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Department of Physiology, Ralph L. Smith Research Center, University of Kansas Medical Center, Kansas City, Kansas 66160-7336
Address all correspondence and requests for reprints to: Dr. S. K. Das, Department of Physiology, Ralph L. Smith Research Center, University of Kansas Medical Center, Kansas City, Kansas 66160-7338.
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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, heparin binding-EGF, amphiregulin, betacellulin,
epiregulin, and heregulins, whereas the receptor family (the
erbB genes) consists of erbB1 (EGF-receptor,
EGF-R), erbB2, erbB3, and
erbB4. Interactions of uterine EGF-R with EGF-like
ligands have been examined, but limited information is available
regarding the status of other receptor subtypes. Thus, we examined the
expression of the erbB2 gene in the mouse uterus during
the periimplantation period (days 1–8 of pregnancy) and after
17ß-estradiol and/or progesterone stimulation. Northern blot
hybridization detected two transcripts (
4.0 and 5.0 kb) of
erbB2 messenger RNA (mRNA) in day 1–8 uterine
polyadenylated RNA samples. In situ hybridization
experiments showed unique uterine cell-specific erbB2 mRNA
distribution. On days 1–4, unlike the full-length erbB1
mRNA which is not expressed in the uterine epithelium, the
erbB2 mRNA was detected primarily in epithelial cells; the
day 1 uterus showed the highest accumulation. On day 5, the epithelium
and the decidualizing stromal cells around the implanting blastocyst
exhibited accumulation of this mRNA. On days 6–8, the accumulation
persisted in the epithelium at both the implantation and
interimplantation sites in addition to modest levels of signals in the
secondary decidual zone. On days 7 and 8, accumulation of the
erbB2 mRNA was also prominent in the trophoblastic giant
cells. Western blotting detected a predicted protein of 185 kDa in day
4 uterine membrane preparations. Results of immunocytochemistry
demonstrated colocalization of the erbB2 protein with its
mRNA in the periimplantation uterus. The uterine ErbB2 underwent
phosphorylation by several members of the EGF family. Treatment of
adult ovariectomized mice with 17ß-estradiol, but not progesterone,
up-regulated the expression of the erbB2 mRNA by more than
3.5-fold, as determined by quantitative reverse transcription-PCR, and
this increase was limited to the epithelium, as revealed by in
situ hybridization. Collectively, the results place ErbB2 as a
potential candidate receptor subtype for interaction with the
EGF-related ligands in epithelial cell proliferation/differentiation
during the preimplantation period and stromal cell
proliferation/decidualization during the postimplantation period. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The EGF family includes EGF, transforming growth factor- (TGF),
amphiregulin (AR), heparin binding-EGF (HB-EGF), betacellulin,
epiregulin, and heregulins/neu differentiation factors (NDFs) (17, 18, 19, 20, 21, 22, 23).
In the rodent, these ligands can interact with the receptor subtypes of
the erbB gene family. This family is comprised of four
receptor tyrosine kinases: ErbB1 (EGF-R), ErbB2, ErbB3, and ErbB4. They
share a common structural feature, but differ in their ligand
specificities and kinase activities (24, 25, 26). EGF, TGF, HB-EGF, AR,
betacellulin, and epiregulin can interact either with ErbB1 via
homodimerization or with other ErbB members through heterodimerization
(27, 28). In this respect, the NDFs, which can stimulate
phosphorylation of the 185-kDa product of the erbB2 gene
(23), require direct binding to either erbB3 or
erbB4 (29). Thus, cross-talk between the receptor subtypes
with various ligands can serve as a potential signaling mechanism (27, 28).
The expression patterns of EGF, TGF, AR, and HB-EGF in the
uterus during the periimplantation period and their responsiveness to
sex steroid hormones have been examined (4, 6, 8, 11). Overall, the
results suggest that AR may be involved in uterine preparation for
implantation, whereas HB-EGF could interact with blastocyst ErbB1/EGF-R
for the attachment reaction (6, 30). The role of TGF in implantation
is questioned, because female mice deficient in TGF are apparently
fertile (31, 32). These studies also suggested that uterine cell
proliferation or differentiation by steroid hormones is mediated
locally by these growth factors. EGF was shown to bind to uterine
epithelial cells and induce their proliferation both in
vitro and in vivo (33, 34). Thus, it was suggested that
EGF-like growth factors can influence epithelial cell functions
directly. However, more rigorous studies document that mouse uterine
epithelial cells exhibit little or no expression of the functional
full-length ErbB1/EGF-R, although a truncated form is expressed in
these cell (7, 35). This controversial issue of how EGF mediates
epithelial cell proliferation could be resolved if it is found that
epithelial cell proliferation directed by EGF-like ligands or steroid
hormones is mediated either indirectly via stromal cell ErbB1 in a
paracrine manner or via other members of the ErbB family in epithelial
cells. However, little or no information is available regarding the
status of these receptor subtypes in the mouse uterus.
As the nature of ligand-receptor signaling with EGF-like growth
factors is complex and varies according to the cell types and receptor
subtypes expressed (36), it is important to examine the expression
profiles of the erbB gene family in the uterus to better
understand their roles in uterine biology and implantation. In this
investigation, we examined whether ErbB2 could be a candidate mediator
of signaling by EGF-like growth factors in the uterus, as TGF,
HB-EGF, and AR, which are expressed in the uterus, have been shown to
induce heterodimerization of erbB1 and erbB2, and
the later is considered an integral subunit of this heterodimerization
(36, 37). Thus, we cloned the mouse erbB2 complementary DNA
(cDNA) and examined its temporal and cell type-specific expression in
the mouse uterus during the periimplantation period and its modulation
by ovarian steroid hormones in ovariectomized mice. Receptor
phosphorylation studies were performed to determine whether uterine
ErbB2 is functional. The results establish that unlike the
erbB1/EGF-R gene, the erbB2 gene is expressed in
the preimplantation or estrogen-treated uterine epithelium and in the
deciduum during the postimplantation period. The findings suggest that
ErbB2 is a potential mediator of signaling by EGF-related growth
factors in the mouse uterus.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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1 and NDFß1 were generous gifts of D. Wen (Amgen,
Thousand Oaks, CA). The antibody used in phosphorylation studies was
antihuman Neu polyclonal antibody (Santa Cruz Biotechnology, Santa
Cruz, CA).
Animals and tissue preparation
CD-1 mice (Charles River Laboratory, Raleigh, NC) were housed in
the animal care facility at the University of Kansas Medical Center
according to NIH and institutional guidelines for the care of
laboratory animals. Adult female mice (20–25 g; 48–60 days old) were
mated with fertile males of the same strain. The morning when a vaginal
plug was found was designated day 1 of pregnancy. Mice were killed
between 0830–0900 h on days 1–8 of pregnancy. Whole uteri were
collected on days 1–7 of pregnancy, whereas the deciduum was
surgically separated on day 8. Early implantation sites on day 5
(0830–0900 h) were visualized by iv injections (0.1 ml/mouse) of a
Chicago blue B dye solution (1% in saline) and killed 5 min later to
identify the blue bands (implantation sites) along the uterus (2).
To determine the effects of estrogen and P4, mice were ovariectomized without regard to the stage of the estrous cycle and rested for 2 weeks. They were given an injection of 17ß-estradiol (E2; 250 ng/mouse; Sigma Chemical Co, St. Louis, MO), P4 (2 mg/mouse; Sigma), or a combination of the same doses of P4 and E2. All steroids were dissolved in sesame oil and injected sc (0.1 ml/mouse). The control animals received the vehicle (0.1 ml/mouse) only. Mice were killed at different times after hormone injections, and their uteri were collected for analysis.
Cloning and sequencing of the mouse erbB2 partial cDNA
Reverse transcription-PCR (RT-PCR) was used to generate the
erbB2 partial cDNA clone. RT-PCR conditions were essentially
as described previously by us (10). Oligonucleotide primers were
synthesized based on the cDNA sequence of the human erbB2
gene for the cytoplasmic domain (38). The primers were
5'-GCTCCCCATATGTCTCCCGC-3' (sense) and 5'-GCCGCTCCCCCTTTTCCAGC-3'
(antisense). The sense strand primer corresponds to 2483–2502
nucleotides (nt), whereas the antisense strand primer encompasses
2852–2971 nt of the human erbB2 gene. Day 4 pregnant mouse
uterine total RNA (1 µg) was reverse transcribed using the antisense
primer as previously described (10). RT products (3 µl) were
amplified by PCR for 45 cycles using the cycle parameters: 94 C, 1.5
min; 55 C, 2 min; 72 C, 2.5 min. The predicted RT-PCR product (489 bp)
was analyzed by gel electrophoresis, and its authenticity was verified
by Southern blot hybridization using a 32P end-labeled
internal primer derived from the human erbB2 sequence.
RT-PCR product was cloned into pCR-Script SK+ cloning
vector (Stratagene, La Jolla, CA). Several colonies were analyzed by
restriction digestion, and finally, the nt sequence of one clone was
determined on both strands by the dideoxy nt chain termination method
(39) and the Sequenase version 2.0 kit (U.S. Biochemical, Cleveland,
OH).
Hybridization probes
For Northern hybridization, 32P-labeled antisense
complementary RNA (cRNA) probes were generated, whereas for in
situ hybridization, sense and antisense 35S-labeled
cRNA probes were generated using the appropriate polymerases. Probes
had specific activities of about 2 x 109
dpm/µg.
Northern blot hybridization
Total RNAs were extracted from whole uteri pooled from 10–15
mice on the indicated days of pregnancy by a modified guanidine
thiocyanate procedure (6, 7). Polyadenylated [poly(A)+]
RNAs were isolated from total RNAs by oligo (deoxythymidine)-cellulose
column chromatography (40). Poly(A)+ RNA (2 µg) was
denatured, separated by formaldehyde-agarose gel electrophoresis,
transferred to nylon membranes, and cross-linked by UV irradiation
(Spectrolinker, XL-1500, Spectronics Corp., Westbury, NY). The blots
were prehybridized, hybridized, and washed as described previously (6).
After hybridization, the blots were washed under stringent conditions,
and the hybrids were detected by autoradiography (6). The blots were
stripped and rehybridized with ß-actin probe as described previously
(7).
In situ hybridization
In situ hybridization was performed as described
previously (6). Uteri were cut into 4- to 6-mm pieces and flash-frozen
in freon. Frozen sections (11 µm) from days 1–4 or days 5–8 of
pregnancy were mounted onto poly-L-lysine-coated slides,
fixed in cold 4% paraformaldehyde solution in PBS, acetylated, and
hybridized at 45 C for 4 h in 50% formamide hybridization buffer
containing the 35S-labeled antisense cRNA probe. After
hybridization and washing, the sections were incubated with
ribonuclease A (RNase A; 20 µg/ml) at 37 C for 15 min. RNase
A-resistant hybrids were detected by autoradiography using Kodak NTB-2
liquid emulsion (Eastman Kodak, Rochester, NY). Parallel sections
hybridized with the sense probe served as negative controls. Slides
were poststained with hematoxylin and eosin.
Construction of competitive templates for erbB2 and ribosomal
protein L7 (rPL7)
To generate the competitive templates for quantitation of
uterine erbB2 and rPL7 messenger RNAs (mRNAs) (41, 42), a
nonspecific DNA fragment (185-bp SspI fragment obtained from
PGEM 72f+ vector) was ligated at the StyI site
of the mouse erbB2 cDNA or at the PstI site of
the mouse rPL7 cDNA. These competitive templates were used to
quantitate mRNAs for these genes. rPL7 served as a housekeeping gene
(43).
RT-PCR quantitation of erbB2 mRNA after steroid hormone
treatments
The oligonucleotide primers were synthesized based on the
sequence of the cloned mouse erbB2 cDNA as follows: sense
strand, 5'-TCTGCCTGACATCCACAGTG-3'; and antisense strand,
5'-AATAGATTCCAATGCCATCC-3'. The primers for mouse rPL7 clone were as
follows: sense strand, 5'-TCAATGGAGTAAGCCCAAAG-3'; and antisense
strand, 5'-CAAGAGACCGAGCAATCAAG-3'. Uterine total RNAs (8.75 µg) from
ovariectomized mice injected with oil, E2, P4,
or E2 plus P4 were subjected to RT reactions
using the antisense primers in a total volume of 14 µl as previously
described (10). A constant amount of the sample cDNA (one tenth of the
total RT product) and increasing amounts of the mutant template were
coamplified by PCR for 35 cycles using the sense and antisense primers
for each specific mRNA. The PCR protocol and cycle parameters were same
as described above for RT-PCR cloning. The predicted sizes of the
sample and mutant DNA templates were 320 and 505 bp for
erbB2 and 246 and 431 bp for rPL7, respectively. The
products were subjected to 1% agarose gel electrophoresis for Southern
blot hybridization using 32P end-labeled internal primers:
5'-ATGCAGATGGGGGCAAGGTG-3' for erbB2 and
5'-GATTGCCTTGACAGATAATTC-3' for rPL7. The quantitation of band
intensities on the autoradiogram was achieved using densitometric
scanning (Personal Densitometric SI, Molecular Dynamics, Sunnyvale,
CA). The ratios of band intensities of the sample bands to those of the
mutant bands were plotted against the amount of the mutant templates.
The amount of sample DNA, reflecting the initial levels of mRNA, was
determined from the zero equivalence point on the logarhythmic
graph.
Generation of antipeptide antibody to ErbB2
Antipeptide antibodies to ErbB2 were raised in rabbits using a
synthetic 14-amino acid peptide representing the cytoplasmic domain of
the mouse erbB2 amino acid sequence, 808–821
(DHVREHRGRLGSQD). The antipeptide antibodies were affinity purified
through Affi-Gel 10 (Bio-Rad Laboratories, Hercules, CA) conjugated
with the corresponding peptide (9) and used for Western blot and
immunostaining.
Western blotting
The method essentially followed the protocol described by us
previously (9). In brief, day 4 mouse uteri were collected into buffer
A [10 mM Tris-HCl (pH 7.4), 250 mM sucrose, 2
mM EGTA, 10 µg/ml leupeptin, 20 µg/ml
phenylmethylsulfonylfluoride, and 10 µg/ml aprotinin]. They were
homogenized in buffer A and centrifuged at 2,000 rpm for 10 min at 4 C.
The supernatants were recentrifuged at 35,000 rpm for 1 h at 4 C.
The pellets were resuspended in the same buffer and spun again for
1 h at 35,000 rpm at 4 C. The pellets were then dissolved in
buffer B [10 mM Tris-HCl (pH 7.4), 0.15 mM
NaCl, 1 mM EGTA, 10 µg/ml leupeptin, 20 µg/ml
phenylmethylsulfonylfluoride, and 10 µg/ml aprotinin], and protein
concentrations were measured. Aliquots of protein (60 µg) were mixed
with sample buffer and boiled for 5 min. The samples were run on a
7.5% SDS-polyacrylamide gel under reduced condition. The proteins on
the gel were transferred onto a nitrocellulose membrane. The membrane
was preincubated with 5% nonfat dry milk in Tris-buffered saline (TBS)
for 2 h to block nonspecific binding. The membrane was incubated
in antipeptide antibody to ErbB2 overnight at 4 C. The membrane was
washed three times for 10 min each in 5% nonfat dry milk in TBS and
incubated with goat antirabbit IgG conjugated with horseradish
peroxidase (1:15,000) for 1 h. The membrane was again washed three
times (10 min each time) in 5% nonfat dry milk in TBS and three times
in TBS. Signals were detected with the ECL kit (Amersham, Arlington
Heights, IL). As a control, the antipeptide antibody was preneutralized
with a 200-fold molar excess of the synthetic peptide that was used as
the immunogen.
Immunohistochemistry
Pieces of uteri from day 1–8 pregnant mice were fixed in cold
4% paraformaldehyde in PBS for 2 h, dehydrated, and embedded in
paraffin. Paraffin sections (7 µm) were mounted onto
poly-L-lysine-coated slides, deparaffinized, rehydrated,
and washed in PBS. The blocking of nonspecific binding was achieved by
incubating sections in 10% normal goat serum for 10 min. The sections
were then incubated in the antipeptide antibody overnight at 4 C.
Immunostaining was performed using a Zymed-Histostain-SP kit (Zymed
Laboratories, San Francisco, CA) containing a biotinylated secondary
antibody, a horseradish peroxidase-streptavidin conjugate, and a
substrate-chromogen mixture (7). The endogenous peroxidase activity was
blocked by 0.23% periodic acid in PBS for 30 sec after incubation with
the secondary antibody. Sections were counterstained with hematoxylin.
Red deposits indicated the sites of positive immunostaining. As a
control, the antipeptide antibody was preneutralized with a 200-fold
molar excess of the antigenic peptide.
Phosphorylation of uterine ErbB2
The phosphorylation of ErbB2 was determined in day 5 pregnant
uterine membranes using the method described by us previously (6, 7, 8).
Membranes (100 µg protein) were suspended in 50 µl reaction buffer
[50 mM PIPES (pH 7.0), 1 mM MnCl2,
and 0.1 mM Na vanadate] and preincubated with or without a
ligand (100 ng/ml) for 10 min at 4 C. The labeling reaction was
initiated by the addition of 5 µCi [
-32P]ATP (1
µM) in the presence of 0.1% Triton X-100 and was
continued for 2 min at 4 C. The reaction was terminated by the addition
of 15 µl of an ice-cold mixture of 1 mM ATP and 0.1%
BSA, followed by an equal volume of 10% trichloroacetic acid (wt/vol).
After incubation on ice for 1 h, the mixture was centrifuged for 5
min in a microcentrifuge at the maximum speed, and the precipitates
were collected. The precipitates were washed three times with a mixture
of diethylether and ethanol (1:1), and dissolved in 50 µl 50
mM Tris buffer (pH 7.5). An equal volume of protein
A-Sepharose-ErbB2 antibody conjugate (3 mg:0.4 µg) was added to this
mixture and incubated for 90 min at 4 C with constant shaking. The
protein A-Sepharose-antibody conjugates were washed sequentially with
buffer A (50 mM HEPES, 0.1% Triton X-100, 0.1% SDS, and 5
mM EGTA, pH 8.0), buffer B (50 mM HEPES, 0.1%
Triton X-100, 0.1% SDS, and 150 mM NaCl, pH 8.0), and
buffer C (10 mM Tris-HCl, pH 8.0). The pellets were heated
in 1 x SDS sample buffer [62.5 mM Tris (pH 6.8), 2%
SDS, 10% glycerol, and 5% ß-mercaptoethanol] to 100 C for 3 min
and centrifuged, and the resultant supernatants were subjected to 10%
SDS-PAGE in parallel with mol wt markers. The gel was transferred to
nitrocellulose membrane, and the products were visualized by
autoradiography.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). This region has 90% sequence
identity with the human or 95% with the rat erbB2 cDNA (38, 44). Further, the deduced amino acid sequence of the mouse
erbB2 is 98% and 99% homologous with those of the human
and rat ErbB2, respectively. The GenBank accession number of this cDNA
is U71126.
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).
Two major transcripts (4.0 and 5.0 kilobases) were detected in whole
uterine RNA samples. The levels of this mRNA were highest on day 1, but
did not show much variation thereafter. The significance of two
transcripts (4.0 and 5.0 kilobases) of the erbB2 mRNA in the
mouse uterus is not yet clear. However, multiple transcripts have also
been identified in the human and rat tissues (38, 44). In addition, in
human breast cancer cell lines, a truncated form of ErbB2, generated by
alternative RNA splicing, has growth regulatory role (45). However, no
information is yet available about whether a truncated form of ErbB2 is
produced in the mouse.
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, A–D). On days 5 and 6 (early
postimplantation), the erbB2 mRNA accumulation occurred in
the epithelium situated at the implantation or interimplantation
regions (Fig. 3, E and F). In addition, this mRNA accumulated in the
stroma surrounding the implanting blastocyst. With the progression of
decidualization on days 7 and 8, the remaining luminal epithelium and
glands in the interimplantation regions still maintained the
erbB2 mRNA accumulation (Fig. 3, G and H). Around the
implanting blastocyst, erbB2 mRNA was localized in the
secondary decidual zone at higher abundance. An accumulation of the
erbB2 mRNA was also noted in the embryo proper and the
trophoblastic giant cells on days 7 and 8.
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).
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, A and B). No positive immunostaining was
obtained when sections were incubated with the primary antibody
preneutralized with the antigenic peptide (data not shown).
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1, NDF-ß1, or
betacellulin, followed by labeling with [-32P]ATP and
immunoprecipitated with an antibody to ErbB2. Autoradiography exhibited
the phosphorylation of the p185erbB2 protein
when the samples were challenged with either EGF, HB-EGF, NDFs, or
betacellulin, although the levels of induction of phosphorylation by
NDFs were lower than those induced by other members (Fig. 6). No phosphorylated 185-kDa band was detected in the
absence of any ligand (control). The specificity of the antibody was
confirmed by the detection of the p185erbB2
product after immunoprecipitation with protein A-Sepharose-ErbB2
antibody conjugate followed by Western blotting; however,
immunoprecipitation using normal rabbit IgG did not detect this product
in uterine membrane preparations (data not shown).
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and Table 1, an injection of
E2 induced uterine erbB2 mRNA levels by more
than 3.5-fold by 24 h. In contrast, an injection of P4
alone down-regulated the constitutive erbB2 mRNA levels that
were observed in oil-treated uterus. Furthermore, coinjection of
P4 with E2 antagonized the E2
induction of uterine erbB2 mRNA levels. The levels of
uterine erbB2 mRNA did not fluctuate appreciably at earlier
time points after each of these steroid treatments (data not shown).
The efficiency of the RT reactions was controlled by measuring the
levels of rPL7 mRNA (a housekeeping gene) in each sample (Fig. 8). The rPL7 mRNA levels did not show much variation
after any of the steroid treatments (4.0 x 107
copies/µg total RNA).
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). The apparent levels of
accumulation in the uterine epithelium after an injection of oil,
P4, or P4 and E2 were consistent
with the RT-PCR results (illustration not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The highlights of the present investigation are that ErbB2 is expressed
in a temporal and cell type-specific manner in the periimplantation
uterus and that this receptor subtype could be influenced by ovarian
steroid hormones. Another important finding is that the
erbB2 gene is expressed predominantly in uterine epithelial
cells. This finding is contrary to the observation that ErbB1 was not
detected in these cells (7, 35). The present results thus point toward
the possibility that uterine epithelial cells could be the direct
targets for EGF-like growth factors and may explain the controversy
regarding the mechanism of EGF-induced uterine epithelial cell
proliferation in vitro and in vivo (7, 33, 34, 35).
However, it should be recognized that for EGF-like ligands to be
effective in mediating signal transduction via ErbB2 apparently
requires the presence of another member of the erbB gene
family (see Fig. 10) (36). The current model for the
signaling of EGF-like growth factors and their receptors is as follows.
Upon ligand binding, either heterodimerization or homodimerization of
the receptors occurs, followed by receptor autophosphorylation. These
autophosphorylation sites then create docking sites for downstream
signal transduction molecules containing Src homology 2 domains (24).
Although ErbB2 was formerly considered an orphan receptor, recent
studies using different mammalian cell lines demonstrate that
heterodimerization of ErbB2 with other subtypes is favored as the
physiological receptors for NDF and EGF (37). Our demonstration of
phosphorylation of ErbB2 in whole uterine membrane preparations by
several ligands suggests interaction of ErbB2 with other known or as
yet unidentified subtypes of the ErbB family coexpressed in specific
uterine cell types or transphosphorylation of ErbB2 by other
membrane-associated nonreceptor kinases. Although ErbB1/EGF-R is not
present in uterine epithelial cells, we have preliminary evidence for
the presence of ErbB4 in these cell types (unpublished), suggesting
heterodimerization of ErbB2 with ErbB4 upon ligand binding that may
influence epithelial cell functions in the absence of ErbB1/EGF-R.
Indeed, we have evidence that the periimplantation mouse uterus
expresses NDF and betacellulin in a cell-specific and temporal manner
(unpublished observations). Thus, it is possible that EGF-like ligands
of epithelial, stromal, or embryonic origin may modulate epithelial
cell functions via interaction with epithelial cell ErbB2 and other
receptor subtypes. However, the stromal cell-mediated modulation of
epithelial cell functions by EGF or estrogen in a paracrine manner
cannot be ruled out. Paracrine interactions between stromal and
epithelial cells in the mouse uterus has again been demonstrated using
estrogen receptor (ER) knock-out (ERKO) mice (52). In this study, it
was shown that E2 treatment induced proliferation of
ER-negative epithelial cells when they were reconstituted with
ER-positive stromal cells. In contrast, E2 failed to induce
proliferation of ER-positive epithelial cells reconstituted with
ER-negative stromal cells. These results strongly suggest indirect
effects of E2 on epithelial cell mitogenesis via modulation
of stromal cell functions.
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The higher levels of erbB2 mRNA expression in the day 1 uterus suggest possible up-regulation of this gene by preovulatory estrogen (3). This is consistent with our observation of up-regulation of this mRNA in the ovariectomized uterine epithelium by E2. It is interesting to note that P4 not only antagonized the E2 induction, but also down-regulated the constitutive (oil-treated) levels of the erbB2 mRNA in the uterus. These results are contrary to our observation of up-regulation of the uterine erbB1 mRNA by E2, P4, or a combination of E2 plus P4 (7). Thus, expression of the erbB2 gene in the uterus from day 4 onward appears not to be under the regulation of rising P4 levels. Whether embryonic or other factors influence the expression of this gene in the uterus at the time of implantation and thereafter will require further investigation.
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Footnotes |
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Received September 19, 1996.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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precursors in the mouse
uterus during periimplantation period and after steroid hormone
treatment. Biol Reprod 50:481–491[Abstract]
in the
mouse uterus during the periimplantation period. Biol Reprod 45:365–372[Abstract]
, and TGF-ß genes in
preimplantation mouse embryos. Science 242:1823–1825
gene have
abnormal skin architecture, wavy hairs and curly whiskers and often
develop corneal inflammation. Cell 73:249–261[CrossRef][Medline]
deficiency results in hair follicle and eye
abnormalities in targeted and waved-1 mice. Cell 73:263–278[CrossRef][Medline]
, heparin-binding epidermal
growth factor-like factor and amphiregulin to Neu, ErbB3 and ErbB4.
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S. K. Das, J. Tan, D. C. Johnson, and S. K. Dey Differential Spatiotemporal Regulation of Lactoferrin and Progesterone Receptor Genes in the Mouse Uterus by Primary Estrogen, Catechol Estrogen, and Xenoestrogen Endocrinology, June 1, 1998; 139(6): 2905 - 2915. [Abstract] [Full Text] [PDF]
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Y. K. Hom, P. Young, J. F. Wiesen, P. J. Miettinen, R. Derynck, Z. Werb, and G. R. Cunha Uterine and Vaginal Organ Growth Requires Epidermal Growth Factor Receptor Signaling from Stroma Endocrinology, March 1, 1998; 139(3): 913 - 921. [Abstract] [Full Text] [PDF]
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S. K. Das, J. A. Taylor, K. S. Korach, B. C. Paria, S. K. Dey, and D. B. Lubahn Estrogenic responses in estrogen receptor-alpha deficient mice reveal a distinct estrogen signaling pathway PNAS, November 25, 1997; 94(24): 12786 - 12791. [Abstract] [Full Text] [PDF]
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