ECM is required for skeletal muscle differentiation independently of muscle regulatory factor expression

Nelson Osses, Enrique Brandan

Abstract

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 β-d-xyloside, were used. Treatment of cultured skeletal muscle cells with each inhibitor substantially abolished the expression of creatine kinase and α-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 β-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

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)-β (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.

MATERIALS AND METHODS

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 Larraı́n et al. (36). For inhibitor treatments, sodium chlorate (final concentration 30 mM), β-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 C2C12cells were radiolabeled by incubation in medium containing 100 μCi35S-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-α-tubulin, were purchased from Sigma. Polyclonal anti-rat glypican was a generous gift from Dr. David J. Carey (Sigfried and Janet Weis Center for Research, Geisinger Clinic, Danville, PA). Polyclonal anti-myogenin antibody was obtained from Santa Cruz Biotechnology.

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 α-dystroglycan antibody (1:500; Upstate Biotechnology, Lake Placid, NY), polyclonal anti-human MEF2A antibody (1:200; Santa Cruz Biotechnology), or monoclonal anti-α-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.

RESULTS

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 β-d-xyloside (23), whereas the sulfation of proteoglycans can be specifically inhibited by sodium chlorate (28). Figure 1 Ashows that the induction of creatine kinase activity was totally inhibited by β-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. 1 B; this figure also indicates tubulin transcript levels as loading controls. Figure1 C shows that in the presence of either β-d-xyloside or sodium chlorate the synthesis of α-dystroglycan, a late skeletal muscle marker, was also inhibited because of the alteration of proteoglycan synthesis.

Fig. 1.

Inhibition of proteoglycan synthesis affects skeletal muscle differentiation. A: C2C12cells were induced to differentiate in the absence (control) or presence of β-d-xyloside (BDX; 1 mM), DMSO (0.1%), or sodium chlorate (30 mM). Creatine kinase activity was quantified as a function of the timing of differentiation. B: Creatine kinase expression in C2C12 cells was evaluated by Northern blot analysis. Total RNA (10 μg) was isolated from growing myoblasts (GM) and myoblasts incubated for 6 days in differentiation medium (DM) in the absence (control) or presence of the proteoglycan synthesis inhibitors indicated in A. RNA was separated by electrophoresis, blotted onto nylon membranes, and hybridized with a 32P-labeled creatine kinase cDNA probe. The membrane was reprobed after stripping with a32P-labeled tubulin cDNA probe that acted as a control of membrane-bound RNA levels. Transcript sizes are indicated.C: Western blot analysis was used to evaluate α-dystroglycan protein expression in GM and after 6 days in DM in the absence (control) or presence of the proteoglycan synthesis inhibitors described in A. Cellular extracts were separated by SDS-PAGE (7.5%), transferred onto nitrocellulose membranes, stained with either anti-α-dystroglycan or anti-α-tubulin antibodies, and detected by enhanced chemiluminescence (ECL). Molecular mass markers are shown onleft.

Table 1 shows that β-d-xyloside treatment produced an increase of almost 19-fold in the incorporation of 35SO4 into the secreted GAG fraction, which was detected in the myoblast medium with no changes observed for the cell extracts. In contrast, the same β-d-xyloside treatment decreased the amount of35SO4-labeled proteoglycans by 77%. Table 1also shows that sodium chlorate treatment strongly inhibited the sulfation of both proteoglycans and GAGs present in the medium and cell extracts. These results suggest that the inhibition of proteoglycan synthesis also inhibits the process of skeletal muscle differentiation.

View this table:
Table 1.

Effect of β-d-xyloside and chlorate treatments on35SO4 incorporation into proteoglycans and glycosaminoglycans in C2C12 myoblasts

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. 2 A) and the induction of myogenin protein expression after the onset of differentiation (Fig. 2 B). 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. 2 A), and MEF2A, a transcriptional activator of muscle-specific genes (Fig. 2 C), were equally unaffected by the proteoglycan inhibitors.

Fig. 2.

Proteoglycan synthesis inhibitors do not affect myogenin, MyoD, and MEF2A expression. A: the expression of myogenin and MyoD was evaluated by Northern blot analysis in GM and myoblasts maintained for 30 h in DM in the absence (control) or presence of BDX (1 mM), DMSO (0.1%), or sodium chlorate (30 mM). Total RNA (10 μg) was separated by electrophoresis, blotted onto nylon membranes, and hybridized with 32P-labeled myogenin, myoD, and tubulin cDNA probes. Transcript sizes are indicated. B: myogenin protein expression was evaluated by Western blot in GM and after 48 h in DM in the absence (control) or presence of the proteoglycan synthesis inhibitors indicated in A. Cellular extracts were separated by SDS-PAGE (10%), transferred onto nitrocellulose membranes, stained with anti-myogenin or anti-α-tubulin, and detected by ECL. Molecular mass markers are shown on left. C: the effect of BDX or sodium chlorate on the expression of MEF2A was determined by Western blot as described in A and B.

In this context, a possible explanation for the inhibition of late skeletal muscle differentiation described above is the lack of a nuclear destination for myogenin because of the alteration of proteoglycan synthesis. Figure3 A shows that after the induction of terminal differentiation myogenin localized to the nuclei of differentiating cells independently of the inhibition of proteoglycan synthesis. The quantitative analysis shown in Fig.3 B indicates that ∼40% of myoblasts were induced to express myogenin under control conditions, results that are concordant with previous findings (61). On the other hand, no effect was observed on the nuclear destination of myogenin for either proteoglycan inhibitor treatment (Fig. 3 B). Together, these results strongly suggest that terminal skeletal muscle differentiation does not occur on alteration of proteoglycan synthesis, although the expression, synthesis, and localization of myogenin remain unaffected.

Fig. 3.

Proteoglycan synthesis inhibitors do not affect the nuclear localization of myogenin. A: GM cell cultures and myoblasts maintained for 48 h in DM in the absence (control) or presence of DMSO (0.1%), BDX (1 mM), or sodium chlorate (30 mM) were fixed, permeabilized, and stained with polyclonal anti-mouse myogenin (A–E) or 1 μg/ml Hoechst 33258 (F–J). FITC-conjugated anti-rabbit IgG was used as secondary antibody. Bar, 50 μm. B: the percentage of myogenin-positive nuclei (myogenin-positive nuclei/Hoechst-stained nuclei) was determined in each experimental condition in 10 independent fields. Values are means ± SD.

β-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 β-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 β-d-xyloside and sodium chlorate treatments almost abolished the anti-glypican staining (Fig. 4, C andD). 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 β-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 β-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.

Fig. 4.

Proteoglycan synthesis inhibitors affect the assembly of extracellular matrix (ECM) proteins. C2C12cells were induced to differentiate for 6 days in the absence (control) or presence of DMSO (0.1%), BDX (1 mM), or sodium chlorate (30 mM). Nonpermeabilized cells were processed for indirect immunofluorescent staining for either anti-glypican (Gly; A–D), anti-laminin (Ln; E–H), or anti-fibronectin (Fn;I–L) antibodies. Permeabilized cells were processed for indirect immunofluorescent staining with anti-α-tubulin antibodies (Tub; M–P). FITC-conjugated secondary antibodies were used. All fields were photographed and printed under identical conditions. Phase contrast (PC) microscopy is shown for indicated treatments (Q–T). Bar, 50 μm.

Exogenous ECM prevents inhibitory action of β-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 5 A 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. 5 B. These results suggest that cell-ECM contact is required for skeletal muscle differentiation and that the inhibitory action of both β-d-xyloside and sodium chlorate stems from their effect on ECM deposition.

Fig. 5.

Exogenous ECM prevents the inhibition of skeletal muscle differentiation by proteoglycan synthesis inhibitors. A: C2C12 cells induced to differentiate for 6 days in the absence (control) or presence of sodium chlorate (30 mM) or BDX (1 mM) are shown on left. C2C12cells induced to differentiate under similar conditions but in the presence of ECM gel are shown on right. Cells were visualized by phase contrast microscopy. Bar, 50 μm. B: C2C12 cells were induced to differentiate and treated as described in A. At the indicated times, cell-associated creatine kinase activity was determined.

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. Figure6 A 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. 6 B), Western blot (Fig. 6 C), and immunocytolocalization (Fig.6 D). 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.

Fig. 6.

RGDS peptides inhibit skeletal muscle differentiation independently of myogenin expression or localization. A: creatine kinase activity was determined in GM and after 6 days in DM in the absence (control) or presence of RGDS peptides (0.2 mg/ml).B: myogenin and MyoD expression in GM and after 30 h in DM in the absence (control) or presence of RGDS peptides (0.2 mg/ml) was evaluated by Northern blot analysis as described in Fig.2 A. Transcript sizes as well as the ratios of myogenin and MyoD levels with respect to tubulin are indicated. C: myogenin protein expression in GM and in myoblasts cultivated for 48 h in DM in the absence (control) or presence of RGDS peptides (0.2 mg/ml) was evaluated by Western blot as described in Fig.2 B. Molecular mass markers are shown on left. D: C2C12 cells were induced to differentiate for 48 h in the absence (control) or presence of RGDS peptides (0.2 mg/ml), and the percentage of myogenin-positive nuclei was determined as described in Fig. 3.

Further studies into ECM and integrin requirements were performed by analyzing the extent of phosphorylation of FAK. Integrin signals are relayed, in part, by the activation of this ubiquitously expressed tyrosine kinase. In the presence of sodium chlorate, a decrease in the autophosphorylation of the FAK tyrosine-397 was observed, with no changes in total FAK expression levels (Fig.7 A). This decrease could be reversed by the addition of the exogenous ECM gel. On the other hand, a minor effect on the autophosphorylation of FAK was detected for β-d-xyloside (Fig. 7 B). These results suggest that inhibition of proteoglycan synthesis, caused by chlorate treatment and β-d-xyloside treatments, led to an inhibition of FAK signaling.

Fig. 7.

Focal adhesion kinase (FAK) phosphorylation decreases with chlorate and BDX treatments in differentiated C2C12 cells: effect of exogenous ECM.A: C2C12 cells from GM and after 6 days in DM, maintained in the absence or presence of sodium chlorate (30 mM) or ECM gel, were harvested in RIPA buffer, and FAK immunoprecipitation (IP) was performed. FAK phosphorylation was determined by immunoblot analysis (WB) with an anti-phosphotyrosine (P-Tyr) antibody (top). Bottom, reprobing of the polyvinylidene difluoride membrane with an anti-FAK antibody. Serum IgG was used as a negative control. Molecular mass markers are shown onleft. B: C2C12 cells from GM and after 6 days in DM, maintained in the absence or presence of BDX (1 mM) or ECM gel, were harvested in RIPA buffer, and FAK phosphorylation was determined as described in A.

DISCUSSION

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-β (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-β 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 α7β1-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 α7-integrin (10,11). In the muscles of Duchenne and Becker dystrophy patients, an increase in the expression of α7 proteins occurs (27), whereas mutations in the α7 gene cause human congenital myopathies (26). Recently, it was shown that enhanced expression of α7β1-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 α- and β-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 β1A-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 β-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 β-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 β-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 β-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 β-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.

Acknowledgments

This work was supported in part by grants from Fondo Nacional de Desarrollo Cientı́fico 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).

Footnotes

  • 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

REFERENCES

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