C2C12 myoblasts

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

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

This study developed biofunctional scaffolds that can promote the proliferation and differentiation of C2C12 myoblasts. The RGD/PLGA nanofiber matrices were successfully fabricated by electrospinning and the char- acteristics of the matrices were investigated. Our results showed that the structure of the RGD/PLGA nanofiber matrices was dimensionally similar to that of the natural ECM and the RGD-M13 phages were homogeneously distributed in the matrices. It was confirmed that the RGD/PLGA matrices were cell compatible and could support the growth and proliferation of C2C12 myo- blasts. In addition, when the matrices were used with GO, the myogenic differentiation of C2C12 myoblasts was effectively stimulated and accelerated. In conclusion, it is suggested that the RGD/PLGA nanofiber matrices are suitable scaffolds with the ability to support cellular behaviors. Moreover, the matrices can be used in com- bination with GO as a novel strategy for skeletal tissue regeneration and treatment of muscle dysfunction by stimulating the differentiation of myoblasts. Further studies with GO-loaded RGD/PLGA nanofiber matrices are needed to employ the approach presented in this study for in vivo applications.

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PRAS40 Regulates Protein Synthesis and Cell Cycle in C2C12 Myoblasts

PRAS40 Regulates Protein Synthesis and Cell Cycle in C2C12 Myoblasts

mTOR regulates cell cycle as evi- denced by the ability of rapamycin to ar- rest cells in the G1 phase (4,32,37–40). Propidium iodide staining revealed that myoblasts with PRAS40 knockdown had a greater proportion of cells in the G1/G0 phase, compared with scramble controls, and fewer cells in the active S phase. Collectively, these data suggest PRAS40 is required for mTOR activity in regulating cell cycle and that knock- down of PRAS40 in myoblasts retarded cell cycle progression. Alternatively, we cannot exclude the possibility that the reduction in PRAS40 alters cell cycle ki- netics by an undetermined mechanism that is mTOR independent. Because PRAS40 knockdown cells were arrested in G1/G0 of the cell cycle, we focused on elucidating potential underlying mechanisms that might produce cell cycle arrest. Regulation of cell cycle pro- gression by the cyclin-dependent kinase (cdk) inhibitor p21 blocks cells from en- tering into the DNA synthesis (or S) phase in many cell types. The opposite role of p21 in skeletal muscle growth and differentiation, compared with its role in HEK293 cells, has received recent attention. In HEK293 cells, AICAR in- creased phosphorylation of p53 with an increased expression of p21 (41,42). While p21 null mice develop normally during embryogenesis (43) because of the presence and activation of another redundant cdk inhibitor (p57) (44), my- ocytes from these mice have difficulty differentiating to myotubes (45). C2C12 myoblasts treated with AICAR were ar- rested in G1, and H9c2 cardiomyocytes had reduced expression of the p21 pro- Figure 9. Effect of PRAS40 knockdown on C2C12 differentiation. Myoblasts were trans-

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Clenbuterol Induces Cell Cycle Arrest in C2C12 Myoblasts by Delaying p27 Degradation through β-arrestin 2 Signaling

Clenbuterol Induces Cell Cycle Arrest in C2C12 Myoblasts by Delaying p27 Degradation through β-arrestin 2 Signaling

opposite reports on cell proliferation [26-29]. Thus, it is of great value to investigate the effect of CLB on myoblast proliferation, a critical step in muscle regeneration, which progresses from activation of satellite cells to proliferation, differentiation, and fusion with each other or with existing fibers [30]. C2C12 are murine myoblast derived from satellite cells and cycling myoblasts are comparable to activated satellite cells in muscle fiber [31]. Here, we examine proliferation of C2C12 myoblasts in response to CLB treatment. We show, for the first time, that CLB induces cell cycle arrest by repressing p27 degradation, via a pathway that depends on β 2 -AR

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A fractionation method to identify qauntitative changes in protein expression mediated by IGF-1 on the proteome of murine C2C12 myoblasts

A fractionation method to identify qauntitative changes in protein expression mediated by IGF-1 on the proteome of murine C2C12 myoblasts

Figure 3 demonstrates that IGF-1 treatment of C2C12 cells induced numerous changes in protein expression in each of the four fractions. To illustrate such changes, the data from the 200 mM NaCl elution of proteins in the cyto- plasmic fraction of C2C12 cells are shown. Overall, the amounts of most of the observed proteins did not change after IGF-1 treatment of C2C12 cells. A minority of pro- teins demonstrate increases or decreases. PDQuest analy- sis of selected spots followed by generation of mass spectrometric data allowed us to identify proteins that were quantitatively increased (Table 1) or decreased (Table 2) after IGF-1 stimulation for each of the four elu- tion conditions. Figure 3 represents a magnified region of two different sections of 2D gels at each of the 5 time points and demonstrates the most frequently observed types of changes in protein expression analyzed in these studies. In the top panels (yellow circles), expression of an unidentified protein appears after 1 hour and is sustained for 8 hours after IGF-1 treatment. In the bottom panels, expression of an unidentified protein decreases after IGF- 1 treatment (red circle). Additionally in these panels, shifts in both the intensity and localization of a group of proteins are observed (blue arrows). Each of these spots was identified as the molecular chaperone Mot66. During the time course of the experiment, the intensity of the individual spots changes with one spot appearing after 2 hours with IGF-1 (marked with an arrow). The observed shifts in position of proteins could represent splice vari- ants and/or posttranslational modifications rather than increases or decreases in absolute amounts of proteins These changes represent a significant challenge to the analysis and interpretation of the data (see discussion). Figure 4A shows changes in protein expression of four selected proteins from different regions of 2D gels from the 200 mM NaCl elution. Western blot analysis of Rho- GDI, cofilin

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Selection of Muscle-Binding Peptides from Context-Specific Peptide-Presenting Phage Libraries for Adenoviral Vector Targeting

Selection of Muscle-Binding Peptides from Context-Specific Peptide-Presenting Phage Libraries for Adenoviral Vector Targeting

Given this “context” problem, we have engineered viral “context-specific” phage libraries by introducing the H and I sheets of the adenovirus knob domain on to the pIII protein of filamentous bacteriophage. A 12-amino-acid (12-mer) random peptide library was constructed by insertion between the H and I sheets in the normal position of the HI loop. Selection of this HI loop context-specific peptide library against C2C12 myoblasts with preclearing against nontarget cells generated a peptide designated 12.51 with binding substantially better than that of the positive control integrin-binding ligand RGD. When this peptide was translated back into the knob domain of an Ad5 vector, this vector was functional, and 12.51 mediated improved muscle cell transduction compared to wild-type Ad5. These data suggest context-specific phage libraries may be used to identify compatible peptide ligands for viral vector targeting.

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Prion Infection of Muscle Cells In Vitro

Prion Infection of Muscle Cells In Vitro

scrapie-infected N2A cells at a ratio of 1:2 (Table 1). The cocultures were grown for 1 week in medium without Zeocin, after which the cocultures were switched to medium containing 200 ␮ g/ml Zeocin in order to eliminate the antibiotic-suscep- FIG. 1. Morphology and prion protein expression in C2C12 myo- blasts and myotubes. (A and B) The morphologies of C2C12 myoblasts (A) and D5 myotubes (B) are illustrated by immunofluorescence stain- ing for the cytoskeletal protein desmin. (C) Lysates from N2A cells (63 ␮ g protein), C2C12 myoblasts (126 ␮ g protein), and C2C12 myotubes (126 ␮ g protein) at days 3, 7, 10, and 14 in vitro were analyzed for total PrP in the absence of PK digestion by Western blotting using anti-PrP 6H4 monoclonal antibody as described in Materials and Methods. Molecular masses are indicated to the left. on November 8, 2019 by guest

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Multiphoton imaging of myogenic differentiation in gelatin-based hydrogels as tissue engineering scaffolds

Multiphoton imaging of myogenic differentiation in gelatin-based hydrogels as tissue engineering scaffolds

Immunofluorescence staining analysis was conducted to examine the morphology of the C2C12 myoblasts in GM and DM, respectively. MHC, a marker for myogenic differentiation, was stained with green fluorescence and the F-actins were stained with red fluorescence [38, 39]. The cells were located at different positions and grown in 3D in the GHPA hydrogels. Therefore, not all the cells were observed at one time by conventional 2D fluores- cence microscopy because the focus was settled only a flat layer. On the other hand, MPM can scan layer by layer in a short time, which leads to the construction of 3D structure images from 2D images by stacking the Z axis [39–41]. In addition, MPM used a longer wave- length than conventional 2D fluorescence microscopy, which in turn means that MPM can obtain clearer and deeper layer images than conventional 2D fluorescence microscopy. As shown in Figs. 5 (a-c), the conventional 2D fluorescence images are blurry except for the center area. In contrast, MPM images are clear and bright overall, as shown in Figs. 5 (d-f). On the other hand, MHC was not expressed in the C2C12 myoblasts when they were cultured in GM (Figs. 5 (a) and (d)). MHC was expressed only in the differentiated myotubes. Differentiated cells were observed in the 3D hydrogels (Figs. 5 (h-n)). The red fluorescence from F-actins was detected at high intensity throughout the cells, but the green fluorescence from MHC was detected at low intensity. Although the MHC was expressed in the cells, it was difficult to detect the green fluorescence of MHC by 2D fluorescence microscopy due to an unclear fluorescence signal and the image was blurry (Fig. 5(h)). On the other hand, the MPM images were not only clear, but also bright. Figures 5 (k-m) shows the differentiated C2C12 myoblasts obtained by MPM. F-actins were connected between the cells and MHC was expressed in the differenti- ated C2C12 myoblasts. Therefore, the MPM can detect the weak fluorescence signal of MHC more easily than conven- tional 2D fluorescence microscopy, and brighter and clearer images are obtained by MPM. Furthermore, 3D images were acquired by MPM to provide functional information of the 3D hydrogels as well as morphological information. Figures 5 (g) and (n) represent 3D images of the C2C12 myoblasts cultured in GHPA hydrogels. Functional infor- mation of the cells and hydrogels can be obtained from these 3D images. These results suggest that the MPM is an effective method for observing 3D scaffolds and biomedical imaging.

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Anti-fatigue activity of a mixture of seahorse (Hippocampus abdominalis) hydrolysate and red ginseng

Anti-fatigue activity of a mixture of seahorse (Hippocampus abdominalis) hydrolysate and red ginseng

Cell viability was estimated using the MTT assay, which is a test of metabolic competence predicated upon the assessment of mitochondrial performance. It is a colori- metric assay, which is dependent on the conversion of yellow tetrazolium bromide to its purple formazan de- rivative by mitochondrial succinate dehydrogenase in the viable cells (Kang et al. 2012). The viabilities of C2C12 myoblasts treated with different concentrations of HH (50, 100, 150, and 200 μg/mL) were expressed to repre- sent 100% viability (the viability of control cells; Fig. 1). In a preliminary experiment, HH concentrations up to 200 μg/mL showed no significant cytotoxicity for 24 h.

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Ternary Aligned Nanofibers of RGD Peptide-Displaying M13 Bacteriophage/PLGA/Graphene Oxide for Facilitated Myogenesis

Ternary Aligned Nanofibers of RGD Peptide-Displaying M13 Bacteriophage/PLGA/Graphene Oxide for Facilitated Myogenesis

quantitatively compare the MHC expression in C2C12 skeletal myoblasts on nanofiber sheets (Figure 4D). The C2C12 skeletal myoblasts on the GO-PLGA/RGD nanofiber sheets exhibited remarkably high MHC- positive area. These results are in accordance with the earlier studies. It has been documented that the graphene and GO can enhance the differentiation of myoblasts, as well as various types of stem cells, such as mesenchymal, neural, embryonic, and induced pluripotent stem cells [16, 73-77]. In particular, GO has been found to stimulate and accelerate the myogenic differentiation of C2C12 myoblasts through increasing serum protein adsorption from culture media via interfacial interactions between serum proteins and the oxygen-containing functional moieties of the GO [16]. Hence, the GO-PLGA/RGD nanofiber sheets could significantly accelerated spontaneous myoblast fusion as well as the myotube maturation of myoblasts. As shown in Figure 4C, the well-formed and mature myotubes were evidently observed on the GO-PLGA/RGD nanofiber sheets. These findings strongly support our hypothesis that the GO-PLGA/RGD nanofiber sheets can not only promote cell adhesion and proliferation, but can also stimulate and accelerate the spontaneous myogenic differentiation. Therefore, the GO-PLGA/RGD nanofiber sheets are verified to be biofunctional

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Culturing C2C12 myotubes on micromolded gelatin hydrogels accelerates myotube maturation

Culturing C2C12 myotubes on micromolded gelatin hydrogels accelerates myotube maturation

Historically, C2C12 myoblasts have been cultured on uncoated cell culture dishes until they near confluence. At this stage, myoblasts are serum withdrawn to induce differentiation and fuse into multinucleated, post-mitotic myotubes [11]. Over the next several days, myotubes develop similarly to embryonic skeletal muscle, but often detach from the cell culture dish after approximately 7 to 10 days due to spontaneous contraction [15, 16]. Because detachment of myotubes leads to cell death and presents obvious challenges for subsequent study, cul- tured myotubes are generally unsuitable for long-term studies. This problem has been addressed by coating cell culture dishes with substrates such as collagen, gelatin, and Matrigel ™ (Corning) that allow enhanced adhesion and/or modulate the stiffness of the surface such that detachment is delayed, but prolonged culture of myo- tubes on these substrates is still not possible [17, 18]. To address these shortcomings, biomedical engineers have developed methods that permit culture of more mature myotubes in vitro, including bioengineered substrates, 3D culture systems, and paradigms that include elec- trical stimulation or mechanical stretching [19–25]. Though some methods have been successful, the result- ant myotubes have not been sufficiently characterized, particularly at the molecular level. Additionally, tech- nical challenges preclude the implementation of many of these methods in basic biology laboratories. For these reasons, many skeletal muscle labs continue to use suboptimal culture substrates for C2C12 studies.

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Negative auto-regulation of myostatin expression is mediated by Smad3 and MicroRNA-27

Negative auto-regulation of myostatin expression is mediated by Smad3 and MicroRNA-27

In the present study we have further characterized the role of miR-27a/b in regulating Mstn expression and activity. Evidence presented here confirms that Mstn is indeed a target of miR-27a/b both in vitro and in vivo. Consistent with previous reports [23,24], we show that over expression of miR-27a results in reduced Mstn 39UTR reporter activity, which is blocked upon mutation of the miR-27a/b binding site in the Mstn 39 UTR. Furthermore, over expression of miR-27a in vivo led to decreased Mstn expression concomitant with myofiber hypertrophy and increased numbers of Pax7 + cells and activated myoblasts (MyoD + ); quite consistent with the fact that loss of Mstn leads to increased muscle mass and Figure 2. Inhibition of miR-27a and miR-27b results in increased Mstn activity. (A) Analysis of C2C12 myoblast proliferation in cultures treated with conditioned medium collected from C2C12 myoblasts transfected with either the negative control AntagomiR (AntagomiR Neg), miR- 27a-specific AntagomiR (AntagomiR-27a) or miR-27b-specific AntagomiR (AntagomiR-27b) for 72 h, as monitored by methylene blue assay. Values represent mean values 6 S.E.M (n = 3). p,0.001 (***). (B) Representative images of H&E stained AntagomiR Neg, AntagomiR-27a or AntagomiR-27b transfected C2C12 myoblasts after 48 h differentiation, followed by a further 72 h differentiation in the absence (Dialysis buffer; DB) or presence of 3 mg/ml sActRIIB. Scale bars = 100 mm. (C) Quantification of average myotube area (mm 2 ) in AntagomiR Neg, AntagomiR-27a or AntagomiR-27b

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Electrically Conductive Polydopamine–Polypyrrole as High Performance Biomaterials for Cell Stimulation in Vitro and Electrical Signal Recording in Vivo

Electrically Conductive Polydopamine–Polypyrrole as High Performance Biomaterials for Cell Stimulation in Vitro and Electrical Signal Recording in Vivo

ABSTRACT: Conductive polymers (CPs) such as polypyrrole (PPY) are emerging biomaterials for use as scaffolds and bioelectrodes which interact with biological systems electrically. Still, more electrically conductive and biologically interactive CPs are required to develop high performance biomaterials and medical devices. In this study, in situ electrochemical copolymerization of polydopamine (PDA) and PPY were performed for electrode modification. Their material and biological properties were characterized using multi- ple techniques. The electrical properties of electrodes coated with PDA/PPY were superior to electrodes coated with PPY alone. The growth and differentiation of C2C12 myoblasts and PC12 neuronal cells on PDA/PPY was enhanced compared to PPY. Electrical stimulation of PC12 cells on PDA/PPY further promoted neuritogenesis. In vivo EMG signal measurements demonstrated more sensitive signals from tibia muscles when using PDA/PPY coated electrodes than bare or PPY coated electrodes, revealing PDA/PPY to be a high performance biomaterial with potential for various biomedical applications.

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Raf kinase inhibitor protein1 is a myogenic inhibitor with conserved function in avians and mammals

Raf kinase inhibitor protein1 is a myogenic inhibitor with conserved function in avians and mammals

Figure 6. Overexpression of RKIP in C2C12 myoblasts inhibits myogenesis. Transgenic populations of C2C12 myoblasts expressing WT and mutant (S153A and S153/7EE) RKIP proteins c-terminally tagged with GFP, plus a GFP control, were established and assessed for RKIP production. A-D show expression of GFP in these cultures indicating that these cells produce either GFP (control) or GFP tagged RKIP. Levels of RKIP transgene expression were also assessed by duplex RT-PCR (E) and these were largely comparable for the different RKIP populations but absent from the GFP control as expected. These populations were subsequently assessed for their ability to differentiate in vitro in response to serum starvation. F-I show representative images from control GFP (F) and RKIP-overexpressing (WT, G; S153A, H; S153/7EE, I) cultures stained for Myosin Heavy Chains. Compared with controls (F) there are fewer myotubes in WT and non-phosphorylated RKIP overexpressing cultures (G,H) but no difference with phosphomimetic RKIP (I). J and K shows a graphical representations of the percentage of nuclei incorporated into MHC positive myotubes and percentage area of myotubes per culture respectively. Results are shown as mean±SD. L shows proliferation rates in populations of myoblasts expressing either GFP (average for 4 different populations) or RKIP proteins (2 different populations for each). Statistical analyses were by Anova followed by a multiple comparison test with p<0.05 considered significant. a, P<0.0001 compared with GFP control; b P<0.0001 comparted with RKIP EE; c, P<0.0002 compared with RKIP EE; d, P<0.0002 compared with GFP; e, P<0.004 compared with GFP; f, P<0.002 compared with RKIP EE. Scale bars in A-D are 100µm and F-I are 200µm.

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The adipose tissue stromal vascular fraction secretome enhances the proliferation but inhibits the differentiation of myoblasts

The adipose tissue stromal vascular fraction secretome enhances the proliferation but inhibits the differentiation of myoblasts

We found that the L6 rat myoblasts did not efficiently differentiate into myotubes (see Additional file 1: Figure S1), so we decided to switch to the more commonly used C2C12 cell line. Already after 4 days in differenti- ation medium (DM), multinucleated tube-like structures, which stained positively for fast type myosin heavy chain protein (MyHC2) (Fig. 5a), were observed. The C2C12 myoblasts cultured in the presence of SVF cells dis- played markedly fewer myotubes as compared to DM alone. The number of myotubes formed in DM supple- mented with HGF was also reduced but not to the same extent as in the presence of SVF (Fig. 5a). Similar obser- vations were made at later time points, and this corre- lated with lower levels of myosin heavy chain isoform 1 (Myhc1) and 2 (Myhc2) mRNA in the presence of SVF after 7 days (Fig. 5b) and 14 days (Fig. 5c) of differenti- ation. The inhibitory effect of SVF cells on MyHC2 pro- tein was confirmed by Western blotting (Fig. 5b, c). The mRNA expression of myogenic regulatory factor 5 (Myf5), one of the earliest markers of myogenesis [18], was inversely related to the myotube formation. The addition of Norleual, an HGF inhibitor, or MEK inhibi- tor to the C2C12 + SVF co-cultures made no significant difference on the expression of fast myosin heavy chain

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Regulation of myotube formation by the actin-binding factor drebrin

Regulation of myotube formation by the actin-binding factor drebrin

Drebrin is a ubiquitously expressed F-actin side-bind- ing protein that is highly abundant in the brain [25,26]. Drebrin contains an actin-depolymerizing factor II/cofi- lin-like domain, an actin-binding domain and two Homer-binding domains [27], and it remodels actin to facilitate the maturation of filopodia into dendritic spines during synaptogenesis in developing neurons (for reviews, see [27,28]). It is localized in lamellipodia and filopodia, at sites of cell-cell contact and in adhesion plaques [27-32]. Furthermore, drebrin associates with several proteins that promote myoblast differentiation and/or fusion, including the microtubule plus-tip bind- ing protein EB3 [33,34]; the scaffold protein Homer [35,36] and, via Homer, the small GTPase Cdc42 [35,37,38]; and the chemokine receptor CXCR4 [39,40]. We report herein that drebrin expression is induced during differentiation of primary and C2C12 myoblasts in a p38 MAPK-dependent manner. Furthermore, deple- tion of drebrin by RNA interference (RNAi) or inhibi- tion of its function with a small-molecule antagonist diminished expression of muscle-specific genes and myotube formation. Drebrin is therefore an actin-regu- lating factor induced during myogenic differentiation that promotes the differentiation process.

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miR-1/206 downregulates splicing factor Srsf9 to promote C2C12 differentiation

miR-1/206 downregulates splicing factor Srsf9 to promote C2C12 differentiation

450a-1, a non-myogenic miRNA which does not have overlapping predicted targets with miR-1/206. Luciferase quantification showed that this miRNA did not cross- react with the selected SR 3′ UTRs or with 3′ UTRs car- rying tandem copies of the complete reverse comple- ment of either miR-206 (2x206) or miR-1 (2x1) (data not shown). However, it efficiently downregulated the positive control construct 2x450a-1, constructed simi- larly to 2x206 and 2x1 (Additional file 3: Figure S3). To determine the sensitivity of the assay, we next assessed the activity of miR-1/206 on the 2x206 and 2x1 con- structs as well as on the 3′ UTR of Ccnd1, previously identified as a miR-206 target in C2C12 cells [42]. We also monitored promoter interference causing potential transcriptional repression of the reporter construct using a miRless construct, which expresses the Renilla lucifer- ase RNA without miRNA target sites. Figure 1a shows the result of this analysis with each luciferase signal measured in the presence of miR-1 or miR-206 normal- ized to the same constructs co-transfected with the con- trol miRNA, miR-450a-1 (see “Methods”). The data, presented as fold change versus miR-450a-1 control, show that miRless expression did not change appreciably in the presence of miR-1 or miR-206 and that miR-1 and miR-206 efficiently targeted 2x1 and 2x206 along with the positive control Ccnd1 (Fig. 1a). Furthermore, both miR-1 and miR-206 also reduced luciferase activity by targeting the 3′ UTRs of Srsf9 and Tra2b fused to the reporter gene while they did not have any effect on the Srsf3 construct (Fig. 1b). While statistically significant, the negative regulation of the Tra2b 3′ UTR was modest in magnitude. Thus, we focused our next set of experi- ments only on Srsf9 activity and its potential role in muscle differentiation. We substantiated the specific miR-1/206 targeting of the Srsf9 3′ UTR by MRE muta- genesis. When we reversed the orientation of the pre- dicted MRE in the Srsf9 3′ UTR, which preserves positioning of any unrecognized flanking elements, this mutant construct (termed Srsf9 MRE Rev) restored re- porter gene activity to levels measured in the presence of the control miR-450a-1 (Fig. 1c). Moreover, endogen- ous Srsf9 mRNA expression decreased in myoblasts when we transfected miR-1 or miR-206 overexpression plasmids (Additional file 4: Figure S4 A). Taken to- gether, these results establish that expression of the miR-1/206 family can directly modulate the level of Srsf9 in C2C12 myoblasts.

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Retinoic acid promotes myogenesis in myoblasts by antagonizing transforming growth factor-beta signaling via C/EBPβ

Retinoic acid promotes myogenesis in myoblasts by antagonizing transforming growth factor-beta signaling via C/EBPβ

In silico analysis of the mouse Smad3 gene and the upstream 5-kb region of the predicted promoter re- vealed no canonical retinoic acid response elements (RARE) by which RA could induce transcription by binding to the retinoic acid receptor: retinoid X recep- tor (RAR:RXR) heterodimer. Indeed, the promoters driv- ing expression of Smad3 in mouse, rat, and humans are quite divergent, with the exception of large CpG islands (Figure 1C). These CG-rich regions are prone to methyla- tion, and it has previously been demonstrated that the co-Smad Smad4 is silenced through this mechanism [33]. Methylation of the Smad3 promoter has also been demonstrated in humans [34]. Despite this, incorpor- ation of 5-azacytidine (AZA), a methylation-resistant cytosine analog, failed to induce Smad3 expression des- pite significantly increasing Rarb expression, a RA tar- get gene that is also regulated through a CpG island [35]. A 24-h treatment with AZA, however, failed to in- duce Smad3, suggesting that methylation of the CpG is- land is not the primary mode of regulation for Smad3 expression (Additional file 2: Figure S2 A,B). Published ChIP-seq data in which RAR occupancy of DNA elements in embryonic stem cells were mapped [GSM482750] confirmed that RARs do not occupy the promoter re- gion of Smad3 in mice, but rather appear to occupy a site in the intron between exons 3 and 4 of the gene (Figure 1C). ChIP analysis of RAR occupancy of the pu- tative intronic RARE revealed a significant 4-fold en- richment when compared to IgG controls in C2C12 myoblasts (Figure 1D), suggesting that occupancy of this site by RARs may play a role in the regulation of Smad3 expression by RA.

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The omega-3 fatty acid, eicosapentaenoic acid (EPA), prevents the damaging effects of tumour necrosis factor (TNF)-alpha during murine skeletal muscle cell differentiation

The omega-3 fatty acid, eicosapentaenoic acid (EPA), prevents the damaging effects of tumour necrosis factor (TNF)-alpha during murine skeletal muscle cell differentiation

Disease states can be both chronic and acute, but both ageing and disease can share some common factors. In particular, inflammation has been suggested to be present in a number of disease states associated with muscle atro- phy, such as cancer, heart failure, rheumatoid arthritis, chronic obstructive pulmonary disease, HIV/AIDS, and also ageing related muscle wasting. Associated with this response are pro-inflammatory cytokines, such as tumour necrosis factor (TNF)-α that are commonly present at ele- vated levels (0.5–10 ng/ml) during disease[2-5]. A bimo- dal response to TNF-α has been reported. Whereas, pathologic levels of TNF-α have been identified as playing a significant role in the mechanisms associated with skel- etal muscle wasting [6], low physiological concentrations (0.05 ng/ml) appear to activate myogenesis [7]. A number of previous studies, both in vitro and in vivo have shown that raised levels of TNF-α causes increased muscle loss [8- 12]. At least two mechanisms may account for the skeletal muscle-wasting effects of TNF-α: inhibition of myogenesis in myoblasts; apoptosis of myoblasts and myotubes. A number of in vitro studies suggest the effects of TNF-α are specific to the stage of myotube differentiation at the time of administration. Thus, delivery of TNF-α to primary human myoblasts or murine C2C12 myoblasts inhibits myosin heavy chain (MyHC) expression and myogenic differentiation [13-16] whereas treatment of differenti- ated myotubes with TNF-α appears to have marginal effects on their total or MyHC protein content [13,16]. However, a more recent finding suggests differentiated (C2C12) myotubes are susceptible to TNF-α-mediated apoptosis [17].

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MECHANISMS OF NOTCH-MEDIATED INHIBITION  OF SKELETAL MYOGENESIS

MECHANISMS OF NOTCH-MEDIATED INHIBITION OF SKELETAL MYOGENESIS

While my array list of Notch-responsive genes in C2C12 myoblasts provided a solid starting point to probe the transcriptional output of the pathway in muscle, it remains possible that important targets were missed due to the selection of an early 6- hour time point. I chose this time point to bias the list of genes towards direct targets of the pathway and indeed captured several known CSL-dependent primary Notch effectors (Nrarp, Hey1, HeyL). To fully flesh out the broader scope of transcriptional changes downstream of ligand-mediated signaling, however, the gene profiling analysis should be extended to a time-course over 12-48 hours. Notch typically functions as a transcriptional cascade, in which primary targets like Hes/Hey proteins act as repressors to shut off expression of a set of secondary targets. However, in some cases, secondary or indirect targets may also be induced, perhaps as a consequence of “feed-forward” collaboration between NICD and one of its primary effectors. For example, in Th2 cells, Notch activates expression of GATA3, which then synergizes with NICD to induce the transcription of the cytokine IL-4 (Fang et al., 2007). In muscle, MyoR induction may reflect an analogous mode of regulation, as its expression is delayed relative to that of Hey1 and HeyL (Figure 4.7). By examining an extended temporal window following ligand stimulation, I would gain a clearer sense of secondary expression changes in muscle, whether positive or negative, which could offer important functional insights into

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Sphingosine kinase/sphingosine 1-phosphate axis: a new player for insulin-like growth factor-1-induced myoblast differentiation

Sphingosine kinase/sphingosine 1-phosphate axis: a new player for insulin-like growth factor-1-induced myoblast differentiation

Figure 7 Role of sphingosine kinase in the mitogenic effect of insulin-like growth factor-1. (A) Serum-starved C2C12 myoblasts were pre-incubated for 30 min in the presence or absence of 1 μ M SKI-2 before being stimulated with 50 ng/ml insulin-like growth factor-1 (IGF-1) for 16 h. [ 3 H]Thymidine (1 μ Ci/well) was added during the last hour of incubation. [ 3 H]Thymidine incorporation in untreated cells was 29893 ± 1584 dpm. Results are reported as -fold change over control (vehicle, no addition; set as 1). Data are mean ± SEM of at least three independent experiments performed in triplicate. The mitogenic effect of IGF-1 in challenged versus unchallenged cells was statistically significant by Student ’ s t test (*P < 0.05). The effect of SK inhibition on the mitogenic effect of IGF-1 in challenged cells versus control (no SKI-2, IGF-1 added) was statistically significant by Student ’ s t test (#P < 0.05). (B) Scrambled (SCR) or specific SK1- (upper panel) or SK2-siRNA (lower panel) transfected C2C12 cells were treated or not treated with 50 ng/ ml IGF-1 for 16 h. [ 3 H]Thymidine (1 μ Ci/well) was added during the last

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