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Cell to Cell Interactions and Normal Mammary Gland Function
Cell to Cell Interactions and Normal Mammary Gland Function S A N D R A Z. HASLAM Department of Anatomy Michigan State University East Lansing 48824 ABSTRACT
The nonepithelial components of the mammary gland are reviewed and their potential for regulatory cell-cell interactions with the epithelial cells are discussed. Studies undertaken to examine the regulatory potential of mammary stromal fibroblasts using an in vitro cell culture system are presented. The influence was examined of epithelial-fibroblast interactions on estrogenic regulation of progesterone receptor concentration in epithelial cells and on epithelial and fibroblast DNA synthesis. Mammary fibroblasts affect estrogen responsiveness in epithelial cells by two different mechanisms. In the case of progesterone receptor regulation, fibroblasts promote estrogen-dependent increases in the receptor via a substratum effect possibly by the production of collagen type I. By contrast, the fibroblast effect promoting estrogen-dependent cell proliferation requires fibroblasts to be metabolically active and in close contact with the epithelium. Additionally, under coculture conditions, estrogen-dependent stimulation of fibroblast DNA synthesis is also observed, indicating a bidirectional, interactive phenomenon between the two types of cells. It is possible that the modulations in epithelial responsiveness to estrogen that are associated with the presence of mammary fibroblasts in vitro reflect regulatory mechanisms that operate in vivo. INTRODUCTION
The mammary gland is composed of epithelial, adipose, and fibrous connective tissues; the
Received July 6, 1987. Accepted November 16, 1987. 1988 J Dairy Sci 71:284-3-2854
relative proportion of each tissue type varies with the species and developmental state of the gland. Under the influence of a wide range of hormones, the gland undergoes dramatic changes in morphology and function (20). The most readily detectable changes occur in the epithelial compartment of the gland, and the proliferation, morphogenesis, and functional differentiation of the epithelium have been studied almost to the exclusion of all other cell types present. In recent years, there has been a growing interest in more detailed analysis of the cellular composition of the gland, and this has led some to consider that the nature of various cell-cell interactions might provide the basis for important regulatory mechanism(s) in normal mammary gland function. This paper provides a brief review of the various nonepithelial cells types present and what is known about their potential for regulatory interactions with mammary epithelium. The main emphasis of this paper will deal with studies (lO, 1 1 ) o f marnmary fibroblast interactions with mammary epithelial cells in culture and their influence on hormona/ regulation. Because rodent mammary gland has been studied extensively as a model for delineating hormone action in normal mammary tissue, studies in mice and rats provide the focus for this paper. In the nonpregnant animal, the epithelial component of the gland is organized into a branching ductat system. The epithelial cells lining the duct are invested with a basal layer of myoepithelial cells and a continuous basement membrane encloses the entire ductal system (Figure 1). In the context of cell-cell interaction, the first nonepithelial cell type to be considered is the myoepithelial cell. The best known function of myoepithelial cells is their facilitation of milk removal during lactation as a result of contractile activity in response to oxytocin (21). However, these cells are directly interposed between the epithelial cells and the basement membrane, i.e., the extracellular
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HASLAM
Figure 1. Representation of the various cells present in normal mammary gland. The epithelial compartment of the gland contains epithelial cells (a) surrounded by myoepithelial cells (b) and separated from the stromal compartment by a basement membrane (c). The stromal compartment consists of the fibroblasts (d) and adipocytes (e); also present are mast cells (f).
matrix. A recent study (6) has shown that the physical relationship of the myoepithelial cell to the epithelium changes dramatically with different developmental states of the gland. In glands of nonpregnant animals, myoepithelial cells form a complete collar around the ductaI epithelium forming linear tracts oriented parallel to the long axis of the ducts, thus effectively separating the epithelium from the basement membrane. By contrast, during pregnancy and lactation, with the development of alveoli, myoepithelial cells assume a stellate appearance, forming loosely woven baskets around individual alveoli, thus leaving large areas of the epithelial cell surface in potential direct contact with the basement membrane. In view of the proposed role of the extracellular matrix in regulation of functional differentiation (3, 15), it is tempting to speculate that myoepithelial cells could have a regulatory role by virtue of their ability to modulate epithelial cell exposure to the extracellular matrix. Journal of Dairy Science Vol. 71, No. 10, 1988
Beyond the basement membrane, mammary stromal fibroblasts form a collagenous sheath around the mammary ducts and separate them from the subadjacent adipose tissue (Figure 1). Thus, fibroblasts are the stromal cells in closest proximity to the epithelium. These cells are also responsible for contributing stromal components such as collagen t y p e I to the extracellular space and firbonectin to the basement membrane (27). One can speculate that mammary fibroblasts may play a regulatory role in epithelial function based upon their contribution to the extracellular matrix composition. The third cell type to be considered is the adipocyte. In the nonpregnant state, the adipose tissue constitutes the major portion of the gland. During pregnancy and lactation, the epithelial component of the gland increases concomitant with a depletion of the adipose tissue. After involution, there is degeneration and loss of the secretory epithlium and the adipose tissue is restored. Thus, cyclic changes in mammary adipose tissue composition occur in relation to mammary gland function. Accumulation of glycogen, the activity of glycogen synthetase, and lipogenic rate in mammary adipocytes are modulated during pregnancy and lactation to channel nutrients to the mammary epithelial cell (2). These changes appear to require the presence of mammary epithelium (2), indicating possible interactive phenomena between the epithelial cells and adipocytes. Recent studies have also shown that linoleate metabolites enhance the in vitro proliferative response of mouse mammary epithelial cells (1). Thus, metabolic cooperation may be a key to adipocyte epithelial cell interactions. One other cell type that may interact with mammary epithelial cells and play a regulatory role in mammary gland function is the mast cell (Figure 1). Mast cells are ubiquitous in connective tissue; they originate in bone marrow and are similar but not identical to basophils (8). They possess numerous granules that contain a variety of bioactive molecules as well as IgE; these cells are generally associated with the mediation of hypersensitivity reactions. Recent studies on mast cells derived from dimethylbenz [a] anthracene-induced rat mammary tumors indicate that these cells may play a role in hormonal regulation (9). Tumor-derived mast cells can concentrate prolactin in their secretory granules and under appropriate con-
SYMPOSIUM: ROLE OF THE EXTRACELLULAR MATRIX IN MAMMARY DEVELOPMENT 2845 ditions prolactin can also be released from the cells. These observations may provide another perspective on mechanisms that could control local concentrations of hormones within mammary tissue. Studies on embryonic development of the mammary gland have demonstrated that mammary mesenchyme, the stromal precursor, directs and specifies epithelial morphogenesis (22). The adult mammary gland is distinct from many other epithelial organs in that its major morphogenesis occurs postnatally, during pregnancy, and is hormonally regulated (20). Several lines of evidence indicate that the morphogenic processes that occur during pregnancy are similar to those operative during fetal development of the organ and may be dependent upon epithelial-stromal interactions (23, 24). At present little is known about epithelialstromal interactions and hormonally regulated growth and function in the adult mammary gland. This question is difficult to approach in vivo in the whole animal, and thus, this problem was approached in vitro using a cell culture system. In vivo, estrogen is an important hormone for regulation of growth and function of mammary gland; it promotes mammary epithelial ceil proliferation and regulates mammary progesterone receptor ( P g R ) c o n centration (12, 13, 20). Hormone receptors for estrogen (ER) are localized in the mammary gland and are present in both the epithelial and stromal compartments of the gland (14). Of further interest, ER concentration varies in stromal and epithelial tissues depending on the
developmental state of the gland (Table 1). For these reasons, attention was focused on estrogen action in relation to mammary epithelialstromal cell interactions. Initial stu dies (10, 11 ) addressed specifically the interaction of the mammary fibroblast with the epithelium and examined the response of these cells to estrogenic regulation of cell proliferation and PgR concentration. MATERIALS AND METHODS Cell Culture
Balb/C mice 2 to 5 mo of age and 14 to 18 d pregnant were the source of mammary tissue. The tissue was dissociated according to previously described methods to obtain mixed cultures containing epithelial cells and fibroblasts (11). The two cell types were separated (10) to produce either epithelial cultures or fibroblast cultures. The epithelial cell and fibroblast cultures were determined to be 97 and 100% pure, respectively, using immunocytochemical methods (11); antibody directed against vimentin was used to identify fibroblasts (17). All cell types were cultured in Medium 199 containing 5% charcoal-stripped fetal calf serum (CS-FCS), .006 /ag/ml insulin, 1/ag/ml prolactin, and 10 4 M cortisol. CuItures were kept at 37°C in a humidified atmosphere of 5% CO2 in air; medium was changed every 2 d. To determine the effect estrogen on PgR concentrations or DNA synthesis cells were cultured with or without 20 nM 173-estradiol. Fibroblasts cultures that were used for coculture experiments were plated 1 wk prior to
TABLE 1. Distribution of estrogen receptors between stromal and epithelial components of adult mouse mammary gland at different states of development.
Developmental state 1
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1In all cases mice were ovariectomized, or ovariectomized plus hysterectomized (pregnant mice) 5 to 7 d prior to assay to remove endogenous estrogens. Thus measured = total cytoplasmic estrogen receptor. Estrogen receptor concentration was determined by Scatchard analysis. For methods see (14). E2 = Estradiol. Journal of Dairy Science Vol. 71, No. 10, 1988
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HASLAM
the addition of the epithelial cells. The fibroblasts received treatments to render them either nonproliferative by irradiation or nonviable by gluteraldehyde fixation as previously described (10). To obtain conditioned medium, fibroblast cultures were incubated for 24 h in Medium 199 without FCS or hormones and processed and stored as described (10). Steroid Hormone Receptor Assay
Intact cells grown for indicated lengths of time were assayed while still attached to culture dishes. To measure ER or PgR the cells were incubated with either [3H] estradiol (1 to 10 nM) or [3H]R5020 (1 to 12 nM) with or without 100-fold excess radioinert estradiol or R5020 and processed as previously described (10). All binding data were analyzed by the method of Scatchard (25). The DNA content was determined according to the method of Ceriotti (4).
cultures were treated with estrogen (20 nM) there was almost a threefold increase in PgR concentration after 3 to 5 d of treatment. This result is very comparable to the two- to threefold stimulation of PgR observed after in vivo administration of estradiol to ovariectomized virgin (12) or pregnant mice (13). The observed effect of estrogen on PgR concentration appears to specific for estrogenic compounds (Figure 4) since estradiol and diethylstilbestrol were effective in increasing PgR concentration and the antiestrogen, tamoxifen (10--6M), was able to block the estrogenic effect; further evidence of specificity is indicated by the lack of effect obtained with 20 nM progesterone or dexamethasone. Having established that mixed cultures exhibit a characteristic PgR response to estrogen,
Measurement of Deoxyribonucleic Acid Synthesis
Cells were plated, in parallel, on glass coverslips, and autoradiography was as described 19) using [3H]thymidine (6.7 Ci/mmol). Labeling was for 1 h. Labeled nuclei were counted in 20 random high power (400x) microscope fields with a minimum of 1000 of each cell type counted (10).
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Initial experiments were carried out with mixed cultures containing both epithelial cells and fibroblasts in order to determine if this model system would exhibit an in vitro response to estrogen. The effects of estrogen treatment on PgR concentration are shown in Figures 2 and 3. Soon after plating the cells (Figure 2), when cultures were still subconfluent, estrogen was only moderatively effective in increasing PgR concentration. However, when confluent Journal of Dairy Science Vol. 71, No. 10, 1988
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Figure 2. Time course of estrogen stimulation of progesterone receptor concentration in mixed cells. Cells were plated and kept in medium with (D) or w i t h o u t (=) 20 nM estradiol and assayed for progesterone receptors (PgR) 1, 3, or 5 d later as described in (13). *P = .005 that in estradiol-treated group PgR value is significantly greater than in untreated controis.
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Figure 3. Time course of estrogen stimulation of progesterone receptor concentration in confluent cultures of mixed cells. Cells were grown to confluence in the absence of estradiol, changed to medium with (n) or without (i) 20 nM estradiol, and assayed for progesterone receptors (PgR) 1, 3, or 5 d later (11). *P = .001 in estrogen-treated groups; PgR are higher than in untreated controls.
it was of interest to d e t e r m i n e if purely epithelial cultures responded similarly. No stimulation of PgR was observed in the epithelial cultures (Figure 5). One possible explanation for the lack of response in epithelial cultures was that the increase in PgR c o n c e n t r a t i o n was occurring in the fibroblast p o p u l a t i o n o f m i x e d cultures. To investigate this possibility, fibroblast cultures were e x a m i n e d for the presence o f PgR and for the ability o f estrogen to increase PgR concentration. In all cases, Scatchard analysis o f [ 3 H ] R 5 0 2 0 binding revealed only low affinity binding that did not satisfy the criteria for receptor binding (Figure 6). F u r t h e r m o r e , t r e a t m e n t with estrogen had no effect on the quality or q u a n t i t y of the binding. Estrogenic regulation of PgR c o n c e n t r a t i o n in m a m m a r y tissue is t h o u g h t to be regulated
Figure 4. Steroid hormone specificity of progesterone receptor stimulation. Confluent mixed cells cultured without estradiol were changed to medium without estradiol (C), or 20 nM estradiol (Ea), or diethylstibestrol (DES), or dexamethasone (DEX), or progesterone (Pg), or 10 -6 M tamoxifen (T), or estradiol plus tamoxifen (E 2 +T).
via an E R - m e d i a t e d m e c h a n i s m (16). Thus, the lack of estrogen responsiveness observed in the epithelial cultures was possibly due to a loss of ER. To test this possibility, epithelial, fibroblast, and m i x e d cultures were assayed for E R at various times after plating. C o n c e n t r a t i o n s of E R in the three t y p e s of cultures were similar, and in all cases, E R c o n c e n t r a t i o n s were maintained equally well over t i m e (Table 2). F r o m these studies we conclude that one specific m a m m a r y epithelial cell response to estrogen in vitro, n a m e l y PgR regulation, is associated with the presence of m a m m a r y stromal fibroblasts.
Analysis of the Fibroblasts Influence on Epithelial Cell Responsiveness to Estrogen
The favorable effect o f fibroblasts on mamm a r y epithelial cells could be explained in several different ways. One possibility is that fibroblasts provide some undefined soluble factor that is required for epithelial response to estrogen. In this c o n t e x t , conditioned media f r o m cultured mouse fibroblasts have been r e p o r t e d to stimulate normal m o u s e m a m m a r y Journal of Dairy Science Vol. 71, No. 10, 1988
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Figure 5. Comparative effect of estrogen treatment on progesterone receptor concentration in mixed versus epithelial cultures. Confluent epithelial (~) or mixed (~) cultures were changed to medium with (E~) or without (C) 20 nM estradiol and assayed for progesterone receptors (PgR) 3 d later (11). *P<.01 that PgR concentration in estrogen-treated group is greater than in controls.
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epithelial cell proliferation in vitro (7). However, the physical nature and chemical composition of the culture substratum are also known to be important for mammary epithelial cell function in vitro (5). Thus, fibroblasts might function to improve the culture substratum. Alternatively, direct contact between epithelial cells and fibroblasts may be required to facilitate estrogen responsiveness (19). The purpose of the following series of experiments was to distinguish among these described possibilities and to elucidate the mechanism(s) by which fibroblasts might influence epithelial cell responsiveness. Observations include fibroblast effects on estrogen dependent epithelial cell DNA synthesis. Progesterone Receptor Regulation
To investigate the nature of fibroblast influence on the PgR response a number of fibroblast conditions were created and they were cocultured with epithelial cells 7 d later. The results are shown in Figure 7. Fibroblasts were either control untreated, lethally irradiated, gluteraldehyde-killed (GK) or epithelial cells were cultured in the presence of conditioned medium (CM) from fibroblasts. All of the conditions tested were about equally effective in promoting estrogen-dependent increases in PgR concentration. F r o m these results one could conclude that fibroblasts need not be alive or even physically present since conditioned medium was also effective. Fibroblasts m a y be influencing the epithelial cells via a substratum effect. Pretreating culture dishes with collagen t y p e I, an extracellular product of fibroblasts, was also effective in promoting the PgR response providing further evidence for a substratum effect (Figure 7). Regulation of Deoxyribonucleic Acid Synthesis
To determine the nature of fibroblast influence on epithelial cell proliferation, DNA synthesis was measured in parallel cocultures, autoradiographically. The results of these studies are shown in Figure 8. When epithelial cells were grown alone, estrogen had no stimulatory effect. In contrast, coculture with live fibroblasts in the presence of estrogen resulted in a significant increase in the labeling index (LI, percent labeled nuclei). Coculture with ir-
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radiated fibroblasts also resulted in a significant increase in LI on d 1; however, after 5 d of culture a stimulatory effect was not observed. Coculture with GK fibroblasts was not effective. Conditioned medium also failed to promote epithelial cell DNA synthesis. It was possible that the presence of estrogen in fibroblast cultures may have been necessary to produce a stimulatory soluble factor, so CM was also obtained from fibroblasts cultured with estrogen; no stimulatory effect was observed with this CM either. Because coculture with either untreated or irradiated fibroblasts were the only conditions that facilitated estrogen-dependent epithelial cell DNA synthesis, this suggested that in addition to being metabolically active, that the fibroblasts needed to be in close contact with the epithelial cells. To determine if this was
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Figure 7. Effect of fibroblast t r e a t m e n t on estrogen-dependent stimulation of progesterone receptor in m a m m a r y epithelial cells. Epithelial cells were either cultured alone (E) or with m a m m a r y fibroblasts that were u n t r e a t e d (F), irradiated (IF), or gluteraldehydekilled (GK), with conditioned m e d i u m (CM), with gluteraldehyde-killed fibroblasts plus conditioned m e d i u m (GK + CM), or in culture dishes pretreated with type I collagen (C). At confluence the cultures
kept without estradiol (-) or treated with 20 nM estradiol (o) and assayed for progesterone receptors (PgR) 3 d later (14). *P<.05 that estrogen-treated cultures have higher PgR than controls. Journal o f Dairy Science Vol. 71, No. 10, 1988
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Figure 8. Effect of fibroblast treatment on estrogen-dependent stimulation of epithelial cell DNA synthesis. Epithelial cells were either cultured alone (E) or with mammary fibroblasts that were untreated (F 1), irradiated (IF) , or gluteraldehyde-killed (GK) or with conditioned medium for fibroblasts cultured with (E + CM) or without (CM) 20 nM estradiol; epithelial cells were also coplated with fibroblasts (F2). In all cases, epithelial ceils were plated with (o) or without (=) 20 nM estradiol and labelling indices were determined 1 (a) or 5 (b) d later (10). **P<.05 that cocultures treated with estrogen have higher LI than do corresponding control cultures.
the case, epithelial cells were coplated with fibroblasts. After 1 d of culture, little direct contact between the two cell types was observed (Figure 9d) and there was no corresponding increase in LI (Figure 8). However, after 5 d of culture, the cells were extensively in contact with each other (Figure 10c), and at this time, estrogen caused a significant increase in the LI (Figure 8). These results support the conclusion that close or direct contact between the two cell types is required for the promotion of estrogen-dependent DNA synthesis. In the course of the experiments, the effect of estrogen and culture conditions on fibroblast DNA synthesis was also monitored. The results presented in Figure 11 demonstrate that estrogen had no stimulatory effect on purely Journal of Dairy Science Vol. 71, No. 10, 1988
fibroblast cultures. However, if fibroblasts were coplated with epithelial ceils, estrogen treatment resulted in a significant stimulation of DNA synthesis, particularly at 3 and 5 d of culture (Figure 11). No stimulation was observed after 1 d of culture, suggesting that contact between the two cell types was also important for the fibroblast response. Interesting differences were observed in the culture morphology of epithelial cells or fibroblasts grown separately as compared with their morphologies when they were cultured together. When epithelial cells were cultured alone, colonies of cells with rounded contours were observed (Figure 9a). As confluence was reached, the colonies coalesced. When epithelial cells were cultured with live or irradiated fibroblasts, there were several notable differences. Colony contours were stellate in shape (Figure 10c, d), and advancing strands of epithelial cells (Figure lOd) were frequently observed. Fibroblasts morphology was also affected by coculture conditions. When grown separately, fibroblasts appeared to have a random orientation at confluence (Figure lOb). By contrast, when cocultured with epithelial cells, fibroblasts appeared more elongated and assumed a characteristic orientation of parallel arrays between the epithelial colonies (Figure 10 c, d). The observations that under coculture conditions, fibroblast response to estrogen and culture morphology are influenced by the epithelial cells suggests there may be a bidirectional interactive phen o m e n o n between the two cell populations. SUMMARY
AND
CONCLUSIONS
Using a cell culture model system, mammary gland cell-cell interactions and their potential regulatory role in normal gland function have been investigated, specifically, the nature of mammary epithelial cell interactions with mammary stromal fibroblasts in relation to estrogen-regulated DNA synthesis and PgR concentration. There appear to be at least two different mechanisms by which mammary fibroblasts affect estrogen specific responses in the epithelial cell. In the first case, estrogen-dependent stimulation of PgR appears to be mediated by fibroblasts via a potential extracellular matrix, substratum effect. Type I collagen, which is produced by fibroblasts, is one potential
SYMPOSIUM: ROLE OF THE EXTRACELLULAR MATRIX IN MAMMARY DEVELOPMENT candidate for the molecular m e d i a t o r o f the observed effect. In recent years, there have been n u m e r o u s reports that expression of differentiated functin of m a m m a r y epithelial cells can be critically influenced b y the culture substratum and as herein, collagen t y p e I has been implicated (26). Epithelial cell shape, p r o d u c t i o n of a basement m e m b r a n e , and
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acquisition of polarity are all influenced by collagenous substrata (5). The e x a c t mechanism(s) b y which collagen or o t h e r extracellular m a t r i x c o m p o n e n t s regulate m a m m a r y epithelial response to m a m m a g e n i c h o r m o n e s such as estrogen remains to be elucidated. In the second case, m a m m a r y fibroblasts need to be metabolically active and either in
Figure 9. Culture morphologies 1 d after plating epithelial cells, a) Epithelial cells alone; b) epithelial cells on confluent monolayers of gluteraldehyde-killed fibroblasts. Note rounded colony contours in both a and b. c) Epithelial cells plated on confluent monolayers of untreated, live fibroblasts; epithelial colonies display stellate contours and fibroblasts are seen in contact with epithelial cells but are randomly oriented, d) Epithelial cells coplated with fibroblasts; fibroblasts are randomly oriented and there is little contact with the epithelial cells. H&E, ×85. Journal of Dairy Science Vol. 71, No. 10, 1988
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close contact with epithelial cells or present in sufficient numbers in order to p r o m o t e estrogen-dependent D N A synthesis. Additionally, under the same conditions, m a m m a r y epithelial cells p r o m o t e e s t r o g e n - d e p e n d e n t D N A synthesis in m a m m a r y fibroblasts. F u r t h e r m o r e , differences in morphologies of the two cells types are observed under c o c u l t u r e c o n d i t i o n s
that are not seen w h e n each cell t y p e is cultured by itself. These results strongly indicate s o m e f o r m of c o o p e r a t i o n b e t w e e n the t w o cell types. The mechanism(s) involved in this interaction also remain to be elucidated. Despite the finding that fibroblast-conditioned m e d i u m was ineffective in this case, the possibility still remains t h a t a soluble factor(s) is p r o d u c e d t h a t
Figure 10. Culture morphologies after 5 d of culture, a) Epithelial cells grown alone at confluence, b) Fibroblasts grown alone at confluence; note random orientation of cells, c) Epithelial cells grown on confluent, untreated fibroblasts; note parallel orientation of fibroblasts and extensive contact with the epithelial cells, d) Epithelial cells coplated with fibroblasts with advancing strand of epithelial cells (arrow). Inset, high magnification of epithelial strand (arrow), a to d, H&E, X 85. Inset X 200. Journal of Dairy Science Vol. 71, No. 10, 1988
SYMPOSIUM: ROLE OF THE EXTRACELLULAR MATRIX IN MAMMARY DEVELOPMENT
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ance a n d J. Bullis a n d B. H o l m e r - H e c k m a n f o r t y p i n g t h e m a n u s c r i p t . This w o r k was supp o r t e d b y NCI R e s e a r c h G r a n t C A - 4 0 1 0 4 .
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Figure 11. Effect of estrogen in fibroblast DNA synthesis in the presence of absence of mammary epithelial cells. Fibroblasts were cultured either alone (o) or coplated with epithelial cells (=) with (% =) or without (=, m) 20 nM estradiol and labelling index (LI) were determined 1, 3, or 5 d later (10). *P<.05 that the estrogen-treated groups have higher LI than the controls.
is effective only over s h o r t d i s t a n c e s or t h a t a n effective c o n c e n t r a t i o n is r e a c h e d o n l y w h e n f i b r o b l a s t s are p r e s e n t in high n u m b e r s . A n o t h e r possibility is t h a t c o m m u n i c a t i o n o c c u r s b y direct transfer of informational molecules across cell m e m b r a n e s b y specialized c h a n n e l s , such as gap j u n c t i o n s (18). In vivo t h e m a m m a r y e p i t h e l i u m exists w i t h in a c o m p l e x s t r o m a l e n v i r o n m e n t . A l o n g w i t h t h e m a m m a r y s t r o m a l f i b r o b l a s t are a n u m b e r of o t h e r cell t y p e s t h a t m a y have p o t e n t i a l r e g u l a t o r y roles in n o r m a l m a m m a r y g / a n d f u n c t i o n . A d v a n c e m e n t of o u r k n o w l e d g e in t h e area o f cell-cell i n t e r a c t i o n s in t h e m a m m a r y gland m a y p r o v i d e i m p o r t a n t insights i n t o m e c h a n i s m s t h a t regulate cell p r o l i f e r a t i o n as well as t h e s y n t h e s i s a n d s e c r e t i o n o f milk constituents during lactation. ACKNOWLEDGMENTS
T h e a u t h o r t h a n k s M. Levely, K. Gale, a n d S. D a c h t l e r f o r t h e i r e x c e l l e n t t e c h n i c a l assist-
1 Bandyopadhyay, G. K., W. Imagawa, D. Wallace, and S. Nandi. 1987. Linoleate metabolites enhance the in vitro proliferative response of mouse mammary epithelial cells to epidermal growth factor. J. Biol. Chem. 262:2750. 2 Bartley, J. C., T. T. Emerman, and M. J. Bissell. 1981. Metabolic cooperativity between epithelial cells and adipocytes of mice. Am. J. Physiol. 241: C204. 3 Bissell, M. J., G. H. Hall, and G. Parry. 1982. How does the extracellular matrix direct gene expression? J. Theoret. Biol. 99:31. 4 Ceriotti, G. 1952. A microchemical determination of desoxyribonucleic acid. J. Biol. Chem. 198:297. 5 Emerman, J. T., J. Enami, D. R. Pitelka, and S. Nandi. 1977. Hormonal effects on intracellular and secreted casein in cultures of mouse mammary epithelial cells on floating collagen membranes. Proc. Natl. Acad. Sci. 74:4466. 6 Emerman, J. T., and A. W. Vogl, 1986. Cell size and shape changes in the myoepithelium of the mammary gland during differentiation. Anat. Rec. 216:405. 7 Enami, J., S. Enami, and M. Koga. 1983. Growth of normal and neoplastic mammary epithelial cells in primary culture: stimulation by conditioned medium from mouse mammary fibroblasts. Gann 74:845. 8 Goldberg, B., and M. Rabinovitch. 1983. Connective tissue. Page 139 in Histology cell and tissue biology. 5th ed. L. Weiss, ed. Elsevier, New York, NY. 9 Hafez, M. M., and M. E. Costlow. 1987. Light and electron microscopic immunolocalization of prolactin in mast cells in rat mammary tumors. Anat. Rec. 218:54. (Abstr.) 10 Haslam, S. Z. 1986. Mammary flbroblast influence on normal mouse mammary epithelial cell responses to estrogen in vitro. Cancer Res. 46:310. 11 Haslam, S. Z., andM. L. Levely. 1985. Estrogen responsiveness of normal mouse mammary cells in prim ary cell culture: association of mammary fibroblasts with estrogenic regulation of progesterone receptors. Endocrinology 116:1835. 12 Haslam, S. Z., and G. Shyamala. 1979. Effect of oestradiol on progesterone receptors in normal mammary glands and its relationship with lactation. Biochem. J. 182: 127. 13 Haslam, S. Z., and G. Shyamala. 1980. Progesterone receptors in normal mammary gland: receptor modulations in relation to differentiation. J. Call Biol. 86:730. 14 Haslam, S. Z., and G. Shyamala. 1981. Relative distribution of estrogen and progesterone receptors among epithelial, adipose and connective tissue components of normal mammary gland. Endocrinology 108:825.
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15 Hay, E. D. 1981. The cell biology of the extraceUular matrix. Plenum Press, New York, NY. 16 Horwitz, K. B., and W. L. McGuire. 1978. Estrogen control of progesterone receptor in h u m a n breast cancer. J. Biol. Chem. 7:2223. 17 Hynes, R. O., and A. T. Destree. 1978. 10 n m filam e n t s in normal and transformed cells. Cell 13: 151. 18 Loewenstein, W. R. 1981. Junctional intercellular c o m m u n i c a t i o n : the cell-to-cell m e m b r a n e channel. Physiol. Rev. 61:829. 19 McGrath, C. M. 1983. A u g m e n t a t i o n of response of normal m a m m a r y epithelial ceils to estradiol by m a m m a r y stroma. Cancer Res. 43:1355. 20 Nandi, S. 1958. Endocrine control of m a m m a r y gland development and function in the C3H/He Crgl mouse. J. Natl. Cancer Inst. 21:1039. 21 Pitelka, D. R. 1983. The m a m m a r y gland. Page 944 in Histology cell in tissue biology. 5th ed. C. Weiss, ed. Elsevier, New York, NY. 22 Sakakura, T., T. Nishizuka, and C. J. Dawe. 1976. Mesenchyme-dependent morphogenesis and epithe-
Journal of Dairy Science Vol. 71, No. 10, 1988
23
24
25
26
27
lium-specific cytodifferentiation in m o u s e m a m m a r y gland. Science 194:1439. Sakakura, T., Y. Nishizuka, and C. J. Dawe. 1979. Capacity of m a m m a r y fat pads of C3H/heMs mice to interact morphogenetically with fetal m a m m a r y epithelium. J. Natl. Cancer Inst. 63:733. Sakakura, T., Y. Sakagami, and Y. Nishizuka. 1979. Persistence of responsiveness of adult m o u s e mamm a r y gland to induction by embryonic mesenchyme. Dev. Biol. 72:201. Scatchard, G. 1947. The attractions of proteins for small molecules and ions. Ann. New York Acad. Sci. 51:660. Sugrue, S. P., and E. D. Hay. 1981. Response of basal epithelial cell surface and cytoskeleton and solubilized extracellular matrix molecules. J. Cell Biol. 91:45. Warburton, M. J., D. Mitchell, E. J. Omerod, and P. Rudland. 1982. Distribution of myoepithelial cells and basement m e m b r a n e proteins in the resting, pregnant, lactating and involuting rat m a m m a r y gland. J. Histochem. Cytochem. 30:667.
The nonepithelial components of the mammary gland are reviewed and their potential for regulatory cell-cell interactions with the epithelial cells are discussed. Studies undertaken to examine the regulatory potential of mammary stromal fibroblasts using an in vitro cell culture system are presented. The influence was examined of epithelial-fibroblast interactions on estrogenic regulation of progesterone receptor concentration in epithelial cells and on epithelial and fibroblast DNA synthesis. Mammary fibroblasts affect estrogen responsiveness in epithelial cells by two different mechanisms. In the case of progesterone receptor regulation, fibroblasts promote estrogen-dependent increases in the receptor via a substratum effect possibly by the production of collagen type I. By contrast, the fibroblast effect promoting estrogen-dependent cell proliferation requires fibroblasts to be metabolically active and in close contact with the epithelium. Additionally, under coculture conditions, estrogen-dependent stimulation of fibroblast DNA synthesis is also observed, indicating a bidirectional, interactive phenomenon between the two types of cells. It is possible that the modulations in epithelial responsiveness to estrogen that are associated with the presence of mammary fibroblasts in vitro reflect regulatory mechanisms that operate in vivo. INTRODUCTION
The mammary gland is composed of epithelial, adipose, and fibrous connective tissues; the
Received July 6, 1987. Accepted November 16, 1987. 1988 J Dairy Sci 71:284-3-2854
relative proportion of each tissue type varies with the species and developmental state of the gland. Under the influence of a wide range of hormones, the gland undergoes dramatic changes in morphology and function (20). The most readily detectable changes occur in the epithelial compartment of the gland, and the proliferation, morphogenesis, and functional differentiation of the epithelium have been studied almost to the exclusion of all other cell types present. In recent years, there has been a growing interest in more detailed analysis of the cellular composition of the gland, and this has led some to consider that the nature of various cell-cell interactions might provide the basis for important regulatory mechanism(s) in normal mammary gland function. This paper provides a brief review of the various nonepithelial cells types present and what is known about their potential for regulatory interactions with mammary epithelium. The main emphasis of this paper will deal with studies (lO, 1 1 ) o f marnmary fibroblast interactions with mammary epithelial cells in culture and their influence on hormona/ regulation. Because rodent mammary gland has been studied extensively as a model for delineating hormone action in normal mammary tissue, studies in mice and rats provide the focus for this paper. In the nonpregnant animal, the epithelial component of the gland is organized into a branching ductat system. The epithelial cells lining the duct are invested with a basal layer of myoepithelial cells and a continuous basement membrane encloses the entire ductal system (Figure 1). In the context of cell-cell interaction, the first nonepithelial cell type to be considered is the myoepithelial cell. The best known function of myoepithelial cells is their facilitation of milk removal during lactation as a result of contractile activity in response to oxytocin (21). However, these cells are directly interposed between the epithelial cells and the basement membrane, i.e., the extracellular
2843
2844
HASLAM
Figure 1. Representation of the various cells present in normal mammary gland. The epithelial compartment of the gland contains epithelial cells (a) surrounded by myoepithelial cells (b) and separated from the stromal compartment by a basement membrane (c). The stromal compartment consists of the fibroblasts (d) and adipocytes (e); also present are mast cells (f).
matrix. A recent study (6) has shown that the physical relationship of the myoepithelial cell to the epithelium changes dramatically with different developmental states of the gland. In glands of nonpregnant animals, myoepithelial cells form a complete collar around the ductaI epithelium forming linear tracts oriented parallel to the long axis of the ducts, thus effectively separating the epithelium from the basement membrane. By contrast, during pregnancy and lactation, with the development of alveoli, myoepithelial cells assume a stellate appearance, forming loosely woven baskets around individual alveoli, thus leaving large areas of the epithelial cell surface in potential direct contact with the basement membrane. In view of the proposed role of the extracellular matrix in regulation of functional differentiation (3, 15), it is tempting to speculate that myoepithelial cells could have a regulatory role by virtue of their ability to modulate epithelial cell exposure to the extracellular matrix. Journal of Dairy Science Vol. 71, No. 10, 1988
Beyond the basement membrane, mammary stromal fibroblasts form a collagenous sheath around the mammary ducts and separate them from the subadjacent adipose tissue (Figure 1). Thus, fibroblasts are the stromal cells in closest proximity to the epithelium. These cells are also responsible for contributing stromal components such as collagen t y p e I to the extracellular space and firbonectin to the basement membrane (27). One can speculate that mammary fibroblasts may play a regulatory role in epithelial function based upon their contribution to the extracellular matrix composition. The third cell type to be considered is the adipocyte. In the nonpregnant state, the adipose tissue constitutes the major portion of the gland. During pregnancy and lactation, the epithelial component of the gland increases concomitant with a depletion of the adipose tissue. After involution, there is degeneration and loss of the secretory epithlium and the adipose tissue is restored. Thus, cyclic changes in mammary adipose tissue composition occur in relation to mammary gland function. Accumulation of glycogen, the activity of glycogen synthetase, and lipogenic rate in mammary adipocytes are modulated during pregnancy and lactation to channel nutrients to the mammary epithelial cell (2). These changes appear to require the presence of mammary epithelium (2), indicating possible interactive phenomena between the epithelial cells and adipocytes. Recent studies have also shown that linoleate metabolites enhance the in vitro proliferative response of mouse mammary epithelial cells (1). Thus, metabolic cooperation may be a key to adipocyte epithelial cell interactions. One other cell type that may interact with mammary epithelial cells and play a regulatory role in mammary gland function is the mast cell (Figure 1). Mast cells are ubiquitous in connective tissue; they originate in bone marrow and are similar but not identical to basophils (8). They possess numerous granules that contain a variety of bioactive molecules as well as IgE; these cells are generally associated with the mediation of hypersensitivity reactions. Recent studies on mast cells derived from dimethylbenz [a] anthracene-induced rat mammary tumors indicate that these cells may play a role in hormonal regulation (9). Tumor-derived mast cells can concentrate prolactin in their secretory granules and under appropriate con-
SYMPOSIUM: ROLE OF THE EXTRACELLULAR MATRIX IN MAMMARY DEVELOPMENT 2845 ditions prolactin can also be released from the cells. These observations may provide another perspective on mechanisms that could control local concentrations of hormones within mammary tissue. Studies on embryonic development of the mammary gland have demonstrated that mammary mesenchyme, the stromal precursor, directs and specifies epithelial morphogenesis (22). The adult mammary gland is distinct from many other epithelial organs in that its major morphogenesis occurs postnatally, during pregnancy, and is hormonally regulated (20). Several lines of evidence indicate that the morphogenic processes that occur during pregnancy are similar to those operative during fetal development of the organ and may be dependent upon epithelial-stromal interactions (23, 24). At present little is known about epithelialstromal interactions and hormonally regulated growth and function in the adult mammary gland. This question is difficult to approach in vivo in the whole animal, and thus, this problem was approached in vitro using a cell culture system. In vivo, estrogen is an important hormone for regulation of growth and function of mammary gland; it promotes mammary epithelial ceil proliferation and regulates mammary progesterone receptor ( P g R ) c o n centration (12, 13, 20). Hormone receptors for estrogen (ER) are localized in the mammary gland and are present in both the epithelial and stromal compartments of the gland (14). Of further interest, ER concentration varies in stromal and epithelial tissues depending on the
developmental state of the gland (Table 1). For these reasons, attention was focused on estrogen action in relation to mammary epithelialstromal cell interactions. Initial stu dies (10, 11 ) addressed specifically the interaction of the mammary fibroblast with the epithelium and examined the response of these cells to estrogenic regulation of cell proliferation and PgR concentration. MATERIALS AND METHODS Cell Culture
Balb/C mice 2 to 5 mo of age and 14 to 18 d pregnant were the source of mammary tissue. The tissue was dissociated according to previously described methods to obtain mixed cultures containing epithelial cells and fibroblasts (11). The two cell types were separated (10) to produce either epithelial cultures or fibroblast cultures. The epithelial cell and fibroblast cultures were determined to be 97 and 100% pure, respectively, using immunocytochemical methods (11); antibody directed against vimentin was used to identify fibroblasts (17). All cell types were cultured in Medium 199 containing 5% charcoal-stripped fetal calf serum (CS-FCS), .006 /ag/ml insulin, 1/ag/ml prolactin, and 10 4 M cortisol. CuItures were kept at 37°C in a humidified atmosphere of 5% CO2 in air; medium was changed every 2 d. To determine the effect estrogen on PgR concentrations or DNA synthesis cells were cultured with or without 20 nM 173-estradiol. Fibroblasts cultures that were used for coculture experiments were plated 1 wk prior to
TABLE 1. Distribution of estrogen receptors between stromal and epithelial components of adult mouse mammary gland at different states of development.
Developmental state 1
Intact mammary gland specific [3H]E2 binding
Epithelium-devoid stroma specific [3H]E2 binding (fmol/mg DNA)
Virgin (2 to 5 mo) Pregnancy (14 d) Lactation (7 to 10 d)
1733 1023 787
SE
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277 153 118
923 920 430
138 120 56
1In all cases mice were ovariectomized, or ovariectomized plus hysterectomized (pregnant mice) 5 to 7 d prior to assay to remove endogenous estrogens. Thus measured = total cytoplasmic estrogen receptor. Estrogen receptor concentration was determined by Scatchard analysis. For methods see (14). E2 = Estradiol. Journal of Dairy Science Vol. 71, No. 10, 1988
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HASLAM
the addition of the epithelial cells. The fibroblasts received treatments to render them either nonproliferative by irradiation or nonviable by gluteraldehyde fixation as previously described (10). To obtain conditioned medium, fibroblast cultures were incubated for 24 h in Medium 199 without FCS or hormones and processed and stored as described (10). Steroid Hormone Receptor Assay
Intact cells grown for indicated lengths of time were assayed while still attached to culture dishes. To measure ER or PgR the cells were incubated with either [3H] estradiol (1 to 10 nM) or [3H]R5020 (1 to 12 nM) with or without 100-fold excess radioinert estradiol or R5020 and processed as previously described (10). All binding data were analyzed by the method of Scatchard (25). The DNA content was determined according to the method of Ceriotti (4).
cultures were treated with estrogen (20 nM) there was almost a threefold increase in PgR concentration after 3 to 5 d of treatment. This result is very comparable to the two- to threefold stimulation of PgR observed after in vivo administration of estradiol to ovariectomized virgin (12) or pregnant mice (13). The observed effect of estrogen on PgR concentration appears to specific for estrogenic compounds (Figure 4) since estradiol and diethylstilbestrol were effective in increasing PgR concentration and the antiestrogen, tamoxifen (10--6M), was able to block the estrogenic effect; further evidence of specificity is indicated by the lack of effect obtained with 20 nM progesterone or dexamethasone. Having established that mixed cultures exhibit a characteristic PgR response to estrogen,
Measurement of Deoxyribonucleic Acid Synthesis
Cells were plated, in parallel, on glass coverslips, and autoradiography was as described 19) using [3H]thymidine (6.7 Ci/mmol). Labeling was for 1 h. Labeled nuclei were counted in 20 random high power (400x) microscope fields with a minimum of 1000 of each cell type counted (10).
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Initial experiments were carried out with mixed cultures containing both epithelial cells and fibroblasts in order to determine if this model system would exhibit an in vitro response to estrogen. The effects of estrogen treatment on PgR concentration are shown in Figures 2 and 3. Soon after plating the cells (Figure 2), when cultures were still subconfluent, estrogen was only moderatively effective in increasing PgR concentration. However, when confluent Journal of Dairy Science Vol. 71, No. 10, 1988
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Figure 2. Time course of estrogen stimulation of progesterone receptor concentration in mixed cells. Cells were plated and kept in medium with (D) or w i t h o u t (=) 20 nM estradiol and assayed for progesterone receptors (PgR) 1, 3, or 5 d later as described in (13). *P = .005 that in estradiol-treated group PgR value is significantly greater than in untreated controis.
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it was of interest to d e t e r m i n e if purely epithelial cultures responded similarly. No stimulation of PgR was observed in the epithelial cultures (Figure 5). One possible explanation for the lack of response in epithelial cultures was that the increase in PgR c o n c e n t r a t i o n was occurring in the fibroblast p o p u l a t i o n o f m i x e d cultures. To investigate this possibility, fibroblast cultures were e x a m i n e d for the presence o f PgR and for the ability o f estrogen to increase PgR concentration. In all cases, Scatchard analysis o f [ 3 H ] R 5 0 2 0 binding revealed only low affinity binding that did not satisfy the criteria for receptor binding (Figure 6). F u r t h e r m o r e , t r e a t m e n t with estrogen had no effect on the quality or q u a n t i t y of the binding. Estrogenic regulation of PgR c o n c e n t r a t i o n in m a m m a r y tissue is t h o u g h t to be regulated
Figure 4. Steroid hormone specificity of progesterone receptor stimulation. Confluent mixed cells cultured without estradiol were changed to medium without estradiol (C), or 20 nM estradiol (Ea), or diethylstibestrol (DES), or dexamethasone (DEX), or progesterone (Pg), or 10 -6 M tamoxifen (T), or estradiol plus tamoxifen (E 2 +T).
via an E R - m e d i a t e d m e c h a n i s m (16). Thus, the lack of estrogen responsiveness observed in the epithelial cultures was possibly due to a loss of ER. To test this possibility, epithelial, fibroblast, and m i x e d cultures were assayed for E R at various times after plating. C o n c e n t r a t i o n s of E R in the three t y p e s of cultures were similar, and in all cases, E R c o n c e n t r a t i o n s were maintained equally well over t i m e (Table 2). F r o m these studies we conclude that one specific m a m m a r y epithelial cell response to estrogen in vitro, n a m e l y PgR regulation, is associated with the presence of m a m m a r y stromal fibroblasts.
Analysis of the Fibroblasts Influence on Epithelial Cell Responsiveness to Estrogen
The favorable effect o f fibroblasts on mamm a r y epithelial cells could be explained in several different ways. One possibility is that fibroblasts provide some undefined soluble factor that is required for epithelial response to estrogen. In this c o n t e x t , conditioned media f r o m cultured mouse fibroblasts have been r e p o r t e d to stimulate normal m o u s e m a m m a r y Journal of Dairy Science Vol. 71, No. 10, 1988
2848
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Journal of Dairy Science Vol. 71, No. 10, 1988
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SYMPOSIUM: ROLE OF THE E X T R A C E L L U L A R MATRIX IN MAMMARY DEVELOPMENT
epithelial cell proliferation in vitro (7). However, the physical nature and chemical composition of the culture substratum are also known to be important for mammary epithelial cell function in vitro (5). Thus, fibroblasts might function to improve the culture substratum. Alternatively, direct contact between epithelial cells and fibroblasts may be required to facilitate estrogen responsiveness (19). The purpose of the following series of experiments was to distinguish among these described possibilities and to elucidate the mechanism(s) by which fibroblasts might influence epithelial cell responsiveness. Observations include fibroblast effects on estrogen dependent epithelial cell DNA synthesis. Progesterone Receptor Regulation
To investigate the nature of fibroblast influence on the PgR response a number of fibroblast conditions were created and they were cocultured with epithelial cells 7 d later. The results are shown in Figure 7. Fibroblasts were either control untreated, lethally irradiated, gluteraldehyde-killed (GK) or epithelial cells were cultured in the presence of conditioned medium (CM) from fibroblasts. All of the conditions tested were about equally effective in promoting estrogen-dependent increases in PgR concentration. F r o m these results one could conclude that fibroblasts need not be alive or even physically present since conditioned medium was also effective. Fibroblasts m a y be influencing the epithelial cells via a substratum effect. Pretreating culture dishes with collagen t y p e I, an extracellular product of fibroblasts, was also effective in promoting the PgR response providing further evidence for a substratum effect (Figure 7). Regulation of Deoxyribonucleic Acid Synthesis
To determine the nature of fibroblast influence on epithelial cell proliferation, DNA synthesis was measured in parallel cocultures, autoradiographically. The results of these studies are shown in Figure 8. When epithelial cells were grown alone, estrogen had no stimulatory effect. In contrast, coculture with live fibroblasts in the presence of estrogen resulted in a significant increase in the labeling index (LI, percent labeled nuclei). Coculture with ir-
2849
radiated fibroblasts also resulted in a significant increase in LI on d 1; however, after 5 d of culture a stimulatory effect was not observed. Coculture with GK fibroblasts was not effective. Conditioned medium also failed to promote epithelial cell DNA synthesis. It was possible that the presence of estrogen in fibroblast cultures may have been necessary to produce a stimulatory soluble factor, so CM was also obtained from fibroblasts cultured with estrogen; no stimulatory effect was observed with this CM either. Because coculture with either untreated or irradiated fibroblasts were the only conditions that facilitated estrogen-dependent epithelial cell DNA synthesis, this suggested that in addition to being metabolically active, that the fibroblasts needed to be in close contact with the epithelial cells. To determine if this was
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Figure 7. Effect of fibroblast t r e a t m e n t on estrogen-dependent stimulation of progesterone receptor in m a m m a r y epithelial cells. Epithelial cells were either cultured alone (E) or with m a m m a r y fibroblasts that were u n t r e a t e d (F), irradiated (IF), or gluteraldehydekilled (GK), with conditioned m e d i u m (CM), with gluteraldehyde-killed fibroblasts plus conditioned m e d i u m (GK + CM), or in culture dishes pretreated with type I collagen (C). At confluence the cultures
kept without estradiol (-) or treated with 20 nM estradiol (o) and assayed for progesterone receptors (PgR) 3 d later (14). *P<.05 that estrogen-treated cultures have higher PgR than controls. Journal o f Dairy Science Vol. 71, No. 10, 1988
2850 20
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Figure 8. Effect of fibroblast treatment on estrogen-dependent stimulation of epithelial cell DNA synthesis. Epithelial cells were either cultured alone (E) or with mammary fibroblasts that were untreated (F 1), irradiated (IF) , or gluteraldehyde-killed (GK) or with conditioned medium for fibroblasts cultured with (E + CM) or without (CM) 20 nM estradiol; epithelial cells were also coplated with fibroblasts (F2). In all cases, epithelial ceils were plated with (o) or without (=) 20 nM estradiol and labelling indices were determined 1 (a) or 5 (b) d later (10). **P<.05 that cocultures treated with estrogen have higher LI than do corresponding control cultures.
the case, epithelial cells were coplated with fibroblasts. After 1 d of culture, little direct contact between the two cell types was observed (Figure 9d) and there was no corresponding increase in LI (Figure 8). However, after 5 d of culture, the cells were extensively in contact with each other (Figure 10c), and at this time, estrogen caused a significant increase in the LI (Figure 8). These results support the conclusion that close or direct contact between the two cell types is required for the promotion of estrogen-dependent DNA synthesis. In the course of the experiments, the effect of estrogen and culture conditions on fibroblast DNA synthesis was also monitored. The results presented in Figure 11 demonstrate that estrogen had no stimulatory effect on purely Journal of Dairy Science Vol. 71, No. 10, 1988
fibroblast cultures. However, if fibroblasts were coplated with epithelial ceils, estrogen treatment resulted in a significant stimulation of DNA synthesis, particularly at 3 and 5 d of culture (Figure 11). No stimulation was observed after 1 d of culture, suggesting that contact between the two cell types was also important for the fibroblast response. Interesting differences were observed in the culture morphology of epithelial cells or fibroblasts grown separately as compared with their morphologies when they were cultured together. When epithelial cells were cultured alone, colonies of cells with rounded contours were observed (Figure 9a). As confluence was reached, the colonies coalesced. When epithelial cells were cultured with live or irradiated fibroblasts, there were several notable differences. Colony contours were stellate in shape (Figure 10c, d), and advancing strands of epithelial cells (Figure lOd) were frequently observed. Fibroblasts morphology was also affected by coculture conditions. When grown separately, fibroblasts appeared to have a random orientation at confluence (Figure lOb). By contrast, when cocultured with epithelial cells, fibroblasts appeared more elongated and assumed a characteristic orientation of parallel arrays between the epithelial colonies (Figure 10 c, d). The observations that under coculture conditions, fibroblast response to estrogen and culture morphology are influenced by the epithelial cells suggests there may be a bidirectional interactive phen o m e n o n between the two cell populations. SUMMARY
AND
CONCLUSIONS
Using a cell culture model system, mammary gland cell-cell interactions and their potential regulatory role in normal gland function have been investigated, specifically, the nature of mammary epithelial cell interactions with mammary stromal fibroblasts in relation to estrogen-regulated DNA synthesis and PgR concentration. There appear to be at least two different mechanisms by which mammary fibroblasts affect estrogen specific responses in the epithelial cell. In the first case, estrogen-dependent stimulation of PgR appears to be mediated by fibroblasts via a potential extracellular matrix, substratum effect. Type I collagen, which is produced by fibroblasts, is one potential
SYMPOSIUM: ROLE OF THE EXTRACELLULAR MATRIX IN MAMMARY DEVELOPMENT candidate for the molecular m e d i a t o r o f the observed effect. In recent years, there have been n u m e r o u s reports that expression of differentiated functin of m a m m a r y epithelial cells can be critically influenced b y the culture substratum and as herein, collagen t y p e I has been implicated (26). Epithelial cell shape, p r o d u c t i o n of a basement m e m b r a n e , and
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acquisition of polarity are all influenced by collagenous substrata (5). The e x a c t mechanism(s) b y which collagen or o t h e r extracellular m a t r i x c o m p o n e n t s regulate m a m m a r y epithelial response to m a m m a g e n i c h o r m o n e s such as estrogen remains to be elucidated. In the second case, m a m m a r y fibroblasts need to be metabolically active and either in
Figure 9. Culture morphologies 1 d after plating epithelial cells, a) Epithelial cells alone; b) epithelial cells on confluent monolayers of gluteraldehyde-killed fibroblasts. Note rounded colony contours in both a and b. c) Epithelial cells plated on confluent monolayers of untreated, live fibroblasts; epithelial colonies display stellate contours and fibroblasts are seen in contact with epithelial cells but are randomly oriented, d) Epithelial cells coplated with fibroblasts; fibroblasts are randomly oriented and there is little contact with the epithelial cells. H&E, ×85. Journal of Dairy Science Vol. 71, No. 10, 1988
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close contact with epithelial cells or present in sufficient numbers in order to p r o m o t e estrogen-dependent D N A synthesis. Additionally, under the same conditions, m a m m a r y epithelial cells p r o m o t e e s t r o g e n - d e p e n d e n t D N A synthesis in m a m m a r y fibroblasts. F u r t h e r m o r e , differences in morphologies of the two cells types are observed under c o c u l t u r e c o n d i t i o n s
that are not seen w h e n each cell t y p e is cultured by itself. These results strongly indicate s o m e f o r m of c o o p e r a t i o n b e t w e e n the t w o cell types. The mechanism(s) involved in this interaction also remain to be elucidated. Despite the finding that fibroblast-conditioned m e d i u m was ineffective in this case, the possibility still remains t h a t a soluble factor(s) is p r o d u c e d t h a t
Figure 10. Culture morphologies after 5 d of culture, a) Epithelial cells grown alone at confluence, b) Fibroblasts grown alone at confluence; note random orientation of cells, c) Epithelial cells grown on confluent, untreated fibroblasts; note parallel orientation of fibroblasts and extensive contact with the epithelial cells, d) Epithelial cells coplated with fibroblasts with advancing strand of epithelial cells (arrow). Inset, high magnification of epithelial strand (arrow), a to d, H&E, X 85. Inset X 200. Journal of Dairy Science Vol. 71, No. 10, 1988
SYMPOSIUM: ROLE OF THE EXTRACELLULAR MATRIX IN MAMMARY DEVELOPMENT
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ance a n d J. Bullis a n d B. H o l m e r - H e c k m a n f o r t y p i n g t h e m a n u s c r i p t . This w o r k was supp o r t e d b y NCI R e s e a r c h G r a n t C A - 4 0 1 0 4 .
3o
REFERENCES x.~_ I&l@
zg
=o
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1
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DAYS IN CULTURE
Figure 11. Effect of estrogen in fibroblast DNA synthesis in the presence of absence of mammary epithelial cells. Fibroblasts were cultured either alone (o) or coplated with epithelial cells (=) with (% =) or without (=, m) 20 nM estradiol and labelling index (LI) were determined 1, 3, or 5 d later (10). *P<.05 that the estrogen-treated groups have higher LI than the controls.
is effective only over s h o r t d i s t a n c e s or t h a t a n effective c o n c e n t r a t i o n is r e a c h e d o n l y w h e n f i b r o b l a s t s are p r e s e n t in high n u m b e r s . A n o t h e r possibility is t h a t c o m m u n i c a t i o n o c c u r s b y direct transfer of informational molecules across cell m e m b r a n e s b y specialized c h a n n e l s , such as gap j u n c t i o n s (18). In vivo t h e m a m m a r y e p i t h e l i u m exists w i t h in a c o m p l e x s t r o m a l e n v i r o n m e n t . A l o n g w i t h t h e m a m m a r y s t r o m a l f i b r o b l a s t are a n u m b e r of o t h e r cell t y p e s t h a t m a y have p o t e n t i a l r e g u l a t o r y roles in n o r m a l m a m m a r y g / a n d f u n c t i o n . A d v a n c e m e n t of o u r k n o w l e d g e in t h e area o f cell-cell i n t e r a c t i o n s in t h e m a m m a r y gland m a y p r o v i d e i m p o r t a n t insights i n t o m e c h a n i s m s t h a t regulate cell p r o l i f e r a t i o n as well as t h e s y n t h e s i s a n d s e c r e t i o n o f milk constituents during lactation. ACKNOWLEDGMENTS
T h e a u t h o r t h a n k s M. Levely, K. Gale, a n d S. D a c h t l e r f o r t h e i r e x c e l l e n t t e c h n i c a l assist-
1 Bandyopadhyay, G. K., W. Imagawa, D. Wallace, and S. Nandi. 1987. Linoleate metabolites enhance the in vitro proliferative response of mouse mammary epithelial cells to epidermal growth factor. J. Biol. Chem. 262:2750. 2 Bartley, J. C., T. T. Emerman, and M. J. Bissell. 1981. Metabolic cooperativity between epithelial cells and adipocytes of mice. Am. J. Physiol. 241: C204. 3 Bissell, M. J., G. H. Hall, and G. Parry. 1982. How does the extracellular matrix direct gene expression? J. Theoret. Biol. 99:31. 4 Ceriotti, G. 1952. A microchemical determination of desoxyribonucleic acid. J. Biol. Chem. 198:297. 5 Emerman, J. T., J. Enami, D. R. Pitelka, and S. Nandi. 1977. Hormonal effects on intracellular and secreted casein in cultures of mouse mammary epithelial cells on floating collagen membranes. Proc. Natl. Acad. Sci. 74:4466. 6 Emerman, J. T., and A. W. Vogl, 1986. Cell size and shape changes in the myoepithelium of the mammary gland during differentiation. Anat. Rec. 216:405. 7 Enami, J., S. Enami, and M. Koga. 1983. Growth of normal and neoplastic mammary epithelial cells in primary culture: stimulation by conditioned medium from mouse mammary fibroblasts. Gann 74:845. 8 Goldberg, B., and M. Rabinovitch. 1983. Connective tissue. Page 139 in Histology cell and tissue biology. 5th ed. L. Weiss, ed. Elsevier, New York, NY. 9 Hafez, M. M., and M. E. Costlow. 1987. Light and electron microscopic immunolocalization of prolactin in mast cells in rat mammary tumors. Anat. Rec. 218:54. (Abstr.) 10 Haslam, S. Z. 1986. Mammary flbroblast influence on normal mouse mammary epithelial cell responses to estrogen in vitro. Cancer Res. 46:310. 11 Haslam, S. Z., andM. L. Levely. 1985. Estrogen responsiveness of normal mouse mammary cells in prim ary cell culture: association of mammary fibroblasts with estrogenic regulation of progesterone receptors. Endocrinology 116:1835. 12 Haslam, S. Z., and G. Shyamala. 1979. Effect of oestradiol on progesterone receptors in normal mammary glands and its relationship with lactation. Biochem. J. 182: 127. 13 Haslam, S. Z., and G. Shyamala. 1980. Progesterone receptors in normal mammary gland: receptor modulations in relation to differentiation. J. Call Biol. 86:730. 14 Haslam, S. Z., and G. Shyamala. 1981. Relative distribution of estrogen and progesterone receptors among epithelial, adipose and connective tissue components of normal mammary gland. Endocrinology 108:825.
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15 Hay, E. D. 1981. The cell biology of the extraceUular matrix. Plenum Press, New York, NY. 16 Horwitz, K. B., and W. L. McGuire. 1978. Estrogen control of progesterone receptor in h u m a n breast cancer. J. Biol. Chem. 7:2223. 17 Hynes, R. O., and A. T. Destree. 1978. 10 n m filam e n t s in normal and transformed cells. Cell 13: 151. 18 Loewenstein, W. R. 1981. Junctional intercellular c o m m u n i c a t i o n : the cell-to-cell m e m b r a n e channel. Physiol. Rev. 61:829. 19 McGrath, C. M. 1983. A u g m e n t a t i o n of response of normal m a m m a r y epithelial ceils to estradiol by m a m m a r y stroma. Cancer Res. 43:1355. 20 Nandi, S. 1958. Endocrine control of m a m m a r y gland development and function in the C3H/He Crgl mouse. J. Natl. Cancer Inst. 21:1039. 21 Pitelka, D. R. 1983. The m a m m a r y gland. Page 944 in Histology cell in tissue biology. 5th ed. C. Weiss, ed. Elsevier, New York, NY. 22 Sakakura, T., T. Nishizuka, and C. J. Dawe. 1976. Mesenchyme-dependent morphogenesis and epithe-
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