Low-Intensity Exercise Suppresses CCAAT/Enhancer-Binding Protein δ/Myostatin Pathway Through Androgen Receptor in Muscle Cells.

BACKGROUND: Androgen production following exercise has been suggested to contribute anabolic actions of muscle. However, the underlying mechanisms of the androgen receptor (AR) in androgen's action are still unclear. OBJECTIVE: In the present study, we examined androgen/AR-mediated action in exercise, especially for the suppression of myostatin, a potent negative regulator of muscle mass. METHODS: To examine the effects of exercise, we employed low-intensity exercise in mice and electric pulse stimulation (EPS) in C2C12 myotubes. Androgen production by C2C12 myotubes was measured by enzyme-linked immunosorbent assay. To block the action of AR, we pretreated C2C12 myotubes with flutamide. Quantitative real-time polymerase chain reaction was used to determine the expression levels of proteolytic genes including CCAAT/enhancer-binding protein delta (C/EBPδ), myostatin and muscle E3 ubiquitin ligases, as well as myogenic genes such as myogenin and PGC1α. The activation of 5'-adenosine-activated protein kinase and STAT3 was determined by Western blot analysis. RESULTS: Both mRNA and protein levels of AR significantly increased in skeletal muscle of low-intensity exercised mice and C2C12 myotubes exposed to EPS. Production of testosterone and dihydrotestosterone from EPS-treated C2C12 myotubes was markedly increased. Of interest, we found that myostatin was clearly inhibited by EPS, and its inhibition was significantly abrogated when AR was blocked by flutamide. To test how AR suppresses myostatin, we examined the effects of EPS on C/EBPδ because the promoter region of myostatin has several C/EBP recognition sites. C/EBPδ expression was decreased by EPS, and this decrease was negated by flutamide. IL-6 and phospho-STAT3 (pSTAT3) expression, the downstream pathway of myostatin, were decreased by EPS and this was also reversed by flutamide. Similar downregulation of C/EBPδ, myostatin, and IL-6 was seen in skeletal muscle of low-intensity exercised mice. CONCLUSIONS: Muscle AR expression and androgen production were increased by exercise and EPS treatment. As a mechanistical insight, it is suggested that AR inhibited myostatin expression transcriptionally by C/EBPδ suppression, which negatively influences IL-6/pSTAT3 expression and consequently contributes to the prevention of muscle proteolysis during exercise.

Palmitate exerts opposite effects on proliferation and differentiation of skeletal myoblasts.

The purpose of the study was to examine mechanisms controlling cell cycle progression/arrest and differentiation of mouse C2C12 myoblasts exposed to long-chain saturated fatty acid salt, palmitate. Treatment of proliferating myoblasts with palmitate (0.1 mmol/l) markedly decreased myoblast number. Cyclin A and cyclin D1 levels decreased, whereas total p21 and p21 complexed with cyclin-dependent kinase-4 (cdk4) increased in myoblasts treated with palmitate. In cells induced to differentiation addition of palmitate augmented the level of cyclin D3, the early (myogenin) and late (α-actinin, myosin heavy chain) markers of myogenesis, and caused an increase of myotube diameter. In conclusion, exposure to palmitate inhibits proliferation of myoblasts through a decrease in cyclin A and cyclin D1 levels and an increase of p21-cdk4 complex formation; however, it promotes cell cycle exit, myogenic differentiation and myotube growth.

Sodium arsenite delays the differentiation of C2C12 mouse myoblast cells and alters methylation patterns on the transcription factor myogenin.

Epidemiological studies have correlated arsenic exposure with cancer, skin diseases, and adverse developmental outcomes such as spontaneous abortions, neonatal mortality, low birth weight, and delays in the use of musculature. The current study used C2C12 mouse myoblast cells to examine whether low concentrations of arsenic could alter their differentiation into myotubes, indicating that arsenic can act as a developmental toxicant. Myoblast cells were exposed to 20 nM sodium arsenite, allowed to differentiate into myotubes, and expression of the muscle-specific transcription factor myogenin, along with the expression of tropomyosin, suppressor of cytokine signaling 3 (Socs3), prostaglandin I2 synthesis (Ptgis), and myocyte enhancer 2 (Mef2), was investigated using QPCR and immunofluorescence. Exposing C2C12 cells to 20 nM sodium arsenite delayed the differentiation process, as evidenced by a significant reduction in the number of multinucleated myotubes, a decrease in myogenin mRNA expression, and a decrease in the total number of nuclei expressing myogenin protein. The expression of mRNA involved in myotube formation, such as Ptgis and Mef2 mRNA, was also significantly reduced by 1.6-fold and 4-fold during differentiation. This was confirmed by immunofluorescence for Mef2, which showed a 2.6-fold reduction in nuclear translocation. Changes in methylation patterns in the promoter region of myogenin (-473 to +90) were examined by methylation-specific PCR and bisulfite genomic sequencing. Hypermethylated CpGs were found at -236 and -126 bp, whereas hypomethylated CpGs were found at -207 bp in arsenic-exposed cells. This study indicates that 20 nM sodium arsenite can alter myoblast differentiation by reducing the expression of the transcription factors myogenin and Mef2c, which is likely due to changes in promoter methylation patterns. The delay in muscle differentiation may lead to developmental abnormalities.

The formation of skeletal muscle myotubes requires functional membrane receptors activated by extracellular ATP.

Skeletal muscle differentiation follows an organized sequence of events including commitment, cell cycle withdrawal, and cell fusion to form multinucleated myotubes. The role of adenosine 5'-triphosphate (ATP)-mediated signaling in differentiation of skeletal muscle myoblasts was evaluated in C(2)C(12) cells, a myoblast cell line. Cell differentiation was inhibited by P2X receptor blockers or by degradation of endogenous ATP with apyrase. However, pertussis toxin, known to block only a group of P2Y receptors, did not alter the differentiation process. Cells were heterogeneous in their expression of functional P2X receptors, evaluated by the uptake of fluorescent permeability tracers (Lucifer yellow and ethidium bromide), and by immunofluorescence of P2X(7) receptors. Moreover, xestospongin C, a selective and membrane-permeable inhibitor of IP(3) receptors, inhibited both myotube formation and myogenin expression. Based on these results, we suggest that the known increase in intracellular Ca(2+) concentration required for differentiation is due at least in part to Ca(2+) influx through P2X receptors and Ca(2+) release from intracellular stores. The possible involvement of P2X receptors and other pathways that might set the intracellular Ca(2+) at the level required for myoblast differentiation as well as the possible involvement of gap junction channels in the intercellular transfer of second messengers involved in coordinating myogenesis is proposed.

A myogenic differentiation checkpoint activated by genotoxic stress.

Cell-cycle checkpoints help to protect the genomes of proliferating cells under genotoxic stress. In multicellular organisms, cell proliferation is often directed toward differentiation during development and throughout adult homeostasis. To prevent the formation of differentiated cells with genetic instability, we hypothesized that genotoxic stress may trigger a differentiation checkpoint. Here we show that exposure to genotoxic agents causes a reversible inhibition of myogenic differentiation. Muscle-specific gene expression is suppressed by DNA-damaging agents if applied prior to differentiation induction but not after the differentiation program is established. The myogenic determination factor, MyoD (encoded by Myod1), is a target of the differentiation checkpoint in myoblasts. The inhibition of MyoD by DNA damage requires a functional c-Abl tyrosine kinase (encoded by Abl1), but occurs in cells deficient for p53 (transformation-related protein 53, encoded by Trp53) or c-Jun (encoded by the oncogene Jun). These results support the idea that genotoxic stress can regulate differentiation, and identify a new biological function for DNA damage-activated signaling network.

Preferential binding of Grb2 or phosphatidylinositol 3-kinase to the met receptor has opposite effects on HGF-induced myoblast proliferation.

Hepatocyte growth factor (HGF) and its receptor, Met, play a crucial role in regulating adult skeletal myoblast proliferation and differentiation. Met signaling is mediated by phosphorylation of two carboxy-terminal tyrosines, which act as docking sites for a number of intracellular mediators. These include Grb2 and p85, which couple the receptor with the Ras and phosphatidylinositol 3-kinase (PI3K) pathways, respectively. In this study, we define the role of these effectors in response to HGF by utilizing Met mutants, designed to obtain preferential coupling of Met to either Grb2 or PI3K or both. We found that relative to the wild-type receptor, enhanced binding to Grb2 further increases the incorporation of bromodeoxyuridine and the expression of Twist, while decreasing that of p27(Kip1) and myogenin. Conversely, preferential coupling with PI3K induced cell-cycle withdrawal and differentiation. Whereas enhanced Grb2 binding increased the phosphorylation of the mitogen-activated protein kinase/extracellular signal-regulated protein kinases (MAPK/ERK) and abrogated that of p38 MAPK, PI3K had the opposite effect. PD098059 reversed the inhibitory effects of Met on cell proliferation and differentiation, while wortmannin had only a very marginal effect. Taken together, these data suggest that coupling of Met with Grb2 is necessary for HGF-mediated inhibition of muscle differentiation. This inhibition occurs only when PI3K signaling downstream of Met is low. Imposing an efficient coupling of PI3K to Met would lead to upregulation of muscle regulatory factors and subsequent cell differentiation.

E2F1 inhibition of transcription activation by myogenic basic helix-loop-helix regulators.

Cellular transcription factor E2F1 is thought to regulate the expression of genes important for cell cycle progression and cell proliferation. Deregulated E2F1 expression induces S-phase entry in quiescent cells and inhibits myogenic differentiation. We show here that E2F1 inhibits the activation of gene transcription by myogenic basic helix-loop-helix proteins myoD and myogenin. Transfection assay using different deletion constructs indicates that both the DNA binding and the transactivation domains of E2F1 are required for its inhibition of myoD transcription activation. However, the retinoblastoma protein (RB) binding domain is not required. Furthermore, co-transfection with the RB, which inhibits the transcription activity of E2F1, can also repress E2F1 inhibition of myoD transactivation. These results suggest an essential role of E2F1-mediated transcription in its inhibition of myogenesis.

Simultaneous myogenin expression and overall DNA hypomethylation promote in vitro myoblast differentiation.

Two clones of the L5 myoblast line (M6 and the fusion-defective M12) were examined for the expression of myogenin, one of the regulatory genes involved in the regulation of differentiation to myofibers after treatment with 3-deazaadenosine, a metabolic inhibitor of methyl transfer reactions. Cultures treated with 3-deazaadenosine showed, using Northern blot hybridization, a conspicuous increase in myogenin expression, which in clone M6 correlated to the extent of cell differentiation under fusing conditions but was evident also in growth medium, although the drug was unable to start the myogenic program. We also tested the extent of total DNA methylation to verify whether the activation of the regulatory cascade could be correlated to the decrease of the overall number of 5-methylcytosines present in the genome. The results show that the loss of 5-methylcytosine from newly synthesized DNA, but not from preexisting DNA, is evident in fusing conditions and enhanced by 3-deazaadenosine. It appears that there is a positive correlation between the passive demethylation of newly synthesized DNA, the activation of the myogenin gene by demethylation, and the differentiation of myoblasts. However, in fusing conditions, the defective clone M12, although it is able to express myogenin and its DNA is hypomethylated, fuses only in the presence of 3-deazaadenosine, suggesting some alternative way of induction.