short arm of the X chromosome, prevents the production of dystrophin, a large cytoskeletal protein of the spectrin superfamily (2, 3). Previous studies have shown that, in muscle, dystrophin is located at the cytoplasmic face of the sarcolemma where it links the intracellular cytoskeleton network to the extracellular matrix via a complex of dystrophin-associated proteins (for reviews, see Refs. 4-7).
Several years ago, an autosomal homologue to dystrophin was identified on chromosome 6q24 (8). This gene, now referred to as utrophin, presents a genomic organization similar to that of the dystrophin gene, indicating that both genes evolved from an ancestral duplication event (9). Cloning of a full-length cDNA and subsequent analysis of its deduced amino acid sequence revealed, in fact, that utrophin shares considerable identity with dystrophin, particularly in the actin binding domain and carboxyl terminus (10). However, in comparison to high molecular mass isoforms of dystrophin, which are predominantly expressed in brain and muscle, utrophin displays a ubiquitous pattern of expression since it can be detected in most tissues (11-13).
In normal skeletal muscle, expression of utrophin is known to be influenced by the state of differentiation and innervation of muscle fibers. In developing myotubes, for example, utrophin is first localized to the entire length of the sarcolemma (14-17). Following the establishment of synaptic contacts, utrophin becomes highly enriched within the postsynaptic membrane domain of the neuromuscular junction (18, 19). However, several studies have shown that in dystrophic muscles, utrophin expression is not restricted to postsynaptic compartments, since it extends well into extrasynaptic regions of adult muscle fibers (14, 20-23). Such modulations in the pattern of expression indicate that distinct cellular and molecular mechanisms must exist to maintain the uneven distribution of utrophin along normal adult muscle fibers and to alter its levels and localization in developing and diseased muscles.
Despite these recent advances, however, our knowledge of the regulatory mechanisms presiding over utrophin expression in muscle is clearly lacking. A better understanding of these mechanisms appears important particularly since up-regulation of utrophin is currently envisaged as a therapeutic strategy to prevent the relentless progression of DMD (24, 25). In this context, we have recently shown that the nerve exerts a profound influence on utrophin gene expression (26). Since our previous experiments also demonstrated that nerve-derived electrical activity is not a key factor regulating utrophin expression (27), we postulated in these initial studies that nerve-derived trophic factors likely mediate the local transcriptional activation of the utrophin gene within nuclei of the postsynaptic membrane domain (26). In the present study, we have therefore determined the effects of nerve-derived trophic factors on utrophin expression in cultured myotubes. A preliminary account of this work has previously appeared in abstract form (28).
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EXPERIMENTAL PROCEDURES |
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Tissue Culture-- C2 cells were cultured on Matrigel-coated (Collaborative Biomedical Products, Bedford, MA) 35-mm culture plates and kept at 37 °C in a water-saturated atmosphere containing 5% CO2. Myoblasts were grown in Dulbecco's modified Eagle's medium supplemented with 20% horse serum, 10% fetal bovine serum, 100 units/ml penicillin-streptomycin, and 292 ng/ml L-glutamine until they reached confluence. At this stage, the concentration of horse serum was reduced to 5%, and fetal bovine serum was eliminated to promote myotube formation. Myoblasts were allowed to fuse into multinucleated myotubes for 3-4 days and were then used for experiments. To examine the effects of nerve-derived trophic factors, 0.1 µM rat CGRP (Sigma) or 10 ng/ml purified Torpedo agrin (29) was added directly to the culture medium for 24-48 h. Additionally, the effects of 1 nM recombinant neural (C-Ag12,4,8) or muscle (C-Ag12,0,0) isoforms of agrin were also examined (30).
Immunofluorescence and Quantitation of Acetylcholine Receptors
(AChR) Clusters--
Differentiated C2 myotubes were treated with 10 ng/ml Torpedo or recombinant agrin for 24-48 h. Cultures
were subsequently fixed for 10 min in 4% paraformaldehyde. Clusters of
AChR were visualized with fluorescein isothiocyanate-conjugated
-bungarotoxin used at a final concentration of 4 ng/ml in
phosphate-buffered saline (PBS). Following thorough washing with PBS,
the myotubes were covered with a glycerol:PBS solution and a coverslip,
and they were then examined by epifluorescence using a Zeiss
photomicroscope. For the determination of agrin-induced AChR clusters,
the numbers of myotubes and AChR aggregates were determined in 10 fields of view per culture at a 400 × magnification as described
in detail in Gee et al. (31). A minimum of four cultures
were quantitated for each experimental condition. Photographs were
taken with Kodak T-MAX 400 black and white films.
Immunoblotting--
C2 myotubes were treated with agrin for
48 h, washed in PBS, and then solubilized in RIPA (1% sodium
deoxycholate, 0.1% SDS, 0.5% Triton X-100, 1 mM
phenylmethylsulfonyl fluoride, 5 mM iodoacetamide, 2 µg/ml aprotinin, 0.01 M Tris-HCl, pH 8.0, 0.14 M NaCl, and 0.025% NaN3) (32). Samples were
centrifuged, and the supernatant was collected and stored at 20 °C
until analysis. The resulting pellet was further solubilized in RIPA
containing 5% SDS. Following centrifugation, the supernatant was
collected and stored at
20 °C. The concentration of
SDS-solubilized protein was determined using the bicinchoninic acid
protein assay reagent protocol (BCA; Pierce). Equivalent amounts of
cell extracts (70 µg) were separated on a 6% polyacrylamide gel and
electroblotted onto a polyvinylidene difluoride membrane (Sigma). To
ensure that equivalent amounts of proteins were loaded for each sample,
membranes were also stained with Ponceau S (Sigma). Membranes were
subsequently incubated with monoclonal antibodies directed against
either utrophin (MANCHO-7; kindly supplied by Dr. Glen Morris, N.E.
Wales Institute, UK),
-actinin (Sigma), or sarcomeric myosin (MF-20)
(33). Bound antibodies were detected by secondary antibodies linked to
horseradish peroxidase and revealed via chemiluminescence using a
commercially available kit (NEN Life Science Products). Membranes were
then exposed onto BioMax autoradiographic films (Eastman Kodak Co.),
developed, and scanned at 200 dots/inch using a Hewlett-Packard Scanjet
4C.
RNA Extraction and Reverse Transcription-Polymerase Chain Reaction (RT-PCR)-- Total RNA was extracted using Trizol as recommended by the manufacturer (Life Technologies, Inc.). Briefly, cells were scraped into 1 ml of Trizol. Following addition of 200 µl of chloroform, the samples were mixed vigorously and centrifuged at 12,000 × g for 15 min at 4 °C. The aqueous layer was then transferred to a fresh tube, and 500 µl of ice-cold isopropanol were added. For RNA precipitation, the isopropanol mixture was spun, and the resultant pellets washed twice with ice-cold 75% ethanol.
For all samples, total RNA was redissolved into 20 µl of RNase-free water. From each of these stocks, the RNA was further diluted to a final concentration of 50 ng/µl, and only 2 µl of this dilution were used for RT-PCR as described in detail in Jasmin et al. (27, 34). Briefly, a RT master mix was prepared containing 5 mM MgCl2, 1 × PCR buffer II (50 mM KCl, 10 mM Tris-HCl, pH 8.3, 1 mM dNTPs, 20 units of RNase inhibitor, 50 units of reverse transcriptase, and 2.5 mM of random hexamers (GeneAmp RNA PCR kit; Perkin-Elmer Corp.). The master mix was aliquoted, and the appropriate RNA sample was subsequently added. Negative controls consisted of RT mixtures in which the RNA sample was replaced with RNase-free water. RT was performed for 45 min at 42 °C, and the mixture was heated to 99 °C for 5 min to terminate the reaction. Complementary DNAs encoding utrophin and dystrophin were specifically amplified using primers designed on the basis of available mouse cDNA sequences (27, 34). Amplification of the selected cDNAs was performed in a DNA thermal cycler (Perkin-Elmer) by adding 4 µl of the RT mixture to 16 µl of a PCR master mix. Each cycle of amplification for utrophin cDNAs consisted of denaturation at 94 °C for 1 min, primer annealing at 60 °C for 1 min, and extension at 72 °C for 1 min. For dystrophin amplification, each cycle consisted of denaturation at 94 °C for 1 min, followed by primer annealing and extension at 72 °C for 3 min. The number of cycles for utrophin and dystrophin was 26 and 44, respectively. PCR products were visualized on a 1.5% agarose gel containing ethidium bromide. The 100-bp molecular mass marker (Life Technologies, Inc.) was used to estimate the molecular mass of the PCR products. Quantitative PCR experiments were performed to determine strictly the relative abundance of transcripts following different experimental treatments. These experiments were carried out using either one of two methods. In one case, 1.5 × 106 cpm per sample of 32P end-labeled primers were added to the PCR master mix. PCR products were visualized and carefully excised from the agarose gel with the use of a scalpel. The level of radioactivity present in these gel bands was determined by Cerenkov counting. Alternatively, PCR products were separated in 1.5% agarose gels containing the fluorescent dye Vistra Green (Amersham Corp.), and the labeling intensity of the PCR product, which is linearly related to the amount of DNA, was quantitated using a Storm PhosphorImager (Molecular Dynamics, Sunnyvale, CA).Expression of Utrophin Promoter-Reporter Gene
Constructs--
Several human utrophin promoter-reporter gene
constructs were used in these experiments (35). These 1.3- and 0.5-kb
promoter fragments were inserted upstream of the reporter gene
LacZ and a nuclear localization signal (26). Additionally,
two other 1.3-kb constructs were generated with mutations of the N-box. The 1.3-kb HindIII human utrophin promoter clone (35) was
digested with XhoI and PstI liberating a 300-bp
fragment containing the N-box, which was then further cloned into
pBSSKII() (Stratagene, Cambridge, UK) generating the clone pBSXP.
Mutagenesis was performed using Quick Change (Stratagene) essentially
following the manufacturer's instructions except for using cloned
Pfu polymerase (Stratagene). Two pairs of complementary
primers were generated with a single or double point mutation in the
N-box (N5F, 5-U-GTG GGG CTG ATC TTC CAG AAC AAA GTT GC; N5R, 5-U-GCA
ACT TTG TGG AAG ATC AGC CCC AC; N34F, 5-U-GGG GCT GAT CTT TTG GAA CAA
AGT TGC TGG G; and N34R, 5-U-CCC AGC AAC TTT CTT CCA AAA GAT CAG CCC
C). pBSXP was used as the template for synthesis of the mutations using
these oligonucleotide primer pairs. Following 15 cycles of 95 °C for
30 s, 56 °C for 1 min, 68 °C for 7 min, the wild-type
plasmid template was destroyed using the methylation-sensitive
restriction endonuclease DpnI. The mutant plasmids were cloned and
sequenced to verify the addition of the mutations in the N-box and to
confirm that no new mutations had been introduced into other sequences.
The 300-bp XhoI/PstI was released and used to
replace the equivalent nonmutated fragment at the same sites in the
plasmid 1.3-kb nlsLacZ (26). The new promoter mutant/reporter
constructs were then sequenced to check for no further mutations. For
transfection and direct gene transfer experiments, plasmid DNA was
prepared using the Qiagen mega-prep procedure (Qiagen, Chatsworth,
CA).
Statistical Analysis-- Paired Student's t tests were performed to evaluate the effects of agrin on utrophin expression. These tests were used to strictly compare the effects of agrin-treated versus nontreated myotubes. The level of significance was set at p < 0.05. Data are expressed as mean ± S.E. throughout.
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RESULTS |
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Agrin Increases Expression of Utrophin in Cultured Myotubes-- In an initial series of experiments, 3-4-day-old myotubes were treated with agrin purified from Torpedo electric tissue or with recombinant agrin isoforms in attempts to identify putative extracellular cues capable of regulating utrophin gene expression. As expected, agrin treatment increased the number of AChR clusters present on the surface of these C2 myotubes (Fig. 1). Quantitative analyses revealed that the number of AChR clusters per myotube increased by approximately 15-fold (p < 0.05) following Torpedo agrin treatment (Fig. 1). Immunofluorescence experiments using the monoclonal antibody MANCHO 7 showed that utrophin was present at these AChR clusters but only at the largest ones (data not shown). As expected, treatment of myotubes with the predominant isoform of agrin expressed in muscle (C-Ag12,0,0) failed to induce the formation of AChR clusters above the levels normally detected in nontreated cultures.
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Agrin Stimulates Transcription of the Utrophin Gene-- To determine if the increase in utrophin following agrin treatment resulted from enhanced transcriptional activation of the utrophin gene, we first examined the levels of utrophin transcripts in agrin-treated versus nontreated myotube cultures by RT-PCR. Quantitative analysis revealed that utrophin mRNA levels increased significantly (p < 0.05) following Torpedo agrin treatment (Figs. 3 and 4). Recombinant neural agrin (C-Ag12,4,8) had a similar effect (Fig. 4) thus ruling out the possibility that the increased expression of utrophin transcripts seen after treatment with Torpedo agrin was caused by contaminants present in this purified extract. Interestingly, treatment of myotubes with the muscle isoform of agrin (C-Ag12,0,0) also increased the expression of utrophin mRNAs by approximately 2-fold (Fig. 4). Myotubes treated for 48 h with Torpedo or recombinant isoforms of agrin showed slightly higher increases in the levels of utrophin transcripts in comparison to those observed following 24 h-treatments (data not shown). In these experiments, agrin did not affect the levels of dystrophin transcripts (Fig. 3).
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Role of the N-box Motif in Regulating Utrophin Gene
Expression--
Based on recent studies, which have shown that the
N-box motif plays a crucial role in regulating the expression of genes encoding the - and
-subunits of the AChR (36, 40), we examined the contribution of this DNA element in the transcriptional regulation of the utrophin gene by agrin. For these studies, site-directed mutagenesis was used to introduce single or double-base pair mutations into the N-box motif contained within the utrophin promoter (26, 35).
Two different mutants were generated and differed from the wild-type
N-box (TTCCGG) by one (N5 = TTCCAG) or two bases (N34 = TTTTGG). The mutant utrophin promoter fragments were inserted upstream
of the nlsLacZ reporter gene.
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The N-box Motif Regulates the Synaptic Expression of the Utrophin Gene in Vivo-- To determine whether the N-box motif participates also in the regulation of the utrophin gene in vivo (26, 36, 40), we injected directly into mouse tibialis anterior muscles constructs containing either the 1.3-kb wild-type utrophin promoter fragment or the N-box mutants. In agreement with our previous findings (26), we observed that ~55% of all blue myonuclei clusters seen in muscles injected with constructs containing the wild-type 1.3-kb promoter fragment coincided with the presence of neuromuscular junctions (Fig. 8). Mutations of the N-box, however, led to a marked reduction in the percentage of synaptic events. In fact, quantitative analysis revealed that, in muscles injected with either one of the mutant constructs, less than 20% of all blue myonuclei clusters were located in the vicinity of neuromuscular junctions (Fig. 8). These results indicate, therefore, that the N-box motif regulates also in vivo expression of the utrophin gene since it modulates its pattern of synaptic expression.
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DISCUSSION |
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In a recent study, we demonstrated that utrophin transcripts accumulate preferentially within the postsynaptic sarcoplasm of muscle fibers and that this accumulation resulted from the local transcriptional activation of the utrophin gene in myonuclei concentrated beneath the neuromuscular junction (26). Induction of ectopic synapses at sites distant from the original neuromuscular junctions further revealed that nuclei located in extrasynaptic regions were capable of expressing utrophin upon receiving appropriate neuronal cues. Together with the demonstration that levels of utrophin in muscle are largely insensitive to elimination of nerve-evoked electrical activity (19, 27), these experiments led us to postulate that nerve-derived trophic factors regulate locally the expression of the utrophin gene (25, 26). Among the molecules known to regulate the expression or localization of AChR (for review, see Refs. 37 and 38), agrin appeared as a plausible candidate for several reasons. For example, detailed analysis of agrin (41)- and muscle-specific kinase (42)-deficient mice has led to the suggestion that, in vivo, agrin may ultimately affect transcription of genes encoding synaptic proteins such as AChR. Moreover, in response to exogenously applied agrin, cultured myotubes show increase numbers of AChR clusters with only large ones containing utrophin (43, 44). Although agrin treatment leads to a redistribution of normally diffusing AChR molecules, it is unlikely that it causes a similar clustering of presynthesized, membrane-attached utrophin. The presence of utrophin in large AChR clusters may thus result from compartmentalized de novo expression of utrophin by nuclei located in the vicinity of the growing clusters. In the present study, we have therefore focused on the effect of agrin on utrophin expression.
In attempts to determine whether agrin treatment induced utrophin
expression, we initially measured levels of utrophin and its mRNA
in cultures of treated versus nontreated myotubes. In addition to causing the clustering of AChR, agrin treatment also increased the levels of utrophin. In these experiments, we observed that utrophin levels increased within an easily dissociated cellular fraction, thereby suggesting that this increase resulted from a newly
synthesized pool of utrophin not yet intertwined within the existing
cytoskeleton. Similarly, we also noted that agrin treatment induced a
significant 2-fold increase in the levels of utrophin transcripts.
Interestingly, both nerve- and muscle-derived isoforms of agrin had a
comparable stimulatory effect on utrophin expression. These increases
are in fact of similar magnitude to those reported recently by Jones
et al. (45) who examined the impact of both muscle and
neural isoforms of agrin on expression of transcripts encoding the AChR
-subunit. However, a major difference between the two studies is
that we were able to observe an effect on utrophin gene expression
without the necessity of agrin being substrate-bound (45). Although the
reason for this difference remains currently obscure, it appears
reasonable to assume that it likely arises from differences in culture
conditions. In particular, recent experiments have revealed that
MatrigelTM is capable of binding agrin (46, 47). Since, in
our experiments, myotube cultures are plated on Matrigel-coated plates,
it appears likely that Torpedo agrin as well as recombinant
agrin fragments may become bound to this substrate via an unknown
mechanism (see Denzer et al. (46, 47) for further
discussion) and therefore do not remain in a "soluble" form (45).
Nonetheless, since the pattern of expression of the utrophin gene along
muscle fibers resembles that of the
-subunit gene (26, 36, 48),
these results are coherent with the notion that expression of genes encoding membrane and cytoskeletal proteins of the postsynaptic membrane are co-regulated and therefore involve a common signal transduction pathway.
Transfection experiments with utrophin promoter-reporter gene
constructs indicated that the increase in utrophin mRNA levels following agrin treatment resulted from the transcriptional activation of the utrophin gene. In agreement with our previous in vivo
studies (26), deletion of 800 bp from the 3 region of the 1.3-kb
promoter fragment significantly reduced the activity of the reporter
gene in transfected cells. More importantly, it also abolished the response to agrin treatment. Together, these results indicate that DNA
elements contained within the deleted 800 bp are not only regulating
the basal level of utrophin gene expression in muscle cells in
vivo (26) and in vitro (this study), but they also
confer to the utrophin promoter its sensitivity to neuronal cues
including agrin. Among the putative elements that may play a crucial
role in this regulatory mechanism is the N-box motif (26, 35, 40),
which was shown recently to be essential for the synapse-specific
expression of AChR
- and
-subunit genes (36, 40). In the present
study, site-directed mutagenesis confirmed that the N-box motif is
indeed essential in this regulatory mechanism. These results further
suggest that the N-box motif may in fact represent the ultimate target
within the utrophin promoter that mediates the agrin effect in cultured
myotubes. In addition, it appears that this DNA element also plays an
essential role in vivo in the regulation of the utrophin
gene, since direct injection of constructs containing mutant utrophin
promoter fragments into tibialis anterior muscles failed to induce
synapse-specific expression of the reporter gene as observed with the
wild-type 1.3-kb utrophin promoter fragment (26).
The molecular mechanism by which nerve- and muscle-derived isoforms of agrin lead to the transcriptional activation of the utrophin gene remains to be established. In this context, however, there are several pathways that may be currently envisaged. One signaling pathway involves binding of agrin to a complex that includes the tyrosine kinase receptor muscle-specific kinase and a myotube-specific accessory component (49). This binding is known to trigger a series of biochemical events that culminate in the clustering of AChR on the surface of myotubes and in a reorganization of the underlying cytoskeleton. However, this pathway is probably not directly involved since only neural agrin activates muscle-specific kinase and induces AChR clustering (49).
A more likely mechanism responsible for the agrin-induced effects on
utrophin gene expression involves not only clustering of AChR but also
of other postsynaptic membrane proteins that, in turn, may directly
participate in the regulation of utrophin. For example, it has been
recently demonstrated that intramuscular injections of plasmid
DNA-encoding agrin into extrasynaptic regions of denervated soleus
muscle fibers induced, in addition to AChR clustering, the aggregation
of muscle-derived ARIA along with its receptors, erbB2 and erbB3 (50).
Since these molecules are known to regulate expression of AChR subunit
genes (51-53), agrin treatment may thus ultimately stimulate
ARIA-dependent gene expression via an autocrine mechanism
involving muscle ARIA and its receptors (45, 50). Accordingly, agrin
may be sufficient for: (i) the initial events underlying AChR
clustering and (ii) the positioning of other molecules involved in
regulating expression of synaptic proteins. Such a role for agrin would
thereby ensure the proper growth of developing postsynaptic membrane
domains as well as their long term maintenance. Furthermore, it could
also explain the presence of utrophin only in large AChR clusters,
since recruitment of all necessary components would parallel the growth
of the clusters. In fact, this mechanism is consistent with our
statistical analysis demonstrating that the agrin effect on the
activity of the reporter gene was caused by a significantly greater
number of nuclei expressing the 1.3-kb construct as opposed to a
similar number of nuclei increasing their level of expression. These
results indicate therefore, that the effect of agrin is to stimulate
transcription of the utrophin gene in normally quiescent nuclei; an
expected effect given that agrin increases the number of clusters
containing AChR and other synaptic proteins on the surface of these
myotubes. In the case of muscle-derived agrin however, the effect on
utrophin gene expression likely occurs via a mechanism altogether
distinct from that involving the muscle-specific
kinase-dependent pathway (45). Finally, it is also
conceivable that the effects of both muscle and neural isoforms of
agrin occurs via a distinct and unique pathway involving therefore a
MuSK-independent mechanism. For example, as a protein of the
extracellular matrix, agrin may activate transcription of synaptic
genes by first binding to other receptors such as the integrins or
-dystroglycan which are known to accumulate at developing
postsynaptic membrane domains (54, 55). We are currently examining
these possibilities using several experimental approaches.
In a recent study, Tinsley et al. (24) showed that expression of utrophin in extrasynaptic regions of muscle fibers from mdx mice functionally compensated for the lack of dystrophin and alleviated the dystrophic pathology. These findings demonstrate that up-regulation of utrophin may indeed represent an effective treatment for DMD. In this context, the next logical step is naturally to identify molecules capable of increasing utrophin gene expression in skeletal muscle fibers. Our observation that agrin increases levels of utrophin protein and mRNA via a transcriptional regulatory mechanism is therefore not only relevant for our basic understanding of the events involved in the assembly and maintenance of the postsynaptic membrane domain of the neuromuscular junction but also, for the potential use of utrophin as a therapeutic strategy for DMD.
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FOOTNOTES |
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* This work was supported by in part by grants from the Muscular Dystrophy Association of America (to B. J. J.), l'Association Française Contre les Myopathies (to B. J. J. and J. C.), and the Medical Research Council of Canada (to B. J. J.) and of the United Kingdom (to K. E. D.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Arthur Minden Predoctoral Fellow of the Muscular Dystrophy Association of Canada.
Supported by an Action Research training fellowship.
§§ Scholar of the Medical Research Council of Canada. To whom all correspondence should be addressed: Dept. of Cellular and Molecular Medicine, Faculty of Medicine, University of Ottawa, 451 Smyth Rd., Ottawa, Ontario, Canada K1H 8M5. Tel.: 613-562-5800 (ext. 8383); Fax: 613-562-5434; E-mail: bjasmin{at}danis.med.uottawa.ca.
1 The abbreviations used are: DMD, Duchenne muscular dystrophy; AChE, acetylcholinesterase; AChR, acetylcholine receptor; C-Ag12,4,8, recombinant neural agrin; C-Ag12,0,0, recombinant muscle agrin; RT, reverse transcription; PCR, polymerase chain reaction, RIPA, radioimmune precipitation buffer; PBS, phosphate-buffered saline; CAT, chloramphenicol acetyltransferase; bp, base pair(s); kb, kilobase pair(s).
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REFERENCES |
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