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Effect of Transforming Growth Factor-β on Decorin and β1 Integrin Expression During Muscle Development in Chickens1
Effect of Transforming Growth Factor-β on Decorin and β1 Integrin Expression During Muscle Development in Chickens1 X. Li,* D. C. McFarland,† and S. G. Velleman*2 *Department of Animal Sciences, Ohio Agricultural Research and Development Center, The Ohio State University, Wooster, Ohio 44691; and †Department of Animal and Range Sciences, Brookings, South Dakota 57007 PCR. The LSN condition has elevated expression of TGFβ2 and TGF-β4 with increased expression of decorin and decreased β1 integrin during myogenic satellite cell proliferation and differentiation. Normal satellite cell cultures were treated with the addition of exogenous TGFβ during differentiation to determine if the altered expression of LSN decorin and β1 integrin was associated with TGF-β expression. The addition of exogenous TGF-β decreased decorin expression during differentiation and reduced β1 integrin expression at 24 and 48 h of differentiation. These results suggested that alteration of decorin expression in the LSN myogenic satellite cells may occur by a mechanism involving factors in addition to TGF-β, but the addition of exogenous TGF-β did affect both decorin and β1 integrin expression. These data, therefore, suggested that TGF-β might play a pivotal role in chicken skeletal muscle formation through modulation of the expression of both extracellular matrix molecules and cellular receptors important in the control of cell migration and growth regulation.
Key words: β1 integrin, decorin, proteoglycan, satellite cell, transforming growth factor-β 2006 Poultry Science 85:326–332
cells (Scott, 1995), and is required for skeletal muscle myogenesis (Melo et al., 1996). Signaling between the extracellular matrix and cells plays a pivotal role in the regulation of cellular behavior. Through interactions with growth factors such as TGF-β and fibroblast growth factor 2, the extracellular matrix is involved in the regulation of cell gene expression, proliferation, migration, adhesion, and differentiation, all of which are essential for muscle development and growth (Yanagishita, 1993). The extracellular matrix proteoglycan decorin is a small, leucine-rich proteoglycan that consists of a central core protein of approximately 45 kDa and a single chondroitin or dermatan sulfate chain covalently attached to the core protein (Krusius and Ruoslahti, 1986; Iozzo, 1998). The decorin core protein interacts with TGF-β and modulates its activity (Yamaguchi et al., 1990; Kresse et al., 1994). Riquelme et al. (2001) demonstrated that TGFβ signaling occurs in a decorin-dependent fashion. As a result, the inhibition of decorin expression decreased myoblast responsiveness to TGF-β and accelerated skeletal muscle differentiation. The extracellular concentration
INTRODUCTION During skeletal muscle development, growth factors and the extracellular matrix environment surrounding the cells are involved in regulating cell proliferation and differentiation. Transforming growth factor-β (TGF-β) is a multifunctional regulator of cell growth and differentiation (Roberts and Sporn, 1985). Transforming growth factor-β is a potent inhibitor of both myoblast proliferation and differentiation (Allen and Boxhorn, 1987). The extracellular matrix is defined as a dynamic network of polysaccharides and proteins that are immobilized outside
2006 Poultry Science Association, Inc. Received August 11, 2005. Accepted October 13, 2005. 1 Salary and research support to S.G.V. were provided by state and federal funds appropriated to the Ohio Agricultural Research and Development Center, The Ohio State University, and the Midwest Poultry Consortium to S.G.V. and D.C.M. 2 Corresponding author: [email protected]
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ABSTRACT Myoblast-extracellular matrix interactions play a pivotal role in skeletal muscle development. Transforming growth factor-β (TGF-β) is a key regulator of muscle cell proliferation and differentiation. The level of TGF-β expressed will affect the concentration of the extracellular matrix proteoglycan decorin and the cell surface β1 integrin subunit. The decorin proteoglycan is a regulator of cell growth as well as the organization of the extracellular matrix. The β1 integrin plays a role in muscle cell attachment, migration, and the formation of multinucleated myotubes. In the current study, chicken myogenic satellite cells isolated from the pectoralis major muscle from the chicken genetic muscle weakness, low score normal (LSN), and normal pectoralis major muscle were used to investigate TGF-β expression as it relates to decorin and β1 integrin mRNA expression. The LSN muscle defect is characterized by altered myotube formation and sarcomere structure, and the satellite cells have reduced proliferation and differentiation. The mRNA expression was measured by real-time quantitative reverse transcription
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Sequence1
Decorin
5′-GAGGGTAGTGAAGGCTGCTG-3′ (forward) 5′-TTGGCACTCTTTCCAGACCT-3′ (reverse) 5′-AGGAATGTGCAGGATAATT-3′ (forward) 5′-ATTTTGGGTGTTTTGCCAA-3′ (reverse) 5′-AGGATCTGCAGTGGAAGTGG-3′ (forward) 5′-CGGCCCACGTAGTAAATGAT-3′ (reverse) 5′-AATTTGTAGCAGGCGTGGTTG-3′ (forward) 5′-CACGGCGCTCTTCTAAATAGG-3′ (reverse) 5′-GAGGGTAGTGAAGGCTGCTG-3′ (forward) 5′-CCACAACACGGTTGCTGTAT-3′ (reverse)
TGF-β2 TGF-β4 β1 integrin GAPDH
Product size (bp) 256 269 300 169 200
1 Primer sequences were designed from the following GenBank accession numbers: decorin: X63797; transforming growth factor (TGF)-β2: X59080; TGF-β4: M31160; β1 integrin: U37029; and glyceralderaldehyde-3phosphate dehydrogenase (GAPDH): U94327.
Figure 1. Gel electrophoresis of real-time PCR products. Lane 1: DNA ladder; lane 2: glyceraldehyde-3-phosphate dehydrogenase; lane 3: decorin; lane 4: β1 integrin; lane 5: transforming growth factor-β4; and lane 6: transforming growth factor-β2.
The expression of the β1 integrin is also affected by TGFβ1 (Ignotz and Massague´, 1987; Heino et al., 1989). It is known that TGF-β is a potent inhibitor of both muscle cell proliferation and differentiation (Allen and Boxhorn, 1987). Transforming growth factor-β may, in part, regulate cell proliferation and differentiation through its effect on decorin and β1 integrin expression. However, to date, the relationship of TGF-β with the extracellular matrix and cell adhesion receptors is not well understood. In chickens, 3 isoforms exist, TGF-β2, β3, and β4, where TGF-β4 is the chicken analog of the mammalian TGF-β1 (Groenen et al., 2000; Schmid et al., 2000; Smith et al., 2000). The chicken genetic muscle weakness, low score normal (LSN) is an abnormal muscle weakness condition that was originally detected in 1997 at the University of Connecticut in F2 progeny in an outcross of chickens with hereditary muscular dystrophy (L. J. Pierro and J. S. Haines, Department of Animal Genetics, University of Connecticut, Storrs, personal communication). The LSN birds are distinguished from normal birds by an impaired ability to right themselves when repeatedly placed on their backs (exhaustion score test). Low score normal birds have an exhaustion score intermediate between that of normal birds and muscular dystrophy birds. Velleman and Nestor (2001) concluded that the LSN trait is influenced primarily by an autosomal dominant gene but is also influenced by other genes, some of which are on the sex chromosome. In addition to decreased righting ability, LSN birds have reduced pectoral muscle mass in posthatch birds as early as 1 wk of age (Velleman et al., 1996). The LSN pectoralis major muscle exhibits altered formation of myotubes in vitro (Velleman and McFarland, 1999) and modified sarcomere structure in vivo (Velleman et al., 1997). Velleman and Coy (1998) showed that TGFβ2 expression is up-regulated in LSN pectoral muscle late in embryonic development through 1 wk posthatch, and TGF-β1 expression is up-regulated at 1 d posthatch. The late embryonic increase in TGF-β2 expression coincides with an increase in decorin protein concentration (Velleman et al., 1996) and mRNA expression (Velleman and Coy, 1997). Furthermore, β1 integrin protein concentra-
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of TGF-β also affects the expression of decorin in many cell types (Bassols and Massague´, 1988). One of the means by which the extracellular matrix will transduce signals to the cells is through the integrin receptors. Integrins are heterodimeric transmembrane cell adhesion receptors containing α and β subunits that span the plasma membrane providing a transmembrane linkage between the extracellular matrix and the cytoskeleton (Hynes, 1992). The integrins, therefore, provide a means for the bidirectional transmission of signal information between the extracellular matrix and cellular cytoskeletal network. The β1 integrin subunit is ubiquitous and plays a pivotal role in cell attachment to the extracellular matrix, cell migration, proliferation, and differentiation (reviewed in Berman et al., 2003). The β1 integrin has further been shown to be involved in regulating muscle contraction through the interaction of the β1 integrin cytoplasmic domain with the cytoskeleton (Belkin et al., 1997).
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MATERIALS AND METHODS
nant human TGF-β1 was administered to normal satellite cell cultures with fresh medium added at 24-h intervals until 96 h. The concentration of recombinant human TGFβ1 was previously determined in a study by Velleman and McFarland (1999). Control cultures were grown without treatment. The cell cultures were collected every 24 h and stored at −70°C until use.
Total RNA Extraction and cDNA Synthesis Total RNA was extracted from the cell cultures by using TRIzol (Invitrogen, Carlsbad, CA) according to the manufacturer’s protocol. Reverse transcription of total RNA to a cDNA was conducted using Moloney murine leukemia virus reverse transcription (M-MLV, Promega, Madison, WI). In brief, an RNA-primer mix [1 g of total RNA, 1 L of oligo (dT), and nuclease-free water up to 13.5 L] was incubated at 80°C for 3 min, followed immediately by incubation on ice. The reaction mix [5 L of 5× FirstStrand buffer (MJ Research, Reno, NV), 1.25 L of 10 mM deoxynucleoside triphosphate mix (MJ Research), 0.5 L of RNasin (40 U/L, MJ Research), 1 L of Moloney murine leukemia virus (200 U/L), and nuclease-free water up to 11.5 L] was added to the mixture. The complete reaction mixture was incubated at 55°C for 60 min, and then heated at 90°C for 10 min for inactivation.
Satellite Cell Culture
Real-Time Quantitative PCR
Satellite cells from both normal and LSN pectoralis major muscle were isolated as described by Li et al. (1997). Satellite cells from the 2 lines were cultured in McCoy’s 5A medium (Life Science Technologies, Inc., Grand Island, NY) with 10% chicken serum (Sigma Chemical Co., St. Louis, MO), 5% horse serum, and 20 ng/mL of fibroblast growth factor 2 (Pepro Tech Inc., Rocky Hill, NY; Li et al., 1997). The cells were plated in gelatin-coated, 35-mm cell culture wells (Corning Costar, Corning, NY) at a density of 36,000 cells per well. The normal and LSN satellite cells were cultured at a cell density permitting the cells to reach 65% confluency at the same time minimize culturing differences. When the cells reached approximately 65% confluency, differentiation was induced by culturing in low-serum media containing Dulbecco’s Modified Eagle Medium (Life Science Technologies), 3% horse serum, 0.01 mg/mL of porcine gelatin (Sigma Chemical Co.), and 1.0 mg/mL of bovine serum albumin (Sigma Chemical Co.). Cell cultures were removed from the incubator at 72 h of proliferation and at 0, 24, 48, 72, and 96 h of differentiation. The culture medium was removed from the plates, and the cells were rinsed with sterile PBS (170 mM NaCl, 3 mM KCl, 10 mM Na2HPO4, and 2 mM KH2PO4, pH 7.08) and 2 mM KH2PO4. All plates were then stored at −70°C until use. To address the effect of TGF-β on decorin and β1 integrin expression, the addition of exogenous TGF-β1 was performed according to the method described by Velleman and McFarland (1999). In brief, at the initiation of differentiation, medium containing 1 ng/mL of recombi-
Real-time quantitative PCR was performed using the DyNAmo Hot Start SYBR Green qPCR kit (MJ Research). The PCR reaction consisted of 2 L of the reverse transcription reaction mixture diluted with 25 L of nucleasefree water, 10 L of 2× master mix (MJ Research) provided by the manufacturer, 250 nM of each of the forward and reverse primers, and nuclease-free water up to 20 L. Reaction components were assembled in low-profile multiplates (MJ Research) and sealed with Microseal B adhesive seals (MJ Research). Primers used in the amplification of decorin, TGF-β2, TGF-β4, β1 integrin, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were designed from published sequences as listed in Table 1. The specificity of the primers was confirmed by DNA sequence analysis of the amplified PCR product. The realtime PCR amplification was done in a DNA Engine Opticon 2 real-time system (MJ Research). For all the amplified genes, the cycling program was initiated with a hotstart step at 95°C for 15 min. The cycling program for decorin and GAPDH consisted of denaturation at 94°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C for 30 s for 34 cycles, with a final elongation of 72°C for 5 min. The cycling conditions for TGF-β2 and TGF-β4 comprised denaturation at 94°C for 30 s, annealing at 54°C for 45 s, and extension at 72°C for 45 s for 34 cycles, with a final elongation for 5 min at 72°C. The PCR cycling conditions for β1 integrin were denaturation at 94°C for 1 min, annealing at 58°C for 1 min, and extension at 72°C for 3 min for 34 cycles with a final elongation at 72°C for 10 min. The final PCR products were analyzed on a 1.5%
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tion is reduced in the LSN pectoral muscle at 16 and 18 d of embryonic development and 1 wk posthatch compared with normal pectoral muscle (Velleman et al., 2000). Due to these changes in muscle structure, extracellular matrix molecules, and cell surface receptors, the LSN condition is a useful model to further evaluate how changes in TGFβ regulation affect muscle cell decorin and β1 integrin expression during the proliferation and differentiation of muscle. It was, therefore, the objective of the present study to further investigate the relationship between TGF-β, decorin, and β1 integrin expression during myogenesis. To address the relationship between TGF-β, decorin, and β1 integrin, their expression was measured in normal and LSN satellite cells cultured with exogenous TGF-β. The expression of decorin and β1 integrin was examined. Satellite cells are quiescent myogenic cells residing between the basement membrane and plasma membrane of muscle fibers (Mauro, 1961), and are largely responsible for postnatal muscle growth and muscle regeneration (Moss and LeBlond, 1971). The satellite cell cultures allow the in vitro monitoring of satellite cell proliferation through the formation and differentiation of multinucleated myotubes.
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agarose gel to check amplification specificity. Standard curves were constructed for decorin, TGF-β2, TGF-β4, β1 integrin, and GAPDH with serial dilutions of the 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 sample concentrations 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 decorin, TGF-β2, TGF-β4, and β1 integrin was normalized to GAPDH expression.
Statistical Analyses Differences between the means of LSN and normal satellite cells at each sampling interval for the relative mRNA expression of the target genes were evaluated with a Student’s t-test. Differences were considered sig-
nificant at P < 0.05. Each experiment was repeated at least 3 times with graphical data showing a representative experiment.
RESULTS Decorin, TGF-β2, TGF-β4, and β1 Integrin mRNA Expression in Chicken Normal and LSN Satellite Cells Real-time PCR was used to measure the relative expression of decorin, TGF-β2, TGF-β4, and β1 integrin mRNA expression during normal and LSN satellite cell proliferation and differentiation. The specificity of the real-time PCR assay was confirmed by melting curve analysis (data not shown) and gel electrophoresis (Figure 1). Gel electrophoresis showed single bands at the appropriate size for decorin, TGF-β2, TGF-β4, β1 integrin, and GAPDH.
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Figure 2. Real-time PCR analysis of decorin (panel A), transforming growth factor-β2 (TGF-β, panel B), transforming growth factor-β4 (TGFβ4, panel C), and β1 integrin (panel D) in normal chicken and low score normal (LSN) chicken satellite cells during proliferation (P) and differentiation (D). The y-axis denotes arbitrary units of expression, which are defined as the ratio of the relative cDNA concentration of the target genes to the cDNA concentration of glyceraldehyde 3-phosphate dehydrogenase. * Indicates a significant different (P < 0.05) between the lines at a sampling interval. Error bars represent the standard error of the mean.
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differentiation than in the normal cells, which expressed more TGF-β4 compared with the LSN cells at 72 and 96 h of differentiation. Expression of β1 integrin was significantly higher at all times measured except 72 h of proliferation in the normal satellite cell cultures compared with the LSN satellite cells (Figure 2D).
The Effect of TGF-β1 on Decorin and β1 Integrin mRNA Expression
DISCUSSION
Figure 3. The effect of transforming growth factor-β1 (TGF-β1) on decorin (panel A) and β1 integrin (panel B) mRNA expression during myogenic satellite cell proliferation (P) and differentiation (D). The yaxis denotes arbitrary units of expression, which are defined as the ratio of the relative cDNA concentration of the target genes to the cDNA concentration of glyceraldehyde 3-phosphate dehydrogenase. *Indicates a significant difference (P < 0.05) between the control and TGF-β1 treatment at a sampling interval. Error bars represent the standard error of the mean.
The expression of decorin was significantly elevated in the LSN satellite cells cultures during both proliferation and differentiation. Decorin expression peaked in both the normal and LSN cultures at 0 and 72 h of differentiation (Figure 2A). Expression of TGF-β2 was elevated in the LSN satellite cell cultures after 72 h of proliferation, and at 48 and 72 h of differentiation (Figure 2B). Expression of TGF-β2 generally decreased with differentiation for both the normal and LSN satellite cells. Expression of TGF-β4 was similar to that for TGF-β2 in that levels were higher for both the normal and LSN cells during proliferation and decreased with differentiation (Figure 2C). Expression of TGF-β4 was significantly higher in the LSN cells from 72 h of proliferation through 24 h of
Satellite cells are myogenic precursors that are required for the process of postnatal muscle growth through hypertrophy. During the period of postnatal muscle growth, satellite cells proliferate, differentiate, and fuse with adjacent muscle fibers or with other satellite cells, and then add additional nuclei to the muscle fibers (Allen et al., 1979). This ultimately increases protein synthesis potential due to the increased number of nuclei, and thus leads to muscle growth through muscle fiber hypertrophy. Certain growth factors are involved in the regulation of muscle hypertrophy by either stimulating or inhibiting cell proliferation and differentiation (Charge´ and Rudnicki, 2004). Many studies have focused on the mechanism of growth factor regulation of satellite cell activity (Vaidya et al., 1989; Brennan et al., 1991). Only recently has the role of the extracellular matrix in the muscle growth process received research attention, in part due to its role in the regulation of growth factor responsiveness (reviewed in Velleman, 1999). Decorin, an extracellular matrix proteoglycan, interacts with TGF-β and regulates muscle cellular responsiveness to TGF-β (Riquelme et al., 2001). During differentiation, muscle cell migration is essential for the alignment of myoblasts and adhesion to form differentiated multinucleated myotubes, which requires the proper cellular adhesion to the extracellular matrix through integrins (Meredith and Schwartz, 1997; Velleman and McFarland, 2004). Myoblasts cultured in the presence of the β1 integrin cell substrate attachment antibody do not form multinucleated myotubes but continue to replicate (Menko and Boettiger, 1987). Low score normal myogenic satellite cells form shorter myotubes with a reduced number of myonuclei per myotube in vitro (Velleman and McFarland, 1999). These changes in LSN myotube morphology in vitro are associated with reduced proliferation and differentiation rates
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Decorin and β1 integrin mRNA expression were measured in normal satellite cell cultures treated with exogenous TGF-β1 during the 96 h of differentiation. The addition of exogenous TGF-β1 decreased decorin expression during differentiation compared with the untreated control satellite cell cultures (Figure 3A). β1 Integrin expression was reduced in the treated cultures at 24 and 48 h of differentiation, and was significantly increased in the TGF-β1 treated cultures by 96 h of differentiation (Figure 3B).
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(Li et al., 1997) as well as reduced concentrations of β1 integrin protein during differentiation (Velleman et al., 2000). In the present study, chicken TGF-β2 and TGF-β4 were expressed at higher levels in the LSN satellite cells than in the normal satellite cells during proliferation. Interestingly, expression of TGF-β2 and TGF-β4 were altered at different times. Transforming growth factor-β2 was mainly affected during the differentiation process, whereas TGF-β4 was increased in the LSN cultures during proliferation and early differentiation. These data suggest that TGF-β2 and TGF-β4 are affecting different aspects of LSN muscle development. In addition, decorin expression was increased and β1 integrin levels were reduced in LSN cultures. The addition of exogenous TGF-β to normal cultures decreased decorin expression compared with the increased expression observed in the LSN satellite cell cultures. β1 Integrin expression was decreased at 24 and 48 h of differentiation and increased by 96 h of differentiation in the normal cell cultures with the addition of exogenous TGF-β, whereas decreased β1 integrin expression was detected throughout proliferation and differentiation in the LSN cell cultures. These data suggest that the mechanism leading to the changes in LSN decorin and β1 integrin expression may result from other factors as well as changes in TGF-β expression. However, the findings from the current study do indicate that expression of decorin and β1 integrin are associated with TGF-β during chicken satellite cell proliferation and differentiation. Future research will need to address the mechanism by which TGFβ regulates decorin and β1 integrin expression as it relates to muscle development and growth.
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Velleman, S. G., and K. E. Nestor. 2001. Mode of inheritance of the low score normal condition in chickens. Poult. Sci. 80:1273–1277. Velleman, S. G., J. D. Yeager, H. Krider, D. A. Carrino, S. D. Zimmerman, and R. J. McCormick. 1996. The avian low score normal muscle weakness alters decorin expression and collagen crosslinking. Connect. Tissue Res. 34:33–39. Yamaguchi, Y., D. M. Mann, and E. Ruoslahti. 1990. Negative regulation of transforming growth factor-beta by the proteoglycan decorin. Nature 346:281–284. Yanagishita, M. 1993. Function of proteoglycans in the extracellular matrix. Acta Pathol. Jpn. 43:283–293.
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Key words: β1 integrin, decorin, proteoglycan, satellite cell, transforming growth factor-β 2006 Poultry Science 85:326–332
cells (Scott, 1995), and is required for skeletal muscle myogenesis (Melo et al., 1996). Signaling between the extracellular matrix and cells plays a pivotal role in the regulation of cellular behavior. Through interactions with growth factors such as TGF-β and fibroblast growth factor 2, the extracellular matrix is involved in the regulation of cell gene expression, proliferation, migration, adhesion, and differentiation, all of which are essential for muscle development and growth (Yanagishita, 1993). The extracellular matrix proteoglycan decorin is a small, leucine-rich proteoglycan that consists of a central core protein of approximately 45 kDa and a single chondroitin or dermatan sulfate chain covalently attached to the core protein (Krusius and Ruoslahti, 1986; Iozzo, 1998). The decorin core protein interacts with TGF-β and modulates its activity (Yamaguchi et al., 1990; Kresse et al., 1994). Riquelme et al. (2001) demonstrated that TGFβ signaling occurs in a decorin-dependent fashion. As a result, the inhibition of decorin expression decreased myoblast responsiveness to TGF-β and accelerated skeletal muscle differentiation. The extracellular concentration
INTRODUCTION During skeletal muscle development, growth factors and the extracellular matrix environment surrounding the cells are involved in regulating cell proliferation and differentiation. Transforming growth factor-β (TGF-β) is a multifunctional regulator of cell growth and differentiation (Roberts and Sporn, 1985). Transforming growth factor-β is a potent inhibitor of both myoblast proliferation and differentiation (Allen and Boxhorn, 1987). The extracellular matrix is defined as a dynamic network of polysaccharides and proteins that are immobilized outside
2006 Poultry Science Association, Inc. Received August 11, 2005. Accepted October 13, 2005. 1 Salary and research support to S.G.V. were provided by state and federal funds appropriated to the Ohio Agricultural Research and Development Center, The Ohio State University, and the Midwest Poultry Consortium to S.G.V. and D.C.M. 2 Corresponding author: [email protected]
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ABSTRACT Myoblast-extracellular matrix interactions play a pivotal role in skeletal muscle development. Transforming growth factor-β (TGF-β) is a key regulator of muscle cell proliferation and differentiation. The level of TGF-β expressed will affect the concentration of the extracellular matrix proteoglycan decorin and the cell surface β1 integrin subunit. The decorin proteoglycan is a regulator of cell growth as well as the organization of the extracellular matrix. The β1 integrin plays a role in muscle cell attachment, migration, and the formation of multinucleated myotubes. In the current study, chicken myogenic satellite cells isolated from the pectoralis major muscle from the chicken genetic muscle weakness, low score normal (LSN), and normal pectoralis major muscle were used to investigate TGF-β expression as it relates to decorin and β1 integrin mRNA expression. The LSN muscle defect is characterized by altered myotube formation and sarcomere structure, and the satellite cells have reduced proliferation and differentiation. The mRNA expression was measured by real-time quantitative reverse transcription
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Sequence1
Decorin
5′-GAGGGTAGTGAAGGCTGCTG-3′ (forward) 5′-TTGGCACTCTTTCCAGACCT-3′ (reverse) 5′-AGGAATGTGCAGGATAATT-3′ (forward) 5′-ATTTTGGGTGTTTTGCCAA-3′ (reverse) 5′-AGGATCTGCAGTGGAAGTGG-3′ (forward) 5′-CGGCCCACGTAGTAAATGAT-3′ (reverse) 5′-AATTTGTAGCAGGCGTGGTTG-3′ (forward) 5′-CACGGCGCTCTTCTAAATAGG-3′ (reverse) 5′-GAGGGTAGTGAAGGCTGCTG-3′ (forward) 5′-CCACAACACGGTTGCTGTAT-3′ (reverse)
TGF-β2 TGF-β4 β1 integrin GAPDH
Product size (bp) 256 269 300 169 200
1 Primer sequences were designed from the following GenBank accession numbers: decorin: X63797; transforming growth factor (TGF)-β2: X59080; TGF-β4: M31160; β1 integrin: U37029; and glyceralderaldehyde-3phosphate dehydrogenase (GAPDH): U94327.
Figure 1. Gel electrophoresis of real-time PCR products. Lane 1: DNA ladder; lane 2: glyceraldehyde-3-phosphate dehydrogenase; lane 3: decorin; lane 4: β1 integrin; lane 5: transforming growth factor-β4; and lane 6: transforming growth factor-β2.
The expression of the β1 integrin is also affected by TGFβ1 (Ignotz and Massague´, 1987; Heino et al., 1989). It is known that TGF-β is a potent inhibitor of both muscle cell proliferation and differentiation (Allen and Boxhorn, 1987). Transforming growth factor-β may, in part, regulate cell proliferation and differentiation through its effect on decorin and β1 integrin expression. However, to date, the relationship of TGF-β with the extracellular matrix and cell adhesion receptors is not well understood. In chickens, 3 isoforms exist, TGF-β2, β3, and β4, where TGF-β4 is the chicken analog of the mammalian TGF-β1 (Groenen et al., 2000; Schmid et al., 2000; Smith et al., 2000). The chicken genetic muscle weakness, low score normal (LSN) is an abnormal muscle weakness condition that was originally detected in 1997 at the University of Connecticut in F2 progeny in an outcross of chickens with hereditary muscular dystrophy (L. J. Pierro and J. S. Haines, Department of Animal Genetics, University of Connecticut, Storrs, personal communication). The LSN birds are distinguished from normal birds by an impaired ability to right themselves when repeatedly placed on their backs (exhaustion score test). Low score normal birds have an exhaustion score intermediate between that of normal birds and muscular dystrophy birds. Velleman and Nestor (2001) concluded that the LSN trait is influenced primarily by an autosomal dominant gene but is also influenced by other genes, some of which are on the sex chromosome. In addition to decreased righting ability, LSN birds have reduced pectoral muscle mass in posthatch birds as early as 1 wk of age (Velleman et al., 1996). The LSN pectoralis major muscle exhibits altered formation of myotubes in vitro (Velleman and McFarland, 1999) and modified sarcomere structure in vivo (Velleman et al., 1997). Velleman and Coy (1998) showed that TGFβ2 expression is up-regulated in LSN pectoral muscle late in embryonic development through 1 wk posthatch, and TGF-β1 expression is up-regulated at 1 d posthatch. The late embryonic increase in TGF-β2 expression coincides with an increase in decorin protein concentration (Velleman et al., 1996) and mRNA expression (Velleman and Coy, 1997). Furthermore, β1 integrin protein concentra-
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of TGF-β also affects the expression of decorin in many cell types (Bassols and Massague´, 1988). One of the means by which the extracellular matrix will transduce signals to the cells is through the integrin receptors. Integrins are heterodimeric transmembrane cell adhesion receptors containing α and β subunits that span the plasma membrane providing a transmembrane linkage between the extracellular matrix and the cytoskeleton (Hynes, 1992). The integrins, therefore, provide a means for the bidirectional transmission of signal information between the extracellular matrix and cellular cytoskeletal network. The β1 integrin subunit is ubiquitous and plays a pivotal role in cell attachment to the extracellular matrix, cell migration, proliferation, and differentiation (reviewed in Berman et al., 2003). The β1 integrin has further been shown to be involved in regulating muscle contraction through the interaction of the β1 integrin cytoplasmic domain with the cytoskeleton (Belkin et al., 1997).
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MATERIALS AND METHODS
nant human TGF-β1 was administered to normal satellite cell cultures with fresh medium added at 24-h intervals until 96 h. The concentration of recombinant human TGFβ1 was previously determined in a study by Velleman and McFarland (1999). Control cultures were grown without treatment. The cell cultures were collected every 24 h and stored at −70°C until use.
Total RNA Extraction and cDNA Synthesis Total RNA was extracted from the cell cultures by using TRIzol (Invitrogen, Carlsbad, CA) according to the manufacturer’s protocol. Reverse transcription of total RNA to a cDNA was conducted using Moloney murine leukemia virus reverse transcription (M-MLV, Promega, Madison, WI). In brief, an RNA-primer mix [1 g of total RNA, 1 L of oligo (dT), and nuclease-free water up to 13.5 L] was incubated at 80°C for 3 min, followed immediately by incubation on ice. The reaction mix [5 L of 5× FirstStrand buffer (MJ Research, Reno, NV), 1.25 L of 10 mM deoxynucleoside triphosphate mix (MJ Research), 0.5 L of RNasin (40 U/L, MJ Research), 1 L of Moloney murine leukemia virus (200 U/L), and nuclease-free water up to 11.5 L] was added to the mixture. The complete reaction mixture was incubated at 55°C for 60 min, and then heated at 90°C for 10 min for inactivation.
Satellite Cell Culture
Real-Time Quantitative PCR
Satellite cells from both normal and LSN pectoralis major muscle were isolated as described by Li et al. (1997). Satellite cells from the 2 lines were cultured in McCoy’s 5A medium (Life Science Technologies, Inc., Grand Island, NY) with 10% chicken serum (Sigma Chemical Co., St. Louis, MO), 5% horse serum, and 20 ng/mL of fibroblast growth factor 2 (Pepro Tech Inc., Rocky Hill, NY; Li et al., 1997). The cells were plated in gelatin-coated, 35-mm cell culture wells (Corning Costar, Corning, NY) at a density of 36,000 cells per well. The normal and LSN satellite cells were cultured at a cell density permitting the cells to reach 65% confluency at the same time minimize culturing differences. When the cells reached approximately 65% confluency, differentiation was induced by culturing in low-serum media containing Dulbecco’s Modified Eagle Medium (Life Science Technologies), 3% horse serum, 0.01 mg/mL of porcine gelatin (Sigma Chemical Co.), and 1.0 mg/mL of bovine serum albumin (Sigma Chemical Co.). Cell cultures were removed from the incubator at 72 h of proliferation and at 0, 24, 48, 72, and 96 h of differentiation. The culture medium was removed from the plates, and the cells were rinsed with sterile PBS (170 mM NaCl, 3 mM KCl, 10 mM Na2HPO4, and 2 mM KH2PO4, pH 7.08) and 2 mM KH2PO4. All plates were then stored at −70°C until use. To address the effect of TGF-β on decorin and β1 integrin expression, the addition of exogenous TGF-β1 was performed according to the method described by Velleman and McFarland (1999). In brief, at the initiation of differentiation, medium containing 1 ng/mL of recombi-
Real-time quantitative PCR was performed using the DyNAmo Hot Start SYBR Green qPCR kit (MJ Research). The PCR reaction consisted of 2 L of the reverse transcription reaction mixture diluted with 25 L of nucleasefree water, 10 L of 2× master mix (MJ Research) provided by the manufacturer, 250 nM of each of the forward and reverse primers, and nuclease-free water up to 20 L. Reaction components were assembled in low-profile multiplates (MJ Research) and sealed with Microseal B adhesive seals (MJ Research). Primers used in the amplification of decorin, TGF-β2, TGF-β4, β1 integrin, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were designed from published sequences as listed in Table 1. The specificity of the primers was confirmed by DNA sequence analysis of the amplified PCR product. The realtime PCR amplification was done in a DNA Engine Opticon 2 real-time system (MJ Research). For all the amplified genes, the cycling program was initiated with a hotstart step at 95°C for 15 min. The cycling program for decorin and GAPDH consisted of denaturation at 94°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C for 30 s for 34 cycles, with a final elongation of 72°C for 5 min. The cycling conditions for TGF-β2 and TGF-β4 comprised denaturation at 94°C for 30 s, annealing at 54°C for 45 s, and extension at 72°C for 45 s for 34 cycles, with a final elongation for 5 min at 72°C. The PCR cycling conditions for β1 integrin were denaturation at 94°C for 1 min, annealing at 58°C for 1 min, and extension at 72°C for 3 min for 34 cycles with a final elongation at 72°C for 10 min. The final PCR products were analyzed on a 1.5%
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tion is reduced in the LSN pectoral muscle at 16 and 18 d of embryonic development and 1 wk posthatch compared with normal pectoral muscle (Velleman et al., 2000). Due to these changes in muscle structure, extracellular matrix molecules, and cell surface receptors, the LSN condition is a useful model to further evaluate how changes in TGFβ regulation affect muscle cell decorin and β1 integrin expression during the proliferation and differentiation of muscle. It was, therefore, the objective of the present study to further investigate the relationship between TGF-β, decorin, and β1 integrin expression during myogenesis. To address the relationship between TGF-β, decorin, and β1 integrin, their expression was measured in normal and LSN satellite cells cultured with exogenous TGF-β. The expression of decorin and β1 integrin was examined. Satellite cells are quiescent myogenic cells residing between the basement membrane and plasma membrane of muscle fibers (Mauro, 1961), and are largely responsible for postnatal muscle growth and muscle regeneration (Moss and LeBlond, 1971). The satellite cell cultures allow the in vitro monitoring of satellite cell proliferation through the formation and differentiation of multinucleated myotubes.
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agarose gel to check amplification specificity. Standard curves were constructed for decorin, TGF-β2, TGF-β4, β1 integrin, and GAPDH with serial dilutions of the 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 sample concentrations 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 decorin, TGF-β2, TGF-β4, and β1 integrin was normalized to GAPDH expression.
Statistical Analyses Differences between the means of LSN and normal satellite cells at each sampling interval for the relative mRNA expression of the target genes were evaluated with a Student’s t-test. Differences were considered sig-
nificant at P < 0.05. Each experiment was repeated at least 3 times with graphical data showing a representative experiment.
RESULTS Decorin, TGF-β2, TGF-β4, and β1 Integrin mRNA Expression in Chicken Normal and LSN Satellite Cells Real-time PCR was used to measure the relative expression of decorin, TGF-β2, TGF-β4, and β1 integrin mRNA expression during normal and LSN satellite cell proliferation and differentiation. The specificity of the real-time PCR assay was confirmed by melting curve analysis (data not shown) and gel electrophoresis (Figure 1). Gel electrophoresis showed single bands at the appropriate size for decorin, TGF-β2, TGF-β4, β1 integrin, and GAPDH.
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Figure 2. Real-time PCR analysis of decorin (panel A), transforming growth factor-β2 (TGF-β, panel B), transforming growth factor-β4 (TGFβ4, panel C), and β1 integrin (panel D) in normal chicken and low score normal (LSN) chicken satellite cells during proliferation (P) and differentiation (D). The y-axis denotes arbitrary units of expression, which are defined as the ratio of the relative cDNA concentration of the target genes to the cDNA concentration of glyceraldehyde 3-phosphate dehydrogenase. * Indicates a significant different (P < 0.05) between the lines at a sampling interval. Error bars represent the standard error of the mean.
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differentiation than in the normal cells, which expressed more TGF-β4 compared with the LSN cells at 72 and 96 h of differentiation. Expression of β1 integrin was significantly higher at all times measured except 72 h of proliferation in the normal satellite cell cultures compared with the LSN satellite cells (Figure 2D).
The Effect of TGF-β1 on Decorin and β1 Integrin mRNA Expression
DISCUSSION
Figure 3. The effect of transforming growth factor-β1 (TGF-β1) on decorin (panel A) and β1 integrin (panel B) mRNA expression during myogenic satellite cell proliferation (P) and differentiation (D). The yaxis denotes arbitrary units of expression, which are defined as the ratio of the relative cDNA concentration of the target genes to the cDNA concentration of glyceraldehyde 3-phosphate dehydrogenase. *Indicates a significant difference (P < 0.05) between the control and TGF-β1 treatment at a sampling interval. Error bars represent the standard error of the mean.
The expression of decorin was significantly elevated in the LSN satellite cells cultures during both proliferation and differentiation. Decorin expression peaked in both the normal and LSN cultures at 0 and 72 h of differentiation (Figure 2A). Expression of TGF-β2 was elevated in the LSN satellite cell cultures after 72 h of proliferation, and at 48 and 72 h of differentiation (Figure 2B). Expression of TGF-β2 generally decreased with differentiation for both the normal and LSN satellite cells. Expression of TGF-β4 was similar to that for TGF-β2 in that levels were higher for both the normal and LSN cells during proliferation and decreased with differentiation (Figure 2C). Expression of TGF-β4 was significantly higher in the LSN cells from 72 h of proliferation through 24 h of
Satellite cells are myogenic precursors that are required for the process of postnatal muscle growth through hypertrophy. During the period of postnatal muscle growth, satellite cells proliferate, differentiate, and fuse with adjacent muscle fibers or with other satellite cells, and then add additional nuclei to the muscle fibers (Allen et al., 1979). This ultimately increases protein synthesis potential due to the increased number of nuclei, and thus leads to muscle growth through muscle fiber hypertrophy. Certain growth factors are involved in the regulation of muscle hypertrophy by either stimulating or inhibiting cell proliferation and differentiation (Charge´ and Rudnicki, 2004). Many studies have focused on the mechanism of growth factor regulation of satellite cell activity (Vaidya et al., 1989; Brennan et al., 1991). Only recently has the role of the extracellular matrix in the muscle growth process received research attention, in part due to its role in the regulation of growth factor responsiveness (reviewed in Velleman, 1999). Decorin, an extracellular matrix proteoglycan, interacts with TGF-β and regulates muscle cellular responsiveness to TGF-β (Riquelme et al., 2001). During differentiation, muscle cell migration is essential for the alignment of myoblasts and adhesion to form differentiated multinucleated myotubes, which requires the proper cellular adhesion to the extracellular matrix through integrins (Meredith and Schwartz, 1997; Velleman and McFarland, 2004). Myoblasts cultured in the presence of the β1 integrin cell substrate attachment antibody do not form multinucleated myotubes but continue to replicate (Menko and Boettiger, 1987). Low score normal myogenic satellite cells form shorter myotubes with a reduced number of myonuclei per myotube in vitro (Velleman and McFarland, 1999). These changes in LSN myotube morphology in vitro are associated with reduced proliferation and differentiation rates
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Decorin and β1 integrin mRNA expression were measured in normal satellite cell cultures treated with exogenous TGF-β1 during the 96 h of differentiation. The addition of exogenous TGF-β1 decreased decorin expression during differentiation compared with the untreated control satellite cell cultures (Figure 3A). β1 Integrin expression was reduced in the treated cultures at 24 and 48 h of differentiation, and was significantly increased in the TGF-β1 treated cultures by 96 h of differentiation (Figure 3B).
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(Li et al., 1997) as well as reduced concentrations of β1 integrin protein during differentiation (Velleman et al., 2000). In the present study, chicken TGF-β2 and TGF-β4 were expressed at higher levels in the LSN satellite cells than in the normal satellite cells during proliferation. Interestingly, expression of TGF-β2 and TGF-β4 were altered at different times. Transforming growth factor-β2 was mainly affected during the differentiation process, whereas TGF-β4 was increased in the LSN cultures during proliferation and early differentiation. These data suggest that TGF-β2 and TGF-β4 are affecting different aspects of LSN muscle development. In addition, decorin expression was increased and β1 integrin levels were reduced in LSN cultures. The addition of exogenous TGF-β to normal cultures decreased decorin expression compared with the increased expression observed in the LSN satellite cell cultures. β1 Integrin expression was decreased at 24 and 48 h of differentiation and increased by 96 h of differentiation in the normal cell cultures with the addition of exogenous TGF-β, whereas decreased β1 integrin expression was detected throughout proliferation and differentiation in the LSN cell cultures. These data suggest that the mechanism leading to the changes in LSN decorin and β1 integrin expression may result from other factors as well as changes in TGF-β expression. However, the findings from the current study do indicate that expression of decorin and β1 integrin are associated with TGF-β during chicken satellite cell proliferation and differentiation. Future research will need to address the mechanism by which TGFβ regulates decorin and β1 integrin expression as it relates to muscle development and growth.
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Velleman, S. G., and K. E. Nestor. 2001. Mode of inheritance of the low score normal condition in chickens. Poult. Sci. 80:1273–1277. Velleman, S. G., J. D. Yeager, H. Krider, D. A. Carrino, S. D. Zimmerman, and R. J. McCormick. 1996. The avian low score normal muscle weakness alters decorin expression and collagen crosslinking. Connect. Tissue Res. 34:33–39. Yamaguchi, Y., D. M. Mann, and E. Ruoslahti. 1990. Negative regulation of transforming growth factor-beta by the proteoglycan decorin. Nature 346:281–284. Yanagishita, M. 1993. Function of proteoglycans in the extracellular matrix. Acta Pathol. Jpn. 43:283–293.
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