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.     

Retrovirally mediated overexpression of insulin-like growth factor binding protein 4: Evidence that insulin-like growth factor is required for skeletal muscle differentiation

Recent studies have indicated that the insulin-like growth factors (IGFs) stimulate skeletal myoblast proliferation and differentiation. However, the question of whether IGFs are required for myoblast differentiation has not been resolved. To address this issue directly, we used a retroviral vector (LBP4SN) to develop a subline of mouse C2 myoblasts (C2-BP4) that constitutively overexpress IGF binding protein-4 (IGFBP-4). A control C2 myoblast subline (C2-LNL6) was also developed by using the LNL6 control retroviral vector. C2-BP4 myoblasts expressed sixfold higher levels of IGFBP-4 protein than C2-LNL6 myoblasts. ¹²⁵I-IGF-I cross linking indicated that IGFBP-4 overexpression reduced IGF access to the type-1 IGF receptor tenfold. At low plating densities, myoblast proliferation was inhibited, and myoblast differentiation was abolished in C2-BP4 cultures compared with C2-LNL6 cultures. At high plating densities in which nuclear numbers were equal in the two sets of cultures, C2-BP4 myoblast differentiation was inhibited completely. Differentiation was restored in C2-BP4 cells by treatment with high levels of exogenous IGF-I or with des(1–3)IGF-I, an analog of IGF-I with reduced affinity for IGFBPs. These findings confirm the hypothesis that positive differentiation signals from the IGFs are necessary for C2 myoblast differentiation, and they suggest that the present model of myogenic differentiation, which involves only negative external control of differentiation by mitogens, may be incomplete. J. Cell. Physiol. 175:109–120, 1998. © 1998 Wiley-Liss, Inc.     

Overexpression of the type-1 insulin-like growth factor receptor increases ligand-dependent proliferation and differentiation in bovine skeletal myogenic cultures

Previous studies demonstrated that overexpression of the type-1 insulin-like growth factor (IGF) receptor (IGF-1R) in skeletal myogenic cell lines increased proliferation and differentiation responses to IGF. However, it was unclear if such manipulations in primary, untransformed skeletal myogenic cells would result in modulation of these responses, which may be more stringently regulated in primary cells than in myogenic cell lines. In this study, low passage untransformed fetal bovine myogenic cultures were infected with a replication-deficient retroviral expression vector (LISN) coding for the human IGF-1R or with a control retroviral vector (LNL6). Bovine myogenic cultures infected with the LISN vector (Bov-LISN) displayed ten times more IGF-1Rs than controls (Bov-LNL6). Bov-LISN myogenic cultures exhibited elevated rates of IGF-I-stimulated proliferation and increased rates of terminal differentiation which were reduced to control levels by the anti-human IGF-1R antibody αIR3. These findings indicate overexpression of the IGF-1R can enhance IGF sensitivity and thereby modify the proliferation and differentiation behavior of untransformed low passage myoblasts. Such manipulations may be useful to increase muscle mass in clinical or agricultural applications. © 1996 Wiley-Liss, Inc.     

Induced Wnt5a expression perturbs embryonic outgrowth and intestinal elongation, but is well-tolerated in adult mice

Embryonic mesoangioblasts are the in vitro counterpart of vessel-associated progenitors, able to differentiate into different mesoderm cell types. To investigate signals recruiting these progenitors to a skeletal myogenic fate, we developed an in vitro assay, based upon co-culture of E11.5 dorsal aorta (from MLC3F-nLacZ transgenic embryos, expressing nuclear beta galactosidase only in striated muscle) with differentiating C2C12 or primary myoblasts. Under these conditions muscle differentiation from cells originating from the vessel can be quantified by counting the number of beta gal+nuclei. Results indicated that Noggin (but not Follistatin, Chordin or Gremlin) stimulates while BMP2/4 inhibits myogenesis from dorsal aorta progenitors; neutralizing antibodies and shRNA greatly reduce these effects. In contrast, TGF-β1, VEGF, Wnt7A, Wnt3A, bFGF, PDGF-BB and IGF1 have no effect. Sorting experiments indicated that the majority of these myogenic progenitors express the pericyte marker NG2. Moreover they are abundant in the thoracic segment at E10.5 and in the iliac bifurcation at E11.5 suggesting the occurrence of a cranio-caudal wave of competent cells along the aorta. BMP2 is expressed in the dorsal aorta and Noggin in newly formed muscle fibers suggesting that these two tissues compete to recruit mesoderm cells to a myogenic or to a perithelial fate in the developing fetal muscle.     

Fibroblast growth factor inducible 14 (Fn14) is required for the expression of myogenic regulatory factors and differentiation of myoblasts into myotubes. Evidence for TWEAK-independent functions of Fn14 during myogenesis

Fibroblast growth factor-inducible 14 (Fn14), distantly related to tumor necrosis factor receptor superfamily and a receptor for TWEAK cytokine, has been implicated in several biological responses. In this study, we have investigated the role of Fn14 in skeletal muscle formation in vitro. Flow cytometric and Western blot analysis revealed that Fn14 is highly expressed on myoblastic cell line C2C12 and mouse primary myoblasts. The expression of Fn14 was decreased upon differentiation of myoblasts into myotubes. Suppression of Fn14 expression using RNA interference inhibited the myotube formation in both C2C12 and primary myoblast cultures. Fn14 was required for the transactivation of skeletal alpha-actin promoter and the expression of specific muscle proteins such as myosin heavy chain fast type and creatine kinase. RNA interference-mediated knockdown of Fn14 receptor in C2C12 myoblasts decreased the levels of myogenic regulatory factors MyoD and myogenin upon induction of differentiation. Conversely, overexpression of MyoD increased differentiation in Fn14-knockdown C2C12 cultures. Suppression of Fn14 expression in C2C12 myoblasts also inhibited the differentiation-associated increase in the activity of serum response factor and RhoA GTPase. In addition, our data suggest that the role of Fn14 during myogenic differentiation could be independent of TWEAK cytokine. Collectively, our study suggests that the Fn14 receptor is required for the expression of myogenic regulatory factors and differentiation of myoblasts into myotubes.     

Interaction of scaffolding adaptor protein Gab1 with tyrosine phosphatase SHP2 negatively regulates IGF-I-dependent myogenic differentiation via the ERK1/2 signaling pathway

Grb2-associated binder 1 (Gab1) coordinates various receptor tyrosine kinase signaling pathways. Although skeletal muscle differentiation is regulated by some growth factors, it remains elusive whether Gab1 coordinates myogenic signals. Here, we examined the molecular mechanism of insulin-like growth factor-I (IGF-I)-mediated myogenic differentiation, focusing on Gab1 and its downstream signaling. Gab1 underwent tyrosine phosphorylation and subsequent complex formation with protein-tyrosine phosphatase SHP2 upon IGF-I stimulation in C2C12 myoblasts. On the other hand, Gab1 constitutively associated with phosphatidylinositol 3-kinase regulatory subunit p85. To delineate the role of Gab1 in IGF-I-dependent signaling, we examined the effect of adenovirus-mediated forced expression of wild-type Gab1 (Gab1(WT)), mutated Gab1 that is unable to bind SHP2 (Gab1(DeltaSHP2)), or mutated Gab1 that is unable to bind p85 (Gab1(Deltap85)), on the differentiation of C2C12 myoblasts. IGF-I-induced myogenic differentiation was enhanced in myoblasts overexpressing Gab1(DeltaSHP2), but inhibited in those overexpressing either Gab1(WT) or Gab1(Deltap85). Conversely, IGF-I-induced extracellular signal-regulated kinase 1/2 (ERK1/2) activation was significantly repressed in myoblasts overexpressing Gab1(DeltaSHP2) but enhanced in those overexpressing either Gab1(WT) or Gab1(Deltap85). Furthermore, small interference RNA-mediated Gab1 knockdown enhanced myogenic differentiation. Overexpression of catalytic-inactive SHP2 modulated IGF-I-induced myogenic differentiation and ERK1/2 activation similarly to that of Gab1(DeltaSHP2), suggesting that Gab1-SHP2 complex inhibits IGF-I-dependent myogenesis through ERK1/2. Consistently, the blockade of ERK1/2 pathway reversed the inhibitory effect of Gab1(WT) overexpression on myogenic differentiation, and constitutive activation of the ERK1/2 pathway suppressed the enhanced myogenic differentiation by overexpression of Gab1(DeltaSHP2). Collectively, these data suggest that the Gab1-SHP2-ERK1/2 signaling pathway comprises an inhibitory axis for IGF-I-dependent myogenic differentiation.     

TIEG1 negatively controls the myoblast pool indispensable for fusion during myogenic differentiation of C2C12 cells

The transforming growth factor (TGF)-β inducible early gene (TIEG)-1 is implicated in the control of cell proliferation, differentiation, and apoptosis in some cell types. Since TIEG1 functioning may be associated with TGF-β, a suppressor of myogenesis, TIEG1 is also likely to be involved in myogenesis. Therefore, we investigated the function of TIEG1 during myogenic differentiation in vitro using the murine myoblasts cell line, C2C12. TIEG1 expression increased during differentiation of C2C12 cells. Constitutive expression of TIEG1 reduced survival and decreased myotube formation. Conversely, knocking down TIEG1 expression increased the number of viable cells during differentiation, and accelerated myoblast fusion into multinucleated myotubes. However, expression of the myogenic differentiation marker, myogenin, remained unaffected by TIEG1 knockdown. The mechanism underlying these events was investigated by focusing on the regulation of myoblast numbers after induction of differentiation. The knockdown of TIEG1 led to changes in cell cycle status and inhibition of apoptosis during the initial stages of differentiation. Microarray and real-time PCR analyses showed that the regulators of cell cycle progression were highly expressed in TIEG1 knockdown cells. Therefore, TIEG1 is a negative regulator of the myoblast pool that causes inhibition of myotube formation during myogenic differentiation.     

Evidence for the participation of nerve growth factor and its low-affinity receptor (p75NTR) in the regulation of the myogenic program

We have studied expression and function of neurotrophins and their receptors during myogenic differentiation of C2C12 cells, a clonal cell line derived from mouse muscle that is capable of in vitro differentiation. The genes coding for nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) and their common low-affinity receptor p75^{neurotrophin receptor} (p75^NTR) were shown to be expressed in C2C12 myoblasts and downregulated during myogenic differentiation and fusion into myotubes. Cocultures with dorsal root ganglia from day 8 chick embryos revealed neurite-promoting activities of C2C12 cells that ceased with myogenic differentiation. These data suggest a temporal and developmental window for the effect of myogenic cell-derived neurotrophins on neuronal as well as on myogenic cell populations. NGF was shown to increase DNA synthesis and cell growth of C2C12 myoblasts and to enhance myogenic differentiation in this cell line. We present evidence that NGF-mediated processes take place at stages preceding myogenic differentiation. Enhanced muscle differentiation was also seen in p75^NTR-overexpressing C2C12 myoblasts which maintained high levels of receptors but ceased to produce NGF during differentiation. In contrast, when exogenous NGF was present at the onset of myogenic differentiation of receptor-overexpressing cells, muscle cell development was strongly repressed. This indicates that downregulation of p75^NTR is necessary for guiding myogenic cells towards terminal differentiation. Since none of the trk high-affinity neurotrophin receptors could be demonstrated in C2C12 cells, we conclude that NGF mediates its nonneurotrophic effect via its low-affinity receptor in an autocrine fashion. J. Cell. Physiol. 176:10–21, 1998. © 1998 Wiley-Liss, Inc.     

Overexpression of insulin-like growth factor-II induces accelerated myoblast differentiation

Previous studies have shown that exogenous insulin-like growth factors (IGFs) can stimulate the terminal differentiation of skeletal myoblasts in culture and have established a correlation between the rate and the extent of IGF-II secretion by muscle cell lines and the rate of biochemical and morphological differentiation. To investigate the hypothesis that autocrine secretion of IGF-II plays a critical role in stimulating spontaneous myogenic differentiation in vitro, we have established C2 muscle cell lines that stably express a mouse IGF-II cDNA under control of the strong, constitutively active Moloney sarcoma virus promoter, enabling us to study directly the effects of IGF-II overproduction. Similar to observations with other muscle cell lines, IGF-II overexpressing myoblasts proliferated normally in growth medium containing 20% fetal serum, but they underwent enhanced differentiation compared with controls when incubated in low-serum differentiation medium. Accelerated differentiation of IGF-II overexpressing C2 cells was preceded by the rapid induction of myogenin mRNA and protein expression (within 1 h, compared with 24–48 h in controls) and was accompanied by an enhanced proportion of the retinoblastoma protein in an underphosphrylated and potentially active form, by a marked increase in activity of the muscle-specific enzyme, creatine phosphokinase, by extensive myotube formation by 48 h, and by elevated secretion of IGF binding protein-5 when compared with controls. These results confirm a role for IGF-II as an autocrine/paracrine differentiation factor for skeletal myoblasts, and they define a model cell system that will be useful in determining the biochemical mechanisms of IGF action in cellular differentiation. © 1996 Wiley-Liss, Inc.     

TGF-β's delay skeletal muscle progenitor cell differentiation in an isoform-independent manner

Satellite cells are a quiescent heterogenous population of mononuclear stem and progenitor cells which, once activated, differentiate into myotubes and facilitate skeletal muscle repair or growth. The Transforming Growth Factor-β (TGF-β) superfamily members are elevated post-injury and their importance in the regulation of myogenesis and wound healing has been demonstrated both in vitro and in vivo. Most studies suggest a negative role for TGF-β on satellite cell differentiation. However, none have compared the effect of these three isoforms on myogenesis in vitro. This is despite known isoform-specific effects of TGF-β1, -β2 and -β3 on wound repair in other tissues. In the current study we compared the effect of TGF-β1, -β2 and -β3 on proliferation and differentiation of the C2C12 myoblast cell-line. We found that, irrespective of the isoform, TGF-β increased proliferation of C2C12 cells by changing the cellular localisation of PCNA to promote cell division and prevent cell cycle exit. Concomitantly, TGF-β1, -β2 and -β3 delayed myogenic commitment by increasing MyoD degradation and decreasing myogenin expression. Terminal differentiation, as measured by a decrease in myosin heavy chain (MHC) expression, was also delayed. These results demonstrate that TGF-β promotes proliferation and delays differentiation of C2C12 myoblasts in an isoform-independent manner.     

miR-22 regulates C2C12 myoblast proliferation and differentiation by targeting TGFBR1

Recently, miR-22 was found to be differentially expressed in different skeletal muscle growth period, indicated that it might have function in skeletal muscle myogenesis. In this study, we found that the expression of miR-22 was the most in skeletal muscle and was gradually up-regulated during mouse myoblast cell (C2C12 myoblast cell line) differentiation. Overexpression of miR-22 repressed C2C12 myoblast proliferation and promoted myoblast differentiation into myotubes, whereas inhibition of miR-22 showed the opposite results. During myogenesis, we predicted and verified transforming growth factor beta receptor 1 (TGFBR1), a key receptor of the TGF-β/Smad signaling pathway, was a target gene of miR-22. Then, we found miR-22 could regulate the expression of TGFBR1 and down-regulate the Smad3 signaling pathway. Knockdown of TGFBR1 by siRNA suppressed the proliferation of C2C12 cells but induced its differentiation. Conversely, overexpression of TGFBR1 significantly promoted proliferation but inhibited differentiation of the myoblast. Additionally, when C2C12 cells were treated with different concentrations of transforming growth factor beta 1 (TGF-β1), the level of miR-22 in C2C12 cells was reduced. The TGFBR1 protein level was significantly elevated in C2C12 cells treated with TGF-β1. Moreover, miR-22 was able to inhibit TGF-β1-induced TGFBR1 expression in C2C12 cells. Altogether, we demonstrated that TGF-β1 inhibited miR-22 expression in C2C12 cells and miR-22 regulated C2C12 cell myogenesis by targeting TGFBR1.     

Role of endogenous TGF-β family in myogenic differentiation of C2C12 cells

The present study evaluated endogenous activities and the role of BMP and transforming growth factor-β (TGF-β), representative members of the TGF-β family, during myotube differentiation in C2C12 cells. Smad phosphorylation at the C-terminal serines was monitored, since TGF-β family members signal via the phosphorylation of Smads in a ligand-dependent manner. Expression of phosphorylated Smad1/5/8, which is an indicator of BMP activity, was higher before differentiation, and rapidly decreased after differentiation stimulation. Differentiation-related changes were consistent with those in the expression of Ids, well-known BMP-responsive genes. Treatment with inhibitors of BMP type I receptors or noggin in C2C12 myoblasts down-regulated the expression of myogenic regulatory factors, such as Myf5 and MyoD, leading to impaired myotube formation. Addition of BMP-2 during the myoblast phase also inhibited myotube differentiation through the down-regulation of Myf5 and MyoD. In contrast to endogenous BMP activity, the phosphorylation of Smad2, a TGF-β-responsive Smad, was higher 8-16 days after differentiation stimulation. A-83-01, an inhibitor of TGF-β type I receptor, increased the expression of Myf5 and MyoD, and enhanced myotube formation. The present results reveal that endogenous activities of the TGF-β family are changed during myogenesis in a pathway-specific manner, and that the activities are required for myogenesis.     

Transforming growth factor-beta isoform expression in insulin-like growth factor stimulated myogenesis

Transforming growth factor betas (TGF-b?s) are the defining members of a superfamily of small proteins that are involved in the regulation of development and morphogenesis in a wide array of systems. Previous studies have demonstrated that TGF-b?s both inhibit and, under specialized conditions, induce the differentiation of myoblasts. TGF-b?s have been shown to be secreted by mouse C2C12 myoblast cultures undergoing differentiation. Insulin-like growth factors (IGFs) have also been shown to be secreted by myoblasts and to induce myogenesis. This study characterizes the effects of IGF treatment on the expression and secretion of TGF-b?s in the IGF-sensitive L6A1 myoblast line. IGF downregulated the expression of TGF-b?3 in a concentration-dependent manner at 24 and 48 hours; TGF-b?1 was not sensitive to IGF treatment at 24 hours but was downregulated by IGFs at 48 hours. This downregulation was mediated by the type I IGF receptor and modulated by IGF binding proteins secreted by the myoblasts. Some reexpression of TGF-b?1 and TGF-b?3 mRNAs was observed after extensive morphological differentiation had occurred. These results support the hypothesis that IGFs act through the IGF type I receptor as part of a concerted mechanism to modulate expression of the TGF-b? genes, as part of a coordinated set of changes associated with terminal myogenic differentiation. © 1995 Wiley-Liss, Inc.     

Stac3 Inhibits Myoblast Differentiation into Myotubes

The functionally undefined Stac3 gene, predicted to encode a SH3 domain- and C1 domain-containing protein, was recently found to be specifically expressed in skeletal muscle and essential to normal skeletal muscle development and contraction. In this study we determined the potential role of Stac3 in myoblast proliferation and differentiation, two important steps of muscle development. Neither siRNA-mediated Stac3 knockdown nor plasmid-mediated Stac3 overexpression affected the proliferation of C2C12 myoblasts. Stac3 knockdown promoted the differentiation of C2C12 myoblasts into myotubes as evidenced by increased fusion index, increased number of nuclei per myotube, and increased mRNA and protein expression of myogenic markers including myogenin and myosin heavy chain. In contrast, Stac3 overexpression inhibited the differentiation of C2C12 myoblasts into myotubes as evidenced by decreased fusion index, decreased number of nuclei per myotube, and decreased mRNA and protein expression of myogenic markers. Compared to wild-type myoblasts, myoblasts from Stac3 knockout mouse embryos showed accelerated differentiation into myotubes in culture as evidenced by increased fusion index, increased number of nuclei per myotube, and increased mRNA expression of myogenic markers. Collectively, these data suggest an inhibitory role of endogenous Stac3 in myoblast differentiation. Myogenesis is a tightly controlled program; myofibers formed from prematurely differentiated myoblasts are dysfunctional. Thus, Stac3 may play a role in preventing precocious myoblast differentiation during skeletal muscle development.     

In vitro characterization of proliferation and differentiation of trout satellite cells

Fish satellite cells have been extracted from various species, but the myogenic characteristics of these cells in culture remain largely unknown. We show here that 60%-70% of the adherent cells are myogenic based on their immunoreactivity for the myogenic regulatory factor MyoD. In DMEM containing 10% fetal calf serum (FCS), trout myoblasts display rapid expression of myogenin (18% of myogenin-positive cells at day 2) combined with rapid fusion into myotubes (50% of myogenin-positive nuclei and 30% nuclei in myosin heavy chain [MyHC]-positive cells at day 7). These kinetics of differentiation are reminiscent of the behavior of fetal myoblasts in mammals. However, not all the myogenic cells differentiate; this subpopulation of cells might correspond to the previously named “reserve” cells. More than 90% of the BrdU-positive cells are also positive for MyoD, indicating that myogenic cells proliferate in vitro. By contrast, less than 1% of myogenin-positive cells are positive for BrdU suggesting that myogenin expression occurs only in post-mitotic cells. In order to maximize either the proliferation or the differentiation of cells, we have defined new culture conditions based on the use of a proliferation medium (F10+10%FCS) and a differentiation medium (DMEM+2%FCS). Three days after switching the medium, the differentiation index (% MyHC-positive nuclei) is 40-fold higher than that in proliferation medium, whereas the proliferation index (% BrdU-positive nuclei) is three-fold lower. Stimulation of cell proliferation by insulin-like growth factor 1 (IGF1), IGF2, and FGF2 is greater in F10 medium. The characterization of these extracted muscle cells thus validates the use of this in vitro system of myogenesis in further studies of the myogenic activity of growth factors in trout.