The next step consisted mainly in answering the following question: what regulates EGFR expression in myoblasts during proliferation and differentiation?
a. Roles of EGFR ligands
Degradation of EGFR was shown to follow the binding of ligands. We thus investigated if the addition of EGFR ligands in differentiation medium resulted in receptor degradation.
Figure 50 : Myoblasts were cultured in growth medium (GM) or 24h in differentiation medium (DM) supplemented or not by EGF (10ng/ml). EGFR expression was assessed, as well as MEF2 and myogenin (one representative experiment out of three).
EGFR expression was detected in GM. EGFR was degraded in DM in presence of EGF as expected (figure 50). However, EGFR was not degraded in absence of EGF. This result confirms that EGFR degradation requires EGFR activation by its ligand, as demonstrated by others (Roepstorff et al., 2009),. Expression of MEF2 and myogenin, two markers of differentiation, was undetectable in GM but found in DM with or without EGF. Thus, the presence of EGF in DM does not affect the differentiation process as assessed by myogenin and MEF2 expression. This is likely due to the fact that EGFR is either inactive (-EGF) or
%MEF2 positive nuclei (compared to control)
* *
A B
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We then wondered whether other EGFR ligands were able to induce EGFR degradation in differentiation medium. By western blot analysis, we observed that, in differentiation medium, presence of three EGFR ligands (EGF, HB-EGF and TGFα) resulted in EGFR degradation (figure 51). Another growth factor, aFGF, was tested as Lim et al. showed that decline of EGFR followed FGF removal (Lim and Hauschka, 1984a). This growth factor cannot bind EGFR and thus, had no effect on the receptor expression.
→ This result confirms that EGFR degradation relies the activation of EGFR by its ligands.
Figure 51 : expression of EGFR was assessed after 24h in differentiation medium condition supplemented or not with growth factors. EGFR ligands (EGF, HB-EGF and TGFα) decreased EGFR expression while aFGF had no effect (one representative experiment out of three performed).
Growth medium (GM) contains EGF and, possibly other EGFR ligands from the fetal bovine serum. However, we observed that EGFR was detectable when myoblasts are cultured in GM (figures 36 and 51). We propose that in proliferation conditions, EGFR is preferentially recycled to the membrane, while during differentiation, EGFR activation by ligands leads to its degradation.
The effects on differentiation of the three EGFR ligands (EGF, HB-EGF and TGFα) were assessed by MEF2 immunostaining. Confirming the results observed in figure 50, EGF did not significantly affect MEF2 expression when added to differentiation medium (figure 52).
However, addition of HB-EGF or TGFα significantly decreased by approximately 30% MEF2
- + EGF + aFGF + HB-EGF + TGFα
EGFR
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expression after 24h (figure 52). HB-EGF and TGFα, after binding to EGFR, were shown to preferentially induce the recycling of the receptor to the membrane whereas EGF preferentially leads to its degradation (Roepstorff et al., 2009). These differences in ligand action on the receptor may explain the decrease in MEF2 expression after 24h in differentiation. However, the recycling phase is probably only transitory as after 24h, EGFR is no more detected and the differentiation process has started (figures 35 and 36).
Figure 52: Myoblasts were cultured in EGFR-ligand free differentiation medium (control). EGF, HB-EGF or TGFα were added to the medium. After 24h, myoblasts were fixed, immunostained for MEF2 and comparison of MEF2 expression was assessed by the ratio of nuclei positive for MEF2 by the total number of nuclei. Result obtained for the control was normalized to 100% (n>5; * p<0.05).
EGF
DM
EGFR GM
- +
MEF2
Myogenin
α tubulin 0
20 40 60 80 100 120
- EGF HB-EGF TGFa
%MEF2 positive nuclei (compared to control)
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b. Effect of calcium on EGFR expression
Figure 53 : Myoblasts were cultured in growth medium (GM) or differentiation medium (DM). Addition of a calcium chelator, BAPTA-AM (15µM) for 24h in GM, 3h or 24h in DM, was used to chelate intra-cellular calcium. EGFR expression was assessed by western blot (n=3).
Darbellay et al. showed that calcium signaling is important at very early stages of differentiation and affects myoblast hyperpolarization (Darbellay et al., 2009). Moreover, we previously showed that calcium signaling is involved in different steps of myoblast differentiation (Arnaudeau et al., 2006). We thus wondered whether EGFR expression could be modulated by cytosolic calcium. Myoblasts were cultured in GM or DM (3h or 24h) with or without BAPTA-AM, a calcium chelator. As shown in figure 53, no change in EGFR expression pattern could be observed after addition of BAPTA-AM at these time points.
→ Although calcium is a major actor in human myoblast differentiation, it is unlikely that calcium is involved in EGFR degradation.
EGFR
α-tubulin
Bapta-AM
- + - + - +
GM DM3h DM24h
BAPTA-AM
80 c. Degradation of EGFR
Our previous results lead us to the hypothesis that EGFR is actively degraded at the onset of myoblast differentiation. Degradation of EGFR was shown to occur via either the lysosomal compartment or via the proteasome-ubiquitin system (see “introduction” section). We studied both systems in human myoblasts.
o Lysosomal compartment
The degradation of EGFR in the lysosomal compartment has been tremendously studied as a model of receptor degradation (see “introduction” section). We thus assessed the role of lysosomal system in EGFR expression during proliferation and differentiation of myoblasts. Three different lysosomal inhibitors were tested: chloroquine, leupeptin and NH4Cl.
In proliferation condition, due to important variations in the results, only leupeptin significantly increased EGFR expression (figure 54A and quantification in 54C). However in differentiation condition, lysosomal drugs did not exhibit remarkable effects on EGFR (figure 54B and quantification in 54C). More precisely, only the treatment with chloroquine resulted in a significantly higher EGFR expression compared to DM (p=0.006). Moreover, addition of lysosomal inhibitors to DM did not inhibit differentiation assessed by MEF2 and myogenin expression (figure 54B).
→ Finally, the huge variations observed between experiments did not permit to raise definitive conclusions on the role of the lysosomal degradation of EGFR in myoblasts.
Further experiments are needed in order to define its role. However, these preliminary data do not point-toward a major role of lysosomal degradation of EGFR at the onset of myoblasts differentiation.
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Figure 54: Western blots of the effects of 24h-treatment with lysosomal inhibitors (chloroquine 10µM;
leupeptin 5µg/ml; NH4Cl 10mM) on EGFR expression in GM (A) and EGFR, MEF2, myogenin expression in DM (B). (C) Quantification of the immunoblots (* p<0.05 comparison to GM; # p<0.05 comparison to DM;
2<n<4).
0.00 0.50 1.00 1.50 2.00 2.50
EGFR expression (AU)
EGFR
α-Tubulin GM
α-Tubulin Myogenin MEF2 EGFR DM
A
C
B
* * *
*
# #
#
#
82 o Proteasome
To evaluate the impact of the proteasome-ubiquitin system on EGFR expression during myoblasts differentiation, inhibitors of proteasome were used. The most common inhibitor of the proteasome is MG132.
Potential effects of MG132 were investigated on proliferating (GM) and differentiating (DM) myoblasts. Western blot revealed that in GM, EGFR was present and its expression was unchanged by addition of MG132 (figure 55A). However, in DM, treatment with MG132 maintained EGFR expression. Efficacy of MG132 treatment was confirmed by the accumulation of ubiquitinylated proteins in both GM and DM conditions (figure 55B). Then, we investigated whether proteasome inhibition in DM had an effect on the differentiation process.
In DM+MG132 where EGFR is maintained, differentiation was inhibited (assessed by MEF2 and myogenin expression) while these markers of differentiation were not detected in GM±MG132. Immunostaining of myoblasts confirmed the effect of MG132 addition on MEF2 expression in differentiation condition (figure 55C/D). In proliferation condition, the number of MEF2 positive cells increased but, even if this result was significant, the proportion of MEF2 positive myoblast remained very low (≈3%) (figure 55C/D). This effect was hardly detectable by western blot (figure 55A). Moreover, Ki67 immunostaining showed that MG132 treatment severely reduced proliferation in GM while it increased it in DM (from 2% to 12% of Ki67 positive cells) (figure 55C/E).
→ Inhibition of the proteasome with MG132 maintains EGFR in differentiation condition.
MG132 also inhibits the differentiation process in myoblast.
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Figure 55 : myoblasts were cultured either in growth medium (GM) or differentiation medium (DM), and treated or not by the proteasome inhibitor MG132 (2µM) for 24h. A, EGFR expression was assessed as well as MEF2 and myogenin as markers of differentiation. B, immunoblot for ubiquitin. C, immunostaining of myoblasts in GM or DM, with or without treatment by MG132 (2µM for 24h). Differentiation was assessed by immunostaining for MEF2 (green) and proliferation by Ki67 (red). Nuclei were stained with DAPI (blue). D and E, quantification of C (n≥3; * p<0.05).
0
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Confirmation of the effects of MG132 was obtained using two other proteasomal drugs:
epoxomycin and lactacystin. Effect on ubiquitinylated protein accumulation and EGFR maintenance in DM was identical to MG132 (figure 56A).
Figure 56 : (A) effects of the treatment by epoxomycin (10µM) and lactacystin (10µM) in GM or DM, on the expression of ubiquitin, EGFR, MEF2 and myogenin by western blot. Quantification of the percentage of myoblasts immunostained positive for MEF2 (B) or Ki67 (C) expression after treatment by epoxomycin or lactacystin in proliferation (GM) and differentiation (DM) conditions. (n=1)
0.0
85
Only a small reduction of myogenin or MEF2 expression was observed by western blot.
Immunostaining analysis also revealed the same effects as MG132 (figure 56B/C); in differentiation condition, percentage of MEF2 positive cells was reduced while Ki67 positive cells number increased. The effect on Ki67 expression in proliferation condition was small, likely due to the very low ratio obtained in control condition (figure 56C).
→ In summary, preliminary results suggest that the proteasome system is indeed involved in the early differentiation of myoblasts and is responsible, at least in part, for the degradation of EGFR during differentiation.
86 d. Variations of the cellular model
o Rhabdomyosarcoma RD cells
Rhabdomyosarcoma are soft tissue tumor arising from skeletal muscle satellite cells.
Recently, EGFR was shown to be over-expressed in these tumors and proposed as a marker of rhabdomyosarcoma. RD cells are a cell line derived from a human rhabdomyosarcoma. We decided to characterize this cell line by comparing it to our primary myoblasts. RD cells were cultured in the proliferation medium or differentiation medium used for human primary myoblasts. After 24h, cells were either fixed for immunostaining analysis (figure 57) or lysed for western blots analysis (figure 58).
Figure 57 : RD cells were fixed after a day in GM or DM and immunostained for different markers (ki67, MEF2, MEF2C, myogenin). DAPI was used for nuclei staining.
MEF2A DAPI
Myogenin merge
DMGM
Ki67 MEF2C DAPI
merge
DMGM
A
B
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Surprisingly, the two techniques revealed the presence in proliferating conditions of muscle transcription factors characteristic of differentiated myoblasts such as MEF2 (in particular MEF2C) and myogenin. In addition, these markers were up-regulated in DM. EGFR expression was also detected in RD cells but was unaffected when cultured in differentiation conditions (figure 57). Moreover, ErbB2 expression was also detected in RD cells while its expression was under detection level in primary myoblasts. Ki67, a marker of proliferation, was expressed at a higher level in differentiation condition (figure 57). As Ki67 was previously shown by others to be actively degraded via the proteasome (Wu et al., 2000), we do not know whether if it is less degraded or highly expressed in DM in these cells.
Figure 58 : RD cells were cultured in GM or DM (24h). Cells extracts were used for western-blot analysis.
EGFR and erbB2 were expressed equally in proliferation and differentiation condition. Myogenin and MEF2 expression increased in differentiation. α-tubulin was used as loading control.
→ Taken together, these data suggest a dysfunction of protein expression or degradation systems, possibly the proteasome.
EGFR
MEF2 ErbB2
Myogenin
α-tubulin
GM DM
88 o Reserve cells
Reserve cells are myoblasts that stay mononucleated and undifferentiated in DM. They represent ≈20% of the nuclei in differentiation condition. Surprisingly, while observing the membrane expression of EGFR during differentiation, it appeared that although punctae of EGFR were absent from myotubes (MT) membranes, the punctae remained present on most of the reserve cells (RC). Figure 59 shows this restricted pattern of expression.
Figure 59 : Myoblasts were maintained in differentiation medium for two days. Nuclei were stained with DAPI (blue) while extra-cellular EGFR was immunostained (red). Groups of nuclei were observed, corresponding to myotubes (MT). The contours of two reserve cells (RC) expressing EGFR are outlined by a dotted line.
We thus decided to compare expression of various proteins in reserve cells and in myotubes. Myotubes were harvested, after three days in differentiation conditions, by washing petri dish several times with PBS. After the treatment, the only cells that remained attached were the reserve cells. These were collected after trypsin incubation. As shown in figure 60,
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only myotubes expressed markers of differentiation such as MEF2, myogenin, troponin T.
Moreover, they over-express STIM1 and STIM2 that were shown to be up-regulated during differentiation (Darbellay et al., 2010;Darbellay et al., 2009). Even the newly identified long form of STIM1 (STIM1L), that was shown to appear during the late steps of myoblast differentiation, was only found in myotubes extracts (Darbellay et al., 2011). In contrast, EGFR was clearly detected in reserve cells but not in myotubes, in accordance to what was seen on immunostaining. Consistently with the absence of markers of differentiation in reserve cells, using electrophysiology technique (patch-clamp), I could not detect active Kir2.1 in reserve cells (n=9; data not shown).
Figure 60 : after three days in differentiation medium, reserve cells were mechanically separated from myotubes. The two populations were then tested by western blot for the expression of MEF2, myogenin, troponin, STIM1, STIM2. α-tubulin was used as loading control (one representative experiment out of two).
EGFR
MEF2
Myogenin
Troponin
STIM1S
STIM2
α-tubulin
Reserve cells Myotubes
STIM1L
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DISCUSSION
Myogenesis is a complex succession of molecular events. After birth, quiescent satellite cells are activated after an injury. In their niche, they receive signals of proliferation. After few days, the resulting pool of proliferating myoblasts undergoes myogenic differentiation. One of the earliest event is the cell membrane hyperpolarization. This hyperpolarization is triggered by the activation of Kir2.1 channels via their de-phosphorylation on tyrosine 242. The drop of membrane potential (to -70mV) generates an increase in driving force for calcium ions.
Subsequent intracellular calcium signalings activate the expression of muscle specific transcription factors (MRFs and MEF2s), which will then turn on the expression of muscle specific proteins.
This work presented the role of a receptor tyrosine kinase, the epidermal growth factor receptor (EGFR), during myogenesis. As previously described in the introduction part, EGFR belongs to a family of four members (EGFR, ErbB2, ErbB3, and ErbB4). Following ligand binding, EGFR auto-phosphorylates and mediates cellular events (such as proliferation, survival, differentiation, migration) until it is degraded. Deregulation of EGFR expression or activation was found in many cancers. In skeletal muscle, its specific role was so far unclear.
This work shows, using human primary myoblasts, that EGFR is down-regulated during differentiation. This mechanism results in cell proliferation arrest and induction of differentiation. More precisely, the knock-down of EGFR by siRNA induces activation of Kir2.1 channels, increases store-operated calcium entry, expression of muscle specific proteins, and myoblasts fusion. Investigation of the regulation of EGFR expression in myoblasts revealed that, during differentiation, EGFR is actively degraded.
91 A. ErbBs and skeletal muscle
My work reveals the physiological down-regulation of the EGF receptor at the onset of human myoblast differentiation. This result is in accordance with the observation made by the group of Hauschka of the disappearance of both EGFR and FGFR in a myoblast mouse cell line of myoblasts (Lim and Hauschka, 1984b;Lim and Hauschka, 1984a). However, in other studies expression of EGFR was observed on mature myofibers where its role could not be related to myogenesis. Recent work provided evidences, although in different models than skeletal muscle, that EGFR can be linked to glucose metabolism. In cancer cells, Weihua et al. (2008) showed that EGFR could stabilize the expression, at the membrane level, of glucose transporter SGLT1 (Weihua et al., 2008;Engelman and Cantley, 2008). The EGFR/SGLT1 interaction, independently from EGFR kinase activity, results in a survival signal for cancer cells by maintaining their glucose level high enough. In mice, addition of EGFR inhibitor to high-fat diet improves the glucose tolerance and insulin action (Prada et al., 2009). However, it is important to note that these results were observed on the whole metabolism of rodents and cannot be directly related to an effect on skeletal muscles. Fukatsu et al. (2009) focused on mouse skeletal muscle and showed an increase in HB-EGF (an EGFR ligand) expression after exercise (Fukatsu et al., 2009). Thus, they generated transgenic mice with skeletal muscle specific overexpression of HB-EGF. These mice presented "increases in glucose tolerance, insulin sensitivity, and glucose uptake by skeletal muscle". The authors proposed that HB-EGF acts as an insulin sensitizer during exercise. Withal, if HB-EGF is a ligand of EGFR, it is important to note that it can also bind erbB3.
EGFR expression is down-regulated during myoblast differentiation. Its presence was however detected on mature skeletal muscle fibers. One hypothesis would be a role of EGFR on glucose metabolism.
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As shown in figure 34, erbB2 was poorly activated during proliferation or during the first 24 hours of differentiation of human myoblast. On the other hand, erbB3 activity increased during early differentiation. Finally, erbB4 activity was even below the detection level. If our work did not reveal a role for these receptors, it is likely a question of study model. Indeed, the other erbB members (erbB2/erbB3/erbB4) were shown to contribute to late steps of skeletal muscle organization. Golding et al. investigated their expression which is not detected on quiescent cells, such as for EGFR (Golding et al., 2007): erbB2 expression starts during differentiation and is high on myotube membranes where it acts mainly as a survival factor (Andrechek et al., 2002;Golding et al., 2007). Zhu et al. showed that erbB3 and erbB4 are found exclusively in myotubes (Zhu et al., 1995). ErbB3 combined with neuregulins was shown to have a differentiation-promoting activity which is, however, unnecessary to the establishment of post-mitotic phenotype as concluded by Kim et al. (Kim et al., 1999).
Expression of erbB2/B3/B4 was detected in adult human skeletal muscles (Lebrasseur et al., 2003;Srinivasan et al., 1998). After exercise, their phosphorylation increase, so does erbB3 expression, revealing a regulation by the contractile activity and an adaptation to training (Lebrasseur et al., 2003;Lebrasseur et al., 2005). Nevertheless, the most important role for erbB2/B3/B4 and neuregulins known so far, in skeletal muscle, is their participation to the establishment of the neuromuscular junction (Zhu et al., 1995;Moscoso et al., 1995;Rimer et al., 1998;Golding et al., 2007). Neuromuscular junction is associated to differentiated fibers in vicinity to neurons that cannot be observed in our in vitro model of human myoblasts.
ErbB2, erbB3 and erbB4 were not studied in this work. Their activity is rather linked to late steps of muscular differentiation and, in particular, to the establishment of the neuro-muscular junction.
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B. EGFR in human myoblasts: stimulator of proliferation and inhibitor of differentiation (Kir2.1, calcium, ligands)
EGFR is a potent mitogen and as expected, I could show that EGFR is involved in myoblast proliferation. Figure 40 shows that EGFR stimulation by ligands (such as EGF) is required for myoblasts to proliferate. However, the combination EGFR and ligands is not sufficient for proliferation to occur in a medium without any other growth factor (except insulin).
As shown in figure 48, in differentiation conditions (low mitogen containing medium), maintenance of EGFR by addition of vitamin K3, in presence of EGF, did not increase the number of myoblasts. Moreover, “reserve cells”, the quiescent myoblasts observed in differentiation conditions present membranous expression of EGFR but do not proliferate even when exposed to EGF (personal observation). Thus, it appears that another growth factor is necessary for myoblast growth. Still it is difficult to postulate which receptor could be involved.
As shown in figure 33, the only RTKs whose activity is modulated during myoblast differentiation are the insulin receptor (IR) and insulin-like growth factor 1 receptor (IGF1R).
More precisely, IGF1R presents a similar timing pattern of activity of EGFR (high during proliferation and reduced during differentiation). In C2 cells, IGF1 was shown to stimulate myoblast proliferation (Foulstone et al., 2001). Interestingly, Napier et al. defined that IGF1 was not sufficient on its own to stimulate proliferation of rat L6 cells, such as EGFR on human myoblasts (Napier et al., 1999). Riedemann et al. showed in a variety of cellular models (human carcinoma) that EGFR could directly interact with IGF1R (Riedemann et al., 2007).
The consequence of this interaction is the stabilization of IGF1R which is protected from proteasomal degradation. Thus, in myoblasts, the decrease of expression of EGFR could
The consequence of this interaction is the stabilization of IGF1R which is protected from proteasomal degradation. Thus, in myoblasts, the decrease of expression of EGFR could