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.     

Neural cell adhesion molecule (NCAM) and myoblast fusion

Considerable evidence points to an involvement of neural cell adhesion molecule (NCAM) in myoblast fusion. Changes in the level of NCAM expression, isoform specificity, and localization in muscle cells and tissues correspond to key morphogenetic events during muscle differentiation and repair. Furthermore, anti-NCAM antibodies have been shown by others to reduce the rate of myoblast fusion, whereas overexpression of NCAM cDNAs increases the rate of myoblast fusion compared to controls. In this study we have used a novel fusion assay based on intracistronic complementation of lacZ, in combination with fluorescent X-gal histochemistry and immunocytochemistry to assess levels of NCAM expression in individual muscle cells. Our results indicate that a substantial proportion of newly fused myoblasts have NCAM expression levels unchanged from the levels of the surrounding unfused population suggesting that increased expression of NCAM is not required for wild-type myoblasts to fuse. Moreover, pure populations of primary myoblasts isolated from mice homozygous null for NCAM and therefore lacking the molecule, when placed in differentiation medium, consistently fused to form contractile myotubes with kinetics equivalent to wild-type primary myoblasts. We conclude that the increase in expression of NCAM, although typically observed during myogenesis, is not essential to myoblast fusion to form myotubes.     

Targeted down-regulation of caveolin-3 is sufficient to inhibit myotube formation in differentiating C2C12 myoblasts. Transient activation of p38 mitogen-activated protein kinase is required for induction of caveolin-3 expression and subsequent myotube formation.

Caveolin-3 is the principal structural protein of caveolae membrane domains in striated muscle cells. Caveolin-3 mRNA and protein expression are dramatically induced during the differentiation of C2C12 skeletal myoblasts, coincident with myoblast fusion. In these myotubes, caveolin-3 localizes to the sarcolemma (muscle cell plasma membrane), where it associates with the dystrophin-glycoprotein complex. However, it remains unknown what role caveolin-3 plays in myoblast differentiation and myotube formation. Here, we employ an antisense approach to derive stable C2C12 myoblasts that fail to express the caveolin-3 protein. We show that C2C12 cells harboring caveolin-3 antisense undergo differentiation and express normal amounts of four muscle-specific marker proteins. However, C2C12 cells harboring caveolin-3 antisense fail to undergo myoblast fusion and, therefore, do not form myotubes. Interestingly, treatment with specific p38 mitogen-activated protein kinase inhibitors blocks both myotube formation and caveolin-3 expression, but does not affect the expression of other muscle-specific proteins. In addition, we find that three human rhabdomyosarcoma cell lines do not express caveolin-3 and fail to undergo myoblast fusion. Taken together, these results support the idea that caveolin-3 expression is required for myoblast fusion and myotube formation, and suggest that p38 is an upstream regulator of caveolin-3 expression.     

Developmental regulation of a cadherin during the differentiation of skeletal myoblasts

Cadherins are a family of integral membrane glycoproteins which mediate calcium-dependent intercellular adhesion in vertebrate species. Here we present evidence that fusion-competent rat L6 myoblasts express a cadherin (Mr 127 kDa). The levels of this cadherin were found to be developmentally regulated. Maximal levels were expressed prior to fusion. The increase in cadherin levels observed during differentiation was prevented by the differentiation inhibitor, 5-bromo-2'-deoxyuridine. L6 myoblasts grown in the presence of anti-cadherin antibodies exhibited an altered morphology in comparison to control cultures, coupled with decreased myoblast fusion. These data indicate that the developmental regulation of cadherin is part of the program of terminal differentiation of skeletal myoblasts, and that cadherins are involved in the process of myoblast fusion.     

Long-term analysis of differentiation in human myoblasts repopulated with mitochondria harboring mtDNA mutations

Short-term analysis of myogenesis in respiration-deficient myoblasts demonstrated that respiratory chain dysfunction impairs muscle differentiation. To investigate long-term consequences of a deficiency in oxidative phosphorylation on myogenesis, we quantitated myoblast fusion and expression of sarcomeric myosin in respiration-deficient myogenic cybrids. We produced viable myoblasts harboring exclusively mtDNA with large-scale deletions by treating wild-type myoblasts with rhodamine 6G and fusing them with cytoplasts homoplasmic for two different mutated mtDNAs. Recovery of growth in transmitochondrial myoblasts demonstrated that respiratory chain function is not required for recovery of rhodamine 6G-treated cells. Both transmitochondrial respiration-deficient cultures exhibited impaired myoblast fusion. Expression of sarcomeric myosin was also delayed in deficient myoblasts. However, 4 weeks after induction of differentiation, one cell line was able to quantitatively recover its capacity to form postmitotic muscle cells. This indicates that while oxidative phosphorylation is an important source of ATP for muscle development, myoblast differentiation can be supported entirely by glycolysis.     

Coordinate regulation of contractile protein synthesis during myoblast differentiation

The synthesis of contractile proteins has been studied during the differentiation of quail skeletal muscle myoblasts in culture. Myoblast differentiation was synchronized by transferring secondary cultures of rapidly dividing myoblasts into medium lacking cell division-promoting factors. Cultures at various stages of differentiation were then pulse-labeled with 35S-methionine, and cell extracts were resolved by electrophoresis on two-dimensional gels. Incorporation into specific proteins was quantitated by autoradiography and fluorography using a scanning densitometer. Contractile proteins synthesized by muscle cultures were identified by their co-electrophoresis on two-dimensional gels with contracile proteins purified from quail breast muscle. Our results show that the synthesis of myosin heavy chain, two myosin light chains, two subunits of troponin and two subunits of tropomyosin is first detected at the time of myoblast fusion and then rapidly increase at least 500 fold to maximum rates which remain constant in muscle fibers. Both the kinetics of activation and the molar rates of synthesis of these contractile proteins are virtually identical. Muscle-specific actin (alpha) synthesis also increases at the time of myoblast fusion, but this actin (alpha) is synthesized at 3 times the rate of other contractile proteins. The synthesis of 30 other muscle cell proteins was quantitated, and most of these are shown to follow different patterns of regulation. From these results, we conclude that the contractile proteins are regulated coordinately during myoblast differentiation.     

Nestin as a regulator of Cdk5 in differentiating myoblasts

Many types of progenitor cells are distinguished by the expression of the intermediate filament protein nestin, a frequently used stem cell marker, the physiological roles of which are still unknown. Whereas myogenesis is characterized by dynamically regulated nestin levels, we studied how altering nestin levels affects myoblast differentiation. Nestin determined both the onset and pace of differentiation. Whereas depletion of nestin by RNAi strikingly accelerated the process, overexpression of nestin completely inhibited differentiation. Nestin down-regulation augmented the early stages of differentiation, at the level of cell-cycle withdrawal and expression of myogenic markers, but did not affect proliferation of undifferentiated dividing myoblasts. Nestin regulated the cleavage of the Cdk5 activator protein p35 to its degradation-resistant form, p25. In this way, nestin has the capacity to halt myoblast differentiation by inhibiting sustained activation of Cdk5 by p25, which is critical for the progress of differentiation. Our results imply that nestin regulates the early stages of myogenesis rather than maintains the undifferentiated state of progenitor cells. In the bidirectional interrelationship between nestin and Cdk5, Cdk5 regulates the organization and stability of its own nestin scaffold, which in turn controls the effects of Cdk5. This nestin-Cdk5 cross-talk sets the pace of muscle differentiation.     

Sonic hedgehog inhibits the terminal differentiation of limb myoblasts committed to the slow muscle lineage

The proliferation, differentiation, and fusion of a small number of myogenic precursor cells must be precisely regulated during development to ensure the proper size, organization, and function of the limb musculature. We have examined the role of Sonic hedgehog (Shh) in these processes by both augmentation and inhibition of the Shh-mediated signaling pathway. Our data show that Shh regulates muscle development by repressing the terminal differentiation of early myogenic precursor cells and does not function as a myoblast mitogen. Shh function in hypaxial muscle appears to be spatially restricted to the early myoblast population within the ventral muscles of the posterior region of the limb. Furthermore, Shh appears to act as a permissive, rather than an inductive, signal for slow MyHC expression in myoblasts. Our data thus provide the foundation for a new hypothesis for Shh function in hypaxial skeletal muscle development.     

Spatial and functional restriction of regulatory molecules during mammalian myoblast fusion

Myoblast fusion is a highly regulated process that is key for forming skeletal muscle during development and regeneration in mammals. Much remains to be understood about the molecular regulation of myoblast fusion. Some molecules that influence mammalian muscle fusion display specific cellular localization during myogenesis. Such molecules can be localized to the contact region between two fusing cells either in both cells or only in one of the cells. How distinct localization of molecules contributes to fusion is not clear. Further complexity exists as other molecules are functionally restricted to myoblasts at later stages of myogenesis to regulate their fusion with multinucleated myotubes. This review examines these three categories of molecules and discusses how spatial and functional restriction may contribute to the formation of a multinucleated cell. Understanding how and why molecules become restricted in location or function is likely to provide further insights into the mechanisms regulating mammalian muscle fusion.     

p75NTR-mediated signaling promotes the survival of myoblasts and influences muscle strength

During muscle development, the p75(NTR) is expressed transiently on myoblasts. The temporal expression pattern of the receptor raises the possibility that the receptor is influencing muscle development. To test this hypothesis, p75(NTR)-deficient mutant mice were tested for muscle strength by using a standard wire gripe strength test and were found to have significantly decreased strength relative to that of normal mice. When normal mybolasts were examined in vivo for expression of NGF receptors, p75(NTR) was detected on myoblasts but the high affinity NGF receptor, trk A, was not co-expressed with p75(NTR). In vitro, proliferating C2C12 and primary myoblasts co-expressed the p75(NTR) and MyoD, but immunofluorescent analysis of primary myoblasts and RT-PCR analysis of C2C12 mRNA revealed that myoblasts were devoid of trk A. In contrast to the cell death functions that characterize the p75(NTR) in neurons, p75(NTR)-positive primary and C2C12 myoblasts did not differentiate or undergo apoptosis in response to neurotrophins. Rather, myoblasts survived and even proliferated when grown at subconfluent densities in the presence of the neurotrophins. Furthermore, when myoblasts treated with NGF were lysed and immunoprecipitated with antibodies against phosphorylated I-kappaB and AKT, the cells contained increased levels of both phospho-proteins, both of which promote cell survival. By contrast, neurotrophin-treated myoblasts did not induce phosphorylation of Map Kinase p42/44 or p38, indicating the survival was not mediated by the trk A receptor. Taken together, the data indicate that the p75(NTR) mediates survival of myoblasts prior to differentiation and that the activity of this receptor during myogenesis is important for developing muscle.     

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 differentiating prefusion myoblasts connexin43 gap junction coupling is upregulated before myoblast alignment then reduced in post-mitotic cells

Previously we have shown that during in vivo muscle regeneration differentiating rat primary myoblasts transiently upregulate connexin43 (Cx43) gap junctions and leave cell cycle synchronously. Here, we studied the temporal regulation of Cx expression in relation to functional dye coupling in allogenic primary myoblast cultures using western blotting, immuno-confocal microscopy and dye transfer assays. As in vivo, Cx43 was the only Cx isotype out of Cx26, 32, 37, 40, 43 and 45 found in cultured rat myoblasts by immunostaining. Cultured myoblasts showed similar temporal regulation of Cx43 expression and phenotypic maturation to those regenerating in vivo. Cx43 protein was progressively upregulated in prefusion myoblasts, first by the cytoplasmic assembly in sparse myoblast meshworks and then in cell membrane particles in aligned cells. Dye injection using either Lucifer Yellow alone, Cascade Blue with a non-junction permeant FITC-dextran revealed an extensive gap junction coupling between the sparse interacting myoblasts and a reduced communication between the aligned, but still prefused cells. The aligned myoblasts, uniformly upregulate p21^waf1/cip1 and p27^kip1 cell cycle control proteins. Taken together, in prefusion myoblasts less membrane-bound Cx43 was found to mediate substantially more efficient dye coupling in the growing cell fraction than those in the aligned post-mitotic myoblasts. These and our in vivo results in early muscle differentiation are consistent with the role of Cx43 gap junctions in synchronizing cell cycle control of myoblasts to make them competent for a coordinated syncytial fusion.     

Ornithine decarboxylase is upregulated by the androgen receptor in skeletal muscle and regulates myoblast proliferation

The aim of this study is to determine if the Odc1 gene, which encodes ornithine decarboxylase (ODC), the rate-limiting enzyme in polyamine biosynthesis, is directly regulated by the androgen receptor (AR) in skeletal muscle myoblasts and if Odc1 regulates myoblast proliferation and differentiation. We previously showed that expression of Odc1 is decreased in muscle from AR knockout male mice. In this study, we show in vivo that Odc1 expression is also decreased >60% in muscle from male muscle-specific AR knockout mice. In normal muscle homeostasis, Odc1 expression is regulated by age and sex, reflecting testosterone levels, as muscle of adult male mice expresses high levels of Odc1 compared with age-matched females and younger males. In vitro, expression of Odc1 is 10- and 1.5-fold higher in proliferating mouse C(2)C(12) and human skeletal muscle myoblasts, respectively, than in differentiated myotubes. Dihydrotestosterone increases Odc1 levels 2.7- and 1.6-fold in skeletal muscle cell myoblasts after 12 and 24 h of treatment, respectively. Inhibition of ODC activity in C(2)C(12) myoblasts by α-difluoromethylornithine decreases myoblast number by 40% and 66% following 48 and 72 h of treatment, respectively. In contrast, overexpression of Odc1 in C(2)C(12) myoblasts results in a 27% increase in cell number vs. control when cells are grown under differentiation conditions for 96 h. This prolonged proliferation is associated with delayed differentiation, with reduced expression of the differentiation markers myogenin and Myf6 in Odc1-overexpressing cells. In conclusion, androgens act via the AR to upregulate Odc1 in skeletal muscle myoblasts, and Odc1 promotes myoblast proliferation and delays differentiation.     

PKCθ signaling is required for myoblast fusion by regulating the expression of caveolin-3 and β1D integrin upstream focal adhesion kinase

Fusion of mononucleated myoblasts to form multinucleated myofibers is an essential phase of skeletal myogenesis, which occurs during muscle development as well as during postnatal life for muscle growth, turnover, and regeneration. Many cell adhesion proteins, including integrins, have been shown to be important for myoblast fusion in vertebrates, and recently focal adhesion kinase (FAK), has been proposed as a key mediator of myoblast fusion. Here we focused on the possible role of PKC, the PKC isoform predominantly expressed in skeletal muscle, in myoblast fusion. We found that the expression of PKC is strongly up-regulated following freeze injury-induced muscle regeneration, as well as during in vitro differentiation of satellite cells (SCs; the muscle stem cells). Using both PKC knockout and muscle-specific PKC dominant-negative mutant mouse models, we observed delayed body and muscle fiber growth during the first weeks of postnatal life, when compared with wild-type (WT) mice. We also found that myofiber formation, during muscle regeneration after freeze injury, was markedly impaired in PKC mutant mice, as compared with WT. This phenotype was associated with reduced expression of the myogenic differentiation program executor, myogenin, but not with that of the SC marker Pax7. Indeed in vitro differentiation of primary muscle-derived SCs from PKC mutants resulted in the formation of thinner myotubes with reduced numbers of myonuclei and reduced fusion rate, when compared with WT cells. These effects were associated to reduced expression of the profusion genes caveolin-3 and β1D integrin and to reduced activation/phosphorylation of their up-stream regulator FAK. Indeed the exogenous expression of a constitutively active mutant form of PKC in muscle cells induced FAK phosphorylation. Moreover pharmacologically mediated full inhibition of FAK activity led to similar fusion defects in both WT and PKC-null myoblasts. We thus propose that PKC signaling regulates myoblast fusion by regulating, at least in part, FAK activity, essential for profusion gene expression.     

Insulin receptor substrate protein 53kDa (IRSp53) is a negative regulator of myogenic differentiation

Fusion of mononucleated myoblasts to generate multinucleated myotubes is a critical step in skeletal muscle development. Filopodia, the actin cytoskeleton based membrane protrusions, have been observed early during myoblast fusion, indicating that they could play a direct role in myogenic differentiation. The control of filopodia formation in myoblasts remains poorly understood. Here we show that the expression of IRSp53 (Insulin Receptor Substrate protein 53kDa), a known regulator of filopodia formation, is down-regulated during differentiation of both mouse primary myoblasts and a mouse myoblast cell line C2C12. Over-expression of IRSp53 in C2C12 cells led to induction of filopodia and decrease in cell adhesion, concomitantly with inhibition of myogenic differentiation. In contrast, knocking down the IRSp53 expression in C2C12 cells led to a small but significant increase in myotube development. The decreased cell adhesion of C2C12 cells over-expressing IRSp53 is correlated with a reduction in the number of vinculin patches in these cells. Mutations in the conserved IMD domain (IRSp53 and MIM (missing in metastasis) homology domain) or SH3 domain of IRSp53 abolished the ability of this protein to inhibit myogenic differentiation and reduce cell adhesion. Over-expression of the IMD domain alone was sufficient to decrease the cell-extracellular matrix adhesion and to inhibit myogenesis in a manner dependent on its function in membrane shaping. Based on our data, we propose that IRSp53 is a negative regulator of myogenic differentiation which correlates with the observed down regulation of IRSp53 expression during myoblast differentiation to myotubes.