1 Department of Pulmonology, Maastricht University, 6202 AZ Maastricht, The Netherlands; and 2 Department of Pathology, University of Vermont, Burlington, Vermont 05405
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ABSTRACT |
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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- (TNF-
) inhibits myogenic differentiation via the
activation of nuclear factor-
B (NF-
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-
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-
on troponin I
promoter activation and only partially restored creatine kinase activity in TNF-
-treated cells. In contrast, the administration of
NAC before treatment with TNF-
almost completely restored the
formation of multinucleated myotubes. NAC decreased TNF-
-induced activation of NF-
B only marginally, indicating that the
redox-sensitive component of the inhibition of myogenic differentiation
by TNF-
occurred independently, or downstream of NF-
B. Our
observations suggest that the inhibitory effects of TNF-
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-B; myotube; myogenic
differentiation
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INTRODUCTION |
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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- (TNF-
) 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-
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-
B (NF-
B).
NF-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-
B resides in a latent form in the cytoplasm, where it is bound to the inhibitory protein of NF-
B,
I
B. Upon stimulation, a rapid and transient activation of I
B
kinases (IKK) occurs, which phosphorylate I
B, resulting in
subsequent ubiquitination and rapid degradation via the 26S proteasome
pathway. The liberation from I
B unmasks the nuclear localization
sequence of NF-
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-B,
based upon many studies demonstrating that antioxidants or metal
scavengers prevent cytokine-induced activation of NF-
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-
-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- and whether this occurs via the activation of
NF-
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-
B in response
to TNF-
(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-
and
that this occurred independently of NF-
B.
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METHODS |
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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- (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-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-
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-
B (Santa Cruz).
Transfections and plasmids.
For the assessment of transcriptional activity of NF-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).
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RESULTS |
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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- 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- was administered at the onset of the differentiation process.
Because TNF-
is known to cause oxidative stress, we next assessed
the reduced GSH content of C2C12 cells in the
presence or absence of TNF-
. 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-
, 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-
(Fig. 1D), further indicative of
oxidative stress after TNF-
treatment.
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We next determined whether the induction of oxidative stress by the
addition of the oxidant H2O2 mimicked the
inhibitory effects of TNF- 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-
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-
(Fig. 3A) and only marginally attenuated TNF-
-dependent
inhibition of CK (Fig. 3B). Surprisingly, the ability to
fuse into multinucleated myotubes was almost completely restored in
TNF-
-treated cells after pretreatment with NAC (Fig. 3C).
These results illustrate that the inhibition of myogenic
differentiation observed after TNF-
exposure is partially dependent
on alterations in cellular thiol pools.
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Because NF-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-
was related to the modulation of NF-
B. We first
determined the ability of NF-
B to bind to its cognate DNA sequence
and transactivate gene expression in cells treated with TNF-
or
H2O2. Results in Fig.
4 demonstrate a marked binding of RelA
and p50 NF-
B subunits to DNA after exposure to TNF-
, which
coincided with transactivation of an NF-
B-dependent luciferase
reporter gene. Surprisingly, H2O2
concentrations that interfered with myogenic differentiation induced
no, or small, increases in NF-
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-
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-
B.
Last, we determined whether NAC was able to inhibit NF-
B activation
in cells exposed to TNF-
. Importantly and consistent with its
inability to fully restore myogenic differentiation in the presence of
TNF-
, NAC only partially prevented NF-
B activation in
TNF-
-exposed cells (Fig. 4C).
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DISCUSSION |
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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- (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-
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- 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-
. However, compared with control cultures, GSH concentrations were reduced the following 48 h in TNF-
-exposed cells. Moreover, the presence of TNF-
increased DCF oxidation, suggesting that the reduction of GSH levels may be the result of increased ROS generation. A potential source of these TNF-
-induced ROS may be the
mitochondria, since a recent study demonstrated that ROS generation
after TNF-
(measured as DCF oxidation) required functional
mitochondria (6). In fact, TNF-
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-
. 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- 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-
. Specifically, TnI
promoter activation was still inhibited by TNF-
, 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-
. In contrast, myotube formation
was almost completely restored in H2O2- or
TNF-
-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- or IL-1 inhibits myogenesis through the
activation of the transcription factor NF-
B. In fact, the activation
of NF-
B is sufficient to inhibit myogenesis, since constitutive
activation of NF-
B by overexpression of IKK
inhibits myogenesis.
The redox sensitivity of the transcription factor NF-
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-
B
and, conversely, whether the use of NAC modulated the activation of
NF-
B by TNF-
, since it restored some aspects of myogenic
differentiation. Surprisingly, H2O2, at
concentrations that inhibited myogenesis, did not induce significant NF-
B DNA binding nor transactivation at any of the time points. The
absence of NF-
B activation was not the result of methodological problems, since TNF-
induced a robust activation in NF-
B DNA binding activity and reporter gene transcription. Our findings are in
contrast with studies by others in which H2O2
induced NF-
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-
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-
did not appear to require the induction of
oxidative stress. In contrast, the inhibition of morphological myogenic differentiation, i.e., myotube formation by TNF-
, 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
-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-, myotube formation is inhibited in a redox-dependent manner
but that the inhibition of other aspects of myogenic differentiation and the activation of NF-
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-
B
activity. Thus, dependent upon the stimulus that myoblasts encounter,
multiple NF-
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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ACKNOWLEDGEMENTS |
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This work was supported by a grant from Numico Research, The Netherlands.
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FOOTNOTES |
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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.
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