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Induction of the Endogenous Whey Acidic Protein (Wap) Gene and a Wap-myc Hybrid Gene in Primary Murine Mammary Organoids CORA-ANN

SCHOENENBERGER,*~’

*Department

ANNA

ZUK,* BERND GRONER,~WALISJONES,*

AND

ANNE-CATHERINE

ANDRES?~

of Anatomy and Cellular Biology, Harvard Medical School, 220 Longwood Avenue, Boston, Massachusetts 02115; $Gluxo Croup Research Limited, Green&-d Road, Grew&n-d, Middlesex, UB6 OHE, England; and TFriedrich Miescher Institute, P.O. B03~ 25U Base& Switzerland Accepted Januam 22, 1990

In rodents, the whey acidic protein (Wap) is the major whey protein expressed in mammary glands in response to lactogenic hormones. The regulation of the Wap gene differs from that of other milk protein genes, with one consequence being that little or no Wap expression is detectable in cell culture. Here we describe the efficient in vitro induction of the Wap gene in mammary organoids isolated from midpregnant mice. Mammary organoids were isolated as intact epithelial subcomponents which retained the glandular microarchitecture. If organoids were cultured in contact with a monolayer of 3T3-Ll adipocytes, significant levels of Wap mRNA were induced upon hormonal stimulation, with the highest level of Wap mRNA being induced by a combination of hydrocortisone, prolactin, and insulin. Dissociation of the three-dimensional organization abrogated Wap inducibility. Organoids cultured on plastic or hydrated type I collagen did not transcribe Wap mRNA even after hormonal stimulation, Addition of hormones was required to maintain low levels of Wap mRNA in organoids cultured on reconstituted basement membrane, however, Wap mRNA was not induced. Organoid-adipocyte interactions as well as cell-cell interactions inherent in the structure of organoids promote hormone-dependent Wap mRNA expression. In order to study the Wap promoter region in vitro, we cocultured organoids from transgenic mice harboring a chimeric Wap-myc gene with 3T3-Ll adipocytes. Lactogenic hormones induced the Wap-myc transgene in vitro. The kinetics of induction were similar for both the transgene and the endogenous Wap gene indicating that the 2.5-kb regulatory Wap region present in the hybrid gene contains the sequence elements required for hormone-induced gene expression in vitro. o is90 Academic PRESS. I~C.

for elucidating mammary differentiation and milk gene regulation. The differentiation of the mammary gland is directed Primary mammary epithelial cells, cultured on tissue by a complex interplay of hormones during pregnancy culture plastic, rapidly lose their differentiated funcand lactation (Rosen et ah, 1980; Topper and Freeman, tions, even in the presence of the lactogenic hormones, 1980). In addition, interactions of the mammary epitheand insulin (Li et al., 1987; hum with surrounding stromal cells are thought to play prolactin, hydrocortisone, Wiens et al., 1987). Substrates other than plastic have an important role in establishing a differentiated phebeen more successful in promoting synthesis of certain notype (Kratochwil, 1969; Sakakura et al, 1979; Daniel milk proteins (Emerman and Pitelka, 1977; Wicha et al, et al, 1984; Kimata et al., 1985; Sakakura, 1987). The 1982; Lee et uL, 1985; Li et uL, 1987; Wiens et uL, 1987). function of the differentiated mammary epithelium is Mammary epithelial cells on released, but not on atto synthesize and secrete milk in response to lactogenic tached type I collagen gels, accumulate and secrete spehormones. Individual milk proteins are induced with cific caseins when lactogenic hormones are present different kinetics and accumulate to different extents, and Pitelka, 1977; Suard et uL, 1983; Lee et suggesting that milk protein gene expression is not co- (Emerman uZ., 1985). It has been proposed that the changes in morordinately controlled (Guyette et ah, 1979; Hobbs et aZ., phology and organization of the mammary epithelial 1982; Ray et aZ., 1986). Studies of the regulation of milk cells, which accompany the contraction of the gel are protein genes have been impeded by the complexity of conducive to differentiation (Emerman et uL, 1977; Bisthe cell-cell interactions and endocrine influences in sell and Hall, 1987). More recently, the importance of the pregnant animal and in mammary gland explants. the extracellular matrix in mediating morphological Thus, defined cell-culture systems that allow induction differentiation and tissue-specific gene expression has and maintenance of differentiation in vitro are critical been recognized (Wicha et uZ., 1982; Blum et ub, 1987; Li et uZ., 1987). Proliferation as well as hormone-dependent ’ To whom correspondence should be addressed. milk protein secretion has also been observed in pri‘Present address: L.G.M.E-C.N.R.S, 11 Rue Humann, Strasbourg, France. mary mammary epithelial cells cultured on 3T3-Ll adiINTRODUCTION

327

0012-1606/90 Copyright All rights

$3.00

0 1990 by Academic Press, Inc. of reproduction in any form reserved.

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pocytes (Levine and Stockdale, 1984,1985; Wiens et al, 1987). This culture system mimics epithelial-mesenchyma1 interactions that may play a role in the differentiation of the mammary gland in viva. In the milk of rodents, the whey acidic protein (Wap) is the predominant whey protein (Hennighausen and Sippel, 1982; Hobbs et aZ., 1982). Like casein genes, the Wap gene appears to be regulated by both hydrocortisone and prolactin in experiments performed in organ explant cultures (Hobbs et al, 1982; Pittius et al, 1988). However, Wap gene expression is virtually undetected in cultured mammary epithelial cells regardless of the nature of the substratum (Lee et ab, 1985; BarcellosHoff and Bissell, 1989; Park et aL, 1989), indicating that additional as yet unrecognized factors may be necessary for the in vitro synthesis and secretion of Wap. Since considerable amounts of Wap mRNA are expressed in the fully differentiated mammary gland, Wap may serve as a valuable marker in assessing mammary epithelial cell differentiation in vitro. It is possible that the expression of Wap is hormoneinducible in intact mammary gland explants because important cell-cell and cell-stromal relationships are maintained. Mammary gland-specific traits are often altered in culture such that complete differentiation may not be attainable. Therefore, we attempted to use in vitro culture conditions that mimic the intact gland. We modified the coculture system of Levine and Stockdale (1984, 1985) to analyze the regulation of the Wap gene. In contrast to their studies, we cultured intact epithelial subcomponents isolated from midpregnant mice and referred to as organoids (Jones et al, 1983), with 3T3-Ll adipocytes. We describe efficient hormone-induced Wap mRNA expression in primary mammary epithelial cells. Using this coculture system, we made three observations: (i) Wap gene expression depended upon both the structural integrity of the organoids and their interaction with 3T3-Ll adipocytes; (ii) efficient Wap mRNA induction was only achieved by the synergistic action of hydrocortisone, prolactin, and insulin; (iii) experiments performed with organoids prepared from transgenic mice bearing a Wap-myc hybrid gene (Schoenenberger et al, 1988) indicated that the promoter region of the Wap gene is sufficient to confer hormone responsiveness to the linked reporter gene in vitro. MATERIAL AND METHODS

Cell Culture, Organoid Preparation, and Hormone Induction 3T3-Ll cells were kindly provided by Dr. H. Green, (Harvard Medical School, Boston, MA) and maintained in Dulbecco’s modified Eagle’s medium (DMEM) + 10%

calf serum at 37°C in a 5% C02-95% air atmosphere. Confluent monolayers were converted to adipocytes as described (Levine and Stockdale, 1985), and sublethally irradiated with 6000 rad before addition of mammary organoids. Basement membrane gel from the EngelbrethHolm-Swarm (EHS) tumor was kindly provided by Dr. H. Kleinman (National Institute of Dental Research, Bethesda, MD). Frozen aliquots were thawed in ice water prior to use. Type I collagen was extracted from adult rat tail tendon and hydrated type I collagen gel prepared as previously described (Zuk et aZ., 1989). Tissue culture dishes (10 cm; Falcon) were coated with 1 ml of either basement membrane or type I collagen gel. The gel was allowed to polymerize at room temperature or 37°C for 30 min. Epithelial subcomponents of mouse mammary glands were isolated according to the procedure of Jones et aL, 1983. Briefly, the fourth inguinal mammary glands from midpregnant (14-16 day) CD-1 or Wap-myc transgenie mice were aseptically removed, minced, and dissociated by limited collagenase digestion [SIGMA, Type IA, 25 U/mg wet tissue, 250 U/ml DMEM + 5% fetal bovine serum (FBS)] and mechanical disruption at 30°C on a rotary mixer. The intact epithelial subcomponents were enriched by filtration through filters with decreasing pore size (840, 420, 250, 149, 105, 53 pm pore size). The multicellular organoids, collected on the 250 to 53-pm filters, were washed with medium at 30°C on a rotary mixer. Pellets of fractions 250 to 53 pm were pooled and resuspended in basal medium defined as DMEM + 5% FBS + insulin (5 pg/ml; SIGMA). Organoids derived from approximately one mammary gland were plated on a lo-cm tissue culture dish containing an irradiated monolayer of 3T3-Ll adipocytes. Alternatively, organoids were plated on basement membrane or collagen type I gels, or plastic. Organoids were also dissociated into single-cell suspensions with trypsinEDTA at 37°C followed by filtration through a 53-pm filter. Dissociation was confirmed by microscopic examination. Cells were plated at an equivalent number to that used for organoid cocultures. From Day 4, basal medium was supplemented with ovine prolactin (5 pg/ml; SIGMA) and hydrocortisone (5 pug/ml; SIGMA). Cultures without prolactin and hydrocortisone served as controls. In some cases, insulin was omitted. The medium was replaced every 24 hr. Photos were taken with Kodak T-MAX, ASA 400 film on a Zeiss Axiophot light microscope. Electron Microscopy Organoid cocultures were rinsed in phosphate-buffered saline, pH 7.4, and fixed for 30 min at room tem-

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perature in 2% glutaraldehyde, 2% paraformaldehyde in 0.1 Mcacodylate buffer, pH 7.4. After osmication (1% OsOl in 0.1 M cacodylate buffer) at 0°C for 30 min, the cultures were stained with 1% uranyl acetate. Specimens were dehydrated in ethanol and embedded in situ in Epon 812 (Coon and Manasek, 1970). Specimens were reembedded so that thin sections could be cut perpendicular to the substratum with a Sorvall MTB-B ultramicrotome (DuPont Instruments). A JEOL 1OOCX electron microscope was used to scan the sections. RNA Analysis Total cellular RNA was prepared by lysing cells in 4 M guanidine isothiocyanate, 25 mM EDTA, 0.5% Nlauroyl-sarcosine, 0.1 M 2-mercaptoethanol. After homogenizing with a Dounce homogenizer, samples were layered onto a cushion of 5.7 M CsCVO.1 M EDTA and centrifuged at 20°C for 16 hr (SW40 rotor, 32,000 rpm). RNA pellets were dissolved in water. Poly(A)+ enriched RNA was then isolated on oligo (dT) columns (Chirgwin et aZ., 1979). Synthesis of 32P-radiolabeled antisense RNA probes and RNase protection analyses were performed as described in Schoenenberger et al. (1988). For Northern blot analysis (Thomas, 1980) a PstI restriction fragment of a rat y actin cDNA clone (Ginzburg et al, 1980) was used. RESULTS

Horwume-Induced Expression of the Wap Gene in Organoids Cocultured with 3T3-Ll Adipocytes In order to achieve Wap gene expression in vitro, we cultured primary mammary epithelial cells under conditions that mimic the organization of the intact mammary gland. The procedure we used to isolate intact organoids (Jones et ab, 1983) preserved the three-dimensional organization and cell-cell interactions inherent in the glandular epithelium. The parenchyma of midpregnant CD-l mouse mammary glands was released as a mixture of multicellular organoids and distinct epithelial subpopulations were enriched for by filtration. The 250-pm fraction (Fig. 1A) consisted of ductal structures, the 105-pm filter (Fig. 1B) retained ductal-lobular structures and lobular units, and the 53-pm fraction (Fig. 1C) was enriched for terminal end buds (Jones et ah, 1983). In order to preserve the heterogeneity of the epithelial subpopulations present in the midpregnant gland, glandular material from the 250- to 53-pm filters was pooled and subsequently used to study Wap induction in vitro. The interactions between the epithelium and the adipose tissue that occur in the mammary gland were

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mimicked by culturing the organoid fractions in contact with a confluent monolayer of irradiated 3T3-Ll adipocytes (Fig. 1D). In this coculture system, organoids display a three-dimensional organization (Fig. lE), reminiscent of mammary gland morphology. Ducts extend from the multicellular structure (ORG, Fig. 1E) to grow out on top of the adipocyte monolayer. Frequently, ducts terminate as alveolar-like end buds. Thus, the two most prominent characteristics of the intact mammary gland, the histiotypic three-dimensional organization of the epithelial cells, and cell-cell interactions with adipocytes, are preserved in this in vitro coculture system. The ultrastructural analysis further supports this conclusion (Figs. lF-1H). Organoids cocultured with 3T3-Ll adipocytes maintain this glandular microarchitecture whether in the presence (Fig. 1F) or absence (Fig. 11) of lactogenic hormones. Luminal epithelial cells are polarized in that their apical plasma membrane is separated from the medium by junctional complexes (J, Fig. lH), whereas basal cell surfaces are exposed to the culture environment. Addition of lactogenic hormones induced specific cytodifferentiation. In the presence of hydrocortisone and prolactin, ductal and alveolar lumina (AL, Fig. 1F) become filled with granular material, consistent with milk protein secretion. The alveolar epithelium surrounding the dilated lumen is flat, extends apical microvilli, and contains a cytoplasm (Figs. lF-1H) characteristic of mammary alveoli during pregnancy and lactation. Dilated profiles of rough endoplasmic reticulum (RER, Fig. 1G) are prominent and a thin continuous basal lamina (arrows, Fig. 1G) surrounds the alveolar-like structure. Well developed Golgi areas (GZ, Fig. 1H) and vesicles are further indications for secretory activity. Before hormonal induction of cytodifferentiation (Fig. lI), proteinaceous material is not present in the ductal (DL, Fig. 11) or terminal end bud lumen (EB, Fig. 11) and the RER is not dilated. Thus, the data indicates that coculturing organoids with 3T3-Ll adipocytes is a viable in vitro system in which to study milk protein expression. To test the regulation of the Wap gene, poly(A)+ RNA was extracted after different culture periods and Wap mRNA expression monitored by RNase protection analyses carried out with an antisense probe protecting exon 1 (110 nucleotides) and exon 2 (135 nucleotides) of the Wap transcripts (Andres et al, 1987). Typically, cultures were maintained in basal medium without hormones for 4 days (-Day 1) and mRNA from these cultures was analyzed. Either medium supplemented with hydrocortisone and prolactin, or basal medium was then added to cultures which were incubated 1 to 20 days before mRNA extraction.

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FIG. 1. Organoids enriched for lobular

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fraction (A) consists of ducts, the 105-em These fractions were pooled and plated

fraction (B) is on a confluent

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Freshly isolated organoids from mammary glands of 14- to l&day pregnant mice express Wap mRNA (data not shown). However, after 4 days of coculture in the absence of the lactogenic hormones, hydrocortisone and prolactin, Wap mRNA is no longer detected (Fig. 2A, lane 1) nor are casein milk proteins (data not shown). The ultrastructure in Fig. 11 confirms the absence of milk protein expression. One day after addition of prolactin and hydrocortisone, cocultures express Wap mRNA (Fig. 2A, lane 2). Wap mRNA levels continue to increase and reach a maximum expression after 5 days of hormone treatment (Fig. 2A, lane 3). Thereafter, the level of Wap transcripts gradually decreases, but is still appreciable after an induction period of 20 days (Fig. 2A, lanes 4-6). Organoids cultured in the absence of prolactin and hydrocortisone for 10 days (Fig. 2A, lane 7) do not express Wap mRNA (Fig. 2A, compare lanes 7 and 4). In addition, Wap mRNA is not expressed in 3T3-Ll adipocytes even in the presence of lactogenic stimuli (data not shown). Northern blot analysis of corresponding RNA samples hybridized to a y actin probe (Fig. 2B) reveals that actin is expressed at comparable levels throughout the time course of induction. Thus, the addition of lactogenic hormones to cocultures does not result in a general induction of total mRNA, but specifically induces Wap gene expression. These results indicate that cocultured organoids retain their competence for differentiated functions in vitro and respond to lactogenic hormones with the rapid induction of Wap gene expression within 1 day. Furthermore, the accumulation of Wap mRNA is not a result of the coculture of organoids and adipocytes, but also depends upon the addition of the lactogenic hormones, hydrocortisone and prolactin. To analyze the importance of the three-dimensional structure in maintaining hormonal responsiveness, the cell-cell interactions provided by the structural integrity of organoids were disrupted by digestion with trypsin-EDTA. If the dissociated mammary epithelial cells were plated on 3T3-Ll adipocytes and cultured following the protocol used with intact organoids, Wap mRNA is not detected even after 10 days of exposure to

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hydrocortisone and prolactin (Fig. 2C). This suggests that the three-dimensional organization and distinct cell-cell interactions provided by the organoids is required for mammary epithelial cells to express the Wap gene in response to hormonal stimuli. Wap Gene Expression in Organoids Cultured Extracellular Matrices

on

Previous studies of mammary epithelial cells have demonstrated that the cytodifferentiation as well as the synthesis and secretion of casein milk proteins are influenced by the substratum (Suard et al, 1983; Lee et al, 1985; Li et al., 1987; Wiens et al., 1987). We compared four different substrates with regard to their ability to promote hormone-dependent Wap gene expression. Organoids were plated on plastic, collagen type I gel, reconstituted basement membrane or irradiated 3T3-Ll adipocytes. After 4 days of culture with only basal medium, the medium was supplemented with hydrocortisone and prolactin for an additional 10 days. Control cultures of organoids on each substrate were maintained in medium without prolactin and hydrocortisone. RNase protection analyses reveal that in organoids cultured on plastic (Fig. 3A, lane 2) or on a collagen substrate (Fig. 3A, lane 4), Wap mRNA expression is not induced by the addition of lactogenic hormones. Organoids that were cultured on reconstituted basement membrane with hormones maintain low levels of Wap mRNA (Fig. 3A, lane 6) even after the initial 4 days (-Day 1) of culture in the absence of hydrocortisone and prolactin (Fig. 3A, lane 7). Moreover, the amount of Wap mRNA expressed before hormone addition does not increase after 10 days of lactogenic stimuli (Fig. 3A, lane 6). Although organoids cultured on reconstituted basement membrane in the presence of hormones maintain baseline levels of Wap mRNA, this substrate does not efficiently promote Wap mRNA induction. The highest levels of Wap mRNA are achieved by organoids cultured on 3T3-Ll adipocytes with hydrocortisone, prolactin, and insulin in the medium (Fig. 3B). In control cultures, the absence of lactogenic hormones during

monolayer of irradiated 3T3-Ll adipocytes (D). Note the cytoplasmic fat droplets typical for adipocytes. (E) Phase contrast micrograph of organoid (ORG) cocultured with 3T3-Ll adipocytes for 10 days in the presence of hydrocortisone, prolactin, and insulin, after an initial culture period of 4 days in basal medium. (F) Ultrastructural analysis reveals that under these conditions, proteinaceous granular material is secreted into the alveolar-like lumen (AL). The cells lining the dilated lumen are flat with numerous luminal microvilli and contain a cytoplasm consistent with differentiated function. Representative sections of the boxed area and the region denoted by the asterisk (*) in (F) is shown at higher magnification in (G) and (H), respectively. (G) The cytoplasm contains an extensive network of dilated RER. A thin basement membrane (arrows) surrounds the alveolus-like structure. (H) The epithelial cells have a well developed Golgi zone (GZ) and display junctional complexes (J) apically. MV, microvilli. (I) Before hormonal induction (-Day l), proteinaceous material is not secreted into the ductal (DL) or terminal end bud lumen (EB). The micrograph shows a terminal end bud that has branched off from the duct. The cuboidal epithelial cells lining the duct and the flat cells of the terminal end bud do not show cytoplasmic evidence of differentiated function. ADP, adipocyte layer. Bar D, E = 50 pm; F, I = 5 pm; G, H = 0.3 pm.

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of the individual hormones prolactin, hydrocortisone, and insulin. Figure 3C shows the analysis of mRNA isolated from mammary organoids cultured on adipocytes for 10 days under various hormonal stimuli. In the absence of insulin (Fig. 3C, lane l), moderate levels of Wap-specific transcripts are detected in cocultures exposed to both prolactin and hydrocortisone. Minimal levels of Wap mRNA are found in organoids exposed to prolactin and insulin (Fig. 3C, lane 2) or hydrocortisone and insulin (Fig. 3C, lane 3). The combination of all three hormones (Fig. 3B) induces the highest level of Wap mRNA expression. Prolactin, hydrocortisone, and insulin appear to act synergistically to induce Wap mRNA in vitro.

1

The Promoter Region of the Wap Gene ConJkrs Hormone Responsiveness to a Linked Reporter -i--

S

P 1

wap exona

2

1a 2

FIG. 2. Wap and actin mRNA expression in vitro. Poly(A)+ RNA was prepared from cocultures prior to or after hormone addition and then analyzed by RNase protection assays (A, C) or Northern blotting (B). The lower panel shows the probe used to detect Wap mRNA. The radiolabeled antisense probe transcribed from this construct protects two fragments of 110 nucleotides (Wap exon 1) and 135 nucleotides (Wap exon 2), respectively. S, SacI; P, PstI. (A) Wap gene expression in cocultured organoids. Cocultures were maintained for 4 days in basal medium without HP [lane 1, -Day (d)l, -HP]. HP was added (+HP) and RNA was extracted after 1 day (lane 2, Day l), 5 days (lane 3, Day 5), 10 days, (lane 4, Day lo), 15 days (lane 5, Day 15), or 20 days (lane 6, Day 20). Control cultures were maintained in the absence of HP for 14 days (lane 7, Day 10, -HP). Each lane represents 2.5 pg poly(A)+ RNA. The migration of HpaII digested, denatured pBR322 DNA size marker is indicated in nucleotides on the right. (B) Actin mRNA expression in cocultures. Each lane represents 5 pg of poly(A)+. Lane 1, 1 day (-Day 1) before hormone addition; lane 2, 5 days +HP; lane 3,10 days +HP, lane 4,15 days +HP; lane 5,20 days +HP; lane 6, 10 days -HP. (C) Absence of Wap gene expression in cocultures of dissociated epithelial cells. 2.5 pg poly(A)+ RNA from cocultures exposed to HP for 10 days were analyzed by RNase protection assay (lane 1, Day 10 +HP). H, hydrocortisone; P, prolactin.

the lo-day culture period abolishes Wap mRNA expression regardless of the substrate (Fig. 2A, lane 7 and Fig. 3A, lanes 1, 3, 5). These experiments demonstrate that, in addition to the cell-cell interactions present in intact glandular subcomponents, interaction of the epithelium with the 3T3-Ll adipocytes is important for effective Wap mRNA induction. Synergistic A&m Expression by

of Hormones in Wap Gene Cocultured with Adipocytes

Organds

To study further the hormone requirements for optimal Wap mRNA expression in vitro, we tested the effect

Gene

We have described elsewhere the mammary glandspecific and lactogenic hormone-dependent expression of the murine c-myc oncogene subjected to the control of the Wap gene promoter in transgenic mice (Schoen-

A. PL /--+-+-+F

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EM

3T3-Ll

3T3-Ll +

1

PH

PI HI

123

FIG. 3. Effect of substrate and hormones on Wap mRNA expression. RNase protection assays with 2.5 pg poly(A)+ RNA hybridized to the Wap-specific antisense probe are shown. (A) Organoids were plated on plastic (PL), on collagen type I (Coll), and on EHS reconstituted basement membrane (BM). Cultures were incubated for 10 days with (lanes 2, 4, 6) or without (lanes 1, 3, 5) hormone induction after an initial culture period of 4 days in basal medium. Wap mRNA expression levels before hormone stimulation are shown for organoids cultured on BM (lane 7, -Day 1). (B) Expression of Wap mRNA in organoids plated on 3T3-Ll adipocytes after 10 days of stimulation with HPI. (C) Organoids cocultured with 3T3-Ll adipocytes were stimulated for 10 days with HP (lane l), PI (lane 2), or HI (lane 3). The size of the protected fragments is indicated on the right. P, prolactin; H, hydrocortisone; I, insulin.

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enberger et a& 1988). In the hybrid gene construct, the expression of the c-rngc gene is governed by 2.5-kb of Wap regulatory sequence. We used organoids from these transgenic mice in our coculture system to analyze whether the elements responsive to prolactin and hydrocortisone were contained within the 2.5-kb 5’Wap regulatory sequence; this would result in a similar induction of the Wap-myc transgene and the endogenous Wap gene upon exposure to lactogenic stimuli. Organoids were prepared from the fourth abdominal mammary glands of midpregnant transgenic Wap-myc mice during their first pregnancy. 250- to 53-pm fractions were cocultured with 3T3-Ll adipocytes either in the presence or absence of laetogenic hormones. Poly(A)+ mRNA was isolated after different periods of coculture and analyzed by RNase protection assays. The time course of induction of the endogenous Wap gene in response to lactogenic stimuli is presented in Fig. 4. Addition of lactogenic hormones induces expression of the endogenous Wap gene and maximal Wap mRNA levels are detected on Day 10 (Fig. 4, lane 4). The decrease in Wap gene expression following maximal in-

P I 5 I

,

4 135

4110

1234567 FIG. 4. In vitro induction of the endogenous Wap gene in organoids from transgenic mice. Poly(A)+ RNA from cocultures of organoids isolated from midpregnant Wap-myc transgenic mice was analyzed in RNase protection assays. Culture and assay conditions were as in Fig. 2 except that 0.5 pg and 1.5 fig poly(A)’ RNA were used in lanes 2 and 7, respectively. The size of the protected fragments is indicated on the right. H, hydrocortisone; P, prolactin.

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duction that is observed in organoids from CD-l mice (Fig. 2, lanes 3-6) does not occur in the transgenic organoids, rather Wap mRNA continues to be expressed at similar levels for the remaining culture period (Fig. 4, lanes 4-6). In the absence of lactogenic hormones, organoids stop expressing the endogenous Wap gene after the initial 4 days of coculture (Fig. 4, lane 1) and Wap mRNA is not induced by extended coculturing (Fig. 4, lane 7). Thus, expression of the endogenous Wap gene depends on lactogenic hormones in cocultured organoids from both CD-1 or transgenic Wap-myc mice but appears to occur with somewhat different kinetics. The expression of the Wap-myc transgene was analyzed using an antisense probe indicative of the first exon of the Wap-myc hybrid gene. The probe protects a fragment of 198 nucleotides. Wap-myc-specific transcripts are not detected after 4 days of coculture in the absence of hormones (Fig. 5, lane 1). Transgene expression is induced by hydrocortisone and prolactin and appreciable amounts of Wap-myc transcripts are detected on Day 5 of hormone exposure (Fig. 5, lane 3). Analagous to the endogenous Wap gene induction, no significant increase or decrease of Wap-myc mRNA levels occurs between Day 5 and Day 20 of hormone treatment (Fig. 5, lanes 4-6). In organoids from transgenic mice, the kinetics of hormone-dependent Wap-myc mRNA and endogenous Wap mRNA induction are comparable. Likewise, transgenic Wap-myc gene expression is effectively induced only by the combination of hydrocortisone, prolactin, and insulin (data not shown). It appears that transcription of a reporter gene regulated by Wap promoter region also requires the synergistic action of all three hormones. We have previously shown that the level of hybrid transcripts in lactating mammary glands of transgenic mice is significantly lower than the level of endogenous Wap mRNA (Schoenenberger et al, 1988). This difference was also observed in organoid cocultures throughout the time course of induction, further indicating that the coculture system reflects the in viva situation. The absence of an apparent Wap-myc signal on Day 1 after hormone stimulation (Fig. 5, lane 2) is probably due to the reduced expression of Wap-myc gene relative to the endogenous Wap gene (Fig. 4, lane 2), resulting in mRNA levels below the limits of detection. The similarities between the expression patterns of the transgenic Wap-myc and the endogenous Wap gene in organoid cocultures suggest that the 2.5-kb Wap promoter region is sufficient to confer hormone responsiveness to a linked reporter gene in vitro. DISCUSSION

In culture, mammary gland-specific traits are often altered such that complete differentiation is not at-

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334 nI

+HP

4198

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Wap-myc

exon 1

probe protected

S \

KP ::

I fragment

I w 198n

FIG. 5. Hybrid Wap-myc mRNA expression in cocultured organoids from transgenic mice. The probe used for RNase protection assays is diagrammed in the lower panel. This probe protects a fragment of 198 nucleotides of the chimeric Wap-myc exon 1. Organoids from transgenie mice were cultured as in Fig. 2 and mRNA samples were loaded correspondingly. The size of the protected fragment is indicated on the right. K, KpnI; P, PstI; S, SacI; H, hydrocortisone; P, prolactin.

tained. Therefore mammary epithelial cells may not be able to express the entire range of the tissue-specific genetic program in response to lactogenic hormones. Several culture systems that are suitable for stimulating casein synthesis and secretion do not promote Wap gene expression (Lee et aL, 1985; Rosen et ak, 1986; Park et al, 1989). In this report we describe efficient, hormone-dependent Wap gene induction in cocultured organoids. The coculture of organoids and 3T3-Ll adipocytes displays distinctive features also present in the intact gland, which appear to be critical for Wap regulation. Organoids retain their three-dimensional glandular architecture in vitro. Ultrastructural analysis reveals

duct-like structures that occasionally branch off and/or terminate in bulbous end-buds. Recent studies have shown that the establishment of multicellular alveolar-like structures on basement membrane gels coincides with an increase in hormone-dependent casein secretion into the luminal compartment (Barcellos-Hoff et aZ., 1989), suggesting that the three-dimensional organization represents an additional aspect of tissuespecific function. Our results support and expand upon this notion in that they indicate that histiotypic organization is important for a more complete functional differentiation including Wap gene expression upon hormone stimulation. It is likely that the cell heterogeneity and cell-cell interactions inherent in the organoids promote Wap gene expression. Disruption of the structural integrity of organoids abrogates hormone-induced Wap mRNA expression. Wiens et aZ. (1987) have shown that hormone-independent morphogenetic changes occur if dissociated mammary epithelial cells are cocultured with 3T3-Ll adipocytes, resulting in duct formation. However, hormone-induced synthesis of 0 casein was detected before duct formation, indicating that duct formation is not a prerequisite for casein induction (Wiens et al., 1987). In contrast, we detect hormone-dependent Wap mRNA only in intact cocultured organoids, and not if organoids are dissociated. Our data suggest that Wap gene regulation is influenced by distinct cell-cell interactions in organoids that do not develop between dissociated cells within 14 days of coculture. Reichmann et al. (1989) have described that coculturing of a mesenchymal cell line with an epithelial mammary cell line induces morphological differentiation which is conducive to hormone-dependent casein synthesis. The absence of Wap mRNA expression in this system (E. Reichmann, personal communication) is further indication that cell heterogeneity and specific epithelial cellcell interactions in organoids are necessary for Wap gene induction. The vectorial secretion of milk in a lactating mammary gland depends on the structural polarization of the epithelium, thus suggesting that polarization may promote functional differentiation. Luminal epithelial cells in cocultured organoids are polarized with apical cell surfaces facing a central lumen and basal surfaces exposed to the culture environment. The possibility that reconstituting this aspect of the in vivo situation could lead to promotion of functional differentiation has been emphasized by studies of Parry et al. (1987). They observed that if the COMMA 1D mammary epithelial cell line is cultured on a permeable support, thus allowing basal uptake of nutrients and hormones, synthesis and polarized secretion of al, a2, and p caseins are promoted. In our coculture system the polarity needs of

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of insulin, hydrocortisone, and prolactin is epithelial cells may be met, in that interaction with the combination medium with its complement of hormones occurs ba- required to induce a 50-fold increase of Wap mRNA in sally. Because accessibility of the basal surface is promurine mammary gland explants initiated at Days 8 to 10 of gestation (Pittius et al, 1988). Consistent with this vided by substrates that do not promote Wap mRNA finding, our coculture experiments show that the three expression, it cannot be the only determinant of comhormones together induce Wap mRNA expression most plete differentiation. efficiently. Along with the accumulation of Wap In a further attempt to approximate the in vivo state, in horwhere the glandular epithelium is surrounded by a pre- mRNA, we also observe cytodifferentiation dominantly adipose stroma, organoids were cultured in mone-stimulated organoids, reminiscient of the secrecontact with 3T3-Ll adipocytes (Levine and Stockdale, tory epithelium of pregnant and lactating mammary 1985). This coculture system features interactions be- glands. Important insights into the molecular mechanisms tween the organoids and the mesenchymal substrate by which hormones control gene expression have been that are involved in Wap gene regulation. These intergained by the transfection of genes and promoter-reactions do not appear to be reproduced by culturing porter gene constructs into cultured cells (Maniatis et organoids on other substrates, even though previous studies have shown that the ability of mammary epi- al, 1987). Hormone response elements in the promoter region of the rat /3 casein gene have recently been dethelial cells to retain their tissue-specific phenotypes and functional characteristics in vitro is influenced by fined by this approach (Doppler et ab, 1989). However, cell-substratum interactions (for review, see Bissell mammary epithelial cell lines transfected with Wap and Barcellos-Hoff, 1987). Our data indicates that hor- gene constructs have so far failed to show hormone-regmone stimulation does not result in Wap mRNA accu- ulated reporter gene expression (Rosen et aL, 1986; our unpublished observations). We have therefore used ormulation in organoids plated on plastic or on collagen ganoids isolated from transgenic Wap-myc mice to obtype I gel. In contrast, reconstituted basement memtain information on the regulatory potential present in brane derived from the EHS tumor has been more effective in eliciting tissue-like morphogenesis and horthe 5’ flanking sequence of the Wap gene. Using the coculture system, we demonstrate hormone-dependent mone-dependent casein production (Li et ab, 1987; Barcellos-Hoff et aL, 1989). We cultured organoids on the expression of a Wap fusion gene in vitro. Induction experiments with organoids from transgenic Wap-myc EHS matrix and did not detect an increase of Wap mRNA levels upon hormone stimulation, although or- mice do not reveal any significant difference in the reganoids maintained a three-dimensional organization quirements for the induction of the endogenous Wap (unpublished observation). Our results provide evidence gene and the Wap-myc gene construct. Our data suggest that 3T3-Ll adipocytes are a particularly effective sub- that the regulatory sequences that mediate the synerstrate in eliciting Wap gene expression. gistic action of insulin, hydrocortisone, and prolactin Interactions between mesenchymal cells and mamupon myc expression are located within 2.5-kb of 5’ Wap mary epithelial cells promote structural and functional flanking sequence. differentiation in vitro (Wiens et aL, 1987; Reichmann et In contrast to the induction experiments performed ak, 1989). Extracellular matrix molecules may mediate with organoids from nontransgenic CD-l mice, Wap the mesenchymal influence on epithelial differentiamRNA levels did not decrease during prolonged cocultion. In this context, it has been suggested that extraturing of organoids from transgenic mice, indicating cellular matrix components deposited by the mesenchythat myc expression interferes with Wap regulation. A ma1 cells provide the initial structural basis for the constitutive expression of the Wap-myc oncogene as assembly of a basal lamina (Reichmann et al, 1989). The well as the endogenous Wap gene has been found in the presence of a continuous basal lamina is a feature commammary tumors that frequently develop in transgenic mon to several experimental conditions that promote Wap-myc mice (Schoenenberger et ah, 1988). These obmammary differentiation (Wiens et ak, 1987; Barcellosservations support the notion that the expression of the Hoff et aZ., 1989), including the coculture system de- myc oncogene is correlated with the deregulation of the endogenous Wap gene. Furthermore, myc oncogene exscribed in this report. pression has not been demonstrated in a chemically inWap gene expression in the pregnant and lactating duced rat mammary carcinoma and expression of the mouse is governed by the complex interaction of steroid Wap gene remains hormone-dependent (Johnson et aL, and peptide hormones. Hydrocortisone and prolactin have been shown to modulate milk protein gene tran1985). scription (Doppler et al, 1989) as well as mRNA stabilThe present study extends the complement of hority (Rosen et aL, 1986), however the role of insulin in mone-responsive milk protein genes inducible in mammammary gland function is poorly understood. The mary epithelial cells in vitro and reflects an interesting

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aspect of mammary gland differentiation. Wap gene expression is dependent upon the three-dimensional organization of the glandular epithelium, on distinct cell-cell and cell-substratum interactions, and on the synergistic action of hormones. The studies address the complex cellular and extracellular signaling that exists in the mammary gland to ensure the full expression of functional differentiation in response to lactogenic hormones. Our coculture system now makes feasible further elucidation of the molecular mechanisms governing Wap gene regulation. Moreover, using organoids from transgenic Wap-myc mice allows one to elucidate the effect of myc oncogene expression on these mechanisms and on the differentiation process of mammary epithelial cells. We acknowledge the technical assistance of Beatrice Dolder (Basel) and Franziska Fltickiger (Bern). We express our appreciation to Drs. Roland Ball (Base]) and Rick Lathe (Strashourg) for inspiring discussions. We also thank Drs. Jean Wilson (Boston) and Peter Hollenbeck (Boston) for helpful criticism and revision of the manuscript. The experiments described in this publication were in part carried out at the Ludwig Institute for Cancer Research in Bern, Switzerland, and supported in part by CA 08380 and HD00143 from National Institutes of Health. A preliminary report of these results has appeared in abstract form (Schoenenberger et al, 1986, Eur. J. Cell Bid). Note added in proc$ During the revision of this manuscript, a publication describing Wap expression in vitro has appeared (Chen, L.-H. and Bissell, M.J., 1989. A novel regulatory mechanism for whey acidic protein gene expression. Cell ReguL 1,45-54). REFERENCES ANDRES, A.-C., SCHOENENBERGER, C.-A., GRONER, B., HENNIGHAUSEN, L., LEMEUR, M., and GERLINGER, P. (1987). Ha-ras oncogene expression directed by a milk protein gene promoter: Tissue specificity, hormonal regulation and tumor induction in transgenic mice. Proc. Natl. Acad Sci USA 74,1299-1303. BARCELLOS-HOFF, M. H., and BISSELL, M. J. (1989). Mammary epithelial cells as a model for studies of the regulation of gene expression. In “Functional Epithelial Cells in Culture” (K. S. Matlin and J. D. Valentich, Eds.), pp. 399-433. A. R. Liss, New York. BARCELLOS-HOFF, M. H., AGGELER, J., RAM, T. G., and BISSELL, M. J. (1989). Functional differentiation and alveolar morphogenesis of primary mammary cultures on reconstituted basement membrane. Development 105,223~235. BISSELL, M. J., and BARCELLOS-HOFF, M. H. (1987). The influence of extracellular matrix on gene expression: Is structure the message? J. Cell Sci SuppL 8,32X%3. BISSELL, M. J., and HALL, H. G. (1987). In “The Mammary Gland: Development, Regulation, and Function” (M. Neville and C. W. Daniel, Eds.), pp. 97-146, Plenum, New York. BLUM, J. L., ZEIGLER, M. E., and WICHA, M. S. (1987). Regulation of rat mammary gene expression by extracellular matrix components. Exp. Cell Res. 173,322-340. CHIRGWIN, J. M., PRZYBYLA, A. E., MACDONALD, R. J., and RUTTER, W. J. (1979). Isolation of biologically active ribonucleic acid from sources enriched in ribonuelease. Biochemistry l&5294-5299. COON,M. G., and MANASEK, F. J. (1970). Electron microscopy of culture cells. Cam Inst. Yearbook 68,540-542.

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