Stimulation and inhibition of myoblast differentiation by hormones

The growth and differentiation of L6 myoblasts are subject to control by two proteins secreted by cells of the Buffalo rat liver line. The first of these, rat insulinlike growth factor-II (formerly designated multiplication stimulating activity) is a potent stimulator of myoblast proliferation and differentiation, as well as associated processes such as amino acid uptake and incorporation into protein, RNA synthesis, and thymidine incorporation into DNA. In addition, this hormone causes a significant decrease in the rate of protein degradation. All of these actions seem to be attributable to a single molecular species, although their time courses and sensitivity to the hormone differ substantially. The second protein, the differentiation inhibitor (DI), is a nonmitogenic inhibitor of all tested aspects of myoblast differentiation, including fusion and the elevation of creatine kinase. Indirect immunofluorescence experiments demonstrated that DI also blocks accumulation of myosin heavy chain and myomesin. Upon removal of DI after 72 h incubation, all of these effects were reversed and normal myotubes containing the usual complement of muscle-specific proteins were formed. Thus, this system makes it possible to achieve specific stimulation or inhibition of muscle cell differentiation by addition of purified proteins to cloned cells in serum-free medium.     

Inhibition of myoblast differentiation in vitro by a protein isolated from liver cell medium

We have recently discovered that cells of Coon's Buffalo rat liver (BRL) line secrete a protein which is a potent inhibitor of skeletal myoblast differentiation in vitro. Using ion exchange and molecular exclusion chromatography, we have prepared this protein, which we designate "differentiation inhibitor" (DI), from the materials secreted by BRL cells maintained in serum-free medium. It is a relatively heat- stable protein which is inactivated by treatment with trypsin and mercaptoethanol and has an apparent molecular weight in the range 30,000--36,000. It exhibits no detectable mitogenic or lectin activity and differs from previously reported inhibitors of myoblast differentiation in several respects. It is active in all skeletal myoblast systems tested (Yaffe's L6 line as well as primary cultures of rat, chick, and Japanese quail myoblasts), and it blocks fusion, elevation of creatine kinase, and increased binding of alpha- bungarotoxin. Parallel fractionation of fetal bovine serum (FBS) and chick embryo extract (CEE) yields a peak of activity which similarly inhibits myoblast differentiation. We suggest that the differentiation inhibitor from BRL cells may correspond to the differentiation- inhibiting component(s) of FBS and CEE, and we call attention to the possibility that such a substance could play a role in embryonic growth of myoblasts and in satellite cell formation.     

Stimulation and inhibition of myoblast differentiation by hormones

Summary The growth and differentiation of L6 myoblasts are subject to control by two proteins secreted by cells of the Buffalo rat liver line. The first of these, rat insulinlike growth factor-II (formerly designated multiplication stimulating activity) is a potent stimulator of myoblast proliferation and differentiation, as well as associated processes such as amino acid uptake and incroporation into protein, RNA synthesis, and thymidine incorporation into DNA. In addition, this hormone causes a significant decrease in the rate of protein degradation. All of these actions seem to be attributable to a single molecular species, although their time courses and sensitivity to the hormone differ substantially. The second protein, the differentiation inhibitor (DI), is a nonmitogenic inhibitor of all tested aspects of myoblast differentiation, including fusion and the elevation of creatine kinase. Indirect immunofluorescence experiments demonstrated that DI also blocks accumulation of myosin heavy chain and myomesin. Upon removal of DI after 72 h incubation, all of these effects were reversed and normal myotubes containing the usual complement of muscle-specific proteins were formed. Thus, this system makes it possible to achieve specific stimulation or inhibition of muscle cell differentiation by addition of purified proteins to cloned cells in serum-free medium. This work was supported by a Muscular Dystrophy Association Postdoctoral Fellowship (M. J. E.-H.), U.S. Public Health Service Grant HL-11551 and AG-00629 (J. R. F.) and AM-28433 (B. M. V.), and a grant from the Muscular Dystrophy Association (J. R. F.).     

Cell adhesion to collagen and decreased myogenic gene expression implicated in the control of myogenesis by transforming growth factor beta

Transforming growth factor beta 1 (TGF-beta 1) is an inhibitor of skeletal muscle myoblast differentiation. Myoblast differentiation is dependent on the expression of certain myogenic differentiation genes and is affected by cell interaction with the extracellular matrix. We have searched for events in the differentiation process of L6E9 rat myoblasts that may be involved in the inhibitory action of TGF-beta 1. Elevated expression of the myogenic differentiation gene, myogenin, which is thought to play a central role in the differentiation process, occurs 10 h after the shift of L6E9 myoblasts to differentiation medium. Elevation of myogenin mRNA is blocked by TGF-beta 1 added at the time of the shift. This effect is preceded by, and might be related to, a rapid up-regulation of junB mRNA observed in TGF-beta 1-treated L6E9 myoblasts. However, TGF-beta 1 can also block myogenic differentiation in cells transfected with the myogenin gene under the control of a constitutive SV40 viral promoter. The nature of a mechanism that could mediate the inhibitory action of TGF-beta 1 without blocking myogenin mRNA expression is suggested by the observations that (a) TGF-beta 1 upregulates type I collagen expression and deposition in L6E9 myoblasts, (b) a fibrillar type I collagen layer inhibits L6E9 myoblast differentiation, and (c) inhibition of L6E9 myoblast differentiation by a type I collagen layer occurs without a block in myogenin expression. Thus, the data suggest that inhibition of L6E9 myoblast differentiation by TGF-beta 1 may be accomplished by at least two mechanisms acting in concert. One mechanism leads to a block in the expression of myogenin whereas the other mechanism may involve TGF-beta 1-induced changes in cell adhesion that either block the action of myogenic differentiation gene products or prevent the function of other as yet unknown components of the myogenic differentiation pathway.     

Effects of the somatomedins and insulin on myoblast differentiation in vitro

The effects of insulin and the somatomedins on differentiation of rat myoblasts were investigated in experiments on cells cloned from Yaffe's L6 line. Incubation for 48 hr with either insulin or Temin's multiplication stimulating activity (MSA), a member of the somatomedin family, caused a dramatic increase in myoblast fusion. This stimulation of differentiation is not a simple consequence of the increased cell density resulting from the effects of these hormones on myoblast proliferation, and the increase in fusion is not an effect common to all mitogens (FGF inhibits the process). Other somatomedins (human somatomedin C and insulin-like growth factor I), were as effective as MSA in stimulating differentiation. The somatomedins were active at concentrations in the range of their levels in fetal blood, in contrast to insulin, which was inactive at concentrations below 10^?7, M. Growth hormone (GH) had no effect on muscle differentiation. In serum-free medium MM-1 (in which myoblasts maintain apparently normal morphology and metabolic activity), the very high levels of insulin required to stimulate differentiation could be replaced entirely by physiological levels (1.0 μg/ml) of MSA, further supporting our view that insulin at high concentrations serves primarily as an analogue of the somatomedins in stimulating the growth and development of muscle cells.     

Recombinant platelet-derived growth factor-BB stimulates growth and inhibits differentiation of rat L6 myoblasts.

We previously found that L6 myoblasts and skeletal muscle isolated from developing rats express the platelet-derived growth factor (PDGF) beta-receptor gene (Jin, P., Rahm, M., Claesson-Welsh, L., Heldin, C.-H., and Sejersen, T. (1990) J. Cell Biol. 110, 1665-1672). We now report that recombinant human PDGF-BB is a mitogen for L6 myoblasts and also a potent inhibitor of myogenic differentiation. Treatment of L6J1 myoblasts with PDGF-BB increased the rate of DNA synthesis and stimulated cell proliferation. In differentiation medium (Dulbecco's modified Eagle's medium/0.5% fetal calf serum or Dulbecco's modified Eagle's medium/insulin), PDGF-BB prevented fusion of confluent myoblasts and suppressed biochemical differentiation in L6J1 cells. Inhibition of myoblast differentiation was, however, reversible. Withdrawal of PDGF-BB from the medium allowed myoblast fusion to occur. Northern blot hybridization showed that the PDGF beta-receptor mRNA was down-regulated to an undetectable level when confluent cultures of L6J1 myoblasts in growth medium (Dulbecco's modified Eagle's medium/5% fetal calf serum) were shifted to differentiation medium. Receptor binding assays further indicated that binding of PDGF-BB to its receptors on L6J1 myoblasts declined rapidly before creatine kinase activity rose. Our results provide the first demonstration that PDGF-BB is a potent regulator of myogenesis of L6 rat myoblasts and suggest that it may regulate muscle differentiation in vivo.     

Insulin and wnt1 pathways cooperate to induce reserve cell activation in differentiation and myotube hypertrophy

During ex vivo myoblast differentiation, a pool of quiescent mononucleated myoblasts, reserve cells, arise alongside myotubes. Insulin/insulin-like growth factor (IGF) and PKB/Akt-dependent phosphorylation activates skeletal muscle differentiation and hypertrophy. We have investigated the role of glycogen synthase kinase 3 (GSK-3) inhibition by protein kinase B (PKB)/Akt and Wnt/beta-catenin pathways in reserve cell activation during myoblast differentiation and myotube hypertrophy. Inhibition of GSK-3 by LiCl or SB216763, restored insulin-dependent differentiation of C2ind myoblasts in low serum, and cooperated with insulin in serum-free medium to induce MyoD and myogenin expression in C2ind myoblasts, quiescent C2 or primary human reserve cells. We show that LiCl treatment induced nuclear accumulation of beta-catenin in C2 myoblasts, thus mimicking activation of canonical Wnt signaling. Similarly to the effect of GSK-3 inhibitors with insulin, coculturing C2 reserve cells with Wnt1-expressing fibroblasts enhanced insulin-stimulated induction of MyoD and myogenin in reserve cells. A similar cooperative effect of LiCl or Wnt1 with insulin was observed during late ex vivo differentiation and promoted increased size and fusion of myotubes. We show that this synergistic effect on myotube hypertrophy involved an increased fusion of reserve cells into preexisting myotubes. These data reveal insulin and Wnt/beta-catenin pathways cooperate in muscle cell differentiation through activation and recruitment of satellite cell-like reserve myoblasts.     

Characterization of proliferating human skeletal muscle-derived cells in vitro: differential modulation of myoblast markers by TGF-beta2

Adult human skeletal muscle-derived cells (HuSkMC) propagated in vitro are under investigation as a cell-based therapy for the treatment of myocardial infarction. We have characterized HuSkMC with respect to cell identity and state of differentiation as a prerequisite to their clinical use. Flow cytometric analysis of propagated HuSkMC revealed a population of cells that expressed the myoblast markers CD56 and desmin. The presence of myoblasts in these cultures was further confirmed by their capacity to form myotubes and increase creatine kinase activity when cultured in low serum conditions. The non-myoblast fraction of these propagated cells expressed TE7, a marker associated with the fibroblast phenotype. Spontaneous differentiation of myoblasts occurred during serial propagation of HuSkMC, as judged by myotube formation, thereby reducing the myoblast representative fraction with continued cell expansion. We examined transforming growth factor beta2 (TGF-beta2) for its utility in controlling this spontaneous differentiation of adult human myoblasts in vitro. Propagation of HuSkMC in the presence of 1 ng/ml TGF-beta2 for 5 days decreased desmin expression within the myoblast population and caused a parallel reduction of creatine kinase activity. CD56 expression was unaffected, indicating a differential regulation of these myoblast markers. The reduction in desmin expression and creatine kinase activity was, however, reversible upon the removal of TGF-beta. These data collectively indicate that TGF-beta2 restrained differentiation of adult human skeletal myoblasts during propagation without causing irreversible loss of the myoblast phenotype, demonstrating the potential utility of using TGF-beta2 during cultivation and expansion of HuSkMC intended for therapeutic implantation.     

Integrin alpha subunit ratios, cytoplasmic domains, and growth factor synergy regulate muscle proliferation and differentiation

The role of integrins in muscle differentiation was addressed by ectopic expression of integrin alpha subunits in primary quail skeletal muscle, a culture system particularly amenable to efficient transfection and expression of exogenous genes. Ectopic expression of either the human alpha5 subunit or the chicken alpha6 subunit produced contrasting phenotypes. The alpha5-transfected myoblasts remain in the proliferative phase and are differentiation inhibited even in confluent cultures. In contrast, myoblasts that overexpress the alpha6 subunit exhibit inhibited proliferation and substantial differentiation. Antisense suppression of endogenous quail alpha6 expression inhibits myoblast differentiation resulting in sustained proliferation. These effects of ectopic alpha subunit expression are mediated, to a large extent, by the cytoplasmic domains. Ectopic expression of chimeric alpha subunits, alpha5ex/6cyto and alpha6ex/5cyto, produced phenotypes opposite to those observed with ectopic alpha5 or alpha6 expression. Myoblasts that express alpha5ex/6cyto show decreased proliferation while differentiation is partially restored. In contrast, the alpha6ex/5cyto transfectants remain in the proliferative phase unless allowed to become confluent for at least 24 h. Furthermore, expression of human alpha5 subunit cytoplasmic domain truncations, before and after the conserved GFFKR motif, shows that this sequence is important in alpha5 regulation of differentiation. Ectopic alpha5 and alpha6 expression also results in contrasting responses to the mitogenic effects of serum growth factors. Myoblasts expressing the human alpha5 subunit differentiate only in the absence of serum while differentiation of untransfected and alpha6-transfected myoblasts is insensitive to serum concentration. Addition of individual, exogenous growth factors to alpha5-transfected myoblasts results in unique responses that differ from their effects on untransfected cells. Both bFGF or TGFbeta inhibit the serum-free differentiation of alpha5- transfected myoblasts, but differ in that bFGF stimulates proliferation whereas TGF-beta inhibits it. Insulin or TGF-alpha promote proliferation and differentiation of alpha5-transfected myoblasts; however, insulin alters myotube morphology. TGF-alpha or PDGF-BB enhance muscle alpha-actinin organization into myofibrils, which is impaired in differentiated alpha5 cultures. With the exception of TGF- alpha, these growth factor effects are not apparent in untransfected myoblasts. Finally, myoblast survival under serum-free conditions is enhanced by ectopic alpha5 expression only in the presence of bFGF and insulin while TGF-alpha and TGF-beta promote survival of untransfected myoblasts. Our observations demonstrate (1) a specificity for integrin alpha subunits in regulating myoblast proliferation and differentiation; (2) that the ratio of integrin expression can affect the decision to proliferate or differentiate; (3) a role for the alpha subunit cytoplasmic domain in mediating proliferative and differentiative signals; and (4) the regulation of proliferation, differentiation, cytoskeletal assembly, and cell survival depend critically on the expression levels of different integrins and the growth factor environment in which the cells reside.     

Trypanosoma cruzi: reversal of inhibition of host muscle differentiation after exposure to elevated temperatures

The L6E9 myoblast cell line can be grown as individual cells in "growth medium," or can be induced to fuse and differentiate to form multinucleated myotubes either at 37 C or at 40.5 C in "differentiation medium." It has previously been shown that myoblasts with infected Trypanosoma cruzi (Brazil strain) cannot differentiate to form myotubes. Moreover, the mRNAs for contractile proteins are not induced in these infected cells. Infected myoblasts grown in "differentiation medium" at 37C were unable to differentiate by 7 days. The infection was maintained at 100%, and the number of trypomastigotes in the supernatant increased with time (peak greater than 10(6)/ml). At 40.5C, however, infected myoblasts gradually eliminated their infection. The percentage of parasitized cells was reduced to less than 1% by the 7th day of observation. There was also a decrease in the number of trypomastigotes in the supernatant. Moreover, significant fusion was observed in these cultures by morphological criteria. Using 32P-labeled recombinant DNA probes, it was shown that, at 37C, there was an inhibition of mRNAs for muscle-specific contractile proteins (myosin heavy chain and alpha-actin), whereas nonspecific mRNAs were not inhibited. Furthermore, infected myoblasts exposed to 40.5C exhibited no inhibition of mRNAs for myosin heavy chain and alpha-actin. Myoblasts cleared of their infection could readily be reinfected. This study demonstrates that the inhibition of muscle differentiation induced by T. cruzi is reversible when cultures are exposed to elevated temperatures.     

Integrin-linked kinase is a positive mediator of L6 myoblast differentiation

Overexpression of ILK in L6 myoblasts results in increased ILK kinase activity, stimulating myotube formation and induction of biochemical differentiation markers. Expression of a dominant negative ILK mutant, ILK(E359K), inhibits endogenous ILK activation and L6 differentiation. Cell cycle analysis of ILK(E359K) cells cultured in serum-free conditions indicates significant apoptosis (11-19% sub-diploid peak) which is not seen in insulin treated cells. Expression of ILK variants does not have significant effects on S-phase transit, however. Known targets of ILK, PKB/Akt or glycogen synthase kinase 3beta are not obviously involved in ILK-induced L6 differentiation. Insulin-stimulated phosphorylation of PKB at Ser473 is unimpaired in the ILK(E359K) cells, suggesting that PKB is not a myogenic target of ILK. Inhibition of GSK3beta by LiCl blocks L6 myogenesis, indicating that ILK-mediated inhibition of GSK3beta is not sufficient for differentiation. Our data do suggest that a LiCl-sensitive interaction of ILK is important in L6 myoblast differentiation.     

The amphoterin (HMGB1)/receptor for advanced glycation end products (RAGE) pair modulates myoblast proliferation, apoptosis, adhesiveness, migration, and invasiveness. Functional inactivation of RAGE in L6 myoblasts results in tumor formation in vivo

We reported that RAGE (receptor for advanced glycation end products), a multiligand receptor of the immunoglobulin superfamily expressed in myoblasts, when activated by its ligand amphoterin (HMGB1), stimulates rat L6 myoblast differentiation via a Cdc42-Rac-MKK6-p38 mitogen-activated protein kinase pathway, and that RAGE expression in skeletal muscle tissue is developmentally regulated. We show here that inhibition of RAGE function via overexpression of a signaling deficient RAGE mutant (RAGE delta cyto) results in increased myoblast proliferation, migration, and invasiveness, and decreased apoptosis and adhesiveness, whereas myoblasts overexpressing RAGE behave the opposite, compared with mock-transfected myoblasts. These effects are accompanied by a decreased induction of the proliferation inhibitor, p21(Waf1), and increased induction of cyclin D1 and extent of Rb, ERK1/2, and JNK phosphorylation in L6/RAGE delta cyto myoblasts, the opposite occurring in L6/RAGE myoblasts. Neutralization of culture medium amphoterin negates effects of RAGE activation, suggesting that amphoterin is the RAGE ligand involved in RAGE-dependent effects in myoblasts. Finally, mice injected with L6/RAGE delta cyto myoblasts develop tumors as opposed to mice injected with L6/RAGE or L6/mock myoblasts that do not. Thus, the amphoterin/RAGE pair stimulates myoblast differentiation by the combined effect of stimulation of differentiation and inhibition of proliferation, and deregulation of RAGE expression in myoblasts might contribute to their neoplastic transformation.     

Inhibition of skeletal muscle satellite cell differentiation by transforming growth factor-beta

Skeletal muscle satellite cells were cultured from mature rats and were treated in vitro with transforming growth factor-beta (TGF-beta). Muscle-specific protein synthesis and satellite cell fusion were used as indicators of muscle differentiation; a dose-dependent inhibition of differentiation was observed in response to TGF-beta. In addition, TGF-beta depressed cell proliferation in a dose-dependent manner. Half-maximal inhibition of differentiation was seen with a TGF-beta concentration of approximately 0.1 ng/ml. Although proliferation was not inhibited, it was depressed and half-maximal suppression of proliferation occurred in response to 0.1-0.5 ng TGF-beta/ml. Neonatal rat myoblasts were also subjected to TGF-beta treatment, and similar results were observed. Neonatal cells, however, were more sensitive to TGF-beta than satellite cells, as indicated by the reduced concentrations of TGF-beta required to inhibit differentiation and reduce the rate of proliferation. Under identical culture conditions proliferation of muscle-derived fibroblasts were also depressed. The differentiation inhibiting effect of TGF-beta on satellite cells was reversible. It has been suggested that TGF-beta could be an important regulator of tissue repair, and its in vitro effects on satellite cells suggest a possible role in regulation of muscle regeneration.     

Actions of transforming growth factor-beta on muscle cells

It has recently been reported by three laboratories that transforming growth factor-beta (TGF-beta) is a potent and reversible inhibitor of differentiation in myogenic cells. To improve our understanding of this inhibition, we investigated the effects of TGF-beta on several other processes in L6 myoblasts, with emphasis on actions of the insulin-like hormones (which stimulate myoblast differentiation). We found that TGF-beta had no effect on the binding of insulin-like growth factors (IGFs) to their receptors on the cell surface, and it had little or no effect on some actions of the IGFs. There was essentially no change in the suppression of proteolysis or the stimulation of cell proliferation by IGFs when TGF-beta was also added to the medium. However, there was an effect of TGF-beta on another process stimulated by the IGFs; TGF-beta was an equally active and more potent stimulator of amino acid uptake than was IGF-I, and the stimulation was additive beyond the maximal response attained with IGF-I, suggesting that the two act by different mechanisms. TGF-beta had significant effects on myoblast morphology, causing the formation of abundant stress fibers containing cytoplasmic (but not myofibrillar) actin. Addition of TGF-beta at various times after initiation of differentiation demonstrated that TGF-beta inhibits an early process in differentiation. Thus it appears that the interactions of TGF-beta and the IGFs in myoblasts are complex; in some instances the effects of IGFs are inhibited and in others they are mimicked or are unaffected. It is clear that TGF-beta does not act by simply interfering with IGF binding or blocking early steps in its action on myoblasts.     

Calpain and calpastatin in myoblast differentiation and fusion: Effects of inhibitors

Myoblast differentiation and fusion to multinucleated muscle cells can be studied in myoblasts grown in culture. Calpain (Ca²⁺-activated thiol protease) induced proteolysis has been suggested to play a role in myoblast fusion. We previously showed that calpastatin (the endogenous inhibitor of calpain) plays a role in cell membrane fusion. Using the red cell as a model, we found that red cell fusion required calpain activation and that fusibility depended on the ratio of cell calpain to calpastatin. We found recently that calpastatin diminishes markedly in myoblasts during myoblast differentiation just prior to the start of fusion, allowing calpain activation at that stage; calpastatin reappears at a later stage (myotube formation). In the present study, the myoblast fusion inhibitors TGF-β, EGTA and calpeptin (an inhibitor of cysteine proteases) were used to probe the relation of calpastatin to myoblast fusion. Rat L8 myoblasts were induced to differentiate and fuse in serum-poor medium containing insulin. TGF-β and EGTA prevented the diminution of calpastatin. Calpeptin inhibited fusion without preventing diminution of calpastatin, by inhibiting calpain activity directly. Protein levels of μ-calpain and m-calpain did not change significantly in fusing myoblasts, nor in the inhibited, non-fusing myoblasts. The results indicate that calpastatin level is modulated by certain growth and differentiation factors and that its continuous presence results in the inhibition of myoblast fusion.     

Insulin-like growth factor binding protein secretion by muscle cells: effect of cellular differentiation and proliferation

Several cell types have been shown to secrete insulin-like growth factor binding proteins (IGF-BP) in vitro. Since IGF-BP influences cell responsiveness to IGF, three muscle cell types were investigated to determine if they produced IGF-BP and to identify factors that regulate IGF-BP secretion. Porcine smooth muscle cells (pSMC), rat L6 skeletal muscle cells, and mouse BC3H-1 myocytes were used. IGF-BP activity in serum-free conditioned media was quantitated with a polyethylene glycol precipitation method. All three cell types secreted IGF-BP activity into the medium. Insulin was a potent stimulant of IGF-BP secretion for each cell type. Specifically, 1 microgram/ml insulin increased the IGF-BP concentration in conditioned media from 10.5 +/- 1.3 to 15.0 +/- 1.5 ng/ml in confluent L6 myotubes, from 42.5 +/- 11.1 to 90.5 +/- 9.8 ng/ml in confluent BC3H-1 cells, and from 2.1 +/- 0.1 to 3.8 +/- 0.1 ng/ml in confluent pSMC. L6 myotubes required more insulin (8 micrograms/ml) to achieve a half-maximal stimulation of IGF-BP secretion than confluent pSMC, differentiation deficient L6.DD cells or BC3H-1 cells, where half-maximal stimulation occurred between 125 and 300 ng/ml. L6 myoblasts were 40-fold more sensitive to insulin stimulation of IGF-BP secretion than L6 myotubes. IGF-I, although it interferes with the assay and thereby lowers the amount of detectable IGF-BP, stimulated the secretion of IGF-BP from all three cell types. Dexamethasone, (10(-7) M) decreased IGF-BP secretion into the media by approximately 50% for all three cell types. Affinity cross-linking and ligand blotting of 125I-IGF-I to conditioned media from each cell type showed (IGF-BP)-(IGF-I) complexes with molecular weights ranging 32-40 kDa (24-32 kDa for IGF-BP and 7.5 kDa for IGF-I). Insulin stimulated cell proliferation for both L6 myoblasts and BC3H-1 myocytes. This cell proliferative response was associated with an increase in IGF-BP secretion/cell in response to insulin. In contrast dexamethasone decreased L6 myoblast proliferation and decreased IGF-BP secretion/cell. We conclude that IGF-BP is secreted by each muscle cell type and that the state of cellular differentiation or quiescence influences its basal and insulin-stimulated secretion. Insulin and IGF-I are stimulators of IGF-BP secretion, whereas dexamethasone inhibits IGF-BP secretion. Because these hormones control muscle cell growth and differentiation, the IGF-BP may play an important regulatory role in these processes.     

Antibodies to 100- and 60-kDa surface proteins inhibit substratum attachment and differentiation of rodent skeletal myoblasts

A rabbit polyclonal antiserum was raised against membrane vesicles shed from the surface of fusing L6 rat myoblasts. In immunoblots the antiserum recognized fibronectin, a protein of approximately 100,000 Da (100-kDa), and a protein of approximately 60,000 Da (60 kDa). If added prior to cellular alignment, immunoglobulins from this serum inhibited fusion of both rat (L6) and mouse (C2) myoblasts in a dose-dependent fashion. To determine which component of this serum was responsible for fusion inhibition, antibodies against fibronectin, the 100- and 60-kDa proteins were microaffinity purified and tested, individually, for their effects on myoblast fusion. Antibodies against fibronectin had no effect on fusion. Antibodies against the 100-kDa protein released most cells from the substratum. Antibodies against the 60-kDa protein completely inhibited fusion. Fusion inhibition was accompanied by a corresponding inhibition of expression of two differentiation markers, creatine phosphokinase and the acetylcholine receptor. The 60-kDa protein was found, by immunoblot analysis, in smooth muscle-like cells (BC3H1 cells) and in variant L6 cells that do not differentiate and do not fuse. However, in the differentiation incompetent cells, the 60-kDa antigen appeared to be present in reduced amount. Indirect immunofluorescence of unpermeabilized L6 cells revealed alterations in the distribution of all three antigens during development. Fibronectin first appeared in long fibrillar arrays above the surface of cells that were beginning to align and fuse; fibronectin was not present on myotubes. The 100-kDa protein was seen initially in prominent fibrillar projections at the tips of prefusion myoblasts. During fusion the antigen was observed at sites of cell-cell contact and on extracellular vesicles. The 100-kDa protein appeared to be less abundant on myotubes. The 60-kDa protein first appeared in regions of cell-cell contact on cells that were beginning to align and fuse. As. fusion progressed, the 60-kDa protein was also found in extracellular vesicles. The 60-kDa protein was not observed on myotubes. As a result of this study we have identified two previously undescribed cell surface proteins involved in rodent skeletal myogenesis. The first is an approximately 100-kDa protein involved in early interactions of skeletal myoblasts with their substratum. The second is an approximately 60-kDa protein involved in myoblast differentiation. Both proteins are shed from the myoblast surface during myotube formation.     

Blunting effect of hypoxia on the proliferation and differentiation of human primary and rat L6 myoblasts is not counteracted by Epo

Objectives: The aim of this study was to evaluate whether hypoxia and/or erythropoietin would be able to modulate proliferation/differentiation processes of rat and human myoblasts. Materials and methods: Rat L6 and primary human myoblasts were grown in 21% or 1% O₂ in the presence or absence of recombinant human erythropoietin (RhEpo). Presence of erythropoietin receptors (EpoR) was assayed using RT‐PCR and Western blotting techniques. Cell proliferation was evaluated by determining the doubling time and kinetics of cultures by counting cells. Cell differentiation was analysed by determining myogenic fusion index using antibodies against the myosin heavy chain. Expression of myogenin and myosin heavy chain (MHC) proteins were evaluated using the Western blotting technique. Results: After 96 h culture in growth medium for 2.5 and 9 h, doubling time of L6 and human primary myoblasts respectively, had increased in 1% O₂ conditions (P < 0.01). Kinetics of culture showed alteration in proliferation at 72 h in L6 myoblast cultures and at 4 days in human primary myoblasts. The myogenic fusion index had reduced by 30% in L6 myoblasts and by 20% in human myoblasts (P < 0.01). Expression of myogenin and MHC had reduced by around 50%. Despite presence of EpoR mRNA and protein, RhEpo did not counteract the effects of hypoxia either in L6 cells or in human myoblasts. Conclusions: The data show that exposure to hypoxic conditions (1% O₂) of rat and human myoblasts altered their proliferation and differentiation processes. They also show that Epo is not an efficient growth factor to counteract this deleterious effect.     

Ligand-dependent inhibition of myoblast differentiation by overexpression of the type-1 insulin-like growth factor receptor

The insulin-like growth factors (IGFs) have paradoxical effects on skeletal myoblast differentiation. While low concentrations of IGF stimulate myoblast differentiation, high concentrations of IGF induce a progressive decrease in myoblast differentiation. The mechanism of this inhibition is unknown. Using a retroviral expression vector, we developed a subline of mouse P2 mouse myoblasts (P2-LISN) which expressed 7.5 times higher levels of type-1 IGF receptors than control (P2-LNL6) myoblasts, which were infected with a virus lacking the type-1 IGF receptor sequence. Overexpression of the type-1 IGF receptor caused the IGF dose-response curves of stimulation and progressive inhibition of differentiation to shift to the left. Additionally, at high insulin and IGF-I concentrations, complete inhibition of P2-LISN myoblast differentiation occurred. These results suggest that inhibition of differentiation at high ligand concentrations was not due to the primary involvement of other species of receptors for IGF. Type-1 IGF receptor downregulation as a mechanism for inhibition of differentiation was also ruled out since P2-LISN myoblasts constitutively expressed high levels of type-1 IGF receptors. Additionally, inhibition of differentiation at high concentrations of IGF-I was not correlated with overt stimulation of proliferation or with IGF binding protein (IGF-BP) release into the culture medium. These results indicate that the type-1 IGF receptor mediates two conflicting signal pathways in myogenic cells, differentiation-inducing and differentiation-inhibitory, which predominate at different ligand concentrations. © 1993 Wiley-Liss, Inc.