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Poult Sci 2008. 87:1834-1840. doi:10.3382/ps.2008-00029
© 2008 Poultry Science Association
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PROCESSING, PRODUCTS, AND FOOD SAFETY: Research Note

The Effect of Fibroblast Growth Factor 2 on the In Vitro Expression of Syndecan-4 and Glypican-1 in Turkey Satellite Cells1

S. G. Velleman*,2, X. Li*, C. S. Coy* and D. C. McFarland{dagger}

* Department of Animal Sciences, The Ohio State University/Ohio Agricultural Research and Development Center, Wooster 44691; and {dagger} Department of Animal and Range Sciences, South Dakota State University, Brookings 57007

2 Corresponding author: Velleman.1{at}osu.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The membrane-associated heparan sulfate proteoglycan families, consisting of the syndecans and glypicans, are low-affinity receptors for fibroblast growth factor 2 (FGF2) that are essential in regulating the cellular response to FGF2. Fibroblast growth factor 2 is a potent stimulator of skeletal muscle cell proliferation and a strong inhibitor of differentiation. The regulation of the expression of the syndecans and glypicans will likely play a role in modulating the effects of FGF2 on cellular growth properties. In the present study, the effect of FGF2 on the expression of syndecan-4 and glypican-1 was measured by real-time PCR during turkey myogenic satellite cell proliferation and differentiation in vitro. Both syndecan-4 and glypican-1 transcription were influenced by the addition of exogenous FGF2. Syndecan-4 mRNA expression was reduced only during proliferation, whereas glypican-1 expression was reduced during both proliferation and differentiation. These results suggest that FGF2 growth factor signaling is, in part, regulated by an autoregulatory loop involving FGF2 regulation of syndecan-4 and glypican-1 expression and will affect the growth of skeletal muscle by modulating the proliferation and differentiation of satellite cells.

Key Words: fibroblast growth factor 2 • glypican-1 • muscle • satellite cell • syndecan-4


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
During early embryonic development, presumptive myoblasts migrate to the appropriate sites for skeletal muscle formation and give rise to myoblasts. The myoblasts proliferate, align with each other, and subsequently fuse to form multinucleated myotubes that develop into mature muscle fibers (Swartz et al., 1994). Another group of myogenic cells that reside between the basement membrane and the plasma membrane of the muscle fibers, the satellite cells, are the primary source of mononucleated cells that contribute to support postnatal muscle hypertrophy and muscle regeneration (Mauro, 1961).

Satellite cells are normally quiescent and must be activated to fuse with existing muscle fibers resulting in the postnatal growth of muscle fibers or the repair of damaged ones. The activation, proliferation, and differentiation of satellite cells are precisely regulated, in part, through cellular interactions with extrinsic factors. These extrinsic ligands include growth factors such as transforming growth factor-ß, platelet-derived growth factor, and fibroblast growth factor 2 (FGF2; McFarland, 1999). For many of these extrinsic ligands to assert their effect on cellular gene expression, proliferation, differentiation, and migration, they must interact with a cellular receptor system.

Cell-associated proteoglycans are a highly complex group of macromolecules that are emerging as key regulators of the interaction of cells with the extracellular matrix and extrinsic ligands. Proteoglycans represent a diverse family of glycosylated proteins that contain a core protein with covalently attached glycosaminoglycans (Hardingham and Fosang, 1992). Glycosaminoglycans attached to the core protein include chondroitin sulfate, dermatan sulfate, keratin sulfate, and heparan sulfate. The heparan sulfate containing proteoglycans are low-affinity receptors for FGF2. Heparan sulfate proteoglycans have been shown to regulate FGF2 binding to its high-affinity tyrosine kinase receptors (Rapraeger et al., 1991). Fibroblast growth factor 2 is a potent stimulator of muscle cell proliferation and a strong inhibitor of differentiation (Dollenmeier et al., 1981). Therefore, the expression of heparan sulfate proteoglycans during muscle cell proliferation and differentiation may play a pivotal role in the development and growth of muscle.

Two major groups of membrane-associated heparan sulfate proteoglycans, the syndecans and glypicans, have been identified in skeletal muscle. The syndecans are transmembrane proteoglycans with 4 members, syndecan-1 through -4, which have all been identified in skeletal muscle. The glypicans are cell surface associated proteoglycans that attach to the cell surface through a glycosylphosphatidylinositol linkage. There are 6 glypican family members with only glypican-1 identified in skeletal muscle. Both of these families of proteoglycans interact with FGF2. However, the mechanism of this interaction between FGF2 and the syndecans and glypicans is not well understood in skeletal muscle. It has been shown that growth factors like fibroblast growth factor can influence the synthesis of glycosaminoglycans, including heparan sulfate (Tzanakakis et al., 1995; Shimabukuro et al., 2008). In the case of syndecan-1, a fibroblast growth factor inducible response element (FiRE) has been identified which augments syndecan-1 expression in epithelial tissue in response to FGF2 (Jaakkola et al., 1997). It is unclear if other proteoglycans involved in regulating the cellular response to FGF2 respond in terms of their expression. If growth factors can influence the expression of proteoglycans, this would in turn modulate the effect of specific growth factors on cell proliferation and differentiation.

In the current study, the effect of FGF2 on the expression of syndecan-4 and glypican-1 has been focused on during the proliferation and differentiation of turkey myogenic satellite cells in vitro. Both of these proteoglycans have been shown to have differential effects on turkey satellite cell proliferation and differentiation, and differ in their responsiveness to FGF2 (Velleman et al., 2007). To date, there are no published reports on how FGF2 affects the expression of individual proteoglycans and the correlation of these effects to muscle cell proliferation and differentiation. Further understanding the mechanisms of FGF2 regulation of skeletal muscle growth properties has significant implications for the poultry industry in obtaining maximal muscle growth while maintaining product quality.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Turkey Myogenic Satellite Cells

Satellite cells were isolated from the pectoralis major muscle of 7-wk-old male randombred control line 2 (RBC2) turkeys. This procedure was described in Velleman et al. (2000).

Effect of Fibroblast Growth Factor 2 on Syndecan-4, and Glypican-1 Expression During Proliferation

The RBC2-line turkey satellite cells were plated at a density of 15,000 cells in gelatin-coated 16-mm plates and grown for 24 h in Dulbecco’s Modified Eagle Medium (DMEM) containing 10% chicken serum (Sigma-Aldrich, St. Louis, MO), 5% horse serum (Invitrogen, Carlsbad, CA), and 1% antibiotic/antimycotic (Invitrogen) in a 37°C 95% air/5% CO2 incubator. After 24 h, the medium was changed to a serum-free defined medium (McFarland et al., 2006) containing 0, 2.5, or 10 ng/mL of FGF2 (Pepro Tech Inc., Rocky Hill, NJ). The serum-free medium developed by McFarland et al. (2006) contains all of the growth factors necessary to provide good proliferation of avian satellite cells. These are platelet-derived growth factor-BB, hepatocyte growth factor, insulin-like growth factor-I, FGF2, and insulin. The cell culture medium was changed every 24 h for 96 h. Cell culture plates were removed every 24 h beginning at 48 h with the wells rinsed 2x with PBS, and the plates stored at –70°C until assayed.

Effect of Fibroblast Growth Factor 2 on Syndecan-4 and Glypican-1 Expression During Differentiation

The RBC2-line turkey satellite cells were plated at a density of 15,000 cells in gelatin-coated 16-mm plates and grown for 24 h in DMEM containing 10% chicken serum (Sigma-Aldrich), 5% horse serum (Invitrogen), and 1% antibiotic-antimycotic (Invitrogen) in a 37°C 95% air/5% CO2 incubator. After 24 h, the medium was changed to McCoy’s 5A (Sigma-Aldrich), 10% chicken serum, 5% horse serum, 1% antibiotics-antimycotics, and 0.1% gentamicin (Invitrogen). The medium was changed every 24 h until the cells reached 65% confluency. Differentiation was initiated by changing the medium to DMEM containing 3% horse serum, 1% antibiotics-antimycotics, 0.1% gentamicin, 1% gelatin, and 1 mg/mL of BSA containing 0, 2.5, or 10 ng/mL of FGF2. The differentiation medium reduces serum levels which results in withdrawal of the cells from the cell cycle and permits differentiation to begin.

Total RNA Extraction and cDNA Synthesis

Total RNA was extracted from the cultured satellite cells with TRIzol Reagent (Invitrogen) according the manufacturer’s procedure. The cDNA from the total RNA was synthesized using Moloney murine leukemia virus reverse transcriptase (Promega, Madison, WI) based on the protocol described by the manufacturer. The reverse transcription (RT) reaction was performed in a 25-µL volume. The RNA primer mix [1 µL of Oligo (dT)20 (50 µM; Operon, Huntsville, AL) and 1 µg of total RNA, and nuclease-free water up to 13.5 µL] was heated to 70°C for 5 min. This mixture was then incubated immediately on ice for 2 min and 11.5 µL of reaction mix [5 µL of 5x first-strand buffer (250 mM Tris-HCl, pH 8.3, 375 mM KCl, 15 mM MgCl2, and 50 mM di-thiothreitol; Promega), 1 µL of 10 mM deoxynucleoside triphosphate mix (Promega), 0.25 µL of RNasin (40 U/ µL; Promega), 1 µL of Moloney murine leukemia virus reverse transcriptase (200 U/µL) and nuclease free H2O up to 11.5 µL] was added. The reaction mixture was incubated at 55°C for 60 min and then heated at 90°C for 10 min to stop the reaction. The synthesized cDNA was diluted with 25 µL of nuclease-free water before performing the real-time quantitative PCR.

Real-Time Quantitative PCR

The real-time quantitative PCR was performed using DyNAmo Hot Start SYBR Green qPCR kit (New England Biolabs, Beverly, MA). The PCR reaction consisted of 2 µL of diluted RT reaction, 10 µL of 2x master mix, 0.4 µL of ROX dye, 0.25 µM of the forward and reverse primers, and nuclease-free water up to 20 µL. Reaction components were assembled in low-profile multiple plates and sealed with Thermal Seal RT (Phenix Research Products, Chandler, NC). Primers (Operon) used for the amplification of syndecan-4, glypican-1, and glyceraldehyde-3-phosphate dehydrogenase (GAP-DH) were designed from published turkey sequences (Table 1Go). Following the reaction assembly, plates were put into a DNA Engine Opticon 2 real-time system (MJ Research, Reno, NV). The cycling program consisted of denaturation (95°C for 15 min), followed by amplification and quantitation with the following conditions for each of the genes: syndecan-4 (34 cycles of 94°C for 30 s, 58°C for 30 s, and 72°C for 30 s), glypican-1 (34 cycles of 94°C for 30 s, 60°C for 30 s, and 72°C for 30 s), and GAPDH (34 cycles of 94°C for 30 s, 55°C for 30 s, and 72°C for 30 s); at the end of the elongation phase a single fluorescence measurement was taken and was followed by a final extension at 72°C for 5 min. The melting curve program was 52°C to 95°C, 0.2°C/read, and a 1-s hold. The final PCR products were checked for specificity by DNA sequence analysis. Standard curves were constructed for all the genes with serial dilutions of purified PCR products from each gene. The PCR products were purified by agarose gel electrophoresis using QIAquick gel extraction kit (Qiagen, Valencia, CA). All the samples fell within the values of the standard curves. The amount of sample cDNA for each gene was interpolated from the corresponding standard curve. The expression of all the genes was normalized to GAPDH expression and calculated as arbitrary units.


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  		Table 1. Primer sequences for real-time PCR
 
Statistical Analysis

The Student’s t-test was performed at each sampling time to evaluate the differences between means. Differences were considered significant at P < 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Effect of Fibroblast Growth Factor 2 on Syndecan-4 and Glypican-1 Expression during Proliferation and Differentiation

The effect of FGF2 on the expression of syndecan-4 and glypican-1 was measured during the period of satellite cell proliferation. Both syndecan-4 and glypican-1 mRNA expression was reduced by higher concentrations of FGF2 (Figures 1Go and 2Go). The effect on syndecan-4 and glypican-1 expression was dependent on the concentration of FGF2. During differentiation, syndecan-4 expression was not affected by the addition of exogenous FGF2 (Figure 3Go). In contrast to the proliferation phase, the differentiation expression of syndecan-4 was independent of FGF2. The mRNA expression of glypican-1 during differentiation was reduced by FGF2 (Figure 4Go). The effect on glypican-1 expression during differentiation was similar with the 2.5 and 10 ng/mL of FGF2 treatments.


Figure 1
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  		Figure 1. Syndecan-4 expression during proliferation of randombred control line 2 (RBC2) turkey satellite cells treated with fibroblast growth factor 2 at 0, 2.5, and 10 ng/mL. The bar represents the standard error of the mean. The mRNA concentrations with no common letter within a sampling time were significantly different (P < 0.05). This experiment was repeated 3 times.

 

Figure 2
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  		Figure 2. Glypican-1 expression during proliferation of randombred control line 2 (RBC2) turkey satellite cells treated with fibroblast growth factor 2 at 0, 2.5, and 10 ng/mL. The bar represents the standard error of the mean. The mRNA concentrations with no common letter within a sampling time were significantly different (P < 0.05). This experiment was repeated 3 times.

 

Figure 3
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  		Figure 3. Syndecan-4 expression during differentiation of randombred control line 2 (RBC2) turkey satellite cells treated with fibroblast growth factor 2 at 0, 2.5, and 10 ng/mL. The bar represents the standard error of the mean. This experiment was repeated 4 times.

 

Figure 4
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  		Figure 4. Glypican-1 expression during differentiation of randombred control line 2 (RBC2) turkey satellite cells treated with fibroblast growth factor 2 at 0, 2.5, and 10 ng/mL. The bar represents the standard error of the mean. The mRNA concentrations with no common letter within a sampling time were significantly different (P < 0.05). This experiment was repeated 4 times.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Fibroblast growth factor 2 is a potent stimulator of muscle cell proliferation and a strong inhibitor of differentiation (Dollenmeier et al., 1981). The cellular response to FGF2 is elicited through FGF2 interacting with a family of tyrosine kinase fibroblast growth factor receptors. For muscle cells to elicit a response to FGF2, FGF2 must interact with a heparan sulfate proteoglycan co-receptor. In other tissues, FGF2 has been shown to affect the transcription of proteoglycans, which are involved in regulation of the growth factor signaling (Tzanakakis et al., 1995; Rautava et al., 2003; Shimabukuro et al., 2008). How FGF2 affects the transcription of individual cell surface-associated heparan sulfate proteoglycans critical to muscle growth and development is not known.

In the present study, the effect of FGF2 on syndecan-4 and glypican-1 expression was measured during muscle satellite cell proliferation and differentiation in vitro. The results from the current study showed that syndecan-4 and glypican-1 expression is decreased during proliferation by the addition of exogenous FGF2. In contrast, during differentiation only glypican-1 expression was affected by the addition of FGF2, and syndecan-4 expression was not altered. These data suggested that glypican-1 expression is dependent on FGF2 during both proliferation and differentiation, and syndecan-4 only during the proliferation phase.

The finding that FGF2 can differentially affect the transcription of an individual proteoglycan has been reported in other tissues (Rautava et al., 2003; Shimabukuro et al., 2008). Syndecan-1 transcription is under the control of FiRE. The activation of FiRE is cell type and growth factor-specific (Jaakkola et al., 1998). In keratinocytes the syndecan-1 gene is induced by epidermal growth factor and in fibroblasts by FGF2. The regulation of the transcription of the syndecan-1 gene by FiRE is dependent upon the cellular state and is well demonstrated in wound healing. After injury to the epithelial cells, FiRE activation precedes the induction of syndecan-1 and is likely responsible for the upregulation in syndecan-1 gene expression (Rautava et al., 2003). In human periodontal ligament cells, FGF2 treatment reduced the expression of syndecan-4 (Shimabukuro et al., 2008).

Unlike syndecan-1, it is not known if the other syndecans or glypicans are regulated by an enhancer similar to FiRE. However, it is clear from the results of the present study that syndecan-4 and glypican-1 expression in turkey satellite cells is modulated by FGF2. This change in the expression of syndecan-4 and glypican-1 will likely have a significant impact on the development and growth of muscle. Syndecan-4 is involved in the formation of focal adhesions (Woods et al., 2000; Woods and Couchman, 2001) which will affect the ability of the muscle cells to migrate. A decrease in syndecan-4 expression during proliferation will likely result in fewer focal adhesions and reduce cell migration altering myotube formation. With regard to glypican-1, glypican-1 is thought to be associated with the differentiation of the muscle (Brandan et al., 1996; Velleman et al., 2004, 2006, 2007), which is supported by the overexpression of glypican-1 resulting in an increase in muscle differentiation (Velleman et al., 2004, 2007). The reduction in glypican-1 during proliferation and differentiation by FGF2 may result in fewer and smaller diameter myotubes. Therefore, the effect of FGF2 on the transcription of genes associated with muscle proliferation and differentiation requires further study as their expression can have a significant impact on the development and growth of muscle in turkeys.


   FOOTNOTES
 
1 Salary and research support was provided by state and federal funds appropriated to the Ohio Agricultural Research and Development Center, The Ohio State University, and to S. G. Velleman from the Cooperative State Research, Education, and Extension Service (CSREES), USDA, under agreement number 2003-35206-13696. Research support to S. G. Velleman and D. C. McFarland was provided by the Midwest Poultry Research Consortium and represents collaborative research between S. G. Velleman and D. C. McFarland as part of USDA CSREES multistate project NC 1131. Back

Received for publication January 17, 2008. Accepted for publication May 2, 2008.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Brandan, E., D. J. Carey, J. Larráin, F. Melo, and A. Campos. 1996. Synthesis and processing of glypican during differentiation of skeletal muscle cells. Eur. J. Cell Biol. 71:170–176.[Web of Science][Medline]

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Jaakkola, P., A. Määtä, and M. Jalkanen. 1998. The activation and composition of FiRE (an FGF-inducible response element) differ in a cell type- and growth factor-specific manner. Oncogene 17:1279–1286.[CrossRef][Web of Science][Medline]

Jaakkola, P., T. Vihinen, A. Määtä, and M. Jalkanen. 1997.Activation of an enhancer on the syndecan-1 gene is restricted to fibroblast growth factor members in mesenchymal cells. Mol. Cell. Biol. 17:3210–3219.[Abstract/Free Full Text]

Mauro, A. 1961. Satellite cell of skeletal muscle fibers. J. Biophys. Biochem. Cytol. 9:493–495.[Medline]

McFarland, D. C. 1999. Influence of growth factors on poultry myogenic satellite cells. Poult. Sci. 78:747–758.[Abstract/Free Full Text]

McFarland, D. C., S. G. Velleman, J. E. Pesall, and C. Liu.2006. Effect of myostatin on turkey myogenic satellite cells and embryonic myoblasts. Comp. Biochem. Physiol. 144A:501–508.

Rapraeger, A., A. Krufka, and B. B. Olwin. 1991. Requirement of heparan sulfate for bFGF-mediated fibroblast growth and myoblast differentiation. Science 252:1705–1708.[Abstract/Free Full Text]

Rautava, J., T. Soukka, K. Heikinheimo, P. J. Miettinen, R.-P. Happonen, and P. Jaakkola. 2003. Different mechanisms of syndecan-1 activation through a fibroblast-growth-factor-inducible response element (FiRE) in mucosal and cutaneous wounds. J. Dent. Res. 82:382–387.[Abstract/Free Full Text]

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Swartz, D. R. S.-S., Lim, T. Fassel, and M. L. Greaser. 1994.Mechanisms of myofibril assembly. Recip. Meat Conf. Proc. 47:141–153.

Tzanakakis, G. N., N. K. Karamanos, J. Klominek, and A. Hjerpe. 1995. Effects of glycosaminoglycan synthesis in cultured human mesothelioma cells of transforming, epidermal, and fibroblast growth factors and their combinations with platelet-derived growth factor. Exp. Cell Res. 220:130–137.[CrossRef][Web of Science][Medline]

Velleman, S. G., C. S. Coy, and D. C. McFarland. 2007. Effect of syndecan-1, syndecan-4, and glypican-1 on turkey muscle satellite cell proliferation, differentiation, and responsiveness to fibroblast growth factor 2. Poult. Sci. 86:1406–1413.[Abstract/Free Full Text]

Velleman, S. G., C. Liu, C. S. Coy, and D. C. McFarland. 2006. Effects of glypican-1 on turkey skeletal muscle cell proliferation, differentiation, and fibroblast growth factor 2 responsiveness. Dev. Growth Differ. 48:271–276.[CrossRef][Web of Science][Medline]

Velleman, S. G., X. Liu, C. S. Coy, and D. C. McFarland. 2004. Effects of syndecan-1 and glypican on muscle cell proliferation and differentiation: Implications for possible functions during myogenesis. Poult. Sci. 83:1020–1027.[Abstract/Free Full Text]

Velleman, S. G., X. Liu, K. E. Nestor, and D. C. McFarland.2000. Heterogeneity in growth and differentiation characteristics in male and female satellite cells isolated from F- and RBC2-line turkeys. Comp. Biochem. Biophys. 125A:503–509.[CrossRef]

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Woods, A., R. L. Longley, S. Tumova, and J. R. Couchman. 2000. Syndecan-4 binding to the high affinity heparin-binding domain of fibronectin drives focal adhesion formation in the fibroblasts. Arch. Biochem. Biophys. 374:66–72.[CrossRef][Web of Science][Medline]





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