Tumor necrosis factor-alpha inhibits myogenesis through redox-dependent and -independent pathways

Ramon C. J. Langen1, Annemie M. W. J. Schols1, Marco C. J. M. Kelders1, Jos L. J. van der Velden1, Emiel F. M. Wouters1, and Yvonne M. W. Janssen-Heininger2

1 Department of Pulmonology, Maastricht University, 6202 AZ Maastricht, The Netherlands; and 2 Department of Pathology, University of Vermont, Burlington, Vermont 05405


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
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DISCUSSION
REFERENCES

Muscle wasting accompanies diseases that are associated with chronic elevated levels of circulating inflammatory cytokines and oxidative stress. We previously demonstrated that tumor necrosis factor-alpha (TNF-alpha ) inhibits myogenic differentiation via the activation of nuclear factor-kappa B (NF-kappa B). The goal of the present study was to determine whether this process depends on the induction of oxidative stress. We demonstrate here that TNF-alpha causes a decrease in reduced glutathione (GSH) during myogenic differentiation of C2C12 cells, which coincides with an elevated generation of reactive oxygen species. Supplementation of cellular GSH with N-acetyl-l-cysteine (NAC) did not reverse the inhibitory effects of TNF-alpha on troponin I promoter activation and only partially restored creatine kinase activity in TNF-alpha -treated cells. In contrast, the administration of NAC before treatment with TNF-alpha almost completely restored the formation of multinucleated myotubes. NAC decreased TNF-alpha -induced activation of NF-kappa B only marginally, indicating that the redox-sensitive component of the inhibition of myogenic differentiation by TNF-alpha occurred independently, or downstream of NF-kappa B. Our observations suggest that the inhibitory effects of TNF-alpha on myogenesis can be uncoupled in a redox-sensitive component affecting myotube formation and a redox independent component affecting myogenic protein expression.

inflammation; glutathione; nuclear factor-kappa B; myotube; myogenic differentiation


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

MUSCLE WASTING (cachexia), a syndrome characterized by the disproportional loss of skeletal muscle, is a frequent complication in many chronic diseases, including chronic obstructive pulmonary disease (28, 34, 39), cystic fibrosis (10), chronic heart failure (1), renal failure (27), acquired immune deficiency syndrome (20), and cancer (2). There is increasing interest in improving the treatment of cachexia, since it is an independent predictor of morbidity and mortality in many of these disease states. Targeted treatment strategies, however, require insight into the etiology of muscle wasting. Imbalances in protein anabolism/catabolism and muscle injury and repair have been hypothesized to be involved. Inflammatory cytokines appear to be critical mediators of cachexia, based on observations that muscle-wasting syndromes are often accompanied by elevated levels of tumor necrosis factor-alpha (TNF-alpha ) and interleukin-1 (IL-1; see Refs. 3, 9, 29, 41). Furthermore, administration of these cytokines is sufficient to induce muscle wasting in rodents (5, 11). Previously, we demonstrated that TNF-alpha or IL-1 inhibit myogenic differentiation in a mouse line of myoblasts (C2C12 cells), which suggested that inhibition of muscle regeneration may contribute to cachexia (21). The cytokine-induced inhibition of myogenic differentiation was causally linked to the activation of the transcription factor, nuclear factor-kappa B (NF-kappa B).

NF-kappa B is considered the master regulator of inflammatory responses. It transcriptionally activates many genes important in inflammation, immune function, anti-apoptotic responses, proliferation, and matrix turnover (30). NF-kappa B resides in a latent form in the cytoplasm, where it is bound to the inhibitory protein of NF-kappa B, Ikappa B. Upon stimulation, a rapid and transient activation of Ikappa B kinases (IKK) occurs, which phosphorylate Ikappa B, resulting in subsequent ubiquitination and rapid degradation via the 26S proteasome pathway. The liberation from Ikappa B unmasks the nuclear localization sequence of NF-kappa B, permitting it to translocate to the nucleus, causing it to bind its recognition sequence and activate transcription (13).

Oxidative stress has also been implicated in the activation of NF-kappa B, based upon many studies demonstrating that antioxidants or metal scavengers prevent cytokine-induced activation of NF-kappa B and that the addition of oxidants to certain cell types, including skeletal muscle, is sufficient to induce its activation (25, 35). In addition, exposure of cultured skeletal myocytes to inflammatory cytokines resulted in the generation of reactive oxygen species (ROS) and reactive nitrogen species (24, 38). Importantly, oxidative stress also may play a role in muscle weakness and wasting, since it has been reported to affect skeletal muscle in various ways. For instance, ROS may contribute to fatigue and decreased force generation through modification of sarcoplasmic reticulum function, alterations in mitochondrial respiration, and even direct oxidation of critical sulfhydryl groups on contractile proteins, which may in turn result in the enhanced degradation of these myofibrillar proteins (32, 36). Moreover, nitric oxide appears to contribute to muscle wasting in an experimental model using the implantation of TNF-alpha -producing Chinese hamster ovary cells that resulted in upregulation of inducible nitric oxide synthase (iNOS). Consequently, chemical inhibitors of iNOS reversed the loss of muscle-specific gene expression observed in cachectic mice (5).

The goal of the present study was to determine whether oxidative stress plays a role in the inhibition of myogenic differentiation after treatment with TNF-alpha and whether this occurs via the activation of NF-kappa B. We explored these questions by using the glutathione (GSH) precursor and antioxidant N-acetyl-L-cysteine (NAC), which is known to reduce the activation of NF-kappa B in response to TNF-alpha (35). Our results demonstrate that morphological aspects of myogenic differentiation, i.e., the formation of myotubes, could be restored by NAC after exposure to TNF-alpha and that this occurred independently of NF-kappa B.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Cell culture. The murine skeletal muscle cell line C2C12 was obtained from the American Type Culture Collection (no. CRL1772; ATCC, Manassas, VA). These cells are capable of differentiating into contracting myotubes upon withdrawal of growth factors after cell cycle exit (40). Myoblasts were cultured in growth medium (GM) consisting of low-glucose DMEM containing antibiotics (50 U/ml penicillin and 50 µg/ml streptomycin) and 9% (vol/vol) FBS (all from Life Technologies, Rockville, MD). For experiments, 60-mm dishes (Becton-Dickinson Labware, Bedford, MA) were coated with Matrigel (Becton-Dickinson Labware), which contains collagen IV, laminin, heparin sulfate proteoglycan, and entactin. Plating on Matrigel resulted in enhanced and more consistent differentiation, compared with frequently used protocols that employ 2% horse serum-containing media (unpublished observation). Cells were plated at 104/cm2 and were cultured in GM for 24 h, allowing them to reach ~60% confluency. To induce differentiation, cells were washed in Hank's balanced salt solution and cultured in differentiation media (DM) containing DMEM low glucose, antibiotics, and 0.5% heat-inactivated FBS. To study the effects on differentiation, one single dose of murine TNF-alpha (Calbiochem, La Jolla, CA) was added to the culture dishes directly after induction of differentiation. To study the effects of oxidative stress per se, cells were treated with 20-200 µM H2O2 (Sigma), which was added as a bolus directly after induction of differentiation. Cells were examined with an inverted phase-contrast microscope (model CH40; Olympus, Melville, NY) equipped with a camera (Nikon N6000).

Treatment with NAC and determination of reduced GSH and oxygen radical generation. For the supplementation of cellular GSH levels, cells were cultured in GM with various concentrations of NAC 16-24 h before induction of differentiation. For determination of cellular GSH, cells were collected by trypsinization, centrifuged for 10 min at 500 g at 4°C, and washed two times in ice-cold PBS. Next, pelleted cells were resuspended in PBS, and an aliquot was taken for determination of total protein. The remaining fraction was centrifuged, and the pelleted cells were lysed in 25% TCA-phosphate buffer. The cell lysate was further diluted in 3 vol of phosphate buffer and centrifuged at 16,000 g at 4°C for 2 min. A 1:20 volume of o-phtalaldehyde (OPT, 1 mg/ml in methanol; Sigma, St. Louis, MO) was added to a suitable volume of supernatant, and total GSH was measured as a fluorescent product of the reaction with OPT, as described by Cohn and Lyle (8). To normalize for potential differences in cell numbers, GSH values were corrected for total protein. ROS production was assessed by the use of the oxidant-sensitive fluorescent probe 2',7'-dichlorofluorescein diacetate (H2DCF-DA; Molecular Probes, Leiden, The Netherlands). Myocytes were loaded with 10 µM H2DCF-DA for 30 min, washed two times in ice-cold PBS, and lysed in the dark in 200 µl of luciferase lysis buffer (Promega, Madison, WI). The lysate was further diluted in 800 µl H2O and centrifuged for 3 min (4°C, 12,000 g). Fluorescence was determined in the supernatants at excitation/emission wavelengths of 500 and 530 nm, respectively.

Assessment of myogenic differentiation. To assess the extent of myogenic differentiation morphologically, the myogenic index, which is defined as the fraction of nuclei residing in cells containing three or more nuclei, was assessed after staining with May-Grunwald-Giemsa (Sigma). Additionally, we determined muscle creatine kinase (CK) activity and the expression of the fast-twitch isotype of myosin heavy chain (MyHCf) or myogenin as markers of differentiation by Western blotting, according to procedures described previously (21). Monoclonal antibodies specific for MyHCf (MY-32) or myogenin (M-225) were obtained from Sigma and Santa Cruz (Santa Cruz, CA), respectively.

Electrophoretic mobility shift analysis. The ability of NF-kappa B to bind to its consensus recognition sequence was evaluated in electrophoretic mobility shift assays, as described previously (21). Nuclear protein (2 µg) was used per binding reaction, and protein-DNA complexes were resolved on a 5% polyacrylamide gel in 0.25× Tris-borate-EDTA buffer at 120 volts for 2 h. Gels were dried and exposed to film (X-Omat Blue XB-1; Kodak, Rochester, NY). Shifted complexes, indicative of NF-kappa B complexes, were evaluated by phosphoImager analysis (Bio-Rad, Hercules, CA), and their subcomposition was verified using antibodies recognizing p50 and RelA subunits of NF-kappa B (Santa Cruz).

Transfections and plasmids. For the assessment of transcriptional activity of NF-kappa B during differentiation, stable cell lines were created, as is described elsewhere (21). Using identical procedures, we also created a cell line that stably expresses the troponin I (TnI) promoter-driven luciferase reporter to assess muscle-specific gene transcription. To determine luciferase activity, cells were lysed in luciferase lysis buffer and stored at -80°C. Luciferase activity was measured according to the manufacturers' instructions (Promega), and values were corrected for total protein content.

Statistical analysis. Raw data were entered into SPSS (version 8.0) for statistical analysis. Values for CK activity, myogenic index, and luciferase activity were subjected to one-way ANOVA, and the various treatment groups were compared post hoc with a Student-Newman-Keuls test (P < 0.05).


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Myogenic differentiation of C2C12 cells is a process characterized by the expression of muscle-specific genes, like myosin heavy chain, CK, myogenin, and the fusion of myocytes into multinucleated myotubes. We previously established that the cytokine TNF-alpha inhibits myogenic differentiation of C2C12 cells (21). To illustrate this effect, CK activity was assessed 72 h after induction of differentiation, since we determined earlier that CK activity is markedly induced at this time point (21). In addition, measuring TnI promoter activation using a C2C12 cell line containing a TnI-luciferase reporter gene provided us with an earlier marker of myogenesis. Potent induction of luciferase activity is already apparent 48 h after induction of differentiation (21.5 ± 4.4 × 105 vs. 3.8 ± 1.0 × 105 relative light units/mg protein in cultures maintained in GM).

The results in Fig. 1 demonstrate an extensive decrease in creatine kinase activity (A) and inhibition of transactivation of the TnI promoter (B) when TNF-alpha was administered at the onset of the differentiation process. Because TNF-alpha is known to cause oxidative stress, we next assessed the reduced GSH content of C2C12 cells in the presence or absence of TNF-alpha . During the first 24 h of differentiation, a threefold increase in GSH levels occurred (Fig. 1C). This increase was transient, and, after 72 h of differentiation, levels of GSH returned to baseline. Importantly, in the presence of 10 ng/ml TNF-alpha , GSH levels also increased during the first 24 h but decreased more rapidly compared with sham controls, almost completely returning to baseline by 48 h. This coincided with increased DCF oxidation in the myocytes differentiated in the presence of TNF-alpha (Fig. 1D), further indicative of oxidative stress after TNF-alpha treatment.


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Fig. 1.   Tumor necrosis factor-alpha (TNF-alpha ) inhibits myogenic differentiation and affects cellular redox status. Myoblasts were allowed to differentiate in the presence or absence of TNF-alpha , administered one time at the indicated concentrations. A: after 72 h, creatine kinase (CK) activity was determined and expressed per mg protein (n = 3; means ± SE, *P < 0.001). B: alternatively, TnI promoter activation was measured in a troponin I (TnI) reporter line (see METHODS) as luciferase activity (n = 2; means ± SE, *P < 0.01) and normalized to protein after 48 h of culture in differentiation medium (DM) with or without TNF-alpha . C: glutathione (GSH) levels were assessed and normalized to protein every 24 h during differentiation (n = 3; means ± SE, *P < 0.001) in parental C2C12 cells that were cultured in the presence or absence of TNF-alpha (10 ng/ml). D: myocytes were loaded with 2',7'-dichlorofluorescein diacetate (DCF; 10 µM for 30 min) after 24 h of differentiation in the presence or absence of TNF-alpha (10 ng/ml). Fluorescence was determined in the supernatants of the cell lysates and expressed as %control (n = 3; means ± SE, *P < 0.02). Shown are representative graphs of 3 or more independent experiments.

We next determined whether the induction of oxidative stress by the addition of the oxidant H2O2 mimicked the inhibitory effects of TNF-alpha on differentiation. As is demonstrated in Fig. 2, H2O2 administered as a bolus in concentrations ranging from 20 to 200 µM decreased MyHCf and myogenin expression (Fig. 2A), inhibited CK activity and TnI gene transcription (Fig. 2, B and C), and blocked myotube formation (Fig. 3C), illustrating that the process of myogenic differentiation is blocked by oxidative stress per se. To assure that the lack of differentiation is not the result of nonspecific toxicity of H2O2, the DM of myocytes treated with H2O2 was replaced after 48 h, and CK activity was evaluated after an additional 72 h in DM. As shown in Fig. 2D, 72 h after replacing the DM medium, CK activity was almost equal to that of control myocytes that had differentiated for 72 h, illustrating that the inhibition of differentiation by H2O2 was not the result of nonspecific cell death. Next, we investigated whether boosting cellular GSH before induction of differentiation would restore myogenic differentiation in cells exposed to TNF-alpha or H2O2. To this end, the myoblasts were incubated in GM for 24 h with the GSH precursor NAC at a 5 mM concentration, which did not modulate myogenesis (Fig. 3) but enhanced intracellular GSH levels (control vs. 5 mM NAC: 8.4 ± 0.8 vs. 28.1 ± 2.5 nmol GSH/mg protein). As demonstrated in Fig. 3, NAC completely restored all aspects of myogenic differentiation in cells treated with H2O2. However, preincubation with NAC did not restore TnI promoter activation after treatment of TNF-alpha (Fig. 3A) and only marginally attenuated TNF-alpha -dependent inhibition of CK (Fig. 3B). Surprisingly, the ability to fuse into multinucleated myotubes was almost completely restored in TNF-alpha -treated cells after pretreatment with NAC (Fig. 3C). These results illustrate that the inhibition of myogenic differentiation observed after TNF-alpha exposure is partially dependent on alterations in cellular thiol pools.


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Fig. 2.   Induction of oxidative stress by H2O2 inhibits myogenic differentiation. A: myocytes were cultured in the presence or absence of increasing concentrations of H2O2, which was added directly after induction of differentiation. After 72 h, lysates were prepared, and equal amounts of protein were separated by SDS-PAGE and subjected to Western blot analysis for myogenin and the fast-twitch isotype of myosin heavy chain (MyHCf) expression. B: separate lysates were prepared for determination of CK activity (n = 3; means ± SE; *P < 0.001), which was normalized to total protein. C: alternatively, myocytes of the TnI reporter line were allowed to differentiate for 48 h in various concentrations of H2O2, after which lysates were prepared to assess TnI promoter activation. Luciferase activity (n = 2; means ± SE; *P < 0.001) was expressed per mg protein. D: myocytes were cultured in DM in the absence or presence of 200 µM H2O2 for 72 h (control and H2O2, respectively). Alternatively, the DM of H2O2-treated myocytes was replaced after 48 h with fresh DM, followed by an additional 72-h incubation (H2O2/wash), after which CK was determined [n = 3; *P < 0.04 and not significant (NS) compared with control]. Shown are representative graphs of 3 or more independent experiments.



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Fig. 3.   N-acetyl-L-cysteine (NAC) reverses certain aspects of the inhibition of myogenesis by TNF-alpha . Cells were cultured in growth medium (GM) in the presence or absence of 5 mM NAC (a concentration that did not affect myogenic differentiation). After 24 h, myocytes were washed and allowed to differentiate in the presence or absence of TNF-alpha (10 ng/ml) or H2O2 (200 µM) as a positive control to test the efficacy of NAC supplementation to prevent oxidative stress. A: for the assessment of TnI promoter activation, myocytes of the TnI reporter line were lysed after 48 h in DM and corrected for total protein (n = 2; means ± SE). B and C: parental C2C12 cells were assessed for CK activity after 72 h in DM (n = 2; means ± SE), or micrographs (×200) were taken and the myogenic index was determined (n = 3; means ± SE). Statistical analysis by 2-way ANOVA demonstrated the following interactions: for TnI-luciferase (luc), *P = 0.019 (A); for CK activity, *P = 0.001 and **P = 0.01 (B); for the myogenic index, *P = 0.003 and **P = 0.003 (C). Shown are representative graphs of 3 or more independent experiments.

Because NF-kappa B has been shown to be redox sensitive, and its activation frequently is preventable by NAC (23), we next determined whether the ability of NAC to reverse the inhibition of myogenic differentiation after exposure to H2O2 or TNF-alpha was related to the modulation of NF-kappa B. We first determined the ability of NF-kappa B to bind to its cognate DNA sequence and transactivate gene expression in cells treated with TNF-alpha or H2O2. Results in Fig. 4 demonstrate a marked binding of RelA and p50 NF-kappa B subunits to DNA after exposure to TNF-alpha , which coincided with transactivation of an NF-kappa B-dependent luciferase reporter gene. Surprisingly, H2O2 concentrations that interfered with myogenic differentiation induced no, or small, increases in NF-kappa B DNA binding (Fig. 4A). Only at 4 and 8 h after H2O2 could a minor induction (1.3- and 2.2-fold over control, respectively) be detected. However, this was not reflected in NF-kappa B transcriptional activation, since luciferase activity demonstrated no changes after H2O2 over the time frame examined (Fig. 4B). These data show that oxidative stress inhibits myogenic differentiation via a pathway independent or downstream of NF-kappa B. Last, we determined whether NAC was able to inhibit NF-kappa B activation in cells exposed to TNF-alpha . Importantly and consistent with its inability to fully restore myogenic differentiation in the presence of TNF-alpha , NAC only partially prevented NF-kappa B activation in TNF-alpha -exposed cells (Fig. 4C).


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Fig. 4.   Nuclear factor (NF)-kappa B activation in C2C12 myocytes is redox independent. A: directly after induction of differentiation, C2C12 cells were treated for the indicated time frames with 10 ng/ml TNF-alpha (T) or 200 µM H2O2 (H) and were compared with controls (C) for DNA binding activity using electrophoretic mobility shift assay (EMSA); ns, nonspecific binding. B: myocytes of an NF-kappa B reporter cell line (6kappa B TK-luciferase) were treated with TNF-alpha or H2O2 and assessed for NF-kappa B transactivation (n = 2; means ± SE, *P < 0.001). C: alternatively, myoblasts were cultured in the presence of NAC for 24 h and subsequently stimulated with TNF-alpha (10 ng/ml). Lysates were prepared to assess NF-kappa B-dependent luciferase activity. Shown are representative graphs of 2 or more independent experiments.


    DISCUSSION
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INTRODUCTION
METHODS
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The etiology of cachexia remains to be elucidated and likely involves multiple processes, including imbalances in anabolism/catabolism and muscle injury and repair. Previous work by our laboratories and other groups revealed that skeletal muscle differentiation is inhibited by TNF-alpha (15, 17, 21, 22, 26, 37), which was confirmed in the present study by the lack of myotube formation, CK activity, and TnI promoter activation. Importantly, this effect of TNF-alpha is not the result of nonspecific toxicity, since the inhibition of differentiation proved to be reversible (21). The implications of these findings are that myogenic differentiation, as part of the regenerative response of skeletal muscle, may be impaired in the presence of inflammatory mediators, ultimately contributing to cachexia.

Many signaling pathways involved in proliferation, apoptosis, and differentiation are modulated by the cellular redox state (7, 18). Therefore, we investigated in the present study whether the inhibition of myogenic differentiation by TNF-alpha was accompanied by alterations in reduced GSH levels. Importantly, we demonstrated a fivefold increase in the content of GSH during the first 24 h of myogenic differentiation. The functional significance of this increase is unclear but may provide necessary protection against oxidative stress associated with enhanced mitochondrial respiratory activity, which occurs during myogenic differentiation (33). In this perspective, it is interesting to note that a fivefold increase in DCF oxidation could be detected in myocytes that had been cultured in DM for 24 h compared with GM (data not shown). The transient increase in GSH observed during the first 24 h of differentiation was not affected by the presence of TNF-alpha . However, compared with control cultures, GSH concentrations were reduced the following 48 h in TNF-alpha -exposed cells. Moreover, the presence of TNF-alpha increased DCF oxidation, suggesting that the reduction of GSH levels may be the result of increased ROS generation. A potential source of these TNF-alpha -induced ROS may be the mitochondria, since a recent study demonstrated that ROS generation after TNF-alpha (measured as DCF oxidation) required functional mitochondria (6). In fact, TNF-alpha is known to elicit the generation of ROS and reactive nitrogen species by several cell types, including skeletal muscle (24, 38). Therefore, we investigated whether the prototypical ROS, H2O2, was able to block myogenic differentiation and mimic the inhibitory effect of TNF-alpha . Expression of MyHCf and myogenin, the activity of CK and the TnI promoter, and the ability of the cells to form multinucleated myotubes were all inhibited by H2O2 in a dose-dependent fashion, illustrating that oxidative stress per se is sufficient to block myogenic differentiation. Importantly, the inhibition of myogenic differentiation by H2O2 was reversible, illustrating that the absence of differentiation markers was not because of nonspecific toxicity.

If the inhibition of myogenic differentiation by TNF-alpha also would depend on the induction of oxidative stress, modulation of the cellular redox status by supplementing reduced GSH levels would be expected to restore myogenesis. We demonstrated in the present study that cellular GSH concentrations were effectively raised using NAC. However, only certain aspects of myogenesis were partially restored by NAC when cells were differentiated in the presence of TNF-alpha . Specifically, TnI promoter activation was still inhibited by TNF-alpha , despite the use of NAC concentrations that completely reversed the inhibitory effect of H2O2. Whereas NAC fully restored CK activity in the presence of H2O2, it only partially rescued CK activity after exposure to TNF-alpha . In contrast, myotube formation was almost completely restored in H2O2- or TNF-alpha -treated cells when intracellular thiol levels were augmented using NAC. These observations suggest that a redox-dependent mechanism is important in the block of myotube formation associated with exposure to oxidants or cytokines, whereas myogenic protein expression can be regulated in a redox-dependent or -independent manner based on the stimulus present.

Recently, our group (21) and others (15) demonstrated that TNF-alpha or IL-1 inhibits myogenesis through the activation of the transcription factor NF-kappa B. In fact, the activation of NF-kappa B is sufficient to inhibit myogenesis, since constitutive activation of NF-kappa B by overexpression of IKKbeta inhibits myogenesis. The redox sensitivity of the transcription factor NF-kappa B has been described in many cell types, including skeletal muscle (25, 35). Therefore, we evaluated whether inhibition of myogenesis by H2O2 coincided with the activation of NF-kappa B and, conversely, whether the use of NAC modulated the activation of NF-kappa B by TNF-alpha , since it restored some aspects of myogenic differentiation. Surprisingly, H2O2, at concentrations that inhibited myogenesis, did not induce significant NF-kappa B DNA binding nor transactivation at any of the time points. The absence of NF-kappa B activation was not the result of methodological problems, since TNF-alpha induced a robust activation in NF-kappa B DNA binding activity and reporter gene transcription. Our findings are in contrast with studies by others in which H2O2 induced NF-kappa B DNA binding in L6 and C2C12 skeletal myocytes (25, 35). However, this discrepancy may be attributed to differences in antioxidant enzyme activity, since NF-kappa B activation by H2O2 in the latter study was assessed in fully differentiated myotubes and antioxidant enzyme activities significantly decrease during myogenic differentiation (12). Therefore, the inhibition of biochemical aspects of myogenesis by TNF-alpha did not appear to require the induction of oxidative stress. In contrast, the inhibition of morphological myogenic differentiation, i.e., myotube formation by TNF-alpha , involved a redox-sensitive component. Fusion into myotubes is a process regulated by cell-matrix and cell-cell interactions and involves the formation of functional beta -catenin-adherin complexes proximate to the cell membrane (14, 19). Potentially, disruption of these complexes, which is shown to occur in response to oxidative stress, is responsible for the inhibition of myotube formation (31).

The signaling pathways that mediate the H2O2-dependent inhibition of myogenic differentiation remain elusive but may involve the members of the mitogen-activated protein kinase family. Activities of extracellular signal-regulated kinase (ERK) and c-Jun NH2-terminal kinase are increased in response to treatment with oxidants (16). In fact, we have detected enhanced ERK phosphorylation after treatment with H2O2 in C2C12 myoblasts (unpublished observations), indicative of ERK activation. Furthermore, it has been demonstrated that ERK is a critical regulator of myogenic differentiation (4).

In summary, we have demonstrated in the present study that, in response to TNF-alpha , myotube formation is inhibited in a redox-dependent manner but that the inhibition of other aspects of myogenic differentiation and the activation of NF-kappa B occurred largely independently of cellular thiol levels. In contrast, NAC completely rescued the inhibition of myogenic differentiation that occurred after treatment with H2O2, without modulation of NF-kappa B activity. Thus, dependent upon the stimulus that myoblasts encounter, multiple NF-kappa B-dependent and -independent and redox-sensitive and -insensitive pathways contribute to the loss of myogenic differentiation, as shown in Fig. 5. These observations may have implications for the treatment of cachexia and suggest the use of combined anti-inflammatory and antioxidant therapies.


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Fig. 5.   Multiple redox-dependent and -independent pathways inhibit myogenic differentiation. Oxidative stress induced by H2O2 inhibits myogenesis at both the level of muscle-specific protein expression [muscle creatine kinase (MCK)] and myotube fusion, in the absence of NF-kappa B activation. In contrast, inhibition of muscle protein expression by TNF-alpha does not depend on oxidative stress but requires NF-kappa B activation. The mechanism by which TNF-alpha blocks myotube formation does involve the generation of reactive oxygen species (ROS), since it can be reversed by NAC, but it remains unclear whether this is independent or downstream of NF-kappa B activation.


    ACKNOWLEDGEMENTS

This work was supported by a grant from Numico Research, The Netherlands.


    FOOTNOTES

Address for reprint requests and other correspondence: Y. Janssen-Heininger, Dept. of Pathology, Univ. of Vermont, Health Science Research Facility 216, Burlington, VT 05405 (E-mail: Yvonne.Janssen{at}uvm.edu).

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.

April 24, 2002;10.1152/ajpcell.00418.2001

Received 28 August 2001; accepted in final form 16 April 2002.


    REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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