§
* Howard Hughes Medical Institute, Center for Cancer Research, Department of Biology, Massachusetts Institute of
Technology, Cambridge, Massachusetts 02139; Department of Biophysics, Faculty of Science, Kyoto University, Kitashirakawa,
Sakyo-ku, Kyoto, 60601, Japan; § Departments of Obstetrics and Gynecology; and
Molecular and Cellular Engineering,
University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104
To investigate the functions of P-cadherin in vivo, we have mutated the gene encoding this cell adhesion receptor in mice. In contrast to E- and N-cadherin- deficient mice, mice homozygous for the P-cadherin mutation are viable. Although P-cadherin is expressed at high levels in the placenta, P-cadherin-null females are fertile. P-cadherin expression is localized to the myoepithelial cells surrounding the lumenal epithelial cells of the mammary gland. The role of the myoepithelium as a contractile tissue necessary for milk secretion is clear, but its function in the nonpregnant animal is unknown. The ability of the P-cadherin mutant female to nurse and maintain her litter indicates that the contractile function of the myoepithelium is not dependent on the cell adhesion molecule P-cadherin. The virgin P-cadherin-null females display precocious differentiation of the mammary gland. The alveolar-like buds in virgins resemble the glands of an early pregnant animal morphologically and biochemically (i.e., milk protein synthesis). The P-cadherin mutant mice develop hyperplasia and dysplasia of the mammary epithelium with age. In addition, abnormal lymphocyte infiltration was observed in the mammary glands of the mutant animals. These results indicate that P-cadherin-mediated adhesion and/or signals derived from cell-cell interactions are important determinants in negative growth control in the mammary gland. Furthermore, the loss of P-cadherin from the myoepithelium has uncovered a novel function for this tissue in maintaining the undifferentiated state of the underlying secretory epithelium.
CLASSICAL cadherins, such as E-, N-, and P-cadherin,
play critical roles in tissue morphogenesis as evidenced by studies in Xenopus and mice (Kintner,
1992 The cadherin cytoplasmic domain interacts with a group
of proteins termed catenins, which link the cadherin to the
actin cytoskeleton. Interaction with both the catenins and
the actin cytoskeleton is necessary for full cadherin adhesive activity (Kemler, 1993 Cell adhesion molecules, including the cadherins, are
known to play important roles in mammary gland morphogenesis. The mammary gland develops postnatally under
the proper hormonal stimuli during puberty and adolescence. The morphogenesis of the mammary ductal tree occurs when the end buds invade the surrounding fatty
stroma until they reach the edge of the fat pad. The end
buds of the mammary ducts represent the growth points
for ductal morphogenesis. The end buds consist of basally
located cap cells and lumenal epithelial cells (Williams and
Daniel, 1983 The mammary duct consists of two main cell types, the
lumenal epithelial cells and a surrounding monolayer of
myoepithelial cells with a closely apposed basement membrane. The myoepithelial cells are thought to differentiate
from the cap cells extending their cell processes laterally
along the duct. In the pregnant animal, the myoepithelium
is present all along the duct and in the alveoli, where myoepithelial cells are basket shaped resulting in space between the cells allowing direct contact between the alveolar epithelial cells and the basal lamina. In contrast with other tissues, the expression pattern of E- and P-cadherin
in the mammary gland is very distinct. In the mouse, cap
cells and myoepithelial cells express P-cadherin while the
lumenal epithelial cells express E-cadherin (Daniel et al.,
1995 The cell-cell and cell-matrix interactions of myoepithelial cells may play an important role in maintaining the
structural integrity of the mammary duct. Myoepithelial
cells are specialized contractile cells, whose ultrastructure
is reminiscent of smooth muscle cells (Deugnier et al., 1995 Recent experiments suggest that myoepithelial cells may
have an important role in branching morphogenesis in the
mammary gland (Niranjan et al., 1995 To understand the role of the P-cadherin adhesion receptor in mouse development, we mutated the P-cadherin
gene in embryonic stem (ES) cells and introduced the
mutation into the mouse germ line (Capecchi, 1989 Derivation of Mutant Mice
The genomic structure of the mouse P-cadherin gene was reported previously (Hatta et al., 1991 Western Immunoblotting
Decidual tissue from wild-type, heterozygous, and homozygous conceptuses was isolated on day 8 of gestation. The protein lysates were subjected to SDS-PAGE (Laemmli, 1970 Morphological and Histological Analysis
The thoracic no. 3 and/or inguinal no. 4 mammary glands were examined.
Whole mount staining of the glands was performed as previously described (Williams and Daniel, 1983 For histology, mammary glands were fixed in 10% formalin, processed
for paraffin sectioning, and stained with hematoxylin and eosin.
Immunohistochemistry
Immunostaining for P- and E-cadherin, Generation of P-Cadherin-deficient Mice
To construct a targeting vector for the P-cadherin gene
(Fig. 1 A), a neomycin phosphotransferase (neo) expression cassette (pMC1neo; Thomas and Capecchi, 1987
The linearized targeting vector was electroporated into
D3 ES cells (Doetschman et al., 1985 P-Cadherin Mutant Mice Are Viable and Fertile
To examine whether animals homozygous for the P-cadherin mutation were viable, heterozygous animals were intercrossed and genotypes of the progeny were determined
by Southern blot or by PCR analysis. Mice homozygous
for the P-cadherin mutation were detected among the intercross progeny (Fig. 1 C). The genotypes of the progeny
showed a good fit to Mendelian distribution (107 +/+:
211 To determine if loss of P-cadherin affected litter size, hybrid 129Sv/C57BL mutant males and females were mated.
The P-cadherin-null females had litter sizes (average 8.8, 114 pups/13 litters) comparable to their wild-type littermates. The ability of the P-cadherin mutant females to
nurse and maintain a normal size litter indicates that the
contractile function of the myoepithelium is not dependent on the cell adhesion molecule P-cadherin.
Loss of P-Cadherin from Myoepithelial Cells Leads to
Alveolar Differentiation in Virgin Females
To examine cadherin expression in the mutant mammary
duct, immunohistochemistry was performed on sections of
mammary tissue. P-cadherin was localized to the myoepithelial cells surrounding the lumenal epithelial cells in the
wild-type, but absent from the mutant duct (Fig. 2, A and
D). The presence of myoepithelium in the mutant duct
was confirmed by smooth muscle actin staining (Fig. 2, B
and E), a marker for myoepithelial cells (Radnor, 1972
During puberty the end buds of the mammary ductal tree
actively penetrate the surrounding adipose stroma, P-cadherin has been shown to be expressed in the cap cells of
the terminal end bud (Daniel et al., 1995
A Mutation in the P-Cadherin Gene Predisposes Mice
to Focal Mammary Hyperplasia and Dysplasia
As demonstrated in various animal models of mammary
tumorigenesis, precocious alveolar differentiation is often
associated with neoplastic lesions, an initial step in tumor
development (Cardiff and Muller, 1993
Gene inactivation of the classical cadherins, E- and N-cadherin, results in embryonic lethality at the pre- and postimplantation stage, respectively (Larue et al., 1994 Ductal morphogenesis in the mammary gland occurs during puberty when the end buds of the ducts invade the surrounding fatty stroma until they reach the edge of the fat
pad. The cells surrounding the end buds, called cap cells,
are thought to play an important role in ductal growth and
branching morphogenesis. P-cadherin is expressed by the
cap cells and function-blocking antibodies can disrupt the
cellular integrity of the end bud (Daniel et al., 1995 How might loss of P-cadherin cause the mammary gland
phenotype? The possibility that P-cadherin is normally
downregulated during early pregnancy leading to alveolar
differentiation is intriguing. However, Northern blot analysis did not detect any change in P-cadherin expression between virgin and early, mid, or late pregnant mammary
glands (D'Cruz, C.M., G.L. Radice, and L.A. Chodosh,
unpublished data). The fact that the P-cadherin-null females lactate and nurse their pups implies that the myoepithelium can still perform its function as a contractile tissue, however, subtle defects in cell adhesion may be
possible. While there is considerable evidence for inductive signals from the stroma and extracellular matrix
(ECM) affecting the differentiation of the lumenal epithelium (Roskelley et al., 1995
In Drosophila, armadillo, the The cadherin/catenin adhesion complex and the Wnt
signaling pathway share a common component, Ectopic or overexpression of several proteins including
Wnt1 (Tsukamoto et al., 1988 P-cadherin is also expressed in myoepithelial cells of the
human mammary gland suggesting that loss of function of
this cell adhesion molecule may promote cell growth and
differentiation in the human breast. While most attention
has focused on the role of E-cadherin in breast cancer, our
data suggest that P-cadherin may also be involved. Loss of
heterozygosity (LOH) of 16q22.1 has been implicated in
many types of cancer including breast (Birchmeier and
Behrens, 1994 Recently, a novel cadherin, H-cadherin, was found to be
expressed in human mammary epithelial cells (Lee, 1996 Several loss of function mutations in the mouse result in
disruption of mammary gland development and function.
Mutations in cyclin D1 (Sicinski et al., 1995 In conclusion, gene targeting of the cell adhesion receptor, P-cadherin, has uncovered a novel function for this
molecule in negative growth control of the mammary gland.
The mechanism by which P-cadherin acts to control cell
growth remains to be determined. No obvious cell adhesion defect has been observed in the myoepithelium of the
mutant animals so far, suggesting the mammary gland phenotype may result from perturbation of cellular signals involved in negative growth control.
; Hermiston and Gordon, 1995
). Cadherins are a family of glycoproteins involved in Ca++-dependent, homotypic cell-cell adhesion (Takeichi, 1995
; Gumbiner, 1996
).
Classical cadherins have five extracellular domains, one transmembrane domain, and a highly conserved cytoplasmic domain. Two subclasses of cadherins, E- and P-cadherin, are detected in various epithelial tissues of mouse
embryos (Nose and Takeichi, 1986
). Antibody perturbation experiments have shown that E- and P-cadherin function cooperatively in the histogenesis of embryonic lung
and lip skin in organ explant cultures (Hirai et al., 1989a
, b
).
In the case of the lung primordia, addition of anti-E-cadherin antibodies resulted in collapsed lobules with little luminal space, while anti-P-cadherin antibodies had a less
dramatic effect. However, a mixture of both antibodies
had a synergistic effect resulting in a severely distorted epithelium that could not undergo the normal branching process. In similar experiments performed on embryonic skin,
P-cadherin appears to be more important compared with
E-cadherin, but again disruption of both E- and P-cadherin produced the more dramatic effect on skin morphogenesis. Taken together, these studies suggest that both
E- and P-cadherin play important roles in maintaining the
structural integrity of epithelial tissues.
). Either
-catenin or plakoglobin, which are members of the Armadillo family of proteins (Peifer, 1995
), binds directly to the cadherin. In
addition to playing structural roles in cell-cell adhesion, these two catenins, along with Armadillo, appear to have
signaling roles, although the exact mechanism is not fully
understood. Armadillo is the product of a Drosophila segment polarity gene and is part of the wingless signaling
pathway, downstream of Zeste-White 3 kinase (Peifer et al.,
1994
).
-catenin and plakoglobin have been implicated in
formation of mesoderm and the anterior-posterior axis in the
Xenopus embryo (Heasman et al., 1994
; Funayama et al.,
1995
). Recently,
-catenin was shown to interact with the transcription factor, LEF-1, providing evidence that
-catenin can regulate gene expression (Behrens et al., 1996
).
-catenin, which shares homology with the cytoskeleton-associated protein vinculin, binds the cadherin indirectly
through
-catenin or plakoglobin. Like vinculin,
-catenin
binds to both
-actinin and actin (Knudsen et al., 1995
;
Rimm et al., 1995
). Thus,
-catenin serves to link the cadherin/catenin complex to the actin cytoskeleton.
). The cap cells are loosely adhering epithelial
cells that lack cytoplasmic polarity and a well organized cytoskeleton. During early pregnancy lateral buds differentiate from the ducts and during the second half of pregnancy
these alveoli develop into fully differentiated secretory lobules. These morphogenetic events are accompanied by
cellular differentiation leading to development of secretory epithelial cells which are capable of synthesizing and
secreting milk proteins.
). Function-blocking antibodies were used in situ to
examine the role of E- and P-cadherin in maintaining the
tissue integrity of the end bud (Daniel et al., 1995
). Antibody to E-cadherin induced disruption of the epithelium resulting in freely floating epithelial cells in the lumen. In contrast, antibody to P-cadherin had no effect on the lumenal layer but partially disrupted the basally located cap
cell layer. These data show that E- and P-cadherin are important for maintaining the integrity of the different cell
layers of the mammary duct.
).
They express smooth muscle contractile and cytoskeletal
proteins such as
-smooth muscle actin (Radnor, 1972
).
However, they are true epithelial cells since cytokeratin is
the major component of the intermediate filament system,
they form desmosomes, hemidesmosomes, and adherens
junctions, and are permanently separated from the connective tissue by the underlying basement membrane (Franke et
al., 1980
; Sonnenberg et al., 1986
; Rasbridge et al., 1993
).
). Hepatocyte growth
factor/scatter factor (HGF/SF)1 is produced by the fibroblasts in the breast. The lumenal epithelium and myoepithelium respond differently to HGF/SF; which acts as a
mitogen for the lumenal cells while it behaves like a morphogenic factor for the myoepithelial cells. Human myoepithelial cells exposed to HGF/SF form extended branching
tubules while lumenal epithelial cells did not show any
morphological changes (Niranjan et al., 1995
). Furthermore, overexpression of HGF/SF in the mammary gland
of transgenic mice leads to precocious alveolar differentiation (Takayama et al., 1997
). These data suggest that myoepithelial cells may play an important role in branching
morphogenesis in the mammary gland.
). In
contrast to E- and N-cadherin knockout mice, the P-cadherin-deficient mice are viable and fertile. However, the
P-cadherin-null females exhibit precocious differentiation of the mammary gland and display mammary hyperplasia
later in life. The P-cadherin-deficient mice provide us with
a valuable model to determine the role of P-cadherin in
cell proliferation and differentiation in the mammary gland.
Materials and Methods
). An EcoRI-EcoRI DNA fragment (8.5 kb) was
isolated from lambda clone, PG21, and subcloned into Bluescript KS
(Stratagene, La Jolla, CA). The neo expression cassette, pMC1neo (Thomas and Capecchi, 1987
), was digested with XhoI-SalI, and subcloned into
a unique XhoI site in exon 11. The BamHI-BamHI DNA fragment was
subcloned into a plasmid containing the pMC1-HSVTK cassette (Mansour et al., 1988
). The construct was linearized with NotI and electroporated into D3 ES cells (Doetschman et al., 1985
), and colonies were selected with G418 and gancyclovir (a gift from Syntex Corp.) as described
(George et al., 1993
). Homologous recombination events were screened
by Southern blot analysis (Sambrook et al., 1989
). An EcoRI-BamHI genomic DNA fragment (0.7 kb) located immediately 5
to the short arm of
the targeting vector was used as a probe. The targeted ES clones were injected into blastocysts from C57Bl/6J mice (Bradley, 1987
). The mice were
genotyped by Southern blot analysis or PCR. One pair of primers corresponding to the intron/exon borders of exon 11 were used in the PCR reactions to amplify the wild-type and mutant alleles: P11F, 5
-TCCTTTCCAGCTACACCAT-3
and P11R, 5
-AAGCTCTCACCACTGTCTGTG-3
.
Unpurified tail DNA was used for the PCR reactions (Hanley and Merlie,
1991
). Temperature cycling conditions for the Perkin Elmer 480 PCR machine were 94°C for 1 min; 65°C for 2 min; and 72°C for 3 min for 40 cycles.
PCR products were resolved on a 1.6% agarose gel.
), and the resolved proteins were
transferred electrophoretically to nitrocellulose. The blot was reacted with
antibodies to P-cadherin (PCD-1; Nose and Takeichi, 1986
) and protein
bands were visualized using the ECL (Amersham Corp., Arlington
Heights, IL) detection system.
). The mammary glands were fixed
overnight in Tellyesniczky's fixative (5% formalin, 5% acetic acid, 70%
EtOH). The fixed glands were defatted in acetone, stained with: hematoxylin (0.65g FeCl3, 67.5 ml H2O, 8.7 ml stock hematoxylin [10% in 95% ethanol], and 1,000 ml 95% ethanol, adjust pH 1.25 with concentrated HCl),
and then rinsed in tap water, dehydrated in increasing concentrations of
ethanol to xylene, and photographed with a dissecting microscope.
-smooth muscle actin, and
caseins was performed on frozen sections of mammary tissue as described
previously (Daniel et al., 1995
). Tissue was embedded in Tissue-Tek OCT
compound (Miles Diagnostic Division, Elkhart, IN) and frozen in an isopentane/dry ice bath. 8-µm sections were cut with a Zeiss cryotome,
placed on Superfrost Plus slides (Fisher Scientific, Pittsburgh, PA), postfixed in 1:1 acetone/methanol at
20°C for 10 min, air dried, and stored at
20°C. Antibodies were diluted in 5% skim milk/PBS except for casein
antibody (1% goat serum/PBS) as follows: rat monoclonal PCD-1 (1:200),
rat monoclonal ECCD-2 (1:500; Shirayoshi et al., 1986
), mouse monoclonal
-smooth muscle actin (clone 1A4, 1:400; Sigma Chemical Co., St. Louis, MO), and rabbit polyclonal casein (1:1,000; a gift from Charles
Daniel, University of California, Santa Cruz). The samples were incubated overnight with primary antibodies except casein antibody (1 h),
washed with PBS, incubated with species specific biotinylated secondary
antibodies (Amersham), washed with PBS, and processed with Vectastain
ABC reagents (Vector Labs, Burlingame, CA), regular or elite, and
washed with PBS. The casein antibody washes contained 1% goat serum.
The peroxidase substrate was applied, sections were lightly counterstained
with hematoxylin, dehydrated, and coverslipped. The samples were photographed with a Nikon Optiphot microscope.
Results
) was
inserted into a unique XhoI site in exon 11 (Nose et al.,
1987
; Hatta et al., 1991
). This disrupts the open reading
frame of the P-cadherin gene in the extracellular domain
of the protein. The neo cassette, containing a polyadenylation signal, was placed in the same transcriptional orientation as the P-cadherin gene thus serving to truncate any P-cadherin/neo fusion transcript. The vector includes 2.5 kb of genomic sequences 5
of the neo gene, 5 kb on the 3
side, and a flanking Herpes simplex virus (HSV) thymidine kinase expression cassette (pMC1-tk; Mansour et al.,
1988
). A similar construct was used successfully to inactivate the N-cadherin gene in mice (Radice et al., 1997
).
Fig. 1.
Targeted disruption of the P-cadherin gene. (A) Schematic representation of the expected gene replacement at the
P-cadherin locus. Exons are represented as closed boxes. The
MC1-neomycin resistance cassette and MC1-thymidine kinase
cassette are designated Neo and HSVtk, respectively; arrows indicate the orientation of the genes. The flanking probe used for
screening ES cell clones and genotyping mice is shown (Probe).
Restriction endonuclease sites are abbreviated as follows: E,
EcoRI; S, SphI; Sa, SacI. The PCR primers, P11F and P11R, used
for genotyping are shown. (B) Southern blot analysis of a targeted ES cell clone. The wild-type (WT) and knockout (KO) genomic fragments are indicated. (C) PCR blot analysis of P-cadherin intercross progeny indicating the genotypes. The wild-type,
0.17 kb, and mutant, 1.2 kb, PCR products are shown. (D) Western analysis of placental lysates from P-cadherin intercross progeny. The arrowhead indicates the 118-kD P-cadherin protein recognized by the mAb PCD-1. No P-cadherin protein was detected
in the homozygous mice. kb, kilobases; m, markers.
[View Larger Version of this Image (35K GIF file)]
), and the electroporated cells were subjected to positive/negative selection
(Mansour et al., 1988
) using G418 and gancyclovir. One of
86 double-resistant ES cell clones (P78) underwent homologous recombination at the P-cadherin locus as determined by Southern blot analysis (Fig. 1 B). Hybridization with a neo probe failed to detect any additional sites of integration (data not shown). The targeted clone was injected into host C57BL/6 blastocysts, and the blastocysts
were transferred to the uteri of pseudopregnant females.
Germline transmission of the mutant allele was achieved
with the targeted ES clone. The heterozygous mice did not
display any obvious abnormalities in comparison with their
wild-type littermates.
/+:114
/
). Homozygous P-cadherin-deficient mice
did not show any overt developmental abnormalities and
were indistinguishable from their heterozygous or wild-type littermates on the basis of size, activity, or fertility. To
determine whether wild-type P-cadherin protein was
present in homozygous mice, we examined the uterine decidua of pregnant mice since P-cadherin protein is very
abundant in this maternal tissue. The decidual tissue without the embryo and extraembryonic membranes was isolated from day 8 conceptuses of wild-type, heterozygous,
and homozygous mice. Western blot analysis of decidual
lysates was performed using monoclonal antibody, PCD-1,
which recognizes the amino terminus of the protein (Nose
et al., 1990
). No full-length or truncated P-cadherin protein was detected in the mutant, while a reduced amount
of P-cadherin protein was present in the heterozygote
(Fig. 1 D). In addition, a pan-cadherin polyclonal antibody
(Takeichi et al., 1990
) which recognizes the conserved
cytoplasmic domain of classical cadherins did not detect
P-cadherin in the mutant decidual tissue (data not shown).
). E-cadherin expression was similar in wild-type and mutant
ducts (Fig. 2, C and F), thus demonstrating that E-cadherin expression was not affected by mutating the closely
linked P-cadherin gene (Hatta et al., 1991
).
Fig. 2.
Cadherin expression in wild-type and mutant mammary ducts. Mammary gland sections of wild-type (A-C) and mutant (D-
F) animals were immunostained for P-cadherin (A and D), smooth muscle actin (B and E), and E-cadherin (C and F). P-cadherin is
present in the myoepithelial cells surrounding the lumenal epithelial cells in the wild-type duct (A) and absent in the mutant duct (D). A diffuse nonspecific background staining was observed in the mutant (D), but the characteristic dark linear staining of the myoepithelial cells (A) was absent in the mutant. Myoepithelial cells are present in both the wild-type (B) and mutant (E) as shown by the smooth muscle actin staining. E-cadherin expression appears similar in wild-type (C) and mutant (F) ducts. The arrowheads indicate lumenal epithelial cells and arrows indicate myoepithelial cells. Original magnification 125x.
[View Larger Version of this Image (79K GIF file)]
). No difference in
the extent of branching morphogenesis was observed in the
mammary glands of mutant mice compared with their wild-type littermates at 5 wk of age, before puberty is complete
(data not shown). However, we observed an unexpected
phenotype when mammary glands from postpubescent
virgin females (10 wk old) were examined by whole-mount
staining. Wild-type animals had normal ductal morphogenesis, while the mutant animals displayed precocious alveolar differentiation resembling an early pregnant gland
(Fig. 3, A and B). The mammary glands from mutant male
mice appeared normal; no alveolar differentiation was observed (data not shown). To determine whether the mutant mammary glands exhibited biochemical changes associated with differentiation, milk protein production was
examined using a polyclonal antibody against caseins. The
wild-type virgin animal did not express casein(s) while the
mutant expressed casein(s) at levels similar to a pregnant animal (Fig. 4, A-C). These data indicate that loss of P-cadherin leads to precocious alveolar differentiation which is
morphologically and biochemically similar to an early pregnant wild-type gland.
Fig. 3.
Structure of normal and mutant mammary glands in
virgin female mice. Whole-gland stain of (A) wild-type and (B)
mutant mammary gland from 10-wk-old virgin animals. P-cadherin-deficient virgin females display precocious alveolar differentiation. Original magnification 10x.
[View Larger Version of this Image (69K GIF file)]
Fig. 4.
Expression of milk protein in P-cadherin-null virgin mice. Mammary gland sections of wild-type virgin (A) , mutant virgin (B),
and wild-type pregnant day 14 (C) animals were immunostained with a polyclonal antibody against caseins. The P-cadherin-deficient virgin mammary gland express casein(s) similarly to a pregnant animal. The wild-type virgin gland was counterstained to distinguish the
ducts. Original magnification 62x.
[View Larger Version of this Image (41K GIF file)]
). Therefore, the
P-cadherin mutant mice and their heterozygous and wild-type littermates were maintained until 2 yr of age. No palpable tumors were observed in the aged P-cadherin mutant animals. However, upon histological examination of
the mammary glands, focal alveolar hyperplasia and ductal
dysplasia were observed in the mutants. Comparison of a
20-mo-old wild-type and mutant virgin female gland reveals
the extent of alveolar hyperplasia observed in the mutants
(Fig. 5, A and B). The clusters of alveoli are shown at higher
magnification (Fig. 5 C). A 24-mo-old virgin mutant animal displays ductal dysplasia and stromal/fibroblastic hyperplasia (Fig. 5 D). Secretory vacuoles were observed in
the alveoli of a 20-mo-old virgin animal consistent with a
differentiated hyperplastic phenotype (Fig. 5 E). In addition, extensive periductal lymphocyte infiltration was observed in the interstitial space of the mutant glands (Fig. 5
F). Furthermore, several multiparous mutant females (15 mo old) exhibited extensive mammary hyperplasia and dysplasia, however, palpable mammary tumors were not observed (data not shown). It should be noted that P-cadherin is not expressed in the epithelium which becomes
hyperplastic, but confined to the surrounding myoepithelium which appears normal in the mutant animals. In conclusion, mutation of the P-cadherin gene predisposes animals to focal hyperplasia and dysplasia as well as leading to an increase in lymphoid cells in the mammary gland.
Fig. 5.
P-cadherin mutant mice develop focal hyperplasia and dysplasia with age. Histology of virgin mammary tissue from wild-type (A) and mutant (B-F) animals, 19-24 mo of age. The mutant mice (B) exhibit extensive alveolar differentiation compared with their control littermates (A). Higher magnification of the alveoli (C). P-cadherin mutant animals develop fibroblastic (D) and secretory hyperplasia (E) and exhibit extensive lymphocyte infiltration (F). The lymphocytes are concentrated around the ducts (arrowhead). A
portion of the lymph node is shown (arrow). Original magnifications: (A and B) 12.5x; (C-F) 31x.
[View Larger Version of this Image (79K GIF file)]
Discussion
; Riethmacher
et al., 1995
; Radice et al., 1997
). Here we report the mutation
of a third classical cadherin, P-cadherin, that is compatible
with normal embryonic development. Although P-cadherin
is expressed at high levels in placenta and testis, P-cadherin-null females and males are both fertile. E- and P-cadherin are often coexpressed in embryonic and adult tissues,
for example, both cadherins are present in the notochord
and basal layer of the epidermis. In these tissues, E-cadherin may functionally substitute for P-cadherin in the mutant animals thus explaining their viability. However, recently, a distinct pattern of expression of E- and P-cadherin
was reported for the mouse mammary gland (Daniel et al.,
1995
). The mammary duct consists of two main cell types,
the lumenal epithelial cells and an outer monolayer of myoepithelial cells with a closely apposed basement membrane.
E-cadherin expression is restricted to the lumenal epithelial cells, while P-cadherin is expressed in the surrounding
myoepithelial cells. We observed an unexpected phenotype in the mammary glands of the mutant animals. Loss of P-cadherin resulted in cell proliferation and differentiation of the
mammary epithelium in virgin female animals.
). However, ductal morphogenesis appears normal in the mutant mice, indicating that P-cadherin is not required by the cap
cells for invasion of the surrounding fat pad. There are
presumably other cell adhesion molecules expressed by the
cap cells which can functionally substitute for P-cadherin
in the mutant mammary glands. Normally, the mammary
gland does not differentiate fully until the onset of pregnancy when lateral buds develop as side branches of the
mammary tree. The alveoli develop further during the second half of pregnancy into fully differentiated secretory lobules capable of synthesizing and secreting milk proteins. The P-cadherin-deficient females display precocious
differentiation of the mammary epithelium similar to an
early pregnant animal. Furthermore, the mutant virgin
glands synthesize milk protein (i.e., casein) indicative of a
differentiated mammary epithelium.
), there is very little information about the role of the myoepithelium in this process, even though it separates the stroma and basal lamina
from the lumenal epithelial cells. The myoepithelium is situated in a unique position to regulate signals from the surrounding ECM and stroma to the underlying epithelium,
the indirect regulation model (Fig. 6 A). The myoepithelium consists of a monolayer of cells that probably does
not represent a physical barrier per se, but it may act as a
filter to transmit specific signals to the underlying epithelium. P-cadherin-mediated adhesion may be important for the assembly of other junctional complexes. A cell adhesion
defect may perturb intercellular communication (i.e., gap
junction assembly) in the myoepithelium leading to altered
growth signals. The loss of P-cadherin may affect the function of other cell adhesion molecules, for example, integrin-
mediated ECM interactions may be compromised leading
to changes in cell morphology of the myoepithelial cells.
Fig. 6.
Possible models for regulation of mammary gland differentiation by P-cadherin. (A) The indirect regulation model is
based on a cell adhesion defect. For example, inductive signals
from the surrounding ECM (basal lamina) and/or stroma may
more easily permeate a less cohesive myoepithelium leading to
differentiation of the epithelium. (B) The direct regulation model
is based on a P-cadherin-mediated cell signaling defect. For example, loss of P-cadherin may cause the myoepithelium to relay a
positive signal(s), or alternatively, it may relieve an inhibitory signal(s) resulting in differentiation of the lumenal epithelium. The
cell adhesion and cell signaling models are not mutually exclusive.
[View Larger Version of this Image (34K GIF file)]
-catenin homologue, is a
segment polarity gene acting in the wingless signaling pathway. As in vertebrates, it also interacts with cadherins and
is localized to adherens junctions. In Drosophila, the functions of armadillo in adherens junctions and wingless signaling are competitive. The segment polarity defect of a
weak armadillo allele can be partially rescued by making
the flies heterozygous for DE-cadherin (Cox et al., 1996
).
The suppression of the armadillo mutation is thought to
occur because there is a reduction in the amount of DE-cadherin thus freeing up some of the wild-type maternal store of armadillo protein, allowing it to function in the
wingless pathway. Similar competitive interactions have
been seen in Xenopus, where increases in cadherin levels
can block action of
-catenin in Wnt signaling as monitored by inhibition of axis duplication (Fagotto et al., 1996
).
In both these cases, there appears to be a competition between the
-catenin bound to the cytoplasmic tail of cadherin and the free cytoplasmic
-catenin involved in the
wingless/Wnt signaling pathway.
-catenin.
Data from transgenic and mutant mice suggest that the
P-cadherin-null phenotype may be due to activation of the
Wnt signaling pathway. Transgenic mice that overexpress
Wnt-1 in the mammary gland develop extensive alveolar
hyperplasia and adenocarcinomas (Tsukamoto et al., 1988
). In addition, mutation of another member of the Wnt signaling pathway, Adenomatous polyposis coli (APC), predisposes mice to focal alveolar hyperplasia and carcinomas
(Moser et al., 1993
). APC is a negative regulator of the intracellular
-catenin pool, hence when APC is active the
cytoplasmic pool of
-catenin is very low. When the Wnt
pathway is activated, APC is turned off, and
-catenin accumulates in the cytoplasm.
-catenin can then interact
with the DNA binding proteins of the T cell factor-lymphoid enhancer factor (Tcf-Lef) family to activate transcription of target genes (Behrens et al., 1996
). The loss of
P-cadherin may increase the available cytoplasmic
-catenin thus activating the Wnt signaling pathway resulting in
transcription of growth factor genes whose gene products
induce differentiation of the neighboring epithelial cells,
the direct regulation model (Fig. 6 B). This scenario assumes that APC cannot efficiently inactivate the excess
-catenin
present in the P-cadherin-deficient mammary gland.
), growth hormone (Bchini
et al., 1991
), HGF/SF (Takayama et al., 1997
), and stromelysin1 (Sympson et al., 1994
) lead to extensive alveolar hyperplasia and mammary tumors in transgenic mice. The
extent of precocious alveolar differentiation observed in
the P-cadherin-deficient mice appears less dramatic in
comparison to these other transgenic strains. No palpable
tumors were observed in the mutant animals, although, focal hyperplastic and dysplastic lesions were observed in
the mutant mammary glands upon necropsy. Hyperplasia
was also observed in the salivary gland where P-cadherin
is normally expressed in the myoepithelial cells (Ferreira-Cornwell, M.C. and G.L. Radice, unpublished data). The finding that disruption of P-cadherin function alone is not
sufficient to induce mammary tumors, suggests that additional genetic lesion(s) are necessary to progress beyond
the hyperplastic phenotype.
). E- and P-cadherin are tandemly arranged
at this locus separated by only 32 kb of genomic DNA
(Bussemakers et al., 1994
). While cadherin expression has
been examined extensively in the tumor tissue itself, perturbation of cadherin function in the surrounding myoepithelium has not been addressed. Germline mutations in
the human P-cadherin gene may predispose women to breast cancer since hyperplastic growth is an early step in
tumor development.
).
H-cadherin lacks a cytoplasmic domain and is most similar
to T-cadherin (Ranscht and Dours-Zimmermann, 1991
). Its
expression was found to be significantly reduced in human
breast carcinoma cell lines and primary breast tumors (Lee,
1996
). Furthermore, transfection of H-cadherin into the breast
cancer cell lines led to a decreased cell growth rate and loss
of anchorage-independent growth in soft agar. These data suggest that two distinct cadherins, H- and P-cadherin, are
involved in negative growth control in the mammary gland.
), activin/inhibin
B (Vassalli et al., 1994
), progesterone (Lydon et al., 1995
),
and prolactin receptors (Ormandy et al., 1997
), and Stat5a
(Liu et al., 1997
) all result in inhibition of lobuloalveolar
outgrowth in the mammary gland. All these gene products
are positive effectors in a signaling pathway(s) leading to
alveolar cell proliferation and differentiation. In contrast,
P-cadherin appears to act as a negative regulator of cell
growth and differentiation in the mammary gland. Its absence leads to precocious alveolar differentiation in virgin animals.
Address correspondence to Glenn Radice, Department of Obstetrics and Gynecology, Division of Reproductive Biology, University of Pennsylvania, Room 778 Clinical Research Building, 415 Curie Blvd., Philadelphia, PA 19104-6142. Tel.: 215-898-0164. Fax: 215-573-5408. e-mail: gradice{at}obgyn.upenn.edu
Received for publication 17 July 1997 and in revised form 28 August 1997.
1. Abbreviations used in this paper: APC, adenomatous polyposis coli; ECM, extracellular matrix; ES, embryonic stem; HGF/SF, hepatocyte growth factor/scatter factor; HSV, Herpes simplex virus; PCD-1, P-cadherin antibody.This work was supported in part by a Japan Society for Promotion of Science Fellowship (G.L. Radice), University of Pennsylvania's Cancer Center Pilot Project Program, IRG-135P from the American Cancer Society, National Institutes of Health (1R21CA66179); the Charles E. Culpeper Foundation (L.A. Chodosh); and a Program of Excellence grant (HL41484) from the National Heart Lung and Blood Institute and by the Howard Hughes Medical Institute. L.A. Chodosh is a Charles E. Culpeper Medical Scholar. R.O. Hynes is an Investigator of the Howard Hughes Medical Institute.
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