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Institut National de la Santé et de la Recherche Médicale (S.R., C.D.), U.142, Hôpital Saint Antoine, 75571 Paris Cedex 12, France; and Laboratoire de développement cellulaire (D.M., C.P.), Unité de Recherche Associée au Centre National de Recherche Scientifique 1947, Institut Pasteur, 75724 Paris Cedex 15, France
Address all correspondence and requests for reprints to: Dr. Catherine Dubois, Institut National de la Santé et de la Recherche Médicale, U.142, Hôpital Saint Antoine, 75571 Paris Cedex 12, France. E-mail: dubois{at}st-antoine.inserm.fr Or, Dr.
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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IGF action is mediated through the type-1 IGF receptor (IGFR-1) (5) and modulated by specific high-affinity binding proteins (IGFBPs). Six species of these ubiquitously secreted IGFBPs have been identified (IGFBP 1–6), which each bind IGF-I and IGF-II with different affinities. IGFBPs can also act independently of IGFs (6, 7, 8). Muscle cells possess receptors for IGFs, produce IGFs, and also express IGFBPs (3, 9, 10, 11, 12, 13). The IGFBPs are highly regulated in the course of myoblast differentiation and IGFBP-5 is strongly up-regulated during differentiation in vitro (12, 14). IGFBP-5 messenger RNA (mRNA) has been detected as early as embryonic day 10.5 in the rat embryo, especially in muscle progenitors, and is present in muscle cells in all regions of the embryo throughout fetal life (15).
In inducible C2 cells, variants of C2 mouse myogenic cells that we have characterized as requiring IGFs to differentiate (16), up-regulation of myogenin and IGFBP-5 are chronologically closely correlated, occurring at the onset of differentiation (17). Because IGFBP-5 gene transcription is rapidly induced following IGF stimulation of myoblasts, we suspected that it may be a consequence of IGFR-1 activation. IGF binding has been shown to result in Ras-dependent activation of mitogen-activated protein kinase (MAP kinase) and phosphoinoside 3-OH kinase (PI 3-kinase) activation (17, 18, 19). However, inhibition of PI 3-kinase suppresses differentiation, but inhibition of MAP kinase does not (20, 21, 22). These observations provided the first clues to be used in elucidating the signaling events required for muscle cell differentiation.
In this study, we have investigated IGFBP expression during IGF-dependent myogenic differentiation using C2 anti-IGF-II myoblasts (23), in which autocrine production of IGF-II is abolished by transfection with a vector generating antisense IGF-II RNA. These cells constitute a useful model to study the effects of de novo IGF or insulin stimulation on muscle cell differentiation. Evidence is provided that up-regulation of IGFBP-5 is mediated via activation of IGFR-1, independently of differentiation.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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RNA preparation and Northern blotting
Whole-cell RNA from cultured cells was prepared, submitted to
gel electrophoresis, and blotted as previously described (16). Ten
micrograms of RNA were analyzed in each case. Homogeneity of RNA
loading and transfer was monitored by ethidium bromide staining of
gels and by hybridizing filters with a probe for ribosomal S26
(25).
Probes
Mouse IGFBP sequences have been published (26), and specific
complementary DNA probes were a generous gift from S. Drop (Erasmus
University, Rotterdam, The Netherlands). Myogenin and atrial myosin
light chain 1 (MLC1A) mRNA specific probes were as previously described
(23). A cDNA probe for ribosomal protein S26 was used to assess amounts
of RNA.
The probes were labeled by random priming in the presence of
-32P-labeled nucleotides (dTTP and dCTP), each at 3000
Ci/mmol (Amersham, Buckinghamshire, UK). Hybridization was performed at
42 C overnight in 50% formamide, 5 x SSC, 10 x Denhart’s
solution, 200 µg/ml denatured salmon sperm DNA, and 50 mM
HEPES, pH 7.
Analysis of secreted IGFBPs
Cell-conditioned serum-free media were collected at various
intervals, clarified by filtration or centrifugation, and used directly
for Western ligand blotting (27). For Western immunoblotting, samples
were desalted on G25 Sephadex columns (PD 10, Pharmacia, France) and
lyophilized before electrophoresis. SDS-12,5% PAGE was run under
nonreducing conditions for ligand blotting and either nonreducing or
reducing conditions for immunoblotting. The proteins were then
transferred onto nitrocellulose membranes (Sartorius, Göttingen,
Germany).
For ligand blotting, the membranes were incubated for 48 h at 4 C with 125I-IGF-I and -II (2 x 105 cpm for each ligand), washed, then exposed to x-ray film at -80 C.
For immunoblotting, the membranes were incubated overnight at 4 C either with a 1:1000 dilution of rabbit polyclonal antihuman IGFBP-5 antiserum (UBI, Euromedex, Souffelweyersheim, France) or rabbit polyclonal antirat IGFBP-2 (gift from Dr. M. M. Rechler, NIH, Bethesda, MD). After incubation with a goat antirabbit antibody coupled to peroxidase (1:10,000), IGFBP-5 was detected by enhanced chemiluminescence (ECL Kit, Amersham).
Mobility shift assay of p70 ribosomal protein S6 kinase (p
70S6k)
C2 anti-IGF-II cells were cultured as described above. Treatment
with rapamycin, LY294002, or wortmannin was performed by preincubating
cells for 1 h in serum-free medium before addition of
10-6 M insulin for a further hour. Cells were
then scraped into cold PBS and centrifuged (6,500 rpm) at 4 C. Cell
pellets were lysed for 30 min at 4 C in 50 mM Tris-HCl, pH
7.4, 1% NP-40, 0.25% sodium deoxycholate, 150 mM NaCl, 1
mM EDTA, 1 mM NaF and a mixture of protease
inhibitors. Homogenates were centrifuged (15,000 rpm) at 4 C for 10
min. Supernatants were submitted to SDS-7.5% PAGE under reducing
conditions. p70S6k was revealed by immunoblotting using
rabbit polyclonal antibodies (Santa Cruz, Tebu, France) at 1:100
dilution.
S6 kinase activity assay
Quiescent myoblasts were treated with insulin and drugs as
described for the mobility shift assay. Cell lysates were prepared
as previously described (28). Briefly, cells were washed twice in
ice-cold PBS and once in lysis buffer containing 120 mM
NaCl, 20 mM NaF, 1 mM EDTA, 15 mM
NaPPi, 0.1 mM phenylmethanesulfonyl fluoride, 1
mM benzamidine, 0.5 mM DTT, and 0.2
mM orthovanadate. Cells were scraped into 0.5 ml of buffer,
after which the extract was sonicated for 30 sec on ice and centrifuged
at 12,000 x g for 10 min at 4 C. 50 µg of protein
lysate were immunoprecipitated with 1 µg of p70S6k
antiserum (UBI) overnight at 4 C, then added to 15 µl (packed volume)
of protein A-sepharose (Pharmacia) for 1 h at room temperature.
Immune complexes were collected and washed five times in lysis buffer
containing lithium chloride at a final concentration of 2
M. S6 kinase activity was measured using, as specific
substrate, the S6 peptide AKRRRLSSLRA. The activities of others kinases
such as pKA, pKC, and calmodulin-dependent kinase, were blocked by
specific inhibitors (S6 kinase kit, UBI). The radioactivity of dried
papers was counted in a Beckman counter (LS 6000 SC).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Differentiation was assessed by Northern blot analysis of myogenin RNA, which is expressed at the onset of differentiation (29). Myogenin transcripts were absent from proliferating myoblasts and barely detectable in quiescent cells, but high levels were present in differentiated cells.
Analysis of IGFBP gene expression revealed changes in the levels of IGFBP-2, -3, and -5 mRNA depending on the state of the cell, whereas IGFBP-6 mRNA levels remained constant. Neither IGFBP-1 nor IGFBP-4 mRNA were detected at any stage.
IGFBP-2 transcripts were present in proliferating myoblasts but severely depressed in quiescent and differentiated cells. Insulin added to the medium of proliferating cells had no effect on IGFBP-2 mRNA levels (data not shown). This indicates that insulin did not affect the expression of IGFBP-2 and that withdrawal of myoblasts from the cell cycle was sufficient to provoke a decrease of IGFBP-2 mRNA accumulation.
IGFBP-3 transcripts were barely detectable in proliferating myoblasts, readily detectable in quiescent cells, and more abundant in differentiated cells.
IGFBP-5 transcripts could not be detected in proliferating myoblasts, even when insulin (10-6 M) was added to the medium (data not shown). Only trace amounts were found in quiescent cells, whereas large amounts of IGFBP-5 mRNA were present in differentiating cells.
Ligand blot analysis of IGFBPs secreted by C2 anti-IGF-II cells (Fig. 2A) revealed a 32-kDa species in the
conditioned media of quiescent cells. Differentiated cells produced
IGFBPs of 30–32 kDa and 39 kDa. In mouse serum, IGFBP-3
characteristically migrates as a doublet of 39–42 kDa, reflecting the
differently glycosylated forms of the protein. The 39-kDa species seen
in the conditioned media of differentiated cells therefore corresponds
to one of the glycosylated forms of IGFBP-3. The 30- to 32-kDa peptide
could correspond to any of several IGFBP species, including IGFBP-5.
Western immunoblot analysis using specific anti-IGFBP-5 antiserum
confirmed the presence of IGFBP-5 in the conditioned medium of
differentiated cells, and its absence in the medium of quiescent cells
(Fig. 2B). Under these reducing conditions, IGFBP-5 had an apparent
molecular mass of 36 kDa. No signal was obtained with the anti-IGFBP-2
in the serum-free media conditioned either by quiescent or
differentiated cells, despite the low levels of IGFBP-2 mRNA in these
cells (data not shown). The 32-kDa IGFBP observed in the media of
quiescent and differentiated cells (Fig. 2A) could correspond to
IGFBP-6, as the cells expressed constant levels of mRNA during all the
stages investigated. No 24-kDa IGFBP species corresponding to
IGFBP-4 could be detected by ligand blotting at any stage (Fig. 2A).
IGFBP-5 was therefore the only IGFBP exclusively expressed in
differentiated muscle cells.
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To determine the possible role of insulin- or IGF-induced IGFR-1
activation in IGFBP-5 expression, we compared the dose-response effects
of insulin with those of des(1, 2, 3) IGF-I, an IGF-I analog with reduced
affinity for IGFBPs but the same affinity for IGFR-1 (30). Predictably,
micromolar concentrations of insulin were required to induce full
expression of IGFBP-5 mRNA, 10 nM insulin being
insufficient (Fig. 3A). This indicated
that the effect of insulin on IGFBP-5 expression is mediated via IGFR-1
activation. At 10 nM, des (1, 2, 3) IGF-I was as effective as
1 µM insulin in stimulating IGFBP-5 expression at both
mRNA and protein levels (Fig. 3B). The same levels of IGFBP-5
expression were obtained with 100 nM IGF-I (data not
shown).
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) revealed induction of IGFBP-5
transcripts 10–15 h after addition of 1 µM insulin.
Moreover, experiments with cycloheximide indicated that the induction
required de novo protein synthesis (data not shown).
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Figure 5A shows that 3 days after
addition of insulin, control cells were fully differentiated, as
confirmed by the presence of myogenin and MLC1A transcripts. Wortmannin
(10-6 M), an inhibitor of PI 3-kinase
activation (31), almost totally inhibited myoblast differentiation, as
did another PI 3-kinase inhibitor, LY294002 (5 µM) (24)
(data not shown). As previously reported, rapamycin (10
nM), an inhibitor of p70S6k (32, 33, 34), also
prevented myoblast differentiation (21).
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). As shown in Fig. 5B, neither
IGFBP-5 mRNA induction nor secretion of the protein into the medium
were affected by wortmannin. However, both were abolished by rapamycin
(Fig. 5C). IGFBP-5 can therefore be induced independently of
differentiation and PI 3-kinase activation. The results
obtained for rapamycin suggested that p70S6k may be
involved in IGFBP-5 induction.
Activation of p70S6k necessitates its phosphorylation,
which occurs within minutes of insulin stimulation. This
phosphorylation reduces electrophoretic mobility, as assessed by
Western blotting. Figure 6 shows that
p70S6k appeared as three bands in insulin-stimulated cells.
The fastest moving band, which was minor in stimulated cells, was
predominant in unstimulated cells and corresponds to an inactive form
of the enzyme. The second intermediate band was present in both
stimulated and unstimulated cells. The slowest moving band appeared
only in stimulated cells. This corresponds to the active form of the
enzyme. Appearance of this active form was abolished not only by
rapamycin, but also by wortmannin and LY 294002. In addition,
measurement of p70S6k activity measured by phosphorylation
of a specific substrate peptide showed that the stimulation by insulin
was totally abolished by all three drugs (Fig. 7). These
findings strongly suggest that p70S6k plays no role in
IGFBP-5 induction because it was equally inhibited by both wortmannin
and rapamycin.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Previous work had established that IGFBP-2 transcripts and proteins are down-regulated during differentiation in cultured muscle cells (10). Our findings provide further evidence that this down-regulation results rather from the arrest of proliferation that precedes differentiation because the decrease occurs in quiescent undifferentiated myoblasts. This contrasts with the pattern of IGFBP-3 expression. IGFBP-3 transcripts become detectable when cells cease proliferating. To date, there has been only one report on the expression of IGFBP-3 by cultured muscle cells (35). Interestingly, we also observed IGFBP-3 expression in both parental C2 cells and inducible C2 myoblasts (data not shown). A link between IGFBP-3 and the arrest of proliferation has previously been established. It was shown that intact IGFBP-3, as well as proteolytic fragments of this IGFBP, possess an intrinsic antiproliferative activity (36, 37, 38, 39). It is tempting to consider that induction of IGFBP-3 could contribute toward the arrest of proliferation that is required for muscle cell differentiation.
As previously reported (40), we have also observed that, in C2 anti-IGF-II myoblasts, IGFBP-5 up-regulation of transcripts and protein occurs at the onset of differentiation and accompanies expression of the myogenic differentiation factor, myogenin. We further addressed the problem of whether the up-regulation of IGFBP-5 results from IGFR-1 activation or whether it forms an integral part of the myogenic program. The results of the dose-response experiments with insulin, IGF-I, and des(1, 2, 3) IGF-I analog indicate that IGFBP-5 induction is dependent upon IGFR-1 activation. Induction occurred 10–15 h after IGFR-1 activation and necessitated de novo protein synthesis.
We tested inhibitors of IGFR-1 tranduction pathways, which also have the advantage of blocking myogenesis. Our observations on the suppression of myogenesis by these inhibitors sheds new light on the regulation of IGFBP-5. IGF induces PI 3-kinase activity, and wortmannin, a specific inhibitor of this enzyme activity, impairs terminal differentiation (22). Strikingly, this drug had no effect on IGFBP-5 induction at either RNA or protein level. These results were confirmed with LY294002, another PI 3-kinase inhibitor. Our data therefore demonstrate that IGFBP-5 induction is divorced from the myogenic program and is, in addition, independent of PI 3-kinase activation.
Rapamycin blocked both myogenic differentiation and the induction of IGFBP-5. The effects of rapamycin are known to be mediated by association with a family of intracellular receptors: the FK506-binding proteins (FKBP) (41). The best documented effect of rapamycin occurs through binding to a species of FKBP, FKBP12, which in turn alters the activity of the putative kinase mTOR (mammalian target of rapamycin) (32). This complex prevents activation of p70S6k by IGF (42). FK 506, like rapamycin, binds to FKBP12 but acts via a different mechanism, involving the phosphatase, calcineurin (41). We observed that neither IGFBP-5 induction nor differentiation were impaired by FK 506. The results of the experiments with rapamycin could be interpreted as support for the notion of p70S6k being involved in the control of IGFBP-5, but those for p70S6k activation would plead against it, this activation being triggered by IGFR-1 stimulation that is totally blocked by wortmannin and rapamycin. It could be considered that p70S6k functions downstream of PI 3-kinase (42, 43, 44, 45, 46) because wortmannin and rapamycin block phosphorylation and hence activation of p70S6k. The effects on IGFBP-5 expression would appear to be paradoxical. Wortmannin had no effect, whereas rapamycin suppressed IGFBP-5 expression. Rapamycin would therefore appear to inhibit IGFBP-5 expression via a mechanism that is independent of p70S6k activation. However, one cannot exclude the possibility that the mode of activation of p70S6k could determine its specificity. Wortmannin prevents the phosphorylation of p70S6k at different sites from those sensitive to rapamycin (47). Therefore, rapamycin could generate a p70S6K form involved in the regulation of IGFBP-5. It is also possible that the p70S6k exerts an effect that is independent of its kinase activity, as has been shown for pp90RSK (48). Furthermore, it is conceivable that the activities of rapamycin are not uniquely devoted to the inhibition of p70S6k and that a rapamycin-sensitive target exists, which would be involved in the control of IGFBP5. The FKBP12-mTOR complex, which is rapamycin sensitive, could interact with and activate another target required for IGFBP-5 expression. Rapamycin also acts through other FKBP proteins, such as FKBP25 (41), which is predominant in the nucleus and well placed to interfere with transcription. Moreover, rapamycin alters the translation of certain transcripts (49, 50), and it seems possible that the induction of IGFBP-5 transcription would require synthesis of some specific factor. This would be compatible with our observations that IGFBP-5 induction requires de novo protein synthesis.
IGF activation of IGFBP-5 has also been observed in other cell types (51, 52, 53) but, in muscle cell lines, it has consistently been related to differentiation (11, 12, 14, 40). Our work shows that IGFBP-5 can be activated independently of differentiation. This is an interesting feature. It gives muscle cells the opportunity to express IGFBP-5 before differentiation. Because overexpression of IGFBP-5 impairs IGF-stimulated myogenesis (54), it can be hypothesized that the role of IGFBP-5 during the course of muscle development would be to prevent premature differentiation. Furthermore, sequestration of IGFs by IGBP-5 in the extracellular matrix (55) could serve as a local source of IGFs to be used to protect cells from apoptosis (56, 57) and to promote future proliferation and differentiation of myoblasts during muscular regeneration.
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Acknowledgments |
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Footnotes |
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Received September 3, 1997.
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