The Mineralocorticoid Receptor May Compensate for the Loss of the Glucocorticoid Receptor at Specific Stages of Mammary Gland Development

Michelle Kingsley-Kallesen, Sudit S. Mukhopadhyay, Shannon L. Wyszomierski, Susan Schanler, Günther Schütz and Jeffrey M. Rosen

Department of Molecular and Cellular Biology (M.K.-K., S.S.M., S.L.W., S.S., J.M.R.), Baylor College of Medicine, Houston, Texas 77030-3498; and Division of Molecular Biology of the Cell I (G.S.), German Cancer Research Center, Im Neuenheimer Feld 280, 69120 Heidelberg, Germany

Address all correspondence and requests for reprints to: Dr. Jeff Rosen, Department of Molecular and Cellular Biology, M638a Debakey Building, Baylor College of Medicine, One Baylor Plaza, Houston, Texas 77030-3498. E-mail: jrosen{at}bcm.tmc.edu.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS AND DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
To study the role of glucocorticoid receptor (GR) at different stages of mammary gland development, mammary anlage were rescued from GR-/- mice by transplantation into the cleared fat pad of wild-type mice. In virgin mice, GR-/- outgrowths displayed abnormal ductal morphogenesis characterized by distended lumena, multiple layers of luminal epithelial cells in some regions along the ducts, and increased periductal stroma. In contrast, the loss of GR did not result in overt phenotypic changes in mammary gland development during pregnancy, lactation, and involution. Surprisingly, despite the known synergism between glucocorticoids and prolactin in the regulation of milk protein gene expression, whey acidic protein and ß-casein mRNA levels were unaffected in GR-/- transplants as compared with wild-type transplants. That mineralocorticoid receptor (MR) might compensate for the loss of GR was suggested by the detection of MR in the mammary gland at d 1 of lactation. This hypothesis was tested using explant cultures derived from the GR-/- transplants in which the mineralocorticoid fludrocortisone was able to synergistically induce ß-casein gene expression in the presence of prolactin and insulin. These studies suggest that MR may compensate for the absence of GR at some, but not at all stages of mammary gland development.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS AND DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
GENE TARGETING AND transgenic techniques have provided powerful tools for elucidating the function of specific genes in mouse development. However, developmental studies are often complicated when the absence of or the introduction of a gene results in perinatal lethality or exerts pleiotropic effects, making it difficult to distinguish between direct and indirect mechanisms of action. This is especially true in the study of the mammary gland development, which occurs primarily postnatally and is highly dependent on the levels of systemic hormones and local growth factors.

In the mouse mammary gland, there are several critical periods of postnatal development ductal morphogenesis, which consists of proliferation and branching during sexual maturity, alveolar budding, and lobular formation that occurs during pregnancy, terminal differentiation and lactation, and involution that is characterized by increased apoptosis and extensive tissue remodeling. These processes are tightly regulated by systemic levels of hormones and interactions with their cognate receptors, such as estrogen (1), progesterone (2, 3), prolactin (Prl) (4, 5), and glucocorticoids (6) and by local expression of growth factors.

Gene targeting experiments have provided considerable insight about the specific function of genes required for mammary gland development (7). For instance, mice that lack either Prl (8) or the Prl receptor (PrlR) (9) fail to undergo lobuloalveolar development and do not lactate. Components of the PrlR signal transduction pathway, STAT5a and STAT5b, are also important for proper mammary gland development. When both STAT5a and STAT5b are deleted, mammary epithelial transplants display a phenotype that is comparable with Prl- and PrlR-deficient mice (10).

The role of glucocorticoid receptor (GR) in mammary gland development is less well defined. Numerous studies have demonstrated that glucocorticoids act synergistically with Prl in mammary epithelial cells to regulate mammary gland differentiation and activate milk protein gene expression [reviewed by Rosen et al. (11)]. Experiments performed using cell culture models have supported the cooperative role of GR and Prl-activated STAT5 in the transcriptional regulation of the milk proteins ß-casein and whey acidic protein (WAP) (12, 13, 14, 15, 16, 17, 18, 19, 20, 21). For ß-casein in particular, these studies showed that GR acts as a transcriptional activator for STAT5 by enhancing and prolonging DNA binding of STAT5, by providing a transcriptional activation domain and by functioning as a bridging molecule to facilitate interactions with other transcription factors and coregulatory molecules [reviewed by Deroo and Archer (22)].

DNA binding also appears to be important for GR and STAT5 cooperativity. Mutation of the STAT5 binding site in the proximal ß-casein promoter inhibits both glucocorticoid- and Prl-induced gene transcription. It has also been shown that although the ß-casein promoter lacks consensus, palindromic glucocorticoid response elements (GREs), it does contain several half-palindromic DNA binding sites (1/2 GREs) capable of interacting weakly with the purified rat liver GR (21). Mutation of several of these half-sites markedly reduces the synergistic effects of glucocorticoids and Prl on ß-casein transcription (18). However, studies using GR mutants defective in binding to a consensus GRE did not initially support a requirement for DNA binding to facilitate glucocorticoid-Prl synergism (23). The interpretation of these experiments was complicated by the use of transient transfection studies in COS cells in which these DNA binding mutants of GR were markedly overexpressed. GR and STAT5 cooperativity was not observed; however, when similar studies were performed in CV-1 cells, in which the levels of the GR DNA-binding mutant were comparable with those of the endogenous GR in mammary epithelial cells (15).

To address the physiological role of GR DNA binding on the regulation of milk protein gene expression, the development and differentiation of the mammary gland was recently examined in mice engineered to express one of these GR DNA-binding mutants (GRdim) (24). In these animals, a decrease in ductal epithelial proliferation in the virgin mammary gland of 10- and 22-wk-old animals was observed. No other mammary phenotype was reported in these mice; lobuloaveolar development appeared normal, and milk protein gene expression was unaffected in the GRdim mice. In addition, no effects on mammary gland involution in the GRdim mice were reported, despite previous studies demonstrating the inhibitory effects of pharmacological glucocorticoid administration during the tissue remodeling phase of involution (25, 26, 27). The authors concluded from these studies that the DNA-binding activity of GR was not required for normal mammary gland development and differentiation during pregnancy and lactation. Furthermore, they indicated that their results supported the model in which GR can act as a coactivator with STAT5 to regulate milk protein gene expression independent of DNA binding.

To validate these conclusions, it is critical to examine the effects of the GR loss on the same phenotypes to rule out compensation by other members of the nuclear receptor family. The GR gene was disrupted in mice by the insertion of a neomycin cassette into the second exon to generate a hypomorphic allele (GRhypo) (28). All of the phenotypic changes that were associated with the GRhypo mutation were also observed in GR mutant mice in which GR was completely inactivated (29). Targeted disruption of GR resulted in perinatal lethality, and thus precluded analysis of the mammary gland in an intact animal (28). The studies presented herein have circumvented this problem by examining the role of GR on the development and differentiation of the mammary gland using transplantation studies to rescue mammary epithelium from GR-deficient mice. This was accomplished by removal of embryonic mammary buds (E14–E15) and transplantation into epithelium-free (cleared) fat pads of 3-wk-old recipient mice. Primary outgrowths were allowed to regenerate for 8 wk, at which time secondary transplants were then performed and mammary gland development was characterized at several time points, including 3 and 8 wk of outgrowth in virgin mice, during midpregnancy, at d 1 of lactation, and finally at d 1, 2, and 4 of involution.


    RESULTS AND DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS AND DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Altered Ductal Morphogenesis during Virgin Development in GR-Deficient Mammary Epithelial Outgrowths
Homozygous deletion of GR results in perinatal lethality primarily due to atelectasis of the lung (28). Therefore, to study the role of GR in the mammary gland, the mammary epithelium from GR-deficient mutants was rescued by transplantation of the mammary anlage at E14–E15 into the cleared fat pad of syngeneic hosts. Two or three embryonic mammary buds from E14–E15 were dissected with skin and placed into the cleared fat pad of 21-d-old syngeneic hosts. A success rate of approximately 50% was obtained for outgrowths of embryonic mammary anlagen, regardless of the genotype of the donor epithelium. However, because of the labor-intensive nature and the variability in the amount of epithelium transplanted, detailed investigation of the mammary phenotypes was performed on secondary transplants where 1-mm2 sections of wild-type, heterozygous, and knockout tissue were transplanted into multiple syngeneic hosts (see Materials and Methods). This facilitated the analysis of the outgrowths at multiple time points during mammary gland development and also allowed a direct comparison of variations among recipients. Furthermore, a direct comparison of the phenotypes of the outgrowths from the wild-type and knockout donors in the same recipient was possible by transplantation into contralateral cleared fat pads, respectively.

Using this approach, it was initially observed that mammary epithelium lacking GR consistently filled the fat pad more rapidly than wild-type donor epithelium. Three weeks after transplantation, GR-deficient outgrowths filled approximately 80% of the fat pad. This was in contrast with wild-type transplants, which filled less than 20% of the fat pad, and heterozygous outgrowths, which have filled less than 50% of the fat pad (Fig. 1Go). This increased rate of filling the fat pad appeared to occur between wk 2 and 3 after transplantation, because at 2 wk the GR-deficient epithelium was still a small mass of cells at the transplantation site, similar to that observed in the wild-type outgrowth 3 wk after transplantation (data not shown). Few, if any, terminal end buds (TEBs) were observed in the GR-deficient epithelial transplants at 3 wk, in contrast to the heterozygous and wild-type transplants. Hence, it was difficult to directly compare the levels of proliferation and apoptosis because the majority of proliferation and apoptosis during ductal morphogenesis is known to occur in the TEBs (30). As expected, when proliferation rates were examined by measuring bromodeoxyuridine incorporation in the ducts, no significant differences were detected among the wild-type, heterozygous, and GR-deficient epithelium (data not shown).



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Figure 1. Whole Mount Analysis of Epithelial Transplants at 3 wk Post Transplantation

The genotype is shown above the size bar for each image. A, Outgrowths from a GR-wild-type donor showing less than 20% of the fat pad filled by 3 wk post transplantation. Five separate transplants were analyzed at this time point. B, Transplanted epithelium from a GR-heterozygous donor showing less than 50% of the fat pad filled. This image is also representative of five separate transplants. C, GR-/- epithelium has filled approximately 80% of the fat pad by 3 wk. Of the three separate transplants examined, only one contained detectable TEBs. Size bars, 1 mm. Note: The percentage of the fat pad filled refers to the extent to which the ducts have grown from the site of transplantation to the edge of the fat pad, not the overall percentage of the fat pad occupied by epithelium; e.g. in a 100% filled fat pad the ducts would have reached the edge of the fat pad.

 
By 8 wk after transplantation, all of the transplants regardless of genotype had completely filled the fat pad. Obvious morphological differences were, however, observed in the GR-deficient transplants. These included a dilated ductal phenotype and an atypical pattern of branching that was antler-like in appearance (Fig. 2Go). Hematoxylin and eosin (H&E) staining of 5-µm sections indicated that the ducts were composed of a single layer of luminal epithelial cells surrounded by a thin layer of condensed stroma in the wild-type and heterozygous mammary epithelium. However, the GR-deficient ducts displayed a distended lumen and in some regions contained multiple epithelial cell layers with an increased amount of periductal stroma, as evidenced by serial sections (Fig. 2Go).



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Figure 2. Morphological and Histological Analysis of Epithelial Transplants 8 wk Post Transplantation

The genotype is shown above the size bar for each image. Images A–C display whole mounted tissue stained with hematoxylin. D–I, Five-micrometer sections stained with H&E. Whole mount analysis (A and B) and H&E-stained sections (D and E) of GR-wild-type and GR-heterozygous epithelium show normal ductal branching and typical histoarchitecture. GR-/- outgrowths show distended ducts in both whole mount images (C) and H&E sections (F). Images G–I show an enlarged view of serial sections of the inset shown in (F). Increased periductal stroma and multiple luminal epithelial cells can be seen. A–C, Size bars, 500 µm; D–F, size bars, 200 µm; G–I, size bars, 100 µm.

 
It is possible that the loss of GR expression in the mammary gland results in a disruption of normal signal transduction pathways required for the establishment of a single layer of luminal epithelium. One possible mechanism responsible for this phenotype might be a disruption of normal interactions of the luminal epithelial cells with the extracellular matrix (ECM). When mammary epithelial cells are not in contact with ECM, they undergo anoikis (31, 32). GR has been reported to negatively influence the expression of ECM perhaps by regulating expression of collagenases and matrix metalloproteases (see below). Accordingly, an increased amount of condensed stroma surrounding the ducts was observed in the GR-deficient as compared with the wild-type transplants using Masson’s trichrome staining (Fig. 3Go, A and B). Examination of the condensed stroma suggested that the GR-deficient transplants contained at least twice as much periductal stroma as compared with wild-type transplant. However, this is an approximation that is based only on the estimated width of the blue-stained collagen layer.



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Figure 3. Increased Periductal Collagen Staining in GR-Null Outgrowths with Normal Myoepthelium

The genotype of the transplanted epithelium is shown at the top of each column. Each image is representative of five separate animals for each genotype. Masson’s trichrome stains collagen blue and nuclei blue-black. A, GR-wild-type epithelium has a thin layer of collagen surrounding the ducts. B, GR-/- outgrowths have approximately two to three times as much collagen surrounding the distended ducts as estimated by the width of the blue-stained collagen layer. C and D, Merged images of DAPI stained nuclei and Texas Red K14 fluorescence. GR-wild-type (C) and GR-/- (D) outgrowths show a single, continuous layer of myoepithelial cells detected with a K14 polyclonal antibody and a Texas Red secondary antibody. DAPI-stained nuclei fluorescing blue are of both ductal and stromal origin. Size bars, 100 µm.

 
Although the involvement of glucocorticoids in regulating ECM during virgin development has not been reported previously, it has been demonstrated that during mammary gland involution glucocorticoids inhibit tissue remodeling. Exogenous administration of hydrocortisone during involution inhibits the induction of a number of enzymes that act on the ECM, such as type I collagenase, stromelysin-1, gelatinase A, and the serine proteinase urokinase-type plasminogen activator (27). This GR-mediated repression has been suggested to be due in part to the interaction of GR with activator protein-1 and/or nuclear factor-{kappa}B, which are required for the transcriptional activation of many of these genes (33, 34). This suggests that GR may also be involved in regulating the cross-talk between the ductal epithelial cells and the surrounding ECM. However, the mechanism by which loss of GR in the mammary epithelium might regulate the activity of these predominantly stromal proteases remains to be established. Presumably, this may involve the production of local cytokines and growth factors. Thus, it appears likely that during virgin development GR is important for the regulation of normal ECM deposition, and that increased ECM in the GR-deficient transplants may in part be responsible for the observed multilayered epithelium.

Furthermore, the observed increase in stroma did not appear to be a result of the absence of the myoepitheial cell layer surrounding the luminal epithelial cells of the mammary duct, as illustrated by cytokeratin-14 (K14) staining (Fig. 3Go, C and D). Only a single layer of elongated and flattened K14-positive myoepithelial cells was observed surrounding the luminal epithelial cells in both the wild-type and GR-deficient transplants as visualized by red fluorescence. 4,6-Diamidino-2-phenylindole (DAPI), which forms fluorescent complexes with double-stranded DNA, is shown fluorescing blue. It indicates nuclear staining in the stroma and of the ductal epithelium, and confirms that the multiple cell layers observed in the H&E-stained serial sections of the GR-deficient ducts (Fig. 2Go, G–I) were luminal epithelial and not myoepithelial cells. It is conceivable that loss of GR in the myoepithelium may directly affect the biosynthesis of ECM components.

GR Does Not Appear to be Required for Lobuloalveolar Development during Pregnancy and Lactation
Because glucocorticoids are thought to play a role in mammary gland differentiation and are known to be important for milk protein gene expression during pregnancy and lactation, the GR-deficient, -heterozygous, and -wild-type outgrowths were examined during these stages of development. Although the distended ductal phenotype that was observed in the GR-deficient outgrowths in virgin animals was retained during pregnancy, neither the differentiation of the epithelial cells nor their ability to form lobuloalveoli during pregnancy (Fig. 4Go) and lactation (Fig. 5Go) appeared to be affected. A direct comparison of whole mounts (Fig. 4Go, A–C) and 5-µm H&E-stained sections (Fig. 4Go, D–F) of the wild-type, heterozygous, and GR-deficient outgrowths did not reveal any apparent phenotypic differences at mid-pregnancy. However, at lactation, the lumens of the GR-deficient outgrowths visually seemed slightly more collapsed than those observed in the wild-type transplants (Fig. 5Go, E and F). Milk lipid synthesis appeared normal in both the wild-type and GR-deficient outgrowths, and a majority of the cells contained large cytoplasmic lipid droplets easily visualized by immunostaining for adipophilin (Fig. 5Go, C and G). Adipophilin is a globule protein found in milk fat and is routinely used as a marker for terminal differentiation of the mammary gland (35, 36). The size and location of these lipid droplets appeared indistinguishable between wild-type and GR-deficient outgrowths.



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Figure 4. Normal Lobuloalveolar Development at Midpregnancy in GR-Wild-Type, -Heterozygous, and -Deficient Transplants

The genotype of the transplanted epithelium is shown at the top of each column. Glands were taken at d 14–15 of pregnancy after 8 wk of initial outgrowth. Whole mount analysis (A–C) and H&E-stained sections (D–F) show no overt differences in morphological and histological development. A–C, Size bars, 1 mm; D–F, size bars, 200 µm.

 


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Figure 5. Outgrowths at d 1 Lactation Are Virtually Indistinguishable Morphologically and in Their Expression of Adipophilin

Sections of GR-wild-type (A–D) and GR-deficient (E–H) transplants display normal alveolar development. Cytoplasmic lipids droplets have developed by d 1 of lactation and stain for adipophilin shown in red (C and G). DAPI-stained nuclei are shown in blue (D and H). Note that adipophilin only stains the cytoplasmic droplets and not stroma or epithelial cells. Bars, 50 µm.

 
No Change Was Observed in the Ability of GR-Deficient Transplants to Undergo Involution
Because it has been established that administration of exogenous hydrocortisone to lactating rodents delays and/or inhibits the onset of mammary involution and partially maintains ß-casein expression, it was postulated that the lack of GR might accelerate involution. Day 1 of involution (Fig. 6Go, A and B) represents a 24-h time point after removal of the pups after birth. At this time point, it was consistently observed that morphologically, the mammary glands of the GR-deficient outgrowths contained approximately 25% less epithelium than the wild-type transplants. However, no change in the level apoptosis at d 1 involution detected by terminal deoxynucleotidyl transferase-mediated deoxy-UTP nick end-labeling (TUNEL) staining was observed between the wild-type and GR-deficient transplants (TUNEL staining detects apoptotic cells by using terminal deoxynucleotidyl transferase to directly label the ends of broken DNA strands). At d 2 (Fig. 6Go, C and D) and 4 (Fig. 6Go, E and F) of involution, the wild-type and GR-deficient transplants were indistinguishable both morphologically and in their levels of apoptosis. Therefore, because GR is thought to play a role in the tissue remodeling phase of involution, the loss of GR in the mammary epithelium may not be compensated for by the presence of mineralocorticoid receptor (MR) (see below) at the beginning of involution. Alternatively, because wild-type GR is present in the mammary stroma of the GR-deficient outgrowths, these results may indicate that glucocorticoids are still able to regulate the expression of stromal proteases during mammary gland involution (26). GR may also affect the expression of proteins, such as surfactant protein A, involved in the recognition and phagocytosis of apoptotic epithelial cells by their neighbors (37). Interestingly, this regulation by glucocorticoids has been reported to occur at the posttranscriptional level in lung type II alveolar cells (38).



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Figure 6. Involution of the Mammary Gland Occurs Normally in Epithelium that Lacks GR

The genotype of the transplanted epithelium is shown at the top of each column. Five-micrometer H&E sections of GR-wild-type and -deficient transplants were histologically similar at d 1 involution (A and B), d 2 involution (C and D), and d 4 involution (E and F). In B, the tissue remodeling phase of involution appears to be increased in the GR-deficient epithelium as compared with GR-wild-type tissue (A). When four separate animals were compared, the amount of the GR-deficient epithelium was reduced by approximately 40–50%. The table shows the percent of nuclei undergoing apoptosis. The number of TUNEL-positive MEC in a given field was expressed as a percentage of total number of DAPI-stained MEC. At least 5000 nuclei were counted per animal and a minimum of three animals examined for each time point. Bars, 250 µm.

 
ß-Casein and WAP Gene Expression Is Unaffected in GR-Deficient Transplants, and this May Be Due to Compensation by MR
Previous studies have established that the glucocorticoid and Prl signal transduction pathways act synergistically in mammary epithelial cells to activate milk protein gene expression. At the morphological level, lactation appeared in general to be unaffected by the loss of GR. However, to determine whether GR was actively involved in the synergism between glucocorticoids and Prl in regulating milk protein gene expression, RNA was isolated from wild-type, heterozygous, and null outgrowths at d 1 of lactation and probed for ß-casein and WAP mRNA expression (Fig. 7AGo). Surprisingly, no detectable difference in either ß-casein or WAP gene expression was observed when normalized to cytokeratin-18 expression.



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Figure 7. ß-Casein and WAP mRNAs and MR Are Expressed Equally in GR-Wild-Type, -Heterozygous, and -Deficient Transplants at d 1 Lactation

A, Northern blot analysis of 1 µg of total RNA isolated from a no. 3 mammary gland, and GR-wild-type, GR-heterozygous, and GR-deficient transplants at d 1 of lactation. RNA blots were probed with ß-casein, stripped, and reprobed with WAP and cytokeratin-18. Images were exposed to a PhosphorImager (Molecular Dynamics, Sunnyvale, CA) and expression levels of ß-casein and WAP mRNA were normalized to K18. No statistically significant differences were observed. At least four individual Northern blots were performed using four different pools of RNA. B and C, Merged images of DAPI-stained nuclei and Texas Red MR fluorescence. MR staining was performed using a monoclonal MR antibody and an antimouse Texas Red secondary antibody. DAPI staining of nuclei is shown in blue and overlapping images are purple. MR is expressed in the majority, but not all nuclei of GR-wild-type (B) and GR-deficient (C) transplants at d 1 lactation. Bars, 100 µm.

 
This is especially surprising in the case of WAP because its expression is more highly dependent on glucocorticoids than ß-casein, and unlike ß-casein, its promoter contains consensus GREs that are bound by GR in vitro (20, 21). Furthermore, administration of dexamethasone to adrenalectomized mice carrying a +2020 rat WAP transgene during lactation demonstrated that glucocorticoids were required to maintain transgene expression in the mammary gland and that these glucocorticoid-induced changes in expression were correlated with the appearance of deoxyribonuclease I hypersensitive sites (20). These results indicated that at least part of glucocorticoid regulation of WAP gene expression was mediated through the direct interaction of GR with GREs in the distal promoter region and resulted in distinct steroid hormone-dependent modifications in chromatin structure. Therefore, this lead to the hypothesis that another member of the steroid hormone receptor family might compensate for the loss of GR, thus explaining the presence of both WAP and ß-casein expression at d 1 of lactation.

In this regard, previous studies have demonstrated that STAT5 can functionally interact with several members of the steroid receptor family, and that GR, MR, and the progesterone receptor (PR) have all been reported to synergize with STAT5 to induce ß-casein gene transcription (39). In contrast, the estrogen receptor diminished STAT5-mediated induction and the androgen receptor had no apparent effect on ß-casein transcription (39). No such analysis has been performed with WAP promoter constructs. Nevertheless, based upon these observations, the expression of MR and PR were examined in wild-type, heterozygous, and GR-deficient outgrowths at d 1 lactation by immunostaining. PR expression was detected in only a few ductal epithelial cells in both wild-type and GR-deficient transplants, as expected from previous studies, which reported the loss of PR expression at the onset of lactation (Ref. 40 , and data not shown). However, MR fluorescing red was detected in the nucleus of virtually every secretory epithelial cell (Fig. 7Go, B and C) in both wild-type and GR-deficient transplants. When merged with DAPI staining, MR did not colocalize with stromal cells or myoepithelial cells but appeared to be specifically expressed in the lobuloalveolar cells. MR staining was confirmed using kidney sections, and nonspecific staining was not observed when the secondary antibody was used in the absence of the primary antibody. These results are consistent with previous reports using immunoelectron microscopic analysis were MR was found to be present in the ducts and secretory tubules but not in the myoepithelium and interstitial cells of the mammary gland (41).

When similar immunofluorescence analysis was performed in the GR outgrowths in virgin mice, MR expression was not readily detected regardless of the genotype of the transplants (data not shown) and thus may be expressed at very low levels in the virgin beyond detection by methods other than immunoelectron microscopy. However, MR expression was observed during midpregnancy in addition to lactation (data not shown). Thus, the ability of MR to compensate for the loss of GR may be dependent on both the stage of development, as well as possibly on the regulation of different gene targets. This might explain why such an overt phenotype was observed in virgin mice, but not during midpregnancy, lactation, and involution.

To determine if MR could functionally compensate for the loss of GR, explant cultures obtained from GR-deficient transplants at midpregnancy were performed under serum-free conditions, and, in the absence of glucocorticoids, the ability of mineralocortiocoids to synergize with Prl and induce ß-casein gene expression was evaluated (Fig. 8AGo). In the presence of insulin and Prl, when compared with insulin alone, only a modest induction of ß-casein mRNA was detected. However, in the presence of insulin, Prl, and fludrocortisone, an approximate 5-fold induction of ß-casein mRNA (over insulin alone) was observed. Fludrocortisone is a ligand for MR similar to aldosterone that is 200 times more potent as a mineralocorticoid than cortisol and binds with the same affinity as aldosterone to MR (42). The same experimental conditions were used with mammary gland transplants from wild-type and heterozygous tissue, and similar results were obtained (data not shown). Furthermore, approximately the same fold induction of ß-casein mRNA was observed using hydrocortisone instead of fludrocortisone and GR wild-type mammary explants in parallel experiments (Fig. 8BGo). Detectable, but low, levels of WAP mRNA were also seen in the Northern blots from the fludrocortisone-treated explant cultures (data not shown).



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Figure 8. Explant Cultures of GR-Deficient Transplants Show that Mineralocorticoids Can Induce ß-Casein Gene Expression

For these studies, transplanted mammary glands from d 14 of pregnancy were removed, diced into 2-mm2 pieces, and placed in chemically defined media on stainless steel grids. The mammary glands were cultured for 48 h in media supplemented with insulin to allow for the removal of endogenous growth factors and hormones. In A, after 48 h, the medium was changed to one of the following conditions: insulin alone (I), insulin and Prl (I + Prl), or insulin, Prl and the mineralocorticoid, fludrocortisone (FC + I + Prl). In B, after 48 h, the medium was changed to one of the following conditions: insulin alone (I), insulin and Prl (I + Prl), or insulin, Prl and hydrocortisone (HC + I + Prl). After an additional 48-h culture period, RNA was isolated from the pooled explants in each group and Northern analysis was performed. Three different concentrations of RNA from each condition were hybridized with a ß-casein-specific probe. The graphs show the normalization of one experiment from an explant culture and are representative of a total of four experiments from four different transplants. The range of fold induction from the GR-deficient cultures was 5- to 8-fold with insulin, Prl, and fludrocortisone and from the GR-wild-type cultures was 7- to 11-fold with insulin, Prl, and hydrocortisone.

 
These results are consistent with experiments performed several decades ago in which aldosterone was shown to regulate mammary gland differentiation (43) and more recent experiments that have demonstrated that MR is able to activate ß-casein gene transcription in the presence of activated STAT5 (39). These experiments also support the hypothesis that in the mammary gland, MR may compensate for the loss of GR in regulating the transcription of milk protein genes. This may explain the failure both to observe overt phenotypic effects during pregnancy, lactation, and involution, as well as to detect any changes in milk protein gene expression.

Ideally, studies performed using MR/GR double null mice will be required to definitively establish the role of MR during mammary gland development, but it is likely that these animals will not survive to a stage that would allow for rescue of the mammary anlage by transplantation. Use of conditional floxed alleles for GR (44) and MR (Schütz, G., unpublished observations) and the tissue-specific Cre recombinase may provide an alternative strategy to elucidate the individual and combined function of these receptors in the future. The present experiments, however, have established that MR may compensate for the absence of GR at specific, but not all stages of mammary gland development. Importantly, when examining the role of specific mutations in mouse models, it is critical to perform parallel studies with the gene-deficient allele at each stage of development. Conclusions based upon the analysis of the GRdim mice in vivo should, therefore, be carefully reevaluated, especially those that involve potential GR/STAT5 interactions. Other functions of GR involving interactions with different transcription factors such as activator protein-1 and nuclear factor-{kappa}B or posttranscriptional effects might not be subject to equivalent compensation by nuclear receptor family members.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS AND DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Animals
Three-week-old C57Bl/6 recipient mice were obtained from Harlan (Indianapolis, IN) for the transplantation studies. Genotyping of the GR animals was performed using PCR and three primers: GR-PG3 5'-AGAATCCTTAGCTCCCCCTGG-3', MUTGR 5' GCCTGCTCTTTACTGAAGGCTC-3' and GR-PG4 5'-CTGCTGCTTGGAATCTGCCTG-3'. The wild-type GR allele generated a 170-bp PCR product, and the null allele a 450-bp PCR product, respectively. The experimental studies described herein were approved by the Baylor College of Medicine Animal Research Committee. All surgical procedures were be performed under anesthesia using an ip injection of 0.1 ml 20 mg/ml Avertin per 10 g animal. The depth of anesthesia was monitored by the absence of withdrawal reflex to toe pinch. Animals were kept on a heating pad during the recovery period and monitored daily after surgery. Euthanasia was performed by cervical dislocation after Avertin anesthesia or CO2 inhalation.

Epithelial Transplantation from Null Embryos and Secondary Transplantation Analysis
Mammary anlagen from E14–E15 were visualized under a dissecting microscope, the nos. 2 and 3 (thoracic) or the nos. 4 and 5 (inguinal) glands were dissected out and transplanted into the no. 4 cleared fat pads of 3-wk-old recipient hosts. Clearing of the endogenous mammary epithelium from the host C57Bl/6 animals involved the surgical excision of the no. 4 inguinal gland from the nipple to the lymph node as described previously (45, 46, 47). The excised portion of the gland was routinely whole mounted and stained with hematoxylin to determine that the epithelium was completely removed from the cleared fat pad. To determine both the genotype and sex of the mice, genomic DNA was isolated from the donor embryos after transplantation, genotyped (as described in Animals), and sexed (by PCR amplification of a 220-bp fragment of the male SRY gene). The SRY primers used were: SRY-F 5'-CGCCCCATGAATGCATTTATG-3' and SRY-R 5'-CCTCCGATGAGGCTGATAT-3'. No differences were detected in the extent of mammary outgrowths in glands from male or female donor embryos, and they appeared morphologically similar. Eight weeks after transplantation, mice were anesthetized and one half of the transplant was removed, fixed, and stained with hematoxylin to assess the extent of outgrowth. The take rate of the mammary anlage from wild-type, heterozygous, and GR-null donor mice was approximately 50%. If outgrowths were observed, 1-mm2 pieces of the transplant were removed and re-transplanted into the cleared fat pads of 3-wk-old C57Bl/6 recipients. This helped provide a sufficient number of transplants to evaluate the effects of the loss of one or both allele of GR throughout mammary gland development and avoided the problems of biological variability due to transplantation of different amounts of mammary epithelium and skin from the embryonic anlage. These were designated secondary transplants and used in the majority of the studies described herein. The take rate of the secondary transplants was approximately 75% and was not influenced by the genotype of the donor epithelium.

Developmental Stages and Whole Gland Morphological Analysis
Developmental stages examined were 3 and 8 wk after transplantation in virgin mice, at mid-pregnancy (d 14–15), at d 1 of lactation, and at d 1, 2, and 4 of involution initiated immediately after d 1 of lactation (see below). For pregnancy, lactation, and involution, mice were mated at least 8 wk after transplantation to ensure complete filling of the mammary fat pad. The percentage of the fat pad filled refers to the extent to which the ducts have grown from the site of transplantation to the edge of the fat pad, not the overall percentage of the fat pad occupied by epithelium, e.g. in a 100% filled fat pad the ducts would have reached the edge of the fat pad. Because the transplanted epithelium has no connection to a nipple, milk stasis occurred in the transplants and induced the first stage of involution. Thus, d 1 lactation glands represent glands taken within 8 h of birth. Glands at d 1–4 of involution were harvested 24, 48, and 96 h, respectively, after the pups were born and removed from the mother.

Mammary glands from virgin mice were surgically removed, divided in half longitudinally, and fixed in ice-cold 4% paraformaldehyde PBS for 2 h. One half of the mammary gland was stained with hematoxylin and examined for developmental abnormalities and the other half was embedded in paraffin, sectioned at 5 µm, and used for H&E staining, Masson trichrome staining, or for immunohistochemical analyses. Trichrome staining was performed using the Accustain Trichrome Stain Kit (HT15, Sigma, St. Louis, MO). Mammary glands from midpregnant, lactating, and involuting animals were divided into three parts; two parts were fixed in ice-cold 4% paraformaldehyde:PBS and used as described above with the virgin glands, and the third part was flash frozen in liquid nitrogen for RNA analysis.

RNA Isolation and Northern Blot Analysis
RNA from frozen tissue was isolated using TRIZOL (Life Technologies, Inc., Grand Island, NY). The 500-bp ß-casein fragment was generated by PCR primers to exon 7, MßCx7F 5' GATGTGCTCCAGGCTAAAGTT-3' and MßCx7R 5'-AGAAACGGAATGTTGTGGAGT-3'. The 620-bp WAP fragment was isolated from the pMWAPI-14 plasmid digested with PstI (48). The 1000-bp keratin-18 (K18) cDNA was isolated from pUC9B1 with EcoRI restriction digestion (49). All probes were labeled using random priming with {alpha}32P-dCTP. Northern blots were performed using the QuikHyb method from Stratagene (La Jolla, CA).

Immunohistochemical Analysis
MR staining was performed using a mouse monoclonal antibody, MA1-620 from Affinity BioReagents, Inc. (Golden, CO) with a 1:150 dilution and the mouse-on-mouse blocking reagent from Vector Laboratories, Inc. (Burlingame, CA). The secondary antibody was a Texas Red-X goat antimouse IgG, T-6390 from Molecular Probes, Inc. (Eugene, OR) diluted 1:1000. The cytokeratin-14 (K14) staining used a rabbit antimouse polyclonal antibody, PRB-155P from Covance Laboratories, Inc. (Richmond, CA) at a 1:5000 dilution with a blocking buffer of 3% BSA in PBS. Adipophilin staining was performed using a rabbit polyclonal antibody developed by T. W. Keenan (Virginia Polytechnic Institute and State University, Blacksburg, VA) at a dilution of 1:500 in 3%BSA:PBS. A Texas Red-X goat antirabbit IgG, T-6391 was used as the secondary antibody for K14 and ADRP at a concentration of 1:1000. For all immunofluorescence studies, the sodium citrate antigen retrieval method was used as described previously (50), and all primary antibodies were incubated overnight at room temperature. After the secondary antibody incubation of 1 h, the sections were mounted in a 3:1 ratio of Vectashield:Vectashield with DAPI (Vector Laboratories, Inc.), coverslipped, and examined under a Carl Zeiss (Jena, Germany) Axiophot fluorescent microscope.

TUNEL Assay
TUNEL was performed to examine the level of apoptosis in the involuting glands using the terminal deoxynucleotidyl kit from Roche (Mannheim, Germany) and the fluorescent uridine triphosphate (UTP) ChromaTide Alexa Fluor 488-5-deoxy-UTP from Molecular Probes, Inc. Briefly, 5-µm serial sections of mammary tissue were deparaffinized and rehydrated, sodium citrate antigen retrieved, blocked in equilibration buffer, and labeled for 2 h at 37 C with 50 µl of TUNEL reaction mixture containing the fluorescent nucleotide deoxy-UTP and terminal deoxynucleotidyl transferase enzyme. After mounting in a 3:1 ratio of Vectashield:Vectashield with DAPI, images were examined using a Carl Zeiss Axiophot fluorescent microscope. The number of TUNEL-positive mammary epithelial cells (MEC) in a given field was expressed as a percentage of total number of DAPI-stained MEC. At least 5000 nuclei were counted per animal and a minimum of three animals examined for each time point.

Culture of Mammary Explants from Wild-Type and Null Transplants
Explant cultures were performed as described in Enami and Nandi (51) and Matusik and Rosen (52). Briefly, mammary glands from d 14/15 pregnant mice were surgically removed under sterile conditions and diced into 2-mm2 pieces. Each piece of mammary tissue were placed on sterile stainless steel grids in Falcon organ culture tissue culture plates (Becton Dickinson, Franklin Lakes, NJ) in Waymouth’s MD 705/1 Medium (BioWhittaker, Inc., Walkersville, MD) supplemented with penicillin G, 35 µg/ml (Sigma), L-glutamine, 35 µg/ml (Life Technologies, Inc.), and gentamicin, 50 µg/ml (Life Technologies, Inc.). Tissues were cultured in a humidified atmosphere of 5% CO2 and 95% O2 for 48 h in media containing bovine insulin, 5 µg/ml (Sigma). Medium was changed and hormonal supplements were added as indicated in the figure legends for an additional 48 h. The concentration of ovine Prl was 5 µg/ml (NIH, P-2-11) and fludrocortisone was 1 µg/ml (Sigma). Tissue was harvested and total RNA was prepared using TRIZOL. RNA was loaded at three different concentrations and the Northern blots were performed using the ß-casein probe in molar excess. The graph represents the normalized level of ß-casein mRNA/µg of RNA and is representative of five separate experiments.


    ACKNOWLEDGMENTS
 


    FOOTNOTES
 
This work was supported by fellowship F32-CA-94579 (to M.K.) and Grant CA-16303 (to J.M.R.), both from the National Cancer Institute.

Abbreviations: DAPI, 4,6-Diamidino-2-phenylindole; ECM, extracellular matrix; GR, glucocorticoid receptor, GRdim, GR DNA binding mutants; GRE, glucocorticoid response element; GRhypo, GR hypomorphic mutant; H&E, hematoxylin and eosin; K-14, cytokeratin-14; K-18, cytokeratin-18; MEC, mammary epithelial cells; MR, mineralocorticoid receptor; PR, progesterone receptor; Prl, prolactin; PrlR, Prl receptor; STAT5, signal transducer and activator of transcription 5; TEB, terminal end bud; TUNEL, terminal deoxynucleotidyl transferase-mediated deoxy-UTP nick end-labeling; UTP, uridine triphosphate; WAP, whey acidic protein.

Received for publication March 13, 2002. Accepted for publication June 4, 2002.


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