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PC4 (pheochromocytoma cell-4) is an immediate early gene related to IFN-gamma, the mRNA of which is induced during the course of neuronal differentiation by nerve growth factor in the PC12 cell line. Here we report that PC4 mRNA is also expressed in the myoblast C2C12 cell line and is regulated during differentiation; its expression decreases within 6 h from the onset of differentiation, attains a minimum after 12 h, and returns to basal level within 36 h. This transient down-regulation of PC4 expression in C2C12 myoblasts is prevented by transforming growth factor beta, a molecule which inhibits the differentiation of muscle. Sense and antisense PC4 cDNA transfection strategies in C2C12 cells were then used to clarify the role of PC4 in muscle differentiation. While no effect was seen by over-expression of PC4, stable transfectants underexpressing PC4 exhibited a delay in attaining the differentiated phenotype, with an impairment of myogenin and myosin expression. Myogenin was also inhibited in C2C12 cells microinjected with the anti-PC4 polyclonal antibody A451. We thus postulate a role for PC4 as a positive regulator during muscle differentiation.

Myogenin, a member of the MyoD family of helix-loop-helix proteins, can induce myogenesis in a wide range of cell types. In addition to activating muscle structural genes, members of the MyoD family can autoactivate their own and cross-activate one another's expression in transfected cells. This has led to the hypothesis that autoregulatory loops among these factors provide a mechanism for amplifying and maintaining the muscle-specific gene expression program in vivo. Here, we make use of myogenin-null mice to directly test this hypothesis. To investigate whether the myogenin protein autoregulates the myogenin gene during embryogenesis, we introduced a myogenin-lacZ transgene into mice harboring a null mutation at the myogenin locus. Despite a severe deficiency of skeletal muscle in myogenin-null neonates, the myogenin-lacZ transgene was expressed normally in myogenic cells throughout embryogenesis. These results show that myogenin is not required for regulation of the myogenin gene and argue against the existence of a myogenin autoregulatory loop in the embryo.

The neurohypophyseal nonapeptide arginine8-vasopressin (AVP) induces phosphoinositide turnover and calcium and pH changes in skeletal myogenic cells in culture. In order to investigate the effect of AVP on skeletal myogenesis, we examined the effect of this hormone on proliferating mononucleated L6 myoblast cultures. Addition of AVP to the medium resulted in the formation of much larger myotubes than those formed in its absence and in a significant increase (2.2-fold) of the percentage of fusion within 3-4 days of treatment. The effect of AVP was dose dependent, in the 10 nM to 1 microM range, and was observed also in primary cultures of mouse satellite cells. The rate of growth of L6 cells was not affected by AVP treatment. The induction of morphological differentiation by AVP correlated with an increased expression of muscle-specific gene products, such as myosin, and an increased number of acetylcholine receptor sites. The accumulation of mRNA transcripts of the acetylcholine receptor subunits was also enhanced by AVP. The mechanism involved in the myogenic action of AVP was investigated. Using AVP-related peptides and antagonists, we found that a specific chemical structure is required and that V1 receptors probably mediate the effect on myogenesis. Expression of muscle-specific transcription factor genes Myf-5 and myogenin and their products are strongly upregulated by AVP. Our findings support the hypothesis that AVP may represent a novel physiological modulator of skeletal muscle differentiation through its action on muscle regulatory genes.

Aprotinin, a Kunitz-type inhibitor of serine proteases, stimulates myotube formation by mouse G8-1 and C2C12 skeletal muscle myoblasts. This stimulation of morphological differentiation is accompanied by accumulation of myogenin transcripts and production of muscle-specific proteins. In contrast, active TGF beta prevents differentiation of G8-1 and C2C12 myoblasts. When active TGF beta and aprotinin are both added to myoblast cultures, differentiation is inhibited, suggesting the active growth factor acts downstream of the protease inhibitor. TGF beta is found in serum as a latent, dimeric propolypeptide that is cleaved by limited proteolysis to release the biologically active carboxy-terminal dimer. To address the possibility that aprotinin may effect myogenesis by inhibiting proteolytic activation of latent TGF beta, levels of the endogenous growth factor were measured in differentiating myoblast cultures. Latent TGF beta is rapidly depleted from control cultures within 24 hours of plating, but remains relatively stable in aprotinin-treated cultures. Consistent with this, aprotinin-treated cultures have reduced levels of active TGF beta. These data indicate that Kunitz-domain containing protease inhibitors may help orchestrate the onset of myogenesis, possibly by regulating the activity of TGF beta-like molecules.

The implantation of bone morphogenetic protein (BMP) into muscular tissues induces ectopic bone formation at the site of implantation. To investigate the mechanism underlying this process, we examined whether recombinant bone morphogenetic protein-2 (BMP-2) converts the differentiation pathway of the clonal myoblastic cell line, C2C12, into that of osteoblast lineage. Incubating the cells with 300 ng/ml of BMP-2 for 6 d almost completely inhibited the formation of the multinucleated myotubes expressing troponin T and myosin heavy chain, and induced the appearance of numerous alkaline phosphatase (ALP)-positive cells. BMP-2 dose dependently induced ALP activity, parathyroid hormone (PTH)-dependent 3',5'-cAMP production, and osteocalcin production at concentrations above 100 ng/ml. The concentration of BMP-2 required to induce these osteoblastic phenotypes was the same as that required to almost completely inhibit myotube formation. Incubating primary muscle cells with 300 ng/ml of BMP-2 for 6 d also inhibited myotube formation, whereas induced ALP activity and osteocalcin production. Incubation with 300 ng/ml of BMP-2 suppressed the expression of mRNA for muscle creatine kinase within 6 h, whereas it induced mRNA expression for ALP, PTH/PTH-related protein (PTHrP) receptors, and osteocalcin within 24-48 h. BMP-2 completely inhibited the expression of myogenin mRNA by day 3. By day 3, BMP-2 also inhibited the expression of MyoD mRNA, but it was transiently stimulated 12 h after exposure to BMP-2. Expression of Id-1 mRNA was greatly stimulated by BMP-2. When C2C12 cells pretreated with BMP-2 for 6 d were transferred to a colony assay system in the absence of BMP-2, more than 84% of the colonies generated became troponin T-positive and ALP activity disappeared. TGF-beta 1 also inhibited myotube formation in C2C12 cells, and suppressed the expression of myogenin and MyoD mRNAs without inducing that of Id-1 mRNA. However, no osteoblastic phenotype was induced by TGF-beta 1 in C2C12 cells. TGF-beta 1 potentiated the inhibitory effect of BMP-2 on myotube formation, whereas TGF-beta 1 reduced ALP activity and osteocalcin production induced by BMP-2 in C2C12 cells. These results indicate that BMP-2 specifically converts the differentiation pathway of C2C12 myoblasts into that of osteoblast lineage cells, but that the conversion is not heritable.

Muscle enhancer factor-2A (MEF2A), a member of the MADS family, induced myogenic development when ectopically expressed in clones of nonmuscle cells of human clones, a function previously limited to the muscle basic helix-loop-helix (bHLH) proteins. During myogenesis, MEF2A and bHLH proteins cooperatively activate skeletal muscle genes and physically interact through the MADS domain of MEF2A and the three myogenic amino acids of the muscle bHLH proteins. Thus, skeletal myogenesis is mediated by two distinct families of mutually inducible and interactive muscle transcription factors, either of which can initiate the developmental cascade.

Acidic fibroblast growth factor (FGF) and related family members regulate differentiation in organisms as diverse as Xenopus laevis and mammals. We utilized a well-characterized model of myogenic development to directly assess the importance of endogenously produced FGF in controlling differentiation. A role for endogenous FGF is suggested by the previous finding that acidic and basic FGF abundance in cultured myocytes decreases during differentiation. In this study we inhibited the endogenous production of FGF in murine Sol 8 myoblasts by using antisense RNA and observed precocious myogenic differentiation. Exogenously supplied acidic FGF rescues this phenotype. Further results suggest that the effect of FGF on myogenic differentiation is mediated in part through inhibition of myogenin expression. These results demonstrate a direct role for endogenously synthesized growth factors in regulating myogenesis and provide support for a general role for related proteins in mammalian development.

The muscle-specific intermediate filament protein, desmin, is one of the earliest myogenic markers whose functional role during myogenic commitment and differentiation is unknown. Sequence comparison of the presently isolated and fully characterized mouse desmin cDNA clones revealed a single domain of polypeptide similarity between desmin and the basic and helix-loop-helix region of members of the myoD family myogenic regulators. This further substantiated the need to search for the function of desmin. Constructs designed to express anti-sense desmin RNA were used to obtain stably transfected C2C12 myoblast cell lines. Several lines were obtained where expression of the anti-sense desmin RNA inhibited the expression of desmin RNA and protein down to basal levels. As a consequence, the differentiation of these myoblasts was blocked; complete inhibition of myoblast fusion and myotube formation was observed. Rescue of the normal phenotype was achieved either by spontaneous revertants, or by overexpression of the desmin sense RNA in the defective cell lines. In several of the cell lines obtained, inhibition of desmin expression was followed by differential inhibition of the myogenic regulators myoD and/or myogenin, depending on the stage and extent of desmin inhibition in these cells. These data suggested that myogenesis is modulated by at least more than one pathway and desmin, which so far was believed to be merely an architectural protein, seems to play a key role in this process.

The terminal stage of myogenic development is marked by the cessation of replication, fusion, and expression of genes which encode the myofibrillar proteins. Prior to terminal differentiation one or more stages of myogenic development take place. Expression of alpha 7 integrin and desmin have been used as markers for these earlier stages of myogenesis. Both proteins are expressed in replicating secondary myoblasts prior to terminal differentiation and when these cells differentiate further, the expression of the alpha 7 integrin and desmin genes is up-regulated. To determine whether the stages of myogenesis which precede terminal development and the factors which regulate them are distinct, the expression of alpha 7 integrin and desmin was assayed in a variety of myogenic cell lines in which terminal differentiation was inhibited. L8E63 and C2 myoblasts in which terminal differentiation was inhibited by SV40 large T antigen, adenovirus E1A protein, or ras and an L6 mutant whose terminal differentiation is sensitive to alpha-amanitin were studied. In all cases, when terminal myogenic differentiation is inhibited the basal levels of desmin and alpha 7 expression are not altered. Under these same conditions expression of the myogenic regulatory genes myogenin and MyoD also were inhibited whereas Myf-5 persisted. These results indicate that expression of the early myogenic phenotype and terminal differentiation are discrete and independent stages of myogenesis and that different transcription factors likely regulate the expression of each stage. In contrast with myoblasts in cultures of newborn rat hindlimb cells and the C2 cell line, myogenic cells derived from C3H10T1/2 cells by treatment with 5-azacytidine or by transfection with MyoD, Myf-5, or MRF4 do not express desmin as replicating myoblasts but do so upon terminal differentiation. This indicates that in vitro, terminal differentiation can proceed in the absence of the phenotypes that normally develop earlier and that the conversion of 10T1/2 cells to myogenic cells can bypass developmental stages which normally occur in vivo. These results are discussed in the context of a model of the myogenic lineage that is based on the expression of desmin.

The gene myogenin encodes a helix-loop-helix protein whose function is critical to the integrity of the mammalian myogenic cascade. In vitro, the expression of this gene immediately precedes terminal differentiation, as measured by the synthesis of those proteins that make up the contractile apparatus. However, during mammalian development, expression of myogenin and the appearance of sarcomeres are separated by at least 1 week. The observation that early embryos (10.5 days post coitum) do not possess myogenin protein despite the fact that transcripts are detected (Cusella-De Angelis et al. (1992) J. Cell Biol. 116, 1243-1255), suggests the possibility of post-transcriptional regulation. Using polymerase chain reaction and in situ analyses, we report here that the mouse embryo accumulates a significant pool of unprocessed myogenin RNA in the developing somites (10.5 days post coitum). These results indicate that post-transcriptional regulation of myogenin may occur at the RNA processing level.

After denervation, mRNA levels of the jun and fos protooncogenes and of the muscular differentiation factors myoD1 and myogenin are increased. Here, immunohistochemistry was used (a) to show that this increase in mRNA is followed by an increase in the transcription factor proteins, and (b) to determine which cell populations in skeletal muscle express these factors after denervation. Rat diaphragms were denervated and analyzed after periods of 90 min-8 days. An increase in Fos and Jun as well as MyoD1 and Myogenin immunoreactivity was found after 2-2.5 days of denervation. Fos, MyoD1, and Myogenin immunoreactivity was mostly confined to muscle cell nuclei, whereas Jun antibodies stained muscle cell and some interstitial cell nuclei. A selective expression of any of the four transcription factors in muscle cell nuclei closely associated with motor endplates could not be detected in either denervated or innervated muscle at any time point examined, indicating that synaptic and extrasynaptic muscle cell nuclei are activated simultaneously after denervation. These results suggest that a genetic program which includes protooncogenes and myogenic differentiation factors is activated in skeletal muscle after denervation.

Transformation of hamster primary myoblasts with the SV40 large T antigen leads to inhibition of terminal differentiation. This process is associated with a block in the transcription of the muscle-specific determinator genes MyoD and myogenin. The effect of SV40 large T antigen on the terminal differentiation is dominant and cannot be bypassed by re-expression of retrovirally encoded MyoD. The intermediate filament protein desmin is normally up-regulated when myoblasts differentiate into myotubes. Surprisingly, desmin is expressed at relatively high levels in transformed hamster muscle cells grown under proliferative conditions. So desmin expression can be independent of the onset of differentiation. This is in accordance with the expression of the protein in fibroblasts, infected with a MyoD-encoding retrovirus and grown under proliferative conditions, when no other muscle-specific proteins are present.

Expression of MyoD, myogenin, MRF4, and Myf-5 converts nonmuscle cells to muscle cells. In an attempt to analyze the roles of these factors, we have investigated their effects on transcription driven by the promoter of the chicken myosin alkaline light-chain (MLC1) gene. The activation by CMD1 or c-myogenin (chicken MyoD or myogenin, respectively) was dependent on the existence of a muscle-specific regulatory region located from positions -2096 to -1743. Its distal half, containing a pair of E boxes (CANNTG), had been previously characterized as an enhancer responsive to CMD1 but not to c-myogenin. In this study, we report the identification of another enhancer in the muscle-specific regulatory region which is preferentially responsive to c-myogenin. Deletion and mutation analyses indicated that this enhancer requires a single E box and its flanking sequences. Furthermore, analysis of chimeric proteins of CMD1 and c-myogenin indicated that regions outside the basic helix-loop-helix domain of c-myogenin are involved in the specificity of the enhancer. These results show that CMD1 and c-myogenin act on the MLC1 gene by recognizing different upstream DNA sequences and that direct or indirect interactions between the regions outside the basic helix-loop-helix domain and flanking sequences of E boxes are involved in the target sequence specificity.

Satellite cells were isolated from human quadriceps and masseter muscles and the phenotype of these cells examined in vitro. The expression of the different isoforms of the myosin heavy chains (embryonic, fetal, fast and slow) and light chain isoforms was used to assay myotube diversification. We found that fused cultures of human satellite cells express adult fast and slow MHCs in addition to the embryonic and fetal isoforms. Only the four fast light chains (MLC1emb, MLC1F, MLC2F and MLC3F) were synthesized. No slow MLCs were ever detected in these cultures. In order to determine if the human satellite cells were committed to distinct fast and slow myogenic lineages, a clonal analysis was carried out on both cell populations. All myogenic clones expressed fast and slow MHCs, suggesting that there is no evidence for different fast and slow satellite cell lineages in human skeletal muscle.

The fast skeletal muscle myosin light chain 2 (MLC2) gene is expressed specifically in skeletal muscles of newborn and adult mice, and has no detectable sequence homology with any of the other MLC genes including the slow cardiac MLC2 gene. The expression of the fast skeletal muscle MLC2 gene during early mouse embryogenesis was studied by in situ hybridization. Serial sections of embryos from 8.5 to 12.5 days post coitum (d.p.c.) were hybridized to MLC2 cRNA and to probes for the myogenic regulatory genes MyoD1 and myogenin. The results revealed different temporal and spatial patterns of hybridization for different muscle groups. MLC2 transcripts were first detected 9.5 d.p.c. in the myotomal regions of rostral somites, already expressing myogenin. Surprisingly, at the same stage, a weak MLC2 signal was also detected in the cardiomyocytes. The cardiac expression was transient and could not be detected at later stages while the myotomal signal persisted and spread to the more caudal somites, very similar to the expression of myogenin. Beginning from 10.5 d.p.c., several extramyotomal premuscle cells masses have been demarcated by MyoD1 expression. MLC2 transcripts were detected in only one of these cell masses. Although, transcripts of myogenin were detected in all these cell masses, the number of expressing cells was significantly lower than that observed for MyoD1. By 11.5 d.p.c., all three hybridization signals colocalized in most extramyotomal muscle-forming regions, with the exception of the diaphragm and the hindlimb buds, where only few cells expressed MLC2 and more cells expressed MyoD1 than myogenin. At 12.5 d.p.c., all three studied genes displayed a similar spatial pattern of expression in most muscle-forming regions. However, in some muscles, the MyoD1 signal spread over more cells compared to myogenin or MLC2. Our results are consistent with the suggestion that multiple myogenic programs exist for myoblasts differentiating in the myotome and extramyotomal regions.

The Role of IGFs in Myogenesis. Thus we are now convinced that the control of myogenesis by IGFs is a general phenomenon that occurs in all skeletal muscle cells, whether or not IGFs are added to the "differentiation" medium. We believe that several medium components contribute to the suppression of IGF-II expression in myoblasts incubated in high serum "growth" medium, and conclude that the IGF-I receptor mediates the feedback inhibition of IGF-II gene expression in muscle cells. Mechanism of Induction of Myogenesis by IGFs. The observations summarized here now permit a reasonably coherent overview of the stimulation of myogenic differentiation by the IGFs. It seems clear that all IGFs act by binding to the Type I IGF receptor, and that this process is inhibited to a significant extent by IGF binding proteins secreted by the target myoblasts. A major, but possibly not the only relevant effect of this binding is the induction of expression of the myogenin gene; this induction appears to require the presence of myf-5 protein, at least during the early part of the response. Cells capable of a mitogenic response undergo a round of division in response to IGF-I, thus delaying their entry into the final processes of postmitotic terminal differentiation. Other laboratories have shown that myogenin complexes with one or more widely occurring proteins such as E12 or E47 to form an active complex that interacts with CAnnTG elements in muscle specific genes, turning on expression of those genes and thus initiating the phenotype associated with terminally differentiated skeletal muscle.