1 Department of Dermatology and Biomolecular Therapeutics (BMT), University of
Vienna, Medical School, Brunnerstr. 59, A-1235 Vienna, Austria
2 Research Institute of Molecular Pathology (IMP), Dr Bohr-Gasse 7, A-1030
Vienna, Austria
3 Division of Molecular Genetics and Centre of Biomedical Genetics, The
Netherlands Cancer Institute, 1066CX Amsterdam, The Netherlands
* Author for correspondence (e-mail: maria.sibilia{at}univie.ac.at)
Accepted 12 June 2003
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SUMMARY |
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Key words: Bone, Hair growth, Heart hypertrophy, Humanised EGFR knock-in mice, Skin
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Introduction |
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Most of the current knowledge about the function of the EGFR and its family
members during normal development derives from the analysis of mutant mice.
Mice lacking Erbb2, Erbb3 and Erbb4 die at midgestation,
because of cardiac dysfunction associated with lack of ventricular
trabeculation, and display abnormal development of the peripheral nervous
system (Gassmann et al., 1995;
Lee et al., 1995
;
Riethmacher et al., 1997
).
Inactivation of the Egfr in mice reveals that mutant mice die at
midgestation (129/Sv), birth (C57BL/6) or can live up to postnatal day 20
(MF1, C3H and CD1) depending on their genetic background. They exhibit
epithelial and neural phenotypes as well as craniofacial malformations
(Miettinen et al., 1995
;
Miettinen et al., 1999
;
Sibilia and Wagner, 1995
;
Threadgill et al., 1995
).
Death in utero results from a placental defect, as the embryonic lethality can
be rescued by generating aggregation chimeras between
Egfr-/- and tetraploid wild-type embryos, the latter
contributing exclusively to extra-embryonic tissues
(Sibilia et al., 1998
).
Independent of the genetic background, surviving Egfr-/-
mice develop a progressive neurodegeneration in the olfactory bulbs and
frontal cortex, which is characterised by massive apoptotic cell death
affecting both neurones and astrocytes
(Kornblum et al., 1998
;
Sibilia et al., 1998
). Ectopic
neurones are always detected in the white matter of the hippocampus,
suggesting that EGFR signalling might be important for neuronal migration
(Sibilia et al., 1998
). The
lack of EGFR does not seem to affect the self-renewing potential of neural
progenitors in response to FGF (Tropepe et
al., 1999
). However, EGFR is crucial for astrocyte development, as
cerebral cortices from Egfr-/- mice contain lower numbers
of astrocytes, which display a severe proliferation defect in vitro
(Kornblum et al., 1998
;
Sibilia et al., 1998
).
Therefore, the EGFR is most probably required for the
proliferation/differentiation of astrocytes and for preserving brain integrity
after birth.
In addition to the neural defects, Egfr-/- mice also
display several abnormalities in epithelial tissues. They are born with open
eyes and show impaired epidermal as well as hair follicle differentiation and
fail to develop a hairy coat most likely because EGFR signalling is necessary
for maintenance of hair follicle integrity
(Miettinen et al., 1995;
Sibilia and Wagner, 1995
;
Threadgill et al., 1995
).
However, Egfr-/- mice do not survive beyond the
termination of the first hair cycle and, therefore, a careful analysis of EGFR
function during skin and hair follicle development could not be performed.
Grafting experiments of Egfr-/- skin explants on athymic
nude mice suggest that Egfr-/- follicles cannot proceed
from the anagen to telogen phase of the hair cycle
(Hansen et al., 1997
). These
alterations eventually lead to necrosis and disappearance of the follicles,
accompanied by strong infiltration of the skin with inflammatory cells
(Hansen et al., 1997
). A
similar but more severe skin and hair phenotype could be detected in
transgenic mice expressing a dominant negative (DN) Egfr (CD533) from
the keratin 5 (K5) promoter (Murillas et
al., 1995
). However, in these mice it could not be excluded that
other Erbb receptors were also inhibited by the DN EGFR, thereby exacerbating
the skin defects. Milder phenotypes characterised by a 'wavy' coat and curly
whiskers are observed in mice deficient for the gene encoding TGF
and
in mice homozygous for the hypomorphic Egfr allele waved 2
(wa2), which carry a point mutation in the kinase domain of the EGFR
resulting in a drastically reduced kinase activity
(Fowler et al., 1995
;
Luetteke et al., 1994
;
Luetteke et al., 1993
;
Mann et al., 1993
).
Numerous studies have documented alterations in EGFR signalling pathways in
the development of human neoplasms
(Olayioye et al., 2000;
Yarden, 2001
). Amplifications,
rearrangements and overexpression of the Egfr gene have been shown to
occur at high frequency in human squamous cell carcinomas and glioblastomas
(Olayioye et al., 2000
;
Yarden, 2001
). The first in
vivo evidence for a direct involvement of the EGFR in epithelial tumour
development stems from the analysis of transgenic mice expressing an activated
form of the Ras activator son of sevenless (Sos) from the K5 promoter
(Sibilia et al., 2000
). All
K5-Sos transgenic mice develop skin tumours only in the presence of a
functional EGFR. K5-Sos transgenic skin papillomas in a EGFR mutant
background show increased apoptosis and are more differentiated indicating
that signalling through the EGFR enhances the survival and inhibits
differentiation of epidermal cells
(Sibilia et al., 2000
). This
is supported by the fact that Egfr is most strongly expressed in the
proliferating compartments of the basal layer of the epidermis and in the
outer root sheath of the hair follicles, and the number of receptors decreases
as keratinocytes enter the pathway of terminal differentiation and migrate to
the suprabasal layers of the epidermis
(Peus et al., 1997
;
Sibilia and Wagner, 1995
;
Stoll et al., 2001
).
The function of the EGFR in adult mice could not be addressed because of the early postnatal lethality of EGFR-deficient mice. Here, we employed a knock-in strategy to generate mice in which the endogenous mouse Egfr is replaced by a human EGFR cDNA that is flanked by loxP sites (hEGFRKI allele). This approach enables the generation of humanised EGFR mice (hEGFRKI/KI mice) to study the functional homologies between the mouse and human EGFR in a conditional way. In addition, the phenotypic consequences of knocking-in well characterised human EGFR mutants can also be addressed. Homozygous hEGFRKI/KI mice are growth retarded but can survive for up to 6 months after birth. However, they display skin and hair growth defects similar to surviving Egfr-/- mice. Interestingly, the neurodegeneration is fully rescued, although hEGFRKI/KI mice develop a severe heart hypertrophy with abnormalities in semilunar valve development. Accelerated chondrocyte and osteoblast differentiation are detected in both hEGFRKI/KI and Egfr-/- mice, suggesting that EGFR signalling negatively regulates bone cell differentiation. The severity of the phenotypes correlates with the expression levels of the hEGFRKI allele in various tissues. In bone cells and epithelial tissues the expression of the hEGFRKI allele is severely reduced, whereas the hEGFRKI allele is expressed at similar levels as the endogenous mouse gene in the brain thereby rescuing the neurodegeneration. Moreover, higher levels of expression of the hEGFRKI allele are detected in the heart and are likely responsible for the development of the heart hypertrophy. These results demonstrate that mice humanised for EGFR display tissue-specific hypomorphic phenotypes thereby uncovering novel functions of the EGFR in bone and heart development.
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Materials and methods |
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Histology
Mice were sacrificed and tissues were fixed in 4% PBS-buffered formaldehyde
and embedded in paraffin wax. Sections (5 µm) were cut and stained with
Haematoxylin and Eosin (Sigma Immunochemicals) according to standard
procedures. Immunohistochemical staining for Ki67 (Novocastra, NCL-Ki67p,
1:1000) was performed using the ABC staining kit (Vector Laboratories)
according to the manufacturer's recommendations. For histomorphometric
analysis, Haematoxylin and Eosin stained cross-sections of hearts from 2.5- to
3.5-month-old hEGFRKI/KI mice (n=4) and
age-matched controls (n=4) were evaluated using an Axioskop
microscope (Carl Zeiss, Oberkochen, Germany) with integrated morphometric
device at 400x magnification. The size of perpendicularly cut
cardiomyocytes was determined in the subendocardial layers of the left
ventricle.
RNAse protection assay
Total RNA was extracted and purified from various organs using TRIzol
reagents (Gibco) and subjected to RNAse protection analysis as previously
described (Fleischmann et al.,
2000). For the generation of the EGFR riboprobe, the region
encompassing the 3' end of the Egfr mouse promoter, the loxP
site and the 5' end of the hEGFR cDNA were cloned into SP64
plasmid, linearised and transcribed in vitro using the in vitro translation
Kit from Stratagene.
Western blot analysis and in vitro EGFR autophosphorylation
assay
Protein lysates were prepared from various tissues, cleared by
centrifugation and either processed for western blot analysis or subjected to
immunoprecipitation employing the rabbit anti EGFR antibody #1001 (Santa Cruz)
as previously described (Sibilia et al.,
2000; Sibilia and Wagner,
1995
). Immunoprecipitated EGFR complexes were resuspended in 20
µl HNTG (20 mM HEPES pH 7.5, 150 mM NaCl, 0.1% Triton X-100, 10% Glycerol)
containing 0.4 µl
32PdATP (10 µC/µl, Amersham) and
incubated for 15 minutes on ice. Kinase reactions were stopped and protein
complexes separated on 10% SDS-PAGE. Gels were dried at 80°C under vacuum
and exposed to X-ray films (Amersham). Western blot analysis was performed
with the anti EGFR antibody #06-129 (Upstate, 1:1000).
Astrocyte cultures
Primary astrocyte cultures were prepared from brains of newborn mice as
previously described (Sibilia et al.,
1998). Cells were plated in DMEM/F12 (1:1, GIBCO) culture medium
containing 10% foetal calf serum (PAA) and after reaching confluency passaged
at a 1:3 split ratio. Before replating, viable cell numbers were determined
using a Neubauer-type haemocytometer with Trypan Blue staining (Sigma). The
primary cultures consisted of >95% GFAP-(Sigma) positive astrocytes.
Osteoblast culture
Primary osteoblasts were isolated from calvariae of neonatal
hEGFRKI/KI, Egfr-/- and wild-type
mice. Calvariae were sequentially digested for 10 minutes at 37°C in
-MEM (GIBCO) containing 0.1% collagenase and 0.2% dispase (Roche).
Cells isolated in fractions 2-5 were combined as an osteoblastic cell
population, expanded twice for 2-3 days in
-MEM with 10% foetal calf
serum (PAA), and either replated in
-MEM for proliferation assays
(105 cells/well in a 6-well plate), or in
-MEM supplemented
with 5 mM ß-glycerolphosphate and 100 µg/ml ascorbic acid (Sigma)
(105 cells/well in a 24-well plate) for bone nodule assays.
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Results |
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Expression analysis in hEGFRKI/KI mice
To compare the expression levels of the hEGFRKI allele
to the endogenous mouse Egfr, total RNA was extracted from various
organs of a hEGFRKI/+ heterozygous mouse. Ribonuclease
(RNAse) protection assay was performed using a riboprobe, which recognises
both the endogenous and the hEGFRKI transcripts, but the
respective protected bands are different in size. Surprisingly, the
hEGFRKI allele was not expressed at lower levels than the
endogenous Egfr in every tissue
(Fig. 2A). Organs, which are
mostly composed of epithelial cells such as liver, lung, skin and stomach
expressed significantly lower levels of the hEGFRKI allele
compared with the endogenous allele (Fig.
2A). By contrast, in brain regions such as cortex and hippocampus,
in kidney and in thymus the hEGFRKI allele was expressed
at similar levels as the endogenous mouse allele
(Fig. 2A). Interestingly, in
the heart the hEGFRKI allele seemed to be expressed at
even higher levels than the endogenous one
(Fig. 2A). These results
suggest that the hEGFRKI allele behaves like a hypomorph
particularly in epithelial tissues.
|
Rescue of the brain phenotypes in hEGFRKI/KI
mice
In the brain, the hEGFRKI allele is expressed at
similar levels than the endogenous Egfr
(Fig. 2A). In order to
investigate whether in hEGFRKI/KI mice the brain
phenotypes were rescued, brains were isolated at different stages after birth
and compared to Egfr-/- and controls. At all stages
analysed, brains of hEGFRKI/KI mice appeared structurally
normal and comparable to controls (Fig.
3A,B). Histological sections through the frontal cortex of a
3-month-old hEGFRKI/KI mouse did not reveal any signs of
degeneration and apoptosis and the cortical tissues appeared normal and
comparable to controls (Fig.
3C,E). By contrast, in the cortex of Egfr-/-
mice morphological changes consisting of nuclear condensations and diminished
cell densities could be observed already at postnatal day 7
(Fig. 3G)
(Sibilia et al., 1998).
Similar to controls, in hippocampal sections of hEGFRKI/KI
mice no ectopic neurones could be observed at any stage, whereas in
Egfr-/- mice nests of ectopic neurones were always present
(Fig. 3D,F,H)
(Sibilia et al., 1998
). In
addition, other brain regions such as the olfactory bulb, thalamus and
cerebellum of hEGFRKI/KI mice appeared normal and
comparable with controls (data not shown).
|
Severe hair follicle and hair cycle defects in
hEGFRKI/KI mice
The hEGFRKI allele is not efficiently expressed in the
skin based on the RNAse protection analysis
(Fig. 2A). Moreover,
hEGFRKI/KI mice displayed curly whiskers and the
development of the first coat hair was impaired
(Fig. 1C). These phenotypes
greatly resemble the ones observed in Egfr-/- mice
(Miettinen et al., 1995;
Sibilia and Wagner, 1995
;
Threadgill et al., 1995
).
However, in contrast to Egfr-/- mice, which do not survive
longer than postnatal day 20, hEGFRKI/KI mice survive up
to 6 months after birth and, therefore, represent a useful model to analyse
how reduced EGFR expression affects hair development. The skin of
hEGFRKI/KI mice was isolated postnatally at different
stages of the hair cycle, after 18 days (end of first cycle, catagen/early
telogen), after 1 month (second cycle, end of anagen/early catagen) and after
3 months (resting phase, telogen). Histological examination of
hEGFRKI/KI skins showed striking alterations in the
morphology and distribution of hair follicles. Around day 18, hair follicles
of control mouse skin go through a telogen stage and are confined to the upper
layer of the dermis (Fig. 4A).
By contrast, at this stage follicles of hEGFRKI/KI mice
were still much longer reaching deep into the subdermal adipose tissue,
suggesting that they had failed to enter into catagen and were still in anagen
(Fig. 4B,G).
hEGFRKI/KI follicles started to appear much larger and
hyperplastic compared with control follicles and this phenotype was even more
pronounced at 1 month when the anagen of the second hair cycle had started
(Fig. 4C,D). After completion
of the second hair cycle, follicles remain for a long period (around 40 days)
in telogen (Fig. 4G). During
this stage (at the age of 3 months), control hair follicles are short and
localised in the upper layers of the dermis
(Fig. 4E). By contrast, in
hEGFRKI/KI mice, only very few structurally abnormal
follicles were present in the upper dermis and most of the follicles, which
had remained stuck in the subdermal fat layers, were degenerated
(Fig. 4F). The cell layers
composing the hair follicles were very thin and partly destroyed
(Fig. 4F). Severe fibrosis of
the dermis and subdermal fat tissue with massive infiltration of inflammatory
cells was observed and had most likely been triggered by the exposure of naked
hair shafts from the degenerating follicles
(Fig. 4F and data not shown).
The inflammatory infiltrate mainly contained macrophages, lymphocytes,
neutrophils and multinucleated giant cells (data not shown). The progressive
degeneration and inflammation led to the loss of most follicles over time and
the majority of hEGFRKI/KI mice were completely bald at
the age of 5-7 months (data not shown). These results indicate that EGFR
signalling is absolutely required for hair cycle progression. Moreover, EGFR
is also essential for proper hair follicle differentiation, orientation and
migration, as well as to preserve hair follicle integrity during the hair
cycle.
|
|
hEGFRKI/KI mice develop heart hypertrophy
hEGFRKI/KI mice can survive up to 6 months after birth,
but often sudden death is observed at earlier times. To investigate the
possible cause of lethality, hEGFRKI/KI mice were
sacrificed and different organ systems were analysed. An increase in heart
size was consistently observed, which was already visible at 3 weeks after
birth and became more pronounced with age
(Fig. 6A). Comparison of heart
weights at 3 months of age showed that hEGFRKI/KI hearts
were about twice as heavy as control hEGFRKI/+ and
EGFR+/+ hearts (Fig.
6B). This difference was even more dramatic when the heart weights
were corrected for the body weights considering that
hEGFRKI/KI mice are smaller and only about half of the
size of control mice (Fig. 6B).
Histological cross-sections through the hearts of
hEGFRKI/KI mice revealed a severe hypertrophy with
dramatically increased thickness of the left ventricular wall and the
interventricular septum at 3 weeks after birth
(Fig. 6D). These defects became
more severe with increasing age (Fig.
6G). Cardiomyocytes of 2.5- to 3.5-month-old
hEGFRKI/KI mice were hypertrophic and measurements of
their mean cross-sectional areas revealed a 1.9-fold increase compared with
controls (mean cross sectional area±s.d.: 309.7±97.6
µm2 versus 166.3±16.3 µm2). None of these
defects was ever observed in control hEGFRKI/+ and
Egfr+/+ mice (Fig.
6C,F; data not shown). In 3-week-old Egfr-/-
mice, the heart size was normal, the heart-to-body weight ratios were
comparable with controls and histological sections did not reveal signs of
hypertrophy (Fig. 6E; data not
shown). Cardiac hypertrophies can occur as primary myocardial diseases or as a
consequence of other conditions such as valve defects leading to valvular
stenosis and/or regurgitation (Katz,
1990). When compared with controls, histological examinations of
hEGFRKI/KI hearts revealed that the cusps of the pulmonary
and aortic valves were thickened and hypercellular, a condition that most
likely resulted from the accumulation of mesenchymal cells
(Fig. 6H,I). By contrast, the
atrioventricular valves were not affected and appeared normal (data not
shown). The same semilunar valve defects as in hEGFRKI/KI
hearts were also observed in Egfr-/- mice (data not shown)
indicating that these defects result from the absence of expression of the
EGFR in valve structures. As Egfr-/- mice do not display
severe myocardial hypertrophy, these results suggest that in
hEGFRKI/KI mice the semilunar valve defects together with
the increased expression of the hEGFRKI allele in the
myocardium (Fig. 2A) exacerbate
the heart hypertrophy thereby contributing to increased lethality of the
mice.
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Discussion |
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In all the brain regions analysed, the hEGFRKI allele
was expressed at similar levels to the endogenous mouse gene. In fact, in
brains of homozygote hEGFRKI/KI mice none of the defects
observed in Egfr-/- mice could be detected.
hEGFRKI/KI mice do not develop the cortical
neurodegeneration and astrocyte proliferation in vitro is comparable with
controls. Only after Cre-mediated deletion of the floxed
hEGFRKI allele a severe proliferation defect was observed
in astrocytes cultured in vitro, demonstrating that the EGFR is required for
proper proliferation of these cells. hEGFRKI/KI mice did
also not contain ectopic neurones in the hippocampus, a phenotype that is
always observed in Egfr-/- mice
(Sibilia et al., 1998).
Because all the brain defects observed in Egfr-/- mice
were rescued in hEGFRKI/KI mice, it can be assumed that
the amount of EGFR protein present in brain cells of
hEGFRKI/KI mice is similar to that in wild-type controls.
Moreover, it appears that the binding affinity of the mouse EGFR ligands to
the human EGFR is not significantly altered, suggesting that impaired receptor
activation is not contributing to the hypomorphic defects seen in other
tissues.
The observed phenotypes are probably determined by the level of
transcription of the hEGFRKI allele in various tissues. In
epithelial tissues such as the skin and hair follicles where the
hEGFRKI allele is poorly transcribed, severe defects were
observed that resemble those observed in Egfr-/- mice
(Miettinen et al., 1995;
Sibilia and Wagner, 1995
;
Threadgill et al., 1995
).
Because Egfr-/- mice do not survive longer than P20, the
skin and hair phenotypes could not be properly analysed directly in mutant
mice. The hEGFRKI/KI mice proved to be extremely useful to
analyse how the absence of the EGFR affects hair follicle differentiation,
migration and cycling. After the first hair cycle, hair follicles of
hEGFRKI/KI mice fail to enter into catagen and remain in
aberrant anagen, indicating that EGFR signalling is needed to regulate hair
cycle progression. The EGFR also seems to be required to preserve hair
follicle integrity over time, because in its absence the follicles are
degraded probably by the infiltrating inflammatory cells. The aberrant
localisation of hEGFRKI/KI hair follicles in the skin
might have triggered an immunological response, which results in the
destruction of the follicles. Alternatively, as the EGFR has been shown to
inhibit differentiation and promote survival of epithelial cells, it is
possible that in the absence of EGFR expression, hair follicles might be
impaired in their survival capacity and/or undergo premature differentiation.
With time, this would lead to hair follicle destruction and release of
follicle material into the dermis, which in turn would trigger the
immunological response. Similar hair abnormalities have also been observed in
grafting experiments with Egfr-/- skin onto
immunodeficient mice and in a skin targeted DN Egfr transgenic mouse
model (Hansen et al., 1997
;
Murillas et al., 1995
).
However, in the latter model it could not be excluded that the severity of the
phenotypes observed was determined not only by inhibition of the EGFR itself
but also by the inhibition of the other Erbb family members which are also
expressed in the epidermis (Stoll et al.,
2001
). As the skin and hair follicle defects observed in
hEGFRKI/KI and the DN EGFR transgenic mice are
very similar, it can be concluded that these phenotypes mainly result from
direct EGFR inhibition.
EGFR signalling does not only seem to inhibit epithelial cell
differentiation, but also the differentiation of bone cells. Bone development
starts with the formation of mesenchymal condensations that first
differentiate into chondrocytes, which become hypertrophic and are then
invaded by blood vessels, bone-forming osteoblasts and bone-resorbing
osteoclasts (Karsenty and Wagner,
2002). Active remodelling ultimately gives rise to a bone with
growth plate and a central marrow cavity
(Karsenty and Wagner, 2002
).
Numerous extracellular factors, such as hormones, growth factors and
cytokines, modulate bone remodelling via differentiation and proliferation of
bone cells, and this process has to be tightly regulated in order to prevent
bone diseases (Karsenty and Wagner,
2002
). The EGFR is expressed in chondroblasts of the developing
ossification centres and the EGFR has also been shown to be expressed in
osteoblasts and osteocytes in vivo
(Davideau et al., 1995
).
However, no bone defects have been reported so far for mice defective in EGFR
signalling. A recent report has shown that transgenic mice expressing EGF
ubiquitously are growth retarded, display defective chondrocyte development in
the growth plate and osteoblasts accumulate in the endosteum and periosteum
(Chan and Wong, 2000
).
Controversial in vitro data regarding the role of EGFR signalling in bone
cells have been reported, although two studies suggest that EGFR might
stimulate osteogenic cell proliferation and suppress the differentiation into
mature osteoblasts and chondrocytes (Chien
et al., 2000
; Yoon et al.,
2000
). In Egfr-/- and
hEGFRKI/KI mice both osteoblasts and chondrocytes display
increased differentiation in vitro and in vivo, respectively. As the
hEGFRKI allele was poorly expressed in the affected cell
types, it is likely that these defects result from the lack of Egfr
expression. In the long bones of hEGFRKI/KI mice, the
region of hypertrophic chondrocytes in the growth plate was significantly
increased, suggesting that chondrocytes had undergone premature
differentiation. This phenotype was even more severe in
Egfr-/- mice, where Egfr expression is completely
absent, indicating that EGFR signalling prevents differentiation of
chondrocytes. Although no overt differences in bone mass could be detected in
newborn Egfr-/- and hEGFRKI/KI mice,
calvarial osteoblasts from these mice proliferate more slowly and display an
increased differentiation capacity in vitro. It is likely that EGFR signalling
is important to keep osteoblasts and chondrocytes in a proliferative state and
inhibit their differentiation, thus allowing proper development of long bones.
In the absence of EGFR expression, accelerated differentiation of chondrocytes
and possibly decreased proliferation of osteoblasts can disturb longitudinal
growth of long bones leading to severe growth retardation in the mice.
hEGFRKI/KI mice develop a severe heart hypertrophy with
dramatically increased thickness of the ventricular walls and the
interventricular septum, which was visible already at 3 weeks after birth and
became more pronounced as age proceeded. Interestingly,
Egfr-/- mice did not display signs of hypertrophy at this
age, suggesting that this phenotype was most probably not due to the lack of
EGFR expression. Cardiac hypertrophy is an adaptive response to many forms of
disorders such as hypertension, myocardial infarction and valve defects aimed
to augment the cardiac output (Katz,
1990). However, sustained hypertrophy usually leads to ventricular
dilatation with consequent heart failure and sudden death
(Katz, 1990
). The response of
cardiomyocytes to hypertrophic signals involves an increase in cell size and
protein synthesis, induction of immediate-early genes such as AP-1 and
re-expression of several foetal myocardial structural proteins
(Sadoshima and Izumo, 1997
).
It has been shown that treatment of rat cardiomyocytes with EGF can trigger a
hypertrophic response resulting in increased protein synthesis and
transcriptional activation of Fos and Jun
(Rebsamen et al., 2000
).
Similarly, G-protein-coupled receptor (GPCR) agonists were shown to
transactivate the EGFR via shedding of HBEGF by the metalloproteinase ADAM12
thereby inducing heart hypertrophy (Asakura
et al., 2002
; Prenzel et al.,
1999
). Moreover, EGF increases adenyl cyclase activity and cAMP
accumulation, thus enhancing the heart contractility and beating rate
(Nair et al., 1993
). As
heart-specific expression of the hEGFRKI allele seemed to
be slightly higher than the endogenous wild-type allele, we speculate that
increased EGFR signalling in cardiomyocytes is contributing to the development
of the heart hypertrophy.
hEGFRKI/KI mice also display semilunar valve defects,
which are known to induce aortic stenosis and regurgitation, and as a
consequence can lead to heart hypertrophy. Heart valves develop from the
endocardium, a specialised endothelium that undergoes complex
epithelial-mesenchymal transformations leading to the formation of mesenchymal
outgrowths (also called the cardiac cushions), from which the mature valve
leaflets originate (Towbin and Belmont,
2000). The pulmonary and aortic, but not the atrioventricular
valves of hEGFRKI/KI hearts were thickened and
hypercellular, which most probably results from the accumulation of
mesenchymal cells. As similar valve defects were also present in
Egfr-/- hearts, it is likely that the
hEGFRKI allele is not expressed in the developing valves.
Although we were unable to detect EGFR expression in wild-type valve
structures by immunohistochemistry, we believe that the valve defects of
hEGFRKI/KI and Egfr-/- hearts result
from lack of EGFR expression in these structures. The other EGFR family
members Erbb2 and Erbb4 are expressed in the endocardium and have been shown
to be involved in the formation of the mesenchymal cushions
(Camenisch et al., 2002
;
Erickson et al., 1997
). It
still remains to be determined how EGFR alone or in combination with these
receptors regulates mesenchyme development and its differentiation into mature
valve structures.
By studying the genetic interaction between EGFR and the protein-tyrosine
phosphatase Shp2, it was shown that EGFR is required for semilunar valve
development (Chen et al.,
2000). Compound mutants between Shp2 and the hypomorphic
EGFRwa2/wa2 mouse strains showed signs of aortic stenosis
and regurgitation with subsequent development of myocardial hypertrophy
(Chen et al., 2000
). Although
these defects were most severe in compound mutants, thickened semilunar valves
were also observed in EGFRwa2/wa2 and
Egfr-/- mice, whereas no cardiac dilatation were reported
for the single EGFR mutants (Chen et al.,
2000
). Similarly, we could not detect any signs of myocardial
hypertrophy in 3-week-old Egfr-/- mice, although semilunar
valve thickening was observed. By contrast, the heart of
hEGFRKI/KI mice at this stage was already enlarged and
displayed a severe thickening of the myocardial walls. Therefore, it is likely
that the severe hypertrophy observed in hEGFRKI/KI mice
results from the malformations of the valves and from the enhanced
hypertrophic response of cardiomyocytes to increased EGFR signalling, as the
hEGFRKI allele is expressed at higher levels in the
myocardium. These defects can lead to a severe heart condition, which is
probably responsible for the lethality of hEGFRKI/KI mice,
whereas the valve defects alone, as seen in Egfrwa2/wa2
mice do not seem to increase the mortality of these mice. Moreover, as
hEGFRKI/+ mice do not develop heart hypertrophy, it seems
unlikely that enhanced expression of the EGFR in the myocardium alone can lead
to the development of this heart condition. The conditional
hEGFRKI/KI mice may provide a useful model not only to
study aortic valve diseases and developmental aspects of bone differentiation,
but also to test novel therapies aimed to inhibit EGFR signalling for the
treatment of cardiac hypertrophies and hyperproliferative bone diseases.
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ACKNOWLEDGMENTS |
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Footnotes |
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Present address: Institute of Clinical Pathology, University Hospital
Zurich, Schmelzbergstrasse 12, CH-8091 Zurich, Switzerland
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REFERENCES |
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