BMP10 positively regulates myogenic differentiation in C2C12 myoblasts via the Smad 1/5/8 signaling pathway

Molecular and Cellular Biochemistry - BMP10 plays an essential role in regulating cardiac growth, chamber maturation, and maintaining normal expressions of several key cardiogenic factors; however,...     

Variation in ACE activity affects myogenic differentiation in C2C12 cells

Variation in ACE activity is related to affect the skeletal muscle function. To elucidate the mechanism by which ACE affects skeletal muscle function, we examined the effects of loss and gain of ACE activity on myogenic differentiation in C2C12 myoblasts. The treatment of captopril, an ACE inhibitor, in differentiating cells significantly induced the up-regulation of myosin heavy chain, and the hypertrophic myotubes. In addition, an AT2 antagonist PD123319, not AT1 antagonist losartan, induced the up-regulation of myosin heavy chain. On the other hand, overexpression of ACE induced the down-regulation of myosin heavy chain. These results suggest that ACE negatively regulate the myogenesis through the mechanism at least in part via production of angiotensin II followed by its binding to AT2 receptor.     

Constitutive overexpression of the integral membrane protein Itm2A enhances myogenic differentiation of C2C12 cells

Integral membrane protein 2A (Itm2A) is a transmembrane protein belonging to a family composed of at least two other members, Itm2B and Itm2C, all of them having a different expression pattern. The protein serves as a marker for early stages in chondrogenesis and T-cell development. Itm2A is also highly expressed in skeletal muscle. In order to understand the role of Itm2A in muscle development, we constitutively overexpressed exogenous Itm2A in C2C12 myoblast cells. Several clones expressing high levels of Itm2a were isolated and characterized. Overexpression was associated with enhanced tube formation and the appearance of multinuclear cells. Gene expression analysis demonstrated that muscle creatin kinase was upregulated in the presence of exogenous Itm2A. Interestingly, proliferation rates were not altered in the undifferentiated myoblast C2C12 cells. These results demonstrate that overexpression of Itm2a in C2C12 enhances myogenic differentiation in vitro.     

TIEG1 negatively controls the myoblast pool indispensable for fusion during myogenic differentiation of C2C12 cells

The transforming growth factor (TGF)-β inducible early gene (TIEG)-1 is implicated in the control of cell proliferation, differentiation, and apoptosis in some cell types. Since TIEG1 functioning may be associated with TGF-β, a suppressor of myogenesis, TIEG1 is also likely to be involved in myogenesis. Therefore, we investigated the function of TIEG1 during myogenic differentiation in vitro using the murine myoblasts cell line, C2C12. TIEG1 expression increased during differentiation of C2C12 cells. Constitutive expression of TIEG1 reduced survival and decreased myotube formation. Conversely, knocking down TIEG1 expression increased the number of viable cells during differentiation, and accelerated myoblast fusion into multinucleated myotubes. However, expression of the myogenic differentiation marker, myogenin, remained unaffected by TIEG1 knockdown. The mechanism underlying these events was investigated by focusing on the regulation of myoblast numbers after induction of differentiation. The knockdown of TIEG1 led to changes in cell cycle status and inhibition of apoptosis during the initial stages of differentiation. Microarray and real-time PCR analyses showed that the regulators of cell cycle progression were highly expressed in TIEG1 knockdown cells. Therefore, TIEG1 is a negative regulator of the myoblast pool that causes inhibition of myotube formation during myogenic differentiation.     

Ebp1 regulates myogenic differentiation of myoblast cells via SMAD2/3 signaling pathway

Regulation of skeletal muscle development requires many of the regulatory networks that are fundamental to developmental myogenesis. ErbB3 binding protein‐1 (Ebp1) is involved in the control of myoblasts development in chicken. However, the expression and biological functions of Ebp1 in the progress of myogenesis are unclear. This study focused on determining the effect of Ebp1 on myogenic proliferation and differentiation using a primary myoblasts culture model. Ebp1 was found to upregulate in proliferating myoblasts and decrease at the early stage of myogenic differentiation. The level of endogenous Ebp1 increased from E9 to E20 chicken leg muscles. Knockdown of Ebp1 had no effect on myoblasts proliferation. However, myogenic differentiation into multinucleated myotubes was significantly reduced. The mRNA and protein expression of MRFs was decreased when Ebp1 was knocked down. Downregulation of Ebp1, accompanied by elevated levels of pSMAD2/3, suggests that Ebp1 is involved in regulating myogenic differentiation via SMAD2/3 inhibition. The phosphorylation of SMAD2/3 was activated and the expression of MYOD and MYOG was reduced in Ebp1 knockdown myoblasts, but addition of LY2109761 (an inhibitor specified to SMAD2/3) blocked these effects. Collectively, these results indicate that Ebp1 promotes myoblast differentiation by inhibition of SMAD2/3 signaling pathway during chicken myogenesis. These data provide new insights into the biological role of Ebp1 in embryonic chicken skeletal muscle development.     

MiR-141-3p regulates myogenic differentiation in C2C12 myoblasts via CFL2-YAP-mediated mechanotransduction

Skeletal myogenesis is essential to keep muscle mass and integrity, and impaired myogenesis is closely related to the etiology of muscle wasting. Recently, miR-141-3p has been shown to be induced under various conditions associated with muscle wasting, such as aging, oxidative stress, and mitochondrial dysfunction. However, the functional significance and mechanism of miR-141-3p in myogenic differentiation have not been explored to date. In this study, we investigated the roles of miR-141-3p on CFL2 expression, proliferation, and myogenic differentiation in C2C12 myoblasts. MiR-141-3p appeared to target the 3’UTR of CFL2 directly and suppressed the expression of CFL2, an essential factor for actin filament (F-actin) dynamics. Transfection of miR-141-3p mimic in myoblasts increased F-actin formation and augmented nuclear Yes-associated protein (YAP), a key component of mechanotransduction. Furthermore, miR-141-3p mimic increased myoblast proliferation and promoted cell cycle progression throughout the S and G2/M phases. Consequently, miR-141-3p mimic led to significant suppressions of myogenic factors expression, such as MyoD, MyoG, and MyHC, and hindered the myogenic differentiation of myoblasts. Thus, this study reveals the crucial role of miR-141-3p in myogenic differentiation via CFL2-YAP-mediated mechanotransduction and provides implications of miRNA-mediated myogenic regulation in skeletal muscle homeostasis.     

Regulation of C2C12 myogenic terminal differentiation by MKK3/p38alpha pathway

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

     

Sprouty-2 overexpression in C2C12 cells confers myogenic differentiation properties in the presence of FGF2

Myoblast C2C12 cells cultured in the presence of FGF2 actively proliferate and showed a differentiation-defective phenotype compared with cells cultured in low serum or in the presence of insulin. These FGF2 effects are associated with sustained activation of p44/p42-MAPK and lack of activation of AKT. Here we demonstrate that Sprouty-2, a protein involved in the negative feedback of receptor tyrosine kinase signaling, when stably overexpressed in C2C12 cells and in the presence of FGF2 produces growth arrest (precluding the expression of PCNA and the phosphorylation of retinoblastoma and inducing the expression of p21(CIP)) and myogenesis (multinucleated myotubes formation, induction of creatine kinase and expression of myosin heavy chain protein). These events were accompanied by repression of p44/p42-MAPK and activation of AKT. When C2C12 cells were stably transfected with a Sprouty-2 (Y55F) mutant defective in inhibiting p44/p42-MAPK activation by FGF, myoblasts in the presence of FGF continue to grow and completely fail to form myotubes. This work is the first evidence of the contribution of sprouty genes to myogenic differentiation in the presence of FGF2.     

Activation of RXR and RAR signaling promotes myogenic differentiation of myoblastic C2C12 cells

Differentiation of embryonic and adult myogenic progenitors undergoes a complex series of cell rearrangements and specification events which are controlled by distinct gene regulatory networks. Delineation of the molecular mechanisms that regulate skeletal muscle specification and formation should be important for understanding congenital myopathies and muscular degenerative diseases. Retinoic acid (RA) signaling plays an important role in development. However, the role of RA signaling in adult myogenic progenitors is poorly understood. Here, we investigate the role of RA signaling in regulating myogenic differentiation of myoblastic progenitor cells. Using the mouse myoblast progenitor C2C12 line as a model, we have found that the endogenous expression of most RAR and RXR isotypes is readily detected. While the nuclear receptor co-repressors are highly expressed, two of the three nuclear receptor co-activators and the enzymes involved in RA synthesis are expressed at low level or undetectable, suggesting that the RA signaling pathway may be repressed in myogenic progenitors. Using the α-myosin heavy chain promoter-driven reporter (MyHC-GLuc), we have demonstrated that either ATRA or 9CRA is able to effectively induce myogenic differentiation, which can be synergistically enhanced when both ATRA and 9CRA are used. Upon ATRA and 9CRA treatment of C2C12 cells the expression of late myogenic markers significantly increases. We have further shown that adenovirus-mediated exogenous expression of RARα and/or RXRα is able to effectively induce myogenic differentiation in a ligand-independent fashion. Morphologically, ATRA- and 9CRA-treated C2C12 cells exhibit elongated cell body and become multi-nucleated myoblasts, and even form myoblast fusion. Ultrastructural analysis under transmission electron microscope reveals that RA-treated myogenic progenitor cells exhibit an abundant presence of muscle fibers. Therefore, our results strongly suggest that RA signaling may play an important role in regulating myogenic differentiation.     

过表达miR-155抑制C2C12成肌分化

为明确miR-155在C2C12成肌分化中的作用及分子机制,本研究构建了miR-155过表达腺病毒载体,运用过表达miR-155的腺病毒感染C2C12,并诱导其成肌分化。通过形态学观察,成肌标志基因mRNA和蛋白表达水平的检测,以及双荧光素酶报告基因系统对预测的miR-155靶基因(TCF4)的验证,结果表明,C2C12细胞分化中,过表达miR-155明显降低了肌管的形成,成肌标志基因MyoG和MyHC的mRNA表达量极显著地下降(P0.01),而MyoD差异不显著(P0.05),成肌标志基因蛋白检测结果与mRNA检测结果一致;进一步研究显示miR-155与预测的TCF4基因的3'UTR 3个靶点(1487-1493,1516-1522,4532-4583)中的1个(4532-4538)结合,并发现过表达miR-155显著降低了TCF4的mRNA水平(P0.05)。表明miR-155可能通过靶向TCF4抑制C2C12成肌分化。     

N-terminal isotope tagging with propionic anhydride: proteomic analysis of myogenic differentiation of C2C12 cells

N-Terminal isotope tagging (NIT) is an important proteomic tool for quantifying proteins in complex mixtures. Here we describe a modified version of the isotope-coded propionylation procedure of Zhang et al. [Zhang et al., Rapid Commun. Mass Spectom. 16 (2002) 2325], which uses 'light' D0 and 'heavy' D10-propionic anhydride. The method has been extensively modified to improve both the kinetics and overall yield of propionylation. Using albumin as a model protein, the overall variation in quantification yields, calculated using several tryptic peptides, was within +/-10% (S.D. +/-0.2) error. The efficacy of the method is demonstrated by the quantitative differences obtained for vimentin in cell lysates of C2C12 myoblasts upon their myogensis to myotubules.     

Micropatterned polyelectrolyte nanofilms promote alignment and myogenic differentiation of C2C12 cells in standard growth media

Alignment of skeletal myoblasts is considered a critical step during myotube formation. The C2C12 cell line is frequently used as a model of skeletal muscle differentiation that can be induced by lowering the serum concentration in standard culture flasks. In order to mimic the striated architectures of skeletal muscles in vitro, micro‐patterning techniques and surface engineering have been proven as useful approaches for promoting elongation and alignment of C2C12 myoblasts, thereby enhancing the outgrowth of multi‐nucleated myotubes upon switching from growth media (GM) to differentiative media (DM). Herein, a layer‐by‐layer (LbL) polyelectrolyte multilayer deposition was combined with a micro‐molding in capillaries (MIMIC) method to simultaneously provide biochemical and geometrical instructive cues that induced the formation of tightly apposed and parallel arrays of differentiating myotubes from C2C12 cells maintained in GM media for 15 days. This study focuses on two different types of patterned/self‐assembled nanofilms based on alternated layers of poly (allylamine hydrochloride) (PAH)/poly(sodium 4‐styrene‐sulfonate) (PSS) as biocompatible but not biodegradable polymeric structures, or poly‐L ‐arginine sulfate salt (pARG)/dextran sulfate sodium salt (DXS) as both biocompatible and biodegradable surfaces. The influence of these microstructures as well as of the nanofilm composition on C2C12 skeletal muscle cells' differentiation and viability was evaluated and quantified, pointing to give a reference for skeletal muscle regenerative potential in culture conditions that do not promote it. At this regard, our results validate PEM microstructured devices, to a greater extent for (PAH/PSS)₅‐coated microgrooves, as biocompatible and innovative tools for tissue engineering applications and molecular dissection of events controlling C2C12 skeletal muscle regeneration without switching to their optimal differentiative culture media in vitro. Biotechnol. Bioeng. 2013; 110: 586–596. © 2012 Wiley Periodicals, Inc.