Epidermal Growth Factor as a Biologic Switch in Hair Growth Cycle*
Kingston K. L. Mak and
Siu Yuen Chan
From the
Department of Paediatrics and Adolescent Medicine, the University of Hong
Kong, Faculty of Medicine Building, 21 Sassoon Road, Hong Kong
Received for publication, November 27, 2002
, and in revised form, April 14, 2003.
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ABSTRACT
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The hair growth cycle consists of three stages known as the anagen
(growing), catagen (involution), and telogen (resting) phases. This cyclical
growth of hair is regulated by a diversity of growth factors. Although normal
expression of both epidermal growth factor and its receptor (EGFR) in the
outer root sheath is down-regulated with the completion of follicular growth,
here we show that continuous expression of epidermal growth factor in hair
follicles of transgenic mice arrested follicular development at the final
stage of morphogenesis. Data from immunoprecipitation and immunoblotting
showed that epidermal growth factor signals through EGFR/ErbB2 heterodimers in
skin. Furthermore, topical application of tyrphostin AG1478 or AG825, specific
inhibitors of EGFR and ErbB2, respectively, completely inhibited new hair
growth in wild type mice but not in transgenic mice. When the transgenic mice
were crossed with waved-2 mice, which possess a lower kinase activity
of EGFR, the hair phenotype was rescued in the offspring. Taken together,
these data suggest that EGFR signaling is indispensable for the initiation of
hair growth. On the other hand, continuous expression of epidermal growth
factor prevents entry into the catagen phase. We propose that epidermal growth
factor functions as a biologic switch that is turned on and off in hair
follicles at the beginning and end of the anagen phase of the hair cycle,
guarding the entry to and exit from the anagen phase.
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INTRODUCTION
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The hair follicle is a regenerating system that undergoes cycles of renewal
in three phases known as anagen, catagen, and telogen. Various signaling
molecules are involved in different phases of hair growth
(1). However, the mechanisms
underlying the switch from phase to phase remain largely unknown. Among the
many growth factors expressed in association with the development of hair
follicles, epidermal growth factor
(EGF)1 is known for
its effects on skin and hair development
(2). Subcutaneous
administration of EGF for 2 weeks into neonatal mice delayed the development
of hair follicles and epidermis. Both the growth of hair bulbs and hair fiber
production were retarded. The proliferative activity of the epidermis at birth
was maintained for 8 days, resulting in a thicker epidermis compared with
control mice. However, the delay in skin development was not prolonged, and
the mitotic index and thickness of the epidermis declined to that of control
values a few days later. By Day 41, final hair length and diameter were not
significantly reduced (3). EGF
also had no effect on back skin when injected into mice with ages ranging from
12 to 20 days (2). The authors
suggested that EGF was only active in skin during the neonatal period
(3). In contrast, EGF induces
follicle regression besides inhibiting hair fiber production and stimulating
mitosis of the basal epidermal cells when infused into adult sheep
(4). Subsequently, in
vitro studies with isolated human hair follicles have shown that EGF
stimulates DNA synthesis in the outer root sheath and hair follicle elongation
but inhibits hair fiber production. DNA synthesis in the matrix cells is
inhibited, and they remain connected to the dermal papilla by a thin strand of
epithelial cells, inducing an artificial catagen-like effect
(5). Despite all these early
studies, the physiological role of EGF on hair development and the signaling
pathways involved remain unclear. It has been shown that administration of
anti-EGF serum can cause accelerated hair growth in newborn mice
(6). This suggests that
endogenous EGF acts as an inhibitory molecule during follicle
morphogenesis.
There are four members of the EGF receptor family namely EGFR or ErbB1,
ErbB2, ErbB3, and ErbB4. EGF, transforming growth factor (TGF)-
,
amphiregulin, and possibly epigen
(7) bind EGFR that can
subsequently form homodimers or heterodimers with any ErbB receptors, whereas
-cellulin, epiregulin, and heparin-binding EGF are ligands for both EGFR
and ErbB4 (8). A null mutation
in Egfr is lethal during embryonic development. Only certain strains
of mutant mice can survive up to 3 weeks after birth, with severe impairment
in development of multiple organs including skin and failure of hair growth
(911).
Hair follicles in the neonatal skin cannot develop normally even when grafted
to nude mice (12). In
transgenic mice expressing a dominant negative mutant of EGFR in the epidermis
and outer root sheath, the hair follicles cannot progress through follicle
morphogenesis and eventually undergo necrosis
(13). A loss of function
mutation in Tgf-
(14,
15) or a specific point
mutation in Egfr (16,
17) results in a less severe
phenotype of curly whiskers and pronounced waviness of first hair coat.
Irregularly distributed hair follicles were found in the dermis and subcutis
layers. Surprisingly, triple null mice lacking EGF, amphiregulin, and
TGF-
demonstrate the same hair and skin phenotype as the TGF-
null mice (18). This suggests
that other members of the EGF family can compensate for the loss of the three
EGFR ligands. Transgenic mice overexpressing the various EGFR ligands in skin
are useful for addressing their specific roles.
Both amphiregulin and heparin-binding EGF are expressed in skin
(19,
20). Upon culture of
keratinocytes, the expression of both molecules increases, and they become the
major autocrine factors for proliferation
(21). Furthermore, trauma can
lead to metalloproteinase-mediated cleavage of amphiregulin, heparin-binding
EGF, and TGF-
from their transmembrane precursors. The evidence
suggests that ectodomain shedding of these growth factors is required for
wound healing (22,
23). These three EGFR ligands
are probably more important in wound healing and in skin pathology.
Overexpression of amphiregulin in transgenic mice induces an epidermal
hyperproliferation and inflammation, which is similar to psoriasis, and is in
agreement with the increased expression of this moiety in psoriasis patients.
TGF-
is expressed in the inner
(14) and outer root sheath
(24) in actively growing
follicles. TGF-
transgenic mice have been reported to exhibit a thicker
epidermis and stunted hair growth, but the epidermal scaliness disappeared,
and hair growth was partially restored at 56 weeks despite a persistent
expression of the transgene. Benign skin papillomas were found in older
animals. EGF is also expressed in the outer root sheath in the growing hair
follicles (25), although the
role of EGF in hair biology has not been clarified using the transgenic
approach. For this purpose we have generated and analyzed a transgenic mouse
line constitutively expressing EGF in skin and hair follicles. Our data
suggest a specific role for EGF in the control of the hair cycle.
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EXPERIMENTAL PROCEDURES
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AnimalsMice were cared for in the Laboratory Animal Unit at
the University of Hong Kong. Waved-2 mice bred with a mixed C57BL/6
and 129/sv background were a generous gift from Dr. Ashley Dunn (Ludwig
Institute for Cancer Research, Royal Melbourne Hospital, Australia).
Waved-2 mice have a hypomorphic allele of Egfr that contains
a point mutation near the tyrosine kinase domain
(16,
17).
Generation of Transgenic MiceThe full-length mouse
Egf cDNA of 4.6 kb was isolated from a C57BL/6 kidney cDNA library
(Invitrogen) and confirmed by sequencing. EGF transgenic mice were established
using the promoter from cytomegalovirus to drive full-length Egf cDNA
joined to an SV40 poly(A). The injection fragment of 6.5 kb was released and
purified using the QIAEX gel extraction kit (Qiagen) and then passed through a
spin-X column (Costar). The purified DNA fragment was introduced at 68
ng/µl into the pronucleus of FVB/N embryos by standard microinjection
procedures (26). Transgenic
mice were identified by PCR of tail DNA using primers for Egf cDNA
and confirmed by Southern blotting (data not shown). The primer sequences
(5' to 3') were gagaatgccgcctgcaccaacac and
agggttctttggggggtttgatag, giving a PCR product of 435 bp.
ImmunohistochemistryThe locations of EGF expression on
dorsal skin in heterozygous animals were determined at birth (Day 1), Days 4,
7, 10, 14, 17, 21, 28, 35, 40, and 50. After the first hair cycle, hair growth
was asynchronized in wild type animals, and therefore no further time points
were examined. Skin samples were orientated and snap-frozen using the Hofmann
technique (27). Cryosections
(10 µm) were stored at -70 °C and equilibrated to room temperature
before use. The sections were fixed in 4% paraformaldehyde for 10 min, washed,
and then treated with 3% hydrogen peroxide, washed thoroughly with distilled
water, and then washed again with Tris-buffered saline (TBS), pH 7.4. After
incubation with 10% normal goat serum, 3.33 µg/ml rabbit antimouse EGF
(Upstate Biotechnology, Inc.) was applied overnight at 4 °C. Then the
sections were washed with TBS and incubated with biotinylated goat anti-rabbit
immunoglobulins (Dako, UK) diluted 1 in 100 for 1 h. The sections were washed
with TBS and incubated with streptavidin-biotinylated peroxidase complex
(Dako) for 30 min. The sections were washed again and incubated for 5 min with
a 1 mg/ml chromogen solution (Dako). After counterstaining with hematoxylin,
the sections were dehydrated and mounted with Permount. As negative controls,
the primary antibody was omitted or replaced with the same concentration of
rabbit IgG (Dako). To confirm signal specificity, the primary antibody was
blocked with 15 µg/ml mouse EGF (PeproTech, Israel) at 4 °C overnight
before use.
Terminal dUTP Nick-end Labeling AssayThe procedures
recommended for the Apoptosis Detection kit (Promega) were followed on fixed
frozen skin sections.
Measurement of Skin ThicknessHematoxylin and eosin
(H&E) staining was performed on frozen sections (three heterozygous mice
per time point, with control mice from nontransgenic littermates). Three
representative sections for each sample were analyzed using a Leica DMRB
microscope. The thickness of epidermal, dermal, and subcutaneous layers was
measured on digital images using the Stereo Investigator 4.0 software (Leica
Ltd., Germany). A total of 50 data sets were collected from the three samples
at each time point. All data were reported as mean ± S.D. and analyzed
using the two-tailed unpaired Student's t test.
Protein Preparation and ImmunoblottingMice were sacrificed
by cervical dislocation, and mid-dorsal skin, liver, and brain were
immediately collected for protein preparation. Tissues were homogenized in 20
mM Hepes, pH 7.4, 2 mM MgCl2, 100 µg/ml
phenylmethylsulfonyl fluoride, 1 mM sodium orthovanadate, and 2
µg/ml aprotinin. The homogenized samples were centrifuged at 4 °C for 5
min, and the supernatants were collected. Samples (80-µg aliquots as
determined by the Coomassie Blue dye binding assay; Bio-Rad) were separated on
a 7.5% polyacrylamide gel and transferred to a Hybond-ECL membrane (Amersham
Biosciences). For immunoblotting, the procedures in the chemiluminescent
detection system (ECL Detection System; Amersham Biosciences) were followed.
The primary antibodies and working concentrations are as follows: rabbit
anti-mouse EGF (Upstate Biotechnology, Inc., 2 µg/ml); mouse anti-human
EGFR (Transduction Laboratories, 1 µg/ml); rabbit anti-mouse ErbB2 (Santa
Cruz Biotechnology, 0.2 µg/ml); rabbit anti-mouse ErbB3 (Santa Cruz
Biotechnology, 0.2 µg/ml); and mouse anti-phosphotyrosine PY20
(Transduction Laboratories, 1 µg/ml). The secondary antibodies and working
dilutions are as follows: goat anti-mouse IgG (Transduction Laboratories,
1:5000) and goat anti-rabbit IgG (Amersham Biosciences, 1:4000).
ImmunoprecipitationSpecific antibody (1 µg) was added to
500 µg of crude protein samples, and the mixture was made up to a final
volume of 0.5 ml with IP buffer (1% Triton X-100, 0.5% Nonidet P-40, 150
mM NaCl, 10 mM Tris, pH 7.4, 1 mM EDTA, 1
mM EGTA, pH 8.0, 1 mM Na3VO4, 100
µg/ml phenylmethylsulfonyl fluoride, and 2 µg/ml aprotinin). Samples
were incubated at 4 °C for 2 h with gentle rocking. Protein A- or
G-agarose (40 µl) was then added, and incubation was continued for 2 h. The
samples were centrifuged for 1 min at 4 °C, and the supernatants were
discarded. The agarose beads were washed with 1 ml of IP buffer at 4 °C
for 5 min. The samples were spun down and the washing procedures repeated 3
more times. Finally, 40 µl of sample buffer with 5%
-mercaptoethanol
were added to the agarose beads and the samples used for immunoblotting.
Topical Application of the ErbB1 Inhibitor AG1478 and ErbB2 Inhibitor
AG825Heterozygous transgenic mice (Day 23, n = 4) and
nontransgenic littermates (n = 4) were each divided into two groups
of treatment and control animals. Tyrphostin AG1478 (10 µg, Sigma) or
tyrphostin AG825 (60 µg, Sigma) was dissolved in 1 ml of Me2SO
and then mixed with 2 g of a neutral aqueous cream base (Orjon, Australia). An
area starting midway along the back of the mice was shaved 1 day before
application of the inhibitor or cream base. AG1478 or AG825 cream was applied
topically every day for 15 consecutive days. In the case of the controls, only
the cream base was administered. Each treatment was repeated on three litters
(n = 8). For large litters of more than 8 mice, 10 µg of AG1478
together with 60 µg of AG825 were added to 1 ml of Me2SO, mixed
with 2 g of cream, and applied to the rest of the litter.
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RESULTS
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Characterization of EGF Transgenic MiceAlthough seven
transgenic lines were generated with the CMV-Egf construct, only one line,
designated 62.7, manifested phenotypic changes, and the heterozygous animals
were used for detailed analysis here. Line 62.7 exhibited thin hair, curly
vibrissae, and stunted growth (Fig. 1,
a and b). Homozygous bred transgenic mice showed
an even more severe phenotype and appeared to be nearly nude. When bred into
different genetic backgrounds, including C57BL/6, ICR, F1 hybrid of C57BL/6
and FVB, transgenic progeny showed similar hair phenotype as the FVB founder
(not shown). From immunohistochemistry, EGF was expressed in the sebaceous
glands, outer root sheath of hair follicles, stratum granulosum, and basal
keratinocytes of the epidermis during follicular morphogenesis in wild type
mice (Fig. 1, c and
d). The EGF expression in hair follicles was switched off
once they had entered the telogen phase
(Fig. 1e). In
contrast, transgenic mice demonstrated constitutive expression of EGF in the
hair follicles (Fig. 1,
fh) which appeared to remain in stage 78 of
follicular morphogenesis according to published guidelines by Paus et al.
(28). Results from reverse
transcriptase-PCR and immunoblotting confirmed that EGF was strongly expressed
in the skin of 62.7 mice with accumulation of the EGF precursor, which was in
contrast to age-matched wild type mice
(Fig. 1i). EGF protein
could also be detected in the liver and brain of transgenic animals, and this
subtle phenotype is still under investigation. Only increased levels of EGF
mRNA, but not of protein, were detected in other transgenic lines that
correlated with the absence of abnormalities in the skin or other organs at
the morphological or histological level.

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FIG. 1. Hair phenotype in EGF transgenic mice. a, representative
transgenic mouse (Tg) at Day 21 with thin fur throughout the whole
body as compared with the wild type (WT) littermate. b,
vibrissae of transgenic mice were curled anteriorly and significantly shorter
than normal. ch, immunohistochemistry of EGF in skin.
c, endogenous EGF expression in wild type skin at Day 4; d,
close-up of a hair follicle showing EGF expression in the outer root sheath.
e, EGF was not detected in hair follicles of wild type mice at Day
21. f, increase in EGF expression in Day 4 transgenic skin;
g, close-up of a hair follicle showing strong EGF expression in the
outer root sheath. h, EGF expression in hair follicles was still
detectable at Day 21 in transgenic skin. i, transgene expression in
seven transgenic lines. Only line 62.7 showed markedly increased expression of
Egf in skin in both reverse transcriptase-PCR and immunoblots.
GAPDH, glyceraldehyde-3-phosphate dehydrogenase. Scale bar,
200 µm.
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Association of EGF Expression with Structural Changes in the Skin and
Hair CyclingFrom H&E-stained sections
(Fig. 2), wild type mice and
transgenic mice had the same skin histology at birth (Day 1,
Fig. 2, a and
b). However, at Day 4, retardation of hair follicle and
skin development was observed in the transgenic mice
(Fig. 2, c and
d). Although more than 90% of hair follicles had grown
into the subcutis layer (stages 7 and 8), an increase in hair follicle size
and total skin thickness was obvious in the wild type mice only. Furthermore,
transgenic skin samples showed a thicker epidermis when compared with wild
type animals at Day 4. There was an increase in the number of cell layers at
the basal layer as well as the stratum corneum, which suggested epidermal
hyperproliferation and a delay in epidermal differentiation
(Fig. 2, c and
d, insets). The hair follicles in wild type
animals were in the dermis layer as the hair cycle proceeded to the
catagen/telogen phase at Day 21 (Fig.
2e). They were in the first anagen at Day 28 and already
at telogen by Day 35. The duration of first anagen was much shorter than that
reported for C57BL/6 (29). For
transgenic animals, hair follicles were found in the subcutis layer when
sampled at Days 7, 10, 14, 17, 21, 28, 35, and 40, and no hair follicles of
catagen or telogen phase were observed at any of these time points.
Furthermore, no apoptotic cells were detected in either the lower bulb or the
root sheath of transgenic follicles by terminal dUTP nick-end labeling
staining at any of the time points studied (not shown). This has been used to
distinguish the late anagen follicles from those in early catagen
(1). At Day 50, some telogen
follicles could be occasionally found in certain transgenic animals. Skin
thickness was measured using computer-assisted morphometric software
(Fig. 3a). In general,
total skin thickness in transgenic samples was less than in controls from
newborn to around Day 14 after birth (p < 0.05). However, as total
skin thickness decreased in wild type mice when progressing through the hair
cycle, transgenic skin became thicker than that of controls (p <
0.05). The reduced skin thickness in transgenic mice in the first 14 days was
mainly contributed by the subcutis (Fig.
3d) (p < 0.05). In contrast, the increase in
thickness observed after Day 21 involved the epidermis, the dermis, and the
subcutis layers (Fig. 3,
bd). Furthermore, the hair shaft diameter of hair
plugged from back skin was only half that of controls, although the percentage
of various types of hair did not change (not shown).

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FIG. 2. Microscopic examination of H&E-stained skin sections. Left
panel, wild type; right panel, transgenic. a and
b, dorsal skin sections of control and transgenic mice at Day 1,
showing no obvious difference. c and d, at Day 4, hair
follicles had entered the subcutis layer. The skin thickness and hair follicle
size in control mice had increased significantly. An increase in thickness of
the epidermis was observed in the transgenic mice as a result of an increase
in the number of cell layers in basal layers and stratum corneum
(arrows and inset). e and f, gross
structural difference of transgenic mouse skin compared with wild type mice at
Day 21. In wild type mice, hair follicles had moved up to the dermis layer and
proceeded to telogen phase. Hair follicles of transgenic mice were still in
the subcutis layer. g and h, at Day 50, wild type hair
follicles were at telogen and transgenic hair follicles remained in the
subcutis. Scale bar, 200 µm.
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FIG. 3. Analysis of skin thickness throughout the hair cycle. The thickness
at each stage was measured for total skin (a), epidermis
(b), dermis (c), and subcutis (d). A reduced total
skin thickness was observed for transgenic mice from birth to Day 14
(p < 0.05). This phenomenon reversed from Day 21 onward with
thicker skin in the transgenic mice (p < 0.05). The reduction in
skin thickness in the first 14 days was contributed mainly by the subcutis
layer (p < 0.05). The increase in skin thickness of transgenic
mice after Day 21 was contributed by all three layers (p < 0.05).
The asterisk represents a significant difference using Student's
t test. The thickness of skin samples from normal mice oscillated
according to the different phases of the hair growth. In contrast, transgenic
skin only showed small changes in skin thickness. Values shown are means
± 1 S.D. and n = 50.
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EGFR Signaling in Hair Development as Revealed by
ImmunoblottingNext, we investigated the pathway of EGF signaling
in wild type and transgenic skin. EGFR was readily detected in crude skin
lysates of both transgenic and wild type mice at Day 7 and Day 14
(Fig. 4a). The
receptors were strongly phosphorylated during this period, which suggested
that there was activation of downstream pathways for hair growth. However, at
Day 21, when hair follicles were at the telogen phase in normal mice, the
expression of EGFR was down-regulated in both transgenic and wild type mice,
although transgenic skin samples indicated a comparatively higher EGFR
expression and stronger phosphorylation than samples from age-matched wild
type animals. By Day 35, when all hair follicles were again at telogen phase
in the wild type mice, the expression of EGFR was barely detectable in both
wild type mice and transgenic mice. In order to determine the involvement of
other EGFR family members, immunoblotting of ErbB2 and ErbB3 was performed.
ErbB4 receptors were excluded since they were not detected in the skin by
immunohistochemistry (30). For
ErbB2, expression was high in both transgenic and wild type skin at Day 7.
However, expression persisted at Day 35 in transgenic mice only and was not
observed in wild type animals. Similar results were obtained for ErbB3
expression.

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FIG. 4. Immunoblots of crude skin samples from specific time points.
a, on both Day 7 and Day 14, expression and phosphorylation of EGFR
were detected in both wild type and transgenic mice. On Day 21 and Day 35,
normally the telogen phase, both the expression level and phosphorylation
level of EGFR were intensively down-regulated. However, samples from
transgenic animals had comparatively higher levels of EGFR expression and
phosphorylation than samples from wild type animals. Complete down-regulation
of ErbB2 and ErbB3 expression was observed on Day 35 in wild type but not in
transgenic skin. Comparable amounts of the mature form of EGF were detected at
each stage in transgenic and wild type samples. b,
immunoprecipitation of ErbB2 and ErbB3. On Day 14, no observable difference
between wild type and transgenic skin was detected with respect to
phosphorylation of ErbB2 and EGFR/ErbB2 heterodimerization. Similar results
were obtained for ErbB3. On Day 35 (telogen), only transgenic skin showed
EGFR/ErbB2 heterodimerization and phosphorylation.
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After relating the expression of various ErbB receptors to follicle
morphogenesis and the hair cycle, we attempted to identify the dimerization
partner(s) of ErbB1 by immunoprecipitation using antibodies specific for
either ErbB2 or ErbB3 (Fig.
4b). For Day 14 samples, both ErbB2 and ErbB3
immunoprecipitates were phosphorylated at a comparable level in both
transgenic and wild type skin samples. EGFR could be detected in both
immunoprecipitates, implying EGFR/ErbB2 and EGFR/ErbB3 heterodimer formation
in Day 14 skin samples. In contrast, for Day 35 skin, ErbB2 activation was
detected in transgenic but not in wild type mice, and EGFR could be detected
in ErbB2 immunoprecipitates. Neither ErbB3 activation nor EGFR/ErbB3
heterodimerization was detected in transgenic or wild type samples at Day 35.
Because ErbB2 has no known ligand and requires a dimerization partner for
phosphorylation, we conclude that ErbB2 activation persisted in the transgenic
skin through heterodimerization with EGFR.
Topical Application of ErbB BlockersOur data suggested that
EGF signaling through EGFR/ErbB2 and EGFR/ErbB3 may be important in follicle
growth and perhaps during the anagen phase of the hair cycle. We tested this
hypothesis in vivo using control and transgenic mice. Either
tyrphostin AG1478 or AG825, selective inhibitors of EGFR and ErbB2 tyrosine
kinase activity, respectively, was administered to the skin of adult mice. An
area on the dorsal side was shaved 1 day before the first inhibitor
application (Fig. 5, a and
b). Shaving could induce anagen
(31) and allow easy assessment
of hair growth. After 15 consecutive days of treatment, wild type mice treated
with vehicle only had a new layer of hair
(Fig. 5e), whereas in
wild type mice treated with either inhibitor, new hair growth was completely
inhibited (Fig. 5c).
Skin histology after treatment showed that hair follicles were in the anagen
phase of the hair cycle (not shown). In contrast, tyrphostin-treated
transgenic mice showed continuous hair growth
(Fig. 5d), although
less dense hair was observed compared with the vehicle-treated transgenic mice
(Fig. 5f). Similar
results were obtained when both inhibitors were applied simultaneously, and
there was a more obvious reduction in hair density. Furthermore, new hair
growth in the transgenic mice treated with both inhibitors was 2 days slower
than that of transgenic mice treated with a single inhibitor. Within the
vehicle-treated group, new hair growth in wild type mice was slower than in
transgenic mice by 2 days. These data suggest that EGFR/ErbB2 signaling is
indispensable for hair growth.

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FIG. 5. Topical application of specific tyrphostin inhibitor. a and
b, an area on the dorsal side of the mouse was shaved 1 day before
the first inhibitor application in wild type (a) and transgenic
littermates (b). c and d, after 15 consecutive days
of treatment with AG1478 in a neutral cream base, transgenic mice showed a new
layer of hair growth. The same result was obtained with application of AG825
(not shown). However, less dense hair growth was observed compared with
transgenic mice treated with cream base. Hair growth in wild type mice was
completely inhibited by AG1478 treatment. e and f, a new
layer of hair growth was observed in both wild type (e) and
transgenic mice (f) treated with cream base only in the same
experiment.
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Rescue by Breeding with Waved-2 MiceTo demonstrate further
that thin fur in line 62.7 was due to EGF overexpression and up-regulated EGFR
signaling, transgenic mice were mated with waved-2 mice. Homozygous
waved-2 mutant mice (Egfrwa-2/wa-2,
Fig. 6a) were bred
with homozygous 62.7 transgenic mice (CMV-Egf/CMV-Egf). All
offspring were therefore double heterozygous for both genes
(Egfrwa-2/+ and CMV-Egf/+). They all
showed normal hair growth throughout the body
(Fig. 6b).
Overexpression of EGF in double heterozygous mice was confirmed by
immunoblotting. As expected, the level of EGF precursor detected in the skin
of such mice was similar to that of heterozygous 62.7 mice (not shown). When
heterozygous waved-2 mice were bred with homozygous 62.7 transgenic
mice, 17 of 37 of the offspring had thin fur, indicating that the genetic
rescue was due to the waved-2 mutation instead of other differences
in the genetic background.

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FIG. 6. Rescue of thin fur phenotype by breeding 62.7 transgenic mice with
Egfrwa-2/wa-2 mice. a, a homozygous
waved-2 mouse at 3 months old. Only the first hair coat is waved, and
older animals such as the one shown had a normal coat phenotype but curly
whiskers. b, offspring of a homozygous waved-2 mutant
intercrossed with a homozygous transgenic mouse from line 62.7. Hair density
appeared normal throughout the whole body, but whiskers remained curly.
c and f, representative H&E-stained skin sections of
wild type mice at Days 7 and 21. d and g, skin sections of
heterozygous transgenic mice at Days 7 and 21. e and h, skin
sections of double heterozygous mice at Days 7 and 21. The skin morphology of
double heterozygous mice was similar to that of wild type mice, with the hair
follicles in the dermis layer at Day 21 indicating that they were in the
telogen phase. Scale bar, 200 µm.
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Histological sections taken from the back skin showed that the hair
follicles of double heterozygous mice were very similar to that of wild type
mice at Day 7 (Fig. 6, c and
e). More importantly, at Day 21 the hair follicles in the
double heterozygous mice were in the dermis layer, and the subcutis layer had
reduced in thickness, in contrast to that of 62.7 transgenic mice
(Fig. 6, fh).
This indicated that hair follicles in the double heterozygous mice had already
entered the telogen phase.
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DISCUSSION
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Based on the earlier studies of EGF in relation to skin and hair
development and given that the EGFR family and ligands play a pivotal role in
skin development, we have investigated the role of EGF and its signaling
mechanism with the aid of a transgenic mouse line constitutively expressing
EGF in skin and hair follicles. To our surprise we have found that
constitutive expression of EGF inhibits entry into the catagen phase.
Furthermore, EGFR/ErbB2 signaling appears to be indispensable for hair
growth.
In mice, follicular morphogenesis began at Day 14.5 of embryonic
development and lasted until around 3 weeks after birth
(32). The present study showed
that even when EGF was overexpressed, the skin histology was normal at birth.
By Day 4, there was an increase in epidermal thickness but a decrease in both
overall skin thickness and hair follicle diameter, consistent with earlier
studies that involved the injection of EGF into newborn animals and showed an
inhibitory role of EGF in skin development
(2,
3). It has also been shown that
although EGFR is expressed continuously in the epidermis, expression in the
hair follicles started a few days after birth. This correlates with the onset
of disorganized hair follicle phenotype in loss-of-function mouse mutants of
EGFR generated by homologous recombination or expression of a dominant
negative Egfr transgene
(12,
13). Some hair follicles even
penetrate the underlying muscle layer a few days later
(13). These data underscore
the importance of EGFR signaling in controlling hair follicle orientation and
elongation. The novel finding here is that continuous expression of EGF leads
to arrest of the hair follicles at stage 8 of follicular morphogenesis. This
conclusion appears to be in contrast to previous reports
(4,
21) of catagen-promoting
activity in sheep, but this effect may be an artifact due to the high doses of
EGF used. At lower doses of EGF, there was a reduction in follicle bulb size
and a reduction in wool fiber diameter, similar to that observed in our
transgenic mice. At higher doses, EGF induces wool follicle regression
(4). This is likely to be due
to the excessive inhibition of EGF in bulb cell division which may
subsequently lead to the observed increase in apoptosis
(33). Also, in cultured human
hair follicles, the stimulation of hair follicle elongation and outer root
sheath proliferation by EGF but inhibition on the proliferation of matrix
cells could lead to an artificial catagen. The matrix cells remained connected
to the dermal papilla by a thin strand of epithelial cells
(5), and this epithelial strand
is also seen in follicles at certain stages of catagen
(29). In summary, all reports
point to a stimulatory effect of EGF on the proliferation of basal
keratinocytes and outer root sheath cells but inhibition on proliferation of
bulb cells.
Is EGF the physiologic ligand in controlling hair cycling? Various
transgenic mouse lines have been produced to address the specific roles of EGF
family ligands in skin development with the aid of the promoter from
keratin 14 to drive transgene expression in the basal epidermis and
outer root sheath. Amphiregulin, heparin-binding EGF, and TGF-
produce
different effects when overexpressed in skin (see Introduction). Arrest in
hair cycle progression has not been reported in these mice. Furthermore, our
EGF transgenic mice were not prone to develop papillomas after skin lesions or
during aging as reported for TGF-
mice (data not shown). From our
immunoblot data it appears that EGF is likely to act through EGFR/ErbB2
dimerization. Furthermore, switching EGF off is associated with progression of
follicle morphogenesis to the catagen phase.
The present data using tyrphostins on mice at Day 23 point to the
indispensable role of EGFR/ErbB2 signaling in the initiation of hair growth in
the hair cycle. In wild type mice treated with the specific EGFR inhibitor, no
new hair growth was seen. The same result was obtained using the specific
inhibitor for ErbB2, indicating that EGFR/EGFR signaling is inadequate for
hair growth. Because ErbB2 does not have its own ligand but signals through
heterodimerization (8), our
results indicate that EGFR/ErbB2 signaling is involved. In contrast,
tyrphostin-treated transgenic mice continued to generate new hair but at a
lower density. It is possible that with continuous overexpression of EGF in
the transgenic mice, the inhibition of EGF signaling with either tyrphostin is
incomplete. Furthermore, in vehicle-treated mice, the transgenic mice were
found to exhibit new hair growth much sooner than the wild type mice,
suggesting that EGF is important for the initiation of hair growth. It is
interesting to note that in TGF-
null mutant mice, hair regrowth after
plucking is faster than that in wild type mice
(15). One intriguing
possibility is that EGF is more important than other EGFR ligands in
stimulating hair regrowth and that TGF-
competes with EGF for available
EGFR. One of the important downstream signaling molecules may be Stat3.
Although hair follicles in Stat3-disrupted mice can progress through
follicle morphogenesis to telogen, they cannot progress to anagen.
Importantly, keratinocytes isolated from these mice lack the normal response
of migration to EGF (34).
These data clearly indicate that the molecular control of hair growth during
morphogenesis, although often referred as the "first anagen," is
different from anagen in subsequent hair cycles. We believe that EGF
expression in the outer root sheath of hair follicles is important for
downward growth of the hair follicle. EGF expression needs to be turned off
before progression of the hair follicles to telogen. In subsequent hair
cycles, EGF is again important for downward growth of the hair follicles and
production of the hair shaft. Although it remains to be determined whether
continuous EGF can lead to anagen arrest in the hair cycle, we propose that
EGF acts as a biologic switch guarding entry to and exit from the anagen phase
of the hair cycle, as depicted in Fig.
7. The expression patterns of both EGF and its receptor are
consistent with such a role. EGF, together with various other signaling
molecules, forms a complex network for the precise control of the hair cycle.
For example, fibroblast growth factor 5 is required for the transition to
catagen. When the gene is mutated in mice, hair follicles fail to progress to
the catagen phase, and this gives rise to an abnormal long hair phenotype
(35).

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|
FIG. 7. Model for EGF as a switch in hair growth. Follicular morphogenesis
begins at 14.5 days post-coitum (dpc). EGF is switched on in the hair
follicles a few days after birth and switched off as hair follicles enter
catagen. EGF is again turned on in anagen and turned off in catagen in
subsequent hair cycles. In both situations, EGF signals through EGFR/ErbB2
heterodimers.
|
|
The findings of this study may be relevant to new therapeutic approaches
for the treatment of abnormally thin hair caused by abnormal EGFR signaling.
On the other hand, prevention of unwanted hair growth can be achieved by
topical application of EGFR/ErbB2 inhibitors. For example, such a strategy may
be of benefit in the treatment of women suffering from hirsutism or for
cosmetic purposes.
 |
FOOTNOTES
|
---|
* This work was supported by the University Block Grant, University of Hong
Kong. The costs of publication of this article were defrayed in part by the
payment of page charges. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section 1734
solely to indicate this fact. 
To whom correspondence should be addressed: Dept. of Paediatrics and
Adolescent Medicine, University of Hong Kong, Faculty of Medicine Bldg., 21
Sassoon Rd., Hong Kong. Tel.: 852 28199352; Fax: 852 28166974; E-mail:
sychan{at}hkucc.hku.hk.
1 The abbreviations used are: EGF, epidermal growth factor; EGFR, epidermal
growth factor receptor; TGF-
, transforming growth factor-
; TBS,
Tris-buffered saline; H&E, hematoxylin and eosin. 
 |
ACKNOWLEDGMENTS
|
---|
We thank Ashley Dunn and Tracy Helman (Ludwig Institute for Cancer
Research, Australia) for the kind gift of wa-2 mice; Malcolm Turner
(University College London, UK) and J. D. Huang (University of Hong Kong) for
comments on the manuscript; and Anthony Chan and Priscilla Mak (University of
Hong Kong) for their interest and technical support.
 |
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