Syndecan-1 Expression Inhibits Myoblast Differentiation through a Basic Fibroblast Growth Factor-dependent Mechanism*

Expression of syndecan-1, a cell-surface heparan sulfate proteoglycan, is down-regulated during skeletal muscle differentiation (Larraı́n, J., Cizmeci-Smith, G., Troncoso, V., Stahl, R. C., Carey, D. J., and Brandan, E. (1997)J. Biol. Chem. 272, 18418–18424). We examined the role of syndecan-1 in basic fibroblast growth factor (bFGF)-dependent inhibition of myogenesis. C2C12 myoblasts were stably transfected with an expression plasmid containing the rat syndecan-1 coding region cDNA. Constitutive syndecan-1 expression resulted in a strongly diminished capacity of the transfected clones to differentiate and to express skeletal muscle-specific markers such as fusion, creatine kinase, and myosin. The expression of myogenin, a master transcription factor for muscle differentiation, was also reduced and delayed. Analysis of the induction of a myogenin promoter-driven reporter revealed that syndecan-1 expression resulted in a 6–7-fold increase in sensitivity to bFGF-dependent inhibition of myogenin expression. Transfecting the cells with a plasmid containing myogenin cDNA reversed the inhibition of myogenin transcriptional activation and myosin expression in syndecan-1-transfected cells; however, cell fusion was not observed. These results demonstrate that syndecan-1 expression enhances cell responsiveness to bFGF and inhibits myoblast fusion and suggest that muscle terminal differentiation is regulated by syndecan-1 expression.

The onset and progression of this process are controlled by a complex set of interactions between myoblasts and their environment. The presence of the extracellular matrix (ECM) 1 is essential for normal myogenesis (4 -6). The involvement of growth factors in the regulation of myoblast proliferation and differentiation is well established in vitro, as well as the modulation of their activities by interaction with ECM constituents such as heparan sulfate proteoglycans. The ability of myoblasts to differentiate is controlled in a negative manner by the extracellular concentration of specific mitogens, such as basic fibroblast growth factor (bFGF), hepatocyte growth factor/scatter factor (HGF/SF), and transforming growth factor type-␤ (7)(8)(9)(10). In contrast, insulin-like growth factor promotes myogenesis (11). In the presence of the inhibitory growth factors, myoblasts continue to proliferate and fail to fuse or to express muscle-specific gene products. Conversely, in cell culture experiments the reduction in the concentration of these growth factors below a critical level results in irreversible withdrawal from the cell cycle and terminal differentiation.
It has been demonstrated that heparan sulfate proteoglycans are necessary for the modulation of terminal myogenesis (12,13), probably by acting as low affinity receptor for some growth factors such as bFGF (14) and HGF/SF (15). We have shown that the expression of heparan sulfate proteoglycans is regulated during terminal skeletal muscle differentiation of the C 2 C 12 myoblast cell line (16). The expression of glypican increases (17,18), whereas expression of perlecan decreases during skeletal muscle differentiation (19). Recently, we have shown that the expression of syndecan-1, a transmembrane heparan sulfate proteoglycan (20,21), is down-regulated during myoblast terminal differentiation. Syndecan-1 mRNA as well as cell-surface syndecan-1 disappear almost completely by 48 h after triggering differentiation (22).
Cell-surface heparan sulfate molecules are critical in the activation of signaling receptors by growth factors (23,24). It has been shown that, depending on the cellular localization, syndecans can either increase or decrease responsiveness to bFGF. Syndecans potentiate bFGF binding to the signaling receptor when they are associated with the membrane but inhibit bFGF receptor binding when added in soluble form (25). Overexpression of syndecan-1 in a cell line in which most of the expressed proteoglycan is shed from the membrane also leads to decreased responsiveness to bFGF (26). The potential to regulate growth factor activity by means of heparan sulfate-dependent interactions provides an attractive system for fine-tuning cellular responses to growth factors in the extracellular environment. Because its potential to act as a growth factor co-receptor (14,21,27), the down-regulation of syndecan-1 might play a critical role in attenuating growth factor activity in myoblasts and thus promote muscle cell differentiation (16,22,28).
To investigate the role of syndecan-1 during skeletal muscle terminal differentiation, we transfected C 2 C 12 myoblasts with expression plasmids containing rat syndecan-1 cDNA. Constitutive expression of syndecan-1 resulted in a strongly diminished capacity of the transfected myoblasts to differentiate, and a 6-7-fold increase in sensitivity to bFGF-dependent inhibition of myogenesis. Thus the sensitivity of myoblasts to bFGF, an inhibitor of myogenesis, is directly modulated by syndecan-1 expression.

EXPERIMENTAL PROCEDURES
Materials-The C 2 C 12 cell line, BALB/c mouse embryo genomic library, and pRSVlacZII phagemid vector (␤-galactosidase reporter) were purchased from ATCC. EMSV-Myo8 is an EMSVscribe vector containing the human myogenin cDNA (a generous gift of Dr. Eric Olson, University of Texas, Houston). pGREEN LANTERN-1 vector was obtained from Life Technologies, Inc. Trizol LS, LipofectAMINE, DMEM, chicken embryo extract (CEE), horse serum, Opti-MEM I, HanksЈ balanced salt solution, and G418 were obtained from Life Technologies, Inc. Wizard Plus Maxipreps, Prime-a-Gene labeling system, pCATbasic vector, and n-butyryl-CoA were from Promega, Madison, WI. Fetal calf serum (FCS), CRC-30 medium, creatine kinase assay kit, monoclonal anti-␣-tubulin, monoclonal anti-myosin, alkaline phosphatase-conjugated goat anti-rabbit IgG, and goat anti-mouse IgG were from Sigma. Affinity purified rabbit polyclonal anti-human FGFR-1 antibody was from Santa Cruz Biotechnology Inc., Santa Cruz. Human bFGF, bFGF enzyme-linked immunosorbent assay system, [ 35  Cell Culture-The mouse skeletal muscle cell line C 2 C 12 was grown as described (22). Three days after plating in growth medium (DMEM supplemented with 10% FCS and 0.05% CEE), differentiation was triggered by changing the medium to differentiation medium (DMEM supplemented with 5% horse serum). Two days later, 0.1 mM cytosine ␤-D-arabinofuranoside was added to the culture medium. Thereafter the incubation medium was changed daily. In some experiments sodium chlorate (final concentration 50 mM) was added to the cultures at the time of plating and maintained during differentiation. Heparin (final concentration 0 -1 mg/ml) was added to differentiation medium containing 5 ng/ml bFGF.
RNA Isolation and Northern Blot Analysis-Total RNA was isolated from cell cultures using Trizol. RNA samples (15 g/lane) were electrophoresed through 1.2% agarose/formaldehyde gels, transferred to Nytran membranes, and hybridized with probes for myogenin and syndecan-1 as described previously (22). Blots were hybridized with random primed labeled probes in a buffer containing 1.0 M NaCl, 1% SDS, 10% dextran sulfate, and 100 g/ml denatured salmon testes DNA at 65°C overnight. Hybridized membranes were washed twice at 65°C in 0.2ϫ SSC, 0.1% SDS for 5 min and exposed to Kodak x-ray film. For quantitative determination of the mRNA levels, the intensity of the hybridization signals was measured by densitometric scanning (GS 300 Scanning Densitometer, Hoefer Scientific Instruments, San Francisco).
Stable Transfection and Isolation of Clones-C 2 C 12 cells were plated at a density of 6,000 cells/cm 2 in 100-mm dishes in growth medium. 24 h later the cells were transfected with the mammalian expression plasmid pCMVneo containing a 1-kb cDNA insert coding for full-length rat syndecan-1 in the sense orientation (29,30). Control cells were transfected with pCMVneo containing no insert. For transfections, 40 g of plasmid DNA and 30 g of LipofectAMINE in a total volume of 600 l of Opti-MEM I were mixed in a glass tube and allowed to sit for 30 min. During this period the cells were washed twice with Opti-MEM I and maintained in this medium. The DNA mixture was added dropwise with swirling to C 2 C 12 cultures. The cells were incubated at 37°C for 6 h with this mixture. FCS and CEE were then added to final concentrations of 10 and 0.5%, and the cells were incubated overnight in this medium. Finally the cells were rinsed twice with HBSS and cultured in normal growth medium. After 3 days the cells were switched to normal growth medium supplemented with G418 (400 g/ml). After 2-3 weeks viable colonies were subcultured using cloning rings.
Labeling of Cultures and Proteoglycan Immunoprecipitation-Metabolic labeling with [ 35 S]Na 2 SO 4 was carried out as described previously (18). The cells were extracted with immunoprecipitation buffer (0.5% Nonidet P-40, 0.5% deoxycholate, 0.1% SDS, 0.05 M Tris-HCl, pH 7.5), and aliquots of the extracts were incubated overnight at 4°C with affinity purified rabbit anti-syndecan-1 (22) or anti-glypican (17) antibodies. The immune complexes were harvested by the addition of protein A-agarose beads followed by centrifugation. The amount of proteoglycans precipitated was determined by liquid scintillation counting. Some aliquots were analyzed by Sepharose-CL 4B for the presence of syndecan-1 (data not shown).
Construction of Myogenin Reporter-For construction of the pMYO-CAT reporter plasmid, a 688-base pair DNA fragment corresponding to a region of the myogenin promoter that contains MEF1 and E box sites (from ϩ62 to Ϫ626 nucleotides (31)) was amplified by nested polymerase chain reaction using a mouse BALB/c genomic library as template. The fragment was subcloned in the pCAT-basic vector. The identity of the DNA insert was verified by DNA sequence analysis using the Sequenase kit.
Transient Transfections-The cells were plated in growth medium 1 day before transfection at a density of 6000 cells/cm 2 in 60-mm plates. For transfection the cells were incubated for 6 h in Opti-MEM I containing 5 g of each plasmid and 15 g of LipofectAMINE. After transfection the cells were incubated for 14 h in Opti-MEM I containing 10% FCS and 0.5% CEE. The cells were then washed twice with HBSS and incubated for 2 days in growth medium followed by 1 or 2 days in differentiation medium or 30 h in differentiation medium containing bFGF (0 -30 ng/ml). The cells were harvested and assayed for chloramphenicol acetyltransferase (CAT) and ␤-galactosidase activities as described previously (22). All transfections were performed at least two times with at least two plasmid DNA preparations.
Analysis of Creatine Kinase Activity-Myoblasts and myoblasts induced to differentiate for 2, 3, or 4 days were washed twice with PBS and lysed with PBS containing 0.1% Triton X-100 for 10 min at 25°C. The cells were scraped, and creatine kinase activity was determined using the creatine kinase assay kit. All data points represent the mean of duplicate determinations from three independent experiments.
Immunofluorescence Microscopy-Cells to be immunostained were grown on coverslips. The medium was removed, and the coverslips were rinsed with PBS. For staining of intracellular myosin, the cells were fixed with 3% paraformaldehyde for 30 min at room temperature, permeabilized with 0.5% Triton X-100, and incubated with the primary antibodies. The cells were then rinsed with BLOTTO and incubated for 1 h at room temperature with affinity purified fluorescein-conjugated secondary antibodies diluted in BLOTTO. After rinsing, the coverslips were mounted and viewed with a Nikon Diaphot inverted microscope equipped for epifluorescence.
bFGF Quantitation-Conditioned medium from 6 plates (100 mm) of cells induced to differentiate for 30 h was collected and loaded onto a heparin-Sepharose column equilibrated with 10 mM Tris, pH 7.5, 0.5 M NaCl. After extensive washing with the equilibration buffer, the column was eluted with the same buffer containing 2 M NaCl. bFGF in the eluted fractions was determined using the bFGF enzyme-linked immunosorbent assay system.
DNA and Protein Determination-DNA was determined in aliquots of cells extracts as described (32). Protein was determined as described (33).

Heparan Sulfate Proteoglycans Modulate Myogenin Activation-
The expression of early myogenic regulators like myogenin is strongly inhibited by growth factors such as bFGF and HGF-SF (7,8). The binding of these growth factors to their signaling receptors and their biological activities are strongly potentiated by binding to heparin or heparan sulfate proteoglycans (15,34). To determine if heparan sulfate proteoglycans could regulate myogenin expression by modulation of heparindependent growth factor activity, we evaluated the effect of chlorate, a metabolic inhibitor of heparan sulfate synthesis, and heparin, a heparan sulfate analogue, on the activation of myogenin expression. Fig. 1 a shows the result of Northern blot analysis using a myogenin cDNA probe that was hybridized to total RNA isolated from C 2 C 12 myoblasts incubated in differentiation medium for 30 h in the presence (lane 1) or absence (lane 2) of 10 ng/ml bFGF. As expected, bFGF inhibited the induction of myogenin expression that occurs during myoblast differentiation (7). When myoblasts were preincubated with 50 mM sodium chlorate (lane 3), bFGF failed to inhibit myogenin expression. The inhibitory activity of bFGF on myogenin expression was partially restored by addition of 10 ng/ml heparin (lane 4). These results demonstrate that inhibition of myogenin expression by bFGF is dependent on the presence of myoblast heparan sulfate proteoglycans.
We have reported previously that the expression of the trans-membrane heparan sulfate proteoglycan syndecan-1 is downregulated during myogenic differentiation of C 2 C 12 cells (22). We hypothesized that after terminal differentiation was initiated, down-regulation of cell-surface heparan sulfate proteoglycans involved in bFGF signaling was necessary to attenuate the strong differentiation-inhibiting activity of bFGF. To determine whether the amount of heparan sulfate present on differentiating myoblasts was indeed limiting bFGF activity, we evaluated the effect of exogenous heparin on bFGF activity in myoblasts induced to differentiate for 30 h. In order to have a more sensitive assay for bFGF-dependent effects on myogenesis, a myogenin reporter plasmid consisting of ϳ700 base pairs of the mouse myogenin promoter (31) linked to a CAT reporter (pMYOCAT) was constructed. pMYOCAT expression was induced in differentiation medium and inhibited by bFGF similarly to myogenin mRNA expression analyzed by Northern blot (data not shown). As shown in Fig. 1B, exogenous heparin produced a significant and concentration-dependent potentiation of the inhibitory effect of bFGF on pMYOCAT activity in differentiating myoblasts. This observation suggests that a decrease in heparan sulfate proteoglycan expression could modulate activation of myogenin expression. Stable Transfection of C 2 C 12 Myoblasts with a Syndecan-1 cDNA-We reasoned if down-regulation of syndecan-1 expression was responsible for the decrease in bFGF activity, then its constitutive expression would inhibit or delay myogenesis. To test this idea a cDNA coding for full-length rat syndecan-1 (29,30) was subcloned into the selectable expression vector pCM-Vneo ( Fig. 2A) and used to stably transfect C 2 C 12 myoblasts. Clones were selected under growth conditions in the presence of G418. As a control, myoblasts were transfected with the same vector containing no cDNA insert. A total of 8 syndecan-1-transfected G418-resistant clones were isolated. Several of these clones were chosen for more detailed analysis.
G418-resistant clones were analyzed by Northern blot for expression of the syndecan-1 transgene. Fig. 2B shows the results of this analysis using a full-length syndecan-1 cDNA probe that was hybridized to total RNA isolated from controltransfected myoblasts (lane1) and four syndecan-1-transfected clones (lanes 2-6). As expected, hybridization signals were detected in all lanes for the endogenous syndecan-1 mRNA FIG. 1. A, bFGF requires sulfated heparan sulfate proteoglycans to inhibit myogenin expression. C 2 C 12 myoblasts were grown for 30 h in differentiation medium in the absence or presence of 10 ng/ml bFGF, 50 mM sodium chlorate, or 10 ng/ml heparin, as indicated. Total RNA was extracted and subjected to Northern blot analysis with a 32 P-labeled myogenin cDNA probe. The resulting autoradiogram (top) and ethidium bromide-stained gel (bottom) are shown. The arrows on the right indicate the sizes of myogenin mRNA and ribosomal RNAs. B, the inhibitory effect of bFGF on myogenin expression is potentiated by heparin during differentiation. C 2 C 12 myoblasts were transiently co-transfected with pMYOCAT and RSV-␤-galactosidase plasmids. The cells were incubated for 48 h in growth medium and then an additional 30 h in differentiation medium containing bFGF (5 ng/ml) and the indicated concentrations of heparin. The cells were harvested, and the CAT and ␤-galactosidase activities were determined. The pMYOCAT/␤-galactosidase activities were as follows: after 48 h in growth medium, 2,808; after 30 h in differentiation medium, 35 (arrows). Three G418-resistant clones (S1, lane 2; S2, lanes 3 and 6; S4, lane 4) also revealed an additional hybridization signal. The size of this band (ϳ1,000 nucleotides) was consistent with the expected size of the polyadenylated product of the syndecan-1 expression vector.
To analyze syndecan-1 synthesis in the stably transfected clones, myoblasts, and myoblasts that were induced to differentiate for 4 days were metabolically labeled with [ 35 S]Na 2 SO 4 , and detergent extracts were immunoprecipitated with polyclonal anti-syndecan-1 (29) or anti-glypican antibodies (17). Fig. 3 shows the results obtained for clones C1, S2, and S4. At the myoblast stage clone S2 showed an increase in the amount of 35 SO 4 -labeled syndecan-1 over the control clone C1. After 4 days of differentiation (when endogenous syndecan-1 expression is down-regulated), clones S2 and S4 showed approximately 2-3-fold increases in 35 SO 4 -labeled syndecan-1 with respect to the control clone. The increased expression of syndecan-1 in the transfected cells was specific for this proteoglycan and was not seen with glypican. As shown in Fig. 3 (inset), the amount of 35 SO 4 -labeled glypican increased in all the clones after triggering of differentiation, consistent with previous findings (18). Clones S2 and S4 synthesized similar amounts of glypican as control cells.
Myogenic Differentiation Is Blocked in Syndecan-1-transfected Myoblasts-The capacity of syndecan-1-transfected clones to differentiate was compared with that of wild type C 2 C 12 cells and with control-transfected cells. Both morphological and muscle-specific biochemical markers were evaluated. Fig. 4 shows the morphology of proliferating myoblasts (left panels) and myoblasts that were induced to differentiate for 4 days (right panels). The syndecan-1-transfected S2 myoblasts spread more extensively than wild type or control-transfected cells and appeared larger and more flattened. This was remi-niscent of the morphology of syndecan-1-transfected Schwann cells reported previously (30). A more striking observation was the essentially complete failure of S2 and S4 myoblasts to undergo fusion to myotubes. In contrast, by 4 days of differentiation many wild type and control-transfected cells had fused to form multinucleated myotubes. Table I shows the expression of the muscle-specific isoform of creatine kinase and myosin heavy chain during differentiation. In wild type and control-transfected cells, creatine kinase-specific activity increases approximately 40-fold over the levels in non-induced myoblasts. In contrast, little increase in creatine kinase activity was detected in syndecan-1-transfected S2 and S4 cells. Myosin heavy chain expression was also diminished in the syndecan-1 transfectants after 4 days in differentiation medium compared with wild type and control-transfected cells.
It is well established that skeletal muscle differentiation, including expression of creatine kinase and myosin, is dependent on the expression of myogenin. Expression of myogenin mRNA in control and syndecan-1-transfected C 2 C 12 cells was measured by Northern blot analysis. As shown in Fig. 5, induction of myogenin mRNA was apparent in control-transfected cells (Fig. 5A) by 12 h after initiation of differentiation. In contrast, in syndecan-1-transfected S2 and S4 cells (Fig. 5, B and C), myogenin induction was delayed and reduced in magnitude. Densitometric analyses of Northern blots (Fig. 5D) indicated that myogenin transcripts in syndecan-1-transfected cells were 4 -5-fold lower than in control cells at 12, 24, and 48 h after initiation of differentiation. The effect of syndecan-1 transfection on myogenin expression was confirmed using the pMYOCAT reporter construct. Fig. 6 a shows the ␤-galactosidase-normalized CAT activities in control (C1) and syndecan-1-transfected (S2) myoblasts that were transiently transfected with pMYOCAT and induced to differentiate for 1 or 2 days. The expression of pMYOCAT was inhibited by approximately 65% in syndecan-1-transfected cells compared with control cells.
To demonstrate that the inhibition of myogenin expression seen in syndecan-1-transfected cells was not caused by a defect in the ability of the cells to activate myogenin expression, we measured the induction of myogenin reporter activity and myosin expression by a mechanism that is independent of growth factor-dependent regulation. Myogenin can activate its own expression by a positive feedback loop (31). We determined whether exogenous myogenin could activate myogenin reporter transcriptional activity to normal levels in syndecan-1-trans-fected cells. Control (clone C1) and syndecan-1-transfected (clone S2) cells were transiently co-transfected with an expression vector containing the full-length myogenin cDNA (EMSV-Myo8), p-MYOCAT, and RSV-␤-galactosidase. The cells were maintained under growth conditions, and CAT activity was measured after 3 days. Fig. 6B shows that exogenous myogenin expression produced a similar increase in ␤-galactosidase-normalized CAT activity in control and syndecan-1-transfected cells. We also determined whether myogenin could activate myosin expression in syndecan-1-transfected cells. As shown in Fig. 7B, myogenin expression induced the expression of myosin in syndecan-1-transfected cells. In contrast, transfection with myogenin cDNA was not able to induce fusion of syndecan-1transfected cells. Transiently transfected cells were visualized in syndecan-1-expressing cells Wild type (WT), control-transfected (clone C1), and syndecan-1-transfected (clones S2 and S4) C 2 C 12 cells were plated and incubated in differentiation medium. On the indicated days the cells were extracted with 0.1% Triton X-100, and creatine kinase activities and DNA content were determined. The results are expressed in IU/ng DNA. The percentage of increase in creatine kinase activity, for each clone, compared to the increase observed in wild type cells after differentiation is indicated in parentheses. Cells were extracted after 4 days in differentiation medium, and steady state levels of myosin heavy chain and tubulin were analyzed by Western blot, and the intensity of the band was determined by densitometry. The results shown are the means Ϯ S.D. from two different experiments performed in duplicate.  by their expression of green fluorescent protein (Fig. 8, A-C). These results demonstrate that intracellular mechanisms for activation of myogenin and myosin expression are intact in syndecan-1-transfected cells. In contrast, myoblast fusion was not restored by myogenin expression in cells that constitutively express syndecan-1.

Syndecan-1 Expression Increases the Sensitivity of Myoblasts to bFGF-dependent Inhibition of Myogenin Expression-To de-
termine whether the diminished expression of myogenin observed in syndecan-1-transfected cells resulted from a higher sensitivity to bFGF-dependent inhibition, we measured the concentration dependence of bFGF inhibition of pMYOCAT expression. Wild type, control-transfected (clone C1), and syndecan-1-transfected (clone S2) C 2 C 12 myoblasts were transiently transfected with pMYOCAT and incubated for 30 h in differentiation medium supplemented with different amounts of bFGF. As shown in Fig. 9 exposure of the cells to bFGF resulted in a significant inhibition of pMYOCAT expression. The syndecan-1-transfected cells, however, showed a marked shift in the dose-response curve, from an IC 50 of approximately 2 ng/ml for wild type and control cells to approximately 0.2 ng/ml for S2 cells (Table II). This shift in sensitivity to bFGF did not appear to result from differences in the levels of expression of the FGFR. Western blot analysis of detergent lysates of wild type, control-transfected, and syndecan-1-transfected myoblasts showed similar amounts of anti-FGFR-1 staining (data not shown).
To determine whether this 6 -7-fold shift in the sensitivity of the cells to bFGF might be relevant to conditions encountered by the cells during in vitro differentiation, we measured the amount of bFGF present in myoblast-conditioned medium by an enzyme-linked immunosorbent assay (Table II). Conditioned medium obtained after 30 h of culture under differentiation conditions contained bFGF at a concentration of approximately 0.2 ng/ml. This value and the bFGF dose-response curve of wild type and control-transfected cells (Fig. 9) are consistent with the ability of these cells to undergo differentiation. In contrast, this concentration of bFGF should be sufficient to maintain the syndecan-1-transfected cells in an undifferentiated state, as was observed.
Heparan Sulfate Proteoglycans Increase during Skeletal Muscle Differentiation-Heparan sulfate proteoglycan synthesis during skeletal muscle terminal differentiation was evaluated. Myoblasts and myoblasts induced to differentiate were labeled for 18 h with 35 SO 4 and extracted with Triton X-100. As shown in Table III there was an increase in the relative amount of cell-associated heparan sulfate, during differentiation, in both detergent-soluble and insoluble forms. This increase likely corresponds to the increase in glypican reported previously by us (18). DISCUSSION In these studies we have shown that the constitutive expression of syndecan-1 in myoblasts induces a strong inhibition of skeletal muscle differentiation, marked by an inhibition of myotube fusion and low levels of expression of the musclespecific proteins creatine kinase and myosin heavy chain. More importantly, we have shown that the constitutive expression of syndecan-1 resulted in decreased and delayed expression of myogenin, an early master gene that governs the expression of several skeletal muscle-specific genes. Analysis of a myogenin reporter revealed that constitutive syndecan-1 expression resulted in a 6 -7-fold increase in sensitivity to bFGF-dependent inhibition of myogenin expression.
Previous studies have shown that soluble heparin/heparan sulfate and heparan sulfate proteoglycans modulate bFGF activity (14, 34 -36). Heparan sulfate-heparin interactions can either increase or decrease growth factor activity, depending on the specific manner in which the proteoglycan interacts with the growth factor and with the cell surface (21). The dependence of skeletal muscle cell differentiation on heparan sulfate proteoglycans was first reported by Rapraeger and co-workers (12), who demonstrated a decrease in myosin expression in myoblasts treated with chlorate, an inhibitor of proteoglycan sulfation. In this work we have extended these observations, and we demonstrate that the inhibitory effect of bFGF on myogenin expression is lost when myoblasts are treated with chlorate and that exogenous heparin potentiates the inhibitory effect of bFGF on myogenin expression in myoblasts induced to differentiate in the presence of a subsaturating concentration of bFGF. These two observations suggest that in myoblasts bFGF activity is dependent on interactions with heparan sulfate proteoglycans and that after induction of skeletal muscle cell differentiation bFGF activity is limited by the amount of available heparan sulfate proteoglycan (heparin).
Syndecan-1 appears to be a good candidate for this function, because it is located on the plasma membrane and its expression decreases after differentiation is triggered. In the present study we determined the effect of syndecan-1 expression on myogenesis by isolating stably transfected cells that constitutively express this proteoglycan. Cells that exhibited increased expression of cell-associated syndecan-1 under conditions that would normally induce differentiation showed a reduced ability to differentiate, as evaluated by the extent of myoblast fusion as well as the induction of creatine kinase activity and myosin expression. Syndecan-1-transfected clones also expressed less myogenin compared with control cells after induction of differentiation. Despite the inability to differentiate exhibited by the syndecan-1-expressing myoblasts, these cells were able to induce myogenin transcriptional activity and the expression of myosin following transfection with a myogenin expression plasmid (31). These results indicate that the intracellular machinery for the induction of myogenin and skeletal muscle-specific markers is intact in these cells and support the conclusion that the developmental defect in these cells results from a change in their response to growth factors.
Our findings strongly suggest that the low levels of myogenin expression exhibited by the syndecan-1-transfected cells and their inability to differentiate are caused by changes in bFGF activity. Cells that constitutively express syndecan-1 are about 6 -7-fold more sensitive to bFGF than wild type or controltransfected cells. The presence of bFGF in myoblast-conditioned medium at a concentration of approximately 0.2 ng/ml, a bFGF concentration required to exert 50% inhibition of myogenin expression in syndecan-1-transfected cells, provides an attractive explanation for the occurrence of this inhibition in syndecan-transfected cells but not in wild type or control-transfected cells.
Previous studies have provided evidence that a factor that is critical for heparan sulfate proteoglycan-dependent modulation of bFGF activity is the location of the proteoglycan with respect to the plasma membrane. It has been shown in hematopoietic cells transfected with FGFR-1 that co-transfection with syndecan-1, syndecan-2, syndecan-4, or glypican produces an increase in binding of bFGF to the receptor (25). Interest-  ingly, the presence of equimolar amounts of soluble syndecan-4 ectodomain has no effect on the binding, suggesting that the heparan sulfate proteoglycans can support bFGF binding to the receptor only when they are located on the cell surface (25). In other cells overexpression of syndecan-1 has been shown to suppress bFGF activity (26). In these cells, however, most of the expressed proteoglycan was shed into the culture medium where it accumulated in a soluble form. In our experiments the syndecan-1-transfected cells expressed syndecan-1 at levels comparable to those in myoblasts, and most of the proteoglycan was associated with the cells.
Because of the ability of syndecan-1 to interact with other polypeptides, one cannot exclude the possibility that sustained expression of syndecan-1 in the transfected cells directly affects myoblast fusion. One of the molecules that participates in myoblast fusion is N-CAM, which co-localizes with N-cadherin. Their expression increases during myoblast differentiation and activates myoblast fusion (43)(44)(45). It has been shown that homophilic N-CAM binding is inhibited by heparan sulfate (46). Therefore, it is possible to speculate that the presence of syndecan-1 might inhibit myoblast fusion. We did not observe any fusion of syndecan-1-transfected myoblasts even after transfection with myogenin cDNA, which induced the expression of myogenin and myosin. It has been shown that fusion is independent of myogenin expression (47)(48)(49). Thus, the inability of myoblasts to fuse is likely to reflect impaired adhesion between the syndecan-1-expressing myoblasts.
As our earlier work has shown, myoblast differentiation produces a concerted pattern of regulation of expression of several heparan sulfate proteoglycans. We have shown that heparan sulfate proteoglycans in conditioned medium of skeletal muscle cells decrease during terminal differentiation (50). In contrast, they accumulate in Triton X-100-soluble and insoluble cellassociated fractions obtained from skeletal muscle cells induced to differentiate. These results are in accordance with a previous report on primary cultures of embryonic chicken skeletal muscle cells (51). It is tempting to speculate that these changes might contribute to regulation of myoblast differentiation. Specifically, perlecan expression is down-regulated after triggering of skeletal muscle differentiation (19). Perlecan is a ubiquitous basal lamina-associated heparan sulfate proteoglycan (37). Its participation in modulation of bFGF activity has been documented (38). Whether perlecan carries out this function in myoblasts remains to be determined. On the other hand, we have shown that glypican increases during differentiation and accounts for 20% of the total proteoglycans associated with myotube membranes (18), being the main heparan sulfate proteoglycan found associated with the cell surface or the ECM (18). Glypican, like syndecan-1, is synthesized as a plasma membrane-associated proteoglycan. The two proteoglycans differ, however, in their modes of attachment to the plasma membrane. We have observed an endogenous processing mechanism for glypican that occurs during skeletal muscle differentiation. This results in the release of most of the proteoglycan from the membrane, to become incorporated into the ECM (18). It is possible that large amounts of glypican in the ECM might sequester growth factors away from the plasma membrane, rendering them unable to interact with transducing receptors.
It is important to point out that other heparin binding growth factors could be involved in the modulation of myogenesis. One of these is HGF/SF, whose ability to bind to its transducing receptor, the c-met receptor (HGF/SF receptor) (39), is also modulated by heparan sulfate proteoglycans (15). We are evaluating if the syndecan-1-transfected cells also exhibit a greater sensitivity toward HGF/SF. If so, alteration of syndecan-1 expression could provide a common regulatory mechanism for modulating the action of different inhibitory growth factors on skeletal muscle cells.
Little is know with respect to the in vivo signals that activate the expression of myogenin to begin terminal differentiation. It has been shown that syndecan-1 is present in the limb mesoderm by day 9 of mouse gestation and decreases by day 11-13 (40). Concomitantly, it has been shown that myogenin mRNA expression begins in the limb muscle cells on day 10.5 to 12 (41,42). These observations, together with the results presented in this paper, suggest syndecan-1 down-regulation as a possible mechanism for modulating muscle differentiation. This idea needs to be tested in vivo, however, in order to be established.
In summary our results support the concept that syndecan-1 plays an important role during skeletal muscle terminal differentiation by modulating the ability of the myogenin inhibitory growth factor bFGF to interact with its signaling receptor.