Inhibits Myoblast Differentiation Research Articles

S100B stimulates myoblast proliferation and inhibits myoblast differentiation by independently stimulating ERK1/2 and inhibiting p38 MAPK

The Ca2+-modulated protein of the EF-hand type, S100B, was shown to inhibit rat L6 myoblast differentiation and myotube formation by interacting with a high affinity with an unidentified receptor (Sorci et al., 2003). We show here that S100B independently inhibits the MKK6-p38 MAPK pathway and stimulates the Ras-MEK-ERK1/2 pathway. The inhibitory effect of S100B on p38 MAPK translates into a defective induction of the muscle-specific transcription factor myogenin and the antiproliferative factor p21(WAF1), while S100B's stimulatory effect on ERK1/2 results in stimulation of myoblast proliferation via cyclin D1 induction and Rb phosphorylation and protection against apoptosis via activation of NF-kappaB transcriptional activity. Also, the S100B's effects that are mediated by the Ras-MEK-ERK1/2 pathway that is, stimulation of proliferation and protection against apoptosis, depend on reactive oxygen species production, being inhibited by antioxidants, while the S100B inhibitory effect on the MKK6-p38 MAPK pathway is not. We propose that S100B might participate in the regulation of myoblast differentiation by stimulating myoblast proliferation, protecting myoblasts against apoptosis, and modulating myotube formation.

Sirt1: A Novel Adipocyte and Myocyte Regulatory Factor

Sirt1 (Sirtuin type 1), a member of the conserved sirtuin family, is a NAD+ -dependent histone deacetylase. It is intimately related to cell proliferation, differentiation, apoptosis, and metabolism. Its role in senescence and metabolism has recently come to light. Sirt1 promotes fat mobilization in white adipocytes by repressing PPARgamma and inhibits myoblast differentiation by down-regulating muscle gene expression. Therefore, Sirt1 is not only an important longevity factor, but also may play a key modulatory role in animal fat deposition and muscle development.

Lipid products of phosphoinositide 3-kinase abrogate genistein-induced fusion inhibition in myoblasts

Genistein (4′,5,7-trihydroxyisoflavone) is a tyrosine kinase inhibitor. Although the agent has shown to inhibit myoblast differentiation, neither intracellular target(s) as a tyrosine kinase inhibitor nor action mechanism of the agent is well known. Here we studied the effect of genistein on the differentiation of myoblasts. Genistein strongly but reversibly blocked both myoblast fusion and synthesis of the muscle-specific proteins. The agent also reversibly reduced the phosphorylation level of focal adhesion kinase (FAK), a cytoplasmic tyrosine kinase, and its interaction with p85, the regulatory subunit of phosphoinositide 3-kinase (PI3-kinase). In addition, genistein indirectly inhibited PI3-kinase activity and blocked calcium influx which is required for myoblast fusion. However, both genistein-induced inhibition of cell fusion and calcium influx were abrogated by the lipid products of PI3-kinase. These results demonstrate that genistein can exert their effect on the signaling pathway from FAK to calcium influx via PI3-kinase in the differentiation of myoblasts.

247. Expressing Full-Length Dystrophin in 50% Cardiomyocytes Corrects Cardiomyopathy in the Mdx Mouse Model for Duchenne Muscular Dystrophy

Muscle wasting, a syndrome characterized by the disproportional loss of skeletal muscle, is a frequent complication in patients with cancer or other chronic diseases. Recent studies in vitro and in vivo have demonstrated that tumor cells produce and secrete cytokines such as TNF- α that are responsible for inhibition of differentiation of myoblast cells into multinucleated myotubes and thereby may prevent muscle repair in cancer patients. Activation of NF-κB triggered by TNF-α interaction with its receptor on muscle cells, is considered a major pathway through which cytokines lead to muscle wasting in cancer patients. Gene therapy offers a potential solution by modulating downstream events within muscle cells. In vitro models of muscle wasting can provide an important resource to understand which pathways to modulate and to test potential therapies. In our In vitro studies, we found that application of TNF-α results in apoptosis of primary C57 mouse myoblast cells at 3 days after treatment. IFN-γ, IL-1β and IL-6 do not lead to apoptosis of myoblast cells, but result in inhibition of myoblast differentiation at 20 ng/ml. In addition, the conditioned media from human prostate cancer cell lines (PC-3 and DU145P) and a human melanoma cell line (Mel) significantly inhibit the differentiation of myoblast cells. NF-κB activity was activated in treated myoblast cells compared to untreated or 293 cell culture supernatant-treated myoblast cells. However we found that myoblasts stably transfected with IKBSR, a mutant IKBα, or cellular FLICE/Caspase-8-inhibitory protein, cFLIP, inhibited NF-κB activation after treatment of myoblast cells with cytokines (TNF-α, IFN-γ, IL-1β or IL-6) or tumor cell conditioned media (PC-3, DU145P or Mel). Inhibition of NF-κB activation was associated with amelioration of the inhibition of muscle cell differentiation. To our knowledge, this is the first report that cFLIP, an important caspase-8-inhibitory protein is capable of inhibiting NF-κB activation and reducing inhibition of myoblast cell differentiation treated with PC-3, DU145P and Mel tumor media. The results encourage us to generate gene transfer virus vectors expressing IKBSR or cFLIP or both, and to determine whether IKBSR or cFLIP or both can ameliorate muscle wasting.

Exogenous hepatocyte growth factor inhibits myoblast differentiation by inducing myf5 expression and suppressing myoD expression in an organ culture system of embryonic mouse tongue.

We examined the effects of exogenous hepatocyte growth factor (HGF) on the differentiation and proliferation of tongue myoblasts by using an organ culture system of tongue obtained from mouse embryos at embryonic day (E) 13. Exogenous HGF induced reductions in the quantities of muscle creatine kinase and myogenin mRNAs and in the number of fast myosin heavy chain-positive myoblasts and myotubes, suggesting that HGF suppressed the differentiation of myoblasts in the cultured E13 tongues. Exogenous HGF induced no significant changes in the percentage of proliferating cell nuclear antigen (PCNA)-positive cell nuclei to total cell nuclei (labeling index) in the muscle portion of the cultured E13 tongue, suggesting that HGF did not affect the proliferation of myoblasts. Exogenous HGF induced the expression of myf5 mRNA but inhibited the expression of myoD mRNA. Since mouse tongue myoblasts are reported to complete proliferation by E13, it appears that exogenous HGF arrests myoblasts in the cell cycle and does not allow them to enter the differentiation process. This is achieved by controlling the expression of myf5 and myoD mRNAs, thus inhibiting the differentiation of tongue myoblasts.

Role of insulin-like growth factor binding protein-3 (IGFBP-3) in the differentiation of primary human adult skeletal myoblasts.

Although muscle satellite cells were identified almost 40 years ago, little is known about the induction of their proliferation and differentiation in response to physiological/pathological stimuli or to growth factors/cytokines. In order to investigate the role of the insulin-like growth factor (IGF)/IGF binding protein (IGFBP) system in adult human myoblast differentiation we have developed a primary human skeletal muscle cell model. We show that under low serum media (LSM) differentiating conditions, the cells secrete IGF binding proteins-2, -3, -4 and -5. Intact IGFBP-5 was detected at days 1 and 2 but by day 7 in LSM it was removed by proteolysis. IGFBP-4 levels were also decreased at day 7 in the presence of IGF-I, potentially by proteolysis. In contrast, we observed that IGFBP-3 initially decreased on transfer of cells into LSM but then increased with myotube formation. Treatment with 20 ng/ml tumour necrosis factor-alpha (TNFalpha), which inhibits myoblast differentiation, blocked IGFBP-3 production and secretion whereas 30 ng/ml IGF-I, which stimulates myoblast differentiation, increased IGFBP-3 secretion. The TNFalpha-induced decrease in IGFBP-3 production and inhibition of differentiation could not be rescued by addition of IGF-I. LongR(3)IGF-I, which does not bind to the IGFBPs, had a similar effect on differentiation and IGFBP-3 secretion as IGF-I, both with and without TNFalpha, confirming that increased IGFBP-3 is not purely due to increased stability conferred by binding to IGF-I. Furthermore reduction of IGFBP-3 secretion using antisense oligonucleotides led to an inhibition of differentiation. Taken together these data indicate that IGFBP-3 supports myoblast differentiation.

Myostatin Inhibits Myoblast Differentiation by Down-regulating MyoD Expression

Myostatin, a negative regulator of myogenesis, is shown to function by controlling the proliferation of myoblasts. In this study we show that myostatin is an inhibitor of myoblast differentiation and that this inhibition is mediated through Smad 3. In vitro, increasing concentrations of recombinant mature myostatin reversibly blocked the myogenic differentiation of myoblasts, cultured in low serum media. Western and Northern blot analysis indicated that addition of myostatin to the low serum culture media repressed the levels of MyoD, Myf5, myogenin, and p21 leading to the inhibition of myogenic differentiation. The transient transfection of C(2)C(12) myoblasts with MyoD expressing constructs did not rescue myostatin-inhibited myogenic differentiation. Myostatin signaling specifically induced Smad 3 phosphorylation and increased Smad 3.MyoD association, suggesting that Smad 3 may mediate the myostatin signal by interfering with MyoD activity and expression. Consistent with this, the expression of dominant-negative Smad3 rescued the activity of a MyoD promoter-reporter in C(2)C(12) myoblasts treated with myostatin. Taken together, these results suggest that myostatin inhibits MyoD activity and expression via Smad 3 resulting in the failure of the myoblasts to differentiate into myotubes. Thus we propose that myostatin plays a critical role in myogenic differentiation and that the muscular hyperplasia and hypertrophy seen in animals that lack functional myostatin is because of deregulated proliferation and differentiation of myoblasts.

Insulin-like growth factors (IGF-I and IGF-II) inhibit C2 skeletal myoblast differentiation and enhance TNF alpha-induced apoptosis.

IGF-I and IGF-II are thought to be unique in their ability to promote muscle cell differentiation. Murine C2 myoblasts differentiate when placed into low serum media (LSM), accompanied by increased IGF-II and IGF binding protein-5 (IGFBP-5) production. Addition of 20 ng/ml TNF alpha on transfer into LSM blocked differentiation, IGF-II and IGFBP-5 secretion and induced apoptosis. We, therefore, wished to assess whether IGFs could protect against the effects of TNF alpha. Neither inhibition of differentiation or induction of apoptosis was rescued by co-incubation with IGF-I or IGF-II. A lower dose of TNF alpha (1 ng/ml) while not inducing apoptosis still inhibited myoblast differentiation by 56% +/- 12, (P < 0.001), indicating that induction of apoptosis is not the sole mechanism by which TNF alpha inhibits myoblast differentiation. Addition of IGF-I or IGF-II alone reduced differentiation by 49% +/- 15 and 33% +/- 20, respectively, (P < 0.001), although neither induced apoptosis. For muscle cells to differentiate, they must arrest in G0. We established that addition of IGF-I, IGF-II or TNF alpha to the myoblasts promoted proliferation. The myoblasts could not exit the cell cycle as efficiently as controls and differentiation was thus reduced. Unexpectedly, co-incubation of IGF-I or IGF-II with 1 ng/ml TNF alpha enhanced the inhibition of differentiation and induced apoptosis. In the absence of apoptosis we show an association between IGF-induced inhibition of differentiation and increased IGFBP-5 secretion. These results indicate that the effects of the IGFs on muscle may depend on the cytokine environment. In the absence of TNF alpha, the IGFs delay differentiation and promote myoblast proliferation whereas in the presence of TNF alpha the IGFs induce apoptosis.

The Ras/Erk Pathway Induces Primitive Endoderm but Prevents Parietal Endoderm Differentiation of F9 Embryonal Carcinoma Cells

The formation of parietal endoderm (PE) is one of the first differentiation processes during mouse development and can be studied in vitro using F9 embryonal carcinoma (EC) cells. Treatment of F9 EC cells with retinoic acid (RA) induces differentiation toward primitive endoderm (PrE), while differentiation toward PE is induced by subsequent addition of parathyroid hormone (PTH) or PTH-related peptide (PTHrP). The signal transduction mechanisms involved in this two-step process are largely unclear. We show that the RA-induced differentiation toward PrE is accompanied by a sustained increase in Ras activity and that ectopic expression of oncogenic Ha-Ras is sufficient to induce PrE differentiation. Ras activity subsequently decreases upon PTH-induced differentiation toward PE. This is a necessary event, since expression of oncogenic Ha-Ras in PrE-like cells prevents PTH-induced PE differentiation. Expression of active PKA in PrE-like F9 cells mimics PTH-induced PE differentiation and is again prevented by oncogenic Ha-Ras. The effect of oncogenic Ras on both differentiation steps is abolished by the MEK inhibitor PD98059 and can be mimicked by constitutively active forms of Raf and MEK. In conclusion, our data suggest that activation of the Ras/Erk is sufficient to induce differentiation to PrE and to prevent subsequent differentiation toward PE. Activation of PKA down-regulates Ras activity, resulting in disappearance of this blockade and transmission of signal(s) triggering PE differentiation.

Up-regulation of insulin-like growth factor binding protein-5 is independent of muscle cell differentiation, sensitive to rapamycin, but insensitive to wortmannin and LY294002.

Skeletal myoblast differentiation is stimulated by insulin-like growth factors (IGFs). The autocrine action of IGFs is mediated through the type-1 IGF receptor (IGFR-1) and modulated by IGF binding proteins (IGFBPs) secreted by the cells. The mouse C2 myoblast cell line stably transfected with a vector producing IGF-II antisense RNA was used to show that specific IGFBP expression changes with the state of the cells: high levels of IGFBP-2 messenger RNA (mRNA) were found only in proliferating myoblasts, whereas IGFBP-3 mRNA was induced in quiescent cells. Secretion of IGFBP5 was strongly stimulated during differentiation. Insulin and IGF dose-response experiments showed that up-regulation of IGFBP-5 resulted from IGFR-1 activation. Drugs interfering with IGFR-1 signaling and inhibiting myoblast differentiation had different effects on IGFBP-5 up-regulation. Two phosphatidylinositol 3-kinase (PI 3-kinase) inhibitors, wortmaninn and LY294002 [2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one], failed to alter IGFBP-5 up-regulation, which persisted in the absence of differentiation. Rapamycin which indirectly prevents activation of the p70 ribosomal protein-S6 kinase (p70S6k), suppressed IGFBP-5 induction. Because the PI3-kinase inhibitors block p70S6k, neither kinase would be required for IGFR-1-dependent IGFBP-5 induction. In C2 anti-IGF-II myoblasts, IGFBP-5 induction is therefore rapamycin-sensitive and independent of differentiation.

Distinct signaling pathways regulate transformation and inhibition of skeletal muscle differentiation by oncogenic Ras

Expression of oncogenic Ras in 23A2 skeletal myoblasts is sufficient to induce both a transformed phenotype and a differentiation-defective phenotype, but the signaling pathways activated by oncogenic Ras in these cells and their respective contribution to each phenotype have not been explored. In this study, we investigated MAP kinase activity in control 23A2 myoblasts and in 23A2 myoblasts rendered differentiation-defective by the stable expression of an oncogenic (G12V)Ha-ras gene (Ras9 cells). The MAP kinase immunoprecipitated from Ras9 cells was 30-40% more active than that from control 23A2 cells. To establish if this elevated MAP kinase activity is essential to the maintenance of the oncogenic Ras-induced phenotype, we utilized the selective MAP kinase kinase 1 (MEK1) inhibitor, PD 098059. PD 098059 decreased the MAP kinase activity in Ras9 cells to the level found in 23A2 cells. PD 098059 did not affect the ability of 23A2 myoblasts to differentiate. PD 098059 reverted the transformed morphology of Ras9 cells but did not restore the ability of these cells to express the muscle-specific myosin heavy chain gene or to form muscle fibers. Treatment with PD 098059 also did not affect the ability of oncogenic Ha-Ras to establish a non-myogenic phenotype in C3H10T1/2 cells co-expressing MyoD. These results demonstrate that the activation of MAP kinase is necessary for the transformed morphology of Ras9 cells but is not required by oncogenic Ras to establish or to maintain a differentiation-defective phenotype. While these data do not rule out the possibility that constitutive signaling by MEK1 or MAP kinase could inhibit myoblast differentiation, they clearly demonstrate that other pathways activated by oncogenic Ras are sufficient to inhibit differentiation.

Zinc and the initiation of myoblast differentiation

Previous studies have indicated that a lack of available zinc inhibited myoblast differentiation as shown by a failure of the cells to fuse and low expression of creatine kinase mRNA and activity. However, the nature of the requirement for zinc and its relationship to the events leading to differentiation have been unclear. The current studies with C 2C 12 cells indicated that the muscle-specific enhancer present in the 5′-flanking region of the creatine kinase gene contributed to the zinc sensitivity of this enzyme. Because this enhancer can be activated by expression of the myogenic factors MyoD and myogenin, their sensitivity to zinc was investigated. The concentrations of both MyoD and, particularly, myogenin mRNA, were decreased by zinc deficiency. In vitro translation experiments suggested that these changes closely corresponded with alterations in their rates of synthesis. Further experiments failed to indicate a major effect of zinc on the stabilities of these mRNAs. Because an induction of myogenin mRNA is one of the earliest known events in myoblast differentiation, its particular sensitivity to lack of zinc suggests that zinc may be required before or during the initiation of myoblast differentiation.

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.

Stimulation of Avian Myoblast Differentiation by Triiodothyronine: Possible Involvement of the cAMP Pathway

In a previous work, we have shown that T3 induces a potent stimulation of avian myoblast differentiation. In this study, we demonstrated that this hormone did not affect MyoD and myogenin expression. As numerous data suggest that T3 could affect the cAMP pathway, we have studied its involvement in the myogenic activity of triiodothyronine on quail myoblast. In agreement with Zalin and Montagues (Cell 2, 103-108 (1974)), we observed a transient rise in myoblast intracellular cAMP level some hours before the onset of terminal differentiation. Interestingly, this rise occurred earlier in T3-treated than in control myoblasts, and cAMP production was significantly increased by the hormone. Moreover, T3 increased CREB transcriptional activity, thus suggesting that the entire cAMP signaling pathway was stimulated by this hormone. In addition, we observed that addition of an inhibitor of adenylate cyclase activity prior to the cAMP rise dramatically inhibited myoblast differentiation. Last, we showed that cAMP mimicked all T3 actions upon myoblast differentiation: (1) T3 and cAMP reduced myoblast proliferation by increasing the number of postmitotic myoblasts at cell confluence; (2) T3 and cAMP increased BTG1 nuclear accumulation; (3) T3 and cAMP stimulated terminal differentiation only when added during the proliferative phasis. These data strongly suggest that the transient rise in cAMP production could be essential for myoblast terminal differentiation. In addition, it appears that, at least in avian myoblasts, T3 stimulation of terminal differentiation involves the cAMP pathway.

Positive and Negative Regulation of D-type Cyclin Expression in Skeletal Myoblasts by Basic Fibroblast Growth Factor and Transforming Growth Factor β: A ROLE FOR CYCLIN D1 IN CONTROL OF MYOBLAST DIFFERENTIATION

Differentiation of skeletal myoblasts in culture is negatively regulated by certain growth factors, including basic fibroblast growth factor (bFGF) and transforming growth factor beta (TGF beta). We investigated the effects of bFGF and TGF beta on D-type cyclin expression in skeletal myoblasts. When myoblasts were induced to differentiate in low mitogen medium, expression of cyclin D1 rapidly fell below detectable levels. In contrast, expression of cyclin D3 increased to levels exceeding those present in myoblasts. Expression of cyclin D1 was induced in myoblasts by bFGF and TGF beta (albeit with different kinetics for each factor), while induction of cyclin D3 expression was inhibited by these growth factors. Although these results are consistent with other reports showing induction of cyclin D1 by growth factors, induction of cyclin D3 expression during terminal differentiation of myoblasts and inhibition of this induction by growth factors is surprising. These results suggest that cyclin D3, previously thought to be only a positive regulator of cell cycle progression, may also function in the cellular context of terminal differentiated muscle. Stable expression of cyclin D1 from an ectopic viral promoter inhibits C2C12 myoblast differentiation, but only in those clones where the level of cyclin D1 expression does not significantly exceed that present in control myoblasts stimulated by bFGF. Together, these result suggest that cyclin D1 expression functions in the inhibition of myoblast differentiation by certain growth factors.

Inhibition of myogenic differentiation in myoblasts expressing a truncated type II TGF-β receptor

Transforming growth factor-beta (TGF-beta) is thought to play a role in mesenchymal cell development and, specifically, in muscle differentiation, yet its precise role in the latter process remains unclear. TGF-beta has been shown to both inhibit and induce myoblast maturation in vitro, depending on the culture conditions. Whether the type I or type II TGF-beta receptor mediates the various TGF-beta effects on myogenesis is not known. In the present study, C2C12 myoblasts were transfected with an expression vector for a truncated type II TGF-beta receptor, which has been shown to act as a dominant negative inhibitor of type II receptor signaling. In contrast to the parental cells, the transfected clones did not efficiently form myotubes or induce expression of MyoD, myogenin and several other differentiation markers following incubation in low serum media. However, some muscle differentiation markers continued to be expressed in the transfected cells suggesting that at least two pathways are involved in muscle cell differentiation. These cells could still growth arrest in low serum media, showing that decreased proliferation can be dissociated from differentiation. Unlike several oncogenes known to block myogenic differentiation, expression of the truncated TGF-beta receptor did not result in myoblast transformation. Injection of the parental or the transfected C2C12 cells into the limb muscle of nude mice revealed quantitative and qualitative differences in their behavior, and suggested that myoblasts expressing the truncated TGF-beta receptor cannot fuse in vivo. Finally, retrovirus-mediated expression of MyoD in the transfected cells restored their ability to form myotubes in vitro, indicating that inhibition of myoblast differentiation by the truncated TGF-beta receptor may depend on decreased MyoD expression. We propose that TGF-beta signaling through the type II receptor is required for several distinct aspects of myogenic differentiation and that TGF-beta acts as a competence factor in this multistep process.

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.

Aberrant expression of high mobility group chromosomal protein 14 affects cellular differentiation

High mobility group (HMG) 14 is a ubiquitous chromosomal protein that binds specifically to nucleosomal DNA and may be involved in a process that confers distinct properties to the chromatin structure of transcriptionally active genes. To explore the involvement of this protein in regulation of gene expression, we studied the effect of aberrant expression of HMG-14 protein on cellular differentiation. We produced stably transfected C2C12 mouse myoblasts expressing the human HMG-14 protein under the control of the mouse mammary tumor virus promoter. Transformed colonies retained their potential do differentiate into myotubes. Induction of human HMG-14 expression by dexamethasone inhibited the myogenic process. Revertant colonies, which lost the ability to express human HMG-14, regained the ability to differentiate into myotubes. Inhibition of myoblast differentiation by aberrantly expressed HMG-14 correlated with down-regulation of myogenic determination factors. The results suggest that proper cellular differentiation requires regulated expression of HMG-14 protein and are consistent with the possibility that this protein may be involved in gene regulation.

Inhibition of myoblast differentiation by lack of zinc

The impact of restricted zinc availability on myoblast differentiation was investigated. Lack of zinc prevented myoblast fusion and the increase in muscle-specific creatine kinase activity. The depression of activity of creatine kinase in the zinc-deficient cultures was accompanied by a similar decrease in the concentration of creatine kinase mRNA and was apparent even when fusion of the myoblasts was inhibited by cytochalasin B. Thus zinc appears to be necessary for the expression of creatine kinase during myoblast differentiation.

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.