| | Effect of differentiation on levels of insulin-like growth factor binding protein mRNAs in cultured porcine embryonic myogenic cells☆Abstract Insulin-like growth factor binding proteins (IGFBPs) have been shown to affect proliferation of several cell types via insulin-like growth factor (IGF)-dependent and IGF-independent mechanisms. The goal of this study was to determine if levels of IGFBP-2, -3, -4 and -5 mRNA changed during differentiation of cultured porcine embryonic myogenic cells. Total RNA was isolated from muscle cultures at various stages of differentiation and Northern blots of this RNA were probed with  -labeled cDNA probes specific for individual IGFBPs. Fusion, myogenin mRNA, and creatine phosphokinase activity were used as markers of differentiation. The level of IGFBP-3 mRNA in differentiating cultures (120 h in culture) was only one-third of the level in myogenin negative, nonfused cultures (72h in culture) (P<0.05, n=4). In contrast, the level of IGFBP-3 mRNA in extensively fused cultures (144h in culture) was increased by three-fold as compared to the level in myogenin negative, nonfused cultures (P<0.05, n=4) and approximately seven-fold as compared to the 120-h cultures (P<0.05, n=4). No significant change in the level of IGFBP-5 mRNA was observed during differentiation of myogenic cultures. IGFBP-2 mRNA levels were not significantly different at 72, 96 and 120h, but at 144h IGFBP-2 mRNA level was increased three-fold as compared to nonfused cultures (72h) (P<0.05, n=4). IGFBP-4 mRNA was not detectable on Northern blots of total RNA from porcine myogenic cultures at any stage of differentiation. Changes in IGFBP-3 and IGFBP-2 mRNA levels are associated with differentiation of embryonic porcine myogenic cells in culture and this may indicate that these IGFBPs play a role in differentiation of these cells.
1. Introduction  Insulin-like growth factor binding proteins (IGFBPs) have the ability to either potentiate or suppress proliferation of numerous cell types via both IGF-dependent and IGF-independent mechanisms [1], [2], [3], [4], [5]. IGFBPs are produced in specific tissues, such as muscle, where they may affect the growth and differentiation of cells in an autocrine/paracrine manner. IGFBP production by immortalized muscle cell lines has been relatively well characterized, however, less is known about the production of IGFBPs by myogenic cells from economically-important domestic animals [6], [7], [8], [9], [10], [11]. This lack of information is due to the unavailability of well-characterized muscle cell culture systems for many domestic animal species and to the fact that the presence of nonfusing cells in primary myogenic cell cultures complicates interpretation of experiments designed to assess IGFBP production by myogenic cells in these cultures. However, we have circumvented these problems by comparing IGFBP production in a well-characterized primary porcine muscle cell culture system with that of porcine muscle-derived fibroblasts isolated from fused muscle cell cultures [12]. Utilizing this system, we have shown that cultured embryonic porcine myogenic cell cultures produce IGFBP-2, -3, -4 and -5, however subcultured porcine muscle-derived fibroblasts produce only IGFBP-2 and -4. Consequently, cultured embryonic porcine myogenic cells are the source of the IGFBP-3 and IGFBP-5 [12], [13], [14] produced by primary porcine myogenic cell cultures. We have previously examined the effects of differentiation on production of IGFBP-3 by primary myogenic cells in serum-free, IGFBP free medium that supports minimal differentiation of porcine myogenic cells. In the current study, we compare levels of IGFBP-2, -3, -4 and -5 mRNA in porcine embryonic myogenic cells during differentiation in serum-containing, differentiation-enhancing medium. 2. Materials and methods 2.1. Porcine embryonic myoblast cell (PEMC) cultures The cell isolation procedure has been described in detail previously [12], [15], [16]. Pregnant Yorkshire gilts were maintained at the University of Minnesota Swine Research Centers. At 50–55 days of gestation, gilts were sacrificed at the University of Minnesota Meat Laboratory by electrical stunning followed by exsanguination. Fetuses were immediately removed by midventral laparotomy under aseptic conditions and rapidly transported to a laminar flow hood where myogenic cells were isolated according to procedures described previously. Isolated cells, suspended in Dulbecco’s Modified Eagle Medium (DMEM) containing 10% (v/v) fetal calf serum (FCS) and 10% (v/v) dimethylsulfoxide (DMSO), were frozen at −80°C and stored in a liquid nitrogen storage tank. To establish cultures from frozen stocks, rapidly thawed cell suspensions were diluted with the appropriate amount of DMEM containing 10% (v/v) FCS and 3% (v/v) chicken embryo extract (CEE) and plated in 100mm dishes coated with Basement Membrane Matrigel® (diluted 1:100 (v/v) in DMEM). All cultures were maintained at 37°C, 5% CO2, 95% air in a water saturated environment. After a 24-h attachment period, cultures were refed with 10ml of DMEM containing 10% FCS and 3% CEE. After a total of 72h in culture, 10ml of a fusion-enhancing medium (3% (v/v) swine serum (SS)/5% CEE/DMEM) was added to the cultures. At 96h, cytosine arabinoside (AraC) was added to give a final concentration 10−6M. Maximal fusion was attained by 144h in culture. These mass cultures of porcine embryonic myogenic cells (PEMCs) routinely yielded maximum fusion percentages of 65–75% at 144h. The University of Minnesota’s Animal Care Committee approved all experimental procedures utilizing animals that were performed in this study. 2.2. Porcine muscle-derived, nonfusing cells (fibroblasts) Porcine muscle-derived, nonfusing cells present in extensively fused porcine myotube cultures were released from the substrata by a 15-min Dispase treatment at 37°C. Cells obtained by this method were plated in DMEM containing 10% FCS and 3% CEE and were grown to confluence to allow fusion of any remaining myogenic cells. Cells were then released from the substrata by a 15-min Dispase treatment at 37°C, plated in DMEM containing 10% FCS and 3% CEE, and allowed to grow to approximately 40% confluence before freezing down in DMEM containing 10% FCS and 10% DMSO. Cells in cultures established from these muscle-derived, nonfusing cells showed typical stellate fibroblastic morphology and did not fuse or produce detectable levels of myogenin mRNA even when grown under optimum conditions for differentiation and fusion of porcine myogenic cells. The predominant cell type in these cultures is probably fibroblasts. Similar procedures have been used to isolate muscle-derived fibroblasts from porcine and bovine embryonic myogenic cell cultures, sheep satellite cell preparations, and fetal mouse muscle preparations [15], [17], [18]. 2.3. Creatine phosphokinase activity Creatine phosphokinase (CPK) activity was determined by using a commercially available kit (Sigma Diagnostics, Catalog no. 47-10) according to the manufacturer’s instructions. 2.5. Northern hybridization Total RNA was isolated from parallel cultures at 72, 96, 120 and 144h using the single step guanidium thiocyanate procedure [19]. Total RNA (75mg) was denatured (24mM HEPES, 6mM Na acetate, 1.2mM EDTA, 50% deionized formamide, and 2.2M formaldehyde) and fractionated on a 1.2% agarose gel containing 2.2M formaldehyde in 20mM phosphate buffer, pH 7.0. RNA was transferred to GeneScreen (NEN Lifescience Products, Boston, MA) by capillary action and hybridized separately with the appropriate IGFBP, myogenin or glyceraldehyde 3-phosphate dehydrogenase (GAPDH) probes. Radiolabeled cDNA probes for myogenin, IGFBP-3 were produced by polymerase chain reaction (PCR) in the presence of appropriate primers and  -labeled dCTP .The plasmid containing rat myogenin was kindly provided by W.E. Wright (clone pB465) [20]. A radiolabeled cDNA probe was produced by polymerase chain reaction (PCR) amplification of a 202-bp sequence within the clone using the forward primer 5′-TAAGACTAACACCCAGCC-3′ and the reverse primer 5′-CACTGTCTCTCAAACTGTCTC-3′. The plasmid containing porcine IGFBP-3 was kindly provided by S. Shimasaki (clone pPBP3ES) [21]. For PCR amplification of a 132-bp sequence within the clone, the primers 5′-TGACTCCAAACTCCACTC-3′ (forward) and 5′-TTGGACTCAGAGGAGAAG-3′ (reverse) were used. Rat IGFBP-2 cDNA was kindly provided by Rechler [22]. Human IGFBP-4 and -5 cDNA were provided by Shimasaki [23]. GAPDH was purchased from Ambion, Inc., Austin, TX and was used to correct for RNA loading. Radiolabeled cDNA probes were produced by random priming (Ambion, Inc., Austin, TX) of these cDNAs. Hybridization (1×106cpm/ml) was for 18h at 42°C. For myogenin and GAPDH, the transfer was washed once in 1× SSC (0.15M NaCl; 0.015M Na citrate), 0.1% SDS at 42°C for 15min and twice in 1× SSC, 0.1% SDS at 55°C for 15min each time, and the extent of hybridization was visualized using autoradiography. For IGFBP-2, -3, -4, and -5, the transfer was washed once in 1× SSC, 0.1% SDS at 55°C for 15min and twice in 0.1× SSC, 0.1% SDS at 60°C for 15min each time, and the extent of hybridization was visualized using autoradiography. The relative amount of RNA was measured by scanning densitometry of autoradiograms, and signal intensities of myogenin, IGFBP-2, -3, -4, and -5 were normalized by expressing them as a proportion of the GAPDH signal intensity to control for differences in loading or transfer efficiency. 2.6. Statistical analysis Data were analyzed by analysis of variance using the General Linear Model procedure of PC SAS™. In cases where main effects were significant, differences between means for preplanned comparisons were tested using Fisher’s least significant differences (LSD) test. Except where specifically indicated, all data were obtained by averaging results from four separate experiments done using cells isolated from fetuses obtained from four separate sows. 3. Results 3.2. Changes in levels of IGFBP-2, -3, -4, and -5 during differentiation of porcine myogenic cultures We have previously reported that porcine embryonic myoblast cultures produce detectable levels of IGFBP-2, -3, -4, and -5 as confirmed by Western ligand blot and immunoprecipitation data [12]. Northern blot hybridization revealed a single IGFBP-3 transcript of 2.6kb in total RNA from porcine embryonic myogenic cells (PEMCs) (Fig. 1). In fusing cultures (120h) the IGFBP-3 mRNA levels were only one-third (P<0.05, n=4) of the levels present in nonfused cultures (72 and 96h) (Fig. 5). However, in extensively fused cultures (144h), the IGFBP-3 mRNA levels were three times greater (P<0.05, n=4) than the levels in 72h cultures (Fig. 5). A dramatic change in IGFBP-3 mRNA level occurred between 120 and 144h in culture. In four separate experiments, we observed a seven-fold increase in IGFBP-3 mRNA level during this 24-h period (P<0.05, n=4) (Fig. 5). At 120h, when fusion is beginning and CPK activity and myogenin expression are maximal (Fig. 2, Fig. 3, Fig. 4), IGFBP-3 mRNA level is suppressed as compared to the level in proliferating myoblast cultures (72h) and differentiated myotube cultures (144h). In contrast to IGFBP-3 mRNA levels, IGFBP-5 mRNA level did not change significantly during differentiation of porcine myogenic cells (Fig. 1, Fig. 6). IGFBP-2 mRNA levels did not change between 72 and 120h (Fig. 7), however, levels in differentiated cultures (144h) were three times greater (P<0.05, n=4) than those in 72-, 96-, or 120-h cultures. We were unable to detect IGFBP-4 mRNA in porcine myogenic cell cultures with Northern hybridization techniques (data not shown). 3.3. Production of IGFBP-2, -3, -4 and -5 mRNA by porcine muscle-derived nonfusing cell cultures To assess the role of nonfusing cells in IGFBP production by porcine muscle myogenic cell cultures, we assessed IGFBP mRNA production by porcine muscle-derived nonfusing cells subcultured from highly fused myogenic cultures (data not shown). These cells did not produce detectable levels of either IGFBP-3 or IGFBP-5 mRNA. These data confirm previous studies showing that these cells do not secrete detectable quantities of IGFBP-3 or IGFBP-5 protein into their medium. Porcine muscle-derived nonfusing cells did produce detectable levels of IGFBP-2 and -4 mRNA (data not shown) and protein [12]. 4. Discussion Several lines of evidence suggest that both IGF-I and -II play an important role in differentiation of muscle cells [24], [25], [26], [27]. IGFBPs may play a critical role in modifying these effects of IGF-I and -II on terminal differentiation of muscle cells. Cultured muscle cells have been shown to produce many of the IGFBPs. The rat L6 muscle cell line produces IGFBP-4, -5, and -6 [6], [7], [11]. Cultured mouse C2 myoblasts produce only IGFBP-5 [8], [9], while the C2C12 subclone produces predominantly IGFBP-2 [10]. Cultured porcine embryonic myogenic cells, in addition to producing IGFBP-2, -4 and -5, also secrete detectable levels of IGFBP-3 and express IGFBP-3 mRNA [12], [13], [14]. In most cases IGFBPs produced locally by muscle cells appear to suppress the ability of IGF to stimulate differentiation. For example, des (1–3) IGF-I, an IGF analog that binds to the type I IGF receptor with normal affinity but binds only weakly to IGFBPs, is a much more potent stimulator of myogenic cell differentiation than is either IGF-I or -II [7], [11], [28]. This suggests that binding IGFs to IGFBPs produced by these muscle cells inhibits the ability of IGFs to stimulate muscle cell differentiation. Additionally, stably transfected C2 myoblasts over expressing IGFBP-5 showed diminished differentiation characteristics while myoblast lines expressing antisense IGFBP-5 exhibited accelerated differentiation [29]. These data suggest that IGFBP-5 inhibits the stimulatory effects of IGFs on differentiation of C2 myoblasts. IGFBP-5 expression has been shown to be induced during differentiation of the mouse C2 cell line [8], [28], [29]. This increase in IGFBP-5 expression appeared to directly parallel changes in myogenin [8]. In contrast to results obtained with C2 cells [8], [28], [29], we observed no change in IGFBP-5 mRNA levels during differentiation of cultured porcine embryonic myogenic cells. Because the IGFBP-5 mRNA level does not change significantly in differentiating porcine embryonic cells, this IGFBP may not affect differentiation in these cells. Levels of IGFBP-2 mRNA remain constant during early and mid-stages of differentiation (72–120h) of porcine myogenic cells, however, IGFBP-2 mRNA levels are increase in highly differentiated cultures (144h). These findings are in contrast to results observed with C2C12 cells in which IGFBP-2 mRNA declined as differentiation proceeded [10]. Although fibroblasts present in the myogenic cultures produce IGFBP-2, it is unlikely that the number of fibroblasts in these cultures is increasing dramatically between 120 and 144h especially in view of the fact that cytosine arabinoside was added to the cultures at 96h. Consequently, the three-fold increase in IGFBP-2 mRNA observed in myogenic cultures between 120 and 144h is likely to result from increased levels in differentiated myogenic cells not from increased numbers of fibroblasts. Thus, our data suggests that highly differentiated porcine myogenic cells have higher concentrations of IGFBP-2 mRNA than do undifferentiated myogenic cells and are in direct contrast to data reported for immortalized murine muscle cell lines. Although the biological function of increased IGFBP-2 in differentiation PEMC cultures is not clear, this binding protein has been shown to affect the biological activity of IGF and thus increased levels in differentiated PEMC cultures may be altering IGF action on these cells [30], [31], [32]. The production of IGFBP-3 by porcine myogenic cells is in contrast to data obtained using immortalized cell lines that do not produce IGFBP-3 mRNA nor secrete IGFBP-3 protein. Moreover, for PEMC cultured for 72, 96 or 120h, our current data show the same general pattern of differentiation-related alterations in IGFBP-3 mRNA in serum-containing, differentiation enhancing medium as we have previously observed in the presence of serum-free media containing no IGFBP-3. In both media alterations in levels of IGFBP-3 mRNA occurred simultaneously with the initiation of fusion/terminal differentiation. At 144h, however, IGFBP-3 mRNA levels in PEMC cultures in differentiation-enhancing, serum-containing media were approximately three times higher than at 72h. In contrast, PEMCs grown in IGFBP-free, serum-free medium exhibited IGFBP-3 mRNA levels at 144h that were equal to those at 72h. Although the reason for increased IGFBP-3 mRNA concentrations at 144h in PEMC cultures grown in differentiation-enhancing, serum-containing medium as compared to PEMC cultures grown in serum free, IGFBP-3-free medium is not clear, it may reflect more extensive differentiation in the cultures treated with differentiation enhancing medium. Since IGFBP-3 has been shown to affect the bioactivity of IGF-I and -II, these changes in IGFBP-3 level may alter the bioactivity of IGF-I or -II during differentiation. In addition to affecting differentiation by altering the bioactivity of IGF, it also is possible that IGFBP-3 has direct, IGF-independent effects on muscle cell differentiation. Numerous studies utilizing nonmuscle cells have shown that IGFBP-3 has IGF-independent affects on proliferation of cultured cells [2], [33], [34], [35], [36], [37], [38], [39]. Additionally, IGFBP-3 reportedly binds to the type V TGF-β receptor [40] and there is a recent report that IGFBP-3 binds specifically to the type II and I TGF-β receptors resulting in phosphorylation of Smad proteins [41], [42]. In light of these reports and our data showing a decrease in IGFBP-3 mRNA levels at the initiation of porcine myogenic cell differentiation, it is possible that reduced IGFBP-3 levels may play an important role in porcine myogenic cell differentiation. This hypothesis is supported by our report that suppression of myogenic cell differentiation by TGF beta prevents the differentiation-related drop in IGFBP-3 mRNA levels while enhancement of differentiation by IGF-I triggers a reduction in IGFBP-3 mRNA levels [13], [14]. In summary, our results show that the levels of IGFBP-2, -3 and -5 mRNA in porcine embryonic myogenic cells are affected differently by differentiation than are the levels of mRNA for these same IGFBPs in immortalized murine muscle cell lines. 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a Animal Growth and Development Laboratory, Department of Animal Science, University of Minnesota, St. Paul, MS 55108, USA b Department of Animal Sciences and Industry, Kansas State University, Manhattan, KS 66506, USA  Corresponding author. Tel.: +1-612-624-2234; fax: +1-612-624-3677.
☆ Published as paper no. of the scientific journal series of the Minnesota Agric. Exp. Sta. on research conducted under Minnesota Agric. Exp. Sta. project no. 16-084. PII: S0739-7240(02)00207-2 © 2002 Elsevier Science Inc. All rights reserved. | |
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