Myogenesis In C2C12 Myoblasts Research Articles

Fibro-adipogenic progenitor cells in skeletal muscle unloading: metabolic and functional impairments

BackgroundSkeletal muscle resident fibro-adipogenic progenitor cells (FAPs) control skeletal muscle regeneration providing a supportive role for muscle stem cells. Altered FAPs characteristics have been shown for a number of pathological conditions, but the influence of temporary functional unloading of healthy skeletal muscle on FAPs remains poorly studied. This work is aimed to investigate how skeletal muscle disuse affects the functionality and metabolism of FAPs.MethodsHindlimb suspension (HS) rat model employed to investigate muscle response to decreased usage. FAPs were purified from m. soleus functioning muscle (Contr) and after functional unloading for 7 and 14 days (HS7 and HS14). FAPs were expanded in vitro, and tested for: immunophenotype; in vitro expansion rate, and migration activity; ability to differentiate into adipocytes in vitro; metabolic changes. Crosstalk between FAPs and muscle stem cells was estimated by influence of medium conditioned by FAP’s on migration and myogenesis of C2C12 myoblasts. To reveal the molecular mechanisms behind unloading-induced alterations in FAP’s functionality transcriptome analysis was performed.ResultsFAPs isolated from Contr and HS muscles exhibited phenotype of MSC cells. FAPs in vitro expansion rate and migration were altered by functional unloading conditions. All samples of FAPs demonstrated the ability to adipogenic differentiation in vitro, however, HS FAPs formed fat droplets of smaller volume and transcriptome analysis showed fatty acids metabolism and PPAR signaling suppression. Skeletal muscle unloading resulted in metabolic reprogramming of FAPs: decreased spare respiratory capacity, decreased OCR/ECAR ratio detected in both HS7 and HS14 samples point to reduced oxygen consumption, decreased potential for substrate oxidation and a shift to glycolytic metabolism. Furthermore, C2C12 cultures treated with medium conditioned by FAPs showed diverse alterations: while the HS7 FAPs-derived paracrine factors supported the myoblasts fusion, the HS14-derived medium stimulated proliferation of C2C12 myoblasts; these observations were supported by increased expression of cytokines detected by transcriptome analysis.Conclusionthe results obtained in this work show that the skeletal muscle functional unloading affects properties of FAPs in time-dependent manner: in atrophying skeletal muscle FAPs act as the sensors for the regulatory signals that may stimulate the metabolic and transcriptional reprogramming to preserve FAPs properties associated with maintenance of skeletal muscle homeostasis during unloading and in course of rehabilitation.

Optomechanically Actuated Hydrogel Platform for Cell Stimulation with Spatial and Temporal Resolution.

Cells exist in the body in mechanically dynamic environments, yet the vast majority of in vitro cell culture is conducted on static materials such as plastic dishes and gels. To address this limitation, we report an approach to transition widely used hydrogels into mechanically active substrates by doping optomechanical actuator (OMA) nanoparticles within the polymer matrix. OMAs are composed of gold nanorods surrounded by a thermoresponsive polymer shell that rapidly collapses upon near-infrared (NIR) illumination. As a proof of concept, we crosslinked OMAs into laminin-gelatin hydrogels, generating up to 5 μm deformations triggered by NIR pulsing. This response was tunable by NIR intensity and OMA density within the gel and is generalizable to other hydrogel materials. Hydrogel mechanical stimulation enhanced myogenesis in C2C12 myoblasts as evidenced by ERK signaling, myocyte fusion, and sarcomeric myosin expression. We also demonstrate rescued differentiation in a chronic inflammation model as a result of mechanical stimulation. This work establishes OMA-actuated biomaterials as a powerful tool for in vitro mechanical manipulation with broad applications in the field of mechanobiology.

Immodulin peptides influence musculoskeletal homeostasis by linking extracellular cues to macrophage and myoblast nuclear receptors.

Immodulins are synthetic peptides derived from the C-terminal domains of insulin-like growth factor binding proteins (IGFBPs). Immodulins from the 3/5/6 (but not 1/2/4) IGFBP evolutionary clade transduce extracellular matrix (ECM) signals to RXR, NR4A1 and PPAR-alpha nuclear receptors (NRs) to stimulate novel macrophage lineages. The rationale of this study was to reconcile physical associations of immodulins with ECM and NRs, effects of siRNAs and chemical inhibitors in vivo, and immodulin-driven pro-differentiation effects in cell culture. When added to THP1D cells, immodulins stimulate CD169+ Clec9a+ and Clec12a+ macrophage lineages via a EP300/RXRγ/Nur77 transcriptional mechanism. This phenomenon is accompanied by the secretion of CCL22, IL-10 and TGFbeta and the ability to stimulate FoxP3+ T-cells in co-culture. ECM ligands of 3/5/6 immodulins include iron, zinc, glycosaminoglycans, transferrin and phosphatidylinositol-4,5,-biphosphate (PIP2), which can influence their pro-differentiation effects. Remarkably, immodulins also stimulate myogenesis in C2C12 myoblasts, thereby revealing a novel link between immune and musculoskeletal homeostasis. Distinct NR agonists stimulate these companion differentiation processes. Using solution NMR to guide design, immodulins with a tripeptide extension near the iron-binding pocket demonstrated higher iron-binding and improved pro-differentiation activities. Transferrin-bound immodulin shows binding preference for both high-molecular-weight hyaluronan (HMWHA) and HMWHA:CD44 complexes at endosomal pH, and interacts with PIP2 at normal physiological pH, offering intriguing mechanistic insights.

Downregulation of Sparc-like protein 1 during cisplatin-induced inhibition of myogenic differentiation of C2C12 myoblasts

Patients with cancer often experience muscle atrophy, which worsens their prognosis. Decreased muscle regenerative capacity plays an important role in the complex processes involved in muscle atrophy. Administration of cisplatin, a cancer chemotherapeutic agent, has been implicated as a cause of muscle atrophy. In this study, we examined whether cisplatin affects the differentiation of myoblasts into myotubes. We treated C2C12 myoblasts with a differentiation medium containing cisplatin and its vehicle during for 8 days and observed the changes in the expression of myosin heavy chain (MyHC) and myogenin in the myoblasts. Cisplatin was injected in mice for 4 consecutive days; on Day 5, the mice quadriceps muscles were sampled and examined. The expression of MyHCs increased and that of myogenin decreased after cisplatin treatment. The secretion of acidic cysteine-rich proteins (e.g., Sparc proteins) reportedly promotes C2C12 myoblast differentiation. Therefore, we investigated the Sparc family gene expression during myogenesis in C2C12 myoblasts after cisplatin treatment. Of all the genes investigated, Sparc-like protein 1 (Sparcl1) expression was significantly suppressed by cisplatin on Days 4–8. Simultaneous treatment with recombinant mouse Sparcl1 almost inhibited the cisplatin-induced suppression of total MyHC and myogenin protein levels. Moreover, Sparcl1 expression decreased in the skeletal muscles of mice, leading to cisplatin-induced muscle atrophy. Our results suggest that cisplatin-induced myogenesis suppression causes muscle atrophy and inhibits the expression of Sparcl1, which promotes C2C12 cell differentiation during myogenesis.

Long acting GLP-1 analog liraglutide ameliorates skeletal muscle atrophy in rodents

BackgroundSkeletal muscle atrophy is characterized by muscle wasting with partial or complete functional loss. Skeletal muscle atrophy severely affects the quality of life and currently, there is no available therapy except for spinal muscular atrophy. ObjectiveDrug repositioning is a promising strategy that reduces cost and time due to prior availability of safety and toxicity details. Here we investigated myogenic and anti-atrophy effects of glucagon-like peptide-1 (GLP-1) analog liraglutide. MethodsWe used several in vitro atrophy models in C2C12 cells and in vivo models in Sprague Dawley rats to study Liraglutide's efficacy. Western blotting was used to assess cAMP-dependent signaling pathways specifically activated by liraglutide. Therapeutic efficacy of liraglutide was investigated by histological analysis of transverse muscle sections followed by morphometry. Myogenic capacity was investigated by immunoblotting for myogenic factors. ResultsLiraglutide induced myogenesis in C2C12 myoblasts through GLP-1 receptor via a cAMP-dependent complex network of signaling events involving protein kinase A, phosphoinositide 3-kinase/protein kinase B, p38 mitogen-activated protein kinase and extracellular signal-regulated kinase. Liraglutide imparted protection against freeze injury, denervation, and dexamethasone -induced skeletal muscle atrophy and improved muscular function in all these models. In a therapeutic model, liraglutide restored myofibrillar architecture in ovariectomy-induced atrophy. Anti-atrophy actions of liraglutide involved suppression of atrogene expression and enhancement in expression of myogenic factors. ConclusionLiraglutide imparted protection and restored myofibrillar architecture in diverse models of muscle atrophy. Given its potent anti-atrophy, and recently reported osteoanabolic effects, we propose liraglutide's clinical evaluation in skeletal muscle atrophy and musculoskeletal disorders associated with diverse pathologies.

Canadine from Corydalis turtschaninovii Stimulates Myoblast Differentiation and Protects against Myotube Atrophy.

Cachexia and sarcopenia are the main causes of muscle atrophy. These result in a reduction in the muscle fiber area, myo-protein content, and muscle strength, with various molecular modulators being involved. Although several reports have proposed potential therapeutic agents, no effective treatments have been found for muscle atrophy. We searched for myogenic modulators from medicinal plants to treat muscle diseases. We isolated six alkaloids from Corydalis turtschaninovii and evaluated their myogenic potential by using the MyoD reporter gene assay in C2C12 cells. Among the tested compounds, canadine showed the strongest transactivation of MyoD and increased MHC expression during myogenesis. The activation of p38 MAP kinase and Akt are major mechanisms that contribute to the myogenesis by canadine. Canadine increased the number of multinucleated and cylinder-shaped myotubes during myogenesis of C2C12 myoblasts. To determine the preventive effect of canadine in cancer-induced muscle wasting, differentiated C2C12 myotubes were treated with conditioned media from CT26 colon carcinoma culture (CT26 CM) in the presence of canadine. Canadine ameliorated the muscle protein degradation caused by CT26-CM by down-regulating the muscle specific-E3 ligases, MAFbx/atrogin-1 and MuRF1. In this study, we found that canadine from C. turtschaninovii stimulates myogenesis and also inhibits muscle protein degradation. Therefore, we suggest canadine as a protective agent against muscle atrophy.

YAP promotes myogenic differentiation via the MEK5-ERK5 pathway.

Yes-associated protein (YAP) is a transcriptional coactivator in the Hippo pathway that regulates cell proliferation, differentiation, and apoptosis. The MEK5/ERK5 MAPK cascade is essential for the early step of myogenesis. In this study, we generated C2C12 stable cell lines that expressed YAP (C2C12-YAP cells) and found that ERK5 and MEK5 were activated in C2C12-YAP cells compared with control C2C12 (C2C12-vector) cells. C2C12-YAP stable cells also differentiated into myotubes better than C2C12-vector cells, and expressed elevated levels of myogenin, a transcription factor that regulates myogenesis, as well as elevated levels of myosin heavy chain, a skeletal muscle marker. Western blot analysis revealed that Src and c-Abl (Abelson murine leukemia viral oncogene homolog 1) activation were enhanced in C2C12-YAP cells. Conversely, treatment of inhibitors of c-Abl, Src, or MEK5 inhibited activation of MEK5 and ERK5 and myogenesis of C2C12 myoblasts. Specific interactions between YAP and proteins in the ERK5 pathway, such as MEK kinase 3 (MEKK3) and ERK5, were illustrated by coimmunoprecipitation experiments. MEKK3 contains the PPGY motif (aa 178-181), which may interact with YAP. Site-directed mutagenesis experiments revealed that expression of MEKK3 Y181F mutant inhibited MEK5/ERK5 activation and myogenic differentiation. These results suggest that YAP promotes muscle differentiation by activating the Abl/Src/MEKK3/MEK5/ERK5 kinase cascade.-Chen, T.-H., Chen, C.-Y., Wen, H.-C., Chang, C.-C., Wang, H.-D., Chuu, C.-P., Chang, C.-H. YAP promotes myogenic differentiation via the MEK5-ERK5 pathway.

Stimulating effect of graphene oxide on myogenesis of C2C12 myoblasts on RGD peptide-decorated PLGA nanofiber matrices.

BackgroundIn the field of biomedical engineering, many studies have focused on the possible applications of graphene and related nanomaterials due to their potential for use as scaffolds, coating materials and delivery carriers. On the other hand, electrospun nanofiber matrices composed of diverse biocompatible polymers have attracted tremendous attention for tissue engineering and regenerative medicine. However, their combination is intriguing and still challenging.ResultsIn the present study, we fabricated nanofiber matrices composed of M13 bacteriophage with RGD peptide displayed on its surface (RGD-M13 phage) and poly(lactic-co-glycolic acid, PLGA) and characterized their physicochemical properties. In addition, the effect of graphene oxide (GO) on the cellular behaviors of C2C12 myoblasts, which were cultured on PLGA decorated with RGD-M13 phage (RGD/PLGA) nanofiber matrices, was investigated. Our results revealed that the RGD/PLGA nanofiber matrices have suitable physicochemical properties as a tissue engineering scaffold and the growth of C2C12 myoblasts were significantly enhanced on the matrices. Moreover, the myogenic differentiation of C2C12 myoblasts was substantially stimulated when they were cultured on the RGD/PLGA matrices in the presence of GO.ConclusionIn conclusion, these findings propose that the combination of RGD/PLGA nanofiber matrices and GO can be used as a promising strategy for skeletal tissue engineering and regeneration.

CXXC5 regulates differentiation of C2C12 myoblasts into myocytes.

CXXC5 is a member of the CXXC-type zinc-finger domain containing protein family, which is suggested to function in gene transcription, cell adhesion and cytoskeleton organization. Previous studies have revealed that CXXC5 is expressed in skeletal muscle, but whether it regulates skeletal myogenesis is yet unknown. Here, we screened for the possible signaling pathways in which CXXC5 might participate using luciferase gene reporters. The results indicated that CXXC5 significantly increased the activities of the promoters of genes involved in skeletal muscle differentiation. We therefore studied the role of CXXC5 during skeletal myogenesis in C2C12 myoblasts. Our findings suggest that overexpression of CXXC5 in C2C12 myoblasts facilitated myocyte differentiation, while RNAi interference of CXXC5 significantly inhibited the differentiation of C2C12 myoblasts. This study suggests that CXXC5 plays a significant role in regulating skeletal myogenesis.

Wnt3a potentiates myogenesis in C2C12 myoblasts through the modulation of intracellular calcium and activation of the β‐catenin signaling pathway (1102.23)

Our research group has been investigating the biochemical crosstalk between bones and muscles. Wnts are secreted by bone cells and have a significant potential to directly influence muscle function. Our previous studies demonstrated potent myogenic effects of Wnt3a at a concentration of 10ng/ml. We now present new evidence that as low as 0.5ng/ml can significantly increase myogenic differentiation, which was confirmed morphologically and immunohistochemically. Wnt3a also promoted translocation of β‐catenin to the nucleus of myocytes/myotubes, suggesting that Wnt3a might lead to the downstream activation of muscle regulatory factors. Intriguingly, the potent effect of Wnt3a on stimulating the activation of the β‐catenin signaling pathway in C2C12 cells strongly correlated with the appearance of intracellular calcium oscillations in myotubes measured with Fura‐2. In agreement, focused qRT‐PCR arrays revealed a 6‐fold increase in IP3‐R1 and IP3R3, also revealed that Wnt3a promoted overexpression of myogenic markers (MyoD, MyoG, MHC), and downstream genes of the β‐catenin pathway (Fhl1 and Numb). We propose that Wnt3a acts through the modulation of intracellular calcium, resulting in translocation of β‐catenin that in turn enhances expression of muscle regulatory factors.Grant Funding Source: NIH‐NIA P01 AG039355 (LB, MB), Dale and Dorothy Thompson Endowment Fund (MB) and Missouri Life Scien

Lactate regulates myogenesis in C2C12 myoblasts in vitro

Satellite cells (SCs) are the resident stem cells of skeletal muscle tissue which play a major role in muscle adaptation, e.g. as a response to physical training. The aim of this study was to examine the effects of an intermittent lactate (La) treatment on the proliferation and differentiation of C2C12 myoblasts, simulating a microcycle of high intensity endurance training. Furthermore, the involvement of reactive oxygen species (ROS) in this context was examined. C2C12 myoblasts were therefore repeatedly incubated for 2h each day with 10mM or 20mM La differentiation medium (DM) and in some cases 20mM La DM plus different antioxidative substances for up to 5days. La free (0mM) DM served as a control. Immunocytochemical staining, Western blot analysis and colorimetric assays were used to assess oxidative stress, proliferation, and differentiation. Results show that La induces oxidative stress, enhances cell-cycle withdrawal, and initiates early differentiation but delays late differentiation in a timely and dose-dependent manner. These effects can be reversed by the addition of antioxidants to the La DM. We therefore conclude that La has a regulatory role in C2C12 myogenesis via a ROS-sensitive mechanism which elicits implications for reassessing some aspects of training and the use of nutritional supplements.

Characterization of myogenesis in C2C12 myoblasts using Flow Cytometry

Impaired myogenesis is linked to the muscle wasting and weakness characteristic of sarcopenia and muscular dystrophies. In the past, fusion index, a key biomarker of the myogenic differentiation process, has provided valuable data, but has required labor intensive techniques that often lack precision. Flow Cytometry (FC) provides an accurate and efficient way to characterize cell size, complexity and DNA content with forward scatter (FSC), side scatter (SSC), and propidium iodide staining (FL2), respectively. We propose that as myogenesis progresses, with the accompanying increase in fusion events, FC can be used to detect increasing mitochondrial biogenesis, DNA content and myosin heavy chain in C2C12 mouse skeletal myoblasts. For a more precise comparison of these parameters, we found it useful to synchronize cell cycle at G0/1 through serum deprivation for 24 hours. While still a developing methodology, our preliminary studies, demonstrate that FC could be a valuable method to quantify variations in cell size, character and DNA content in myoblasts and myotubes. This method promises to provide a more reliable characterization of myogenic differentiation to advance this field of research. Support: NIH‐National Institutes of Aging P01 AG039355 and Missouri Life Sciences Research Board (MB).

Multiple‐staged Regulation of Myogenic Differentiation by Prostaglandin E2

We showed that conditioned media from osteocytes accelerates myogenesis in C2C12 myoblasts, which suggests signaling from bone to muscle cells. Since osteocytes secrete Prostaglandin E2 (PGE2), one of the major PGs synthesized from arachidonic acid through cyclooxygenase pathways, we started a systematic investigation of PGE2 roles in myogenesis. C2C12 myoblasts were used to investigate the effect and related molecular mechanisms of PGE2 on myogenic differentiation. Expression of PGE2 receptors (EP1‐EP4) was determined by real‐time PCR. EP1 and EP4 were highly expressed, whereas EP2 and EP3 were not detectable. Specific inhibitors SC 51322 for EP1 or L161,982 for EP4 significantly attenuated myogenesis. EP1 inhibition reduced the number of early myotubes, and subsequently affected the development of mature myotubes. On the other hand, EP4 inhibition did not affect the generation of early myotubes, but the fusion of early myotubes was dramatically inhibited. Interestingly, expression of two key myogenic regulatory factors, MyoD and Myogenin, was downregulated by EP1, but not by EP4 inhibition. These data provide evidence for the regulation of myogenesis by PGE2 via EP1 and EP4. The different effects of EP1 and EP4 inhibition suggest PGE2 signaling in myogenic differentiation is complex and multiple‐faceted. Support: NIH RC2 AR058962, Missouri Life Sciences Research Board(MB), and NIDCR DE021888(OJI).

Stau1 regulates Dvl2 expression during myoblast differentiation

Post-transcriptional regulation of gene expression by RNA-binding proteins has pivotal roles in many biological processes. We have shown that Stau1, a conserved RNA-binding protein, negatively regulates myogenesis in C2C12 myoblasts. However, its target mRNAs in regulation of myogenesis remain unknown. Here we describe that Stau1 positively regulates expression of Dvl2 gene encoding a central mediator of Wnt pathway in undifferentiated C2C12 myoblasts. Stau1 binds to 3′ untranslated region (UTR) of Dvl2 mRNA and Stau1 knockdown shortened a half-life of the mRNA containing Dvl2 3′ UTR. After induction of myogenic differentiation, association of Stau1 with 3′ UTR of Dvl2 mRNA was decreased. Correlated with the decrease in the association, the Dvl2 mRNA level was reduced during myogenesis. A forced expression of Dvl2 markedly inhibited progression of myogenic differentiation. Our results suggest that Dvl2 has an inhibitory role in myogenesis and Stau1 coordinates myogenesis through the regulation of Dvl2 mRNA.

Low-magnitude vertical vibration enhances myotube formation in C2C12 myoblasts

Whole body vibration training is widely used in rehabilitation and sports activities to improve muscle strength, balance, and flexibility. However, the molecular mechanisms of vertical vibration (VV) training and their effect on the myogenesis of myoblasts remain undefined. This study was undertaken to address the hypothesis that VV can enhance the expression of ECM proteins and myogenic regulatory factors (MRFs) in myoblasts and, in turn, increase myotube formation. Using real-time PCR, Western blot analysis, and immunofluorescence studies, we examined the effect of VV treatment with frequencies of 5, 8, or 10 Hz on the expression of ECM proteins and MRFs as well as myotube formation in C2C12 myoblasts. We showed that VV stimulation is safe and effective at stimulating myogenesis in C2C12 myoblasts. The levels of expression of the ECM proteins type I collagen and decorin were the highest after VV treatment at frequencies of 8 and 10 Hz. Expression of the MRFs MyoD and myogenin increased after VV stimulation in a time- and dose-dependent manner. The total number of myotubes formed, as well as the length and the average area of myotubes, were substantially increased following VV treatment at frequencies of 8 to 10 Hz. In conclusion, VV treatment at frequencies of 8 to 10 Hz can stimulate the expression of ECM proteins and MRFs in myoblasts and, in turn, increase myotube formation.

Stau1 negatively regulates myogenic differentiation in C2C12 cells

Sequential expression of myogenic regulatory factors (MRFs) such as MyoD and myogenin drives myogenic differentiation. Besides transcriptional activation of MRFs, this process is also coordinated by post-transcriptional regulation; MyoD and myogenin mRNAs are stabilized by RNA-binding protein HuR. Stau1 is known to regulate mRNA stability in a complex with Upf1, which is termed Stau1-mediated mRNA decay (SMD). We describe here that Stau1 is involved in the regulation of myogenesis. We found that knockdown of Stau1 promotes myogenesis including the expression of a muscle-specific marker protein, myoglobin, in C2C12 myoblasts. MyoD induces myogenin expression in response to induction of myogenesis, which is a key step to start myogenesis. The level of MyoD protein was not affected when Stau1 was depleted by siRNA, whereas the levels of myogenin mRNA and protein were increased in Stau1-knockdown cells. Interestingly, myogenin promoter activity was also increased in Stau1-knockdown cells in the absence of induction of myogenesis. Furthermore, Stau1-knockdown cells spontaneously progressed myogenesis including the expression of muscle-specific protein. Although Stau1 is involved in mRNA decay together with Upf1, Upf1-knockdown did not affect progression of myogenesis. Our results suggest that Stau1 negatively regulates myogenesis in C2C12 myoblasts through a mechanism that is different from SMD.

Tumor necrosis factor-α exerts pro-myogenic action in C2C12 myoblasts via sphingosine kinase/S1P 2 signaling

In this study, we report that low doses of tumor necrosis factor-α (TNFα) promote myogenesis in C2C12 myoblasts. Moreover, the cytokine increased sphingosine kinase (SphK) activity and induced SphK1 translocation to membranes. The inhibition of SphK functionality by various approaches abrogated the pro-myogenic effect of TNFα. Moreover, silencing of S1P 2 impaired the positive action of TNFα on myogenesis. These results represent the first evidence that SphK/S1P 2 axis is required for the regulation of myogenesis by TNFα. In view of the physiological role of TNFα in muscle regeneration, the present finding reinforces the notion that SphK/S1P 2 signaling is critically implicated in myogenesis.

N6-Methyldeoxyadenosine, a nucleoside commonly found in prokaryotes, induces C2C12 myogenic differentiation

N 6-methyl-2 ′-deoxyadenosine (MedAdo) is a nucleoside naturally found in prokaryotic DNA. Interestingly, the N 6-methylation of adenine in DNA seems to have been counter-selected during the course of evolution since MedAdo has not been detected in mammalian DNA until now. We show here that treatment with MedAdo induces myogenesis in C2C12 myoblasts. The presence of MedAdo in C2C12 DNA was investigated using a method based on HPLC coupled to electrospray ionization tandem mass spectrometry which is several thousand fold more sensitive than assays used previously. By this procedure, MedAdo is detected in the DNA from MedAdo-treated cells but remains undetectable in the DNA from control cells. Furthermore, MedAdo regulates the expression of p21, myogenin, mTOR, and MHC. Interestingly, in the pluripotent C2C12 cell line, MedAdo drives the differentiation towards myogenesis only. Thus, the biological effect of MedAdo is suppressed in the presence of BMP-2 which transdifferentiates C2C12 from myogenic into osteogenic lineage cells. Taken together these results point to MedAdo as a novel inducer of myogenesis and further extends the differentiation potentialities of this methylated nucleoside. Furthermore, these data raise the intriguing possibility that the biological effects of MedAdo on cell differentiation may have led to its counter-selection in eukaryotes.

Insulin produces myogenesis in C2C12 myoblasts by induction of NF-kappaB and downregulation of AP-1 activities.

In the present study, we have examined the insulin-signaling pathways involved in myogenesis in mouse C2C12 skeletal muscle cell line, a cellular system that expresses high number of high affinity insulin receptors. Insulin (50 nM) rapidly (5 min) stimulated beta-chain insulin receptor, activated the phosphatidylinositol (PI) 3-kinase/Akt/p70S6-kinase signaling pathway, as well as phosphorylated both p44/p42- and p38-mitogen-activated protein kinases (MAPKs). Preconfluent cells were differentiated in a serum-free medium in response to 50 nM insulin for 72 h, as revealed by the formation of multinucleated myotubes and the induction of the creatine kinase activity. This differentiation process was also monitored by the inhibition of the PCNA content and induction of the cell cycle inhibitor p21. Furthermore, insulin induced nuclear factor-kappaB (NF-kappaB) DNA binding activity and down-regulated activating protein-1 (AP-1) DNA binding activity throughout the differentiation process. The use of specific inhibitors of the insulin-signaling pathways indicated that myogenesis was precluded by treatment for 72 h with LY294002 (an inhibitor of PI 3-kinase), rapamycin (a p70S6-kinase blocker), and SB203580 or PD169316 (p38-MAPK inhibitors). These inhibitors abolished insulin induction of NF-kappaB DNA binding activity and kappaB-chloramphenicol acetyltransferase (CAT) promoter activity, maintaining expressed cytosolic IkappaB-alpha protein, and increased AP-1 DNA binding activity and TRE-CAT promoter activity. These data suggest that insulin induces myogenesis in C2C12 through PI 3-kinase/ p70S6-kinase and p38-MAPK pathways, the signaling through p44/p42-MAPK being inhibited.

Notch signaling imposes two distinct blocks in the differentiation of C2C12 myoblasts.

Notch signal transduction regulates expression of downstream genes through the activation of the DNA-binding protein Su(H)/CBF1. In Drosophila most of Notch signaling requires Su(H); however, some Notch-dependent processes occur in the absence of Su(H) suggesting that Notch signaling does not always involve activation of this factor. Using constitutively active forms of Notch lacking CBF1-interacting sequences we identified a Notch signaling pathway that inhibits myogenic differentiation of C2C12 myoblasts in the absence of CBF1 activation. Here we show that ligand-induced Notch signaling suppresses myogenesis in C2C12 myoblasts that express a dominant negative form of CBF1, providing additional evidence for CBF1-independent Notch signal transduction. Surprisingly mutant forms of Notch deficient in CBF1 activation are unable to antagonize MyoD activity, despite the fact that they inhibit myogenesis. Moreover, Notch-induced antagonism of MyoD requires CBF1 suggesting that the CBF1-dependent pathway mediates a cell-type-specific block in the myogenic program. However, Notch signaling in the absence of CBF1 activation blocks both myogenesis and osteogenesis, indicative of a general block in cellular differentiation. Taken together our data provide evidence for two distinct Notch signaling pathways that function to block differentiation at separate steps during the process of myogenesis in C2C12 myoblasts.