Thesis
Reference
Epidermal growth factor receptor (EGFR)expression and role during human myoblast differentiation
LEROY, Marina
Abstract
Within skeletal muscles, myogenic stem cells - satellite cells - will proliferate and then, differentiate to build/re-build muscle fibers. In vitro, myoblasts derived from human satellite cells can be induced to differentiate after medium switch. Using this cellular model, we previously showed that one of the first steps of myoblast differentiation is a hyperpolarization of myoblasts' membrane. This change of membrane potential is followed by a calcium entry and the expression of myogenic transcription factors. More precisely, myoblast hyperpolarization relies on a potassium channel, Kir2.1 which is maintained inactive during proliferation. Here, EGF receptor, was identified as a stimulator of proliferation and an inhibitor of differentiation. EGFR expression is physiologically regulated in myoblasts and decreases during differentiation, in particular via the proteasome. Absence of EGFR results in a decrease of Kir2.1 phosphorylation, activation of this channel favoring calcium entry, expression of muscle-specific transcription factors and proteins.
LEROY, Marina. Epidermal growth factor receptor (EGFR)expression and role during human myoblast differentiation. Thèse de doctorat : Univ. Genève, 2013, no. Sc. 4647
URN : urn:nbn:ch:unige-355529
DOI : 10.13097/archive-ouverte/unige:35552
Available at:
http://archive-ouverte.unige.ch/unige:35552
Disclaimer: layout of this document may differ from the published version.
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UNIVERSITÉ DE GENÈVE
Département de Biologie cellulaire FACULTÉ DES SCIENCES Professeur Didier Picard Département de Neurosciences FACULTÉ DE MÉDECINE
Professeur Laurent Bernheim ___________________________________________________________________
Epidermal Growth Factor Receptor (EGFR)
Expression and Role during Human Myoblast Differentiation
THÈSE
Présentée à la Faculté des sciences de l’Université de Genève pour obtenir le grade de Docteur ès sciences, mention Biologie
par Marina LEROY
de France
Thèse N°4647
GENÈVE 2014
1
ABBREVIATIONS ... 3
RESUME ... 5
INTRODUCTION ... 7
A. SKELETALMUSCLEANDREGENERATION ... 7
a. Skeletal Muscle ... 7
i. Different types of muscles ... 7
ii. Origin and development of skeletal muscle ... 7
iii. Organization and structure ... 8
iv. Excitation - contraction coupling... 10
b. Post natal Myogenesis - Regeneration ... 11
i. Degeneration and the role of inflammation ... 11
ii. Muscle stem cells: satellite cells ... 12
iii. Stem cell niche ... 14
c. Regeneration - Molecular myogenesis ... 17
i. Hyperpolarization ... 17
ii. Calcium ... 20
iii. Transcription factors ... 21
iv. Intracellular signaling pathways ... 24
v. Our model ... 26
B. EGFR(EPIDERMALGROWTHFACTORRECEPTOR) ... 28
a. Receptor Tyrosine Kinases (RTKs)... 28
b. ErbB family ... 29
c. EGFR ... 34
i. Activation and activity ... 36
ii. Degradation internalization ... 38
iii. Associated pathologies: cancer ... 41
iv. Nuclear EGFR, a role as a transcription factor ... 42
v. EGFR and skeletal muscle ... 43
Rhabdomyosarcomas ... 43
AIM OF THE THESIS ...45
MATERIAL AND METHODS ...46
A. CELLCULTURE ... 46
B. SIRNA ... 48
C. ELECTROPHYSIOLOGY ... 50
D. CALCIUMIMAGING ... 52
E. WESTERN-BLOT ... 54
F. IMMUNO-FLUORESCENCE ... 55
G. RTKASSAY ... 55
H. PROLIFERATIONASSAY ... 57
I. STATISTICALANALYSIS ... 57
2
RESULTS ...58
A. PART1: THE ROLE OF EGFR DURING HUMAN MYOBLAST DIFFERENTIATION ... 58
a. EGFR expression is down-regulated at the onset of human myoblast differentiation ... 58
b. Compared efficiency of three different siRNAs against EGFR ... 62
c. Silencing EGFR inhibits myoblast proliferation ... 64
d. EGFR silencing induces Kir2.1 activation. ... 66
e. Myoblast differentiation induced in GM by EGFR knockdown... 68
f. EGFR silencing accelerates differentiation ... 72
g. Presence of EGFR inhibits myoblast differentiation ... 72
h. Effects on proliferation and differentiation by EGFR silencing are independent. ... 75
B. PART2:REGULATION OF EGFR EXPRESSION ... 76
a. Roles of EGFR ligands ... 76
b. Effect of calcium on EGFR expression ... 79
c. Degradation of EGFR ... 80
d. Variations of the cellular model ... 86
DISCUSSION ...90
A. ERBBS AND SKELETAL MUSCLE ... 91
B. EGFR IN HUMAN MYOBLASTS: STIMULATOR OF PROLIFERATION AND INHIBITOR OF DIFFERENTIATION (KIR2.1, CALCIUM, LIGANDS) ... 93
C. SIGNALING PATHWAYS: ... 97
D. DEGRADATION ... 99
E. OVEREXPRESSION AND RMS ... 102
F. PERSPECTIVES... 104
APPENDIX ... 106
ACKNOWLEDGEMENT ... 112
FIGURES LIST... 113
REFERENCE LIST ... 116
3
ABBREVIATIONS
ADP: adenosine di-phosphate ATP: adenosine tri-phosphate bHLH: basic helix loop helix
BNDF: brain derived neurotrophic factor BSA: bovine serum albumin
CaT: T-type calcium channel Cdk: cyclin dependant kinase CR: cystein-rich domain CTX: cardiotoxin
DM: differentiation medium EGF: epidermal growth factor
EGFR: epidermal growth factor receptor FACS: fluorescence-activated cell sorting FCS: fetal calf serum
FGF: fibroblast growth factor
FGFR: fibroblast growth factor receptor GFP: green fluorescent protein
GM: growth medium
Grb2: growth factor receptor bound protein 2 HB-EGF: heparin-binding EGF-like growth factor HEK cells: human embryonic kidney cells
hERG: human ether à gogo related gene HGF: hepatocyte growth factor
IGF: insulin-like growth factor
IGF1R: insulin-like growth factor type 1 receptor JAK: janus kinase
Kir: inwardly rectifying potassium channels KO: knock out
MADS: MCM1, agamous, deficient, SRF MB: myoblast
MCK: muscle-specific creatine kinase MEF2: myocyte enhancer factor 2 miRNA: micro-RNA
4 MRF: muscle regulatory factor
mRFP: monomeric red fluorescent protein Myf5: myogenic factor 5
MyoD: myogenic determinant MyHC: myosin heavy chain NFMB: non-fusing myoblast NLS: nuclear localization domain NRG: neuregulin
Pax: paired homeobox transcription factor PBS: phosphate-buffered saline
PFA: paraformaldehyde Pi: inorganic phosphate
PIP2: phosphatidyl inositol diphosphate PI3K: phosphoinositide 3 kinase
PLC: phospholipase C PTK: protein tyrosine kinase
PTP1B: protein tyrosine phosphatase 1B PTP1C: protein tyrosine phosphatase 1C RING finger: really interesting new gene finger RNA: ribonucleic acid
RNAi: RNA interference
RT-PCR: reverse-transcription polymerase chain reaction RTK: receptor tyrosine kinase
SH2: src homology 2
siEGFR: small interfering RNA targeting EGFR siRNA: small interfering RNA
SOCE: store-operated calcium entry
STAT: signal transducer and activator of transcription STIM1L: stromal interaction molecule 1, long isoform STIM1S: stromal interaction molecule 1, short isoform TGFα: transforming growth factor alpha
TM: transmembrane domain WT: wild type
Y242: tyrosine 242 Y1045: tyrosine 1045
5
RESUME
(English version below)
Le muscle strié squelettique représente près de 40% de la masse du corps humain. En son sein, des cellules souches myogéniques, les cellules satellites, permettent la croissance ainsi que la régénération musculaire. Lorsqu’elles sont stimulées, ces cellules satellites prolifèrent puis se différencient pour former/reformer des fibres musculaires.
Notre laboratoire a pour objectif d’étudier les mécanismes moléculaires mis en jeu lors de la régénération musculaire. Des myoblastes dérivés de cellules satellites humains sont utilisés in vitro. Ces myoblastes prolifèrent puis sont induits à se différencier par un changement de milieu. En utilisant ce modèle cellulaire, notre groupe a montré que l’une des premières étapes de la différenciation est une hyperpolarisation des membranes des myoblastes. Ce changement de potentiel induit une augmentation de la force électromotrice sur les ions calcium favorisant ainsi leur entrée dans la cellule. Des facteurs de transcription myogéniques (MRFs, MEF2s) sont ensuite exprimés et vont à leur tour induire l’expression de protéines spécifiques du muscle (par exemple : myosine, troponine). Plus précisément, l’hyperpolarisation des myoblastes dépend d’un canal potassique, Kir2.1. Ce canal est maintenu inactif pendant la phase de prolifération par une phosphorylation sur la tyrosine 242.
Lors de la différentiation, le canal n’est plus phosphorylé et s’active.
Le but de mon travail était de trouver la kinase responsable de la phosphorylation de Kir2.1. En utilisant un kit pour identifier les kinases actives dans notre système, j’ai ainsi identifié le récepteur à l’EGF (epidermal growth factor), EGFR, comme étant à la fois un stimulateur de la prolifération et, plus important, un inhibiteur de la différentiation des myoblastes. L’expression d’EGFR est physiologiquement régulée dans les myoblastes et décroit durant leur différentiation, notamment via une dégradation. L’absence d’EGFR résulte en une diminution de la phosphorylation de Kir2.1, l’activation de ce canal, une entrée de calcium, l’expression de facteurs de transcription et protéines spécifiques du muscle.
L’introduction présentera le muscle strié squelettique et sa régénération, du point de vue moléculaire principalement, puis le récepteur à l’EGF. La partie suivante décrira les résultats obtenus lesquels seront ensuite discutés.
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Skeletal muscle represents about 40% of human body weight. It contains myogenic stem cells (satellite cells) that allow both growth and regeneration of the muscle. Upon stimulation, these cells will proliferate and then, differentiate to build/re-build muscle fibers.
Our laboratory’s goal is to study molecular mechanisms involved during muscle regeneration. Myoblasts derived from human satellite cells proliferate in vitro and can be induce to differentiate after medium switch. Using this cellular model, our group showed that one of the first steps of myoblast differentiation is a hyperpolarization of the membrane of myoblasts. This change of membrane potential is followed by an increase of the driving force on calcium ions favoring a calcium entry. Myogenic transcription factors (MRFs, MEF2s) are then expressed and will in turn induce expression of muscle specific proteins (myosin, troponin). More precisely, myoblast hyperpolarization relies on a potassium channel, Kir2.1.
This channel is maintained inactive during proliferation by a phosphorylation on tyrosine 242.
During differentiation, the channels are no more phosphorylated and they activate.
The goal of my thesis was to study Kir2.1 regulation. Using a kit to identify the active kinases in our model, I identify EGF receptor (epidermal growth factor), EGFR, as a stimulator of proliferation and, more important, an inhibitor of differentiation of myoblasts. EGFR expression is physiologically regulated in myoblasts and decreases during differentiation, in particular via the proteasome. Absence of EGFR results in a decrease of Kir2.1 phosphorylation, activation of this channel favoring calcium entry, expression of muscle- specific transcription factors and proteins.
The introduction part will present skeletal muscle and its regeneration, essentially from a molecular point of view, and then, EGFR. The next part will describe the results obtained which will be further discussed.
7
INTRODUCTION
A. SKELETAL MUSCLE AND REGENERATION a. Skeletal Muscle
i. Different types of muscles
There are three types of muscles, all sharing the contractile property. Smooth muscles are found within the walls of vessels (blood and lymphatic) and organs (gastro-intestinal, urinary, reproductive tracts) where they are involved in shape and diameter changes. Striated muscles are characterized by a recognizable striation pattern, described further, due to the alignment of sarcomeres; they comprise cardiac and skeletal muscles. Cardiac muscle is responsible by its contraction of heart function and thus of blood circulation in the body.
Skeletal muscles are involved both in the maintenance of body posture, body temperature and in the generation of movements. In the human body, there are approximately 639 skeletal muscles accounting for 35-40% of its weight.
ii. Origin and development of skeletal muscle
In the amniotes (human, mouse, chicken), skeletal muscles originate from the somites (Mok and Sweetman, 2011); these structures result of the condensation of paraxial mesoderm, flanking the notochord and the axial tube (figure 1A). The somites then divide in two parts:
ventral part is the sclerotome which will later define bones and cartilages; dorsal part is the dermomyotome which will give rise to the dermatome and the myotome containing myogenic precursor cells. These cells express a specific marker, Pax3 (paired homeobox transcription factor 3), which concentration is particularly high in the lips of the myotome (Relaix et al., 2005) (figure 1B). Pax7, a paralogue of Pax3, appears during somite maturation and is preferentially expressed in the medial part of the myotome (Kassar-Duchossoy et al., 2005). Pax3/7 expressions are induced by signals from the surrounding tissues (Otto et al., 2006). While the
8
dorsal part of the myotome will evolve as back muscles, the ventral part will later correspond to limbs muscles, diaphragm and body wall muscles (figure 1B).
Figure 1: A/ Successive steps in embryonic muscle development (transversal view) leading to myotome formation. B/ Dermomyotome: Pax concentrations and muscle arising from dorsal and ventral part.
iii. Organization and structure
In adults, skeletal muscles are attached to the bones by the tendons (figure 2). Each muscle is composed of several fascicles surrounded by the perimysium. A fasciculus comprises myofibers but also blood vessels (veins and arteries). The endomysium is the structure which delimits myofibers notably because it composes the basal membrane.
Myofibers are pluri-nucleated cells covering the whole length of the muscle. They contain myofibrils, responsible for the contractile property of the muscle, which are surrounded by a tubular network, the sarcoplasmic reticulum. Myofibrils are constituted of repetitive elements, the sarcomeres, which are considered as the functional units of skeletal muscle. Sarcomeres present banding patterns due to the organization of the myofilaments. There are two categories
somite axial tube
sclerotome dermomyotome
- Dorsal muscles - Limb muscles
- Diaphragm - Body wall muscles
Pax7 concentration Pax3 concentration
notochord
myotome A
B
9
of myofilaments: thick filaments of myosin and thin filaments F-actin. Nuclei are located at the periphery of myofibrils. Interestingly, between the basal membrane and the plasmalemma, a special type of cells, the satellite cells, remain in a quiescent state. These cells are stem cells involved in the regeneration of the muscle. Moreover, several types of skeletal muscle fibers exist. They differ in their speed and duration of contraction, and in their metabolism (aerobic versus anaerobic).
Figure 2 : Scheme of skeletal muscle and associated structures. Image in upper right is a section of the tibialis anterior of a mouse depicting the presence of satellite cells in vicinity of vessels. Three connective tissue layers can be distinguished in skeletal muscle: the epimysium ensheating the muscle, the perimysium ensheating fascicles and the endomysium ensheating myofibers. Myofibers comprise the myofibrils organized as successions of sarcomeres. (From (Tajbakhsh, 2009)).
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iv. Excitation - contraction coupling
Arrival of an action potential from a motoneuron on muscle fibers leads to contraction.
Muscle contraction results from the shortening of sarcomeres via filaments sliding. As depicted in figure 2, motor-neurons innervate myofibers. Propagation of an action potential along the myofiber induces a massive intracellular calcium rise. Calcium binds troponin which results in a conformational change allowing the binding of myosin to actin (figure 3). Hydrolysis of ATP induces the bending of myosin cross bridge and, as a consequence, the sliding of the filaments over each other. Products of hydrolysis, ADP+Pi, are released. Binding of another molecule of ATP detaches myosin cross bridge from actin. If calcium concentration if still high enough (over 0.1µM), another cycle starts. If not, muscle will relax.
Figure 3: the different components of the excitation-contraction coupling mechanism and their interactions (from Physiology, Bullock; third edition).
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b. Post natal Myogenesis - Regeneration
Skeletal muscle has a very low turnover rate, as it was estimated that about 1-2%
myonuclei are replaced weekly in the rat (Schmalbruch and Lewis, 2000). However, skeletal muscle exhibits impressive regenerative properties. After a trauma, an acute exercising or because of a genetic disease, myofibers can be injured. Two processes will succeed one another: a phase of degeneration of the injured fibers followed by their regeneration.
Figure 4: Skeletal muscle repair process. A/ section of a tibialis anterior muscle injured by cardiotoxin injection (CTX) results in the rapid necrosis of myofibers. B/ myofiber regeneration is characterized by the activation of proliferation, differentiation, and fusion of myoblasts. Regenerating fibers are characterized by their small caliber and their centrally located myonuclei (arrows). From (Charge and Rudnicki, 2004).
i. Degeneration and the role of inflammation
In vivo study of skeletal muscle degeneration/regeneration was made possible using different strategies: 1/ animal models in which degeneration and/or regeneration are impaired, 2/ direct injury of the fibers (crushing, freezing, denervation) and, 3/ injection of myotoxins such as cardiotoxin (CTX). As shown in figure 4, four days after injection of CTX in a muscle, an important necrosis is observed; a week later, muscle has regenerated. Both phases involve inflammatory cells recruitment (Contreras-Shannon et al., 2007). In the case of acute exercising, Fielding et al. demonstrated that neutrophils invade muscle very quickly (within the first hour post-injury) (Fielding et al., 1993). These neutrophils have different roles; they eliminate muscle debris from the injured fibers by phagocytosis and by the release of
A B
12
proteases. Furthermore, they promote muscle invasion by macrophages. During muscular injury, macrophages have the ability to switch their phenotype from pro-inflammatory to anti- inflammatory (Arnold et al., 2007). These two phenotypes reflect the successive role of macrophages in the stimulation of proliferation of muscle precursor cells, in the inhibition of apoptosis, and then, in the stimulation of differentiation (Cantini and Carraro, 1995;Chazaud et al., 2003;Sonnet et al., 2006;Arnold et al., 2007;Vierck et al., 2000).
ii. Muscle stem cells: satellite cells
Satellite cells were first observed in 1961 by Mauro on skeletal muscle fibers of Xenopus laevis (figure 5) (MAURO, 1961). They were initially described as cells “wedged between the plasma membrane of the muscle fiber and the basement membrane, which invests the fiber throughout its length in close association with the plasma membrane”. The authors already hypothesize their role as “dormant myoblasts (…) ready to recapitulate the embryonic development of skeletal muscle fiber when the main multinucleated cell is damaged”. Indeed, skeletal muscle regeneration relies on the presence of these satellite cells (Zammit et al., 2002).
Figure 5: Longitudinal view of a satellite cell in the periphery of the skeletal muscle fiber of the tibialis anticus. The extreme poles of the cell are indicated (sc). From (MAURO, 1961).
.Among different hypotheses, Mauro proposed that satellite cells could be “remnants from the embryonic development of the multinucleated muscle cell” (MAURO, 1961). It is now admitted that satellite cells, at least a fraction of them, derive from the dermomyotome (Gros et al., 2005).
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Satellite cells, responsible for the post-natal myogenesis, account for 3-5% of myofiber nuclei (Gibson and Schultz, 1983;Zammit et al., 2002). They express, as the embryonic myogenic precursors, paired homeobox transcription factors (Pax). If all satellite cells are characterized by the presence of Pax7, only a subset also expresses Pax3 (Relaix et al., 2006).
There are 9 Pax factors in vertebrates, all of them involved in development. Pax7 was shown to promote peri-natal survival of satellite cells (Lepper et al., 2009). Indeed, in Pax7-/- animals, increased apoptosis of satellite cells is observed after birth, leading to the depletion of the pool of satellite cells and the impairment of muscle regeneration (Kassar-Duchossoy et al., 2005).
In this case, Pax3 cannot compensate Pax7 function.
Figure 6: Satellite cells, proliferation and self renewal mechanisms. Quiescent satellite cells are characterized by the expression of Pax7. After activation, they start to express MyoD and Myf5. Asymetrical division leads to self-renewal of one quiescent satellite cell and one myoblast, while symmetrical division leads to two myoblasts. Myoblasts express MyoD and Myf5 but not Pax7, such as the nuclei present in myofibers.
4 / MYOFIBER 1 / QUIESCENT SATELLITE CELL
Pax7 + MyoD -/ Myf5-
Nucleus of the myofiber MyoD+ / Myf5+
Pax7 Activation
Self-renewal
Regeneration
(differentiation, fusion) 2 / ACTIVATED
SATELLITE CELL Pax7+ / MyoD+ / Myf5+
3 / MYOBLASTS MyoD+ / Myf5+
Pax7-
Asymetrical division
Symetrical
division Proliferation
14
Satellite cells suit the definition of stem cells as they give rise to lineage-specific cells and can also self-renew (Collins et al., 2005;Sacco et al., 2008). Quiescent satellite cells express Pax7. Upon activation, these cells will start expressing MyoD and Myf5, two myogenic determining factors (Halevy et al., 1995a;Fukada et al., 2007). The role of these two factors will be described further (see “C-3 / transcription factors”). Subsequent division can occur either symmetrically (identical daughter cells) or asymmetrically (different daughter cells) (Kuang et al., 2007) (figure 6). Usually, symmetrical division corresponds to planar division. In this case, daughter cells will keep on expressing MyoD and stop expressing Pax7; then, they will evolve and proliferate to generate a sufficient pool of myoblasts which will participate directly in the regenerative process by fusing together or with pre-existing myofibers (Olguin and Olwin, 2004;Collins and Partridge, 2005). However, in the case of asymmetric division (generally corresponding to an apical-basal oriented division), satellite cell can give birth to one daughter cell that will keep expressing Pax7 (not MyoD), and thus reconstitute the pool of stem cells (Yoshida et al., 1998;Zammit et al., 2002).
iii. Stem cell niche
Satellite cells fate depends on their environment, their “niche”, which corresponds to the surrounding tissues and the substance they secrete (figure 7). As previously mentioned, inflammatory cells, in particular macrophages, stimulate satellite cells so that they can proliferate or differentiate, depending on the cytokines and growth factors secreted. Another important partner is the neighboring vasculature. Satellite cells were shown to localize preferentially in close vicinity to vessels so that they can reciprocally act on each other (Christov et al., 2007); hence, endothelial cells supply growth factors necessary for proliferation while satellite cells stimulate angiogenesis (Christov et al., 2007). In addition, satellite cells secrete growth factors that will act in an autocrine manner. Thus, we can discriminate three types of factors: (i) activators of the satellite cells, (ii) stimulators of proliferation and, (iii) stimulators of differentiation (Boonen and Post, 2008;Karalaki et al., 2009). Among them, some growth factors will be described below (figure 8).
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Figure 7: The satellite cell niche, growth factors and hormones released from the surrounding tissues. From (Hawke and Garry, 2001).
After a muscular lesion, HGF (hepatocyte growth factor) is provided by the vasculature and secreted by immune cells. HGF was shown to activate satellite cells (Tatsumi et al., 1998).
As soon as they are activated, these satellite cells also produce HGF which will reinforce their expansion (Jennische et al., 1993;Sheehan et al., 2000;Miller et al., 2000;Yamada et al., 2010).
Moreover, HGF is secreted by satellite cells when activated by stretch or nitric oxide (Tatsumi et al., 2002). Auto-secretion of fibroblast growth factor (FGF1, FGF2, FGF4 and FGF6) by the satellite cells has a double-effect: stimulation of proliferation and inhibition of differentiation (Soulet et al., 1994;Kastner et al., 2000). These effects are amplified by BDNF (brain-derived neurotrophic factor) which works as an inhibitor of differentiation (Mousavi and Jasmin, 2006).
Insulin-like growth factors (IGFs) are secreted by activated satellite cells and myoblasts (Florini et al., 1991). IGFs are positive regulators of muscle growth as they exhibit the interesting
16
property of promoting proliferation as well as differentiation of satellite cells(Philippou et al., 2007). These effects were shown to occur via different signaling pathways (Coolican et al., 1997) and depend on the cytokines present, such as IL1beta (Foulstone et al., 2001;Broussard et al., 2004). Other studies pointed out that, rather than showing a proliferative effect, IGFs exert anti-apoptotic properties (Napier et al., 1999;Chakravarthy et al., 2001;Conejo and Lorenzo, 2001). Epidermal growth factor (EGF), a growth factor provided to satellite cells by the vasculature, has still an unclear role concerning myoblast differentiation. Investigation of the roles of EGF and its cognate receptor, EGFR, on skeletal muscle regeneration are the goal of this work.
Growth factor Proliferation Differentiation
HGF ↑ ↓
FGF ↑ ↓
BDNF ↓
IGFs ↑ ↑
EGF ↑?
Figure 8 : Selected growth factors and their role on myoblast proliferation and differentiation. Adapted from (Hawke and Garry, 2001).
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c. Regeneration - Molecular myogenesis
The following section focuses on molecular events occurring at the onset of myoblast differentiation. The cascades described thereafter correspond to our model of investigation.
We used human satellite-cells derived myoblasts. These myoblasts, after a proliferative phase, can be induced to differentiate by medium switch. Before fusing together, they will undergo a suite of finely orchestrated molecular events. One of the first steps of this process known so far is a hyperpolarization of the myoblasts.
i. Hyperpolarization
During the last two decades, research conducted in our laboratory has been focused on the early stages of myoblast differentiation using in vitro techniques and, in particular, electrophysiology. Therefore, we highlighted the important role of ion channels and membrane potential throughout the differentiation process of human myoblasts (Cooper, 2001).
Electrophysiology techniques revealed that several potassium channels are activated in differentiating myoblasts. The first potassium channels activated are eag (ether a gogo) and herg (human ether a gogo related gene) (Bijlenga et al., 1998;Occhiodoro et al., 1998).
Afterwards, another potassium channel was found to be involved in the process. This channel exhibited a strong inward rectification (Liu et al., 1998a) and was identified as K+ inward rectifier (Kir2.1) (Fischer-Lougheed et al., 2001). Activation of Kir2.1 led to the hyperpolarization to - 70mV of human myoblasts at the onset of differentiation, before fusion (Liu et al., 2003).
Satellite cells derived myoblasts
Proliferating
myoblasts Differentiating
myoblasts Myotubes
proliferation differentiation fusion
18 Kir2.1
Inwardly rectifying potassium channels (Kir) are a large family of membrane potential modulators. High homology between the members allows them to work as homo- or hetero- tetramers (figure 9A). Second subfamily of Kir channels (Kir2x channels) is involved in muscular and neuronal excitability via hyperpolarizing effect. These channels exhibit a strong inward rectification, resulting from the physical block of K+ permeation by cations such as Mg2+
or polyamines (Hibino et al., 2010;Reimann and Ashcroft, 1999).
Figure 9: A/ Degree of identity between Kir family members; From (Hibino et al., 2010). B/ Representation of Kir2.1 subunit structure; Adapted from (Reimann and Ashcroft, 1999).
Kir2.1 was the first of these channels to be cloned (Raab-Graham et al., 1994). Its cDNA encodes a 427 amino acids protein. The protein structure comprises two transmembrane domains (TM1 and TM2), a pore loop and cytoplasmic tails (-NH2 and -COOH) (figure 9B). Kir2.1 channel activity is complexly regulated and was shown to involve several partners and mechanisms such as ATP, G-proteins (Firth and Jones, 2001;Jones, 2003;Boyer et al., 2009), PIP2 (Huang et al., 1998;Xie et al., 2008), phosphorylation (Wischmeyer and Karschin, 1996;Wischmeyer et al., 1998;Karle et al., 2002;Scherer et al., 2007;Zhang et al., 2011a;Alesutan et al., 2011), pH (Dahlmann et al., 2004), shear stress (Hoger et al., 2002).
Kir2.1 transcripts were found in heart, skeletal muscle, brain, placenta, lung and kidney.
Mutations in KCNJ2 gene, encoding for Kir2.1 subunit, usually lead to the establishment of the TM
1 TM
Cell membrane Pore loop 2
A B
rectification PIP2
19
Andersen-Tawill syndrome (Haruna et al., 2007;Tristani-Firouzi and Etheridge, 2010). Most of the mutations result in a loss of function and the establishment a dominant negative channel (see details in “appendix”). Symptoms are, in the heart, lengthening of QT and, in skeletal muscle, episodes of paralysis and muscular weakness.
Kir2.1 and skeletal muscle
In our model of human myoblasts, Kir2.1 currents are observed at the onset of differentiation. Study of their regulation revealed that Kir2.1 channels are present at the plasma membrane of myoblasts even during proliferation (Hinard et al., 2008). However, in proliferating myoblasts, these channels are inactive. Use of kinase or phosphatase inhibitors and Kir2.1 specific mutant expression revealed that Kir2.1 inactivation was due to its phosphorylation on a specific residue, Y242 (Wischmeyer et al., 1998;Hinard et al., 2008). This tyrosine, close to the rectification zone, was not described as mutated in Andersen-Tawill patients (Zhang et al., 2005). In summary, Kir2.1 channels are phosphorylated on Y242 during myoblasts proliferation, maintaining them inactive. At the onset of differentiation, which corresponds to roughly 6 hours after a medium switch in our system, Kir2.1 channels are not phosphorylated anymore and thus get activated, inducing myoblast membrane hyperpolarization.
20 ii. Calcium
As a consequence of a membrane hyperpolarization, the driving force for calcium ions increases. In addition, Kir2.1 driven hyperpolarization permanently activates for T-type calcium channels (CaT) (reviewed by Bernheim and Bader (Bernheim and Bader, 2002)) which are involved in myoblast differentiation (Bijlenga et al., 2000). Later, Arnaudeau et al. showed that calcium was not only provided via T-type channels but also by internal stores and via store operated calcium entry (SOCE) (figure 10) (Arnaudeau et al., 2006). Recently, our work highlighted the importance of the SOCE and described the proteins involved: the Ca2+ channel is constituted of Orai proteins and the calcium sensor of STIM proteins (stromal interaction molecule). Indeed, silencing of either Orai1, Orai3, STIM1 or STIM2 considerably inhibited myoblasts differentiation (Darbellay et al., 2010;Darbellay et al., 2009). Moreover, an intracellular calcium rise was shown to activate intracellular pathways such as calcineurin and induce the expression of transcription factors (Konig et al., 2006).
Figure 10: Model of the different calcium sources used by human myoblasts during differentiation: CaT channel activated by the Kir2.1-induced hyperpolarization, internal calcium stores (reticulum endoplasmic), and store-operated channels activated by calcium store depletion; Adapted from (Arnaudeau et al., 2006).
[Ca 2+ ] i ↗
Kir2.1 K
+CaT Ca
2+Ca
2+STIM
Orai
SOCE
21 iii. Transcription factors
Two families of transcription factors play a determining role in myoblast differentiation:
the muscle regulatory factors (MRFs) and the myocyte enhancer factors (MEF2s).
MRFs (muscle regulatory factors)
The MRF family comprises four members - Myf5, MyoD, Myogenin (=Myf4) and MRF4 – which are sequentially expressed during myoblast differentiation (Cornelison and Wold, 1997). They belong to the basic-helix-loop-helix (bHLH) family of transcription factors, and more specifically to the class II of this family, because of their tissue-specificity. They function as heterodimer with the class I bHLH factors, the E proteins. Their dimerization occurs via the bHLH domain. MRFs regulate transcription via the binding to a specific DNA sequence named E-box (CANNTG).
The first member identified, MyoD (myogenic determinant), was discovered and extensively studied by the group of Weintraub (Lassar et al., 1986). They showed that forced expression of MyoD in fibroblasts could convert them into myoblasts (Weintraub et al., 1989).
Over-expression of MyoD in other cell lines also induced the expression of muscle specific proteins such as myogenin, myosin heavy chain (MyHC), muscle-specific creatine kinase (MCK)…; even the characteristic striated pattern was observed after forced expression of MyoD after differentiation (Choi et al., 1990) and the myogenic characteristics maintained after cell transplantation in nude mice (Qin et al., 2007).
As previously mentioned, upon satellite cell activation, the first MRFs to be expressed are MyoD and Myf5, which are also important during embryogenesis and considered as the myogenic determinant factors (Olson and Klein, 1994). Then, myogenin is expressed and will, in its turn, induce the transcription/activation of MEF2s and muscle specific proteins. Finally, MRF4, which timing of expression is complex, is rather involved in primary myogenesis as well as late stages of myoblast differentiation (Kassar-Duchossoy et al., 2004). Using transgenic
22
animals, MRFs were shown to have overlapping roles (Rudnicki et al., 1992) (figure 11). While null animals for MyoD or Myf5 present a moderate muscular phenotype, animals knocked out for these two determinant myogenic factors do not have muscles (Braun and Arnold, 1996).
Meadows et al. demonstrated that myogenin was important during peri-natal development;
however, adult satellite cells myogenin-/- exhibit normal muscle regeneration probably because of compensation by the other MRFs (Meadows et al., 2008).
Figure 11 : Phenotypes of knocked out animals for MRFs and combination of these factors; From (Megeney and Rudnicki, 1995).
In addition to their role in the induction of MEF2 protein expression, MRFs were involved in cell cycle arrest. Withdrawal from the cell cycle is a necessary step for a proper differentiation. Myogenin expression was shown to be followed by the expression of p21 (Halevy et al., 1995a;Andres and Walsh, 1996), a well known cdk (cyclin dependant kinase) inhibitor whose expression results in post mitotic state establishment.
23
MEF2 (myocyte enhancer factor 2)
MEF2s, myocyte enhancer factors 2, belong to the MADS (MCM1, agamous, deficient, SRF) family of transcription factors. In the vertebrates, there are four MEF2 genes (A, B, C and D). All MEF2 gene products present in their N-ter region a highly conserved MADS box (recognizing A/T rich sequence on DNA) and the MEF2 domain (figure 12A). These two specific regions provide dimerization, DNA binding and co-factor binding properties of the MEF2s (Potthoff and Olson, 2007). MEF2s participate to the differentiation process of several cell types (figure 12B). MEF2A/B/D are ubiquitously expressed while MEF2C is preferentially expressed in cardiac and skeletal muscles, spleen and brain (Potthoff and Olson, 2007).
Figure 12 : A/ Sequence conservation of the different protein domains between the MEF2 member and from different species. B/ expression of MEF2 in several human cell types; from (Potthoff and Olson, 2007).
Black and Olson reviewed that all the MEF2s were expressed in muscle lineage during myogenesis. MEF2D is already expressed in proliferation, MEF2A appears during early stage of differentiation, and MEFC during the late stages. Expression of MEF2B was not shown to be modulated (Black and Olson, 1998).
MEF2C null mice die because of cardiac myogenesis defect (Lin et al., 1997;Lin et al., 1998;Bi et al., 1999); however, their skeletal phenotypes differ depending on the strain of the
A B
A B
A B
24
animal from the absence of muscle defect to a loss of sarcomeres integrity (Potthoff et al., 2007). In addition, transfection of myoblasts with a dominant-negative form of MEF2A inhibits myogenesis (Ornatsky et al., 1997).
In contrast to MRFs, MEF2s are not sufficient but necessary for muscle development and regeneration. MEF2s expressions are induced by MRFs. Furthermore, MEF2A/C/D bind bHLH proteins (figure 13) via their MEF2 domain and synergistically activate the myogenic program (Molkentin et al., 1995;Black et al., 1998). Such cooperation is involved for the expression of desmin and MCK (Ohkawa et al., 2007). MEF2s can also stimulate bHLH expression (in particular myogenin), thus creating a positive feedback loop (Cheng et al., 1993). Finally, MEF2s with the exception of MEF2C were shown to auto-regulate their own expression (Dodou et al., 2003;Cripps et al., 2004).
Figure 13 : Potential mechanisms for activation of skeletal muscle transcription by MEF2 and myogenic bHLH factors. Transfection assays have revealed four potential mechanisms for synergistic activation of transcription by MEF2 and myogenic bHLH proteins. (1) MEF2 can interact with MyoD/E12 heterodimers bound to DNA. (2) MyoD/E12 heterodimers can interact with MEF2 bound to DNA. (3) MEF2 and MyoD/E12 heterodimers can bind adjacent sites to activate transcription synergistically. (4) MEF2 and MyoD/E12 heterodimers can bind non-adjacent sites and cooperatively activate transcription through protein-protein interactions. From (Black and Olson, 1998)
iv. Intracellular signaling pathways
25
Several intracellular signaling pathways have been involved in myoblast differentiation.
Konig et al. showed that calcineurin, p38-MAPK and PI3K regulatory pathways are involved in human myoblasts differentiation (Konig et al., 2006). However, among them, only the calcineurin pathway could be linked to the Kir2.1 driven hyperpolarization. Moreover, Bakkar and Guttridge reviewed the complex role of another pathway involved in myogenesis, NF-kB, acting either as a positive or a negative regulator of myogenesis (Bakkar and Guttridge, 2010).
However, this section will focus mainly on the JAK/STAT pathway as it appears promising in our system.
JAK/STAT
JAKs (janus kinases) are cytoplasmic tyrosine kinases. They bind to activated (phosphorylated) membrane receptors (such as EGFR, IGF1R, FGFR3, interleukin receptors, interferon receptor…). Then, they auto-phosphorylate. STATs (Signal Transducer and Activator of Transcription) are recruited by JAKs; Then STATs bind to the activated membrane receptor. This will result in their activation and subsequent translocation to the nucleus. There, STATs bind DNA and activate transcription of genes involved in proliferation, migration, immunity… (reviewed in (Rawlings et al., 2004)). The JAK-STAT pathway is evolutionarily conserved. JAK/STAT system comprises 4 JAK proteins (JAK1/2/3+tyk2) and 7 STAT members (STAT1/2/3/4/5a/5b/6). JAK/STAT exert pleitropic effects (proliferation, migration, differentiation…) and it was shown that different members can induce opposite effects. For instance, while STAT3 and STAT5 are tumorigenous, STAT1 has anti-proliferative properties (Bromberg, 2000;Bromberg and Darnell, Jr., 2000). Indeed, STAT1 was shown to induce p21 expression which will in turn stop the cell cycle (Chin et al., 1996). Moreover, STAT1 also blocks proliferation via a direct interaction with cyclin D1/cdk4 complex (Dimco et al., 2010).
However, in skeletal muscle, STAT1 coupled to JAK1 promoted myoblast proliferation and inhibited differentiation (Sun et al., 2007a;Diao et al., 2009). Another study using small interfering RNA (siRNA) revealed that JAK2/STAT2 signaling in myoblasts promoted their
26
differentiation (Wang et al., 2008). Finally, STAT3 was shown to be necessary both for proliferation and differentiation of myoblasts (Kami and Senba, 2002;Yang et al., 2009;Caldow et al., 2011), and to interact with MyoD (Yang et al., 2009). Recently, Trenerry investigated the patterns of expression of JAK/STAT proteins during human myoblast differentiation in vitro (Trenerry et al., 2011). Their data confirmed a preferential role of active JAK1/STAT1 during proliferation and of JAK2 during differentiation (figure 14).
Figure 14: JAK/STAT expression during human skeletal myoblast differentiation. Phosphorylation of JAK2 (Tyr1007/1008) and STAT3 (Tyr705) increases during differentiation, while JAK1 (Tyr1022/1023) and STAT1 (Tyr701) were only apparent during proliferation. From (Trenerry et al., 2011).
v. Our model
27
To summarize the first steps of human myoblast differentiation, we propose the following model (Figure 15). A tyrosine kinase phosphorylates Kir2.1 on Y242 during myoblast proliferation, and maintains the channel inactive. At the onset of differentiation, Kir2.1 channels get de-phosphorylated. Thus Kir2.1 channels activate and membrane hyperpolarization occurs (Hinard et al., 2008). This Kir2.1 driven hyperpolarization increases the driving force on calcium ions which can enter cells via different mechanisms previously described (Arnaudeau et al., 2006). Intracellular calcium rise is necessary for the induction of transcription factors (Konig et al., 2004). The first transcription factors induced are the MRFs, which role is to induce the expression/activation of MEF2s. Together, MRFs and MEF2 drive the expression of muscle specific protein necessary for muscle regeneration.
Figure 15 : Chronology of the molecular events involved during human myoblast differentiation.
ProliferationDifferentiation
T0 (medium switch)
T +6h: Kir2.1 activation / hyperpolarization T +12h: Myogenin expression detected
T +24h: MEF2 expression detected
T +48h: Start of the fusion process
Time
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B. EGFR (EPIDERMAL GROWTH FACTOR RECEPTOR) a. Receptor Tyrosine Kinases (RTKs)
The human kinome is constituted of more than 520 kinases. Among them, more than 90 are protein tyrosine kinases (PTK) including 58 receptor tyrosine kinases (RTK) (figure 16).
RTKs have been shown to be involved in many intracellular signaling pathways, regulating proliferation, migration, survival, differentiation (reviewed in (Schlessinger, 2000))…
Figure 16: receptor tyrosine kinase families, structural organization in specific domains. From (Hubbard and Till, 2000).
29 b. ErbB family
The erbB family comprises four members: erbB1 (=EGFR), erbB2 (=c-neu, HER-2), erbB3 and erbB4. The first member identified was EGFR, epidermal growth factor receptor, soon after the discovery of its cognate ligand, EGF. The other receptors were found by sequence homology. Thus, the four erbBs share a close organization. However, because of their innate properties and their ligand affinity, they differ in their cellular role (Citri and Yarden, 2006). Furthermore, erbBs can function either as homo- or hetero-dimers which increases the complexity of the signaling network.
EGFR is the most studied because of its ubiquitous expression and its role in cancer development. EGFR structure, functioning and signaling will be described further.
ErbB2 (=neu in rodents) was identified in the 1980’s, first as an oncogene and then as a homologue of EGFR (Shih et al., 1980;Schechter et al., 1985). Klapper et al. showed howerver that erbB2 was unable to bind any EGF-like ligand (Klapper et al., 1999). Actually, it is the only member for which no ligand was found so far. Thus, erbB2 is thought to function independently from direct ligand binding. It was proposed that ErbB2 hetero-dimerizes with the other erbBs and potentiates their effects (King et al., 1988;Olayioye, 2001) (figure 17).
ErbB3 was isolated simultaneously by two groups (Kraus et al., 1989;Plowman et al., 1990) from human mammary epithelial cell and breast carcinoma cell line. Its expression was then found in many other tissues (placenta, skin, skeletal muscle, lung, colon, kidney, brain…) and tumors. Studies on its function revealed that erbB3 possesses an impaired intrinsic kinase, making impossible an intracellular signal transduction except when it forms a hetero- dimer with another erbB (Carraway, III et al., 1994;Guy et al., 1994).
ErbB4 was the last identified member of the erbB family and thus, the less studied. It is mostly expressed in the nervous system and striated muscles (fetal and adult) where it exhibits a heterogenous pattern (Srinivasan et al., 1998). In vivo and in vitro studies revealed
30
its major importance in heart, brain but also mammary glands development (Bersell et al., 2009). However, its role in cancers is not clear. Depending on the tumors, it was found either over-expressed or down-regulated. This is likely related to the existence of at least four isoforms of erbB4 (Junttila et al., 2000).
Figure 17: All of the ErbB-stimulatory ligands are presented along with their ErbB preference. Interactions with the indicated ErbB homodimers (above diagonals) and the corresponding heterodimers with ErbB-2 (below diagonals) are indicated by using a color code: The most mitogenic interactions of each ligand are shown in black whereas white areas indicate absence of mitogenic signals. From (Klapper et al., 1999).
31 Ligands
ErbBs signaling requires an imput, the ligand binding (Wong, 2003). ErbBs are activated by a subset of ligands which bind with different affinity. EGF (epidermal growth factor), TGFα and amphiregulin only bind EGFR whereas HB-EGF (heparin-binding EGF like growth factor), β-cellulin and epiregulin can bind either EGFR or erbB4 (figure 18). Finally, neuregulins (NRGs) bind erbB3 and/or erbB4. All these pre-mentioned ligands are synthesized as transmembrane precursors that can act in a juxtacrine way (Harris et al., 2003). After cleavage by proteases, they are released as free ligands for an autocrine or paracrine action (Singh and Harris, 2005).
Figure 18: ErbB family and their ligands
Only EGFR most studied ligands (EGF, TGFα and HB-EGF) will be described below.
EGF
In 1962, Cohen et al. injected purified extracts of submaxillary glands to new born mice and observed an interesting phenotype, ”earlier development of the incisors and eyelid”, as well as a hair development inhibition (COHEN, 1962). Inversely, after sialadenectomy (submaxillary glands removal), animals exhibit smaller and/or thinner organs (epidermis, mammary glands, etc). Later, the peptide involved in these effects was identified and named epidermal growth factor, due to its ability to induce proliferation of epidermal cells. Its mitogenic
EGFR ErbB2 ErbB3 ErbB4
EGF TGFα amphiregulin
HB-EGF βcellulin epiregulin
NRGs
32
properties also applied to many cell types (Carpenter, 1979a). Its chemical and physical properties were extensively studied by the group of Cohen (Taylor et al., 1972). The human gene encoding EGF is located on chromosome 4 (4q25-7) and presents 24 exons. EGF mRNA sequence identified in 1983 revealed that EGF was first expressed as a large precursor, pro- EGF (Scott et al., 1983). After cleavage by proteases, active EGF is released as a 53 amino acids peptide (Savage, Jr. et al., 1972;Savage, Jr. and COHEN, 1972). Expression analysis of EGF by RT-PCR showed the presence of EGF in many tissues such as kidney, salivary glands, prostate, cerebrum (Groenestege et al., 2007).
TGFα (transforming growth factor α)
Using a cDNA clone, TGFα gene was identified on chromosome 13 (2p13) (Derynck et al., 1984;Brissenden et al., 1985). The first effect observed to be linked to TGFα was eye opening as for EGF (Smith et al., 1985). Later, its ability to confer a transformed phenotype to normal cells in vitro was described and, as a consequence, its name given. TGFα is also involved in cell proliferation (Luetteke and Lee, 1990). Thus, its over-expression is associated to hyper-proliferative phenotypes such as skin hypertrophy (Vassar and Fuchs, 1991), renal cysts (Lowden et al., 1994) or hyperplasia of mammary glands (Sandgren et al., 1990;Parham and Jankowski, 1992). On the contrary, null animals for TGFα exhibit defects in the development of the skin, the eyes, hair follicles and dopaminergic neurons (Mann et al., 1993).
HB-EGF
HB-EGF was discovered as a peptide secreted by macrophages and exhibiting high affinity for heparin (Higashiyama et al., 1991). High expression is found in lung, heart, brain, and skeletal muscle (Abraham et al., 1993). It binds EGFR with a higher affinity than EGF and is thus a more potent mitogen. It activates proliferation of smooth muscle cells, fibroblasts but not endothelial cells. In its transmembrane form, HB-EGF exhibits a unique property as it is
33
the receptor for diphtheria toxin (Mitamura et al., 1995). In skeletal muscle, its expression was shown to be induced by MyoD (Chen et al., 1995).
Finally, it is important to know that the EGFR ligands differ in their affinity for the receptor. These differences have a major role on the fate of the receptor: (i) bound by high affinity ligands, EGFR will be preferentially degraded while (ii) bound by low affinity ligands, it will be preferentially recycled to the plasma membrane (Figure 19). Moreover, depending on pH conditions, the affinity between ligands and receptor changes. Thus, the availability of ligands will have an impact on the EGFR’s fate after activation.
Figure 19 : fate of EGFR after ligand binding; adapted from (Roepstorff et al., 2009) Endosomal
compartment
Endosomal compartment
Activated EGFR (ligand bound) Plasma membrane
EGF HB-EGF betacellulin
HB-EGF TGFα Epiregulin Amphiregulin
degradation
34 c. EGFR
In 1979, Carpenter et al. showed that EGF was able to bind to carcinoma cell membranes and to an increase membrane protein phosphorylation (O'Keefe et al., 1974;Carpenter, 1979b;Carpenter, 1979a). Soon after, the receptor for EGF, EGFR, was discovered and its concentration on carcinoma cell membranes was estimated of 40000 to 100000 receptors per cell (Carpenter, 1979b;Carpenter and COHEN, 1979). EGFR is synthesized as a precursor of 1210 amino acids. After cleavage, the 1186 amino acid receptor has a molecular weight of 170kDa, 20% of which due to N-glycosylations. EGFR possesses a hydrophobic domain spanning the cell membrane (figure 20A). Its extra-cellular domain comprises two cystein-rich domains (CR1 and CR2) overlapped by the ligand-binding domain (Saxon and Lee, 1999). Intra-cellular domain possesses an active kinase domain and an actin binding domain (den Hartigh et al., 1992). Moreover, juxtamembrane zone presents a NLS sequence which could target the receptor to the nucleus (Hsu and Hung, 2007;Liao and Carpenter, 2007). Finally, series of tyrosines involved in protein-protein interactions and signaling is observed in the C-terminal part of the receptor, there is a (Riedel et al., 1989).
35
Figure 20: A/ schematic structure of EGFR depicting the main domains of the proteins. B/ intracellular domain of EGFR present many tyrosines involved in protein-protein interaction and cellular signaling.
Figure 1
CR1
CR2
Kinase domain
Ligand binding domain (294543)
151
312
481
622644 687 TM domain
NLS sequence (645657) Fragment missing in EGFR VIII
(6273)
Actin binding domain (984996)
955
1186
Extracellular domain
Intracellular domain
A
Kinase domain
K721 ATP binding
Y845 STAT5
Y920
Y992
Y1045 Y1068 Y1086 Y1148 Y1173 Y891
Y701 STAT1/3
PI3K/Akt
PLCγ
Cbl
Grb2 / Akt-MAPK Grb2
Y1101 Y976
PTP1B / Grb2 / MAPK SHP1 / PLCγ PI3K / Akt
Auto-phosphorylated residues
Src-phosphorylated residues
B
36 i. Activation and activity
Upon ligand binding, EGFR conformation changes, allowing a dimerization loop to form (Yarden and Schlessinger, 1987;Schlessinger, 1988). Dimerization of the receptor is followed by its auto-phosphorylation. Phosphorylated tyrosines in the intracellular domain of EGFR are docking sites for many proteins leading to signal transduction. In particular, phospho-EGFR provides binding sites for SH2- (src homology) and PTB- (phospho-tyrosine binding) domain containing proteins (figure 20B); among them, enzymes (PLCγ, src, Cbl, PTP-1) and adaptor proteins (i.e. Shc, Grb2) were identified (Jorissen et al., 2003). Most of these proteins mediate signal transduction but some are involved in EGFR activation regulation. For example, PTP- 1B and PTP-1C (=SHP1) dephosphorylate the receptor (Tomic et al., 1995) and Src can phosphorylate EGFR on several residues and possibly also on auto-phosphorylation sites (Stover et al., 1995). As Src and EGFR are both kinases which share many substrates (JAKs or NFkB, for example), it is difficult to clearly discriminate Src- or EGFR-mediated signaling.
Moreover, it is important to note that EGFR can activate STATs in a kinase-dependant / JAK- independent manner (Quelle et al., 1995;Leaman et al., 1996). PLCγ/PKC and PI3K pathways mediated cell survival. MAPK cascade is induced after the binding of SOS/Ras to Grb2 and mediates cellular events such as proliferation, differentiation or survival. Thus, the diversity of partner proteins and signaling pathways reflect the diversity of cellular effects mediated by EGFR.
The main and most obvious role of EGFR is the stimulation of cell proliferation (LeVea et al., 2004). This property was identified via its cognate ligand EGF. Stimulation of proliferation by EGFR was also showed to involve mainly the Akt pathway. Mitogenic activity of EGFR was showed in various cell types such as fibroblasts, epithelial cells (Schneider and Wolf, 2008), keratinocytes (Rheinwald and Green, 1977), preodontoblasts (Topham et al., 1987), hepatocytes (Kim and Akaike, 2007;Reinehr and Haussinger, 2009), pro-osteoblast (Kumegawa et al., 1983;Hata et al., 1984), neuron (Gonzalez-Perez et al., 2009) and glial cell precursors (Hayakawa et al., 2007)… EGFR is also an important modulator of cell migration
37
(keratinocytes (Barrandon and Green, 1987;Chen et al., 1993), midgut precursors in Drosophila (Jiang and Edgar, 2009), neurons (Gonzalez-Perez et al., 2009), osteoblasts (Wang et al., 2004)). This effect was further shown to be independent from proliferation (Chen et al., 1994). Moreover, EGFR is involved in cell differentiation either by stimulating (hepatocytes (Kim and Akaike, 2007)) or inhibiting it (keratinocytes (Peus et al., 1997), osteoblast (Zhu et al., 2011)).
The ubiquitous expression of EGFR and its multiple roles explain the lethality of null animals for EGFR. Depending on the genetic background, KO (knock out) animals die at different stages: during implantation, during embryogenesis (mid-gestation) or peri-natally (Threadgill et al., 1995;Sibilia and Wagner, 1995). Some mice survive up to 3 weeks after birth but suffer from multi-organ defects (skin, lung, gastrointestinal tract, brain, liver, mammary glands, eyelid, kidney and heart) (Sibilia and Wagner, 1995;Miettinen et al., 1995).
38
ii. Degradation internalization
Figure 21 : From activation to degradation of EGFR.
Among receptor tyrosine kinase, EGFR is probably the most studied for the understanding of activation-internalization-degradation mechanisms (figure 21). After ligand binding, EGFR dimerizes and auto-phosphorylates on several tyrosine residues (figure 20B).
An ubiquitin ligase, c-Cbl, then binds EGFR on a specific residue, Y1045 (Levkowitz et al., 1999), and negatively regulates the receptor auto-phosphorylation (Ueno et al., 1997) (figure 22). C-cbl presents a RING finger motif and a proline-rich domain necessary for ubiquitination of the receptor and its subsequent degradation (Waterman et al., 1999;Levkowitz et al., 1999);
when these domains are absent (v-Cbl) binding to EGFR results in its recycling to the plasma membrane (Levkowitz et al., 1998). Using, a mutant of EGFR (Y1045F), Waterman et al.
revealed that c-Cbl could also act on the receptor indirectly, via the adaptor protein grb2 (Waterman et al., 2002). Grb2 preferably binds EGFR on Y1068 and Y1086, and recruits it and c-Cbl (via an interaction with its proline rich domain) to clathrin coated pits (Jiang and Sorkin, 2003;Jiang et al., 2003;Stang et al., 2004); However, EGFR internalization was also shown to occur via clathrin independent pathway (Myromslien et al., 2006). Recently, Sigismund et al.
(Sigismund et al., 2008) showed that clathrin mediated internalization of EGFR was important for EGFR signaling and recycling rather than degradation. What really regulate EGFR endocytosis and what role plays ubiquitination are still controversial. Some showed that ubiquitination of EGFR was important for its sorting to endosomes but not for its internalization (Longva et al., 2002;Duan et al., 2008). This was supported by Huang (Huang et al., 2006) results which suggest that poly-lysines mutants of EGFR, poorly ubiquitinated, were
EGFR activation (ligand binding):
Auto-phosphorylation Internalization
Recycling
Degradation
Lysosomal system
Proteasomal system
39
internalized but not properly degraded. On the other hand, mono-ubiquitination of EGFR by c- Cbl was pointed out as a prerequisite for its endocytosis (Mosesson et al., 2003). Even the role of kinase activity during endocytosis is unclear. Sorkina et al. (Sorkina et al., 2002) demonstrated that activation of the receptors was necessary for its recruitment to clathrin coated pits. Using kinase inhibitors or kinase-dead receptor expression, Wang et al. (Wang et al., 2005) showed that EGFR kinase activity was not necessary for either dimerization or internalization but that dimerization (via the loop domain) was indeed necessary for the endocytosis to occur. Later, Huang et al. (Huang et al., 2007), concluded that internalization required an active kinase, Cbl and grb2 while ubiquitination was involved but not necessary.
Another protein, Sprouty, inhibits EGFR internalization and ubiquitination by the inhibition of c- Cbl, thus inducing a recycling of the receptor to the membrane (Wong et al., 2002;Stang et al., 2004;Haglund et al., 2005). After internalization, EGFR transits from early to late endosomes where it still signals (Sorkina et al., 2002) and then, to multivesicular bodies (MVB) where it is degraded by lysosomes (Repetto et al., 2007). Even if lysosomal degradation is the commonly admitted pathway, these past decades, several groups showed that EGFR could also be degraded via the proteasome. So far, many studies showed that inhibition of the proteasome by inhibitors (MG132, bortezomib) decreased EGFR degradation (Longva et al., 2002;Sloss et al., 2008;Feng et al., 2007), increased its activation (Sloss et al., 2008;Sloss et al., 2010) and increased its recycling to the membrane (Levkowitz et al., 1998). Levkowitz et al. also showed that EGFR could be degraded by the 26S, a component of the proteasome, in vitro (Levkowitz et al., 1999). Recently, Zhou et al. (Zhou and Yang, 2011) characterized degradation of activated EGFR via the proteasome as c-Cbl independent.
40
Figure 22: a model of EGFR internalization and degradation mechanism (from (Thien and Langdon, 2005));
(a) Growth factor (GF) binding induces RTK tyrosine phosphorylation and the recruitment of Cbl to the activated receptor by adaptor proteins such as Grb2, which is required to support receptor endocytosis.
This allows the TKB domain to engage a specific phosphotyrosine on the RTK. Activation of Src kinases after GF binding induces the tyrosine phosphorylation of Cbl and other proteins, including Sprouty. The association of Sprouty with the RING finger domain initially inhibits Cbl's recruitment of Ubc enzymes (E2s), but tyrosine phosphorylation of Sprouty removes this inhibition by displacing it from the RING finger to now interact with the TKB domain (b). This allows the RING finger to recruit an E2 conjugating enzyme which promotes the polyubiquitylation of Sprouty (c) and its subsequent proteasomal degradation (d). The TKB domain is freed to target the receptor (pTyr1045 of the EGF receptor) and the E3 ligase function of Cbl can then catalyse the transfer of a ubiquitin molecule from the RING-finger-bound E2 to the RTK (e). This process has been found to be sufficient to mediate receptor internalization. Continued addition of ubiquitin moieties leads to multi-ubiquitylation which marks the RTK for intracellular trafficking to the lysosome, where the receptor is degraded. Tyrosine phosphorylation of Cbl also enhances the recruitment of a CIN85–
endophilin complex through a novel proline-arginine motif (shown as PR). This protein complex helps to promote receptor internalization by causing the membrane to invaginate. Cbl tyrosine phosphorylation also recruits SH2-domain-containing proteins, such as Crk and p85 (not shown), and this recruitment can enhance signalling responses from the receptor. For simplicity, the Grb2–Cbl interaction is not shown in (e), although it still contributes to maintaining Cbl's association with the receptor. Ub, ubiquitin.
