Mammary Gland Development Is Mediated by Both Stromal and Epithelial Progesterone Receptors
Robin C. Humphreys,
John Lydon,
Bert W. OMalley and
Jeffrey M. Rosen
Department of Cell Biology, Baylor College of Medicine,
Houston, Texas 77030
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ABSTRACT
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A combination of a knockout mouse model, tissue
transplantation, and gene expression analysis has been used to
investigate the role of steroid hormones in mammary gland development.
Mouse mammary gland development was examined in progesterone receptor
knockout (PRKO) mice using reciprocal transplantation experiments to
investigate the effects of the stromal and epithelial PRs on ductal and
lobuloalveolar development. The absence of PR in transplanted donor
epithelium, but not in recipient stroma, prevented normal
lobuloalveolar development in response to estrogen (E) and progesterone
(P) treatment. Conversely, the presence of PR in the transplanted donor
epithelium, but not in the recipient stroma, revealed that PR in the
stroma may be necessary for ductal development. Members of the Wnt
growth factor family, Wnt-2 and Wnt-5B, were employed as molecular
markers of steroid hormone action in the mammary gland stroma and
epithelium, respectively, to investigate the systemic effects of E and
P. Hormonal treatment of intact, ovariectomized, and
PR-/- mice and mice after transplantation of
PR-/- epithelium into wild type
(PR+/+) stroma demonstrated that these two
locally acting growth factors are regulated by independent mechanisms.
Wnt-2 is acutely repressed by E alone, while Wnt-5B gene expression is
induced only after chronic treatment with both E and P. Wnt 5B appears
to be one of the few molecular markers of P action in the mammary
epithelium. This study suggests that the regulation of mammary gland
development by steroid hormones is mediated by distinct effects of the
stromal and epithelial PR and differential growth factor expression.
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INTRODUCTION
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Aberrant regulation of normal developmental pathways plays an
important role in initiating and supporting mammary gland
transformation. Both hormonal and developmental status are known to be
important factors in the etiology of breast cancer. These hormonal and
developmental cues are often mediated at the molecular level by a
combination of systemic hormones and locally acting growth factors.
Synergism among locally acting growth factors enhances and augments the
diversity of potential signals transmitted to the epithelial and
stromal components of the gland. This synergism can be stimulatory or
inhibitory and can affect gene expression in the epithelium and
mesenchyme.
Estrogen (E) and progesterone (P), in cooperation with pituitary
hormones, are the primary systemic hormones required for the induction
of proliferation and differentiation of epithelial and stromal cells
leading ultimately to the formation of ductal and alveolar structures
during mammary gland development. The interaction of E and P with
GH, PRL, and insulin in regulating this differentiative process has
been well documented (1). Steroid hormones also regulate the expression
of a number of different locally acting growth factors, including
members of the epidermal growth factor, insulin-like growth factor, and
fibroblast growth factor (FGF) families (2, 3). Most of these growth
factors exhibit localized effects due to protein stability, adhesion
and residence in extracellular matrix, transport and secretion, and
availability of receptor molecules. For this reason they are believed
to act as local mediators of the differentiative and proliferative
signals of the systemic hormones. Systemic regulation of locally acting
growth factor activity allows for fine regulation of large-scale
developmentally associated proliferative and differentiative
functions.
Mammary gland development is dependent on physical, molecular, and
often reciprocal, interactions between the stromal and epithelial
compartments (4). The ability to recapitulate fully differentiated
structures from a fragment of syngeneic parenchyma, and to separate and
recombine epithelial and stromal compartments in vivo, makes
the mammary gland an excellent model system in which to study these
interactions. Evidence for this reciprocal dependence has been
demonstrated in classic recombination experiments between the
epithelial and stromal androgen receptor pathways (4). The specific
role of the epithelial and stromal PR in the development and
differentiation of the mammary gland is unclear (5, 6).
Wnt-1, the progenitor of a family of related growth factors, was
discovered in mouse mammary tumors as a result of proviral activation
(7). Members of the Wnt gene family are expressed in invertebrates and
vertebrates where they regulate cell fate and pattern formation
(8). Wnt genes, other than Wnt-1, are expressed in the mammary glands
of mice in a developmentally specific pattern (9, 10, 11). The function of
these endogenous Wnt genes during mammary gland development is unknown.
From these studies it is apparent that Wnt gene expression is tightly
regulated and is dependent on the developmental state of the mammary
gland. In BALB/c mice, Wnt-2 is expressed primarily during early ductal
development, 58 weeks postnatally, coincident with time of PR
induction by E, and is markedly down-regulated at the onset of
pregnancy. Conversely, Wnt-5B transcripts are detectable in the late
virgin gland at 612 weeks of age but increase markedly during
pregnancy, reaching a peak at day 18. Wnt-5B expression is localized
primarily in the ductal and lobuloalveolar cells, while Wnt-2
expression is detected in the stroma (9, 11). These results suggest
that E and P may play a role in regulating Wnt-2 and Wnt-5B gene
expression in both the stroma and epithelium. This restricted pattern
of gene expression is indicative of molecules that may be involved in
the developmental processes of the gland.
In this study the progesterone receptor knockout (PRKO) mouse (12) has
been used for reciprocal transplantation experiments in syngeneic mice
to investigate the distinct roles of the stromal and epithelial PR in
mammary ductal and alveolar development. Wnt-2 and -5B provided
specific molecular markers of steroid hormone action in the mammary
gland stroma and epithelium, respectively. The PRKO mouse permitted
definition of the unique effects of P distinct from those mediated by E
on Wnt gene expression. This experimental approach should facilitate
the identification of other steroid-mediated local growth factors on
mammary gland development.
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RESULTS
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Epithelial and Stromal PRs Have Separate Roles in Mammary Gland
Development
The mammary gland has the unique ability to recapitulate the
complete ductal and alveolar structures from a transplanted fragment of
syngeneic mammary epithelium (13). This characteristic allows the
analysis of interactions between epithelium and stroma in
vivo. The PR is present in both epithelial and stromal
compartments of the murine mammary gland (5, 14). To establish the role
of the PR in each of these compartments, reciprocal transplantation
experiments were performed using epithelium and stroma derived from
syngeneic 129SvEv PR-/- and PR+/+
mice, respectively. PR-/- epithelium
transplanted into the cleared fat pads of PR+/+ mice
penetrated and filled the stroma with ductal structures (Fig. 1A
). Interestingly, the PR-/-
epithelium failed to develop alveoli and to display an increase in the
number of secondary ductal branches in response to steroid hormone
treatment (Fig. 1B
). The control ipsilateral glands from the host
animal responded as expected to steroid hormone treatment with alveolar
proliferation (Fig. 1
, D vs. C). This result demonstrates
that the PR in the epithelium is required for normal lobuloalveolar
formation and differentiation of the epithelium. In addition, the
presence of PR-regulated signaling pathway in the stroma cannot
compensate for the lack of PR in the epithelium. In contrast,
PR+/+ epithelium transplanted into the cleared fat pad of
PR-/- hosts and treated with E and P
exhibited lobuloalveolar development (Fig. 2D
). An
increase in secondary branching in these E- and P-treated transplants
can be clearly seen under higher magnification (Fig. 2F
, arrow). However, an unexpected, marked reduction in the
extent of ductal outgrowth was observed in these transplants after 10
weeks of growth (Fig. 2C
), as compared with the PR+/+ (Fig. 2
, A and B) and the PR-/- (Fig. 1A
)
epithelium transplanted into the PR+/+ stroma. The same
PR+/+ epithelium transplanted in PR+/+ stroma
responded as expected to steroid hormone treatment with extensive
alveolar growth and an increase in secondary branching (Fig. 2B
). The
outgrowths from the PR+/+ epithelium transplanted into
the PR-/- stroma also displayed unusual
terminal endbuds (Fig. 2
, C and E).

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Figure 1. Absence of Lobuloalveolar Development in
Transplanted PR-/- Epithelium
PR-/- epithelium was transplanted into cleared fat pads
of PR+/+ 129SvEv mice. After 10 weeks of growth, the mice
were injected subcutaneously daily with E and P, and the mammary glands
were collected at day 0 (A and C) and day 8 (B and D). The
arrows in panels A and B denote the site of
transplantation. Note that in panel A the fat pad has been penetrated
with ductal epithelium after 10 weeks of growth in vivo.
Also note the increase in alveolar development in the ipsilateral
PR+/+ glands (C and D) after hormonal stimulation (compare
panels C and D). Bar = 1.4 mm.
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Figure 2. The Morphological Response of PR+/+
Epithelium Transplanted into PR-/- and
PR+/+ Stroma to Steroid Hormone Treatment
PR+/+ epithelium was transplanted into
PR+/+ (A and B) and PR-/- (C, D,
E, and F) stroma. After 10 weeks of growth, the mice were injected
daily with E and P subcutaneously, and the mammary glands were
collected at day 0 (A, C, and E) and day 8 (B, D, and F). Alveolar
formation is evident in PR+/+ (epithelium)
PR-/- (stroma) after 8 days of E and P
treatment (arrow in F). Note the reduction in ductal
development in PR-/-/PR+/+ (D)
compared with both PR+/+ (B) and
PR-/- epithelium (Fig. 1 , A and B).
PR+/+ epithelium in PR+/+ stroma responds to
steroid hormone treatment with extensive alveolar growth and an
increase in secondary branching (B). The arrows in A and
B define the site of transplantation. Magnification in panels A, B, C,
and D is defined by bar in A = 2 mm. Magnification
in panels E and F is defined by bar in E = 0.75 mm.
(PR-/-/PR+/+, n = 4 and
PR+/+/PR+/+, n = 4).
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E and P Treatment Represses Wnt-2 Gene Expression in the Mammary
Glands of Ovariectomized (ovx) and Intact Mice
The role of E and P in regulating Wnt gene
expression has been implied from the pattern of Wnt gene expression
observed in normal mammary gland development (9, 10, 11). In particular,
Wnt-2 and Wnt-5B display dramatic and inverse changes in gene
expression levels at the onset of pregnancy. Wnt-2 appears to be
expressed primarily in the mammary gland stroma, and Wnt-2 transcripts
have been detected in the cleared mammary fat pad (9, 11), whereas
Wnt-5B is expressed specifically in ductal and lobuloalveolar cells
(11). Thus, these locally acting growth factors provide excellent
molecular markers to investigate the role of steroid receptors on
ductal and lobuloalveolar development. First, however, it was necessary
to establish whether E and P either alone or in combination could
regulate the expression of Wnt-2 and Wnt-5B in a manner analogous to
that observed during mammary gland development. BALB/c mice were
treated with E and P to mimic the onset of pregnancy. RNA from the
mammary glands of hormonally treated and untreated, ovx, and intact
mice were examined for changes in gene expression using a quantitative,
RT-PCR method (9). A decrease of approximately 4-fold relative to the
untreated (time zero) group in Wnt-2 gene expression was observed after
E and P treatment of intact BALB/c mice (n = 3, P
< 0.001, Fig. 3A
). A 2-fold decrease (P
< 0.002) in Wnt-2 gene expression is observed after only 2 days of E
and P treatment. Wnt-2 gene expression decreased progressively with
daily E and P treatment and remained low to day 12 (data not shown).
This decrease in gene expression of Wnt-2 is not, however, as dramatic
(20-fold) as that observed after the onset of pregnancy (9).

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Figure 3. The Response of Wnt-2 Gene Expression in Intact (A)
and ovx (B) BALB/c Mice to E and P Treatment
A, Quantitative RT-PCR analysis of RNA from mammary glands of BALB/c
mice injected daily with E and P, subcutaneously, for 8 days. EP refers
to treatment with E and P for the number of days designated. Values:
Molecules/nanogram RNA(MOL/NG) represent the mean ±
SEM. The star denotes that EP 4 is
statistically different from EP 0, P < 0.001,
n = 3. B, Quantitative RT-PCR analysis of RNA from mammary glands
of BALB/c mice treated with E and P, E, and vehicle beeswax implants
for 1, 3, and 14 days. E refers to treatment with E alone for the
number of days designated. The absolute values for the entire control
group (C) were low in this experiment, possibly due to an effect of the
vehicle, but did not change significantly with time. Values represent
the mean ± SEM. The stars denote that
EP 14 and E 14 are statistically different from EP 1 and E 1,
respectively; P < 0.001, n = 3.
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Circulating E and P can cause cyclical repression and induction of PR
expression levels and possibly attenuate the molecular effects of
pharmacological doses of E and P. To eliminate the effects of
endogenous ovarian hormones, three groups of ovx mice were implanted
subcutaneously for 14 days with beeswax pellets that contained E and P
together, E alone, or carrier. The thoracic mammary glands were
collected from three animals at each time point within each treatment
group at 1, 3, and 14 days after implantation of the pellets. A 3-fold
decrease relative to the day 1 E and P treatment group was observed in
Wnt-2 expression after 3 days, increasing to 5-fold at 14 days compared
with control (n = 3, P < 0.001, Fig. 2B
).
Interestingly, mice treated with E alone showed the same 5-fold
decrease relative to day 1 E alone mice in Wnt-2 gene expression after
14 days (n = 3, P < 0.001).
Wnt-5B Expression Is Induced by E and P in ovx and Intact Mice
During normal mammary gland development, Wnt-5B expression is
observed initially at 68 weeks in the virgin mouse and increases at
the onset of pregnancy with maximal expression observed at day 1618
of pregnancy (9, 10, 11). Wnt-5B expression increased 4-fold by day 8 of E
and P treatment of intact mice as compared with the untreated (time
zero) mice and was maximally induced by day 16 (P <
0.004, n = 3) as illustrated in Fig. 4A
. Thus, the
increase in Wnt-5B gene expression, which parallels that observed
during midpregnancy, requires chronic E and P treatment. The pattern of
Wnt-5B expression in the ovx mice (Fig. 4B
) was similar to that
observed in the intact animal but displayed a more dramatic response.
Wnt-5B expression remained low at day 1 and day 3 but increased 9-fold
at day 14 relative to the day 1 E- and P-treated group
(P < 0.001, n = 3). In contrast to the regulation
of Wnt-2, there was no significant effect of E alone on Wnt-5B
expression in ovx mice. The large increase observed in Wnt-5B
expression in ovx mice may reflect the sensitization of the gland to P
due to the absence of endogenous hormones and a rapid induction of PR
gene expression.

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Figure 4. The Response of Wnt-5B Gene Expression in Intact
(A) and ovx (B) BALB/c Mice to E and P Treatment
A, Quantitative RT-PCR analysis of RNA from mammary glands of BALB/c
mice injected daily with E and P, subcutaneously, for 18 days. Values
represent mean ± SEM. The star denotes
that EP 16 is statistically different from EP 0, P
< 0.004, n = 3. B, Quantitative RT-PCR analysis of RNA from
mammary glands of BALB/c mice treated with E and P beeswax implants for
1, 3, and 14 days. Values represent mean ± SEM. The
star denotes that EP 14 is statistically different from
EP 1; P < 0.001, n = 3.
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Morphological Changes in Normal and Steroid Hormone-Treated Mice
Correlate with the Changes in Wnt Gene Expression
Changes in Wnt-2 and Wnt-5B gene expression are coincident with
morphological changes observed in the mammary gland in response to E
and P treatment (Fig. 5
). Mammary glands of 12-day E-
and P-treated BALB/c mice (Fig. 5H
) exhibit a morphology similar to
that observed in an 8-day pregnant mouse. Several differences were
observed, however, between the steroid hormone-treated glands and those
from the normal midpregnant mouse, especially when comparing the first
few days after hormone administration. For example, on the second day
of treatment, transient alveolar proliferation was observed (Fig. 5F
).
However, by the fourth day these alveoli were no longer detectable, and
a decrease in the amount of secondary branches was observed (Fig. 5
).
This transient alveolar budding has not been reported in mice treated
with pharmacological doses of E and P but is similar to the effect
observed in some strains of mice who respond to ovarian cycling by
producing a transient alveolar proliferation in the mature virgin
gland. This phenomena has, however, not been observed in BALB/c mice
(1, 15). Surprisingly, at day 8 some major ducts displayed a ductal
hypertrophy (Fig. 5G
, arrow). This hypertrophy has been
observed in mammary glands of mice implanted with hepatocyte-growth
factor (HGF) and treated with E and P (16). Unlike the alveolar
budding, this ductal hypertrophy was not transient and was still
detectable in some glands at day 1216 (Fig. 5H
). Permanent alveoli
appeared at day 12 and increased in number and density throughout the
remainder of the treatment. This progression of morphological changes
can be compared with the normal gland (1). During pregnancy, alveoli
and secondary branching appear by day 4 and increase in density and
number with the progression of pregnancy (Fig. 5
, C and D).

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Figure 5. Morphology of Mammary Glands from Hormone-Treated
and Pregnant Mice
The progression of morphological changes in control mammary glands in
response to the onset of pregnancy (panel A, 10-week virgin; B, day 2
pregnancy; C, day 4 pregnancy; D, day 12 pregnancy) and in E- and
P-treated (panel E, untreated; panel F, E + P, 2 days of treatment;
panel G, E + P, 4 days of treatment; panel H, E + P, 12 days of
treatment). Mammary glands isolated from control and hormonally treated
mice were stained with hematoxylin as described in Materials and
Methods. Note the transient alveoli in panel F. The
arrow in panel G denotes the hypertrophic duct.
Bar = 100 µm.
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These morphological alterations observed in hormonally treated and
normal mammary glands can be correlated with the E- and P-induced
changes in Wnt-2 and Wnt-5B gene expression. The appearance of
transient alveoli at day 2 coincides with the decrease in Wnt-2
expression observed in E- and P-treated and normal glands. Conversely,
permanent and functionally capable alveoli appear after 48 days of E
and P treatment, preceding the increase in Wnt-5B expression. P
concentrations increase gradually during pregnancy, affecting the
formation of alveoli and the induction of the differentiated alveolar
phenotype (6, 17). Appropriately, the increase in Wnt-5B expression in
the E- and P-treated mouse requires long-term treatment of steroid
hormones mimicking the pattern of morphological and gene expression
changes, observed in the pregnant gland.
Differential Regulation of Wnt-2 and Wnt-5B Gene Expression in
PR-/- Mammary Glands and
in PR-/-
Epithelium Transplanted into PR+/+ Stroma after
E and P Treatment
The response of Wnt-2 and Wnt-5B gene expression to the onset of
pregnancy and exogenous E and P suggested that the P-signaling pathway
might play a primary role in regulating Wnt gene expression in the
mammary gland. To examine the role of the PR in Wnt gene regulation,
Wnt gene expression levels were determined in
PR-/- mice (12) after treatment with E and P.
Wnt-5B gene expression did not change in response to E and P treatment
in PR-/- mice (n = 3, Fig. 6A
). However, the E and P repression of Wnt-2 expression
was still observed but was not significant until day 8 of hormone
treatment (P < 0.003, n = 3, Fig. 6B
). This
E-induced decrease in Wnt-2 gene expression was observed in the absence
of any detectable changes in mammary gland morphology in the
PR-/- mice.

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Figure 6. The Response of Wnt Gene Expression in
PR-/- Mammary Glands and in Transplanted
PR-/- Epithelium into PR+/+
Stroma to Steroid Hormone Treatment
A, Quantitative RT-PCR analysis of Wnt-5B gene expression from
PR-/- mammary glands after daily injections
with E and P, subcutaneously, for 0, 4, and 8 days (n = 3). B,
Quantitative RT-PCR analysis of Wnt-2 gene expression from
PR-/- mammary glands after daily injections
with E and P, subcutaneously, for 0, 4, and 8 days. The
star denotes that EP 8 is statistically different from
EP 0; P < 0.001, n = 3. C and D,
PR-/- epithelium was transplanted into cleared fat pads
of PR+/+ 129SvEv mice. After 10 weeks of growth, the mice
were treated with E and P. C, Quantitative RT-PCR analysis of Wnt-5B
gene expression in transplanted mammary glands after daily injections
with E and P, subcutaneously, for 0, 4, and 8 days. Values represent
the mean ± SEM (n = 6). D, Quantitative RT-PCR
analysis of Wnt-2 gene expression in transplanted mammary glands after
daily injections with E and P, subcutaneously., for 0, 4, and 8 days.
Values represent the mean ± SEM. The
star denotes that EP 8 is statistically different from
EP 0; P < 0.013, n = 6.
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To determine whether stromal or epithelial PRs were involved in
differential Wnt gene response, the levels of Wnt transcripts were
quantitated in RNA isolated from the previously described transplants.
The role of the epithelium in the induction of Wnt-2 and Wnt-5B gene
regulation was examined using PR-/-
epithelium, transplanted into the stromal fat pad of cleared
PR+/+ hosts. In these transplants no increase in the level
of P-dependent Wnt-5B expression was observed after 8 days of E and P
treatment (n = 6, Fig. 6C
). To confirm that the low level of
Wnt-5B expression seen in the different samples was due to the response
in the epithelium and not due to absence or degradation of RNA, the
expression of glyceraldehyde-3-phosphate dehydrogenase (G3PDH) was also
analyzed by RT-PCR. G3PDH transcripts are readily detectable and
constant throughout the treatment period (data not shown). The same
129SvEv mice containing transplanted PR-/-
epithelium and treated with E and P for 8 days still possessed the
ability to repress Wnt-2 expression when subjected to steroid hormone
treatment (P < 0.013, n = 3, Fig. 6D
).
Interestingly, the reduction of Wnt-2 gene expression was again delayed
requiring 8 days of steroid hormone treatment as observed previously in
PR-/- mice.
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DISCUSSION
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PR Deficiency in the Stroma and Epithelium Has Distinct Effects on
Mammary Gland Development
The absence of lobuloalveolar development in the
PR-/- transplants into the PR+/+
fat pad after steroid hormone treatment indicates that the PR in the
epithelium is required for this stage of mammary gland development.
These results are consistent with previous studies showing that the
absence of the PR influences lobuloalveolar development (12).
Furthermore, in the reciprocal transplants, PR+/+
epithelium into PR-/- stroma (Fig. 2
, D and
E), lobuloalveolar development was observed in response to E and P,
indicating that the absence of the PR in the stroma does not
significantly impair the ability of the epithelium to undergo alveolar
differentiation. However, ductal growth was impaired, as the
PR+/+ epithelium failed to fill the
PR-/- fat pad. In addition to the limited
ductal development, unusual distended endbuds were present in these
outgrowths. This restricted ductal growth may be due to disruption of
reciprocal interactions between the stromal and epithelial compartments
required for this stage of development. Less dramatic effects on ductal
morphogenesis have been observed in the PR-/-
mouse (12). This suggests that the PR-/-
stroma, when recombined with PR+/+ epithelium in
vivo, lacks the mechanism to correctly interact with and stimulate
growth in the epithelium and that the stroma fails to generate signals
upon which the ductal growth of the epithelium is dependent. In
contrast, in the PR-/- mouse, the
PR-/- epithelium, in the presence of
PR-/- stroma, has adapted to the absence of
reciprocal signals, and the epithelium has become independent of these
inputs required for ductal growth. This hormone-dependent reciprocity
of growth regulation has been observed previously in recombination
experiments with wild type and TfM androgen-insensitive mammary glands.
These experiments revealed that the epithelium can induce the
expression of mesenchymal androgen receptors. In turn, the mesenchyme
condenses around the epithelium and causes an epithelial regression
(18, 19).
The PR in the stroma is expressed in a temporally distinct pattern from
the epithelial receptor, has a different signaling mechanism, and
affects a separate group of target genes (5). Therefore, these data
support the theory of two separate functional effects of mammary gland
PR based on their compartmentalization and roles in development. The
unexpected results observed in transplants of PR+/+
epithelium in the PR-/- stroma suggest that
there is a P-dependent stromal signal for ductal development. Recent
in vivo studies of murine mammary glands treated with HGF in
the presence of E and P suggest that HGF is a potential candidate
second messenger for ductal growth in the mammary gland (16, 20).
HGF-treated mammary glands respond to E and P by stimulating ductal
growth. Interestingly, this growth factor has also been shown to
regulate Wnt-5A expression (21). If this hypothesis is valid, it may be
possible to rescue the PR-/- stroma defect by
the direct addition of HGF. Unfortunately, because HGF knockouts are
embryonic lethal and die before E16.5, no information has been obtained
to date on mammary ductal development in these knockout mice (22).
Wnt-2 and Wnt-5B Gene Expression Are Regulated Independently by
Steroid Hormones
This study demonstrates that two developmentally regulated
Wnt genes are regulated by distinct mechanisms. The unique temporal and
spatial patterns of expression of Wnt-2 and Wnt-5B suggest that these
genes may play some role in the development of the mammary gland. The
response of Wnt-2 and Wnt-5B to E and P treatment indicates that these
genes are useful markers for the action of E in the virgin mammary
gland, and for P during pregnancy, respectively. Wnt-2 gene expression
is highest in the immature virgin gland of BALB/c mice and declines
rapidly at the onset of pregnancy (9). In ovx and intact BALB/c mice
this effect can be mimicked with the addition of pharmacological doses
of E. This acute repression of Wnt-2 gene expression is correlated with
the appearance of lobuloalveolar structures and the termination of
ductal development. Conversely, an increase in Wnt-5B gene expression
in ovx and intact mice requires chronic treatment with E and P.
Ovariectomy enhances the magnitude of this response. Without the
stimulation from the ovaries, the basal levels of Wnt-5B expression are
probably significantly reduced, thereby allowing an enhanced response.
Wnt 5B provides one of the few molecular endpoints for the action of P,
and changes in Wnt 5B are coincident with lobuloalveolar
development.
The results from the PR-/- studies
demonstrate that Wnt-5B gene expression is induced by P and dependent
on the presence of PR specifically in the epithelial compartment. E
alone has no effect on Wnt-5B expression. Interestingly, the PR present
in the stroma cannot compensate for the absence of PR in the epithelium
for lobuloalveolar development or for induction of Wnt 5B gene
expression. This result implies that P acts directly on the epithelium
to induce lobuloalveolar development and, either directly or
indirectly, to activate Wnt-5B gene expression. E is required for
induction and maintenance of PR expression in the mammary gland (6).
Therefore, it is unlikely that the regulation of Wnt-5B expression is
independent of E.
Wnt-2 gene expression was inhibited by administration of E in
both PR-/- mice and in transplanted
PR-/- epithelium. These results suggest that
Wnt-2 gene expression is not primarily regulated by PR signaling. In
the PR-/- studies there was a delay in the
kinetics of the Wnt-2 response. The absence of the PR may have
restricted the development of the gland in these mice and slowed the
appearance of the ER, which normally appears at 4 weeks of age (23, 24). Alternatively, the absence of the PR could influence reciprocal
interactions between the stroma and the epithelium, thereby preventing
proper induction of ERs. ER gene expression is affected by feedback
controls between E and other hormones including P (14).
Previous studies performed in Parks mice demonstrated Wnt-2 expression
through midpregnancy and a repression of Wnt-2 and Wnt-5B expression
after ovariectomy (11). Parks mice possess virgin lobuloalveolar
development, which is absent in BALB/c mice, and it is possible that
this epithelial sensitivity to estrous-associated hormones alters
the regulation of Wnt-2 and Wnt-5B gene expression.
The rapid repression of Wnt-2 expression suggests the ER may be
directly regulating Wnt-2. The ER is expressed in both the stroma and
epithelium including the endbud (5, 14, 23, 24, 25). Interestingly, PR is
induced by the ER-signaling pathway in the epithelial compartment
48 h after initial addition of E (26). This temporal delay in
receptor response coincides exactly with the initial decrease observed
in Wnt-2 gene expression after hormonal treatment. Therefore, the
timing of this E-induced gene expression and localization of some Wnt-2
transcripts in the epithelium suggests that Wnt-2 could be regulated
directly by E.
The Wnts Are Growth Factors with Pleiotropic Effects on
Development
The development of the mammary gland is dependent on the
interaction and cooperation of growth factors and hormones functioning
through the stromal and epithelial compartments. Studies of PRL,
epidermal growth factor, FGF, TGF
and ß, insulin-like growth
factor, and HGF action reveal that they are regulated in specific
spatial and temporal patterns and have effects on proliferation and
differentiation in mammary gland development (1, 27, 28, 29, 30). The
developmentally associated expression pattern, their role in the
development of other organisms, biochemical characteristics, and
hormonal regulation of the Wnts suggest that they are members of this
complex family of locally acting growth factors.
The function of the Wnt genes in the development of the mammary gland
can only be inferred from limited expression studies in vivo
and in vitro and functional studies in other organisms.
Wingless, the Drosophila homolog of Wnt-1, has
proliferative, inductive, and cell fate determination functions (8). In
addition, Wnt genes have demonstrated functional roles in
Xenopus, mouse, and chicken (31, 32, 33, 34, 35, 36). These diverse studies
revealed that Wnt genes can possess inductive, growth-stimulatory, and
growth-restrictive functions all within a single organism.
In the mammary gland, overexpression of Wnt-1 influences the
proliferation of mammary epithelium (37). The expression of Wnt-4 and
Wnt-5A has been inversely correlated with proliferation in mammary
epithelial cells (38). Because of its localization both within and
around the highly proliferative terminal endbud, it is possible that
Wnt-2 has a role in regulating proliferation in the virgin gland. The
pattern of Wnt-5B expression and its dependence on the PR suggests that
it interacts with cells in a more differentiated state. Interestingly,
proliferation is high in the pregnant gland coincident with the
increase in Wnt-5B expression. Localization of Wnt-5B transcripts to
the ductal epithelium reveals it is expressed in the proper cellular
location to be involved in regulating proliferation in these cells. The
localization of Wnt-5B and Wnt-2 transcripts to the ductal epithelial
and stromal compartments of the mammary gland (9, 11), respectively,
suggest that although these two genes may have separate or even
overlapping functional roles, their temporal and spatial expression
patterns restrict their activity to specific stages of development.
Therefore, it is probable that the expression of these Wnt genes is
regulated in a specific manner to restrict their functional activities
to particular developmental stages in the mammary gland.
Alteration of Wnt Gene Expression Can Transform Mammary
Epithelium
In the mammary gland, ectopic expression of the Wnt genes has
dramatic consequences on the transformation and development of the
gland. Inappropriate expression of Wnts either temporally or spatially
may result in mammary tumorigenesis. For example, Wnt-1 and Wnt-4 have
been demonstrated to affect the development and transformation of the
gland in vivo (37, 39, 40). Numerous other Wnts, including
Wnt-2 and Wnt-5B, have in vitro transforming effects (41, 42). These in vitro transfection experiments have revealed
that separate classes of Wnts exist that are distinguished by their
transforming ability (43), although the properties defined in these
in vitro assays do not always correspond to their effects
in vivo (R. C. Humphreys and J. M. Rosen, submitted for
publication).
In addition, overexpression of Wnt genes, including Wnt-2 and Wnt-5B,
has been found associated with tumors in the breast and intestinal
epithelium (44, 45, 46, 47). Thus, loss of regulatory control on these two Wnt
genes, as with other growth factor molecules like TGF-ß and FGF (48, 49), has deleterious consequences for the development of the mammary
gland. Interestingly, compartment switching of Wnt-2 expression from
breast fibroblasts to tumor epithelium has been observed recently in
human breast tumors (50). Therefore, there is evidence for a critical
role of Wnt-2, and possibly Wnt-5B, in the transformation of the gland.
Since most of the Wnt knockouts are embryonic lethals resulting in
neural or kidney defects, the precise functional roles of these and
other Wnt family members on normal mammary gland development will
require the use of tissue-specific or regulated knockouts.
To summarize, the Wnt genes act in a cell-autonomous manner in
cooperation with other growth factors and have pleuripotent effects on
various developmental processes within the same organism (8). Wnt gene
expression can be differentially regulated by steroid hormones in the
mammary stroma and epithelium where they may act as locally acting
growth factors to influence ductal and lobuloalveolar development.
Hopefully, with the recent discovery of the Wnt receptor in
Drosophila (51), the mechanism of Wnt action and the
function of the individual Wnt family members in mammary gland
development will begin to be illuminated.
 |
MATERIALS AND METHODS
|
---|
Animals
BALB/c mice were acquired from Charles River Laboratories
(Wilmington, MA) or from a breeding colony at Baylor College of
Medicine, courtesy of Dr. Daniel Medina. Mice carrying the PRKO
mutation in the 129SvEv inbred genetic background were used in these
studies. All animals were maintained according to IACUC approved
guidelines.
Isolation of Mammary Glands and RNA
Number 4 (thoracic) mammary glands were removed from aged
matched 6- and 8-week-old virgin BALB/c and C3H mice using standard
surgical techniques. The thoracic and inguinal mammary glands from
11-week-old wild type control mice, 6-week-old 129SvEv
PR-/- mice, 129SvEv PR+/+, and
129SvEv PR-/- mice with transplanted
PR-deficient epithelium or wild type epithelium, respectively, were
removed using standard surgical techniques. For morphological analysis,
mammary glands were fixed in Tellyesniczkys solution for 5 h and
stained with hematoxylin as described previously (52). For isolation of
RNA, mammary glands were homogenized in a PT2000 Polytron (Brinkmann,
Westbury, NY) with RNazol (Biotecx, Houston, TX) as described by the
manufacturer or homogenized in 4 M guanididium
isothiocynate (Sigma, St. Louis MO) and isolated by CsCl centrifugation
method. RNA was quantitated spectrophotometrically and stored at -20 C
in 70% ethanol.
Construction and Transcription of cRNA Templates
Quantitative noncompetitive RT-PCR was performed as previously
described (9). Complementary DNAs from Wnt-5B and Wnt-2 were prepared
according to standard bacterial plasmid isolation protocols, and the
DNA was purified on Qiagen (Qiagen, Chatsworth, CA) columns according
to the manufacturer and isolated from the vector using unique
restriction enzymes. To construct the Wnt-2 cDNA deletion template,
StyI (New England Biolabs, Beverly, MA) was used to excise a
fragment from bases 493580. Digestion products were separated from
the small internal fragment, religated, and subcloned into the vector
pBKSII (Stratagene, La Jolla, CA). Clones were analyzed for a size
difference and sequenced to confirm the location of the deletion. The
Wnt-5B template was constructed in the same manner with an
AvaI (New England Biolabs, Beverly MA) deletion of bases
501576. Both templates were sequenced to confirm the orientation in
the vector and the presence of an internal deletion. These constructs
were used as templates for in vitro transcription reactions
as described in Promega Protocols and Applications Guide, ed 2
(Promega, Madison WI). The cRNA reactions were treated with 1 U of
ribonuclease-free RQ1 deoxyribonuclease in deoxyribonuclease buffer
(Promega) for 60 min at 37 C and then extracted with phenol-chloroform
twice and precipitated with 3 M NaAc and 100% ethanol at
-20 C. The cRNA was resuspended in Tris-EDTA, quantitated
spectrophotometrically, and stored at -20 C in 70% ethanol. Each
template was assayed by PCR to confirm the absence of contaminating
cDNA template. Optimum RT-PCR conditions for each of the templates were
developed that allowed a linear response with respect to the RNA input
and exhibited noncompetitive PCR.
Quantitative RT-PCR
Isolated RNA was transcribed in a reaction consisting of 1x
Taq polymerase buffer (Promega), 3 mM
MgCl2, 100 pmol hexanucleotide random primers (Boehringer
Mannheim, Indianapolis, IN) 1.25 U of RT (GIBCO BRL, Gaithersburg MD),
1 mM of each of four deoxynucleoside triphosphates
(Pharmacia, Milwaukee WI), and 20 U of RNasin (Pharmacia, Milwaukee WI)
in a final reaction volume of 20 µl. Fifty nanograms, 100 ng, and 150
ng of sample RNA were added to separate RT reactions. A constant amount
of cRNA template (
10,000 molecules) was added to each RT reaction as
an internal standard to control for differences in RT and PCR reaction
efficiency.
The primer sequences for the Wnt-2 and Wnt-5B amplifications,
respectively, were:
forward: 5'-AGTCGGGAATCGGCCTTTGTTTACG-3' and reverse:
5'-AAAGTTCTTCGCGAAATGTCGGAAG-3'; forward: 5'-GACAGCGCCGCGGCCATGCGC-3'
and reverse: 5'-CATTTGCAGGCGACATCAGC-3'. PCR conditions were 94 C for 1
min, 60 C for 2 min, and 72 C for 3 min, for 30 cycles and 94 C for 1
min, 65 C for 2 min, and 72 C for 3 min for 32 cycles for Wnt-2 and
Wnt-5B, respectively. Primers for G3PDH were: forward:
5'-AGAGGCCTTTGCTCGAACTGGAAAG-3' and reverse:
5'-CACCAAGACGTCTGTCGCCTACTTA-3. PCR conditions were 94 C for 1 min, 60
C for 2 min, and 72 C for 3 min, for 30 cycles. All PCRs were followed
by an extension at 72 C for 5 min. PCR was performed with 10 µl of
each RT reaction, 2 mM magnesium chloride, 1xPCR buffer
(Promega), 0.1 µCi [
-32P]dCTP (NEN DuPont,
Boston, MA), 1 U of Taq polymerase (Promega, Madison WI) in
a final reaction volume of 50 µl. Ten microliters of the RT-PCR
products were separated on a 2% Nusieve agarose (FMC Bioproducts,
Rockland, ME) gel and transferred overnight in 0.4 M NaOH
to Hybond N+ nylon membrane (Amersham, Buckinghamshire,
UK), and the radioactive signal was quantitated with 48 h exposure on
a PhosphoImager (Molecular Dynamics, Sunnyvale, CA).
Steroid Hormone Treatment of Mice
All groups of mice were treated with 1 mg of P (Steris, Phoenix,
AZ) and 1 µg of 17 ß-estradiol (Sigma) per day in 60 µl of sesame
seed oil (Sigma) subcutaneously. Mammary glands were collected at days
0, 1, 2, 4, 8, 12, and 18. Animals were ovariectomized and allowed to
regress for 4 weeks before hormone treatments were begun. Beeswax
implants containing 20 µg of E and/or 20 mg of P were synthesized by
adding the powdered form of the hormones to melted beeswax. The
suspended hormone mixture was dropped onto dry ice to form pellets. The
pellets, synthesized to deliver 1 mg P and 1 µg E/day, respectively,
were implanted subcutaneously in the neck of the mice for 2 weeks.
Inguinal mammary glands were collected at days 1, 3, and 14 and
analyzed as described.
Transplantation Studies
Tissue fragments of 10-week-old virgin
PR-/- mammary epithelium were isolated and
implanted into six PR-positive 129SvEv hosts using the technique
described by DeOme et al. (13). Epithelium from 129SvEv
hosts was removed as described (13). In addition, tissue fragments from
a 10-day pregnant 129SvEv PR+/+ mammary epithelium were
isolated and implanted into four PR-/-
129SvEv hosts. Due to the limited number of
PR-/- homozygote recipients and the limited
extent of ductal outgrowth, these glands could not be examined for
changes in Wnt gene expression. Mammary gland epithelial transplants
were allowed to proliferate and penetrate the stromal fat pad for 10
weeks and then treated with steroid hormones as described above.
Control experiments with wild type 129SvEv PR+/+ epithelium
into cleared fat pads of three wild type 129SvEv PR+/+ mice
were performed in the same manner.
Whole Mount Staining and Sectioning
The whole gland staining was carried out essentially as
described (25) except that glands were stained for only 2 h in
hematoxylin.
 |
ACKNOWLEDGMENTS
|
---|
The authors thank Dr. Susanne Krnacik for providing the RNA for
the ovariectomy experiments and for critical reading of the
manuscript.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Jeffrey M. Rosen, Baylor College of Medicine, Houston, Texas 77030.
This work was supported by NIH Grant CA-64255 and Grant
DAMD17-94-J-4253 from the Department of Defense (to J.M.R.).
Received for publication September 9, 1996.
Revision received November 14, 1996.
Accepted for publication November 21, 1996.
 |
REFERENCES
|
---|
-
Imagawa W, Yang J, Guzman R, Nandi S 1994 Control of
Mammary Gland Development. In: Knobrl E, Neill JD (eds) The Physiology
of Reproduction. Raven Press, New York, pp 10331063
-
Daniel CW, Silberstein GB 1987 Postnatal development of the
rodent mammary gland. In: Neville MC, Daniel CW (eds) The Mammary
Gland. Plenum Press, New York, pp 336
-
Vonderhaar BK 1988 Regulation of development of the normal
mammary gland by hormones and growth factors. Cancer Treat Res 40:251266[Medline]
-
Cunha GR, Hom YK 1996 Role of mesenchymal-epithelial
interactions in mammary gland development. J Mammary Gland Biol
Neoplasia 1:2135[Medline]
-
Haslam SZ, Shyamala G 1981 Relative distribution of estrogen
and progesterone receptors among the epithelial, adipose, and
connective tissue components of the normal mammary gland. Endocrinology 108:825830[Abstract]
-
Haslam SZ 1988 Acquisition of estrogen-dependent progesterone
receptors by normal mouse mammary gland. Ontogeny of mammary
progesterone receptors. J Steroid Biochem 31:913[CrossRef][Medline]
-
Nusse R, van OA, Cox D, Fung YK, Varmus H 1984 Mode of
proviral activation of a putative mammary oncogene (int-1) on mouse
chromosome 15. Nature 307:131136[Medline]
-
Klingensmith J, Nusse R 1994 Signaling by wingless in
Drosophila. Dev Biol 166:396414[CrossRef][Medline]
-
Bühler TA, Dale TC, Kieback C, Humphreys RC, Rosen JM 1993 Localization and quantification of Wnt-2 gene expression in mouse
mammary development. Dev Biol 155:8796[CrossRef][Medline]
-
Gavin BJ, McMahon AP 1992 Differential regulation of the wnt
gene family during pregnancy and lactation suggests a role in postnatal
development of the mammary gland. Mol Cell Biol 12:24182423[Abstract]
-
Weber-Hall SJ, Phippard DJ, Niemeyer CC, Dale TC 1994 Developmental and hormonal regulation of Wnt gene expression in the
mouse mammary gland. Differentiation 57:205214[CrossRef][Medline]
-
Lydon JP, DeMayo FJ, Funk CR, Mani SK, Hughes AR, Montgomery
CJ, Shyamala G, Conneely OM, OMalley BW 1995 Mice lacking
progesterone receptor exhibit pleiotropic reproductive abnormalities.
Genes Dev 9:22662278[Abstract]
-
DeOme KB, Faulkin LJ, Bern HA, Blair PB 1958 Development of
mammary tumors from hyperplastic alveolar nodules transplanted into
gland-free mammary pads of female C3H mice. Cancer Res 19:515519
-
Haslam SZ 1989 The ontogeny of mouse mammary gland
responsiveness to ovarian steroid hormones. Endocrinology 125:27662772[Abstract]
-
Vonderhaar BK 1984 Hormone and growth factors in mammary gland
development. In: Veneziale CM (ed) Control of Cell Growth and
Proliferation. Van Nostrand-Reinhold, Princeton, NJ, pp 1133
-
Jones FE, Jerry JJ, Guarino BC, Andrews GC, Stern DF 1996 Heregulin induces in vivo proliferation and differentiation
of mammary epithelium into secretory lobuloalveoli. Cell Growth Differ 7:10311038[Abstract]
-
Murr SM, Stabenfeldt GH, Bradford GE, Geschwind II 1974 Plasma
progesterone during pregnancy in the mouse. Endocrinology 94:12091211[Medline]
-
Heuberger B, Fitzka I, Wasner G, Kratochwil K 1982 Induction
of androgen receptor formation by epithelium-mesenchyme interaction in
embryonic mouse mammary gland. Proc Natl Acad Sci USA 79:29572961[Abstract]
-
Kratochwil K 1987 Tissue combination and organ culture studies
in the development of the embryonic mammary gland. In: Gwatkin RBL (ed)
Developmental Biology: A Comprehensive Synthesis. Plenum Press, New
York, pp 315334
-
Yang Y, Spitzer E, Meyer D, Sachs M, Niemann C, Hartmann
G, Weidner KM, Birchmeier C, Birchmeier W 1995 Sequential requirement
of hepatocyte growth factor and neuregulin in the morphogenesis and
differentiation of the mammary gland. J Cell Biol 131:215226[Abstract]
-
Huguet EL, Smith K, Bicknell R, Harris AL 1995 Regulation of
Wnt5a mRNA expression in human mammary epithelial cells by cell shape,
confluence, and hepatocyte growth factor. J Biol Chem 270:1285112856[Abstract/Free Full Text]
-
Schmidt C, Bladt F, Goedecke S, Brinkmann V, Zschlesche W,
Sharpe M, Gherardl E, Birchmeier C 1995 Scatter factor/hepatocyte
growth factor is essential for liver development. Nature 373:699702[CrossRef][Medline]
-
Muldoon TG 1978 Characterization of mouse mammary tissue
estrogen receptors under conditions of differing hormonal backgrounds.
J Steroid Biochem 9:485494[CrossRef][Medline]
-
Hunt ME, Muldoon TG 1977 Factors controlling estrogen receptor
levels in normal mouse mammary tissue. J Steroid Biochem 8:181186[CrossRef][Medline]
-
Daniel CW, Silberstein GB, Strickland P 1987 Direct action of
17 beta-estradiol on mouse mammary ducts analyzed by sustained release
implants and steroid autoradiography. Cancer Res 47:60526057[Abstract]
-
Shyamala G, Ferenczy A 1984 Mammary fat pad may be a potential
site for initiation of estrogen action in normal mouse mammary glands.
Endocrinology 115:10781081[Abstract]
-
Knight CH, Peaker M 1982 Development of the mammary gland. J
Reprod Fertil 65:521536[CrossRef][Medline]
-
Nandi S 1959 Hormonal control of mammogenesis and lactogenesis
in the C3H/He Crgl mouse. In: Stern C, Benson S, Quay W (eds)
University of California Berkeley Publications in Zoology. University
of California Press, Berkeley, pp 1128
-
Topper YJ, Freeman CS 1980 Multiple hormone interactions in
the developmental biology of the mammary gland. Physiol Rev 60:10491106[Free Full Text]
-
Plaut K, Ikeda M, Vonderhaar BK 1993 Role of growth hormone
and insulin-like growth factor-I in mammary development. Endocrinology 133:18431848[Abstract]
-
Cui Y, Brown JD, Moon RT, Christian JL 1995 Xwnt-8b: a
maternally expressed Xenopus Wnt gene with a potential role in
establishing the dorsoventral axis. Development 121:21772186[Abstract/Free Full Text]
-
Du SJ, Purcell SM, Christian JL, McGrew LL, Moon RT 1995 Identification of distinct classes and functional domains of Wnts
through expression of wild-type and chimeric proteins in Xenopus
embryos. Mol Cell Biol 15:26252634[Abstract]
-
Augustine KA, Liu ET, Sadler TW 1995 Interactions of Wnt-1 and
Wnt-3a are essential for neural tube patterning. Teratology 51:107119[Medline]
-
Hollyday M, McMahon JA, McMahon AP 1995 Wnt expression
patterns in chick embryo nervous system. Mech Dev 52:925[CrossRef][Medline]
-
Yoshioka H, Ohuchi H, Nohno T, Fujiwara A, Tanda N, Kawakami
Y, Noji S 1994 Regional expression of the Cwnt-4 gene in developing
chick central nervous system in relationship to the diencephalic
neuromere D2 and a dorsal domain of the spinal cord. Biochem Biophys
Res Commun 203:15811588[CrossRef][Medline]
-
McMahon AP, Bradley A 1990 The wnt-1 (int-1) proto-oncogene is
required for development of a large region of the mouse brain. Cell 62:10731085[Medline]
-
Edwards PA, Hiby SE, Papkoff J, Bradbury JM 1992 Hyperplasia
of mouse mammary epithelium induced by expression of the Wnt-1 (int-1)
oncogene in reconstituted mammary gland. Oncogene 7:20412051[Medline]
-
Olson DJ, Papkoff J 1994 Regulated expression of Wnt family
members during proliferation of C57mg mammary cells. Cell Growth Differ 5:197206[Abstract]
-
Lin TP, Guzman RC, Osborn RC, Thordarson G, Nandi S 1992 Role
of endocrine, autocrine, and paracrine interactions in the development
of mammary hyperplasia in Wnt-1 transgenic mice. Cancer Res 52:44134419[Abstract]
-
Bradbury JM, Edwards PA, Niemeyer CC, Dale TC 1995 Wnt-4
expression induces a pregnancy-like growth pattern in reconstituted
mammary glands in virgin mice. Dev Biol 170:553563[CrossRef][Medline]
-
Bradley RS, Brown AM 1995 A soluble form of Wnt-1 protein with
mitogenic activity on mammary epithelial cells. Mol Cell Biol 15:46164622[Abstract]
-
Blasband A, Schryver B, Papkoff J 1992 The biochemical
properties and transforming potential of human wnt-2 are similar to
wnt-1. Oncogene 7:153161[Medline]
-
Wong GT, Gavin BJ, McMahon AP 1994 Differential transformation
of mammary epithelial cells by Wnt genes. Mol Cell Biol 14:62786286[Abstract]
-
Huguet EL, McMahon JA, McMahon AP, Bicknell R, Harris AL 1994 Differential expression of human Wnt genes 2, 3, 4, and 7B in
human breast cell lines and normal and disease states of human breast
tissue. Cancer Res 54:26152621[Abstract]
-
Iozzo RV, Eichstetter I, Danielson KG 1995 Aberrant expression
of the growth factor Wnt-5A in human malignancy. Cancer Res 55:34953499[Abstract]
-
Lejeune S, Huguet EL, Hamby A, Poulsom R, Harris AL 1995 Wnt5a
cloning, expression, and upregulation in human primary breast cancers.
Clin Cancer Res 1:215222[Abstract]
-
Vider BZ, Zimber A, Chastre E, Prevot S, Gespach C, Estlein D,
Wolloch Y, Tronick SR, Gazit A, Yaniv A 1996 Evidence for the
involvement of the Wnt 2 gene in human colorectal cancer. Oncogene 12:153158[Medline]
-
MacArthur CA, Shankar DB, Shackleford GM 1995 Fgf-8, activated
by proviral insertion, cooperates with the Wnt-1 transgene in murine
mammary tumorigenesis. J Virol 69:25012507[Abstract]
-
Shackleford GM, MacArthur CA, Kwan HC, Varmus HE 1993 Mouse
mammary tumor virus infection accelerates mammary carcinogenesis in
Wnt-1 transgenic mice by insertional activation of int-2/Fgf-3 and
hst/Fgf-4. Proc Natl Acad Sci USA 90:740744[Abstract]
-
Dale TC, Weber-Hall SJ, KS, Huguet EL, Jayatilake H, Gusterson
BA, Shuttleworth G, OHare M, Harris AL 1996 Compartment switching of
WNT-2 expression in human breast tumors. Cancer Res 56:43204323[Abstract]
-
Bhanot P, Brink M, Samos CH, Hsieh J-C, Wang Y, Macke JP,
Andrew D, Nathans J, Nusse R 1996 The new member of the
frizzled family from Drosophila functions as a
wingless receptor. Nature 382:225230[CrossRef][Medline]
-
Humphreys RC, Krajewska M, Krnacik S, Jaeger R, Weiher H,
Krajewski S, Reed JC, Rosen JM 1996 Apoptosis in the terminal endbud of
the murine mammary gland: a mechanism of ductal morphogenesis.
Development 122:40134022[Abstract/Free Full Text]