C2C12 Myoblast Fusion Research Articles

Reduced myotube diameter induced by combined inhibition of transforming growth factor-β type I receptors Acvr1b and Tgfbr1 is associated with enhanced β1-syntrophin expression.

Simultaneous inhibition of transforming growth factor-β (TGF-β) type I receptors Acvr1b and Tgfbr1 signalling has been associated with excessive skeletal muscle hypertrophy in vivo. However, it remains unclear whether the increased muscle mass in vivo is a direct result of inhibition of intracellular TGF-β signalling or whether this is an indirect effect of an altered extracellular anabolic environment. Here, we tested whether individual or simultaneous knockdown of TGF-β type I receptors in C2C12 myotubes was sufficient to induce muscle hypertrophy. The expression levels of TGF-β type I receptors Acvr1b and Tgfbr1 in myotubes were knocked down individually or in combination in the absence or presence of TGF-β1 and myostatin. Knocking down either Acvr1b or Tgfbr1 did not significantly change cell phenotype. Unexpectedly, simultaneous knockdown of both receptors reduced C2C12 myotube diameter, mRNA expression levels of Hgf, Ccn2 and Mymx with or without TGF-β1 and myostatin administration. In spite of decreased phosphorylation of Smad2/3, phosphorylation of P70S6K was reduced. In addition, the gene expression level of β1-syntrophin (Sntb1), which encodes a protein associated with the dystrophin-glycoprotein complex, was increased. Parallel experiments where Sntb1 gene expression was reduced showed an increase in myotube diameter and fusion of C2C12 myoblasts. Together, these results indicate that the knockdown of both TGF-β type I receptors reduced myotube diameter. This atrophic effect was attributed to reduced protein synthesis signalling and an increased expression of β1-syntrophin. These results have implications for our fundamental understanding of how TGF-β signalling regulates skeletal muscle size.

Early tension regulation coupled to surface myomerger is necessary for the primary fusion of C2C12 myoblasts

Here, we study the time-dependent regulation of fluctuation–tension during myogenesis and the role of the fusogen, myomerger. We measure nanometric height fluctuations of the basal membrane of C2C12 cells after triggering differentiation. Fusion of cells increases fluctuation–tension but prefers a transient lowering of tension (at ~2–24 h). Cells fail to fuse if early tension is continuously enhanced by methyl-β-cyclodextrin (MβCD). Perturbing tension regulation also reduces fusion. During this pre-fusion window, cells that finally differentiate usually display lower tension than other non-fusing cells, validating early tension states to be linked to fate decision. Early tension reduction is accompanied by low but gradually increasing level of the surface myomerger. Locally too, regions with higher myomerger intensity display lower tension. However, this negative correlation is lost in the early phase by MβCD-based cholesterol depletion or later as differentiation progresses. We find that with tension and surface-myomerger’s enrichment under these conditions, myomerger clusters become pronouncedly diffused. We, therefore, propose that low tension aided by clustered surface-myomerger at the early phase is crucial for fusion and can be disrupted by cholesterol-reducing molecules, implying the potential to affect muscle health.

Nec-1 alleviated the deleterious effect of CoCl2 on C2C12 myoblast differentiation and fusion via the mTOR pathway

Myoblast differentiation and fusion are vital for muscle development and repair in mammals. We previously showed that necrostatin-1(Nec-1) protects C2C12 myotubes from cobalt chloride (CoCl2)-induced pseudo-hypoxia. However, the function of Nec-1 in mouse C2C12 myoblast differentiation and fusion was still unknown. In this study, we found that CoCl2 substantially impaired C2C12 myoblast differentiation and fusion, and reduced the expression of Myh1, Myh2, Myh4, Myh7, myomaker, and myomerger. Nec-1 treatment rescued C2C12 myoblast differentiation and fusion and promoted the expression of myomaker and myomerger. Mechanistically, Nec-1 promoted C2C12 myoblast differentiation and fusion by inhibiting mTOR-mediated autophagy. Rapamycin abolished the increases in expression of muscle fusion-related genes, indicating that the upregulation of differentiation and fusion is mTOR-dependent. These results suggest that Nec-1 inhibited autophagy in an mTOR-dependent mechanism that is crucial for mTOR-induced C2C12 myoblast differentiation and fusion.

Fer1L5, a Dysferlin Homologue Present in Vesicles and Involved in C2C12 Myoblast Fusion and Membrane Repair.

Simple SummaryFer1L5 is a dysferlin and myoferlin homologue and has been implicated in muscle membrane fusion events; myoblast fusion and membrane repair respectively during C2C12 skeletal muscle development. The role of Fer1L5 was analyzed by immunoblot analysis, biochemical fractionation, confocal microscopy and electroporation method. We demonstrated that Fer1L5 is present in low density vesicles and resistant to non-ionic detergent and shows overlapping properties with dysferlin and myoferlin. The expression of Fer1L5 was highly observed at the fusing myoblasts membranes and its expression level is gradually increase at the early stages multinucleated myotube formation. Fusion defects were observed in the Fer1L5 deficient C2C12 cells. Fer1L5 shows impaired membrane repair. Our data provide evidence that Fer1L5 is involved in aligning the adjacent myotubes close to each other for membrane—membrane fusion to increase the muscle mass for contraction during muscle development. Our data for Fer1L5 will be of great importance in the dysferlinopathy research in near future.Fer1L5 is a dysferlin and myoferlin related protein, which has been predicted to have a role in vesicle trafficking and muscle membrane fusion events. Mutations in dysferlin and otoferlin genes cause heredity diseases: muscular dystrophy and deafness in humans, respectively. Dysferlin is implicated in membrane repair. Myoferlin has a role in myogenesis. In this study, we investigated the role of the Fer1L5 protein during myoblast fusion and membrane repair. To study the functions of Fer1L5 we used confocal microscopy, biochemical fractionation, Western blot analysis and multiphoton laser wounding assay. By immunolabelling, Fer1L5 was detected in vesicular structures. By biochemical fractionation Fer1L5 was observed in low density vesicles. Our studies show that the membranes of Fer1L5 vesicles are non-resistant to non-ionic detergent. Partial co-staining of Fer1L5 with other two ferlin vesicles, respectively, was observed. Fer1L5 expression was highly detected at the fusion sites of two apposed C2C12 myoblast membranes and its expression level gradually increased at D2 and reached a maximum at day 4 before decreasing during further differentiation. Our studies showed that Fer1L5 has fusion defects during myoblast fusion and impaired membrane repair when the C2C12 cultures were incubated with inhibitory Fer1L5 antibodies. In C2C12 cells Fer1L5 vesicles are involved in two stages, the fusion of myoblasts and the formation of large myotubes. Fer1L5 also plays a role in membrane repair.

LRRC8 channel activation and reduction in cytosolic chloride concentration during early differentiation of C2C12 myoblasts

Leucine-rich repeat containing family 8 (LRRC8) proteins form the volume-regulated anion channel (VRAC). Recently, they were shown to be required for normal differentiation and fusion of C2C12 myoblasts, by promoting membrane hyperpolarization and intracellular Ca2+ signals. However, the mechanism by which they are involved remained obscure. Here, using a FRET-based sensor for VRAC activity, we show temporary activation of VRAC within the first 2 h of myogenic differentiation. During this period, we also observed a significant decrease in the intracellular Cl− concentration that was abolished by the VRAC inhibitor carbenoxolone. However, lowering the intracellular Cl− concentration by extracellular Cl− depletion did not promote differentiation as judged by the percentage of myogenin-positive nuclei or total myogenin levels in C2C12 cells. Instead, it inhibited myosin expression and myotube formation. Together, these data suggest that VRAC is activated and mediates Cl− efflux early on during myogenic differentiation, and a moderate intracellular Cl− concentration is necessary for myoblast fusion.

Thermal stimulation at 39°C facilitates the fusion and elongation of C2C12 myoblasts.

The aim of this study was to determine the effect of thermal stimulation at 39°C on the fusion and elongation of skeletal muscle cells. During a 5day differentiation process of C2C12 cells, nine groups subjected to varying lengths of thermal stimulation at 39°C were established. Afterward, all groups were immunostained using anti-muscle heavy-chain antibody to test for myotube formation. Quantification of the myotube area demonstrated a significant increase in the group subjected to thermal stimulation at 39°C during the latter half of the differentiation compared with the control group, but the fusion index was significantly higher in the group that received hyperthermic treatment during the first half of the differentiation period. Moreover, the longitudinal length of myotubes was significantly increased in the groups that were subjected to thermal stimulation at 39°C during the latter half of the differentiation period. The distance between the center of myotubes and the nucleus farthest away from the center was substantially extended in the group receiving thermal stimulation at 39°C only on the fourth day of the differentiation. Together, these results demonstrate that thermal stimulation at 39°C facilitates myoblast fusion and elongation.

Continuous exposure of isoprenaline inhibits myoblast differentiation and fusion through PKA/ERK1/2-FOXO1 signaling pathway

AimThe objective of this study is to determine if exuberant sympathetic nerve activity is involved in muscle satellite cell differentiation and myoblast fusion.Methods and resultsBy using immunoassaying and western blot analyses, we found that β1 and β2-adrenergic receptors (AdR) were expressed in C2C12 cells. The differentiated satellite cells exhibited an increased expression of β2-AdR, as compared with the proliferating cells. Continuous exposure of isoprenaline (ISO), a β-AdR agonist, delayed C2C12 cell differentiation, and myoblast fusion in time- and dose-dependent manner. ISO also increased short myotube numbers while decreasing long myotube numbers, consistent with the greater reduction in MyHC1, MyHC2a, and MyHC2x expression. Moreover, continuous exposure of ISO gradually decreased the ratio of PKA RI/RII, and PKA RI activator efficiently reversed the ISO effect on C2C12 cell differentiation and myoblast fusion while PKA inhibitor H-89 deteriorated the effects. Continuous single-dose ISO increased β1-AdR expression in C2C12 cells. More importantly, the cells showed enhanced phospho-ERK1/2 levels, resulting in increasing phospho-β2-AdR levels while decreasing β2-AdR levels, and the specific effects could be abolished by ERK1/2 inhibitor. Furthermore, continuous exposure of ISO induced FOXO1 nuclear translocation and increased the levels of FOXO1 in nuclear extracts while reducing pAKT, p-p38MAPK, and pFOXO1 levels. Conversely, blockade of ERK1/2 signaling partially abrogated ISO effects on AKT, p38MAPK, and FOXO1signaling, which partially restored C2C12 cell differentiation and myoblast fusion, leading to an increase in the numbers of medium myotube along with the increased expression of MyHC1 and MyHC2a.ConclusionContinuous exposure of ISO impedes satellite cell differentiation and myoblast fusion, at least in part, through PKA-ERK1/2-FOXO1 signaling pathways, which were associated with the reduced β2-AdR and increased β1-AdR levels.

Naa15 knockdown enhances c2c12 myoblast fusion and induces defects in zebrafish myotome morphogenesis.

The understanding of muscle tissue formation and regeneration is essential for the development of therapeutic approaches to treat muscle diseases or loss of muscle mass and strength during ageing or cancer. One of the critical steps in muscle formation is the fusion of muscle cells to form or regenerate muscle fibres. To identify new genes controlling myoblast fusion, we performed a siRNA screen in c2c12 myoblasts. The genes identified during this screen were then studied in vivo by knockdown in zebrafish using morpholino. We found that N-alpha-acetyltransferase 15 (Naa15) knockdown enhanced c2c12 myoblast fusion, suggesting that Naa15 negatively regulates myogenic cell fusion. We identified two Naa15 orthologous genes in the zebrafish genome: Naa15a and Naa15b. These two orthologs were expressed in the myogenic domain of the somite. Knockdown of zebrafish Naa15a and Naa15b genes induced a "U"-shaped segmentation of the myotome and alteration of myotome boundaries, resulting in the formation of abnormally long myofibres spanning adjacent somites. Taken together, these results show that Naa15 regulates myotome formation and myogenesis in fish.

Glucocorticoids Improve Myogenic Differentiation In Vitro by Suppressing the Synthesis of Versican, a Transitional Matrix Protein Overexpressed in Dystrophic Skeletal Muscles.

In Duchenne muscular dystrophy (DMD), a dysregulated extracellular matrix (ECM) directly exacerbates pathology. Glucocorticoids are beneficial therapeutics in DMD, and have pleiotropic effects on the composition and processing of ECM proteins in other biological contexts. The synthesis and remodelling of a transitional versican-rich matrix is necessary for myogenesis; whether glucocorticoids modulate this transitional matrix is not known. Here, versican expression and processing were examined in hindlimb and diaphragm muscles from mdx dystrophin-deficient mice and C57BL/10 wild type mice. V0/V1 versican (Vcan) mRNA transcripts and protein levels were upregulated in dystrophic compared to wild type muscles, especially in the more severely affected mdx diaphragm. Processed versican (versikine) was detected in wild type and dystrophic muscles, and immunoreactivity was highly associated with newly regenerated myofibres. Glucocorticoids enhanced C2C12 myoblast fusion by modulating the expression of genes regulating transitional matrix synthesis and processing. Specifically, Tgfβ1, Vcan and hyaluronan synthase-2 (Has2) mRNA transcripts were decreased by 50% and Adamts1 mRNA transcripts were increased three-fold by glucocorticoid treatment. The addition of exogenous versican impaired myoblast fusion, whilst glucocorticoids alleviated this inhibition in fusion. In dystrophic mdx muscles, versican upregulation correlated with pathology. We propose that versican is a novel and relevant target gene in DMD, given its suppression by glucocorticoids and that in excess it impairs myoblast fusion, a process key for muscle regeneration.

E-cadherin cytoplasmic domain inhibits cell surface localization of endogenous cadherins and fusion of C2C12 myoblasts.

ABSTRACTMyoblast fusion is a highly regulated process that is essential for skeletal muscle formation during muscle development and regeneration in mammals. Much remains to be elucidated about the molecular mechanism of myoblast fusion although cadherins, which are Ca2+-dependent cell–cell adhesion molecules, are thought to play a critical role in this process. Mouse myoblasts lacking either N-cadherin or M-cadherin can still fuse to form myotubes, indicating that they have no specific function in this process and may be functionally replaced by either M-cadherin or N-cadherin, respectively. In this study, we show that expressing the E-cadherin cytoplasmic domain ectopically in C2C12 myoblasts inhibits cell surface localization of endogenous M-cadherin and N-cadherin, as well as cell–cell fusion. This domain, however, does not inhibit myoblast differentiation according to microarray-based gene expression analysis. In contrast, expressing a dominant-negative β-catenin mutant ectopically, which suppresses Wnt/β-catenin signaling, did not inhibit cell–cell fusion. Therefore, the E-cadherin cytoplasmic domain inhibits cell–cell fusion by inhibiting cell surface localization of endogenous cadherins and not by inhibiting Wnt/β-catenin signaling.

A hydrogel derived from decellularized dermal extracellular matrix

The ECM of mammalian tissues has been used as a scaffold to facilitate the repair and reconstruction of numerous tissues. Such scaffolds are prepared in many forms including sheets, powders, and hydrogels. ECM hydrogels provide advantages such as injectability, the ability to fill an irregularly shaped space, and the inherent bioactivity of native matrix. However, material properties of ECM hydrogels and the effect of these properties upon cell behavior are neither well understood nor controlled. The objective of this study was to prepare and determine the structure, mechanics, and the cell response in vitro and in vivo of ECM hydrogels prepared from decellularized porcine dermis and urinary bladder tissues. Dermal ECM hydrogels were characterized by a more dense fiber architecture and greater mechanical integrity than urinary bladder ECM hydrogels, and showed a dose dependent increase in mechanical properties with ECM concentration. In vitro, dermal ECM hydrogels supported greater C2C12 myoblast fusion, and less fibroblast infiltration and less fibroblast mediated hydrogel contraction than urinary bladder ECM hydrogels. Both hydrogels were rapidly infiltrated by host cells, primarily macrophages, when implanted in a rat abdominal wall defect. Both ECM hydrogels degraded by 35 days in vivo, but UBM hydrogels degraded more quickly, and with greater amounts of myogenesis than dermal ECM. These results show that ECM hydrogel properties can be varied and partially controlled by the scaffold tissue source, and that these properties can markedly affect cell behavior.

CKIP-1 REGULATES MAMMALIAN AND ZEBRAFISH MYOBLAST FUSION

Multinucleated muscle fibres arise by fusion of precursor cells called myoblasts. We previously showed that CKIP-1 ectopic expression in C2C12 myoblasts increased cell fusion. In this work, we report that CKIP-1 depletion drastically impairs C2C12 myoblast fusion in vitro and in vivo during zebrafish muscle development. Within developing fast-twich myotome, Ckip-1 localises at the periphery of fast precursor cells, closed to the plasma membrane. Unlike wild-type myoblasts that form spatially arrayed multinucleated fast myofibres, Ckip-1-deficient myoblasts show a drastic reduction in fusion capacity. A search for CKIP-1 binding partners identified the ARPC1 subunit of Arp2/3 actin nucleation complex essential for myoblast fusion. We demonstrate that CKIP-1, through binding to plasma membrane phosphoinositides via its PH domain, regulates cell morphology and lamellipodia formation by recruiting the Arp2/3 complex at the plasma membrane. These results establish CKIP-1 as a regulator of cortical actin that recruits the Arp2/3 complex at the plasma membrane essential for muscle precursor elongation and fusion.

GEP constitutes a negative feedback loop with MyoD and acts as a novel mediator in controlling skeletal muscle differentiation.

Granulin-epithelin precursor (GEP) is an autocrine growth factor that has been implicated in embryonic development, tissue repair, tumorigenesis, and inflammation. Here we report that GEP was expressed in skeletal muscle tissue and its level was differentially altered in the course of C2C12 myoblast fusion. The GEP expression during myoblast fusion was a consequence of MyoD transcription factor binding to several E-box (CANNTG) sequences in the 5'-flanking regulatory region of GEP gene, followed by transcription. Recombinant GEP potently inhibited myotube formation from C2C12 myoblasts whereas the knockdown of endogenous of GEP via a siRNA approach accelerated the fusion of myoblasts to myotubes. Interestingly, the muscle fibers of GEP knockdown mice were larger in number but noticeably smaller in size when compared to the wild-type. Mechanistic studies revealed that during myoblast fusion, the addition of GEP led to remarkable reductions in the expressions of muscle-specific transcription factors, including MyoD. In addition, the regulation of myotube formation by GEP is mediated by the anti-myogenic factor JunB, which is upregulated following GEP stimulation. Thus, GEP growth factor, JunB, and MyoD transcription factor form a regulatory loop and act in concert in the course of myogenesis.

TGF-β isoforms inhibit IGF-1-induced migration and regulate terminal differentiation in a cell-specific manner

Following muscle injury, the damaged tissue and influx of inflammatory cells stimulate the secretion of growth factors and cytokines to initiate repair processes. This release of chemotactic signaling factors activates resident precursor cells and stimulates their mobilization and migration to the site of injury where terminal differentiation can occur. The three transforming growth factor-β (TGF-β) isoforms, and insulin-like growth factor-1 (IGF-1) are among the known regulatory factors released following muscle damage. We investigated the effect of recombinant active TGF-β1, -β2, -β3 and IGF-1 on C2C12 skeletal muscle satellite cell and P19 embryonal carcinoma cell terminal differentiation and migration. C2C12 myoblast fusion as well as P19 embryoid body formation and myogenic differentiation was assessed following 72h TGF-β treatment (5ng/ml), whereas the effect of the TGF-β isoforms on migration was determined following 7h incubation. Our results showed that TGF-β decreases C2C12 myoblast fusion in an isoform-independent manner, whereas in the P19 cell lineage, results demonstrate that TGF-β1 specifically and significantly increased P19 embryoid body formation, but not expression of Connexin-43 or Myosin Heavy Chain. IGF-1 significantly increased migration compared to TGF-β isoforms, which, on their own, had no significant effect on the mobilization of either C2C12 or P19 cells. TGF-β isoforms decreased IGF-1-induced migration of both cell lineages. By distinguishing the factors involved in, and the molecular signals required for, myoblast recruitment during repair processes, strategies can be developed towards improved cell-mediated therapies for muscle injury.

ADP-Ribosylation Factor 6 Regulates Mammalian Myoblast Fusion through Phospholipase D1 and Phosphatidylinositol 4,5-Bisphosphate Signaling Pathways

Myoblast fusion is an essential step during myoblast differentiation that remains poorly understood. M-cadherin-dependent pathways that signal through Rac1 GTPase activation via the Rho-guanine nucleotide exchange factor (GEF) Trio are important for myoblast fusion. The ADP-ribosylation factor (ARF)6 GTPase has been shown to bind to Trio and to regulate Rac1 activity. Moreover, Loner/GEP(100)/BRAG2, a GEF of ARF6, has been involved in mammalian and Drosophila myoblast fusion, but the specific role of ARF6 has been not fully analyzed. Here, we show that ARF6 activity is increased at the time of myoblast fusion and is required for its implementation in mouse C2C12 myoblasts. Specifically, at the onset of myoblast fusion, ARF6 is associated with the multiproteic complex that contains M-cadherin, Trio, and Rac1 and accumulates at sites of myoblast fusion. ARF6 silencing inhibits the association of Trio and Rac1 with M-cadherin. Moreover, we demonstrate that ARF6 regulates myoblast fusion through phospholipase D (PLD) activation and phosphatidylinositol 4,5-bis-phosphate production. Together, these data indicate that ARF6 is a critical regulator of C2C12 myoblast fusion and participates in the regulation of PLD activities that trigger both phospholipids production and actin cytoskeleton reorganization at fusion sites.

The mTORC2 Complex Regulates Terminal Differentiation of C2C12 Myoblasts

Rapamycin, a selective inhibitor of mTORC1 signaling, blocks terminal myoblast differentiation. We found that downregulation of rictor, a component of the mTORC2 complex, but not downregulation of raptor, a component of the mTORC1 complex, prevented terminal differentiation (fusion) of C2C12 myoblasts. Both rapamycin and rictor downregulation suppressed the phosphorylation of AKT(S(473)), and rapamycin treatment of C2C12 myoblasts disrupted the mTORC2 complex. Importantly, downregulation of rictor inhibited TORC2 signaling without inhibiting mTORC1 signaling, suggesting that inhibition of mTORC1 by rapamycin may not be the cause of arrested differentiation. In support of this, expression of a phosphomimetic mutant AKT(S473D) in rictor-deficient cells rescued myoblast fusion even in the presence of rapamycin. mTORC2 signaling to AKT appears necessary for downregulation of the Rho-associated kinase (ROCK1) that occurs during myogenic differentiation. Rapamycin treatment prevented ROCK1 inactivation during differentiation, while suppression of ROCK1 activity during differentiation and myoblast fusion was restored through expression of AKT(S473D), even in the presence of rapamycin. Further, the ROCK inhibitor Y-27632 restored terminal differentiation in rapamycin-treated myoblasts. These results provide the first evidence of a specific role for mTORC2 signaling in terminal myogenic differentiation.

Comparative expression analysis of the murine palladin isoforms

Palladin fulfils a crucial function as a molecular scaffold in organizing and stabilizing the actin cytoskeleton. At least four major palladin isoforms exist due to different promoter usage and alternative splicing: a 200-kDa isoform, a 140-kDa isoform, and two isoforms with a size of 90-92 kDa. Here, we describe their expression during mouse development and in adult tissues. The 200-kDa isoform is predominantly expressed in developing heart and skeletal muscle. The 140-kDa isoform is expressed in various mesenchymal tissues, and also represents the major isoform of the brain. The 90-92-kDa isoforms are almost ubiquitously expressed with the highest levels in smooth muscle-rich tissues. Immunohistochemical and immunofluorecence staining with an anti-200-kDa isoform-specific antiserum localizes the large isoform to the Z-discs of cardiac and skeletal muscle cells. Interestingly, the expression of this isoform is initiated and increasing during in vitro differentiation and fusion of C2C12 myoblasts, suggesting that the 200-kDa palladin isoform may play a scaffolding role during sarcomeric organization.

Wnt3a promotes β‐catenin signaling and myotube formation during myogenic differentiation

Satellite cells are muscle precursor cells required for postnatal skeletal muscle growth and regeneration. Glycogen Synthase Kinase 3beta (GSK‐3beta) has been implicated in negative regulation of skeletal muscle growth and muscle differentiation. Two distinct cellular pools of GSK‐3beta are selectively inhibited by IGF‐I/Akt‐or canonical Wnt signaling, respectively. Since canonical Wnt signaling and subsequent stabilization of beta‐catenin is involved in embryonic muscle formation, the effects of Wnt3a on differentiating myoblasts were investigated, and compared to those resulting from GSK‐3beta inactivation by LiCl or IGF‐I.Wnt3a promoted C2C12 myoblast fusion, and induced beta‐catenin signaling in a time and dose dependent fashion. In addition, beta‐catenin‐dependent transcriptional activation was also induced after GSK‐3beta inhibition using LiCl, but not by IGF‐I. Furthermore, stimulation of muscle gene expression was only observed in response to LiCl but not Wnt3a, which uncouples the promoting effects of Wnt3a on myoblast fusion from muscle specific gene expression.These data suggest that myoblast fusion and muscle specific gene expression are controlled by distinct cellular pools of GSK‐3beta, and provide evidence for a role of GSK‐3beta as a central regulator of muscle differentiation.This work was supported by a research grant of the Netherlands Astma Foundation (NAF 3.2.02.63).

Cooperation of the Metalloprotease, Disintegrin, and Cysteine-rich Domains of ADAM12 during Inhibition of Myogenic Differentiation

The extracellular domain of the mature form of ADAM12 consists of the metalloprotease, disintegrin, cysteine-rich, and epidermal growth factor (EGF)-like domains. The disintegrin, cysteine-rich, and EGF-like fragments have been shown previously to support cell adhesion via activated integrins or proteoglycans. In this study, we report that the entire extracellular domain of mouse ADAM12 produced in Drosophila S2 cells supported efficient adhesion and spreading of C2C12 myoblasts even in the absence of exogenous integrin activators. This adhesion was not mediated by beta1 integrins or proteoglycans, was myoblast-specific, and required the presence of both the metalloprotease and disintegrin/cysteine-rich domains of ADAM12. Analysis of the recombinant proteins by far-UV circular dichroism suggested that the secondary structures of the autonomously expressed metalloprotease domain and the disintegrin/cysteine-rich/EGF-like domains differ from the structures present in the intact extracellular domain. Furthermore, the intact extracellular domain (but not the metalloprotease domain or the disintegrin/cysteine-rich/EGF-like fragment alone) decreased the expression of the cell cycle inhibitor p21 and myogenin, two markers of differentiation, and inhibited C2C12 myoblast fusion. Thus, the novel protein-protein interaction reported here involving the extracellular domain of ADAM12 may have important biological consequences during myoblast differentiation.

The Homeodomain Protein Barx2 Promotes Myogenic Differentiation and Is Regulated by Myogenic Regulatory Factors

The homeobox protein Barx2 is expressed in both smooth and skeletal muscle and is up-regulated during differentiation of skeletal myotubes. Here we use antisense-oligonucleotide inhibition of Barx2 expression in limb bud cell culture to show that Barx2 is required for myotube formation. Moreover, overexpression of Barx2 accelerates the fusion of MyoD-positive limb bud cells and C2C12 myoblasts. However, overexpression of Barx2 does not induce ectopic MyoD expression in either limb bud cultures or in multipotent C3H10T1/2 mesenchymal cells, and does not induce fusion of C3H10T1/2 cells. These results suggest that Barx2 acts downstream of MyoD. To test this hypothesis, we isolated the Barx2 gene promoter and identified DNA regulatory elements that might control Barx2 expression during myogenesis. The proximal promoter of the Barx2 gene contained binding sites for several factors involved in myoblast differentiation including MyoD, myogenin, serum response factor, and myocyte enhancer factor 2. Co-transfection experiments showed that binding sites for both MyoD and serum response factor are necessary for activation of the promoter by MyoD and myogenin. Taken together, these studies indicate that Barx2 is a key regulator of myogenic differentiation that acts downstream of muscle regulatory factors.