Targeted Expression of a Dominant Negative Epidermal Growth Factor Receptor in the Mammary Gland of Transgenic Mice Inhibits Pubertal Mammary Duct Development
Wen Xie,
Andrew J. Paterson,
Edward Chin,
Lisle M. Nabell and
Jeffrey E. Kudlow
Departments of Medicine and Cell Biology Division of
Endocrinology and Metabolism University of Alabama at
Birmingham Birmingham, Alabama 35294
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ABSTRACT
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The epidermal growth factor (EGF) system has been
thought to play an important role in normal mammary development and
carcinogenesis. To study the role of the EGF receptor (EGFR) in mammary
development, we developed a transgenic mouse model in which a
C-terminal truncated mouse EGFR (EGFR-TR) was expressed in the mouse
mammary epithelium under the control of the mouse mammary tumor virus
long terminal repeat. The EGFR-TR lacks most of the cytoplasmic domain
of the receptor, including the entire protein tyrosine kinase domain.
In cultured cells, we show that it acts in a dominant negative manner
in EGF-signaled EGFR autophosphorylation. Several lines of mice were
characterized and shown to express the transgene at the mRNA and
protein levels not only in the mammary gland but also in the salivary
glands, epididymis, and prostate. In postpubertal virgin female mice,
the expression of the EGFR-TR in the mammary glands was greater than
the expression of the endogenous wild type EGFR. In these virgin mice,
inhibition in mammary ductal development and a decrease of mammary
epithelial DNA synthesis were observed beginning at 56 weeks. The
inhibition of duct development was most apparent by 1516 weeks,
resulting in a significant defect in ductal branching and outgrowth and
an apparent overall decrease in the size of the mammary glands.
However, during pregnancy, expression of the endogenous wild type EGFR
was markedly increased relative to the EGFR-TR and, at this stage,
normal presecretory alveoli developed from the hypoplastic duct tree.
Postpartum, normal lactation occurred. Despite EGFR-TR expression in
other tissues, no morphological abnormalities were observed. This model
demonstrates that the EGFR-TR behaves as a dominant negative regulator
of the EGFR system in vivo and that the EGFR system plays
an important role in mammary ductal development.
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INTRODUCTION
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The epidermal growth factor receptor (EGFR) and its ligands are
believed to play a vital role in mammary gland development and
carcinogenesis (for review, see Ref.1). However, the role of EGFR
signaling in mammary development has not been fully explored, partly
because EGFR-devoid animals are not viable postnatally (2, 3, 4) when
mammary development occurs. Furthermore, there appears to be
considerable redundancy in the ligands for the EGFR. These ligands
include EGF itself (5), transforming growth factor-
(TGF
) (6),
amphiregulin (AR) (7), heparin-binding EGF-like growth factor (8), and
betacellulin (9). Thus, strategies to eliminate any of these ligands
singly could be overcome by this redundancy, a problem suggested by the
TGF
"knockout" studies (10, 11).
We approached blocking EGFR signaling in the mammary gland using a
dominant negative strategy. The wild type receptor consists of a
ligand-binding extracellular domain, a transmembrane domain, and an
intracellular protein tyrosine kinase domain (12). Upon ligand binding,
EGFRs oligomerize, the cytoplasmic tyrosine kinase is activated, and
the receptor undergoes autophosphorylation. While an intramolecular
mechanism of EGFR autophosphorylation has not been ruled out (13), a
few lines of evidence suggest that EGFR autophosphorylation occurs in
trans as a result of the oligomerization process (for review, see Ref.12). This autophosphorylation of the receptor is a critical component
of the signaling process in that the phosphotyrosines in the
carboxyl-terminal tail are recognized by a variety of proteins that
contain Src homology 2 (SH2) domains, such as phospholipase C-
(5),
SH2-containing sequence (14), or growth factor receptor-bound protein 2
(15). The binding of these SH2-containing proteins to the
phosphorylated receptor creates a submembranous signaling complex whose
activation leads to the pleiotropic response to the growth factors. In
addition, the dimeric EGFR exhibits increased binding affinity for EGF
even in the absence of a protein kinase domain (16, 17). We made use of
this mechanism of EGFR signaling by overexpressing, in the mammary
glands of transgenic mice, a truncated form of the EGFR that lacks the
protein kinase and carboxyl-terminal domains. We hypothesized that this
mutant receptor would either form nonsignaling dimers with the
endogenous wild type receptor or sequester ligands, thereby impairing
signaling by all ligands utilizing this receptor. Studies in cultured
cells using similarly mutated EGFRs (18, 19) had indicated that this
strategy might have an effect in vivo (20).
There is considerable indirect evidence for the role of the EGFR system
in mammary ductal development. Most of this development occurs
postnatally with a rudimentary ductal pattern having been patterned by
the age of 67 weeks. At this stage, these minimal ducts terminate in
an end bud, which shows evidence of dichotomous branching, while by age
13 weeks the ductal pattern has become quite extensive, forming a
tree-like pattern. It is upon these ducts that the secretory alveolar
lobules develop during pregnancy. This ductal expansion occurs as a
result of cell division of the ductal epithelium in the end buds. EGFR
is present in the normal ductal system. The observation that the EGFR
concentration in the mammary gland varies through stages of development
suggests a role for this receptor in ductal development. Receptor
levels are high in the immature pubertal mammary gland and then
decrease with age. Receptor levels increase again with the onset of
pregnancy, reaching a peak level at 10 days followed by a rapid decline
to very low levels during lactation (21). EGF/TGF
stimulate ductal
cell proliferation, but inhibit functional differentiation of mouse
mammary epithelial cells in culture (22). Indeed, overexpression of
TGF
in transgenic mice results in mammary carcinoma (23, 24, 25) and
impaired milk secretion, partly as a result of the inhibition of the
expression of the milk-specific whey acidic protein gene (26). The role
of this receptor system in mammary carcinoma is also suggested by the
finding of increased TGF
expression in human breast cancers (27) and
amplification of the EGFR gene in some breast cancer cell lines (28)
and in some primary breast cancers (29).
To further elucidate the in vivo role of the EGFR system in
mammary development, we created a transgenic mouse model in which a
C-terminal truncated EGFR was expressed in the mammary ductal system
under the influence of the mouse mammary tumor virus (MMTV) promoter.
We found that these mice exhibited considerable suppression of mammary
ductal development that was very similar to the phenotype exhibited in
mice expressing an activated transforming growth factor-ß1 (TGFß1)
under the MMTV promoter (30). These results strongly suggest that the
EGFR system is involved in the proliferation and branching that occurs
in the ductal system during pubertal development.
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RESULTS
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Generation and in Vitro Functional Characterization of
a C-Terminal Truncated EGFR
A C-terminal truncated EGFR (EGFR-TR) cDNA was generated by
inserting a double-stranded oligonucleotide into the SacI
site of the mouse EGFR cDNA sequence (31) and, by doing so, introducing
a stop codon (TGA) after the L690 codon. To test the function of this
mutant in vitro, the EGFR-TR and the wild type EGFR
(EGFR-WT) (32) cDNAs were placed downstream of the cytomegalovirus
promoter. These expression vectors, encoding the wild type or truncated
EGF receptors, were transiently transfected at various ratios into
Chinese hamster ovary (CHO) cells, a cell line that does not express
detectable levels of the endogenous EGFR (33). In cells expressing both
the EGFR-TR and EGFR-WT, we could detect heterodimer formation between
the EGFR-WT and EGFR-TR as revealed by bis(sulfosuccinimidyl)suberate
(BS3)-mediated covalent cross-linking experiments with
[125I]EGF (data not shown).
EGF-stimulated EGFR autophosphorylation is an essential component of
EGFR signaling (for review, see Ref.12). To examine the effects of the
EGFR-TR on EGFR autophosphorylation in response to EGF, CHO cells were
transiently transfected with EGFR-WT alone or together with increasing
amounts of EGFR-TR. After EGF treatment or mock treatment, the EGFR-WT
was immunoprecipitated from the transfected cells using an anti-EGFR
monoclonal antibody H9B4 (34) that is specific for EGFR intracellular
domain. The immunoprecipitates were subjected to immunoblotting to
determine the recovery of the wild type EGFR and its degree of tyrosine
phosphorylation. The recovery of the receptor was determined by probing
the blot with H9B4. Regardless of the ratio of EGFR-WT to EGFR-TR, the
recovery of the wild type receptor was uniform (Fig. 1
). However, when the blot was stripped
and reprobed with an anti-phosphotyrosine antibody, 4G10, the level of
autophosphorylation of the EGFR-WT was down-regulated, even in the
presence of a 1:1 ratio of EGFR-TR to EGFR-WT (Fig. 1
). At a ratio of
2:1 of EGFR-TR to EGFR-WT, the EGFR-WT autophosphorylation was largely
abolished. This down-regulation of EGF-stimulated EGFR
autophosphorylation by the EGFR-TR indicated that this
C-terminus-truncated EGFR mutant could exert a dominant negative effect
on the EGFR-WT auto-phosphorylation.

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Figure 1. The Truncated EGF Receptor (EGFR-TR) Inhibits
EGF-Stimulated Autophosphorylation of the Wild-Type EGFR
CHO cells were transfected with various ratios of the expression
plasmids encoding the EGFR-WT and the EGFR-TR. The transfected cells
were subsequently treated with 10-8 M EGF or
mock treated as described in Materials and Methods. Cell
lysates were prepared, and the EGFR-WT was immunoprecipitated with a
monoclonal antibody, H9B4, directed to the intracellular domain of the
EGFR. The precipitated proteins were separated by SDS-PAGE and analyzed
by immunoblotting. The filter was first probed with H9B4 and
subsequently stripped and reblotted with the monoclonal
anti-phosphotyrosine antibody, 4G10. The immunoblotted signals were
observed using the ECL detection system. In this assay, untransfected
MDA468 human breast cancer cells (28) and empty vector-transfected CHO
cells were used as EGFR-positive and -negative control, respectively.
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Generation of MMTV-EGFR-TR Transgenic Mice
Transgenic mice expressing EGFR-TR under the control of the MMTV
long terminal repeat (MMTV-LTR) were generated by injecting one-cell
B6xSJL mouse zygotes with the transgene construct shown in Fig. 2
. Before these injections, we tested the
transgene construct in cultured cells. Transfection of the plasmid
containing the MMTV-EGFR-TR construct into CHO cells resulted in the
expression of the expected 115-kDa EGFR-TR protein as revealed by
BS3-mediated chemical cross-linking experiments with
[125I]EGF (data not shown). This in vitro
study indicated that the ß-globin intron splicing and MMTV promoter
functioned normally in conjunction with the EGFR-TR cDNA.

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Figure 2. Schematic Representation of the MMTV-EGFR-TR
Transgene Construct
The cross-hatched region corresponds to the 1.5 kb of the MMTV LTR. The
solid regions correspond to the second and third exons
of the rabbit ß-globin gene. The open region
corresponds to the second intron and the 3'-flanking region of the
ß-globin gene. The stippled region corresponds to the
2.3-kb segment of the mouse EGF receptor cDNA that encodes codons
1690 of the receptor. A stop codon was inserted after codon 690,
resulting in the expression of the C-terminal truncated mouse EGF
receptor (EGFR-TR). The EGFR-TR cDNA (stippled region)
was inserted into the third exon of the rabbit ß-globin gene.
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A total of 10 gene-positive founders, five females and five males, were
identified by PCR using a pair of transgene-specific oligonucleotides
(data not shown). The integration status of the transgene was tested by
Southern blot analysis. There is only one XbaI site in the
transgene construct. XbaI digestion of the genomic DNA
from the transgenic mice released the predicted 4.9-kb transgene,
detected by hybridization with the transgene-specific rabbit ß-globin
exon 3 probe (Fig. 3
). This result
indicates that the integrated transgene is intact, and that the
multiple copies of the transgene are in a head-to-tail array. Of the 10
founders, one male (31-3) was infertile for unknown reasons, and one
male (31-5) was unable to transmit the transgene to its offspring,
probably as a result of mosaicism (35). The eight other founders (7-6,
7-8, 21-6, 20-1, 21-5, 31-1, 31-4, 31-7) successfully transmitted the
gene in a Mendelian manner, thereby allowing the establishment of eight
independent transgenic mouse lines.

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Figure 3. Southern Blot Analysis of MMTV-EGFR-TR Transgenic
Mice
Eight micrograms of genomic DNA extracted from a tail biopsy from each
transgenic (7-6-6, 31-7-4) or nontransgenic (21-7-4) mouse were
digested with XbaI and subjected to Southern blot
analysis. The transgene fragment (25 pg) derived from the original
plasmid in which it was constructed was loaded as a positive control.
The filter was probed with a radiolabeled rabbit ß-globin exon 3
probe that is specific for the transgene. The transgene (4.9-kb) bands
were detected in transgenic but not in nontransgenic control mice.
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Expression of the Transgene
Expression of the transgene was investigated by Northern blot
analysis of RNA from the mouse mammary glands using the
transgene-specific rabbit ß-globin exon 3 as a probe. Initial
Northern blotting of RNA from pregnant female mouse mammary glands
revealed that lines 7-6, 31-4, 20-1, and 21-6 had relatively high
expression of the transgene. The 2.8-kb transcript was of the predicted
size for the transgene and was detected with the ß-globin probe in
the mammary glands of transgenic but not wild-type mice. When a cDNA
probe encoding the extracellular domain of the EGFR was used, both the
endogenous and transgene EGFR transcripts were detected (data not
shown). Because these four lines of mice had the highest transgene
expression, they were characterized further. Northern blot analysis
carried out in line 21-6 using a cDNA probe encoding the extracellular
domain of the EGFR revealed that the 2.8-kb transgene was expressed in
the mammary glands of both virgin and pregnant transgenic females but
not in their nontransgenic siblings (Fig. 4A
). Of note, in the virgin transgenic
females, the steady state level of the EGFR-TR transcripts was higher
than that of the endogenous EGFR transcripts (10 and 6 kb). However, in
late pregnancy, while no significant change in the level of the
transgene transcript was detected, the level of the endogenous EGFR
transcripts increased significantly, causing the ratio of EGFR-TR
transcripts to the endogenous EGFR transcripts to decrease. When using
the rabbit ß-globin exon 3 probe, only the transgene transcripts were
detected in the mammary glands of transgenic females (Fig. 4B
). In line
21-6, expression of the transgene was also detected in salivary glands,
lungs, and epididymis (Fig. 4B
), which is consistent with previous MMTV
promoter-based transgenic studies (for review, see Ref.36). Relatively
low levels of transgene expression were observed in the placenta and
prostate (data not shown). No transgene expression was detected in
brain, liver, or kidney. There was no clear correlation between the
transgene copy number and the level of transgene expression as judged
by Northern blot analysis.

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Figure 4. Northern Blot Analysis of the MMTV-EGFR-TR
Expression in Tissues from Female and Male Transgenic Mice
Twenty micrograms of total cellular RNA from each of the indicated
tissues from transgenic or nontransgenic control mice were subjected to
Northern blot analysis. The filters were hybridized with a
[32P]-labeled 2.3-kb EGFR-TR cDNA probe or the rabbit
ß-globin exon 3 probe. The filters were subsequently stripped and
reprobed with the GAPDH cDNA for the purpose of a loading control. A,
Northern blot of mammary gland RNA using the EGFR cDNA probe. Both
transgene and endogenous EGFR transcripts were detected. Lane 1 and 2
are control and transgenic (line 21-6) virgin females, respectively;
lane 3 and 4 are control and transgenic (line 21-6) pregnant females,
respectively. EGFR-TR or endogenous wild type EGFR transcripts are
indicated. B, Northern blot of mammary gland RNA using ß-globin
probe. Only the transgene transcripts were detected. Lanes 1 and 2
contain mammary gland RNA from control and transgenic (line 21-6)
virgin females, respectively. Lanes 37 show transcripts from an adult
male transgenic mouse (line 21-6); lane 3, epididymis; lane 4, seminal
vesicle; lane 5, liver; lane 6, lung; lane 7, salivary gland.
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The EGFR-TR protein, encoded by the transgene, was expressed in tissues
of the transgenic animals. To detect the EGFR-TR,
BS3-mediated chemical cross-linking was performed on
microsomes purified from the examined tissues. This approach had the
advantage of detecting the functional ligand-binding domain of the
receptor and its dimerization partners while adding only 6 kDa to the
molecular mass of the receptors. Figure 5
shows that, in addition to the endogenous EGFR (170 kDa), the 115 kDa
EGFR-TR protein was detected in the mammary glands of pregnant
transgenic mice. EGFR-TR protein was also detected in the salivary
gland (Fig. 5
), prostate, and epididymis of transgenic animals (data
not shown). Cross-link labeling of these EGF receptor proteins by
[125I]EGF was completely inhibited in the presence of
excess unlabeled EGF. We were not able to detect either EGFR-TR or the
endogenous wild type EGFR protein in the mammary glands of virgin
animals, probably because of the low mammary epithelial cellularity at
this stage.

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Figure 5. Expression of the Truncated EGF Receptor (EGFR-TR)
Protein in Transgenic Mice
Microsomes prepared from the indicated mouse tissues were analyzed for
EGF binding using a cross-linking assay with [125I]EGF.
The positions corresponding to the EGFR-TR and the endogenous wild type
EGFR are indicated. Lanes 1 and 2 show the EGF receptors in the mammary
glands of control and transgenic (line 20-1) pregnant females,
respectively. Lanes 3 and 4 show the receptors in transgenic male mouse
tissues (line 7-6); lane 3, testis; lane 4, epididymis.
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Consequences of MMTV-EGFR-TR Transgene Expression in Female
Mice
The mammary glands of the MMTV-EGFR-TR transgenic mice were
examined to determine whether the expression of the truncated receptor
resulted in developmental changes in this tissue. Comparison of
whole-mount preparations of the fourth inguinal mammary glands from
virgin transgenic and wild type mice indicated that the expression of
EGFR-TR was associated with the inhibition of mammary duct development.
The first signs of ductal development inhibition could be seen in the
virgin transgenic mice as early as 56 weeks of age when puberty
begins. As shown in Fig. 6
, A and B,
there were dramatically fewer end buds in a 5-week-old transgenic
animal (Fig. 6B
) as compared with its control littermate (Fig. 6A
). The
end buds show clear signs of dichotomous branching in the control, but
not in the transgenic mice. Furthermore, the size of the end buds was
considerably smaller in the transgenics. At this early stage, however,
no significant differences were observed in the rudimentary duct
pattern or density. The ductal inhibition in the transgenics progressed
with age. By 10 weeks, the transgenic mammary glands exhibited a much
simpler, sparser duct system, with a dramatic reduction in both side
branching and terminal branching (Fig. 6
, C and D). The reduction in
side- and terminal branching was even more striking at higher
magnification as depicted in Fig. 6
, E and F. Figure 6
, G and H, shows
the mammary whole-mounts of 17-week-old mice. At this age, while the
wild type virgin animals exhibited extensive dichotomous branching,
complete ductal arborization and extension of the duct tree to the
limit of the mammary fat pad (Fig. 6G
), the transgenic virgin animals
exhibited a profound defect in dichotomous branching (Fig. 6H
).
Beginning at 10 weeks, the size of the fourth inguinal mammary glands
was also apparently smaller in the transgenics than in the control
animals (Fig. 7
), probably as a result of
lesser fad pad invasion by the ductal tree. This inhibition of ductal
development was observed consistently in multiple transgenic mice,
indicating that the observed inhibition was not due to sampling at
different stages of the estrous cycle. Similar inhibition of mammary
duct development was also observed in each of the four transgenic mouse
lines (7-6, 31-4, 21-6, and 20-1), in which the expression of the
transgene in the mammary glands had been proven at both the mRNA and
protein levels. Therefore, the phenotype was not dependent on the site
of transgene integration. A correlation between the level of transgene
expression and the extent of mammary duct inhibition was sustained. The
expression of the EGFR-TR in line 31-7 was one third of that observed
in line 31-4 as determined by Northern blot analysis normalized to the
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA. Correspondingly,
the low-expression line 31-7 exhibited only subtle inhibition of
mammary duct development as compared with the high-expression line 31-4
(data not shown).

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Figure 6. Whole Mounts of Mammary Glands from Transgenic
Females and Nontransgenic Control Mice
Whole mounts of fourth inguinal mammary glands from control (A, C, E,
and G) or transgenic (line 21-6) (B, D, F, and H) virgin females were
prepared and stained with hematoxylin. Mice from three age groups were
examined. A and B, 5-week-old; notice the difference in number and size
of the end buds (larger arrowhead indicates a typical
normal end bud in wild type animal) and degree of dichotomous branching
(smaller arrowhead); C and D, 10-week-old; notice the
difference in side- and terminal branching; E and F, higher
magnification of the periphery of the glands shown in C and D; G and H,
17-week-old; notice the difference in mammary duct outgrowth, density,
and branching. The dark, elliptical masses in E and F
are lymph nodes (arrowheads). Original magnification A
to D, 50x; E and F, 200x; G and H, 15x.
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Figure 7. Gross Morphology of the Mammary Glands from
Transgenic and Nontransgenic Control Mice
The fourth inguinal mammary glands from a 10-week-old transgenic (line
7-6) (bottom) and its nontransgenic litter mate
(top). Notice the smaller size of the transgenic mammary
gland.
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Despite the inhibition of mammary duct development in virgin mice,
normal alveolar development was observed in the transgenic mice during
pregnancy as judged by whole-mount preparations (data not shown). All
transgenic females, including those mice that were homozygous for the
transgene, were able to suckle their young successfully even with
litters of as many as 12 pups. Consistent with this observation, no
significant difference in the lobuloalveolar structure was observed on
mammary gland whole-mount examination between transgenic mice and
wild type animals during lactation (data not shown).
Analysis of paraffin sections of fourth inguinal mammary glands
confirmed that expression of the truncated receptor impaired ductal
development in the postpubertal animals. Compared with the wild type
virgin mice (Fig. 8A
), the duct density
in the virgin transgenic animals (line 314) (Fig. 8B
) was
significantly reduced at the age of 17 weeks. Under higher
magnification, the apparent mammary duct branching observed in wild
type animals (Fig. 8C
) was markedly reduced in the transgenic mice
(Fig. 8D
). Consistent with the whole-mount results, the mammary glands
from pregnant transgenic animals (Fig. 8F
) exhibited only subtle
histological differences from pregnant wild type animals (Fig. 8E
). The
alveoli in the transgenic animals appeared somewhat hypertrophied,
perhaps in compensation for the hypoplastic ductal tree. Similarly,
during lactation, slightly more secretory alveoli in the wild type
animal (Fig. 8G
) and slightly compensatory enlargement of the alveoli
in the transgenic female were observed (Fig. 8H
).

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Figure 8. Histological Sections from the Mammary Glands of
Transgenic and Nontransgenic Controls
Mammary glands from control (A, C, E, and G) or transgenic (line 7-6)
(B, D, F, and H) female animals were fixed, paraffin-embedded, and
sectioned, followed by routine hematoxylin and eosin staining. Mice in
different physiological stages were examined. A and B, 17-week-old
virgins; C and D, higher magnification views of 17-week-old virgins; E
and F, late pregnancy (day 1920); G and H, lactation day 1. Original
magnification, A and B, 100x; C and D, 400x, E to H, 100x.
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Finally, no consistent structure or function changes were observed in
transgenic mice in tissues other than the mammary glands, despite high
levels of transgene expression. The examined tissues included salivary
glands, lungs, epididymis, and prostates. No tumor has been found in
MMTV-EGFR-TR transgenic mice after observation for 1.5 yr.
Effects of Transgene Expression on Mammary Epithelial Cell DNA
Synthesis
We performed in vivo bromodeoxyuridine (BrdU)
incorporation assay to determine whether the inhibition of mammary duct
development in MMTV-EGFR-TR mice resulted from the inhibition
of mammary epithelial cell proliferation. Inhibition of the EGF-induced
mitogenic response by a similarly truncated EGFR has been reported in
cultured cells (19). Ten-week-old wild type and transgenic animals were
injected intraperitoneally with BrdU, and paraffin sections of the
fourth inguinal mammary glands were prepared for immunostaining with an
anti-BrdU antibody. Three transgenic virgin females from line 21-6 and
two of their nontransgenic litter mates were examined. Figure 9
shows representative ducts stained with
BrdU. It is significant that consistently more BrdU-positive mammary
epithelial cells were observed in control (Fig. 9A
) than transgenic
(Fig. 9B
) animals. At this age, because end buds were not
readily seen in the sections, only the terminal ducts were scored. Five
representative mammary terminal ducts from each transgenic animal or
control mouse were scored for the BrdU-labeling index. This index is
the percentage of BrdU-positive epithelial cells in relation to the
total cells scored of individual duct. Table 1
shows that the BrdU labeling index in
the 10-week-old transgenic virgin females of line 21-6 was about one
third of that seen in the control wild type mice, indicating that
expression of the EGFR-TR does inhibit mammary ductal epithelial cell
proliferation in vivo. A similar degree of inhibition of
mammary epithelial BrdU labeling was also observed in transgenic line
31-4 (data not shown). The inhibition of BrdU labeling in the
transgenic animals was specific for the mammary duct epithelial cells,
in that the transgene had no impact on the BrdU staining in lymphocytes
in the mammary lymph nodes (data not shown).

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Figure 9. In vivo BrdU Labeling of the Mammary
Glands of Transgenic and Nontransgenic Controls
Ten-week-old transgenic mice (line 216) or their nontransgenic litter
mates were subjected to in vivo BrdU labeling. The
animals received an intraperitoneal injection of BrdU 2 h before
sacrifice. Paraffin sections of the fourth inguinal mammary gland were
examined by immunostaining with an anti-BrdU monoclonal antibody
detected using the peroxidase system. The sections were counterstained
with hematoxylin. Sections are from the control animal (A) and from the
transgenic animal (B). Original magnification 400x.
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Table 1. BrdU Labeling Index in Mammary Terminal Duct
Epithelial Cells of Wild Type and MMTV-EGFR-TR Transgenic (Line 21-6)
Mice at 10 Weeks
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DISCUSSION
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Expression of a C-terminal truncated EGFR in transgenic animals
under the control of the MMTV promoter caused moderate inhibition of
mammary ductal development. This observation is compatible with the
notion that the truncated receptor behaves in a dominant negative
manner, thereby blocking signaling through this receptor. Our own data
with the EGFR-TR and published data using a similarly truncated
receptor (18, 19) indicate that the EGFR-TR, when expressed in cultured
cells at sufficiently high levels, result in a blockade of EGF
signaling through the wild type receptor. Both EGF-stimulated receptor
autophosphorylation and EGF induction of c-fos gene
expression are down-regulated by the EGFR-TR. More recently, expression
of a truncated EGFR in the skin under the control of a keratin promoter
was shown to result in developmental abnormalities in the skin (20)
presumed to result from the dominant negative action of the truncated
receptor. However, interpretation of this skin model was equivocal, in
that areas of hyperplasia were also observed, raising the possibility
of a compensatory response from another growth factor system or an
action of the truncated receptor other than signal blockade. In the
mammary gland system described in this paper, only a hypoproliferative
response was observed in the ductal cells, completely compatible with a
dominant negative mechanism for the EGFR-TR. This dominant negative
strategy using receptor truncation or kinase domain mutation has also
been applied to study the role of some other receptor systems including
the fibroblast growth factor receptor (37, 38), vascular endothelial
growth factor receptor Flk-1 (39), and insulin receptor (40).
The phenotype of the MMTV-EGFR-TR transgenic animals has features in
common with animals overexpressing activated TGFß1 in the mammary
gland (30) and animals in which the estrogen receptor gene has been
disrupted (for review, see Ref.41). In the case of the MMTV-TGFß1
mice, it was demonstrated that the lack of ductal elongation and
branching in the postpubertal, nonpregnant animals resulted from the
ability of the TGFß1 to block duct epithelial cell proliferation
(30). The inhibition of mammary ductal epithelial proliferation by
TGFß1 is consistent with the known ability of TGFß1 to block the
proliferation of most nontransformed epithelial cells (for review, see
Ref.42). This antiproliferative effect of TGFß1 could either be
direct (43) or result, in part, from TGFß1 down-regulation of
expression of the EGFR and TGF
(44). That ductal development and
BrdU labeling of ductal cells were also partially blocked by the
expression of the EGFR-TR in the mammary epithelium suggests that the
truncated receptor also inhibited epithelial cell proliferation,
thereby resulting in a similar phenotype. Another similarity shared
between the MMTV-TGFß1 and MMTV-EGFR-TR models was the developmental
stage at which the phenotype was most apparent. In both models, it was
the postpubertal elaboration of the ductal system that was most
affected (30). The ability of the EGFR-TR to block some degree of
postpubertal growth of the ductal system implies that the EGFR and its
ligands play an important role in this stage of mammary gland
development.
Most mammary gland development occurs postpubertally, implying an
important role for gonadal steroids. Classic endocrinological
experiments using gonadectomy and hormone replacement had indicated a
crucial role for estrogen and progesterone in this process. Estrogen
promotes proliferation of normal (45, 46) and cancerous mammary
epithelial cells. More recently, the role for estrogen was further
supported by the development of an animal model in which the endogenous
estrogen receptor gene was disrupted (41). These animals display very
limited mammary ductal growth and branching, implying that estrogen
plays a crucial role in the control of the proliferation of the mammary
epithelium. There is considerable evidence that estrogen and the EGFR
signaling interact in the control of this proliferation. TGF
, a
ligand for the EGFR, is expressed in the normal mammary gland (27) and
in other estrogen- responsive tissues such as the pituitary lactotrophs
(47) and uterus (48). In each of these tissues, TGF
gene expression
has been shown to be stimulated by estrogen. Furthermore, the TGF
gene promoter contains an atypical estrogen response element that
confers an estrogen response to a reporter gene (49, 50). Thus,
estrogen might stimulate growth of the ductal cells by increasing the
local expression of TGF
and, consequently, the autocrine stimulation
of the EGFR in the ductal epithelial cells. There is also some evidence
that estrogen and EGF act synergistically in the activation of
estrogen-responsive genes (51, 52, 53). Thus, this transgenic animal model,
with impaired EGFR signaling in the mammary gland, may exhibit
suboptimal estrogen action, thereby impairing other actions of estrogen
that are required for ductal development. However, the inhibition of
duct development by the EGFR-TR is unlikely to relate just to the
estrogen effects. Mammary duct development also depends on many other
factors. Indeed, estrogen replacement in prepubertally ovariectomized
mice is not sufficient to allow normal duct development, and other
hormones, such as progesterone, GH, PRL, and adrenal steroids, also
contribute to this process (54, 55, 56). Whether the EGFR-TR also affects
the action of these other hormones or whether the partial development
of the mammary duct system, despite the EGFR-TR, results from continued
action of these other hormones remains to be seen.
In addition to controlling mammary epithelial proliferation, the EGFR
system may also play a role in the control of mammary
gland-differentiated function. Transgenic animals overexpressing TGF
in the mammary gland exhibited a failure to lactate (24). This
antilactogenic effect of EGF/TGF
may relate to the observation that
EGFR activation inhibits the expression of milk-specific ß-casein and
whey acidic protein both in cultured cells and in transgenic animals
(26). In concert with these known effects of EGF/TGF
, the
MMTV-EGFR-TR animals were functionally normal during lactation. These
animals exhibited normal alveolar outgrowth during pregnancy and were
able to sustain sufficient lactation to support large litters. This
result suggests that either the EGFR system is not crucial for alveolar
development during pregnancy and lactation or that the level of
expression of the EGFR-TR was insufficient to block these stages of
mammary development. In midgestation, maternal expression of the
endogenous EGFR in the mammary gland has been shown to be at its
highest (21). Since the dominant negative effect of the EGFR-TR largely
relies on a relatively high ratio of mutant over wild type (Fig. 1
and
Refs. 17 and 36), the mammary gland could escape from this EGFR
blockade in midgestation. Indeed, Northern blot analysis and
[125I]EGF cross-linking studies performed on the mammary
glands of pregnant transgenic mice indicated that the expression of the
endogenous receptor had increased greatly while the expression of the
truncated receptor did not. Therefore, this observed change in the EGFR
to EGFR-TR ratio could explain why mammary alveolar development during
pregnancy is practically normal in this transgenic model. The
involvement of estrogen, progesterone, and PRL in alveolar development
has also been well established (55, 56). Alveolar development is
markedly impaired in animals in which the progesterone (54) or PRL (57)
receptor gene has been disrupted. The role of EGF signaling in
conjunction with these hormones has not been resolved by this model
because of the failure of the MMTV promoter to drive expression of the
EGFR-TR to levels greater than that of the endogenous receptor.
However, the high level of expression of the endogenous EGFR during
pregnancy does suggest some role for this receptor at this maternal
developmental stage.
In contrast to the situation in pregnancy, during lactation, endogenous
EGFR expression is low (21). This low receptor number and the lack of
an observed effect of the EGFR-TR during lactation suggests that EGFR
signaling at this time plays only a minor role and that other hormones
or other growth factors dominate in the establishment of lactation.
Interestingly, the waved-2 mouse, a line that expresses a mutant EGFR
with partial loss of protein tyrosine kinase activity, does exhibit
impaired lactation (58). While this lactation defect might result from
a direct effect of the mutant EGFR on mammary development, it also
remains possible that the defect in the EGFR gene of waved-2 mice,
which occurs in all cell types including the other endocrine glands,
exerts its effect on lactation indirectly by altering the complex
hormonal milieu that is required to establish and maintain
lactation.
In addition to directing expression to the mammary gland, the MMTV-LTR
has been reported to direct transgene expression to salivary epithelial
cells, lung, kidney, and seminal vesicles (for review, see Ref.36).
Expression of oncogenes in transgenic animals under the control of the
MMTV-LTR therefore often results in hyperplasia or neoplasia at these
nonmammary sites (36). In the present study, we observed high levels of
extramammary EGFR-TR expression in the lung (line 21-6), salivary
gland, epididymis, and prostate. However, thus far, no significant
phenotype has been observed in these tissues. Interestingly, the
MMTV-TGF
mice, with similar extramammary transgene expression, also
exhibited no histological abnormalities in these extramammary tissues
(24). The lack of effect of EGFR-TR and TGF
expression in these
tissues suggests that the EGFR system is less important for development
in these tissues than it is in the mammary gland.
Other growth factor receptor systems, such as other members of the erbB
family of receptor tyrosine kinases, may play a role in mammary
development. The neu/erbB2 receptor tyrosine kinase, when overexpressed
in the mammary gland, results in carcinogenesis (59, 60). Furthermore,
abnormalities in erbB2 have been detected in spontaneous human breast
carcinomas (61). Recently, there is some evidence that erbB2 interacts
with the EGFR through the formation of signaling heterodimers (62, 63).
It remains possible that the EGFR-TR could also interact with erbB2,
perhaps impairing signaling through this receptor. Thus, the phenotype
observed in the MMTV-EGFR-TR transgenic mice may also relate to
interactions, not only with the endogenous EGFR, but other growth
factor receptor systems. The transgenic model described in this
manuscript could be used to explore these interactions.
 |
MATERIALS AND METHODS
|
---|
Generation of EGFR-TR Expression Vector
A 2.3-kb C-terminal truncated mouse EGFR (EGFR-TR) cDNA encoding
amino acid 1 to 690 (L) was generated by inserting a double-stranded
oligonucleotide 5'-GAGCTG-TGAATTCTGATCACAGCTC-3' into
the SacI site at nucleotide 2224 of the mouse EGFR cDNA (31)
and, by doing so, introduced a stop codon (TGA) after L690 followed by
an EcoRI site. EGFR-TR or EGFR-WT cDNA (32) was placed
downstream of the cytomegalovirus promoter of plasmid pGFP (Clontech,
Palo Alto, CA) after removal of the GFP cDNA using standard cloning
strategies.
Transient Transfection and Western Blot Assay
Electroporation-mediated transient transfection was carried out
as described (64). CHO cells were transfected with EGFR-WT alone (7.5
µg plasmid) or together with increasing amounts of EGFR-TR (7.5 µg
or 15 µg plasmid). Thirty six hours after transfection, the cells
were treated with 10-8 M EGF for 5 min at 37 C
or mock treated. Cells were subsequently collected and lysed in RIPA
buffer [50 mM HEPES, pH 7.2, 150 mM NaCl, 1.5
mM MgCl2, 1.5 mM EGTA, 1% Triton
X-100, 0.1% SDS, 0.1% deoxycholic acid, 10 µg/ml leupeptin, 1
mM phenylmethylsulfony fluoride (PMSF), 1 mM
sodium vanadate, 50 mM NaF]. Equivalent amounts of lysate
were immunoprecipitated with an anti-EGFR monoclonal antibody H9B4
(34), which is specific for the EGFR intracellular domain. The
immunoprecipitates were resolved in 7.5% SDS-PAGE, followed by Western
transfer, and blotted with H9B4 using enhanced chemiluminsescence (ECL)
detection system (Amersham, Arlington Heights, IL). The same filter was
subsequently stripped and reblotted with an anti-phosphotyrosine
antibody, 4G10 (Upstate Biotechnology, Inc, Lake Placid, NY).
Generation of MMTV-EGFR-TR Transgenic Mice
The MMTV-LTR transgene cassette was kindly provided by Dr.
H. L. Moses and was described in detail elsewhere (30). The
EGFR-TR cDNA encoding amino acids 1 to 690 (L) was inserted into the
EcoRI site of ß-globin exon 3. The 4.9-kb XhoI
fragment containing the entire transgene cassette was isolated by
electroelution from a 1.0% agarose gel followed by purification with
GlassMax (GIBCO-BRL, Grand Island, NY). All mice were handled in an
accredited university facility in accordance with the institutional
animal care policies. Microinjection into one-cell B6xSJL mouse zygotes
at a concentration of 2 ng/ml was carried out at the University of
Alabama at Birmingham transgenic animal facility under the direction of
Dr. Carl Pinkert.
Identification of Transgenic Mice
Genomic DNA was isolated by phenol/chloroform extractions as
described (65). The PCR was used to screen for transgene-positive mice
with one oligonucleotide designed to anneal to the EGFR-TR cDNA,
5'-CTGGGCACAGATGATTT-3' (corresponding to nucleotides 795 to 811) (31),
and the other oligonucleotide was designed to anneal to the rabbit
ß-globin intron between exon 2 and exon 3 in the transgene cassette,
5'-CACTGTTTGAGATGAGG-3' (corresponding to Il -70 to -86)
(66) The PCR was carried out on a DNA thermal cycler
(Perkin-Elmer/Cetus, Norwalk, CT) using the following program: 94 C for
1 min, 57 C for 2 min, and 72 C for 3 min. PCR products were analyzed
by electrophoresis on a 1% agarose gel.
PCR identification of the transgenic animals was confirmed by Southern
blot analysis. Eight micrograms of mouse tail genomic DNA was digested
with KpnI or XbaI restriction enzymes and
subjected to Southern blotting and hybridization using Hybond-N+ nylon
membrane (Amersham), following the protocol of the membrane
manufacturer. The probes used were a 2.3-kb EGFR-TR cDNA
(EcoRI-EcoRI fragment) or the 526 bp
EcoRI-XhoI fragment of rabbit ß-globin exon
3.
Northern Blot Analysis
Total RNA was prepared from the tissues using acid guanidinium
thiocyanate-phenol-chloroform extraction as described (65). RNA was
separated on 1.25% agarose-6% formaldehyde gel and transferred to a
Hybond-N+ nylon membrane (Amersham). To detect specific
transcripts, [32P]cDNA probes labeled by Deca Prime II
kit from Ambion (Austin, TX) were used for hybridization on the
membranes. The probes used were the same as described in Southern blot.
The filters were cohybridized or subsequently rehybridized with a
plasmid containing the murine GAPDH cDNA for normalization of
loading.
Tissue Microsome Preparation and
[125I]EGF-Mediated EGF Receptor Cross-Linking
Assay
The tissues were homogenized in buffer A (5 ml/g tissue)
containing 10 mM HEPES, pH 7.5, 1 mM PMSF, 1
mM dithiothreitol, 0.3 M sucrose using a
Polytron. After centrifugation at 10,000 x g for 30
min at 4 C, the supernatant was transferred to a new tube and further
centrifuged at 95,000 x g for 75 min at 4 C. The crude
microsome pellet was resuspend in 5 ml of buffer B (buffer A containing
0.6 M KCl), followed by centrifugation at 95,000 x
g for 75 min at 4 C. The microsomes were subjected to the
cross-linking assay, or resuspended in 200 µl of buffer A and stored
in liquid nitrogen until use. For the cross-linking assay, the
microsome pellet was resuspended in 200 µl of binding buffer (PBS
with 0.1% BSA and 1 mM PMSF), and incubated with
[125I]EGF (final concentration about 1.4 x
10-8 M EGF) at 4 C for 5 h. The
microsomes were recovered by centrifugation at 50,000 x
g for 60 min at 4 C. After washing with 1 ml of binding
buffer twice, the microsomes were resuspended in 250 µl PBS
containing 2.0 mM BS3 (Pierce, Rockford, IL).
Cross-linking was carried out at room temperature for 30 min. The
cross-linking reaction was quenched with 250 mM glycine,
and the microsomes were recovered by centrifugation. The
125I-labeled proteins were subjected to 4.5% SDS-PAGE and
visualized by autoradiography.
Mammary Gland Whole-Mount and Histological Analysis
Inguinal mammary fat pads were removed from transgene-positive
and control animals and fixed in formaldehyde (4% in PBS) at 4 C for
at least 24 h, followed by whole-mount preparation and hematoxylin
staining as described (30). The whole mounts were analyzed in methyl
salicylate under a dissecting microscope. Parts of the fixed tissues
were embedded in paraffin, sectioned, and stained with hematoxylin and
eosin for regular histological examination.
BrdU Labeling and Immunohistochemistry
In vivo BrdU labeling was performed by
intraperitoneal injection of BrdU as described (30). Paraffin sections
were deparaffinized, hydrated, pretreated with 2 N HCl for
20 min at 37 C, and exposed to 0.01% trypsin at 37 C for 3 min,
followed by BrdU immunostaining using a rat monoclonal anti-BrdU
antibody MSA250p (Accurate, Westbury, NY) at 1:100 dilution and
Vectastain Elite ABC Kit (Vector, Burlingame, CA).
3,3'-Diaminobenzidine tetrahydrochloride was used as chromogen, and
sections were counterstained with Gills Hematoxylin (Vector).
 |
ACKNOWLEDGMENTS
|
---|
The authors wish to thank Dr. H. L. Moses for the MMTV-LTR
transgene cassette, Drs. A. Aviv and D. Givol for the mouse EGF
receptor cDNA, and Dr. A. Wells for wild type human EGF receptor cDNA.
We also gratefully acknowledge Drs. S. J. Frank and A. Wells for
helpful discussions.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Jeffrey E. Kudlow, Division of Endocrinology and Metabolism, University of Alabama at Birmingham, Birmingham, Alabama 35294.
This work was supported by Public Health Service Grant DK-43652 and NIH
Grant DK-48882.
Received for publication June 19, 1997.
Revision received August 1, 1997.
Accepted for publication August 5, 1997.
 |
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