Sema4C participates in myogenic differentiation in vivo and in vitro through the p38 MAPK pathway

Sema4C is a member of transmembrane semaphorin proteins which regulate axonal guidance in the developing nervous system. The expression of Sema4C was dramatically induced not only during differentiation of C2C12 mouse myoblasts, but also during injury-induced skeletal muscle regeneration. C2C12 cells stably or transiently expressing Sema4C both showed increased myogenic differentiation reflected by accelerated myotube formation and expression of muscle-specific proteins. Overexpression of Sema4C elicited p38 phosphorylation directly, and the effects of Sema4C during myogenic differentiation could be abolished by the p38alpha-specific inhibitor SB203580. Knockdown of Sema4C by siRNA transfection during C2C12 myoblasts differentiation could suppress the phosphorylation of p38 followed by dramatically diminished myotube formation. Sema4C could activate the myogenin promoter during myogenic differentiation. This activation could be abolished by p38 inhibitor SB203580. Taken together, these observations reveal novel functional potentialities of Sema4C which suggest that Sema4C promotes terminal myogenic differentiation in a p38 MAPK-dependent manner.     

Myogenesis defect due to Toca-1 knockdown can be suppressed by expression of N-WASP

Skeletal muscle formation is a multistep process involving proliferation, differentiation, alignment and fusion of myoblasts to form myotubes which fuse with additional myoblast to form myofibers. Toca-1 (Transducer of Cdc42-dependent actin assembly), is an adaptor protein which activates N-WASP in conjunction with Cdc42 to facilitate membrane invagination, endocytosis and actin cytoskeleton remodeling. Expression of Toca-1 in mouse primary myoblasts and C2C12 myoblasts was up-regulated on day 1 of differentiation and subsequently down-regulated during differentiation. Knocking down Toca-1 expression in C2C12 cells (Toca-1^KD cells) resulted in a significant decrease in myotube formation and expression of shRNA-resistant Toca-1 in Toca-1^KD cells rescued the myogenic defect, suggesting that the knockdown was specific and Toca-1 is essential for myotube formation. Toca-1^KD cells exhibited elongated spindle-like morphology, expressed myogenic markers (MyoD and MyHC) and localized N-Cadherin at cell periphery similar to control cells suggesting that Toca-1 is not essential for morphological changes or expression of proteins critical for differentiation. Toca-1^KD cells displayed prominent actin fibers suggesting a defect in actin cytoskeleton turnover necessary for cell–cell fusion. Toca-1^KD cells migrated faster than control cells and had a reduced number of vinculin patches similar to N-WASP^KO MEF cells. Transfection of N-WASP-expressing plasmid into Toca-1^KD cells restored myotube formation of Toca-1^KD cells. Thus, our results suggest that Toca-1^KD cells have defects in formation of myotubes probably due to reduced activity of actin cytoskeleton regulators such as N-WASP. This is the first study to identify and characterize the role of Toca-1 in myogenesis.     

Stimulation of fascin spikes by thrombospondin-1 is mediated by the GTPases Rac and Cdc42

Cell adhesion to extracellular matrix is an important physiological stimulus for organization of the actin-based cytoskeleton. Adhesion to the matrix glycoprotein thrombospondin-1 (TSP-1) triggers the sustained formation of F-actin microspikes that contain the actin-bundling protein fascin. These structures are also implicated in cell migration, which may be an important function of TSP-1 in tissue remodelling and wound repair. To further understand the function of fascin microspikes, we examined whether their assembly is regulated by Rho family GTPases. We report that expression of constitutively active mutants of Rac or Cdc42 triggered localization of fascin to lamellipodia, filopodia, and cell edges in fibroblasts or myoblasts. Biochemical assays demonstrated prolonged activation of Rac and Cdc42 in C2C12 cells adherent to TSP-1 and activation of the downstream kinase p21-activated kinase (PAK). Expression of dominant-negative Rac or Cdc42 in C2C12 myoblasts blocked spreading and formation of fascin spikes on TSP-1. Spreading and spike assembly were also blocked by pharmacological inhibition of F-actin turnover. Shear-loading of monospecific anti-fascin immunoglobulins, which block the binding of fascin to actin into cytoplasm, strongly inhibited spreading, actin cytoskeletal organization and migration on TSP-1 and also affected the motility of cells on fibronectin. We conclude that fascin is a critical component downstream of Rac and Cdc42 that is needed for actin cytoskeletal organization and cell migration responses to thrombospondin-1.     

The LIM-only protein FHL2 interacts with beta-catenin and promotes differentiation of mouse myoblasts

FHL2 is a LIM-domain protein expressed in myoblasts but down-regulated in malignant rhabdomyosarcoma cells, suggesting an important role of FHL2 in muscle development. To investigate the importance of FHL2 during myoblast differentiation, we performed a yeast two-hybrid screen using a cDNA library derived from myoblasts induced for differentiation. We identified beta-catenin as a novel interaction partner of FHL2 and confirmed the specificity of association by direct in vitro binding tests and coimmunoprecipitation assays from cell lysates. Deletion analysis of both proteins revealed that the NH2-terminal part of beta-catenin is sufficient for binding in yeast, but addition of the first armadillo repeat is necessary for binding FHL2 in mammalian cells, whereas the presence of all four LIM domains of FHL2 is needed for the interaction. Expression of FHL2 counteracts beta-catenin-mediated activation of a TCF/LEF-dependent reporter gene in a dose-dependent and muscle cell-specific manner. After injection into Xenopus embryos, FHL2 inhibited the beta-catenin-induced axis duplication. C2C12 mouse myoblasts stably expressing FHL2 show increased myogenic differentiation reflected by accelerated myotube formation and expression of muscle-specific proteins. These data imply that FHL2 is a muscle-specific repressor of LEF/TCF target genes and promotes myogenic differentiation by interacting with beta-catenin.     

Mutant actins that stabilise F-actin use distinct mechanisms to activate the SRF coactivator MAL

Nuclear accumulation of the serum response factor coactivator MAL/MKL1 is controlled by its interaction with G-actin, which results in its retention in the cytoplasm in cells with low Rho activity. We previously identified actin mutants whose expression promotes MAL nuclear accumulation via an unknown mechanism. Here, we show that actin interacts directly with MAL in vitro with high affinity. We identify a further activating mutation, G15S, which stabilises F-actin, as do the activating actins S14C and V159N. The three mutants share several biochemical properties, but can be distinguished by their ability to bind cofilin, ATP and MAL. MAL interaction with actin S14C is essentially undetectable, and that with actin V159N is weakened. In contrast, actin G15S interacts more strongly with MAL than the wild-type protein. Strikingly, the nuclear accumulation of MAL induced by overexpression of actin S14C is substantially dependent on Rho activity and actin treadmilling, while that induced by actin G15S expression is not. We propose a model in which actin G15S acts directly to promote MAL nuclear entry.     

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.     

Myoblast structure affects subsequent skeletal myotube morphology and sarcomere assembly

Skeletal myogenesis is a precise procedure marked by specific changes in muscle cell morphology and cytoarchitecture. Cessation of proliferation by skeletal muscle precursor cells (myoblasts) coincides with the induction of fusion to form multinucleated myotubes and the initiation of differentiation, the process through which sarcomeres are formed. Concurrently, there is a distinct upregulation in expression of muscle-specific isoforms and an extreme downregulation of non-muscle-specific cytoskeletal isoforms. The sarcomere is the contractile unit of the cell and is comprised of a number of different proteins aggregated and aligned in very ordered arrays along the myotube. It is this rigorously controlled alignment that gives striated muscle its characteristic "striped" appearance. Previous studies, conducted predominantly in cardiac muscle, propose models for the development of the sarcomere that attribute little of the differentiative process to the myoblast morphology and cytoskeletal arrangement. In this study, perturbation of myoblast morphology and cytoskeletal arrangement by transfection with nonmuscle actin genes in the mouse skeletal muscle cell line C2 resulted in myotubes of both varied morphology and sarcomeric structure. The results presented herein not only provide novel insights into the formation of the sarcomere in skeletal muscle, but also suggest a role for myoblast morphology and cytoskeletal structure in the subsequent differentiation of the myotube.     

A WNT/beta-catenin signaling activator, R-spondin, plays positive regulatory roles during skeletal myogenesis

R-spondins (RSPOs) are a recently characterized family of secreted proteins that activate WNT/β-catenin signaling. In this study, we investigated the potential roles of the RSPO proteins during myogenic differentiation. Overexpression of the Rspo1 gene or administration of recombinant RSPO2 protein enhanced mRNA and protein expression of a basic helix-loop-helix (bHLH) class myogenic determination factor, MYF5, in both C2C12 myoblasts and primary satellite cells, whereas MYOD or PAX7 expression was not affected. RSPOs also promoted myogenic differentiation and induced hypertrophic myotube formation in C2C12 cells. In addition, Rspo2 and Rspo3 gene knockdown by RNA interference significantly compromised MYF5 expression, myogenic differentiation, and myotube formation. Furthermore, Myf5 expression was reduced in the developing limbs of mouse embryos lacking the Rspo2 gene. Finally, we demonstrated that blocking of WNT/β-catenin signaling by DKK1 or a dominant-negative form of TCF4 reversed MYF5 expression, myogenic differentiation, and hypertrophic myotube formation induced by RSPO2, indicating that RSPO2 exerts its activity through the WNT/β-catenin signaling pathway. Our results provide strong evidence that RSPOs are key positive regulators of skeletal myogenesis acting through the WNT/β-catenin signaling pathway.     

Expression of phospholipase C beta family isoenzymes in C2C12 myoblasts during terminal differentiation

In the present work, we have analyzed the expression and subcellular localization of all the members of inositide-specific phospholipase C (PLCbeta) family in muscle differentiation, given that nuclear PLCbeta1 has been shown to be related to the differentiative process. Cell cultures of C2C12 myoblasts were induced to differentiate towards the phenotype of myotubes, which are also indicated as differentiated C2C12 cells. By means of immunochemical and immunocytochemical analysis, the expression and subcellular localization of PLCbeta1, beta2, beta3, beta4 have been assessed. As further characterization, we investigated the localization of PLCbeta isoenzymes in C2C12 cells by fusing their cDNA to enhanced green fluorescent protein (GFP). In myoblast culture, PLCbeta4 was the most expressed isoform in the cytoplasm, whereas PLCbeta1 and beta3 exhibited a lesser expression in this cell compartment. In nuclei of differentiated myotube culture, PLCbeta1 isoform was expressed at the highest extent. A marked decrease of PLCbeta4 expression in the cytoplasm of differentiated C2C12 cells was detected as compared to myoblasts. No relevant differences were evidenced as regards the expression of PLCbeta3 at both cytoplasmatic and nuclear level, whilst PLCbeta2 expression was almost undetectable. Therefore, we propose that the different subcellular expression of these PLC isoforms, namely the increase of nuclear PLCbeta1 and the decrease of cytoplasmatic PLCbeta4, during the establishment of myotube differentiation, is related to a spatial-temporal signaling event, involved in myogenic differentiation. Once again the subcellular localization appears to be a key step for the diverse signaling activity of PLCbetas.     

The roles of integrin-linked kinase in the regulation of myogenic differentiation

Myogenic differentiation is a highly orchestrated, multistep process that is coordinately regulated by growth factors and cell adhesion. We show here that integrin-linked kinase (ILK), an intracellular integrin- and PINCH-binding serine/threonine protein kinase, is an important regulator of myogenic differentiation. ILK is abundantly expressed in C2C12 myoblasts, both before and after induction of terminal myogenic differentiation. However, a noticeable amount of ILK in the Triton X-100-soluble cellular fractions is significantly reduced during terminal myogenic differentiation, suggesting that ILK is involved in cellular control of myogenic differentiation. To further investigate this, we have overexpressed the wild-type and mutant forms of ILK in C2C12 myoblasts. Overexpression of ILK in the myoblasts inhibited the expression of myogenic proteins (myogenin, MyoD, and myosin heavy chain) and the subsequent formation of multinucleated myotubes. Furthermore, mutations that eliminate either the PINCH-binding or the kinase activity of ILK abolished its ability to inhibit myogenic protein expression and allowed myotube formation. Although overexpression of the ILK mutants is permissive for the initiation of terminal myogenic differentiation, the myotubes derived from myoblasts overexpressing the ILK mutants frequently exhibited an abnormal morphology (giant myotubes containing clustered nuclei), suggesting that ILK functions not only in the initial decision making process, but also in later stages (fusion or maintaining myotube integrity) of myogenic differentiation. Additionally, we show that overexpression of ILK, but not that of the PINCH-binding defective or the kinase-deficient ILK mutants, prevents inactivation of MAP kinase, which is obligatory for the initiation of myogenic differentiation. Finally, inhibition of MAP kinase activation reversed the ILK-induced suppression of myogenic protein expression. Thus, ILK likely influences the initial decision making process of myogenic differentiation by regulation of MAP kinase activation.     

Cytoplasmic nuclear transfer of the actin-capping protein tropomodulin

Tropomodulin (Tmod) is a cytoskeletal actin-capping protein that interacts with tropomyosin at the pointed end of actin filaments. E-Tmod is an isoform that expresses predominantly in cardiac cells and slow skeletal muscle fibers. We unexpectedly discovered significant levels of Tmod in nuclei and then defined peptide domains in Tmod responsible for nuclear import and export. These domains resemble, and function as, a nuclear export signal (NES) and a pattern 4 nuclear localization signal (NLS). Both motifs are conserved in other Tmod isoforms and across species. Comparisons of wild-type Tmod and Tmod carrying mutations in these peptide domains revealed that Tmod normally traffics through the nucleus. These observations logically presuppose that Tmod functions may include a nuclear role. Indeed, increasing Tmod in the nucleus severely hampered myogenic differentiation and selectively suppressed muscle-specific gene expression (endogenous p21, myosin heavy chain, myogenin, and Tmod) but did not affect endogenous glyceraldehyde-3-phosphate dehydrogenase or expression from a transfected E-GFP vector. These results suggest that, at least in myogenic cells, nuclear Tmod may be involved in the differentiation process.     

The SWI/SNF Subunit/Tumor Suppressor BAF47/INI1 Is Essential in Cell Cycle Arrest upon Skeletal Muscle Terminal Differentiation

Myogenic terminal differentiation is a well-orchestrated process starting with permanent cell cycle exit followed by muscle-specific genetic program activation. Individual SWI/SNF components have been involved in muscle differentiation. Here, we show that the master myogenic differentiation factor MyoD interacts with more than one SWI/SNF subunit, including the catalytic subunit BRG1, BAF53a and the tumor suppressor BAF47/INI1. Downregulation of each of these SWI/SNF subunits inhibits skeletal muscle terminal differentiation but, interestingly, at different differentiation steps and extents. BAF53a downregulation inhibits myotube formation but not the expression of early muscle-specific genes. BRG1 or BAF47 downregulation disrupt both proliferation and differentiation genetic programs expression. Interestingly, BRG1 and BAF47 are part of the SWI/SNF remodeling complex as well as the N-CoR-1 repressor complex in proliferating myoblasts. However, our data show that, upon myogenic differentiation, BAF47 shifts in favor of N-CoR-1 complex. Finally, BRG1 and BAF47 are well-known tumor suppressors but, strikingly, only BAF47 seems essential in the myoblasts irreversible cell cycle exit. Together, our data unravel differential roles for SWI/SNF subunits in muscle differentiation, with BAF47 playing a dual role both in the permanent cell cycle exit and in the regulation of muscle-specific genes.