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Departament de Bioquímica i Biologia Molecular, Facultat de Biologia, Universitat de Barcelona; and Departament de Neurologia (E.G., I.I.), Hospital Universitari de la Sta. Creu i Sant Pau 08028 Barcelona, Spain
Address all correspondence and requests for reprints to: Dr. Perla Kaliman, Departament de Bioquímica i Biologia Molecular, Facultat de Biologia, Universitat de Barcelona, Avinguda Diagonal 645, 08028 Barcelona, Spain. E-mail: perlak{at}porthos.bio.ub.es
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Introduction |
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During the last few years, the intracellular myogenic signaling process
initiated by IGFs has begun to be elucidated. In this context, it has
recently been shown that phosphatidylinositol 3-kinase (PI 3-kinase) is
an essential second messenger for the myogenic actions of IGFs (8). The
PI 3-kinase inhibitors, wortmannin and LY294002, potently block the
differentiation program of rat and mouse skeletal muscle cell lines
(9, 10, 11, 12). Moreover, the overexpression in L6E9 muscle cells of a mutant
p85 regulatory subunit of PI 3-kinase (
p85) lacking the ability to
bind and activate the p110 catalytic subunit (L6E9-p85) showed that
the heterodimeric p85-p110 is the PI 3-kinase isoform essential for
IGF-induced myogenesis (10). Interestingly, L6E9-p85 and both
wortmannin- and LY294002-treated L6E9 myoblasts proliferate normally,
indicating that although PI 3-kinase is essential for muscle
differentiation, it is not a key element for mitogenesis (9, 10).
Currently, there is scarce information regarding the downstream elements activated by IGFs/PI 3-kinase or its phosphatidylinositol 3-phosphate (3-P) products during myogenesis. It has recently been demonstrated that the serine/threonine kinase p70 S6 kinase acts downstream of PI 3-kinase (13, 14). Rapamycin is a potent immunosuppressant that inhibits p70 S6 kinase and the subsequent phosphorylation of its main target, the ribosomal protein S6 (13, 15, 16). By using rapamycin as a specific inhibitor, p70 S6 kinase has been shown to be a key regulator of proliferation in a variety of mammalian cells, including T and B cells (17, 18, 19), hepatoma cells (13), rhabdomyosarcoma cells (20), osteosarcoma cells (21), and myogenic cells (11, 22). In contrast, the role of p70 S6 kinase in differentiation varies greatly among the different cell types: 1) rapamycin inhibits differentiation in 3T3-L1 cells (23), human B lymphocytes (19), L6A1 skeletal muscle cells (11), and, partially, fetal brown adipocytes (24); 2) rapamycin induces differentiation in BC3H1 skeletal muscle cells (22) and B16 melanoma cells (25); and 3) rapamycin has no effect on the differentiation of J2E erythroid cells (26) or monocytic U-937 myeloid leukemia cells (27).
As stated above, contradictory observations have been reported concerning the role of the p70 S6 kinase in skeletal muscle differentiation; although the p70 S6 kinase inhibitor rapamycin induced myogenesis in BC3H1 mouse cells (22), it has been shown to inhibit differentiation in L6A1 rat skeletal muscle cells (11). In an attempt to clarify the role of p70 S6 kinase in myogenesis, here we have studied the effect of rapamycin on rat, mouse, and human skeletal muscle cells. We show that although p70 S6 kinase inactivation potently inhibited serum-induced proliferation of L6E9 and Sol8 myoblasts, rapamycin did not alter IGF-II-induced proliferation or differentiation of these cells, as determined by myotube formation and expression of muscle-specific biochemical markers [i.e. myogenin, myosin heavy chain, and GLUT4 (glucose transporter-4)]. Furthermore, we have extended our studies to human myoblasts derived from skeletal muscle biopsies. Here we show that exogenous addition of IGF-II to human muscle cells induced myoblast fusion into myotubes; as previously shown for rat and mouse skeletal muscle cells, IGF-II-induced human myotube formation was totally blocked by LY294002, a specific PI 3-kinase inhibitor, but remained unaffected in the presence of the p70 S6 kinase inhibitor rapamycin.
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Materials and Methods |
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Cell culture
Rat L6E9 and mouse Sol8 myoblasts were grown in monolayer
culture in DMEM containing 10% (vol/vol) FBS and 1% (vol/vol)
antibiotics (10,000 U/ml penicillin G and 10 mg/ml streptomycin).
Confluent myoblasts were differentiated by serum depletion in DMEM
containing 0.5 mg/ml BSA and antibiotics with or without IGFs (40
nM) and rapamycin (1–20 ng/ml) when indicated. Images
shown are representative of 10–20 microscope fields analyzed at random
from each of three independent experiments. Cells were photographed
after staining the nuclei with Mayer’s hemalum solution for microscopy
(Merck & Co., Darmstadt, Germany). To quantify cell
proliferation, cells were plated in multiwell culture dishes, grown
from 1–4 days in 10% FBS-containing medium in the absence or presence
of 10 ng/ml rapamycin, and counted after pancreatinization.
A total of 6 human muscle normal biopsies were minced into small pieces and cultured in Eagle’s MEM (Mediatech, Reston, VA) supplemented with 10% heat-inactivated FCS (M. A. Bioproducts, Springville, MD), 2% chick embryo extracts (Life Technologies, Bethesda, MD), 50 mM glutamine, and gentamicin, as described previously (30). Cultures were grown to subconfluence and examined to assess the development of myotubes after 4 days in DMEM containing 0.5 mg/ml BSA and antibiotics without or with IGFs (40 nM) together with LY294002 (15 µM) or rapamycin (20 ng/ml) when indicated. Images shown are representative of 10–20 microscope fields analyzed at random from each one of 3 independent experiments, in which each condition was tested in duplicate. Cells were photographed after staining the nuclei with Mayer’s hemalum solution for microscopy (Merck & Co.). For each condition, cell fusion was quantified by counting the percentage of nuclei in myotubes; 300–900 nuclei were counted in a total of 10–20 randomly selected microscopic fields from 3 independent experiments (at least 3 fields were analyzed from each of 3 independent experiments).
Electrophoresis and immunoblotting of membranes
Cells were lysed in 50 mM HEPES (pH 7.4), 150
mM NaCl, 0.1% Triton X-100, 0.5 mM
phenylmethylsulfonylfluoride, 2 mM leupeptin, and 2
mM pepstatin. Cell extracts were centrifuged for 15 min at
13,000 rpm at 4 C, and 30 µg of the solubilized proteins were loaded.
SDS-PAGE was performed according to the method of Laemmli (31). Gels
were blotted into Immobilon-P (Millipore, Bedford, MA) in buffer
consisting of 20% methanol, 200 mM glycine, and 25
mM Tris, pH 8.3. After transfer, the filters were blocked
with 5% nonfat dry milk in PBS for 1 h at 37 C and then incubated
overnight at 4 C with primary antibodies in PBS containing 1% nonfat
dry milk and 0.02% sodium azide. ß1 integrin was
detected using [125I]protein A for 3 h at room
temperature. GLUT4, myogenin, myosin heavy chain, and p70 S6 kinase
were detected using the enhanced chemiluminiscence system (ECL,
Amersham, Aylesbury, UK).
Detection of p70 S6 kinase activation by electrophoretic mobility
assay
Cells were starved in DMEM containing 0.2% BSA for 24 h
before treatment with 1 µM insulin or 40 nM
IGF-II in the absence or presence of different doses of rapamycin
(0–20 ng/ml; 30 min at 37 C). After washing twice in PBS solution,
cells were lysed in a buffer containing 50 mM Tris (pH 8),
120 mM NaCl, 1 mM EDTA, 6 mM EGTA,
15 mM Na4P2O7, 20
mM NaF, 30 mM PNPP, 0.1 mM
4-(2-aminoethyl)-benzenesulfonyl fluoride, 0.5
mM dithiothreitol, 0.5 mM
phenylmethylsulfonylfluoride, 2 mM leupeptin, and 2
mM pepstatin supplemented with 1% Nonidet-P40. The lysates
were centrifuged at 13,000 rpm for 20 min at 4 C, and 80 µg of the
supernatants were loaded onto 10% polyacrylamide gels (30:0.1,
acrylamide-bisacrylamide). Western blotting was performed as described
above.
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Results |
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, a and b). The addition of rapamycin
inhibited the induction of low electrophoretic mobility forms by
insulin and IGF-II. The faster migrating forms detected in the presence
of 1 ng/ml rapamycin showed the same electrophoretic mobility as the
enzyme in 24-h starved control cells.
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). This serum-induced
proliferation was potently inhibited by 10 ng/ml rapamycin. Rapamycin
inhibited the L6E9 myoblast growth rate by 65% on day 2 and by 80% on
day 3 compared with that of untreated cells. Sol8 myoblast
proliferation was decreased by 65% on day 2 and by 61% on day 3
compared with that in untreated cells.
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). IGF-II was 1.6-fold more potent than
10% serum in inducing proliferation on day 2 (3.6 ± 0.3-
vs. 2.2 ± 0.3-fold increase in cell number over that
on day 1). However, in clear contrast to the effect of 10% serum
treatment, in the presence of IGF-II, cell number remained unchanged
from days 2–4, and confluence was not reached. We ruled out the
possibility that this effect was due to a depletion of IGF-II by the
proliferating cells, as we added fresh medium to the cells on day 2
without any increase in cell number. In contrast to its inhibitory
effect on serum-induced proliferation, rapamycin had no significant
effect on IGF-II-induced proliferation over the 72-h period analyzed
(Table 2).
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, c and g,
respectively). As observed for IGF-II-dependent cell proliferation
(Table 2
), rapamycin had no effect on IGF-II-induced myotube formation
in L6E9 or Sol8 cells; in the presence of 20 ng/ml rapamycin, the
percentage of nuclei in myotubes induced by IGF-II was 71% (534 nuclei
were analyzed in a total of 14 randomly selected microscopic fields) in
L6E9 cells and 58.9% (916 nuclei were analyzed in a total of 20
randomly selected microscopic fields) in Sol8 cells (Fig. 2, d and h,
respectively). For these experiments, we included controls of cells
incubated with IGF-II in the presence of the PI 3-kinase inhibitor
LY294002, which completely blocked myotube formation, as we have
previously described (9, 10) (data not shown).
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, a and b, Mb). After 4 days in
serum-free medium supplemented with IGF-II, GLUT4 expression was
potently induced in L6E9 (Fig. 3a) and Sol8 (Fig. 3b) cells, as we have
previously described (10). The effect of IGF-II was specific for the
muscle protein, as it did not modify the expression of the
nonmuscle-specific plasma membrane protein ß1 integrin.
As shown for myotube formation, no effect on IGF-II-induced GLUT4
expression was observed for rapamycin concentrations ranging from 1–10
ng/ml. The percentage of GLUT4 expression after a 2-day IGF-II
treatment in the presence of rapamycin with respect to the expression
in cells treated with IGF-II alone was 117.8 ± 17.2% (n =
4) and 100.0 ± 9.1% (n = 3) for L6E9 and Sol8 cells,
respectively. We also analyzed the effect of rapamycin on the
expression of two other muscle-specific proteins in L6E9 and Sol8
cells: the myogenic transcription factor myogenin and the myosin heavy
chain (Fig 4). These proteins were
induced in response to differentiation by IGF-II, and again, the
addition of rapamycin (10 ng/ml) to the differentiation medium had no
significant effect on the IGF-II-induced expression of these
muscle-specific markers.
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). In a serum-free
medium, myoblasts did not fuse into multinucleated myotubes (Fig. 5b).
When IGF-II (40 nM) was present during the differentiation
period (6 days), large multinucleated myotubes were formed (Fig. 5c).
As previously described for rat and mouse muscle cells, IGF-induced
differentiation was totally blocked by the PI 3-kinase inhibitor
LY294002 (15 µM; Fig. 5d). Finally, as observed for rat
and mouse myoblasts, rapamycin (20 ng/ml) did not significantly alter
the ability of IGF-II to induce differentiation of human myoblasts
[66% (n = 492 nuclei in myotubes) and 68.5% (n = 475
nuclei in myotubes) for IGF-II without and with rapamycin,
respectively; Fig. 5e].
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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p85)
that lacks the ability to bind and activate the p110 catalytic subunit
(10). Taking into account the primary myogenic role of PI 3-kinase in
human cells, the use of specific chemical activators of this enzyme as
possible routes to new therapies to enhance muscle growth or repair
could be of clinical interest. However, although two highly specific
chemical inhibitors of PI 3-kinase activity have been well
characterized (35, 36), to date no direct chemical activator of PI
3-kinase has been described.
On the other hand, there is scarce information regarding the downstream
elements activated by PI 3-kinase or its phosphatidylinositol 3-P
products. It has recently been demonstrated that the serine/threonine
kinase p70 S6 kinase acts downstream of PI 3-kinase (12, 13). In this
study we analyzed the involvement of p70 S6 kinase in myogenesis. We
show that in L6E9 and Sol8 myoblasts, p70 S6 kinase activation was
induced by both insulin and IGF-II and was inhibited by rapamycin.
However, rapamycin did not alter the IGF-II-induced differentiation
program of rat L6E9, mouse Sol8, or human skeletal muscle cells, thus
indicating that p70 S6 kinase is not a downstream effector in the
myogenic cascade. Indeed, IGF-II-induced cell fusion and expression of
muscle-specific biochemical markers (i.e. myogenin, myosin
heavy chain and GLUT4) remained unaltered by rapamycin at doses as high
as 20 ng/ml (1 ng/ml was sufficient to totally inhibit p70 S6 kinase
activation by insulin or IGF-II). Our results are in direct contrast to
those reported by Coolican et al. (11), who have recently
concluded that p70 S6 kinase is an essential mediator of IGF-induced
myogenesis. In the cited study, rapamycin treatment (1 ng/ml) of L6A1
myoblasts completely abolished IGF-induced differentiation. These
observations also contrast with those described for mouse BC3H1 muscle
cells, in which high levels of rapamycin (100 ng/ml) actually induced
expression of 
-actin, a muscle-specific biochemical marker (22).
When we analyzed the effect of concentrations of rapamycin as high as
100 ng/ml on L6E9 cell differentiation, we observed that the
IGF-induced myoblast ability to fuse into myotubes was slightly
increased compared with that of untreated cells (data not shown).
However, as 1 ng/ml rapamycin was sufficient to totally inhibit p70 S6
kinase activation in L6E9 cells, we interpret this result as indicating
that such high concentrations of the drug could alter cellular elements
other than p70 S6 kinase.
IGFs are the only known growth factors that stimulate both
proliferation and differentiation of skeletal muscle cells (37). It is
well established that IGFs cause myoblasts to go through the S phase
and to complete a single round of the cell cycle at 24 h of
treatment; thereafter, IGFs induce an accelerated progression of
myogenesis (38). In this context, IGF-induced proliferation during the
first 24 h of treatment could be considered a preliminary step in
the IGF-dependent myogenic program (38, 39, 40). Here we show that both
10% serum and IGF-II induced L6E9 myoblast proliferation after 24
h of treatment. However, although 10% serum continued to stimulate
cell growth to confluence, IGF-II-treated cells stopped proliferating
after a 24-h treatment and then initiated a series of morphological
changes associated with differentiation, i.e. elongation and
alignment (Table 2 and data not shown).
In both yeast and mammalian cells, rapamycin has been shown to block cell cycle progression by causing G1 arrest and abrogating the activation of p34 cdc2 and p33 cdk2 kinases (21, 41, 42). In accordance with these observations, here we show that rapamycin potently inhibited serum-induced cell proliferation in L6E9 and Sol8 myoblasts, indicating that serum-induced cell proliferation involved a p70 S6 kinase-dependent pathway. In contrast, IGF-II-induced myoblast cell growth occurred via a p70 S6 kinase-independent pathway, as it was not altered by rapamycin.
We conclude from these results that p70 S6 kinase activation plays a
primary role in serum-induced muscle cell proliferation but is not
required for the differentiating program initiated by IGF-II comprising
both the initial mitogenic phase and the subsequent myogenic actions.
These results together with previously published data seem to indicate
that p70 S6 kinase and PI 3-kinase play distinct roles in the
regulation of skeletal muscle cell fate: 1) p70 S6 kinase has a primary
role in serum-induced myoblast proliferation, whereas PI 3-kinase is
not a key element in this process (9); and 2) IGF-induced myogenesis is
totally dependent on PI 3-kinase activity, but does not require p70 S6
kinase activation as a downstream signaling element (9, 10, 11, 12). From this,
p70 S6 kinase does not appear to be an essential downstream element in
the IGF/PI 3-kinase-induced myogenic cascade. Among the possible PI
3-kinase downstream proteins involved in IGF-II-induced myogenesis are
the Ser/Thr kinases protein kinase B and some protein kinase C
isoforms (
, 
, 
, and 
), which have been reported to interact
directly with PI 3-kinase or its PI 3-P products in a variety of
mammalian cell types (43, 44, 45, 46). We are at present focusing our efforts
on further defining the differentiation signaling cascade induced by
IGFs in mouse, rat, and human myoblasts. Indeed, identification of the
molecular elements that signal myogenesis in human skeletal muscle
cells will be of particular interest in clinical investigation, as it
would extend the possible targets to be considered in the design of
therapeutic strategies for muscle regeneration.
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Acknowledgments |
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Footnotes |
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Received March 10, 1998.
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References |
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isozyme of protein kinase C by phosphatidylinositol
3,4,5-triphosphate. J Biol Chem 268:13–16 specifically associates with phosphatidylinositol 3-kinase
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