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Laboratoire de Différenciation Cellulaire et Croissance, Institut National de la Recherche Agronomique (M.N., B.B., V.G., H.B.), 34060 Montpellier, France; and the Department of Molecular Biology, Max Planck Institut für Biochemie (J.S.), Martinsried, Germany
Address all correspondence and requests for reprints to: Dr. Henri Bernardi, Laboratoire de Differenciation Cellulaire et Croissance, Institut National de la Recherche Agronomique, place Viala, 34060 Montpellier Cedex 01, France. E-mail: bernardi{at}ensam.inra.fr
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Abstract |
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Inducible myoblasts were transfected with either the eukaryotic expression vector pNTK or pNTK containing the human type I IGF-R complementary DNA, and we isolated two clones named Ind-Neo and Ind-R, respectively. Binding and autophosphorylation experiments indicate that Ind-R cells express about 10 times as much type I IGF-R compared with Ind-Neo control cells and that the transfected type I IGF-R is functional in Ind-R cells.
We show that overexpression of the human type I IGF-R makes inducible myoblasts able to differentiate spontaneously, as assessed by expression of the myogenic transcription factors MyoD and myogenin, detection of the muscle-specific protein troponin T, and myotube formation. Moreover, when exposed to IGF-I, Ind-R cells lose contact inhibition, grow in the presence of a low level of growth factors and form colonies in soft agar, which is characteristic of a ligand-dependent transformed phenotype.
It emerges from this study that 1) the type I IGF-R is strongly involved in the phenotypic differences between inducible and permissive cells with respect to the differentiation program; and 2) overexpression causes this receptor to act as a ligand-dependent transforming protein in muscle cells. We suggest that type I IGF-R abundance and level of activation may determine the efficiency of the autocrine mode of action of IGFs and discriminate their biological functions.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-subunits and two ß-subunits. The
ß-subunit includes a tyrosine kinase domain responsible for
autophosphorylation and initiation of the signaling pathways through
the receptor (9, 11). Several type I IGF-R variants, which differ in
electrophoretic mobility (12), binding properties (13), or
immunoreactivity (14), have been reported in various tissues, arising
from splice variants (15), posttranslational modifications (16), or
hybrid formation with the insulin receptor (17). It has been well
established that muscle cells express all the components of the IGF
system (18, 19, 20) and that IGFs stimulate both proliferation and
differentiation of muscle cells (21). Therefore, muscle cell lines
provide a suitable model to study and discriminate the different IGF
effects and their mechanism of action. They also prove to be of
particular interest in the study of mutually exclusive cellular
programs, such as proliferation and differentiation. During skeletal
muscle differentiation, proliferating undifferentiated myoblasts cease
dividing and fuse to form multinucleated myotubes. This process is
accompanied by the coordinate expression of several muscle-specific
genes under the control of the members of the myogenic transcription
factor family (Myf 5, MyoD, myogenin, and MRF4) (22). It has been
demonstrated that IGFs can induce the expression of myogenin and MyoD
genes (23, 24); however, the particular mechanism by which type I IGF-R
activation modulates the expression of myogenic genes is not precisely
known. To further investigate the role of the type I IGF-R in myoblast
differentiation, we took advantage of the inducible variant of the C2
murine muscle cell line (25), which exhibits interesting features with
respect to the IGF system. Unlike the parental cells, referred to as
permissive, which are autonomous for differentiation, inducible
myoblasts require exogenous IGFs to differentiate (26). Two differences
at the molecular level have been implicated in this phenotype: 1)
inducible myoblasts produce 200 and 10 times less IGF-I and IGF-II than
permissive myoblasts, respectively (27); and 2) although expression of
MyoD is constitutive in permissive cells, this gene is only induced
during the differentiation program in inducible myoblasts (28).
Interestingly, we have shown that inducible and permissive cells also
differ with respect to their IGF-Rs. Indeed, we observed an atypical
2ß2 IGF-R in quiescent inducible, but not
permissive myoblasts. This receptor has higher mol wt - and
ß-subunits than the classical type I IGF-R and exhibits higher
affinity for IGF-II than for IGF-I (29, 30). In this study we
overexpressed the classical human type I IGF-R in inducible myoblasts
and examined the effects of this overexpression on differentiation and
proliferation. We found that type I IGF-R overexpression abrogates the
exogenous IGF requirement for differentiation and induces a
ligand-dependent transformed phenotype in Ind-R cells, but not in
control cells. |
Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Proliferating myoblasts were routinely maintained in a growth medium consisting of DMEM-Ham’s F-12 (1:1) supplemented with 20% FCS (Life Technologies, Cergy Pontoise, France) and incubated at 37 C under 5% CO2.
To achieve differentiation, myoblasts grown for 3 days in proliferation medium supplemented with dexamethasone (10-6 M) were switched to low serum differentiation medium consisting of DMEM supplemented with 0.5% FCS with or without IGF-I (10-8 M; gift from Dr. H. H. Peter, Ciba Geigy, Basel, Switzerland). The initial cell density was 640 cells/cm2.
Monoclonal anti-type I IGF-R antibody IR3 was obtained from
Calbiochem and used in culture medium at 1 µg/ml.
Transfection of C2 cells with human type I IGF-R-expressing
vector
The pNTK eukaryotic expression vector containing or lacking the
4.4-kilobase (kb) human type I IGF-R-coding sequence was used.
Transcription of the receptor complementary DNA (cDNA) is under the
control of the thymidine kinase promotor, and the vector contains the
neomycin phosphotransferase gene that confers resistance to the
antibiotic G418. Inducible cells were transfected using Lipofectamine
reagent (Life Technologies) as described by the supplier. Stably
transfected cells were selected in the presence of 1 mg/ml G418
(Geneticin, Life Technologies). Individual colonies were isolated after
5 days, passaged into stable cell lines, and cloned by limiting
dilution. Two clones, named Ind-R (expressing human type I IGF-R) and
Ind-Neo (control cells, transfected with pNTK), were used for the
following studies.
Binding studies
For type I IGF-R binding assays, membrane samples (150 µg
protein, determined by the Bio-Rad protein assay, Hercules, CA),
prepared as previously described (29), were incubated overnight at 4 C
with [125I]IGF-I (50 pM) and an appropriate
dilution of unlabeled IGF-I in a final volume of 0.3 ml buffer A (20
mM Tris-Cl, pH 7.5, and 10 mM
MgCl2) containing 0.2% BSA (Sigma Chemical Co., St. Louis,
MO). Nonspecific binding was determined by adding an excess of IGF-I
(5 x 10-8 M). Membrane-bound and free
hormone were separated by adding 3 ml ice-cold buffer A and
centrifuging at 5000 x g for 30 min at 4 C. The
supernatant was removed, and the radioactivity of the pellets was
measured in a 
-counter (Kontron, St. Quentin en Yvelines,
France).
Autophosphorylation of type I IGF-R
Microsomal membranes were solubilized, and IGF-R were partially
purified using a wheat germ agglutinin (WGA)-Sepharose column, as
previously described (31). WGA-purified receptors (5–30 µg protein)
were preincubated for 30 min at 4 C in 50 µl 50 mM HEPES
(pH 7.8)-2.5 mM MnCl2 buffer in the presence or
absence of IGF-I (10-9 M). The kinase reaction
was initiated by the addition of 1 µM
[-32P]ATP and 50 µM sodium orthovanadate
and, after 10-min incubation at 20 C, was stopped by the addition of
200 µl chilled stop mix [5 mM HEPES (pH 7.8), 2% Triton
X-100, 150 mM NaCl, 0.4 mM sodium
orthovanadate, 4 mM EDTA, 4 mM EGTA, and 0.4
mM phenylmethylsulfonylfluoride containing 40 µg each of
leupeptin, aprotinin, and soybean trypsin inhibitor/ml]. The mixture
was incubated overnight at 4 C in the presence of polyclonal antibody
P2, specific for the tyrosine kinase domain of the receptor (32).
Immune complexes were recovered by centrifugation after incubation for
1 h at 4 C with protein A-Sepharose beads and washed as previously
described (31). Autophosphorylated receptors were eluted by the
addition of denaturing Laemmli buffer containing 1 M
2ß-mercaptoethanol and 0.2 M dithiothreitol, boiled for 3
min, and subjected to electrophoresis (6.5% SDS-PAGE). Then, the gel
was exposed to autoradiography film, and the radiolabeled band
corresponding to the ß-subunit of the receptor was cut out for
Cerenkov counting.
Immunofluorescence assays
Cells were fixed for 5 min in PBS containing 3.7% formaldehyde
(wt/vol), followed by a 30-sec extraction in glacial acetone.
Expression of troponin T was assayed using a 1-h incubation of cells
with 1:100 dilution of a mouse monoclonal antibody against troponin T
(Sigma, T-6277) followed by a 30-min incubation with
fluorescein-conjugated antimouse antibodies (Cappel, Organon Technica,
Fresnes, France). Cells were stained with Hoechst B2883 dye and mounted
in Airvol 205 [15% Airvol 205 (Air Products, Utrecht, The
Netherlands), 33% glycerol, and 0.1% NaN3 in PBS, pH 7].
Stained cells were observed on a microscope (Axiophot, Carl Zeiss,
Thornwood, NY) using a planapochromat x40 objective. Fluorescent
images were recorded onto TriXpan 400 film (Eastman Kodak, Rochester,
NY).
RNA extraction and Northern blot analysis
Total RNA was prepared using the guanidinium thiocyanate method,
as previously described (33). For Northern analysis, total RNA (20
µg) was separated on a 2 M formaldehyde-containing 1%
agarose gel, transferred, and bound to nylon membranes (Hybond-N,
Amersham, Arlington Heights, IL) as recommended by the supplier.
Filters were prehybridized for at least 2 h in a buffer containing
50% formamide, 5 x SSPE (1X SSPE = 0.15 M NaCI, 0.01
NaH2PO4·H2O, 1 mM
EDTA), 0.1% SDS, 100 µg/ml denatured DNA salmon sperm, and 5 x
Denhardt’s solution and hybridized in the same medium containing the
appropriated random-primed 32P-labeled probe
(106 cpm/ml) at 42 C for 6 h. Filters were washed at
room temperature in 2 x SSC (standard saline citrate)
buffer-0.1% SDS for 5 min and twice at 65 C for 20 min each time in
0.2 x SSC-0.1% SDS. Filters were then exposed to X-Omat Kodak
x-ray films at -70 C.
RT-PCR amplification of MyoD
First strand cDNA synthesis of RNA was carried out at 37 C for
1 h in a final volume of 50 µl containing 1 µg total RNA, 2
µg random primers, 5 U RNasin (Promega, Madison, WI), 1
mM of each deoxy-NTP, and 200 U reverse transcriptase
Moloney murine leukemia virus ribonuclease H-
(Promega).
PCR amplification of a 144-bp fragment of MyoD was performed using 1 x standard PCR reaction buffer [50 mM KCl, 10 mM Tris-Cl (pH 9 at 25 C), 0.1% Triton X-100, 2 mM MgCl2], 200 µM deoxy-NTPs, 2.5 U Taq polymerase (Promega), and 20 pmol of each primer in a 50-µl reaction. The oligonucleotide primers used were MyoD identical and MyoD complementary, previously described by Montarras et al. (34). Amplification of 5 µl of the template first strand cDNA was carried out in ultrathin-walled tubes in a model PTC-150 DNA thermocycler (MJ Research, Watertown, MA) with the following program: 1 min at 93 C and 30 cycles of 1 min at 93 C, 1 min at 50 C, and 1 min at 72 C. Amplified cDNA was analyzed after electrophoretic migration in 2% agarose gels by alkaline Southern blotting onto positively charged nylon membrane (Hybond-N+, Amersham) and hybridization with a random primed 32P-labeled MyoD probe (35).
Soft agar assay
To test the ability of cells to form colonies in soft agar, a
double layer culture technique was used. Per 60-mm dish, 1.8 x
104 cells were seeded in 3 ml proliferation medium
containing 0.2% agar (Life Technologies) with or without IGF-I
(10-8 M) and placed over a bottom agar
consisting of 4 ml proliferation medium containing 0.5% agar. Cells
were fed once a week with 200 µl proliferation medium with or without
IGF-I (10-8 M). Two weeks after seeding,
colonies were scored.
When used to inhibit colony formation, IR3 was included in top agar
medium at a final concentration of 1 µg/ml.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The overexpression of the human type I IGF-R in Ind-R cells was
characterized by Northern blot analysis (Fig. 1A
), [125I]IGF-I binding
studies (Fig. 1B) and autophosphorylation experiments (Fig. 1C).
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). In addition, Ind-R cells and Ind-Neo cells express a
transcript of about 7 kb and a minor transcript of about 11 kb (which
appears after longer exposure). These two latter transcripts correspond
to the endogenous type I IGF-R mRNA, in agreement with the multiple
type I IGF-R transcripts previously reported in mouse cells (36).
Densitometric analysis of autoradiographs showed that mRNAs expressed
from the pNTK expression vector are 20 times more abundant than mRNAs
transcribed from endogenous gene.
Binding displacement curves of [125I]IGF-I (Fig. 1B) and
Scatchard analysis of data (Fig. 1B, inset) indicated that
the maximal binding capacity is about 9 times as much in Ind-R cells (6
pmol/mg protein) as that in Ind-Neo cells (0.7 pmol/mg protein) and
that the transfected human type I IGF-R exhibits an affinity for IGF-I
of 3 x 10-10 M. This value is in the
same range as the affinity for the type I IGF-R in Ind-Neo cells (Fig. 1B) as well as in Per and Ind cells (29). The affinity for IGF-II is
10-9 M (not shown).
Autophosphorylation experiments (Fig. 1C) showed that the ß-subunits
of the type I IGF-R are labeled in Ind-R and Ind-Neo cells. In both
cell lines we observed a 106-kDa band corresponding to the atypical
type I IGF-R previously described in Ind cells (30). Ind-R cells also
exhibited a major band at 101 kDa, which represents the ß-subunit of
the transfected receptor. The two bands cannot be distinguished in Fig. 1C, but they are visible in experiments in which less protein is loaded
(data not shown). The 101-kDa major band in Ind-R cells is labeled
10-fold more strongly than the 106-kDa band in Ind-Neo cells. This
agrees with the 9-fold higher IGF-I binding level present in Ind-R
cells. In the absence of IGF-I stimulation, the labeled ß-subunit
moieties show that the endogenous type I IGF-R as well as the
transfected human type I IGF-R display a basal autophosphorylation
activity. IGF-I (10-9 M) induces a 2-fold
increase in the phosphorylation of the ß-subunit moieties in both
Ind-R and Ind-Neo receptor preparations. This indicates that the
transfected human type I IGF-R and the endogenous type I IGF-R respond
similarly to this ligand.
Taken together, these data demonstrate that Ind-R myoblasts express about 10 times as much type I IGF-R as control Ind-Neo myoblasts and that the transfected human type I IGF-R is fully functional.
Differentiation of Ind-R cells occurs without any IGF-I
addition
Because C2 inducible cells exhibit an atypical
2ß2 IGF-R and require addition of IGFs to
undergo terminal differentiation, we hypothesized that overexpression
of the classical human type I IGF-R in these cells could abrogate their
IGF requirement for differentiation. Therefore, we examined the
kinetics of differentiation of Ind-R cells in the absence of added
IGF-I, which is usually termed spontaneous differentiation. Ind-R and
Ind-Neo myoblasts were grown for 3 days in proliferation medium (20%
FCS), then switched to differentiation medium (0.5% FCS) and assayed
for the appearance of myotubes at 24-h intervals for 4 days. Per cells,
which differentiate spontaneously, were used as a positive control. We
confirmed the validity of Ind-Neo cells as a control by also testing
untransfected Ind cells in each experiment. Throughout this study,
similar results were obtained with Ind-Neo and Ind cells.
To be certain that our results were not due to differences in cell number at the time of induction of differentiation, cell counts were performed on day 3 in proliferation medium. No difference in cell density in Ind-R and Ind-Neo cell cultures was observed.
Activation of muscle-specific genes is controlled by the MyoD family of
transcription factors. Among these transcription factors, two were of
interest in this study: MyoD (which is not expressed in Ind myoblasts,
but is constitutive in Per myoblasts) and myogenin (as an early
differentiation marker). Their expression was analyzed during the time
course of spontaneous differentiation by RT-PCR and Northern blot for
MyoD and myogenin, respectively (Fig. 2).
At the time of differentiation medium switch, Ind-Neo and Ind-R cells
did not express MyoD or myogenin. The two transcription factors
appeared in Ind-R cells from 24 h onward, whereas they were not
expressed in Ind-Neo cells even after 72 h in differentiation
medium. As expected, MyoD was present at all times in Per cells, while
myogenin appeared within 24 h in differentiation medium.
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). This protein
was detected in Ind-R and Per cells after 48 h in differentiation
medium (Fig. 3A). In contrast, Ind-Neo cells failed to exhibit any
positive staining for this protein during the entire differentiation
time course. Hoechst staining showed that cell density was similar in
the three cell lines and that the cells expressing troponin T were
fused and multinucleated cells (Fig. 3A). Consistent with the
expression of troponin T, a large number of fused and terminally
differentiated muscle cells appeared in Ind-R and Per cell cultures
after 72 h in differentiation medium. In contrast, no myotubes
were formed in Ind-Neo cell cultures (data not shown).
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To further characterize the phenotype of Ind-R cells with regard to
differentiation, cells were stimulated with 10-8
M IGF-I, a concentration inducing optimal differentiation
of C2 cells (26). Under these conditions, instead of differentiating,
Ind-R cells continued to proliferate and formed cellular aggregates
that failed to express troponin T (Fig. 3B). Using lower concentrations
of IGF-I (10-9 and 10-10 M),
Ind-R cells differentiated normally.
These data suggest that type I IGF-R signals may induce differentiation or proliferation depending on the level of activation of the receptor.
Overexpression of type I IGF-R promotes a ligand-dependent
transformation of Ind-R cells
Type I IGF-R overexpression has been shown to promote neoplastic
transformation of fibroblasts (4). Likewise, Quinn et al.
(37, 38) have shown that myoblasts overexpressing type I IGF-R exhibit
some properties similar to those of transformed cells. Therefore, we
studied the proliferation of Ind-R cells and evaluated the
transformation potential of type I IGF-R overexpression in inducible C2
myoblasts.
In proliferation medium (20% FCS) supplemented or not with IGF-I, Ind-R and Ind-Neo cells grew at the same rate until they became confluent. This shows that the growth rate of Ind cells in proliferation medium is not affected by type I IGF-R overexpression (data not shown).
Next, we evaluated the effect of type I IGF-R overexpression on the three common characteristics of transformed cells, i.e. loss of contact inhibition, growth in the presence of low level of growth factors, and ability to grow and form colonies in soft agar.
Under proliferation conditions (20% FCS) and in the absence of IGF-I,
Ind-R cells continued to proliferate after reaching confluence, and
occasional areas of multilayered cells were observed (data not shown).
In the presence of IGF-I (10-8 M), the loss of
contact inhibition was further stimulated, and multilayering became
uniform in the culture (Fig. 4A). This
was not observed in Ind-Neo cells, which grew as a monolayer and
stopped proliferating when they reached confluence in the presence or
absence of IGF-I. In low serum differentiation medium supplemented with
IGF-I (10-8 M), as described above, Ind-R
cells failed to differentiate. In contrast, they proliferated and lost
contact inhibition, forming large multilayered focal aggregates (Fig. 4B). This was not observed with Ind-Neo cells, which differentiated
normally.
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). In contrast, Ind-Neo
cells did not grow in low serum in the presence or absence of
IGF-I.
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). Cells were plated in soft agar in the
presence of 20% FCS, with (Fig. 6B) or without (Fig. 6A) IGF-I, and
plates were examined 2 weeks later. Under these conditions, we were
able to clearly distinguish small colonies containing less than 100
cells from large colonies of several hundred cells. Ind-R cells
exhibited anchorage-independent growth that was strongly stimulated by
IGF-I. Indeed, in the absence of IGF-I about 5% of seeded Ind-R cells
formed colonies, but the proportion increased to 40% with IGF-I
stimulation. Moreover, large size colonies predominated in the presence
of IGF-I, whereas most colonies were small in its absence. In contrast,
Ind-Neo cells failed to form colonies even in the presence of
IGF-I.
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Antibody IR3 inhibits the effects of human type I IGF-R
overexpression
To make sure that the phenotypic characteristics of Ind-R cells
are due to the forced expression of human type I IGF-R, we incubated
the cells with IR3, a monoclonal antibody specific for the human
type I IGF-R. This antibody recognizes the -subunit of human type I
IGF-R, but not the murine type, prevents IGF-I binding, and thus blocks
IGF-I-mediated receptor activation (39, 40). The effects of IR3 were
studied in differentiation and transformation assays (Fig. 7).
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IR3 strongly inhibited spontaneous
formation of myotubes in Ind-R cell cultures (Fig. 7A, upper right). Furthermore,
the addition of IR3 abolished the IGF-I-induced Ind-R cell
transformation. Indeed, Ind-R cells differentiated in differentiation
medium containing IGF-I plus IR3 (Fig. 7A, lower right),
whereas in the absence of IR3, they proliferated and lost contact
inhibition (Fig. 7A, lower left). Colony formation of Ind-R
cells in soft agar was nearly completely inhibited by IR3 in both
the presence and absence of IGF-I (Fig. 7B). Control Ind-Neo cells were
not affected by the presence of antibody IR3, which did not bind to
murine type I IGF-R.
Thus, antibody IR3 strongly inhibits the phenotypic changes observed
in Ind-R cells, indicating that these modifications are mediated
through the transfected human type I IGF-R.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The type I IGF-R overexpression level in Ind-R cells indicates that the
thymidine kinase promotor of the pNTK expression vector is active in
the C2 myogenic cell line and provides a transcription rate similar to
that observed with other promoters used in different muscle cell lines
(38, 42). Binding experiments and autophosphorylation assays indicate
that the transfected human type I IGF-R is fully functional in Ind-R
cells and exhibits affinities for IGF-I and IGF-II that agree with
values usually reported for the type I IGF-R (43). We have previously
demonstrated that inducible cells exhibit an atypical
2ß2 type I IGF-R that differs from the
classical type I IGF-R by its higher affinity for IGF-II compared with
IGF-I and the higher mol wt of its ß-subunits (29, 30). This atypical
type I IGF-R may result from specific posttranslational processing of
the type I IGF-R in inducible cells. Therefore, we looked at the
molecular size of the ß-subunit of the transfected receptor in Ind-R
cells. Ind-Neo and Ind-R cells exhibit a 106-kDa ß-subunit
corresponding to the endogenous atypical type I IGF-R previously found
in inducible cells (30). In addition, Ind-R cells exhibit a 101-kDa
band that represents the ß-subunit of the transfected receptor. This
101-kDa moiety may correspond to one of the type I IGF-R ß-subunits
previously described in C2 permissive cells (30). Taken together, these
results and the higher affinity of the transfected receptor for IGF-I
than for IGF-II indicate that the human type I IGF-R is processed as a
classical type I IGF-R in murine C2 inducible cells. Further
transfection experiments using the murine type I IGF-R cDNA will be
performed to definitively determine whether posttranslational
processing is responsible for the properties of the atypical type I
IGF-R in these cells. However, Ind-R cells overexpressing a fully
functional human type I IGF-R constitute a suitable tool to study the
role of the type I IGF-R on the control of differentiation and
proliferation of muscle cells.
Forced expression of the classical human type I IGF-R allows C2
inducible myoblasts to differentiate spontaneously as do parental C2
permissive myoblasts. The inhibitory effect of the monoclonal antibody
IR3 confirms that spontaneous differentiation is mediated through
the transfected human type I IGF-R and that activation of this receptor
by IGF-I binding is necessary for induction of cell differentiation.
Quinn et al. (38) previously observed that differentiation
of C2 cells overexpressing human type I IGF-R was faster than that in
control cells. The present study provides evidence that the type I
IGF-R is more than a modulator and is a major effector of myoblast
differentiation. However, Ind-R myoblasts do not differentiate until
they are switched to low serum differentiation medium. This suggests
that overexpression of type I IGF-R is not sufficient to abrogate the
effects of some serum growth factors inhibiting myoblast
differentiation (2).
The spontaneous differentiation of Ind-R cells may be closely related to the level of type I IGF-R activation. Indeed, the IGFs present in the medium bind to the overexpressed human type I IGF-R and thus contribute to its high endogenous kinase activity. Consequently, the signaling provided by the human type I IGF-R in response to endogenously produced IGFs may be sufficient to promote cell differentiation in the absence of exogenous IGF when Ind-R cells are switched to low serum differentiation medium. In addition, the molecular form of the transfected IGF-R may be also involved in supporting spontaneous differentiation of myoblasts. Indeed, overexpression of the human type I IGF-R introduces into inducible myoblasts a classical type I IGF-R, whereas these cells endogenously express an atypical one (29, 30). Thus, to accurately determine the relative importance of the molecular form and the number of type I IGF-R for inducible myoblast differentiation, the classical murine type I IGF-R should be transfected into inducible cells in which the gene of the endogenous type I IGF-R has been disrupted.
Although Ind-R myoblasts undergo spontaneous differentiation, they do not exhibit all the characteristics of the permissive phenotype. In particular, MyoD mRNA is not detectable at the myoblast stage in Ind-R cells. Moreover, Ind-R myoblasts produce 200 and 10 times less IGF-I and IGF-II than Per myoblasts, respectively (not shown). The same difference in IGF production has been observed between Ind and Per myoblasts (27). Thus, overexpression of the human type I IGF-R is not sufficient to induce MyoD expression and increase the secretion of IGFs. This suggests that the expression of the MyoD gene and the production of IGFs are not regulated solely by the type I IGF-R (24), but require other pathways that are active in permissive, but not in inducible or Ind-R, myoblasts.
We observed that Ind-R cell differentiation is stimulated by IGF-I concentrations up to 10-9 M, whereas it is inhibited by higher concentrations. This is in agreement with previous observations reporting that low concentrations of IGF-I stimulate whereas higher concentrations cause progressive inhibition of myoblast differentiation (2). However, compared with Per and Ind-Neo cells, Ind-R cells display an increased sensitivity to the ligand. Indeed, at the same dose of IGF-I (10-8 M), differentiation is inhibited in Ind-R cells, whereas it is stimulated in Per and Ind-Neo cells. This increased sensitivity to the ligand in Ind-R cells is probably due to amplified signaling through much more abundant type I IGF-R. Indeed, we find that Ind-R cells express about 10 times as much type I IGF-R as Ind-Neo cells. The number of type I IGF-R in Ind-R cells is also 10 times higher than that reported in Per cells (29).
Previous studies have shown that overexpression of tyrosine kinase
receptors such as the epidermal growth factor receptor, insulin
receptor, and type I IGF-R induces a ligand-dependent transformed
phenotype in fibroblasts (4, 44, 45). The involvement of the type I
IGF-R in the expression of the transformed phenotype was clearly
demonstrated by Sell et al. (46) using a fibroblast cell
line generated from mouse embryos homozygous for a targeted disruption
of the Igf1r gene. These findings led us to evaluate the
transformation potential of type I IGF-R overexpression in C2 inducible
myoblasts. We found that in the presence of IGF-I (10-8
M), Ind-R cells exhibit the characteristics of a cellular
transformed phenotype, i.e. growth in 0.5% FCS medium, loss
of contact inhibition, and anchorage-independent growth. We also
observed that IGF-I is 100-fold more potent than insulin in promoting
colony formation in soft agar, a ratio that parallels the difference in
affinities of the two ligands for the type I IGF-R. This and the fact
that IR3 antibody blocks colony formation indicate clearly that
IGF-I-induced transformation is mediated through the overexpressed
human type I IGF-R. Thus, overexpression causes the type I IGF-R to
function as a ligand-dependent transforming protein in C2 inducible
myoblasts. Furthermore, we observed that in the absence of type I IGF-R
overexpression, high levels of IGFs do not promote transformation of
inducible or permissive cells. This suggests that a sufficient type I
IGF-R density is required to invoke transformation-related signaling
pathways. Thus, in the present Ind-R cells as well as in murine
fibroblasts (47), the rate-limiting feature for transformation seems to
be the number of type I IGF-Rs and the associated level of kinase
activation. It is of interest that Ind-R cells lose contact inhibition
and form colonies in soft agar in the presence of 20% FCS and IGF-I
(10-8 M), although they exhibit no change in
their rate of growth. This result further suggests that the control of
cellular proliferation and transformation may involve distinct
signaling pathways (46, 48).
We cannot positively exclude that the human receptor overexpressed in murine cells behaves differently from the murine receptor. Therefore, the effects observed in transfected cells could be due to the specific properties of the human type I IGF-R rather than to its overexpression per se. However, our conclusions agree with previous statements obtained in several cell types and species on the role of the type I IGF-R in cellular differentiation, proliferation, and transformation (4, 43, 49, 50, 51). In addition, we found that the two C-terminus regions of the human type I IGF-R that were proven to be specifically implicated in its transforming activity (i.e. Y1251 and the residues around H1293) (52) are identical in the human and murine receptors (personal data). Therefore, we can reasonably consider that the effects observed in transfected cells are not artifacts due to species specificity, but are actually due to overexpression of the type I IGF-R.
We conclude from this study that 1) the type I IGF-R is strongly involved in the phenotypic differences between inducible and permissive C2 cells with respect to the differentiation program; and 2) the type I IGF-R can mediate signals for both differentiation and transformation of muscle cells. It seems that discrimination among IGF-I-induced differentiation, proliferation, or transformation is dependent upon IGF-R number and the corresponding level of kinase activation. Discrimination among these cellular programs may also be related to the molecular form of the type I IGF-R. Thus, these quantitative and qualitative characteristics may direct the interaction of the type I IGF-R with different intracellular substrates to trigger specific pathways for cellular differentiation, proliferation, or transformation. Further studies with the Ind-R cell line may help to distinguish the different signal transduction pathways activated in response to type I IGF-R stimulation in muscle cells.
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Acknowledgments |
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2 Recipient of a fellowship financed by the Association
Française contre les Myopathies and the Ministère de la
Recherche et de la Technologie.
Received May 28, 1997.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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2ß2 IGF-II receptor type in C2 myoblasts.
Eur J Biochem 208:273–279[Medline]
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) and Ind-R (•) cells. B, Specific binding;
nonspecific binding represented 7% of total binding.
Inset, Scatchard analysis of the data concerning
[125I]IGF-I binding displacement by IGF-I to microsomes
from Ind-Neo and Ind-R cells. Dissociation constants were 3 x
10-10 and 5 x 10-10 M for
Ind-R and Ind-Neo, respectively. B/F, Bound to free ratio. C,
Autophosphorylation response of WGA-purified type I IGF-R from Ind-Neo
and Ind-R cells. Samples were autophosphorylated in the absence (-) or
presence (+) of IGF-I (10-9 M),
immunoprecipitated with antihuman type I IGF-R antipeptide antibody P2,
and subjected to 6.5% SDS-PAGE under reducing conditions as described
in Materials and Methods.
and 
) and Ind-R (
