J. Anim Sci. 2007. 85:143-150. doi:10.2527/jas.2006-351
© 2007 American Society of Animal Science
ANIMAL GROWTH, PHYSIOLOGY, AND REPRODUCTION |
Growth factor messenger ribonucleic acid expression during differentiation of porcine embryonic myogenic cells1
G. Xi,
M. R. Hathaway,
W. R. Dayton and
M. E. White2
Animal Growth and Development Laboratory, Department of Animal Science, University of Minnesota, 350 ABLMS, 1354 Eckles Avenue, St. Paul 55108
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Abstract
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The growth factors, IGF-I and II, their binding proteins, IGFBP, and members of the transforming growth factor (TGF) superfamily (myostatin and TGFß1) are known to regulate proliferation and differentiation of myogenic cells. We hypothesized that changes in the relative expression of members of the IGF and TGFß systems play a significant role in regulating myogenesis in porcine embryonic myogenic cell (PEMC) cultures. Therefore, determining the expression patterns of these factors during PEMC myogenesis is important. Consequently, we used real-time PCR to explore the pattern of IGF-I; IGF-II; IGFBP-2, -3, and –5; IGF-type-I receptor; myogenin; myostatin; and TGFß1 mRNA expression during PEMC myogenesis. The progression of differentiation was assessed using creatine kinase activity and myogenin mRNA expression. As anticipated, creatine kinase activity was low in PEMC cultures at 48 h and increased 20-fold (P < 0.0001) between 48 h and its peak at 144 h. Similarly, myogenin mRNA was low at 48 h and increased approximately 5-fold (P < 0.0001) as differentiation progressed, peaking at 120 h and decreasing at 144 h. The patterns of IGF-I and IGFBP-2 mRNA expression were similar and were relatively lower in 48-h PEMC cultures, increasing approximately 5-fold (P < 0.0001) to their greatest levels at 120 h. In contrast, IGF-II and IGFBP-5 mRNA levels were relatively high at 48 h, peaking at 72 h, and steadily decreasing by 60 and 80%, respectively (P < 0.001), at 144 h. The level of IGF-type-I receptor mRNA was relatively high until 96 h of culture, after which it decreased 40% (P < 0.01), reaching a low at 144 h. Levels of IGFBP-3 mRNA were relatively high at 48 h, dropped approximately 40% to their lowest level at 72 h (P < 0.001), and then increased approximately 60% (P < 0.001) to their greatest levels at 144 h. Levels of TGFß1 mRNA decreased approximately 30% (P < 0.0001) between 48 and 96 h, then quickly rebounded to a peak at 120 h, and by 144 h had dropped to the levels seen at 72 h. Myostatin mRNA was at its greatest level at 48 h and declined rapidly between 72 and 96 h, finally decreasing by approximately 80% at 144 h (P < 0.0001). Our data demonstrate that these factors are differentially regulated during PEMC myogenesis and provide new information about their pattern of mRNA expression in cultured porcine muscle cells.
Key Words: insulin-like growth factor system • myogenesis • myostatin • porcine myoblast • transforming growth factor ß1
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INTRODUCTION
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The IGF system and transforming growth factor (TGF) ß superfamily members, TGFß1 and myostatin, are known to regulate skeletal muscle growth and development. Insulin-like growth factors stimulate proliferation and differentiation of cultured muscle cells, and IGF is important for skeletal muscle growth in vivo (Sarbassov et al., 1995
; Florini et al., 1996; Tureckova et al., 2001). Myostatin is a skeletal muscle-specific, TGFß superfamily member that suppresses myogenic cell proliferation and differentiation and impairs satellite cell activation and self-renewal (Thomas et al., 2000; Langley et al., 2002; McCroskery et al., 2003) Furthermore, we have previously reported that TGFß1 and myostatin inhibit porcine embryonic myogenic cell (PEMC) proliferation, and this inhibition is partially mediated by IGFBP-3 and -5 (Kamanga-Sollo et al., 2003; Kamanga-Sollo et al., 2005).
It has been postulated that primary myoblast cultures isolated directly from muscle tissue recapitulate muscle development in vivo more precisely than immortal myogenic cell lines (Blanco-Bose et al., 2001). We hypothesized that myogenesis in PEMC is controlled in part by a coordinated regulation of components of the IGF and TGFß systems. However, the endogenous expression patterns of IGF system components and TGFß1 or myostatin during myogenic differentiation of PEMC is not known. Therefore, to understand their relative roles in porcine muscle development and differentiation, it is important to explore the expression patterns of these factors during PEMC myogenesis.
In this study, we used real-time PCR (RT-PCR) to explore the patterns of IGF-I; IGF-II; IGFBP-2, -3, and -5; IGF-type-I receptor; myogenin; myostatin; and TGFß1 mRNA expression during myogenesis in PEMC cultures.
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MATERIALS AND METHODS
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Porcine Embryonic Myogenic Cell Cultures
All procedures involving animals were approved by the local institutional animal care and use committee.
Chicken embryo extract (CEE) was prepared in our laboratory according to procedures described previously (Hembree et al., 1991), and swine serum (SS) was collected in our laboratory from 6- to 7-wk-old pigs from the university swine herd. Porcine embryonic myoblasts were isolated from 53- to 54-d-old porcine fetuses and stored in liquid nitrogen, as described previously (Pampusch et al., 1990; Hembree et al., 1991, 1996). The frozen cells were quickly thawed at 37°C and diluted with Dulbecco’s Modified Eagle Medium (DMEM; Gibco, Invitrogen, Grand Island, NY) containing 7% (vol/vol) SS and 3% (vol/vol) CEE (Hembree et al., 1991). The cells were plated on culture dishes coated with reduced growth factor matrigel (BD Biosciences, Chicago, IL) diluted 1:60 (vol/vol) in DMEM and incubated at 37°C in a 5% CO2, 95% air, and water-saturated environment.
After a 24-h attachment period, cells were fed with fresh growth medium (DMEM containing 7% SS and 3% CEE) and then incubated for an additional 48 h. The culture medium was switched to differentiation medium (DMEM containing 3% SS and 5% CEE) at 72 h. At 24 h later, cytosine arabinoside (Sigma, St. Louis, MO) was added to the medium to a final concentration of 1 x 10–5 M to inhibit DNA synthesis of proliferating, nonmyogenic cells and thereby promoting PEMC differentiation. After another 48 h in culture with no media changes, maximal fusion was reached.
Giemsa Cell Staining
At each time point indicated, the medium was removed, and the cultures were washed 3 times with PBS followed by incubation with ice-cold 85% methanol for 8 min at 4°C. After allowing the plates to air dry, the cultures were incubated for 20 min at room temperature with buffered formalin solution (3.7% formalin, pH adjusted to 7.0 with 1 N KOH). The buffered formalin solution was aspirated away, and the plates were allowed to air dry. Modified Giemsa-stain solution (Sigma, St. Louis, MO) was added to each plate and left in contact with the cells for 4 min at room temperature. The plates were rinsed twice with water and then examined under an inverted microscope (Nikon Microscope, Fryer Company Inc., Edina, MN), and pictures were taken of 4 random fields on each plate (Figure 1).
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Figure 1. Giemsa staining of porcine embryonic myogenic cells (PEMC) on different days in culture. Cells were plated in 9.6-cm2 culture dishes in growth medium (GM). After a 24-h attachment, fresh GM was applied to each culture for another 48 h. At 72 h after plating, cells were rinsed, and differentiation medium was added to each culture. Twenty-four hours later, 1 x 10–5 M cytosine arabinoside was added to each culture. At the time points indicated, PEMC cultures were stained and photographed. Magnification for each image is 300x.
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Creatine Kinase Activity
The PEMC were cultured as described above. At the time points indicated, medium was removed, cells were rinsed with PBS, and 0.5 mL of lysis buffer (50 mM Tris-MES, 1% Triton X-100, pH 7.8) was added to release creatine kinase (CK) from the cells. The CK activity was then determined utilizing a commercially available kit (Pointe Scientific, Lincoln Park, MI) according to the protocol recommended by the manufacturer. The CK values were the average of 3 separate experiments, each of which contained triplicate cultures for each time point.
Preparation of Total RNA and RT-PCR
Total RNA was isolated at the time points indicated by using an Absolutely RNA Microprep Kit (Stratagene, La Jolla, CA). After 2 phenol chloroform extractions of the cell lysate, RNA was isolated following the protocol recommended by the manufacturer. Samples were treated with DNase while bound to the fiber matrix during the isolation.
Quantitative RT-PCR was used to measure the quantity of each specific mRNA relative to the quantity of porcine glyceraldehyde-3-phosphate dehydrogenase mRNA in the total RNA isolated from the cells. Total RNA (1 µL) was reverse transcribed to cDNA with Taq-Man reverse transcription Reagents (Applied Biosystems, Foster City, CA) using random hexamer primers. Measurements were carried out using 1 µL of the cDNA mixture, SYBR Green PCR Master Mix (Applied Biosystems), and 300 nM of the appropriate forward and reverse primers. Because primers arrive from the manufacturer at variable concentrations, a set volume is not added. The primers are added to a final concentration of 300 nM, and that is the only correct unit of measure here. Volume is variable, but concentration is not. The primer pairs used for the specific amplification of porcine IGF-I; IGF-II; IGF-type-I receptor; IGFBP-2, -3, and -5; myogenin; TGFß1; myostatin; and glyceraldehyde-3-phosphate dehydrogenase are shown in Table 1.
Assays were performed in the GeneAmp 5700 Sequence Detection System (Applied Biosystems) using thermal cycling parameters recommended by the manufacturer (40 cycles of 15 s at 95°C and 1 min at 60°C). Titration of primer pairs for each growth factor (300 nM forward and reverse primers) against increasing amount of cDNA gave a linear response.
Statistical Analysis
All data were analyzed using the MIXED procedure (SAS Inst. Inc., Cary, NC). In each case, data from at least 2 independent assays, each containing triplicate cell cultures, were statistically pooled and analyzed. When significant interactions were detected (P < 0.05), least squares means were separated using LSD tests.
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RESULTS
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Characterization of Porcine Embryonic Myogenic Cell Differentiation
Giemsa staining was used to observe the morphological changes of PEMC cultures during differentiation (Figure 1
). At 48 h, cells were small and mononucleated. At 72 h, differentiation medium was applied. By 96 h, cell density was increased and small, multinucleated myotubes could be detected. Myotubes increased in size and number between 96 and 144 h, with cultures routinely reaching approximately 70% fusion.
The CK activity and myogenin mRNA levels were determined in order to monitor differentiation. The CK activity was low in proliferating PEMC and increased 20-fold (P < 0.0001) between 48 h and its peak at 144 h. Similarly, low levels of myogenin mRNA were detected in proliferating PEMC (48 h) and increased approximately 5-fold (P < 0.0001) as differentiation progressed, peaking at 120 h and dropping at 144 h (Figure 2).
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Figure 2. Relative creatine kinase (CK) activity and steady-state myogenin mRNA level at 48, 72, 96, 120, and 144 h of porcine embryonic myogenic cell (PEMC) differentiation in vitro. Total RNA was isolated, and porcine myogenin mRNA level relative to glyceraldehyde-3-phosphate dehydrogenase mRNA was determined using real time-PCR. The CK activity is representative of 2 independent assays, each containing triplicate cell cultures. To compare relative levels, all values are presented as a percentage of the maximal value observed for each data set. For myogenin, all data points in individual assays were the average obtained from triplicate cultures. Data from 3 separate assays were statistically pooled and analyzed to yield the values shown in the graph and to test for significance. a–eWithin an individual data set (e.g., myogenin), points with different superscript letters were significantly different (P < 0.05). Pooled SE was 0.89 for CK and 5.7 for myogenin.
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IGF System mRNA Expression in Differentiating PEMC Cultures
Components of the IGF system play a significant role in the proliferation and differentiation of skeletal muscle cells. Therefore we measured the mRNA expression patterns of various components of the IGF system in PEMC throughout differentiation. The IGF-I mRNA levels were relatively low at 48 h and increased approximately 5-fold (P < 0.0001) to their greatest levels at 120 h (Figure 3). In contrast, IGF-II mRNA levels began relatively high and increased approximately 40% (P < 0.05) to a peak at 72 h and steadily decreased 65% (P < 0.001) by 144 h (Figure 3). Relative IGF-type-I receptor (IGF-1R) mRNA levels remained consistently high until 96 h in culture after which they decreased 40% (P < 0.01) by 144 h (Figure 3). The IGFBP-2 mRNA levels paralleled those of IGF-I mRNA, progressively increasing approximately 5-fold (P < 0.0001) between 48 and 120 h (Figure 4). Conversely, IGFBP-5 mRNA levels paralleled those of IGF-II beginning relatively high and progressively decreasing by 80% (P < 0.0001) as differentiation progressed reaching its lowest level at 144 h (Figure 4). The IGFBP-3 mRNA levels were relatively high at 48 h, dropped approximately 40% to their lowest level at 72 h (P < 0.001), and then increased approximately 60% (P < 0.001), reaching their greatest level at 144 h (Figure 4). We have previously reported IGFBP-3 mRNA expression patterns in PEMC (Johnson et al., 1999, 2003); however, the previous studies were performed using different culture conditions and were assessed using different mRNA detection methods and therefore would not be comparable with the other factors measured in this study. In combination, data presented here clearly demonstrate that components of the IGF/IGFBP system are differentially regulated during myogenic differentiation in primary porcine embryonic skeletal muscle myoblasts.
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Figure 3. Relative porcine IGF-I, IGF-II, and IGF-type-1 receptor (IGF-1R) mRNA levels at 48, 72, 96, 120, and 144 h of porcine embryonic myogenic cell (PEMC) differentiation in vitro. The mRNA levels relative to glyceraldehyde-3-phosphate dehydrogenase mRNA were measured using real time-PCR. To compare relative levels, all values are presented as a percentage of the maximal value observed for each data set. Data are representative of 2 independent assays, each containing triplicate cell cultures, and were statistically pooled and analyzed to yield the values shown in the graph and to test for signifi-cance. a–cWithin an individual data set (e.g., IGF-I), points with different superscript letters were significantly different (P < 0.05). Pooled SE for IGF-I, IGF-II, and IGF-1R were 6.15, 6.28, and 6.54, respectively.
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Figure 4. Relative porcine IGFBP-2, IGFBP-3, and IGFBP-5 mRNA levels at 48, 72, 96, 120, and 144 h of porcine embryonic myogenic cell (PEMC) differentiation in vitro. The mRNA levels relative to glyceraldehyde-3-phosphate dehydrogenase mRNA were measured using real time-PCR. To compare relative levels, all values are presented as a percentage of the maximal value observed for each data set. Data are representative of 2 independent assays, each containing triplicate cell cultures, and were statistically pooled and analyzed to yield the values shown in the graph and to test for significance. a–cWithin an individual data set (e.g., IGFBP-2), points with different superscript letters were significantly different (P < 0.05). Pooled SE for IGFBP-2, -3, and -5 were 2.6, 8.0, and 4.6, respectively.
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Myostatin and TGFß1 mRNA Expression in Differentiating PEMC Cultures
The TGFß1 and myostatin are members of the TGFß superfamily and have been shown to negatively modulate proliferation and differentiation of skeletal muscle cells and antagonize many of the stimulatory effects of IGF (Thomas et al., 2000; Langley et al., 2002; McCroskery et al., 2003). Therefore, we investigated the mRNA expression pattern of TGFß1 and myostatin during PEMC differentiation in order to determine whether the expression of these factors is differentially regulated and to compare these patterns with components of the IGF system. The levels of TGFß1 mRNA decreased 30% (P < 0.0001) between 48 and 96 h and then quickly rebounded to a peak level at 120 h, returning to 72 h levels at 144 h (Figure 5). In contrast, myostatin mRNA level was at its greatest level at 48 h and rapidly declined between 72 and 96 h, decreasing approximately 80% by 144 h (P < 0.0001).
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Figure 5. Relative porcine transforming growth factor ß1 (TGFß1) and myostatin mRNA levels at 48, 72, 96, 120, and 144 h of porcine embryonic myogenic cell (PEMC) differentiation in vitro. The mRNA levels relative to glyceraldehyde-3-phosphate dehydrogenase mRNA were measured using real time-PCR. To compare relative levels, all values are presented as a percentage of the maximal value observed for each data set. Data are representative of 2 independent assays, each containing triplicate cell cultures, and were statistically pooled and analyzed to yield the values shown in the graph and to test for significance. a–dWithin an individual data set (e.g., TGFß1), points with different superscript letters were significantly different (P < 0.05). Pooled SE for TGFß1 and myostatin were 4.3 and 6.5, respectively.
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DISCUSSION
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Because of the broad differences reported in the expression patterns of IGF and TGFß1 system components during myogenic differentiation, it is not possible to extrapolate the results observed in other studies to PEMC cultures. Therefore, because the IGF system and the TGF profoundly affect myogenesis, it is necessary to investigate the endogenous expression patterns of IGF system components as well as myostatin and TGFß1 in PEMC cultures. It has been reported that some myogenic cell systems such as the Sol8, C2 cell lines (Florini et al., 1991), and primary mouse skeletal myoblasts (Galvin et al., 2003) are insensitive to exogenous IGF-I, whereas others such as L6 cells (Florini et al., 1991), primary rat myoblasts (Allen and Boxhorn, 1989; Galvin et al., 2003), and chicken myoblasts (Schmid et al., 1983) are sensitive to exogenous IGF-I. It has been hypothesized that this sensitivity or lack thereof is dependent on the level of endogenous IGF production in these different cell types. Most muscle lines and primary muscle cell cultures have been reported to produce IGF-I, IGF-II, or both. However, the expression patterns depend on the origin of the cells and state of development (Tollefsen et al., 1989b; Rosenthal et al., 1991; Florini et al., 1991). In the current study, we detected IGF-I and IGF-II mRNA expression using RT-PCR in PEMC cultures. The level of IGF-I mRNA was relatively low in 48 h cultures and increased during differentiation, whereas IGF-II mRNA level was relatively high in 48 and 72 h cultures and decreased with the progression of differentiation. These expression patterns are generally consistent with in vivo studies using pig fetuses (Gerrard et al., 1998). Because the biological functions of IGF-I and -II are primarily mediated by the IGF-1R, we investigated the expression pattern of IGF-1R during PEMC differentiation. The mRNA levels of IGF-1R in PEMC cultures dropped by 40% between 96 and 144 h. This is similar to reports for L6 cells (Magri et al., 1994), BC3H-1 cells (Rosenthal and Brown, 1994), and turkey satellite cells (Minshall et al., 1990; Ernst et al., 1996). However, this is in direct contrast to reports for C2 cells (Tollefsen et al., 1989a), trout satellite cells (Castillo et al., 2002), and human satellite cells (Crown et al., 2000) that showed an upregulation of the IGF-1R during differentiation. Therefore, it appears that the pattern of the IGF-1R varies with myogenic cell type, species of origin, and stage of development. Although no cause and effect determination can be made, the reduction in IGF-1R mRNA expression in PEMC occurs over the same time frame as when IGF-ImRNA reaches its greatest level. This is similar to studies using BC3H1 cells where pre-incubation with high levels of exogenous or endogenous IGF-I, IGF-II, or both caused a downregulation of IGF-1R expression (De Vroede et al., 1984; Rosenthal et al., 1991; Rosenthal and Brown, 1994).
The biological actions of IGF are regulated by 6 IGFBP termed IGFBP-1–6. The expression of these IGFBP varies by muscle cell model (McCusker et al., 1989; Ernst et al., 1992; Ewton and Florini, 1995). We have previously shown that the myogenic cells in PEMC cultures produce IGFBP-3 and -5 and the nonmyogenic cells in these cultures produce IGFBP-2 and -4 (Hembree et al., 1996; Yang et al., 1999; Johnson et al., 1999; Johnson et al., 2003). We have previously shown that IGFBP-3 mRNA and protein in PEMC were relatively high at 48 h, dropped significantly, and increased again at 144 h (Johnson et al., 1999; Johnson et al., 2003). This is similar to our observations of IGFBP-3 mRNA expression in PEMC in the current study. Because PEMC were cultured using different sera and mRNA expression was measured using a different method than in previous studies, in the current study it was important to measure and report the IGFBP-3 mRNA pattern here for purposes of comparison with those of the other factors measured in this study. The expression pattern of IGFBP-3 appeared to be the reverse of that for IGF-II mRNA such that when IGFBP-3 decreased, IGF-II increased. Furthermore, IGFBP-2 mRNA expression exhibited a pattern similar to that of IGF-I and was relatively low at 48 h and increased to the greatest level at 120 h. This is in contrast with the downregulation of IGFBP-2 mRNA in differentiating C2C12 cells (Ernst and White, 1996). Interestingly, our current study shows that in PEMC cultures, IGFBP-5 mRNA expression is opposite of that for IGF-I. The level of IGFBP-5 mRNA was relatively high at 48 and 72 h, followed by a rapid decrease, whereas IGF-I mRNA was lowest at 48 and 72 h, followed by a rapid increase. The expression of IGFBP-5 mRNA in PEMC is very different from that reported for L6 and C2 muscle cells where IGFBP-5 mRNA is undetectable during proliferation but is upregulated during differentiation (Ewton and Florini, 1995; Rotwein et al., 1995; Rousse et al., 1998). It had been hypothesized earlier that this increase in IGFBP-5 may enhance myogenic differentiation. Later studies demonstrated, however, that when IGFBP-5 was overexpressed in C2 cells, differentiation was inhibited, and that differentiation was enhanced in cells expressing antisense IGFBP-5 (James et al., 1996). This has been supported by recent in vivo studies that demonstrated that the overexpression of IGFBP-5 in mice led to whole body growth inhibition and retardation of muscle development (Salih et al., 2004). Therefore, the decrease in IGFBP-5 expression along with the increase in IGF-I during PEMC differentiation may facilitate myogenesis in these cells.
Myostatin and TGFß1 are potent negative regulators of muscle growth and differentiation. Myostatin is the muscle-specific member of the TGFß superfamily and mysotatin knock-out studies in mice have shown that both muscle cell proliferation and differentiation are enhanced resulting in significant increase in number and enlargement of muscle fibers (McPherron et al., 1997). Similar enhancement of muscle fiber number and size have been observed in naturally occurring myostatin mutations in double muscled cattle where the biological activity of myostatin is eliminated (Grobet et al., 1997; Kambadur et al., 1997; McPherron and Lee, 1997). Although TGFß1 can be found in the muscle and has similar negative effects on in vitro muscle growth, TGFß1 knock-out experiments in mice showed that both primary and secondary myofibers appear normal in the newborn pups (McLennan et al., 2000). These disparate findings among myostatin and TGFß1 knockout studies may indicate differing roles from myostatin and TGFß1 in muscle development. It is important to note that although myostatin and TGFß1 mRNA are differentially regulated during PEMC differentiation, both of these factors require significant posttranslational modification for biological activity. Therefore, changes observed in their mRNA may not reflect changes in biologically functional protein. In the current study TGFß1 mRNA expression in PEMC fluctuated throughout differentiation, whereas myostatin mRNA expression was relatively high at 48 h and rapidly decreased by approximately 80% by 96 h reaching its lowest level at 144 h. Investigators using C2C12 and L6 cells have shown differentiation-associated decreases in TGFß mRNA (Lafyatis et al., 1991; Bosche et al., 1995). Others have shown little change in myostatin mRNA is differentiating bovine primary myoblasts (McFarlane et al., 2005), whereas differentiation-associated increases in myostain were observed using chicken satellite cells and in murine muscle and cell lines (Mendler et al., 2000; Kocamis et al., 2001). These differences in TGFß1 and mysotatin mRNA expression during differentiation are likely due to differences among species, differences between primary and immortalized cells, as well as differences between satellite cells and embryonic myoblasts.
In summary, our data demonstrate that the mRNA expression of members of the IGF system and members of the TGFß superfamily are differentially regulated during PEMC differentiation. These factors are known to affect myogenesis, and these data provide new information about their regulation and potential interaction during myogenic differentiation and muscle development in porcine muscle cells. Given the significant role of the IGF system, myostatin, and TGFß1 in regulating muscle growth, this basic information about the regulation of these factors during porcine muscle development will enhance our understanding of muscle growth and could ultimately lead to the development of targeted strategies to increase rate and efficiency of muscle growth in pigs.
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Footnotes
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1 This research was supported by National Research Initiative Competitive Grants No. 2000-35206-9342 and 2006-35206-16632 from the USDA Cooperative State Research, Education and Extension Service Program and by the Minnesota Agricultural Experiment Station. 
2 Corresponding author: mwhite{at}umn.edu
Received for publication June 1, 2006.
Accepted for publication August 25, 2006.
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Abstract
INTRODUCTION
