c-myc inhibition of MyoD and myogenin-initiated myogenic differentiation.

In vertebrate development, a prominent feature of several cell lineages is the coupling of cell cycle regulation with terminal differentiation. We have investigated the basis of this relationship in the skeletal muscle lineage by studying the effects of the proliferation-associated regulator, c-myc, on the differentiation of MyoD-initiated myoblasts. Transient cotransfection assays in NIH 3T3 cells using MyoD and c-myc expression vectors demonstrated c-myc suppression of MyoD-initiated differentiation. A stable cell system was also developed in which MyoD expression was constitutive, while myc levels could be elevated conditionally. Induction of this conditional c-myc suppressed myogenesis effectively, even in the presence of MyoD. c-myc suppression also prevented up-regulation of a relative of MyoD, myogenin, which is normally expressed at the onset of differentiation in all muscle cell lines examined and may be essential for differentiation. Additional experiments tested whether failure to differentiate in the presence of myc could be overcome by providing myogenin ectopically. Cotransfection of c-myc with myogenin, MyoD, or a mixture of myogenin and MyoD showed that neither myogenin alone nor myogenin plus MyoD together could bypass the c-myc block. The effects of c-myc were further dissected by showing that c-myc can inhibit differentiation independently of Id, a negative regulator of muscle differentiation. These results lead us to propose that c-myc and Id constitute independent negative regulators of muscle differentiation, while myogenin and any of the other three related myogenic factors (MyoD, Myf-5, and MRF4/herculin/Myf-6) act as positive regulators.     

Regulation of C2C12 myogenic terminal differentiation by MKK3/p38alpha pathway

The signal transduction pathwaysconnecting cell surface receptors to the activation of muscle-specificpromoters and leading to myogenesis are still largely unknown.Recently, a contribution of the p38 mitogen-activated protein kinase(MAPK) pathway to this process was evoked through the use ofpharmacological inhibitors. We used several mutants of the kinasescomposing this pathway to modulate the activity of the muscle-specificmyosin light chain and myogenin promoters in C2C12 cells by transienttransfections. In addition, we show for the first time, using a stableC2C12 cell line expressing a dominant-negative form of the p38activator MAPK kinase (MKK)3, that a functional p38 MAPK pathway isindeed required for terminal muscle cell differentiation. The mostobvious phenotype of this cell line, besides the inhibition of theactivation of p38, is its inability to undergo terminaldifferentiation. This phenotype is accompanied by a drastic inhibitionof cell cycle and myogenesis markers such as p21, p27, MyoD, andtroponin T, as well as a profound disorganization of the cytoskeleton.

     

Insulin-like growth factor-I stimulates terminal myogenic differentiation by induction of myogenin gene expression.

Stimulation of myogenic differentiation by the insulin-like growth factors (IGFs) has been established for many years, but our attempts to elucidate the mechanism of that stimulation have been successful only in eliminating some likely possibilities. The recent discovery of a family of muscle determination genes has opened a new approach to this question, allowing specific focus on those genes that might play central roles in controlling myogenesis. We now report that IGF-I stimulates terminal myogenic differentiation in L6A1 cells by inducing a large increase in expression of the myogenin gene. This conclusion is supported by the following observations. 1) Myogenin mRNA is elevated by IGF-I, with a concentration dependency that parallels the stimulation of differentiation, including a decrease in stimulation at higher concentrations. 2) The time course of elevation of myogenin mRNA is consistent with its acting as an intermediate in the signalling pathway between occupancy of the IGF-I receptor and induction of expression of muscle-specific genes. 3) Inhibitors of myogenesis also inhibit elevation of myogenin mRNA in response to IGF-I. 4) An antisense oligonucleotide to the N-terminus of myogenin prevents the stimulation of differentiation by IGF-I and IGF-II, but has no effect on other actions of IGF-I on myoblasts. MyoD has been reported not to be expressed in L6 cells, and the expression of myf-5 and herculin/myf-6/MRF4 is reportedly low or undetectable. Thus, the stimulation of differentiation by IGF-I can be attributed largely, if not entirely, to increased expression of the myogenin gene. However, the relatively long time period between addition of the IGFs and elevation of myogenin mRNA as well as the inhibition of this process by several inhibitors indicate that increased myogenin mRNA levels are not a simple direct result of occupation of the IGF-I receptor.     

Rb is required for progression through myogenic differentiation but not maintenance of terminal differentiation

To investigate the requirement for pRb in myogenic differentiation, a floxed Rb allele was deleted either in proliferating myoblasts or after differentiation. Myf5-Cre mice, lacking pRb in myoblasts, died immediately at birth and exhibited high numbers of apoptotic nuclei and an almost complete absence of myofibers. In contrast, MCK-Cre mice, lacking pRb in differentiated fibers, were viable and exhibited a normal muscle phenotype and ability to regenerate. Induction of differentiation of Rb-deficient primary myoblasts resulted in high rates of apoptosis and a total inability to form multinucleated myotubes. Upon induction of differentiation, Rb-deficient myoblasts up-regulated myogenin, an immediate early marker of differentiation, but failed to down-regulate Pax7 and exhibited growth in low serum conditions. Primary myoblasts in which Rb was deleted after expression of differentiated MCK-Cre formed normal multinucleated myotubes that did not enter S-phase in response to serum stimulation. Therefore, Rb plays a crucial role in the switch from proliferation to differentiation rather than maintenance of the terminally differentiated state.     

Neural control of early myogenic differentiation in cultures of mouse somites

Neural tubes, with flanking somite streaks, were isolated from mouse embryos ranging in age from 8 to 11 days post coitus (dpc). The somites were further dissected along the neural tube to obtain one somite streak associated with the neural tube and the other free of nerve cells. The two groups of somites (with and without neural tubes) were dissociated to single cell suspension by a brief incubation with EDTA. High-density micro-mass cultures were established from these two groups of cells and the extent of cell differentiation was assayed by staining the cultures with an anti-myosin antibody. The results obtained indicated that during early somitogenesis (8.5 dpc) the presence of cells from neural tube is necessary for the emergence of myosin-positive cells in culture. At later stages (10.5 dpc), however, the total number of myosin-positive cells appearing in culture is largely independent from the presence of nerve cells. At these later stages, the presence of nerve cells inhibited the occurrence of fusion in myogenic cells. Many multinucleated myotubes appeared in cultures of somitic cells in the absence of nerve cells, but very few in their presence. The possible relationship of these data with the appearance of mononucleated differentiated cells in myotomes in vivo and the possible neural control of this stage of myogenesis are discussed.     

ADAM12 and alpha9beta1 integrin are instrumental in human myogenic cell differentiation

Knowledge on molecular systems involved in myogenic precursor cell (mpc) fusion into myotubes is fragmentary. Previous studies have implicated the a disintegrin and metalloproteinase (ADAM) family in most mammalian cell fusion processes. ADAM12 is likely involved in fusion of murine mpc and human rhabdomyosarcoma cells, but it requires yet unknown molecular partners to launch myogenic cell fusion. ADAM12 was shown able to mediate cell-to-cell attachment through binding alpha9beta1 integrin. We report that normal human mpc express both ADAM12 and alpha9beta1 integrin during their differentiation. Expression of alpha9 parallels that of ADAM12 and culminates at time of fusion. alpha9 and ADAM12 coimmunoprecipitate and participate to mpc adhesion. Inhibition of ADAM12/alpha9beta1 integrin interplay, by either ADAM12 antisense oligonucleotides or blocking antibody to alpha9beta1, inhibited overall mpc fusion by 47-48%, with combination of both strategies increasing inhibition up to 62%. By contrast with blockade of vascular cell adhesion molecule-1/alpha4beta1, which also reduced fusion, exposure to ADAM12 antisense oligonucleotides or anti-alpha9beta1 antibody did not induce detachment of mpc from extracellular matrix, suggesting specific involvement of ADAM12-alpha9beta1 interaction in the fusion process. Evaluation of the fusion rate with regard to the size of myotubes showed that both ADAM12 antisense oligonucleotides and alpha9beta1 blockade inhibited more importantly formation of large (> or =5 nuclei) myotubes than that of small (2-4 nuclei) myotubes. We conclude that both ADAM12 and alpha9beta1 integrin are expressed during postnatal human myogenic differentiation and that their interaction is mainly operative in nascent myotube growth.     

Up-regulation of nuclear PLCbeta1 in myogenic differentiation

Phospholipase C beta(1) (PLCbeta(1)) signaling in both cell proliferation and differentiation has been largely investigated, but its role in myoblast differentiation is still unclear. The C2C12 myogenic cell line has been used in this study in order to find out the role of the two subtypes of PLCbeta(1), i.e., a and b in this process. C2C12 myoblast proliferate in response to mitogens and upon mitogen withdrawal differentiates into multinucleated myotubes. We found that differentiation of C2C12 skeletal muscle cells is characterized by a marked increase in the amount of nuclear PLCbeta(1)a and PLCbeta(1)b. Indeed, treatment with insulin induces a dramatic rise of both PLCbeta(1) subtypes expression and activity, as determined by immunochemical and enzymatic assays. Immunofluorescence experiments with anti-PLCbeta(1) specific monoclonal antibody showed a low level of cytoplasmatic and nuclear staining during the initial 12 h of differentiation whilst a massive nuclear staining is appreciable in differentiating cells. The time course of PLCbeta(1) expression versus Troponin T expression clearly indicates that the increase in the amount of PLCbeta(1) takes place 24 h earlier than that of Troponin T. Moreover, the overexpression of the PLCbeta(1)M2b mutant, lacking the nuclear localization signal and entirely located in the cytoplasm, represses the formation of mature multinucleated myotube. Taken together these results suggest that nuclear PLCbeta(1) is a key player in myoblast differentiation, functioning as a positive regulator of this process.     

Participation of multiple factors, including proliferin, in the inhibition of myogenic differentiation.

Proliferin (PLF) is a secreted glycoprotein in the prolactin-growth hormone family in mice. PLF expression was detected in C3H 10T1/2 fibroblasts, but not in two 10T1/2-derived myogenic cell lines, and was restored in two nondifferentiating variants of one of these myogenic cell lines. Transient expression of one form of PLF (PLF1) inhibited expression from a muscle-specific gene promoter; a second form of PLF, which differed at three amino acid residues, displayed no activity in this transient assay. Introduction of a PLF1 expression construct into both muscle- and 10T1/2-derived myoblasts resulted in cell lines that were no longer myogenic or that differentiated only partially. Analysis of these cell lines revealed that differentiation could be obstructed at several steps and by one or more factors in addition to PLF. Although expected to function in vivo as an extracellular hormone, PLF did not appear to be acting through a cell surface receptor to inhibit differentiation in these cultured myoblasts.     

Regulation of the M-cadherin-beta-catenin complex by calpain 3 during terminal stages of myogenic differentiation

The cysteine protease calpain 3 (CAPN3) is essential for normal muscle function, since mutations in CAPN3 cause limb girdle muscular dystrophy type 2A. Previously, we showed that myoblasts isolated from CAPN3 knockout (C3KO) mice were able to fuse to myotubes; however, sarcomere formation was disrupted. In this study we further characterized morphological and biochemical features of C3KO myotubes in order to elucidate a role for CAPN3 during myogenesis. We showed that cell cycle withdrawal occurred normally in C3KO cultures, but C3KO myotubes have an increased number of myonuclei per myotube. We found that CAPN3 acts during myogenesis to specifically control levels of membrane-associated but not cytoplasmic beta-catenin and M-cadherin. CAPN3 was able to cleave both proteins, and in the absence of CAPN3, M-cadherin and beta-catenin abnormally accumulated at the membranes of myotubes. Given the role of M-cadherin in myoblast fusion, this finding suggests that the excessive myonuclear index of C3KO myotubes was due to enhanced fusion. Postfusion events, such as beta1D integrin expression and myofibrillogenesis, were suppressed in C3KO myotubes. These data suggest that the persistence of fusion observed in C3KO cells inhibits subsequent steps of differentiation, such as integrin complex rearrangements and sarcomere assembly.     

Thrombin-induced inhibition of myoblast differentiation is mediated by Gbetagamma

Thrombin has been shown to inhibit skeletal muscle differentiation. However, the mechanisms by which thrombin represses myogenesis remain unknown. Since the thrombin receptor couples to G(i), G(q/11) and G(12), we examined which subunits of heterotrimeric guanine nucleotide-binding regulatory proteins (Galpha(i), Galpha(q/11), Galpha(12) or Gbetagamma) participate in the thrombin-induced inhibition of C2C12 myoblast differentiation. Galpha(i2) and Galpha(11) had no inhibitory effect on the myogenic differentiation. Galpha(12) prevented only myoblast fusion, whereas Gbetagamma inhibited both the induction of skeletal muscle-specific markers and the myotube formation. In addition, the thrombin-induced reduction of creatine kinase activity was blocked by the C-terminal peptide of beta-adrenergic receptor kinase, which is known to sequester free Gbetagamma. These results suggest that the thrombin-induced inhibition of muscle differentiation is mainly mediated by Gbetagamma.     

Differential expression of the Na,K-ATPase alpha 1 and alpha 2 subunit genes in a murine myogenic cell line. Induction of the alpha 2 isozyme during myocyte differentiation

Expression of Na,K-ATPase catalytic alpha isoform (alpha 1, alpha 2, and alpha 3) and beta subunit genes in rodent muscle was investigated using the murine C2C12 myogenic cell line. RNA blot analyses of myoblasts revealed expression primarily of the alpha 1 mRNA and low levels of alpha 2 mRNA. Fusion of the proliferating myoblasts to form myotubes was accompanied by an approximate 12-fold induction of the alpha 2 mRNA. In contrast, expression of alpha 1 mRNA remained constant throughout myogenesis. The alpha 3 mRNA was not detected in either myoblasts or myotubes. The beta mRNA abundance also increased 2-3-fold during myotube formation. In rodent tissues, low and high affinity cardiac glycoside (e.g. ouabain) receptors have been shown to be associated with the Na,K-ATPase catalytic alpha 1 and alpha 2 isoform subunits, respectively. The existence of these two functional classes of Na,K-ATPase in myoblasts and myotubes correlated with the biphasic ouabain inhibition of Na,K-ATPase activity. Confluent myoblasts expressed primarily the alpha 1 isozyme (IC50 = 3.6 X 10(-5) M; 95% of total activity) and lesser amounts of the alpha 2 isozyme (IC50 = 1.1 X 10(-7) M; 5% of total activity). In contrast, the myotubes showed significant levels of the alpha 1 isozyme (IC50 = 4.0 X 10(-5) M; 68% of total activity) and, in addition, showed a 6-fold increase in the relative levels of the alpha 2 isozyme (IC50 = 1.1 X 10(-7) M; 32% of total activity). To quantitate further the expression of the high affinity, ouabain-sensitive alpha 2 isozyme, a whole cell [3H]ouabain-binding assay was used. Results revealed that myotubes have an approximately 6-fold greater concentration of [3H]ouabain-binding sites than myoblasts with an apparent dissociation constant (Kd) of 1.4 X 10(-7) M. The results indicate that muscle cells can express multiple isozymes of Na,K-ATPase and that expression of the alpha 2 isozyme is developmentally regulated during myogenesis.     

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.     

Per1/Per2–Igf2 axis–mediated circadian regulation of myogenic differentiation

Circadian rhythms regulate cell proliferation and differentiation, but circadian control of tissue regeneration remains elusive at the molecular level. Here, we show that proper myoblast differentiation and muscle regeneration are regulated by the circadian master regulators Per1 and Per2. Depletion of Per1 or Per2 suppressed myoblast differentiation in vitro and muscle regeneration in vivo, demonstrating their nonredundant functions. Both Per1 and Per2 were required for the activation of Igf2, an autocrine promoter of myoblast differentiation, accompanied by Per-dependent recruitment of RNA polymerase II, dynamic histone modifications at the Igf2 promoter and enhancer, and the promoter–enhancer interaction. This circadian epigenetic priming created a preferred time window for initiating myoblast differentiation. Consistently, muscle regeneration was faster if initiated at night, when Per1, Per2, and Igf2 were highly expressed compared with morning. This study reveals the circadian timing as a significant factor for effective muscle cell differentiation and regeneration.     

Selective inhibition of mutant Ha-ras mRNA expression by antisense oligonucleotides.

A biological reporter gene assay was employed to determine the crucial parameters for maximizing selective targeting of a Ha-ras codon 12 point mutation (G----T) using phosphorothioate antisense oligonucleotides. We have tested a series of oligonucleotides ranging in length between 5 and 25 bases, each centered around the codon 12 point mutation. Our results indicate that selective targeting of this point mutation can be achieved with phosphorothioate antisense oligonucleotides, but this selectivity is critically dependent upon oligonucleotide length and concentration. The maximum selectivity observed in antisense experiments, 5-fold for a 17-base oligonucleotide, was closely predicted by a simple thermodynamic model that relates the fraction of mutant to wild type target bound as a function of oligonucleotide concentration and affinity. These results suggest thermodynamic analysis of oligonucleotide/target interactions is useful in predicting the specificity that can be achieved by an antisense oligonucleotide targeted to a single base point mutation.