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INTRODUCTION |
The IGFs1 are the only
known growth factors that are crucial to myogenesis (1). IGF-I and
IGF-II switch on the myogenic program through the IGF-I receptor (2),
activating the expression of myogenic transcription factors, cell cycle
arrest, muscle-specific protein expression, and cell fusion to form
multinucleated myotubes (3, 4). PI3K is an essential second messenger
for myogenesis (5-9). We have recently described a myogenic signaling
cascade initiated by IGF-II that leads to biochemical and morphological skeletal muscle cell differentiation and that involves PI3K activation, NF-
B activation, and inducible nitric-oxide synthase expression and
activation (10). In this report, we further analyze the role of the
NF-
B-activating signaling cascade in myogenesis. NF-
B
transcription factors are key mediators of inflammatory responses,
immune system functioning, transformation, oncogenesis, and
anti-apoptotic signaling (11-13). NF-
B exists in the cytoplasm in
an inactive form by virtue of its association with inhibitory proteins
termed I
B (11-15). NF-
B translocation to the nucleus and
activation are most frequently achieved through the signal-induced proteolytic degradation of I
B in the cytoplasm. Two kinases, IKK
and IKK
, which are contained in a high-molecular-weight multiprotein
complex, show inducible I
B kinase activity and play a key role in
NF-
B activation by a variety of stimuli (16-19). Despite their high
sequence similarity, IKK
and IKK
have different regulatory and
functional roles. In mice lacking IKK
, the activation of NF-
B by
cytokines is abolished, and mouse embryos die on days 12-13 of
gestation due to massive liver apoptosis (20). In contrast, IKK
is
dispensable for pro-inflammatory responses, but plays an essential role
in embryonic development. Mice lacking IKK
exhibit defective
proliferation and differentiation of epidermal keratinocytes and
defective limb and skeletal patterning (21, 22). IKK
and IKK
are
themselves phosphorylated and activated by one or more upstream
kinases, like NIK, which is a member of the mitogen-activating protein
kinase kinase kinase family (23-25).
We report here that I
B
phosphorylation at Ser-32 and Ser-36 is
required for both IGF-II-dependent NF-
B activation and
differentiation in L6E9 myoblasts. We show that IKK
is involved in
IGF-II-dependent multinucleated myotube formation and
muscle-specific gene expression, whereas IKK
is not essential
for these processes. Our data suggest that NIK activation triggers
myogenin expression and multinucleated myotube formation in the absence
of IGF-II.
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EXPERIMENTAL PROCEDURES |
Materials--
IGF-II was kindly given by Lilly. The rat
skeletal muscle cell line L6E9 was kindly provided by Dr. B. Nadal-Ginard (Harvard University). Low-passage 293 cells were from
Microbix (Ontario, Canada). The PI3K inhibitor LY294002 was from BIOMOL
Research Labs Inc. (Plymouth Meeting, PA). The NF-
B probe for
electrophoretic mobility shift assay was kindly given by Dr.
Jean-François Peyron (INSERM U364, Nice, France). The cDNAs
encoding FLAG-IKK
, FLAG-IKK
(K44A), FLAG-IKK
, and
FLAG-IKK
(K44A) were provided by Dr. D. Goeddel (Tularik, Inc.). The
cDNA encoding GST-I
B
-(1-54) was provided by Dr. M. Karin
(University of California, San Diego, CA). Recombinant adenoviral
vectors expressing I
B
(S32A/S36A), kinase-proficient FLAG-tagged
NIK, LacZ, and green fluorescent protein (GFP) were provided by Dr.
Yibin Wang (University of Maryland, Baltimore, MD). Polyclonal antibody
C38320 raised against caveolin-3 was from Transduction Laboratories
(Lexington, KY). Mouse monoclonal antibody MF20, which stains all
sarcomeric myosin heavy chain isoforms, and mouse anti-rat myogenin
monoclonal antibody F5D were from the Developmental Studies Hybridoma
Bank. Polyclonal antibodies against I
B
(C-15), IKK
,
IKK
, and NIK were from Santa Cruz Biotechnology (Santa Cruz, CA).
Anti-
-actin (clone AC-15) and anti-FLAG M2 monoclonal antibodies
were from Sigma.
Cell Culture--
Rat L6E9 myoblasts were cultured as described
previously (5). Subconfluent myoblasts were differentiated by serum
depletion in DMEM plus antibiotics with or without IGF-II (40 nM) in the absence or presence of other compounds, as
indicated for each experiment. Cells were photographed after staining
the nuclei with Mayer's hemalum solution for microscopy (Merck,
Darmstadt, Germany), and cell fusion was quantified by counting nuclei
in myotubes from a total of at least 1000 nuclei from 10-20 randomly selected microscope fields for each condition. For adenoviral transduction, subconfluent L6E9 myoblasts were transduced at a multiplicity of 50-100 particles/cell and then cultured for an additional 36 h before inducing differentiation with or without IGF-II. To compare the impact of IKK
and IKK
on myoblast
differentiation, experimental conditions were selected to ensure
similar levels of expression of IKK
and IKK
constructs (data not shown).
Construction of Recombinant Adenoviruses--
Recombinant
adenoviruses expressing FLAG-tagged versions of either wild-type or
dominant-negative mutant K44A human IKK
and IKK
were generated by
homologous recombination as described by Graham and Prevec (38).
cDNAs were cloned into the shuttle plasmid pAdl1/RSV and
cotransfected with pJM17 into 293 cells to achieve homologous
recombination. Individual plaques were isolated and checked for
recombinant protein expression after infection of 293 cells.
Recombinant adenoviruses were further amplified in 293 cells; purified
by cesium chloride gradient centrifugation; dialyzed against 1 mM MgCl2, 10 mM Tris (pH 7.4), and
10% glycerol; and stored at
80 °C (39). Viral stocks were
titrated by infecting 293 cells with serial dilutions of the
preparation and observing the cytopathic effect on the cells 48 h
after infection. An infectious titer was given assuming that a
multiplicity of infection of 10 is required to cause a complete
cytopathic effect at 48 h. In addition, the
A260 of the preparation was measured to estimate the particle titer (1 A260 unit = 1012 particles/ml).
Electrophoresis and Immunoblotting of Membranes--
Cells were
lysed for 30 min at 4 °C in 50 mM Tris (pH 7.5), 120 mM NaCl, 1 mM EDTA, 6 mM EGTA, 15 mM Na4P2O7, 20 mM NaF, 0.1% phenylmethylsulfonyl fluoride, and 0.1%
aprotinin supplemented with 1% Nonidet P-40. Cell extracts were
centrifuged at 10,000 × g for 15 min at 4 °C, and
50 µg of the solubilized proteins was loaded. SDS-polyacrylamide gel
electrophoresis and immunoblot analysis were performed as described
previously (5).
Immunoprecipitation and Kinase Assays--
Cells were washed in
PBS and resuspended at 106 cells/10 µl in hypotonic
solution (10 mM Hepes (pH 7.8), 10 mM KCl, 2 mM MgCl2, 1 mM dithiothreitol, 0.1 mM EDTA, 15 mM
Na4P2O7, 20 mM NaF,
0.1% phenylmethylsulfonyl fluoride, and 0.1% aprotinin). After 10 min at 4 °C, Nonidet P-40 was added to 1%, and cells were centrifuged in a microcentrifuge at 10,000 × g for 15 min. The
supernatant containing the cytoplasmic fraction was recovered.
For immunoprecipitation, antibodies were preadsorbed on protein
G-Sepharose at 4 °C for 1 h and washed twice in hypotonic solution and 1% Nonidet P-40 before being incubated with the protein extracts for 2 h at 4 °C. The immunopellets were washed six
times in the same buffer and once in kinase buffer (20 mM
Hepes, 10 mM MgCl2, 100 µM
Na3VO4, 20 mM
-glycerophosphate,
2 mM dithiothreitol, and 50 mM NaCl (pH 7.5)).
Kinase reactions were carried out for 30 min at 30 °C using 5 µCi
of [
-32P]ATP and GST-I
B
-(1-54) as substrate
(except for NIK kinase assays, in which autophosphorylation of
immunoprecipitated NIK was analyzed). The reaction products were
analyzed on 10% polyacrylamide gels and revealed by autoradiography.
Electrophoretic Mobility Shift Assay--
NF-
B DNA-binding
activity was analyzed in total cell extracts made in Totex lysis buffer
(20 mM Hepes, 350 mM NaCl, 20% glycerol, 1%
Nonidet P-40, 1 mM MgCl2, 0.5 mM
EDTA, 0.1 mM EGTA, 1 mM dithiothreitol, 0.1%
phenylmethylsulfonyl fluoride, and 0.1% aprotinin) (10). Supernatants
from a 15-min 10,000 × g centrifugation were
collected. The NF-
B probe was a synthetic double-stranded
oligonucleotide containing the NF-
B-binding site of the
interleukin-2 gene promoter (5'-GATCCAAGGGACTTTCCATG-3').
Samples (70 µg) were incubated for 10 min at 4 °C with 30 ng of
poly(dI·dC) and 5 µl of 5× reaction buffer (50 mM Tris (pH 7.5), 500 mM NaCl, 5 mM
dithiothreitol, 5 mM EDTA, 20% glycerol, and 0.4 mg/ml
salmon sperm DNA) in a final volume of 25 µl. The end-labeled probe
was added for a further incubation of 25 min at 25 °C. The
specificity of the bands detected was verified by adding a 10-100-fold
excess of competing unlabeled NF-
B probe. NF-
B-unrelated
oligonucleotide probe controls did not show any specific binding
activity (data not shown).
Immunofluorescence--
Cells grown on coverslips were fixed for
20 min with 3% paraformaldehyde in PBS, washed three times in PBS, and
then treated as follows: (a) 10 min in PBS containing 50 mM NH4Cl, (b) 10 min in PBS
containing 20 mM glycine, and (c) 30 min in PBS
containing 10% fetal bovine serum. Subsequently, coverslips were
incubated with primary antibodies (anti-NIK polyclonal antibodies, 1 µg/ml; anti-myogenin monoclonal antibody, undiluted culture
supernatant) for 1 h at room temperature. After washing in PBS,
coverslips were incubated with fluorochrom-conjugated antibodies
(Oregon Green or Texas Red) for 45 min. Cells were washed three times in PBS and then mounted in immunofluor medium (ICN Biomedicals Inc.,
Aurora, OH). Images were obtained using a Leica TCS 4D laser confocal
fluorescence microscope with a 40× objective.
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RESULTS |
IGF-II-induced Skeletal Muscle Cell Differentiation Involves
NF-
B Signaling Cascade Activation--
IGF-II induces NF-
B
DNA-binding activity as an early event during L6E9 myoblast
differentiation (10). Most extracellular stimuli that activate the
NF-
B pathway induce phosphorylation of the NF-
B repressor
I
B
at Ser-32 and Ser-36 as a requisite for its degradation (19).
However, alternative pathways for NF-
B activation have been
described (26, 27). To analyze the mechanism required by IGF-II for
NF-
B activation during differentiation, L6E9 myoblasts were
transduced with an adenovirus expressing an I
B
mutant with Ser-32
and Ser-36 replaced by alanine residues (adv/I
B
(S32A/S36A)). This mutant inhibits NF-
B in
pathways involving serine phosphorylation and subsequent proteasome
degradation of I
B
. L6E9 myoblasts differentiated for 24 h
with IGF-II exhibited an induction of NF-
B DNA-binding activity
(Fig. 1A), which was blocked
in myoblasts overexpressing I
B
(S32A/S36A) (Fig. 1B). Myoblasts infected with an adenovirus expressing green fluorescent protein (adv/GFP) were used as control (Fig. 1B). Next, we
analyzed the impact of I
B
phosphorylation on
IGF-II-dependent myoblast differentiation. After 4 days of
IGF-II treatment, the expression of muscle-specific proteins such as
myosin heavy chain and caveolin-3 was highly decreased in myoblasts
overexpressing the I
B
(S32A/S36A) mutant (50 ± 12%,
n = 3) compared with non-transduced control cells or
cells transduced with adv/GFP, whereas the expression of the
non-muscle-specific protein
-actin was similar under all conditions
(Fig. 1C). Moreover, cells overexpressing the
I
B
(S32A/S36A) mutant did not fuse to myotubes, whereas control
cells (adv/GFP) showed 82% of the nuclei in myotubes from a total of
1661 nuclei randomly counted (Fig. 1D). These data suggest
that IGF-II requires NF-
B activation through a mechanism that
involves I
B
phosphorylation to trigger skeletal muscle cell
differentiation.

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Fig. 1.
IGF-II-induced skeletal muscle cell
differentiation requires I B
phosphorylation at Ser-32 and Ser-36 for
NF- B activation. A, total cell
extracts from L6E9 myoblasts differentiated with or without 40 nM IGF-II for 24 h were incubated with a
32P-labeled NF- B probe corresponding to the
NF- B-binding site in the interleukin-2 gene promoter and analyzed by
electrophoretic mobility shift assay. The specificity of the bands was
verified by adding a 50-fold excess of competing unlabeled NF- B
probe (cold probe) or unrelated oligonucleotide probes (data
not shown). B, total cell extracts from L6E9 myoblasts
transduced with adv/I B (S32A/S36A)
(adv/I B AA) or adv/GFP and
differentiated with IGF-II (40 nM) for 24 h were
incubated with a 32P-labeled NF- B probe and analyzed by
electrophoretic mobility shift assay. C, expression of
I B , myosin heavy chain (MHC), and caveolin-3
(Cav-3) was analyzed by immunoblotting of total cell lysates
from non-transduced myoblasts (nt) or myoblasts transduced
with adv/GFP or adv/I B (S32A/S36A) and differentiated for 4 days
with IGF-II. -Actin content was analyzed as a control of relative
amounts of proteins in each sample. D, L6E9 myoblasts
non-transduced or transduced with adv/GFP or adv/I B (S32A/S36A)
were grown to confluence and then allowed to differentiate in the
presence of IGF-II for 4 days. Morphological differentiation was
assessed by myotube formation. Images shown are representative of
15-25 microscope fields taken at random. The scale is the same for all
panels. All infections were performed in duplicate, and the results
shown are representative of three independent experiments.
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IGF-II Induces PI3K-dependent IKK
and IKK
Activation during Myoblast Differentiation--
The data presented
above indicating that I
B
phosphorylation was required by IGF-II
to induce myoblast differentiation led us to analyze the activity and
expression of IKK
and IKK
during this process. L6E9 myoblasts
exhibited a peak of IKK
activity after 24 h in
IGF-II-containing differentiation medium (Fig.
2A, upper panel).
The kinetics of IKK
activation was consistent with that of
IGF-II-dependent NF-
B DNA-binding activation, which we have previously shown to be dependent on PI3K activity (10). To analyze
whether IGF-II-dependent IKK
activation involves PI3K, L6E9 myoblast differentiation was induced with or without IGF-II in the
absence or presence of the PI3K inhibitor LY294002. The PI3K inhibitor
(20 µM) blocked the ability of IKK
to phosphorylate GST-I
B
-(1-54) in response to IGF-II (Fig. 2B,
upper panel). Neither IGF-II nor LY294002 altered IKK
protein expression (Fig. 2C, upper panel);
however, the 24-h delay between the start of the IGF-II treatment and
the activation of IKK
seemed to indicate that IKK
was not the
direct target of the IGF-induced phosphorylation cascade. This appears
to be the case, as IKK
activation by IGF-II was blocked in the
presence of 5 µg/ml cycloheximide (Fig. 2D, upper
panel, CH), indicating that it depends on de
novo protein synthesis.

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Fig. 2.
Endogenous IKK activation during
IGF-II-induced myoblast differentiation. A, cell
extracts (50 µg) from L6E9 myoblasts differentiated with IGF-II (40 nM) were immunoprecipitated (IP) with
anti-IKK (upper panel) or anti-IKK (lower
panel) antibodies (10 µg) preadsorbed on protein G-Sepharose
beads, and kinase assay was performed as described under
"Experimental Procedures" using GST-I B -(1-54) as a
substrate. Nonspecific kinase activity was analyzed using a nonimmune
antibody (data not shown). B, kinase assay was performed as
described for A with cells differentiated for 24 h in
the absence or presence of IGF-II (40 nM) with or without
LY294002 (20 µM). C, IKK expression was
analyzed by immunoblotting (IB) of cell lysates obtained
from myoblasts differentiated for 24 h in the absence or presence
of IGF-II with or without LY294002. The relative amounts of proteins in
each sample were checked by expression of the non-muscle-specific
protein -actin. D, subconfluent L6E9 myoblasts were
differentiated for 24 h in DMEM in the absence or presence of
IGF-II (40 nM) with or without cycloheximide
(CH; 5 µg/ml). Kinase assay was performed as described
above. The results shown are representative of three to four
independent experiments.
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Phosphorylation of the GST-I
B
-(1-54) substrate by IKK
was
also induced 24 h after triggering differentiation with IGF-II (Fig. 2A, lower panel). IKK
activation by
IGF-II was blocked by the PI3K inhibitor LY294002 (Fig. 2B,
lower panel). Neither IGF-II nor LY294002 treatment modified
the level of IKK
expression in differentiating myoblasts (Fig.
2C, middle panel), indicating that changes in
activity are caused by activation of the kinase rather than variations
in IKK
protein content. As for IKK
, the activation of IKK
by
IGF-II was blocked by cycloheximide (Fig. 2D, lower
panel).
IGF-II Requires IKK
, but Not IKK
, to Induce Skeletal Muscle
Cell Differentiation--
To determine whether the activation of IKKs
by IGF-II is functionally linked to myogenic differentiation, we
generated replication-deficient adenoviral vectors expressing
FLAG-tagged wild-type (adv/FLAG-IKK
and adv/FLAG-IKK
) or
dominant-negative (adv/FLAG-IKK
(K44A) and adv/FLAG-IKK
(K44A)
forms of IKK
and IKK
. Subconfluent L6E9 myoblasts were transduced
with the different recombinant adenoviruses using adv/GFP as a control;
and 36 h later, differentiation was induced by placing the cells
in an IGF-II-containing medium.
Expression and kinase activity of the transduced proteins were
evaluated 24 h after inducing differentiation with IGF-II. Cells
transduced with adv/FLAG-IKK
exhibited GST-I
B
-(1-54) phosphorylation activity in immunoprecipitates with an anti-FLAG monoclonal antibody, whereas no specific I
B
phosphorylation activity was detected in immunoprecipitates from cells transduced either with adv/GFP or adv/FLAG-IKK
(K44A) (Fig.
3A, upper panel). Under these conditions, similar levels of wild-type and
dominant-negative forms of IKK
were expressed, as verified by
immunoblotting using anti-FLAG antibody (Fig. 3A,
lower panel). After 4 days of differentiation in the
presence of IGF-II, cells overexpressing FLAG-IKK
(K44A) remained
highly undifferentiated, as measured by caveolin-3 and myosin heavy
chain expression, compared with cells transduced with either adv/GFP or
adv/FLAG-IKK
. The expression of the non-muscle-specific protein
-actin was not altered by FLAG-IKK(K44A) overexpression (Fig.
3B).

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Fig. 3.
IGF-II requires IKK
(but not IKK ) activation to induce
skeletal muscle cell differentiation. Subconfluent L6E9 myoblasts
were transduced with adv/GFP, adv/FLAG-IKK , or adv/FLAG-IKK (K44A)
(A and B) or with adv/GFP, adv/FLAG-IKK , or
adv/FLAG-IKK (K44A) (C and D). After 36 h,
cells were induced to differentiate with IGF-II. A and
C: upper panels, kinase activity of the
transduced proteins was measured in anti-FLAG immunoprecipitates
(IP) after 24 h in IGF-II differentiation medium by
in vitro phosphorylation of GST-I B -(1-54) fusion
protein. Nonspecific (ns) I B phosphorylation activity
was analyzed using a nonimmune antibody. Lower panels,
expression of transduced proteins was checked by immunoblotting using
anti-FLAG antibody. B and D, shown is the
muscle-specific protein expression in total cell lysates from
undifferentiated myoblasts (mb) or from myoblasts
differentiated for 4 days with IGF-II 36 h after being transduced
with adv/GFP, adv/FLAG-IKK , adv/FLAG-IKK (K44A), adv/FLAG-IKK ,
or adv/FLAG-IKK (K44A) or left non-transduced (nt). The
relative amounts of proteins in each sample were checked by expression
of the muscle-specific protein -actin. All infections were done in
duplicate, and the results shown are representative of three
independent experiments. MHC, myosin heavy chain.
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To analyze the role of IKK
activity in myoblast differentiation,
cells were transduced with adv/FLAG-IKK
or adv/FLAG-IKK
(K44A). Both proteins were expressed to similar levels as detected on anti-FLAG
immunoblots (Fig. 3C, lower panel), and
adv/FLAG-IKK
(K44A)-transduced cells exhibited no I
B
kinase
activity on anti-FLAG immunoprecipitates (upper panel). In
contrast to IKK
, IKK
does not seem to play an essential role in
myoblast differentiation, as cells transduced with
adv/FLAG-IKK
(K44A) expressed skeletal muscle-specific proteins as
efficiently as non-transduced cells or cells transduced with adv/GFP or
adv/FLAG-IKK
(Fig. 3D). At the morphological level, overexpression of FLAG-IKK
(K44A) did not alter the ability of myoblasts to fuse in response to IGF-II (78% of nuclei in myotubes from a total of 1488 nuclei randomly counted) compared with
non-transduced control cells (78%, total of 1616), cells transduced
with adv/GFP (82%, total of 1661), or cells transduced with
adv/FLAG-IKK
(80%, total of 1011) (Fig.
4; arrows show large
accumulations of nuclei in myotubes). Conversely, when
FLAG-IKK
(K44A) was overexpressed, only thin, spindle-like myotubes
were formed compared with the large myotubes observed in non-transduced
cells, cells transduced with adv/GFP, or cells transduced with
adv/FLAG-IKK
(Fig. 4). Indeed, after 4 days in IGF-II-containing
differentiation medium, only 25% of the nuclei (total of 1367) in
cells overexpressing FLAG-IKK
(K44A) were in myotubes with >10
nuclei. Under the same culture conditions, 77% of the nuclei from
cells overexpressing FLAG-IKK
(total of 2987) were in myotubes with
>10 nuclei. Taken together, these results suggest that IKK
plays a
relevant role in IGF-II-dependent morphological and
biochemical differentiation of skeletal muscle cells, whereas IKK
is
not essential to this process.

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Fig. 4.
Overexpression of dominant-negative
FLAG-IKK (K44A) in L6E9 myoblasts impairs
IGF-II-induced myotube formation. Subconfluent myoblasts
transduced with adv/GFP, adv/FLAG-IKK , adv/FLAG-IKK (K44A),
adv/FLAG-IKK , or adv/FLAG-IKK (K44A) were differentiated with
IGF-II for 4 days. Cells were photographed after nuclear staining, and
myotube formation was quantified. Proliferating myoblasts are also
shown. Images are representative of 25 microscope fields taken at
random from each of at least five independent experiments. The scale is
the same for all panels.
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NIK Is Activated in Differentiating Myoblasts--
NIK is a common
mediator in the NF-
B signaling cascades, and IKK
has been
reported to be a better substrate than IKK
for phosphorylation by
NIK (24). The endogenous kinase activity and protein expression of NIK
in differentiating L6E9 myoblasts were studied. Induction of NIK
autophosphorylation activity was detected after 24 h in
IGF-II-containing differentiation medium (Fig.
5A). As observed for NF-
B
DNA-binding activation (10) and IKK
and IKK
activities (Fig.
2B), the activation of NIK was totally blocked by LY294002,
indicating that IGF-II requires PI3K to activate NIK in differentiating
myoblasts (Fig. 5A). No changes were detected in NIK protein
expression in response to IGF-II or LY294002 (Fig. 5B). As
for IKK
and IKK
, NIK activation by IGF-II was blocked by
cycloheximide (Fig. 5C), indicating that it requires
de novo protein synthesis.

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Fig. 5.
NIK activation during IGF-II-induced
differentiation. A, cell extracts (50 µg) from
L6E9 myoblasts differentiated for 24 h in DMEM in the absence or
presence of IGF-II (40 nM) with or without LY294002 (20 µM) were immunoprecipitated (IP) with anti-NIK
antibody (5 µg) preadsorbed on protein G-Sepharose beads.
Autophosphorylation assay was performed as described under
"Experimental Procedures," and nonspecific (ns) NIK
activity was tested using a nonimmune antibody. B, NIK
expression was analyzed by Western blotting of cell lysates obtained at
24 h of differentiation, and the relative amounts of proteins in
each sample were checked by expression of -actin. C,
subconfluent L6E9 myoblasts were differentiated for 24 h in DMEM
in the absence or presence of IGF-II (40 nM) with or
without cycloheximide (CH; 5 µg/ml). Kinase assay was
performed as described above. D, subconfluent L6E9 myoblasts
were differentiated for 24 h with IGF-II (40 nM) with
or without LY294002 (20 mM), added either for the whole
24 h or during the last 6 h or the last 1 h of IGF-II
treatment. Kinase assay was performed at 24 h as described above.
The data shown are representative of three independent
experiments.
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Interestingly, LY294002 inhibited NIK activation only when it was
present together with IGF-II during the whole 24-h treatment. In
contrast, when LY294002 was added during the last 6 h or the last
1 h of IGF-II incubation (at 18 or 23 h of differentiation, respectively), no inhibition of NIK activity was observed (Fig. 5D). These results suggest that PI3K is most probably
involved in the de novo synthesis of the factor(s) required
for NIK activation rather than being a direct upstream element of the
NIK-activating cascade.
To test whether the activation of NIK by IGF-II was a key event during
differentiation, we transduced L6E9 myoblasts with recombinant
adenovirus expressing kinase-proficient FLAG-tagged wild-type NIK that
exhibited a high degree of basal autophosphorylation activity on
anti-FLAG or anti-NIK immunoprecipitates (Fig.
6A; the lower band
that was detected with anti-NIK antibodies in cells transduced with
adv/FLAG-NIK is probably due to cross-activation of endogenous NIK by
the overexpressed kinase). After 2 days in the absence of IGF-II
(DMEM), myoblasts infected with control adv/GFP fused poorly (12% from
a total of 1713 nuclei counted at random were found in multinucleated
myotubes) (Fig. 6B). In contrast, transduction with
adv/FLAG-NIK promoted myoblast fusion (48% of nuclei in myotubes from
a total of 1789 nuclei counted at random) (Fig. 6B).
Co-immunofluorescence assays showed that NIK-overexpressing cells
highly expressed myogenin in their nuclei (Fig. 6C). The
number of nuclei expressing myogenin was 6-fold higher in cells
overexpressing NIK (adv/NIK) than in control cells (adv/LacZ) (15 randomly selected fields were analyzed from each of two independent
experiments with each condition performed in triplicate).

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Fig. 6.
NIK overexpression induces myoblast
differentiation in the absence of IGF-II. A, 293 cells
were transduced with adv/FLAG-NIK. Non-transduced (nt) cells
were used as controls. Cell extracts obtained 24 h after infection
were immunoprecipitated (IP) with antibodies against NIK or
FLAG preadsorbed on protein G-Sepharose beads. Autophosphorylation
assay was performed as described under "Experimental Procedures."
Nonspecific (ns) NIK autophosphorylation activity was tested
using nonimmune antibodies. B, subconfluent L6E9 myoblasts
were transduced with adv/FLAG-NIK or control adv/GFP. 36 h after
infection, cells were allowed to differentiate for 2 days in the
absence of IGF-II (DMEM). Morphological differentiation was assessed by
myotube formation, and cells were photographed after nuclear staining.
Images shown are representative of 25 microscope fields taken at random
from each of three independent experiments. The scale is the same for
both panels. C, shown is myogenin expression in L6E9 cells
transduced with adv/FLAG-NIK or control adv/LacZ. Cells were grown to
subconfluence on glass coverslips and maintained for 2 days in
serum-free medium in the absence of exogenous IGF-II (DMEM). For
immunofluorescence detection, cells were fixed and simultaneously
probed for myogenin and NIK as described under "Experimental
Procedures." Cells showing myogenin nuclear staining were counted and
averaged from a minimum of 15 randomly selected fields. All infections
were done in triplicate, and the results shown are representative of
two independent experiments.
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|
 |
DISCUSSION |
We describe a pathway by which IGF-II modulates skeletal muscle
cell differentiation through activation of the IKK complex. The
activation of the NF-
B cascade (NIK, IKK
, and IKK
activation; I
B degradation; and NF-
B DNA-binding activation) was detected 24 h after placing subconfluent myoblasts in IGF-II-containing differentiation medium. These are early events during the
differentiation program induced by IGF-II, but they differ greatly in
their kinetics compared with the rapid activation of this cascade,
which can be detected within minutes of exposure of cells to cytokines
or other stimuli. These observations indicate that IKK might not be a
direct target of the IGF-induced phosphorylation cascade, an assumption
reinforced by the fact that IGF-mediated IKK and NIK activation
requires de novo protein synthesis. The nature of the
factor(s) induced by IGF-II to trigger NF-
B cascade activation during myogenesis remains undefined. Among the possibilities to be
considered is that IGF-II could induce the secretion of an autocrine
factor by differentiating myoblasts. In this context, we did not detect
expression of tumor necrosis factor-
(a classical activator of
NF-
B) by reverse transcription-polymerase chain reaction studies in
myoblasts induced to differentiate by IGF-II (data not shown). Another
possibility is that the newly synthesized protein is a
differentiation-induced kinase that may be a target for IGF-II itself
or for an alternative factor generated in response to IGF-II.
The involvement of NF-
B in myogenic signaling has been described in
rat, human, and chick embryonic myoblasts (10, 28, 29), although
differentiation of C2C12 skeletal muscle cells in a 2% horse
serum-containing medium seems to occur through a signaling cascade in
which NF-
B plays a negative regulatory role (30). We have previously
established that PI3K, NF-
B, and inducible nitric-oxide synthase are
elements of a common myogenic cascade in which IGF-II induces, through
a PI3K-dependent pathway, a decrease in I
B
protein
content that correlates with a decrease in the amount of I
B
associated with p65 NF-
B, NF-
B DNA-binding activation, and NO
production (10). PI3K is a key mediator of myogenesis (5-9), and the
role of PI3K in NF-
B cascade activation during myogenesis is
reinforced by data presented here showing that activation of both NIK
and IKK by IGF-II in differentiating myoblasts is blocked by inhibiting
PI3K. PI3K is known to be directly involved in the activation of
NF-
B in processes like anti-apoptotic platelet-derived growth factor
signaling (31) and tumor necrosis factor-mediated immune and
inflammatory responses (32). However, in view of our results, PI3K does
not seem to directly activate NIK phosphorylation (LY294002 inhibited
NIK activation only when it was present together with IGF-II during the
whole 24-h treatment, but not during the last 6 h or the last
1 h). These data suggest that PI3K is most probably involved in
the de novo synthesis of protein(s) required for NIK
activation rather than being an upstream element in the activation of
NIK. In contrast, our results do not rule out a biphasic role for
IGF-II: first, in initiating the de novo synthesis of a
NIK-activating factor and, second, in promoting NIK activation by the
newly synthesized protein that (in view of the results presented in
Fig. 5D) could occur only through a PI3K-independent pathway.
Although the stimuli that activate IKK
and the substrates that
mediate its biological activity are known, the stimuli and the relevant
substrates for IKK
are less well characterized. IKK
and IKK
appear to exert different and non-interchangeable physiological roles.
Gene targeting experiments revealed that although IKK
is not
involved in the activation of NF-
B by pro-inflammatory stimuli, it
is involved in morphogenesis (20-22). In this context, our results
show that although IGF-II induced both IKK
and IKK
activities
early during the differentiation program, the overexpression of a
kinase-deficient mutant of IKK
did not alter the expression of
muscle-specific proteins or the formation of multinucleated myotubes.
The differentiation process was, however, blocked by a kinase-deficient
mutant of IKK
, suggesting that endogenous IKK
cannot substitute
for IKK
in myogenic signaling. Interestingly, skeletal muscle poorly
expresses IKK
, whereas it is one of the tissues with the higher
expression levels of IKK
(33).
Proliferation precedes differentiation in IGF-stimulated myogenesis,
and the opposing early and late effects of IGF during myogenesis are
reflected in the phosphorylation state of the cell cycle regulatory
retinoblastoma protein in skeletal myoblasts (34, 35). Before exiting
from the cell cycle, IGFs induce a last round of proliferation in which
NF-
B is required to increase cyclin D1 expression and pRb
hyperphosphorylation (33, 35, 37). Then, a decrease in NF-
B activity
followed by a decrease in cyclin D1 levels seems to be required to
allow the exit from the cell cycle that precedes differentiation (3,
36). A causative relation between NF-
B down-regulation and
myogenesis was initially proposed, without considering that the NF-
B
activity detected after 24 h in differentiation medium (even if it
was lower than that observed during proliferation) could exert a
myogenic role (36). Indeed, this appears to be the case, as, consistent
with data reported here, NF-
B activity was shown to be required by human, mouse, and chicken myoblasts to fuse (10, 31, 32). We show here
that IGF-II-dependent differentiation triggers a delayed
induction of the NF-
B-activating cascade, which requires PI3K
activity and de novo synthesis of still undefined factors. Our data suggest that the activation of NIK and IKK
and the
subsequent phosphorylation of I
B
at Ser-32 and Ser-36 are key
events in skeletal muscle differentiation induced by IGF-II.