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


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS AND DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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 8–12 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.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS AND DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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 6–8 weeks of age to a nonuniform pattern by 11–12 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.


    RESULTS AND DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS AND DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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. 1AGo, c–d). 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. 1Go 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. 1BGo). After acute E+P exposure, a 3-fold increase in PR+ MEC was observed in the contralateral glands of the C/EBPß-/- mice (Fig. 1CGo). In C/EBPß+/+ mice, the percentage of PR+ MEC in both treatment groups averaged 25% (Fig. 1Go, 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).

 
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. 1AGo, 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. 1DGo, 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 (7–9 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. 2cGo) and was more concentrated in the inner cell layers of the TEBs than in the outer, more proliferative cells (Fig. 2aGo) (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. 2eGo), 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. 2cGo). 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).

 
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 6–8 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 6–8 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 11–12 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. 3AGob and Fig. 1CGo vs. Fig. 3CGo). 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. 3AGoa, 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. 3BGo, 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.

 
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. 4AGo). 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 1–4) or after 21 days of E+P treatment (21d E+P, lanes 5–8); n = 2 per genotype per treatment).

 
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. 4BGo).

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. 4CGo). As previously reported, C/EBPß mRNA levels increase in response to steroid hormones (lanes 1–4 vs. 5–8) (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. 5Go). 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.

 
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. 5Go), 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){alpha}, 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.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS AND DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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 (48–50 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, 11–20 weeks of age; C/EBPß-/- mice, n = 8, 11–32 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 90–120 min or in buffered formalin for 6 h at room temperature (RT). Paraffin sections (5–7 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 (1:5–1:10; 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 manufacturer’s 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, 11–32 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 [{alpha}32P]dATP by the Strip-EZ DNA kit (Ambion, Inc., Austin, TX) corresponding to PR (34), nucleotides (nt) 1–2740 or C/EBPß, nt 1–1480, 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 2383–2778 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-77530–1 (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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