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B
1
* Department of Pulmonology, Maastricht University, Maastricht, The Netherlands, and
Department of Pathology, University of Vermont, Burlington, Vermont 05405, USA
1Correspondence: Department of Pathology, University of Vermont, Medical Alumni Building A143, Burlington VT 05405, USA. E-mail: yjanssen{at}zoo.uvm.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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(TNF-) has been implicated as a major
mediator of cachexia, its effects on C2C12 myocytes were examined.
TNF- activated nuclear factor-
B (NF-B) and interfered with the
expression of muscle proteins in differentiating myoblasts.
Introduction of a mutant form of inhibitory protein B (IB)
restored myogenic differentiation in myoblasts treated with TNF- or
interleukin 1ß. Conversely, activation of NF-B by overexpression
of IB kinase was sufficient to block myogenesis, illustrating the
causal link between NF-B activation and inhibition of myogenic
differentiation. The inhibitory effects of TNF- on myogenic
differentiation were reversible, indicating that the effects of the
cytokine were not due to nonspecific toxicity. Treatment of
differentiated myotubes with TNF- did not result in a striking loss
of muscle-specific proteins, which shows that myogenesis was
selectively affected in the myoblast stage by TNF-. An important
finding was that NF-B was activated to the same extent in
differentiating and differentiated cells, illustrating that once
myocytes have differentiated they become refractory to the effects of
NF-B activation. These results demonstrate that inflammatory
cytokines may contribute to muscle wasting through the inhibition of
myogenic differentiation via a NF-B-dependent pathway.—Langen,
R. C. J., Schols, A. M. W. J., Kelders,
M. C. J. M., Wouters, E. F. M.,
Janssen-Heininger, Y. M. W. Inflammatory cytokines inhibit
myogenic differentiation through activation of nuclear factor-B.
Key Words: cachexia • muscle wasting • TNF- • NF-B • myogenic differentiation
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, cancer (2)
, chronic heart failure
(3)
, and chronic obstructive pulmonary disease
(4
5
6)
, and is an independent predictor of mortality.
Remarkably, in many of these conditions muscle wasting is associated
with chronic elevations in circulating inflammatory cytokines, in
particular tumor necrosis factor (TNF-) (7
8
9
10)
.
Muscle wasting is generally believed to result from disturbances in the
energy or anabolism-catabolism balance. For example, imbalances in
myofibrillar protein synthesis and proteolysis have been demonstrated
in experimental models of cancer cachexia (11
, 12)
and
sepsis (13)
. Elevated levels of circulating inflammatory
mediators, such as TNF- and interleukin 1 (IL-1), were believed to
trigger the events leading to muscle wasting in these models. In
support of this, chronic administration of TNF- or IL-1 induced
weight loss and skeletal muscle wasting in rats (14)
.
Muscle wasting may alternatively result from a decrease of the number
of fibers. In support of this proposal, an increased prevalence of
apoptotic myonuclei has been associated with the loss of skeletal
muscle tissue in muscular dystrophies and inflammatory myopathies
(15
16
17)
. In addition, satellite cells form a reservoir of
myogenic stem cells, which are essential in the replacement of damaged
or degenerated myofibers. If inflammatory mediators interfere with the
function of satellite cells by decreasing or blocking their ability to
fuse with or replace damaged myofibers, this action could ultimately
result in the loss of skeletal muscle tissue.
Evidence for a direct role of inflammatory mediators in muscle wasting
has been documented in in vitro studies. For example, administration of
TNF- to primary cultures of differentiating human myoblasts
inhibited the expression of -actin and myosin heavy chain (MyHC)
(18)
. Others, using the murine skeletal muscle cell line
C2C12 and primary cultures of rat skeletal muscle, demonstrated the
depletion of MyHC in differentiated myotubes in response to TNF-
(19)
.
One well-studied pathway activated by TNF- involves signaling to the
transcription factor nuclear factor-B (NF-B). Under normal
conditions, NF-B is present within the cytoplasm in an inactive
state, bound to its inhibitory protein B (IB). Stimulation
with TNF- initiates an intracellular signaling cascade, resulting in
the phosphorylation of IB on serine residues 32 and 36 by IB
kinase (IKK). Phosphorylation and subsequent ubiquitination of these
residues target IB for degradation by the 26S-proteasome complex.
Once liberated from its inhibitory protein, NF-B translocates to the
nucleus, where it orchestrates the transcription of a number of
cytokine and chemokine, growth regulatory, and survival genes
(20
, 21)
.
The goals of the present study were to evaluate the effects of the
inflammatory cytokines TNF- and IL-1ß on C2C12 myocytes and to
explore the role of NF-B activation therein. We compared the effects
of TNF- on myogenesis during and after the differentiation of
myoblasts into myotubes, as studies have demonstrated that this
cytokine can affect both undifferentiated myoblasts and differentiated
myotubes (18
, 19
, 22)
. In addition to the effects of
TNF-, the effects of IL-1ß on muscle-specific gene expression were
investigated, because a role for this cytokine as a mediator of
cachexia has been suggested (14
, 23)
. Finally, we
evaluated whether the effects of TNF- and IL-1ß on myogenic
differentiation were mediated through the activation of NF-B.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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.
Myoblasts were cultured in low-glucose Dulbecco’s modified Eagle’s
medium (DMEM) containing antibiotics (50 U/ml penicillin and 50 µg/ml
streptomycin) and 9% (v/v) FBS (all from Life Technologies, Rockville,
MD), referred to as growth media (GM). For experiments, 60-mm dishes
(Becton Dickinson Labware, Bedford, MA) were coated for 1 h at
37°C with diluted (1:50 in DMEM) Matrigel (Becton Dickinson Labware),
which contains collagen IV, laminin, heparan sulfate proteoglycan, and
entactin, which are constituents of the natural extracellular matrix of
skeletal muscle fibers (25)
. This plating protocol
resulted in enhanced and more consistent differentiation, compared with
protocols that use 2% horse serum containing media to induce
differentiation (unpublished results). Cells were plated at
104/cm2 and cultured in GM
for 24 h, allowing them to reach 
60% confluency. To induce
differentiation, cells were washed in HBSS and further cultured in
low-glucose DMEM containing antibiotics and 0.5% heat-inactivated FBS,
referred to as differentiation media (DM). To study the effects on
differentiation, murine TNF- or IL-1ß (Calbiochem, La Jolla, CA)
was added to the culture dishes directly after induction of
differentiation. To study the susceptibility of differentiated myotubes
to TNF-, myoblasts were cultured in DM for 5 days and received fresh
media every other day, prior to addition of the cytokine. Cells were
examined with an inverted phase-contrast microscope (Olympus CH40,
Melville, NY) equipped with a camera (Nikon N6000).
Assessment of myogenic differentiation
As a morphological parameter of differentiation, the myogenic
index was defined as the fraction of nuclei residing in cells
containing three or more nuclei after staining with May-Grünwald
Giemsa (Sigma, St. Louis, MO). Myogenic differentiation was assessed
biochemically via determination of muscle creatine kinase (CK)
activity. Cells were washed twice in cold PBS, lysed in 0.5% Triton
X-100, and scraped off the dish with a rubber policeman. Lysates were
centrifuged for 2 min at 16,000g, and the supernatant was
stored in separate aliquots at -80°C for determination of protein
content or CK activity. CK activity was measured by using a
spectrophotometric-based (26)
kit from Sigma (Sigma
Diagnostics, St. Louis, MO). Specific CK activity was calculated after
correction for total protein, which was assessed by the Bradford method
(27)
. Expression of the fast-twitch isotype of MyHC
(MyHCf) or myogenin was evaluated by Western blotting. Cells were
washed in PBS, and whole-cell lysates were prepared by addition of
lysis buffer, composed of 40 mM Tris, 300 mM NaCl, 2% (v/v) Nonidet
P-40, 1 mM dithiothreitol (DTT), 1 mM
Na3VO4, 1 mM
phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, and 1% (v/v)
aprotinin. Lysates were incubated on ice for 30 min, followed by 30-min
centrifugation at 16,000g. A fraction of the supernatant was
saved for protein determination, and 2x Laemmli sample buffer (2%
[w/v] sodium dodecyl sulfate, 10% [v/v] glycerol, 0.1 M DTT, and
0.01% [w/v] bromophenol blue) was added. Samples were then boiled
for 5 min and stored at -20°C. Approximately 10 µg of protein was
loaded per lane and separated on a 7 or 10% polyacrylamide gel (Mini
Protean System, Bio-Rad, Hercules, CA), followed by transfer to a
nitrocellulose membrane (Schleicher & Schuell, Keene, NH) by semidry
electroblotting. The membrane was blocked overnight for nonspecific
binding in 5% (w/v) nonfat dried milk at 4°C. Nitrocellulose blots
were washed in PBS-Tween20 (0.05% v/v), followed by a 1-h incubation
with monoclonal antibodies specific for MyHCf (MY-32, Sigma) or
myogenin (M-225) (Santa Cruz, Santa Cruz, CA). After three wash steps
of 20 min each, the blots were probed with a peroxidase-conjugated
secondary antibody and were visualized by chemiluminescence according
to the manufacturer’s instructions (KPL, Gaithersburg, MD).
Assessment of cell death
Cell death was determined by measuring lactate dehydrogenase
(LDH) activity in the supernatant of the culture media, using a
spectrophotometric assay according the manufacturer’s instructions
(Promega, Madison, WI). Activity was expressed as a percentage of
maximal acitivity, i.e., the LDH activity after total lysis of the
cells of a control dish. Apoptosis was evaluated after pooling detached
and adherent cells from each dish. Detached cells were collected from
the culture media by centrifugation at 500g for 10 min and
were resuspended in 200 µl of HBSS. Adherent cells were trypsinized,
centrifuged (10 min, 500g, 4°C), and resuspended in 1 ml
of HBSS. A sample of 200 µl was saved to determine adherent cell
number, and the remaining 800 µl was pooled with the cells collected
from the culture media. Cells were permeabilized in 0.1% v/v Triton
X-100 (in PBS) and stained with PBS solution containing 50 µg/ml
propidium iodide (Sigma). After a 30-min incubation on ice, cell cycle
distribution was assessed to evaluate the
sub-G0/G1 fraction, using
flow cytometry (Coulter, EPICS Elite, Miami, FL). The total adherent
cell number was determined by using a Coulter particle counter
(Coulter, Miami) after dilution of a suitable volume of cells in 10 ml
of Isoton and was expressed as the number of adherent cells per plate.
Electrophoretic mobility shift analysis (EMSA)
To determine DNA binding activity of NF-B, complexes binding
to an oligonucleotide containing a B consensus sequence were
analyzed (Santa Cruz). Nuclear extracts were prepared as described
previously (28)
. A sample of 2 µg of nuclear protein was
used per binding reaction, and protein-DNA complexes were resolved on a
5% polyacrylamide gel in 0.25x Tris borate-EDTA buffer at 120 V for
2 h. Gels were dried and exposed to film (X-Omat Blue XB-1, Kodak,
Rochester, NY). Shifted complexes were quantitated by phosphoimager
analysis (Bio-Rad). To determine the subcomposition of the complexes,
supershift reactions were performed by preincubation of the nuclear
extracts with antibodies specific to the RelA or p50 subunits of
NF-B (Santa Cruz).
Transfections and plasmids
For the assessment of NF-B transcriptional activity during
differentiation, stable cell lines were created to contain either the
6B-TK-luciferase (NF-B reporter) plasmid or the empty
TK-luciferase plasmid, both kindly provided by Dr. Patrick Baeuerle
(Micromet, Germany). Cells, 8 x 105/400
µl in GM, were transfected by electroporation (Genepulser, Bio-Rad)
at 240 V in the presence of 2.5 µg of the plasmid DNA of interest,
and 0.7 µg of a plasmid containing the neomycin resistance gene
(pSV2-Neo, Stratagene, La Jolla, CA). For selection of positive clones,
cells were cultured in GM containing 850 µg/ml geneticin (Life
Technologies). Transient transfections were performed by calcium
phosphate precipitation according to the manufacturer’s instructions
(Specialty Media, Phillipsburg, NJ). The total amount of plasmid DNA
was kept constant at 25 µg. pBIISK (Stratagene) was used as carrier
DNA, and pSV-ß-gal (1 µg/transfection) (Promega) was employed to
correct for differences in transfection efficiency. The
6B-TK-luciferase and the troponin I (TnI)-luciferase plasmid, the
latter kindly provided by Dr. Albert Baldwin (University of North
Carolina, Chapel Hill, NC), were used as reporters for transcriptional
activity of NF-B or muscle-specific transcription factors,
respectively (1 µg/transfection). To inhibit NF-B activation,
cells were transiently transfected (6 µg/transfection) with a plasmid
encoding IB-SR, which was constitutively expressed under control
of the Friend spleen focus-forming virus 5'-long terminal repeat
(SFFV-LTR) (pSFFV-NEO IB-SR), kindly provided by Dr. Rosa Ten
(Mayo Clinic, Rochester, MN), or an empty vector as a control. For
constitutive activation of NF-B, cells were transiently transfected
with a plasmid encoding IKK-ß, kindly provided by Dr. Michael Karin
(University of California, San Diego, La Jolla, CA). To determine
luciferase and ß-galactosidase acitivity, cells were lysed in 1x
luciferase lysis buffer and were stored at -80°C. Luciferase
(Promega) and ß-galactosidase (Tropix, Bedford, MA) were measured
according to manufacturer’s instructions.
Immunofluorescence
To assess the extent of myogenic differentiation in cells
transfected with IB-SR or control vector after treatment with
TNF-, the expression of myogenin and IB-super-repressor
(SR)-FLAG was assessed by using immunofluorescence. Cells were grown,
manipulated, and fixed on glass coverslips. A biotinylated antibody (10
µg/ml, M2, Sigma) directed against the FLAG epitope, in combination
with a streptavidin-conjugated Alexa fluorophore (10 µg/ml, Alexa
488, Molecular Probes, Eugene, OR), was used to identify
IB-SR-transfected cells. Simultaneously, an antibody recognizing
myogenin (10 µg/ml, M225, Santa Cruz) combined with an anti-rabbit
allophycocyanin-conjugated antibody (10 µg/ml, Molecular Probes) was
applied. Immunoreactivity was evaluated with fluorescent microscopy
(Olympus BX50) and was analyzed for colocalization by confocal scanning
laser microscopy (Bio-Rad MRC 1024 ES).
Statistical analysis
Raw data were entered into SPSS (version 8.0) for statistical
analysis. Values for CK activity, myogenic index, relative DNA binding
activity, LDH activity, 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).
Time-dependent decrements in luciferase activity for
6B-TK-luciferase and TK-luciferase were assessed by using multiple
regression analysis. A two-way ANOVA was employed to address whether
myoblasts and myotubes activated NF-B (assessed by EMSA and
6B-TK-luciferase activity) similarly in response to TNF-.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, was found to increase with differentiation
(Fig. 1A
). Small increases in CK activity became apparent within 2
days after induction of differentiation, and a striking augmentation
was observed between days 2 and 9. Increases in CK activity
corresponded with increases in expression of MyHCf, a major
myofibrillar protein (29)
, and followed the expression of
myogenin, a key muscle-specific transcription factor (30)
(Fig. 1
B
, C
, respectively).
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Effect of TNF- on differentiated myotubes
To investigate the effect of the inflammatory cytokine TNF- on
the abundance of muscle-specific proteins in differentiated myotubes,
cells were cultured in DM for 5 days, followed by an additional 24- or
72-h incubation in the presence or absence of TNF-. Because
catabolic effects of TNF- are well documented (19)
, we
determined the total protein content per dish after exposure to the
cytokine. However, the total protein content was not altered after
24 h of culture in the presence of the cytokine (data not shown).
In fact, 72 h after the addition of TNF- to the myotube
cultures, a dose-dependent increase in total protein content became
apparent (Table 1
), indicating that TNF- did not exert a catabolic effect in myotubes.
CK activity was decreased 72 h after exposure to TNF-
(Fig. 2A
). In addition, slight reductions in MyHCf content were
detected in lysates of myotubes treated with TNF- for 24 h
(Fig. 2B
). Overall, the effects of TNF- on differentiated
myotubes were marginal and occurred only at the highest dose (50 ng/ml)
of cytokine.
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Effect of TNF- on differentiating myoblasts
We next assessed the effects of TNF- during the myogenic
differentiation process. As shown in Fig. 3A
, addition of TNF- immediately after induction of
differentiation markedly inhibited the formation of myotubes.
This effect of the cytokine was further evidenced by a dose-dependent
decrease of the myogenic index (Fig. 3B
). In addition, the
total protein content per dish was lower when myoblasts were cultured
in DM for 72 h in the presence of TNF- (Table 1)
. The
appearance of biochemical differentiation markers was also blocked by
TNF-. For instance, CK activity (Fig. 3C
), MyHCf content
(Fig. 3D
), and myogenin expression (Fig. 3E
) were
suppressed in a dose-dependent manner in differentiating myocytes
treated with the cytokine, even though all of these parameters were
normalized for total protein. These results illustrate that TNF-
preferentially affects myogenesis in differentiating myoblasts, whereas
differentiated myotubes are more refractory to the effects of TNF-
on muscle protein expression. Concentrations of TNF- that interfere
with myogenic differentiation here have been documented in the
circulation of patients with cachexia (9)
.
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TNF-: cytotoxicity and reversibility
To investigate whether the loss of myogenesis in differentiating
myoblasts was the result of cytotoxicity by TNF-, cell death was
assessed. LDH activity in the culture media, resulting from its release
by necrotic cells, was determined as a measure of cell death.
Increasing LDH activity was found in control cells during the
differentiation process, reflecting a population of cells that appeared
to be incapable of survival under growth-restricting conditions. The
presence of TNF- increased LDH release in a dose-dependent manner
during differentiation (Fig. 4A
). Analysis of cell death by flow cytometry, using the
fluorimetric exclusion dye Sytox, confirmed the rise in cell death
inherent to differentiation and the additional increase after TNF-
(data not shown). Further investigation revealed that the observed
necrosis could be secondary to an apoptotic mechanism, as myoblasts
treated with TNF- for 48 and 72 h demonstrated a dose-dependent
elevation in the sub-G0/G1
fraction, as shown by staining with propidium iodide and analyzed by
flow cytometry (Fig. 4B
). Surprisingly, the total number of
adherent cells increased after 48 h of exposure to TNF-,
illustrating that this cytokine causes proliferation in the first
48 h of myogenic differentiation (Fig. 4C
). After
72 h, however, cell numbers after TNF- treatment were equal to
control cell numbers again, which had remained constant, suggesting
that additional cell death observed in the presence of TNF- during
this period (Fig. 4A
, B
) may in fact result from
apoptosis of these myocytes that are initially proliferating. We next
tested whether the effect of TNF- on differentiation was reversible,
which would not expected to be the case for a nonspecific cytotoxic
signal. TNF- was washed out after 48 h of culture, and fresh DM
was added to the myocytes. Although LDH activity continued to increase
after removal of the cytokine (Fig. 5C
), myogenesis was restored completely and rapidly, as was
demonstrated by CK activity (Fig. 5A
) and myogenin (Fig. 5B
). These results confirm that the inhibitory effects of
TNF- on myogenic differentiation are not due to a nonspecific toxic
effect.
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NF-B in myoblasts and myotubes
To investigate potential mechanisms responsible for the observed
inhibition of myogenic differentiation, we focused on NF-B, a
transcription factor that has a central role in inflammatory responses
and is known to be activated by inflammatory cytokines such as TNF-
and IL-1ß (21)
. Exposure to TNF- caused a marked and
sustained activation of NF-B DNA binding activity (Fig. 6A
) and transactivation (Fig. 6B
) in response to
TNF-. Analysis of the subcomposition of the retarded complexes by
supershift assay, using antibodies directed against the RelA and p50
subunits of NF-B, revealed that the major complex induced by TNF-
(upper band) was composed of a RelA-p50 heterodimer, and possibly
another RelA hetero- or homodimer, because preincubation with the p50
antibody did not result in a complete shift of this band (Fig. 6A
). As expected, NF-B DNA binding activity and
transactivation in response to TNF- were preceded by rapid
degradation of IB (Fig. 6C
), as determined by Western
blotting using an antibody directed against IB (sc371, Santa
Cruz).
|
We next determined how NF-B activity correlated with myogenic
differentiation. Results in Fig. 7
demonstrate baseline DNA binding activity of NF-B complexes composed
of both RelA-p50 and p50 homodimers in unstimulated, undifferentiated
cells. Basal levels of NF-B DNA binding activity decreased during
myogenic differentiation, after an initial increase in binding by the
p50 homodimer (Fig. 7A
). The use of C2C12 cells that were
stably transfected with 6B-TK-luciferase revealed that basal
transcriptional activity of NF-B also decreased during muscle
differentiation. In contrast, no changes were observed in a cell line
containing the (control) TK-luciferase construct (Fig. 7B
).
The decrease in NF-B activity was accompanied by increasing levels
of IBß, whereas IB abundance remained constant during
differentiation (Fig. 7C
). However, the inducibility of
NF-B was the same during or after differentiation, as exposure to
TNF- increased NF-B DNA binding activity (Fig. 7D
,
upper panel) and transcriptional activity (Fig. 7D
, lower
panel) to a similar extent in myoblasts and differentiated myotubes.
These results demonstrated an inverse relation between basal NF-B
activity and myogenic differentiation. Because IL-1, like TNF-, is
known to activate NF-B, differentiating myoblasts were treated with
this cytokine (IL-1ß, 0.1–10 ng/ml). As observed for TNF-,
IL-1ß inhibited myogenic differentiation in a dose-dependent manner,
as was revealed by measurement of CK activity and assessment of MyHCf
expression (data not shown).
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NF-B inhibits myogenic differentiation
To determine whether NF-B activation was causally involved in
the inhibition of myogenic differentiation in response to TNF- or
IL-1ß, cells were transiently transfected with IB-SR, a
nondegradable mutant of IB, and a troponin promoter-luciferase
reporter plasmid (TnI-luciferase), which is expressed only in
differentiated muscle. As expected, expression of IB-SR reduced
NF-B transactivation by TNF- (Fig. 8A
) and IL-1ß (Fig. 8B
). It is important to note
that overexpression of IB-SR enhanced myogenic differentiation
(Fig. 9A
) and restored the loss of TnI-luciferase expression (Fig. 9B
) in response to exposure to these cytokines. The causal
role of NF-B in the TNF--induced inhibition of myogenic
differentiation was also illustrated by immunofluorescence, which
revealed that only IB-SR-positive cells expressed myogenin when
cultured in DM in the presence of TNF- (Fig. 10
). Finally, overexpression of IKKß, which resulted in constitutive
activation of NF-B (Fig. 8B
), dose dependently blocked
TnI-luciferase expression (Fig. 9C
), further emphasizing the
causal role of NF-B activation in the inhibition of myogenic
differentiation.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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(7
8
9
10
, 31)
. Furthermore, chronic administration
of TNF- or IL-1ß (14)
, or the implantation of a
TNF--producing tumor (12)
, results in muscle wasting.
Because the involvement of catabolic hormones or other mediators could
not be ruled out in those studies, it is not clear whether muscle
wasting is mediated through a direct effect of these inflammatory
cytokines on skeletal muscle. In the present study, the effects of
TNF- on C2C12 cells were analyzed during and after their
differentiation. Undifferentiated C2C12 cells, or myoblasts, are the in
vitro equivalent of satellite cells, which constitute the myogenic stem
cell population in postnatal skeletal muscle (32)
.
Although present as a low percentage of total myonuclei in adult
skeletal muscle, satellite cells are indispensable in the maintenance
of functional muscle because they possess the capacity to proliferate
and differentiate to replace lost or injured muscle fibers. Our
findings demonstrate dramatically different responses to TNF- in
differentiating myoblasts vs. fully differentiated myotubes. First,
when cultured in the presence of TNF-, differentiating myoblasts
demonstrated a decreased protein content per dish compared with
controls, whereas differentiated myotubes contained more protein per
dish. The latter result is in contrast to that from a study in which a
catabolic effect of TNF- on total protein content of differentiated
myotubes was reported (19)
. Second, although some loss in
MyHCf content and CK activity was observed after exposure of myotubes
to TNF-, these decreases were marginal. In contrast, addition of
TNF- to differentiating myoblasts completely inhibited myogenic
differentiation at concentrations of 1–10 ng/ml, which have been
reported in the circulation in patients with chronic inflammatory
diseases (31
, 33
, 34)
. In support of our findings, others
have also reported marked inhibitory effects of TNF- on myogenic
differentiation (18
, 22
, 35
, 36)
and have failed to
demonstrate effects in differentiated myotubes (18
, 35)
. A
recent study also demonstrated this differential effect of TNF- but
showed that myotubes became susceptible to TNF- once interferon-
was administered in addition to TNF- (37)
. This result
is a strong indication that a secondary signaling event is required in
the sensitization of myotubes to TNF- and could involve the
activation of STAT1 (38)
.
TNF- has been shown to evoke toxic effects in a variety of cell
types (39
, 40)
and proliferative responses in other cell
types (41)
. Our study also demonstrates that in the
presence of TNF-, a subpopulation of myoblasts undergoes cell death
when cultured in DM, which is evidenced by increased LDH levels in the
medium. However, the inhibition of myogenesis was completely reversible
after removal of the cytokine, indicative of a specific inhibitory
effect of TNF-. We speculate that the observed cell death here may
involve apoptosis of the fraction of myoblasts, which initially
responded to TNF- by inappropriate proliferation.
Similar to treatment with TNF-, exposure of differentiating myocytes
to IL-1ß (0.1–10 ng/ml) impaired myogenesis, as indicated by
decreased CK activity and MyHCf expression (data not shown). We
therefore propose that the inhibition of myogenic differentiation of
satellite cells, resulting from a sustained elevation in the levels of
circulating inflammatory cytokines, may constitute an important
mechanism leading to muscle wasting in chronic inflammatory conditions,
through impairment of the regeneration process.
Binding of TNF- and IL-1 to their respective receptors can trigger
multiple pathways that, depending on the adaptor complexes that are
associated with the cytoplasmic domain, can converge at the level of
the IB-kinase (IKK) complex, resulting in the activation of NF-B
(20)
. We tested here whether TNF-- or IL-1-induced
activation of NF-B was causally involved in the inhibition of
myogenic differentiation. We first demonstrated that baseline DNA
binding and transcriptional activity of NF-B markedly decreased
during differentiation, in agreement with findings of others (42
, 43)
. More detailed analysis revealed that the overall decrease
in DNA binding activity was preceded by an initial increase in p50
homodimer binding acitivity. Such a transient rise in NF-B DNA
binding activity during the initial phase of myogenic differentiation
has been described (44)
. In that study it was concluded,
on the basis of additional data demonstrating a loss of myogenesis
following the addition of nonspecific NF-B inhibitors, that NF-B
activation is required for myogenic differentiation to occur. However,
NF-B transactivation was not assessed during any phase of
differentiation, nor was the composition of the NF-B complex
contributing to the increased DNA binding activity identified. Because
the DNA binding activity of the p50 homodimer was selectively increased
in the initial phase of differentiation in our system, we believe that
it may in fact function as a repressor of NF-B-dependent
transcription, as the p50 subunit does not contain a transactivation
domain but can still occupy NF-B binding sites (45)
.
This idea is further supported by the observation that NF-B
transactivation decreased during differentiation (Fig. 7B
).
In addition, IBß levels increased during differentiation, whereas
IB abundance remained constant. The decrease in NF-B DNA
binding and transcriptional activity during differentiation coincided
with increasing expression of muscle-specific proteins and fusion of
cells into myotubes. Others have reported that reduction of basal
NF-B activity, via retroviral transfer of IB-SR or by addition
of the NF-B inhibitor curcumin, enhanced myogenic differentiation
(43
, 46)
.
These observations led us to further investigate the link between
TNF-- or IL-1ß-induced NF-B activation and the observed
inhibition of myogenic differentiation. We report here that expression
of IB-SR in differentiating myoblasts abrogated TNF-- or
IL-1ß-induced NF-B transactivation and, more important, restored
myogenic differentiation in the presence of these cytokines,
demonstrating that TNF- and IL-1ß block myogenic differentiation
through the activation of NF-B. Despite equivalent activation of
NF-B in myoblasts and myotubes (Fig. 7D
), only myoblasts
appear to be adversely affected by TNF-, illustrating that the cells
can respond to TNF- in both stages and that the lack of effects of
TNF- in myotubes is not the result of a loss of TNF- signaling.
These observations contrast with a report that demonstrated enhanced
activation of NF-B in C2C12 myotubes compared with myoblasts,
presumably because of the higher expression of TNF receptor-associated
factor 2 (TRAF-2) in the myotubes (47)
. Possibly, these
discrepancies can be attributed to the different protocols used to
allow differentiation of the C2C12 cells, or to differences in the
transfection procedures that were used.
An explanation of why myocytes are uniquely susceptible to TNF-
during differentiation may be linked to the cell cycle machinery. For
example, it has been demonstrated that cyclin D1 is transcriptionally
regulated by NF-B (43)
. Initiation of myogenic
differentiation is preceded by exit from the cell cycle, an event that
requires down-regulation of cyclin D1. Therefore, activation of NF-B
by TNF- and subsequent expression of cyclin D1 may inhibit
myogenesis in myoblasts by preventing cell cycle exit, whereas
expression of cyclin D1 is irrelevant in myotubes, because they
irreversibly reside in
G0/G1 (48)
. In
support of this, we observed an increase in cell number after 24 and
48 h of culture in DM when TNF- was present (Fig. 4D
), which coincided with an increased fraction of cells
residing in the S and G2/M phases of the cell
cycle (data not shown). Alternatively, the activity of the
muscle-specific basic-helix-loop-helix transcription factors MyoD and
Myf-5, which have proved to be indispensable for myogenic
differentiation (49)
, could be repressed as a direct
consequence of NF-B activation. In support of the latter
alternative, it has been shown that the catalytic subunit of protein
kinase A (PKAc) is associated with active NF-B (50)
,
whereas another study demonstrated that PKA can inactivate MyoD and
Myf-5 (51)
. Another mechanism by which MyoD may be
inhibited by NF-B has been described very recently. This study
demonstrated that the MyoD mRNA half-life decreased in differentiating
myoblasts following TNF- exposure and that this depended on
NF-B-regulated gene expression (37)
.
In summary, we have demonstrated that the inflammatory cytokines
TNF- and IL-1ß interfere with myogenic differentiation. Once
skeletal muscle cells have differentiated, they appear to be become
relatively resistant to these cytokines. Therefore, it is possible that
the loss of skeletal muscle associated with chronic inflammatory
disease states is the result of an inability of satellite cells to
differentiate into functional fibers after damage or degeneration.
Although therapeutic interventions for skeletal muscle wasting are
usually designed to restore imbalances in protein anabolism and
catabolism, our results suggest that intervention strategies should
also focus on stimulation or restoration of satellite cell
differentiation, because this process may be compromised under
conditions of chronic inflammation. Besides the administration of
agents that have been demonstrated to increase muscle mass in healthy
individuals, such as specific nutritional supplements, hormonal
treatment, and insulin-like growth factor I, specific intracellular
pathways such as the activation of NF-B may have to be
modulated in cachectic patients for these agents to have an
effect.
|
ACKNOWLEDGMENTS |
|---|
Received for publication August 9, 2000.
Revision received November 20, 2000.
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