Max-Delbrück-Center for Molecular Medicine, D-13122 Berlin, Germany
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 ![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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.
![]() |
Materials and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Recombinant HGF/SF and Neuregulin
Recombinant HGF/SF was produced in Sf9 insect cells using the baculovirus expression system as described (Weidner et al., 1993; Brinkmann et
al., 1995
). For production of neuregulin, the coding sequence (corresponding to amino acids 20-239) of a mouse
2 isoform (Wen et al., 1992
; Meyer
and Birchmeier, 1994
) was fused in frame to the signal sequence of the
baculovirus expression vector pAcGP67 (Dianova, Hamburg, Germany)
followed by bulk transfection of Sf21 insect cells using calcium phosphate.
Recombinant baculovirus was selected by plaque screening and biological
assay, and high-titer virus was used to infect Sf21 cells in serum-free Excell 401 medium (Seralab, Crowley Down, UK) supplemented with 0.1%
Pluronic F-68 (Life Technologies, Gaithersburg, MD). Conditioned medium was clarified after 3 d of culture, and neuregulin was purified on a
heparin-Sepharose column (Pharmacia, Uppsala, Sweden) by the elution
by a 0.4-1.2-M NaCl gradient. The fractions were tested for biological activity of neuregulin in the morphology assay on MDA MB 453 cells (Plowman et al., 1993
) and for protein on a silver-stained gel. Neuregulin with
highest biological activity eluted at 0.8-1.0 M NaCl.
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 × 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
-casein (Binas et al., 1992
) and
-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-
-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).
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Effect of HGF/SF on Morphogenesis and Functional Differentiation of EpH4 Mammary Epithelial Cells
EpH4/K6 mouse mammary epithelial cells (Reichmann et
al., 1989; refer to Materials and Methods) cultured on
matrigel in the presence of hormones grow slowly, form
small spheroids and secrete milk proteins. Lumina in these
structures are surrounded by a single layer of epithelial
cells (Fig. 1 a). This resembles the appearance of primary
or CID9 mammary epithelial cells grown in matrigel (Bissell and Ram, 1989
; Wicha et al., 1982
; Wilde et al., 1984
;
Hahm and Ip, 1990a
,b; Schmidhauser et al., 1992
). When HGF/SF was added to the EpH4/K6 cell cultures, fast
growing, long tubular structures that can reach a length of
several millimeters were observed (Fig. 1 b). 44% of the
HGF/SF-induced aggregates were tubular, of which 26%
were branched (two to seven branches per structure, Fig. 2
B). In control cultures, 90% of the aggregates were small
and round, and only 3% showed an elongated structure (Fig. 2 A). The K6 subline of EpH4 cells produces particularly pronounced tubular structures in the presence of
HGF/SF; however, these structures were also observed
with the original EpH4 cells, albeit at lower frequency. 20 ng/ml of HGF/SF produced an optimal morphogenic response of EpH4/K6 cells. Histological analysis confirmed that in the absence of HGF/SF, EpH4/K6 cells form loose
aggregates or small spheroids, and that the lumina are
lined by a layer of flattened cells (Fig. 1 d; see also below).
The tubular structures induced by HGF/SF consist of several layers of cuboidal cells lining the elongated lumina
(Fig. 1 e). We also tested other growth factors: epidermal
growth factor (EGF) stimulated growth but did not induce
branched tubules, whereas keratinocyte growth factor (KGF) had a moderate effect on growth but did not elicit
morphogenic responses.
|
|
EpH4/K6 cells were transfected with the cDNA of the
recently identified new substrate of c-met, Gab1 (Weidner
et al., 1996; refer to Materials and Methods). When grown
in matrigel, the transfected cells produced tubular and
branched structures, now in the absence of HGF/SF (Fig. 1
c). This was not observed with cells transfected with control plasmids (pSV2neo). The histology of the Gab-1-
induced structures was identical to those observed in the
presence of HGF/SF (Fig. 1 f). Branched tubular structures could also be produced in EpH4/K6 cells transfected
with a trk/c-met receptor hybrid (Sachs et al., 1996
) when
the cells were stimulated by NGF (data not shown). EpH4/
K6 cells transfected with trk/KGFR or trk did not produce
branched tubules in the presence of NGF. Together, these
data demonstrate that the formation of branching structures of EpH4 cells requires specific signals provided by
the c-met receptor tyrosine kinase.
The effect of HGF/SF on the expression of -casein during formation of tubules was analyzed by Northern blot
hybridization (Fig. 3). HGF/SF strongly inhibited
-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.
|
Effect of Neuregulin on the Morphogenesis of EpH4 Mammary Epithelial Cells
Neuregulin produces an entirely different response in EpH4/K6 cells: fast growing, large alveolar-like structures are formed (Fig. 4 a, compare with the small aggregates in the control in Fig. 1 a). The concentration of 3 nM of neuregulin was more effective than higher or lower concentrations. Histological analysis demonstrated that the alveoli consisted of single-layered, highly columnar epithelial cells (Fig. 4 c, see also below). Statistical analysis revealed that 75% of the structures had an alveolar-like morphology and contained a lumen (Fig. 2 c): the aggregates were generally larger than 100 µm, and 15% were larger than 200 µm. The original EpH4 cells produced the same structures, but at lower frequencies. In contrast, only 5% of the aggregates formed by cells grown in the absence of neuregulin exhibited large alveolar structures (Fig. 2 A). Other growth factors tested, like EGF or KGF, did not induce alveolar structures (data not shown).
|
EpH4/K6 cells were then transfected with the cDNAs of
receptor hybrids consisting of extracellular trk (the NGF
receptor) and intracellular c-erbBs (Sachs et al., 1996; see
also Materials and Methods). Remarkably, stable clones
of EpH4/K6 mammary epithelial cells which expressed the
hybrid trk/c-erbB2 receptor produced alveolar-like structures in the presence of NGF (Fig. 4 b, see also the statistics in Fig. 5 A). These structures were histologically identical to the neuregulin-induced structures of EpH4/K6
cells (Fig. 4 d). Control transfectants with pSV2neo, trk, or
a trk/KGFR hybrid (Sachs et al., 1996
) did not show a
morphogenic response in matrigel (data not shown). Similarly, stable cell clones expressing a trk/c-erbB4 hybrid receptor did not form alveolar structures in response to
NGF, although NGF-dependent tyrosine phosphorylation of the receptor was observed (data not shown). We then
examined EpH4/K6 cells expressing mutants of trk/c-erbB2
to identify essential docking sites in the erbB2 receptor
that elicit this morphogenic response: a mutant of c-erbB2
that contains only the ultimate tyrosine residue P1 (Y1253;
Ben-Levy et al., 1994
) was inactive (Fig. 5 B), whereas a
mutant containing the residual tyrosine residues (Y1028,
Y1144, Y1201, and Y1226/27) produced alveoli (Fig. 5 C).
This indicates that tyrosine residue Y1028, Y1144, Y1201,
and Y1226/27 of c-erbB2, but not Y1253, are important for mediating the neuregulin-induced morphogenic response.
Moreover, these findings demonstrate that an activated
trk/erbB2 receptor is capable of eliciting a specific morphogenic response in mammary epithelial cells, formation
of alveolar-like structures, in the absence of active c-erbB3
and c-erbB4 coreceptors.
|
Effect of Inhibitors of PI3 Kinase and MAPK Kinase on the Formation of Morphogenic Structures of EpH4 Cells
Addition of wortmannin and LY294002 to EpH4/K6 cells significantly reduced the production of tubular structures by HGF/SF, whereas PD9805 was without effect (Fig. 6). In contrast, PD98059 quenched the induction of large alveoli, whereas wortmannin was without effect. These results suggest that the formation of tubular structures by HGF/SF/c-met requires pathways involving PI3 kinase, whereas alveoli formation depends on the activity of MAP kinase pathways.
|
The effects of HGF/SF and neuregulin seen on matrigel
reveal striking similarities to the results obtained in whole
organ culture (Yang et al., 1995). Since HGF/SF and neuregulin are expressed sequentially during mammary gland
development, we also added the factors in a consecutive
manner to EpH4 cells. We thus cultured the cells on matrigel for 4 d first in the presence of HGF/SF, removed HGF/
SF and cultured the cells in the presence of neuregulin for
an additional 4 d. Complex structures were formed consisting of branched tubules with adherent alveoli along the
tubes and at branching points (data not shown). In these
sequential experiments, expression of
-casein was examined: HGF/SF inhibited
-casein expression in the first
phase; removal of HGF/SF allowed re-expression of
-casein (Fig. 7, A and B). Addition of neuregulin increased
-casein expression slightly compared with control
levels.
|
Differential Effects of HGF/SF and Neuregulin on the Polarity of EpH4 Cells and on Expression of Cell Adhesion Molecules
The ultrastructure of the HGF/SF and neuregulin-induced
aggregates was examined, and the distribution of the cell
adhesion molecule E-cadherin and the expression of the
differentiation marker -casein were visualized by immunofluorescence analysis. In the control aggregates, cells that
surround lumina contain flat nuclei and many vesicles with
electron-dense material, likely representing casein-containing vesicles (Fig. 8 a). Immunofluorescence analysis
revealed that
-casein expression was high in the controls; E-cadherin was evenly distributed along all cell surfaces,
suggesting only moderate polarization of the cells (Fig. 9, a
and arrows in d);. The HGF/SF-induced tubular structures
were composed of more cuboidal cells with round nuclei
(Fig. 8 b).
-Casein production was reduced by HGF/SF,
as assessed by the reduction in numbers of vesicles and by
the reduction of immunofluorescence-staining intensity
(Fig. 8 b and Fig. 9, b and e). E-cadherin was largely distributed along the whole cell surface (Fig. 9 b, arrows in e).
However, the cells in neuregulin-induced alveoli showed
higher degree of polarization, as revealed by the columnar
cell shape and the elongation of nuclei along the apical-
basal axis (Fig. 8 c). E-cadherin was now predominantly
located at lateral membranes, in accordance with the more
pronounced polarization of the cells (Fig. 9, c and arrows
in f).
-Casein was strongly expressed, as assessed by the
increased numbers of vesicles and the staining intensity
observed by immunofluorescence analysis (Fig. 8 c and Fig. 9, c and f).
|
|
Morphogenesis Responses to HGF/SF and Neuregulin on Explants of Human Breast Carcinoma Cells
To analyze whether the different morphogenic responses
are restricted to EpH4 cells, we examined morphogenic
responses in explant culture of human breast carcinomas
obtained as xenografts. Xenografts of estrogen-responsive
and -nonresponsive tumors were dissected into 1 × 1-mm
pieces and cultured in the presence or absence of HGF/SF
or neuregulin (refer to Materials and Methods for details).
In the absence of factors, the explants from estrogen-dependent tumor 3366 (which was grown at minimal estrogen concentration in nude mice; Naundorf et al., 1992)
were mainly composed of stromal tissue containing scattered epithelial islands that showed little mammary gland-specific differentiation (Fig. 10, a and b and Fig. 11, a-c).
In the presence of HGF/SF, epithelial proliferation was strongly promoted and epithelial cells formed multilayered, cribiform, and papillary structures (Fig. 10, c and d
and c' and d'). Duct-like structures were also observed.
Thus, a similar multicellular organization was observed in
HGF/SF-treated tumor tissue as in EpH4 cells grown on
matrigel. No morphogenic response to HGF/SF was observed in the estrogen-independent human breast carcinoma 4151 (data not shown) (Naundorf et al., 1993
). In
contrast, addition of neuregulin to explants of the estrogen-responsive tumor 3366 produced an entirely different
response: growth of epithelial cells was moderately promoted, epithelial sheets were predominantly single layered and cells lining the newly formed lumina were well polarized (Fig. 11, d-f), Thus, tumor explants and EpH4 cells on
matrigel both form alveoli-like structures when exposed to
neuregulin. No morphological response of tumor 4151 was
detected in response to neuregulin (data not shown).
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We show here that EpH4 mammary gland epithelial cells
on matrigel respond in distinct manners when stimulated
with HGF/SF or neuregulin. HGF/SF induces branched
multilayered tubes and inhibits the production of milk
proteins. In contrast, neuregulin induces alveolar-like aggregates which often consist of a monolayered epithelium,
and allows -casein expression. HGF/SF and neuregulin can thus induce complex morphogenic programs in mammary gland epithelial cells, which are also observed in the
mammary gland during postnatal development. We used
this cell culture system for the biochemical analysis of signals elicited by HGF/SF and neuregulin: the formation of
branched tubules was found to be dependent on c-met and
a pathway involving Gab1 and PI3 kinase, whereas alveolar formation required c-erbB2 and components of the ras/
MAPK kinase pathway.
Morphogenic activities of various factors on mammary
gland development have been examined; for instance,
EGF (or TGF-) and TGF-
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
-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
-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.
![]() |
Footnotes |
---|
Received for publication 21 March 1997 and in revised form 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
V. Brinkmann's present address is Max-Planck-Institute for Infection
Biology, Monbijoustrasse 2, 10117 Berlin, Germany.
E. Spitzer's present address is Institute for Medicine of Molecular Diagnostic, Frankfurter Allee 65, 10247 Berlin, Germany.
G. Hartmann's present address is Glaxo Wellcome Research and Development, Gunnelswood Road, Stevenage SG1 2NY, UK.
The authors would like to thank C. Birchmeier (Max-Delbrück-Center, Berlin, Germany) for critically reading the manuscript, E. Reichmann (ISREC, Lausanne, France) for providing the EpH4 cell line, Y. Yarden (Weizmann Institute of Science, Rehovot, Israel) for providing the mutant c-DNAs of c-erbB2, and H.R. Gelderblom (Robert Koch Institute, Berlin) for using his electron microscopic facilities. We also thank R. Franke, R. Vogel, and B. Büttner for excellent technical assistance (all three from Max-Delbrück-Center). We dedicate this work to the memory of the late Prof. W. Zschiesche (Max-Delbrück-Center) who contributed invaluably to our work in mammary gland biology.
This work was supported by grants of the Deutsche Krebshilfe (Bonn, Germany).
![]() |
Abbreviations used in this paper |
---|
EGF, epidermal growth factor; HGF/ SF, hepatocyte growth factor/scatter factor; KGF, keratinocyte growth factor; NGF, nerve growth factor.
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. |
Alessi, D.R.,
A. Cuenda,
P. Cohen,
D.T. Dudley, and
A.R. Saltiel.
1995.
PD
098059 is a specific inhibitor of the activation of mitogen-activated protein
kinase kinase in vitro and in vivo.
J. Biol. Chem.
270:
27489-27494
|
2. | Bacus, S.S., A.V. Gudkov, C.R. Zelnick, D. Chin, R. Stern, I. Stancovski, E. Peles, N. Ben-Baruch, H. Farbstein, R. Lupu, et al . 1993. Neu differentiation factor (heregulin) induces expression of intercellular adhesion molecule 1: implications for mammary tumors. Cancer Res 53: 5251-5261 [Abstract]. |
3. | Ben-Levy, R., H.F. Paterson, C.J. Marshall, and Y. Yarden. 1994. A single autophosphorylation site confers oncogenicity to the Neu/ErbB-2 receptor and enables coupling to the MAP kinase pathway. EMBO (Eur. Mol. Biol. Organ.) J 13: 3302-3311 [Abstract]. |
4. |
Berdichevsky, F.,
D. Alford,
B. D'souza, and
J. Taylor-Papadimitriou.
1994.
Branching morphogenesis of human mammary epithelial cells in collagen
gels.
J. Cell. Sci
107:
3557-3568
|
5. | Binas, B., E. Spitzer, W. Zschiesche, B. Erdmann, A. Kurtz, T. Müller, C. Niemann, W. Blenau, and R. Grosse. 1992. Hormonal induction of functional differentiation and mammary-derived growth inhibitor expression in cultured mouse mammary explants. Cell. Dev. Biol 18: 625-634 . |
6. | Birchmeier, C., and W. Birchmeier. 1993. Molecular aspects of mesenchymal-epithelial interactions. Annu. Rev. Cell Biol 9: 511-540 . |
7. | Bissell, M.J., and T.G. Ram. 1989. Regulation of functional cytodifferentiation and histogenesis in mammary epithelial cells: role of the extracellular matrix. Environ. Health Perspect 80: 61-70 . |
8. | Bladt, F., D. Riethmacher, S. Isenmann, A. Aguzzi, and C. Birchmeier. 1995. Essential role for the c-met receptor in the migration of myogenic precursor cells into the limb bud. Nature 376: 768-771 |
9. | Boccaccio, C., M. Ando, L. Tamagnone, A. Bardelli, P. Michieli, C. Battistini, and P.M. Comoglio. 1998. Induction of epithelial tubules by growth factor HGF depends on the stat pathway. Nature 391: 285-288 |
10. | Bottaro, D.P., J.S. Rubin, D.L. Faletto, A.M. Chan, T.E. Kmiecik, G.F. Vande-Woude, and S.A. Aaronson. 1991. Identification of the hepatocyte growth factor receptor as the c-met proto-oncogene product. Science 251: 802-804 |
11. | Brinkmann, V., H. Foroutan, M. Sachs, K.M. Weidner, and W. Birchmeier. 1995. Hepatocyte growth factor/scatter factor induces a variety of tissue-specific morphogenic programs in epithelial cells. J. Cell Biol 131: 1573-1586 [Abstract]. |
12. | Carraway, K. III, and L.C. Cantley. 1994. A neu acquaintance for erbB3 and erbB4: a role for receptor heterodimerization in growth signalling. Cell 78: 5-8 |
13. | Carraway, K. III, and S.J. Burden. 1995. Neuregulins and their receptors. Curr. Opin. Neurobiol 5: 606-612 |
14. |
Carraway, K. III,
M.X. Sliwkowski,
R. Akita,
J.V. Platko,
P.M. Guy,
A. Nuijens,
A.J. Diamonti,
R.L. Vandlen,
L.C. Cantley, and
R.A. Cerione.
1994.
The erbB3 gene product is a receptor for heregulin.
J. Biol. Chem
269:
14303-14306
|
15. | Chomczynski, P., and N. Sacchi. 1987. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem 162: 156-159 |
16. | Coleman, S., G.B. Silberstein, and C.W. Daniel. 1988. Ductal morphogenesis in the mouse mammary gland: evidence supporting a role for epidermal growth factor. Dev. Biol 127: 304-315 |
17. | Cunha, G.R., and Y.K. Hom. 1996. Role of mesenchymal-epithelial interactions in mammary gland development. J. Mamm. Gl. Biol. Neopl 1: 21-35 . |
18. | Cunha, G.R., P. Young, S. Hamamoto, R. Guzman, and S. Nandi. 1992. Developmental response of adult mammary epithelial cells to various fetal and neonatal mesenchymes. Epithelial Cell. Biol 1: 105-118 |
19. | D'souza, B., and J. Taylor-Papadimitriou. 1994. Overexpression of ERBB2 in human mammary epithelial cells signals inhibition of transcription of the E-cadherin gene. Proc. Natl. Acad. Sci. USA 91: 7202-7206 [Abstract]. |
20. | Daniel, C.W., and S.D. Robinson. 1992. Regulation of mammary growth and function by TGF-beta. Mol. Reprod. Dev 32: 145-151 |
21. | Dankort, D.L., Z. Wang, V. Blackmore, M.F. Moran, and W.J. Muller. 1997. Distinct tyrosine autophosphorylation sites negatively and positively modulate neu-mediated transformation. Mol. Cell. Biol. 17: 5410-5425 [Abstract]. |
22. | Derman, M.P., M.J. Cunha, E.J.G. Barros, S.K. Nigam, and L.G. Cantley. 1995. HGF-mediated chemotaxis and tubulogenesis require activation of the phosphatidylinositol 3-kinase. Am. J. Physiol. 268: 1211-1217 . |
23. | Durnberger, H., and K. Kratochwil. 1980. Specificity of tissue interaction and origin of mesenchymal cells in the androgen response of the embryonic mammary gland. Cell 19: 465-471 |
24. | Falls, D.L., K.M. Rosen, G. Corfas, W.S. Lane, and G.D. Fischbach. 1993. ARIA, a protein that stimulates acetylcholine receptor synthesis, is a member of the neu ligand family. Cell 72: 801-815 |
25. | Feinberg, A.P., and B. Vogelstein. 1984. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Addendum. Anal. Biochem 137: 266-267 . |
26. | Fixman, E.D., M.A. Naujokas, G.A. Rodrigues, M.F. Moran, and M. Park. 1995. Efficient cell transformation by the Tpr-Met oncoprotein is dependent upon tyrosine 489 in the carboxy-terminus. Oncogene 10: 237-249 |
27. |
Fixman, E.D.,
M. Holgado-Madruga,
L. Nguyen,
D.M. Kamikura,
T.M. Fournier,
A.J. Wong, and
M. Park.
1997.
Efficient cellular transformation by the
Met oncoprotein requires a functional Grb2 binding site and correlates with
phosphorylation of the Grb2-associated proteins, Cbl and Gab1.
J. Biol.
Chem.
272:
20167-20172
|
28. | Gassmann, M., F. Casagranda, D. Orioli, H. Simon, C. Lai, R. Klein, and G. Lemke. 1995. Aberrant neural and cardiac development in mice lacking the ErbB4 neuregulin receptor. Nature 378: 390-394 |
29. | Grobstein, C.. 1953. Morphogenetic interaction between embryonic mouse tissues separated by a membrane filter. Nature 172: 869-871 . |
30. | Groner, B., S. Altiok, and V. Meier. 1994. Hormonal regulation of transcription factor activity in mammary epithelial cells. Mol. Cell. Endocrinol 100: 109-114 |
31. | Hahm, H.A., and M.M. Ip. 1990a. Primary culture of normal rat mammary epithelial cells within a basement membrane matrix. I. Regulation of proliferation by hormones and growth factors. Cell. Dev. Biol 26: 791-802 . |
32. | Hahm, H.A., M.M. Ip, K. Darcy, J.D. Black, W.K. Shea, S. Forczek, M. Yoshimura, and T. Oka. 1990b. Primary culture of normal rat mammary epithelial cells within a basement membrane matrix. II. Functional differentiation under serum-free conditions. Cell. Dev. Biol 26: 803-814 . |
33. | Happ, B., and B. Groner. 1993. The activated mammary gland specific nuclear factor (MGF) enhances in vitro transcription of the beta-casein gene promoter. J. Steroid. Biochem. Mol. Biol 47: 21-30 |
34. |
Hartmann, G.,
K.M. Weidner,
H. Schwarz, and
W. Birchmeier.
1994.
The motility signal of scatter factor/hepatocyte growth factor mediated through the
receptor tyrosine kinase met requires intracellular action of Ras.
J. Biol.
Chem.
269:
21936-21939
|
35. | Holgado-Madruga, M., D.R. Emlet, D.K. Moscatello, A.K. Godwin, and A.J. Wong. 1996. A Grb2-associated docking protein in EGF- and insulin-receptor signaling. Nature 379: 560-564 |
36. | Jhappan, C., C. Stahle, R.N. Harkins, N. Fausto, G.H. Smith, and G.T. Merlino. 1990. TGF alpha overexpression in transgenic mice induces liver neoplasia and abnormal development of the mammary gland and pancreas. Cell 61: 1137-1146 |
37. | Jhappan, C., A.G. Geiser, E.C. Kordon, D. Bagheri, L. Hennighausen, A.B. Roberts, G.H. Smith, and G. Merlino. 1993. Targeting expression of a transforming growth factor beta 1 transgene to the pregnant mammary gland inhibits alveolar development and lactation. EMBO (Eur. Mol. Biol. Organ.) J 12: 1835-1845 [Abstract]. |
38. |
Karunagaran, D.,
E. Tzahar,
N. Liu,
D. Wen, and
Y. Yarden.
1995.
Neu differentiation factor inhibits EGF binding. A model for trans-regulation within
the ErbB family of receptor tyrosine kinases.
J. Biol. Chem
270:
9982-9990
|
39. | Keely, P.J., J.K. Westwick, I.P. Whitehead, C.J. Der, and L.V. Parise. 1997. Cdc42 an Rac1 induce integrin-mediated cell motility and invasiveness through PI(3)K. Nature. 390: 632-636 |
40. |
Khwaja, A.,
P. Rodriguez-Viciana,
S. Wennström,
P.H. Warne, and
J. Downward.
1997.
Matrix adhesion and Ras transformation both activate a phosphoinositide 3-OH kinase and protein kinase B/Akt cellular survival pathway.
EMBO (Eur. Mol. Biol. Organ.) J.
16:
2783-2793
|
41. | Kita, Y.A., J. Barff, Y. Luo, D. Wen, D. Brankow, S. Hu, N. Liu, S.A. Prigent, W.J. Gullick, and M. Nicolson. 1994. NDF/heregulin stimulates the phosphorylation of Her3/erbB3. FEBS (Fed. Eur. Biochem. Soc.) Lett 349: 139-143 . |
42. | Kratochwil, K., and P. Schwartz. 1976. Tissue interaction in androgen response of embryonic mammary rudiment of mouse: identification of target tissue for testosterone. Proc. Natl. Acad. Sci. USA 73: 4041-4144 [Abstract]. |
43. | Kratochwil, K. 1987. Tissue combination and organ culture studies in the development of the embryonic mammary gland. In Developmental Biology: A Comprehensive Synthesis. R.B.L. Gwatkin, editor. Plenum Press, New York. 315-334. |
44. | Kraus, M.H., W. Issing, T. Miki, N.C. Popescu, and S.A. Aaronson. 1989. Isolation and characterization of ERBB3, a third member of the ERBB/epidermal growth factor receptor family: evidence for overexpression in a subset of human mammary tumors. Proc. Natl. Acad. Sci. USA 86: 9193-9197 [Abstract]. |
45. | Lee, K.F., H. Simon, H. Chen, B. Bates, M.C. Hung, and C. Hauser. 1995. Requirement for neuregulin receptor erbB2 in neural and cardiac development. Nature 378: 394-398 |
46. | Lopez-Barahona, M., I. Fialka, J.M. Gonzalez-Sancho, M. Asuncion, M. Gonzalez, T. Iglesias, J. Bernal, H. Beug, and A. Munoz. 1995. Thyroid hormone regulates stromelysin expression, protease secretion and the morphogenetic potential of normal polarized mammary epithelial cells. EMBO (Eur. Mol. Biol. Organ.) J 14: 1145-1155 [Abstract]. |
47. | Marchionni, M.A., A.D. Goodearl, M.S. Chen, O. Bermingham-McDonogh, C. Kirk, M. Hendricks, F. Danehy, D. Misumi, J. Sudhalter, K. Kobayashi, et al . 1993. Glial growth factors are alternatively spliced erbB2 ligands expressed in the nervous system. Nature 362: 312-318 |
48. | Marikovsky, M., S. Lavi, R. Pinkas-Kramarski, D. Karunagaran, N. Liu, D. Wen, and Y. Yarden. 1995. ErbB-3 mediates differential mitogenic effects of NDF/heregulin isoforms on mouse keratinocytes. Oncogene 10: 1403-1411 |
49. | Marte, B.M., M. Jeschke, D. Graus-Porta, D. Taverna, P. Hofer, B. Groner, Y. Yarden, and N.E. Hynes. 1995. Neu differentiation factor/heregulin modulates growth and differentiation of HC11 mammary epithelial cells. Mol. Endocrinol 9: 14-23 [Abstract]. |
50. | Meyer, D., and C. Birchmeier. 1994. Distinct isoforms of neuregulin are expressed in mesenchymal and neuronal cells during mouse development. Proc. Natl. Acad. Sci. USA 91: 1064-1068 [Abstract]. |
51. | Meyer, D., and C. Birchmeier. 1995. Multiple essential functions of neuregulin in development. Nature 378: 386-390 |
52. |
Meyer, D.,
T. Yamaai,
A. Garratt,
E. Riethmacher-Sonnenberg,
D. Kane,
L.E. Theill, and
C. Birchmeier.
1997.
Isoform-specific expression and function of
neuregulin.
Development (Camb.).
124:
3575-3586
|
53. | Montesano, R., K. Matsumoto, T. Nakamura, and L. Orci. 1991a. Identification of a fibroblast-derived epithelial morphogen as hepatocyte growth factor. Cell 67: 901-908 |
54. | Montesano, R., G. Schaller, and L. Orci. 1991b. Induction of epithelial tubular morphogenesis in vitro by fibroblast-derived soluble factors. Cell 66: 697-711 |
55. | Moore, M.W., R.D. Klein, I. Farinas, H. Sauer, M. Armanini, H. Phillips, L.F. Reichardt, A.M. Ryan, K. Carver-Moore, and A. Rosenthal. 1996. Renal and neuronal abnormalities in mice lacking GDNF. Nature 382: 76-79 |
56. | Naldini, L., K.M. Weidner, E. Vigna, G. Gaudino, A. Bardelli, C. Ponzetto, R.P. Narsimhan, G. Hartmann, R. Zarnegar, G.K. Michalopoulos, et al . 1991. Scatter factor and hepatocyte growth factor are indistinguishable ligands for the MET receptor. EMBO (Eur. Mol. Biol. Organ.) J 10: 2867-2878 [Abstract]. |
57. | Naundorf, H., I. Fichtner, B. Buttner, and J. Frege. 1992. Establishment and characterization of a new human oestradiol- and progesterone-receptor-positive mammary carcinoma serially transplantable in nude mice. J. Cancer. Res. Clin. Oncol 119: 35-40 |
58. | Naundorf, H., I. Fichtner, G.J. Saul, W. Haensch, and B. Buttner. 1993. Establishment and characteristics of two new human mammary carcinoma lines serially transplantable in nude mice. J. Cancer. Res. Clin. Oncol 119: 652-656 |
59. |
Nguyen, L.,
M. Holgado-Madruga,
C. Maroun,
E.D. Fixman,
D. Kamikura,
T. Fournier,
A. Charest,
M.L. Tremblay,
A.J. Wong, and
M. Park.
1997.
Association of the multisubstrate docking protein Gab1 with the hepatocyte
growth factor receptor requires a functional Grb2 binding involving tyrosine
1356.
J. Biol. Chem.
272:
20811-20819
|
60. | Orr-Urtreger, A., L. Trakhtenbrot, R. Ben-Levy, D. Wen, G. Rechavi, P. Lonai, and Y. Yarden. 1993. Neural expression and chromosomal mapping of Neu differentiation factor to 8p12-p21. Proc. Natl. Acad. Sci. USA 90: 1867-1871 [Abstract]. |
61. | Peles, E., S.S. Bacus, R.A. Koski, H.S. Lu, D. Wen, S.G. Ogden, R.B. Levy, and Y. Yarden. 1992. Isolation of the neu/HER-2 stimulatory ligand: a 44 kd glycoprotein that induces differentiation of mammary tumor cells. Cell 69: 205-216 |
62. | Peters, K., S. Werner, X. Liao, S. Wert, J. Whitsett, and L. Williams. 1994. Targeted expression of a dominant negative FGF receptor blocks branching morphogenesis and epithelial differentiation of the mouse lung. EMBO (Eur. Mol. Biol. Organ.) J 13: 3296-3301 [Abstract]. |
63. | Pichel, J.G., L. Shen, H.Z. Sheng, A.C. Granholm, J. Drago, A. Grinberg, E.J. Lee, S.P. Huang, M. Saarma, B.J. Hoffer, H. Sariola, and H. Westphal. 1996. Defects in enteric innervation and kidney development in mice lacking GDNF. Nature 382: 73-76 |
64. | Pierce, D. Jr., M.D. Johnson, Y. Matsui, S.D. Robinson, L.I. Gold, A.F. Purchio, C.W. Daniel, B.L. Hogan, and H.L. Moses. 1993. Inhibition of mammary duct development but not alveolar outgrowth during pregnancy in transgenic mice expressing active TGF-beta 1. Genes. Dev 7: 2308-2317 [Abstract]. |
65. | Pinkas-Kramarski, R., L. Soussan, H. Waterman, G. Levkowitz, I. Alroy, L. Klapper, S. Lavi, R. Seger, B.J. Ratzkin, M. Sela, and Y. Yarden. 1996. Diversification of neu differentiation factor and epidermal growth factor signaling by combinatorial receptor interaction. EMBO (Eur. Mol. Biol. Organ.) J 15: 2452-2467 [Abstract]. |
66. | Plowman, G.D., J.M. Green, J.M. Culouscou, G.W. Carlton, V.M. Rothwell, and S. Buckley. 1993. Heregulin induces tyrosine phosphorylation of HER4/ p180erbB4. Nature 366: 473-475 |
67. | Ponzetto, C., A. Bardelli, Z. Zhen, F. Maina, P. dalla-Zonca, S. Giordano, A. Graziani, G. Panayotou, and P.M. Comoglio. 1994. A multifunctional docking site mediates signalling and transformation by the hepatocyte growth factor/scatter factor receptor family. Cell. 77:261-271. |
68. | Press, M.F., C. Cordon-Cardo, and D.J. Slamon. 1990. Expression of the HER-2/neu proto-oncogene in normal human adult and fetal tissues. Oncogene 5: 953-962 |
69. | Prigent, S.A., N.R. Lemoine, C.M. Hughes, G.D. Plowman, C. Selden, and W.J. Gullick. 1992. Expression of the c-erbB-3 protein in normal human adult and fetal tissues. Oncogene 7: 1273-1278 |
70. | Reichmann, E., R. Ball, B. Groner, and R.R. Friis. 1989. New mammary epithelial and fibroblastic cell clones in coculture form structures competent to differentiate functionally. J. Cell Biol 108: 1127-1138 [Abstract]. |
71. | Riese, D. II, T.M. van-Raaij, G.D. Plowman, G.C. Andrews, and D.F. Stern. 1995. The cellular response to neuregulins is governed by complex interactions of the erbB receptor family. Mol. Cell. Biol 15: 5770-5776 [Abstract]. |
72. | Riethmacher, D., E. Sonnerberg-Riethmacher, V. Brinkmann, T. Yamaai, G.R. Lewin, and C. Birchmeier. 1997. Severe neuropathies in mice with targeted mutations in the erbB3 receptor. Nature 389: 725-730 |
73. | Rivera, E.M.. 1971. Mammary gland culture. Methods Mammal. Embryol 31: 442-471 . |
74. | Robinson, S.D., G.B. Silberstein, A.B. Roberts, K.C. Flanders, and C.W. Daniel. 1991. Regulated expression and growth inhibitory effects of transforming growth factor-beta isoforms in mouse mammary gland development. Development (Camb.). 113: 867-878 [Abstract]. |
75. |
Royal, I., and
M. Park.
1995.
Hepatocyte growth factor-induced scatter of Madin-Darby canine kidney cells requires phosphatidylinositol 3-kinase.
J. Biol.
Chem.
270:
27780-27787
|
76. | Royal, I., T.M. Fournier, and M. Park. 1997. Differential requirement of Grb2 and PI3-kinase in HGF/SF-induced cell motility and tubulogenesis. J. Cell Physiol. 173: 196-201 |
77. | Sachs, M., K.M. Weidner, V. Brinkmann, I. Walther, A. Obermeier, A. Ullrich, and W. Birchmeier. 1996. Motogenic and morphogenic activity of epithelial receptor tyrosine kinases. J. Cell Biol 133: 1095-1107 [Abstract]. |
78. | Sakakura, T., Y. Nishizuka, and C.J. Dawe. 1976. Mesenchyme-dependent morphogenesis and epithelium-specific cytodifferentiation in mouse mammary gland. Science 194: 1439-1441 |
79. | Sakakura, T.. 1991. New aspects of stroma-parenchyma relations in mammary gland differentiation. Int. Rev. Cytol 125: 165-202 |
80. | Sanchez, M.P., I. Silos-Santiago, J. Frisen, B. He, S.A. Lira, and M. Barbacid. 1996. Renal agenesis and the absence of enteric neurons in mice lacking GDNF. Nature 382: 70-73 |
81. | Saxen, L. 1987. Organogenesis of the kidney. In Developmetal and Cell Biology Series. Vol. 19. Cambridge University Press, Cambridge, UK. 184 pp. |
82. | Schmidhauser, C., G.F. Casperson, C.A. Myers, K.T. Sanzo, S. Bolten, and M.J. Bissell. 1992. A novel transcriptional enhancer is involved in the prolactin- and extracellular matrix-dependent regulation of beta-casein gene expression. Mol. Biol. Cell 3: 699-709 [Abstract]. |
83. | Schmidt, C., F. Bladt, S. Goedecke, V. Brinkmann, W. Zschiesche, M. Sharpe, E. Gherardi, and C. Birchmeier. 1995. Scatter factor/hepatocyte growth factor is essential for liver development. Nature 373: 699-702 |
84. | Schuchardt, A., V. D'Agati, L. Larsson-Blomberg, F. Costantini, and V. Pachnis. 1994. Defects in the kidney and enteric nervous system of mice lacking the tyrosine kinase receptor Ret. Nature 367: 380-383 |
85. | Segatto, O., G. Pelicci, S. Giuli, G. Digiesi, P.P. Di-Fiore, J. McGlade, T. Pawson, and P.G. Pelicci. 1993. Shc products are substrates of erbB-2 kinase. Oncogene 8: 2105-2112 |
86. | Shah, N.M., M.A. Marchionni, I. Isaacs, P. Stroobant, and D.J. Anderson. 1994. Glial growth factor restricts mammalian neural crest stem cells to a glial fate. Cell 77: 349-360 |
87. | Silberstein, G.B., and C.W. Daniel. 1987. Reversible inhibition of mammary gland growth by transforming growth factor-beta. Science 237: 291-293 |
88. |
Sliwkowski, M.X.,
G. Schaefer,
R.W. Akita,
J.A. Lofgren,
V.D. Fitzpatrick,
A. Nuijens,
B.M. Fendly,
R.A. Cerione,
R.L. Vandlen, and
K. Carraway III..
1994.
Coexpression of erbB2 and erbB3 proteins reconstitutes a high affinity
receptor for heregulin.
J. Biol. Chem
269:
14661-14665
|
89. | Snedeker, S.M., C.F. Brown, and R.P. DiAugustine. 1991. Expression and functional properties of transforming growth factor alpha and epidermal growth factor during mouse mammary gland ductal morphogenesis. Proc. Natl. Acad. Sci. USA 88: 276-280 [Abstract]. |
90. | Songyang, Z., S.E. Shoelson, M. Chaudhuri, G. Gish, T. Pawson, W.G. Haser, F. King, T. Roberts, S. Ratnofsky, R.J. Lechleider, et al . 1993. SH2 domains recognize specific phosphopeptide sequences. Cell 72: 767-778 |
91. | Sonnenberg, E., K.M. Weidner, and C. Birchmeier. 1993. Expression of the met-receptor and its ligand, HGF-SF during mouse embryogenesis. EMBO (Eur. Mol. Biol. Organ.) J 65: 381-394 . |
92. |
Soriano, J.V.,
M.S. Pepper,
T. Nakamura,
L. Orci, and
R. Montesano.
1995.
Hepatocyte growth factor stimulates extensive development of branching
duct-like structures by cloned mammary gland epithelial cells.
J. Cell Sci
108:
413-430
|
93. | Spooner, B.S., and N.K. Wessells. 1970. Mammalian lung development: interactions in primordium formation and bronchial morphogenesis. J. Exp. Zool 175: 445-454 |
94. | Staebler, A., C. Sommers, S.C. Mueller, S. Byers, E.W. Thompson, and R. Lupu. 1994. Modulation of breast cancer progression and differentiation by the gp30/heregulin [correction of neregulin]. Breast Cancer Res. Treat 31: 175-182 |
95. | Stein, D., J. Wu, S.A. Fuqua, C. Roonprapunt, V. Yajnik, P. D'Eustachio, J.J. Moskow, A.M. Buchberg, C.K. Osborne, and B. Margolis. 1994. The SH2 domain protein GRB-7 is co-amplified, overexpressed and in a tight complex with HER2 in breast cancer. EMBO (Eur. Mol. Biol. Organ.) J 13: 1331-1340 [Abstract]. |
96. |
Streuli, C.H.,
G.M. Edwards,
M. Delcommenne,
C.B. Whitelaw,
T.G. Burdon,
C. Schindler, and
C.J. Watson.
1995.
Stat5 as a target for regulation by extracellular matrix.
J. Biol. Chem
270:
21639-21644
|
97. | Sutherland, D., C. Samakovlis, and M.A. Krasnow. 1996. Branchless encodes a Drosophila FGF homolog that controls tracheal cell migration and the pattern of branching. Cell 87: 1091-1101 |
98. | Sympson, C.J., R.S. Talhouk, C.M. Alexander, J.R. Chin, S.M. Clift, M.J. Bissell, and Z. Werb. 1994. Targeted expression of stromelysin-1 in mammary gland provides evidence for a role of proteinases in branching morphogenesis and the requirement for an intact basement membrane for tissue-specific gene expression. J. Cell Biol 125: 681-693 [Abstract]. |
99. |
Tzahar, E.,
G. Levkowitz,
D. Karunagaran,
L. Yi,
E. Peles,
S. Lavi,
D. Chang,
N. Liu,
A. Yayon,
D. Wen, et al
.
1994.
ErbB-3 and ErbB-4 function as the
respective low and high affinity receptors of all Neu differentiation factor/
heregulin isoforms.
J. Biol. Chem
269:
25226-25233
|
100. | Uehara, Y., O. Minowa, C. Mori, K. Shiota, J. Kuno, T. Noda, and N. Kitamura. 1995. Placental defect and embryonic lethality in mice lacking hepatocyte growth factor/scatter factor. Nature 373: 702-705 |
101. | Vestweber, D., and R. Kemler. 1985. Identification of a putative cell adhesion domain of uvomorulin. EMBO (Eur. Mol. Biol. Organ.) J 4: 3393-3398 [Abstract]. |
102. | Vonderhaar, B.K.. 1987. Local effects of EGF, alpha-TGF, and EGF-like growth factors on lobuloalveolar development of the mouse mammary gland in vivo. J. Cell. Physiol 132: 581-584 |
103. | Wakao, H., F. Gouilleux, and B. Groner. 1995. Mammary gland factor (MGF) is a novel member of the cytokine regulated transcription factor gene family and confers the prolactin response. EMBO (Eur. Mol. Biol. Organ.) J 14: 854-855 |
104. | Weidner, K.M., N. Arakaki, G. Hartmann, J. Vandekerckhove, S. Weingart, H. Rieder, C. Fonatsch, H. Tsubouchi, T. Hishida, Y. Daikuhara, et al . 1991. Evidence for the identity of human scatter factor and human hepatocyte growth factor. Proc. Natl. Acad. Sci. USA 88: 7001-7005 [Abstract]. |
105. | Weidner, K.M., M. Sachs, and W. Birchmeier. 1993. The Met receptor tyrosine kinase transduces motility, proliferation, and morphogenic signals of scatter factor/hepatocyte growth factor in epithelial cells. J. Cell Biol 121: 145-154 [Abstract]. |
106. | Weidner, K.M., M. Sachs, D. Riethmacher, and W. Birchmeier. 1995. Mutation of juxtamembrane tyrosine residue 1001 suppresses loss-of-function mutations of the met receptor in epithelial cells. Proc. Natl. Acad. Sci. USA 92: 2597-2601 [Abstract]. |
107. | Weidner, K.M., S. Di Cesare, M. Sachs, V. Brinkmann, J. Behrens, and W. Birchmeier. 1996. Interaction between Gab1 and the c-Met receptor tyrosine kinase is responsible for epithelial morphogenesis. Nature 384: 173-176 |
108. | Wen, D., E. Peles, R. Cupples, S.V. Suggs, S.S. Bacus, Y. Luo, G. Trail, S. Hu, S.M. Silbiger, R.B. Levy, et al . 1992. Neu differentiation factor: a transmembrane glycoprotein containing an EGF domain and an immunoglobulin homology unit. Cell 69: 559-572 |
109. | Wicha, M.S., G. Lowrie, E. Kohn, P. Bagavandoss, and T. Mahn. 1982. Extracellular matrix promotes mammary epithelial growth and differentiation in vitro. Proc. Natl. Acad. Sci. USA 79: 3213-3217 [Abstract]. |
110. | Wilde, C.J., H.R. Hasan, and R.J. Mayer. 1984. Comparison of collagen gels and mammary extracellular matrix as substrata for study of terminal differentiation in rabbit mammary epithelial cells. Exp. Cell. Res 151: 519-532 |
111. | Yang, Y., E. Spitzer, D. Meyer, M. Sachs, C. Niemann, G. Hartmann, K.M. Weidner, C. Birchmeier, and W. Birchmeier. 1995. Sequential requirement of hepatocyte growth factor and neuregulin in the morphogenesis and differentiation of the mammary gland. J. Cell Biol 131: 215-226 [Abstract]. |