 |
INTRODUCTION |
Skeletal muscle cell differentiation is a highly ordered multistep
process that involves the expression of myogenic transcription factors,
followed by cyclin kinase inhibitor p21 protein induction, cell cycle
arrest, muscle-specific protein expression, and cell fusion to form
multinucleated myotubes (1-4). The commitment to differentiate into
myotubes is influenced negatively by several factors. Treatment of
myoblasts with fetal bovine serum, basic fibroblast growth factor 2, or
transforming growth factor
1 is known to inhibit differentiation of
myoblasts (5, 6). Myogenesis is also regulated negatively by oncogenes
such as c-fos, Ha-ras, and E1a (7-9). The
insulin-like growth factors
(IGFs)1 are the only known
growth factors that are crucial to myogenesis (10, 11). IGF expression
is increased during myoblast differentiation in response to serum
withdrawal (12-15). The amount of IGF-II secreted correlates with the
rate of spontaneous differentiation that, in the absence of exogenous
IGF-II, can be inhibited by antisense oligonucleotides complementary to
IGF-II mRNA (16). Because of their myogenic actions, IGFs have been
postulated as potential therapeutic tools in conditions characterized
by muscle myopathy, atrophy, or muscle injury. In this context, IGFs
have been implicated in the regulation of satellite cell function
during regeneration, a characteristic response of adult muscle to
injury (17, 18). However, IGFs are pleiotropic growth factors, and they
also affect the growth of several tissues other than skeletal muscle.
This has led to the analysis of the intracellular myogenic process initiated by IGFs. It is known that IGF-I and IGF-II switch on the
myogenic program by activating the IGF-I receptor (19). During the last
2 years, the phosphatidylinositol (PI) 3-kinase has emerged as an
essential second messenger for skeletal muscle cell differentiation
(20-22). Moreover, by overexpressing a mutant p85 regulatory subunit
of PI 3-kinase (
p85) lacking the ability to bind and activate the
p110 catalytic subunit (L6E9-
p85), we showed that the heterodimeric
p85-p110 is the PI 3-kinase isoform essential for IGF-induced
myogenesis in L6E9 muscle cells (23). Currently, there is no
information regarding the downstream signals activated by IGFs and PI
3-kinase or its PI 3-phosphate products during myogenesis, and we have
recently shown that the serine/threonine p70 S6 kinase, a downstream
element in several PI 3-kinase-dependent signaling cascades
(24, 25), is not involved in the myogenic actions of IGFs in rat,
mouse, or human cells (26).
Among the signaling events involved in myogenesis, the induction of
chick embryonic myoblast fusion in low serum conditions requires NO
production and NF-
B activity (27, 28). However, the mechanisms that
trigger NF-
B and NOS activation in differentiating myoblasts and the
involvement of these molecules in biochemical differentiation
(i.e. expression of structural and functional muscle
markers) remain to be defined. In the present study, we attempted to
further characterize the myogenic intracellular pathway depending on
IGF-II and PI 3-kinase. We describe here a myogenic signaling cascade
initiated by IGF-II that leads to biochemical and morphological
skeletal muscle cell differentiation and that involves (i) PI 3-kinase
activation, (ii) I
B-
degradation and dissociation from p65
NF-
B, (iii) NF-
B activation, and (iv) iNOS expression and
activation. Moreover, we show the ability of the NOS substrate
L-Arg to induce myogenesis in the absence of IGFs in
both rat and human skeletal muscle cells.
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EXPERIMENTAL PROCEDURES |
Materials--
IGF-II was kindly given by Eli Lilly
(Indianapolis, IN). L6E9 rat skeletal muscle cell line was kindly
provided by Dr. B. Nadal-Ginard (Harvard University). Human muscle
biopsies were obtained from the Departament de Neurologia, Hospital
Universitari de la Santa Creu i de Sant Pau, Barcelona. Dulbecco's
modified Eagle's medium (DMEM), fetal bovine serum, and pancreatin
were from Whittaker (Walkersville, Belgium). PI 3-kinase inhibitor LY294002 (29) was from BioMol Research Laboratories (Plymouth, MA). The
NF-
B probe for electrophoretic mobility shift assay was kindly given
by Dr. Jean-François Peyron (Inserm U364, Nice, France).
Sodium salicylate was from Merck (Darmstadt, Germany);
L-arginine monohydrochloride was from Fluka (Buchs,
Switzerlamd); L-lysine monohydrochloride and
N
-nitro-L-arginine were from
Sigma; pyrrolidinedithiocarbamic acid was from Alexis Corporation
(Laufelfingen, Switzerland). Nitrate/nitrite colorimetric assay kit was
from Cayman Chemical Company (Ann Arbor, MI).
The polyclonal antibody OSCRX was raised against the C terminus of
GLUT4 (30). A rabbit polyclonal antibody against
1-integrin was kindly given by Dr. Carles Enrich
(University of Barcelona) (31). Polyclonal antibody C38320 against
caveolin 3 was from Transduction Laboratories (Lexington, KY). Mouse
monoclonal antibody MF 20, which stains all sarcomeric myosin heavy
chain isoforms, was from the Developmental Studies Hybridoma Bank
(Baltimore, MD). Polyclonal antibodies against p21 (C-19),
-I
B
(C-15), and p65 NF-
B (C-20) were from Santa Cruz Biotech (Santa
Cruz, CA). The nitric-oxide synthase antibody set containing iNOS,
cNOS, and eNOS polyclonal antibodies was from Calbiochem (La Jolla, CA).
Cell Culture--
Rat L6E9 myoblasts were grown in monolayer
culture in DMEM containing 10% (v/v) fetal bovine serum and 1% (v/v)
antibiotics (10,000 units/ml penicillin G and 10 mg/ml streptomycin).
Confluent 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. Images
shown are representative of 10-20 randomly selected microscope fields from each one of at least 10 independent experiments. Cells were photographed after staining the nuclei with Mayer's hemalum solution for microscopy (Merck). Cell fusion was quantified by counting the
percentage of nuclei in myotubes from a total of at least 2500 nuclei
from 15-25 independent randomly selected microscope fields for each
condition tested.
Human muscle normal biopsies were minced into small pieces and cultured
in Eagle's minimal essential medium (Mediatech, Reston, VA)
supplemented with 10% heat-inactivated fetal calf serum (MA Bioproducts, Springville, MD), 2% chick embryo extracts (Life Technologies, Inc., Bethesda, MD), 50 mM glutamine and
gentamycin, as described previously (26). Cultures were grown to
subconfluence and examined to assess the development of myotubes after
6 days in DMEM and antibiotics without or with IGF-II (40 nM), together with other compounds when indicated. Images
shown are representative of 10-20 randomly selected microscope fields
from each one of three independent experiments, in which each condition
was tested in duplicate.
Immunofluorescence Analysis--
For immunofluorescence
labeling, cells were grown on 0.1% gelatin-pretreated glass
coverslips. Myoblasts or 2-day-old IGF-II induced myotubes were fixed
on coverslips and incubated with 30 µl of a 1:100 dilution of primary
antibody in phosphate-buffered saline (PBS) containing 0.5% bovine
serum albumin for 1 h at room temperature. Coverslips were washed
three times in PBS, the last one for 15 min, before incubating with the
rodamine-conjugated secondary antibody (1:100 dilution in 0.5% bovine
serum albumin) for 1 h at room temperature. Nonspecific controls
were carried out in the absence of primary antibody (data not shown).
Coverslips were then washed three times in PBS, the last one for 15 min. Finally, coverslips were mounted with immunofluorescence medium.
Electrophoresis and Immunoblotting of Membranes--
Cells were
lysed for 30 min at 4 °C in a buffer containing 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, 0.1% aprotinin, supplemented with
1% Nonidet-P40. Cell extracts were centrifuged for 15 min at
10,000 × g at 4 °C, and 50 µg of the solubilized proteins was loaded. SDS-polyacrylamide gel electrophoresis was performed according to Laemmli (32). Gels were blotted into Immobilon
in buffer consisting of 20% methanol, 200 mM glycine, 25 mM Tris, pH 8.3. Following transfer, the filters were
blocked with 5% nonfat dry milk in PBS for 1 h at 37 °C and
then incubated overnight at 4 °C with primary antibodies in PBS
containing 1% nonfat dry milk and 0.02% sodium azide. Proteins were
detected by ECL chemiluminiscence system (Amersham Pharmacia Biotech)
except for
1-integrin, which was detected using
125I-protein A for 3 h at room temperature.
Quantification of protein expression was performed by scanning
densitometry of at least three independent experiments for each condition.
-I
B/p65 NF-
B Association--
Cells were scraped and
solubilized for 30 min at 4 °C in a buffer containing 50 mM Hepes, 150 mM NaCl, 10 mM EDTA,
10 mM Na4P2O7, 100 mM NaF, 2 mM vanadate, 0.1%
phenylmethylsulfonyl fluoride, 0.1% aprotinin, supplemented with 1%
Nonidet P-40. The cell lysates were centrifuged at 10,000 × g for 15 min at 4 °C, and 0.5 mg of the supernatants were
immunoprecipitated with 10 µg of anti-p65 NF-
B or nonimmune
controls (not shown). Antibodies were preadsorbed on protein
G-Sepharose at 4 °C for 1 h and washed twice in 50 mM Hepes, 150 mM NaCl, 10 mM EDTA,
10 mM Na4P2O7, 100 mM NaF, 1% Nonidet P-40, before being incubated with the
solubilized proteins for 2 h at 4 °C. The immunopellets were
washed three times in the same buffer before being resuspended in
SDS-polyacrylamide gel electrophoresis sample buffer and analyzed by
Western blot using polyclonal antibodies against anti-I
B-
, as
described above.
Electrophoretic Mobility Shift Assay--
NF-
B-DNA binding
activity was analyzed in total cellular 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, 0.1% aprotinin)
(33). 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'). Totex extract samples (70 µg) were
incubated for 10 min at 4 °C with 30 ng of poly(dI·dC) and 5 µl
of 5× NF buffer (50 mM Tris, pH 7.5, 500 mM
NaCl, 5 mM dithiothreitol, 5 mM EDTA, 20%
glycerol 0.4 mg/ml salmon sperm DNA) in a final volume of 25 µl. The
end-labeled probe was then added for a further incubation of 25 min at
25 °C. The specificity of the bands detected was verified by adding
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).
Nitrite/Nitrate Assay--
The NO production was determined by
assaying for nitrite and nitrate accumulation in the culture media.
Briefly, following treatment of the cells with the indicated agents,
culture media were filtered with 0.2-µm filters, and 80 µl of each
sample was treated with nitrate reductase and its cofactors to convert
all of the nitrate to nitrite before applying 100 µl of the Griess reagent (0.5% naphthylethylenediamine dihydrochloride, 1%
sulfanylamidein, 2.5% phosphoric acid). Absorbance was measured at 543 nm, and nitrite concentration was determined using a standard curve of sodium nitrite concentrations ranging from 0 to 50 nmol/ml.
 |
RESULTS |
IGF-II Induces NF-
B and iNOS Activation as Early Events during
Myogenesis--
During the process of L6E9 myoblast differentiation,
IGF-II caused the induction of skeletal muscle protein markers such as the insulin-sensitive glucose transporter GLUT4, the isoform 3 of
caveolin, which is a component of the distrophin complex, and the
myosin heavy chain (Fig. 1a).
The expression of these proteins increased with the time of exposure to
IGF-II, being maximal at day 4, when a fully morphological
differentiation is achieved (23). We observed that IGF-II induced as an
early myogenic marker the expression of iNOS (Fig. 1a). At
differentiation day 1, IGF-II induced 2.3 ± 0.2-fold
(n = 3) increase in iNOS expression compared with
myoblasts maintained in DMEM alone, and a progressive decrease in its
expression was observed from day 1 to day 4. As a control, we examined
the expression levels of
1-integrin, a
nonmuscle-specific plasma membrane protein, that remained unaltered
during IGF-II-induced differentiation (Fig. 1a).

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Fig. 1.
IGF-II induces NF- B
and iNOS activation as early events during myogenesis.
a, confluent L6E9 myoblasts were allowed to differentiate in
serum-free medium (DMEM) for 4 days in the absence or presence IGF-II
(40 nM). iNOS and muscle-specific protein expression
(caveolin 3, insulin-sensitive glucose transporter GLUT 4, and myosin
heavy chain (MHC) were analyzed by Western blot of total
cell lysates (50 µg) from each condition. Relative amounts of
proteins in each sample were checked by expression of the
nonmuscle-specific protein 1-integrin. b,
total cell extracts from cells differentiated in the absence (day 1) or
presence of IGF-II (days 1-4) were incubated with a
32P-labeled NF- B probe corresponding to the B site in
the interleukin-2 gene promoter and analyzed by electrophoretic
mobility shift assay. The specificity of the bands was verified by
adding 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). c, total cell
extracts from cells differentiated for 24 h in the absence (DMEM)
or presence of IGF-II with or without 10 mM NaSal were
incubated with a 32P-labeled NF- B probe corresponding to
the B site in the interleukin-2 gene promoter and analyzed by
electrophoretic mobility shift assay. d, iNOS expression was
analyzed in total cell lysates from myoblasts differentiated for 1 day
in the absence or presence of IGF-II with or without NF- B inhibitors
NaSal (10 mM) or PDTC (100 µM) (upper
panel). NOS activity was determined as nitrite and nitrate
accumulation in the culture media at differentiation day 1 in the
absence (bar a) or presence of IGF-II without (bar
b) or with NaSal (10 mM, bar c) or PDTC
(100 µM, bar d) and expressed as percentage of
nitrite/nitrate accumulation in serum-free medium (DMEM) (lower
panel).
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|
Taking into account that iNOS expression is tightly regulated by the
transcription factor NF-
B, we analyzed the DNA binding activity of
NF-
B on total cell extracts from myoblasts differentiated in the
absence or presence of IGF-II. Fig. 1b shows that the
NF-
B DNA binding activity was increased by IGF-II in a way that
correlated in time with the profile of iNOS expression. Maximal
activation was detected at 24 h, when the level of NF-
B DNA
binding activity induced by IGF-II was 2.6 ± 0.3-fold
(n = 3) over controls (DMEM-treated cells). No
significant differences in NF-
B DNA binding activity were observed
at shorter differentiation times (data not shown and see Fig.
3a). NF-
B activation by IGF-II was sensitive to inhibitors of I
B phosphorylation or degradation such as sodium salicylate (10 mM) (Fig. 1c) or PDTC (100 µM) (data not shown) (34, 35). By using these NF-
B
inhibitors, we determined that IGF-II-induced iNOS expression was
totally dependent on NF-
B activation (Fig. 1d,
upper panel). Moreover, IGF-II-induced iNOS expression at
day 1 correlated with IGF-II-dependent NO production, as
measured by nitrite/nitrate accumulation in the cell culture media; the
IGF-II-induced NO production was dependent on NF-
B activity, because
it was completely blocked by NaSal or PDTC (Fig. 1d,
lower panel).
IGF-II Requires NF-
B and iNOS Activation to Signal
Myogenesis--
Results presented above indicate that
NF-
B-dependent iNOS expression and activity are early
events during myogenesis induced by IGF-II. We next tested the
relevance of this IGF-II-induced pathway for muscle differentiation.
L6E9 myoblast differentiation was induced with IGF-II (40 nM) in the absence or presence of NF-
B or NOS
inhibitors, and after 4 days the expression of differentiation parameters was analyzed (Fig. 2,
a-c). The IGF-II induction of muscle markers such as GLUT4
or caveolin 3 was inhibited in a dose-dependent manner by
NF-
B inhibitors PDTC (Fig. 2a) or NaSal (Fig.
2b) and by NOS inhibitors NNA (Fig. 2c) or
NG-nitro-L-arginine-methyl ester
(data not shown).

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Fig. 2.
IGF-II-induced myogenesis is blocked by
NF- B and NOS inhibitors. a-c,
confluent L6E9 myoblasts were differentiated for 4 days in the absence
( ) or presence (+) of 40 nM IGF-II without or with
increasing amounts of PDTC (a), NaSal (b), or NNA
(c). The content of the skeletal muscle-markers GLUT4 and
caveolin 3 was analyzed by Western blot in cell lysates from each
condition (50 µg). Relative amounts of proteins in each sample were
checked by expression of the nonmuscle-specific protein
1-integrin. d, L6E9 myoblasts were grown to
confluence in a 10% fetal bovine serum-containing medium
(Mb) and then allowed to differentiate in a serum-free
medium (DMEM) or DMEM supplemented with 40 nM IGF-II in the
absence or presence of NF- B inhibitors PDTC (100 µM)
or NaSal (10 mM) or NOS inhibitor NNA (5 mM).
After 4 days in each condition, morphological differentiation was
assessed by myotube formation. Images shown are representative of
15-25 microscope fields taken at random from each one of at least 10 independent experiments. e, human myoblasts (Mb)
derived from skeletal muscle normal biopsies were grown to
subconfluence and examined to assess the development of myotubes after
6 days in DMEM without or with IGF-II (40 nM) in the
absence or presence of NaSal (10 mM). Images are
representative of 10-20 microscope fields analyzed at random from each
one of three independent experiments, in which each condition was
tested in duplicate. Scale bars, 30 µm (the scale is the
same for all panels).
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At the morphological level, subconfluent myoblasts incubated for 4 days
in a medium containing DMEM supplemented with IGF-II formed large
multinucleated myotubes (Fig. 2d). In these conditions, the
percentage of nuclei in myotubes induced by IGF-II was 66 ± 3%
(a total of 2555 nuclei were counted from 19 randomly selected microscopic fields), whereas in the presence of DMEM alone only 9 ± 2% nuclei were in myotubes (a total of 2538 nuclei were counted from 21 randomly selected microscopic fields). When IGF-II-induced cell
fusion was analyzed in the presence of NF-
B inhibitors such as NaSal
(10 mM) or PDTC (50 µM) or in the presence of
nitric-oxide synthase inhibitors such as NNA (5 mM) or
NG-nitro-L-arginine-methyl ester
(data not shown), a total blockade of the IGF-II myotube formation
capacity was observed (no myotube formation was seen in any of at least
10 independent experiments performed under each of these conditions)
(Fig. 2d). The involvement of NF-
B activation in
IGF-II-induced myogenesis was also detected in myoblast primary
cultures derived from human skeletal muscle biopsies; human myoblasts
responded to IGF-II by activating cell fusion, and this effect was
blocked by the addition of sodium salicylate (Fig. 2e) or
PDTC (data not shown).
L-Arginine Mimics IGF-II-induced Morphological and
Biochemical Skeletal Muscle Cell Differentiation--
As very little
or no expression of iNOS is detected in L6E9 cells in the absence of
IGF-II, we analyzed whether L6E9 cells expressed other NOS isoforms
because both cNOS and the eNOS isoforms have been detected in other
skeletal muscle models (36, 37). We detected the expression of cNOS in
both L6E9 myoblasts and myotubes by immunofluorescence (Fig.
3a), whereas very low levels of type III NOS (eNOS) were detected (data not shown). As a control, we
analyzed the expression of p21 cyclin-dependent kinase
(Cdk) inhibitor expression, which as expected, was only detected in nuclei from myotubes (Fig. 3a). Based on these observations,
we analyzed whether the NOS substrate L-Arg was able to
mimic IGF-II ability to induce biochemical and morphological myoblast
differentiation. We incubated subconfluent L6E9 myoblasts with the NOS
substrate, L-Arg (5 mM), to generate NO in the
absence of exogenous IGF-II addition. We observed that after 24 h
of incubation, L-Arg induced a NO production similar to
that induced by IGF-II, which was totally blocked by the NOS inhibitor
NNA (Fig. 3b).

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Fig. 3.
L-Arginine mimics IGF-II-induced
morphological and biochemical skeletal muscle cell
differentiation. a, cNOS expression in L6E9 cells.
Cells were grown to subconfluence on gelatin-treated glass coverslips
and differentiated or not (myoblasts or myotubes, respectively) for 2 days in serum-free medium supplemented with 40 nM IGF-II.
For immunofluorescence detection of cNOS and Cdk inhibitor p21
expression, cells were fixed with methanol and incubated with anti-cNOS
or anti p21 antibodies. Finally, cells were incubated with a
rodamine-conjugated secondary antibody, as described under "Materials
and Methods." Scale bars, 10 µm. b, confluent
L6E9 myoblasts were allowed to differentiate for 1 day in serum-free
medium alone (DMEM) or supplemented with (i) 40 nM IGF-II,
(ii) 5 mM L-Arg, or (iii) 5 mM
L-Arg plus 5 mM NNA. NOS activity was estimated
as nitrite and nitrate accumulation in the culture media and expressed
as percentage of nitrite/nitrate accumulation in serum-free medium
(DMEM). c, L6E9 myoblasts were grown to confluence in a 10%
fetal bovine serum-containing medium and then allowed to
differentiate in a serum-free medium supplemented either with 10 mM L-Arg or 10 mM
L-Lys. After 4 days in each condition, morphological
differentiation was assessed by myotube formation. Images shown are
representative from 15-25 microscope fields taken at random from each
one of at least 10 independent experiments. Scale bars, 75 µm (the scale is the same for all panels). d, human
myoblasts derived from skeletal muscle normal biopsies were grown to subconfluence and then allowed to differentiate for
6 days in a serum-free medium supplemented with either 10 mM L-Arg or 10 mM
L-Lys. Images are representative of 10-20 microscope
fields analyzed at random from each one of three independent
experiments in which each condition was tested in duplicate.
Scale bars, 30 µm (the scale is the same for all panels).
e, confluent L6E9 myoblasts were allowed to differentiate
for 4 days in serum-free medium alone DMEM or supplemented with either
(i) 40 nM IGF-II, (ii) 5 mM L-Arg,
(iii) 5 mM L-Lys, or (iV) 5 mM
L-Arg plus 5 mM NNA. Muscle-specific protein
expression (caveolin 3, insulin-sensitive glucose transporter GLUT 4, and myosin heavy chain) was analyzed by Western blot of total cell
lysates (50 µg) from each condition. Relative amounts of proteins in
each sample were checked by expression of the nonmuscle-specific
protein 1-integrin.
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L-Arginine (5 mM) induced a high level of
morphological differentiation of L6E9 cells (46 ± 4% of nuclei
into myotubes from a total of 2099 nuclei in 18 randomly selected
microscopic fields) (Fig. 3c). This effect was specific for
the NOS substrate, because in the same conditions the basic amino acid
L-Lys did not induce additional cell fusion to that
observed with DMEM alone (12 ± 2% of nuclei into myotubes from a
total of 3158 nuclei in 22 randomly selected microscopic fields).
Similar effects were observed in myoblast primary cultures derived from
human skeletal muscle biopsies, where L-Arg but not
L-Lys induced multinucleated myotube formation (Fig.
3d). L-Arg also led to the expression of
muscle-specific protein markers such as caveolin 3, myosin heavy chain,
and GLUT4, at levels comparable with those observed with IGF-II (IGF-II
and L-Arg induced 2.6 ± 0.3- and 2.2 ± 0.4-fold
increase over DMEM controls, respectively, in muscle-specific protein
expression, n = 3) (Fig. 3e). Moreover, the
effect of L-Arg on biochemical differentiation was
dependent on NOS activity because it was totally blocked by the NOS
inhibitor NNA, whereas no induction of muscle-specific proteins was
observed in the presence of L-Lys (5 mM) in the
same conditions (Fig. 3e).
PI 3-Kinase Is Required for IGF-II Activation of NF-
B and
iNOS--
We have recently reported that the p85/p110
phosphatidylinositol 3-kinase is an essential mediator of
IGF-II-myogenic actions (23). Indeed, either overexpression of a
dominant negative mutant of p85 PI 3-kinase regulatory subunit or
treatment of L6E9 myoblast with the PI 3-kinase inhibitor LY294002
blocked the IGF-II-dependent myogenic program. Results
presented above indicate that NF-
B and iNOS activation are also
critical for IGF-II-induced myogenic program. Therefore, we next
evaluated whether the differentiation cascade involving
IGF-II/NF-
B/iNOS was dependent on PI 3-kinase activity. To this end,
L6E9 myoblast differentiation was induced with IGF-II in the absence or
presence of the PI 3-kinase inhibitor LY294002. Fig.
4 shows that the presence of the PI
3-kinase inhibitor (20 µM) blocked the IGF-II-induced
peak of NF-
B activation (Fig. 4a), the IGF-II induction
of iNOS expression (Fig. 4b), and the IGF-II-induced NO
production (Fig. 4c). In all, these results indicate that
IGF-II requires PI 3-kinase to induce NF-
B and iNOS activation.

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Fig. 4.
PI 3-kinase is required for IGF-II induction
of NF- B DNA binding activity, iNOS expression,
and NOS activity. a, total cell extracts were obtained
at different times (12, 24, and 48 h) after differentiation in the
absence or presence of 40 nM IGF-II without or with 20 µM LY294002. Cell extracts were incubated with a
32P-labeled NF- B probe corresponding to the B site in
the interleukin-2 gene promoter and analyzed by electrophoretic
mobility shift assay. The specificity of the bands was verified by
adding 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). b, confluent
L6E9 myoblasts were allowed to differentiate in serum-free medium
(DMEM) for 24 h in the absence or presence 40 nM
IGF-II without or with 20 µM LY294002. iNOS protein
expression was analyzed by Western blot of total cell lysates (50 µg)
from each condition. c, NOS activity was determined as
nitrite and nitrate accumulation in the culture media at
differentiation day 1 in the absence (DMEM) or presence of IGF-II
without or with 20 µM LY294002 and expressed as
percentage over nitrite/nitrate accumulation in serum-free medium
(DMEM).
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IGF-II Induces I
B-
Degradation and Dissociation from p65
NF-
B through a PI 3-Kinase-dependent Pathway--
We
next analyzed the mechanism involved in the activation of NF-
B by
IGF-II and PI 3-kinase. A primary level of control for NF-
B is
through interaction with the inhibitory protein I
B, and most of
NF-
B activators tested induced I
B-
degradation (38).
Therefore, we analyzed the effect of IGF-II on I
B-
protein levels
in L6E9 myoblasts. We observed an up-regulation in the expression of
both I
B-
and the p65 subunit of NF-
B after 12 h of IGF-II
treatment compared with cells incubated in DMEM alone. The
IGF-II-induced increase in protein levels was 2.0 ± 0.1- and 1.5 ± 0.1-fold (n = 3) for I
B-
and p65,
respectively (Fig. 5a, IGF-II
versus DMEM at 12 h).

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Fig. 5.
IGF-II decreases
-I B protein levels and
I B- dissociation from
p65 NF- B through a PI
3-kinase-dependent pathway. a, confluent
L6E9 myoblasts were allowed to differentiate in serum-free medium
(DMEM) for 1 day in the absence or presence IGF-II (40 nM).
I B- and p65 NF- B protein content was analyzed at different
times by Western blot of total cell lysates (50 µg) from each
condition. b, confluent L6E9 myoblasts were allowed to
differentiate for 24 h in serum-free medium supplemented with 40 nM IGF-II without or with 20 µM LY294002.
I B- protein content was analyzed at different times by Western
blot of total cell lysates (50 µg) from each condition. c,
confluent L6E9 myoblasts were differentiated for 24 h in
serum-free medium supplemented with 40 nM IGF-II without or
with 20 µM LY294002. Cells were solubilized and proteins
(0.5 mg) were incubated with 10 µg of antiserum against p65 NF- B
and protein G-Sepharose. Immune complexes were analyzed by Western blot
with an antibody against I B- .
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From 12 to 24 h of treatment, IGF-II induced a 39 ± 4%
(n = 3) decrease in I
B-
total cell content
without any detectable alteration in the expression levels of p65 (Fig.
5a, IGF-II, 24 h versus 12 h).
IGF-II-induced decrease in I
B-
levels was inhibited when
differentiation was carried out in the presence of the PI 3-kinase
inhibitor LY294002. Thus, at 24 h of IGF-II treatment, IkB-
protein content was 1.7 ± 0.3-fold (n = 3) higher
in the presence than in the absence of PI 3-kinase inhibitor (Fig.
5b, IGF-II+LY294002 versus IGF-II at 24 h).
Immunoprecipitation of myoblast lysates with an anti-p65 antibody
co-immunoprecipitated I
B-
(Fig. 5c). After 16 h
of IGF-II treatment, the amount of I
B-
associated with p65 was
similar in the absence or presence of LY294002 (data not shown).
However at 24 h, the amount of I
B-
associated with p65 was
1.8 ± 0.2-fold (n = 3) higher in the presence of
IGF-II and LY294002 than in the presence of IGF-II alone.
It is interesting to note that the increase in I
B-
and p65
expression detected at 12 h of IGF-II treatment remained
unaffected by LY294002 in three independent experiments (for I
B-
,
Fig. 5, a and b, 12 h lanes; for p65,
data not shown). These results suggest that during the first 12 h
of treatment, IGF-II up-regulated the expression of I
B-
and p65
NF-
B subunit in a PI 3-kinase-independent manner (Fig. 5,
a and b, 12 h lanes); during the
following 12 h, IGF-II induced a decrease in I
B-
protein
content and the activation of NF-
B by dissociation of p65 from
I
B-
through a PI 3-kinase-dependent pathway (Fig. 5,
b and c, 24 h lanes).
 |
DISCUSSION |
Results presented here define a model for the IGF-II-induced
myogenic pathway, in which PI 3-kinase, NF-
B, and NOS activities are
critical elements. Our data show that IGF-II-induced myogenesis can be
blocked by either PI 3-kinase inhibitors, NF-
B inhibitors, or NOS
inhibitors. Several lines of evidence indicate that PI 3-kinase,
NF-
B, and iNOS are elements of a common myogenic cascade: (i)
IGF-II-induced NF-
B activation was blocked by PI 3-kinase inhibition, (ii) IGF-II-induced iNOS expression correlated in time with
the activation of NF-
B and was blocked by either PI 3-kinase or
NF-
B inhibitors, and (iii) IGF-II-induced iNOS expression was
accompanied by an IGF-II-induced NO production that was blocked by PI
3-kinase, NF-
B, or NOS inhibitors.
An early event in the IGF-II-induced differentiation program was the
increase in I
B-
and the p65 NF-
B subunit protein content. These data are consistent with the evidence that NF-
B and I
B are
components of a mutual regulatory system in which the levels of one
component control the activity or quantity of the other (39). After
24 h of IGF-II treatment, a transient peak of NF-
B DNA binding
activity was observed that correlated with a decrease in I
B-
protein levels. It has been reported that differentiation of skeletal
muscle cells in a medium containing low serum concentration induced a
down-regulation of the activity of NF-
B and other proliferating transcription factors (40). The authors did not consider the effects of
IGFs in their systems, and they established from their data that there
was a causative relation between NF-
B down-regulation and
myogenesis. However, the authors did not examine whether the NF-
B
activity detected after 24 h in a low serum differentiation medium, even if it was lower than that observed during proliferation, was necessary for differentiation. Indeed, this appears to be the case,
because consistent with our observations for rat and human myoblasts
differentiated with IGF-II, Lee et al. (28) reported that
chick embryonic myoblasts require NF-
B activity to fuse in a low
serum-containing differentiation medium.
The effect of IGF-II in modulating NF-
B activity in L6E9 myoblasts
appears to be tightly regulated by PI 3-kinase activity. Our results
indicate that PI 3-kinase is required for NF-
B activation during
myoblast differentiation through a mechanism that involves I
B-
degradation. The peak of NF-
B DNA binding activity after 24 h
of IGF-II treatment was blocked by PI 3-kinase inhibition, and this
correlated in time with higher amounts of I
B-
protein and higher
amounts of inactive p65 NF-
B complexed with I
B-
in the
presence of LY294002 than those detected in control myoblasts.
Our results suggest a biphasic effect of IGF-II in modulating NF-
B
activity in L6E9 myoblasts: during the first 12 h, IGF-II induced
the up-regulation of I
B-
and p65 expression in a PI 3-kinase-independent way, and from 12 to 24 h, IGF-II induced the
dissociation of I
B-
from p65 and the degradation of I
B-
through PI 3-kinase-dependent mechanisms. We interpret the
complexity of these results as an example of the diversity of signals
that can be elicited when a growth factor activates a tyrosine kinase receptor and generates a set of distinct second messengers in the cell.
In this particular case, the expression of p65 and IkB-
is induced
by IGF-II through yet undefined signaling pathways that do not involve
PI 3-kinase activity. In contrast, the degradation of IkB-
and its
dissociation from p65 require PI 3-kinase activity. In this latter
case, it is tempting to hypothesize that PI 3-kinase could be acting
upstream from the IkB kinases responsible for NF-
B activation.
Indeed, many if not all activators of NF-
B have been reported to
induce degradation of I
B-
(41, 42) by activating I
B kinases
that phosphorylate I
B-
on serines 32 and 36 within the N-terminal
regulatory domain (43-47). This is the most probable mechanism by
which IGF-II and PI 3-kinase induce NF-
B activation in
differentiating myoblasts, because we did not detect any IGF-II-induced
I
B-
tyrosine phosphorylation, which represents a
proteolysis-independent mechanism for NF-
B activation (data not
shown) (48). Potential kinases acting between PI 3-kinase and IkB
kinases during IGF-II myogenesis could be the protein kinase B and some
PKC isoforms that are activated by PI 3-kinase products (49-51). In
this context,
PKC and
PKC have been implicated in NF-
B
activation (52, 53).
We show here that iNOS expression and activation are early events
during IGF-II-induced myogenesis. Moreover, we describe here the
ability of the NOS substrate L-Arg to induce myogenesis in
the absence of IGFs in both rat and human skeletal muscle cells. The
identification of direct targets for NO action in myogenesis is a
matter of current interest, and one gene that may be regulated by NO in
skeletal muscle cells is the Cdk inhibitor p21. It has been reported
that NO blocks the cell cycle progression at the G1/S
transition by inhibiting Cdk2-mediated phosphorylation of the
retinoblastoma protein and that the p21 induction is involved in the
Cdk2 inhibition (54). A causative role for NO action in nerve growth
factor-induced cell cycle exit and differentiation of PC12 neuronal
cells has been reported (55). It has been proposed that NO diffusion
from the producer cell could promote cessation of growth in adjacent
cells, contributing to synchronization of development from precursor
cells. The expression of Cdk inhibitor p21 and cell cycle exit are
prerequisites for myogenesis, and these events, which are both induced
by IGF-II in L6E9 cells, may be mediated by NO (1, 26, 23). Results
presented here suggest that IGF-II-dependent myoblast
differentiation can be mimicked by L-Arg treatment, both at
the biochemical and at the morphological level, and this ability of
L-Arg to induce myogenesis in the absence of IGFs may
contribute to the development of new strategies for the treatment of myopathies.