Mouse C2C12 Cells Research Articles

Identification of the Immunoproteasome as a Novel Regulator of Skeletal Muscle Differentiation

While many of the molecular details of myogenesis have been investigated extensively, the function of immunoproteasomes (i-proteasomes) in myogenic differentiation remains unknown. We show here that the mRNA of i-proteasome subunits, the protein levels of constitutive and inducible proteasome subunits, and the proteolytic activities of the 20S and 26S proteasomes were significantly upregulated during differentiation of skeletal muscle C2C12 cells. Knockdown of the i-proteasome catalytic subunit PSMB9 by short hairpin RNA (shRNA) decreased the expression of both PSMB9 and PSMB8 without affecting other catalytic subunits of the proteasome. PSMB9 knockdown and the use of i-proteasome-specific inhibitors both decreased 26S proteasome activities and prevented C2C12 differentiation. Inhibition of the i-proteasome also impaired human skeletal myoblast differentiation. Suppression of the i-proteasome increased protein oxidation, and these oxidized proteins were found to be more susceptible to degradation by exogenous i-proteasomes. Downregulation of the i-proteasome also increased proapoptotic proteins, including Bax, as well as cleaved caspase 3, cleaved caspase 9, and cleaved poly(ADP-ribose) polymerase (PARP), suggesting that impaired differentiation is likely to occur because of significantly increased apoptosis. These results demonstrate for the first time that i-proteasomes, independent of constitutive proteasomes, are critical for skeletal muscle differentiation of mouse C2C12 cells.

Effects of CMV Enhancer on Activity and Specificity of Bovine MyoG Gene Promoter

Connected a segment of CMV enhancer to the front of MyoG gene promoter and then constructed the corresponding dual luciferase expression vector pGL3-CMV-MyoGpro. We set four eukaryotic expression vectors including pGL3-CMV, pGL3-MyoGpro, pGL3-CMV-MyoGpro, and pGL3-Basic which contained CMV promoter, MyoG promoter, CMV-MyoG synthesis promoter, and a promoterless negative control, respectively. Then the four vectors and internal control Renilla luciferase report gene vector phRL-TK were transfected into bovine skeletal muscle satellite cells, mouse C2C12 cells and bovine fetal fibroblast cells to detect the promoter activity with dual luciferase report system. The results showed that CMV enhancer could significantly improve the transcription activity of bovine MyoG gene promoter in muscle satellite cells and mouse C2C12 cells, and it had certain specificity. This study provided experimental materials for increasing the high expression of exogenous gene in bovine muscle cells, and also laid the molecular theoretical basis for obtaining the high specific promoter of bovine muscle and the transgenic beef cattle.

Assessment of Reference Genes for Real-Time Quantitative PCR Gene Expression Normalization During C2C12 and H9c2 Skeletal Muscle Differentiation

Skeletal muscle differentiation occurs during muscle development and regeneration. To initiate and maintain the differentiated state, a multitude of gene expression changes occur. Accurate assessment of these differentiation-related gene expression changes requires good quality template, but more specifically, appropriate internal controls for normalization. Two cell line-based models used for in vitro analyses of muscle differentiation incorporate mouse C2C12 and rat H9c2 cells. In this study, we set out to identify the most appropriate controls for mRNA expression normalization during C2C12 and H9c2 differentiation. We assessed the expression profiles of Actb, Gapdh, Hprt, Rps12 and Tbp during C2C12 differentiation and of Gapdh and Rps12 during H9c2 differentiation. Using NormFinder, we validated the stability of the genes individually and of the geometric mean generated from different gene combinations. We verified our results using Myogenin. Our study demonstrates that using the geometric mean of a combination of specific reference genes for normalization provides a platform for more precise test gene expression assessment during myoblast differentiation than using the absolute expression value of an individual gene and reinforces the necessity of reference gene validation.

DNA fragmentation induced by all‐trans retinoic acid and its steroidal analogue EA‐4 in C2C12 mouse and HL‐60 human leukemic cells in vitro

We have recently shown that retinoic acid induces micronucleation mainly via chromosome breakage (Alakhras et al. Cancer Lett 2011; 306: 15-26). To further study retinoic acid clastogenicity and evaluate DNA damaging potential we investigated the ability of (a) all-trans retinoic acid and its steroidal analogue EA-4 to induce DNA fragmentation by using Comet assay under alkaline unwinding and neutral condition electrophoresis, and (b) the retinoids under study to induce small (0-1 kb) DNA fragments. Two cell lines, C2C12 mouse cells and HL-60 human leukemic cells were used in this study. We found that all-trans retinoic acid and its steroidal analogue EA-4 (a) provoke DNA migration due to DNA fragmentation as it is shown by the increased values of Comet parameters, and (b) induce significantly small-size fragmented genomic DNA as indicated by the quantification of necrotic/apoptotic small DNA segments in both cell systems. A different response between the two cell lines was observed in relation to retinoid ability to increase the percentage of DNA in the tail as well as break DNA in to small fragments. Our findings confirm the ability of retinoic acid to provoke micronucleation by disrupting DNA into fragments, among which small pieces of double-stranded DNA up to 1 kb are identified.

DUX4 differentially regulates transcriptomes of human rhabdomyosarcoma and mouse C2C12 cells.

Facioscapulohumeral muscular dystrophy (FSHD) is linked to the deletion of the D4Z4 arrays at chromosome 4q35. Recent studies suggested that aberrant expression of double homeobox 4 (DUX4) from the last D4Z4 repeat causes FSHD. The aim of this study is to determine transcriptomic responses to ectopically expressed DUX4 in human and mouse cells of muscle lineage. We expression profiled human rhabdomyosarcoma (RD) cells and mouse C2C12 cells transfected with expression vectors of DUX4 using the Affymetrix Human Genome U133 Plus 2.0 Arrays and Mouse Genome 430 2.0 Arrays, respectively. A total of 2267 and 150 transcripts were identified to be differentially expressed in the RD and C2C12 cells, respectively. Amongst the transcripts differentially expressed in the RD cells, MYOD and MYOG (2 fold, p<0.05), and six MYOD downstream targets were up-regulated in RD but not C2C12 cells. Furthermore, 13 transcripts involved in germline function were dramatically induced only in the RD cells expressing DUX4. The top 3 IPA canonical pathways affected by DUX4 were different between the RD (inflammation, BMP signaling and NRF-2 mediated oxidative stress) and the C2C12 cells (p53 signaling, cell cycle regulation and cellular energy metabolism). Amongst the 40 transcripts shared by the RD and C2C12 cells, UTS2 was significantly induced by 76 fold and 224 fold in the RD and C2C12 cells, respectively. The differential expression of MYOD, MYOG and UTS2 were validated using real-time quantitative RT-PCR. We further validated the differentially expressed genes in immortalized FSHD myoblasts and showed up-regulation of MYOD, MYOG, ZSCAN4 and UTS2. The results suggest that DUX4 regulates overlapped and distinct groups of genes and pathways in human and mouse cells as evident by the selective up-regulation of genes involved in myogenesis and gametogenesis in human RD and immortalized cells as well as the different molecular pathways identified in the cells.

Shape-dependent cell migration and focal adhesion organization on suspended and aligned nanofiber scaffolds

In the body, cells dynamically respond to chemical and mechanical cues from the extracellular matrix (ECM), yet precise mechanisms by which biophysical parameters (stiffness, topography and alignment) affect cell behavior remain unclear. Here, highly aligned and suspended multilayer polystyrene (PS) nanofiber scaffolds are used to study biophysical influences on focal adhesion complex (FAC) arrangement and associated migration behavior of mouse C2C12 cells arranged in specific shapes: spindle, parallel and polygonal. Furthermore, the role of cytoskeletal-altering drugs including blebbistatin, nocodazole and cytochalasin-D on FAC formation and migratory behavior is investigated. For the first time, this work reports that cells on suspended fiber networks, including cells with administered drugs, elongated along the fiber axes and developed longer (∼ 4×) and more concentrated FAC clusters compared to cells on flat PS control substrates. Additionally, substrate designs which topographically restrict sites of cell attachment and align adhesions were found to promote higher migration speeds (spindle: 52μmh−1, parallel: 39μmh−1, polygonal: 25μmh−1, flat: 32μmh−1). This work demonstrates that suspended fiber topography-induced concentration of FACs along fiber axes generates increased migration potential as opposed to flat surfaces, which diffuse and randomly orient adhesions.

Requirement of decreased O-GlcNAc glycosylation of Mef2D for its recruitment to the myogenin promoter

Previously, we demonstrated that the expression of myogenin, a critical transcription factor for myogenesis, is negatively regulated by O-linked β-N-acetylglucosamine (O-GlcNAc) glycosylation in mouse C2C12 cells. In this study, we found that Mef2 family proteins, especially Mef2D which is a crucial transcriptional activator of myogenin, are O-GlcNAc glycosylated. Between the two splice variants of Mef2D, Mef2D1a rather than Mef2D1b appears to drive the initiation of myogenin expression in the early stage of myogenesis. A deletion mutant analysis showed that Mef2D1a is glycosylated both in its DNA-binding and transactivation domains. A significant decrease in the glycosylation of Mef2D was observed in response to myogenic stimulus in C2C12 cells. Inhibition of the myogenesis-dependent decrease in the glycosylation of Mef2D suppressed its recruitment to the myogenin promoter. These results indicate that the expression of myogenin is regulated, at least in part, by the decreased glycosylation-dependent recruitment of Mef2D to the promoter region, and this is one of the negative regulatory mechanisms of skeletal myogenesis by O-GlcNAc glycosylation.

Apoptosis induced by copper oxide quantum dots in cultured C2C12 cells via caspase 3 and caspase 7: a study on cytotoxicity assessment

We report herein the synthesis and characterization of copper oxide quantum dots and their cytotoxic impact on mouse C2C12 cells. The utilized CuO quantum dots were prepared by the one-pot wet chemical method using copper acetate and hexamethylenetetramine as precursors. The physicochemical characterization of the synthesized CuO quantum dots was carried out using X-ray diffraction, energy-dispersive X-ray analysis, and transmission electron microscopy. To examine the in vitro cytotoxicity, C2C12 cell lines were treated with different concentrations of as-prepared quantum dots and the viability of cells was analyzed using Cell Counting Kit-8 assay at regular time intervals. The morphology of the treated C2C12 cells was observed under a phase-contrast microscope, whereas the quantification of cell viability was carried out via confocal laser scanning microscopy. To gain insight into the mechanism of cell death, we examined the effect of CuO quantum dots on the candidate genes such as caspases 3 and 7, which are key mediators of apoptotic events. In vitro investigations of the biological effect of CuO quantum dots have shown that it binds genomic DNA, decreases significantly the viability of cells in culture in a concentration (10-20μg/mL) dependent manner, and inhibits mitochondrial caspases 3 and 7. To sum up, the elucidation of the pathways is to help in understanding CuO quantum dot-induced effects and evaluating CuO quantum dot-related hazards to human health.

BMP Induces Cochlin Expression to Facilitate Self-renewal and Suppress Neural Differentiation of Mouse Embryonic Stem Cells

BMP4 maintains self-renewal of mouse embryonic stem cells (ESCs) in collaboration with LIF. Here, we report the identification of a novel key BMP target gene, cochlin (Coch) in mouse ESCs. Coch can be significantly up-regulated by BMP4 specifically in ESCs but not in somatic differentiated cells, and this up-regulation is dependent on the BMP signaling mediators Smad1/5 and Smad4. Overexpression of Coch can partially substitute BMP4 to promote self-renewal of mouse ESCs together with LIF, whereas knockdown of Coch impairs self-renewal marker gene expression even in the presence of both BMP4 and LIF. Further studies showed that COCH could mimic BMP4 in repressing neural differentiation of mouse ESCs upon LIF withdrawal and the inhibitory effect of BMP4 on neural differentiation is compromised by Coch knockdown. Taken together, our data suggest that COCH is a part of the downstream target network of BMP signaling and serves as another important effector to fine-tune mouse ESC fates.

Wdr68 Requires Nuclear Access for Craniofacial Development

Wdr68 is a highly conserved scaffolding protein required for craniofacial development and left-right asymmetry. A Ras-Map3k-Wdr68-Dyrk1 signaling relay may mediate these and other diverse signaling events important in development and disease. While the sub-cellular localization of Wdr68 has been shown to be dependent on that of its interaction partners, it is not clear where Wdr68 activity is required during development. Here we show that while a GFP-Wdr68 fusion functionally substituted for craniofacial development in the zebrafish, that a Nuclear Export Signal (NES) fusion protein (GFPNESWdr68) failed to support craniofacial development. As control for NES activity, we show that while GFP-Wdr68 exhibited a pan-cellular distribution in C2C12 cells, the GFPNESWdr68 fusion predominantly localized to the cell cytoplasm, as expected. Interestingly, while GFP-Wdr68 and RFP-Dyrk1a co-localized to the cell nucleus as expected based on the known sub-cellular localization for Dyrk1a, we found that the GFPNESWdr68 fusion redistributed RFP-Dyrk1a to the cell cytoplasm potentially disconnecting the Ras/Dyrk1 signal relay from further downstream targets. Consistent with a nuclear role in gene regulation, we also found that while a transcriptional activation domain fusion, CebpFlagWdr68, functionally substituted for endogenous Wdr68 for craniofacial development, that a transcriptional repression domain fusion, MadFlagWdr68, failed to support craniofacial development. Dyrk1b is required for myogenin (myog) expression in differentiating mouse C2C12 cells and here we report that wdr68 is also important for myog expression in differentiating C2C12 cells. Using a C2C12 cell myog promoter-reporter system, we found that Wdr68 overexpression increased reporter activity while moderate expression levels of MadFlagWdr68 interfered with reporter activity. Taken together, these findings support a nuclear role for Wdr68-containing complexes.

Hydrogen Improves Glycemic Control in Type1 Diabetic Animal Model by Promoting Glucose Uptake into Skeletal Muscle

Hydrogen (H2) acts as a therapeutic antioxidant. However, there are few reports on H2 function in other capacities in diabetes mellitus (DM). Therefore, in this study, we investigated the role of H2 in glucose transport by studying cultured mouse C2C12 cells and human hepatoma Hep-G2 cells in vitro, in addition to three types of diabetic mice [Streptozotocin (STZ)-induced type 1 diabetic mice, high-fat diet-induced type 2 diabetic mice, and genetically diabetic db/db mice] in vivo. The results show that H2 promoted 2-[14C]-deoxy-d-glucose (2-DG) uptake into C2C12 cells via the translocation of glucose transporter Glut4 through activation of phosphatidylinositol-3-OH kinase (PI3K), protein kinase C (PKC), and AMP-activated protein kinase (AMPK), although it did not stimulate the translocation of Glut2 in Hep G2 cells. H2 significantly increased skeletal muscle membrane Glut4 expression and markedly improved glycemic control in STZ-induced type 1 diabetic mice after chronic intraperitoneal (i.p.) and oral (p.o.) administration. However, long-term p.o. administration of H2 had least effect on the obese and non-insulin-dependent type 2 diabetes mouse models. Our study demonstrates that H2 exerts metabolic effects similar to those of insulin and may be a novel therapeutic alternative to insulin in type 1 diabetes mellitus that can be administered orally.

Genetic manipulation of myoblasts and a novel primary myosatellite cell culture system: comparing and optimizing approaches

The genetic manipulation of skeletal muscle cells in vitro is notoriously difficult, especially when using undifferentiated muscle cell lines (myoblasts) or primary muscle stem cells (myosatellites). We therefore optimized methods of gene transfer by overexpressing green fluorescent protein (GFP) in mouse C2C12 cells and in a novel system, primary rainbow trout myosatellite cells. A common lipid-based transfection reagent was used (Lipofectamine 2000) along with three different viral vectors: adeno-associated virus serotype 2 (AAV2), baculovirus (BAC) and lentivirus. Maximal transfection efficiencies of 49% were obtained in C2C12 cells after optimizing cell density and reagent : DNA ratio, although the GFP signal rapidly dissipated with proliferation and was not maintained with differentiation. The transduction efficiency of AAV2 was optimized to 65% by extending incubation time and decreasing cell density, although only 30% of cells retained expression after passing. A viral comparison revealed that lentivirus was most efficient at transducing C2C12 myoblasts as 97% of cells were transduced with only 10(6) viral genomes (vg) compared to 54% with 10(8) vg AAV2 and 23% with 10(9) vg BAC. Lentivirus also transduced 90% of primary trout myosatellites compared to 1-10% with AAV2 and BAC. The phosphoglycerate kinase 1 (pgk) promoter was 10-fold more active than the cytomegalovirus immediate-early promoter in C2C12 cells and both were effective in trout myosatellites. Maximal transduction of C2C12 myotubes was achieved by differentiating myoblasts previously transduced with lentivirus and the pgk promoter. Thus, our optimized protocol proved highly effective in diverse muscle cell systems and could therefore help overcome a common technological barrier.

Specification of skeletal muscle differentiation by repressor element-1 silencing transcription factor (REST)-regulated Kv7.4 potassium channels

Changes in the expression of potassium (K(+)) channels is a pivotal event during skeletal muscle differentiation. In mouse C(2)C(12) cells, similarly to human skeletal muscle cells, myotube formation increased the expression of K(v)7.1, K(v)7.3, and K(v)7.4, the last showing the highest degree of regulation. In C(2)C(12) cells, K(v)7.4 silencing by RNA interference reduced the expression levels of differentiation markers (myogenin, myosin heavy chain, troponinT-1, and Pax3) and impaired myotube formation and multinucleation. In K(v)7.4-silenced cells, the differentiation-promoting effect of the K(v)7 activator N-(2-amino-4-(4-fluorobenzylamino)-phenyl)-carbamic acid ethyl ester (retigabine) was abrogated. Expression levels for the repressor element-1 silencing transcription factor (REST) declined during myotube formation. Transcript levels for K(v)7.4, as well as for myogenin, troponinT-1, and Pax3, were reduced by REST overexpression and enhanced upon REST suppression by RNA interference. Four regions containing potential REST-binding sites in the 5' untranslated region and in the first intron of the K(v)7.4 gene were identified by bioinformatic analysis. Chromatin immunoprecipitation assays showed that REST binds to these regions, exhibiting a higher efficiency in myoblasts than in myotubes. These data suggest that K(v)7.4 plays a permissive role in skeletal muscle differentiation and highlight REST as a crucial transcriptional regulator for this K(+) channel subunit.

Interplay between two myogenesis-related proteins: TBP-interacting protein 120B and MyoD

Gene expression in myogenesis is governed by multiple myogenic factors including MyoD. Previously, we demonstrated that TBP-interacting protein 120B (TIP120B) promotes in vitro myogenesis through its anti-ubiquitination ability. In this study, we investigated interplay between MyoD and TIP120B. Mouse C2C12 cells subjected to myotube differentiation contained increased amounts of TIP120B and MyoD. Dexamethasone, which inhibits myogenic signaling, decreased the amounts of those proteins. Mouse and human TIP120B promoters, which carry multiple E-box motifs, were potentiated by MyoD. In the human TIP120B, a proximal E-box binds to MyoD in vitro and exhibits MyoD-dependent transcription activation function. Expression of the endogenous TIP120B gene was correlated with the level of MyoD in different types of muscle-related cells. Furthermore, MyoD binds specifically to a proximal E-box-carrying promoter region in chromatin. Proteasome-sensitive MyoD was increased and decreased by overexpression and knockdown of TIP120B, respectively. Moreover, stability of MyoD was increased by TIP120B. The results suggest that MyoD and TIP120B potentiate each other at gene expression and post-translation levels, respectively, which may promote myogenesis cooperatively.

Carvedilol suppresses fatty acid oxidation and stimulates glycolysis in C2C12 cells

Beta adrenergic receptor blocking drugs (β-blockers) are used chronically in many cardiovascular diseases such as hypertension, ischemic heart disease, arrhythmia, and heart failure. Beneficial effects are associated with the inhibition of symphathetic nervous system hyperactivity, reduction of heart rate, and remodeling by blocking the mitogenic activity of catecholamines. A possible effect of β-blockers on substrate metabolism has also been suggested. The direct effects of β-blockers on mouse C2C12 cells were investigated in this study. C2C12 cells were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and differentiated into myotubes in the same medium that contained 1% FBS. Palmitic acid oxidation and glycolysis were measured by using [9,10-(3)H]palmitate and [5-(3)H]glucose, respectively. The amount of (3)H(2)O was measured as an indicator of substrate usage. Carvedilol (100 µmol/L) inhibited palmitate oxidation and increased glycolysis by nearly 50%. Prazosin altered substrate metabolism in a similar fashion as carvedilol, whereas propranolol or bisoprolol were devoid of metabolic effects. When added to mimic sympathetic activation, epinephrine stimulated glycolysis but did not alter fatty acid oxidation. Based on these results, carvedilol appears to have direct effects on substrate metabolism that are related to the blockade of α1 adrenergic receptors.

Cohesin-SA1 deficiency drives aneuploidy and tumourigenesis in mice due to impaired replication of telomeres

Cohesin is a protein complex originally identified for its role in sister chromatid cohesion, although increasing evidence portrays it also as a major organizer of interphase chromatin. Vertebrate cohesin consists of Smc1, Smc3, Rad21/Scc1 and either stromal antigen 1 (SA1) or SA2. To explore the functional specificity of these two versions of cohesin and their relevance for embryonic development and cancer, we generated a mouse model deficient for SA1. Complete ablation of SA1 results in embryonic lethality, while heterozygous animals have shorter lifespan and earlier onset of tumourigenesis. SA1-null mouse embryonic fibroblasts show decreased proliferation and increased aneuploidy as a result of chromosome segregation defects. These defects are not caused by impaired centromeric cohesion, which depends on cohesin-SA2. Instead, they arise from defective telomere replication, which requires cohesion mediated specifically by cohesin-SA1. We propose a novel mechanism for aneuploidy generation that involves impaired telomere replication upon loss of cohesin-SA1, with clear implications in tumourigenesis.

Regulation of myotube formation by the actin-binding factor drebrin

BackgroundMyogenic differentiation involves cell-cycle arrest, activation of the muscle-specific transcriptome, and elongation, alignment and fusion of myoblasts into multinucleated myotubes. This process is controlled by promyogenic transcription factors and regulated by signaling pathways in response to extracellular cues. The p38 mitogen-activated protein kinase (p38 MAPK) pathway promotes the activity of several such transcription factors, including MyoD and MEF2, thereby controlling the muscle-specific transcription program. However, few p38-regulated genes that play a role in the regulation of myogenesis have been identified.MethodsRNA interference (RNAi), chemical inhibition and immunofluorescence approaches were used to assess the role of drebrin in differentiation of primary mouse myoblasts and C2C12 cells.ResultsIn a search for p38-regulated genes that promote myogenic differentiation, we identified Dbn1, which encodes the actin-binding protein drebrin. Drebrin is an F-actin side-binding protein that remodels actin to facilitate the change of filopodia into dendritic spines during synaptogenesis in developing neurons. Dbn1 mRNA and protein are induced during differentiation of primary mouse and C2C12 myoblasts, and induction is substantially reduced by the p38 MAPK inhibitor SB203580. Primary myoblasts and C2C12 cells depleted of drebrin by RNAi display reduced levels of myogenin and myosin heavy chain and form multinucleated myotubes very inefficiently. Treatment of myoblasts with BTP2, a small-molecule inhibitor of drebrin, produces a phenotype similar to that produced by knockdown of drebrin, and the inhibitory effects of BTP2 are rescued by expression of a mutant form of drebrin that is unable to bind BTP2. Drebrin in myoblasts is enriched in cellular projections and cell cortices and at regions of cell-cell contact, all sites where F-actin, too, was concentrated.ConclusionsOur findings reveal that Dbn1 expression is a target of p38 MAPK signaling during myogenesis and that drebrin promotes myoblast differentiation.

Reduction of myoblast differentiation following multiple population doublings in mouse C2C12 cells: A model to investigate ageing?

Ageing skeletal muscle displays declines in size, strength, and functional capacity. Given the acknowledged role that the systemic environment plays in reduced regeneration (Conboy et al. [2005] Nature 433: 760-764), the role of resident satellite cells (termed myoblasts upon activation) is relatively dismissed, where, multiple cellular divisions in-vivo throughout the lifespan could also impact on muscular deterioration. Using a model of multiple population doublings (MPD) in-vitro thus provided a system in which to investigate the direct impact of extensive cell duplications on muscle cell behavior. C(2) C(12) mouse skeletal myoblasts (CON) were used fresh or following 58 population doublings (MPD). As a result of multiple divisions, reduced morphological and biochemical (creatine kinase, CK) differentiation were observed. Furthermore, MPD cells had significantly increased cells in the S and decreased cells in the G1 phases of the cell cycle versus CON, following serum withdrawal. These results suggest continued cycling rather than G1 exit and thus reduced differentiation (myotube atrophy) occurs in MPD muscle cells. These changes were underpinned by significant reductions in transcript expression of: IGF-I and myogenic regulatory factors (myoD and myogenin) together with elevated IGFBP5. Signaling studies showed that decreased differentiation in MPD was associated with decreased phosphorylation of Akt, and with later increased phosphorylation of JNK1/2. Chemical inhibition of JNK1/2 (SP600125) in MPD cells increased IGF-I expression (non-significantly), however, did not enhance differentiation. This study provides a potential model and molecular mechanisms for deterioration in differentiation capacity in skeletal muscle cells as a consequence of multiple population doublings that would potentially contribute to the ageing process.

Amylin and leptin activate overlapping signalling pathways in an additive manner in mouse GT1-7 hypothalamic, C2C12 muscle and AML12 liver cell lines

It has been suggested that amylin amplifies leptin's effects and affects energy homeostasis synergistically with leptin in animals and humans. However, no previous study has reported on amylin and leptin signalling in hypothalamic, muscle and liver cells. Leptin and amylin signalling studies were performed in vitro in mouse GT1-7 hypothalamic, C₂C₁₂ muscle and AML12 liver cell lines. Treatment of mouse GT1-7 and C₂C₁₂ cells with leptin or amylin increased signal transducer and activator of transcription 3 (STAT3) phosphorylation in a dose- and time-dependent manner. In mouse AML12 cells, leptin and amylin produced a biphasic response of STAT3 activity. Importantly, all leptin and amylin signalling pathways were saturable at leptin and amylin concentrations of ∼100 and ∼50 to 100 ng/ml, respectively. Leptin and amylin in combination activated STAT3, AMP-activated protein kinase (AMPK), extracellular signal-regulated kinase (ERK) 1/2 and Akt signalling pathways in an additive manner, effects that were abolished under endoplasmic reticulum (ER) stress. Leptin, but not amylin, increased IRS-1 phosphorylation in GT1-7 hypothalamic, but not in C₂C₁₂ muscle and AML12 liver cell lines. Our data suggest that leptin and amylin have overlapping and additive, but not synergistic, effects in the activation of intracellular signalling pathways. ER stress may induce leptin and amylin resistance in hypothalamic, muscle and liver cell lines. These novel insights into the mode of action of leptin and amylin suggest that these hormones may play an additive role in regulating energy homeostasis in humans.

HMGA1 down-regulation is crucial for chromatin composition and a gene expression profile permitting myogenic differentiation

BackgroundHigh mobility group A (HMGA) proteins regulate gene transcription through architectural modulation of chromatin and the formation of multi-protein complexes on promoter/enhancer regions. Differential expression of HMGA variants has been found to be important for distinct differentiation processes and deregulated expression was linked to several disorders. Here we used mouse C2C12 myoblasts and C2C12 cells stably over-expressing HMGA1a-eGFP to study the impact of deregulated HMGA1 expression levels on cellular differentiation.ResultsWe found that induction of the myogenic or osteogenic program of C2C12 cells caused an immediate down-regulation of HMGA1. In contrast to wild type C2C12 cells, an engineered cell line with stable over-expression of HMGA1a-eGFP failed to differentiate into myotubes. Immunolocalization studies demonstrated that sustained HMGA1a-eGFP expression prevented myotube formation and chromatin reorganization that normally accompanies differentiation. Western Blot analyses showed that elevated HMGA1a-eGFP levels affected chromatin composition through either down-regulation of histone H1 or premature expression of MeCP2. RT-PCR analyses further revealed that sustained HMGA1a expression also affected myogenic gene expression and caused either down-regulation of genes such as MyoD, myogenin, Igf1, Igf2, Igfbp1-3 or up-regulation of the transcriptional repressor Msx1. Interestingly, siRNA experiments demonstrated that knock-down of HMGA1a was required and sufficient to reactivate the myogenic program in induced HMGA1a over-expressing cells.ConclusionsOur data demonstrate that HMGA1 down-regulation after induction is required to initiate the myogenic program in C2C12 cells. Sustained HMGA1a expression after induction prevents expression of key myogenic factors. This may be due to specific gene regulation and/or global effects on chromatin. Our data further corroborate that altered HMGA1 levels influence the expression of other chromatin proteins. Thus, HMGA1 is able to establish a specific chromatin composition. This work contributes to the understanding of how differential HMGA1 expression is involved in chromatin organization during cellular differentiation processes and it may help to comprehend effects of HMGA1 over-expression occurring in malign or benign tumours.