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Endocrinology Vol. 143, No. 8 2953-2960
Copyright © 2002 by The Endocrine Society

ARTICLE

Hepatocyte Growth Factor Is Required for Progestin-Induced Epithelial Cell Proliferation and Alveolar-Like Morphogenesis in Serum-Free Culture of Normal Mammary Epithelial Cells

N. Sunil, Jessica M. Bennett and Sandra Z. Haslam

Department of Physiology, Michigan State University, East Lansing, Michigan 48824

Address all correspondence and requests for reprints to: Sandra Z. Haslam, Ph.D., Department of Physiology, 108 Giltner Hall, Michigan State University, East Lansing, Michigan 48824. E-mail: . shaslam{at}msu.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The steroid hormones, estrogen and progesterone, are required for mammary epithelial cell proliferation and alveolar morphogenesis in vivo. We have developed a minimally supplemented, serum-free medium, collagen gel primary mammary culture system to determine the mechanism of progestin-induced proliferation and alveolar morphogenesis. In epithelial cells cultured alone, treatment with progestin (R5020) alone produced a lumen within the epithelial organoids, but did not stimulate epithelial cell proliferation. The formation of lumens was associated with increased apoptosis, targeted within the organoids. We have previously reported that in our culture system hepatocyte growth factor (HGF) increases epithelial cell proliferation and induces a tubulo-ductal morphological response. In the present report we show that treatment with HGF and progestin (R5020) further increases epithelial proliferation above that with HGF alone and also produces an alveolar-like morphology similar to that observed in vivo in response to progestin treatment. To the best of our knowledge this is the first in vitro demonstration of both progestin-induced proliferation and alveolar-like morphogenesis of normal nonpregnant mouse mammary epithelial cells in vitro. These results suggest that HGF may play a crucial role in progestin-induced proliferation and morphogenesis in vivo.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
PROGESTERONE has important mitogenic activity in adult human, monkey, and mouse mammary glands (1, 2, 3). Based upon extensive in vivo studies in ovariectomized mice, progesterone (P) acts primarily on the sexually mature mammary gland to induce ductal side-branching and alveolar bud formation. During pregnancy, P plays a major role in the extensive epithelial cell proliferation and alveologenesis that precede lactation (2). The specificity of the role of P in mammary gland growth and development has been confirmed in P receptor (PR) knockout mouse (4). In the PR knockout mouse, ductal morphogenesis occurs normally during puberty; however, there is an absence of ductal side-branching and alveolar morphogenesis in the adult mammary gland and during pregnancy. Thus, P acting through its cognate receptor is required for epithelial cell proliferation and alveolar morphogenesis in the adult gland.

Mammary epithelial cell culture models have been useful to elucidate the effects and mechanisms of action of mammogenic hormones on epithelial cell proliferation, morphogenesis, and function. The use of extracellular matrix gel preparations (collagen I and Matrigel) has allowed mammary epithelial cells to be cultured as three-dimensional structures that more closely resemble their architecture in vivo and provide a more physiological context to study growth and lactational function (5). The use of defined serum-free medium in these culture studies has helped limit the confounding effects of growth stimulatory and inhibitory components of serum. However, many previous serum-free cell culture studies were carried out in the presence of growth factors and/or high concentrations of insulin and supplements (i.e. serum albumin, fetuin, pituitary extracts, crude soybean lectin, trypsin inhibitor, {alpha}-tocopherol succinate, and cholesterol), many of which were required for sustained growth (6). How these various factors interact with growth factors and/or steroid hormones has not been delineated.

In the present studies we used a minimally supplemented, serum-free medium, collagen gel primary culture system to address the issue of progestin-induced proliferation and morphogenesis. As described previously (7), the minimally supplemented serum-free medium used herein produced a low basal proliferative state and an absence of morphological response, similar to those observed in ovariectomized mice. This aspect of our culture system is particularly advantageous, because a majority of studies to define the growth-promoting and morphological effects of hormones and growth factors in vivo have been carried out in ovariectomized mice. Furthermore, the short (3-d) period in which the in vitro studies are performed is similar to the period in which the acute proliferative and morphological effects of hormones and growth factors have been observed in vivo in ovariectomized mice (8, 9, 10). Using this culture model, we show for the first time in vitro that mammary epithelial cells treated with hepatocyte growth factor (HGF) and progestin undergo alveolar-like morphogenesis similar to that observed in ovariectomized mice treated with estrogen (E) and progestin in vivo. Although E alone had no proliferative or morphological effect, a greater proliferative response was observed in cells treated with E, HGF, and progestin.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Regents and chemicals
Collagenase III and pronase were obtained from Worthington Biochemical Corp. (Freehold, NJ) and Calbiochem (La Jolla, CA), respectively. DMEM/Ham’s nutrient mixture F-12 (1:1; DMEM/F12), Hanks’ Balanced Salt Solution, epidermal growth factor (EGF), trypsin, anti-HGF neutralizing antibody, and 17ß-estradiol (E2) were purchased from Sigma (St. Louis, MO). Neutralizing antibodies to EGF and IGF-I were obtained from BD Biosciences (Bedford, MA) and R\|[amp ]\|D Systems, Inc. (Minneapolis, MN), respectively. L-Glutamine, penicillin, and streptomycin were purchased from Life Technologies, Inc. (Gaithersburg, MD). Human recombinant HGF was purchased from Calbiochem (San Diego, CA). Human recombinant IGF-I was obtained from GroPep Pty. Ltd. (Adelaid, Australia), and EGF was obtained from Sigma. Rat collagen I (>90% pure) was purchased from BD Biosciences. The synthetic progestin, R5020 (promegestone), was purchased from NEN Life Science Products (Boston, MA). ICI 182,780 was a gift from ICI Pharmaceuticals (Macclesfield, UK).

Cell culture
Mammary epithelial cells and fibroblasts were isolated from adult virgin (10- to 14-wk-old) BALB/c female mice using enzymatic dissociation methods as previously described (11). Purified fibroblasts were obtained in the supernatant fraction by differential centrifugation at 80 x g for 30 sec. Cell viability was about 95% as determined by trypan blue exclusion. Fibroblasts were plated in 5% fetal bovine serum-DMEM/F12 and allowed to attach for 2 h, and any contaminating epithelial cells were removed by gently rinsing with DMEM/F12. The purity of fibroblast cultures was determined to be greater than 95% (11). Fibroblast contamination of epithelial cultures was less than 5%, as determined by immunocytochemical assay with antivimentin antibody (11).

Freshly isolated epithelial cells (1 x 105/well) were suspended and plated in 96-well culture dishes in neutralized collagen I (2 mg/ml, 75 µl/well), and allowed to gel for 30 min at 37 C. Preparation of collagen gel was according to the manufacturer’s instructions. All serum-free cultures were carried out in basal medium: serum- and phenol red-free DMEM/F12 supplemented with 0.1 mM nonessential amino acids, 2 mM L-glutamine, 100 ng/ml insulin, 1 mg/ml fatty acid-free BSA (fraction V), 100 µg/ml penicillin, and 50 µg/ml streptomycin. Cultures were kept in 5% CO2 at 37 C for 3 d. Treatments with conditioned media, growth factors, and hormones were added at the time of plating and included 50 ng/ml HGF, 25 ng/ml EGF, 100 ng/ml IGF-I, 20 nM E2, and 10 nM of the synthetic progestin, promogestone (R5020). R5020 was used instead of P because it is resistant to metabolic inactivation.

Preparation of conditioned medium
Freshly isolated mammary fibroblasts were plated in 100-mm plates in basal medium containing 5% fetal bovine serum as described above and allowed to reach confluence. The cultures were then rinsed twice and cultured in serum-free basal medium, and conditioned medium was collected 48 h later. Conditioned medium was immediately centrifuged at 900 x g for 5 min to remove the cell debris, filtered through a 0.22-µm filter, and kept at 4 C for up to 1 month. Four-fold concentration of conditioned medium was obtained using a 3-kDa cut-off membrane (YM3, Millipore Corp., Marleborough, MA). Conditioned medium was diluted 1:1 with basal medium containing 0.2 µg/ml insulin before assay in epithelial cultures. Medium was changed every other day.

[3H]Thymidine incorporation assay and analysis of cell proliferation
After 3 d of treatment, cells were incubated with 0.1 µCi/well [3H]thymidine (specific activity, 50 Ci/mmol; ICN Pharmaceuticals, Inc., Irvine, CA) at 37 C for 6 h (12). Collagen gels containing epithelial cells were removed, dissolved with 3.5 mM acetic acid, and transferred to GF/C filters (Whatman, Clifton, NJ), followed by washing with Hanks’ balanced salt solution, ice-cold 10% trichloroacetic acid, and 90% ethanol. Radioactivity was determined by liquid scintillation counting. Organoid sizes under various culture conditions were determined by computer-assisted morphometry. Digitized images of organoids were captured using an inverted microscope at a magnification of x40, and the area per organoid was determined using NIH Image software as previously described (13).

Terminal deoxynucleotidyltransferase-mediated deoxy-UTP nick end labeling (TUNEL) assay for apoptosis
To determine the apoptotic index of cultured cells, collagen gels containing cultured cells were fixed on d 2–3 of culture, embedded in paraffin, sectioned, mounted on coverslips, and assayed for apoptotic cells using the TUNEL assay method (Intergen, Purchase, NY) according to the manufacturer’s instructions. The percentage of apoptotic cells was determined with the aid of a light microscope from cell counts of at least 500 cells in each treatment group.

Statistical analysis
Data were expressed as the mean ± SEM, and statistical significance was determined using t test or ANOVA.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effects of HGF, E, and R5020 on epithelial cell proliferation
The ability of E plus progestin to stimulate extensive epithelial cell proliferation and alveolar morphogenesis in the adult mammary gland in vivo has been well established, but has not been demonstrated in vitro in normal cells. We recently reported that in serum-free monolayer cultures only epithelial cells cultured on the extracellular matrix proteins, collagen IV and fibronectin, exhibit a proliferative response to the synthetic progestin, R5020 (12). We had hypothesized that the absence of a response to E and R5020 was due to the lack of an additional soluble factor(s) that might be produced by mammary fibroblasts. In this regard the addition of the growth factors EGF and IGF-I to the monolayer cultures was not effective in promoting a proliferative response to either E or E plus R5020 (14).

We had previously observed that E stimulates HGF production in mammary stromal fibroblasts in vitro and via HGF indirectly mediates epithelial cell proliferation (7). Therefore, we asked whether HGF might play a role in mediating progestin-dependent epithelial cell proliferation. To test this possibility the effects of R5020 were analyzed in the presence or absence of HGF (Fig. 1Go). When either E2 or R5020 was added to epithelial cells alone, no increase in cell proliferation was observed. When R5020 was added with HGF, proliferation was increased 1.5-fold above that obtained with HGF alone (P < 0.05). The addition of E2 plus HGF did not increase cell proliferation above that with HGF alone; however, when E2 was added with HGF plus R5020, proliferation was increased over that with HGF plus R5020 (P < 0.05).



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  		Figure 1. Effect of R5020 plus HGF on epithelial cell proliferation. Mammary epithelial cells were suspended in collagen I gels and cultured alone in BM, E2 (10 nM), R5020 (20 nM), or E2 (10 nM) plus R5020 (20 nM) or in the same combinations in the presence of HGF (50 ng/ml) of FCM. [3H] Thymidine incorporation into DNA was assayed after 3 d of culture. Each bar represents the mean ± SEM of triplicate values from a representative experiment. The data are expressed for BM groups as [3H]thymidine incorporated per well and for HGF- and FCM-treated groups as the fold increase over the BM control. *, P = 0.05, proliferation is greater in the HGF plus R5020 group than in the HGF or HGF plus E2 group. **, P = 0.01, fold increases in proliferation in HGF, E2, plus R5020 and FCM, E2, plus R5020 groups are greater than in all other groups within the same experiment.

 
Fibroblast-conditioned medium (FCM) has also been shown to stimulate epithelial cell proliferation via HGF. Similar to exogenous HGF, increased proliferation was obtained with FCM plus R5020 and FCM, E2, and R5020 (Fig. 1Go). When R5020 was added alone or in combination with E2 and FCM, proliferation was increased 1.8- and 2.2-fold, respectively. These results indicate that the enhanced proliferative response to E plus progestin in vitro may require the presence of HGF.

HGF plus R5020 induce alveolar-like morphology in vitro
In addition to the increased proliferation obtained in the presence of HGF plus R5020 and HGF, E2, and R5020, a novel morphological response was also observed. The long tubular projections observed with HGF were attenuated to short, rounded projections, similar to alveolar bud formation, which is observed in vivo in the adult mammary gland in response to E and P treatment (Fig. 2Go). Histological analysis of the cultured structures revealed cellular organization with multiple lumens similar to lobuloalveolar organization observed in vivo (Fig. 3Go). These structures were different from the tubules produced by HGF treatment alone (Fig. 2Go). The differences in proliferative and morphological responses to HGF alone vs. R5020 alone vs. HGF plus R5020 could also be quantified by morphometric analysis. As shown in Table 1Go, the organoid size in cultures treated with HGF or HGF plus R5020 was significantly increased 2-fold over that in basal medium (BM)- or R5020-treated cultures and was a reflection of the proliferative response observed in HGF- and HGF- plus R5020-treated cultures. Also, the tubule length of HGF- plus R5020-treated cultures was significantly reduced by 48%.



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  		Figure 2. Phase contrast photomicrographs of epithelial cell organoid morphology in collagen gel cell culture. Mammary epithelial cells were suspended in collagen I gels and cultured for 3 d in BM, E2, PRL (1 µg/ml), R5020, HGF, or R5020 plus HGF. Gross organoid morphology was visualized in situ in collagen gels with the aid of an inverted microscope (magnification, x100) and in histological sections of collagen gels (E and F; magnification, x400). Note the solid appearance of organoids from BM, E2, and PRL, whereas lumens (L), tubules (T), and alveolar buds (AB) are visible in R5020, HGF, and R5020 plus HGF cultures, respectively. Sections through a tubule and alveolar structure are also shown.

 


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  		Figure 3. Effect of culture treatments on apoptotic index. ii, Epithelial cultures were treated with BM or R5020 (20 nM); after 2–3 d, the cultures were fixed, sectioned, and stained for apoptotic cells by the TUNEL assay method. The percentage of apoptotic cells was quantified by microscopic analysis based on counts of at least 500 cells/treatment. *, P = 0.05, percent apoptotic cells in the R5020-treated group was greater than that in the BM group. ii, Photomicrographs of R5020-treated organoids. Note that in R5020-treated cells (B and D), the dark staining apoptotic cells are centrally located where the lumens are forming. In BM-treated cells, the dark-staining apoptotic cells are located at the periphery of the organoids (A and B). Magnification, x 400.

 

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  		Table 1. Morphometric analysis

 
R5020-induced lumen formation is associated with increased apoptosis
A distinct morphological response, lumen formation, was observed in cultures treated with R5020 alone (Fig 2Go). As organogenesis during embryonic development results from the net effect of the precise spatial patterning of proliferation and apoptosis, it was possible that spatially localized apoptosis was causing lumen formation. To address this question, TUNEL assays were carried out on histological sections of organoids within the collagen gels under different treatment conditions. Figure 3Go shows that the apoptotic index of R5020-treated cultures was 3-fold higher than that of control cultures. Thus, R5020 had a pronounced proapoptotic effect on the cultured cells. Histological analysis (Fig. 3Go, B and D) revealed that R5020-induced apoptosis was spatially localized within the organoids and centrally within luminal structures. In contrast, in BM-treated cultures, apoptotic cells were localized at the periphery of the organoids (Fig. 3Go, A and C). The spatial localization of apoptotic cells within the organoids in R5020-treated cultures indicates that this response to R5020 is a key event in lumen formation.

Specificity of the progestin-induced response
To establish the steroid specificity of the progestin response obtained with R5020, the effect of the progestin antagonist, RU486, on proliferation and morphogenesis was examined. As shown in Fig. 4Go, the combination of HGF, R5020, and RU486 counteracted the enhanced proliferation response observed with HGF and R5020. RU486 alone or in combination with R5020 or HGF had no stimulatory or inhibitory effect on proliferation. The effects of the antiprogestin on HGF-, R5020-, and HGF- plus R5020-induced changes in morphology were also investigated. Figure 4AGo shows that treatment with RU486 alone did not cause lumen formation in the cultured organoids. In addition, RU486 inhibited R5020-induced lumen formation (Fig. 4BGo). The antiprogestin also failed to reduce tubule length in HGF-treated cultures and blocked R5020-induced alveolar-like morphogenesis in cultures treated with HGF, R5020, and RU486 (Fig. 4Go).



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  		Figure 4. Effects of the antiprogestin, RU486, on HGF- and R5020-induced proliferation and morphogenesis. Mammary cultures were treated with BM or the indicated combinations of HGF (50 ng/ml), R5020 (20 nM), and RU486 (20 nM). Cultures were assayed for [3H]thymdine incorporation (A) and morphological responses (B) on d 3. Each bar represents the mean ± SEM of triplicate values from a representative experiment. The data are expressed as [3H]thymidine incorporated per culture well. *, P < 0.05, proliferation in the HGF-treated group was significantly greater than that in the BM-, R5020-, RU486-, or R5020- plus RU486-treated group. **, P < 0.05, proliferation in the HGF- plus R5020-treated group was greater than with all other treatments.

 
Lack of PRL effect
It had previously been reported that progestin plus PRL produce a synergistic proliferative response of adult mammary epithelial cells in serum-free collagen gel primary culture (15). Thus, it was of interest to determine the effect of PRL in our culture system. Treatment with PRL alone produced neither an increase in proliferation (Fig. 5Go) nor any morphological response, and the appearance of the organoids was the same as that in BM-treated cultures (Fig. 2Go). Treatment with PRL plus R5020 also did not increase proliferation. However, lumen formation in these cultures was similar to that in cultures with R5020 alone (data not shown). Thus, in contrast to previous reports, in our culture system PRL alone or in combination with progestin had no effect on proliferation or morphogenesis.



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  		Figure 5. Effect of PRL and R5020 on proliferation. Mammary cultures were treated with BM, PRL (1 µg/ml), R5020 (20 nM), PRL (1 µg/ml) plus R5020 (20 nM), or HGF (50 ng/ml). Cultures were assayed for [3H]thymdine incorporation on d 3. Each bar represents the mean ± SEM of triplicate determinations from a representative experiment. Only HGF significantly increased proliferation.

 
Interactions among E2, R5020, and EGF and IGF-I
EGF and IGF-I have also been shown to play important roles in normal mammary gland development (16). Addition of either EGF or IGF-I with E2, R5020, or E2 plus R5020 did not have a stimulatory effect on proliferation above that obtained with either growth factor alone (Fig. 6Go). Furthermore, neither growth factor in combination with R5020 or E2 plus R5020 produced an alveolar-like morphology (data not shown). The combination of IGF-I and R5020 caused a significant (40%) decrease in proliferation compared with IGF-I alone (Fig. 6Go).



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  		Figure 6. Effect of R5020 on epithelial cell proliferation in collagen gels of cells treated with EGF or IGF-I. Mammary epithelial cells in collagen gels were cultured alone in the presence HGF (50 ng/ml), EGF (50 ng/ml), or IGF-I (100 ng/ml). For each of these conditions cultured cells also received treatment with E2 (20 nM), R5020 (10 nM), or E2 (20 nM) plus R5020 (10 nM). [3H]Thymidine incorporation was assayed on d 3. The data are expressed as the fold increase over [3H]thymidine incorporation in cells cultured only in BM. Each bar represents the mean ± SEM of triplicate determinations from three to five separate experiments. For IGF-I treatment groups: *, P = 0.05, groups receiving R5020 had significantly less proliferation than all other groups.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this report we have shown that treatment of mammary epithelial organoids in serum-free, collagen primary culture elicits a proliferative response to HGF plus progestin and produces mammary structures similar to alveoli that are observed in vivo in response to E plus P treatment. The earliest events in alveologenesis in vivo result in the morphological changes of ductal side-branching and alveolar bud formation. In our culture model system the assay period is 3 d. We speculate that the morphological changes that we observed in vitro may correspond to the very earliest occurrence of ductal side-branching and alveologenesis such as is induced in vivo in ovariectomized mice after 3 d of treatment with exogenous E plus P. At present no molecular markers have been identified that are specific to cells undergoing ductal side-branching and early alveologenesis (17). It has been suggested that decreased expression of Na-K-Cl cotransporter (NKCC1) in alveolar cells compared with epithelial cells in ducts may be a suitable marker (18). However, this is based upon the published observation of decreased expression of the cotransporter in alveoli on d 12 of pregnancy. However, the same study showed that signal transducer and activator of transcription-5 and PRL receptor-null mice both developed alveolar structures without a down-regulation of NKCC1. Using the same antibody to NKCC1 we found that there was only sporadic down-regulation of NKCC1 in alveoli of early pregnant BALB/c mice, the strain used in our present studies. In addition, we used this antibody to assess NKCC1 down-regulation in early alveologeneis in adult virgin mice, ovariectomized and injected for 3 d with E plus P Analysis of NKCC1 expression in these mammary glands revealed the same expression in ducts, side-branches, and alveolar buds (Haslam, S. Z., unpublished observations). Thus, NKCC1 does not appear to be a marker of early alveologenesis in BALB/c strain mice. Other markers of cell differentiation, such as casein expression, are not suitable, because ductal cells also express this protein under appropriate hormonal conditions. Thus, in the absence of specific molecular markers for early events in alveologenesis that can distinguish between ductal and alveolar cells in vivo, we have termed the morphological changes induced in our culture system alveolar-like morphogenesis.

Additional support for a role for HGF in alveologenesis in vivo comes from studies of the temporal pattern of HGF expression in the mouse mammary gland in vivo (19, 20). In the mouse mammary gland HGF is expressed only in mammary fibroblasts, and the HGF receptor, c-Met, is expressed only in epithelial cells (19, 20). HGF expression starts to rise toward the end of puberty (6 wk), when end-bud proliferation is nearing completion. HGF is maximally expressed at sexual maturity (12 wk) when the mammary gland is poised for P-induced ductal side-branching and alveolar development. HGF is also present during pregnancy when maximal alveolar development occurs and declines to prepubertal levels during lactation.

Ductal side-branching and alveolar development in vivo are critically dependent upon the action of P. This has been convincingly demonstrated by the observation that PR-null mice produce normal mammary ducts, but fail to develop side-branches or undergo alveologenesis (4). Alveologenesis in vivo is also critically dependent upon the action of E (2). One of the roles proposed for E in alveologenesis is the up-regulation of PR. We have recently reported that E treatment of mammary fibroblast cultures results in increased production of HGF (7). Our current observation that alveolar-like morphogenesis can be induced in culture by HGF plus R5020 in the absence of E suggests that another potential role of E in vivo is the up-regulation of HGF by mammary stromal fibroblasts, which then can act in concert with P to induce alveologenesis.

In the mouse, E has been implicated to be an indirect acting mitogen in mammary epithelium. Support for this hypothesis comes from in vivo tissue recombination studies with epithelium from E receptor (ER)-null mice combined with stroma from ER-positive wild-type mice. In this case E induces proliferation and ductal development. The converse tissue recombination, ER-positive epithelium plus ER-negative stroma does not exhibit E-induced proliferation in the epithelium (21). Thus, it appears that E action in the stroma is required for mammary ductal development. Our studies on in vitro alveolar-like morphogenesis with R5020 and HGF suggest that E may also play an indirect role in alveologenesis via up-regulation of stromal HGF.

In our culture system, treatment with HGF alone induced proliferation and a tubular morphology. Treatment with R5020 alone produced luminal structures, but no proliferation. The development of R5020-induced luminal structures coincided with increased apoptosis that was spatially targeted to the interior of the epithelial organoids. In vivo, alveologenesis and lobuloalveolar development result in multiluminal structures. The transition from a single lumen present in ducts to multiple lumens present in lobules of alveoli is poorly understood. Our results lead us to speculate that one of the roles of progestin in alveologenesis may be the formation of multiple lumens and that this process may occur through progestin-induced apoptosis. Thus, the morphological changes associated with alveolar morphogenesis may be the net result of cell proliferation and concomitant spatially targeted apoptosis similar to the proliferative and apoptotic processes that direct organogenesis during embryonic development. The study of apoptosis in vivo is complicated by the highly dynamic nature of the process and the rapid removal of apoptotic cells by macrophages. In this regard our cell culture model, which lacks macrophages to remove apoptotic cells, allows us a unique opportunity to observe the temporal and spatial patterns of apoptosis in epithelial organoids that can form epithelial structures that reflect tubular and alveolar morphogenesis in vivo.

PRL is an important hormone required for lactational function of the mammary gland. Thus, it was of interest to investigate the role of PRL in alveologenesis in our culture system. PRL alone or in combination with R5020 had neither a stimulatory effect on proliferation nor any morphological effect. Previously it had been reported that P and PRL alone and in combination increased proliferation of mouse mammary epithelial cells in primary collagen gel cultures under serum-free conditions (15). However, these serum-free cell culture studies were carried out in the presence of growth factors (EGF) and/or high concentrations of insulin (10 µg/ml) (15). It has since been shown that at these concentrations insulin has a potent proliferative effect, which is most likely mediated through the IGF-I receptor (22). Furthermore, IGF-I can interact with EGF and other mammogenic hormones in serum-free medium to stimulate proliferation (14). In addition to high insulin concentrations, numerous supplements have been added to serum-free cultures (i.e. serum albumin, fetuin, pituitary extracts, crude soybean lectin, trypsin inhibitor, {alpha}-tocopherol succinate, and cholesterol), many of which were required for sustained growth (6). How these various factors interact with growth factors and/or steroid hormones has not been delineated. In those studies control medium also supported some growth and limited tubulogenesis. In the presence of P and/or PRL (and insulin), although epithelial colonies were larger, the morphology of the colonies was the same in all media and was characterized by tubulogenesis and cell spreading. No morphological correlate of alveologenesis was observed. Thus, previous cell culture models have not achieved the responses of both proliferation and morphological changes that are induced by P in vivo. Why this is the case is not known. However, this may be attributable to the confounding variables of a high insulin concentration, the presence of numerous supplements in the serum-free medium, and the lack of HGF.

With regard to other steroid hormone/growth factor interactions, we found that progestin treatment inhibited the proliferative response to IGF-I. We have made a similar observation in serum-free monolayer culture of adult mammary epithelial cells on collagen I or fibronectin (14). It has been previously reported that both IGF-I and IGF-R mRNAs are expressed in the mouse mammary gland at high levels in the end buds of the pubertal gland, are undetectable in the adult gland, and are reexpressed in late pregnancy and during lactation (23). Previous studies from our laboratory, based upon receptor ligand-binding activity, have demonstrated that PR levels are low at puberty, are highest in the adult gland and during early pregnancy, and are reduced again at late pregnancy and during lactation (2). Furthermore, we found that ovariectomized pubertal and lactating mammary glands are nonresponsive to the proliferative effect of progestins (8). A major proliferative response to progestins is ductal side-branching and alveolar morphogenesis. Thus, the IGF-I and progestin appear to be important during different stages of proliferation: IGF-I during ductal elongation and lactation, and progestins during alveolar development. In this context, the present finding that R5020 reduces the proliferative response to IGF-I lends further credence to the hypothesis that progestins may inhibit IGF-I effects in vivo (14).

In summary, we have demonstrated in vitro under minimally supplemented, serum-free conditions that in the presence of HGF, progestin causes morphological changes reminiscent of alveolar morphogenesis in vivo. Our results demonstrated that an enhanced proliferative response to E plus progestin in adult mammary epithelial cells in vitro, similar to that obtained in vivo, was obtained only in the presence of HGF; neither EGF nor IGF-I could replace HGF to obtain this response. These results indicate that the growth factors produced by the mammary gland are not likely to be merely interchangeable or redundant. Our results support the concept that the temporal and cell type-specific expression of individual growth factors in coordination with steroid hormone receptor expression and function are required for the developmental stage-specific processes of ductal morphogenesis vs. alveolar morphogenesis. Advancing our understanding of the complexities of growth regulation by E and progestin in relation to epithelial-stromal cell interactions in the normal mammary gland may provide important information about the nature of changes in hormone-regulated growth that occur in mammary cancer. Furthermore, identification of the specific responses of epithelial vs. stromal cells to steroid hormones may lead to new therapeutic strategies in breast cancer treatment.


   Acknowledgments
 


   Footnotes
 
This work was supported by NIH Grant R01-CA-40104 (to S.Z.H.).

Abbreviations: BM, Basal medium; E, estrogen; E2, 17ß-estradiol; EGF, epidermal growth factor; ER, estrogen receptor; FCM, fibroblast-conditioned medium; HGF, hepatocyte growth factor; NKCC1, Na-K-Cl cotransporter; P, progesterone; PR, progesterone receptor; TUNEL, terminal deoxynucleotidyltransferase-mediated deoxy-UTP nick end labeling.

Received February 26, 2002.

Accepted for publication April 29, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Cline JM, Solderkvist G, Von Schoultz E, Skoog L, Von Schoultz B 1996 Effects of hormone replacement therapy on the mammary glands of surgically postmentopausal cynmolgus macaques. Am J Obstet Gynecol 174:93–100[CrossRef][Medline]
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