AJP - Endo Information on EB 2010
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Endocrinol Metab 294: E241-E250, 2008. First published October 30, 2007; doi:10.1152/ajpendo.00129.2007
0193-1849/08 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
294/2/E241    most recent
00129.2007v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Plaisance, I.
Right arrow Articles by Brink, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Plaisance, I.
Right arrow Articles by Brink, M.

TNF-{alpha} increases protein content in C2C12 and primary myotubes by enhancing protein translation via the TNF-R1, PI3K, and MEK

Isabelle Plaisance, Christian Morandi, Claire Murigande, and Marijke Brink

Institute of Physiology, Department of Biomedicine, University and University Hospital of Basel, Basel, Switzerland

Submitted 26 February 2007 ; accepted in final form 29 October 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Recent evidence supports that TNF-{alpha}, long considered a catabolic factor, may also have a physiological function in skeletal muscle. The catabolic view, mainly based on correlative studies in human and in vivo animal models, was challenged by experiments with myoblasts, in which TNF-{alpha} induced differentiation. The biological effects of TNF-{alpha} in differentiated muscle, however, remain poorly understood. In the present study, we tested whether TNF-{alpha} has growth-promoting effects in myotubes, and we characterized the mechanisms leading to these effects. Treatment of C2C12 myotubes with TNF-{alpha} for 24 h increased protein synthesis (PS) and enhanced cellular dehydrogenase activity by 22 and 26%, respectively, without changing cell numbers. These effects were confirmed in myotubes differentiated from primary rat myoblasts. TNF-{alpha} activated two signaling cascades: 1) ERK1/2 and its target eIF4E and 2) Akt and its downstream effectors GSK-3, p70S6K, and 4E-BP1. TNF-{alpha}-induced phosphorylation of Akt, and ERK1/2 was inhibited by an antibody against TNF-{alpha} receptor 1 (TNF-R1). PD-98059 pretreatment abolished TNF-{alpha}-induced phosphorylation of ERK1/2 and eIF4E, whereas PS was only partially inhibited. LY-294002 completely abolished TNF-{alpha}-induced stimulation of PS as well as phosphorylation of Akt and its downstream targets GSK-3, p70S6K, and 4E-BP1. Rapamycin inhibited TNF-{alpha}-induced phosphorylation of the mTOR C1 target p70S6K without altering TNF-{alpha}-induced PS and 4E-BP1 phosphorylation. In conclusion, our results provide evidence that TNF-{alpha} enhances PS in myotubes and that this is based on enhanced protein translation mediated by the TNF-R1 and PI3K-Akt and MEK-ERK signaling cascades.

tumor necrosis factor-{alpha}; tumor necrosis factor-{alpha} receptor 1; phosphatidylinositol 3-kinase; mitogen-activated protein kinase/extracellular signal-related kinase

MUSCLE LOSS OCCURS AFTER INJURY as a result of aging and in a variety of cachectic conditions associated with severe disease states such as AIDS, cancer, and congestive heart failure. A change in lean muscle mass is often attributed to a changed balance between protein synthesis (PS) and degradation rates in the myofibers (29). On the other hand, muscle loss or reduced muscle growth may be related to reduced capacity of regeneration from satellite cells. Regulation of either of these biological processes involves complex interactions among multiple growth factors and cytokines (reviewed in Ref. 5). Tumor necrosis factor (TNF)-{alpha}, originally known as cachectin, is considered to be a catabolic factor that mediates the development of skeletal muscle wasting. In patients, elevated circulating levels of this cytokine are generally associated with catabolic states (reviewed in Ref. 4), and muscle wasting as a result of changed protein metabolism has been observed after acute or chronic administration of TNF-{alpha} to rats (13, 15, 27), although it was at the same time reported that no change in protein metabolism occurred in isolated muscle preparations (15). In attempts to analyze the TNF-{alpha}-induced cellular mechanisms, several investigators mimicked the in vivo situation by using differentiated myotube cultures, but results obtained from such studies remain controversial. The cytokine was shown to decrease protein content by enhancing protein degradation (26), others reported that protein loss happens predominantly via decreased synthesis of contractile proteins (16), whereas others again provided evidence for the contrary, namely enhanced protein synthesis in myotube models (1, 10).

Similarly, studies with myoblasts instead of myotubes have reported seemingly contradictory results. It has been proposed that TNF-{alpha} induces muscle loss by inhibiting differentiation (7, 25, 32), and NF-{kappa}B was shown to mediate this effect by decreasing transcription of contractile proteins (16, 22, 24). Recent work with myoblasts, however, showed that endogenously produced TNF-{alpha} is crucial for myogenesis because it promotes the expression of myocyte enhancer factor (MEF)-2C and the myosin heavy chain (6). The view that the cytokine may contribute in a positive way to regeneration of muscle was supported by several other studies. Expression of TNF-{alpha} and its receptor in muscle fibers in human inflammatory myopathies was correlated with regeneration (9, 21), and expression was increased in muscle after injury (34, 39). Another study showed that TNF/dystrophin double-knockout mice have decreased muscle mass and accelerated pathological progression compared with TNF+/dystrophin– mice, indicating a positive role for TNF-{alpha} in muscle (31).

Taken together, in several myoblast and myotube models, beneficial as well as deleterious effects of TNF-{alpha} have been reported. The reasons for these diverse responses remain poorly understood but certainly include the high complexity of the signaling induced by TNF-{alpha}. At the cell membrane, the cytokine may find, depending on which cell type is studied, one or both of its two different cell membrane receptors, TNF-{alpha} receptor 1 (TNF-R1) or TNF-R2. These differ in their cytoplasmic structure and initiate multiple distinct and overlapping intracellular pathways, which each can be influenced by other simultaneous input stimuli that the cell receives from its environment. In cardiac pathologies, e.g., after myocardial infarct, deleterious effects appear to be mediated through the TNF-R1, as ablation of this receptor attenuated the development of heart failure in TNF-{alpha}-overexpressing mice (17, 30). By contrast, ablation of the TNF-R2 actually increased mortality in mice overexpressing TNF-{alpha}, suggesting that pathways downstream of TNF-R2 might be cardioprotective (17). Far less is known about the role of each TNF-R subtype in mediating protective or harmful pathways in skeletal muscle.

The present study was designed to obtain mechanistic insights into the actions of TNF-{alpha} in skeletal muscle cells, with special focus on the receptor subtype involved and on the signaling molecules normally implicated in protection. Knowledge of these pathways may contribute to novel strategies to improve the muscle protein balance. We used myotubes after their differentiation from C2C12 or rat primary myoblasts to examine TNF-{alpha}-induced signaling pathways and their effects on protein metabolism and cell viability. TNF-{alpha} increased protein content, PS, and cell viability in both myotube models, effects that were mediated by the TNF-R1. The effects of TNF-{alpha} on PS were due to enhanced initiation of translation, and this was mediated by MEK/ERK and PI3K/Akt signaling.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Antibodies and reagents. Recombinant mouse TNF-{alpha} was from R&D Systems (Minneapolis, MN). The inhibitors LY-294002 (LY), PD-98059 (PD), rapamycin (Rap), cycloheximide (CHX), and actinomycin D (Act D)were all from Calbiochem (Merck Bioscience, Darmstadt, Germany). Antibodies against phospho-ERK1/2 (Thr202/Tyr204), ERK1/2, phospho-Akt (Ser473), phopho-Akt (Thr308), Akt, phospho-glycogen synthase kinase-3 (GSK-3; Ser9/21), GSK-3, eukaryotic initiation factor (eIF)-binding protein-1 (4E-BP1), phospho-eIF4E (Ser209), phospho-p70S6K (ribosomal protein p70 S6 kinase; Thr389), and p70S6K were all purchased from Cell Signaling Technology (Danvers, MA). The anti-TNF-R1 and anti-TNF-R2 antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA) and the anti-actin antibody was from Sigma (St. Louis, MO). All the media for cell cultures were purchased from GIBCO (Invitrogen, Auckland, NZ). Type 2 collagenase was from Biochrom (Berlin, Germany), and trypsin and collagen were from Sigma. The primers used for real-time PCR were designed with assistance of the software Primers Express (Applied Biosystems, Foster City, CA) and synthesized by Microsynth (Balgach, Switzerland). The sequences of the primers are the following: TNF-R1 forward 5'-CTG GCC TGC TGC TGT CAC T-3' and reverse 5'-TCC AGA CCC CTG ATG GAT GTA-3'; TNF-R2 forward 5'-TGC CGC TGG TCT TCG AA-3' and reverse 5'-CCT GTG AGA TCT GGC ACT CGT A-3'.

Myogenic cell cultures. Myoblasts from the mouse muscle-derived C2C12 cell line were obtained from American Type Culture Collection (Manassas, VA). The seeding density used throughout the experiments was 104 cells/cm2 surface. Undifferentiated cells were grown in Dulbecco's modified Eagle's medium (DMEM, 4.5 mg/ml glucose) supplemented with 10% heat-inactivated fetal calf serum at 37°C in the presence of 5% CO2. The myoblasts were fused into myotubes by shifting them to differentiation medium (DM), consisting of DMEM supplemented with 2% heat-inactivated horse serum (HS). The DM was changed after 48 h (day 2), and the differentiation was allowed to continue for 96 more hours. All experiments were performed at 6 days after the beginning of differentiation (t = 0). For primary cultures, limb skeletal muscles were excised from neonatal rats (2–4 days old), subsequently minced with razor blades in ice-cold PBS, and enzymatically dissociated in buffer containing 0.1% trypsin and 0.1% collagenase type 2 for three or four successive dissociations with continuous stirring (20 min, 37°C). The supernatants with released cells were collected, filtered through a nylon mesh, and centrifuged (350 g, 5 min). The pellet was resuspended in growth medium (DMEM-F-12 supplemented with 20% of FBS and 2.5 ng/ml β-fibroblast growth factor), and cells were plated in plastic Petri dishes for 1 h (37°C, 5% CO2). The unattached cells were seeded in collagen-coated Petri dishes (Sigma). After 1 or 2 days, cells were released by trypsinization and preplated in noncoated dishes for 30 min to remove contaminating fibroblasts. The remaining cells were plated on collagen-coated dishes in growth medium. The preplating procedure was repeated once to obtain a pure myoblast culture. The density used for the experiments was 105 cells/cm2 surface, and differentiation was induced by shifting the growth medium to DMEM-F-12 supplemented with 2% HS. The medium was changed every day for 5 days. To preserve the characteristics of cells, splitting of the cells was done up to a maximum of seven and five times for the C2C12 and primary cells, respectively.

The inhibitors LY (20 µM), PD (10 µM), and Rap (20 ng/ml) were added prior to TNF-{alpha}, which was used at 10 ng/ml or 0.5 ng/ml for C2C12 and primary cells, respectively. Time periods are indicated in the figure legends.

PS and protein content. To determine the rate of PS, cells were incubated with TNF-{alpha} in the presence of radiolabeled [3H]phenylalanine (Amersham Biosciences, Buckinghamshire, UK) at a final activity of 5 µCi/ml when incubated for 4 h, or 1 µCi/ml when incubated for 24 h. The reaction was stopped by washing the cell culture twice with ice-cold PBS, and then the cells were precipitated with 10% trichloroacetic acid, rinsed with chilled ethanol (95%), and dried. The cells were solubilized by incubation with 0.2 M NaOH for 45 min under constant agitation. A part of the resulting lysates was mixed with scintillation liquid and counted in a β-counter, while the other part was used to measure protein content with the Micro BCA protein assay kit (Pierce, Rockford, IL).

Real-time PCR. Total RNA was extracted using Tri Reagent (Sigma) and treated with DNAse I (Ambion, Austin, TX). RNA concentrations, purity, and quality were determined by spectrophotometry and agarose gel electrophoresis. cDNA was synthesized with the reverse transcriptase Omniscript RT kit (Qiagen, Valencia, CA) using 0.5 µg/µg RNA of random hexamers (Promega, WI). Real-time PCR was performed on a Light-Cycler apparatus (Applied Biosystems, Foster City, CA) using 5 µl of cDNA from the Omniscript reaction, a concentration of 0.5 x ITaQ SYBR Green Supermix Kit (Bio-Rad, Reinach, Switzerland), and a final concentration of 300 nM for forward and reverse primers in a total volume of 20 µl. Thermal cycling was as follows: activation for 2 min at 95°C followed by 40 cycles of 15 s at 95°C and and 45 s at 60°C for each cycle. The mRNA level was based on the critical threshold (CT) value and normalized for β-tubulin.

Measurement of dehydrogenase activity. Cell viability was quantified using a colorimetric assay (Cell Proliferation reagent WST-1; Roche Diagnostics, Basel, Switzerland), which measures mitochondrial dehydrogenase activity (DHA). The WST-1 substrate was added to the medium during the two last hours before optical density (OD) measurements (450 nm). After a reading of the 96-well plates, cells were rinsed with PBS, fixed with 4% formaldehyde, and after washing incubated in crystal violet solution for 30 min at room temperature, and then extensively rinsed and lysed in 1% SDS solution under constant agitation for 1 h. OD was read at 595 nm to evaluate the DNA amount. The relative DHA was obtained by calculation of OD450/OD595 ratios, this to normalize for the number of cells.

Western blotting. Cells were washed in PBS containing 1 µM orthovanadate and lysed in RIPA buffer containing 50 mM Tris·HCl, pH 7.4, 1% NP-40, 150 mM NaCl, 1 mM NaF, 1 mM orthovanadate, 1 µg/µl pepstatin, and 1 mM PMSF plus and "Mini-Complete" protease inhibitor cocktail (Roche Diagnostics). Equal amounts of 15 µg were resolved by SDS-polyacrylamide gel electrophoresis and transferred to PVDF membranes (Millipore, Volketswil, Switzerland). The membranes were blocked with 5% nonfat dry milk in Tris-buffered saline-0.1% Tween (TBST) and probed overnight with primary antibodies in TBST supplemented with 5% bovine serum albumin. After reaction in TBST with horseradish peroxidase-conjugated secondary antibodies (Jackson ImmunoResearch Europe, Cambridgeshire, UK), bands were visualized using enhanced chemiluminescence reagents (Supersignal West Pico, Pierce) and exposure to autoradiography film. Membranes were first probed with anti-phosphopeptide antibodies. To control for loading, membranes were stripped and reprobed with antibodies that recognize the same protein independently of its phosphorylation state or with anti-actin antibodies. For quantification, the intensity of each phosphorylated protein band was measured by densitometry using the NIH Image 1.62 software and normalized to the corresponding value obtained for the total protein band.

Measurement of Akt activity. Measuring Akt activity was achieved using an Akt kinase kit (Cell Signaling Technology, Danvers, MA). Briefly, Akt was immunoprecipitated from cytosolic extracts by overnight incubation with an anti-Akt mouse monoclonal antibody immobilized on agarose hydrazide beads. Beads were harvested by centrifugation and resuspended in a kinase buffer containing 200 µM ATP and 1 µg of GSK-3 fusion protein. The reaction, performed at 37°C for 30 min, was stopped by addition of SDS sample buffer. Beads were pelleted by microcentrifugation and the supernatants subjected to SDS-PAGE. Detection of the phosphorylated protein was performed using routine procedures described above under Western blotting, using the phospho-GSK-3 antibody.

Statistics. All data are expressed as means ± SD (error bars). Comparisons between TNF-{alpha} and time- or medium-matched controls were made using one-way analysis of variance with Dunnett's or Bonferroni's multiple comparison test of post hoc analysis where appropriate.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
TNF-{alpha} increases PS and DHA in C2C12 myotubes. To examine the effects of TNF-{alpha} on PS, differentiated C2C12 myotubes were incubated for different time periods with TNF-{alpha} and [3H]Phe. For each time point, measurements of radiolabeled amino acid incorporation are shown in Fig. 1A and those of total protein content in Fig. 1B. In serum-free medium (SF) without TNF-{alpha}, [3H]Phe incorporation increased over time, an increase that was accentuated in the presence of 2% HS. Compared with time-matched control myotubes in SF, the addition of TNF-{alpha} increased [3H]Phe incorporation by 27.5 ± 9.8 and 128.4 ± 24% at 24 and 48 h, respectively (P < 0.001 for both time points). In HS, PS was enhanced by TNF-{alpha} in a more pronounced manner (67.2 ± 15.7 and 185.2 ± 21% at 24 and 48 h, respectively, P < 0.001). Consistent with these PS data, total protein content was increased by TNF-{alpha} in both media (Fig. 1B).


Figure 1
View larger version (18K):
[in this window]
[in a new window]

  		Fig. 1. TNF-{alpha} increases protein synthesis (PS) and dehydrogenase activity (DHA) in differentiated C2C12 myotubes. Myotubes cultured in serum-free (SF) or 2% horse serum (HS) medium were incubated with [3H]phenylalanine (3H-Phe) in the absence or presence of TNF-{alpha} (10 ng/ml) for 12, 24, or 48 h. PS, measured in cpm/well (A) and protein content, measured in mg/well (B) are expressed as percentage of control cells in SF at 12 h. Representative example of 2 independent experiments is shown. C: myotubes in 96-well plates were incubated with TNF-{alpha} (10 ng/ml) for 12, 24, or 48 h, and DHA was assessed with the WST-1 assay. Data are expressed as percentage of control at 0 h (n ≥ 3). D: C2C12 myotubes were incubated for 24 h with increasing concentrations of TNF-{alpha} as indicated. Data represent the average of 3 independent experiments and are expressed as percentage of untreated controls. For all graphs, #P < 0.05 and ##P < 0.01 vs. SF at 12 h, *P < 0.05 and **P < 0.01 vs. time-matched SF or HS controls.

 
To test for potential effects of TNF-{alpha} on C2C12 myotube viability, we assessed cellular DHA using the WST-1 assay. Figure 1C shows that in the absence of TNF-{alpha} baseline C2C12 viability was decreased in SF at 24 and 48 h compared with controls at 0 h (P < 0.05). At 12 h after TNF-{alpha} addition, the C2C12 myotubes displayed enhanced DHA compared with time-matched SF controls (10 ± 2%, P < 0.01) and the increase reached a plateau at 24 h (27 ± 3.5%, P < 0.001). The effects of TNF-{alpha} were slightly more pronounced when applied to cultures in HS. In HS there was no decreased DHA under baseline conditions. The increases in response to TNF-{alpha} were 16 ± 3 and 31.5 ± 2.5% at 12 and 24 h, respectively (P < 0.001). The WST-1 is often used to measure proliferation when enhanced DHA is directly proportional to increased cell numbers. To test whether TNF-{alpha} affected cell numbers, we stained the DNA of our myotube preparations with crystal violet, and the results indicated that DNA content did not change in response to TNF-{alpha} (data not shown). Taken together, our results show that TNF-{alpha} increases PS as well as viability in C2C12 myotubes irrespective of the presence or absence of serum in the medium and without changing cell numbers. As viability decreased over time in SF, we used HS for all subsequent experiments.

The concentration-dependence of the effects of TNF-{alpha} on PS was assessed at 24 h (Fig. 1D). The increase in incorporation of radioactive label compared with untreated controls reached significance at 1 ng/ml of TNF-{alpha} (6.7 ± 2.6%, P < 0.05) and increased further to a plateau of 26.8 ± 3.5% at 50–100 ng/ml (P < 0.01). Half-maximal stimulation of PS occurred at 4.9 ng/ml. The following experiments were therefore performed at 10 ng/ml.

Analysis of the mechanism by which TNF-{alpha} changes PS. Increases in PS require activation of the translational machinery and in addition may consist of indirect effects after stimulation of gene transcription. To assess the role of gene transcription in the TNF-{alpha}-induced increase in PS, we measured [3H]Phe in the presence of the transcriptional inhibitor Act D. The response to TNF-{alpha} was significantly diminished from 22% in the absence of Act D to 14.2 ± 2.9% in the presence of Act D (P < 0.05; Fig. 2A). Additional experiments consisted of myotubes pretreated for 30 min with CHX, followed by incubation with TNF-{alpha} for 4 h. CHX abrogated completely the TNF-{alpha}-induced PS, confirming that the observed effects required translational mechanisms and incorporation of amino acids into peptides and excluding effects such as nonspecific internalization of the radiolabel. Taken together, our findings indicate that TNF-{alpha} enhances PS via a direct increase in protein translation and, in part, also indirectly via a transcriptional mechanism.


Figure 2
View larger version (20K):
[in this window]
[in a new window]

  		Fig. 2. Effect of specific inhibitors on TNF-{alpha}-induced PS and DHA. A: C2C12 myotubes were pretreated with vehicle (DMSO), actinomycin D (Act D, 0.5 µM), or cycloheximide (CHX, 10 ng/ml) for 30 min before addition of TNF-{alpha} (10 ng/ml, gray bars) together with [3H]Phe (5 µCi/ml) for 4 h. Data represent the average of 3 independent experiments. B: C2C12 myotubes were preincubated with vehicle (DMSO), PD-98059 (PD, 10 µM, 1 h), LY-294002 (LY, 20 µM, 30 min), or rapamycin (Rap, 20 ng/ml, 30 min), followed by addition of [3H]Phe (1 µCi/ml) and TNF-{alpha} (10 ng/ml, gray bars) for 24 h. Data represent the average of 3 independent experiments. C: C2C12 myotubes were treated with inhibitors and TNF-{alpha} for 24 h as described for (B), and DHA was assessed by WST-1. Data represent average results of 3 independent experiments. #P < 0.05 and ##P < 0.01 vs. DMSO and *P < 0.05 and **P < 0.01 vs. corresponding inhibitor control condition.

 
To further identify the direct signaling pathways by which TNF-{alpha} enhances PS, we used the inhibitors PD, LY, and Rap, which block MEK, PI3K, and mTOR activity, respectively. In time course experiments, we had already established that direct downstream molecules of these kinases, namely ERK1/2, Akt, and p70S6K, were indeed phosphorylated in response to TNF-{alpha} (data not shown). The effect of the inhibitors on [3H]Phe incorporation at 24 h is shown in Fig. 2B. In absence of TNF-{alpha}, treatment with PD, LY, and Rap decreased baseline PS to 81 ± 3.6, 37 ± 6.7, and 54 ± 3.8% of untreated controls, respectively (P < 0.01). These results indicate a strong impact of PI3K and mTOR as well as a modest impact of MEK on baseline PS. The TNF-{alpha}-induced increase in [3H]Phe incorporation was reduced by PD from 22 ± 4.6 to 11 ± 3.2% (P < 0.05) above the corresponding control conditions, indicating that MEK contributes to TNF-{alpha}-induced PS. LY treatment completely abolished the TNF-{alpha}-induced [3H]Phe incorporation, implying the requirement of PI3K activation for this response. Similar results were obtained with the alternative PI3K inhibitor wortmannin followed by 4 h of TNF-{alpha} (data not shown). Intriguingly, Rap preincubation had no effect at all on TNF-{alpha}-induced [3H]Phe incorporation. In parallel myotube cultures, WST-1 assays after PD, LY, or Rap pretreatment showed that TNF-{alpha} increased viability in C2C12 myotubes via PI3K and that the response is not sensitive to Rap (Fig. 2C), mirroring our results obtained on protein synthesis.

Analysis of phosphorylation cascades induced by TNF-{alpha}. Our next experiments were aimed at determining which signaling molecules mediate the actions of TNF-{alpha}. In preliminary time course experiments, we established that 30 min of TNF-{alpha} incubation significantly increased phosphorylation of all molecules of interest, and this time point was therefore chosen to study the effects of specific inhibitors. In all experiments, TNF-{alpha} phosphorylated Akt at its two sites Ser473 and Thr308 in an identical manner, and therefore only Ser473 will be presented here. Fig. 3A shows that PD pretreatment completely abrogated TNF-{alpha}-induced phosphorylation of ERK1/2 and of eIF4E, whereas that of Akt was unchanged. The role of eIF4E in translational regulation is well established (20). This cap-binding protein is obligatory for the start of cap-dependent translation initiation, and its activity is regulated in two ways: directly by its own phosphorylation, which is related to the rate of PS, or indirectly by its release from 4E-BP1, which allows eIF4E to form the eIF4F complex, leading to initiation of protein translation (20). The complete inhibition of TNF-{alpha}-induced eIF4E phosphorylation by PD shows that this factor is downstream of MEK (Fig. 3A). Together with the fact that PD partially blocks PS and fully blocks ERK1/2 phosphorylation, this supports that the MEK-ERK-eIF4E signaling pathway is involved in TNF-{alpha}-induced PS but also that an additional pathway is implicated.


Figure 3
View larger version (28K):
[in this window]
[in a new window]

  		Fig. 3. Effects of PD, LY, or Rap on TNF-{alpha}-induced phosphorylation. Differentiated C2C12 myotubes were deprived of serum for 3 h and pretreated with vehicle or pharmacological inhibitors as for Fig. 2B, followed by TNF-{alpha} (10 ng/ml) for 30 min. Cellular protein extracts were subjected to SDS-PAGE and immunoblots analyzed for total and phosphorylated proteins as indicated. A kinase assay with fusion protein glycogen synthase kinase-3 (fGSK-3) was performed as described in MATERIALS AND METHODS (B, middle). For each protein, a representative blot of 3 or more independent experiments is presented. Graphs represent the level of phosphorylation of the indicated molecule, expressed as percentage of untreated control. #P < 0.05, ##P < 0.01 vs. vehicle; *P < 0.05 and **P < 0.01 vs. PD, LY, or Rap.

 
In Fig. 2B it was shown that LY fully blocked TNF-{alpha}-induced PS. Figure 3B shows that LY completely abolished TNF-{alpha}-induced Akt phosphorylation. To ensure that Akt phosphorylation at Ser473 and Thr308 is correlated with enhanced Akt kinase activity and that LY indeed blocks this activity, we performed in vitro kinase assays with a GSK-3 fusion protein (fGSK-3), GSK-3 being a downstream effector of Akt (Fig. 3B). The fGSK-3 was added to protein extracts of TNF-{alpha}-treated (30 min) and control C2C12 myotubes, and the phosphorylation state of fGSK-3 detected by immunoblotting corresponded with Akt activity. Akt activity in protein extracts from TNF-{alpha}-treated cells was 189 ± 23% higher than that from untreated controls (P < 0.001), and this difference was fully abrogated by LY (Fig. 3B). In additional experiments, we showed that TNF-{alpha} induced phosphorylation of endogenous GSK-3 and that this effect was again completely abolished by LY (Fig. 3C). Finally, LY did not decrease ERK1/2 phosphorylation; in fact, ERK phosphorylation in response to TNF-{alpha} was increased by LY (Fig. 3B). Taken together, our findings strengthen the idea that in C2C12 myotubes the PI3K/Akt signaling cascade is required for the effects of TNF-{alpha} on PS and indicate that GSK-3 is one of the molecules downstream of Akt that mediates the actions of TNF-{alpha}.

An alternative mediator of protein translation is mTOR, but surprisingly, Rap did not block the effects of TNF-{alpha} on PS (Fig. 2B). To further evaluate this finding, we examined the effects of Rap on phosphorylation of the mTOR substrate p70S6K. We found that TNF-{alpha} increased p70S6K phosphorylation by 133 ± 18%, an effect that was fully inhibited by LY and Rap (Fig. 3C). As Rap did not inhibit TNF-{alpha}-induced PS, this could mean that p70S6K is not absolutely required for this biological effect or that Rap induces a feedback loop that maintains PS via a different route. Rap did not modify TNF-{alpha}-induced phosphorylation of GSK-3 (Fig. 3C) and eIF4E (data not shown).

4E-BP1 is a known mTOR target, and, as explained above, it participates in the regulation of protein translation (20). Whereas the hypophosphorylated of form of 4E-BP1 binds efficiently to eIF4E, its phosphorylation abrogates this interaction and makes more eIF4E available for eIF4F formation, which is the complex that activates translation (20). We assessed 4E-BP1 phosphorylation by examining changes in its electrophoretic mobility. 4E-BP1 has multiple phosphorylation sites, and the various phosphorylated forms of the protein resolve as three bands in 15% SDS-PAGE, named {alpha}, β, and {gamma}, from the least to the most phosphorylated form (Fig. 3C). After TNF-{alpha} stimulation, increased density of the highest molecular weight band {gamma} was observed. LY abolished the increased intensity of the {gamma} band and shifted it to the β and {alpha} forms. Rap increased baseline phosphorylation of the {alpha} and β forms of 4E-BP1, but TNF-{alpha} induced a significant further increase compared to Rap alone. Thus, 4E-BP1 does not seem to be downstream of TNF-induced mTOR activation. Under baseline conditions, Rap decreased PS, suggesting that in this case 4E-BP1 and eIF4E phosphorylation are compensatory mechanisms in an attempt to stimulate translation but by themselves are not sufficient to increase PS. In conclusion, our data suggest that TNF-{alpha} increases PS in C2C12 myotubes via three signaling cascades: MEK-ERK-eIF4E, PI3K-Akt-GSK-3, and PI3K-Akt-4E-BP1.

TNF-R1 mediates TNF-{alpha}-induced PS in C2C12 myotubes. To assess which TNF receptor mediates the effects on PS, we analyzed gene expression of TNF-R1 and TNF-R2 in C2C12 myotubes by real-time PCR at 6 days of differentiation, as well as at 24 and 48 h afterward. Both TNF-R1 and TNF-R2 mRNAs were abundantly present in C2C12 myotubes, and Fig. 4A shows that baseline levels of TNF-R1 slightly decreased during the first 24 h of incubation. This was at 48 h followed by an increase in the expression of TNF-R1 and -R2 by 40 ± 6 and 76 ± 3% over controls at 0 h, respectively (P < 0.01). Western blotting confirmed that TNF-R1 protein was higher at 48 h than at 0 h and that TNF-{alpha} or LY did not change TNF-R1 levels at any of the time points analyzed (Fig. 4B).


Figure 4
View larger version (15K):
[in this window]
[in a new window]

  		Fig. 4. Analysis of the role of TNF-{alpha} receptor 1 (TNF-R1) and TNF-R2 in TNF-induced PS in C2C12 myotubes A: changes in TNF-R1 and TNF-R2 mRNA expression over time. Starting at 6 days of differentiation (t = 0), total RNA was isolated from myotubes at 0, 4, 24, and 48 h, and TNF-R1 or TNF-R2 mRNA levels were measured by real-time PCR (n = 3). B: Effect of TNF-{alpha} and LY on TNF-R1 protein levels. Differentiated C2C12 were pretreated with vehicle or LY (20 µM) followed by TNF-{alpha} (10 ng/ml). At 0, 24, and 48 h, myotubes were lysed, and protein extracts were subjected to SDS-PAGE and immunoblotting for subsequent analysis with anti-TNF-R1 and actin antibodies. Representative example of 2 experiments is shown. C: effect of receptor blockade on TNF-{alpha}-induced Akt activation. Differentiated myotubes were incubated for 1 h with antibodies against TNF-R1 or TNF-R2 at indicated concentrations prior to incubation with TNF-{alpha} (30 min, 10 ng/ml). Immunoblots of protein extracts were incubated with antibodies against phosphorylated and total Akt. Representative example is shown (n = 2). D: effect of TNF-R1 blockade on TNF-{alpha}-induced PS. Differentiated myotubes were incubated for 1 h with anti-TNF-R1 prior to addition of [3H]Phe and TNF-{alpha} (10 ng/ml, gray bars) for 24 h; n = 3, ##P < 0.01.

 
To determine which TNF-{alpha} receptor is involved in TNF-{alpha}-induced Akt phosphorylation, differentiated cells were incubated for 1 h with antibodies against TNF-R1 or TNF-R2 prior to the addition of TNF-{alpha}. Fig. 4C shows that the anti-TNF-R1 antibody abrogated TNF-{alpha}-induced Akt phosphorylation in a concentration-dependent manner, whereas the anti-TNF-R2 antibody had no effect. In an additional experiment, we confirmed that TNF-{alpha}-induced [3H]Phe incorporation was also blocked after preincubation with the anti-TNF-R1 antibody (Fig. 4D). These data support that TNF-R1 mediates TNF-{alpha}-induced Akt activation and PS in C2C12 myotubes.

Effects of TNF-{alpha} on PS in primary rat myotubes. To investigate whether similar responses occur in primary muscle cells, we isolated myoblasts from neonatal rat limb muscle. After 5 days of differentiation, we stimulated these cells with TNF-{alpha} for different time periods and measured PS and total protein content as for the C2C12 cells. The time course experiment showed that TNF-{alpha} increased PS at 24 h irrespectively of the absence or presence of HS in the medium (Fig. 5A). The increases over control were 77 ± 15.5 and 79.3 ± 10.3% in SF and HS, respectively (P < 0.001 for both). At 48 h, the cumulatively incorporated [3H]Phe in the presence of TNF-{alpha} was diminished but still significantly higher than in controls (26.2 ± 6.4 and 33.5 ± 9.1% for SF and HS, respectively, P < 0.001). Measurements of total protein content (Fig. 5B) and viability (Fig. 5C) show that the responses parallel the changes in PS in the primary rat myotubes.


Figure 5
View larger version (19K):
[in this window]
[in a new window]

  		Fig. 5. TNF-{alpha} increases PS and DHA in primary myotubes. After 5 days of differentiation, rat primary myotubes in SF or 2% HS were incubated with [3H]Phe in the absence or presence of TNF-{alpha} (0.5 ng/ml) for 12, 24, or 48 h. PS (A) and protein content (B) were measured as for Fig. 1. One of two independent experiments, each performed in triplicate, is shown. #P < 0.05 and ##P < 0.01 vs. SF at 12 h; *P < 0.05 and **P < 0.01 vs. time-matched SF or HS. C: primary myotubes in 96-well plates were incubated for 24 h with TNF-{alpha} (0.5 ng/ml), and DHA was assessed as for Fig. 1. One of two independent experiments, performed in sextuplet, is shown. D: primary myotubes were incubated for 24 h with increasing concentrations TNF-{alpha}. Representative experiment with triplicate measurements is shown (n = 2). *P < 0.05 and **P < 0.01.

 
As a function of its concentration, the effects of TNF-{alpha} on PS were bimodal in primary myotubes (Fig. 5D). At 0.1 ng/ml TNF-{alpha}, the incorporation of radioactive label was enhanced by 16.2 ± 2.4% compared with untreated cells (P < 0.01), an increase that became maximal at 1 ng/ml (28.4 ± 4.8% P < 0.01). At higher TNF-{alpha} concentrations, the incorporated [3H]Phe started to decrease and was not above untreated controls anymore at 50 ng/ml. The WST-1 assay revealed that at 100 ng/ml the cytokine is toxic for the primary cells (data not shown). Taken together, our data indicate that the primary myotubes are more sensitive to TNF-{alpha} than C2C12 myotubes.

Analysis of the role of TNF-R1 on the TNF-{alpha}-induced effects in primary myotubes. Like in C2C12 myotubes, we confirmed that in the primary myotubes TNF-{alpha} caused the increase in PS via Akt activation, because PI3K inhibition by LY completely abolished the TNF-{alpha}-stimulated Akt phosphorylation (Fig. 6A) and [3H]Phe incorporation (Fig. 6B). Preincubation with anti-TNF-R1 antibodies prior to TNF-{alpha} addition completely blocked the Akt activation, whereas the anti-TNF-R2 antibody had no effect (Fig. 7A). Consistently, the anti-TNF-R1 antibody fully abrogated the TNF-{alpha}-induced-PS in the C2C12 myotubes (Fig. 7B). To test whether the difference in responsiveness of two cell types was due to variable TNF-receptor expression, receptor levels were assessed semiquantitatively after Western blotting. Figure 7D shows that, at baseline, TNF-R1 is expressed in primary myotubes at a level comparable to that in C2C12 myotubes. However, expression is transiently increased at 24 h in the primary myotubes. At this time point, receptor protein levels are comparable to those in mouse gastrocnemius muscle (Fig. 7D).


Figure 6
View larger version (19K):
[in this window]
[in a new window]

  		Fig. 6. Effect of LY on TNF-{alpha}-induced Akt phosphorylation and PS in primary mytubes. A: At 5 days of differentiation, cells were depleted of serum for 3 h and incubated with LY (20 µM) for 30 min prior to treatment with TNF-{alpha} (0.5 ng/ml) for 30 min. Protein lysates were analyzed as for Fig. 4C. Representative blot is shown (n = 2). B: primary myotubes were incubated with vehicle or LY (20 µM) for 30 min prior to treatment with TNF-{alpha} (gray bars, 0.5 ng/ml) for 24 h. PS was measured as for Fig. 1A. ##P < 0.01 vs. DMSO.

 

Figure 7
View larger version (18K):
[in this window]
[in a new window]

  		Fig. 7. Analysis of the role of TNF-R1 and TNF-R2 in TNF-induced PS in primary myotubes. A: effects of receptor blockade on TNF-{alpha}-induced Akt phosphorylation. Differentiated primary myotubes were treated like C2C12 in Fig. 4C but with TNF-{alpha} at 0.5 ng/ml. Blot shows representative example of 2 experiments. B: effects of TNF-R1 blockade on TNF-{alpha}-induced PS. Primary myotubes were treated like C2C12 in Fig. 4D but with TNF-{alpha} at 0.5 ng/ml (gray bars, n = 3). ##P < 0.01. C: time course of TNF-R1 expression in rat primary myotubes. Myotubes were lysed at 5 days of differentiation (t = 0) and at 24 or 48 h afterward and prepared as for Fig. 4B. A similarly prepared protein extract from gastrocnemius muscle of an adult Wistar rat was loaded for comparison. Immunoblots were analyzed with TNF-R1 and actin antibodies.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The present study was designed to analyze the effects of TNF-{alpha} on PS in myotubes and to characterize the cellular mechanisms involved. With our experiments we established that TNF-{alpha} increases protein content in cultured C2C12 myotubes by enhancing protein translation via PI3K-Akt-GSK-3, PI3K-Akt-4E-BP1, and MAP-ERK-eIF4E signaling and that TNF-R1 transduces these effects. To the best of our knowledge, our study is the first to provide extensive mechanistic data on these protective pathways activated by TNF-{alpha} in differentiated myotubes.

Our conclusion that TNF-{alpha} increases protein content and de facto is able to specifically induce a hypertrophic response is valid for C2C12 as well as for primary rat myotubes and is supported by multiple experiments performed with both models. First, TNF-{alpha} significantly increased PS measured as [3H]Phe incorporation; second, it did not change protein breakdown, measured as the release of preincorporated [3H]Phe (data not shown), and, consistent with these first two findings, TNF-{alpha} increased total protein content without changing cell numbers. The presence of HS in the medium did not explain the enhanced PS, as similar effects were obtained in SF medium. Measurement of DHA confirmed that TNF-{alpha} indeed acts in a beneficial manner in both myotube models, since viability was enhanced at 24 h after addition of the cytokine to a similar extent as PS and protein content. In fact, in C2C12, the magnitude of the increase in DHA obtained with TNF-{alpha} was as important as that induced by IGF-I at 20 ng/ml (data not shown). Increased protein translation is normally associated with enhanced phosphorylation of eIF4E, 4E-BP1, and p70S6K (14, 20). Indeed, increased phosphorylation of all three factors confirmed that protein translation was initiated by TNF-{alpha}. Finally, our experiments with the translational inhibitor CHX provided further support for a translational mechanism.

Our finding that TNF-{alpha} can increase protein content in myotube models is consistent with earlier reports on L6 and C2C12 myotubes (1, 10), and we have now provided the specific mechanism behind this effect. Several important differences between the C2C12 myotubes and the rat primary myotubes should, however, be pointed out here, because they may help us to understand some of the apparently controversial results reported to date. In the primary myotubes, the maximal increase in PS was already attained at 1 ng/ml TNF-{alpha} and then started to decline, whereas in the C2C12 myotubes the maximal PS response was obtained only at ~50 ng/ml and maintained afterward. When analyzed over time, it was apparent that in primary myotubes the increase in PS and protein content happened mainly during the first 24 h of TNF-{alpha} incubation and thereafter did not increase any further. Instead, a slight decrease in [3H]Phe at later time points indicated loss of proteins from the primary myotubes, and this was confirmed by trends toward decreased protein content and viability. In contrast, in the C2C12 myotubes, PS continued to increase up to the latest time point measured. Our data on the expression of the TNF-{alpha} receptors in the two models may provide a mechanism for the observed phenomena. We showed that TNF-R1 mediates the TNF-{alpha}-induced PS in both types of myotubes but that receptor levels were differentially regulated over time between the two models. At the start of TNF-{alpha} incubation, TNF-R1 levels were similar, but during the following 24 h the primary myotubes significantly increased their TNF-R1 levels. The C2C12 myotubes increased receptor expression to a much lesser extent and only after 48 h of incubation. Their higher receptor levels may explain the higher sensitivity of the primary myotubes, reflected in enhanced PS at lower concentrations of TNF-{alpha}. It may, on the other hand, also explain the transient character of the increase over time. The blunted increase in PS at later time points could be due to induction of feedback loops, such as secretion of soluble TNF-R, which sequesters the cytokine, or a decrease in autocrine IGF-I production (11, 12), which reduces the PS response.

In C2C12 myotubes, indirect transcriptional effects contributed to the TNF-induced PS in a positive manner, as was shown after Act D treatment. This effect might be related to NF-{kappa}B, which was rapidly activated by TNF-{alpha} in our C2C12 cells (data not shown). Previous in vitro studies have demonstrated that TNF-{alpha} can dramatically increase expression of cytokines in muscle cells, in particular IL-6 and IFN-{gamma} (2, 3). It is of note that differentiating cultures produce multiple autocrine factors that contribute to the final response of the cells, including TNF-{alpha} itself (6). Thus, positive and/or negative transcriptional relay effects occur that may differ depending on the myotube model studied, the culture methods used, and the differentiation state of the cells.

By means of specific inhibitors, we further analyzed the direct transcription-independent pathways that transduce the signal from TNF-R1 toward enhanced protein translation, and the major conclusions drawn from our results are summarized in Fig. 8. First, PD abolished TNF-{alpha}-stimulated phosphorylation of ERK1/2 and eIF4E, whereas it only partly inhibited the increase in PS, supporting that ERK1/2 and eIF4E contribute to the PS response. The mechanism by which ERK1/2 regulates PS likely involves the phosphorylation of eIF4E through MAPK signal-integrating kinase-1 (Mnk1) (33, 35). Early reports have already described that TNF-{alpha} can activate PS via MAPK in a variety of other, mainly proliferative cells (28). Consistent with our own findings, studies with L6 and C2C12 myotubes have reported that TNF-{alpha} activates ERK1/2 (10), that PD blocks an increase in protein content (1), and that TNF-{alpha} transiently increases eIF4E phosphorylation (36); however, causal relationships between the different molecules have not been established. We have now shown that eIF4E phosphorylation is downstream of MEK-ERK and that this pathway enhances protein synthesis in differentiated C2C12 myotubes.


Figure 8
View larger version (10K):
[in this window]
[in a new window]

  		Fig. 8. Diagram showing signaling pathways involved in TNF-{alpha}-induced PS in C2C12 myotubes. Arrows represent phosphorylation events stimulated by TNF-{alpha}. Inhibitors are underlined. Two initiation factors (shown in boxes) are implicated in the activation of protein translation by TNF-{alpha}: eukaryotic initiation factor (eIF)2B after activation of the PI3K-Akt-GSK-3 signaling cascade and eIF4E after stimulation of the MEK-ERK-Mnk1 and PI3K-Akt-4E-BP1 pathways. WM, wortmannin; mTOR, mammalian target of rapamycin; TORC1/2, mTOR C1 and C2.

 
The second major pathway that we found to be necessary for TNF-{alpha}-induced PS in myotubes is the PI3K-Akt cascade. This is the first report on activation of this pathway by TNF-{alpha} in myotubes, and our conclusion is supported by multiple experiments in which Akt activation and [3H]Phe incorporation were fully abrogated by the PI3K inhibitors LY and wortmannin. Similar observations have been made in neonatal cardiac myocytes (18) and fibroblasts (19). It is well established that Akt signaling is involved in the differentiation of muscle cells (37). A recent study shows that TNF-{alpha} enhances differentiation of myoblasts, but involvement of Akt in this response was not demonstrated (6). Consistent with this study, TNF-{alpha} did not phosphorylate Akt when we used C2C12 myoblasts at the first day of differentiation for our experiments (data not shown). After 6 days of differentiation, however, we show that Akt activation contributes to PS, suggesting that this pathway causes myotube growth by enhancing synthesis of contractile proteins.

GSK-3 was activated in the TNF-{alpha}-treated myotubes, this as a response downstream of Akt activation because LY blocked GSK-3 phosphorylation, and as a response parallel to ERK activation, because phosphorylation of GSK-3 was not inhibited by PD. GSK-3 is known to regulate eIF2B, which in turn activates protein translation initiation (8), and this pathway therefore likely contributes to PS in our model (Fig. 8). Independently of GSK-3, mTOR is generally associated with PS and growth as part of the PI3K-Akt pathway (38). In our experiments, Rap completely inhibited the TNF-{alpha}-induced increase in phosphorylation of p70S6K, consistent with published data that mTOR is upstream of this kinase (38). Surprisingly, Rap did not diminish TNF-{alpha}-induced PS and 4E-BP1 phosphorylation. These findings indicate that this pathway is not required for the PS caused by TNF-{alpha} and that a Rap-insensitive protein kinase causes 4E-BP1 phosphorylation. Our data are in favor of an involvement of mTOR as part of mTOR C2, in which it is not sensitive to Rap (38). Whether mTOR C2 or a different kinase activates 4E-BP1 remains to be established.

In summary, our study is the first to show in skeletal myotubes that TNF-{alpha} specifically activates pathways that are important for protein synthesis and viability, and we have provided insights into the mechanisms implicated in these effects, including identification of the TNF-R1 as the major mediator. In general, in vivo rat studies have shown catabolic responses to TNF-{alpha}. These effects may be related to the high concentration of the applied cytokine and/or, as suggested previously (15), to the systemic release of additional factors from for example macrophages, which either by themselves or together with TNF-{alpha} adversely affect protein metabolism in muscle. Similarly, muscle wasting that occurs during pathologies such as cancer or heart failure, generally associated with elevated TNF-{alpha} levels, is due to the final balance between levels of multiple cytokines and growth factors together with expression of the respective receptors. Under "physiological" conditions, for example when an appropriate balance of growth factors such as IGF-I is present, TNF-{alpha} is not necessarily detrimental, as exemplified by our study. The new knowledge provided by the present study on the growth-promoting pathways activated by TNF-{alpha} in myotubes has consequences for patients with, for example, rheumatoid arthritis, in which TNF-{alpha} inhibition strategies are used. Careful monitoring of therapeutic consequences at the level of muscle in these patients would be appropriate.


   FOOTNOTES
 

Address for reprint requests and other correspondence: M. Brink, CardioBiology Laboratories, DKBW Dept. of Research, Univ. of Basel, Hebelstrasse 20, CH-4031 Basel, Switzerland (e-mail: marijke.brink{at}unibas.ch)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Alvarez B, Quinn LS, Busquets S, Lopez-Soriano FJ, Argiles JM. Direct effects of tumor necrosis factor alpha (TNF-alpha) on murine skeletal muscle cell lines. Bimodal effects on protein metabolism. Eur Cytokine Netw 12: 399–410, 2001.[Web of Science][Medline]
  2. Alvarez B, Quinn LS, Busquets S, Lopez-Soriano FJ, Argiles JM. TNF-alpha modulates cytokine and cytokine receptors in C2C12 myotubes. Cancer Lett 175: 181–185, 2002.[CrossRef][Web of Science][Medline]
  3. Alvarez B, Quinn LS, Busquets S, Quiles MT, Lopez-Soriano FJ, Argiles JM. Tumor necrosis factor-alpha exerts interleukin-6-dependent and -independent effects on cultured skeletal muscle cells. Biochim Biophys Acta 1542: 66–72, 2002.[Medline]
  4. Argiles JM, Busquets S, Lopez-Soriano FJ. The pivotal role of cytokines in muscle wasting during cancer. Int J Biochem Cell Biol 37: 2036–2046, 2005.[CrossRef][Web of Science][Medline]
  5. Brink M, Anwar A, Delafontaine P. Neurohormonal factors in the development of catabolic/anabolic imbalance and cachexia. Int J Cardiol 85: 111, 2002.[CrossRef][Web of Science][Medline]
  6. Chen SE, Jin B, Li YP. TNF-{alpha} regulates myogenesis and muscle regeneration by activating p38 MAPK. Am J Physiol Cell Physiol 292: C1660–C1671, 2007.[Abstract/Free Full Text]
  7. Coletti D, Yang E, Marazzi G, Sassoon D. TNFalpha inhibits skeletal myogenesis through a PW1-dependent pathway by recruitment of caspase pathways. EMBO J 21: 631–642, 2002.[CrossRef][Web of Science][Medline]
  8. Cross DA, Alessi DR, Cohen P, Andjelkovich M, Hemmings BA. Inhibition of glycogen synthase kinase-3 by insulin mediated by protein kinase B. Nature 378: 785–789, 1995.[CrossRef][Medline]
  9. De Bleecker JL, Meire VI, Declercq W, Van Aken EH. Immunolocalization of tumor necrosis factor-alpha and its receptors in inflammatory myopathies. Neuromuscul Disord 9: 239–246, 1999.[CrossRef][Web of Science][Medline]
  10. el-Naggar EA, Kanda F, Okuda S, Maeda N, Nishimoto K, Ishihara H, Chihara K. Direct effects of tumor necrosis factor alpha (TNF-alpha) on L6 myotubes. Kobe J Med Sci 50: 39–46, 2004.[Medline]
  11. Fernandez-Celemin L, Pasko N, Blomart V, Thissen JP. Inhibition of muscle insulin-like growth factor I expression by tumor necrosis factor-{alpha}. Am J Physiol Endocrinol Metab 283: E1279–E1290, 2002.[Abstract/Free Full Text]
  12. Frost RA, Nystrom GJ, Lang CH. Tumor necrosis factor-alpha decreases insulin-like growth factor-I messenger ribonucleic acid expression in C2C12 myoblasts via a Jun N-terminal kinase pathway. Endocrinology 144: 1770–1779, 2003.[Abstract/Free Full Text]
  13. Garcia-Martinez C, Lopez-Soriano FJ, Argiles JM. Acute treatment with tumour necrosis factor-alpha induces changes in protein metabolism in rat skeletal muscle. Mol Cell Biochem 125: 11–18, 1993.[CrossRef][Web of Science][Medline]
  14. Gingras AC, Raught B, Sonenberg N. eIF4 initiation factors: effectors of mRNA recruitment to ribosomes and regulators of translation. Annu Rev Biochem 68: 913–963, 1999.[CrossRef][Web of Science][Medline]
  15. Goodman MN. Tumor necrosis factor induces skeletal muscle protein breakdown in rats. Am J Physiol Endocrinol Metab 260: E727–E730, 1991.[Abstract/Free Full Text]
  16. Guttridge DC, Mayo MW, Madrid LV, Wang CY, Baldwin AS Jr. NF-kappaB-induced loss of MyoD messenger RNA: possible role in muscle decay and cachexia. Science 289: 2363–2366, 2000.[Abstract/Free Full Text]
  17. Higuchi Y, McTiernan CF, Frye CB, McGowan BS, Chan TO, Feldman AM. Tumor necrosis factor receptors 1 and 2 differentially regulate survival, cardiac dysfunction, and remodeling in transgenic mice with tumor necrosis factor-alpha-induced cardiomyopathy. Circulation 109: 1892–1897, 2004.[Abstract/Free Full Text]
  18. Hiraoka E, Kawashima S, Takahashi T, Rikitake Y, Kitamura T, Ogawa W, Yokoyama M. TNF-{alpha} induces protein synthesis through PI3-kinase-Akt/PKB pathway in cardiac myocytes. Am J Physiol Heart Circ Physiol 280: H1861–H1868, 2001.[Abstract/Free Full Text]
  19. Kim BC, Lee MN, Kim JY, Lee SS, Chang JD, Kim SS, Lee SY, Kim JH. Roles of phosphatidylinositol 3-kinase and Rac in the nuclear signaling by tumor necrosis factor-alpha in rat-2 fibroblasts. J Biol Chem 274: 24372–24377, 1999.[Abstract/Free Full Text]
  20. Kleijn M, Scheper GC, Voorma HO, Thomas AA. Regulation of translation initiation factors by signal transduction. Eur J Biochem 253: 531–544, 1998.[Web of Science][Medline]
  21. Kuru S, Inukai A, Kato T, Liang Y, Kimura S, Sobue G. Expression of tumor necrosis factor-alpha in regenerating muscle fibers in inflammatory and non-inflammatory myopathies. Acta Neuropathol (Berl) 105: 217–224, 2003.[Medline]
  22. Ladner KJ, Caligiuri MA, Guttridge DC. Tumor necrosis factor-regulated biphasic activation of NF-kappa B is required for cytokine-induced loss of skeletal muscle gene products. J Biol Chem 278: 2294–2303, 2003.[Abstract/Free Full Text]
  23. Lang CH, Frost RA, Nairn AC, MacLean DA, Vary TC. TNF-{alpha} impairs heart and skeletal muscle protein synthesis by altering translation initiation. Am J Physiol Endocrinol Metab 282: E336–E347, 2002.[Abstract/Free Full Text]
  24. Langen RC, Schols AM, Kelders MC, Wouters EF, Janssen-Heininger YM. Inflammatory cytokines inhibit myogenic differentiation through activation of nuclear factor-kappaB. FASEB J 15: 1169–1180, 2001.[Abstract/Free Full Text]
  25. Layne MD, Farmer SR. Tumor necrosis factor-alpha and basic fibroblast growth factor differentially inhibit the insulin-like growth factor-I induced expression of myogenin in C2C12 myoblasts. Exp Cell Res 249: 177–187, 1999.[CrossRef][Web of Science][Medline]
  26. Li YP, Reid MB. NF-{kappa}B mediates the protein loss induced by TNF-{alpha} in differentiated skeletal muscle myotubes. Am J Physiol Regul Integr Comp Physiol 279: R1165–R1170, 2000.[Abstract/Free Full Text]
  27. Llovera M, Lopez-Soriano FJ, Argiles JM. Effects of tumor necrosis factor-alpha on muscle-protein turnover in female Wistar rats. J Natl Cancer Inst 85: 1334–1339, 1993.[Abstract/Free Full Text]
  28. Marino MW, Feld LJ, Jaffe EA, Pfeffer LM, Han HM, Donner DB. Phosphorylation of the proto-oncogene product eukaryotic initiation factor 4E is a common cellular response to tumor necrosis factor. J Biol Chem 266: 2685–2688, 1991.[Abstract/Free Full Text]
  29. Mitch WE, Goldberg AL. Mechanisms of muscle wasting. The role of the ubiquitin-proteasome pathway. N Engl J Med 335: 1897–1905, 1996.[Free Full Text]
  30. Monden Y, Kubota T, Inoue T, Tsutsumi T, Kawano S, Ide T, Tsutsui H, Sunagawa K. Tumor necrosis factor-{alpha} is toxic via receptor 1 and protective via receptor 2 in a murine model of myocardial infarction. Am J Physiol Heart Circ Physiol 293: H743–H753, 2007.[Abstract/Free Full Text]
  31. Spencer MJ, Marino MW, Winckler WM. Altered pathological progression of diaphragm and quadriceps muscle in TNF-deficient, dystrophin-deficient mice. Neuromuscul Disord 10: 612–619, 2000.[CrossRef][Web of Science][Medline]
  32. Stewart CE, Newcomb PV, Holly JM. Multifaceted roles of TNF-alpha in myoblast destruction: a multitude of signal transduction pathways. J Cell Physiol 198: 237–247, 2004.[CrossRef][Web of Science][Medline]
  33. Ueda T, Watanabe-Fukunaga R, Fukuyama H, Nagata S, Fukunaga R. Mnk2 and Mnk1 are essential for constitutive and inducible phosphorylation of eukaryotic initiation factor 4E but not for cell growth or development. Mol Cell Biol 24: 6539–6549, 2004.[Abstract/Free Full Text]
  34. Warren GL, Hulderman T, Jensen N, McKinstry M, Mishra M, Luster MI, Simeonova PP. Physiological role of tumor necrosis factor alpha in traumatic muscle injury. FASEB J 16: 1630–1632, 2002.[Abstract/Free Full Text]
  35. Waskiewicz AJ, Flynn A, Proud CG, Cooper JA. Mitogen-activated protein kinases activate the serine/threonine kinases Mnk1 and Mnk2. EMBO J 16: 1909–1920, 1997.[CrossRef][Web of Science][Medline]
  36. Williamson DL, Kimball SR, Jefferson LS. Acute treatment with TNF-{alpha} attenuates insulin-stimulated protein synthesis in cultures of C2C12 myotubes through a MEK1-sensitive mechanism. Am J Physiol Endocrinol Metab 289: E95–E104, 2005.[Abstract/Free Full Text]
  37. Wilson EM, Tureckova J, Rotwein P. Permissive roles of phosphatidyl inositol 3-kinase and Akt in skeletal myocyte maturation. Mol Biol Cell 15: 497–505, 2004.[Abstract/Free Full Text]
  38. Wullschleger S, Loewith R, Hall MN. TOR signaling in growth and metabolism. Cell 124: 471–484, 2006.[CrossRef][Web of Science][Medline]
  39. Zador E, Mendler L, Takacs V, de Bleecker J, Wuytack F. Regenerating soleus and extensor digitorum longus muscles of the rat show elevated levels of TNF-alpha and its receptors, TNFR-60 and TNFR-80. Muscle Nerve 24: 1058–1067, 2001.[CrossRef][Web of Science][Medline]




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
294/2/E241    most recent
00129.2007v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Plaisance, I.
Right arrow Articles by Brink, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Plaisance, I.
Right arrow Articles by Brink, M.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2008 by the American Physiological Society.