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Molecular and Cellular Biology, April 1999, p. 3115-3124, Vol. 19, No. 4
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Maturation of the Myogenic Program Is Induced by
Postmitotic Expression of Insulin-Like Growth Factor I
Antonio
Musarò and
Nadia
Rosenthal*
Cardiovascular Research Center, Massachusetts
General Hospital
East, Charlestown, Massachusetts 02129
Received 22 September 1998/Returned for modification 1 December
1998/Accepted 29 December 1998
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ABSTRACT |
The molecular mechanisms underlying myogenic induction by
insulin-like growth factor I (IGF-I) are distinct from its
proliferative effects on myoblasts. To determine the postmitotic role
of IGF-I on muscle cell differentiation, we derived L6E9 muscle cell
lines carrying a stably transfected rat IGF-I gene under the control of
a myosin light chain (MLC) promoter-enhancer cassette. Expression of
MLC-IGF-I exclusively in differentiated L6E9 myotubes, which express
the embryonic form of myosin heavy chain (MyHC) and no endogenous
IGF-I, resulted in pronounced myotube hypertrophy, accompanied by
activation of the neonatal MyHC isoform. The hypertrophic myotubes
dramatically increased expression of myogenin, muscle creatine kinase,
-enolase, and IGF binding protein 5 and activated the myocyte
enhancer factor 2C gene which is normally silent in this cell line.
MLC-IGF-I induction in differentiated L6E9 cells also increased the
expression of a transiently transfected LacZ reporter driven by the
myogenin promoter, demonstrating activation of the differentiation
program at the transcriptional level. Nuclear reorganization,
accumulation of skeletal actin protein, and an increased expression of
1D integrin were also observed. Inhibition of the phosphatidyl
inositol (PI) 3-kinase intermediate in IGF-I-mediated signal
transduction confirmed that the PI 3-kinase pathway is required only at
early stages for IGF-I-mediated hypertrophy and neonatal MyHC induction
in these cells. Expression of IGF-I in postmitotic muscle may therefore
play an important role in the maturation of the myogenic program.
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INTRODUCTION |
During mammalian embryogenesis,
skeletal muscle undergoes a series of developmental changes that are
reflected in a progression of structural gene expression patterns, such
that the specific contractile protein composition of a myocyte is
considered a measure of its maturity. The orchestration of this
progression presumably involves a complex interplay between intrinsic
programs of lineage specification and extrinsic controls such as
innervation, extracellular milieu, and humoral factors in the embryonic
environment (46). In the adult, muscle injury and subsequent
regeneration result in a partial recapitulation of these embryonic
programs of maturation, underscoring the enduring plasticity of the
muscle phenotype in the face of changing external conditions.
The insulin-like growth factors (IGFs) have been implicated in the
control of skeletal muscle growth and differentiation in both embryonic
development and in the muscle regeneration process (20, 22, 29,
36). Recent studies of the effects of IGFs on the myogenic
program in muscle cell culture have focused specifically on IGF-I,
since it is the predominant form in mature muscle tissue. In cultured
myoblasts, as in other cell types, IGF-I induces cell proliferation
(14, 37). However, unlike other growth factors, IGF-I also
stimulates myogenic differentiation and generates pronounced myocyte
hypertrophy (14, 18, 37), suggesting that this growth factor
can regulate both proliferative and differentiative responses in muscle cells.
In previous reports, we and others have established that proliferation
precedes differentiation in IGF-I-stimulated myogenesis in culture
(14, 41). From these studies, it is clear that the effects
of IGF-I on proliferation and differentiation are temporally separated.
IGF-I plays its roles step by step, first acting upon myoblast
replication, increasing the expression pattern of factors involved in
the cell cycle progression (14, 41), and subsequently
promoting myogenic differentiation by induction of myogenic regulatory
factors (MRFs). The effects of IGF-I are also modulated by a family of
six IGF binding proteins (IGFBPs) (25), which can either
inhibit or potentiate the IGF-stimulated actions in vivo and in vitro.
Recent experimental evidence suggests that IGF-I exerts its functions
by activating two different intracellular signal transduction pathways,
conveying either proliferative or differentiative signals (10,
26). The proliferative response is mediated by the
mitogen-activated protein (MAP) kinase pathway, whereas the pathway
leading to differentiation involves the activation of
phosphatidylinositol (PI) 3-kinase. The phosphorylated products of this
lipid kinase, PI 3,4-bisphosphate and PI 3,4,5-trisphosphate activate
downstream effectors, ultimately leading to induction of muscle gene
expression. The alternate activation of these two pathways presumably
underlies the observed pleiotropic effects of IGF-I on muscle cells.
To determine whether IGF-I can stimulate myogenesis in the absence of
proliferation, we established an experimental cell culture system where
the action of IGF-I on proliferation and on differentiation could be
uncoupled. The neonatal rat L6E9 myogenic line has been used
extensively to study the role of IGF-I in myogenesis (33, 47), since it does not express this growth factor itself and can
respond to IGF-I stimulation through presentation of IGF-I receptors
(39). Although the L6 cells fail to express a number of
characteristic myogenic regulators such as MyoD (5, 38) or
myocyte enhancer factor (MEF) 2C (30), they are still able to undergo fusion and to activate a subset of muscle structural genes
in response to exogenous stimuli (19). Treatment of L6E9 myoblasts with exogenous IGF-I in defined serum-free medium induces a
transient increase in cell cycle markers and cell proliferation (14, 41). This response is rapidly followed by withdrawal from the cell cycle and activation of myogenic factors, resulting in a
net increase in structural gene expression and larger myotubes (14).
To bypass the proliferative effects of IGF-I, we stably transfected
L6E9 cultures with a muscle-specific IGF-I expression vector, myosin
light chain (MLC)-IGF-I, which is activated only after myoblasts have
withdrawn from the cell cycle and have committed to differentiation. In
this way, the influence of IGF-I on myogenic differentiation could be
dissected and manipulated independently of its role in myoblast
proliferation. Studies of the MLC-IGF-I transfectants suggest that
postmitotic expression of IGF-I potentiates and accelerates the
myogenic program, induces dramatic myotube hypertrophy, activates a new
battery of genes, and initiates a shift towards a more mature fiber
type. Moreover, disruption of specific signal transduction pathways in
these cells reveals a potential third PI 3-kinase-independent signal
transduction pathway that presumably participates in later stages of
myogenic differentiation. These studies in cell culture support a role
for IGF-I in the establishment and maintenance of the mature muscle
phenotype in normal and regenerating muscle tissue.
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MATERIALS AND METHODS |
Cell culture and transfection.
L6E9 cell cultures (33,
47) were maintained in growth medium (GM) consisting of
Dulbecco's modified Eagle's medium (DMEM) supplemented with 20%
fetal bovine serum (FBS).
For stable transfection, L6E9 cells were grown in 100-mm-diameter
plates to 70% confluence and then washed with serum- and antibiotic-free medium. Cells were transfected with an MLC-IGF-I plasmid (pMLC/IGF-I) and with a puromycin-selectable vector (pPUR; Clontech) (10:1) according to the Lipofectamine transfection method (Gibco-BRL). Briefly, DNA (10 µg of pMLC/IGF-I and 1 µg of pPUR), 50 µl of Lipofectamine, and 1.6 ml of serum- and antibiotic-free medium were incubated at room temperature for 45 min and then added to
the plate for 5 h. Following incubation, 10 ml of GM was added. At
24 h following the start of transfection, the transfection mixture
was removed and replaced with DMEM plus 20% FBS. At 48 h after
transfection, the cells were passaged at a 1:10 dilution into selection
medium containing 20% FBS and 3 µg of puromycin per ml. Stable
myoblasts were maintained in DMEM selection medium, which was replaced
every 2 days, for 12 days. Fifteen drug-resistant clones (L6MLC/IGF-I)
were isolated by using cloning rings and characterized morphologically
and by Northern blot analysis for IGF-I expression.
For transient transfections, L6E9 and L6MLC/IGF-I cells were
transfected with Lipofectamine by using either a
myogenin-LacZ-
-Gal
(Myo1565LacZ) or a CMV-
-Gal plasmid. The
CMV-
-Gal plasmid was
included to assess transfection efficiency in
both L6E9 and L6MLC/IGF-I
cells. To activate myogenic differentiation,
and the expression
of MLC-IGF-I, myoblasts were switched to
differentiation medium
(DM; DMEM plus 1% bovine serum albumin).
Myogenin promoter activity
was analyzed, by determining relative
numbers of LacZ-positive
cells, after either 0 (80% confluence) or 3 days in
DM.
RNA preparation, Northern blotting, and hybridization.
Total
RNA from L6E9 and L6MLC/IGF-I cells was obtained by RNA-TRIzol
extraction (Gibco-BRL). Fifteen micrograms of total RNA was separated
on 1.3% agarose gels containing 2.2 M formaldehyde and transferred
directly from the gel to Nytran membranes (GeneScreen Plus; NEN) in
10× SSC (1× SSC is 0.15 M NaCl plus 1.5 mM sodium citrate) overnight.
After transfer, RNA was UV cross-linked (120,000 µJ of UV) and
the membranes were baked at 80°C for 2 h.
Hybridization was carried out at 42°C in a mixture containing 50%
formamide, 10% dextran sulfate, 1% sodium dodecyl sulfate
(SDS), and
100 µg of salmon sperm DNA (Sigma) per ml. The cDNA
probes were
32P labeled by random priming (
16) to a specific
activity of 10
8 cpm/mg of DNA. Filters were washed at high
stringency with 0.2×
SSC-0.5% SDS at 65°C and exposed for
autoradiography on RP-X-OMAT
film
(Kodak).
Immunohystochemical analysis.
L6E9 and L6MLC/IGF-I myotubes
(after 4 days in DM) were fixed in 4% paraformaldehyde and then
preincubated in phosphate-buffered saline (PBS) containing 1% BSA and
a 1:30 dilution of goat serum for 30 min. Myotubes were treated with
monoclonal antibody (MAb) against either skeletal actin (Sigma) (1:6),
the myosin heavy chain (MyHC) embryonic subunit (MAb BF-45)
(1) (1:100), or the MyHC neonatal subunit (MAb BF-34)
(1) (1:100) overnight at 4°C.
For actin analysis, after primary antibody incubation, cells were
rapidly washed twice with PBS and once with PBS containing
1% BSA for
15 min and incubated with biotinylated goat anti-mouse
antibody (Sigma)
at a 1:800 dilution for 1 h at room temperature,
washed again as
described above, and incubated with avidin-alkaline
phosphatase
conjugate at a 1:100 dilution for 1 h at room temperature.
The
cells were washed with PBS containing 1% BSA and processed
for
alkaline phosphatase activity (Sigma kit
B5655).
After incubation with the MyHC embryonic subunit or the MyHC neonatal
subunit primary antibody, the myotubes were rapidly
washed twice with
PBS and once with PBS containing 1% BSA for
15 min and incubated with
goat anti-mouse rhodamine-conjugated
antibody. The cells were washed
again as described above and mounted
in 90% glycerol in PBS (pH 8).
The nuclei of myogenic cells were
visualized by Hoechst
staining.
Biotinylation and immunoprecipitation.
Control L6E9 and
L6MLC/IGF-I myoblasts were grown to approximately 80% confluence in GM
and then switched to DM (day 0) and cultured for 3 days. The membrane
proteins of myoblasts and myotubes were labeled with 0.5 mg of
Sulfo-NHS-biotin (Pierce) in buffer A (1.3 mM CaCl2, 0.4 mM
MgSO4, 5 mM KCl, 138 mM NaCl, 5.6 mM D-glucose, 25 mM HEPES [pH 7.4]) at 4°C for 30 min on a rocker. The labeled reaction mixture was blocked by washing with DMEM containing 0.6% BSA
and 20 mM HEPES (pH 7.4).
For integrin immunoprecipitation, L6E9 and L6MLC/IGF-I myoblasts and
myotubes were extracted with 0.5% Triton X-100 in 50
mM HEPES (pH
7.5)-150 mM NaCl containing protease and phosphatase
inhibitors (2 mM
phenylmethylsulfonyl fluoride, 100 U of aprotinin
per ml, 10 µg of
leupeptin and pepstatin per ml, 20 mM sodium
vanadate) and centrifuged
at 12,000 ×
g for 30 min at 4°C to remove
cellular
debris. The supernatant was incubated with protein A-Sepharose
beads to
effect a preclearing for 1 h at 4°C on a rocker. The
protein
concentration was determined by using a protein assay
reagent kit
(Bio-Rad). Samples containing 1 mg of total protein
extract were
immunoprecipitated with rabbit polyclonal anti-
1D
integrin antibody
(kindly provided by G. Tarone) for 2 h at 4°C
on a rocker and
then incubated with protein A-Sepharose beads
at 4°C for 1 h.
The beads were extensively washed with 0.5% Triton
X-100 in 50 mM
HEPES (pH 7.5)-150 mM
NaCl.
For electrophoresis, proteins were separated by use of a 7%
polyacrylamide gel (SDS-polyacrylamide gel electrophoresis [PAGE])
(
28) and transferred onto an Immobilon membrane (Amersham)
as
described by Towbin et al. (
44). The blot was blocked
with 5%
nonfat dry milk-0.1% Tween 20 in Tris-buffered saline (TBS;
50
mM Tris-HCl, 150 mM NaCl [pH 7.5]) for 1 h at room
temperature
and probed with ExtrAvidin-peroxidase-conjugated (dilution,
1:2,000).
The blots were washed extensively with TBS plus 0.1% Tween
20
and then with TBS and finally developed by using enhanced
chemiluminescence
ECL reagents
(Amersham).
LY294002 treatment.
Cells were grown as described above and
treated with a 10 µM concentration of a PI 3-kinase inhibitor,
LY294002, at different times in the differentiation program: during
growth in GM, at 80% of confluence (day 0), and at days 1, 2, and 3 in
DM. Cells that were treated during growth in GM with LY294002 in
dimethyl sulfoxide were switched to DM without PI 3-kinase inhibitor
when they were at 80% confluence. Cells that did not receive inhibitor received the control vehicle dimethyl sulfoxide. After 4 days in DM,
the cells were analyzed morphologically by eosin-Wright's stain or
analyzed by Northern blotting or immunohistochemistry analysis.
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RESULTS |
Postmitotic expression of IGF-I in L6E9 cultures stimulates the
myogenic program.
To uncouple the proliferative and
differentiative effects of IGF-I on muscle cells, we generated a
myogenic cell line in which the IGF-I expression was forced only after
myoblast withdrawal from the cell cycle and induction of
differentiation. The L6E9 line is a subclone of the parental rat
neonatal myogenic line which does not express IGF-I but which presents
IGF-I receptors and is therefore responsive to the growth factor. L6E9
myoblasts were stably transfected with an expression vector in which
rat IGF-I cDNA was driven by regulatory elements from the MLC1/3 locus. These elements (an MLC1 promoter and the downstream MLC enhancer) have
been extensively characterized both in cell culture and in transgenic
mice (13, 40) and are activated only after differentiation has been initiated. Therefore, the transfected MLC-IGF-I transcription unit was expressed only in L6E9 cultures that have permanently withdrawn from the cell cycle and that have committed to myogenic differentiation.
MLC-IGF-I expression was evaluated by Northern blot analysis of total
RNA isolated both from untransfected L6E9 cultures and
from MLC-IGF-I
transfectants (L6MLC/IGF-I). An IGF-I cDNA probe
confirmed that IGF-I
expression was undetectable in control L6E9
cells, whereas it
accumulated to high levels in L6MLC/IGF-I cells
after removal of growth
medium and induction of differentiation
(Fig.
1).
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FIG. 1.
Postmitotic expression of IGF-I enhances expression of
the markers of muscle terminal differentiation. Control L6E9 myoblasts
and L6E9 myoblasts stably transfected with the MLC-IGF-I expression
vector (L6MLC/IGF-I) were grown to approximately 80% confluence in GM
and then switched to DM (day 0). Total RNA was isolated from
proliferative myoblasts (lanes GM) and at 0, 1, 2, 3, and 4 days after
switching to DM. Northern blots of total RNA samples (15 µg) were
analyzed with the indicated 32P-labeled probes. Ethidium
bromide (EtBr) staining was used to verify equal loading of the RNA
sample.
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It has been previously demonstrated that myogenin expression increases
in L6 cultures treated with IGF-I (
14,
19). To
verify
whether the induction of transfected MLC-IGF-I in postmitotic
myotubes
correlated with an increase in myogenin levels, we analyzed
the same
RNA samples with a myogenin probe. Similar levels of
myogenin
transcript were observed in proliferative (GM) myoblasts
of both
control L6E9 and L6MLC/IGF-I cell lines (Fig.
1). However,
a marked
increase in myogenin transcripts was observed in L6MLC/IGF-I
cells as
early as 1 day after serum withdrawal (Fig.
1, day 1).
In addition, the
levels of MRF4 transcripts were modulated by
postmitotic expression of
IGF-I (Fig.
1).
To verify whether the increased levels of myogenin transcript
accumulation correlated with an increase of myogenin promoter
activity,
we transfected both L6E9 and L6MLC/IGF-I myoblasts with
a myogenin
promoter-driven LacZ reporter construct (myogenin-LacZ).
As shown in
Fig.
2, MLC-IGF-I expression resulted in
a dramatic
increase in myogenin promoter activity, as determined by the
numbers
of LacZ-positive cells relative to those of parental L6E9
cells.
In contrast, analysis of the myogenin promoter activity at day
0 (80% confluence), before MLC-IGF-I expression was induced, did
not
show any significant difference between the numbers of LacZ-positive
cells in L6E9 and L6MLC/IGF-I cultures (data not shown).
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FIG. 2.
IGF-I enhances myogenin promoter activity. L6E9 and
L6MLC/IGF-I myoblasts were transiently transfected with a
myogenin-LacZ--Gal vector. Cell cultures were shifted 24 h
after transfection to serum-free medium and cultured for an additional
72 h. Fifty separate randomly selected fields per each cell line
were counted to quantitate LacZ-expressing cells. The numbers of total
cells in each field per each cell line were approximately the same. The
number of -Gal-positive cells per field are indicated on the
y axis.
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The effects of IGF-I, both in cell culture and in vivo, are modulated
by a family of serum binding proteins, IGFBPs, with
different roles
(
25). Specifically, IGFBP4 expression is associated
with
myoblast proliferation (
31), IGFBP6 plays a role in
quiescence
(
15), whereas the positive modulator of the
differentiative
action of IGF-I, IGFBP5, is expressed during myoblast
differentiation
(
32). Analysis of the IGFBP5 transcript in
L6E9 and L6MLC/IGF-I
cultures during differentiation revealed an
increase during myoblast
differentiation in response to IGF-I induction
(Fig.
1), suggesting
that the IGFBP5 gene is itself controlled by
IGF-I and that IGFBP5
positively modulates the differentiative effect
of IGF-I.
To determine whether expression of MLC-IGF-I affected only those genes
expressed after differentiation, we analyzed genes
expressed in
proliferating L6E9 myoblasts. The myogenic regulatory
factor Myf-5
(
6) was expressed at very low levels, only in
the
proliferative L6E9 myoblasts, and this expression pattern
was
maintained in both L6E9 and L6MLC/IGF-I cells (data not shown).
Expression of another member of the helix-loop-helix family of
transcriptional regulators, Id1 (
4), was repressed similarly
during the differentiation of both L6E9 and L6MLC/IGF-I cells
(Fig.
1).
Thus, the expression of the early genes myf-5 and Id1,
which are
presumably involved in myoblast proliferation, is not
affected by
postmitotic expression of IGF-I.
IGF-I potentiates the myogenic program by induction of MEF-2C
expression.
It has been demonstrated that myogenin expression is
necessary but clearly not sufficient for the enhanced myogenesis caused by IGF-I (21). In fact, the induction of myogenic
differentiation by the MRFs is achieved by interaction with multiple
additional transcription factors. Among these, the MEF-2 family is
involved in a complex regulatory network with the MRFs (24,
30). MEF-2A and MEF-2D are active during proliferation, whereas
MEF-2C expression is accumulated during myogenic differentiation
(30). The L6E9 line lacks MEF-2C transcripts (Fig.
3), confirming that the MEF-2C gene is
not activated in these cells.
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FIG. 3.
IGF-I promotes the myogenic program by induction of
MEF-2C expression. Northern blots of total RNA (15 µg) isolated from
L6E9 and L6MLC/IGF-I myogenic cultures at 0, 1, 2, 3, and 4 days after
switching to DM and probed with MEF-2C (30), MCK
(8), and -enolase (35) 32P-labeled
cDNA probes are shown. Ethidium bromide (EtBr) staining was used to
verify equal loading of the RNA sample.
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|
We analyzed the expression pattern of MEF-2C in L6MLC/IGF-I
transfectants to determine whether IGF-I could activate the expression
of silent genes, which are normally involved in regulating the
differentiation program in other muscle cells. Northern blot analysis
revealed that MEF-2C expression was undetectable in control L6E9
proliferating myoblasts (data not shown) and quiescent myoblasts
(Fig.
3, day 0), as well as during all stages of differentiation
(Fig.
3,
1
to
4 days after serum withdrawal). In contrast L6MLC/IGF-I
cultures
activated MEF-2C transcription after only 1 day of serum
withdrawal
(Fig.
3). This result establishes a link between the
action of IGF-I
and the MEF-2C gene, whose expression it
activates.
To verify whether known targets of MEF-2C were also activated during
differentiation of L6MLC/IGF-I cells, we examined expression
of the
muscle creatine kinase (MCK) and
-enolase genes, which
are normally
activated to very low levels in the parent L6E9 line.
The MCK gene
promoter contains, in addition to several MRF E-box
targets, a MEF-2
binding site which is essential for its activity
(
12), and
-enolase presents in its enhancer a G-rich box that
interacts with
ubiquitous factors and a MEF-2 binding site (
35).
MCK and
-enolase expression levels were dramatically enhanced
in L6MLC/IGF-I
differentiating cultures, in contrast to expression
levels found in the
control L6E9 cultures (Fig.
3). This suggests
that the activation of
MEF-2C gene expression by IGF-I reinforces
and amplifies its effect on
the myogenic
program.
Morphological changes induced by postmitotic expression of
IGF-I.
We and others have previously reported that exogenous IGF-I
induces hypertrophy of skeletal myofibers in muscle cell cultures (14, 45). To examine whether transfected MLC-IGF-I
expression in L6E9 cells could induce a similar hypertrophy, we
analyzed the morphological phenotype of control L6E9 and L6MLC/IGF-I
cultures as they differentiated. As shown in Fig.
4B, expression of MLC-IGF-I generated
pronounced myotube hypertrophy. More detailed morphological analysis
revealed an unusual nuclear organization in L6MLC/IGF-I cells, grouped
in characteristic rings located within the middle of the myofibers
(Fig. 4D). That this phenomenon may be related to sarcomeric
reorganization in the hypertrophied fibers is suggested by the
localization of skeletal actin in these cells. Immunohistochemical analysis (Fig. 4E and F) revealed that skeletal actin protein was
uniformly distributed in control L6E9 myotubes, whereas it accumulated
in a perinuclear pattern in L6MLC/IGF-I myotubes.
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FIG. 4.
Postmitotic expression of IGF-I induces myotube
hypertrophy and cytoskeletal reorganization. L6E9 and L6MLC/IGF-I cell
lines were grown to approximately 80% confluence in GM and then
switched to DM and cultured for 4 days. (A and B) Eosin-Wright's stain
(phase contrast) of L6E9 and L6MLC/IGF-I differentiated muscle cells;
(C and D) Hoechst nuclear staining; (E and F) differentiated cultures
immunostained with mouse anti- actin MAb, processed for alkaline
phosphatase activity, and then photographed at the same
magnification.
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Patterns of integrin expression are altered by postmitotic IGF-I
expression.
Integrins play a decisive role in cell adhesion,
modulating many cellular functions as well as cell growth,
differentiation, programmed cell death, and tissue repair. Recently, a
new muscle-specific isoform of 1 integrin 1D, has been
characterized (3); the expression of this isoform is
undetectable in proliferative myoblasts but is induced upon myoblast
fusion. The 1D integrin is associated with 7A and 7B subunits
and localizes to various adhesive structures of muscle cells, as well
as to costameres, and to myotendinous neuromuscular junctions
(3).
To determine whether IGF-I modulates integrin expression, we analyzed
the expression pattern of
1D integrin which is associated
with
myogenic differentiation, as well as its
7 heterodimeric
partner,
which in contrast is expressed during muscle proliferation
and
differentiation. Immunoprecipitation analysis with anti-
1D
integrin
antibody (Fig.
5) revealed that
1D
integrin was undetectable
in L6E9 (data not shown) and in L6MLC/IGF-I
myoblasts (Fig.
5,
lane 1), was expressed at low levels in control L6E9
myotubes
(Fig.
5, lane 2), but was conspicuously accumulated in
L6MLC/IGF-I
myotubes (Fig.
5, lane 3). In contrast, levels of
7
subunits
were not affected by IGF-I expression, since they remained
unchanged
in L6MLC/IGF-I myotubes (Fig.
5). This result suggests that
the
1D integrin, a marker of terminal myogenic differentiation, is
induced by IGF-I, potentially to regulate cytoskeleton reorganization
and to mediate the IGF-I activity on muscle-specific gene expression
and hypertrophy.
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FIG. 5.
IGF-I induces accumulation of 1D integrin.
Biotinylated total cell extracts from L6E9 and L6MLC/IGF-I
proliferative myoblasts and differentiated myotubes were
immunoprecipitated with anti-1D integrin antibody. Proteins were
resolved by SDS-PAGE, blotted onto nitrocellulose membrane, and probed
with ExtrAvidin-peroxidase. Lane 1, L6MLC/IGF-I proliferative myoblasts
plus antibody (Ab); lane 2, L6E9 myotubes plus Ab; lane 3, L6MLC/IGF-I
myotubes plus Ab; lane 4, L6MLC/IGF-I myotubes without Ab. Arrows
labeled 1D and 7 indicate the bands corresponding to 1D and
7 integrins, respectively.
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Postmitotic expression of IGF-I induces a structural marker of
muscle maturation in L6E9 cells.
During development, skeletal
muscle tissue undergoes a succession of changes to more mature
phenotypes (7). This is accomplished by shifts in gene
expression patterns, which includes a switch from embryonic MyHC to
more mature MyHC isoforms. The L6E9 line most closely represents an
immature myogenic stage, in which the embryonic form of MyHC is still
expressed. To explore whether the hypertrophic muscle phenotype induced
by IGF-I in vitro mimics the muscle maturation process in vivo, we
scored the expression of embryonic and neonatal MyHC isoforms in L6E9
and L6MLC/IGF-I myotubes by immunohistochemical analysis. The data in
Fig. 6 show that whereas L6E9 myotubes
express only the embryonic MyHC isoform, L6MLC/IGF-I myotubes express
both embryonic and neonatal MyHCs. This confirms that in L6E9 myotubes,
expression of IGF-I induces a shift towards a more mature muscle type.
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FIG. 6.
IGF-I activates expression of the neonatal isoform of
MyHC. L6E9 and L6MLC/IGF-I cell lines were grown to approximately 80%
confluence in GM and then switched to DM and cultured for 4 days.
Differentiated cultures were immunostained with MAb against either the
embryonic or neonatal isoform of MyHC (Immunofluor) or were subjected
to nuclear staining (Hoechst).
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IGF-I-mediated muscle hypertrophy is only transiently dependent on
the PI 3-kinase pathway.
The activation of a specific
developmental program requires the integration of multiple extrinsic
signals from the cell membrane that culminate in changes of nuclear
gene expression patterns. Among the known signal transduction
intermediates in muscle cells, the PI 3-kinase pathway has been shown
to modulate myogenic differentiation (10, 26).
To define the specific signal transduction pathway through which IGF-I
elicits the morphological and molecular changes observed
in
differentiated L6MLC/IGF-I cultures, we analyzed the requirement
for PI
3-kinase in myogenic differentiation of this line. Using
a specific PI
3-kinase inhibitor, LY294002, we blocked kinase
activity in
proliferating L6E9 and L6LMLC/IGF-I myoblast cultures
and then
challenged them to differentiate in the absence of inhibitor.
Proliferating L6E9 and L6MLC/IGF-I myoblasts cultured in GM were
sensitive to LY294002 and were not able to form myotubes, as analyzed
with eosin-Wright's stain, even after 4 days in serum-free medium
without the inhibitor (Fig.
7, GM
panels). This indicates a requirement
for PI 3-kinase in the transition
from the proliferative to the
myogenic program.
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|
FIG. 7.
Morphological effect of PI 3-kinase inhibitor on
IGF-I-induced hypertrophy. Cells were grown as described in the legend
to Fig. 1 and treated with a 10 µM concentration of the PI 3-kinase
inhibitor LY294002 at different times of culture: during growth in GM,
at 80% of confluence (d0), and at days 1, 2, and 3 in DM. Cells
treated with LY294002 in GM were switched to DM without PI 3-kinase
inhibitor when they were at 80% of confluence. After 4 days, the
cultures were washed with PBS, fixed with methanol, and stained with
eosin-Wright's stain.
|
|
To determine whether later stages of MLC-IGF-I-induced differentiation
were equally dependent upon the PI 3-kinase signal
transduction
pathway, we blocked kinase activity in differentiating
L6E9 and
L6MLC/IGF-I cultures with LY294002 at progressively later
times after
serum withdrawal and then analyzed their morphology
with
eosin-Wright's stain after 4 days in these differentiation-promoting
conditions, in the continuous presence of inhibitor. As expected,
both
L6E9 and L6MLC/IGF-I cultures treated with LY294002 immediately
upon
serum withdrawal (Fig.
7, day 0 panels) failed to differentiate
and
remained myoblastic even after 4 days in serum-free medium.
However,
when LY294002 was added to cells already cultured in
serum-free medium
for 1 or 2 days (Fig.
7, day 1 and day 2 panels),
myogenic
differentiation proceeded and the hypertrophic morphology
of
L6MLC/IGF-I myotubes was similar to that of myotubes differentiated
in
serum-free medium without inhibitor (Fig.
7, upper two panels).
As
shown in the Hoechst-stained cultures (see Fig.
9, upper panels),
characteristic circular localization of nuclei induced by IGF-I
occurred both in untreated cultures (panel U) and in cultures
treated
with inhibitor 2 days after serum withdrawal (panel d2+LY294002).
In
contrast, cultures treated with inhibitor as myoblasts (see
Fig.
9,
upper GM+LY294002 panel) or immediately upon serum withdrawal
(see Fig.
9, upper d0+LY294002 panel) did not exhibit the same
characteristic
nuclear pattern. These results indicate that PI
3-kinase is required
for the initiation but not for the maintenance
of the hypertrophic
phenotype induced by IGF-I.
Since the activity of the transfected IGF-I gene in the L6MLC/IGF-I
line is under the control of differentiation-specific
regulatory
elements with target sites for myogenic factors, MLC-IGF-I
expression
as well as myogenin gene expression should be sensitive
to the
inhibitory effects of LY294002. This was verified by analyzing
the
prevalence of IGF-I and myogenin transcripts in L6MLC/IGF-I
cultures
untreated with LY294002 and differentiated in serum-free
medium for 4 days (Fig.
8, lanes U) in comparison to
L6MLC/IGF-I
cultures treated with inhibitor as myoblasts (Fig.
8, lane
GM)
or differentiated in the presence of inhibitor after 0, 1, 2,
or 3 days in serum-free medium (Fig.
8, lanes 0, 1, 2, and 3).
IGF-I
expression was undetectable in L6MLC/IGF-I cultures treated
during
growth in GM or at day 0, although myogenin transcripts
were present,
suggesting that the levels of myogenin were not
sufficient to activate
MLC-IGF-I expression. By contrast, IGF-I
transcripts as well as
myogenin transcripts were present at high
expression levels in
L6MLC/IGF-I cultures subjected to LY294002
even after only 1 day in
differentiating conditions. This accounts
for the morphology of
L6MLC/IGF-I cultures in Fig.
7, which are
capable of activating the
MLC-IGF-I gene and thereby respond to
the hypertrophic action of IGF-I
in the absence of PI 3-kinase
signaling, once the cells have withdrawn
from the cell cycle.
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|
FIG. 8.
Late inhibition of PI 3-kinase does not affect myogenic
differentiation. Northern blots of total RNA samples (15 µg) isolated
from untreated (lanes L6E9 or L6MLC/IGF-I cells (lanes U) or from cells
treated with 10 µM LY294002 at different times of differentiation are
shown: during growth in GM, at 80% of confluence (day 0), and at days
1, 2, and 3 in DM. RNAs were probed with IGF-I or myogenin
32P-labeled probes. Ethidium bromide (EtBr) staining was
used to verify equal loading of the RNA samples.
|
|
To determine whether the molecular changes elicited by postmitotic
expression of IGF-I in the L6E9 background could also be
sustained in
the absence of PI 3-kinase, we analyzed the profile
of MyHC isoforms in
L6MLC/IGF-I cultures treated with inhibitor
as myoblasts (Fig.
9, panels GM+LY294002) or differentiated
in
the presence of inhibitor added immediately upon serum withdrawal
(panels d0+LY294002) or 2 days in serum-free medium (panels
d2+LY294002).
All cultures were analyzed 4 days after serum withdrawal,
and
the effects of PI 3-kinase inhibition on the IGF-I-mediated shift
in MyHC isoforms was assessed by immunohistochemical analysis.
As shown
in Fig.
9 (lower panels), accumulation of the neonatal
MyHC isoform was
inhibited by LY294002 when added at the time
of serum withdrawal
(panels d0+LY294002) but not by LY294002 when
added after 2 days in
serum-free medium (panels d2+LY294002).
Thus, the ability of IGF-I to
maintain a more mature muscle phenotype
in differentiated L6E9 cultures
is independent of PI 3-kinase
activity.
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|
FIG. 9.
PI 3-kinase is not required in maintenance of muscle
phenotype induced by MLC-IGF-I. (Lower panels) Immunofluorescence
analysis of the neonatal isoform of MyHC in L6MLC/IGF-I cultures
treated with 10 µM LY294002 inhibitor as myoblasts (panels GM) or
differentiated in the presence of inhibitor added immediately upon
serum withdrawal (panels d0+LY294002) or 2 days after serum withdrawal
(panels d2+LY294002) is shown. U, untreated cultures. Upper panels show
Hoechst nuclear staining. All cultures were analyzed 4 days after serum
withdrawal.
|
|
| |
DISCUSSION |
The IGFs play an important and persistent role in skeletal
muscle development and homeostasis. In this study, we focused on the role of IGF-I in the regulation of the differentiated muscle phenotype. The confounding effects of this growth factor on myoblast proliferation were eliminated from the test system by placing an IGF-I
transcription unit under the control of muscle-specific regulatory
elements from the MLC1/3 locus (13). By uncoupling the
proliferative and differentiative roles of IGF-I in muscle cell
cultures that are deficient in IGF-I themselves, we were able to
determine the extent to which this factor affects the myogenic
differentiation program in the postmitotic milieu and to manipulate the
signal transduction pathways induced in response to IGF-I. Our findings
support a model wherein postmitotic expression of IGF-I facilitates
maturation of the myogenic program, through the induction of novel
genetic pathways in the IGF-deficient L6E9 background.
Cell morphology affected by IGF-I.
The postmitotic expression
of IGF-I in differentiated L6E9 cultures induced dramatic morphological
changes, generating pronounced hypertrophic myotubes accompanied by
accumulation of skeletal actin at juxtaposed myotubes, and increased
expression of 1D integrin. This suggests that the effects of IGF-I
include cyotoskeletal reorganization leading to a hypertrophic
phenotype. The formation of nuclear rings within the body of
hypertrophic fibers observed in these cultures may be due to
cytoskeletal disruption in a microtubule-deficient setting, since
similar nuclear arrangements are observed in muscle cultures treated
with cytocalasin B (43a).
Molecular mechanisms of IGF-I action.
It has been previously
demonstrated that enhancement of differentiation in cell cultures by
exogenous IGF-I application is mediated by myogenin (19). In
the present study, we demonstrated that the postmitotic expression of a
stably transfected IGF-I gene had similar effects, inducing hypertrophy
and displaying dramatic increases in the expression of myogenic
differentiation markers, such as myogenin, MRF4, MCK, and IGFBP5.
Moreover, the elevation of myogenin transcript in L6MLC/IGF-I cells was
associated with a dramatic increase in myogenin promoter activity,
suggesting that IGF-I modulates the myogenin expression at the
transcriptional level. This conclusion is supported by our ability to
generate the L6MLC/IGF-I experimental model itself. In L6E9 cells,
endogenous MLC1 transcription is normally undetectable (23,
34). The fact that the transfected IGF-I mRNA is expressed at
high levels during L6MLC/IGF-I differentiation suggests that the
initial elevated expression of myogenin and/or MEF-2C is sufficient to
activate transcriptional activity of the MLC promoter-enhancer, which
in the absence of IGF-I remains undetectable. Activation of MLC-IGF-I expression presumably catalyzes a positive feedback loop that maintains
elevated myogenin expression levels, which in turn guarantees persistent expression of MLC-IGF-I. Although myogenin is the major IGF-I-stimulated differentiation mediator, it is not sufficient to
promote the formation of larger myotubes (14, 15). In
contrast to these previous studies, the postmitotic expression of a
stably transfected IGF-I gene analyzed here induced the expression of novel myogenic markers, such as MEF-2C, which appears to amplify and
reinforce the differentiative effects of the growth factor, possibly by
contributing to the sustained activity of MLC-IGF-I. Our results
support previous models of IGF action (43) in which the IGF
system participates in a feed-forward loop that may modulate the rate
and extent of terminal differentiation and may be part of the molecular
mechanism leading to skeletal muscle hypertrophy.
Postmitotic expression of IGF-I shifts differentiated muscle cells
to a more mature phenotype.
Skeletal muscle development is
characterized by an orderly progression of molecular signals leading to
generation of heterogeneous muscle fibers. The orchestration of this
program involves different populations of myoblasts that, expressing
different combination of muscle-specific genes, define the embryonic
and fetal phenotype (11, 42). L6E9 cells recapitulate some
aspects of immature muscles which express the embryonic, but not
neonatal, isoform of MyHC and support very low expression levels of MCK
and -enolase. In MLC-IGF-I transfectants, the postmitotic
expression of IGF-I induces the expression of the neonatal isoform of
MyHC, normally silent in L6E9 cells (Fig. 6), up-regulation of MCK,
normally silent in embryonic muscle (17), and up-regulation
of -enolase, normally expressed at very low levels in embryonic
stages (2, 27), suggesting that postmitotic IGF-I expression
promotes a maturation of the myogenic program. This view is consistent
with the phenotype of knockout mice nullizygous either for IGF-I or the
IGF-I receptor, each of which suffer a decline in growth rate beginning
at embryonic day 13.5 and exhibit marked muscle hypoplasia at birth
(29, 36).
IGF-I-mediated signal transduction pathways.
In
differentiation of muscle, as in other tissues, the effects of
extracellular stimuli are mediated by signal molecules, such as hormone
and growth factors, that transduce the information from cell membrane
to the nucleus, eliciting changes in gene expression. It has been
recently demonstrated that the proliferative and differentiative effects of IGF-I are mediated by MAP kinase and PI 3-kinase pathways, respectively (10, 26). In the present study, we sought to define further the role of PI 3-kinase in the hypertrophic response to
postmitotic expression of IGF-I in differentiating muscle cultures. From the results presented here, it appears that PI 3-kinase is transiently involved in MLC-IGF-I-mediated signal transduction, leading to enhanced cell differentiation as well as the hypertrophic response. However, after myoblast commitment to the differentiation program, PI 3-kinase is not required for the maintenance of
MLC-IGF-I-mediated phenotype changes, as indicated by activation of
the MLC1 promoter and accumulation of neonatal MyHC protein in the
presence of PI 3-kinase inhibitors.
Several possible scenarios can be invoked to account for the transient
dependence of the IGF-I-mediated response upon PI 3-kinase
in this
system. A positive-feedback loop may be established downstream
of the
PI 3-kinase action in the signal transduction pathway that
perpetuates
the initial response to IGF-I once it is set in motion.
Alternatively,
a third, PI 3-kinase-independent signal transduction
pathway comes into
play after the initial phases of commitment
to the differentiated
phenotype, which mediates the hypertrophic
action of IGF-I in
postmitotic skeletal muscle cells. Current
studies are aimed at
distinguishing between these
possibilities.
Prospects for IGF-I as a therapeutic agent.
One of the most
severe characteristics of muscular dystrophies is the progressive loss
of muscle tissue due to chronic degeneration, accompanied by the
exhaustion of satellite cells necessary to replace damaged fibers. The
persistent protein degradation observed in neuromuscular diseases is
considered an accelerated form of the progressive atrophy which occurs
in skeletal muscles during normal aging and in both cases reflects an
increase in muscle catabolism. Designing approaches directed towards
the attenuation of muscle degeneration in both aging and neuromuscular
pathologies therefore requires the identification of factors involved
in normal anabolic pathways, in order to promote stabilization and
maintenance of muscle tissues.
The present study establishes IGF-I as an important regulator of muscle
cell differentiation, through its ability to potentiate
maturation of
the myogenic program, induce myotube hypertrophy,
and increase
expression of muscle-specific genes. The pleiotropic
actions of IGF-I
in normal muscle growth and regeneration suggest
a crucial role for
this growth factor in multiple steps of the
myogenic program. In mature
muscle, IGF-I is transiently induced
to orchestrate the necessary
responses of muscle cells to changing
functional requirements after
injury or atrophy, or during aging,
making it an attractive candidate
for gene therapeutic approaches
to the attenuation of muscle
degeneration.
The fact that postmitotic expression of IGF-I enhances rather than
inhibits myogenic differentiation in cell culture indicates
that
undesirable hyperplastic side effects may be circumvented
in
therapeutic settings by restricting the expression of exogenously
administered IGF-I genes to postmitotic cells. Evidence from transgenic
mice expressing IGF-I localized to skeletal muscle suggests this
to be
the case, since to date no neoplastic growth has been observed
in these
animals, which undergo varying degrees of muscle hypertrophy
(
9,
32a). It is also possible that persistent forced expression
of
IGF-I in mature postmitotic muscle may provide a milieu which
serves to
maintain or expand the stem cell population, increasing
the
regeneration potential of atrophied or degenerating muscle.
Preliminary
results from our laboratory support this idea, as
dramatic increases in
satellite cell proliferation have been documented
in cell cultures from
the hypertrophic muscles of mice expressing
the MLC-IGF-I cassette as
a transgene (
32a). Our recent demonstration
that
virus-mediated expression of IGF-1 prevents age-related loss
of muscle
mass and function, accompanied by a relative increase
in DNA content
(
2a), lends additional support to a role for
IGF-1 in
satellite cell proliferation. Further elucidation of
the mechanisms by
which IGF-1 augments the regenerative capacity
of adult muscle fibers
will be necessary to determine the efficacy
of IGF-I in the maintenance
of muscle metabolism, morphology,
and function and to define specific
therapeutic courses for the
treatment of neuromuscular
diseases.
| |
ACKNOWLEDGMENTS |
We thank A. Musacchio, members of the Rosenthal laboratory, and
the reviewers for critical evaluation of the manuscript. We also thank
G. Tarone and F. Balzac for providing the 1D antibody, S. Schiaffino
for MyHC antibodies, and A. Giallongo, W. Wright, H. Arnold, E. Olson,
and J. Florini for providing the -enolase, myogenin, myf-5, MEF-2C,
myogenin-LacZ, and IGFBP5 probes, respectively. We are grateful to L. Sweeney and J. Florini for helpful discussion. We acknowledge
José Gonzalez for generating MLC-IGF-I transgenic mice and Esfir
Slonimsky and Serafima Zaltsman for managing the mouse colony.
This work was supported by grants to N.R. from the National Institute
on Aging.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Cardiovascular
Research Center, Massachusetts General HospitalEast, 149 13th St., 4th Floor, Charlestown, MA 02129-2060. Phone: (617) 724-9560. Fax:
(617) 724-9561. E-mail:
rosentha{at}helix.mgh.harvard.edu.
| |
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Molecular and Cellular Biology, April 1999, p. 3115-3124, Vol. 19, No. 4
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Abstract
Introduction
