Centro de Regulación Celular y Patología, Departamento de Biología Celular y Molecular, Facultad de Ciencias Biológicas, Millennium Institute for Fundamental and Applied Biology, Pontificia Universidad Católica de Chile, Santiago, Chile
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ABSTRACT |
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Transcription of specific skeletal
muscle genes requires the expression of the muscle regulatory factor
myogenin. To assess the role of the extracellular matrix (ECM) in
skeletal muscle differentiation, the specific inhibitors of
proteoglycan synthesis, sodium chlorate and -D-xyloside,
were used. Treatment of cultured skeletal muscle cells with each
inhibitor substantially abolished the expression of creatine kinase and
-dystroglycan. This inhibition was totally reversed by the addition
of exogenous ECM. Myoblast treatment with each inhibitor affected the
deposition and assembly of the ECM constituents glypican, fibronectin,
and laminin. These treatments did not affect MyoD, MEF2A, and myogenin
expression and nuclear localization. Differentiated myoblast treatment
with RGDS peptides completely inhibited myogenesis without affecting the expression or nuclear localization of myogenin. Integrin-mediated signaling of focal adhesion kinase was partially inhibited by chlorate
and
-D-xyloside, an effect reversed by the addition of
exogenous ECM gel. These results suggested that the expression of
myogenin is not sufficient to successfully drive skeletal muscle formation and that ECM is required to complete the skeletal muscle differentiation process.
myogenin; proteoglycans; extracellular matrix; proteoglycan inhibitors; satellite cells
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INTRODUCTION |
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THE GROWTH AND REPAIR PROCESSES of skeletal muscle tissue are normally mediated by the satellite cells that surround muscle fibers. These cells are induced to differentiate by signals arising from the damaged fibers and/or infiltrating cells (58). Transplantation of genetically marked bone marrow into immunodeficient mice revealed that marrow-derived cells migrated into areas of induced muscle degeneration, underwent myogenic differentiation, and participated in the regeneration of the damaged fibers (17). Recently, cloned neural stem cells were shown to fuse and form seemingly well-differentiated myotubes (20). A key question arising from these studies was the molecular identity of the differentiation and fusion induction signals, because muscle formation seemed to require cell contact. Experimental outcomes suggested that the induction signal was membrane bound rather than secreted (20).
The process of skeletal muscle cell differentiation is governed by a
network of muscle regulatory factors (62). One such factor, myogenin, is responsible for the induction of terminal differentiation and, as a transcription factor of the basic
helix-loop-helix family, activates the expression of skeletal
muscle-specific products, such as creatine kinase, myosin heavy chain,
and acetylcholine receptor, among others (38, 40). The
ability of myoblasts to differentiate in vitro is negatively controlled
by the extracellular concentration of specific mitogens such as basic
fibroblast growth factor (FGF-2), hepatocyte growth factor/scatter
factor (HGF/SF), and transforming growth factor (TGF)- (2, 8,
16, 18). In the presence of these inhibitory growth factors,
myoblasts continue to proliferate and fail to fuse or to express
muscle-specific gene products. Although skeletal muscle is terminally
differentiated, a small number of cells escape the differentiation
process. These cells, termed satellite cells, lie between the muscle
fiber and the basal lamina (29, 54).
In addition, several lines of evidence have demonstrated the importance of extracellular matrix (ECM) molecules as part of the myogenesis signaling mechanism (9). For instance, an inhibitor of collagen synthesis has been shown to inhibit the differentiation of cultured myoblasts (45, 55). Similarly, the addition to myoblast cultures of either RGDS peptides or antibodies against the integrin receptor was seen to inhibit fusion and further differentiation (43). Studies have also shown that the presence of proteoglycans as modulators of growth factor activities seems to be critical in the control of normal myogenesis (19, 35, 52).
Many cellular events take place during skeletal muscle formation: migration of precursor myoblast cells, proliferation of myoblasts, cessation of the proliferative stage, and induction of the expression of muscle regulatory factors, followed by transcriptional activation of specific skeletal muscle genes and myoblast cell fusion (62). In many of these steps, cell-cell and cell-ECM interactions are required (4). Integrin receptors facilitate cell attachment to the ECM, giving rise to interactions that subsequently generate cell survival, proliferation, and motility signals. Integrin signals are relayed in part by the activation of focal adhesion kinase (FAK) and the formation of a transient signaling complex, initiated by Src-homology 2 (SH2)-dependent binding of Src-family protein-tyrosine kinases to the FAK Tyr-397 autophosphorylation site. FAK, a ubiquitously expressed tyrosine kinase, has been shown to act as the initiator of focal adhesion formation in adherent cells after its binding to integrins and autophosphorylation induction (56, 57). However, FAK can also be activated by a great variety of stimuli, influencing different intracellular signaling pathways (60).
In this report, we describe that neither the expression of myogenin alone nor its localization to myoblast nuclei was sufficient to drive skeletal muscle differentiation. The presence of the ECM and its induction of cell receptor signaling (presumably through the integrin family) were also found to be requisites.
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MATERIALS AND METHODS |
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Cell culture.
The skeletal muscle cell line C2C12 from adult
mouse leg (American Type Culture Collection) was grown and induced to
differentiate as described by Larraín et al.
(36). For inhibitor treatments, sodium chlorate
(final concentration 30 mM), -D-xyloside (final concentration 1 mM), or vehicle solution (DMSO; final concentration 0.1%) was added to the cell cultures at the time of plating. RGDS peptides were added at 0.2 mg/ml as described previously
(43). ECM gel (Sigma, St. Louis, MO) was added after 2 days of growth, when the cells were switched to differentiation medium
(34). The medium was then removed, and 30 µl/cm2 of ECM gel (diluted 1:5 in DME-Ham's F-12) was
added over the cells and allowed to polymerize for 2 h at 37°C.
Fresh differentiation medium was finally added to the plates containing
the polymerized gel.
Labeling of cultures and proteoglycan analysis. Dishes (55 cm2) containing C2C12 cells were radiolabeled by incubation in medium containing 100 µCi 35S-labeled H2SO4 (carrier free; NEN, Boston, MA) for 18 h. Conditioned media were collected, and the cells were lysed with 0.5% Triton X-100 in PBS (0.15 M NaCl, 0.05 M sodium phosphate, pH 7.5). Incorporation of [35S]H2SO4 into macromolecules was measured by cetyl pyridinium chloride precipitation (6).
Immunofluorescence microscopy. Cells to be immunostained were grown on glass coverslips. The medium was removed, and the plates were rinsed with PBS. For staining of ECM proteins, the cells were incubated with primary antibodies for 1 h at room temperature before fixation (laminin 1:100, fibronectin 1:100, glypican-1 1:150, tubulin 1:1,000). After rinsing, the cells were fixed with 3% paraformaldehyde for 30 min at room temperature. For staining of intracellular proteins, the cells were fixed with paraformaldehyde and then permeabilized with 0.05% Triton X-100 in PBS. The cells were rinsed with Blotto and then incubated for 30 min at room temperature with affinity-purified fluorescein-conjugated secondary antibodies (Sigma) diluted in Blotto. After rinsing, the coverslips were mounted on glass slides. Fluorescein was visualized using a Nikon Diaphot inverted microscope equipped for epifluorescence. For nuclear staining, fixed cells were incubated in 1 µg/ml Hoechst 33258 in PBS for 5 min.
Polyclonal anti-laminin and anti-fibronectin antibodies, as well as monoclonal anti-Analysis of creatine kinase activity. Myoblast cells and myoblasts induced to differentiate were washed twice with PBS, lysed by incubation with PBS containing 0.1% Triton-X 100 for 10 min at 4°C, and harvested by scraping. Creatine kinase activity was determined using the creatine phosphokinase assay kit (Sigma). All data points represent the means of triplicate determinations from at least two independent experiments.
Immunoprecipitation and Western blot analysis.
Cells were lysed in RIPA buffer (20 mM Tris · HCl, pH 7.4, 150 mM NaCl, 0.5% Nonidet P-40, 1% Triton X-100) containing 10 µg/ml
aprotinin, 5 µg/ml leupeptin, 10 µg/ml phenylmethylsulfonyl fluoride, 1 µg/ml pepstatin, 2 mM EDTA, 2 mM EGTA, 2 mM sodium orthovanadate, 30 mM sodium pyrophosphate, and 100 mM sodium fluoride. Equal amounts of protein (300 µg) from precleared extracts were immunoprecipitated for 2 h at 4° C with a 1:50 dilution of
polyclonal anti-FAK antibody (Santa Cruz Biotechnology, Santa Cruz,
CA), followed by incubation for 2 h at 4°C with 10 µl of
Affi-Prep protein A support (Bio-Rad, Hercules, CA). Equal volumes of
immunoprecipitated protein were electrophoresed by 7.5%
SDS-polyacrylamide gel electrophoresis and electrotransferred onto
polyvinylidene difluoride. Filters were blocked for 1 h at room
temperature in Blotto and incubated with a monoclonal
anti-phosphotyrosine antibody (1:3,000, Santa Cruz Biotechnology).
Blots were reprobed after stripping with the anti-FAK (1:500)
polyclonal antibodies. Blots of proteins from total cell extracts were
incubated with monoclonal anti-rat myogenin antibody (1:1,000, F5D;
Southwestern Medical Center, University of Texas, TX), monoclonal
anti-rabbit -dystroglycan antibody (1:500; Upstate Biotechnology,
Lake Placid, NY), polyclonal anti-human MEF2A antibody (1:200; Santa
Cruz Biotechnology), or monoclonal anti-
-tubulin (1:1,000) after
stripping. Bound antibodies were visualized with horseradish
peroxidase-coupled secondary antibodies (Pierce, Rockford, IL) followed
by development with an enhanced chemiluminescence system (Pierce).
RNA isolation and Northern blot analysis. Total RNA was isolated from cell cultures using TRIzol (Life Technologies, Grand Island, NY). RNA samples were electrophoresed in 1.2% agarose-formaldehyde gels, transferred onto Nytran membranes, and hybridized with probes for creatine kinase, myogenin, myoD, and tubulin, as described previously (52). Blots were hybridized with random-primed labeled probes in a hybridization buffer at 65°C (18). Where indicated, the intensity of hybridization signals was measured by densitometric scanning (Epson scanning densitometer).
DNA and protein determination. DNA was determined in aliquots of cell extracts by the method of Labarca and Paigen (33). Protein was determined with the bicinchoninic acid protein assay kit (Pierce) with BSA as standard.
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RESULTS |
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Inhibitors of proteoglycan synthesis strongly affect expression of
late skeletal muscle differentiation markers.
To evaluate the effect of specific proteoglycan inhibitors on the
expression of late skeletal muscle-specific differentiation markers,
the expression of creatine kinase was determined at the onset of muscle
differentiation by incubating skeletal muscle myoblasts under
conditions that affected proteoglycan synthesis. The addition of
glycosaminoglycans (GAGs) to proteoglycans can be perturbed by
-D-xyloside (23), whereas the sulfation of proteoglycans can be specifically inhibited by sodium chlorate (28). Figure 1A
shows that the induction of creatine kinase activity was totally
inhibited by
-D-xyloside treatment and diminished to
25% of control values by sodium chlorate treatment. Similar results
were obtained when levels of creatine kinase mRNA were analyzed by
Northern blot, as shown in Fig. 1B; this figure also indicates tubulin transcript levels as loading controls. Figure 1C shows that in the presence of either
-D-xyloside or sodium chlorate the synthesis of
-dystroglycan, a late skeletal muscle marker, was also inhibited
because of the alteration of proteoglycan synthesis.
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Inhibition of terminal skeletal muscle differentiation by
inhibitors of proteoglycan synthesis does not affect expression of
muscle-specific transcription factors.
It is well known that skeletal muscle differentiation is under the
control of early myogenic regulatory genes such as the transcription
factor myogenin. To evaluate whether proteoglycan inhibitors could
affect the expression of myogenin, myoblasts were plated in the
presence of each inhibitor and triggered to differentiate for 48 h. Figure 2 shows that myogenin induction was unaffected by the inhibitors and independent of the nature of the
proteoglycans or GAGs present in the cell, as determined from myogenin
mRNA levels (Fig. 2A) and the induction of myogenin protein
expression after the onset of differentiation (Fig. 2B). In
both cases, myogenin expression was clearly induced under
differentiation conditions, irrespective of the synthesis of
proteoglycans. The expression profiles of MyoD, another myogenic
regulatory gene (Fig. 2A), and MEF2A, a transcriptional
activator of muscle-specific genes (Fig. 2C), were equally
unaffected by the proteoglycan inhibitors.
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-D-Xyloside inhibits ECM deposition in skeletal
muscle cells.
Proteoglycans are critical macromolecules for the structural and
functional organization of the ECM. To determine whether the inhibition
of proteoglycan synthesis, induced by
-D-xyloside or
chlorate treatment, could decrease the deposition of ECM components, three ECM constituents were assessed using indirect immunofluorescence staining: laminin, fibronectin, and the proteoglycan glypican-1, which
is present on the cell surface as well as associated with the ECM
(6). Control cultures induced to differentiate and stained
with anti-glypican antibody showed a bright and specific fibrillar
staining of the ECM (Fig. 4). Conversely,
both
-D-xyloside and sodium chlorate treatments almost
abolished the anti-glypican staining (Fig. 4, C and
D). Similar results were obtained after staining with
antibodies for laminin and fibronectin (Fig. 4, E-L).
On the other hand, the intracellular distribution of tubulin was
unaffected by either treatment, as revealed by a specific anti-tubulin
antibody (Fig. 4, M-P). Phase contrast microscopy of
cells treated with either
-D-xyloside or sodium chlorate
showed a marked inhibitory effect on both the amount and length of the myotubes formed, as shown in Fig. 4. It was shown previously that
-D-xyloside and sodium chlorate treatments do not affect
the synthesis of ECM components (23, 42). These results
demonstrate that the inhibition of proteoglycan synthesis, by two
specific inhibitors, leads to a decreased deposition of both
proteoglycan and glycoproteins in the ECM, affecting the number and
length of the myotubes that are induced to differentiate.
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Exogenous ECM prevents inhibitory action of
-D-xyloside and sodium chlorate on creatine kinase
expression.
Given that the lack of ECM deposition was responsible for the
inhibition of creatine kinase expression, it should be possible to
prevent this inhibitory effect by providing the cells with exogenous
ECM. Figure 5 describes experiments using
ECM gel, a basement membrane-like ECM obtained from mouse
Engelbreth-Holm-Swarm sarcoma, on skeletal muscle
differentiation. The effect of this gel on myotube formation
and creatine kinase activity was tested in the presence of the
proteoglycan inhibitors. Figure 5A shows that the ECM gel
induced the appearance of a significant number of myotubes in the
presence of the inhibitors. This was particularly evident for cells
incubated in the presence of the exogenous ECM and treated with sodium
chlorate. Similar results were observed for creatine kinase activity.
Moreover, the ECM gel was able to induce creatine kinase activity above
the levels observed for control cells, as shown in Fig. 5B.
These results suggest that cell-ECM contact is required for skeletal
muscle differentiation and that the inhibitory action of both
-D-xyloside and sodium chlorate stems from their effect
on ECM deposition.
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ECM-integrin interaction is required for successful terminal
skeletal muscle differentiation.
The above-described results suggest that ECM-skeletal muscle
contact is required for terminal skeletal differentiation. RGDS peptides, a peptide motif recognized by integrins, were therefore used
to study these contact requirements directly. Figure
6A shows that the induction of
creatine kinase activity during differentiation was almost completely
blocked by the presence of the peptides. However, under these
experimental conditions, the expression of myogenin was found to be
unaffected, as evaluated by Northern blot (Fig. 6B), Western
blot (Fig. 6C), and immunocytolocalization (Fig.
6D). These results suggest that an interference in the
interaction between integrins and ECM components can affect terminal
differentiation without inhibiting myogenin expression or affecting its
localization.
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DISCUSSION |
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In this study, inhibitors of proteoglycan synthesis that affect ECM assembly were shown to have a strong effect on skeletal muscle differentiation. However, in myoblasts triggered to differentiate, these inhibitors did not affect the expression of muscle regulatory factors such as myogenin or MyoD or their localization in the nuclei. The process of myogenic development is known to involve an ordered sequence of molecular events that includes the commitment of muscle precursor cells driven by MyoD expression (51), the cessation of cell division, myoblast terminal differentiation commanded by myogenin, and the formation of myotubes expressing muscle-specific genes required for the specialized functions of the myofiber (62). Myogenin, a transcription factor that activates skeletal muscle-specific products such as creatine kinase, myosin heavy chain, and acetylcholine receptor (37, 40), is a key factor in the induction of skeletal muscle terminal differentiation, which in turn is a critical process for muscle formation and muscle regeneration after injury (22).
The ability of myoblasts to differentiate has been shown to be
negatively controlled by the extracellular concentration of specific
mitogens such as FGF-2, HGF/SF, and TGF- (2, 8, 16, 18)
that directly affect the expression of myogenin. Interestingly, it has
been demonstrated that the binding of these factors to proteoglycans
can regulate their activities (3, 53). In particular, cell
surface heparan sulfate proteoglycans have been implicated in the
modulation of terminal myogenesis, possibly by acting as low-affinity
receptors for FGF-2 (19, 35). Recently, the responsiveness of myoblasts to TGF-
was shown to be directly modulated by the expression of decorin, a chondroitin/dermatan sulfate proteoglycan (52).
The presence of ECM is known to be essential for normal myogenesis (4, 25, 42, 55). Here, the presence of an organized ECM was shown to be a requisite for specific differentiation to occur, independent of the expression of MyoD and myogenin in response to stimuli that triggered skeletal muscle differentiation. Apparently, the interaction of specific receptors, present in the plasma membrane, with the ECM generated the signals needed to drive skeletal muscle differentiation. Integrin receptors represent the most likely candidates, because they are known to act as critical components of the process by which cells assimilate mechanical signals from their surrounding environment (30, 60). In fact, blocking the function of integrins with specific antibodies or peptides has been found to inhibit myogenic differentiation (43). Our data showed that the inhibition of integrin activity with RGDS peptides was independent of myogenin expression and localization. Overall, these results suggest that the myogenin-independent inhibition of muscle differentiation, observed as a result of proteoglycan synthesis inhibition, is likely due to the absence of cell-ECM contact mediated by integrins.
The expression regulation of several ECM constituents, such as
proteoglycans (6, 13, 36, 48), fibronectin
(41), laminin (49), and their
7
1-integrin receptor (15),
has been shown to occur during skeletal muscle differentiation.
Alterations in the expression of these ECM constituents have also been
reported for several skeletal muscle pathologies. The expression of
proteoglycans has been found to increase in animals undergoing active
skeletal muscle regeneration (5, 12), suggesting that
these macromolecules play an important role during this process. The
absence or reduction of laminin, as seen in Fukuyama and other
laminin-related congenital dystrophies, is accompanied by a concomitant
decrease in the expression of
7-integrin (10,
11). In the muscles of Duchenne and Becker dystrophy patients,
an increase in the expression of
7 proteins occurs
(27), whereas mutations in the
7 gene cause
human congenital myopathies (26). Recently, it was shown
that enhanced expression of
7
1-integrin
reduces muscular dystrophy and restores viability in dystrophic mice,
extending their longevity (11).
Integrins can be described as a family of heterodimeric transmembrane
glycoproteins, which consist of - and
-chains. On ligand binding,
signals are transduced into the cell through the single
membrane-spanning regions of each chain and their respective cytoplasmic domains. This mechanism likely involves interactions between the integrin protein, the cell cytoskeleton, and additional signal transduction molecules. A vast array of signaling molecules and
cascades have been implicated in integrin signaling, including FAK,
protein kinase C, mitogen-activated protein (MAP) kinase, Ras, and Rho,
to name a few. FAK is recruited to focal adhesion complexes, and the
clustering of integrins and formation of the focal adhesion complex has
been shown to induce the tyrosine phosphorylation of FAK, as occurs
with other stimuli (56, 57). The degree of FAK activation
during myogenesis, shown by
1A-integrin signaling, was
found to regulate myoblast progression through proliferation and
differentiation (59). Here, a decrease in the
autophosphorylation of the FAK tyrosine-397 was observed in the
presence of sodium chlorate and
-D-xyloside, with no
changes in total FAK expression. This decrease was reverted by
coaddition of exogenous ECM, suggesting that in the absence of ECM
assembly, integrin signaling through FAK is inhibited, consequently
impeding skeletal muscle differentiation. Interestingly, a minor effect
on the autophosphorylation of FAK was noted in the presence of
-D-xyloside, although free GAGs in the cell medium were
elevated almost 19-fold. It has been shown that fibronectin is composed
of several domains that mediate multiple cell functions through cell
surface integrin and proteoglycan receptors. One such domain is a
heparin-binding domain that modulates FAK levels (32). On
the other hand, chondroitin sulfate proteoglycans have been shown to
mediate apoptotic mechanisms in fibroblasts, which can be prevented
by intact fibronectin (32). Thus augmented levels of
myoblast GAGs, as a consequence of
-D-xyloside
treatment, likely permitted the correct presentation of fibronectin to
the integrin receptor, with a minor inhibitory effect on FAK signaling. Nevertheless, further experiments are required to resolve this point.
One of the most important cytoplasmic responses to integrin-ECM
interactions is the reorganization of cytoskeletal proteins (30). The absence of an assembled ECM, caused by the
application of chlorate, could lead to the inhibition of integrin
signaling. Although no such inhibition was observed for
-D-xyloside, the overall effect could be the same,
resulting in a negative impact on cytoskeletal organization
(23). The lack of an inhibitory effect on focal contact
formation has been reported previously for this proteoglycan synthesis
inhibitor (1).
Another cell response to integrin-ECM interactions is activation of
intracellular protein kinases including MAP kinase (14). MEF2 proteins cooperate in the control of myoblast differentiation, and
its transcriptional activity is stimulated after phosphorylation by MAP
kinase in muscle cells (46). Although we did not detect changes in expression of MEF2A in -D-xyloside- or
chlorate-treated cultures, differences in the expression of MEF2C or
MEF2D or their level of phosphorylation could account for the skeletal
muscle differentiation inhibition in the absence of cell-ECM contact. Furthermore, MEF2 localization at the nucleus is necessary for skeletal
muscle differentiation, and in the absence of cell-ECM contact a MEF2
sequestration in the cytoplasm could not be rejected.
The process of skeletal muscle formation involves sequential molecular events that include the commitment of muscle precursor cells, the cessation of cell division, myoblast terminal differentiation, and the formation of myotubes expressing muscle-specific genes for specialized myofiber functions (62). Studies have shown the expression requirements of either Myf-5 or MyoD, as well as myogenin and Mrf-4, to commit and successfully differentiate myoblasts (51); however, as shown in this study, the expression of the muscle regulatory transcription factors is not enough to differentiate the cells.
The extracellular signals involved in activating the myogenic program in muscle precursor cells are as yet unknown. Therefore, the manner in which myogenic differentiation is activated and completed remains an open question. The elucidation of such mechanisms is further complicated by the presence of significant levels of various inhibitory myogenic growth factors (24, 31, 39, 47) as well as myogenesis inductors such as retinoic acid and insulin-like growth factor (21, 44, 50) in the region where muscle formation occurs during either muscle development or regeneration processes. The results presented here indicate that, in addition to the expression of myogenic transcription factors, an ECM signaling dependence arises in the induction of terminal skeletal muscle differentiation. Therefore, the presence of the ECM is essential for the conduction of normal myogenesis (25, 42, 55) and can exert its influence through direct interactions with integrin receptors and by modulating growth factor activity (7). These functions are critical for inducing the expression of muscle regulatory factors that are essential but insufficient for skeletal muscle differentiation. Therefore, the ECM and its receptors provide an appropriate and permissive environment for specific differentiation to occur.
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ACKNOWLEDGEMENTS |
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This work was supported in part by grants from Fondo Nacional de Desarrollo Científico y Technológico to E. Brandan (1990151) and to N. Osses (2990088) and by the Fondos de Estudios Avanzados en Areas Prioritarias grant in biomedicine. The research of E. Brandan was supported in part by an International Research Scholars grant from the Howard Hughes Medical Institute and by a Presidential Chair in Science from the Chilean Government. The Millennium Institute for Fundamental and Applied Biology (MIFAB) is financed in part by the Ministerio de Planificación y Cooperación (Chile).
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FOOTNOTES |
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Address for reprint requests and other correspondence: E. Brandan, Dept. of Cell and Molecular Biology, Faculty of Biological Sciences, Pontifical Catholic Univ. of Chile, PO Box 114-D, Santiago, Chile (E-mail: ebrandan{at}bio.puc.cl).
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.
First published October 3, 2001; 10.1152/ajpcell.00322.2001
Received 12 September 2001; accepted in final form 10 October 2001.
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