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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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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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p85). IGF-I, des(1–3)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. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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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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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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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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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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in L6E9 Myoblasts 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, D–F). Figure 3 also shows that the subcellular distribution of p85
under basal conditions was mostly intracellular in both transfected and
untransfected cells (Fig. 3, A–C). In contrast, ß1-integrin
exhibited a typical distribution pattern of a plasma membrane marker
(Fig. 3, D–E).
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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).
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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).
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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).
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p85 Cellsp85-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 10–20 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 (0–100
nM IGF-II). Figure 6 shows the morphological changes
undergone by L6E9-Wp85 (Fig. 6, b–e) and L6E9-p85 cells (Fig. 6, g–j) 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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p85 Cellsp85 and L6E9-Wp85 cells were grown to confluence. Cells
were then incubated at increasing doses of IGF-II (0–100
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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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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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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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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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 10–20 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 (40–50% 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
supplier’s 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 1–4 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).
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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 94–1040), 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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REFERENCES |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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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 following
cytokine stimulation. J Biol Chem 271:14514–14518This article has been cited by other articles:
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  A. Klajn, C. Ferrai, L. Stucchi, I. Prada, P. Podini, T. Baba, M. Rocchi, J. Meldolesi, and R. D'Alessandro The Rest Repression of the Neurosecretory Phenotype Is Negatively Modulated by BHC80, a Protein of the BRAF/HDAC Complex J. Neurosci., May 13, 2009; 29(19): 6296 - 6307. [Abstract] [Full Text] [PDF]
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 T. Koyama, Y. Nakaoka, Y. Fujio, H. Hirota, K. Nishida, S. Sugiyama, K. Okamoto, K. Yamauchi-Takihara, M. Yoshimura, S. Mochizuki, et al. Interaction of Scaffolding Adaptor Protein Gab1 with Tyrosine Phosphatase SHP2 Negatively Regulates IGF-I-dependent Myogenic Differentiation via the ERK1/2 Signaling Pathway J. Biol. Chem., August 29, 2008; 283(35): 24234 - 24244. [Abstract] [Full Text] [PDF]
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 T. Nedachi, A. Kadotani, M. Ariga, H. Katagiri, and M. Kanzaki Ambient glucose levels qualify the potency of insulin myogenic actions by regulating SIRT1 and FoxO3a in C2C12 myocytes Am J Physiol Endocrinol Metab, April 1, 2008; 294(4): E668 - E678. [Abstract] [Full Text] [PDF]
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 A.-L. Wu, J.-H. Kim, C. Zhang, T. G. Unterman, and J. Chen Forkhead Box Protein O1 Negatively Regulates Skeletal Myocyte Differentiation through Degradation of Mammalian Target of Rapamycin Pathway Components Endocrinology, March 1, 2008; 149(3): 1407 - 1414. [Abstract] [Full Text] [PDF]
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C. De Alvaro, I. Nieto-Vazquez, J. M. Rojas, and M. Lorenzo Nuclear Exclusion of Forkhead Box O and Elk1 and Activation of Nuclear Factor-{kappa}B Are Required for C2C12-RasV12C40 Myoblast Differentiation Endocrinology, February 1, 2008; 149(2): 793 - 801. [Abstract] [Full Text] [PDF]
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 M.-S. Yoon and J. Chen PLD regulates myoblast differentiation through the mTOR-IGF2 pathway J. Cell Sci., February 1, 2008; 121(3): 282 - 289. [Abstract] [Full Text] [PDF]
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 A. Mandl, D. Sarkes, V. Carricaburu, V. Jung, and L. Rameh Serum Withdrawal-Induced Accumulation of Phosphoinositide 3-Kinase Lipids in Differentiating 3T3-L6 Myoblasts: Distinct Roles for Ship2 and PTEN Mol. Cell. Biol., December 1, 2007; 27(23): 8098 - 8112. [Abstract] [Full Text] [PDF]
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 M. J. Lim, K. J. Choi, Y. Ding, J. H. Kim, B. S. Kim, Y. H. Kim, J. Lee, W. Choe, I. Kang, J. Ha, et al. RhoA/Rho Kinase Blocks Muscle Differentiation via Serine Phosphorylation of Insulin Receptor Substrate-1 and -2 Mol. Endocrinol., September 1, 2007; 21(9): 2282 - 2293. [Abstract] [Full Text] [PDF]
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 S. Ciarmatori, D. Kiepe, A. Haarmann, U. Huegel, and B. Tonshoff Signaling mechanisms leading to regulation of proliferation and differentiation of the mesenchymal chondrogenic cell line RCJ3.1C5.18 in response to IGF-I J. Mol. Endocrinol., April 1, 2007; 38(4): 493 - 508. [Abstract] [Full Text] [PDF]
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 J. Castillo, I. Ammendrup-Johnsen, M. Codina, I. Navarro, and J. Gutierrez IGF-I and insulin receptor signal transduction in trout muscle cells Am J Physiol Regulatory Integrative Comp Physiol, June 1, 2006; 290(6): R1683 - R1690. [Abstract] [Full Text] [PDF]
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 J. L. J. van der Velden, R. C. J. Langen, M. C. J. M. Kelders, E. F. M. Wouters, Y. M. W. Janssen-Heininger, and A. M. W. J. Schols Inhibition of glycogen synthase kinase-3{beta} activity is sufficient to stimulate myogenic differentiation Am J Physiol Cell Physiol, February 1, 2006; 290(2): C453 - C462. [Abstract] [Full Text] [PDF]
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 C. de Alvaro, N. Martinez, J. M. Rojas, and M. Lorenzo Sprouty-2 Overexpression in C2C12 Cells Confers Myogenic Differentiation Properties in the Presence of FGF2 Mol. Biol. Cell, September 1, 2005; 16(9): 4454 - 4461. [Abstract] [Full Text] [PDF]
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N. Tiffin, S. Adi, D. Stokoe, N.-Y. Wu, and S. M. Rosenthal Akt Phosphorylation Is Not Sufficient for Insulin-Like Growth Factor-Stimulated Myogenin Expression but Must Be Accompanied by Down-Regulation of Mitogen-Activated Protein Kinase/Extracellular Signal-Regulated Kinase Phosphorylation Endocrinology, November 1, 2004; 145(11): 4991 - 4996. [Abstract] [Full Text] [PDF]
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 I. E. Sasson and M. J. Stern FGF and PI3 kinase signaling pathways antagonistically modulate sex muscle differentiation in C. elegans Development, November 1, 2004; 131(21): 5381 - 5392. [Abstract] [Full Text] [PDF]
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F. Vinals and F. Ventura Myogenin Protein Stability Is Decreased by BMP-2 through a Mechanism Implicating Id1 J. Biol. Chem., October 29, 2004; 279(44): 45766 - 45772. [Abstract] [Full Text] [PDF]
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I. Gonzalez, G. Tripathi, E. J. Carter, L. J. Cobb, D. A. M. Salih, F. A. Lovett, C. Holding, and J. M Pell Akt2, a Novel Functional Link between p38 Mitogen-Activated Protein Kinase and Phosphatidylinositol 3-Kinase Pathways in Myogenesis Mol. Cell. Biol., May 1, 2004; 24(9): 3607 - 3622. [Abstract] [Full Text] [PDF]
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 E. Erbay, I.-H. Park, P. D. Nuzzi, C. J. Schoenherr, and J. Chen IGF-II transcription in skeletal myogenesis is controlled by mTOR and nutrients J. Cell Biol., December 8, 2003; 163(5): 931 - 936. [Abstract] [Full Text] [PDF]
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M. L. Hribal, J. Nakae, T. Kitamura, J. R. Shutter, and D. Accili Regulation of insulin-like growth factor-dependent myoblast differentiation by Foxo forkhead transcription factors J. Cell Biol., August 18, 2003; 162(4): 535 - 541. [Abstract] [Full Text] [PDF]
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S. R. Broussard, R. H. MCCusker, J. E. Novakofski, K. Strle, W. Hong Shen, R. W. Johnson, G. G. Freund, R. Dantzer, and K. W. Kelley Cytokine-Hormone Interactions: Tumor Necrosis Factor {alpha} Impairs Biologic Activity and Downstream Activation Signals of the Insulin-Like Growth Factor I Receptor in Myoblasts Endocrinology, July 1, 2003; 144(7): 2988 - 2996. [Abstract] [Full Text] [PDF]
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V. De Arcangelis, D. Coletti, M. Conti, M. Lagarde, M. Molinaro, S. Adamo, G. Nemoz, and F. Naro IGF-I-induced Differentiation of L6 Myogenic Cells Requires the Activity of cAMP-Phosphodiesterase Mol. Biol. Cell, April 1, 2003; 14(4): 1392 - 1404. [Abstract] [Full Text] [PDF]
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H. Lu, P. Shah, D. Ennis, G. Shinder, J. Sap, H. Le-Tien, and I. G. Fantus The Differentiation of Skeletal Muscle Cells Involves a Protein-tyrosine Phosphatase-alpha -mediated C-Src Signaling Pathway J. Biol. Chem., November 22, 2002; 277(48): 46687 - 46695. [Abstract] [Full Text] [PDF]
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Y. Yoshiko, K. Hirao, and N. Maeda Differentiation in C2C12 myoblasts depends on the expression of endogenous IGFs and not serum depletion Am J Physiol Cell Physiol, October 1, 2002; 283(4): C1278 - C1286. [Abstract] [Full Text] [PDF]
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S. Sumitani, K. Goya, J. R. Testa, H. Kouhara, and S. Kasayama Akt1 and Akt2 Differently Regulate Muscle Creatine Kinase and Myogenin Gene Transcription in Insulin-Induced Differentiation of C2C12 Myoblasts Endocrinology, March 1, 2002; 143(3): 820 - 828. [Abstract] [Full Text] [PDF]
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S. Adi, B. Bin-Abbas, N.-Y. Wu, and S. M. Rosenthal Early Stimulation and Late Inhibition of Extracellular Signal-Regulated Kinase 1/2 Phosphorylation by IGF-I: A Potential Mechanism Mediating the Switch in IGF-I Action on Skeletal Muscle Cell Differentiation Endocrinology, February 1, 2002; 143(2): 511 - 516. [Abstract] [Full Text] [PDF]
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J. Tureckova, E. M. Wilson, J. L. Cappalonga, and P. Rotwein Insulin-like Growth Factor-mediated Muscle Differentiation. COLLABORATION BETWEEN PHOSPHATIDYLINOSITOL 3-KINASE-Akt-SIGNALING PATHWAYS AND MYOGENIN J. Biol. Chem., October 12, 2001; 276(42): 39264 - 39270. [Abstract] [Full Text] [PDF]
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M. I. Kontaridis, X. Liu, L. Zhang, and A. M. Bennett SHP-2 complex formation with the SHP-2 substrate-1 during C2C12 myogenesis J. Cell Sci., January 6, 2001; 114(11): 2187 - 2198. [Abstract] [Full Text] [PDF]
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M. A. Lawlor and P. Rotwein Coordinate Control of Muscle Cell Survival by Distinct Insulin-like Growth Factor Activated Signaling Pathways J. Cell Biol., December 11, 2000; 151(6): 1131 - 1140. [Abstract] [Full Text] [PDF]
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M. A. Lawlor and P. Rotwein Insulin-Like Growth Factor-Mediated Muscle Cell Survival: Central Roles for Akt and Cyclin-Dependent Kinase Inhibitor p21 Mol. Cell. Biol., December 1, 2000; 20(23): 8983 - 8995. [Abstract] [Full Text]
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U. Delling, J. Tureckova, H. W. Lim, L. J. De Windt, P. Rotwein, and J. D. Molkentin A Calcineurin-NFATc3-Dependent Pathway Regulates Skeletal Muscle Differentiation and Slow Myosin Heavy-Chain Expression Mol. Cell. Biol., September 1, 2000; 20(17): 6600 - 6611. [Abstract] [Full Text]
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J. R. Singleton, B. L. Baker, and A. Thorburn Dexamethasone Inhibits Insulin-Like Growth Factor Signaling and Potentiates Myoblast Apoptosis Endocrinology, August 1, 2000; 141(8): 2945 - 2950. [Abstract] [Full Text] [PDF]
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Z. Wu, P. J. Woodring, K. S. Bhakta, K. Tamura, F. Wen, J. R. Feramisco, M. Karin, J. Y. J. Wang, and P. L. Puri p38 and Extracellular Signal-Regulated Kinases Regulate the Myogenic Program at Multiple Steps Mol. Cell. Biol., June 1, 2000; 20(11): 3951 - 3964. [Abstract] [Full Text]
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 L. Wei, W. Zhou, L. Wang, and R. J. Schwartz beta 1-Integrin and PI 3-kinase regulate RhoA-dependent activation of skeletal alpha -actin promoter in myoblasts Am J Physiol Heart Circ Physiol, June 1, 2000; 278(6): H1736 - H1743. [Abstract] [Full Text] [PDF]
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M. A. Lawlor, X. Feng, D. R. Everding, K. Sieger, C. E. H. Stewart, and P. Rotwein Dual Control of Muscle Cell Survival by Distinct Growth Factor-Regulated Signaling Pathways Mol. Cell. Biol., May 1, 2000; 20(9): 3256 - 3265. [Abstract] [Full Text]
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K. L. Haugk, H.-M. P. Wilson, K. Swisshelm, and L. S. Quinn Insulin-Like Growth Factor (IGF)-Binding Protein-Related Protein-1: An Autocrine/Paracrine Factor That Inhibits Skeletal Myoblast Differentiation but Permits Proliferation in Response to IGF Endocrinology, January 1, 2000; 141(1): 100 - 110. [Abstract] [Full Text] [PDF]
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R Hooshmand-Rad, L Hajkova, P Klint, R Karlsson, B Vanhaesebroeck, L Claesson-Welsh, and C. Heldin The PI 3-kinase isoforms p110(alpha) and p110(beta) have differential roles in PDGF- and insulin-mediated signaling J. Cell Sci., January 1, 2000; 113(2): 207 - 214. [Abstract] [PDF]
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P. Kaliman, J. Canicio, X. Testar, M. Palacin, and A. Zorzano Insulin-like Growth Factor-II, Phosphatidylinositol 3-Kinase, Nuclear Factor-kappa B and Inducible Nitric-oxide Synthase Define a Common Myogenic Signaling Pathway J. Biol. Chem., June 18, 1999; 274(25): 17437 - 17444. [Abstract] [Full Text] [PDF]
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D. Li, G. Sweeney, Q. Wang, and A. Klip Participation of PI3K and atypical PKC in Na+-K+-pump stimulation by IGF-I in VSMC Am J Physiol Heart Circ Physiol, June 1, 1999; 276(6): H2109 - H2116. [Abstract] [Full Text] [PDF]
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 B.-H. Jiang, M. Aoki, J. Z. Zheng, J. Li, and P. K. Vogt Myogenic signaling of phosphatidylinositol 3-kinase requires the serine-threonine kinase Akt/protein kinase B PNAS, March 2, 1999; 96(5): 2077 - 2081. [Abstract] [Full Text] [PDF]
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D. D. Sarbassov and C. A. Peterson Insulin Receptor Substrate-1 and Phosphatidylinositol 3-Kinase Regulate Extracellular Signal-Regulated Kinase-Dependent and -Independent Signaling Pathways during Myogenic Differentiation Mol. Endocrinol., December 1, 1998; 12(12): 1870 - 1878. [Abstract] [Full Text]
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J. Canicio, E. Gallardo, I. Illa, X. Testar, M. Palacin, A. Zorzano, and P. Kaliman p70 S6 Kinase Activation Is Not Required for Insulin-Like Growth Factor-Induced Differentiation of Rat, Mouse, or Human Skeletal Muscle Cells Endocrinology, December 1, 1998; 139(12): 5042 - 5049. [Abstract] [Full Text] [PDF]
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B.-H. Jiang, J. Z. Zheng, and P. K. Vogt An essential role of phosphatidylinositol 3-kinase in myogenic differentiation PNAS, November 24, 1998; 95(24): 14179 - 14183. [Abstract] [Full Text] [PDF]
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L. Wei, W. Zhou, J. D. Croissant, F.-E. Johansen, R. Prywes, A. Balasubramanyam, and R. J. Schwartz RhoA Signaling via Serum Response Factor Plays an Obligatory Role in Myogenic Differentiation J. Biol. Chem., November 13, 1998; 273(46): 30287 - 30294. [Abstract] [Full Text] [PDF]
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Q. Xu and Z. Wu The Insulin-like Growth Factor-Phosphatidylinositol 3-Kinase-Akt Signaling Pathway Regulates Myogenin Expression in Normal Myogenic Cells but Not in Rhabdomyosarcoma-derived RD Cells J. Biol. Chem., November 17, 2000; 275(47): 36750 - 36757. [Abstract] [Full Text] [PDF]
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Y. Tamir and E. Bengal Phosphoinositide 3-Kinase Induces the Transcriptional Activity of MEF2 Proteins during Muscle Differentiation J. Biol. Chem., October 27, 2000; 275(44): 34424 - 34432. [Abstract] [Full Text] [PDF]
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L. L. Tortorella, D. J. Milasincic, and P. F. Pilch Critical Proliferation-independent Window for Basic Fibroblast Growth Factor Repression of Myogenesis via the p42/p44 MAPK Signaling Pathway J. Biol. Chem., April 20, 2001; 276(17): 13709 - 13717. [Abstract] [Full Text] [PDF]
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J. Canicio, P. Ruiz-Lozano, M. Carrasco, M. Palacin, K. Chien, A. Zorzano, and P. Kaliman Nuclear Factor kappa B-inducing Kinase and Ikappa B Kinase-alpha Signal Skeletal Muscle Cell Differentiation J. Biol. Chem., June 1, 2001; 276(23): 20228 - 20233. [Abstract] [Full Text] [PDF]
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Q. Kang, Y. Cao, and A. Zolkiewska Direct Interaction between the Cytoplasmic Tail of ADAM 12 and the Src Homology 3 Domain of p85alpha Activates Phosphatidylinositol 3-Kinase in C2C12 Cells J. Biol. Chem., June 29, 2001; 276(27): 24466 - 24472. [Abstract] [Full Text] [PDF]
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