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B mediates the protein loss induced by TNF-
in
differentiated skeletal muscle myotubes
Department of Medicine, Baylor College of Medicine, Houston, Texas 77030
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Nuclear factor-
B (NF-B)
regulates the transcription of a variety of genes involved in immune
responses, cell growth, and cell death. However, the role of NF-B in
muscle biology is poorly understood. We recently reported that tumor
necrosis factor-
(TNF-) rapidly activates NF-B in
differentiated skeletal muscle myotubes and that TNF- acts directly
on the muscle cell to induce protein degradation. In the present study,
we ask whether NF-B mediates the protein loss induced by TNF-. We
addressed this problem by creating stable, transdominant negative
muscle cell lines. C2C12 myoblasts were transfected with viral plasmid
constructs that induce overexpression of mutant I-B proteins that
are insensitive to degradation via the ubiquitin-proteasome pathway.
These mutant proteins selectively inhibit NF-B activation. We found
that differentiated myotubes transfected with the empty viral vector
(controls) underwent a drop in total protein content and in fast-type
myosin heavy-chain content during 72 h of exposure to TNF-. In
contrast, total protein and fast-type myosin heavy-chain levels were
unaltered by TNF- in the transdominant negative cell lines. TNF-
did not induce apoptosis in any cell line, as assessed by DNA ladder
and annexin V assays. These data indicate that NF-B is an essential
mediator of TNF--induced catabolism in differentiated muscle cells.
cachexia; cytokine; free radicals; signal transduction; inflammation
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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SKELETAL MUSCLE IS OFTEN IMPAIRED by diseases of other organs. Signs of muscle atrophy or wasting are frequently seen in inflammatory disorders that include cancer (27, 30), acquired immunodeficiency syndrome (33), chronic obstructive pulmonary disease (10), and congestive heart failure (2). Loss of muscle mass contributes importantly to morbidity and mortality in individuals with such diseases (2, 10, 27, 30, 33).
Among the various humoral factors that are altered in inflammatory
disease, tumor necrosis factor- (TNF-) has been widely implicated
as a possible mediator of muscle catabolism (2, 9, 10, 30,
33). TNF- is a polypeptide cytokine that is present in the
serum of healthy individuals at undetectable-to-low, picogram-per-milliliter levels (24, 26, 32). Circulating TNF- levels are elevated by inflammatory disease, reaching values as
high as 2.8 ng/ml in rheumatoid arthritis (32) and 6 ng/ml in cancer (24). Such clinical values fall within the range
of serum concentrations that induce muscle wasting in experimental animals (13, 25, 29, 31). Despite a long-standing
association with catabolic pathology, the role of TNF- in muscle
wasting remains poorly understood and somewhat controversial
(9).
In previous studies, we used cell culture techniques to evaluate
TNF- effects on differentiated skeletal muscle myotubes (20). We found that prolonged exposure to clinically
relevant levels of TNF- (1-6 ng/ml) stimulates muscle protein
loss without causing significant cell death, a situation similar to
muscle atrophy in vivo. We further determined that TNF- stimulates
the degradation of muscle-specific proteins, including fast-type myosin heavy chains (MHCf). Studies of [35S]methionine
incorporation into MHCf showed that protein loss is not due to a
reduction in MHCf synthesis. Rather, TNF- appears to act directly on
differentiated myotubes to stimulate protein degradation
(20). These findings challenged previous conclusions that
TNF- acts via indirect mechanisms to stimulate muscle wasting (13) and provided evidence that the cytokine exerts direct
catabolic effects on muscle cells.
Previous studies have also evaluated TNF- signal transduction in
differentiated myotubes. TNF- binding to surface receptors stimulates a stereotypical cascade of events that results in
proteasomal degradation of I-B (20), the protein
that inhibits nuclear factor-B (NF-B). TNF- thereby activates
NF-B and causes its translocation to the nucleus (20).
This process appears to depend on TNF--induced reactive oxygen
species (ROS) (20) that derive from mitochondrial electron
transport and are essential for NF-B activation (19).
NF-B regulates the transcription of genes involved in immune
responses, cell growth, and cell death (3, 18). Recent evidence indicates that NF-B influences cellular proliferation and
exiting of the cell cycle by undifferentiated myoblasts
(14). However, the functional importance of NF-B in
differentiated skeletal muscle has not been evaluated.
The present study was conducted to assess the putative involvement of
NF-B in TNF--induced muscle wasting. Using a standardized cell
culture model, we tested the hypothesis that NF-B activation causes
net protein loss in differentiated myotubes. To evaluate cause and
effect, we developed transdominant negative skeletal muscle cell lines
in which NF-B signaling was selectively inhibited. Stable
transfection of C2C12 myoblasts was used to induce overexpression of
mutant I-B proteins. These I-B variants are insensitive to
degradation via ubiquitin-proteasome activity and, therefore, inhibit
NF-B activation. We found that selective blockade of NF-B
prevented protein loss in myotubes challenged with TNF-. These
findings indicate that NF-B mediates the catabolic response of
differentiated muscle cells to TNF- and suggest a central role for
NF-B in the regulation of cachexia.
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METHODS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Myogenic cell culture and transfection.
Myoblasts from the mouse muscle-derived C2C12 cell line were obtained
from American Type Culture Collection (Rockville, MD). As described
previously (20), undifferentiated cells were grown in DMEM
supplemented with 20% newborn calf serum and gentamicin at 37°C in
the presence of 5% CO2. Stable transfection was carried out using Lipofectamine reagent (GIBCO BRL, Gaithersburg, MD) according
to the manufacturer's protocol. Myoblasts were transfected with
plasmid constructs of I-B
N (truncation of amino acids 1-36) or I-BS32/A36 (point mutations of Ser32
and Ser36 to alanine); each mutant protein lacks
Ser32 and Ser36, phosphorylation sites that are
required for I-B degradation (5, 8). Plasmid
constructs of I-BN and I-BS32/A36 under control of the
cytomegalovirus (CMV) promoter were gifts of Dr. Dean Ballard
(Vanderbilt University), as was the empty pCMV4 vector used for the
control cell line. Plasmid pSVneo was cotransfected for selection by
use of neomycin. The selected colonies were pooled.
(Boehringer Mannheim, Indianapolis, IN) was added
to differentiated myotubes at 24-h intervals.
Electrophoresis mobility shift assay.
Electrophoresis mobility shift assay was carried out as previously
described (20). Briefly, the binding assay buffer
contained 5 mM Tris · HCl (pH 7.5), 100 mM NaCl, 0.3 mM
dithiothreitol, 5 mM MgCl2, 10% glycerol, 2 µg of BSA,
0.2% NP-40, and 1 µg of poly(dI-dC). Nuclear extracts were prepared
according to Andrews and Faller (1). In each reaction,
4-5 µg of nuclear extract were combined with 1 ng
(10,000-15,000 cpm) of NF-B-binding DNA probe
(5'-AGTTGAGGGGACTTTCCCAGGC-3') labeled with
[-32P]dATP (3,000 Ci/mmol; Amersham Life Science,
Arlington Heights, IL) by use of the Klenow fragment. After 30 min of
incubation on ice, the reaction mixtures were resolved on 4.5%
polyacrylamide gels. The optical density of bands detected on the X-ray
film was quantified using commercial densitometry software (Pharmacia, Piscataway, NJ). Protein concentration of the nuclear extracts was
determined with the Bio-Rad (Hercules, CA) protein assay kit.
Western blot analysis.
As described previously (20), cell lysates were prepared
by boiling harvested cells in Laemmli buffer for 5 min and then were
separated using SDS-PAGE and transferred to nitrocellulose membranes.
Membranes were incubated in the presence of a monoclonal antibody to
MHCf (Novocastra Laboratories, Newcastle, UK) or the FLAG tag (Sigma
Chemical, St. Louis, MO) used to identify I-BN. Horseradish
peroxidase-conjugated secondary antibodies were used to locate the
primary antibodies. Antibodies were visualized by the enhanced
chemiluminescence method (Amersham). Bands detected on the X-ray films
were quantified using commercial software (Pharmacia). Protein
concentration in the cell lysates was determined using the Bio-Rad
Dc protein assay kit.
Analysis of apoptosis.
For DNA ladder detection, DNA of C2C12 myotubes or fibroblast-derived
L929 cells (American Type Culture Collection) was extracted using the
Quick Apoptosis DNA Ladder Detection Kit (BioVision, Palo Alto, CA) and
was separated on 1% agarose gel containing ethidium bromide. For
annexin V detection, C2C12 myotubes or L929 cells were grown on
coverslips. C2C12 myotubes were differentiated for 96 h and then
treated with TNF- (6 ng/ml) for an additional 72 h; L929 cells
were treated with TNF- (1 ng/ml) for 24 h. The medium then was
removed, the coverslips were washed twice with PBS, and the cells were
analyzed using the Annexin V-FITC Apoptosis Detection Kit (BioVision).
Briefly, the cells were incubated on the coverslips in 500 µl of 1×
binding buffer, 5 µl of annexin V-FITC, and 5 µl of propidium
iodide for 5 min at room temperature in the dark. Fluorescence
microscopy was used to detect emissions from apoptotic cells that
stained positive for annexin V.
Statistics.
Data were analyzed for differences among groups with use of commercial
software (SigmaStat, Jandel Scientific, Corte Madera, CA).
Concentration-dependent decrements in total protein content and MHCf
levels were assessed using linear regression analysis (34). Differences in optical densities of NF-B bands
were evaluated using Student's t-test (34).
Differences were considered significant at P < 0.05.
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Transdominant negative cell lines.
To determine whether NF-B mediates TNF--induced catabolism, we
established C2C12 cell lines that overexpressed either of two dominant
negative I-B mutants. The phosphorylation sites required for
degradation of I-B (Ser32 and Ser36) are
absent from I-BN (truncation of amino acids 1-36) and I-BS32/36A (point mutations of Ser32 and
Ser36 to alanine), preventing ubiquitin conjugation and
proteolysis of either protein (5, 8). Overexpression of
mutant I-B was confirmed by Western blot analysis (Fig.
1).
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BN or I-BS32/36A inhibited
activation and nuclear translocation of NF-B in response to TNF-
(Fig. 2). TNF- activated NF-B in
control myotubes transfected with the pCMV4 vector. This response was
indistinguishable from responses previously observed in C2C12 myotubes
and in primary myotubes cultured from rat limb muscle (19,
20). In contrast, NF-B activation by TNF- was largely
blocked in myotubes that overexpressed I-BN (Fig. 2) or
I-BS32/36A (data not shown). Densitometry showed that TNF-
increased NF-B content of vector-transfected control myotubes by
13.2 ± 3.7-fold (mean ± SE) relative to untreated controls.
In myotubes that overexpressed I-BN, TNF- produced only a
2.2 ± 1.0-fold increase. On average, therefore, TNF-
activation of NF-B was inhibited by >80% in transdominant negative
myotubes (P < 0.05, n = 3/group).
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NF-B in TNF--induced catabolism.
Chronic exposure to TNF- causes dose-dependent reductions in MHCf
levels and total protein content of differentiated myotubes (20). The transdominant negative cell lines were used to
test NF-B mediation of this response. Control myotubes transfected with the pCMV4 vector were compared with myotubes that overexpressed either I-B mutant. Figure 3 shows
Western blots that illustrate the effect of TNF- on MHCf protein
levels. Treatment with TNF- for 72 h caused a dose-dependent
reduction of MHCf levels in control myotubes but had no effect on
myotubes that overexpressed I-BN. Figure
4 depicts averaged data. TNF-
significantly diminished MHCf and total protein content of control
myotubes. In contrast, myotubes that overexpressed I-BN were
unaffected by TNF-; neither MHCf levels nor total protein content
was altered. Myotubes that overexpressed I-BS32/36A exhibited a
similar insensitivity to TNF- (data not shown). The capacity of
mutant I-B to inhibit TNF--induced protein loss indicates that
this response depends on NF-B signaling.
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does not induce apoptosis in
C2C12 myotubes under the present experimental conditions (20). Similarly, we obtained no evidence that TNF-
stimulates apoptosis in transdominant negative myotubes by use of
assays for DNA laddering (Fig. 5) or
annexin V (data not shown). In contrast, apoptotic changes were
detectable in positive control studies. DNA laddering was evident after
TNF- treatment of L929 fibroblasts (Fig. 5), a nonmuscle cell line
with known sensitivity to TNF- (4). Also, apoptotic
changes were observed in differentiated C2C12 myotubes treated with a
more potent stimulus (staurosporine plus cycloheximide; data not
shown).
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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The present study shows that activation of NF-B is required for
the loss of skeletal muscle protein induced by TNF-. NF-B has
been studied extensively because of its involvement in biological processes that include immune and inflammatory responses, regulation of
cell growth, and apoptosis (3, 18). The present study provides the first direct evidence that NF-B regulates adaptive responses of differentiated skeletal muscle cells.
TNF- and muscle wasting.
Muscle wasting and negative nitrogen balance are the hallmarks of
inflammatory diseases that range from cancer (27, 30) to
emphysema (10) and from congestive heart failure
(2) to acquired immunodeficiency syndrome
(33). Loss of muscle mass contributes importantly to the
mortality and morbidity associated with these disease states.
Accordingly, the pathological mechanisms responsible for such losses
are of major clinical interest. A primary factor thought to mediate
inflammatory catabolism in these and other conditions is TNF-, which
was originally termed "cachectin" because of the strong association
between this cytokine and cachexia (9). Circulating
TNF- levels are elevated in inflammatory disease, with serum levels
as high as 3-6 ng/ml reported in humans (24, 32).
Animal studies clearly demonstrate that exogenous TNF- stimulates
loss of muscle mass and contractile function (13, 25, 29,
31). It previously was believed that systemic administration of
TNF- stimulated muscle catabolism via indirect humoral or behavioral
effects (13). However, the present observations and those
in our previous studies (20) indicate that TNF- acts directly on differentiated muscle cells to stimulate net protein loss.
Studies of MHCf metabolism indicate that TNF- does not alter protein
synthesis (20), suggesting that the cytokine accelerates protein degradation. These observations bolster the biological relevance of TNF- effects on skeletal muscle and the signaling mechanisms that regulate such effects.
TNF-/NF-B signaling.
Studies in nonmuscle cell types have identified three major pathways
that transduce the TNF- signal (16, 21). Briefly, these
include a proapoptotic pathway regulated by interaction of the
TNF--receptor complex with the Fas-associated protein with death
domain. A second pathway activates the transcription factor activator
protein-1 via Jun-NH2-terminal kinases. The third pathway
leads to activation of NF-B. This last pathway represents a major
mechanism of transcriptional control by TNF- (21) and has been a primary focus of research for our laboratory over the past
few years.
B is constitutively expressed and exists in the cytosol as part
of a heterotrimeric complex. This complex typically comprises the
DNA-binding proteins p50 and p65 plus the inhibitory protein I-B.
Activation of NF-B requires phosphorylation of I-B at Ser32 and Ser36, followed by ubiquitin
conjugation and proteolysis of I-B by the 26S proteasome. The
activated NF-B dimer is then translocated to the cell nucleus, where
it regulates gene expression in a manner that is cell type specific.
TNF- rapidly activates NF-B in skeletal muscle cells, including
differentiated myotubes (19, 20) and undifferentiated myoblasts (14, 28). Much of the cascade that transduces
the TNF- signal in differentiated muscle has recently been
elucidated. Events are triggered by TNF- binding to sarcolemmal
receptors, with the type 1 TNF- receptor being most likely to
regulate protein loss (17). Receptor activation stimulates
mitochondrial ROS production, an event that appears to be essential for
NF-B activation in differentiated muscle (19). TNF-
stimulation increases the activity of redox-sensitive kinases,
including protein kinase C (19), and causes rapid
conjugation of ubiquitin to muscle proteins (20). These
events result in proteasomal degradation of I-B and translocation
of activated NF-B to the nucleus within 15 min of TNF- exposure
(20).
NF-B undoubtedly stimulates protein loss via effects on muscle gene
expression. The most likely targets are genes that regulate the
ubiquitin-proteasome pathway. Animal studies indicate that TNF-
increases ubiquitin mRNA and ubiquitin protein levels in intact
skeletal muscle tissue (11, 12). Ubiquitin mRNA also is
increased in excised muscle after 3 h of incubation with TNF- in vitro (22), indicating a direct effect of the cytokine
on differentiated muscle fibers. The specific genes that respond to
NF-B and the proteins that regulate the ubiquitin-proteasome pathway
under these conditions have not been determined.
Muscle cell death.
Inflammatory cytokines such as TNF- can be strongly proapoptotic,
and NF-B is known to regulate apoptosis in nonmuscle cells (18, 21). However, after 72 h of TNF- exposure, we
found no evidence that TNF- induced apoptosis in transdominant
negative cell lines or in control myotubes transfected with the empty
pCMV vector. Resistance to apoptosis may have been conferred on the transdominant negative myotubes by the leak in NF-B signaling (~17% control) that persisted in these cells. The present data are
consistent with our previous findings that TNF- fails to induce
apoptosis in differentiated C2C12 myotubes or in myotubes from rat
primary cultures (20); nor do TNF- concentrations <10
ng/ml stimulate necrotic cell death (20). Data in this
study and in our previous reports have been obtained using a
standardized 72-h protocol and TNF- concentrations in the clinical
range. We cannot rule out the possibility that apoptosis is induced by higher TNF- concentrations or longer exposure times.
NF-B and oxidative stress.
NF-B activation by TNF- appears to be a redox-sensitive process
(28) that involves mitochondrial ROS generation as an essential intermediate step (19). In the absence of
TNF-, exogenous ROS can activate NF-B directly (20).
The present findings suggest that NF-B activation is a novel
mechanism by which ROS may stimulate muscle wasting. This model is
consistent with observations that antioxidants inhibit TNF--induced
muscle wasting in vivo (6) and provides a mechanism
whereby oxidative stress may diminish muscle mass in the absence of
overt cell death. For example, ionizing radiation activates NF-B
(15), and radiation therapy causes oxidative stress and
muscular weakness (17). Perhaps therapeutic levels of
ionizing radiation generate sufficient ROS within muscle fibers to
activate NF-B and thereby stimulate muscular atrophy.
Perspectives
This is the first report of a specific transcription factor that regulates loss of skeletal muscle protein. NF-B activation is only
one component of the postreceptor signaling cascade triggered by
TNF-, but it appears to be essential for the catabolic effect of
this cytokine on differentiated muscle cells. These findings highlight
the importance of determining NF-B effects on skeletal muscle gene expression.
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ACKNOWLEDGEMENTS |
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We thank Jim Agan and Juan Chen for technical assistance and Melanie Moody for assistance with graphics.
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FOOTNOTES |
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This study was supported by National Heart, Lung, and Blood Institute Grant HL-59878.
Address for reprint requests and other correspondence: M. B. Reid, Pulmonary Medicine, Suite 520B, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030 (E-mail: reid{at}bcm.tmc.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.
Received 21 December 1999; accepted in final form 1 May 2000.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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W. J. Durham, Y.-P. Li, E. Gerken, M. Farid, S. Arbogast, R. R. Wolfe, and M. B. Reid |
; n = 3/data point) and total protein
content (
; n = 4/data point) in C2C12
myotubes transfected with the empty pCMV4 vector (A) and
those that overexpressed I-
); in contrast, DNA laddering was
evident in L929 cells treated with TNF-