Insulin-like Growth Factors Require Phosphatidylinositol 3-Kinase to Signal Myogenesis: Dominant Negative p85 Expression Blocks Differentiation of L6E9 Muscle Cells
Perla Kaliman,
Judith Canicio,
Peter R. Shepherd,
Carolyn A. Beeton,
Xavier Testar,
Manuel Palacín and
Antonio Zorzano
Departament de Bioquímica i Biologia Molecular (P.K.,
J.C., X.T., M.P., A.Z.) Facultat de Biologia Universitat de
Barcelona 08028 Barcelona, Spain
Department of
Biochemistry and Molecular Biology (P.R.S., C.A.B.) University
College London, United Kingdom W1P8BT
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ABSTRACT
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Phosphatidylinositol 3 (PI 3)-kinases are potently
inhibited by two structurally unrelated membrane-permeant reagents:
wortmannin and LY294002. By using these two inhibitors we first
suggested the involvement of a PI 3-kinase activity in muscle cell
differentiation. However, several reports have described that these
compounds are not as selective for PI 3-kinase activity as assumed.
Here we show that LY294002 blocks the myogenic pathway elicited by
insulin-like growth factors (IGFs), and we confirm the specific
involvement of PI 3-kinase in IGF-induced myogenesis by overexpressing
in L6E9 myoblasts a dominant negative p85 PI 3-kinase-regulatory
subunit (L6E9-
p85). IGF-I, des(13)IGF-I, or IGF-II induced L6E9
skeletal muscle cell differentiation as measured by myotube formation,
myogenin gene expression, and GLUT4 glucose carrier induction. The
addition of LY294002 to the differentiation medium totally inhibited
these IGF-induced myogenic events without altering the expression of a
non-muscle-specific protein, ß1-integrin. Independent clones of L6E9
myoblasts expressing a dominant negative mutant of the p85-regulatory
subunit (
p85) showed markedly impaired glucose transport activity
and formation of p85/p110 complexes in response to insulin, consistent
with the inhibition of PI 3-kinase activity. IGF-induced myogenic
parameters in L6E9-
p85 cells, i.e. cell fusion and
myogenin gene and GLUT4 expression, were severely impaired compared
with parental cells or L6E9 cells expressing wild-type p85. In all,
data presented here indicate that PI 3-kinase is essential for
IGF-induced muscle differentiation and that the specific PI 3-kinase
subclass involved in myogenesis is the heterodimeric p85-p110 enzyme.
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INTRODUCTION
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Growth factors are generally considered to inhibit myogenesis.
However, it is well documented that insulin-like growth factors (IGFs)
are crucial to this process (1). IGF-I and IGF-II are potent
stimulators of muscle differentiation, and they are potential
candidates for regulation of satellite cell function during
regeneration, a characteristic response of adult muscle to exercise or
injury (2, 3). It has been shown that IGF expression is increased
during myoblast differentiation in response to serum withdrawal (4, 5, 6, 7, 8).
Furthermore, the level of IGF-II secreted from muscle cells correlates
with the rate of spontaneous differentiation, and antisense
oligonucleotides complementary to IGF-II mRNA inhibited differentiation
in the absence but not in the presence of exogenous IGF-II (6). The
biological significance of the IGFs has also been analyzed by
recombinant ablation studies. A common observation in mouse lines
lacking IGF-I or its receptor is that embryos are viable, but embryonic
development is impaired and neonates die immediately after birth
because they cannot breathe (9, 10). Furthermore, expression of IGF-I
in skeletal muscle results in myofiber hypertrophy (11), and
overexpression of IGF-I in the heart leads to cardiomegaly mediated by
an increased number of cells in the heart (12).
Much information has recently been gained on the role of IGFs in
myogenesis (reviewed in Ref.13). However, the intracellular myogenic
signaling process dependent on IGFs is poorly understood. We have
recently reported that the phosphatidylinositol 3 (PI 3)-kinase
inhibitors, wortmannin and LY294002, block differentiation of skeletal
muscle cells, suggesting that phosphatidylinositol 3-kinase is
essential for the terminal differentiation of muscle cells (14). In
this context, it has recently been reported that LY294002 inhibits L6A1
muscle cell differentiation induced by IGF-I (15). Indeed, during the
last few years, much insight has been gained on the cellular functions
of PI 3-kinase by the use of wortmannin (for review see Ref.16) and
LY294002 (17), both of which inhibit all PI 3-kinase subclasses so far
described in the nanomolar or low micromolar range. However, several
reports have described that these compounds are not as selective for PI
3-kinase activity as assumed. Indeed, wortmannin and its structural
analog demethoxyviridin inhibit stimulated phospholipase A2 activity
with an IC50 of 2 nM (18). Moreover, wortmannin
has also been reported to inhibit phosphatidylinositol 4-kinase (19),
phospholipase C and D (20), and myosin light chain and pleckstrin
phosphorylation (21) albeit at concentrations greater than those
required to inhibit PI 3-kinase. On the other hand, the specificity of
LY294002 for other lipid-metabolizing enzymes has not been examined.
Accordingly, the assignation of a role for PI 3-kinase in a particular
cellular pathway on the sole basis of its chemical inhibition may lead
to incorrect conclusions.
In an attempt to identify a signaling intermediate for the myogenic
actions of IGFs, here we analyze the effects of 1) LY294002 and 2) the
expression of a dominant negative mutant of p85 PI 3-kinase-regulatory
subunit in IGF-induced L6E9 muscle cell differentiation. We show that
PI 3-kinase is an essential element for IGF-induced muscle
differentiation and that the specific PI 3-kinase subclass involved in
myogenesis is the heterodimeric p85-p110 enzyme.
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RESULTS
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IGFs Induce Biochemical and Morphological Differentiation of L6E9
Myoblasts: Blockade by the PI 3-Kinase Inhibitor LY294002
Confluent L6E9 myoblasts were incubated in a serum-free medium
supplemented with increasing concentrations of IGF-II. After 2 days in
these conditions, cells expressed myogenin mRNA with an
ED50 for IGF-II of
20 nM, which was
consistent with the activation of the IGF-I receptor (Fig. 1a
). IGF-II increased myogenin mRNA
levels up to 6-fold compared with cells maintained in serum-free medium
alone (basal conditions). Fig 1b
shows that IGF-I and its more potent
analog des(1, 3)IGF-I also induced myogenin mRNA and they were at least
10-fold more potent than IGF-II. The ability of all three IGFs to
induce myogenin gene expression was completely abolished by the PI
3-kinase inhibitor LY294002 (20 µM), suggesting that PI
3-kinase is an essential downstream effector for IGF induction of L6E9
muscle cell differentiation (Fig. 1b
).

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Figure 1. IGFs-Induced Biochemical and Morphological
Differentiation in L6E9 Myoblasts Is Blocked by the PI 3-Kinase
Inhibitor LY294002
Confluent L6E9 myoblasts were allowed to differentiate in
serum-free medium for 2 days in the absence or presence of IGFs with or
without LY294002 (20 µM). (a) Myogenin mRNA was analyzed
by Northern blots and quantitated by densitometry. Myogenin mRNA
abundance in the absence of IGF-II was considered as basal expression,
and data are expressed as fold-stimulation over basal. (b) Myogenin
mRNA expression was analyzed in myoblasts (Mb) and after 2 days in
serum-free medium without (0) or with 3 nM IGF-I, 3
nM des(1, 3)IGF-I, or 40 nM IGF-II
supplementation, in the absence or presence of 20 µM
LY294002. Representative autoradiograms from three separate experiments
are shown. The integrity and relative amounts of RNA in each sample
were checked by ethidium bromide staining (rRNA). (c) GLUT4 glucose
transporter and ß1-integrin content was analyzed by Western blot of
total cell lysates. GLUT4 level after 2 days in serum-free medium in
the absence of IGF-II was considered as basal expression, and data are
expressed as percentage over basal. (d) GLUT4 glucose transporter and
ß1-integrin content was analyzed in myoblasts (Mb) and after 2 days
in serum-free medium without (0) or with 3 nM IGF-I, 3
nM des(1, 3)IGF-I, or 40 nM IGF-II
supplementation, in the absence or presence of 20 µM
LY294002. Representative autoradiograms from three independent
experiments are shown. (e) Cells were grown to confluence in a 10%
FBS-containing medium and then allowed to differentiate in a serum-free
medium (0.5 mg/ml BSA containing DMEM) (left),
supplemented with 40 nM IGF-II (center), or
supplemented with 40 nM IGF-II and 20 µM
LY294002 (right). After 2 days in each condition, cells
were photographed. Images shown are representative of 1020
microscopic fields taken at random from each one of at least 10
independent experiments. Scale bars, 30 µm (the scale is the same for
all panels).
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Among the functional markers of skeletal muscle terminal
differentiation is the insulin-sensitive glucose transporter GLUT4
(22). After 2 days in serum-free medium supplemented with IGF-II, L6E9
cells expressed GLUT4 with an IGF-II dose dependency reflecting the
activation of IGF-I receptor (ED50 of
20 nM)
(Fig. 1c
). The effect of IGF-II was specific for the muscle protein as
it did not modify the expression of the ubiquitous membrane protein
ß1-integrin (Fig. 1c
, inset). IGF-I and des(1, 3)IGF-I
also induced the expression of GLUT4, but they were at least 10-fold
more potent than IGF-II (Fig. 1d
). As observed for myogenin expression,
PI 3-kinase inhibitor LY294002 blocked the effect of IGF-I (3
nM), des(1, 3)IGF-I (3 nM), and IGF-II (40
nM) on GLUT4 expression (Fig. 1d
). LY294002 did not alter
the expression of ß1-integrin, indicating that the inhibitor did not
affect the protein levels of a structural component of the cells and
that it specifically blocked the IGF-induced expression of GLUT4 (Fig. 1d
).
At the morphological level, confluent L6E9 myoblasts incubated in a 2%
serum containing medium initiate a differentiation program that
consists, at the morphological level, in myoblast elongation and
alignment during the first 24 h, followed by multinucleate myotube
formation (14). The presence of low serum concentrations in the
differentiation medium was found to be essential for terminal
differentiation of L6E9 cells since after 2 days in a serum-free medium
(DMEM containing 0.5 mg/ml BSA), cells aligned to each other and showed
an elongated morphology, although they did not fuse or fused very
poorly into myotubes (Fig. 1e
, left). Supplementation of
serum-free medium with IGFs led to a potent induction of cell fusion.
Fig 1e
(center) shows large multinucleated myotubes induced
by 40 nM IGF-II. IGF-I (3 nM) or des(1, 3)IGF-I
(3 nM) induced cell fusion comparable to that induced by 40
nM IGF-II (data not shown). As observed for myogenin and
GLUT4, after 2 days in serum-free medium supplemented with 40
nM IGF-II and 20 µM LY294002, L6E9 cells
remained largely unfused (Fig. 1e
, right). PI 3-kinase
inhibitor also blocked the cell fusion induced by IGF-I (3
nM) or des(1, 3)IGF-I (3 nM) (data not shown).
All these results suggest that PI 3-kinase activity is essential for
IGF-induced biochemical and morphological differentiation of L6E9
cells.
p85
Is the Predominant PI 3-Kinase Adapter Subunit Isoform
Expressed in L6E9 Cells
Fully differentiated muscle expresses a number of splice variants
of p85
adapter subunit of PI 3-kinase, all of which are regulated by
insulin and could therefore potentially be involved in IGF-mediated
processes (23). In an effort to determine whether any of these isoforms
was important in the differentiation of L6E9 muscle cells, we first
analyzed, by Western blot, lysates and total membranes of L6E9
myoblasts and myotubes using a previously described antibody that
recognizes p85ß and all the splice variant forms of p85
(23). In
human muscle lysates, the antibody recognized four identified major
bands of 87 kDa (p85ß), 85 kDa (p85
), 53 kDa (p55
/AS53), and 48
kDa (p50) (Fig. 2
) (23). However, in both
L6E9 myoblasts and myotubes the full-lengh p85
was the predominant
adapter subunit expressed.

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Figure 2. Characterization of PI 3-Kinase Regulatory Subunits
Present in L6E9 Skeletal Muscle Cells
L6E9 myoblasts (Mb) and myotubes (Mt) lysates or total membrane
fractions (75 µg) and a control of human muscle lysates (300 µg)
were analyzed by Western blot with a polyclonal antibody raised against
glutathione S-transferase fusion protein, corresponding to the N-SH2
domain of human p85 , and visualyzed by [125I]protein
A.
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Expression of a Dominant-Negative p85
in L6E9 Myoblasts
All PI 3-kinase isoforms so far described are potently inhibited
in the nanomolar or low micromolar range by two structurally unrelated
membrane- permeant reagents: wortmannin (16) and LY294002 (17). By
using these two compounds we first suggested the involvement of a PI
3-kinase activity in muscle cell differentiation (14). Furthermore, the
results presented above seem to indicate that PI 3-kinase is essential
for IGF-induced myogenesis. However, one question remains: whether the
inhibition of myogenesis by wortmannin and LY294002 is a specific
reflection of PI 3-kinase involvement. In an effort to clarify this
aspect and taking into account that p85
is the predominant PI
3-kinase adapter subunit form expressed in L6E9 cells, we stably
overexpressed in L6E9 myoblasts a p85
lacking a binding site for the
p110 catalytic subunit of PI 3-kinase (L6E9-
p85) and a wild-type
p85
as a control (L6E9-Wp85) (24).
Screening of positive clones overexpressing p85 (Wp85 or
p85) was
performed by immunofluorescence assays using polyclonal rabbit
antibodies against rat p85 PI 3-kinase. We selected five independent
clones for each Wp85- or
p85-transfected cells in which the level of
expression of
p85 was comparable to the level of expressed Wp85.
Transfected proteins were 2- to 3 times overexpressed compared with the
level of endogenous p85 in untransfected cells (Fig. 3
, B and C vs. A). As a
control, we analyzed the level of expression of ß1-integrin, which
was essentially identical for untransfected and transfected cells (Fig. 3
, DF). Figure 3
also shows that the subcellular distribution of p85
under basal conditions was mostly intracellular in both transfected and
untransfected cells (Fig. 3
, AC). In contrast, ß1-integrin
exhibited a typical distribution pattern of a plasma membrane marker
(Fig. 3
, DE).
We compared the growth rate of the selected clones of Wp85- and
p85-transfected cells to analyze the impact of p85 dominant negative
expression on L6E9 cell proliferation. Consistent with our previous
observations in L6E9 myoblasts grown in the presence of wortmannin
(14),
p85-transfected cells proliferated normally in response to
serum, and no differences in cell growth were detected when compared to
Wp85-transfected cells (Table 1
) and untransfected cells (data not
shown).
As a functional assay to test the inhibition of PI 3-kinase activity in
L6E9-
p85 cells, we analyzed the glucose transport activity. We and
others have previously shown that PI 3-kinase activity is crucial to
the regulation of glucose transport in L6E9 myoblasts (25) and other
mammalian cell types (26, 27, 28, 29, 30). Moreover, studies from Hara et
al. (24) showed that glucose uptake is markedly impaired in
Chinese hamster ovary (CHO) cells overexpressing
p85. We analyzed
three independent clones of both L6E9-
p85 and L6E9-Wp85 cells for
glucose transport activity (Fig. 4
). In
L6E9-Wp85 maximal glucose transport activity was observed in the
absence of insulin. This seems to indicate that the overexpression of
Wp85 saturated the endogenous cell machinery sensitive to insulin which
did not cause any further enhancement of glucose uptake. In contrast,
cells overexpressing
p85 showed a marked decrease in both basal and
insulin-stimulated 2-deoxyglucose uptake compared with either
untransfected or L6E9-Wp85 cells (Fig. 4
). The fact that glucose uptake
by L6E9-
p85 cells remained sensitive to insulin is consistent with
our observation that, in L6E9 and Sol8 myoblasts, wortmannin produced a
parallel decrease in basal and insulin-stimulated glucose uptake, but
that insulin action is abolished only at very high wortmannin
concentrations (1 µM) (25).
To further characterize the dominant negative effect of
p85
transfection in L6E9 cells, we compared the ability of p85 to bind to
the catalytic p110
PI 3-kinase subunit after insulin stimulation in
untransfected,
p85 and Wp85 L6E9 myoblasts (Fig. 5
). Consistent with the glucose transport
experiment, in which insulin showed no further effect on L6E9-Wp85
cells compared with untransfected cells, the level of p110
complexed
with p85 in Wp85-cells was essentially the same as in untransfected
cells. In contrast,
p85-transfected cells showed a 2-fold decrease
in the level of p110 coimmunoprecipitated with p85 (Fig. 5
).
Myotube Formation Is Impaired in L6E9-
p85 Cells
Cell differentiation was analyzed in five independent clones of
both Wp85- and
p85-transfected L6E9 cells. L6E9-Wp85 cells were
morphologically indistinguishable from L6E9 parental cells at all the
conditions tested, i.e. proliferation and differentiation
(data not shown). Images shown are representative of 1020 microscopic
fields taken at random from each one of four independent experiments in
which L6E9 parental cells and Wp85- and
p85-L6E9 clones were
cultured in parallel under identical conditions.
L6E9-
p85 myoblasts proliferated normally in a 10% FBS-containing
medium and were morphologically similar to L6E9-Wp85 (Fig. 6
, a and f). Confluent cells were allowed
to differentiate in a serum-free medium with or without IGF-II (0100
nM IGF-II). Figure 6
shows the morphological changes
undergone by L6E9-Wp85 (Fig. 6
, be) and L6E9-
p85 cells (Fig. 6
, gj) during a 4-day differentiation period. After 2 days in serum-free
medium without IGF-II, L6E9-Wp85 and L6E9-
p85 cells were aligned to
each other and elongated compared with myoblasts (Fig. 6
, b
vs. a and g vs. f, respectively), but little or
no fusion was observed in these conditions. In the presence of IGF-II,
myotube formation was observed in L6E9-Wp85 cells (Fig. 6
, c and d, for
20 and 100 nM IGF-II, respectively), whereas under the same
conditions, L6E9-
p85 did not fuse or fused very poorly (Fig. 6
, h
and i, for 20 and 100 nM IGF-II, respectively). Large
multinucleated myotubes were observed in L6E9-Wp85 cells after 4 days
in the presence of 100 nM IGF-II (Fig. 6e
) while
L6E9-
p85 remained aligned and elongated, but fusion was largely
prevented (Fig. 6j
).

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Figure 6. Myotube Formation Is Impaired in L6E9- p85 Cells
Cell differentiation was analyzed in five independent clones of both
Wp85- and p85-transfected L6E9 cells. Results shown are
representative of 1020 microscopic fields taken at random from each
of four independent experiments in which L6E9 parental cells and Wp85-
and p85-L6E9 clones were cultured in parallel under identical
conditions. L6E9-Wp85 cells were indistinguishable from L6E9 parental
cells in all conditions assayed (data not shown), and in all the
experiments, they were both considered as controls for proliferation
and differentiation. Control (ae) and L6E9- p85 (fj) cells were
grown to confluence in a 10% FBS-containing medium (a and f) and then
allowed to differentiate in a serum-free medium (0.5 mg/ml BSA
containing DMEM) without (b and g) or with IGF-II at concentrations of
20 nM (c and h) or 100 nM (d, e, i, and j).
Cells were photographed after 2 days (b, c, d, g, h, and i) or after 4
days (e and j). Scale bars, 30 µm (the scale is the same for all
panels).
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Myogenin and Glucose Transporter GLUT4 Expression Is Decreased in
L6E9-
p85 Cells
L6E9-
p85 and L6E9-Wp85 cells were grown to confluence. Cells
were then incubated at increasing doses of IGF-II (0100
nM). The IGF-II dose-dependence for myogenin expression in
L6E9-Wp85 cells was similar to that observed for untransfected cells
(Fig. 7
vs. Fig. 1a
). In
L6E9-
p85 cells, the maximal response to IGF-II for myogenin gene
induction was reduced by 62 ± 13% (n = 3) compared with
L6E9-Wp85 cells (Fig. 7
). Figure 8
shows
glucose transporter GLUT4 expression in L6E9-
p85 and L6E9-Wp85 after
4 days in a serum-free medium with or without IGF-II. Little or no
induction of GLUT4 was observed in L6E9-
p85 or L6E9-Wp85 cells in
the absence of IGF-II. As determined for untransfected cells (Fig. 1
c), maximal expression of GLUT4 was detected at 50
nM IGF-II concentrations. However, the maximal expression
of the glucose transporter was decreased by 80 ± 7% (n = 3)
in L6E9-
p85 cells compared with control cells (Fig. 8
). The
expression of ß1-integrin, a ubiquitous plasma membrane component,
remained unaltered in all the conditions tested, indicating that
IGF-II-induced GLUT4 protein expression was specifically associated
with muscle differentiation and that PI 3-kinase was required for this
process.

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Figure 7. IGF-II-induced Myogenin Gene Expression Is
Decreased in L6E9- p85 Cells
Confluent L6E9-Wp85 and L6E9- p85 cells were allowed to differentiate
in serum-free medium for 2 days in the absence or presence of
increasing concentrations of IGF-II (10, 50 and 100 nM).
Total RNA was obtained from the different experimental groups, and 10
µg of RNA were laid on gels. After blotting, myogenin mRNA was
detected by hybridization with a 1,100 bp EcoRI fragment
as a cDNA probe. Representative autoradiograms after 30 min of exposure
from three separate experiments are shown. The integrity and relative
amounts of RNA in each sample were checked by ethidium bromide staining
(rRNA).
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Figure 8. IGF-II-induced Glucose Transporter GLUT4 Expression
Is Decreased in L6E9- p85 Cells
Confluent L6E9-Wp85 and L6E9- p85 cells were allowed to differentiate
in serum-free medium for 4 days in the absence or presence of IGF-II
(50 and 100 nM). GLUT4 glucose transporter and
ß1-integrin content were analyzed by immunoblotting 30 µg of
solubilized proteins from the different experimental groups.
Representative autoradiograms from three independent experiments are
shown.
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DISCUSSION
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In a previous study, the use of the cell-permeant inhibitors,
wortmannin and LY294002, suggested that PI 3-kinase was essential for
terminal differentiation of muscle cells (14). In this study, we show
that PI 3-kinase is an essential second messenger for the myogenic
actions of insulin-like growth factors (IGFs), and we identify the
heterodimeric p85-p110 PI 3-kinase as the PI 3-kinase subclass involved
in myogenesis.
We have previously shown that PI 3-kinase activity in L6E9 cells is
stimulated by insulin at concentrations that correlate with the
activation of the IGF-I receptor, this stimulation being inhibited in a
dose-response manner by wortmannin (25). Indeed, most of insulin
actions in these cells are mainly signaled through the IGF-I receptor
since L6 cells express insulin and IGF-I receptors in a ratio about
1:400 in myoblasts and 1:50 in myotubes (31). Here we show that the
effect of IGFs on cell fusion and myogenin and GLUT4 expression was
totally blocked by the PI 3-kinase inhibitor LY294002. Wortmannin was
not used in this study because of its short half-life in aqueous
solution (32), which renders it unsuitable for experiments involving 2-
to 4-day incubations. The dose-response studies presented here seem to
indicate that the IGF-II receptor is not relevant for GLUT4 or myogenin
expression in L6E9 cells. However, the contribution of IGF-II receptor
to myogenesis cannot be ruled out since in mouse BC3H-1 muscle cells an
IGF-II receptor-selective analog of IGF-II promoted cell
differentiation (33).
A family of distinct PI 3-kinase enzymes has been cloned and
characterized in mammals, and these can be distinguished on the basis
of structure, function, and mechanisms of activation (reviewed in Ref.34). A well characterized class of PI 3-kinases are heterodimers
composed of a regulatory p85 subunit (isoforms:
, ß, p55PIK, and
other p85 splice variants) (35, 36, 37, 38, 39, 40) and a catalytic p110 subunit
(isoforms
and ß) (40, 41), which possesses a Ser/Thr protein
kinase activity in addition to its lipid kinase activity (42, 43, 44). This
group of enzymes is regulated by cell surface receptors via intrinsic
or associated tyrosine-kinase activities. Here, we stably transfected
L6E9 cells with a dominant negative p85
-subunit (
p85) that lacks
the binding site for the p110 catalytic subunit of PI 3-kinase. As
expected,
p85-cells showed impaired ability to form p85/p110
complexes in response to insulin, and they also showed reduced basal
and insulin-stimulated glucose transport activity, which is known to be
dependent on intact PI 3-kinase activity in L6E9 cells (25). However,
probably due to the low level of overexpression of transfected
proteins,
p85-cells remained insulin-sensitive for both parameters,
although to a much lesser extent than untransfected cells.
IGF-induced myogenic parameters in L6E9-
p85, i.e. cell
fusion, myogenin gene, and GLUT4 expression, were severely impaired
compared with parental cells or L6E9-Wp85 cells. As for glucose
transport activity, the effect of maximal doses of PI 3-kinase chemical
inhibitors on cell differentiation blockade was more dramatic than the
effect of a 2- to 3-fold overexpression of
p85. However, the absence
of large multinucleated myotubes and the reduction by 62% in myogenin
mRNA and by 80% in GLUT4 protein expression in
p85-transfected
cells indicate that the heterodimeric PI 3-kinase is essential for
IGF-induced L6E9 cell differentiation. In this context, several splice
variants of p85 are present in fully differentiated human muscle, and
each of these is stimulated by insulin to a different extent,
indicating that they could have distinct roles in insulin and IGF-I
signaling (23). However, in the current study we find that p85
is
the predominant PI 3-kinase adapter subunit expressed in both L6E9
myoblasts and myotubes. This, together with the inhibition of
differentiation by
p85, indicates that IGF stimulation of
full-length p85
is sufficient to activate the PI 3-kinase required
for myogenesis. However, in human muscle, other adapter subunits that
are also abundant (23) may play a role in cell differentiation. Indeed,
observations from our laboratory show that PI 3-kinase is essential for
myotube formation in human skeletal muscle cells (our unpublished
observations).
There is scarce information regarding the downstream elements activated
by PI 3-kinase or its PI 3-phosphate products. It has recently been
shown that p70S6k activity is increased substantially
during skeletal muscle cell diffentiation in the absence, but not in
the presence, of LY294002 and that rapamycin, an inhibitor of
p70S6k activity, abolishes IGF-I-induced differentiation
(15). These results strongly suggest that p70S6k is
involved in the IGF/PI 3-kinase myogenic pathway. Other putative
downstream elements of this pathway may include the Ser/Thr protein
kinase PKB (also known as Akt/RAC) and some protein kinase C (PKC)
isoforms. PKB is activated by insulin in L6 myotubes, and this
activation is prevented by PI 3-kinase inhibitors (45). Furthermore,
the relationship of PKB and PKC kinase families is particularly
interesting in light of the ability of novel and atypical PKC isoforms
(PKC
, -
, -
, and -
) to interact with PI 3-kinase products
PI 3,4,5-triphosphate and PI 3,4-diphosphate (46, 47). Moreover, PKC
specifically associates wih PI 3-kinase after cytokine stimulation
(48). In the context of these findings combined with our results, it is
tempting to hypothesize that PKB and/or PKC isoforms could be targets
of PI 3-kinase in the myogenic signaling pathway. Moreover, it has
recently been described that ERK6, a mitogen-activated protein kinase,
is involved in C2C12 myoblast differentiation (49). ERK6 seems to be
specifically expressed in skeletal muscle and to signal differentiation
through phosphotyrosine-mediated pathways distinct from those
activating other members of the mitogen-activated protein kinase family
such as ERK1 and ERK2. It would be of interest to determine whether
ERK6 and PI 3-kinase are convergent signals for myogenesis or whether
ERK6 defines an alternative myogenic pathway.
Overall, the results presented here provide evidence that p85-p110 PI
3-kinase is an essential mediator for IGF-induced muscle cell
differentiation through the IGF-I receptor. Additional work is required
to identify the downstream elements involved in the myogenic signaling
cascade.
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MATERIALS AND METHODS
|
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Materials
IGF-I and IGF-II were kindly provided by Eli Lilly
(Indianapolis, IN). des(1, 2, 3)IGF-I was from Angelika F. Schutzdeller
(Tubingen, Germany). LY294002 was from BIOMOL Research Laboratories
(Plymouth, Meeting, PA). L6E9 rat skeletal muscle cell line was kindly
provided by Dr. B. Nadal-Ginard (Harvard University, Boston, MA). The
polyclonal antibody OSCRX was raised against the C terminus of GLUT4
(50). A rabbit polyclonal antibody against ß1-integrin was kindly
given by Dr. Carles Enrich (University of Barcelona, Barcelona, Spain)
(51). Polyclonal antibodies against rat p85 and p110
subunits of PI
3-kinase were from Upstate Biotechnology, Inc. (Lake Placid, NY). A
polyclonal antibody was raised to a glutathione S-transferase fusion
protein corresponding to the N-SH2 (p85
-NSH2) domain of human p85
as described previously (23).
cDNA encoding for myogenin was kindly given by Dr. Eric Olson
(University of Texas, Houston, TX).
Cell Culture
Rat skeletal muscle L6E9 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. IGFs and/or LY294002 were
added at the concentrations and times indicated for each experiment.
Images shown are representative of 1020 microscopic fields taken at
random from each of at least four independent experiments.
Plasmids and Expression of Wild-Type and Mutant p85
in L6E9
Myoblasts
SR
-Wp85 and SR
-
p85 were kindly provided by Dr. Masato
Kasuga (Kobe University, Kobe, Japan). Wp85 was the entire coding
sequence of bovine p85
.
p85 encompasses a deletion mutant bovine
p85
that lacks a binding site for the p110 catalytic subunit of PI
3-kinase. Both cDNAs were subcloned into SR
expression vector (24).
The mutant p85
has a deletion of 35 amino acids (residues 479 to
513) and an insertion of two amino acids (Ser-Arg) replacing the
deleted sequence. To obtain L6E9 myoblasts stably overexpressing Wp85
or
p85, L6E9 cells were cotransfected with pcDNA3, a plasmid
conferring geneticin resistance and either the SR
-Wp85 or the
SR
-
p85 plasmid.
For transfections and clone selection, subconfluent L6E9 cell
monolayers (day 0) were pancreatinized and seeded 1:7 in two
25-cm2 flasks. On day 1 (4050% of confluence) cells were
washed three times and then covered with 3 ml of serum-free medium.
Cells were then transfected by adding dropwise 120 µl of
DNA-Lipofectin mixture to each flask and swirling gently.
DNA-Lipofectin mixture (1:1, vol/vol) was prepared with pcDNA3 together
with Wp85 or
p85 constructs in a 1:15 concentration ratio (45 µg
total DNA/60 µl) and Lipofectin (30 µg/60 µl), following the
suppliers protocol (Life Technologies, Inc). Cells were incubated
with DNA-Lipofectin-containing medium for 16 h under standard cell
culture conditions. Medium was removed and replaced by complete medium
(i.e. with 10% serum). Cells were grown to subconfluence,
pancreatinized, and seeded in the presence of 0.4 mg/ml Geneticin
(G418; Life Technologies, Inc, Gaithersburg, MD) to a very low density
(1:200) so that single clones could be isolated by picking the clones
with sterile pancreatin-embedded cotton swabs. G418-resistant clones
were continuously grown in the presence of G418 (0.4 mg/ml). The
culture time for transfected cells did not exceed the time for which
the ability of L6E9 cells to differentiate is preserved. Screening of
positive clones overexpressing p85 (Wp85 or
p85) was performed by
immunofluorescence assays using polyclonal rabbit antibodies against
rat p85 PI 3-kinase (1:100) as primary antibody and rodamine-conjugated
goat anti-rabbit Igs (1:100) as secondary antibody, as described
below.
To quantify cell proliferation, cells were plated in multiwell culture
dishes, grown from 14 days in 10% FBS-containing medium, and counted
after pancreatinization.
Cell differentiation was analyzed in five independent
immunofluorescence-positive clones of both Wp85- and
p85-transfected
L6E9 cells.
RNA Isolation and Northern Blot Analysis
Total RNA from cells was extracted using the phenol/chloroform
method as described by Chomczynski and Sacchi (52). All samples had a
260:280 absorbance ratio above 1.7.
After quantification, total RNA (10 µg) was denatured at 65 C in the
presence of formamide, formaldehyde, and ethidium bromide (53). RNA was
separated on a 1% agarose/formaldehyde gel and blotted on Hybond
N+ filters. The RNA in gels and filters was visualized with
ethidium bromide and photographed by UV transillumination to ensure the
integrity of RNA, to check the loading, and to confirm proper transfer.
RNA was transferred in 10 x standard saline citrate (0.15
M NaCl and 0.015 M sodium citrate, pH 7.0).
Blots were probed with fluorescein-labeled probes prepared with the
Gene Image (Amersham, Buckinghamshire, U.K.) random prime labeling
module and were detected with the CDP-Star detection module (Amersham,
Buckinghamshire, U.K.). The mouse cDNA probe for myogenin was a
1,100-bp EcoRI fragment.
Electrophoresis and Immunoblotting of Membranes
SDS-PAGE was performed as described by Laemmli (54). Proteins
were transferred to Immobilon in buffer consisting of 20% methanol,
200 mM glycine, 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 antibodies
against GLUT4 (1:400) and ß1-integrin (1:1000) in PBS containing 1%
nonfat dry milk and 0.02% sodium azide. ß1-integrin and PI
3-kinase-adapted subunits were detected using
[125I]protein A for 3 h at room temperature. GLUT4
and p110 were detected by ECL chemiluminiscence system (Amersham).
p85-p110
Complex Formation in Untransfected and Transfected
L6E9 Cells
Cells were incubated in DMEM containing 0.2% BSA for 2 h
before treatment with insulin to a final concentration of 1
µM (10 min at 37 C). After being washed twice in PBS
solution, cells were scraped and solubilized for 30 min at 4 C in a
buffer containing 50 mM HEPES, 150 mM NaCl, 10
mM EDTA, 10 mM
Na4P2O7, 100 mM NaF, 2
mM vanadate, 0.5 mM PMSF, 2 mM
leupeptin, and 2 mM pepstatin, supplemented with 1% NP40
(buffer A). The solubilizates were centrifuged at 10,000 x
g for 20 min at 4 C and 2.5 mg of the supernatants were
immunoprecipitated with 5 µl of polyclonal antibodies against rat p85
or nonimmune serum as controls (not shown). Antibodies were preadsorbed
on protein-G-Sepharose at 4 C for 1 h and washed twice in 30
mM HEPES, 30 mM NaCl, 0.1% Triton X-100, pH
7.4, before being incubated with the solubilized proteins for 90 min at
4 C. The immunopellets were washed three times in buffer A before being
resuspended in SDS-PAGE sample buffer under reduction conditions and
analyzed by Western blot using polyclonal antibodies against rat
p110
as described above.
Immunofluorescence Analysis
For immunofluorescence labeling, cells were grown on glass
coverslips. Coverslips were rinsed in PBS, fixed with methanol (-20 C)
for 2 min, washed twice in PBS, and processed. Cells fixed on
coverslips were incubated with 30 µl of primary antibody (1:100
anti-ß1-integrin, 1:100 polyclonal antibodies against rat
p85 or 1:100 nonimmune serum controls in PBS containing 0.5% BSA) for
45 min at 37 C. Coverslips were washed three times in PBS, the last one
for 15 min, before incubating with the secondary antibody (1:100
rodamine-conjugated goat anti-rabbit Igs in PBS containing 0.5% BSA)
for 30 min at 37 C. Coverslips were then washed three times in PBS; the
third wash was for 15 min in the presence of nuclear stain Hoechst
33342. Finally, coverslips were mounted with immunofluorescence medium.
Confocal microscopy was performed at the confocal microscopy facility
of the Serveis Cinentífico Tècnics of the Universitat de
Barcelona.
Glucose Transport Measurements
Before transport experiments, cells were starved for 2 h in
DMEM containing 0.5 mg/ml BSA and then treated or not with 1
µM insulin for 30 min. Cells were then washed and
transport solution was added (20 mM HEPES, 150
mM NaCl, 5 mM KCl, 5 mM
MgSO4, 1.2 mM KH2PO4,
2.5 mM CaCl2, 2 mM pyruvate, pH
7.4), together with 100 µM
2-deoxy-D-[3H]glucose (96 mCi/mmol). After 20
min, transport was stopped by addition of 2 vol of ice-cold 50
mM glucose in PBS. Cells were washed three times in the
same solution and lysed with 0.1 N NaOH, 0.1% SDS.
Radioactivity was determined by scintillation counting. Protein was
determined by the Pierce method. Each condition was run in triplicate,
and the nonspecific uptake (time zero) was determined by incubation of
the 2-deoxy-D-[3H]glucose in stop solution
(50 mM glucose in PBS) instead of transport solution. In
all cases, time zero represented 4% of the basal transport activity at
t = 20 min. Glucose transport under basal and stimulated
conditions was linear over the time period assayed (data not
shown).
 |
ACKNOWLEDGMENTS
|
---|
We thank Mr. Robin Rycroft for his editorial support and Dr.
Marta Camps, Dr. Ricardo Casaroli, and Susana Castel (Servei
Científico-Tècnics, University of Barcelona) for expert
advice in microscopy techniques. We are grateful to Mr. Quinzaños
for art work.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Antonio Zorzano, Departament de Bioquímica i Biologia Molecular, Facultat de Biologia, Universitat de Barcelona, Avinguda Diagonal 645, 08028 Barcelona, Spain. E-mail: azorzano{at}porthos.bio.ub.es
This work was supported by research grants from the Dirección
General de Investigación Científica y Técnica
(PB92/0805; PB95/0971) from "Fondo de Investigación
Sanitaria" (97/2101), Cost Action B5 and Generalitat de Catalunya
(GRQ 941040), Spain. P.K. is supported by a postdoctoral fellowship
from Comissió Interdepartamental i Innovació Tecnologica,
Generalitat de Catalunya.
Received for publication June 16, 1997.
Revision received September 29, 1997.
Accepted for publication October 8, 1997.
 |
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