Department of Cell Biology, Research Institute for Microbial Diseases, Osaka University, 3-1, Yamadaoka, Suita, Osaka 565-0871, Japan
* Author for correspondence (e-mail: riwamoto{at}biken.osaka-u.ac.jp)
Accepted 2 August 2005
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SUMMARY |
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Key words: HB-EGF, TGF, EGFR, ERK, Eyelid, Leading edge, Epithelial cell migration, Mouse
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Introduction |
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The importance of ERBB signaling during early mouse development has been
demonstrated by gene targeting studies
(Holbro and Hynes, 2004).
Disruption of the EGFR locus results in embryonic or perinatal lethality,
depending on the genetic background
(Miettinen et al., 1995
;
Sibilia and Wagner, 1995
;
Threadgill et al., 1995
). The
phenotypes observed in these mice suggest several physiological roles for
EGFR, including epithelial development. Newborn homozygous null EGFR mice
exhibit immature development of epithelial cells in the skin, lung,
gastrointestinal tract, tooth and eyelid
(Miettinen et al., 1995
;
Sibilia and Wagner, 1995
;
Threadgill et al., 1995
).
Eyelid closure represents a typical model for epidermal development.
Although TGFß/activin signaling is known to be essential for this
process, EGFR signaling also is required
(Xia and Karin, 2004).
EGFR-deficient mice have an open eye at birth (EOB) phenotype
(Miettinen et al., 1995
;
Sibilia and Wagner, 1995
;
Threadgill et al., 1995
), and
mice lacking the EGFR ligand TGF
occasionally exhibit an EOB phenotype
(Luetteke et al., 1993
).
HB-EGF is a member of the EGF family of growth factors that binds to and
activates EGFR and ERBB4 (Elenius et al.,
1997; Higashiyama et al.,
1991
). HB-EGF is synthesized as a type I transmembrane protein
(proHB-EGF) and, like other EGF family members
(Massague and Pandiella,
1993
), is cleaved at the juxtamembrane domain, resulting in the
shedding of soluble HB-EGF (sHB-EGF)
(Goishi et al., 1995
). sHB-EGF
is a potent mitogen and chemoattractant for a number of cell types
(Raab and Klagsbrun, 1997
),
while proHB-EGF acts as a juxtacrine growth factor that signals to neighboring
cells in a non-diffusible manner (Iwamoto
and Mekada, 2000
). HB-EGF has been implicated in a number of
physiological and pathological processes
(Raab and Klagsbrun, 1997
).
Importantly, analysis of HB-EGF-null mice has shown that HB-EGF is a crucial
factor for proper heart development and function
(Jackson et al., 2003
;
Iwamoto et al., 2003
), and for
skin wound healing (Shirakata et al.,
2005
).
Here, we show a novel role for HB-EGF in the process of eyelid closure. Our
data indicate that HB-EGF functions synergistically with TGF in leading
edge formation during eyelid closure, by promoting epithelial sheet migration
through activation of the EGFR-ERK signaling cascade.
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Materials and methods |
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Hematoxylin/eosin staining and lacZ detection
Hematoxylin/eosin staining and lacZ detection were performed as
previously described (Iwamoto et al.,
2003). The rates of eyelid closure and the eyelid formation were
measured by microscopy as indicated in Fig.
2C and in Fig. 4A,
respectively.
BrdU incorporation
E15.5 pregnant mice were injected with BrdU (Nakalai) (100 µg/g of body
weight). Two hours after injection, embryos were harvested, fixed with 4% PFA,
dehydrated and embedded in paraffin. Sections (4 µm) were stained with
anti-BrdU monoclonal antibody (ABCAM). The BrdU-positive cells in serial
sections were measured by microscopy.
Phalloidin staining
Embryos were fixed with 4% PFA, washed with PBS and incubated for overnight
with FITC-phalloidin (Molecular Probes), as previously described
(Zhang et al., 2003).
Immunohistochemistry
Mouse anti-mouse HB-EGF ectodomain monoclonal antibody (clone 4D9) was
prepared by immunizing HBdel/del mice with an abdominal injection
of the recombinant ectodomain of mouse HB-EGF, prepared by the baculovirus
expression system. Lymphoid node cells from the immunized mice were fused with
P3U1 myeloma cells, as previously described
(Iwamoto et al., 1991), and
the hybridoma producing an antibody reacting to mouse HB-EGF was selected.
Purified 4D9 mAb was biotinylated. Rabbit anti-phosphorylated EGFR antibody,
anti-EGFR antibody, anti-phosphorylated ERK antibody and anti-phosphorylated
JNK antibody were purchased from Cell Signaling Technology. For
immunohistochemistry, embryos were fixed by 4% PFA, dehydrated and embedded in
paraffin. Sections (5 µm) were incubated with each antibody in the blocking
solution Block Ace (Dainihon Seiyaku), permeabilized with 0.1% Triton X-100,
and then incubated with Alexa fluorophore-labeled streptavidin or Alexa
fluorophore-labeled secondary antibodies (Molecular Probes). After incubation,
sections were washed with PBS and observed by fluorescence microscopy.
Data analysis
Statistical significance was assessed with the Student's t-test. A
value of P<0.05 was considered statistically significant.
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Results |
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Soluble form of HB-EGF is required for the progress of eyelid closure
HB-EGF is first synthesized as a membrane-anchored form (proHB-EGF), and
the soluble form (sHB-EGF) is subsequently released from the cell surface by
ectodomain shedding (Goishi et al.,
1995). ProHB-EGF acts as a juxtacrine growth factor that signals
to neighboring cells in a non-diffusible manner
(Iwamoto and Mekada, 2000
). To
investigate which form of HB-EGF is involved in eyelid closure, we analyzed
knock-in mice expressing an uncleavable form of proHB-EGF (HBuc)
(Yamazaki et al., 2003
). As
was the case with HB-EGF-null embryos, the average progression of eyelid
closure in HBuc/uc embryos was approximately one-third that in
HBuc/+ embryos (Fig.
2E), indicating that the soluble form of HB-EGF is required for
the progress of eyelid closure.
HB-EGF does not contribute to cell proliferation during eyelid closure
The process of eyelid closure coordinates both cell proliferation and
migration. We therefore examined the cell proliferation during eyelid closure
in HBdel/del embryos and their heterozygous littermates at E15.5 by
measuring BrdU incorporation. BrdU-positive cells were mainly detected in the
eyelid dermis of the root region, but not in epidermal cells at the leading
edge (Fig. 3A-D). There was no
significant difference between the number of BrdU-positive cells in
HBdel/+ and HBdel/del eyelids
(Fig. 3E). These results
indicate that HB-EGF is not involved in cell proliferation during eyelid
closure, and may instead play a role in cell migration.
HB-EGF contributes to the leading edge extension during eyelid closure
Two primary morphological changes occur during eyelid formation: extension
of the root region (root formation) and extension of the leading edge (leading
edge formation). We therefore compared the progression of total eyelid
formation, root formation and leading edge formation in HBdel/+ and
HBdel/del embryos (Fig.
4A).
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HB-EGF is involved in F-actin polymerization in the developing eyelid epithelium
The role of HB-EGF in leading edge extension during eyelid closure strongly
suggests that HB-EGF functions in epithelial cell sheet migration. Previous
studies have shown that migrating epithelial cells exhibit actin
reorganization (Zhang et al.,
2003). During eyelid closure, F-actin in the form of actin cables
are detected at the margin of the migrating epithelial sheet, and radial
F-actin fibers align with the axis of extension of the leading edge
(Xia and Karin, 2004
). To
examine whether epithelial cell migration during eyelid closure is normal in
HBdel/del embryos, we used FITC-phalloidin staining as a marker for
actin polymerization.
At E15.0, although no overt morphological differences were detected in the eyelids of HBdel/+ and HBdel/del embryos, actin cable formation at the margin of the eyelid epithelium was lower in HBdel/del embryos than in HBdel/+ embryos (Fig. 4C, parts a,c,e,g). Moreover, at E15.5, when the leading edge is extending, progressive eyelid closure was accompanied by both actin cable formation and radial F-actin fiber formation in HBdel/+ eyelids (Fig. 4C, parts b,d), while in HBdel/del embryos, eyelid closure was retarded and both actin cable and radial F-actin fiber formation were quite low (Fig. 4C, parts f,h). These results suggest that HB-EGF plays a role in epithelial cell sheet migration during leading edge formation by promoting F-actin polymerization.
HB-EGF activates the EGFR-ERK signaling pathway during leading edge formation
To investigate the molecular mechanism underlying HB-EGF-mediated eyelid
closure, we performed immunohistochemistry for HB-EGF protein in the
developing eyelid using an anti-HB-EGF ectodomain monoclonal antibody. As
shown in Fig. 5C,E, HB-EGF
protein was detected broadly in the leading edge of HBdel/+
eyelids, although HB-EGF mRNA was at the tip of the leading edge
(Fig. 1,
Fig. 5A). As expected, no
HB-EGF protein was detected in HBdel/del eyelids
(Fig. 5D,F), although the
lacZ expression pattern resembled that of HBdel/+ embryos
(Fig. 5B). These results
indicate that sHB-EGF is secreted at the tip of the leading edge, and diffuses
only in this region.
EGFR is a receptor for HB-EGF
(Higashiyama et al., 1991) and
is essential for eyelid closure (Miettinen
et al., 1995
; Sibilia and
Wagner, 1995
; Threadgill et
al., 1995
). To investigate whether HB-EGF activates EGFR during
eyelid closure, we examined HBdel/+ and HBdel/del
eyelids for phosphorylated EGFR. In HBdel/+ embryos, phosphorylated
EGFR protein was detected predominantly in the extending leading edge
(Fig. 5G), while total EGFR
protein was detected more broadly in the epithelial cells of the leading edge
as well as the root region (Fig.
5I). Phosphorylated EGFR was detected in the same region as HB-EGF
(Fig. 5E,G). By contrast, the
level of phosphorylated EGFR was drastically reduced in HBdel/del
embryos, especially in the leading edge
(Fig. 5H), while total EGFR
protein appeared normal (Fig.
5I,J). These results suggest that HB-EGF activates EGFR in the
leading edge and is restricted to this region.
Next, we used antibody staining to examine the phosphorylation state of
ERK, a major downstream effector of EGFR signaling
(Jorissen et al., 2003). In
HBdel/+ embryos, phosphorylated ERK protein was detected
predominantly in the extending leading edge
(Fig. 5K), as was
phosphorylated EGFR (Fig. 5G).
By contrast, the level of phosphorylated ERK was decreased in
HBdel/del eyelids, especially in the leading edge
(Fig. 5L). Thus, activation of
ERK in the leading edge is correlated with HB-EGF-mediated EGFR activation
during eyelid closure.
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|
EGFR mediates HB-EGF-dependent eyelid closure
Since EGFR phosphorylation was decreased in leading edge of the eyelid in
HBdel/del embryos, we used ZD1839, an EGFR-specific kinase
inhibitor (Tanno et al., 2004;
Von Pawel, 2004
), to examine
whether loss of EGFR function would phenocopy loss of HB-EGF. We administrated
ZD1839 to E14.5 wild-type embryos in utero, and harvested and analyzed them 24
hours later. Embryos with reduced EGFR activity had a similar phenotype to
HBdel/del and HBuc/uc embryos (data not shown). In
ZD1839-treated embryos, eyelid formation was significantly impaired owing
primarily to defects in leading edge formation, but BrdU incorporation was
normal, indicating that EGFR activity does not promote proliferation of dermal
cells in the root region. In addition, formation of actin cables and radial
F-actin fibers was drastically decreased in ZD1839-treated embryos. These
results indicate that from E15.0 to E16.0, EGFR activity is correlated with
HB-EGF activity during leading edge formation. EGFR signaling does not appear
to regulate cell proliferation during leading edge formation, but probably
instead functions to control epithelial cell sheet migration.
In order to determine whether EGFR acts as a receptor for HB-EGF in eyelid
closure, we used a hypomorphic EGFR mutant, waved 2, and tested for a genetic
interaction with HB-EGF. In waved 2 mice, the kinase activity of EGFR is
decreased to less than 10% that of wild-type EGFR, owing to a point mutation
in the kinase domain (Fowler et al.,
1995; Luetteke et al.,
1994
). Although HBdel/del and waved 2
(wa2/wa2) mice did not exhibit an EOB phenotype,
HBdel/+; wa2/wa2 (66.6%) and
HBdel/del; wa2/wa2 (100%) mice did
(Fig. 6). These results suggest
that eyelid closure relies on dose-dependent HB-EGF-EGFR signaling, and that
EGFR mediates HB-EGF-controlled eyelid closure.
TGF functions synergistically with HB-EGF in eyelid closure
TGF, another EGFR ligand, also has been reported to be a critical
factor in eyelid closure (Luetteke et al.,
1993
; Wong et al., 2003). As expression of TGF
is regulated
by HB-EGF in an autocrine manner in keratinocytes
(Hashimoto et al., 1994
;
Piepkorn et al., 1998
), we
examined whether the defects in eyelid closure in HBdel/del embryos
resulted from downregulation of TGF
expression. TGF
mRNA was
expressed in the tip of the leading edge in both HBdel/+ and
HBdel/del eyelids, and no significant difference in the level of
TGF
expression was detected between HBdel/+ and
HBdel/del eyelids (data not shown), indicating that the expression
of TGF
is not affected by the presence or absence of HB-EGF.
To clarify the functional relationship between HB-EGF and TGF in
eyelid closure, we investigated this process in TGF
-null mice. As was
the case with HB-EGF null mice, the BrdU incorporation in TGF
mutant
eyelids appeared normal (Fig.
7A-C). We next compared the progression of eyelid closure in
Tgfa+/- and Tgfa-/- embryos. The
average progression of total eyelid formation, root formation and leading edge
formation were significantly lower in Tgfa-/- embryos than
in Tgfa+/- embryos (2.0-, 3.0- and 1.8-times,
respectively, Fig. 7D), with
the progression of total eyelid formation in Tgfa-/-
embryos similar to that of HBdel/del embryos
(Fig. 4B,
Fig. 7D). Thus, as with the
HB-EGF mutants, the reduction in the progression of leading edge formation
contributed more strongly to the reduced progression of total eyelid formation
than did root formation in TGF
embryos.
Finally, to test for genetic interactions between HB-EGF and TGF in
eyelid closure, we examined double mutants of TGF
and HB-EGF. As shown
in Fig. 7E-G, both
HBdel/del; Tgfa+/- and HBdel/+;
Tgfa-/- embryos exhibited similar frequencies of EOB (10%
and 11%, respectively), while HBdel/del and
Tgfa-/- single homozygotes did not have an EOB phenotype.
Moreover, homozygous double mutants (HBdel/del;
Tgfa-/-) had an even higher frequency of EOB (50%). These
results strongly suggest that TGF
and HB-EGF contribute equally to
leading edge formation and function synergistically in this process.
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Discussion |
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HB-EGF-EGFR signaling functions in cell migration but not proliferation during eyelid closure
We demonstrate here using HB-EGF-null embryos that HB-EGF-mediated
activation of EGFR promotes epithelial cell sheet migration, but not cell
proliferation, during eyelid closure. Among our conclusions are the following:
(1) In HB-EGF-deficient embryos, eyelid closure was significantly retarded;
(2) the level of cell proliferation was not significantly different between
HBdel/del and HBdel/+ eyelids; (3) retardation of eyelid
formation in HBdel/del embryos was mainly due to defects in leading
edge extension rather than root region extension; (4) the level of cell
migration, judged by the detection of actin bundle formation in epithelial
cells of the leading edge, was decreased in HBdel/del eyelids; (5)
phosphorylated EGFR was dramatically decreased in the leading edge lacking
HB-EGF; (6) inhibition of EGFR activity by the kinase inhibitor ZD1839
phenocopied the loss of HB-EGF; and (7) reduction of EGFR activity in mice
lacking both HB-EGF and EGFR function accelerated the defect of eyelid
closure.
|
Recently, we demonstrated that in skin wound healing, HB-EGF promotes
epithelial cell sheet migration, but not cell proliferation, in the process of
re-epithelialization (Shirakata et al.,
2005). In adult mice, HB-EGF is not usually expressed in the skin,
but wounding induces expression of HB-EGF at the migrating leading edge of the
wound. The parallels in both the domain of expression and function of HB-EGF
between eyelid formation and skin wound healing suggest that HB-EGF functions
to promote cell motility, but not proliferation in epithelial
keratinocytes.
Role of EGFR and downstream signaling in eyelid closure
In this study, we present evidence that activation of EGFR by HB-EGF is
important for eyelid formation. Although HB-EGF can also bind to and activate
ERBB4 receptor, ERBB4 expression is not detectable in the murine epidermis
(Kiguchi et al., 2000;
Xian et al., 1997
) or the
human epidermis (Plowman et al.,
1993
), ruling out a role in eyelid closure. By contrast, both EGFR
knockout mice (Miettinen et al.,
1995
; Sibilia and Wagner,
1995
; Threadgill et al.,
1995
) and the spontaneous EGFR mutant mice velvet
(Du et al., 2004
) showed an EOB
phenotype. However, it remained unclear whether EGFR functions in cell
proliferation or migration. In the present study, we show that EGFR is
essential for epithelial cell sheet migration and promotes leading edge
formation during eyelid closure process. These results suggest that the
defects in eyelid closure resulting from loss of EGFR activity are mainly
caused by retarded epithelial sheet migration.
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JNK signaling, which can be activated by TGFß/activin
(Xia and Kao, 2004;
Xia and Karin, 2004
;
Zhang et al., 2003
), may also
play a role in eyelid closure. Mice that lack MEKK1 showed defects in leading
edge formation, owing to loss of JNK activation
(Zhang et al., 2003
). In
addition, mice that lack the JNK target JUN showed defects in leading edge
formation (Grose, 2003
;
Li et al., 2003
;
Zenz et al., 2003
).
Furthermore, mutant Jnk1-/- Jnk2+/-
pups exhibited an EOB phenotype (Weston et
al., 2004
). Interestingly, it has been reported that expression of
HB-EGF depends on JUN activation in keratinocytes
(Zenz et al., 2003
). However,
in our experiments, there was no difference in JNK activation between
HBdel/+ and HBdel/del animals. Thus, JNK may function as
an upstream factor for HB-EGF-EGFR-ERK signaling pathway in eyelid
closure.
Relationship between HB-EGF and TGF in eyelid closure
TGF is also known to be required for normal eyelid closure, with
TGF
deficient mice occasionally exhibiting an EOB phenotype
(Luetteke et al., 1993
;
Wong, 2003
). However, the
TGF
-null mice used in the present study did not show an EOB phenotype.
This difference could be due to differences in the genetic background of our
mice and the previously reported mice. Although it has been reported that
TGF
functions upstream of HB-EGF signaling pathway involved in eyelid
closure (Hayashi et al.,
2005
), our data suggest that TGF
and HB-EGF function at
equal levels and synergistically in eyelid closure, based on the following
findings: (1) expression of TGF
was not affected by HB-EGF expression;
(2) TGF
-null mice phenocopy HB-EGF mutants, with defects in epithelial
sheet migration but not proliferation; (3) mice that are homozygous null for
HB-EGF and heterozygous for a TGF
null mutation showed a variably
penetrant EOB phenotype; likewise, mice that are homozygous null for
TGF
and heterozygous for a HB-EGF null mutation had a similar
phenotype, while both single null mutant mice failed to show any EOB
phenotype; and (4) HB-EGF and TGF
doubly homozygous null mice had
dramatically increased penetrance of the EOB phenotype. However, in contrast
to the complete penetrance of the EOB phenotype in EGFR-null mice
(Miettinen et al., 1995
;
Sibilia and Wagner, 1995
;
Threadgill et al., 1995
), only
50% of these double homozygotes were born with EOB. This suggests that other
EGFR ligand(s) might also be involved in eyelid closure. Interestingly, it has
been reported that triple null mice lacking EGF, amphiregulin, and TGF
exhibited a more severe EOB phenotype than did TGF
single null mice.
However, even in these triple null mice, the EOB phenotype is not fully
penetrant (Luetteke et al.,
1999
). This indicates that the EGFR ligands HB-EGF, TGF
,
EGF and amphiregulin may cooperate in eyelid closure.
Mode of action of HB-EGF in the process of eyelid formation
We show using the HBdel/+ lacZ reporter allele that
HB-EGF is specifically expressed at the tip of the leading edge of the
migrating epithelial sheet, and only between E15.0 and E16.5. By contrast,
HB-EGF protein was detected throughout the leading edge of HBdel/+
developing eyelids. Moreover, embryos with an uncleavable mutant version of
proHB-EGF (HBuc/uc) displayed defects in eyelid closure. Together,
these findings indicate that sHB-EGF, but not proHB-EGF, functions in eyelid
closure, and that ectodomain shedding of proHB-EGF is essential for this
process, as is the case in cardiomyocytes and in the valvulogenesis
(Yamazaki et al., 2003).
Previous cell culture studies indicate that ectodomain shedding of HB-EGF
is regulated by multiple signaling cascades
(Izumi et al., 1998; Prentzel
et al., 1999; Takenobu et al.,
2003
; Umata et al.,
2001
). Interestingly, activation of RTKs stimulates HB-EGF
shedding via the RAS-MAPK pathway (Umata
et al., 2001
). Thus, activation of EGFR by HB-EGF may induce
shedding of HB-EGF, forming an autocrine positive feedback loop. As shown in
the present study, HB-EGF, activated EGFR and activated ERK were colocalized
in the leading edge during eyelid closure, consistent with the existence of a
feedback loop that stimulates HB-EGF shedding.
Among the proteases (convertases) that might induce ectodomain shedding of
HB-EGF, ADAM17/TACE, a member of the ADAM family metalloproteases, is a prime
candidate for functioning in this process in vivo
(Lee et al., 2003).
ADAM17-null mice had enlarged cardiac valves resembling those of HB-EGF-null
mice (Jackson et al., 2003
)
and also exhibited an EOB phenotype (Sahin
et al., 2004
). ADAM17 has also been shown to be the sheddase for
TGF
(Lee et al., 2003
).
These results suggest that ADAM17 may function in eyelid closure as a sheddase
for HB-EGF and TGF
.
Although the precise mechanism is still unclear, we propose the following
model for the mode of function of HB-EGF in mouse eyelid closure
(Fig. 8). Before E15.0, the
leading edge is not yet formed, and HB-EGF expression is not yet detected
(Fig. 8A). At E15.0, the
leading edge starts to extend, and the expression of HB-EGF appears in a few
cells at the tip of the leading edge (Fig.
8B). Between E15.0 and E16.0
(Fig. 8C), HB-EGF is
constitutively expressed at the tip of the extending leading edge. On the
surface of the cells located in this region, ectodomain shedding of proHB-EGF
may be mediated by ADAM17 directly or indirectly, and liberated sHB-EGF
diffuses throughout the leading edge region. sHB-EGF then binds to and
activates EGFR, activating the ERK pathway. This results in actin
polymerization and promotion of epithelial cell sheet migration. TGF
also functions synergistically with HB-EGF, possibly in a similar manner to
HB-EGF.
Detection of the endogenous proteins is important for the analysis of growth factor function in vivo; however, it is very difficult to obtain suitable antibodies to detect endogenous proteins in mouse tissues. In the present study, we were able to show the pattern of HB-EGF protein localization in the leading edge of eyelids using a monoclonal anti-mouse HB-EGF mAb (4D9), which was generated by immunizing HB-EGF null mice with recombinant mouse HB-EGF protein. Although many previous reports have examined HB-EGF localization using other anti-HB-EGF antibodies, this represents the first clear and specific detection of endogenous HB-EGF protein in mouse tissue. Thus, our new antibody will be a very useful tool for the analysis of HB-EGF function in mice in vivo.
In conclusion, in the present study we have demonstrated for the first time
that HB-EGF signaling contributes to eyelid development. HB-EGF functions
synergistically with TGF in leading edge formation during eyelid
closure, by promoting epithelial sheet migration through activation of the
EGFR-ERK signaling cascade. We have demonstrated also that gene-expression,
processing and protein localization of HB-EGF are strictly regulated
temporally and spatially during eyelid closure process. Molecular mechanisms
regulating these processes are remained unclear. Future study will be
necessary to solve these issues.
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ACKNOWLEDGMENTS |
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