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First published online 10 July 2006
doi: 10.1242/dev.02479
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1 Department of Clinical Neurosciences, University Hospital, Geneva,
Switzerland.
2 Department of Basic Neurosciences, University Medical Center, Rue Michel
Servet 1, 1211 Geneva 4, Switzerland.
* Author for correspondence (e-mail: stephane.konig{at}medecine.unige.ch)
Accepted 6 June 2006
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Myogenesis, Calcineurin, Hyperpolarization, Human myoblasts
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Liu et al.,
2003). The goal of the present work was to uncover the molecular
link between the Kir2.1-induced hyperpolarization and the expression of
myogenin and MEF2, two major transcription factors of the differentiation
process.
We are using primary human myoblast cultures derived from single satellite
cells. Human myoblasts can proliferate for several months in culture, and
terminal differentiation and fusion into myotubes can be induced by serum
withdrawal. At the molecular level, the induction of the differentiation
process is associated with the expression of an early marker, myogenin.
Myogenin belongs to the family of myogenic basic helix-loop-helix (bHLH)
transcription factors, which includes MYOD, MYF5 and MRF4
(Braun et al., 1989;
Davis et al., 1987;
Rhodes and Konieczny, 1989;
Wright et al., 1989). These
factors are implicated in the specification and in the differentiation of
myogenic cells. During myoblast differentiation, activation of muscle-specific
genes by myogenic bHLH proteins also requires their interaction with
transcription factors of the MEF2 family
(Black and Olson, 1998). MEF2
family has four members (MEF2A-D) that bind to a consensus sequence present in
several muscle-specific promoters.
Differentiation of human myoblasts requires a hyperpolarization of their
membrane resting potential to approximately -70 mV
(Fischer-Lougheed et al.,
2001; Liu et al.,
2003). Preventing this hyperpolarization impedes both expression
and activity of myogenin and MEF2, indicating that it is a prerequisite for
differentiation (Konig et al.,
2004). We proposed that Kir2.1-linked hyperpolarization initiates
the differentiation process by increasing cytoplasmic free Ca2+
(Arnaudeau et al., 2006;
Bijlenga et al., 2000;
Liu et al., 2003). The
question is what are the signal transduction pathways downstream of this
cytoplasmic Ca2+ signal that initiate human myoblast
differentiation?
In mouse myoblasts, myogenin expression, an early marker for
differentiation, has been suggested to be regulated by at least four different
pathways: p38 mitogen-activated protein kinase (p38-MAPK),
phosphatidyl-inositol 3-kinase (PI3K), Ca2+-calmodulin-dependent
kinase (CaMK) and calcineurin (Cuenda and
Cohen, 1999; Friday et al.,
2003; Xu et al.,
2002; Zetser et al.,
1999). The p38-MAPK, CaMK and calcineurin pathways appear capable
of inducing the transcriptional activity of MEF2
(Tamir and Bengal, 2000).
Although it is well known that during myoblast differentiation CaMK
(Chin, 2005) and calcineurin
(Stiber et al., 2005) activity
is strongly controlled by cytoplasmic Ca2+, the role of
Ca2+ in the activation of p38-MAPK and PI3K is less clear.
Activation of p38-MAPK is linked to direct phosphorylations by MKK3 and MKK6
(Derijard et al., 1995;
Han et al., 1996), and
activation of PI3K is coupled to insulin growth factor (IGF1) tyrosine kinase
receptor (Jiang et al., 1998;
Kaliman et al., 1996;
Kandel and Hay, 1999). The
principal downstream target of PI3K is AKT (protein kinase B). Full activation
of AKT by insulin or IGF1 requires a phosphorylation at two sites by two
separate kinases that both depend on PI3K activity
(Alessi et al., 1996;
Sarbassov et al., 2005;
Stokoe et al., 1997). Whether
these four signaling pathways are involved in human myoblast differentiation,
and whether they are modulated be the membrane hyperpolarization, however, is
not known.
In the present study, we show that the Kir2.1-induced hyperpolarization controls the onset of the differentiation process through the selective activation of the calcineurin pathway, although p38-MAPK, PI3K and CaMK pathways are also required for a full expression of myogenin and MEF2. We find, in addition, that p38-MAPK and PI3K are already activated during myoblast proliferation, and that CaMK activation can be induced during myoblast proliferation through a Ca2+-dependent mechanism not related to the hyperpolarization. We propose that the differentiation process in human myoblasts is initiated by a membrane hyperpolarization that acts as a molecular switch, forcing differentiation by generating a Ca2+ signal responsible for the specific activation of the calcineurin signaling pathway.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Muscle
samples were obtained from children during corrective orthopedic surgery
according to the guidelines of the local ethical committee. Single satellite
cells obtained after muscle enzymatic dissociation were manually collected
under the microscope on a size criteria, transferred into single wells (one
cell/well) of a 96 wells container (Becton Dickinson) using a micropipette
(clonal culture). Myoblasts were amplified in serum-containing medium (growth
medium) and induced to differentiate into myotubes in a serum-free medium
(differentiation medium, DM). When indicated, differentiation medium was
complemented with 10 µM SB202190 (Calbiochem), 50 µM LY194002
(CellSignaling), 30 µM KN-93 (Calbiochem), 7 µM CsA (Calbiochem) or 5
µM FK-506 (A.G. Scientific).
Immunostaining
Immunostaining was performed as previously described
(Konig et al., 2004). Myogenin
was revealed using a mouse monoclonal antibody (1/1000, BD Biosciences) and
MEF2 using a rabbit polyclonal antibody (1/300, Sc-313 SantaCruz
Biotechnology). Immunostaining fluorescence from myoblasts plated on 25 mm
coverslips was imaged with a Zeiss Axiovert S100TV microscope using a
40x Fluar 1.3 NA oil-immersion objective (Carl Zeiss AG, Feldbach,
Switzerland). DAPI, Alexa 488 and Alexa 546 were respectively excited by the
360±10 nm, 488±10 nm and 546±10 nm line from an Optoscan
Monochromator (Cairn Research, Faversham, UK) through a XF2050 dichroic mirror
(Omega Optical, Brattleboro, VT) and the fluorescence emission were
respectively acquired at 460±30 nm, 520±40 nm and 602±50
nm (XF3063 Omega Optical, Brattleboro, VT) using a cooled, 12 bits TE/CCD
interlined Coolsnap HQ camera (Photometrics, Roper Scientific, Trenton, NJ).
Image acquisition and analysis was performed with the Metamorph 5.0 software
(Universal Imaging, West Chester, PA).
Western blots
Myoblasts were lyzed using PhosphoSafe Extraction Buffer (Novagen). After
adding an equivalent volume of Laemmli 2x, extracts were boiled for 3
minutes. Western blots were performed as previously described
(Konig et al., 2004), except
that saturation with 5% non-fat milk in TTBS was carried out in the presence
of phosphatase inhibitors (50 mM NaF and 1 mM sodium orthovanadate).
Antibodies were diluted in TTBS with 5% BSA. Antibodies used: mouse monoclonal
antibody against myogenin (1/2000, BD Biosciences) and p38-MAPK (1/1000,
CellSignaling Technology #9212); rabbit monoclonal antibody against
phospho-p38-MAPKThr180/Tyr182 (1/1000, CellSignaling Technology
#9215); phopho-AKTSer473 (1/1000, CellSignaling Technology #4058);
phopho-AKTThr308 (1/1000, CellSignaling Technology, #4056); and
p42/44 MAPKThr202/Tyr204 (1/1000, CellSignaling Technology,
#4377).
CaMKII assay
CaMK activity was quantified using the radioactive assay SignaTECT
Ca2+/Calmodulin-Dependent Protein Kinase Assay System (Promega).
Myoblasts were lyzed using 100 µl PhosphoSafe Extraction Buffer (Novagen)
and spun for 5 minutes (13,000 g) at 4°C. Five microlitres
of supernatant were collected for each assay and incubated 2 minutes at
30°C in a buffer solution containing the specific peptide for CaMKII.
Endogenous activity (which represents the level of CaMKII activity of
myoblasts in each conditions of culture) was assessed in a buffer solution
containing 5 mM EGTA and 0.5 µCi of 
32P-ATP. Total CaMKII
activity (which represents the maximum CaMKII activity of the myoblast sample)
was assessed from 1 µl of supernatant (to avoid saturation) in a buffer
solution containing nonlimiting amounts of Ca2+ and calmodulin to
allow maximal CaMKII activation in vitro (5 mM CaCl2, 5 µM
calmodulin and 0.5 µCi of 32P-ATP). Specific activation
of CaMKII was calculated as the ratio between the endogenous activity in
cultured myoblasts and the total activity. Variation of the specific
activation of CaMKII throughout the experiments reflects a modification of the
CaMKII activity in cultured myoblasts as the total CaMKII remains nearly
constant (not shown). For each experiment, the ratio obtained with myoblasts
maintained in differentiation medium containing 15 µM BAPTA-AM was set to
1.
p38-MAPK assay
p38-MAPK activity was assessed using the non-radioactive p38-MAPKinase
Assay Kit (Cell Signaling Technology, #9820). At the indicated times, cells
lysis was carried out with 500 µl of provided lysis buffer. The active form
of p38-MAPK was immunoprecipitated (overnight at 4°C) from 200 µl cell
extracts with 20 µl phospho-p38-MAPK (Thr180/Tyr182) monoclonal antibody.
An in vitro kinase assay was performed directly on the immunoprecipitated
phospho-p38-MAPK in presence of 200 µM ATP and using recombinant ATF-2
(recATF-2) as a substrate. Phosphorylated recATF-2 was detected by
immunoblotting using a phospho-ATF2 (Thr71)-specific antibody.
Luciferase assay
Using electroporation (Espinos et al.,
2001), 2x106 human myoblasts were transfected
with 2 pmol firefly luciferase encoding plasmid (3MEF2-luc or 9NFAT-luc)
together with 1 pmol control plasmid encoding the Renilla luciferase
[phRL-TK-luc, Promega (Konig et al.,
2004)]. At the indicated times, cells were processed with the
Dual-Luciferase reporter assay kit (Promega) as recommended by the
manufacturer.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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CaMK, PI3K, calcineurin and p38-MAPK regulatory pathways have been
suggested to regulate myogenin and MEF2 expression during murine myoblast
differentiation (Xu et al.,
2002). We first investigated whether any of these regulatory
pathways were involved in human myoblast differentiation
(Fig. 1B). Myoblast
differentiation was assessed using the percentage of nuclei expressing
myogenin or MEF2 over the total number of nuclei. In proliferating myoblasts,
very few nuclei were positive for myogenin (6%) or MEF2 (7%). After 3-4 days
in differentiation medium, 69% and 71% of the nuclei were positive for
myogenin and MEF2, respectively. Inhibition of the hyperpolarization with 10
mM Cs+ (a Kir2.1 blocker) reduced myogenin and MEF2 expression by
1.9-fold (Konig et al., 2004).
Fig. 1B also shows that
inhibiting p38-MAPK (with SB202190) and CaMK (with KN-93) reduced myogenin and
MEF2 expression to the same level as that obtained by treating cells with 10
mM Cs+. However, inhibition of PI3K (with LY294002) had a stronger
effect on myoblast differentiation, reducing the percentage of positive nuclei
by a 3.8 and 4.1-fold for myogenin and MEF2, respectively. To inhibit the
calcineurin pathway, we used cyclosporin A and FK506 mixed together to reduce
the toxicity of each drug. Concentrations used were those required to inhibit
calcineurin activity fully (see below). Using this strategy, myogenin and MEF2
expression was reduced by 3.7 and 2.6-fold, respectively.
Taken together, these results suggested that p38-MAPK, CaMK, PI3K and calcineurin pathways are all involved in the early steps of human myoblast differentiation. What is less clear is whether one or more of these pathways are controlled by membrane hyperpolarization generated by Kir2.1 channels.
Early activation of calcineurin during human myoblast differentiation requires a membrane hyperpolarization
Our previous work on human myoblasts suggested that membrane
hyperpolarization generated by Kir2.1 channels increases intracellular
Ca2+ and that this step is essential to allow myoblast
differentiation to proceed (Arnaudeau et
al., 2006; Bijlenga et al.,
2000; Liu et al.,
2003). As calcineurin is a Ca2+-dependent phosphatase
involved in human myoblast differentiation, we tested whether its activity is
controlled by a Ca2+ signal that could be induced by the
hyperpolarization. To assess calcineurin activity we used its ability to
dephosphorylate and thereby activate the transcription factor NF-AT. For this
purpose, human myoblast were electroporated with a plasmid encoding the
luciferase protein under the control of a specific promoter inducible by NF-AT
transcription factors (9NF-AT-luc plasmid). The electroporated myoblasts were
either maintained in proliferation condition (growth medium) or induced to
differentiate in differentiation medium for 1 to 4 days. Kinetics of
activation of NF-AT transcription factor in electroporated myoblasts
expressing the 9NF-AT-luc plasmid are illustrated in
Fig. 2A. It can be seen that
NF-AT activity is absent in proliferating myoblasts, is induced during the
first 24 hours of differentiation, and then increases during the following
days (4.7-fold after 3 days and 10.0-fold after 4 days). Kinetics of
activation of NF-AT (calcineurin) was then compared with that of myogenic bHLH
and MEF2 (myoblasts were electroporated with a luciferase plasmid controlled
by either myogenic bHLH or MEF2 transcription factors). Interestingly,
kinetics of activation of NF-AT are similar to that of myogenic bHLH,
suggesting that, like myogenic bHLH, calcineurin pathway is activated at the
very beginning of the differentiation process. Activation of MEF2 is, however,
slower. From these results, we conclude that calcineurin is activated during
the very early steps of myoblast differentiation, at the same time as myogenic
bHLH and before MEF2.
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), we
examined whether an inhibition of calcineurin affects these two activities in
human myoblasts. Fig. 2C shows
that the inhibition of calcineurin by cyclosporin A and FK-506 together
reduced myogenic bHLH and MEF2 transcriptional activity by 3.0- and 17.5-fold,
respectively. Together, these results show that suppression of the Kir2.1-induced membrane hyperpolarization inactivates myogenin and MEF2 activities through the inhibition of the calcineurin pathway.
The role of CaMKII in human myoblast differentiation is not linked to the Kir2.1-induced membrane hyperpolarization
As seen in Fig. 1B, CaMK
regulatory pathway is a second Ca2+-dependent pathway involved in
the control of human myoblast differentiation. We, thus, examined whether this
pathway was activated by Ca2+ signals induced by the Kir2.1-linked
hyperpolarization, in the same way as calcineurin was. Endogenous CaMKII
activity was evaluated during myoblast differentiation in control conditions
and in conditions that prevent membrane hyperpolarization (10 mM
Cs+ or 116 mM K+). In order to compare experiments, we
normalized the results to the CaMKII activity obtained in presence of 15 µM
BAPTA-AM (background). Fig. 3A
shows that low levels of CaMKII activity (40% above background) are present in
proliferating conditions, and that this activity is increased by 2.3-fold
after 24 hours of differentiation. However, this increased activity is
insensitive to depolarizing agents (10 mM Cs+ or 116 mM
K+). This indicates that, unlike calcineurin, CaMKII is not
regulated by the Kir2.1-linked hyperpolarization. In agreement with this
result, a maximum CaMKII activation could already be detected after a 1-hour
exposure to differentiation medium, i.e. before the differentiation-linked
hyperpolarization, which occurs after 6 hours of differentiation
(Konig et al., 2004).
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)]. In
this low Ca2+-containing differentiation medium, CaMKII activity
was reduced to the background level after 1 hour
(Fig. 3A). Furthermore, when
myoblasts were induced to differentiate in a differentiation medium containing
the same Ca2+ concentration as in the growth medium (0.7 mM
Ca2+), the CaMKII activity was similar to that of proliferating
myoblasts kept in growth medium (P=0.78). However, the CaMKII
activity in myoblasts proliferating in growth medium containing 1.8 mM
Ca2+, was not statistically different to that obtained in control
differentiation medium after 1 hour (P=0.14) or after 24 hours
(P=0.55), but significantly different to that obtained in control
growth medium (P=0.002). Taken together, these results suggest that,
in human myoblasts, CaMK activity is directly linked to extracellular
Ca2+ concentration and that it is unaffected by
hyperpolarization. We then tested the capacity of human myoblast to differentiate (i.e. to express myogenin and MEF2) in differentiation medium containing 0.7 mM Ca2+ (CaMKII activity is low) or in growth medium containing 1.8 mM Ca2+ (CaMKII activity is increased). As expected, when CaMK is maintained at a low activity in differentiation medium containing 0.7 mM Ca2+, the percentage of positive nuclei for both myogenin and MEF2 was reduced by 2.4 and 1.8-fold respectively (Fig. 3B). This reduction is comparable with that obtained with the CaMK inhibitor KN-93 (Fig. 1A). However, activation of the CaMKII in growth medium containing 1.8 mM Ca2+ did not allow any induction of myogenin or MEF2 expression. We also verified that myoblasts maintained in growth medium containing 1.8 mM Ca2+ (myoblasts with CaMK activity increased) were still able to proliferate actively. Myoblasts (105) were seeded in growth medium containing either 0.7 (control) or 1.8 mM Ca2+, and counted after 48 hours. We found that the proliferation rate was not affected by CaMK activation (4.7±0.3x105 versus 4.6±0.3x105 myoblasts after 48 hours in 0.7 and 1.8 mM Ca2+, respectively, P=0.74).
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We also verified directly phospho-p38-MAPK activity by evaluating the ability of immunoprecipitated phospho-p38-MAPK from proliferating and differentiating human myoblasts to phosphorylate recombinant ATF2. Fig. 4B shows that recombinant ATF2 is phosphorylated by phospho-p38-MAPK immunoprecipitated from proliferating myoblasts, as well as by myoblasts induced to differentiate for 4 and 24 hours. The same kinase activity was observed in presence of 10 mM Cs+, confirming by a direct assessment of phospho-p38-MAPK activity that the Kir2.1-linked hyperpolarization is not involved in the activation or maintained activity of the p38-MAPK regulatory pathway during human myoblast differentiation.
Activation of the PI3K regulatory pathway depends upon insulin/IGF1
signaling (Jiang et al., 1998;
Kaliman et al., 1996;
Kandel and Hay, 1999). In
order to avoid unwanted stimulation of PI3K, insulin was omitted from both
growth and differentiation media in the experiments involving this
pathway.
A key downstream molecule activated by PI3K is AKT (protein kinase B). Thus, as readout for PI3K activity, we used AKT phosphorylation. When the PI3K pathway is activated, AKT is phosphorylated at two sites, Ser473 and Thr308. Fig. 4C shows that AKT is already phosphorylated at the two sites in proliferating myoblasts, and that the phosphorylation level is comparable with that of myoblasts induced to differentiate for 24 hours. This result shows that, like p38-MAPK, PI3K is already fully active in proliferating human myoblast. Treating myoblast with 10 mM Cs+ to inhibit Kir2.1-induced hyperpolarization had no effect on AKT phosphorylation (Fig. 4C), indicating that the hyperpolarization is not modulating the PI3K regulatory pathway. In the same experiment, a downregulation of myogenin expression indicated that Cs+ was efficient at inhibiting differentiation. Fig. 4C also shows that the AKT pathway is not regulated by Ca2+. In the presence of 15 µM BAPTA-AM, AKT phosphorylation was not affected although myogenin expression was markedly inhibited.
Taken together, these results show that PI3K and p38-MAPK pathways are activated during myoblast proliferation. They are not controlled by the Kir2.1-linked hyperpolarization, but they are required for a full myoblast differentiation to take place.
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Our model is that the Kir2.1-linked hyperpolarization and the resulting calcineurin activation constitute the molecular switch that forces myoblast to differentiate and fuse to form myotubes (Fig. 5).
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).
Calcineurin is a serine/threonine phosphatase that is activated during
sustained elevations of intracellular Ca2+
(Klee et al., 1998), as occur
in muscle fibers by slow type motoneuron firing
(Schiaffino and Serrano,
2002). In vivo, the downstream substrates for calcineurin
implicated in the fast-to-slow fiber switch include the transcription factors
NFAT and MEF2 (Chin et al.,
1998; Wu et al.,
2000a). A role for calcineurin at early steps of differentiation
in mouse myoblasts has also been suggested
(Friday et al., 2000;
Xu et al., 2002). However, the
trigger of a change in calcineurin activity at the beginning of the
differentiation process, i.e. prior to innervation, remained to be
explained.
In human myoblasts induced to differentiate, we showed that calcineurin
activation is linked to the Kir2.1 linked membrane hyperpolarization. In
presence of either Cs+ or high K+, calcineurin activity
is reduced to a level close to the basal level measured in proliferating
myoblast. In a previous work, we showed that blockade of Kir2.1 channels with
10 mM Cs+ depolarizes myoblast induced to differentiate to
approximately -35 mV (Konig et al.,
2004), while high external K+ (116 mM) clamps the
resting membrane potential in the vicinity of 0 mV. The remaining potential of
about -35 mV in presence of 10 mM Cs+ is probably due to activity
of ether-à-gogo and/or ether-à-gogo-related gene
K+channels (Bijlenga et al.,
1998; Liu et al.,
2003). Here, we show that calcineurin activity is already fully
inactivated by 10 mM Cs+, which suggests that, during the early
stages of human myoblast differentiation, calcineurin activation is strictly
controlled by the activity of Kir2.1 channels and the signals they induce.
There are evidences that L-type Ca2+ channels are essential to
initiate calcineurin activation in neurons and in rat ventricular myocytes
(Graef et al., 1999;
Perrier et al., 2004). In
addition, in differentiating C2C12 myoblasts, it was clearly shown that
calcineurin is a downstream mediator of IGF-1-induced signaling through L-type
Ca2+ channels (Spangenburg et
al., 2004). In human myoblasts, however, inhibition of L-type
Ca2+ channels has no effect on the early steps of the
differentiation process, whereas T-type Ca2+ channels activity
induced by the Kir2.1 linked hyperpolarization plays an important role
(Bijlenga et al., 2000).
Specifically, we could show that activation of the T-type Ca2+
channels at hyperpolarized potentials allows a sustained low amplitude
increase of intracellular Ca2+
(Bijlenga et al., 2000) that is
compatible with the Ca2+ signals that activate calcineurin
(Klee et al., 1998). However,
we recently showed that all clones of human myoblasts do not exclusively use
T-type Ca2+ channels to differentiate
(Arnaudeau et al., 2006). We
found that, as they differentiate, different clones of human myoblasts can
use, in addition to or as substitutes for T-type Ca2+ channels,
others sources of Ca2+ such as an influx through store operated
channels or a release of Ca2+ from the endoplasmic reticulum via
IP3 receptors. However, all these Ca2+ signals are suppressed when
the hyperpolarization is blocked.
Thus, we propose that, in differentiating human myoblasts and prior to the innervation of myotubes, there is already a first activation of calcineurin that links the early activation of the Kir2.1-induced membrane hyperpolarization and its associated Ca2+ signals to the expression/activity of myogenin and MEF2 transcription factors.
CaMK regulatory pathway
CaMK is another Ca2+-dependent pathway involved in human
myoblast differentiation. CaMKII activation is, however, not linked to
Kir2.1-induced hyperpolarization. Indeed, CaMKII activities measured at 1 hour
in differentiation medium (before activation of Kir2.1 channels) or at 24
hours (long after membrane hyperpolarization took place) are statistically
identical. Furthermore, blockade of Kir2.1 channels with 10 mM Cs+
or inhibition of the hyperpolarization with 116 mM K+ have no
effect on the CaMKII activity. These results suggest that the Ca2+
signals that are linked to the hyperpolarization do not control the CaMKII
activity, and that at least two different Ca2+ signals must exist
to activate sequentially CaMK and calcineurin. This is in agreement with
previous work suggesting that these two enzymes are activated by different
Ca2+ signals, i.e. transient high amplitude Ca2+ spikes
preferentially activate CaMKII, whereas sustained low-amplitude
Ca2+ increases rather induce calcineurin activity
(McKinsey et al., 2002).
Our results also show that CaMKII activation depends on extracellular Ca2+ concentration (influx). We found that the CaMK activity measured in myoblasts kept for 1 hour in differentiation medium containing only 15 µM Ca2+ is massively reduced, and that CaMK activity is similar in myoblasts kept in either growth or differentiation medium containing the same Ca2+ concentration (this was tested for 0.7 and 1.8 mM Ca2+). CaMKII activation can thus be induced either in proliferation or in differentiation conditions. It seems to depend only on extracellular Ca2+ concentration, and its activation does not affect the rate of myoblast proliferation. In vivo, it is possible that CaMKII is activated in proliferating myoblasts according to extracellular Ca2+ concentration. Indeed, we expect that during a massive myofiber degeneration or after a muscle injury, Ca2+ concentration could be locally increased. It is worth recalling that CaMK activation on its own does not induce myoblast differentiation but is required for an optimal differentiation to take place. It is possible that a graded CaMK activation, depending on extracellular Ca2+ concentration, could optimize myoblast differentiation and muscle regeneration.
p38-MAPK and PI3K regulatory pathways
Several authors have proposed that p38-MAPK is activated after the
induction of myoblast differentiation and that this activation promotes
myoblast differentiation (Cuenda and Cohen,
1999; Wu et al.,
2000b; Zetser et al.,
1999). Inhibition of p38-MAPK in human primary myoblasts decreases
myoblast differentiation, which confirms an implication of this pathway in
human cells. However, using two different approaches, we found that p38-MAPK
is already activated in proliferating myoblasts, and that this pathway is not
regulated during the early steps of the differentiation process. Our results
are in agreement with a recent work, suggesting that p38-MAPK plays a major
role in triggering differentiation of quiescent satellite cells into
proliferating myoblasts rather than myoblasts into myotubes
(Jones et al., 2005). However,
as p38-MAPK activity is required for a full myoblast differentiation to
proceed, it was important to show that this pathway was not affected
(downregulated) by the differentiation-linked hyperpolarization. We found,
indeed, that p38-MAPK activity is Ca2+-independent and insensitive
to the change in potential generated by Kir2.1 channel activation.
Kinetics of activation of PI3K during human myoblast differentiation is
similar to that of p38-MAPK. We found that PI3K is active in proliferating
myoblasts and that its activity is neither Ca2+-dependent nor
hyperpolarization sensitive. It should be noted, however, that, both in
proliferating and differentiating myoblasts, PI3K is activated in the absence
of added insulin in the culture media. As differentiation medium does not
contain any serum and as our cultures are purely composed of myoblasts (see
Materials and methods), we hypothesize that either human myoblasts are able to
secrete IGF1, or that PI3K is activated via an IGF1-independent mechanism
(Rauch and Loughna, 2005). It
should also be mentioned that, in contrast to what occurs in mouse cell lines
(Gonzalez et al., 2004;
Li et al., 2000;
Wu et al., 2000b), concerted
activation of both PI3K and p38-MAPK pathways is not sufficient in itself to
induce human myoblast differentiation.
Taken together, our results highlight the importance of the calcineurin activation in the control of the early steps of myoblast differentiation. We give for the first time the sequence and the respective roles of the different signaling pathways involved in the differentiation process. We show that p38-MAPK, PI3K and CaMK activity are required for a full human myoblast differentiation but that these three pathways, even together, do not trigger the differentiation process. We also show that these three pathways are or can be activated during proliferation without affecting the proliferation rate. As CaMK is activated before calcineurin, it thus appears that at least two different Ca2+ signals may be required for a full differentiation to take place, and that only the signal activating calcineurin is directly linked to the induction of the differentiation leading to myoblast fusion. In our model, the triad constituted by the Kir2.1-linked hyperpolarization, its associated Ca2+ signals and the resulting calcineurin activation controls the initiation of myoblast fusion.
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Alessi, D. R., Andjelkovic, M., Caudwell, B., Cron, P., Morrice, N., Cohen, P. and Hemmings, B. A. (1996). Mechanism of activation of protein kinase B by insulin and IGF-1. EMBO J. 15,6541 -6551.[Medline]
Arnaudeau, S., Holzer, N., Konig, S., Bader, C. R. and Bernheim, L. (2006). Calcium sources used by post-natal human myoblasts during initial differentiation. J. Cell. Physiol. 208,435 -445.[CrossRef][Medline]
Bijlenga, P., Occhiodoro, T., Liu, J. H., Bader, C. R.,
Bernheim, L. and Fischer-Lougheed, J. (1998). An
ether-a-go-go K+ current, Ih-eag, contributes to the hyperpolarization of
human fusion-competent myoblasts. J. Physiol.
512,317
-323.
Bijlenga, P., Liu, J. H., Espinos, E., Haenggeli, C. A.,
Fischer-Lougheed, J., Bader, C. R. and Bernheim, L. (2000).
T-type alpha 1H Ca2+ channels are involved in Ca2+
signaling during terminal differentiation (fusion) of human myoblasts.
Proc. Natl. Acad. Sci. USA
97,7627
-7632.
Black, B. L. and Olson, E. N. (1998). Transcriptional control of muscle development by myocyte enhancer factor-2 (MEF2) proteins. Annu. Rev. Cell Dev. Biol. 14,167 -196.[CrossRef][Medline]
Braun, T., Buschhausen-Denker, G., Bober, E., Tannich, E. and Arnold, H. H. (1989). A novel human muscle factor related to but distinct from MyoD1 induces myogenic conversion in 10T1/2 fibroblasts. EMBO J. 8,701 -709.[Medline]
Chin, E. R. (2005). Role of
Ca2+/calmodulin-dependent kinases in skeletal muscle plasticity. J.
Appl. Physiol. 99,414
-423.
Chin, E. R., Olson, E. N., Richardson, J. A., Yang, Q.,
Humphries, C., Shelton, J. M., Wu, H., Zhu, W., Bassel-Duby, R. and Williams,
R. S. (1998). A calcineurin-dependent transcriptional pathway
controls skeletal muscle fiber type. Genes Dev.
12,2499
-2509.
Cuenda, A. and Cohen, P. (1999).
Stress-activated protein kinase-2/p38 and a rapamycin-sensitive pathway are
required for C2C12 myogenesis. J. Biol. Chem.
274,4341
-4346.
Davis, R. L., Weintraub, H. and Lassar, A. B. (1987). Expression of a single transfected cDNA converts fibroblasts to myoblasts. Cell 51,987 -1000.[CrossRef][Medline]
Derijard, B., Raingeaud, J., Barrett, T., Wu, I. H., Han, J.,
Ulevitch, R. J. and Davis, R. J. (1995). Independent human
MAP-kinase signal transduction pathways defined by MEK and MKK isoforms.
Science 267,682
-685.
Espinos, E., Liu, J. H., Bader, C. R. and Bernheim, L. (2001). Efficient non-viral DNA-mediated gene transfer to human primary myoblasts using electroporation. Neuromuscul. Disord. 11,341 -349.[CrossRef][Medline]
Fischer-Lougheed, J., Liu, J. H., Espinos, E., Mordasini, D.,
Bader, C. R., Belin, D. and Bernheim, L. (2001). Human
myoblast fusion requires expression of functional inward rectifier Kir2.1
channels. J. Cell Biol.
153,677
-686.
Friday, B. B., Horsley, V. and Pavlath, G. K.
(2000). Calcineurin activity is required for the initiation of
skeletal muscle differentiation. J. Cell Biol.
149,657
-666.
Friday, B. B., Mitchell, P. O., Kegley, K. M. and Pavlath, G. K. (2003). Calcineurin initiates skeletal muscle differentiation by activating MEF2 and MyoD. Differentiation 71,217 -227.[CrossRef][Medline]
Gonzalez, I., Tripathi, G., Carter, E. J., Cobb, L. J., Salih,
D. A., Lovett, F. A., Holding, C. and Pell, J. M. (2004).
Akt2, a novel functional link between p38 mitogen-activated protein kinase and
phosphatidylinositol 3-kinase pathways in myogenesis. Mol. Cell.
Biol. 24,3607
-3622.
Graef, I. A., Mermelstein, P. G., Stankunas, K., Neilson, J. R., Deisseroth, K., Tsien, R. W. and Crabtree, G. R. (1999). L-type calcium channels and GSK-3 regulate the activity of NF-ATc4 in hippocampal neurons. Nature 401,703 -708.[CrossRef][Medline]
Han, J., Lee, J. D., Jiang, Y., Li, Z., Feng, L. and Ulevitch,
R. J. (1996). Characterization of the structure and function
of a novel MAP kinase kinase (MKK6). J. Biol. Chem.
271,2886
-2891.
Jiang, B. H., Zheng, J. Z. and Vogt, P. K.
(1998). An essential role of phosphatidylinositol 3-kinase in
myogenic differentiation. Proc. Natl. Acad. Sci. USA
95,14179
-14183.
Jones, N. C., Tyner, K. J., Nibarger, L., Stanley, H. M.,
Cornelison, D. D., Fedorov, Y. V. and Olwin, B. B. (2005).
The p38alpha/beta MAPK functions as a molecular switch to activate the
quiescent satellite cell. J. Cell Biol.
169,105
-116.
Kaliman, P., Vinals, F., Testar, X., Palacin, M. and Zorzano,
A. (1996). Phosphatidylinositol 3-kinase inhibitors block
differentiation of skeletal muscle cells. J. Biol.
Chem. 271,19146
-19151.
Kandel, E. S. and Hay, N. (1999). The regulation and activities of the multifunctional serine/threonine kinase Akt/PKB. Exp. Cell Res. 253,210 -229.[CrossRef][Medline]
Klee, C. B., Ren, H. and Wang, X. (1998).
Regulation of the calmodulinstimulated protein phosphatase, calcineurin.
J. Biol. Chem. 273,13367
-13370.
Konig, S., Hinard, V., Arnaudeau, S., Holzer, N., Potter, G.,
Bader, C. R. and Bernheim, L. (2004). Membrane
hyperpolarization triggers myogenin and myocyte enhancer factor-2 expression
during human myoblast differentiation. J. Biol. Chem.
279,28187
-28196.
Li, Y., Jiang, B., Ensign, W. Y., Vogt, P. K. and Han, J. (2000). Myogenic differentiation requires signalling through both phosphatidylinositol 3-kinase and p38 MAP kinase. Cell. Signal. 12,751 -757.[CrossRef][Medline]
Liu, J., Bijlenga, P., Fischer-Lougheed, J., Occhiodoro, T.,
Kaelin, A., Bader, C. R. and Bernheim, L. (1998). Role of an
inward rectifier K+ current and of hyperpolarization in human myoblast fusion.
J. Physiol. 510,467
-476.
Liu, J. H., Konig, S., Michel, M., Arnaudeau, S.,
Fischer-Lougheed, J., Bader, C. R. and Bernheim, L. (2003).
Acceleration of human myoblast fusion by depolarization: graded Ca2+ signals
involved. Development
130,3437
-3446.
McKinsey, T. A., Zhang, C. L. and Olson, E. N. (2002). MEF2: a calciumdependent regulator of cell division, differentiation and death. Trends Biochem. Sci. 27, 40-47.[CrossRef][Medline]
Perrier, E., Perrier, R., Richard, S. and Benitah, J. P.
(2004). Ca2+ controls functional expression of the cardiac K+
transient outward current via the calcineurin pathway. J. Biol.
Chem. 279,40634
-40639.
Rauch, C. and Loughna, P. (2005). C2C12 skeletal muscle cells exposure to phosphatidylcholine triggers IGF-1 like-responses. Cell Physiol. Biochem. 15,211 -224.[CrossRef][Medline]
Rhodes, S. J. and Konieczny, S. F. (1989).
Identification of MRF4: a new member of the muscle regulatory factor gene
family. Genes Dev. 3,2050
-2061.
Sarbassov, D. D., Guertin, D. A., Ali, S. M. and Sabatini, D.
M. (2005). Phosphorylation and regulation of Akt/PKB by the
rictor-mTOR complex. Science
307,1098
-1101.
Schiaffino, S. and Serrano, A. (2002). Calcineurin signaling and neural control of skeletal muscle fiber type and size. Trends Pharmacol. Sci. 23,569 -575.[CrossRef][Medline]
Spangenburg, E. E., Bowles, D. K. and Booth, F. W.
(2004). Insulin-like growth factor-induced transcriptional
activity of the skeletal alpha-actin gene is regulated by signaling mechanisms
linked to voltage-gated calcium channels during myoblast differentiation.
Endocrinology 145,2054
-2063.
Stiber, J. A., Tabatabaei, N., Hawkins, A. F., Hawke, T., Worley, P. F., Williams, R. S. and Rosenberg, P. (2005). Homer modulates NFAT-dependent signaling during muscle differentiation. Dev. Biol. 287,213 -224.[Medline]
Stokoe, D., Stephens, L. R., Copeland, T., Gaffney, P. R.,
Reese, C. B., Painter, G. F., Holmes, A. B., McCormick, F. and Hawkins, P.
T. (1997). Dual role of
phosphatidylinositol-3,4,5-trisphosphate in the activation of protein kinase
B. Science 277,567
-570.
Tamir, Y. and Bengal, E. (2000).
Phosphoinositide 3-kinase induces the transcriptional activity of MEF2
proteins during muscle differentiation. J. Biol. Chem.
275,34424
-34432.
Wright, W. E., Sassoon, D. A. and Lin, V. K. (1989). Myogenin, a factor regulating myogenesis, has a domain homologous to MyoD. Cell 56,607 -617.[CrossRef][Medline]
Wu, H., Naya, F. J., McKinsey, T. A., Mercer, B., Shelton, J. M., Chin, E. R., Simard, A. R., Michel, R. N., Bassel-Duby, R., Olson, E. N. et al. (2000a). MEF2 responds to multiple calcium-regulated signals in the control of skeletal muscle fiber type. EMBO J. 19,1963 -1973.[CrossRef][Medline]
Wu, Z., Woodring, P. J., Bhakta, K. S., Tamura, K., Wen, F.,
Feramisco, J. R., Karin, M., Wang, J. Y. and Puri, P. L.
(2000b). p38 and extracellular signalregulated kinases regulate
the myogenic program at multiple steps. Mol. Cell.
Biol. 20,3951
-3964.
Xu, Q., Yu, L., Liu, L., Cheung, C. F., Li, X., Yee, S. P.,
Yang, X. J. and Wu, Z. (2002). p38 Mitogen-activated protein
kinase-, calcium-calmodulin-dependent protein kinase-, and
calcineurin-mediated signaling pathways transcriptionally regulate myogenin
expression. Mol. Biol. Cell
13,1940
-1952.
Zetser, A., Gredinger, E. and Bengal, E.
(1999). p38 mitogen-activated protein kinase pathway promotes
skeletal muscle differentiation. Participation of the Mef2c transcription
factor. J. Biol. Chem.
274,5193
-5200.
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