Epimorphin mediates mammary luminal morphogenesis through control of C/EBPbeta.
ABSTRACT We have shown previously that epimorphin (EPM), a protein expressed on the surface of myoepithelial and fibroblast cells of the mammary gland, acts as a multifunctional morphogen of mammary epithelial cells. Here, we present the molecular mechanism by which EPM mediates luminal morphogenesis. Treatment of cells with EPM to induce lumen formation greatly increases the overall expression of transcription factor CCAAT/enhancer binding protein (C/EBP)beta and alters the relative expression of its two principal isoforms, LIP and LAP. These alterations were shown to be essential for the morphogenetic activities, since constitutive expression of LIP was sufficient to produce lumen formation, whereas constitutive expression of LAP blocked EPM-mediated luminal morphogenesis. Furthermore, in a transgenic mouse model in which EPM expression was expressed in an apolar fashion on the surface of mammary epithelial cells, we found increased expression of C/EBPbeta, increased relative expression of LIP to LAP, and enlarged ductal lumina. Together, our studies demonstrate a role for EPM in luminal morphogenesis through control of C/EBPbeta expression.
- SourceAvailable from: pnas.org[show abstract] [hide abstract]
ABSTRACT: Extracellular matrix (ECM) profoundly influences the growth and differentiation of the mammary gland epithelium, both in culture and in vivo. Utilizing a clonal population of mouse mammary epithelial cells that absolutely requires an exogenous ECM for function, we developed a rapid assay to study signal transduction by ECM. Two components of the cellular response to a basement membrane overlay that result in the expression of the milk protein beta-casein were defined. The first component of this response involves a rounding and clustering of the cells that can be physically mimicked by plating the cells on a nonadhesive substratum. The second component is biochemical in nature, and it is associated with beta 1 integrin clustering and increased tyrosine phosphorylation. The second component is initiated in a morphology-independent manner, but the proper translation of this biochemical signal into a functional response requires cell rounding and cell clustering. Thus, physical and biochemical signal transduction events contribute to the ECM-dependent regulation of tissue-specific gene expression in mouse mammary epithelial cells.Proceedings of the National Academy of Sciences 01/1995; 91(26):12378-82. · 9.74 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: Eight cows in early lactation were used to study the effect of milk accumulation on the state of mammary tight junctions and to examine alpha-lactalbumin as an indicator of tight junction permeability in vivo. During three successive periods, the cows were milked twice (4 days), once (6 days), and twice daily (4 days). Plasma lactose, alpha-lactalbumin, and milk sodium concentrations were used as indicators of tight junction permeability. Furthermore, four cows were used to study the clearance of lactose and alpha-lactalbumin from the blood. Milk yield during once-daily milking decreased by 15.4% (P < 0.001). All indicators of mammary tight junction patency increased (P < 0.05) transiently during once-daily milking and indicated that tight junctions opened after approximately 18 h. Plasma alpha-lactalbumin and lactose were highly correlated (r = 0.82, P < 0.001), indicating the suitability of plasma alpha-lactalbumin as an indicator of tight junction status in vivo. Clearance of alpha-lactalbumin and lactose from the blood was best described by a biexponential model. Elimination half-lives for lactose and alpha-lactalbumin were 44 and 40 min, respectively. This study showed that milk stasis during early established lactation induces tight junctions to switch to a leaky state after approximately 18 h and to revert to the closed state shortly after milking.The American journal of physiology 07/1997; 273(1 Pt 2):R379-86. · 3.28 Impact Factor
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ABSTRACT: We developed a novel promoter system, designated SR alpha, which is composed of the simian virus 40 (SV40) early promoter and the R segment and part of the U5 sequence (R-U5') of the long terminal repeat of human T-cell leukemia virus type 1. The R-U5' sequence stimulated chloramphenicol acetyltransferase (CAT) gene expression only when placed immediately downstream of the SV40 early promoter in the sense orientation. The SR alpha expression system was 1 or 2 orders of magnitude more active than the SV40 early promoter in a wide variety of cell types, including fibroblasts and lymphoid cells, and was capable of promoting a high level of expression of various lymphokine cDNAs. These features of the SR alpha promoter were incorporated into the pcD-cDNA expression cloning vector originally developed by Okayama and Berg.Molecular and Cellular Biology 02/1988; 8(1):466-72. · 5.37 Impact Factor
The Journal of Cell Biology, Volume 153, Number 4, May 14, 2001 785–794
The Rockefeller University Press, 0021-9525/2001/05/785/10 $5.00
Epimorphin Mediates Mammary Luminal Morphogenesis
through Control of C/EBP
*Life Science Division, Lawrence Berkeley National Laboratory, University of California, Berkeley, California
Osaka R&D Laboratory (Yokohama-lab), Sumitomo Electric Industries Ltd., Yokohama 244, Japan
Derek Radisky,* Rosanne Boudreau,* Marina Simian,* Mary E. Stevens,*
Kyoko Takebe, Shinichiro Niwa, and Mina J. Bissell*
(EPM), a protein expressed on the surface of myoepi-
thelial and fibroblast cells of the mammary gland, acts
as a multifunctional morphogen of mammary epithelial
cells. Here, we present the molecular mechanism by
which EPM mediates luminal morphogenesis. Treat-
ment of cells with EPM to induce lumen formation
greatly increases the overall expression of transcription
factor CCAAT/enhancer binding protein (C/EBP)
and alters the relative expression of its two principal
isoforms, LIP and LAP. These alterations were shown to
be essential for the morphogenetic activities, since con-
stitutive expression of LIP was sufficient to produce lu-
We have shown previously that epimorphin
men formation, whereas constitutive expression of LAP
blocked EPM-mediated luminal morphogenesis. Fur-
thermore, in a transgenic mouse model in which EPM
expression was expressed in an apolar fashion on the
surface of mammary epithelial cells, we found increased
expression of C/EBP
, increased relative expression of
LIP to LAP, and enlarged ductal lumina. Together, our
studies demonstrate a role for EPM in luminal morpho-
genesis through control of C/EBP
morphogenesis • epithelial–stromal interactions • CCAAT/
enhancer binding protein • transgenic mice
transcription factor balance • mammary
The majority of mammary gland development occurs post-
natally. During puberty, a system of branching ducts pene-
trates the fatty stroma; in pregnancy, the epithelium con-
tinues to proliferate, developing additional complexity and
lobuloalveolar structures. Many of these developmental
processes are dependent on reciprocal communication be-
tween the stroma and the epithelium (Hennighausen and
Robinson, 1998; Wiesen et al., 1999), although the signal-
ing molecules that mediate the reciprocal interactions are
only beginning to be identified.
was originally characterized as a
stromal cell surface molecule involved in embryonic epi-
thelial morphogenesis (Hirai et al., 1992; Hirai, 1993); a
later study also showed that the same gene encoded a
member of the syntaxin family (Bennett et al., 1993; Pel-
ham, 1993). In its capacity as an extracellular morphogen,
EPM has been shown to control developmental processes
in endothelial (Oka and Hirai, 1996), liver parenchymal
(Hirose et al., 1996; Watanabe et al., 1998), embryonic
lung (Koshida and Hirai, 1997), and pancreatic carcinoma
cells (Lenhert et al., 2001). In the mammary gland, EPM is
present at the surface of both stromal fibroblasts and myo-
epithelial cells (Hirai et al., 1998) and thus is poised to play
a role in mammary morphogenesis. Previously, we used a
three-dimensional collagen assay to characterize the func-
tion of EPM in normal mammary epithelial cell morpho-
genesis (Hirai et al., 1998). When presented in a polar
basal fashion, EPM produced branching morphogenesis,
whereas apolar presentation triggered formation of struc-
tures with large central lumina. However, the mechanism
of its morphogenic activities remained to be identified.
The transcription factor CCAAT/enhancer binding pro-
has been implicated in many developmen-
tal processes (for review see Lekstrom-Hines and Xantho-
polous, 1998); however, no upstream effector of C/EBP
has, as yet, been identified (Robinson et al., 1998). C/EBP
is found in several isoforms that possess altered transacti-
vation potentials: LAP, the full-length 34-kD isoform and
Address correspondence to Mina J. Bissell, Life Science Division,
Lawrence Berkeley National Laboratory, University of California,
Berkeley, CA 94720. Tel.: (510) 486-4365. Fax: (510) 486-5586. E-mail:
M.E. Stevens’ present address is Bayer Corp., Berkeley, CA 94701.
Abbreviations used in this paper:
; CMV, cytomegalovirus; EPM, epimorphin; MMP, metallopro-
teinase; rEPM, recombinant EPM; RT, reverse transcription; WAP, whey
C/EBP, CCAAT/enhancer binding
The Journal of Cell Biology, Volume 153, 2001
LIP, a truncated isoform of 20 kD (Descombes and Schibler,
1991). The relative expression of these isoforms changes
throughout development of the mammary gland (Raught
et al., 1995). Investigations of mice deficient for expression
have revealed that this transcription factor is
essential for normal mammary gland morphogenesis (Rob-
inson et al., 1998; Seagroves et al., 1998). Among other de-
mice were found to have bloated ducts
with enlarged central lumina, reminiscent of the luminal
structures formed in our culture assays when mammary
epithelial cells were presented with EPM in an apolar
fashion. We hypothesized that this transcription factor
might participate in the luminal morphogenesis triggered
by apolar presentation of EPM to cultured mammary epi-
thelial cells and that EPM may play a similar role in vivo.
This hypothesis led to several specific predictions. First,
presentation of EPM to mammary epithelial cells in vitro
should regulate C/EBP
, and the morphogenic effects of
EPM should be reproduced by appropriate artificial regu-
lation of C/EBP
in cultured mammary epithelial cells.
Second, EPM should be present at the apical surface of the
mammary epithelial cells in vivo during the development
of alveolar lumina, and apolar presentation of EPM to
mammary epithelial cells in vivo should regulate C/EBP
and produce enlarged lumina. Here, we present experi-
mental results that confirm these predictions.
Materials and Methods
Cells and Tissue Culture
SCp2 cells, a normal mammary epithelial cell line (Desprez et al., 1993;
Roskelley et al., 1994; Hirai et al., 1998), and primary mammary epithelial
cells were maintained in growth medium (DME/F12 [GIBCO BRL] sup-
plemented with 5% FBS [Hyclone], 5
g/ml gentamycin). SCp2 tetracycline supresses epimorphin cells, gen-
erated as described in Hirai et al. (1998), were maintained in growth me-
dium supplemented with 5
g/ml of tetracycline. For generation of SCp2
cells expressing LIP and LAP under control of a tetracycline-repressible
promoter, cDNAs were isolated from cytomegalovirus (CMV)–LIP and
CMV–LAP constructs and were cloned into the EcoRI site of the eukary-
otic expression vector pTetT-Splice (Life Technologies). SCp2 cells (5
10) were transfected with 5
g of this vector, 5
(Life Technologies), and 0.5
g of pSV40neo (Schmidhauser et al., 1992)
using lipofectamine (Life Technologies) according to the manufacturer’s
instructions. After selection for neomycin-resistant clones in the continu-
ous presence of tetracycline, the expression of LIP and LAP was analyzed
by Western blotting in the presence and absence of 5
SCp2/LIP1, SCp2/LIP2, SCp2/LIP3, and SCp2/LAP cell lines were iso-
lated using this procedure.
g/ml insulin [Sigma-Aldrich], and
g of pTet.tTAK vector
Assays for formation of luminal structures were performed essentially as
described previously (Hirai et al., 1998). In brief, cells were grown in se-
rum-free medium either containing 100
(rEPM) (for SCp2 cells) or lacking tetracycline (for PTSE cells). In some
wells, rEPM (H123) was coated before seeding the cells (Hirai et al.,
1998). The level of protein expression of C/EBP
sured by immunoblotting the total cell lysates at day 3 or as described.
Morphogenesis assays were performed using clustered cells that were em-
bedded in collagen gels as described previously (Hirai et al., 1998) with
slight modifications. In brief, cell clusters were prepared on agarose gels
and suspended in a mixture of 8.5 vol of collagen type I (I-PC; Koken
Corp.) and 1 vol of 10
serum-free medium, adjusted to pH 7.4 with 0.5
vol of alkaline solution. After addition of 1 vol of PBS, cell clusters were
suspended in the collagen solution (50–100 clusters in 100
onto basal collagen gels in individual wells of 48-well plates. Growth me-
dium containing 50 ng/ml EGF and the appropriate amount of tetracy-
g/ml soluble recombinant EPM
gene products was mea-
l) and poured
cline was added to each well. As indicated, soluble rEPM was added to a
final concentration of 50
Generation of rEPM
All rEPM proteins could be solubilized in 1.5 mM HCl, whereas solubility
of the proteins was increased by COOH-terminal truncation. By removal
of all of domain 3 (amino acids 189–264), rEPM (termed H12, amino acids
1–188) is freely soluble in PBS or growth medium. By three-dimensional
collagen assay (Hirai et al., 1998), the truncated proteins retained all origi-
nal morphogenic activities. All rEPM polypeptides were produced in
BL21 and purified in the presence of 8 M urea as described
previously (Oka and Hirai, 1996) with slight modifications. Deletion mu-
tants of EPM tagged with a 6
His sequence were generated by PCR, in-
serted into the prokaryotic expression vector PET3a (Novagen), and intro-
duced into bacterial cells. All the recombinant proteins purified from the
bacteria with Ni-NTA–agarose beads (QIAGEN) were dialyzed either di-
rectly against 1.5 mM HCl or gradually against 1, 0.8, 0.5, 0.25, and 0.125 M
urea in ice-cold PBS, followed by urea-free PBS. The soluble fraction in
each sample was sterile filtered and assayed for protein concentration.
To prepare antibodies specific for individual domains of EPM, antiserum
raised against untagged EPM (Hirai, 1994) was affinity purified with nitro-
cellulose membranes precoated with purified rEPM fragments (1, amino
acids 1–104; 2, amino acids 105–188; 3, amino acids 189–264) as described
previously (Hirai et al., 1998). To prepare antibodies recognizing just the
COOH-terminal sequence of EPM, affinity purified anti-H123 antibodies
were absorbed with a column (mixture of Affigel 10 and 15; Bio-Rad Lab-
oratories) immobilized with histidine-tagged rEPM deletion mutant H1–
230 (amino acids 1–230). The antibodies bound to the column were eluted
with 0.25 M glycine-HCl (pH 2.7), immediately neutralized with 1 M phos-
phate buffer (pH 8.0), dialyzed against PBS, and used as anti–1–230.
These antibodies were used for immunoblotting at a concentration of 10
g/ml. For preparation of the anti-LAPonly antibody, a construct contain-
ing the nucleotide sequence of C/EBP
ATG and a 6
CAT sequence (for 6
bacterial expression vector pet 3C
and expressed in
The recombinant LAPonly peptide was purified on Ni-NTA gel in the
presence of 4 M urea, dialyzed against PBS (final purity was
used for injecting rats. Antibodies to C/EBP
were from Santa Cruz Biotechnology, Inc., and antibody to
from Sigma-Aldrich. These reagents were used for immunoblotting at the
dilution of 1:200. The antibody to T7 peptide (Novagen) was used at a
His) at the 5
from 190–582 was fused with an
end was cloned into
Analysis of EPM Cleavage
Lactating mammary gland tissue (1 g) was sonicated in 5 ml of 20 mM
Tris-HCl (pH 8.0), 0.5 mM CaCl
, and 25 mM NaCl on ice. After centrifu-
gation at 200
for 1 min, the supernatant was collected and centrifuged at
for 30 min at 4
C. The pellet was then washed several times with
PBS and resuspended in 500
l serum-free medium. A 100-
this membrane-enriched fraction was mixed with 10
full-length EPM (isoform I), tagged with 6
(Oka and Hirai, 1996), and incubated at 37
urea (pH 8.0) was added to the reaction to dissolve all the insoluble mate-
rials. Ni-NTA–agarose beads (QIAGEN) were then added to the super-
natant to collect the His-tagged products. After washing the beads several
times with 8 M urea (pH 8.0), the collected products were analyzed by im-
munoblotting with anti-EPM antibodies. Untagged rEPM was used as a
control. For transfection experiments, the expression vector SR
(Takebe et al., 1988), containing the full-length cDNA for EPM isoform I
tagged with T7 peptide at the NH
was electrophoretically transfected into SCp2, SCp2
mary epithelial cells using a Bio-Rad Laboratories GenePulser. Cells were
incubated in growth medium for 24 h, then in serum-free DME/F12 sup-
plemented with 5
g/ml insulin (Sigma-Aldrich), 3
tional Institutes of Health, Bethesda, MD), and 1
(Sigma-Aldrich) for an additional 2 d. The cDNA products in the cells
were analyzed by immunoblotting using an anti-T7 tag monoclonal anti-
body (Novagen). Supernatants from cultured cells were concentrated by
immunoprecipitation using anti-EPM antibodies and protein A–Sepha-
rose (Bio-Rad Laboratories) and analyzed by immunoblot.
l portion of
g of recombinant
histidine at the NH
C. After 48 h, 800
l of 8 M
TEPM) (Hirai, 1994),
, and primary mam-
g/ml prolactin (Na-
Hirai et al.
Involvement of Epimorphin in Luminal Morphogenesis
Estimation of LIP/LAP Ratios in Cells
Cells were directly dissolved in SDS sample buffer, electrophoresed in
SDS-PAGE gels, and blotted onto polyvinylidene difluoride filter mem-
gene products were visualized with anti-C/EBP
ies. Nuclear extracts were prepared as described by Deryckere and Gan-
non (1994). The estimation of intensity of LIP and LAP signals was
carried out with the ChemiImager 4000 low light imaging system (Alpha
Innotech). The relative amount of LIP/LAP was compared with control,
which was loaded on the same blot.
For generation of whey acidic protein (WAP)–EPM mice, EPM cDNA
was tagged with a mouse IL-2 signal peptide sequence (5
AGC ATG CAG CTC GCA TCC TGT GTC ACA TTG ACA CTT
GTG CTC CTT GTC AAC AGC GCT CCC-3
scribed previously (Hirai et al., 1998). The construct was subcloned into
the HindIII site of the CA10 vector, which contains a WAP gene pro-
moter and a sequence for the 3
untranslated region of WAP protein
(Sympson et al., 1994). After linearization with NotI, the target gene was
purified with glass milk (Bio-Rad Laboratories) and microinjected into
fertilized eggs. The injected embryos were then transferred to
pseudopregnant Friend virus B mice. To determine the presence and in-
tegrity of the transgene, DNA was isolated from the tail of offspring of
transgenic mice and analyzed by PCR using 5
(from the WAP promoter sequence) as 5
primer. For the positive lines, the expression of the transgene was
further analyzed by reverse transcription (RT)-PCR. In brief, total RNA
from pregnant mice was isolated, treated with DNase, reverse transcribed,
and analyzed using 5
IL-2 signal peptide sequence) as 5
(from the EPM sequence) as 3
) generated by PCR as de-
(from the EPM sequence)
primer and 5
Apolar Presentation of EPM Regulates LIP
When PTSE mammary epithelial cells, which express
EPM under control of the tetracycline transactivator, were
cultured in the absence of tetracycline for 4 d, they ex-
pressed EPM in an apolar fashion and formed structures
composed of a single cell layer and a central lumen (Fig. 1
A, a). Addition of tetracycline at this point repressed the
EPM transgene and caused the luminal structures to col-
lapse (Fig. 1 A, c). However, addition of soluble and
rEPM (H12) along with tetracycline caused the luminal
structures to continue to expand and to develop even
larger central lumina (Fig. 1 A, b). Using this assay, we
found that EPM elevated the overall levels of C/EBP
increased the relative ratio of the 20-kD isoform (LIP) to
the 34-kD isoform (LAP) (Fig. 1 B). A similar effect was
observed in primary mammary epithelial cells (Fig. 1 C).
EPM also increased the expression of C/EBP
the truncated form of this protein was not observed (Fig. 1
D), suggesting that the alteration in the LIP/LAP ratio was
not due to increased expression of nonspecific proteases.
EPM-induced LIP Is Not Produced by Proteolysis
To verify that increased LIP did not result from specific in
vitro proteolysis, we generated an antiserum against the
-terminal 14 kD of C/EBP
within LAP that is absent in LIP (Fig. 2 A). This antiserum
was used to probe a blot containing extracts of control
SCp2 cells, SCp2 cells treated with rEPM, SCp2 cells tran-
siently transfected with LAP (SCp2/LAP cells), or SCp2/
LAP cells treated with rEPM (Fig. 2 B). We found the ex-
, the region contained
pected increase in 34-kD LAP in both the rEPM-treated
samples and in the LAP transfectants but did not find any
14-kD band that would have resulted from proteolytic
cleavage of LAP. A parallel blot probed with a commer-
cial antibody directed against a region of C/EBP
mon to LAP and LIP (Fig. 2 C) showed a comparable in-
crease in LIP expression in both EPM-treated samples.
Despite the considerably higher expression of LAP in the
SCp2/LAP cells, there was only a very faint band corre-
sponding to LIP. A third blot containing extracts from
SCp2 cells transiently transfected with either LAP or LIP
constructs was probed with the commercial antibody, and
no significant degradation of LAP was observed (Fig. 2 C).
Together, these experiments show that our extraction pro-
tocol produces little or no proteolysis of C/EBP
LIP Mimics and LAP Blocks the Effects of EPM
We found that regulation of C/EBP
was sufficient to mediate luminal morphogenesis of cul-
tured mammary epithelial cells using constructs with tetra-
cycline-regulated expression of LIP or LAP (Fig 3 A). In
collagen assays, untransfected or mock-transfected cells
formed aggregates. Moderate expression of LIP achieved
through attenuation of expression in SCp2/LIP1 and
SCp2/LIP2 cells and through incomplete repression in
SCp2/LIP3 cells promoted luminal morphogenesis. How-
ever, high expression of LIP caused apoptosis, assayed by
accumulation of fragmented DNA (data not shown). In
several experiments, we found that increases in the LIP/
LAP ratio between
2–10 triggered luminal morphogene-
sis (Fig. 3 B, c [0.5
g/ml tetracycline] and C, b), and in-
creases in ratios that were
10 led to apoptosis (Fig. 3 B, b
g/ml tetracycline] and C, c). By contrast, overex-
pression of LAP led to formation of compact colonies with
no lumina that were resistant to EPM-mediated luminal
morphogenesis (Fig. 3 D, c), possibly through the ability of
LAP to neutralize LIP (Descombes and Schibler, 1991;
Buck et al., 1994). Taken together, these results demon-
strate that EPM upregulates the relative expression of LIP
to LAP, constitutive expression of LIP is sufficient to pro-
duce luminal morphogenesis, and constitutive expression
of LAP can block EPM from producing this morphogenic
Soluble EPM Is Present in Milk
In mammary glands from nulliparous animals, we had pre-
viously detected EPM only on the stromal fibroblasts and
myoepithelial cells (Hirai et al., 1998). However, as apolar
presentation of EPM was important for luminal morpho-
genesis in culture, we investigated the possibility that en-
dogenous EPM might also be present at the apical surface
of luminal epithelial cells during normal development.
When unfixed lactating glands from wild-type mice were
stained with a gentle washing protocol, EPM could be de-
tected also in the lumina (Fig. 4 A). This localization sug-
gested a soluble form of EPM, and Western blot analysis
of normal mouse milk with anti-EPM antibodies identified
30-kD band that was smaller than full-length 34-kD
EPM (Fig. 4 B, b). The presence of this protein was not
due to simple cell lysis, since
cells (Mather and Keenan, 1998), was not found in the
-actin, a diagnostic for burst
The Journal of Cell Biology, Volume 153, 2001
milk (Fig. 4 B, a). Western analyses using antibodies spe-
cific for domains within EPM suggested that the
protein is a COOH-terminal truncation (Fig. 4 C).
We reasoned that the soluble form of EPM might result
from proteolysis of membrane-bound EPM. Consistent
with this hypothesis, when full-length rEPM containing an
His tag was incubated with a membrane
extract derived from whole lactating mammary tissue, the
protein was cleaved to form a single
5 B, b). Although it was not possible to isolate sufficient
30-kD product (Fig.
EPM. (A) EPM activates luminal morphogene-
sis. PTSE cell clusters were cultured in collagen
for 4 d in the absence of tetracycline (EPM ON)
(a), then cultured for an additional 4 d in the
presence of tetracycline (EPM OFF) and with
(b) or without (c) H12 form of EPM in the me-
dium. The luminal diameter of ?20 clusters in
each category was measured; graph bar indi-
cates SD. (B) EPM increases C/EBP? expres-
sion and the LIP/LAP ratio. PTSE cell clusters
were cultured on plastic in the presence or ab-
sence of tetracycline, and SCp2 cells were cul-
tured on recombinant full-length EPM (rEPM)
or collagen (Hirai et al., 1998) or in the pres-
ence of rEPM/H12. Analyses of unclustered
SCp2 cells incubated on tissue culture plastic or
plastic coated with collagen are shown as con-
trols. CRM, cross-reactive material (Seagroves
et al., 1998). Estimated LIP/LAP ratios relative
to the control samples are indicated. (C) LIP
and LAP are dramatically upregulated by both
EPM transfection (sig-EPM) and addition of
rEPM to primary mammary epithelial cells. The
identity of the band between CRM and LAP is
unknown. (D) SCp2 cells cultured in the pres-
ence of rEPM also upregulate C/EBP?. For B,
C, and D, the results are typical of three inde-
pendent experiments. Bar, 100 ?m.
Effects of apolar presentation of
LAPonly antibody and of the commercial anti-C/EBP? antibody. (B) Western blot probed with the anti-LAPonly antibody. (C) West-
ern blots probed with commercial anti-C/EBP? antibody. Left: Parallel blot to B; asterisk, cross-reactive material. Right: Blot of SCp2
cells transiently transfected with LAP or LIP expression plasmids. Results shown are typical of two independent experiments.
Minimal proteolysis of LAP occurs during sample preparation. (A) Diagram depicting the targeted location of the anti-
Hirai et al.
Involvement of Epimorphin in Luminal Morphogenesis
quantities of the
other COOH-terminal truncations are fully functional in
morphogenesis assays (Hirai et al., 1998).
When an expression construct containing full-length
EPM with an NH
-terminal T7 tag was transfected into
primary mammary epithelial cells derived from pregnant
animals, the cells produced both
(Fig. 5 A, a). Although the general population of SCp2
cells when transfected with the expression construct did
not normally produce the soluble
(Fig. 5 A, b), a subclone of transfectants named SCp2
sessed the ability to do so (Fig. 5 A, c). Cleavage of tagged
EPM by SCp2
cell extracts was inhibited by the metallo-
proteinase (MMP) inhibitor GM6001 (Fig. 5 B, b), sug-
gesting that an MMP could mediate the conversion to the
soluble ?30-kD species. The identity of this protease is
30-kD species to verify its activity,
30- and 34-kD products
30-kD EPM species
WAP–EPM Transgenic Mice Showed
Upregulation of LIP
To investigate EPM-mediated mammary luminal morpho-
genesis in vivo, we generated transgenic mice that ex-
pressed membrane-tethered EPM on the surface of lumi-
nal epithelial cells. This was accomplished by fusing the
interleukin 2 signal peptide to the EPM cDNA and then
sandwiching this construct between the promoter and 3?
untranslated region of the WAP gene (Pittius et al., 1988;
Sympson et al., 1994) to generate the WAP–EPM con-
struct (Fig. 6 A). We had previously used a similar con-
struct to localize the EPM to the surface of cultured mam-
mary epithelial cells (Hirai et al., 1998).
Two out of six founder mice, EP4 and EP6, showed in-
corporation of the transgene (Fig. 6 B). Nulliparous het-
erozygotes from these lines displayed no detectable in-
transfectants cultured in the presence (5 ?g/ml) or absence of tetracycline (tet). Results shown are typical of three independent experi-
ments. (B and C) Behavior of LIP-transfected cells cultured in various concentrations of tetracycline. Clusters of SCp2 controls (B, a)
and of SCp2/LIP1 and SCp2/LIP2 in 0.5 ?g/ml tet (B, b) formed compact colonies in collagen (shown in C, a for SCp2/LIP1; 0.5 ?g/ml
tetracycline). SCp2/LIP3 expresses moderate levels of LIP and forms lumina in the presence of tetracycline (B c and C b). Reduction
of tetracycline in medium of SCp2/LIP1 and SCp2/LIP2 cells results in apoptotic cell death (B, b; shown in C, c for SCp2/LIP1; 0.02 ?g/
ml tetracycline). (D) Induction of LAP transgene inhibits luminal morphogenesis. (a) LAP expression in clustered parental SCp2 cells.
(b and c) Clustered parental Scp2 cells and SCp2/LAP cells cultured in collagen gels in the absence of tetracycline (tet) and in the pres-
ence and absence of rEPM. For B, C, and D, ?20 colonies from each condition were examined. Bar, 100 ?m.
Characterization of clones that conditionally express LIP and LAP. (A) Analysis of C/EBP? gene products in LIP and LAP
The Journal of Cell Biology, Volume 153, 2001
crease in EPM expression and had no distinctively aberrant
phenotype (data not shown). Pregnant and lactating het-
erozygotes did express EPM and also had dramatically en-
larged ducts (Fig. 6 C, b). In addition, transgenic animals
had large and disorganized secretory alveoli. A similar
phenotype with varying severity was observed in all of the
WAP–EPM mice but not in any of the transgene-negative
littermates examined (n ? 25), and the phenotype of all
the transgenic mice became stronger as animals aged (data
not shown). Lactating transgenic mice displaying the stron-
gest phenotype had problems producing milk and would
cannibalize their young.
When analyzed by Western blot, mammary glands from
WAP–EPM mice showed a dramatic increase in C/EBP?
relative to wild-type mice, both in total protein and in the
relative ratio of LIP to LAP (Fig. 6 D). Thus, the WAP–
EPM mice paralleled the culture studies, since apolar ex-
pression of EPM on mammary epithelial cells upregulated
the LIP/LAP ratio and led to enlarged ductal lumina.
In this study, we have identified a functional relationship
between EPM-triggered luminal morphogenesis and in-
creased relative expression of the LIP isoform of C/EBP?.
This was both necessary and sufficient for the morpho-
genic activity in cultured cells as expression of LIP in-
duced luminal morphogenesis, whereas expression of LAP
blocked luminal morphogenesis induced by EPM. Since
apical presentation of EPM seemed to be important for
this morphogenic process, we examined EPM expression
in vivo and found a truncated soluble form present in
vivo. (A) Frozen sections of prefixed (a) and unfixed
(b) lactating mammary glands labeled with anti-
EPM antibodies. The tissue in b was fixed on the
slide immediately after sectioning, and the staining
was carried out with mild washing so as not to re-
move soluble proteins in the lumina (asterisk). (B)
Immunoblot analysis of the lactating mammary
gland tissue (T) and milk (M) with anti–?-actin and
anti-EPM antibodies. 5 (?5) or 1 ?g protein samples
of mammary gland extract (T) or milk (M) collected
from lactating wild-type mice were probed with
anti–?-actin (a) and EPM (b) antibodies. (C) 30-kD
soluble EPM reacts with all anti-EPM antibodies ex-
cept those directed against the COOH terminus. (a)
Schematic diagram of affinity purified antibodies
targeted to different domains of EPM. (b) Immuno-
blot analyses of milk and recombinant full-length
34-kD EPM (r-EPM) using the affinity purified anti-
bodies. Anti-1, -2, -3, -1–230, and -c are specific to
EPM amino acids 1–104, 105–188, 189–264, 1–230,
and 231–264, respectively. Bar, 30 ?m.
Identification of ?30-kD soluble EPM in
in transfected primary mammary cells (a), SCp2 cells (b), and SCp2? cells (c) were analyzed by immunoblot with monoclonal anti-T7
antibody. Cells (C) and supernatants (S) were analyzed separately. For detection in the supernatant, an immunocomplex with anti-EPM
antibodies was collected with protein A–Sepharose beads. Untagged rEPM was used as a control. (B) rEPM-tagged with 6? His at the
NH2 terminus was incubated with membranes derived from lactating mammary glands (a) or from either SCp2 or SCp2? cells (b). Prod-
ucts were collected with a Ni-agarose column and analyzed by immunoblotting with anti-EPM antibodies. For a, untagged rEPM was
used as a control. In b, the MMP inhibitor GM6001 or the inactive structural homologue C1004 was added to a final concentration of 10
?M. Results are typical of three independent experiments.
Production of ?30-kD soluble EPM in vitro. (A) The products of EPM cDNA tagged with T7 peptide at the NH2 terminus
Hirai et al. Involvement of Epimorphin in Luminal Morphogenesis
mouse milk. Analysis of the WAP–EPM transgenic mice
supported the role of EPM as an effector of LIP-mediated
luminal morphogenesis. These animals express EPM in an
apolar fashion on luminal epithelial cells and also have in-
creased LIP/LAP ratios and greatly enlarged ductal lu-
mina in pregnant and lactating animals.
It is unclear if EPM-induced luminal morphogenesis is
caused by a positive activity of LIP or by the downregula-
tion of LAP by LIP. The latter possibility is supported by
the similarities between mammary ductal morphology of
WAP–EPM and C/EBP??/? mice. However, it should be
noted that the phenotype of the WAP–EPM mice is signif-
icantly different from the phenotype of the C/EBP??/?
mice. The latter have severe ovarian dysfunction and con-
sequent female sterility (Sterneck et al., 1997), defective
differentiation of myeloid cells (Screpanti et al., 1995;
Tanaka et al., 1995), adipocytes (Tanaka et al., 1997),
hepatocytes (Lee et al., 1997), and keratinocytes (Zhu et
al., 1999), and aberrant expression of lactogenic hormone
receptors (Seagroves et al., 2000). Even when comparisons
are limited to the mammary gland, there are still substan-
tial differences, since lobuloalveolar development was in-
hibited in the C/EBP??/? mice (Seagroves et al., 1998).
These differences may be partially attributed to the spa-
tiotemporal modulation of EPM expression by the WAP
promoter and the consequent limitation of the affected
cell population. Furthermore, differences in mouse strain
backgrounds may also be relevant.
Soluble EPM in Murine Ductal Lumina
Examination of normal mouse milk revealed a soluble
molecule of ?30 kD that was cross-reactive with all anti-
bodies to EPM except those generated against the COOH-
terminal sequence. The hypothesis that the truncated EPM
could result from proteolytic cleavage at the cell surface
was examined by reconstitution experiments in which mem-
brane fractions from mammary glands of lactating mice
were incubated with tagged recombinant EPM. We found
that this treatment resulted in selective generation of the
?30-kD form (Fig. 6 B). Several other signaling mole-
cules, including the kit ligand and TGF-?, are released by
membrane-bound MMPs (Blobel, 1997; Werb, 1997), and
we found that a specific inhibitor of MMPs inhibited the
conversion of full-length EPM to the ?30-kD form in
It is unclear how soluble extracellular EPM, if produced
by stromal cells, is transported to the ductal lumina. Possi-
bly, after removal of the membrane-anchoring sequence
by proteolysis EPM could be delivered by transcytosis
across the epithelial membrane, or it could pass through
temporary junctions between luminal epithelial cells. Such
mechanisms are involved in the delivery of other stromal
proteins to the luminal space (Linzell and Peaker, 1973;
Grosvenor et al., 1992; Stelwagen et al., 1997; Ollivier-
Bousquet, 1998). Alternatively, since some mammary epi-
thelial cell lines are able to express EPM in culture (Hirai
et al., 1998) it is possible that a subpopulation of luminal
epithelial cells produce soluble EPM during development.
In this case, secretion of the large volume of milk proteins
and lipid droplets by the lactating gland (Burgoyne and
Duncan, 1998; Mather and Keenan, 1998) may act as a ve-
hicle for efficient distribution of EPM. Currently, there
are no data to distinguish between these possibilities.
Regulation of the LIP/LAP Ratio
LAP and LIP are mutually antagonistic isoforms of C/EBP?
(Descombes and Schibler, 1991; Buck et al., 1994). In our
culture system, moderate expression of LIP led to lumen
formation, whereas expression of LAP blocked this pro-
cess. That high expression of LIP produced apoptosis is an
intriguing observation in light of the role of apoptosis in
the formation of lumina in terminal endbuds and alveolar
transgene construct. N, NotI; H, HindIII. (B) PCR analysis of the integration of the transgene into genomic DNA and RT-PCR analysis
of EPM transgene expression. As a control for RT-PCR, endogenous stromelysin 1 (SL-1) was analyzed in the same samples. Lower
bands (asterisk) are unreacted primers that remained in control samples after RT-PCR. (C) Phenotypic appearance of the mammary
gland (circled) from midpregnant (7 d) normal (a) and transgenic (b) mice. Whole-mount stained mammary gland of normal (c) and
transgenic (d) mice. (D) Analysis of C/EBP? gene products in mammary tissue of normal and transgenic mice using commercial anti-C/
EBP? antibody. CRM, cross-reactive material. Bars: (B) 5 ?m; (C) 300 ?m.
WAP–EPM transgenic mice have enlarged ductal lumina and altered C/EBP? expression. (A) Schematic diagram of the
The Journal of Cell Biology, Volume 153, 2001
structure in culture (Humphreys et al., 1996). The possibil-
ity that LIP may contribute to this process warrants care-
ful analysis in three-dimensional cultures and in vivo.
However, as morphogenic processes are likely to be or-
chestrated within microdomains of the mammary gland,
specific antibodies and quantitative real time imaging may
LIP has been implicated in other physiological pro-
cesses as diverse as inflammation (An et al., 1996), liver
regeneration (Timchenko et al., 1998; Welm et al., 2000),
development (Diehl et al., 1994; Darlington, 1999), and
aging (Hseih et al., 1998; Timchenko et al., 1998). LIP
was originally proposed to arise through alternative
translation by leaky ribosome scanning (Descombes and
Schibler, 1991; Ossipow et al., 1993). Subsequent investi-
gations implicated a small, out-of-frame ORF in the 5? se-
quence of C/EBP? as a regulator of LIP/LAP ratios and
determinant of cell differentiation (Calkhoven et al., 2000
and references therein). Some studies have suggested that a
specific RNA-binding protein may control the alternative
translation initiation (Timchenko et al., 1999; Welm et al.,
2000). Other investigations have shown that LIP can re-
sult from a mechanism that is regulated by C/EBP? (Welm
et al., 1999), and in this regard it is relevant that apolar
presentation of EPM leads to upregulation of C/EBP?
as well (Fig. 1 D). In some cases, observation of the LIP
isoform may be due to in vitro proteolysis of LAP (Baer
et al., 1998; Lincoln et al., 1998; Baer and Johnson, 2000),
but we excluded this possibility in our system (Fig. 2).
EPM Is a Member of the Syntaxin Family
The gene that encodes EPM also encodes syntaxin-2, a
protein that has been shown to function during targeting/
fusion of intracellular vesicles with the plasma membrane
(Bennett et al., 1993; Pelham, 1993). Although syntax-
ins have been shown to act at the cytoplasmic face of
membranes (Brose, 1993; Pelham, 1993; Rothman, 1994;
Wheeler et al., 1996; Jagadish et al., 1997), the subcellular
localization of syntaxins can be modulated through vari-
ous mechanisms (Hui et al., 1997; Jagadish et al., 1997;
Rodger et al., 1998; Quiñones et al., 1999), and syntaxins
have been found at the cell surface at least transiently (Hi-
rai et al., 1993, 1998; Smirnova et al., 1993a,b; Butt et al.,
1996; Nagamatsu et al., 1997; Guo et al., 1998). Evidently,
these complex and versatile proteins can perform multiple
functions, and it will be a considerable challenge to dis-
cover how these diverse roles are related. However, func-
tional relationships of known protein motifs within EPM/
syntaxin-2 may provide some clues. The SNARE motif is a
highly conserved domain within the syntaxin family that
is located between amino acids 188 and 253 of EPM
(Weimbs et al., 1997). Although domain analyses of syn-
taxins demonstrate the SNARE motif is essential for for-
mation of competent membrane fusion complexes (Sutton
et al., 1998; Jahn and Südhof, 1999), we have found that
this motif is not required for the morphogenic activity of
EPM in mammary epithelial cells, since a genetically engi-
neered EPM (H12) lacking the SNARE motif is fully com-
petent in our assays (Fig. 1; Hirai et al., 1998). These find-
ings support a model in which syntaxin/EPM molecules
harbor distinct functional domains that allow the mole-
cules to function in more than one cellular context.
The mechanism by which EPM is translocated to the
outside of the plasma membrane is not known, although
cell surface localization of other membrane-associated
molecules lacking signal peptides has been reported (Wen
et al., 1992; Skach et al., 1993; Ostapchuk et al., 1994; Guo
et al., 1998). In such cases, the membrane topology depends
on the three-dimensional conformation of the NH2-termi-
nal sequence (Denzer et al., 1995; Spiess, 1995; Wahlberg
and Spiess, 1997). Similarly, intramolecular interactions
within the EPM sequence (Hirai, 1994) may allow a sub-
population to orient itself on the outer surface of the plasma
In conclusion, we have shown that EPM can mediate lu-
minal morphogenesis of mammary epithelial cells in vitro by
control of C/EBP?, and we have implicated this mechanism
in mammary morphogenesis. The conclusive link will re-
quire further analyses of WAP–EPM, WAP–LIP, and
WAP–LAP transgenic mice, generated in the same strain
background, and a knockout of C/EBP? in our assay sys-
tem. Through such experiments, it would be possible to de-
termine if C/EBP? is the only mediator of EPM-triggered
luminal morphogenesis in vivo. Characterization of the mo-
lecular mechanism by which EPM controls C/EBP? isoform
levels will require analysis of the epithelial receptor for ex-
tracellular EPM; it should be easier to identify this receptor
by using the soluble form of EPM, identified in this study as
a probe. These investigations are currently underway.
We thank Jinger Xie and Dewight Williams for help in breeding the trans-
genic mice, William Johansen and Amy Ukena for their administrative
support, and Zena Werb, Eva Turley, Joan Brugge, John Muschler, Karen
Schmeichel, and Jimmie Fata for critical reading of the manuscript. The
eukaryotic expression vectors CMV–LIP and CMV–LAP were generous
gifts from Ueli Schibler.
This work was supported by the U.S. Department of Energy, Office of
Biological and Environmental Research (contract DE-AC03-76SF00098),
and the National Institutes of Health (grant CA57621), by a Distinguished
Hollaender Postdoctoral fellowship to D. Radisky, and by support from
the Science and Technology Agency of Japan to Y. Hirai.
Submitted: 12 February 2001
Revised: 30 March 2001
Accepted: 30 March 2001
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