C/EBPß (CCAAT/Enhancer Binding Protein) Controls Cell Fate Determination during Mammary Gland Development
Tiffany N. Seagroves,
John P. Lydon,
Russell C. Hovey,
Barbara K. Vonderhaar and
Jeffrey M. Rosen
Department of Cell Biology (T.N.S., J.P.L., J.M.R.) Baylor
College of Medicine Houston, Texas 77030-3498
Laboratory
of Tumor Immunology and Biology (R.C.H., B.V.) National Cancer
Institute National Institute of Health Bethesda, Maryland
20892-1402
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ABSTRACT
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Deletion of the transcription factor
CCAAT/enhancer binding protein (C/EBP)ß results in a severe
inhibition of lobuloalveolar development in the mouse mammary gland.
Because progesterone receptor (PR) is requisite for alveolar
development, the expression of PR was investigated in
C/EBPß-/- mice. Unexpectedly, the number of
PR-positive cells, as well as the levels of PR mRNA, were elevated
3-fold in the mammary glands of C/EBPß-/-
mice. Furthermore, in contrast to wild-type nulliparous mice, in which
PR distribution shifted from a uniform to nonuniform pattern between
812 weeks of age, C/EBPß-/- mice
exhibited uniform PR distribution throughout all stages of mammary
development analyzed. No change in C/EBPß mRNA levels was observed in
the mammary glands of PR-/- mice, suggesting
that PR acts in a pathway either in parallel to or downstream of
C/EBPß. The overexpression and disrupted cellular distribution of PR
in C/EBPß-/- mice were coincident with a
striking 10-fold decrease in cell proliferation after acute steroid
hormone treatment, assayed by incorporation of
bromodeoxyuridine. In wild-type mice, PR and
bromodeoxyuridine-positive cells were adjacent to each other and rarely
colocalized. No differences in the level or pattern of PR
expression were observed in the uterus, suggesting that C/EBPß
influences PR in a mam-mary-specific fashion. Together, these data
suggest that C/EBPß may control cell fate decisions in the mammary
gland through the appropriate temporal and spatial expression of
molecular markers, such as PR, that induce the proliferation of
alveolar progenitor cells via juxtacrine mechanisms.
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INTRODUCTION
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A key question in the study of breast cancer is the
mechanism by which hormonally regulated signaling pathways that promote
normal development in response to pregnancy become altered to result in
aberrant proliferation. Progesterone (P) has been implicated as a
mitogen for the mammary gland during pregnancy (1, 2) when alveolar
secretory units of the gland bud from a simple tree of ductal
epithelium. Since breast tumors are initially steroid hormone
dependent, the progesterone receptor (PR) has been extensively used as
a molecular marker. Recent reports suggest that the spatial
distribution of steroid receptors is critical to normal lobuloalveolar
development. For example, while PR and estrogen receptors (ER)
colocalize in more than 96% of normal breast epithelial cells,
proliferating cells are steroid receptor-negative (3, 4). However, very
little is known about the mechanisms that regulate the expression and
spatial distribution of PR in the normal breast or in mouse mammary
epithelium. Furthermore, the molecular mechanisms of steroid
hormone-mediated regulation of target genes that control normal mammary
epithelial cell (MEC) proliferation remain poorly defined.
Previous studies have demonstrated that the transcription factor
CCAAT/enhancer binding protein ß (C/EBPß) is required for normal
ductal morphogenesis and for the proliferation and differentiation of
mammary epithelial cells in response to estrogen (E) and P during
pregnancy (5, 6). C/EBPß belongs to a family of basic leucine-zipper
(bZIP) DNA-binding proteins that regulate transcription by binding as
homo- or heterodimers with other C/EBPs to a common nucleotide
consensus sequence. C/EBPß has been implicated as a critical
regulator of proliferation vs. differentiation in multiple
tissues including liver, adipose tissue, ovary, immune system, and skin
(7, 8, 9, 10, 11). Alternative translation of the intronless C/EBPß transcript
produces proteins that differ in their activities based on inclusion of
the N-terminal transactivation domain. The ratio of activating to
repressing protein isoforms is critical in mediating expression of
target genes (12). The expression of the dominant-negative C/EBPß
isoform is tightly regulated during mouse mammary gland development (5)
and during the progression of breast cancer (13, 14).
Studies in mice lacking the PR have confirmed that PR is required for
the initiation of alveolar budding from the ductal tree in response to
E+P (15). However, alveolar development can be rescued if PR
-/- MEC mixed with PR +/+
MEC are reconstituted in close proximity within the cleared fat pads of
RAG1-/- hosts, suggesting a juxtacrine
mechanism of PR action (16). Recombination of PR
-/- stroma and PR+/+
epithelium indicates that the stroma does not play a critical role in
alveolar morphogenesis, further emphasizing the importance of
epithelial-epithelial juxtacrine interactions, rather than
epithelial-stromal interactions, in PR action (16). Similarly,
transplantation of MEC from the C/EBPß-/-
mouse into the cleared fat pads of C/EBPß++ hosts has
also demonstrated that C/EBPß, like PR, acts in an epithelial
cell-autonomous manner (5, 6).
Coupled with the marked inhibition of lobuloalveolar development,
a transient decrease in proliferation of
C/EBPß-/- epithelium transplanted into the
cleared fat pads of C/EBPß+/+ mice has been
observed during pregnancy (6). Based on these observations, the
expression and localization of PR and the relationship to proliferation
were determined in wild-type and C/EBPß-/-
mice over the course of mammary gland development. These studies
revealed that C/EBPß acts either upstream of, or parallel to, PR in
the normal mammary gland to regulate proliferation in response to
steroid hormones in a mammary-specific fashion. Unexpectedly, the lack
of C/EBPß in the mammary gland resulted in increased levels of PR
mRNA per cell and an increase in the total number of PR-positive
(PR+) MEC compared with wild-type controls.
Furthermore, the cellular distribution of PR shifted in wild-type mice
from a uniform pattern at 68 weeks of age to a nonuniform pattern by
1112 weeks of age. In contrast, at all stages of development
analyzed, C/EBPß-/- mice exhibited a uniform
pattern of cellular distribution of PR. The increased expression and
mislocalization of PR in C/EBPß-/- mice was
concomitant with a marked inhibition of epithelial cell proliferation
in response to E+P treatment. These results support the hypothesis that
C/EBPß controls cell fate decisions of putative alveolar progenitor
cells through regulation of the expression and cellular distribution of
molecular markers such as PR.
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RESULTS AND DISCUSSION
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Increased Expression of PR in the
C/EBPß-/- Mammary Gland
The expression and spatial distribution of PR were examined
by indirect immunofluoresence (IF) in mammary tissue biopsied from
intact mature virgin mice (at 11 weeks to 8 months of age) and in the
contralateral mammary glands of the same cohort of females after acute
E+P treatment. Acute steroid treatment was chosen to simulate alveolar
development in early pregnancy when alveoli begin to proliferate from
ductal progenitor cells. A marked impairment of alveolar development
was observed in C/EBPß -/- mice in response
to E+P (Fig. 1A
, cd). Mice lacking
C/EBPß possessed only a simple network of enlarged ducts as reported
previously (5). Surprisingly, these studies revealed an increase in the
percentage of PR+ MEC in
C/EBPß-/- compared with
+/+ mice (Fig. 1
A, b and f vs. a and
e). In mature virgins, the percentage of PR+ MEC
was approximately 2.5-fold greater in C/EBPß
-/- mice (Fig. 1B
). After acute E+P exposure, a
3-fold increase in PR+ MEC was observed in the
contralateral glands of the C/EBPß-/- mice
(Fig. 1C
). In C/EBPß+/+ mice, the percentage of
PR+ MEC in both treatment groups averaged 25%
(Fig. 1
, B and C), and, in general, individual cells in
C/EBPß+/+ mice stained less intensely for PR
than in mice lacking C/EBPß. No specific IF signal corresponding to
PR was detected in mammary glands isolated from mice lacking PR (PRKO)
mice (data not shown).

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Figure 1. Increased Expression and Aberrant Localization of
PR in the Mammary Epithelium of C/EBPß-/- Mice
A, Sections were stained for PR via indirect IF. The number and
intensity of PR+ cells increased in mature virgin
C/EBPß-/- mice (b) compared with
C/EBPß+/+ mice (a). The increased number of MEC
expressing PR in C/EBPß-/- mice (f) persists after
acute E+P treatment compared with +/+ controls (e).
Histological sections reveal that no alveoli form in response to E+P in
C/EBPß-/- mice (d) whereas clusters of alveoli develop
in C/EBPß+/+ mice (c). B and C, The mean percentage of
PR+ MEC (±SEM) in C/EBPß +/+
(solid bars) and -/- mice (hatched
bars) from either mature virgins (B) or after 2 days of E+P
(C). D, In situ hybridization reveals an increase in the
PR mRNA levels in individual cells as well as an increase in the number
of cells expressing PR mRNA in C/EBPß-/- mice (b)
compared with controls (a). No signal was observed with a sense probe
in C/EBPß -/- mice (c).
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The elevated number of PR+ MEC in
C/EBPß-/- mice also corresponded with a
change in their spatial distribution from the normal scattered pattern
(20) to a more uniform pattern in C/EBPß-/-
mice (Fig. 1A
, a and e vs. b and f). The increased
expression of PR in individual MEC, as well as the increased percentage
of MEC expressing PR protein, was confirmed at the mRNA level by
in situ hybridization (Fig. 1D
, a and b). Similar to PR
protein, PR mRNA is expressed in a nonuniform pattern in
C/EBPß+/+ mice (>11 weeks of age) and in a
uniform pattern in C/EBPß-/- mice. Therefore,
the lower levels of PR IF staining observed in wild-type animals do not
result from lack of sensitivity during antibody staining. The combined
results of these analyses demonstrate that up-regulated expression and
altered distribution of PR in the absence of C/EBPß corresponds to an
inability to initiate alveolar development. The defect in alveolar
development in C/EBPß-/- mice is MEC
autonomous. Since the stroma does not contribute to the defects
observed in alveolar development, they may result from a deficiency in
epithelial-epithelial juxtacrine signaling pathways. Recently Shyamala
et al. (17) demonstrated the involvement of such
interactions in PR-regulated development where transgenic mice
overexpressing PR-A lose cell-cell junctions and expression of
E-cadherin along cell borders. In light of this observation, we
analyzed the expression of E-cadherin in
C/EBPß-/- mice. Immunostaining for E-cadherin
revealed appropriate basolateral localization in MEC of
C/EBPß-/- mice (data not shown), suggesting
that any juxtacrine-mediated signaling mechanism dependent on this type
of cell-cell interaction should be intact despite the absence of
C/EBPß.
Marked Changes in PR Expression and Localization Occur during
Normal Mammary Gland Development in Virgin Mice
Intact mice of at least 11 weeks of age were initially studied
because younger animals (79 weeks old) exhibited considerably less
alveolar development after E+P treatment (18). Therefore, it was
hypothesized that the maximal responsiveness of a nulliparous female to
E+P may correlate with the expression and/or spatial distribution of PR
in the mammary gland. The expression of PR during normal mammary
development in nulliparous mice was determined by IF at 6, 8, and 12
weeks of age in intact C57BL/6 mice. This period of development
includes two phases of ductal proliferation: 1) penetration of the fat
pad by ducts from 3 to 8 weeks of age through proliferation at distal
tips within specialized structures known as terminal end buds (TEBs),
and 2) cessation of proliferation between 9 and 12 weeks of age as
ducts approach the edges of the fat pad and TEBs disappear. PR was
present in almost every MEC of previously formed ducts in 6- to 8-week
virgins (Fig. 2c
) and was more
concentrated in the inner cell layers of the TEBs than in the outer,
more proliferative cells (Fig. 2a
) (19). However, by 12 weeks of age,
the pattern of PR distribution in the majority of the ducts was
restricted to a subset of MEC (Fig. 2e
), as previously reported (20).
When mammary glands from 8-week C/EBPß+/+ and
-/- mice were stained for PR, the expression
and localization of PR were very similar to the uniform pattern
observed in the C57BL/6 8-week-old female mice (data not shown, and
Fig. 2c
). Therefore, between 8 and 12 weeks of age, as wild-type female
mice approach the age of maximal response to exogenous E+P, the
cellular distribution of PR changes dramatically.

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Figure 2. PR Localization Changes Markedly during Normal
Virgin Mammary Development
In 6-week old females the expression of PR is confined to the innermost
cell layers in the TEB (a). In previously formed ducts of 8-week
females, PR is expressed in the majority of MEC (c). By 12 weeks of age
(e), PR localized to a subset of MEC. DAPI-stained sections are shown
in parallel (b, d, and f).
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These observations suggest that the temporally regulated switch in
spatial distribution of PR from a uniform to a scattered pattern may be
required to facilitate maximal alveolar proliferation in response to
steroid hormones. At 68 weeks of age, when PR is expressed uniformly
in a majority of the epithelial cells, the MEC population may not be
spatially defined to assume an alveolar cell fate program. At this age,
the ducts have not yet reached the edges of the fat pad so this
mechanism may exist to reduce extensive premature alveolar development
before formation of the entire ductal tree. Mice at 68 weeks of age
are more refractory to the acute effects of E+P (18), although
wild-type females impregnated as early as 5 weeks of age are capable of
some alveolar development. Subsequent restriction of PR to a subset of
MEC in the mammary gland of the mature nulliparous female depends on
C/EBPß, since aberrant localization and expression of PR persists in
C/EBPß-/- females as old as 32 weeks of age
and in C/EBPß -/- females treated acutely or
chronically with E+P. Once pregnancy is initiated in wild-type mice,
growth factors may act as juxtacrine mediators to stimulate
proliferation of alveoli from the adjacent steroid receptor-negative
cells.
Although the results demonstrate that the shift in PR localization
occurs in C/EBPß+/+ nulliparous mice between 8
and 1112 weeks of age, it should be noted that the spatial
distribution of PR may be differentially temporally regulated in other
strains of mice. For example, mammary glands of pure C57BL/6 mice do
not form alveolar buds or fine side branching in response to the estrus
cycle, and they exhibit minimal alveolar development in response to
exogenous hormone treatment compared with other strains of mice such as
C3H (21). However, C/EBPß+/+ females, which are
maintained as a mixed strain, do exhibit alveolar budding from the
ducts as early as 9 weeks of age, and in these females PR is expressed
in the nonuniform pattern (T. Seagroves and J. Rosen, personal
observations). Haslam has reported that the responsiveness to P is
acquired at 7 weeks of age in Balb/C females; therefore, it would be
interesting to determine whether the spatial distribution of PR was
coincident with response to P in this strain of mice (18). Although
some strains of mice do exhibit minimal MEC alveolar budding as
virgins, there are relatively few alveoli present in virgin compared
with pregnant females, and there are relatively low levels of
proliferation of MEC until the onset of pregnancy or the administration
of exogenous hormones.
Deletion of C/EBPß Inhibits Alveolar Proliferation from Ductal
Progenitor Cells
Recent studies have suggested the steroid receptor-positive cells
are not the proliferative population of cells in the normal human
breast (3, 4). These observations led to the hypothesis that the
increased number of PR+ MEC in
C/EBPß-/- mice might prevent alveolar
development if steroid receptor expression and proliferation are
mutually exclusive. Analysis of bromodeoxyuridine (BrdU)-labeled MEC
and their association with PR revealed that in
C/EBPß-/- mice an inverse relationship
existed between the expression of PR by MEC and their proliferation
(Fig. 3A
b and Fig. 1C
vs. Fig. 3C
). Very few MEC exhibited positive BrdU staining in
C/EPBß-/- mice (1.5%, decreased 10-fold
compared with C/EBPß+/+ mice), whereas a
majority of MEC were PR+ (68%). In contrast, in
C/EBPß+/+ controls, 15.8% of cells were in
S-phase, and PR was expressed in a subset of ductal MEC, approximately
25%, in the nonuniform pattern observed previously (Figs. 3A
a, 3C, and
1C). As in the human breast, the steroid receptor-positive and
proliferating cells rarely colocalized to the same cell in the mammary
glands of either C/EBPß+/+ (Fig. 3B
, a and b)
or -/- mice (data not shown). In the normal
gland, the PR+ and BrdU+
MEC were adjacent to each other, suggesting that PR regulates alveolar
proliferation in a juxtacrine fashion. These unique observations of
perturbed PR+ and proliferating MEC populations
in C/EBPß-/- mice are, therefore, consistent
with recent data for the human breast wherein steroid receptor-negative
MEC are the proliferative cells in the normal mammary gland of sexually
mature females (3, 4). In addition, the results indicate that this
aspect of steroid receptor physiology of the mouse is similar to the
human.

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Figure 3. Inhibition of Alveolar Proliferation in
C/EBPß-/- Mice Acutely Stimulated with E+P
A, Double IF detection of PR (TR) and BrdU (FITC) was performed on
paraffin sections of mammary glands of C/EBPß+/+ (a) and
-/- (b) mice. The green arrow in Aa
indicates background FITC signal consistently observed in blood vessels
of mammary glands. B, Localization of PR+ and
BrdU+ cells in a duct (a) and alveoli (b) from mammary
glands of C/EBPß+/+ mice. The white arrow
in Bb indicates a rare colocalizing cell. C, MEC proliferation (%
BrdU+ cells ± SEM) in C/EBPß
+/+ (solid bar) and
C/EBPß-/- (hatched bar) mice.
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Alterations in PR Expression in Mice Lacking C/EBPß Are
Specific to the Mammary Gland
Northern blot analyses revealed that PR mRNA levels were increased
3- (6.9- and 3.5-kb transcripts) to 5-fold (8.7-kb transcript) in the
mammary glands of virgin and acutely stimulated
C/EBPß-/- mice as compared with those from
C/EBPß+/+ mice, after normalization to
cyclophilin (Fig. 4A
). In contrast, after
acute E+P treatment, the expression of PR in the uterus was not changed
significantly by deletion of C/EBPß. When double IF was performed on
sections of uterus isolated from the same animals, no differences in
the pattern or expression of PR or the amount of proliferation were
noted between genotypes (data not shown). These results are intriguing
since opposing effects of steroid hormones and their antagonists have
been reported for the mammary gland and uterus (22). Since C/EBPß is
expressed in both the mammary gland and in the uterus (data not shown),
it is possible that C/EBPß may interact with a mammary gland-specific
transcriptional coregulator to achieve this tissue-specific
response.

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Figure 4. Mammary Gland-Specific Overexpression of PR mRNA in
Mice Lacking C/EBPß.
A, The expression of PR mRNA was analyzed in pooled samples of mammary
glands (mg, lanes 1 and 2) or uterine horns (ut, lanes 3 and 4) after
acute E+P treatment (2d E+P) and in mammary glands from mature virgin
animals before treatment (lanes 5 and 6). Lanes 1, 3, and 5
(+/+ = C/EBPß+/+) and lanes 2, 4, and 6
(-/- = C/EBPß-/-) contain 4 µg of
poly(A) RNA (upper panel). Induction of PR mRNA in
mammary glands of C/EBPß-/- mice was normalized to
cyclophilin to correct for RNA loading (lower panel). B,
Northern blot [2 µg of poly (A) RNA per lane] analysis of PR mRNA
isolated from pooled mammary glands of mice chronically treated with
E+P (21d E+P); C/EBPß +/+ mice (+/+) and
C/EBPß -/- mice (-/-). Approximately
equal loading is indicated by the cyclophilin control. C, Expression of
C/EBPß mRNA in total RNA (15 µg/lane) isolated from the mammary
glands of individual nulliparous PR+/+ (lanes 1, 2, 5, and
6) or PR-/- mice (lanes 3, 4, 7, and 8) at 14 weeks of
age (virgin, lanes 14) or after 21 days of E+P treatment (21d E+P,
lanes 58); n = 2 per genotype per treatment).
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Increased levels of circulating P during pregnancy normally result in
the down-regulation of PR mRNA and protein in MEC (23). Since PR is not
down-regulated in mature mice treated acutely with E+P, mice were then
treated chronically (21 days) with E+P to determine whether longer
exposure to E and P would decrease the level of PR expression. However,
in mammary glands of C/EBPß-/- mice treated
chronically with E+P, the 6.9- and 3.5-kb PR transcripts remained
elevated approximately 3-fold compared with
C/EBPß+/+ controls (Fig. 4B
).
Therefore, no significant difference in the percentage of
PR+ cells or the level of PR mRNA for either
genotype was observed before and subsequent to administration of E+P.
These results are consistent with previous observations that
administration of E+P does not alter the overall percentage of
epithelial cells expressing PR (24), suggesting that alveolar
progenitor cells are permanently marked in the sexually mature female.
A 3-fold induction of PR mRNA after E treatment has been reported in
the mammary glands of ovariectomized mice (23). Since P suppresses the
inductive effect of E on PR mRNA during pregnancy (25), the
combinatorial effect of E+P may not significantly alter PR mRNA levels
as observed in these studies.
In summary, the overexpression of PR is persistent in C/EBPß mice
whether or not they are administered exogenous E+P and whether they are
treated acutely or chronically with E+P. The deletion of C/EBPß
impairs the normal spatial distribution and expression of PR in a
temporal fashion independent of the levels of circulating E+P.
Deletion of PR Has No Effect on C/EBPß mRNA Levels
To determine whether C/EBPß acts upstream or downstream of PR,
the level of C/EBPß mRNA was quantitated in mammary glands of
individual PR-/- mice by Northern blotting. At
14 weeks of age (untreated adult virgin) or after 21 days of treatment
with E+P, no significant change in C/EBPß mRNA levels was detected
after correction for loading by cyclophilin (Fig. 4C
). As previously
reported, C/EBPß mRNA levels increase in response to steroid hormones
(lanes 14 vs. 58) (6). Therefore, PR appears to be
regulated either in parallel or downstream of C/EBPß via either
direct or indirect mechanisms.
A Testable Model of the Juxtacrine Mechanisms of Alveolar
Morphogenesis
The increased expression and uniform localization of PR observed
in MEC of virgin mice lacking C/EBPß persisted upon stimulation with
E+P and correlated with an inhibition of alveolar development. These
observations are opposite to the anticipated decrease in PR in the
mammary epithelium of mice lacking C/EBPß given that alveolar
development is impaired in both PR-/- and
C/EBPß-/- mouse models.
Based on these results, and those reported previously by other
laboratories (3, 4, 15, 16, 24), we propose a model in which two cell
populations may coexist in the normal mammary gland in a delicate
balance; the spatially restricted, steroid receptor-positive cells and
the subset of steroid receptor-negative cells that will proliferate in
response to juxtacrine signals generated by pregnancy (Fig. 5
). The aberrant uniform expression of PR
in C/EBPß-/- MEC may disrupt this balance.
The increased proportion of PR+ MEC could
effectively decrease the steroid receptor-negative "target"
subpopulation capable of proliferation in response to
pregnancy-associated signals. The steroid receptor-negative population
of MEC may be growth arrested due to exclusive expression of
cyclin-dependent kinase inhibitors (CKIs) such as p27 (R. Clarke and E.
Anderson, personal communication).

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Figure 5. C/EBPß Mediates Juxtacrine Signaling in the
Normal Mammary Gland to Induce Alveolar Proliferation of the
Steroid-Receptor-Negative Subpopulation of MEC: A Testable Model
In the normal mammary gland, PR+ cells do not appear to
proliferate but may instead be responsible for the expression and/or
secretion of locally acting growth factors such as IGF-II that
stimulate proliferation of adjacent PR- epithelial cells.
C/EBPß mediates this process in part through regulation of PR
expression and localization. In breast cancer, this normal juxtacrine
mechanism may be perturbed by a switch to an autocrine mechanism of
cell growth in the PR+ population with concomitant loss of
CKIs such as p27.
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Although it is proposed in this model that growth factors are
synthesized in the steroid receptor-positive cells and are secreted to
act on adjacent cells, further experiments are required to substantiate
this proposal. In some breast cancers, or in MEC lines derived from
breast tumors, alterations in this temporal and spatial program may
result in the utilization of autocrine signaling pathways in the
steroid receptor-positive cells (Fig. 5
), leading to inappropriately
regulated proliferation in hormone-dependent breast cancer (26). In
fact, we propose that the switch to autocrine regulation of MEC
proliferation may occur early in the progression of breast disease
before the formation of overt breast tumors. Colocalization of PR with
markers of proliferation in preneoplastic lesions may help support this
hypothesis.
One potential candidate juxtacrine effector of proliferation is
insulin-like growth factor (IGF)-II. IGF-II mRNA is expressed in the
mature virgin and midpregnant mammary gland in a restricted pattern
similar to, but not necessarily coincident with, the pattern of
BrdU+ cells observed in serially sectioned
tissues (27). In addition, the overexpression of IGF-II in transgenic
mice results in mammary tumors, suggesting that inappropriate
regulation of IGF-II may occur in the progression to breast cancer
(28). Alternatively, members of the EGF family of growth factors may be
involved, since several members of this family have been shown to play
a role in normal mammary gland development and are amplified in some
breast cancers (29, 30).
C/EBPß as a Mediator of Cell Fate Decisions in the Normal Mammary
Gland
Several lines of preliminary evidence indicate that the inhibition
of alveolar development in mice lacking C/EBPß is not restricted to
the overexpression and altered cellular distribution of PR, leading to
the hypothesis that C/EBPß plays a more global role in cell fate
decisions. First, recent experiments have indicated that deletion of
C/EBPß also results in the up-regulation of several other key players
implicated in alveolar morphogenesis, including estrogen receptor
(ER)
, and PRL receptor (PrlR) (31, 32) (T. Seagroves, S. Grimm, R.
Hovey, B. Vonderhaar, and J. Rosen, personal observations). Second,
suppressive subtraction hybridization screens have identified several
novel genes as well as a molecular marker of stratified epithelium that
are up-regulated in response to deletion of C/EBPß (T. Seagroves, S.
Grimm, and J. Rosen, personal observations). The observed changes in
the expression and patterning of these multiple genes occur in
nulliparous, cycling females in a temporal fashion. By 8 weeks of age
in most mouse strains, TEBs are no longer present and the MECs within
ducts are essentially quiescent. Therefore, the dramatic switch in the
patterning of these factors does not appear to be dependent upon a
partitioning mechanism requiring cell division.
The analysis of PR as a marker of alveolar cell fate in wild-type and
C/EBPß-/- mice has provided novel insight
into mechanisms controlling normal mammary gland lobuloalveolar
development. First, in mice that still contain TEBs, the uniform
distribution of genes that will later control lobuloalveolar
development may serve to inhibit the premature formation of alveoli.
Second, the switch to a nonuniform pattern of distribution of these
molecular markers that occurs between 8 and 12 weeks of age may
facilitate the maximal proliferation of alveolar progenitor cells
induced by steroid hormones. Third, an exact cellular address for each
individual gene, such as PR, may be required to activate proliferation
of a neighboring epithelial cell to prevent autocrine stimulation of
proliferation. Disruption of any of these mechanisms may result in
either inhibition of proliferation of MEC, as observed in the
C/EBPß-/- mouse model, or result in mammary
tumors. Unraveling the molecular mechanism of the C/EBPß-mediated
switch in cellular distribution of these genes awaits further gene
discovery and continued investigation of the cellular distribution
and/or colocalization of molecular markers in additional knockout mouse
models.
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MATERIALS AND METHODS
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Animals
C/EBPß-/- mice were originally
provided by Dr. Valeria Poli (70% C57BL/6:20% 129-Sv:10% MF-1).
C57BL/6 mice (Taconic Farms, Inc., Germantown, NY) were
used to perform IF analysis of PR expression during virgin development
(n = 3 per age group). PR-/- samples were
isolated from mice in the 129-Sv:C57BL/6 background. Animal studies
were conducted in accord with NIH standards for the care and use of
experimental animals.
Steroid Treatment Protocols
One inguinal mammary gland from each ovary-intact, mature virgin
C/EBPß+/+ or -/- mouse
was surgically removed to serve as untreated controls (day 0, n =
5/genotype). These cohorts of mice and three additional mice per
genotype were then treated acutely (4850 h) with estradiol benzoate
(E, 1 µg, Sigma, St. Louis, MO) and P (1 mg,
Sigma) in 100 µl sesame oil via a single interscapular
subcutaneous injection behind the neck. After acute treatment, the
contralateral inguinal gland was removed
(C/EBPß+/+ mice, n = 8, 1120 weeks of
age; C/EBPß-/- mice, n = 8, 1132 weeks
of age at day 0 of experiment). For chronic E+P treatment, mice of at
least 12 weeks of age were treated 21 days with E+P as previously
described (5),
(C/EBPß+/+, n = 6,
C/EBPß-/-, n = 8). Two hours before
sacrifice, all E+P-treated animals were injected with 0.3 mg BrdU per
10 g body weight (Amersham Pharmacia Biotech,
Arlington Heights, IL).
IF
Tissues were fixed in either chilled 4% paraformaldehyde in PBS
for 90120 min or in buffered formalin for 6 h at room
temperature (RT). Paraffin sections (57 µm) were cut onto Probe-On
Plus-charged slides (Fisher Scientific, Pittsburgh, PA).
Sections were dewaxed and subjected to microwave antigen retrieval in
10 mM citrate buffer, pH 6.0 (33). After blocking in 5%
BSA/0.5% Tween-20 for 4 h at RT, sections were incubated
simultaneously with anti-BrdU-fluorescein isothiocyanate
(FITC)-conjugated antibody (1:51:10; Becton Dickinson and Co., Franklin Lakes, NJ) and a rabbit polyclonal antiserum to PR
(1:50; catalog no. A00809, DAKO Corp., Carpinteria, CA) in
blocking solution overnight at RT. Slides were washed in PBS and
incubated with antirabbit IgG-Texas Red (1:1000; TR, Molecular Probes, Inc., Eugene, OR) for 1 h at RT in blocking
solution. After PBS washes, slides were mounted in Vectashield + 4',
6-diamidino-2-phenylindole (DAPI) medium (Vector Laboratories, Inc., Burlingame, CA).
Cell Counting and Analysis
At least six individual 600x microscopic fields per sample were
digitally captured using the appropriate FITC, TR, and DAPI filters. At
least five animals per genotype were used for each experiment
(untreated, n = 5/genotype, 2 days E+P, n = 8/genotype). The
increase in staining intensity of PR protein (PR) was compared by
observing the capture time during digital imaging. The capture time was
on average 3- to 5 times shorter for the
C/EBPß-/- vs.
+/+ mice. The number of
PR+ and BrdU+ MEC in a
given field was expressed as a percentage of total number of
DAPI-stained MEC. Statistical significance was determined by
Mann-Whitney paired t test.
Northern Blot Analysis
Total RNA (RNAzol B, Tel-Test, Friendswood,
TX) and poly (A) -selected RNA (PolyATract, Promega Corp., Madison, WI) were prepared according to the
manufacturers instructions. Mammary tissues were collected from a
subset of the cohorts of C/EBPß+/+ and
-/- animals used for IF analysis. Mammary
tissues biopsied before and after acute treatment with E+P
(C/EBPß+/+, n = 5, all 20 weeks of age;
C/EBPß-/- mice, n = 5, 1132
weeks of age) were pooled by genotype and treatment before RNA
preparation. Subsequent to chronic treatment with E+P, total, then poly
(A)-selected RNA was prepared from pooled mammary tissues of
C/EBPß+/+ or -/- mice.
For analysis of C/EBPß expression, total RNA was prepared from
mammary tissues of individual PR+/+ or
-/- mice at 14 weeks of age or from a separate
cohort of females treated chronically with E+P. For each blot, RNA was
resolved on a 1.2% formaldehyde gel before transfer to nylon membrane.
The blots were hybridized with cDNA probes labeled with
[
32P]dATP by the Strip-EZ DNA kit
(Ambion, Inc., Austin, TX) corresponding to PR (34),
nucleotides (nt) 12740 or C/EBPß, nt 11480, stripped and reprobed
for cyclophilin (Ambion, Inc.). After exposure in
PhosphorImager (Molecular Dynamics, Inc., Sunnyvale, CA)
cassettes, the blots were quantitated by Image Quant 1.1
(Molecular Dynamics, Inc.) analysis. The fold
induction of PR or C/EBPß mRNA was determined by normalization to
cyclophilin.
In Situ Hybridization
Sections prepared as described above were treated with 0.2
M HCl, digested with proteinase K (5 µg/ml), postfixed in
4% paraformaldehyde, and acetylated (0.25% acetic anhydride in 0.1
M triethanolamine buffer, pH 8.0. Sections were
prehybridized for 1 h in hybridization buffer [50% formamide,
0.75 M NaCl, 0.075 M Na3 citrate
(5x SSC), 10% dextran sulfate, 2% SDS, 100 µg/ml salmon sperm DNA,
1 mg/ml yeast soluble tRNA, 100 mM dithiothreitol] at 55
C. Riboprobes for PR were transcribed from a 395-bp fragment
corresponding to nt 23832778 of the mouse PR cDNA (34) subcloned into
PCRScript. Hybridization buffer containing
35S-labeled cRNA probe (5 x
104 cpm/µl) was added to sections that were
then incubated in a humidified chamber overnight at 55 C. Coverslips
were removed in 2xSSC, 50% formamide for 20 min and the sections were
washed in 2xSSC, 50% formamide at 60 C for 30 min. Digestion with
RNAse A (20 µg/ml) was performed before washes with 2x and 0.1xSSC
at 37 C. Sections were exposed to emulsion (NTB-2, Eastman Kodak Co., Rochester, NY) for 4 weeks and then counterstained with
Nuclear Fast Red.
 |
ACKNOWLEDGMENTS
|
---|
We would like to thank Dr. Valeria Poli for initially providing
the C/EBPß-/- mice, Mr. Wilmer Roberts for
performing the E-cadherin staining, Dr. Cindy Zahnow for providing the
C/EBPß probe, Ms. Liz Hopkins for histology support, Ms. Shirley
Small for excellent animal care, and Drs. David Rowley and Sandy Grimm
for help in construction of the model.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Dr. Jeffrey M. Rosen, Department of Cell Biology, M638A, Baylor College of Medicine, One Baylor Plaza, Houston, Texas 77030-3498.
These studies were supported by Grants CA-775301 (J.P.L.) and
CA-16303 (J.M.R) from the National Cancer Institute.
Received for publication October 14, 1999.
Accepted for publication December 15, 1999.
 |
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