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© The Rockefeller University Press,
0021-9525/1998//533 $5.00
The Journal of Cell Biology, Volume 143, Number 2,
, 1998 533-545
Regular Articles |
Reconstitution of Mammary Gland Development In Vitro: Requirement of c-met and c-erbB2 Signaling for Branching and Alveolar Morphogenesis
We have established a cell culture system that reproduces morphogenic processes in the developing mammary gland. EpH4 mouse mammary epithelial cells cultured in matrigel form branched tubules in the presence of hepatocyte growth factor/scatter factor (HGF/SF), the ligand of the c-met tyrosine kinase receptor. In contrast, alveolar structures are formed in the presence of neuregulin, a ligand of c-erbB tyrosine kinase receptors. These distinct morphogenic responses can also be observed with selected human mammary carcinoma tissue in explant culture. HGF/SF-induced branching was abrogated by the PI3 kinase inhibitors wortmannin and LY294002. In contrast, neuregulin- induced alveolar morphogenesis was inhibited by the MAPK kinase inhibitor PD98059. The c-met–mediated response could also be evoked by transfection of a c-met specific substrate, Gab1, which can activate the PI3 kinase pathway. An activated hybrid receptor that contained the intracellular domain of c-erbB2 receptor suffices to induce alveolar morphogenesis, and was observed in the presence of tyrosine residues Y1028, Y1144, Y1201, and Y1226/27 in the substrate-binding domain of c-erbB2. Our data demonstrate that c-met and c-erbB2 signaling elicit distinct morphogenic programs in mammary epithelial cells: formation of branched tubules relies on a pathway involving PI3 kinase, whereas alveolar morphogenesis requires MAPK kinase.
Key Words: development of mammary gland • hepatocyte growth factor • neuregulin • PI3 kinase • MAPK kinase
Abbreviations used in this paper: EGF, epidermal growth factor; HGF/ SF, hepatocyte growth factor/scatter factor; KGF, keratinocyte growth factor; NGF, nerve growth factor.
GROWTH and morphogenesis of epithelia are essential processes in the development of many organs, which are driven not only by an intrinsic genetic program but also by signals provided by neighboring cells and tissues. Accordingly, studies performed in organ culture and transplantation experiments have demonstrated that growth and morphogenesis of epithelia from the salivary gland, kidney, lung, or the mammary gland are controlled by mesenchymal–epithelial interactions (Grobstein, 1953; Spooner and Wessells, 1970; Saxen 1987; Sakakura, 1991). The identification of molecules that provide the essential signals exchanged in mesenchymal–epithelial interactions is an area of active research. Recent evidence suggests that morphogenic programs of epithelia can be triggered by mesenchymal factors that signal via tyrosine kinase receptors (Montesano et al., 1991a,b; Schuchardt et al., 1994; Sutherland et al., 1996; for review see Birchmeier and Birchmeier, 1993). This is also supported by genetic experiments in mice that illustrate the importance of mesenchymal ligands of epithelial tyrosine kinases during development of the kidney, lung, and liver (Peters et al., 1994; Schuchardt et al., 1994; Bladt et al., 1995; Schmidt et al., 1995; Uehara et al., 1995; Moore et al., 1996; Pichel et al., 1996; Sanchez et al., 1996). Similarly, branching of trachea in Drosophila is controlled by a fibroblast growth factor-like molecule produced in surrounding mesenchymal cells (Sutherland et al., 1996).
We have previously identified two mesenchymal ligands of epithelial receptor tyrosine kinases, hepatocyte growth factor/scatter factor (HGF/SF)1 and neuregulin, to be important for development of the mammary gland (Yang et al., 1995). In whole organ cultures of mouse mammary glands, HGF/SF promotes branching of ductal trees and inhibits terminal differentiation, as assessed by the expression of milk proteins. In contrast, neuregulin stimulates lobulo-alveolar differentiation and the production of milk proteins. Thus, different growth factors elicit distinct responses in organ culture of the mammary gland. This finding raises the question whether different cellular populations react to these factors, and how these different responses are evoked on a molecular level.
In collagen matrix, HGF/SF induces the formation of branched tubules from epithelial cells derived from the kidney (MDCK), mammary gland, and other organs (Montesano et al., 1991a,b; Berdichevsky et al., 1994; Brinkmann et al., 1995; Soriano et al., 1995). HGF/SF signals are mediated by the receptor tyrosine kinase c-met; in vivo, c-met is expressed by various epithelial cells, whereas HGF/SF transcripts are found in the mesenchymal compartment (Bottaro et al., 1991; Naldini et al., 1991; Sonnenberg et al., 1993; Weidner et al., 1993). The phosphorylated c-met receptor binds substrates such as PI3K, PLC-
, Grb2, and others (Ponzetto et al., 1994; Fixman et al., 1997). A recently identified c-met-specific substrate, Gab1 (Weidner et al., 1996; Nguyen et al., 1997) suffices to elicit branching morphogenesis in kidney epithelial cells.
Neuregulin has been shown to affect growth and differentiation of epithelial and other cell types in vitro (Peles et al., 1992; Bacus et al., 1993; Falls et al., 1993; Marchionni et al., 1993; Shah et al., 1994; Staebler et al., 1994; Marte et al., 1995). Neuregulin signals are mediated by direct interaction with the high affinity receptors c-erbB4 or c-erbB3; in addition, c-erbB2 acts as an essential coreceptor for the transmission of neuregulin signals (Plowman et al. 1993; Carraway and Cantley, 1994; Carraway et al., 1994; Sliwkowski et al., 1994). Neuregulin is expressed in neuronal and mesenchymal cells during mouse development (Orr-Urtreger et al., 1993; Meyer and Birchmeier, 1994; Yang et al., 1995), whereas the receptors are found in epithelial and other cell types (Kraus et al., 1989; Press et al., 1990; Prigent et al., 1992; Plowman et al., 1993; Meyer et al., 1997).
Previously, analyses of HGF/SF and neuregulin-evoked responses in epithelial cells were mainly conducted with cells grown in monolayers; such culture conditions do not allow the formation of the complex three-dimensional structures observed during organ development. In contrast, organ culture systems provide a good model for the analysis of morphogenic events. However, they preclude a biochemical analysis of signaling cascades activated by morphogenic factors due to their complex cellular composition. Obviously, a lack of appropriate systems exists which allows the study of signaling cascades responsible for morphogenic responses, for instance in mammary epithelial cells.
Here we use EpH4 mammary epithelial cells grown on matrigel and observe that two growth factors that signal via tyrosine kinase receptors, HGF/SF and neuregulin, elicit fundamentally distinct morphogenic responses in these pluripotent cells: HGF/SF induces the formation of branched tubules, and neuregulin evokes the formation of alveolar structures. The complex structures generated in culture resembles the ones formed during mammary gland development. The HGF/SF-induced tubular structures were also observed after transfection of a c-met substrate, Gab1. The alveolar structures produced by neuregulin could also be induced by nerve growth factor in cells transfected with a trk/c-erbB2 hybrid receptor; this indicates that the signals provided by c-erbB2 suffice to induce alveoli. By the use of mutant receptors and specific inhibitors, we could show that PI3 kinase provides an essential signal for branching morphogenesis; in contrast, the MAP kinase pathway plays an essential role for the formation of alveolar structures. Thus, in this cell culture system one single cell type, EpH4, responds by two entirely different morphogenic programs upon stimulation of distinct tyrosine kinase receptors. The distinct responses can moreover be correlated with the activation of different essential signaling cascades.
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Cell Culture on Matrigel
EpH4 cells are a derivative of IM-2 and were originally isolated from mammary tissue of a mid-pregnant mouse (Reichmann et al., 1989; Brinkmann et al., 1995; Lopez-Barahona et al., 1995). From EpH4 cells we subcloned the variant K6 (called EpH4/K6) which exhibits a pronounced morphogenic potential on matrigel.
Matrigel (basement membrane from Engelbreth-Holm-Swarm tumor) was obtained from Collaborative Biomedical Products (Serva, Germany), and 6-cm culture dishes were coated with 400 µl of this solution on ice. After incubation for 1 h at 37°C to allow the matrix to gel, 3.5 x 105 EpH4 cells were plated in DME containing 10% fetal bovine serum and the following hormones: 3 µg/ml bovine prolactin (Sigma Chemical Co., St. Louis, MO); 5 µg/ml insulin (Sigma Chemical Co.); 1 µg/ml hydrocortisone (Merck, Darmstadt, Germany), and neuregulin or HGF/SF at concentrations of 20 ng/ml (100 U/ml) or 3 nM (1 U/ml), respectively. After 1 d of culture, medium was replaced by serum-free DME containing factors and hormones and exchanged daily. Experiments were terminated after 6 d of culture. Inhibitors of the PI3 kinase (10 nM wortmannin and 25 µM LY294002) and MAPK kinase (50 µm PD98059) were added twice a day and experiments were terminated after 3 d of culture (Alessi et al., 1995; Derman et al., 1995; Keely et al., 1997; Khwaja et al., 1997).
Transfection of EpH4 Cells with cDNAs
Hybrid receptors containing the extracellular portion of trk (the nerve growth factor receptor) and the transmembrane and cytoplasmic region of epithelial receptor tyrosine kinases (trk/c-met, trk/KGFR, and trk/c-erbB2) have been described (Sachs et al. 1996). cDNA encoding a new hybrid receptor containing the cytoplasmic domain of c-erbB4, trk/c-erbB4, was constructed. Mutations were introduced into the kinase domain of trk/ c-erbB2 by an exchange of wild-type and mutant c-erbB2 sequences (Ben-Levy et al., 1994): trk/c-erbB2-P1 lacks all COOH-terminal substrate-binding domain of c-erbB2 but the sequences around the ultimate tyrosine residue, Y1253, which is directly fused to the c-erbB2 kinase domain; trk/ c-erbB2Y1253F contains the complete substrate-binding region except the fifth tyrosine residue, which is mutated to phenylalanine (Y1253F). The cDNAs of these hybrid receptors and of Gab1 (Weidner et al., 1996) were cloned into the pBAT expression vector; the expression plasmids were cotransfected with pSV2neo into EpH4/K6 mammary epithelial cells by the calcium phosphate technique. Cell clones producing the trk-hybrid receptors were identified (Sachs et al., 1996) and characterized for their morphogenic response on matrigel after addition of nerve growth factor (NGF). Control transfections were performed with pSV2neo only.
Explant Cultures of Human Mammary Tumors
Human breast tumors cultivated as xenografts in the mammary glands of nude mice (Naundorf et al., 1992) were dissected and cut into 1-mm pieces in medium 199 (GIBCO BRL, Eggenstein, Germany) supplemented with 5 µg/ml insulin and recombinant HGF/SF or neuregulin at a concentration of 20 ng/ml (100 U/ml) and 3 nM (1 U/ml), respectively. The tissue pieces were placed into 25 cm2 Falcon plastic flasks at a density of 30 explants per flask (Falcon Plastics, Cockeysville, MD). The explants were cultured for 8 d under an atmosphere of 5% CO2, with daily medium changes (Binas et al., 1992). For histological analysis, explants were fixed in 4% formalin, embedded in paraffin, sectioned (5 µm), and then stained with hematoxylin/eosin (Rivera, 1971).
Northern Hybridization
Total RNA was isolated from dispase-treated EpH4 cell aggregates as described (Chomczynski and Sacchi, 1987). 5–20 µg RNA were electrophoresed in 1% agarose–formaldehyde gels and transferred to Hybond-C-extra membranes (Amersham, Little Chalfont, UK) and hybridized to 32P-labeled cDNA probes for β-casein (Binas et al., 1992) and β-actin, respectively. The probes were labeled by random priming (109 cpm/µg; Feinberg and Vogelstein, 1984). Hybridization signals were autoradiographed and analyzed with a phosphoimager.
Light, Confocal, and Electron Microscopy
Cell aggregates of EpH4 cells after 6 d of culture were visualized using a Zeiss Axiovert 135 inverse microscope equipped with Nomarski optics (Carl Zeiss Inc., Thornwood, NY). For histological examination, organoids were fixed in situ with 2.5% glutaraldehyde, postfixed with OsO4, and contrasted with tannic acid and uranyl acetate. Specimens were dehydrated in a graded ethanol series and embedded in Epon 812. For light microscopy, semithin sections (0.5 µm) were stained with toluidin blue and analyzed in a Zeiss Axiophot light microscope. Ultrathin sections (50–70 nm) were contrasted with lead citrate and analyzed in a Zeiss EM 10 electron microscope.
For immunohistological analysis, organoids were fixed with 4% formaldehyde in PBS and treated with 0.5% Triton X-100. Antibodies used were monoclonal rat anti–E-cadherin (DECMA-1, Vestweber and Kemler, 1985), rabbit anti–β-casein (Binas et al., 1992), CY3-conjugated goat anti– rat and CY5-conjugated goat anti–rabbit antibodies. Nuclei were stained with quinacrine mustard or Sytox (Molecular Probes, Eugene, OR). The whole mounts were analyzed using a Leica TCS confocal microscope (Leica, St. Gallen, Switzerland).
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The effect of HGF/SF on the expression of β-casein during formation of tubules was analyzed by Northern blot hybridization (Fig. 3). HGF/SF strongly inhibited β-casein mRNA expression in a concentration-dependent fashion; inhibition was maximal at 20 ng/ml HGF/SF (Fig. 3 C). Apparently, formation of branching tubules of EpH4 mammary epithelial cells does not allow concomitant functional differentiation.
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Morphogenic activities of various factors on mammary gland development have been examined; for instance, EGF (or TGF-
) and TGF-β influence ductal and alveolar development (Silberstein and Daniel, 1987; Vonderhaar, 1987; Coleman et al., 1988; Jhappan et al., 1990, 1993; Robinson et al., 1991; Snedeker et al., 1991; Daniel and Robinson, 1992; Pierce et al., 1993); these factors are predominantly produced in an autocrine fashion by epithelial cells and are expressed throughout mammary gland development. Since mesenchymal–epithelial interactions are essential for pre- and postnatal development of the mammary gland (Kratochwil, 1976, 1987; Sakakura et al., 1976, 1991; Durnberger et al., 1980; Cunha et al., 1992; Cunha and Hom, 1996), we have focused on the functional analysis of mesenchymal ligands (HGF/SF and neuregulin) and epithelial tyrosine kinase receptors (c-met and c-erbBs) in this process. In vivo, HGF/SF and neuregulin are expressed specifically during puberty and pregnancy in the mesenchyme of the mammary gland, and promote branching and lobulo-alveolar morphogenesis in whole organ culture, respectively (Yang et al., 1995). The HGF/SF receptor c-met and the neuregulin receptors (erbB2, erbB3, and erbB4) are produced in the outer epithelial cells of the ducts (Yang et al., 1995). Here we show that HGF/SF induces growth and morphogenesis of EpH4 mammary epithelial cells in matrigel; long multilayered tubes are formed from loose aggregates or small spheroids. Interestingly, HGF/SF inhibits the expression of β-casein, indicating that a morphogenic program without concomitant functional differentiation is activated. This is consistent with processes that are observed in vivo: milk production is blocked during tubular branching. HGF/SF-induced tubule formation and branching of mammary gland cells was also previously reported in collagen matrix (Berdichevsky et al., 1994; Brinkmann et al., 1995; Soriano et al., 1995); concomitant inhibition of functional differentiation had not been observed and might be a specific response to the specific substrate used here, i.e., matrigel. Neuregulin exerts an entirely different response of EpH4 mammary epithelial cells in matrigel: alveolar-like structures are induced which consist of single-layered epithelia. Thus, we assign here a morphogenic role to neuregulin in mammary epithelial cells; such a function is in accordance with previous whole organ culture experiments (Yang et al., 1995). Our data thus show that signaling of two different tyrosine kinase receptor types, c-met and c-erbB, elicits distinct morphogenic responses in a single mammary gland epithelial cell line. In whole organ cultures (Yang et al., 1995), HGF/SF and neuregulin stimulated preexisting structures, i.e., the factors enhanced either tubular or alveolar morphogenesis. Here we show that HGF/SF and neuregulin can induce these two morphogenic programs from cell aggregates that are apparently pluripotent.
Epithelial cells derived from the kidney, breast and other organs respond to SF/HGF and c-met by the formation of branched tubules when grown in a collagen matrix (Montesano et al., 1991a,b; Weidner et al., 1993; Berdichevsky et al., 1994; Soriano et al., 1995; Brinkmann et al., 1995). Furthermore, several tyrosine kinase receptors were reported to affect epithelial cells (e.g., trk, c-ros, the KGF-R) but only c-met induces tubulogenesis (Sachs et al., 1996). Although a variety of substrates were found to bind to the tyrosine phosphorylation sites in the COOH terminus of c-met (Ponzetto et al., 1994; Fixman et al., 1995; Weidner et al., 1995), a substrate which can mediate the signal responsible for branching morphogenesis, Gab1, has only recently been identified (Weidner et al., 1996). Gab1 binds specifically to c-met but not to various other receptor tyrosine kinases. We show here that Gab1 also promotes the formation of branched tubules from EpH4 mammary epithelial cells. Thus, Gab1 is an important substrate that transduces this morphogenic signal in various epithelial cell types. In a recent report it was shown that Stat 3 is also required for the formation of branching structures (Boccaccio et al., 1998). The production of milk constituents in the mammary gland is under tight hormonal control and influenced by extracellular matrix components. Stat 5 is an essential factor for the prolactin-controlled β-casein gene expression (Happ and Groner, 1993; Schmidhauser et al., 1992; Sympson et al., 1994; Groner et al., 1994; Wakao et al., 1995; Streuli et al., 1995). c-met signaling might thus interfere with the activity of Stat 5.
Neuregulin signaling requires the c-erbB3 or c-erbB4 receptors, which bind neuregulin with high affinity, and c-erbB2, that acts as an essential coreceptor (Kita et al., 1994; Carraway et al., 1994; Tzahar et al., 1994; Carraway and Burden, 1995; Marikovsky et al., 1995; Karunagaran et al., 1995; Meyer and Birchmeier, 1995; Lee et al., 1995; Gassmann et al., 1995; Riese et al., 1995; Pinkas-Kramarski et al., 1996; Riethmacher et al., 1997). The c-erbB2 receptor binds substrates like Shc (Segatto et al., 1993), Grb2 (D'souza and Taylor-Papadimitriou, 1994), PLC- (Songyang et al., 1993; Ben-Levy et al., 1994), and Grb7 (Stein et al., 1994). It has recently been shown that of the phosphorylated tyrosine residues found in the COOH-terminal substrate-binding domain of c-erbB2, tyrosine Y1144 (tyrosine 2) binds Grb2, and Y1227 (tyrosine 4) binds Shc (Dankort et al., 1997); both substrates signal through the ras pathway. The signaling capacities of Y 1201 (tyrosine 3) and Y1253 (tyrosine 5) are unknown. Tyrosines 1–4 were found to be necessary for the mitogenic response of c-erbB2 (Dankort et al., 1997); others observed that tyrosine residue 5 was required for this activity (Ben-Levy et al., 1994). By the use of a trk/c-erbB2 hybrid receptor, we demonstrate here that c-erbB2 induces alveolar-like differentiation in mammary epithelial cells without activation of coreceptors. Furthermore, a hybrid receptor of c-erbB2 containing tyrosines 1–4 is sufficient to induce alveolar structures; a hybrid containing only tyrosine 5 is not.
We have examined inhibitors that specifically interfere with signaling pathways activated by HGF/SF and neuregulin to analyze their effect on the morphogenic response evoked by these factors. The c-met receptor can potentially signal through ras and PI3 kinase (Hartmann et al., 1994; Ponzetto et al., 1994; Royal and Park, 1995; Royal et al., 1997). Signaling through ras and the resulting cellular growth require the substrate Grb2, which binds to tyrosine residue Y1354 of c-met (i.e., the second tyrosine residue of the bidentate docking site) (Ponzetto et al., 1994; Fixman et al., 1995; Nguyen et al., 1997). Motility and morphogenic responses evoked by c-met require the substrate Gab1, which binds strongly to Y1347 (the first residue of the bidentate docking site; Weidner et al., 1996; Nguyen et al., 1997). Gab1 can also bind to Y1354, but in the presence of Grb2 binding to Y1347 is preferred and stabilized (Nguyen et al., 1997). Furthermore, Gab1-binding blocks the SH3 domain of Grb2 (Holgado-Madruga et al., 1996), and Gab1 harbors three PI3 kinase-binding sites. Indeed, we find that inhibitors of PI3 kinase (wortmannin and LY294002) specifically reduce HGF/SF-induced formation of tubules. This suggests that branching morphogenesis depends on the activity of pathways requiring PI3 kinase. In contrast, alveolar morphogenesis of EpH4 cells by c-erbB2 could be blocked by inhibitors of MAPK kinase, i.e., PD98059. Thus, components of the ras/MAP kinase pathways play an essential role in alveolar morphogenesis. It will now be important to identify which substrates (or substrate combinations) elicit the alveolar morphogenesis that is induced by the c-erbB2 receptor.
The morphogenic activities of HGF/SF and neuregulin can be used to reconstitute further steps of postnatal mammary gland development in vitro. In the matrigel system established here, interactions of morphogenic factors with other mediators of growth and differentiation such as hormones, extracellular matrix components, or other mesenchymal factors can be studied in the future. Furthermore, downstream signaling processes that elicit the different morphogenic programs and eventually result in changes in gene expression are now amenable to investigation.
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This work was supported by grants of the Deutsche Krebshilfe (Bonn, Germany).
Submitted: 21 March 1997
Revised: 26 August 1998
Address all correspondence to W. Birchmeier, Max-Delbrück-Center for Molecular Medicine, Robert-Rossle-Strasse 10, D-13122 Berlin, Germany. Tel.: (49) 30-9406-3800. Fax: (49) 30-9406-2656. E-mail: wbirch{at}mdc-berlin.de
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) or presence of HGF/ SF (•), as analyzed by Northern blotting. Radioactive counts are expressed using a phosphoimager. (C) Concentration dependence on HGF/SF of the inhibition of β-casein expression. Control in the absence of HGF/SF was set to 100%.
