RTK ASSAY - Epidermal growth factor receptor (EGFR)expression and role during human myoblast di

Receptor tyrosine kinase (RTK) assay was performed in order to screen the activity, i.e. the phosphorylation state, of 43 membrane receptors (see appendix for the list of receptors). This assay is based on the ELISA technique. Receptors present in cellular extracts bound their cognate antibodies attached to the assay membrane at specific locations (figure 32). Unbound proteins are then washed away. A pan anti-phospho-tyrosine antibody is then used for the detection of phosphorylated tyrosine residues.

Figure 32 : principle of the RTK assay

o Experimental procedure

Screening of the activated receptor tyrosine kinases was performed with the Human Phospho-RTK array (R&Dsystems) following the manufacturer’s instructions. Briefly, array membranes presenting the capture antibodies were put in a blocking buffer. Then, they were incubated overnight with the cell lysates (≈ 500µg proteins). After several washes, arrays were incubated with the detection antibody (pan tyrosine antibody conjugated with horseradish peroxidase). Afterwards, membranes were treated with chemiluminescent reagents and exposed to X-ray films for 5 minutes (see the western-blot section for details). Pixel density was analyzed with the image analysis software Optiquant®.

Secondary antibody (pan-tyrosine) Cellular extract

Negative control Receptor specific antibodies Membrane

57 H. PROLIFERATION ASSAY

o Experimental procedure

Proliferation was assessed with the CellTiter 96 Aqueous one solution cell proliferation assay from Promega. Cells were cultured in 96-well plates. At different time-points, 20µl of reagent were added to each well and absorbances (490nm) were read after 1h incubation.

I. STATISTICAL ANALYSIS

Results are expressed as the mean ±s.e.m. Statistical analyses were performed using Student’s t-test (asterisk in figures indicates P<0.05).

RESULTS

A. PART 1: the role of EGFR during human myoblast differentiation

a. EGFR expression is down-regulated at the onset of human myoblast differentiation

Kir2.1 channels are regulated by a tyrosine phosphorylation, maintaining them inactive in proliferative myoblasts. We thus looked for a receptor tyrosine kinase (RTK) that could be active in proliferating conditions and inactive in differentiation condition. Figure 33 shows the activity of various RTK in proliferation and differentiation conditions.

Figure 33: Receptor Tyrosine Kinases (RTKs) were tested for their activities in proliferation (GM) and differentiation condition (DM 24h). Panel A: Immunoblots of the membranes of the RTK profiler array. Each pair of spots corresponds to a phospho-RTK. Panel B: List of the RTKs tested.

EGF R FGF R4 MSP R ROR1 VEGF R1 EphA4 ErbB2 Insulin R PDGF Rα ROR2 VEGF R2 EphA6 ErbB3 IGF1 R PDGF Rβ Tie-1 VEGF R3 EphA7

ErbB4 Axl SCF R Tie-2 MuSK EphB1

FGF R1 Dtk Flt-3 TrkA EphA1 EphB2

FGF R2α Mer M-CSF R TrkB EphA2 EphB4 FGF R3 HGF R c-Ret TrkC EphA3 EphB6

A

B

The level of activity (corresponding to the phosphorylation state) of 43 RTKs both in proliferation (GM) and after 24hours in differentiation (DM24h) media was assessed using a RTK proteome profiler array based on the ELISA technique (figure 33).

As expected, insulin receptor (IR) and insulin-like growth factor receptor (IGFR) activities were regulated between proliferation and differentiation (Coolican et al., 1997). More precisely, while IR activity decreases, IGF1R activity increases between proliferation and differentiation conditions. Intriguingly, the fibroblast growth factor receptors (FGFR), which were implicated in the inhibition of rodent myoblast differentiation (Olwin and Hauschka, 1988), did not exhibit any activity in human primary myoblasts either in proliferating or in differentiating condition. However, among all the receptors tested, the epidermal growth factor receptor (EGFR) appeared as the most active in proliferation.

Figure 34: Cells were cultured in either growth medium (GM) or differentiation medium for 24 h (DM24h).

Phospho-RTK arrays were hybridized with 500µg proteins of whole cell lysates and detected with an anti-phosphotyrosine antibody. Quantification of each spot was performed and expressed in arbitrary units (AU). The results for the ErbB receptors are shown (n=2). Top of the figure corresponds to a zoom in figure 33.

Figure 34 illustrates that phosphorylation of EGFR was intense in GM and decreased by ≈62% after 24h in differentiation. In comparison, ErbB2 activity was poorly modified between

A

GM and DM, and ErbB3 activity tends to increase during differentiation. We then investigated whether the variation of EGFR activity was due to a difference in the expression of the receptor.

Presence of the receptor at the plasma membrane was assessed by immuno-fluorescence in situ on non-permeabilized cells (figure 35). The EGF receptor appeared as punctae widely distributed on the cell membrane. These membrane punctae of EGFR were quantified.

Figure 35: A,EGFR expression at the plasma membrane (extracellular epitope) was detected by immuno-fluorescence in situ (red), and nuclei are stained with DAPI (blue). B, punctae of EGFR were quantified at different time in differentiation condition. For each time point, the number of punctae was divided by the number of nuclei, and normalized to the control (time 0) (n=5).

Results were normalized to the expression in proliferating conditions (100%). A decrease of 50.7% of the membrane punctae was detected after 6h in differentiation medium, reaching 90.3% after 2 days.

To confirm these results, western blots were performed with whole cell lysates (figure 36) and showed that, in well differentiated myoblasts, i.e. expressing both myogenin and MEF2

DM 24h

A

0.0 20.0 40.0 60.0 80.0 100.0

0 1 3 6 24 48

Granules of EGFR / nuclei (%control)

Hours in differentiation medium (DM)

B

(DM24h), there was nearly no more EGFR protein detectable whereas it was well expressed in proliferating myoblasts.

Figure 36: EGFR expression was confirmed by western blot (whole cell lysates). Myogenin and MEF2 expressions were used as differentiation markers and α-tubulin as loading control.

→ Taken together these results show that EGFR is downregulated during myoblast differentiation.

EGFR Myogenin

MEF2

α-Tubulin DM 24h

b. Compared efficiency of three different siRNAs against EGFR

Because EGFR expression decreased during early differentiation, we decided to use silencing RNA (siRNA) strategy to mimic this down-regulation. For this purpose, three siRNAs were selected (siEGFR from Qiagen: #5, #10 and #12). Efficacy of silencing was assessed by western blot (figure 37A). EGFR expression was detected in control and to a lesser extend when siEGFR #5 was used. With siEGFR #10 and #12, EGFR expression was below detection level. EGFR expression was also assessed by FACS (fluorescence-activated cell sorter), confirming the previous results (figure 37B). As shown in figure 37B, siEGFR #10 and #12 reduced the membrane expression of EGFR of ≈80% while #5 had little effect (≈15%

reduction). Thus, except when mentioned, all results presented thereafter were performed with siEGFR #10 and confirmed by siEGFR #12.

Figure 37 : Myoblasts were transfected with a control siRNA or with different siRNAs against EGFR (#5,

#10, and #12 from Qiagen). One day after transfection, myoblasts were either (A) lysed to assess EGFR expression by western blot analysis (anti-EGFR: 15F8 from Cell Signaling); or (B) analyzed by flow cytometry to measure the membrane expression of EGFR without permeabilization (anti-EGFR: GR01 from Calbiochem). Results were normalized to control condition (n=1).

EGFR

control #5 #10 #12

0 20 40 60 80 100

% of positivecells

control #5 #10 #12 B

To confirm these results, EGFR expression at the plasma membrane was assessed with an antibody recognizing an extracellular epitope on non-permeabilized cells (figure 38).

Figure 38: Myoblasts were transfected either with a control siRNA or with a siEGFR (#10), and then maintained in proliferation condition for 2 days. EGFR expression at the membrane was determined by immuno-fluorescence in situ (EGFR extracellular epitope in red; nuclei in blue)

→ These results show that two out of three siRNAs (#10 and #12) reduce EGFR expression

Control siEGFR

c. Silencing EGFR inhibits myoblast proliferation

When EGFR was silenced in myoblasts cultured in GM, we observed a strong inhibition of myoblast proliferation. As EGFR is a known mitogen, this effect was expected. We nevertheless quantified the proliferation rate of human primary myoblasts after EGFR silencing. Figure 39 shows that siEGFR clearly reduced the proliferation rate of myoblasts. Six days after transfection, EGFR-silenced cells only multiplied by 2.7 whereas control myoblasts by 8.

Figure 39 : Myoblasts were transfected either with siControl or siEGFR, and then maintained in proliferation conditions for up to 6 days. Proliferation was assessed by using a proliferation assay based on absorbance measurements (CellTiter from Promega) (n=3; * p<0.05).

0 1 2 3 4 5 6 7 8 9 10

0 1 2 3 4 5 6 7

proliferation rate (fold increase)

time (days) control

siEGFR

* *

*

We then evaluated EGFR activity on myoblast proliferation by increasing EGF, one of the major ligand for EGFR (Wells, 1999) in the culture medium. Our proliferation medium includes 15% fetal calf serum that contains an unknown amount of EGF to which we added 10 ng/ml of EGF (Baroffio et al., 1996). As shown in figure 40, more than 40% of myoblasts treated with a medium supplemented with 10ng/ml of EGF were positive for a nuclear proliferation marker, Ki67. The percentage of Ki67 positive myoblasts felt to 10% when myoblasts were maintained in a medium not supplemented with EGF. These results confirm that EGFR activity is essential for myoblast proliferation. In the remaining experiments, the proliferation medium was supplemented with 10 ng/ml EGF.

Figure 40 : myoblast proliferation relies on the presence of EGF in the culture medium. Proliferation was assessed using Ki67 (red), a nuclear marker of proliferation (n=3; p<0.05).

→ These results confirm that proliferation of human primary myoblast relies on EGFR activity.

-EGF

mergeKi67DAPI

0 10 20 30 40 50

- EGF + EGF

% Ki67 positive cells

*

+EGF

d. EGFR silencing induces Kir2.1 activation.

As the initial aim of the study was to identify a tyrosine kinase implicated in Kir2.1 channel inactivation, we tested whether siEGFR transfection was able to increase kir2.1 channel activity. Kir2.1 channel activation was previously defined as one of the earliest events of myoblast differentiation (Konig et al., 2004;Hinard et al., 2008). Figure 41 shows that downregulation of EGFR is associated with a premature Kir2.1 channel activation in myoblasts maintained in proliferation conditions.

Figure 41:Myoblasts were transfected, either with siControl or with siEGFR. A, percentages of myoblasts (control or siEGFR) with functional Kir2.1 channels; B, 48 hours post-transfection whole cell current densities of the total population of myoblasts (including myoblasts with no current, i.e. <5 pA). C current-voltage relationships of a myoblast transfected with siEGFR. Voltage-steps were to -40, -60, -80, -100, -120 and -140 mV from a holding potential at -60 mV. The inset shows a control myoblast with no Kir2.1 current, a typical Kir2.1 current recorded from a myoblast, 48h after transfection with siEGFR. Addition of 500µM Barium inhibited this current.

-80

myoblasts with functional Kir2.1 channels (%)

*

As previously demonstrated, in control condition, only 9% of proliferating myoblasts exhibited Kir2.1 activity and, in these cells, Kir2.1 current density was low (Kir2.1 measured at -120mV: -0.3±0.27 pA/pF, n=11; figure 41). Interestingly, while still in proliferation conditions, 64% of siEGFR-transfected myoblasts exhibited typical large Kir2.1 currents with current densities comparable to those obtained with myoblasts induced to differentiate for 24 hours (measured at -120mV: -2.2pA/pF±0.5; n=18; figure 41) (Fischer-Lougheed et al., 2001). As expected, addition of 500µM barium completely blocked this current, and the current-to-voltage relationships exhibited the classic inward rectification of Kir currents (figure 41).

Kir2.1 channel activation is responsible for a myoblast hyperpolarization induced in the early steps of differentiation (Liu et al., 1998b;Konig et al., 2004). Hyperpolarization increases the driving force for calcium (Arnaudeau et al., 2006;Darbellay et al., 2009). We might thus expect an increased SOCE in hyperpolarized cells. Accordingly, in EGFR-silenced proliferating myoblasts (expressing Kir2.1 channel and thus hyperpolarized at -70 mV), a larger store-operated calcium-entry (SOCE) should be observed.

Figure 42: A, cytoplasmic Ca²⁺ was assessed with Fura-2 on proliferating myoblasts, 2 days after siRNA transfection. Intracellular Ca²⁺ stores were depleted with 10µM Tg in a medium containing 250nM Ca²⁺, and 1.8mM Ca²⁺ was subsequently added to reveal SOCE. The first part of the experiment was performed with a medium containing 30mM KCl in order to clamp cells at ≈-40mV. The second part was performed with a medium containing 5mM KCl allowing cells to hyperpolarize. B, quantification of peak SOCE (n=6; * p<0.05).

0.3

In figure 42, SOCE was induced by a thapsigargin application to deplete calcium stores, and assessed (using Fura-2AM) during re-addition of extracellular calcium (1.8 mM). In our protocol, calcium re-addition was performed twice. The first calcium re-addition (arrow A) was made in presence of 30 mM external potassium. This high potassium concentration was chosen as it precludes the hyperpolarization to -70mV and, according to the Nernst equation, maintains the resting potential around -40 mV. It can be seem that, in myoblasts not able to hyperpolarize to -70mV, SOCE was not affected by EGFR silencing. On the other hand, when calcium was re-added (arrow B) in presence of a potassium concentration (5 mM) allowing the hyperpolarization to -70mV, SOCE was clearly larger in EGFR-silenced myoblasts. This result suggests that, in myoblasts silencing EGFR triggers a hyperpolarization that can increase calcium entry.

→ In proliferating conditions, myoblasts silenced for EGFR exhibit an active Kir2.1 current and an increased hyperpolarization-related SOCE.

e. Myoblast differentiation induced in GM by EGFR knockdown

As a Kir2.1-induced hyperpolarization is essential for the initiation of myoblast differentiation (Konig et al., 2004), we investigated in proliferating myoblasts the effect of EGFR silencing on the expression of myogenin and MEF2, two well-known myogenic early markers, and MyHC, a muscle specific protein. As shown in figure 43A, proliferating myoblasts silenced for EGFR clearly expressed these three markers of differentiation. Expression of these proteins after EGFR knockdown was confirmed by immuno-fluorescence (figure 43B and quantification in figure 43C). MEF2 expression increased 5.7 fold, myogenin 4.8 fold and MyHC 15 fold after EGFR silencing in myoblasts. It is important to note that in myoblasts transfected with siEGFR, MyHC staining revealed the presence of multinuclear myotubes demonstrating that EGFR knockdown induced myoblast differentiation in proliferation medium (figure 43B, white arrows).

Addition of 10mM cesium, a known inhibitor of potassium channels that we previously used to inhibit myoblast hyperpolarization to -70 mV, counteracted the effect of EGFR knockdown on differentiation (Konig et al., 2004).

Figure 43: Myoblast differentiation is induced by EGFR knockdown. Proliferating myoblasts were transfected either with siControl or with siEGFR, and then maintained in proliferation conditions for 5 days.

A, total cell lysates were analyzed by western-blot. Myogenin and MEF2 expressions were used as early markers of differentiation, Myosin heavy chain (MyHC) as a late marker of differentiation, and α-tubulin as loading control. B, differentiation of myoblasts was observed by the staining of MEF2 (red) and MyHC (green). Nuclei are stained by DAPI (blue). Cesium (Cs 10mM) was added to the medium to block the hyperpolarization. Small myotubes resulting from myoblast fusion were observed (white arrows). C, quantification of the immuno-fluorescence shown in B (n>5).

These results were obtained 5 days post siEGFR transfection, while figure 41 shows that Kir2.1 channels are activated within 24 to 48 hours post transfection. We thus assessed the expression of the early marker MEF2, 48 hours after siEGFR transfection. Figure 44 shows that, 48 hours post transfection, MEF2 expression (8% of EGFR- silenced myoblasts) was far below Kir2.1 expression (64% of EGFR-silenced myoblasts; figure 41).

Figure 44 : Two days after transfection, myoblasts (siControl and siEGFR) in proliferation condition, were fixed and stained for MEF2 (red) and nuclei with DAPI (blue). Results were quantified as percentage of MEF2 positive cells (right panel).

Thus, this result proposes that Kir2.1 activation precedes the induction of myogenic transcription factors.

0 5 10 15 20 25 30

control siEGFR

%MEF2 positive cells

Control siEGFR

DAPIMEF2

The progressive expression of MEF2 and MyHC after EGFR silencing is illustrated in figure 45. After EGFR knockdown, expression of MEF2 and MyHC in GM was observed at different time points: 24h, 48h, 72h and 144h (figure 45).

Figure 45: Myoblasts were transfected either with siControl or siEGFR, and maintained in proliferation medium (GM). Cells were then fixed at different time points (24h, 48h 72h and 144h) and differentiation was assessed by the immunostaining of MEF2 (red) and myosin heavy chain (green). Nuclei were stained with DAPI (blue).

In control proliferation conditions, myoblasts expressing MEF2 and/or MyHC remained very rare and their expression did not increase over time. However, when transfected with siEGFR, the number of myoblasts expressing both MEF2 and MyHC progressively increased.

→ This result suggests that expression of muscle specific proteins is induced after the activation of Kir2.1 channels, by EGFR silencing.

24h 48h 72h 144h

control

siEGFR

f. EGFR silencing accelerates differentiation

From our previous results, we hypothesized that silencing EGFR in differentiating myoblasts should accelerate the differentiation process. Figure 46 shows that, indeed, EGFR knockdown increased MEF2 expression after 24h of differentiation (23% of EGFR-silenced myoblasts versus 6% of control myoblasts; p=0.02; n=3).

Figure 46: Myoblasts were transfected with either siEGFR or siControl, and induced to differentiate (DM) for one day. Differentiation was assessed by MEF2 quantification(n=4; p<0.05).

g. Presence of EGFR inhibits myoblast differentiation

The next step was to assess the effects of EGFR over-expression. According to our model, over-expression of EGFR should inhibit or, at least, slow down differentiation of myoblasts in DM. Over-expression of EGFR-WT (wild type), EGFR-GFP, and EGFR-mRFP were evaluated.

Transfection was performed by electroporation (Amaxa system). Unfortunately, we did not manage to force the expression of EGFR in human myoblasts (figure 47). Not only the percentage of GFP positive cells was extremely low using the EGFR-GFP construct (8±4% vs.

87±4% for the control EGFP) but the few positive cells expressed low amount of EGFR-GFP

Control siEGFR

MEF2 DAPI

0 5 10 15 20 25 30

control siEGFR

% MEF2 positive nuclei

*

(illustrated by the fluorescence intensity; figure 47C). On the other hand, in HEK cells, EGFR transfection was easy and efficient. We thus propose that EGFR is tightly regulated at the protein level in myoblasts, and that a mechanism inhibiting uncontrolled and potentially oncogenic expression of EGFR may exist.

Figure 47: Myoblasts were transfected with a plasmid encoding the fusion protein EGFR-EGFP or the empty plasmid EGFP-N3. Fluorescence was quantified 48 hours post-transfection using FACS (Accuri C6). (A) Representative analysis showing the intensity of green fluorescence (FL1-A). (B) Quantification of the percentage of EGFP positive cells. (C) Quantification of the green fluorescence intensity (mean). From 3 independent experiments expressed as mean ± standard deviation.

→ In our model of human myoblasts cultured in vitro, EGFR cannot be over-expressed

Abdelmohsen et al. showed that vitamin K3 (menadione) could, by blocking the activity of phosphatases that dephosphorylate EGFR, maintain the receptor in an active state (Abdelmohsen et al., 2003). Figure 48A shows that, after 48 hours in differentiation conditions, control myoblasts clearly expressed MEF2 and MyHC. As predicted, addition of 10µM vitamin K3 strongly prevented MEF2 and MyHC expression (figure 48A/B). In control cultures, 57%±4 and 65%±5 of myoblasts expressed MEF2 and MyHC whereas, in vitamin K3-treated cultures, only 18%±5 and 3%±0.4 of myoblasts expressed MEF2 and MyHC (p<0.01, n=4). Figure 48C confirmed that addition of vitamin K3 (10µM) inhibited the EGFR degradation usually observed in myoblasts induced to differentiate (see Figure 36).

Moreover, as shown in figure 48D the number of cells in Vitamin K3-treated condition is not significantly different from the untreated condition. These results suggest that EGFR activity inhibits myoblast differentiation by mechanisms independent from the activation of proliferation. Furthermore, we can conclude that EGFR is necessary but not sufficient to maintain the myoblast proliferation.

Figure 48: Vitamin K3 maintains EGFR in differentiation condition, and thus prevents differentiation without inducing proliferation. A, myoblasts were cultured either in GM or in DM (± 10µM vitamin K3). Differentiation was assessed by immunostaining (MEF2 in red; MyHC in green; nuclei in blue with DAPI). B, quantification of the immuno-fluorescence in situ shown in A (n=4). C, western blot reveals the maintenance of EGFR in myoblasts treated with vitamin K3 in differentiation condition (α-tubulin used as a loading control) (n=3). D, vitamin K3 in differentiation conditions for 24h does not increase cell proliferation as assessed by the average of the number of nuclei (DAPI) per field (from 3 independent experiments; p=0.4).

→ Maintenance of EGFR in DM inhibits myoblast differentiation.

DM +10µM K3

h. Effects on proliferation and differentiation by EGFR silencing are independent.

Finally, we excluded that the effect of EGFR silencing in differentiation conditions could be a consequence of the cell cycle arrest. It is known that myoblasts induced to differentiate stop their cell cycle in G1 (Halevy et al., 1995b). We used a cdk4 inhibitor in proliferation conditions to block the cell cycle in G1. As expected this inhibitor strongly affected proliferation (figure 49A). However, blocking the cell cycle did not induce any differentiation, as the percentage of MEF2 positive cells was unaffected by five days of cdk4 inhibitor treatment (figure 49B).

Figure 49 : Cell cycle arrest does not induce myoblast differentiation. A, myoblast proliferation was inhibited by 600nM of the cdk4 inhibitor (Calbiochem) (n=3; * p<0.05). B, addition of 600nM cdk4 inhibitor in proliferation conditions does not induce myoblast differentiation, assessed by MEF2 expression (n=3).

→ Induction of myoblast differentiation, observed in EGFR downregulated myoblasts, does not result from the inhibition of myoblast proliferation.

A B

B. PART 2: Regulation of EGFR expression

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

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