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INTRODUCTION |
Rel/NF-
B
(NF-
B)1 is a dynamic
transcription factor family involved in the regulation of innate immune
response, cellular proliferation and differentiation, and cell survival
(1-3). In mammalian cells this family consist of RelA/p65, c-Rel,
RelB, p50 (p105 precursor) and p52 (p100 precursor). These proteins are
distinguished by a REL homology domain contained in the amino terminus,
which specify protein dimerization, DNA binding, and the nuclear
localization signal (NLS) (1-3). This family can be further subdivided
into proteins that contain a transactivation domain at their carboxyl
terminus (p65, c-Rel, and RelB) and those that do not (p50 and p52).
Although in vitro, each of these proteins possesses the
ability to homo- or heterodimerize, the prototypic form of NF-
B
consists of the p50/p65 heterodimer. In most cells, the majority of
NF-
B resides in the cytoplasm, bound to the I
B inhibitory protein
family, which include I
B
, I
B
, I
B
, Bcl-3, p100, and
p105. These proteins function as inhibitors through ankyrin repeats,
which bind to the REL domain of NF-
B and mask the NLS site thus
preventing NF-
B nuclear translocation (1-3).
Activation of NF-
B is mediated through the I
B kinase complex
(IKK), which functions to phosphorylate two serine residues on I
B
proteins (4). Phosphorylation of these residues causes the
ubiquitination and subsequent degradation of I
B proteins by the 26 S
proteasome complex (4). Upon loss of I
B, NF-
B is free to
translocate to the nucleus where it binds to its cognate DNA sequence
and interacts with the basal transcription machinery and
transcriptional co-activators to stimulate gene expression (5). One of
the numerous genes induced by NF-
B is its own inhibitor, I
B
(6-8). Once resynthesized, usually within 1 h of NF-
B
activation depending on the activating signal and cell type, I
B
is transported to the nucleus where it binds and inhibits NF-
B DNA
binding (9). NF-
B is sequestered back to the cytoplasm through a
nuclear export signal located in the amino terminus of I
B
(10,
11). In this fashion, the rapid resynthesis of I
B
ensures the
equally rapid turnover of NF-
B activity.
NF-
B activation is stimulated by a wide variety of both intra- and
extracellular stimuli, including inflammatory cytokines, viral and
bacterial products, growth factors, and pro-oncogenic signals (12).
Among the proinflammatory cytokines, tumor necrosis factor
(TNF
)
is one of the most potent activators of NF-
B in cell types
possessing TNF receptors (13). TNF is produced primarily from
macrophages and functions in the early phases of infection by
activating and recruiting other immune cells through the production of
chemokines and other proinflammatory cytokines (14). Overproduction of
TNF is also thought to contribute to the pathophysiology of several
diseases including septic shock, cancer, AIDS, diabetes, and rheumatoid
arthritis (14). In cancer and AIDS, chronic production of TNF has been
further linked to the degeneration of skeletal muscle associated with
tissue wasting or cachexia (15, 16).
Skeletal muscle wasting involves the proteolytic degradation of
myofibrillar proteins as well as the inability to synthesize new muscle
gene products (15, 16). In vitro, TNF has been shown to
function as a potent inhibitor of skeletal myogenesis (17, 18). This
activity is mediated through NF-
B (17), which promotes the
down-regulation of the myogenic bHLH transcription factor, MyoD (19).
Absence of MyoD is known to impair skeletal myogenesis (20), and
compromise the efficiency of muscle regeneration in response to injury
(21). In contrast, TNF activity alone is not sufficient to cause full
degradation of mature muscle (22), which has prompted the notion that
TNF functions in concert with other proinflammatory cytokines, such as
interleukin-1
(IL-1), interleukin-6 (IL-6), and interferon
(IFN
) to induce wasting (16, 23). Consistent with this
thinking, recent evidence showed that the combined
treatment of skeletal muscle with TNF and IFN
induced strong
down-regulation of muscle-specific gene products (17). Importantly,
cytokine-mediated muscle loss was prevented in myotubes lacking NF-
B
activity, suggesting that this transcription factor may function as a
critical regulator of skeletal muscle integrity.
To gain greater insight on the role of NF-
B in cytokine-induced
muscle wasting, the present study was undertaken to elucidate the
regulation of NF-
B in response to TNF and IFN
signaling in
differentiated skeletal muscle. Since IFN
has not been demonstrated on its own to activate NF-
B, our initial goal was to determine whether IFN
signaling could synergize with TNF to activate NF-
B to a threshold level required for muscle decay. Utilizing
differentiated C2C12 muscle cultures, results showed that IFN
did
not potentiate TNF-induced activation of NF-
B, suggesting that the
synergistic action of TNF and IFN
to induce muscle loss functions
downstream from the initial point of NF-
B activation. However, in
the course of this analysis, we observed that treatment of C2C12
myotubes with TNF alone caused a clear and pronounced biphasic activity of NF-
B. In contrast to the first phase, which was potent but transient, the activity of the second phase was equally potent but
persisted, lasting an additional 24-36 h following TNF treatment. This
type of NF-
B activity profile is unlike the classical scheme represented by a single transient phase. It also differs from the more
commonly described chronic or persistent activity of NF-
B. Thus, in
this study differentiated skeletal muscle cells were utilized to
characterize the biphasic activation pattern of NF-
B in response to
TNF, and to determine its biological significance with respect to
cytokine-induced muscle wasting.
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EXPERIMENTAL PROCEDURES |
Reagents--
Human and murine TNF
were purchased from
Promega and Roche Molecular Biochemicals, respectively. Recombinant
IL-1 and IL-6 were purchased from Promega, IFN
from Invitrogen and
LPS from Alexis. Antibodies p50 (NLS), I
B
(C-21), I
B
(C-20), I
B
(G-4), and Myf-5 (C-20) were from Santa Cruz
Biotechnology. Antibody p65 was obtained from Rockland,
anti-phospho-I
B
(Ser-32) from Cell Signaling, anti-
-tubulin
(B-5), and anti-myosin heavy chain (MY-32) from Sigma. A Cdk4 antibody
was generously provided by Y. Xiong (University of North Carolina,
Chapel Hill). Secondary anti-rabbit and anti-mouse IgG antibodies
conjugated to horseradish peroxidase were purchased from Promega.
Oregon Red goat anti-mouse IgG was purchased from Molecular Probes. The
proteasome inhibitor MG-132 and p38 MAP kinase inhibitor SB203580 were
obtained from Calbiochem. IKK inhibitors Bay 11-7085 was obtained from
Biomol and PS-1145 was generously provided by J. Adams (Millennium
Pharmaceuticals). Protease and phosphatase inhibitor cocktails were
purchased from Sigma.
Cell Culture--
Murine C2C12 myoblasts were obtained from ATCC
and primary murine myoblasts were provided by J. Samulski (University
of North Carolina, Chapel Hill). Cells were grown in Dulbecco's
modified Eagle's medium with high glucose (DMEM-H), supplemented with
20% fetal bovine serum and antibiotics (Invitrogen). Cells were
passaged every 2-3 days. For the induction of differentiation, cells
were trypsinized and replated at 60-70% confluence in growth medium. The following day, cells were washed once in phosphate-buffered saline
(PBS), and then switched to DMEM-H supplemented with 2% horse serum
and 10 µg/ml insulin.
EMSAs and Western Blot Procedures--
Nuclear extracts in EMSAs
were performed as previously described (24). Briefly, 5 µg of nuclear
extract was incubated with 1 mM phenylmethylsulfonyl
fluoride and 1 µg of poly(dI-dC)-poly(dI-dC) (Amersham Biosciences)
for 10 min at room temperature. To this mixture 2 × 104 cpm of a 32P-labeled oligonucleotide probe
corresponding to the promoter of the class I major histocompatibility
complex gene (5'-CAGGGC TGGGGATTCCCCATCTCCACAGTTTCACTTC- 3') was added
(NF-
B binding site is underlined) in a buffer consisting of 10 mM Tris-HCl, pH 7.7, 50 mM NaCl, 0.5 mM EDTA, 1 mM dithiothreitol, and 10% glycerol. Complexes were resolved on a non-denaturing 5%
polyacrylamide gel, and then subsequently exposed for 4-8 h on X-OMAT
film (Kodak). For supershifts, antibodies raised against specific
subunits of NF-
B were preincubated with nuclear extracts for 10 min
at room temperature before the addition of phenylmethlsulfonyl fluoride and poly(dI-dC)-poly(dI-dC).
For Western blot analysis, extracts were prepared as previously
described in the presence of protease inhibitors (24). For detection of
phospho-I
B
, phosphatase inhibitors were also included in extract
buffers. Following protein fractionation on SDS-polyacrylamide gels and
protein transfer to nitrocellulose membranes (Gelman Corp.), blocking
was performed for overnight in 5% nonfat dry milk in a buffer
consisting of 25 mM Tris-HCl, pH 8.0, 125 mM NaCl, and 0.1% Tween 20 (TBST). Primary and secondary antibodies were
diluted in 0.5% nonfat dry milk in TBST, and incubated from 1-2 h
room temperature. The only exception was the anti-phospho-I
B
antibody, which was diluted in 5% milk and incubated overnight at
4 °C. Membranes were treated with enhanced chemiluminescence (PerkinElmer Life Sciences), and proteins were visualized either by
exposing to X-OMAT film or by using the ChemiDoc gel documentation system (Bio-Rad Laboratories, CA).
Northern and RT-PCR Analyses--
Total RNA was isolated with
TRIzol (Invitrogen), fractionated on a 1.4% agarose gel and
subsequently transferred overnight to a charged nylon membrane
(Biodyne, Gelman Corporation). RNA was cross-linked with a UV
crosslinker (Stratagene), and prehybridized with QuickHyb (Stratagene).
Random prime probes were generated with Rediprime II (Amersham
Biosciences) and [
-32P]dCTP. Probes were boiled with
100 µg/ml salmon sperm DNA and hybridized for 1 h at 68 °C.
Membranes were washed two times in 2× SSC (1× is 0.15 M
NaCl, 0.015 M sodium citrate, pH 7.0), 0.1% SDS at room
temperature for 10 min. followed by one wash in 0.1× SSC, 0.1% SDS at
65 °C for 30 min. For semiquantitative RT-PCR analysis, 2 µg of
total RNA from differentiated C2C12 myotubes were used in reactions
with Access RT-PCR according to the manufacturer (Promega). Forward
(CGGCATGGATCTCAAAGACAACC) and reverse (GAAGACTCCTCCCAGGTATATGG) primers
were used to amplify a 252-bp fragment from the murine TNF
mRNA.
Forward (GCTGTGAAGATACGAGAGAGAACC) and reverse
(CACATGTACTGACAAGCTGCATGC) primers were used to amplify a 290-bp
fragment from the murine A20 gene.
Generation of Cell Lines--
C2C12 myoblasts stably expressing
a wild type (3x
B-Luc) or mutated (3x
B-Mut-Luc) NF-
B-responsive
luciferase reporter, and cells expressing the I
B
-SR transgene
were previously described (24). The stable expression of the A20 gene
in C2C12 cells was performed by transiently transfecting 2 µg of a
human A20 expression plasmid in myoblasts grown on a 60-mm culture
dishes. 48 h post-transfection, cells were passaged at 1:50 and
1:100 of their density in the presence of 1 mg of Geneticin. Resistant
clones were expanded and pooled and tested for their expression of A20
by semiquantitative RT-PCR analysis using forward
(GTGAAGATACGGGAGAGAACTCC) and reverse (GTACCAAGTCTGTGTCCTGAACG)
primers, which amplifies a 310-bp product. The generation of
MyoD-expressing fibroblasts was accomplished by infecting I
B
+/+
and
/
fibroblasts with a MyoD-expressing retrovirus as previously
described (17).
Immunofluorescence--
Staining experiments were performed as
previously described (24). Briefly, following incubations cells were
gently rinsed 3× with PBS. C2C12 myotubes were fixed in 2%
formaldehyde/1× PBS solution for 30 min. Cells were permeabilized with
0.5% Nonidet P-40, PBS for 5 min at room temperature and then blocked
with horse serum 1:100 dilution in PBS. Cells were incubated for 1 h with anti-skeletal myosin heavy chain diluted at 1:500 in 3% bovine
serum albumin, PBS followed by a 1-h incubation in the dark with an
anti-mouse Oregon Red IgG (Molecular Probes) diluted at 1:250 in 3%
bovine serum albumin, PBS. Following additional washes with PBS and
water, cells were mounted with coverslips and examined on a Nikon
fluorescent microscope equipped with a SPOT digital camera.
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RESULTS |
Loss of Muscle-specific Gene Products by TNF
and IFN
Is
Dependent on NF-
B Activity--
TNF is strongly linked with muscle
wasting associated with cancer-induced cachexia (15). Studies indicate,
however, that activities of other proinflammatory cytokines, including
IL-1, IL-6, or IFN
, are required along with TNF to induce loss of
skeletal muscle proteins (25-27). Specifically, combinatorial
treatment of mature muscle with TNF and IFN
was recently shown to
promote pronounced decreases in the levels of the muscle-specific
transcription factor MyoD (17). Consistent with these previous
findings, we again found that treatments of C2C12 myotube cultures with
TNF + IFN
led to the dramatic down-regulation of MyoD protein
expression (Fig. 1A). In
contrast, treatment of differentiated C2C12 cells with TNF and IL-1 or
TNF and IL-6 produced no changes in MyoD expression. These effects by
TNF + IFN
treatment were specific, since similar decreases in
protein levels were not observed with the closely related muscle
transcription factor, Myf-5 (19). In addition, treatments with both TNF + IFN
led to a significant decrease in the expression of the
myofibrillar protein, myosin heavy chain (MHC) (Fig. 1, B
and C). Importantly, loss of MyoD and MHC was prevented in
myotubes stably expressing the NF-
B transdominant inhibitor,
I
B
-SR (non-degradable form of I
B
), supporting the notion
that NF-
B activity is required for cytokine-induced muscle loss.

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Fig. 1.
NF- B is required for
cytokine-induced muscle protein loss. A, C2C12 vector
or I B SR stably expressed myoblasts were differentiated in
DM for a period of 3 days. At this time, cultures were refed
with fresh DM supplemented with either no addition (untreated) or
treated for two 24-h periods with TNF (20 ng/ml) with either IL-1 (20 ng/ml), IL-6 (20 ng/ml), or IFN (100 units/ml). Whole cell extracts
were prepared, and 50 µg of total protein was used in Western blot
analysis to probe for I B and I B SR (which is epitope-tagged
and therefore is a slower migrating protein), MyoD, Myf-5, and
-tubulin used as a loading control. B, vector control or
I B SR-expressing myotubes were treated as described above, and
subsequently stained by immunofluorescence to detect for MHC
expression. Images are shown at 20× magnification digitally captured
by a SPOT camera. C, from immunostained myotubes as
described in B, MHC expression was quantitated using BioRad
Quantity One software. The data were calculated from the area of
MHC staining from a minimum of 20 randomly chosen fields of
cells, under different treatment conditions, visualized at 4×
magnification.
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TNF Induces a Biphasic Activation of NF-
B in Skeletal
Muscle--
Although IFN
is not known to induce NF-
B activity
directly, it has been demonstrated to potentiate the activation of
NF-
B in response to TNF (28). We therefore questioned whether the synergy observed between TNF and IFN
signaling that led to the loss
of MyoD and MHC resulted from such a potentiation in NF-
B activity.
To address this question, we treated C2C12 myotube cultures with either
cytokine alone or in combination and monitored for NF-
B activity.
Treatment with TNF alone caused the expected rapid but transient
induction of NF-
B (Fig.
2A). Activation levels peaked
at 30 min post-TNF treatment and returned near basal levels by 1 h. In contrast, IFN
treatment alone did not induce NF-
B activity,
nor did IFN
potentiate the activity of NF-
B in the presence of
TNF (Fig. 2A). Similar results were obtained when treatments
were repeated for longer time periods (Fig. 2B). These results suggest that the ability of IFN
to synergize with TNF to
induce muscle loss is not associated with an enhanced NF-
B activity.

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Fig. 2.
TNF induces a biphasic activation of
NF- B in C2C12 myofibers. A and
B, IFN does not potentiate TNF-induced activation of
NF- B. C2C12 myotubes were treated with either TNF (20 ng/ml) or
IFN (100 units/ml) or a combination of both cytokines, and at
indicated times, nuclear extracts were prepared and NF- B was
monitored by EMSA. C, EMSA was performed, monitoring NF- B
binding activity from myotubes treated with TNF for up to 48 h.
D, extracts obtained from treated samples, as described in
C, were used in supershift EMSA by incubating with
antibodies raised against either the p50 or p65 subunit of NF- B.
Arrowheads denote supershifted complexes. E,
NF- B biphasic activation correlates with p65 nuclear translocation.
Nuclear extracts (30 µg) prepared from myotubes treated with TNF, as
described in C were used in Western blot analyses probing
for p65, cdk4, or Myf-5.
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During the course of this analysis we made the observation that
following the initial activation phase of NF-
B in response to TNF, a
second NF-
B activation phase developed. This second phase of
activity was nearly as potent as the first, but unlike the first phase
whose signal terminated within 1 h, second phase activity
persisted for an additional 24-36 h (Fig. 2C). To identify NF-
B subunits contributing to DNA binding activities, supershift EMSAs were performed. Myotube nuclei were observed to contain predominantly p50 and p65 subunits, and as previously determined (24),
no binding activity was detected with other NF-
B subunits, p52,
c-Rel, or RelB (data not shown). Furthermore, equivalent binding of
p50/p65 subunits were observed in both phases of NF-
B activity in
response to TNF, demonstrating that no apparent changes in binding
complexes occurred during the biphasic activation (Fig. 2D).
To address whether the biphasic profile identified by EMSA analysis
reflected simply changes in NF-
B DNA binding activity, or rather
represented changes in nuclear translocation events, nuclear Western
analyses were performed. As expected, a clear increase in nuclear p65
was observed within 30 min of TNF treatment, reflecting NF-
B nuclear
translocation during the first phase of activation (Fig.
2E). Levels of nuclear p65 decreased within 1 h, but
then increased a second time beginning at 6 h out to 48 h
post-TNF treatment, suggestive of a second nuclear translocation event.
This effect was specific to p65 since no change in nuclear protein
expression was detected for the cyclin-dependent kinase, cdk4, or the muscle-specific transcription factor, Myf-5.
Next we addressed the specificity of this biphasic activation profile
with respect to TNF. Since IL-1 and LPS are also potent inducers of
NF-
B (1, 2), we used these agents to treat differentiated C2C12
muscle cultures. Results showed that similar to TNF, both IL-1 and LPS
could induce the first phase of NF-
B activity with comparable
kinetics, but neither agent promoted the second activation phase (Fig.
3), suggestive that, in differentiated skeletal muscle, the biphasic activation of NF-
B may be specific to
TNF-mediated signaling.

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Fig. 3.
NF- B biphasic
activation may be specific to TNF signaling. C2C12 myotubes were
treated with TNF (20 ng/ml), IL-1 (20 ng/ml), or LPS (10 µg/ml) for
various times, and NF- B activity was monitored by EMSA.
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One of the caveats in using the C2C12 culture system is that these
cells are immortalized, and thus under differentiation conditions one
typically observes a substantial number of satellite cells that fail to
fuse into multinucleated myotubes (Fig.
4A). Given this limitation,
the possibility existed that the biphasic activation profile of NF-
B
that we had observed was occurring in the undifferentiated satellite
cells rather than in differentiated myotubes. To address this point, we
utilized primary muscle cells, which in contrast to C2C12 cells are
capable of full myotube conversion when induced to differentiate (Fig.
4A). Results showed that in TNF-treated primary myotube
cultures, NF-
B was also activated in a biphasic manner similar to
that seen in C2C12 differentiated cells (Fig. 4B).
Supershift EMSA analysis was also consistent with results obtained in
C2C12 cells, demonstrating the presence of both p50 and p65 subunits of
NF-
B during both phases of DNA binding activity (Fig.
4C). These data thus demonstrate that the biphasic
activation of NF-
B occurs in differentiated skeletal muscle.

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Fig. 4.
TNF induces the biphasic activation of
NF- B in primary myotubes. A,
C2C12 (upper panel) or primary myoblasts (lower
panel) were differentiated for 2 or 3 days, respectively in DM. In
C2C12 cultures, arrowheads denote cells that were unable to
complete their differentiation and that remain as myoblasts.
B, primary myotube cultures were treated with TNF for
indicated times and NF- B activity was monitored by EMSA.
C, supershift EMSA was performed with nuclear extracts
prepared for EMSA in B and incubated with antibodies raised
against NF- B subunits p50 or p65.
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The Biphasic Activity of NF-
B in Response to TNF Is
Transcriptionally Competent--
Next we addressed whether NF-
B DNA
binding activity correlated with transcriptional competency. To make
this determination, reporter assays were performed using a C2C12 cell
line containing a stably integrated NF-
B-responsive heterologous
promoter (3x
B-Luc). These cells were induced to differentiate into
myotubes and subsequently treated with TNF. Results showed that NF-
B
transcriptional activity was regulated in a similar biphasic fashion
(Fig. 5A). In contrast, no
activation was observed by TNF when the assay was repeated with C2C12
cells containing a mutant version of the NF-
B promoter (3x
B-Mut-Luc), indicating that this biphasic regulation of
transcriptional activity is specific to NF-
B. To further assess
NF-
B transcriptional activity, we monitored the expression of a
known NF-
B-regulated gene. Consistent with reporter data, results
showed that I
B
gene expression was maximally induced after 1 h of TNF treatment, which then decreased over the next few hours, but
increased again steadily over a 24-h period (Fig. 5B). Taken
together, these results demonstrate that NF-
B is transcriptionally
competent during each activation phase in skeletal muscle in response
to TNF.

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Fig. 5.
TNF induces NF- B
biphasic transcriptional activity. A, C2C12 myoblasts
containing a stable NF- B-responsive promoter (3x B-Luc), or a
mutated NF- B promoter (3x B-Mut-Luc) were differentiated into
myotubes, and treated with TNF. At indicated times cell extracts were
prepared, and luciferase assays were performed. B, RNA
prepared from TNF-treated myotubes was used in Northern blot analysis
probing for I B . The blot was stripped and reprobed for
glyceraldehyde-3-phosphate dehydrogenase to control for RNA
loading.
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The Biphasic Activity of NF-
B Is Regulated by I
B
Proteins--
In response to most inducing stimuli, classical
activation of NF-
B occurs from the stimulation of the IKK complex,
leading to the direct phosphorylation of two serine residues contained on each of I
B
, I
B
, or I
B
proteins (4). In response to TNF and other pro-inflammatory stimuli, this activity is mediated specifically by the
-subunit of IKK. The phosphorylation of I
B proteins leads to their ubiquitination and subsequent degradation via
the 26 S proteasome. Degradation of I
B proteins unmasks the nuclear
localization signal of NF-
B allowing for nuclear translocation and
NF-
B-dependent transcription (4). Termination of NF-
B DNA binding and transcriptional activity is largely mediated by NF-
B-dependent resynthesis of I
B
, which binds and
exports NF-
B from the nucleus back to the cytosol (10, 11).
Having partially characterized the biphasic activation profile of
NF-
B in skeletal muscle, we next set out to address its regulatory
mechanism. To initiate this analysis we first asked whether the
biphasic regulation was dependent on proteasome activity. Pre-incubation of C2C12 myotube cultures with the proteasome inhibitor, MG-132 completely blocked TNF-mediated activation of NF-
B (Fig. 6A), indicative that the first
phase of activity is proteasome-dependent. To assess the
requirement of proteasome activity in the second phase of NF-
B
activation, the proteasome inhibitor was added to cells 1 h
post-TNF treatment, a time shown to precedes the initiation of the
second phase, as demonstrated by EMSA and reporter data. In comparison
to vehicle-treated myotubes, MG-132 strongly inhibited NF-
B activity
in the second activation phase (Fig. 6B), demonstrating that
the proteasome is required throughout the biphasic regulation of
NF-
B. Next, we assessed IKK activity during biphasic regulation by
monitoring the phosphorylated state of I
B
. TNF treatment of C2C12
myotubes caused the rapid phosphorylation of I
B
, which decreased
within 30 min in conjunction with the degradation of the inhibitor
protein (Fig. 6C). At 1 h post-TNF treatment,
resynthesized I
B
was again highly phosphorylated, suggesting that
IKK activity is maintained throughout the first transient activation
phase of NF-
B. To examine the status of IKK during the second
activation phase, C2C12 myotubes were treated with TNF for 1 h,
and subsequently supplemented with proteasome inhibitor to inhibit the
degradation of I
B
. Results showed that in MG132-treated cells,
I
B
phosphorylation was highly maintained during the second phase
of NF-
B activity (Fig. 6D), indicating that biphasic
activation of NF-
B is mediated through a
IKK/proteasome-dependent pathway.

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Fig. 6.
Biphasic activation of
NF- B activation by TNF is regulated by the
IKK/proteasome pathway and I B
proteins. A and B, both phases of
NF- B activity in response to TNF is
proteasome-dependent. C2C12 myotubes were incubated with
either vehicle alone (DMSO, 0.1%) or the proteasome
inhibitor, MG-132 (50 µM) for 1 h prior to the
addition of TNF (A), or added at 1 h post-TNF treatment
(B). At indicated times, nuclear extracts were prepared, and
NF- B activity was determined by EMSA. C and D,
IKK is active during both phases of NF- B activity. Myotubes were
treated with TNF throughout (C) or at 1 h supplemented
with either vehicle or MG-132 (D). At indicated times,
cytoplasmic extracts were prepared, and Western blot analyses were
performed probing for the phosphorylation of Ser-32 on I B or
total I B expression. E, TNF induces the
down-regulation of I B proteins. Myotubes were treated with TNF, and
at indicated times cytoplasmic extracts were prepared. Western blot
analyses were subsequently performed with 30 µg of extracts probing
for I B , I B , and I B .
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|
In light of the above results, we next examined levels of cytoplasmic
I
B proteins during TNF treatment. With respect to I
B
, initial
rapid degradation and resynthesis was followed by a second degradative
regulation starting at 2 h (Fig. 6C), and levels
remained down-regulated throughout a 48-h treatment period (Fig.
6E). With I
B
, expression also decreased at 30 min in
response to cytokine treatment, but unlike I
B
, was not
resynthesized and instead was persistently down-regulated. In contrast
to
and
forms, we repeatedly observed that I
B
was not
degraded by TNF treatment, but rather levels of this protein increased
over time. Thus, biphasic activation of NF-
B correlates with loss of
both I
B
and I
B
proteins.
To more closely examine the role of I
B
in the regulation of
NF-
B biphasic activity, TNF treatments were performed on myotubes stably expressing the NF-
B transdominant inhibitor, I
B
-SR, which is incapable of degradation in response to an NF-
B stimuli. Results showed that in contrast to vector control myotubes, which displayed a pronounced biphasic activation pattern of NF-
B,
I
B
-SR-expressing myotubes lacked both first and second phases
(Fig. 7A). Consistent with
this finding, levels of I
B
-SR protein was unchanged during TNF
treatment, in comparison to I
B
in vector control cells (Fig. 7B). These results not only demonstrate that the first
activation phase is a prerequisite for the second, but they also
confirm that I
B
degradation is essential for the induction of the
first transient phase. Interestingly, under these experimental
conditions, I
B
down-regulation was observed in both vector
control and I
B
-SR-expressing myotubes (Fig. 7B),
suggestive that regulation of the first activation phase is specific to
I
B
.

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Fig. 7.
I B regulates
the first transient phase of NF- B
activity. A, degradation of I B is required for
the induction of NF- B. Vector control or I B -SR-expressing
myotubes were treated with TNF, and at indicated times nuclear extracts
were prepared to analyze NF- B activity by EMSA (A), and
cytoplasmic extracts were prepared to probe for levels of I B and
I B (B). C-E, I B resynthesis is
required for the termination of the first activation phase.
C, I B +/+ and / fibroblasts were infected with
retrovirus expressing the MyoD transgene (pBabeMyoD). Cells were
selected by puromycin treatment, clones were pooled, and MyoD
expression was verified by Western blotting. D,
MyoD-expressing I B +/+ and / fibroblasts were grown to near
confluency and either maintained in growth medium (GM) or
induced to differentiate (DM). Myotube phenotype was
identified by morphology and by immunofluorescence staining for MHC.
E, I B +/+ and / myotube cultures were treated with
TNF, and at selected times, nuclear and cytoplasmic extracts were
prepared to analyze NF- B activity (upper panel) or levels
of I B (lower panel).
|
|
Based on the above results, we next examined the requirement of
I
B
resynthesis in the termination of this first activation phase.
To make this determination, I
B
+/+ and
/
fibroblasts (29)
were converted to skeletal muscle cells by expression of the MyoD
transgene (Fig. 7C). Under differentiation conditions, both
I
B
wild type and null cells were readily converted to
myosin-expressing myotubes (Fig. 7D), although consistently
fewer and smaller myotubes were detected in null cells. TNF treatment
of I
B
+/+ skeletal muscle cultures induced typical NF-
B
transient activity, correlating with the degradation and resynthesis of
I
B
(Fig. 7E). In contrast, induced NF-
B activity
was sustained in I
B
/
-treated muscle cultures, indicating
that I
B
resynthesis is required for the inactivation of NF-
B
within the first phase.
Having identified that I
B
is a required factor regulating both
the activation and termination of NF-
B activity within the transient
first phase in response to TNF, we next set out to gain insight on the
role of I
B
and I
B
in the second activation phase of
NF-
B. Examination of cytoplasmic extracts had showed that both
I
B
and I
B
were persistently down-regulated during the
second activation phase (Fig. 6E), indicating that I
B
proteins may also be involved in the regulation of this latter phase.
In support of this notion, we observed that removal of TNF following the first activation phase abolished the induction of the second phase,
both at the level of NF-
B DNA binding and transactivation function
(Fig. 8, A and B,
compare wash versus TNF lanes).
Importantly, inhibition of the second phase correlated with restored
levels of both I
B
and I
B
proteins (Fig. 8C),
suggesting that chronic TNF signaling may be required for the
persistent down-regulation of I
B proteins in order to maintain
second activation phase of NF-
B.

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Fig. 8.
Second activation phase of
NF- B correlates with down-regulation of
I B proteins, dependent on continuous TNF
signaling. A, the second phase of NF- B activity is
dependent on exogenous TNF. A and B, C2C12
myotubes were treated with TNF for 1 h and either left alone
(TNF) or washed extensively with PBS and subsequently refed
with fresh differentiation medium (wash). At indicated
times, nuclear extracts were preparedm and NF- B DNA binding activity
was determined (A), or total RNA was prepared from
cytoplasmic extracts and NF- B transactivation function was assessed
by Northern analysis probing for I B (B). C,
second phase activation correlates with down-regulation of I B
proteins. C2C12 myotubes were treated as in A, and at
indicated times cytoplasmic extracts were prepared and probed for I B
proteins by Western analyses.
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|
A20 Contributes to the Regulation of NF-
B Biphasic Activity in
Response to TNF--
In addition to the role of I
B
in the
regulation of NF-
B in differentiated muscle cells, we sought to
expand our analysis of this regulatory mechanism by examining other
potential factors that function upstream of this inhibitory protein.
One such candidate is the zinc finger protein A20, whose expression is
induced by TNF in an NF-
B-dependent manner (30). A20
functions as an inhibitor of NF-
B activity by interfering with TNF
receptor signaling at multiple points in the transduction pathway
(31-33). To analyze the potential regulatory role of A20 in
differentiated muscle cells, we first examined A20 expression in
response to TNF. Consistent with findings in other cell types (34), TNF
treatment of myotubes induced maximal expression of A20 gene expression
within 30 min to 1 h, and expression returned to near basal levels
by 2 h (Fig. 9A). This
was in sharp contrast to the biphasic expression profile of both an
NF-
B-responsive reporter and the I
B
gene, as seen in Fig. 5.
To examine whether induction of A20 contributes to the regulation of
NF-
B biphasic activity, skeletal muscle cells were generated to
overexpress the A20 gene (Fig. 9B). Vector control and A20
expressing C2C12 cells were differentiated and subsequently treated
with TNF. In comparison to vector control cells, overexpression of A20
in myotubes caused a substantial reduction of NF-
B activation (Fig.
9C). Consistent with the inhibitory function of A20 on IKK activity, the phosphorylation state of I
B
was also strongly down-regulated in A20-expressing myotubes, which correlated with a
reduction in I
B
degradation. These results therefore demonstrate that, in addition to the resynthesis I
B
, TNF-mediated induction of A20 leads to the regulation of NF-
B biphasic activity.

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Fig. 9.
A20 regulates biphasic activation of
NF- B. A, C2C12 myotubes were
treated with TNF. At indicated times, total RNA was prepared and
Northern analysis was performed probing for A20 (upper
panel). These results were confirmed by repeating the experimental
conditions outlined in A and by performing semiquantitative
RT-PCR for A20 gene expression (lower panel). B,
C2C12 myoblasts were transfected with a vector control plasmid or an
expression plasmid for the human A20 gene. Drug-resistant clones were
pooled and A20 expression was verified by RT-PCR. C,
A20-expressing myoblasts were induced to differentiate into myotube
cultures, and subsequently treated with TNF. At indicated times,
nuclear extracts were prepared to analyze NF- B activity (upper
panel), and cytoplasmic extracts were prepared to probe for the
phosphorylated and unphosphorylated states of I B (lower
panel)
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|
Biphasic Activity of NF-
B Is Required for Cytokine-induced
Skeletal Muscle Protein Loss--
Having characterized the biphasic
activity of NF-
B in skeletal muscle, and analyzed the regulatory
mechanism, we concluded this study by addressing its physiological
significance. Specifically, we wanted to investigate whether both
phases of NF-
B activity were required for cytokine-mediated damage
of skeletal muscle. As shown in Fig. 1, inhibition of NF-
B activity
through the stable expression of the I
B
-SR transgene in C2C12
myotubes strongly blocked TNF/IFN-dependent muscle protein
loss. Stable expression of the I
B
-SR in these cells was also
shown to inhibit NF-
B activation in response to TNF (Fig.
7A), confirming that at least the first phase of NF-
B
activity is required for cytokine-mediated loss of muscle specific
proteins. To address whether the second phase also plays a role in this
regulation, we sought to selectively inhibit NF-
B activity during
this activation phase. Our initial attempt to transiently express the
I
B
-SR transgene in myotubes by use of an adenoviral gene delivery
system was unsuccessful (data not shown). As an alternative approach,
NF-
B inhibition was selectively inhibited by the use of IKK
inhibitor compounds, Bay 11-7085 and PS-1145. Consistent with previous
findings (35), IKK compounds inhibited NF-
B activation in C2C12
myotube cultures in a dose-dependent manner (Fig.
10A). These inhibitors were
then used to treat myotubes 1 h following the addition of TNF + IFN
(thereby allowing the first phase of NF-
B activity to occur), and the levels of muscle-specific gene products were subsequently analyzed. Our analysis showed that both NF-
B inhibitor compounds significantly blocked cytokine-mediated loss of MHC and MyoD expression (Fig. 10, B and C). The fact that these
inhibitors were not able to completely restore MyoD expression most
likely results from the gradual inactivation of these compounds in
conditioned medium, as well as from the regulatory events that occurred
during the first phase of NF-
B activity. To address the specificity
of these inhibitors, C2C12 myotubes were treated with increasing doses of SB203580, an inhibitor of the p38 stress-activated mitogen-activated protein kinase. In contrast to the IKK inhibitors, p38 inhibition did
not abrogate the ability of cytokines to decrease MyoD expression (Fig.
10C). Taken together, these results indicate that
cytokine-mediated loss of muscle-specific proteins is dependent on both
the first and second phases of NF-
B activity in response to TNF
signaling.

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Fig. 10.
Biphasic activity of
NF- B is required for cytokine-induced loss of
muscle proteins. A, Bay 11-7085 and PS-1145 block
TNF-induced NF- B activation in C2C12 myotubes. Myotube cultures were
preincubated with increasing concentrations of IKK inhibitor compounds
for 90 min prior to a 15-min treatment of TNF (20 ng/ml). Nuclear and
cytoplasmic extracts were prepared and either assayed for NF- B
activity by EMSA (upper panel) or probed for I B
expression by Western blotting (IB, lower panel).
IKK inhibitors block cytokine-induce loss of skeletal muscle proteins.
B, C2C12 myotubes were treated with TNF + IFN for 1 h and subsequently supplemented with either no additional treatment
(untreated) or treated with dimethyl sulfoxide, Bay 11-7085 (10 µM), or PS-1145 (20 µM) for two 24-h
periods. At this time, cells were fixed, and immunofluorescence was
performed probing for MHC as described under "Experimental
Procedures." C, myotube cultures were treated with TNF + IFN as in B and subsequently supplemented with increasing
doses of Bay 11-7085 (5 and 10 µM), PS-1145 (10 and 20 µM), or the p38 inhibitor compound SB203580 (1 and 5 µM). Cell lysates were prepared, and Western analyses
were performed probing for MyoD and -tubulin, used as a loading
control.
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DISCUSSION |
The basis of this study was founded on previous results showing
that skeletal muscle protein loss depended on the combined signaling
activities of inflammatory cytokines TNF and IFN
(17). Results also
demonstrated that NF-
B activity was required for cytokines to induce
muscle damage (17). To gain further insight into how NF-
B
potentially functions in cytokine-induced muscle wasting, we sought to
first examine how these cytokines signal to NF-
B in differentiated
muscle. Unlike the earlier described synergistic actions of TNF and
IFN
on NF-
B signaling in endothelial and neuronal cell types
(28), our current results demonstrate that addition of IFN
does not
potentiate TNF-induced activity of NF-
B. However, in the course of
this analysis we discovered that in skeletal muscle, NF-
B activity
was regulated in a biphasic manner by TNF. In contrast to the first
phase of NF-
B activity, which was potent but transient, the second
identified phase was nearly as potent, and it persisted out to nearly 2 days following initial TNF treatment. Importantly, two other known
inducers of NF-
B, IL-1 or LPS, did not induce this biphasic
activity, suggesting that this regulation on NF-
B may be specific to
certain NF-
B inducing signals.
Biochemical and genetic analyses have soundly established that NF-
B
is an inducible transcription factor, where the majority is maintained
as an inactive complex through the cytoplasmic retention by I
B
inhibitors (1, 2, 36). Stimulation of cells by a wide variety of
signals leads to proteolytic degradation of I
B proteins, and
subsequent nuclear translocation of NF-
B. It has also been well
accepted that activation of NF-
B, by most physiological inducing
signals, is a transient response, reaching its peak usually within 30 min followed by a return to basal levels within 1-4 h. The transient
nature of NF-
B activity is in part due to the resynthesis of
I
B
(6-8), which binds and exports NF-
B from the nucleus back
to the cytoplasm (10, 11). Depending on the inducer and/or cell type,
however, other activation profiles of NF-
B have been described. By
far the most common of these profiles is the described persistent or
constitutive activity of NF-
B that occurs when NF-
B activity does
not return back to its basal state following its initial induction
phase. Ghosh and co-workers (37) had earlier described such a
phenomenon in B cells treated with LPS. In these cells, the persistent
activity of NF-
B was derived from its binding to a
hypophosphorylated form of I
B
, which protects NF-
B from
I
B
binding and inactivation, thereby facilitating NF-
B nuclear
import. A similar mechanism of protection by I
B
was attributed to
the persistent activity of NF-
B observed in myeloid cells infected
with the HIV virus (38). Other mechanisms have been presented to
account for such a persistent activity. For example, the human T-cell
leukemia virus type 1 protein, Tax, has been shown to bind and
stimulate the IKK complex resulting in chronic NF-
B activity (39,
40). In mature B cells, evidence suggests that a calcium-mediated
signaling pathway accounts for the chronic degradation of I
B
,
leading to constitutively high levels of c-Rel activity (41). In
addition, continual loss of I
B
has been attributed to the
prolonged activation state of NF-
B in glial cells treated with IL-1
(42). Furthermore, persistent activity of NF-
B is often associated
with pathophysiological states such as cancer (12).
In contrast to these types of activation profiles, biphasic activity of
NF-
B, as described in this report, is a type of regulatory profile
less well described, and whose regulatory mechanisms and biological
relevance remain poorly understood. With respect to TNF signaling, Han
and Brasier (43) described such a phenomenon in cultured hepatocytes,
in which the duration of the second phase of NF-
B activity was
measured for 6 h correlating with the loss of I
B
. Kemler and
Fontana (44) also detected biphasic activity of NF-
B in glial and
neuroblastoma cell lines for up to 4 h post-TNF treatment, and in
this case activation was shown to result from the combined losses of
both I
B
and I
B
proteins. Similar biphasic activity has been
described in response to other stimuli including viral infection (45),
and most recently, LPS signaling (46). Given these results, in
conjunction with our current data acquired in skeletal muscle, it would
appear that the ability of NF-
B to undergo biphasic activation is
not cell type- or stimuli-specific, nor is it restricted to a single
regulatory mechanism. In addition, the recent citing that NF-
B
undergoes biphasic activation in rat skeletal muscle in response to
ischemia/reperfusion, suggest that biphasic regulation of NF-
B is
relevant in this tissue in vivo (47).
Unclear to this point however is the significance of this regulatory
profile. Are the changes in NF-
B DNA binding activity reflected in
its transcriptional activity, and what is the biological relevance of
the second phase? At least in skeletal muscle, we have revealed that
NF-
B is transcriptionally competent in both phases of its DNA
binding activation profile. This was demonstrated by a reporter-based
assay and also confirmed by analyzing the gene expression profiles of
NF-
B responsive genes, I
B
and TNF
(Fig. 5 and data not
shown, respectively). In regard to the biological relevance of the
second phase, our data demonstrate that this may be associated with the
strength and duration of activation. In skeletal muscle, the strength
of activity of this second phase was seen to nearly match that of the
first, and in sharp contrast to other cell types (43, 44), remained
active for nearly 48 h following cytokine treatment.
Interestingly, this time coincides with the same period at which
significant decreases of muscle specific proteins were detected in
cytokine treated myotubes (Fig. 1). Data showing that the inhibition of
NF-
B activity during this second phase blocked cytokine-mediated
decreases in both MyoD and MHC would suggest that NF-
B biphasic
activity is required for skeletal muscle decay. Although it has not yet
been elucidated, it is likely that the second phase of NF-
B activity
will be found to regulate multiple biological functions in various cell types.
Use of the differentiated skeletal muscle cell system has revealed much
regarding this biphasic activity of NF-
B in response to TNF
signaling. Our results demonstrate that both activation phases are
derived from independent nuclear translocation events, which contain
NF-
B complexes that, as stated above, are transcriptionally competent. The results obtained in both C2C12 and primary skeletal myotubes also reveal that the subunit composition of NF-
B is seemingly identical during each activation phase. This indicates that
following the termination of the first phase, where the p65 subunit is
exported from the nucleus, a similar transcription factor complex is
poised once again to be activated and undergo a second round of nuclear
translocation. The data also argue that both activation phases were
induced through the IKK/26 S proteasome signaling pathway.
Collectively, these results portray a profile of NF-
B activity,
which would appear to be defined by two identical regulatory mechanisms.
Despite these similarities, our results revealed distinguishing
features between each activation phase. The most obvious is the
duration of NF-
B activity during each phase. While the first phase
resembled a typical transient activation profile, where NF-
B
activity is quickly terminated, the second phase persisted for nearly
an additional 48 h. Our results using the I
B
-SR showed that
the induction of the first phase is dependent on the rapid degradation
of I
B
. The additional use of I
B
-null muscle cultures demonstrated that the termination of this first phase is also dependent
on the resynthesis of I
B
within the first hour of TNF treatment.
Although a similar analysis was not conducted with A20-null myotubes,
our results support the notion that the observed rapid induction of A20
in response to TNF treatment functions in conjunction with I
B
to
terminate the first phase of NF-
B. Termination of this phase most
likely occurs both through the nuclear export of NF-
B by I
B
,
as well as through the inhibition of TNF receptor signaling and IKK
activation by A20. Results further demonstrated that induction of the
first phase is a prerequisite for the second more persistent phase of
NF-
B. In the presence of TNF, both I
B
and I
B
proteins
are persistently down-regulated. In addition, removal of TNF from
myotube cultures (as shown in Fig. 8) caused the termination of this
second phase and correlatively restored levels of I
B proteins. These
results support the conclusion that regulation of both I
B
and
I
B
proteins define the duration of the second phase of NF-
B
activity. However, results also indicated that down-regulation of
I
B
occurred even in the absence of the second phase of NF-
B
activity (Fig. 7), bringing into question the essentialness of
degradation during this regulatory process. The development of an
NF-
B transdominant inhibitor form of I
B
(analogous to
I
B
-SR) will be required to formally address the role of this
I
B protein in the first and second phases of NF-
B. Another
contributing factor regulating the second activation phase may be
related to the expression of A20. Interestingly, in contrast to the
biphasic expression pattern observed with an NF-
B responsive reporter and the I
B
gene, A20 expression was not found to be biphasic. In light of previous findings that absence of A20 in fibroblasts leads to chronic down-regulation of I
B
and persistent activity of NF-
B (48), it is tempting to speculate that the persistent second phase of NF-
B activity results from the absence of
A20 resynthesis.
Despite these current findings, it remains unknown at this point how
inflammatory cytokines IFN
and TNF signal in skeletal myotubes to
promote muscle loss. With respect to TNF signal transduction, we have
elucidated that the biphasic activation of NF-
B is required in order
for both TNF and IFN
to mediate the loss of muscle specific gene
products. Importantly, biphasic activation of NF-
B required that
myotube cultures be continuously treated with exogenous TNF (Fig. 8).
Elevated levels of TNF are often associated with chronic inflammatory
conditions (14). This suggest that the biphasic regulation of NF-
B
is likely to reflect a pathophysiological condition where TNF levels
remain high in the circulation, as is often the case in cancer cachexia
(15). Our results also showed that IFN
did not potentiate the
ability of TNF to activate NF-
B (Fig. 2). This would imply that the
synergism exhibited by these cytokines occurs as a result of
independent activation of their respective downstream effectors such as
NF-
B for TNF and STATs/IRFs for IFN
(49). As seen in other
instances (50), these signaling molecules may converge further
downstream, possibly on a common promoter to regulate the expression of
a yet unidentified gene that may potentially be a critical regulator of
skeletal muscle wasting associated in cancer-induced cachexia.