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and ERß in Primate Mammary Gland
Department of Medical Nutrition (G.C., Y.O., T.B., M.N., M.W., Y.-S.P., J.-Å.G.), Karolinska Institute, Novum, S-141 86 Huddinge, Sweden; Biocenter Oulu and Research Center for Molecular Endocrinology (Y.L., P.V.), University of Oulu, Oulu FIN-90014, Finland; and State Key Laboratory of Reproductive Biology (Y.W., Y.-S.P.), Institute of Zoology, Chinese Academy of Sciences, Beijing 100080, China
Address all correspondence and requests for reprints to: Yun-Shang Piao, Department of Medical Nutrition, Karolinska Institute, NOVUM, S14186, Huddinge, Sweden. E-mail: Yun-Shang.Piao{at}mednut.ki.se.
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and ERß) and the two progesterone receptor isoforms (PR-A and PR-B) in the normal human breast. What we know comes from rodent studies, which are not always pertinent for the human breast. We report now on regulation of gonadal hormone receptors during the menstrual cycle, pregnancy, and lactation in rhesus monkey mammary gland and on the relationship of these receptors to proliferation. We found that ER but not ERß is down-regulated when E2 levels increase and when cells enter the cell cycle. PR-B but not PR-A is expressed in proliferating cells. Thus under normal conditions, the ratio of ER to ERß in the breast depends on plasma concentrations of E2. Elevated expression of ER (as occurs in postmenopausal women) is a normal response to loss of E2 and indicates nonproliferating cells. As selective receptor ligands become available, they will be helpful in delineation of the functions of these receptors. |
Introduction |
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Development of mammary gland occurs predominantly after birth, under the control of steroid and peptide hormones (6). At puberty, the combined action of E2 and locally acting growth factors regulates proliferation of the terminal end buds located at the distal ends of the ductal epithelium. In the adult human breast, the highest proliferative activity is during the luteal phase of the menstrual cycle, a time when levels of both E2 and P are high. E2 promotes growth of the ductal epithelium and induces expression of P receptor (PR). P in turn promotes alveolar development (1, 7). At pregnancy, exposure to P and prolactin (PRL) results in extensive epithelial proliferation and increased side branching (2, 8). P, placental lactogens, and PRL signal alveolar proliferation and differentiation during pregnancy and possibly lactation (6).
Apoptosis plays important roles in mammary development from early embryonic formation of the mammary gland to the regression that follows cessation of cycling. The most dramatic occurrence of apoptosis is found during mammary involution (9). During the estrous cycle in rodents and the menstrual cycle in humans, the apoptotic events were confirmed by morphological and molecular analysis (7, 10, 11, 12). It was found that E2 increased the antiapoptotic proteins BCL-2 and BCLXL, whereas P drastically decreased BCL-2 expression (12, 13).
The sex steroid receptors are ligand-activated transcription factors that regulate gene expression. There are two estrogen receptor (ER) genes and two functionally distinct proteins, ER and ERß (14), both of which are expressed in normal breasts of rodent (14, 15), cow (16, 17), monkey (18), and human (19, 20) as well as in breast cancer (21, 22, 23, 24). Cells that express ER are found within the luminal epithelium but not in the myoepithelium or stroma in the human breast (19, 25, 26). ERß, on the other hand, is expressed not only in the luminal epithelial cells but also in myoepithelial cells, stromal cells, and in passenger lymphocytes (23, 24). This widespread distribution of ERß suggests multiple roles for ERß in the mammary gland (19, 20, 27).
PR is also expressed as two different protein isoforms, PR-A and PR-B, which are transcribed by different promoter usage from a single gene. PR-B is the longer version containing an additional 165 amino acids at the N terminus of the protein. In vitro data suggest that PR-B is the active PR, whereas PR-A is either inactive or acts as an inhibitor of PR-B. However, in the normal physiology of breast epithelium, both PR-A and PR-B appear to be coexpressed in the same subset of cells and at similar levels (8, 28, 29).
Information about the hormonal regulation of steroid receptors in the normal breast of healthy women during the menstrual cycle and pregnancy is necessary for understanding how steroid hormones affect proliferation. Such studies are limited by the availability of samples. In the available human breast studies, fine-needle biopsy samples have been used. These samples are not sufficiently large for morphological studies and contain too few epithelial cells to be representative of the breast epithelium. Another source of normal breast is tissue adjacent to benign or malignant breast tumors, which of course may not represent a normal environment. By using these materials, it was found that the highest ER expression is during the early follicular phase of the menstrual cycle, and levels decline as E2 levels increase. In contrast, PR is not down-regulated during the luteal phase and remains constant throughout the cycle (1, 11, 30). In this respect, the hormonal regulation of the normal breast differs from that of the normal endometrium, where both ER and PR in the luminal epithelial cells are down-regulated by E2 (31, 32). So far, there are no published studies on ERß or PR isoform expression in normal breast of human or nonhuman primate during the menstrual cycle and pregnancy.
Our current understanding of the role of steroids in mammary gland biology is mostly derived from studies using mice and rats. However, there are multiple differences between human and rodent. For example, in women, if the ovum is not fertilized, the corpus luteum undergoes atrophy within approximately 14 d after ovulation. However, in rodents, corpora lutea are present at all stages of the estrous cycle. The menstrual cycle in healthy women lasts approximately 28 d, whereas the estrous cycle is 4–5 d in rodents. In women, E2 and P are mainly produced from placenta after 7 wk gestation, but the rodent placenta does not express aromatase, so corpora lutea are the main source of E2 during pregnancy. In addition, ER is expressed in less than 20% of the epithelial cells of normal breast in healthy women, but it is expressed in more than 40% of epithelial cells in the mouse mammary gland. The rhesus monkey, a primate close to human beings, has a menstrual cycle of 26–28 d (33, 34), a gestation period of 155–172 d (35, 36), and its ERß shares more than 97% identity with human ERß (18). Circulating levels of E2 and P in rhesus monkeys during the menstrual cycle and pregnancy have been studied since the 1970s (33, 34, 35, 36, 37). The plasma levels of E2 increased from low levels during the early follicular phase to a midcyclic peak (100–350 pg/ml) followed by a decline for a few days and a subsequent increase to levels averaging 50 pg/ml. The plasma P levels during the follicular phase were less than 0.5 ng/ml but increased dramatically to 2–11 ng/ml in the luteal phase (33). The plasma concentrations of E2 ranged between 200 and 1300 pg/ml throughout the gestation period, and P ranged between 0.5 and 22 ng/ml (38). In this study, we investigated the expression of steroid receptors, including ER, ERß, PR-A, PR-B, and androgen receptor (AR), and proliferation markers in the mammary gland from rhesus monkeys at different stages of the menstrual cycle and during pregnancy and lactation. The results may help to improve our understanding of the roles of steroid receptors in regulating human mammary gland function.
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Materials and Methods |
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The mammary glands were taken from rhesus monkeys (Macaca mulatta) in a project approved by the local ethics committee at the State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences (39). Adult female rhesus monkeys with regular menstrual cycles were housed at the Fujian Experimental Center of Non-Human Primate, Fujian Institute of Planned Parenthood (Fuzhou, China), and Kunming Institute of Zoology, Chinese Academy of Sciences (Kunming, China). At least two menstrual cycles were recorded for each animal before the experiment. The first day of menstruation was defined as d 1. The specimens were taken from six animals at each time point, including early follicular phase (d 2–6), late follicular phase (d 10–14), and luteal phase (d 18–24). Eighteen animals were successfully mated from 5 d before and after the day of ovulation, which was estimated from records of two previous normal menstrual cycles. The presumed day of ovulation was considered d 0 of gestation, and from d 13 on, the actual gestational day was determined by ultrasound diagnosis, mainly based on the size of the uterus and conceptus. At d 15, 25, 50, and 100, at term, and during lactation, mammary glands were taken from three animals at each time point. They were divided into groups of early gestation period (d 15 and 25), middle gestation period (d 50 and 100), and late gestation period (d 160). The samples from lactating mammary gland were called Lac. Each sample was washed twice with PBS buffer and fixed immediately in 4% paraformaldehyde at 4 C for 16 h. Fixed tissues were then gradually dehydrated in ethanol and embedded in paraffin (39).
Antibodies
The monoclonal antibodies raised against human ER (1D5) and Ki67 (Mib-1, M7240) as well as Ki67 rabbit polyclonal (A0047) antibodies were from Dako (Glostrup, Denmark). Rabbit antihuman ERß polyclonal (06-629) antibody was from Upstate Biotechnology (Lake Placid, NY). Monoclonal antibodies against human PR (PGR-312) and PR-B (clone SAN27) were obtained from Novocastra Laboratories (Newcastle upon Tyne, UK). Cyclin A rabbit polyclonal (H432) and rabbit polyclonal anti-AR antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). Biotinylated secondary antibodies (goat antimouse IgG and goat antirabbit IgG) and avidin-biotin kits were obtained from Vector Laboratories (Burlingame, CA). Fluorescein isothiocyanate (FITC)- or Cy3-conjugated antirabbit and FITC- or Cy3-conjugated antimouse antibodies were from Jackson ImmunoResearch (West Grove, PA).
Immunohistochemistry
Paraffin sections (4 µm) were dewaxed in xylene and rehydrated through graduated ethanol to water. Antigens were retrieved by microwaving sections in 10 mM citrate buffer (pH 6.0) for 20 min at 650 W. Endogenous peroxidase was blocked by incubation for 30 min with a solution of 1% hydrogen peroxide. Tissue sections were incubated with 1% Triton X-100 at room temperature for 30 min and followed with normal goat serum diluted at 1/10 in PBS for 1 h at 4 C. Antibodies were diluted individually in PBS containing 3% BSA. Dilutions for antibodies were 1:30 for ER; 1:100 for ERß, PR-A, PR-B, Ki67, and cyclin A; and 1:200 for AR. Sections were incubated with antibodies overnight at 4 C. For negative controls, the primary antibodies were replaced with PBS alone or with primary antibodies after absorption with the corresponding antigen. Before and after the incubation of the secondary antibody, sections were rinsed in PBS. Sections were incubated in biotinylated goat antirabbit or goat antimouse Ig (1:200 dilution) for 2 h at room temperature. The sections were incubated in avidin-biotin-horseradish peroxidase (Vector) for 1 h at room temperature. After thorough washing in PBS, sections were developed with 3,3'-diaminobenzidine tetrahydrochloride (Dako), slightly counterstained with Mayer’s hematoxylin, and dehydrated through an ethanol series, followed by exposure to xylene and mounting.
Double-fluorescence immunostaining
Tissue sections, after antigen retrieval, were incubated with 1% Triton X-100 at room temperature for 30 min, followed by blocking with normal donkey serum (Sigma Chemical Co., St. Louis, MO) diluted at 1:10 in PBS for 1 h at 4 C. This was followed by an overnight incubation at 4 C with a mixture composed of antibodies to either ER and Ki67, ERß and Ki67, PR-A and Ki67, or PR-B and Ki67. PBS alone was used in place of these mixtures in the negative controls. Before addition of secondary antibodies, sections were washed with PBS. Slides were incubated for 1 h with a mixture of FITC-conjugated donkey antirabbit (1:100) and Cy3-conjugated donkey antimouse (1:400). For ERß and Ki67 (M7240), Cy3-conjugated donkey antirabbit (1:400) and FITC-conjugated donkey antimouse (1:100) were used. After washing with PBS for 30 min, the slides were incubated with 0.1 µg/ml of 4',6-diamidino-2-phenylindole dihydrochloride in PBS for 30 sec, washed three times in PBS, and mounted with Vectashield (Vector).
Preparation of 35S-labeled probes
Plasmids were constructed with vector pBluescript II SK± and ER ligand-binding domain (LBD) sequence (M12674, 1142–1580, 439 bp) or ERß LBD sequence (NM_001437, 1495–1982, 488 bp), respectively. In the 5' or 3' direction of the inserted sequence, there was T3 promoter or T7 promoter in the constructed plasmid. Plasmids were linearized by BamHIII or PstI, respectively. Antisense or sense RNA fragments were generated from linearized templates (2 µg) using T7 or T3 RNA polymerases (Promega, Madison, WI), respectively, in a transcription reaction containing 10x transcription buffer (Promega), 10 mM NTP (GTP, UTP, and ATP), RNase inhibitor, 5 µl [35S]UTP (1 mCi/100ml; Amersham, Arlington Heights, IL) for 2 h at 37 C. Samples were then incubated with DNase I (Sigma) at 37 C for 15 min to remove template. The reaction was stopped with 0.2 M EDTA. Unincorporated isotope was removed using NucTrap Probe purification columns (Stratagene, La Jolla, CA). Probes were diluted in STE (100 mM NaCl, 20 mM Tris-HCl PH 7.5, 10 mM EDTA) and were counted as 3.1 x 105 to 5.5 x 105 cpm. Labeled probes were stored at –80 C until applied to slides.
In situ hybridization
Paraffin-embedded sections were routinely deparaffinized and rehydrated. After treatment with 0.1 M glycine/0.2 M Tris-HCl (pH 7.4) for 10 min, slides were digested with 1 µg/ml of proteinase K (Roche, Mannheim, Germany) for 15 min, postfixed in 4% paraformaldehyde at room temperature for 10 min, and washed with PBS for 5 min. Sections were then acetylated in 0.1 M triethanolamine (pH 8.0) with 0.25% acetic anhydride (10 min), rinsed in distilled water, dehydrated through a graded series of alcohol (50–100%; 5 min each), and subsequently air dried. 35S-labeled cRNA probe was diluted to 2 x 105 cpm/µl. Hybridization mixture contained 10% probe, 50% deionized formamide (Sigma), and 40% hybridization buffer [300 mM NaCl, 20 nM Tris-HCl (pH 8.0), 5 mM EDTA, and 10% dextran sulfate (Pharmacia, Uppsala, Sweden); 1x Denhardts and 0.5 mg/ml tRNA from Escherichia coli (Boehringer, Mannheim, Germany); and 100 mM dithiothreitol (Sigma)]. Slides were hybridized for 16 h at 58 C. On each slide were two pieces of tissue, which were separated by tape and hybridized with hybridization mixture containing either sense or antisense probe. The sections were coverslipped and placed in plastic trays moistened with 50% formamide. The next day, coverslips were lifted with 4x standard saline citrate (SSC), and slides were rinsed three times in 4x SSC and incubated in RNase A (20 µg/ml) for 30 min at 37 C. Slides were then washed in 2x SSC, 1x SSC, 0.5x SSC, and 0.1x SSC (15 min each at 60 C). Finally, the sections were rinsed in distilled water and dehydrated in a graded series of alcohols. Slides were then dipped in 50% Kodak NTB2 emulsion in 0.6 M ammonium acetate, dried, and exposed in the dark for 4 wk at –20 C. The emulsion was developed in developer D19 (Kodak, Rochester, NY). Sections were counterstained with Mayer’s hematoxylin and eosin (HE) and then mounted with DPX (EM Sciences, Fort Washington, PA).
Apoptosis by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay
The detection of apoptosis-related DNA fragmentation by TUNEL was performed using the cell death detection kit (Roche) according to the manufacturer’s instructions with the exception that proteinase K digestion was replaced by microwave treatment (700 W for 1min in citrate buffer, pH 6.0) (7). After incubation with TUNEL reaction mixture (enzyme solution and labeling solution) for 60 min at 37 C and washing with PBS, the samples were mounted with Vectashield (Vector) and analyzed by fluorescence microscope. Positive controls were performed on human breast cancer. Negative controls were treated identically, omitting either terminal deoxynucleotidyl transferase or FITC-dUTP in the reaction mixture.
Statistical analysis
The percentage of positively stained cells is an average after counting the stained and the total number of cells from four high-magnification fields (
2000–3000 cells for each sample). Statistical differences between groups were analyzed with Student’s t test and ANOVA test using SPSS (SPSS Inc., Chicago, IL). A value of P < 0.05 was considered significant.
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The expression of ER, ERß, PR-A, PR-B, and AR at the protein level during the menstrual cycle, gestation, and lactation are illustrated in Fig. 1
and Table 1. Immunolocalization of ER revealed a strong positive staining in epithelial cells. The percentages of ER+ epithelial cells in the lobular epithelium at various stages of the menstrual cycle were 12.5%, at the early follicular phase, 3.3% at late follicular phase, and 1.2% at the luteal phase (P < 0.01). In the ductal epithelium, the values were 7.5, 1.4, and 0.4% (P < 0.01), respectively. Myoepithelial cells, lipocytes, and cells of the vascular system (endothelial cells, pericytes, and smooth muscle cells) were consistently ER– at all stages of the menstrual cycle. During pregnancy and lactation, immunostaining for ER could be detected in less than 1% of cells of mammary tissue.
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. In the early follicular phase, ERß was detected in very few epithelial cells. However, a striking increase of ERß expression in lobular epithelial cells was found at late follicular (62%) and luteal (80%) phases. In the ducts, the corresponding figures were 57% and 82%, respectively. In addition, ERß was expressed in myoepithelial cells and fibroblasts at the late follicular and luteal phases. This expression pattern did not change in the early stage of gestation, when 81% of epithelial cells in lobules and 78% in ducts were ERß+. However, by middle pregnancy, only 10% of the lobular epithelial cells and 8% of ductal cells expressed ERß. Very few ERß+ cells were detectable during late pregnancy and lactation. The monoclonal antibody PR312 recognizes both PR-A and PR-B on Western blots; however, it does not detect PR-B in immunohistochemical analysis. This is thought to be because of masking of epitopes of PR-B protein in formalin-fixed samples (40). Thus, positive staining with antibody PR312 is regarded as expression of PR-A.
A comparison of PR-A and PR-B expression is illustrated in Fig. 1
. It seems that PR-A and PR-B are expressed in a quite similar pattern at different stages of the menstrual cycle and during gestation. They were mainly present in epithelial cells in both ducts and lobules. Very few stromal cells were positive for either PR-A or PR-B. Double labeling was not done to investigate whether PR-A and PR-B were expressed in the same cells, because both antibodies were monoclonal. In the lobules, the percentage of PR+ cells increased from 4% at the early follicular phase to 13% at late follicular phase and 17% at the luteal phase. On the other hand, the percentage of PR+ ductal cells remained 12–15% without significant changes during the menstrual cycle. In pregnant monkeys, PR expression decreased as pregnancy progressed. At early, mid, and late gestation, the percentage of PR+ cells was 5, 9, and 0.3% in lobules and 3, 7, and 1% in ducts, respectively. Neither PR-A nor PR-B could be detected in lactating mammary gland.
AR was expressed in 59–75% of epithelial cells in both lobules and ducts throughout the menstrual cycle and in early and mid gestation. AR nuclear staining was also found in myoepithelial cells and stromal cells. However, in mammary glands at late gestation and lactation, no AR+ staining was detectable.
ER and ERß mRNA expression by in situ hybridization
ER and ERß mRNA expression was detected by in situ hybridization (Fig. 2). Probes were complementary to either ER or ERß LBD sequences. Sense probe hybridizations on the same slide were used as controls. The HE staining was used to show the morphology. The small black dots are positive signals of mRNA expression. ER mRNA expression was found in both lobular and ductal epithelium (picture not shown) in the mammary gland at different stages of menstrual cycle and gestation. ER mRNA level appeared to be highest at the early follicular phase and was much reduced in late pregnancy. In the lactating mammary gland, ER mRNA could not be detected.
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The expressions of ER and ERß at the mRNA level were not consistent with their expressions at the protein level. During menstrual cycle, ER mRNA level was unchanged whereas ER protein levels decreased. For ERß, mRNA increase paralleled the increase in protein. During pregnancy, ER protein was only detected in very few samples. ER mRNA, however, was present throughout pregnancy. ERß mRNA was present throughout pregnancy, but high ERß protein level was detected only during early pregnancy.
Cell turnover during the menstrual cycle, gestation, and lactation
The proliferation markers Ki67 and cyclin A were evaluated by immunohistochemistry (Fig. 3). Ki67+ staining indicates that cells are not resting in the G0 phase of the cell cycle, whereas cyclin A expression identifies the cells in the S phase. A double-fluorescence immunostaining confirmed that all cyclin A+ cells expressed Ki67, whereas approximately two thirds of Ki67+ cells were cyclin A negative (picture not shown).
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Apoptotic cells were detected by TUNEL reaction under a fluorescence microscope (Fig. 3). At early follicular phase, the percentage of TUNEL-positive cells was 12.8%, which is significantly higher than the 2.2% at late follicular and 4.3% at luteal phase. During pregnancy and lactation, the percentage of TUNEL-positive cells remained between 1 and 3% (Table 2).
Expression of ER, ERß, and PR isoforms in relation to proliferation
Colocalization of ERs or PRs and Ki67 was assessed by double-fluorescence immunostaining in nonpregnant monkey mammary gland samples (Fig. 4). ER was not detected in proliferating cells. Some Ki67+ cells were adjacent to the ER+ cells, but many were not. ERß, on the other hand, was colocalized with Ki67; 47% of the Ki67+ cells expressed ERß. The expression pattern of PR-A was the same as that of ER. No cells expressed both PR-A and Ki67. However, 21% of the Ki67+ cells expressed PR-B.
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Discussion |
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The proliferation of epithelial cells is balanced by apoptosis during the menstrual cycle and involution at the end of lactation (1). In the present study, cyclic deletion of mammary epithelial cells was demonstrated, apparently balancing cyclic cellular proliferation. The number of apoptotic cells was high at early follicular phase and low during late follicular phase and early luteal phase. During pregnancy, the apoptotic index was low, although proliferation rate was high. Thus, proliferation/apoptosis in the monkey is similar to that found in the rat (1). It is known that the antiapoptosis gene, BCL-2, is increased by E2 and down-regulated by P (13) and PRL (42). In addition, BCL-2 levels are high in the late follicular phase of the menstrual cycle, in line with our findings regarding apoptosis in monkey mammary gland (13).
The expression level of steroid receptors is regulated by their cognate ligands. During the menstrual cycle, as E2 levels increased, ER was down-regulated, whereas ERß was up-regulated. In the luteal phase, there was a further decrease in ER but no obvious change in ERß expression. These results are consistent with our recent finding that in adult ovariectomized mice, ER expression in the mammary gland is lost 4 h after a dose of E2, whereas ERß expression is up-regulated (27).
Comparison of ER and ERß at mRNA and protein levels during the menstrual cycle indicated that these two receptors are regulated in opposite directions by E2 and that two distinct regulatory mechanisms are involved. ER mRNA level was unchanged during the menstrual cycle, whereas ER protein levels decreased as E2 levels increased. This indicates a posttranscriptional regulation for ER expression. It is very likely that there is rapid degradation of ER protein as E2 drives the cells into the cell cycle. For ERß, the mRNA increase paralleled the increase in protein, indicating that the regulation of ERß expression in mammary gland during the menstrual cycle is at the transcriptional level. With fine-needle human breast biopsy samples, a similar ER expression at the protein level was found during the menstrual cycle (43), but the mechanism of regulation of ERß has not previously been examined.
During pregnancy, ER protein could be detected in very few cells. Such a down-regulation of ER was also found in pregnant rat (14), mouse (26), and bovine mammary gland (17). In the present study, however, we found that ER mRNA was present throughout pregnancy, although the circulating E2 level was very high. The down-regulation of ER during pregnancy could be via the same mechanism as in the menstrual cycle, i.e. degradation of ER protein. In the case of ERß, regulation during pregnancy is different from that during the menstrual cycle. ERß mRNA was present throughout pregnancy, but high ERß protein level was detectable only during early pregnancy. A high level of ERß during early pregnancy supports our idea that E2 increases transcription of ERß. The mechanism behind the loss of ERß protein in late pregnancy remains to be investigated.
The expression of PR, and therefore sensitivity to progestins, is under the control of E2. PR expression is increased by E2 but decreased by P in most target tissues (8, 44). In the present study, both PR-A and PR-B levels were low in early follicular phase and significantly increased thereafter. Surprisingly, despite the higher P levels in the luteal phase, PR levels were similar in the late follicular and luteal phases. In the present study, it is clear that the expression of PR in epithelial cells in lobules, but not in ducts, responds to changes in circulating levels of E2. Increase in PR is coincident with the increases in proliferation markers Ki67 and cyclin A in lobules but not in ducts.
The lactating gland has been described as estrogen insensitive because E2 does not elicit either proliferation or induction of PR during lactation. The reason for this insensitivity is not clear. We found that neither mRNA nor protein for either ER or ERß could be detected in lactating monkey mammary gland. Furthermore, neither PR nor AR is expressed. The mechanism for the loss of expression of all sex steroid receptors is not clear. This loss of ERs during lactation in the monkey is different from what has been observed in rats where high levels of both ER and ERßins (an ERß variant with 18-aminal acid insert in LBD) are expressed in lactating mammary gland, and where ERßins is thought to quench ER function (45).
The observation that proliferating mammary epithelial cells express neither ER nor PR (8, 15, 46) has been interpreted as evidence that ovarian steroids stimulate proliferation via paracrine signals secreted by steroid receptor-positive cells (2, 8, 47). This separation between steroid receptor expression and proliferation observed in the normal mammary gland is thought to be disrupted at an early stage in human breast tumorigenesis (48). As an alternative interpretation of the lack of coexpression of ER with proliferation markers, we have presented evidence that the signal to proliferate is received by ER-expressing cells but that ER is rapidly lost from these cells upon receipt of the signal. ER reappears in daughter cells 24 h later (27). In contrast, ERß expression was not affected by administration of E2, and it is expressed in proliferating cells (27). In the present study, we found neither ER nor PR-A in Ki67+ cells. In contrast, in the mammary gland during the menstrual cycle, 47% of Ki67+ cells expressed ERß and 21% expressed PR-B.
Henrich et al. (49) found that ERK7 is involved in the degradation of ER and that there is loss of ERK7 in breast cancer. In such ERK7-negative cancers, ER-expressing cells should not be capable of proliferation. Under these circumstances, E2-induced proliferation may be mediated by ERß. It is evident that ERß can mediate the proliferation signal because ductal elongation and lobuloalveolar development is restored in ovariectomized ER–/– mice upon E2/P treatment (50). Because more proliferation occurred if mice received combined E2/P treatment compared with E2 alone or P only (50), it is likely that ERß induces the expression of PR and, subsequently, that PR-B mediates the proliferation.
Although PR-A and PR-B are commonly coexpressed in cells (8), in the present study, PR-A was not found in proliferating cells, whereas 21% Ki67+ cells expressed PR-B. Studies in transgenic mice have confirmed that PR-B mediates proliferation and PR-A is an inhibitor of proliferation. Furthermore, the ratios of PR isoforms determine cell fate during normal mammary gland development (51). The reason for exclusive expression of PR-B in proliferating cells is under investigation. It could result from selective loss of PR-A or selective transcription from the PR-B promoter. A role for ERß in regulating the ratio of PR isoforms is possible because in MCF-7 cells, introduction of ERßcx, a splice variant of ERß, reduced PR-A and increased PR-B expression (52).
In addition to E2 and P, the ovary produces androgen, but the role of androgen in mammary gland proliferation is still unclear (53). In ovariectomized rhesus monkeys, testosterone treatment inhibited E2-induced proliferation in the mammary gland (54, 55), and in intact monkeys, the AR antagonist flutamide increased proliferation 2-fold (54). These data suggest that androgen may act as an antiproliferation factor in the mammary gland. In the present study, there was substantial expression of AR in 60–75% of cells at all phases of the menstrual cycle and from the early to middle stage of pregnancy. The function of this receptor in the mammary gland deserves much more attention.
In summary, the ER/ERß ratio in the mammary gland is determined by E2 levels. Possibly, ERß regulates the ratio of PR-A to PR-B, which may determine whether a cell will proliferate or not. It is clear that selective ER, ERß, PR-A, and PR-B agonists and antagonists are needed to clarify the various pathways by which hormones induce cell proliferation and perhaps to suggest better strategies for hormone replacement therapy and breast cancer treatment.
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Footnotes |
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First Published Online October 26, 2004
Abbreviations: AR, Androgen receptor; E2, estradiol; ER, estrogen receptor; FITC, fluorescein isothiocyanate; HE, hematoxylin and eosin; LBD, ligand-binding domain; P, progesterone; PR, progesterone receptor; PRL, prolactin; SSC, standard saline citrate; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling.
Received May 10, 2004.
Accepted October 14, 2004.
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References |
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and ß in the rodent mammary gland. Proc Natl Acad Sci USA 97:337–342 and ERß status. J Histochem Cytochem 47:1323–1330 and ERß mRNA in intrauterine tissues of the pregnant rhesus monkey. Am J Physiol Cell Physiol 278:C190–C198
and ERß in normal human mammary gland. J Clin Pathol 55:371–374 and ß differ in normal human breast and breast carcinomas. J Pathol 198:450–457[CrossRef][Medline]
and PR and associated with nodal status, grade, and proliferation rate in breast cancer. Am J Pathol 156:29–35 and ERß in proliferation in the rodent mammary gland. Proc Natl Acad Sci USA 101:3739–3746 in the uterus. Proc Natl Acad Sci USA 97:5936–5941-hydroxyprogesterone during gestation in the rhesus monkey. Acta Endocrinol (Copenh) 74:348–360 knockout mice. Endocrinology 141:2982–2994)-positive breast cancer: specific correlation with progesterone receptor. Cancer Res 62:4849–4853This article has been cited by other articles:
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 G. B Silberstein, K. Van Horn, E. Hrabeta-Robinson, and J. Compton Estrogen-triggered delays in mammary gland gene expression during the estrous cycle: evidence for a novel timing system. J. Endocrinol., August 1, 2006; 190(2): 225 - 239. [Abstract] [Full Text] [PDF]
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 H. Wang, L. Zhou, A. Gupta, R. R. Vethanayagam, Y. Zhang, J. D. Unadkat, and Q. Mao Regulation of BCRP/ABCG2 expression by progesterone and 17beta-estradiol in human placental BeWo cells Am J Physiol Endocrinol Metab, May 1, 2006; 290(5): E798 - E807. [Abstract] [Full Text] [PDF]
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 T. Hatsumi and Y. Yamamuro Downregulation of Estrogen Receptor Gene Expression by Exogenous 17{beta}-Estradiol in the Mammary Glands of Lactating Mice. Experimental Biology and Medicine, March 1, 2006; 231(3): 311 - 316. [Abstract] [Full Text] [PDF]
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