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Progesterone, in Addition to Estrogen, Induces Cyclin D1 Expression in the Murine Mammary Epithelial Cell, in Vivo¹

Department of Cell Biology, Baylor College of Medicine, Houston, Texas 77030

Address all correspondence and requests for reprints to: John P. Lydon, Baylor College of Medicine, Department of Cell Biology, Room M523A, 1 Baylor Plaza, Houston, Texas 77030. E-mail: jlydon{at}bcm.tmc.edu

	Abstract

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Previous investigations, in vitro, have demonstrated that progestins can induce the transcription of the cell cycle regulator, cyclin D1, thereby suggesting that cyclin D1 may mediate, at the molecular level, the proposed mitogenic effects of progesterone during mammary epithelial cell proliferation. To extend these initial studies into an in vivo context, comparative cyclin D1 Northern and immunohistochemical analyses were performed on mammary gland tissue isolated from wild type (WT) females as well as from the recently reported progesterone receptor knockout (PRKO) mouse model. Northern analysis revealed that estrogen induced cyclin D1 expression, 5- to 7-fold over control levels, both in the WT and PRKO female. Immunohistochemistry demonstrated that, for both test groups, the number of mammary epithelial cells expressing cyclin D1 increased significantly as compared with control values, in response to estrogen. In the case of estrogen plus progesterone treatment, Northern analysis revealed that, in the WT gland, cyclin D1 transcription increased approximately 3-fold over estrogen induced levels, an increase that was paralleled by an equivalent increase in the number of mammary epithelial cells expressing cyclin D1. Conversely, under the same hormone regimen, the PRKO mammary gland did not exhibit a further increase in cyclin D1 induction over estrogen only levels. Finally, these studies not only demonstrate that in the mammary epithelial cell, both estrogen and progesterone can induce the expression of cyclin D1 but also show that this induction correlates with mammary gland proliferation in the mouse.

	Introduction

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MAMMARY gland development is regulated by the interplay of systemic hormones, local growth factors, and the reciprocal relay of cell-cell interactions between the epithelium and the surrounding stroma (1). Until recently, it was generally assumed that the normal proliferation of the mammary gland epithelium, as well as the initiation and progression of mammary tumorigenesis, were dependent on the ovarian steroid, estrogen (E). This assumption was based largely on the established E-induced proliferative effects on the endometrial luminal and glandular epithelial cell; conversely, progesterone (P), based on its antiestrogenic effects in the endometrium, was assumed, by extension, to have antiproliferative effects in the mammary gland, (reviewed in Ref. 2 and references therein).

Although a number of previous rodent studies have implicated P-induced proliferative effects in the murine virgin (3) and pregnant (4, 3) mammary gland, as well as during mammary tumorigenesis in the rat and mouse (5, 6, 7), current reports exist suggesting that in the human gland P exhibits insignificant proliferative effects (8).

To define further the role of P in murine mammary gland proliferation and differentiation, we recently generated a progesterone receptor knockout (PRKO) mouse model in which the functional activity of the progesterone receptor (PR) was ablated through gene targeting techniques (9). Comparative whole mount analysis of mammary glands isolated from the ovariectomized PRKO and WT female, previously treated with exogenous E and P, revealed a striking phenotype in mammary epithelial ductal development and differentiation in the PRKO mouse. Specifically, the PRKO mammary gland failed to develop the typical pregnancy-associated epithelial ductal morphogenesis that consists of extensive dichotomous branching with attendant interductal lobuloalveolar development (1). These initial gross morphological studies unequivocally demonstrated a proliferative role, in addition to a differentiative role, for P in this tissue.

The downstream molecular targets and mechanisms by which P exerts these proliferative effects in the mammary gland epithelium are unknown. Previous studies in cultured T-47D cells have revealed that exogenous P can induce the transcription of the gene for cyclin D1, a cell cycle regulatory protein (10). Although these in vitro studies did not demonstrate that P-induced cyclin D1 expression resulted in sustained cell proliferation, these results were, nonetheless, the first to provide preliminary support for the proposal that the proliferative effects of P observed in the murine mammary epithelia in vivo (9) may be mediated, in part, by influencing cell cycle progression through modulation of cyclin D1 expression. In support of this proposal, the mammary gland phenotype of the cyclin D1 null mutant mouse (11, 12) exhibited a striking similarity to the PRKO mammary phenotype (9). Furthermore, recent cyclin D1 in situ localization studies on the normal murine mammary gland have demonstrated that the highest levels of cyclin D1 expression occur during midpregnancy (13), a time period that correlates with the highest levels of serum P (14). Together, these observations implicate extensive overlapping functions between PR and cyclin D1 in mammary gland development and suggest that, during pregnancy, cyclin D1 may mediate, in part, the P-induced proliferative signal in the murine mammary gland.

To substantiate these observations in an in vivo context, we evaluated the comparative levels of cyclin D1 induction in the PRKO and WT type mouse, both at the RNA and protein level to determine whether E and/or P can modulate cyclin D1 expression in the proliferating murine mammary gland.

	Materials and Methods

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Animals and steroid hormone treatment
Two test groups, the 12-week-old PRKO and aged matched WT female mouse were used in these experiments. Two weeks before steroid hormone treatment (see below), animals in both test groups were bilaterally ovariectomized. Mammary glands were stimulated to proliferate with either a daily sc injection of either 1 µg of E or 1 µg of E plus 1 mg of P (E + P) for either one or 20 day(s) as described previously (9). Corresponding controls for both test groups at each time point consisted of daily administration of sesame oil (hormone vehicle). For Northern and histological analysis (see below), at each time point, six mice per test group were used for each hormone treatment. A corresponding number of control treated mice were also used. In all cases, animals were euthanized by anesthetizing the animal with a triple anesthetic combination: (ketamine: 37.5 mg/ml; xylazine: 1.9 mg/ml; and acepromazine: 0.37 mg/ml) (5 µl of anesthetic per gram of body weight). Finally, all animal surgical procedures and experimentation, described herein, met with the highest humane animal care in accordance with the National Institutes of Health guidelines for the care and use of laboratory animals.

Northern analysis
At a given time point (see above), animals were killed and both inguinal glands were removed and pooled, before total RNA was isolated using the RNAzol B extraction method (Cinna/Biotecx, Laboratories Inc., Houston, TX). Fifteen micrograms of total RNA were electrophoresed through a denaturing 2.2 M formaldehyde gel of 1.2% agarose before transfer to Zetaprobe GT membranes (BioRad Laboratories, Hercules, CA) that were subsequently hybridized with a [-³²P] dCTP radiolabeled random primed murine cyclin D1 probe. The full-length mouse cyclin D1 complementary DNA, (CYL-1), (15), was used as probe template, which was kindly provided by Dr. Charles J. Sherr. Subsequent hybridization and washing conditions have been previously described (16). To control for unequal loading and transfer of RNA, filters were routinely hybridized with a probe for glyceraldehyde-3-phosphate dehydrogenase (GAPDH). To quantitate for cyclin D1 messenger RNA (mRNA) induction, densitometric analysis was performed on filters containing hybridization signals for cyclin D1 and subsequently for GAPDH using a Betascope 603 blot analyzer (Betagen, Inc., Waltham, MA).

Immunohistochemistry
Administration of 5-bromo-2-deoxyuridine (BrdU).
Two hours before sacrifice, each animal received an ip injection of BrdU (70 µg of BrdU (Sigma)/g BW). Following BrdU labeling, animals were killed and both inguinal glands were dissected out, carefully flattened on glass slides, fixed in 10% buffered formalin for 8 h, followed by a 3-min wash in ordinary tap water, before long-term storage in 70% ethanol. Following fixation, mammary tissue was embedded in paraffin before being sectioned (4 µm) for either standard hematoxylin and eosin staining, or for immunohistochemical staining (see below).

BrdU and cyclin D1 immunostaining
Before immunostaining, tissue sections were deparaffinized and blocked as described earlier (17). BrdU immunohistochemistry was performed using the Cell Proliferation Kit from Amersham Life Science Inc. (Arlington Heights, IL) and by following the manufacturer’s protocol. For each tissue section, cell counting consisted of counting the number of BrdU staining cells in a random field of 1000 cells. The average number of BrdU staining cells in a given tissue section was obtained by taking the average obtained from counting three separate fields of 1000 cells per section. Representative sections from each inguinal gland were used in these studies.

Following deparaffinization and blocking, cyclin D1 immunostaining was performed by incubating sections with a rabbit anticyclin D1 polyclonal antibody (Upstate Biotechnology Inc., Lake Placid, NY) (1:50 dilution) for 30 min, in a humidified chamber, at 40 C. Sections were subsequently washed three times in Tris buffer (Tris-HCl, pH 7.5, 0.9% sodium chloride and Tween-20) before incubation with an antirabbit biotinylated second antibody (1:500 dilution) for 15 min, at 40 C. Following three washes with Tris buffer, sections were incubated with the Vectastain ABC reagent (Vector Laboratories Inc., Burlingame, CA) (1:80 dilution) for 12 min, at 40 C. After three washes in Tris buffer, tissue sections were incubated in 3,3'-diaminobenzidine (Vector Laboratories Inc.) for 8 min, in the dark, at room temperature. Sections were subsequently counterstained with 0.1% methyl green for 20 seconds, followed by two washes with distilled water, before sequential dehydration in 95%, 100% ethanol, and xylene. Finally, sections were mounted with Permount and coverslipped. Control sections consisted of a similar protocol as above, except that the primary antibody was excluded. The average number of cells expressing cyclin D1 per section were scored as described above. For cell counting, cyclin D1 immunostaining was classified as either low or high intensity; only high intensity immunopositive cells were scored in this study.

	Results

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Induction of cyclin D1 transcription by E and P
To determine whether cyclin D1 transcription was modulated by E and/or E + P treatment, Northern analysis was performed on mammary tissue RNA isolated from the WT and PRKO female. Figure 1 shows that after 1 day of either E or E + P treatment, the level of cyclin D1 expression did not significantly differ between the WT and PRKO treatment groups. Following 20 days of hormone treatment, in the case of the WT and PRKO mouse, E alone was shown to induce cyclin D1 expression 5- to 7-fold over control values in both WT and PRKO mice (Table 1). In the case of E + P stimulation, for the WT gland, cyclin D1 RNA levels were further augmented 3- to 4-fold over levels attained by E stimulation alone. Although the levels of cyclin D1 induction in the E-treated PRKO mouse did not differ significantly from the E treated WT, P failed to increase the level of cyclin D1 transcription over E only values in the PRKO mouse.

View larger version (20K): [in this window] [in a new window]   Figure 1. Cyclin D1 mRNA induction in the WT and PRKO mammary gland, in response to sesame oil (C); estrogen (E); or estrogen plus progesterone (E + P), at day 1 and after day 20 of treatment. Following an overnight exposure to x-ray film, filters containing the cyclin D1 signal were stripped and subsequently probed with GAPDH. Typical GAPDH signals (see above) were achieved after 1.5 h of autoradiography. Using densitometric analysis GAPDH was used to normalize for variations in signal intensity. Each lane of the above Northern result represents an individual mouse and this result was typical of five other Northern blots that were performed in which the RNA samples were derived from a different set of individual mice, in each case.

View larger version (147K): [in this window] [in a new window]   Figure 2. Mammary epithelial ductal proliferation in the WT and PRKO ovariectomized mouse, after 20 days of hormone treatment. The panels show histological sections of the inguinal fat pad with the lymph node (LN) proximal to the nipple as a reference point. A and B, Lack of ductal proliferation in the WT and PRKO mouse respectively, after treatment with sesame oil (control). C and D, Typical transverse sections of mammary glands derived from WT and PRKO mice respectively, following E treatment alone. Note the increase in the number of ductal structures (arrowhead) in both test groups, as compared with corresponding controls (A and B). E and F, Degree of ductal proliferation in the WT and PRKO mammary gland, following E + P treatment. Note the striking increase in epithelial ductal proliferation in the WT (arrowhead) as compared with the PRKO gland. All sections (4 µm) were stained with hematoxylin and eosin, the scale bar represents 300 µm.

View larger version (102K): [in this window] [in a new window]   Figure 3. Cyclin D1 immunohistochemistry of mammary glands derived from ovariectomized WT and PRKO mice, following 20 days of hormone treatment. Mammary glands isolated from WT (A) and PRKO (B) animals did not exhibit cyclin D1 protein induction, following sesame oil administration (control). C, Degree of background staining, in the absence of primary antibody, of epithelial ducts of a WT gland treated withE + P for 20 days. However, following E treatment, both WT (D) and PRKO (E) mammary glands revealed a significant number of cyclin D1 immunoreactive ductal epithelial cells (arrowhead). E + P treatment resulted in a further increase in the number of cyclin D1 expressing cells in the WT (F) but not in the PRKO (G) mammary gland. All sections were lightly counterstained with 0.1% methyl green. Scale bars in A and C represent 25 µm; the scale bar shown in A should be used as a reference magnification for the histology represented in panels B, D, E, F, and G.

View larger version (113K): [in this window] [in a new window]   Figure 4. BrdU incorporation into mammary epithelial cells of the WT and PRKO mammary gland, following 20 days of hormone treatment. Cells staining for BrdU incorporation were not detected in the WT (A) nor the PRKO (B) mammary gland, following sesame oil treatment (controls). Administration of E resulted in the appearance of a significant number of BrdU containing cells both in the WT (C) and PRKO (D) mammary epithelial cell layer. E + P treatment induced a further 3- to 4-fold increase in the number of BrdU staining cells for WT (E) mammary glands but not for the PRKO gland (F). Tissue sections were routinely lightly stained with hematoxylin following BrdU immunocytochemistry; scale bar, 25 µm.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

In the case of E + P treatment, the inclusion of P resulted in a 3- to 4-fold further increase in the number of epithelial cells expressing cyclin D1. In contrast, the addition of P did not further increase the number of cells expressing cyclin D1 in the PRKO mammary gland, thereby underscoring a requirement for PR. Unlike previous in vitro investigations, which failed to show a close correspondence between P-induced cyclin D1 induction and cell proliferation (10, 20), the in vivo studies described herein establish a strong correlation between P-stimulation of cyclin D1 expression and mammary epithelial cell proliferation. Obviously, a future research goal will be to unequivocally prove that the P-induced proliferative effects observed in vivo are dependent on cyclin D1 expression. As with most studies involving knockout mouse models, it could be argued that the PRKO mammary phenotype may be due, in part, to removal of PR function from progestin-target tissues other than the mammary gland. We have recently employed the mammary gland transplantation technique to address this question (21) and have shown that PRKO mammary epithelia transplanted into epithelia-free WT mammary stroma exhibits the same phenotypic responses to E and E + P as the intact PRKO gland, suggesting that the PRKO mammary gland phenotype is due to removal of PR function exclusively from the mammary gland.

In conclusion, although the proliferative effects of P on cultured breast cancer cells (10) and in the human mammary gland (8, 22) have yet to be established, we provide in vivo support for a significant proliferative role for P, in addition to E, in the murine mammary gland. Northern and immunohistochemical analyses revealed that cyclin D1 induction was stimulated by E and was further augmented by P. These observations suggest that induction of cyclin D1 could be responsible for coupling the E and/or the P extracellular signal(s) to the nuclear components of the cell cycle clock responsible for orchestrating mammary epithelial cell progression through the G1 phase of the cycle. Indeed, recent studies have shown that through direct physical association, cyclin D1 can specifically stimulate ER transactivation that, in turn, might induce PR expression (23). In combination with the studies described herein, these observations suggest an important cycle of regulation between the ER, the PR, cyclin D1, and the ER/cyclin D1 complex, the perturbation of which would be predicted to lead to undesirable mammary epithelial cell proliferation. Future investigations will consist of determining (a) whether those mammary epithelial cells exhibiting proliferation also express cyclin D1, ER and PR; and (b) whether ER and PR regulate cyclin D1 expression by direct interaction with promoter elements on the cyclin D1 gene, or indirectly, through intermediary factor(s) that have yet to be identified.

	Acknowledgments

 
We extend special thanks to Marisela Mendoza, Gouqing Ge, and Liz Hopkins for their technical expertise. The secretarial assistance of Laura Birkens is gratefully acknowledged.

	Footnotes

 
¹ This work was supported by grants from the National Institutes of Health, HD-07858 (to B.W.O.) and CA-11944 (to D.M.); and from the Department of Defense, DAMD17–94-J-4254 (to O.M.C.).

Received March 7, 1997.

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