Differentiation Of L6 Myoblasts Research Articles

Calsarcin-2 May Play a Compensatory Role in the Development of Obese Sarcopenia.

Although obese sarcopenia is a major public health problem with increasing prevalence worldwide, the factors that contribute to the development of obese sarcopenia are still obscure. In order to clarify this issue, a high-fat-diet-induced obese sarcopenia mouse model was utilized. After being fed with a high-fat diet for 24 weeks, decreased motor functions and muscle mass ratios were found in the C57BL/6 mice. In addition, the expression of calsarcin-2 was significantly increased in their skeletal muscle, which was determined by a microarray analysis. In order to clarify the role of calsarcin-2 in muscle, lentiviral vectors containing the calsarcin-2 gene or short hairpin RNA targeted to calsarcin-2 were used to manipulate calsarcin-2 expressions in L6 myoblasts. We found that an overexpression of calsarcin-2 facilitated L6 myoblast differentiation, whereas a calsarcin-2 knockdown delayed myoblast differentiation, as determined by the expression of myogenin. However, the calsarcin-2 knockdown showed no significant effects on myoblast proliferation. In addition, to clarify the relationship between serum calsarcin-2 and sarcopenia, the bilateral gastrocnemius muscle mass per body weight in mice and appendicular skeletal muscle mass index in humans were measured. Although calsarcin-2 facilitated myoblast differentiation, the serum calsarcin-2 concentration was negatively related to skeletal muscle mass index in mice and human subjects. Taken together, calsarcin-2 might facilitate myoblast differentiation and appear to play a compensatory role in sarcopenia.

β2-Adrenergic receptor (β2-AR) agonist formoterol suppresses differentiation of L6 myogenic cells by blocking PI3K–AKT pathway

ABSTRACTβ2-Adrenergic receptor (β2-AR) is implicated in muscle metabolic activities such as glycogen metabolism, glucose uptake, lipolysis and muscle growth. However, the functional role of β2-AR in the differentiation of skeletal muscle is largely unknown. Here, we examined the functional role of β2-AR in L6 myoblast differentiation using the long-term-acting β2-AR-specific agonist formoterol. We observed that formoterol treatment strongly suppressed L6 myoblast differentiation and the expression of myosin heavy chain (MHC) in a dose- and time-dependent manner. Showing that both long-acting agonist (formoterol) and short-acting agonist (terbutaline) inhibited the induction of MHC protein, whereas β2-AR antagonist (ICI-118,551) upregulated MHC expression, we clearly demonstrated that β2-AR is involved in L6 myoblast differentiation. Furthermore, our pharmacological inhibition study revealed that the PI3K–AKT pathway is the main signaling pathway for myotube formation. Formoterol inhibited the activation of PI3K–AKT signaling, but not that of ERK signaling. Moreover, formoterol selectively inhibited AKT activation by IGF-I, but not by insulin. Collectively, our findings reveal a previously undocumented role of β2-AR activation in modulating the differentiation of L6 myoblasts.

MEF2A regulates Calpain 3 expression in L6 myoblasts

Calpain 3 (Capn3), a skeletal muscle-specific member of the calpain family, executes some non-proteolytic functions besides its role as a Ca2+-regulated proteolytic enzyme. Previously, we found that changes in Capn3 expression were linearly correlated with the degree of muscular atrophy following reversible sciatic nerve injury and that knockdown of Capn3 gene expression promoted myoblast differentiation. While the regulation of capn3 gene expression is interesting, transcriptional regulation of Capn3 is still unclear. In the present study, we provided experimental data showing that the myogenic enhancer factor 2A (MEF2A) regulated Capn3 gene expression. Firstly, the luciferase reporter assay and EMSA were performed and showed that ectopic expression of the Mef2a gene could bind to the predicted site of the Capn3 promoter region. Furthermore, in the L6 myoblast differentiation model in vitro, Capn3 gene expression was shown to be positively associated with the level of Mef2a by qRT-PCR, western-blotting, and immunocytochemistry. The Capn3 protein level decreased as MEF2A decreased when induced by Mef2a siRNA transfection in L6 myoblasts. Finally, the results of ChIP indicated that MEF2A occupied the promoter region of the Capn3 gene in rat denervated gastrocnemius muscle tissue. Based on these results, we proposed that MEF2A is a transcriptional regulator for Capn3 gene expression.

Role of Intracellular Calcium in Vitamin D‐Treated L6 Myoblast Differentiation and Fiber Type Specification

Emerging evidence suggests that vitamin D plays a role in skeletal muscle differentiation and fiber type‐specific gene expression. It has been demonstrated that vitamin D receptor (VDR) and/or intracellular calcium are involved in this process. The present study was conducted to examine the role of intracellular calcium in vitamin D‐mediated L6 myoblast differentiation. An intracellular calcium chelator, 1,2‐Bis(2‐aminophenoxy)ethane‐N,N,N′,N′‐tetraacetic acid acetoxymethyl ester (BAPTA), was treated to the cells and incubated for 24 hrs prior to differentiation. Then, 100 pM 1,25‐dihydroxyvitamin D3 was treated to the cells for 8 days of differentiation. A total of 4 treatments (a 2×2 factorial design) used in this study were: CON, BAPTA, 1,25‐dihydroxyvitamin D3 (VitD), BAPTA+1,25‐dihydroxyvitamin D3 (BAPTA‐D). Ca2+‐chelating effect of BAPTA was confirmed after BAPTA incubation by using fluorescent Ca2+ indicator, Indo‐1 AM. On day 8 after differentiation, mRNA levels of myogenic regulatory factors (MRFs) and skeletal muscle fiber type‐specific markers were analyzed by qPCR. In VitD treatment, mRNA levels of two MRFs, MyoD and Myogenin, were suppressed in comparison with those of CON (0.8 and 0.6 folds, respectively, p<0.05). In contrast, slow fiber type‐specific markers, including PGC‐1α, ATP2a2 and Tnni1, were upregulated (1.8, 1.7, and 2.9 folds, respectively, p<0.05), while Tnni2, a fast fiber type‐specific gene, was downregulated (0.49 folds, p<0.05) in VitD treatment compared to those of CON. In BAPTA treatment, mRNA levels of Myogenin and Mrf4 were decreased when compared to those of CON (0.75 and 0.42 folds, respectively, p<0.05). However, it was shown that slow fiber type‐specific gene mRNA levels (PGC‐1α, ATP2a2, and Tnni1) were not affected by intracellular calcium level. In BAPTA‐D treatment, decreased gene expression levels of Myogenin and Mrf4 were similar to those of BAPTA treatment, while MyoD was at further lower level compared to that of VitD treatment (0.5 folds, p<0.05). In addition, Tnni1, which was increased in VitD treatment, was not increased in BAPTA‐D treatment. This results may indicate that 1,25‐dihydroxyvitamin D3 modulated Tnni1 mRNA expression via intracellular calcium level. At gene expression level, the inhibitory effect in VitD treatment on MyoD gene expression was amplified in BAPTA‐D treatment. However, the enhancing effect in VitD treatment on Tnni1 mRNA level was abolished in BAPTA‐D treatment. Therefore, intracellular calcium level partially mediates the effects of 1,25‐dihydroxyvitamin D3 on gene expression during L6 myoblast differentiation.

1,25‐Dihydroxyvitamin D3 Mediates L6 Myoblast Differentiation via Vitamin D Receptor (VDR)

In our previous study, it was demonstrated that skeletal muscle fiber type composition was changed by dietary supplementation of 25‐hydroxyvitamin D3 in growing rats. Potential mechanisms of 25‐hydroxyvitamin D3, a precursor of 1,25‐dihydroxyvitamin D3 (active form), have been suggested in previous studies that VDR was involved with mouse C2C12 myoblasts. However, it has not been explained in rat L6 myoblasts. In this study, we examined the role of VDR in skeletal muscle differentiation and fiber type‐specific gene expression by using short hairpin RNA (shRNA) for VDR knockdown in rat L6 myoblast. L6 myoblasts transfected either with scrambled or shVDR vector were treated with vehicle (CON), 1 pM, 10 pM, or 100 pM 1,25‐dihydroxyvitamin D3 (VitD) for 8 days of differentiation. VDR knockdown was confirmed by Western blot. And myogenic regulatory markers and fiber type‐specific gene expression levels were determined by qPCR. VDR protein was increased by VitD at 10 pM and 100 pM compared to that of CON (1.46 and 1.89 folds, respectively, p<0.05). However, shVDR was effective in suppressing VDR up to 10 pM as VDR protein levels were decreased to 34 %, 24 %, and 39 % (CON, 1 pM, and 10 pM, respectively, p<0.05) compared to scrambled vector. And at 100 pM VitD, VDR was not repressed by shVDR compared to that of scrambled vector. Relative gene expression levels of both MyoD and Myogenin were downregulated by VDR knockdown in CON (0.56 and 0.36 folds, respectively, p<0.05), however, VitD treatment abolished the effects. Fiber type‐specific genes were also modulated by VDR knockdown. Gene expression level of Myh7, dominantly expressed gene in slow‐twitch fiber, was suppressed to 37 %, 29 %, and 43 % (CON, 1 pM, and 10 pM, respectively, p<0.05) by VDR knockdown compared to scrambled vector, but was restored at 100 pM. Myh2, which consists of fast‐twitch fiber, was also downregulated by VDR knockdown at CON (0.57 folds, p<0.05), but its level was restored by VitD treatment. The present study demonstrates that VDR affects mRNA levels of the genes involved in L6 myoblast differentiation and fiber type specificity. Also, the VDR knockdown effect was partially modulated by VitD. It could suggest that the effects of VitD on myogenic differentiation might be partially mediated through VDR.

Branched‐chain Amino Acid Catabolism is Required for Muscle Cell Differentiation

The importance of branched‐chain amino acids (BCAAs) in promoting skeletal muscle anabolism has been well studied. BCAAs isoleucine, leucine, and valine have been shown to have a profound effect on activating anabolic signaling pathways in skeletal muscle. This occurs in part by the upregulation in activity of the mammalian target of rapamycin complex‐1 (mTORC1), resulting in increased protein synthesis. However, the regulation of branched‐chain amino acids during the development of muscle remains yet to be elucidated. Here, we studied BCAA metabolism during a 5‐day differentiation of L6 myoblasts. Although no change in intracellular BCAA concentrations was observed during differentiation, L6 cells cultured in the absence of leucine were severely impaired in their ability to differentiate as expression of myosin heavy chain (MHC) was completed abrogated at day 5. Two enzymes which are critical for BCAA metabolism are the branched‐chain amino transferase‐2 enzyme (BCAT2) and the branched chain α‐keto acid dehydrogenase complex (BCKD). BCAT2 catabolizes BCAAs to their corresponding alpha‐keto acids, which are then irreversibly decarboxylated by the BCKD complex. The abundance of BCAT2 did not change during differentiation, whereas levels of the E1α subunit of BCKD was increased 7x on day 5 compared to day 1 (p<0.05). Finally, when either BCAT2 or BCKDE1α was knocked down in the presence of leucine, myoblast differentiation was abrogated (BCKDE1α RNAi: MHC decreased 4.5x from day 3 to day 5 (p<0.05); BCAT2 RNAi: MHC levels completely abolished). Our findings suggest that BCAA catabolism may be a critical process in facilitating muscle differentiation.

Bioinformatic identification of connective tissue growth factor as an osteogenic protein within skeletal muscle.

Aging is associated with increasing incidence of osteoporosis; a skeletal disorder characterized by compromised bone strength that may predispose patients to an increased risk of fracture. It is imperative to identify novel ways in which to attenuate such declines in the functional properties of bone. The purpose of this study was to identify, through in silico, in vitro, and in vivo approaches, a protein secreted from skeletal muscle that is putatively involved in bone formation. We performed a functional annotation bioinformatic analysis of human skeletal muscle‐derived secretomes (n = 319) using DAVID software. Cross‐referencing was conducted using OMIM, Unigene, UniProt, GEO, and CGAP databases. Signal peptides and transmembrane residues were analyzed using SignalP and TMHMM software. To further investigate functionality of the identified protein, L6 and C2C12 myotubes were grown for in vitro analysis. C2C12 myotubes were subjected to 16 h of glucose deprivation (GD) prior to analysis. In vivo experiments included analysis of 6‐week calorie restricted (CR) rat muscle samples. Bioinformatic analysis yielded 15 genes of interest. GEO dataset analysis identified BMP5, COL1A2, CTGF, MGP, MMP2, and SPARC as potential targets for further processing. Following TMHMM and SignalP processing, CTGF was chosen as a candidate gene. CTGF expression level was increased during L6 myoblast differentiation (P <0.01). C2C12 myotubes showed no change in response to GD. Rat soleus muscle samples exhibited an increase in CTGF expression (n = 16) in response to CR (35%) (P <0.05). CTGF was identified as a skeletal muscle expressed protein through bioinformatic analysis of skeletal muscle‐derived secretomes and in vitro/in vivo analysis. Future study is needed to determine the role of muscle‐derived CTGF in bone formation and remodeling processes.

D-31 Free Communication/Poster - Physical Activity Promotion Programming/Intervention Strategies in Youth

PURPOSE: Aging is associated with declines in bone functional properties such as osteoporosis, and it is imperative to identify novel ways in which to attenuate these declines. The purpose of this study was to identify, through in silico and in vitro approaches, a protein secreted from skeletal muscle which is putatively involved in bone formation. METHODS: We performed a functional annotation bioinformatic analysis of human skeletal muscle-derived secretomes (N=321) previously identified through a genome-wide prediction study. (Bortoluzzi et al., Proteins, 62:776-792, 2006). We used DAVID software v 6.7 (NCBI, NIH) for an initial analysis, and cross-referencing was conducted using OMIM, Unigene, UniProt, GEO, and CGAP databases. Signal peptides and transmembrane residues were analyzed using SignalP and TMHMM software. To further investigate functionality of the identified protein, L6 and C2C12 myoblasts were grown until confluency, whereupon they were differentiated into myoutubes for analysis. C2C12 myotubes were subjected to 16 hr of glucose deprivation (GD) prior to analysis. In vivo experimentation included analysis of 8-week calorie restricted (CR) rat muscle samples. Comparisons were made using 1-way ANOVA and independent/paired t-tests. RESULTS: Bioinformatic analysis yielded CTGF as a potential target involved in 11 osteogonic processes such as ossification and bone development. GEO Dataset analysis indicated CTGF transcriptome levels were significantly increased by 48.2% in vastus lateralis m. 6 hr after an acute resistance leg exercise (p=.05). A cleavage site was determined between Gly26 and Gln27, and no transmembrane residue was detected confirming secretory property of CTGF. CTGF protein levels were gradually increased during L6 myoblast differentiation up to day 3 (p<.05). C2C12 myotubes showed a marked increase in the expression level of CTGF, but no change in response to GD. Rat soleus m. samples exhibited CTGF expression (n=16), but no change was found in response to 8-week CR. CONCLUSIONS: CTGF was identified as a skeletal muscle expressed protein through bioinformatic analysis of skeletal muscle derived secretomes and in vitro/ in vivo analysis. Future experiments will be to observe the role of muscle-derived CTGF in bone formation and remodeling processes.

Opposite roles of myocardin and atrogin‐1 in L6 myoblast differentiation

L6 rat myoblasts undergo differentiation and myotube formation when cultured in medium containing a low-concentration of serum, but the underlying mechanism is not well understood. The role of atrogin-1, an E3 ligase with well-characterized roles in muscle atrophy, has not been defined in muscle differentiation. Myocardin is a coactivator of serum response factor (SRF), which together promotes smooth muscle differentiation. Myocardin is transiently expressed in skeletal muscle progenitor cells with inhibitory effects on the expression of myogenin and muscle differentiation. It remains unknown whether myocardin, which undergoes ubiquitination degradation, plays a role in L6 cell differentiation. The current study aimed to investigate the potential roles of myocardin and atrogin-1 in differentiation of L6 cells. As reported by many others, shifting to medium containing 2% serum induced myotube formation of L6 cells. Differentiation was accompanied by up-regulation of atrogin-1 and down-regulation of myocardin, suggesting that both may be involved in muscle differentiation. As expected, over-expression of atrogin-1 stimulated the expression of troponin T and myogenin and differentiation of the L6 myoblasts. Co-expression of myocardin with atrogin-1 inhibited atrogin-1-induced myogenin expression. Over-expression of atrogin-1 decreased myocardin protein level, albeit without affecting its mRNA level. Small-interfering RNA-mediated knockdown of atrogin-1 increased myocardin protein. Consistently, ectopic expression of myocardin inhibited myogenic differentiation. Unexpectedly, myocardin decreased the expression of atrogin-1 without involving Foxo1. Taken together, our results have demonstrated that atrogin-1 plays a positive role in skeletal muscle differentiation through down-regulation of myocardin.

Biphasic Regulation of Intracellular Calcium by Gemfibrozil Contributes to Inhibiting L6 Myoblast Differentiation: Implications for Clinical Myotoxicity

Gemfibrozil is the most myotoxic fibrate drug commonly used for dyslipidemia, but the mechanism is poorly understood. The current study revealed that gemfibrozil inhibits myoblast differentiation through the regulation of intracellular calcium ([Ca(2+)]i) as revealed in L6 myoblasts by use of laser scan confocal microscopy and flow cytometry using Fluo-4 AM as a probe. Gemfibrozil at 20-400 μM, could regulate [Ca(2+)]i in L6 cells in a biphasic manner, and sustained reduction was observed when the concentration reached 200 μM. Inhibition of L6 differentiation by gemfibrozil was concentration-dependent with maximal effect noted between 200 and 400 μM, as indicated by creatine kinase activities and the differentiation index, respectively. In differentiating L6 myoblasts, gemfibrozil at concentrations below 400 μM led to no significant signs of apoptosis or cytotoxicity, whereas differentiation, inhibited by 200 μM gemfibrozil, was only partially recovered. A good correlation was noted between gemfibrozil concentrations that regulate [Ca(2+)]i and inhibit L6 myoblasts differentiation, and both are within the range of total serum concentrations found in the clinic. These data suggest a potential pharmacodynamic effect of gemfibrozil on myogenesis as a warning sign, in addition to the complex pharmacokinetic interactions. It is also noteworthy that mobilization of [Ca(2+)]i by gemfibrozil may trigger complex biological responses besides myocyte differentiation. Information revealed in this study explores the mechanism of gemfibrozil-induced myotoxicity through the regulation of intracellular calcium.

Mechanism underlying long‐term regulation of 3‐hydroxy‐3‐methylglutaryl coenzyme A reductase during L6 myoblast differentiation

3-Hydroxy 3-methylglutaryl Coenzyme A reductase (HMG-CoAR) and its end-products are crucial for insulin-induced differentiation of fetal rat myoblasts (L6) both at early and terminal stages of development. Inhibition of HMG-CoAR activity and reduction of the enzyme levels impair the expression of L6 differentiation markers and prevent myoblast fusion into multinucleated syncytia. The mechanism underlying the modulation of this crucial enzyme so that muscular differentiation can occur is poorly understood. Thus, the aim of this work was to explore the long-term regulation of HMG-CoAR in an attempt to provide a new molecular basis for the control of muscle development. All experiments were performed in L6 rat myoblasts induced to differentiate utilizing insulin. The results indicate the following: (i) at early stages of L6 differentiation, the increase in HMG-CoAR protein levels is probably due to transcriptional induction and a decrease in the enzyme degradation rate; (ii) the subsequent reduction of HMG-CoAR protein levels is related both to an increased degradation rate and reduced gene transcription, as indicated by the rise of Insig-1 levels and the subsequent decrease in the amount of n-SREBP-1; (iii) in the terminal stages of myogenesis, reduced protein levels of HMG-CoAR could be ascribed to the decrease in gene transcription while its degradation rate is not affected. By highlighting the mechanisms involved in HMG-CoAR long-term regulation during myogenesis, this work provides useful information for searching for tools to improve the regenerative ability of muscle tissue and for the development of new pharmacological treatments of myopathies.

P53 Continuous activation of IGF signaling inhibits myogenic differentiation of L6 myoblasts

P53 Continuous activation of IGF signaling inhibits myogenic differentiation of L6 myoblasts

Blunting effect of hypoxia on the proliferation and differentiation of human primary and rat L6 myoblasts is not counteracted by Epo

The aim of this study was to evaluate whether hypoxia and/or erythropoietin would be able to modulate proliferation/differentiation processes of rat and human myoblasts. Rat L6 and primary human myoblasts were grown in 21% or 1% O(2) in the presence or absence of recombinant human erythropoietin (RhEpo). Presence of erythropoietin receptors (EpoR) was assayed using RT-PCR and Western blotting techniques. Cell proliferation was evaluated by determining the doubling time and kinetics of cultures by counting cells. Cell differentiation was analysed by determining myogenic fusion index using antibodies against the myosin heavy chain. Expression of myogenin and myosin heavy chain (MHC) proteins were evaluated using the Western blotting technique. After 96 h culture in growth medium for 2.5 and 9 h, doubling time of L6 and human primary myoblasts respectively, had increased in 1% O(2) conditions (P < 0.01). Kinetics of culture showed alteration in proliferation at 72 h in L6 myoblast cultures and at 4 days in human primary myoblasts. The myogenic fusion index had reduced by 30% in L6 myoblasts and by 20% in human myoblasts (P < 0.01). Expression of myogenin and MHC had reduced by around 50%. Despite presence of EpoR mRNA and protein, RhEpo did not counteract the effects of hypoxia either in L6 cells or in human myoblasts. The data show that exposure to hypoxic conditions (1% O(2)) of rat and human myoblasts altered their proliferation and differentiation processes. They also show that Epo is not an efficient growth factor to counteract this deleterious effect.

The Amphoterin (HMGB1)/Receptor for Advanced Glycation End Products (RAGE) Pair Modulates Myoblast Proliferation, Apoptosis, Adhesiveness, Migration, and Invasiveness: FUNCTIONAL INACTIVATION OF RAGE IN L6 MYOBLASTS RESULTS IN TUMOR FORMATION IN VIVO

We reported that RAGE (receptor for advanced glycation end products), a multiligand receptor of the immunoglobulin superfamily expressed in myoblasts, when activated by its ligand amphoterin (HMGB1), stimulates rat L6 myoblast differentiation via a Cdc42-Rac-MKK6-p38 mitogen-activated protein kinase pathway, and that RAGE expression in skeletal muscle tissue is developmentally regulated. We show here that inhibition of RAGE function via overexpression of a signaling deficient RAGE mutant (RAGE delta cyto) results in increased myoblast proliferation, migration, and invasiveness, and decreased apoptosis and adhesiveness, whereas myoblasts overexpressing RAGE behave the opposite, compared with mock-transfected myoblasts. These effects are accompanied by a decreased induction of the proliferation inhibitor, p21(Waf1), and increased induction of cyclin D1 and extent of Rb, ERK1/2, and JNK phosphorylation in L6/RAGE delta cyto myoblasts, the opposite occurring in L6/RAGE myoblasts. Neutralization of culture medium amphoterin negates effects of RAGE activation, suggesting that amphoterin is the RAGE ligand involved in RAGE-dependent effects in myoblasts. Finally, mice injected with L6/RAGE delta cyto myoblasts develop tumors as opposed to mice injected with L6/RAGE or L6/mock myoblasts that do not. Thus, the amphoterin/RAGE pair stimulates myoblast differentiation by the combined effect of stimulation of differentiation and inhibition of proliferation, and deregulation of RAGE expression in myoblasts might contribute to their neoplastic transformation.

S100B stimulates myoblast proliferation and inhibits myoblast differentiation by independently stimulating ERK1/2 and inhibiting p38 MAPK

The Ca2+-modulated protein of the EF-hand type, S100B, was shown to inhibit rat L6 myoblast differentiation and myotube formation by interacting with a high affinity with an unidentified receptor (Sorci et al., 2003). We show here that S100B independently inhibits the MKK6-p38 MAPK pathway and stimulates the Ras-MEK-ERK1/2 pathway. The inhibitory effect of S100B on p38 MAPK translates into a defective induction of the muscle-specific transcription factor myogenin and the antiproliferative factor p21(WAF1), while S100B's stimulatory effect on ERK1/2 results in stimulation of myoblast proliferation via cyclin D1 induction and Rb phosphorylation and protection against apoptosis via activation of NF-kappaB transcriptional activity. Also, the S100B's effects that are mediated by the Ras-MEK-ERK1/2 pathway that is, stimulation of proliferation and protection against apoptosis, depend on reactive oxygen species production, being inhibited by antioxidants, while the S100B inhibitory effect on the MKK6-p38 MAPK pathway is not. We propose that S100B might participate in the regulation of myoblast differentiation by stimulating myoblast proliferation, protecting myoblasts against apoptosis, and modulating myotube formation.

L6 myoblast differentiation is modulated by Cdk5 via the PI3K-AKT-p70S6K signaling pathway.

Cdk5 regulates myogenesis but the signaling cascade through which Cdk5 modulates this process remains to be characterized. Here, we investigated whether PI3K, Akt, p70S6K, p38 MAPK, p44/42 MAPK, and Egr-1 serve as upstream regulators of Cdk5 during L6 myoblast differentiation. Upon serum reduction, we found that besides elevated expression of Cdk5 and its activator, p35, and increased Cdk5/p35 activity, Egr-1, Akt, p70S6K, and p38 MAPK activity were upregulated in differentiating L6 cells. However, p44/42 MAPK was downregulated and SAPK/JNK was unaffected. LY294002, a PI3K inhibitor, blocked the activation of Akt and p70S6K, indicating that Akt and p70S6K activation is linked to PI3K activation. The lack of LY294002 effect on p38 MAPK suggests that p38 MAPK activation is not associated with PI3K activation. Rapamycin, a specific inhibitor of FRAP/mTOR (the upstream kinase of p70S6K), also blocked p70S6K activation, indicating the involvement of FRAP/mTOR activation. LY294002 and rapamycin also blocked the enhancement of Egr-1 level, Cdk5 activity, and myogenin expression, suggesting that upregulation of these factors is coupled to PI3K-p70S6K activation. Overexpression of dominant-negative-Akt also reduced Cdk5/p35 activity and myogenin expression, indicating that the PI3K-p70S6K-Egr-1-Cdk5 signaling cascade is linked to Akt activation. SB2023580, a p38 MAPK inhibitor, had no effect on p70S6K, Egr-1, or Cdk5 activity, suggesting that p38 MAPK activation lies in a pathway distinct from the PI3K-Akt-p70S6K-Egr-1 pathway that we identify as the upstream modulator of Cdk5 activity during L6 myoblast differentiation.

S100B causes apoptosis in a myoblast cell line in a RAGE-independent manner.

S100B, a Ca(2+)-modulated protein with both intracellular and extracellular regulatory roles, is most abundant in astrocytes, is expressed in various amounts in several non-nervous cells and is also found in normal serum. Astrocytes secrete S100B, and extracellular S100B exerts trophic and toxic effects on neurons depending on its concentration, in part by interacting with the receptor for advanced glycation end products (RAGE). The presence of S100B in normal serum and elevation of its serum concentration in several non-nervous pathological conditions suggest that S100B-expressing cells outside the brain might release the protein and S100B might affect non-nervous cells. Recently we reported that at picomolar to nanomolar doses S100B inhibits rat L6 myoblast differentiation via inactivation of p38 kinase in a RAGE-independent manner. We show here that at >or=5 nM in the absence of and at >100 nM in the presence of serum S100B causes myoblast apoptosis via stimulation of reactive oxygen species (ROS) production and inhibition of the pro-survival kinase, extracellular signal-regulated kinase (ERK)1/2, again in a RAGE-independent manner. Together with our previous data, the present results suggest that S100B might participate in the regulation of muscle development and regeneration by two independent mechanism, i.e., by inhibiting crucial steps of the myogenic program at the physiological levels found in serum and by causing elevation of ROS production and myoblast apoptosis following accumulation in serum and/or muscle extracellular space. Our data also suggest that RAGE has no role in the transduction of S100B effects on myoblasts, implying that S100B can interact with more than one receptor to affect its target cells.

Integrin-linked kinase is a positive mediator of L6 myoblast differentiation

Overexpression of ILK in L6 myoblasts results in increased ILK kinase activity, stimulating myotube formation and induction of biochemical differentiation markers. Expression of a dominant negative ILK mutant, ILK(E359K), inhibits endogenous ILK activation and L6 differentiation. Cell cycle analysis of ILK(E359K) cells cultured in serum-free conditions indicates significant apoptosis (11–19% sub-diploid peak) which is not seen in insulin treated cells. Expression of ILK variants does not have significant effects on S-phase transit, however. Known targets of ILK, PKB/Akt or glycogen synthase kinase 3β are not obviously involved in ILK-induced L6 differentiation. Insulin-stimulated phosphorylation of PKB at Ser473 is unimpaired in the ILK(E359K) cells, suggesting that PKB is not a myogenic target of ILK. Inhibition of GSK3β by LiCl blocks L6 myogenesis, indicating that ILK-mediated inhibition of GSK3β is not sufficient for differentiation. Our data do suggest that a LiCl-sensitive interaction of ILK is important in L6 myoblast differentiation.

S100B Inhibits Myogenic Differentiation and Myotube Formation in a RAGE-Independent Manner

S100B is a Ca(2+)-modulated protein of the EF-hand type with both intracellular and extracellular roles. S100B, which is most abundant in the brain, has been shown to exert trophic and toxic effects on neurons depending on the concentration attained in the extracellular space. S100B is also found in normal serum, and its serum concentration increases in several nervous and nonnervous pathological conditions, suggesting that S100B-expressing cells outside the brain might release the protein and S100B might exert effects on nonnervous cells. We show here that at picomolar to nanomolar levels, S100B inhibits myogenic differentiation of rat L6 myoblasts via inactivation of p38 kinase with resulting decrease in the expression of the myogenic differentiation markers, myogenin, muscle creatine kinase, and myosin heavy chain, and reduction of myotube formation. Although myoblasts express the multiligand receptor RAGE, which has been shown to transduce S100B effects on neurons, S100B produces identical effects on myoblasts overexpressing either full-length RAGE or RAGE lacking the transducing domain. This suggests that S100B affects myoblasts by interacting with another receptor and that RAGE is not the only receptor for S100B. Our data suggest that S100B might participate in the regulation of muscle development and regeneration by inhibiting crucial steps of the myogenic program in a RAGE-independent manner.

The Adhesion and Differentiation-inhibitory Activities of the Immunoglobulin Superfamily Member, Carcinoembryonic Antigen, Can Be Independently Blocked

The external domains of Ig superfamily members are involved in multiple binding interactions, both homophilic and heterophilic, that initiate molecular events leading to the execution of diverse cell functions. Human carcinoembryonic antigen (CEA), an Ig superfamily cell surface glycoprotein used widely as a clinical tumor marker, undergoes homophilic interactions that mediate intercellular adhesion. Recent evidence supports the view that deregulated overexpression of CEA has an instrumental role in tumorigenesis through the inhibition of cell differentiation and the disruption of tissue architecture. The CEA-mediated block of the myogenic differentiation of rat L6 myoblasts depends on homophilic binding of its external domains. We show here that L6 transfectant cells expressing CEA can "trans-block" the myogenesis of juxtaposed differentiation-competent L6 transfectant cells expressing a deletion mutant of CEA (DeltaNCEA). This result implies the efficacy of antiparallel CEA-CEA interactions between cells in the differentiation block. In addition, DeltaNCEA can acquire differentiation blocking activity by cross-linking with specific anti-CEA antibodies, thus implying the efficacy of parallel CEA-CEA interactions on the same cell surface. The myogenic differentiation blocking activity of CEA was demonstrated by site-directed mutations to involve three subdomains of the amino-terminal domain, shown previously to be critical for its intercellular adhesion function. Monovalent Fab fragments of monoclonal antibodies binding to the region bridging subdomains 1 and 2 could both inhibit intercellular adhesion and release the myogenic differentiation block. Amino acid substitutions Q80A, Q80R, and D82N in subdomain 3, QNDTG, however, were found to completely ablate the differentiation blocking activity of CEA but had no effect on intercellular adhesion activity. A cyclized peptide representing this subdomain was the most effective at releasing the differentiation block.