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Cell Motility and the Cytoskeleton 40:87–100 (1998)
Plakoglobin Induces Desmosome Formation
and Epidermoid Phenotype
in N-Cadherin-Expressing Squamous
Carcinoma Cells Deficient in Plakoglobin
and E-Cadherin
Henry R. Parker,1 Zhi Li,1 Hannah Sheinin,2 Gille Lauzon,2 and Manijeh Pasdar1*
1Department
of Cell Biology and Anatomy, University of Alberta,
Edmonton, Alberta, Canada
2Department of Dermatology, University of Alberta, Edmonton, Alberta, Canada
Pg is a homologue of b-catenin and Armadillo, the product of the Drosophila
segment polarity gene and has been shown to have both adhesive and signaling
functions. It interacts with both classic and desmosomal cadherins. Pg interaction
with the desmosomal cadherins is essential for desmosome assembly. Its precise
role in the classic cadherin complexes is unclear, although Pg-E–cadherin
interaction appears to be necessary for the formation of desmosomes. In addition to
cadherins in adhesion complexes, Pg interacts with a number of proteins involved
in regulation of cell differentiation and proliferation such as Lef-1/Tcf-1 transcription factors and the tumor suppressor protein APC.
In this study, we have introduced Pg cDNA into SCC9 cells, a Pg- and
E-cadherin-deficient squamous cell carcinoma line, which also lacks desmosomes.
These cells have both a-catenin and b-catenin, display unusual expression of
N-cadherin, and have the typical fibroblastic phenotype of transformed cells.
Pg-expressing SCC9 cells (SCC9P) formed desmosomes. Desmosome formation
coincided with the appearance of an epidermoid phenotype, with increased
adhesiveness and a contact-dependent decrease in growth. Biochemical characterization of SCC9P cells showed an increase in the expression and stability of
N-cadherin and a decrease in level and stability of b-catenin, without any apparent
effects on a-catenin. These results show that, in the absence of E-cadherin, Pg can
efficiently use N-cadherin to induce desmosome formation and epidermoid
phenotype. They also suggest a role for Pg as one of the regulators of the
intracellular b-catenin levels and underscore the pivotal role of this protein in
regulating cell adhesion and differentiation. Cell Motil. Cytoskeleton 40:87–100,
1998. r 1998 Wiley-Liss, Inc.
Key words: plakoglobin; b-catenin; cadherins; tumorigenesis; epidermoid; squamous carcinomas
INTRODUCTION
Plakoglobin (Pg, 83 kDa) is a multifunctional
protein that was first studied as a cytoplasmic component
common to both kinds of epithelial adhering junctions
(desmosomes and adherens junctions) [Cowin et al.,
1986]. Adherens junctions and desmosomes connect cells
to each other and anchor the cytoskeleton to the plasma
membrane [for reviews, see Geiger and Ayalon, 1992;
r 1998 Wiley-Liss, Inc.
Contract grant sponsor: Medical Research Council of Canada; Contract
grant sponsor: Leo Laboratories Canada Ltd
Dr. Parker is currently at the Department of Biochemistry, University
of Alberta, Edmonton, Alberta, Canada.
*Correspondence to: Manijeh Pasdar, Department of Cell Biology and
Anatomy, University of Alberta, Edmonton, Alberta T6G 2H7, Canada;
E-mail: mpasdar@anat.med.ualberta.ca
Received 9 December 1997; accepted 12 February 1998
88
Parker et al.
Garrod et al., 1996; Schmidt et al., 1994]. The transmembrane domain of adherens junctions is enriched in
E-cadherin, which interacts with the actin cytoskeleton
via the cytoplasmic catenins [Aberle et al., 1996; Kemler,
1993]. b-Catenin is structurally similar to Pg and interacts directly with the classic cadherins. a-Catenin shows
some structural similarity to vinculin, exhibits actinbinding activity, interacts with cadherins through b-catenin
or Pg, and connects the cadherin–catenin complex to the
actin cytoskeleton [Rimm et al., 1995; Knudsen et al.,
1995]. g-Catenin is identical to Pg [Knudsen et al., 1992;
Peifer et al., 1992]. Pg interacts with both classic and
desmosomal cadherins [for a recent review, see Cowin,
1994]. Pg is an indispensable structural element of the
desmosome. At this junction, desmosomal cadherins,
desmogleins (Dsg) and desmocollins (Dsc) constitute the
transmembrane domain and Pg is essential to mediate the
interaction between these cadherins and the cytokeratin
intermediate filaments via desmosomal plaque proteins,
desmosplakins (DP) [Mathur et al., 1994; Troyanovsky,
1994; Bierkamp et al., 1996; Ruiz et al., 1996; Palka and
Green, 1997; Kowalczyk et al., 1997]. Pg and b-catenin
interact with classic cadherins in a mutually exclusive
manner [Hinck et al., 1994]. The cytoplasmic domains of
desmosomal and classic cadherins share sequence similarity in a small region which is the Pg site of interaction
[Mathur et al.; 1994; Jou et al., 1995; Aberle et al., 1994].
Although Pg is not an essential structural component of
adherens junction, its interaction with E- or P-cadherin in
epithelial cells appears to be necessary for the formation
of desmosomes [Lewis et al., 1997].
In addition to interaction with cadherins and its
structural role in junctional complexes, Pg, as is also true
of its closely related protein b-catenin, interacts with a
number of proteins involved in pathways regulating cell
fate determination and cellular growth and proliferation
[for a review, see Peifer, 1996, and references therein].
One such protein is the product of the tumor suppressor
gene APC, which has been shown to be the major
regulator of the cytoplasmic b-catenin levels. APC binds
to b-catenin and Pg and via either of these two proteins to
acatenin. This protein is also a component of the wnt
signaling pathway. The vertebrate wnt and its Drosophila
homologue wingless (wg) are involved in the determination of embryonic segment polarity [Nusse, and Varmus,
1992; Parkin et al., 1993; Perrimon, 1994; Burrus, 1994;
Moon et al., 1997]. In vertebrates, overexpression of wnt
proteins causes duplication of dorsal axis. Certain tissue
culture cells exposed to wnt show increased levels of Pg
and b-catenin [Bradley et al. 1993; Hinck et al., 1994].
Increased expression of Pg by itself has also been shown
to induce axis duplication in Xenopus [Karnovsky and
Klymkowsky, 1994]. Pg also interacts with transcription
factors Lef-1/Tcf-1 and, if overexpressed, Pg and b-catenin
can be translocated into the nucleus [Karnovsky and
Klymkowsky, 1994; Behrens et al., 1996; Huber et al.,
1996b].
The adhesive and signaling functions of Pg are
regulated by its interacting protein partners. To gain
further knowledge into the functions of Pg, we have used
a squamous carcinoma cell line (SCC9) that lacks both Pg
and E-cadherin and does not assemble desmosomes.
These cells have both a- and b-catenins but display
unusual expression of N-cadherin and have typical fibroblastoid morphology of transformed cells. We have
shown that transfection of SCC9 cells with L-CAM or
E-cadherin cDNAs induces a morphologic transformation from fibroblast to epidermoid which coincides with
downregulation of the endogenous N-cadherin and increased synthesis and stability of the catenins [Li et al., in
press]. Here, we show that the introduction of Pg cDNA
into SCC9 cells enables them to form desmosomes and
induces a fibroblast to epidermoid transition, similar to
that observed for L-CAM. However, unlike the effects of
E-cadherin expression, these Pg-induced changes coincide with increased stability and steady-state level of
N-cadherin and decreased level and stability of b-catenin
without any significant effects on a-catenin.
MATERIALS AND METHODS
Cells and Culture Conditions
The human squamous carcinoma cells SCC9,
SCC15, and SCC25, all derived from carcinomas of the
tongue (American Type Culture Collection [ATCC],
Rockville, MD) were provided by Dr. A. Klein-Santoz
(Fox Chase Cancer Center, Philadelphia, PA). These lines
were maintained in minimum essential medium (MEM)
(Sigma Chemical Co., St. Louis, MO) supplemented with
10% fetal bovine serum (FBS). SCC9 cells that were
transfected with the Pg expression plasmid (hereafter
referred to as SCC9P) were maintained in the same
medium containing 200 µg/ml geneticin (G418, Life
Technologies, Grand Island, NY). SCC9L, L-CAMexpressing SCC9 cells have been described previously
[Li et al., in press] and were maintained under the same
conditions as described for SCC9P cells. Primary cultures
of keratinocytes from neonatal foreskins were established
in low Ca21-containing keratinocyte medium (Life Technologies) supplemented with epidermal growth factor
(EGF), pituitary extract, and 20% FBS. Before metabolic
labeling (30 min–2 h), and to facilitate methionine
uptake, all cultures were incubated in MEM, with calcium levels adjusted to 5 µM (low Ca21 concentration
medium [LCM]).
Plakoglobin Induces Fibroblast to Epidermoid Transition
Plasmid Construction and Transfection
A full-length cDNA for human Pg was produced
using standard polymerase chain reaction (PCR) protocols based on the published sequences [Franke et al.,
1989] with the incorporation of BamHI restriction sites in
the primers. The authenticity of the PCR product was
verified by sequencing. The entire coding sequence was
digested with BamHI and cloned into the mammalian
episomal expression vector, pREP9 (Invitrogen, San
Diego, CA) creating pREP9Pg. Positive clones were
selected for proper orientation relative to the vector’s
promoter, Rous sarcoma virus (RSV), long terminal repeat
enhancer/promoter. This plasmid was used to transfect SCC9
cells using LipofectAMINE (Gibco-BRL, Grand Island, NY)
reagent, according to the manufacturer’s protocol.
Briefly, SCC9 cells were grown in 60-mm culture
dishes to 80% confluency, at which time the medium was
replaced with LCM. Transfection was performed 12–16 h
later using 25 µg (per dish) of either pREP9 or pREP9Pg.
At 48 h post-transfection, medium was replaced with
MEM containing 400 µg/ml G418. Resistant colonies
were selected and screened for Pg expression, using
immunofluorescence and immunoblotting assays (see
below). Positive colonies (SCC9P) were subcultured by
limiting dilution to obtain single-cell isolated colonies.
Antibodies and cDNA Probes
Rabbit polyclonal anti-a-catenin, b-catenin, and
pan-cadherin were purchased from Sigma. We recently
showed that the single cadherin detected by the pancadherin antibodies in SCC9 cells is N-cadherin [Li et al,
in press]; therefore, it is referred to as such throughout
this report. Rabbit polyclonal anti-Pg antibodies have
been described [Pasdar et al., 1995a]. The secondary
antibodies used in immunofluorescence studies were
purchased from Boehringer-Mannheim Biochemicals (Indianapolis, IN).
The N-cadherin cDNA probe was provided by Dr.
John Hemperly (Becton Dickinson Research Center,
Research Triangle Park, NC) and corresponds to the 38
EcoR1 fragment (1.1 kb) of the human N-cadherin cDNA
[Reid and Hemperly, 1990]. A 0.6-kb BamH1 fragment of
mouse a-catenin cDNA and a 1.3 -kb EcoR1 fragment of
mouse b-catenin cDNA were provided by Dr. Lionel
Larue (Curie Institute, Orsay, France). The Pg probe
corresponds to the 1.5-kb Sac1 fragment of the human
cDNA.
Metabolic Labeling
Turnover studies were performed as described [Pasdar, 1992]. Briefly, confluent cultures of SCC9 and
SCC9P cells were grown in MEM in 35-mm culture
dishes. The medium was removed, cells washed, and
LCM added for 1 h, followed by removal, wash, and
89
addition of methionine-free LCM for another hour. Cultures were metabolically labeled with 125 µCi of
35S-methionine (1,189 Ci/mmol, ICN Biochemicals, Irvine, CA) in methonine-free LCM for 30 min, then chased
for different times (0–24 h) in .10,000-fold excess of
unlabeled methionine in MEM. For protein interaction
studies, cells were grown on 60-mm culture dishes and
processed as above except the labeling was with 250 µCi
35S-methionine for 1 h, at which time Ca21 was added to
the media, bringing the concentration to 1.8 mM, and
labeling continued for another 3 h.Upon completion of
labeling or chasing, cells were kept at 4°C, rinsed twice
with phosphate-buffered saline (PBS) and processed for
extraction, followed by immunoprecipitation (see below).
Cell Fractionation and Immunoprecipitation
Cells were extracted in the culture dish with 1 ml of
cytoskeleton extraction (CSK) buffer containing 300 mM
sucrose, 10 mM Pipes, pH 6.8, 50 mM NaCl, 3 mM
MgCl2, 0.5% (v/v) Triton X-100, 1.2 mM PMSF, 0.1
mg/ml DNase, and 0.1 mg/ml RNase [Pasdar, 1992]. This
extraction buffer allows the separation of the cytoskeletonassociated (insoluble) from the soluble pool of cadherin
and catenins. Cells were removed from the dishes with a
rubber policeman and centrifuged at 48,000g for 10 min.
The soluble fraction was removed from the insoluble
pellet. The pellet was resuspended in 100 µl of sodium
dodecyl sulfate (SDS) buffer (1% SDS, 10 mM Tris-HCl,
pH 7.5, 2 mM EDTA, 0.5 mM DTT, 0.5 mM PMSF) and
boiled for 10 min. Before immunoprecipitation, the SDS
concentration was reduced to 0.1% by the addition of 900
µl CSK buffer. The soluble and insoluble fractions were
precleared for 30 min by incubation with 10 µl of
preimmune sera and 35 µl of Pansorbin cells (Calbiochem, La Jolla, CA) as described [Pasdar, 1992]. After a
7-min centrifugation at 14,000g, precleared extracts were
divided into 4 equal aliquots and processed for immunoprecipitation with 2–3 µl of different antibodies, either
Pg, pan-cadherin, a-catenin, or b-catenin. To ensure
complete depletion, each aliquot was immunoprecipitated
three times.
Protein interactions were analyzed in the soluble
fractions. These analyses did not include the insoluble
fractions because the insoluble proteins have to be
solubilized before immunoprecipitation and the solubilization conditions (1% SDS at 100°C) causes dissociation
of the complex. The soluble proteins of metabolically
labeled cells were first immunoprecipitated with anti-pancadherin under low stringency conditions as described
[Pasdar et al., 1995a]. The resulting immune complexes
were eluted from the protein A-Sepharose beads by
boiling for 20 min followed by centrifugation at 14,000g
for 5 min. Supernatants were then transferred to new
tubes and the SDS concentration adjusted to 0.1% by the
90
Parker et al.
addition of 900 µl CSK buffer. The second immunoprecipitations were performed under high stringency conditions
with anti-a- and b-catenins, and Pg antibodies as described above.
Immunoprecipitates (200 µg total protein for each
cell line and/or time point, unless otherwise stated) were
boiled in 65 µl SDS sample buffer and separated by
electrophoresis on SDS 6% polyacrylamide gels, followed by fluorography. For protein turnover studies, the
relative amount of radioactivity in the protein bands
corresponding to Pg, N-cadherin, and a- and b-catenins
was determined by scanning densitometry from multiple
exposures of resulting fluorograms [Pasdar, 1992]. Each
experiment was repeated 3–5 times; the results are
presented for one typical experiment.
Immunoblotting
Total cell extracts were prepared by directly solubilizing cells in SDS sample buffer. Total and CSK soluble
and insoluble cell extracts were resolved in SDS polyacrylamide gels, and the proteins electrophoretically transferred onto nitrocellulose membranes as described previously [Towbin et al., 1979; Pasdar and Nelson, 1988].
Membranes were incubated sequentially with 1/1,000
dilution of primary antibodies (Pg, pan-cadherin,
a-catenin, and b-catenin) for 1.5 h, followed by a 30-min
incubation with 125I-protein A (1 mCi/ml, ICN) and were
exposed at 280°C to XAR-5 x-ray films (Kodak) using
two intensifying screens. Unless otherwise stated equivalent of 50 mg total protein from each cell line were
analyzed in immunoblots.
Northern Blotting
RNA was isolated from cells of various cell lines
and primary keratinocytes using Trizol reagent (Gibco),
following the manufacturer’s protocol. Approximately 25
µg of the resulting RNA was electrophoresed in formaldehyde-containing 1% agarose gels, and transferred to
Hybond N membranes (Amersham, Arlington Heights,
Ill.). Prehybridization and hybridization conditions were
as described [Church and Gilbert, 1984]. After hybridization with 32P-labeled cDNA probes, membranes were
processed for autoradiography.
Immunofluorescence and Electron Microscopy
SCC9 and SCC9P cells were grown in MEM on
collagen-coated coverslips, extracted with CSK buffer,
fixed with formaldehyde, and processed for indirect
immunofluorescence and confocal microscopy as described [Pasdar et al., 1995a]. All antibodies were diluted
in PBS, Pg (1/100), catenins, and pan-cadherin (1/250),
secondary rhodamine-conjugated goat anti-rabbit IgG
(1/100). Coverslips were mounted in elvanol containing
0.2% paraphenylene diamine (PPD), pH 8.6, viewed with
a 1003 objective ,using a laser scanning confocal microscope (Leica Lasertechnic, Heidelberg). Optical sections
through the middle of the cells were recorded on optical
disks and viewed on a Silicon Graphics imager (SGI).
For electron microscopy, cultures were grown to
confluency on collagen-coated coverslips. Cells were
fixed in a freshly made solution containing 1.25% glutaraldehyde, 1% OsO4 in 50 mM Pipes, pH 7.2, for 5 min at
room temperature and processed for electron microscopy
as described [Pasdar et al., 1995b] and examined using a
Philips 410 transmission electron microscope.
Cell Growth and Aggregation Assays
To measure growth, 3 3 104 SCC9 and SCC9P cells
were each plated in replicate 35-mm culture dishes. At 1,
3, 5, 7, 9, and 11 days after plating, cultures were
trypsinized and cells counted.
Aggregation assays were performed as described
[Li et al., in press] and entailed triplicate confluent
60-mm culture dishes of SCC9 and SCC9P cells as stocks
for single-cell suspensions. After trypsinization (0.04%
trypsin in PBS containing 1 mM Ca21) of each culture,
replicates of 2 3 106 single cells were resuspended into 2
ml aliquots. Cells were incubated at 37°C with constant
shaking at 90 rpm for a total of 2 h. At 30-min intervals,
200-µl aliquots were withdrawn and the number of single
cells counted using a hemocytometer. The percentage of
aggregation represents the percent decrease in the number
of single cells at each time point.
RESULTS
SCC9 Cells Lack Plakoglobin
Figure 1A shows the immunofluorescence staining
of Pg in control primary keratinocytes (Fig.1A; KC), the
cells of SCC9 and two other, tongue-derived, squamous
cell carcinoma lines; SCC15 and SCC25. Pg was absent
in SCC9 and expressed in all the other cell lines. In
keratinocytes, most of the Pg was present in a punctate
peripheral distribution typical of desmosomal staining,
whereas in SCC15 and SCC25 Pg was distributed both
intracellularly and at the cell surface. The absence of Pg
protein in SCC9 cells was supported by immunoblotting
of the TX-100-soluble and -insoluble extracts of all cells
with anti-Pg antibodies that detected Pg, in both the
soluble and insoluble pools, in primary keratinocytes and
SCC15 and SCC25, and not in SCC9 (Fig.1B). Furthermore, Northern blotting using a human Pg probe detected
mRNAs of the appropriate size (,3.5 kb) in the primary
keratinocytes and SCC15 and 25 and not in SCC9 cells
(Fig.1C). These three independent assays showed that Pg
is not expressed in SCC9 cells and, therefore, that this cell
line can be used as a useful model to study the function
of Pg.
Plakoglobin Induces Fibroblast to Epidermoid Transition
91
Morphology, Growth, and Adhesive Properties
of SCC9 and SCC9P Cells
Fig. 1. SCC9 cells do not express plakoglobin. A: Primary cultures of
neonatal foreskin keratinocytes and cells of human squamous carcinoma cell lines SCC9, SCC15, and SCC25 were established on
coverslips, extracted with CSK buffer, and processed for indirect
immunofluorescence with a rabbit anti-Pg antibody, as described under
Materials and Methods. Bar 5 20 µm. B: The same cultures were
grown to confluency and extracted with CSK buffer to separate the
CSK soluble (S) and insoluble (P) fractions, followed by immunoblot
analysis using the anti-Pg antibodies. C: A total of 25 µg of total RNA
from the same cultures was processed for Northern blot analysis, using
32P-labeled cDNA probes for Pg, as described under Materials and
Methods.
First, SCC9 cells were subcloned by limiting dilution to develop homogeneous fibroblastoid lines. A number of lines were characterized for expression of the
epithelial marker proteins, cytokeratins, and various
desmosomal proteins, and were all positive. One such
clone was used for transfection with pREP9Pg containing
the full-length human Pg cDNA. A number of Pgexpressing clones were isolated and used for the studies
reported here. All Pg-expressing clones had similar
properties. Three clones were fully characterized and the
results of the studies performed on one such clone are
presented here.
SCC9 cultures showed the typical fibroblastoid
morphology of transformed cells (Fig. 2A,a). Electron
microscopic examination of these cells showed the absence of any well-organized adhering junctions, although
the plasma membranes of adjacent cells were closely
opposed (Fig.2A,b). Growth rate analysis showed that at
single-cell density, these cells grew slowly until they
became subconfluent (5 days), after which the rate of
growth increased significantly. Upon reaching confluency
(day 7), the growth rate of these cells accelerated further,
and they continued to grow at a high rate up to 11 days
(Fig. 2B; growth, open squares).
The aggregation assay showed about 35% decrease
in the number of single cells within the first 30 min. There
was a small increase in the number of aggregates within
the next 90 min, so that by the end of the assay time, 45%
of cells were present in aggregate forms (Fig. 2C; open
squares).
In contrast to the untransfected SCC9 cells, Pgexpressing cells became epidermoid, formed desmosomes and showed decreased growth rate and increased
adhesiveness (Fig. 2A,c,d; Fig. 2B,C; solid squares).
SCC9P cells were flat and developed tight epidermoid
monolayers (Fig. 2A,c). Ultrastructural examination of
these cells showed the presence of well formed desmosomes (Fig. 2A,d, arrows). Growth and aggregation
characteristics of these cells also underwent changes. Pg
expression in these cells led to contact inhibition of
growth as once SCC9P cells reached confluency (7 days),
they began to die (Fig. 2B; solid squares)
The epidermoid morphology of SCC9P cells was
associated with increased adhesivity of these cells (Fig.
2C; solid squares). There was a significant increase in
aggregation of SCC9P cells from the beginning, which
persisted throughout the assay. At 30 min, there were
approximately twice as many aggregates in SCC9P as
that detected in SCC9 cultures (68% vs. 35%). Within the
next 60 min, there was a further increase (to 77%), which
remained unchanged for the rest of the assay period (Fig.
2C; solid squares). Together, these results showed that Pg
expression in SCC9 cells promoted desmosome formation which coincided with a fibroblast to epidermoid
morphological transition associated with contact inhibition of growth and increased adhesiveness.
Exogenously Expressed Pg Shows Biological
Properties of Pg Expressed in Normal
Epithelial Cells
SCC9 and SCC9P cells were grown to confluency
on collagen-coated coverslips, extracted with CSK buffer,
fixed, and stained with anti-Pg antibodies, followed by
rhodamine-conjugated goat-anti-rabbit IgG (Fig. 3A).
92
Parker et al.
Fig. 2. Induction of desmosome assembly
and epidermoid phenotype by the exogenous
Pg in SCC9 cells. A: Phase-contrast (left) and
electron micrographs (right) of untransfected
(SCC9; a and b) and Pg-transfected SCC9
cells (SCC9P, c and d). Arrows, desmosomes.
Bars (a and c) 5 10 µm; (b) 5 0.5 µm; (d) 5
0.25 µm. B: Growth and aggregation properties of SCC9 and SCC9P cells. Growth measurements were taken by establishing replicate cultures at single cell density and counting
cells at 2-day intervals after trypsinization as
described under Materials and Methods. Each
time point represents the average of three
independent experiments. For the aggregation
assays, single cell suspensions were prepared
and cells allowed to aggregate under constant
shaking as described under Materials and
Methods. Aliquots were taken at 30-, 60-, 90-,
and 120-min intervals and the number of
single cells counted. The rate of aggregation
was calculated as the percentage of decrease
in the number of single cells. Each value is the
average of at least three independent experiments; standard errors are indicated by error
bars.
The SCC9 cells were negative, whereas SCC9P cells
showed Pg staining, most of which was detected at the
cell surface in a punctate pattern typical of desmosomes.
Immunoblot analysis of the steady-state distribution of Pg
indicated a ratio of 40:60 for the CSK soluble (S) and
insoluble (P) Pg expressed in SCC9P cells (Fig. 3B), in
agreement with similar ratios reported for various cell
types previously [Kapprell et al., 1987; Pasdar, 1995a]. A
faster migrating band was also detected in the soluble Pg
immunoblots (and immunoprecipitates), the identity of
Plakoglobin Induces Fibroblast to Epidermoid Transition
93
Fig. 3. Characterization of the exogenously expressed Pg in SCC9
cells. A: (SCC9) and Pg-expressing SCC9 cells (SCC9P) were
established on glass coverslips and processed for indirect immunofluorescence with Pg antibodies. Bar 5 10 µm. B: Immunoblot analysis of
total (T) and CSK soluble (S) and insoluble (P) fractions of SCC9 and
SCC9P cells using anti-Pg antibodies. C: Fate of the newly synthesized
exogenously expressed Pg in SCC9 cells. SCC9P cells were grown to
confluency, labeled with 35S-methionine for 30 min and chased up to 24
h, as described under Materials and Methods. After cell fractionation,
the soluble (S) and insoluble (P) fractions were processed for immunoprecipitation with anti-Pg antibodies and analyzed by PAGE followed
by fluorography. D: To estimate the Pg turnover rate, relative amounts
of radioactivity in Pg bands from multiple exposures of the fluorograms
in C were scanned using a scanning densitometer. E: The exogenously
expressed Pg associates with the N-cadherin expressed in SCC9 cells.
Confluent cultures of SCC9P cells were 35S-labeled for a total of 4 h, as
described under Materials and Methods, and the soluble cell extracts
were immunoprecipitated with pan-cadherin antibodies. In a duplicate
aliquot, immune complexes were eluted and reprecipitated with Pg
antibodies as described under Materials and Methods. The position of
N-cadherin, a-catenin, b-catenin, and Pg are indicated in E.
which is unclear. It is noteworthy that this protein has also
been detected in other cell types [Piepenhagen and
Nelson, 1993; Pasdar, 1995a].
Immunoprecipitation of the metabolically labeled
SCC9P cell extracts with anti-Pg antibodies indicated a
40:60 ratio for newly synthesized soluble (S) and insoluble (P) Pg at the termination of the 30-min labeling
period (Fig. 3C; 0S, 0P), which remained throughout the
chase period. The soluble Pg was relatively stable (Fig.3D;
open circles; half-life [t1/2] ,9 6 2 h). There was an
initial rapid decrease in the amount of the insoluble Pg
within the first 2 h of the chase (Fig. 3C; 2S, 2P); after this
time point, the protein was degraded gradually with a t1/2
of ,16 6 1.8 h (Fig. 3C,3D, solid circles).
Sequential immunoprecipitation showed that the
exogenously expressed soluble Pg associated with the
N-cadherin expressed in SCC9 cells (Fig. 3E, N-cad -.
Pg), consistent with the previous reports on the associa-
94
Parker et al.
Fig. 4. Expression of N-cadherin and catenins in SCC9P cells. A:
SCC9 and Pg-expressing (SCC9P) cells were processed for indirect
immunofluorescence with antibodies to pan-cadherin detecting Ncadherin, a-catenin, and b-catenin. Bar 5 10 µm. B: Immunoblot
analysis of the total (T) or CSK soluble (S) and insoluble (P) fractions
of SCC9 and SCC9P cells using pan-cadherin detecting N-cadherin and
a-catenin, and b-catenin antibodies. C: Quantitation of the amounts of
N-cadherin and catenins from three independent experiments, as
represented in B. Error bars indicate standard errors. D: SCC9 and
SCC9P cells were 35S-labeled and chased as described in Fig. 3C. After
cell fractionation, the soluble (S) and insoluble (P) fractions were
processed for N-cadherin immunoprecipitation. Immune complexes
were analyzed by PAGE, followed by fluorography. Arrows (D),
position of N-cadherin precursor.
tions between Pg and N- as well as other classic cadherins
[Knudsen and Wheelock, 1992; Hamagouchi et al., 1993;
Sacco et al., 1995; Simcha et al., 1996; Hertig et al., 1996;
Aberle et al., 1996]. Together, the results of these
experiments indicated that the exogenously expressed Pg
follows the same biosynthetic fate as the Pg normally
expressed by epithelial cells.
SCC9 and SCC9P cells fixed and processed for indirect
immunofluorescence. Staining of SCC9 cells with a
pan-cadherin antibody, which detects N-cadherin in SCC9
cells, and antibodies to a- and b-catenins detected both
intracellular and cell surface distributions of these proteins (Fig. 4A). N-Cadherin and the catenins were
distributed in a similar manner in SCC9 and SCC9P cells
(Fig. 4A), although in SCC9P cells, the staining appeared
more intense and/or more concentrated at the periphery
(Fig. 4A; SCC9P). The redistribution and/or increase in
staining was particularly apparent for N-cadherin (Fig.
4A; cf. SCC9P and SCC9 for N-cadherin). We then
Characterization of the N-Cadherin and Catenins
in SCC9P Cells
SCC9 cells express both a- and b-catenins and
N-cadherin (Fig. 4). Figure 4A shows CSK extracted
Plakoglobin Induces Fibroblast to Epidermoid Transition
95
Fig. 5. Comparison of the fate of newly synthesized adhesion molecules in SCC9 and SCC9P cells. Cultures of SCC9 and SCC9P cells
were grown to confluency, labeled with 35S-methionine for 30 min and
chased up to 24 h, as described under Materials and Methods. After cell
fractionation, the soluble (S) and insoluble (P) fractions were processed
for immunoprecipitation with anti-pan-cadherin detecting N-cadherin,
a-catenin, and b-catenin antibodies. Immune complexes were analyzed
by PAGE, followed by fluorography. The relative amount of radioactivity for each protein band were quantitated by scanning densitometry
from multiple exposures of autoradiograms and graphs were plotted
using Cricket Graph software.
analyzed the steady-state levels of the total (T), and
TX-100 soluble (S) and insoluble (P) N-cadherin, a- and
b-catenins in SCC9 and SCC9P cells by immunoblotting,
followed by quantitation by scanning densitometry (Fig.
4B,C). The results showed increased N-cadherin and
decreased b-catenin levels in SCC9P cells, whereas there
was very little or no difference between the two lines for
a-catenin. (Fig. 4B,C).
respectively). We then determined whetherthe Pg expression in SCC9 cells had any effects on the synthesis or
metabolic stability of the catenins.
Metabolic labeling and turnover analysis of
a-catenin in SCC9 and SCC9P cells detected very little or
no difference in the amount of newly synthesized soluble
and insoluble a-catenin or their metabolic stability between the two lines (Fig. 5; a-catenin, (S) t1/2: 12 6 1.2 h
and 10 6 2 h and (P): 5.3 1 0.5 h and 6 6 1.2 h for SCC9
and SCC9P, respectively).
b-Catenin turnover studies showed both decreased
synthesis and stability for newly synthesized soluble and
insoluble b-catenin protein in SCC9P cells. In SCC9P
cells, the soluble b-catenin degraded faster (t1/2 ,7 6 1.3
h vs. ,15 6 0.8 h in SCC9 cells; Fig. 5, b-catenin).
Similarly, the insoluble b-catenin was degraded faster in
SCC9P cells (t1/2 ,6 6 3 h vs. 4 6 1.75 h for SCC9 and
SCC9P, respectively).
The results of synthesis and turnover studies are
consistent with the steady-state levels of N-cadherin and
catenins in SCC9 and SCC9P cells. We then sought to
determine whether the observed changes in newly synthesized N-cadherin and b-catenin were also reflected in the
levels of mRNAs of these proteins in SCC9 and SCC9P
cells. In Figure 6, total RNA from SCC9, SCC9P, and
Pg Expression Changes the Level and Metabolic
Stability of N-Cadherin and b-Catenin
in SCC9P Cells
SCC9 and SCC9P cells were metabolically labeled
and processed for N-cadherin immunoprecipitation as
described under Materials and Methods (Fig. 4D). The
amount of newly synthesized N-cadherin was significantly more (,4-fold) in SCC9P relative to SCC9 cells,
indicating increased N-cadherin synthesis in Pg-expressing cells. Turnover rate analysis showed that in SCC9P
cells the soluble N-cadherin degraded faster (cf. SCC9
with SCC9P in Fig. 4D and, in Fig. 5, N-cadherin; (S) t1/2
,14 6 3.5 and 8 6 1.5 h for SCC9 and SCC9P,
respectively), whereas the insoluble N-cadherin became
more stable (Figs. 4D and Fig. 5, N-cadherin; (P) t1/2
,4 6 0.5 h and 12 6 1.6 h for SCC9 and SCC9P,
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Parker et al.
Fig. 6. Northern blot analysis of N-cadherin, a-catenin, and b-catenin
mRNAs in SCC9 and SCC9P cells. Total RNA (,25 µg) isolated from
SCC9 and SCC9P cells was processed for Northern blot analysis with
32P-labeled N-cadherin, a-catenin and b-catenin cDNA probes as
described under Materials and Methods.
MDCK (Madin-Darby canine kidney epithelial cells,
used as control) cells were hybridized with cDNA probes
for a-catenin, b-catenin, and N-cadherin. The results
showed a slight decrease in b-catenin in Pg-expressing
cells (ratio of b-catenin/total RNA, 0.7 and 0.9 for
SCC9P and SCC9, respectively), and an approximately
twofold increase in N-cadherin RNA levels in SCC9P
cells (ratio of N-cadherin/total RNA, 1.1 and 0.6 for
SCC9P and SCC9, respectively), whereas a-catenin RNA
levels remained unchanged between the two lines.
The Cadherin/Catenin Complex Is Not Altered
in SCC9P Cells
Figure 7 shows the results of the sequential immunoprecipitation of N-cadherin, followed by either
a-catenin, b-catenin, or Pg. In SCC9 cells, N-cadherin,
a-catenin, and b-catenin were detected (Fig. 7; SCC9;
N-cad). The presence of the catenins in N-cadherin
immunoprecipitates was further confirmed by the second
immunoprecipitation with the respective antibodies,
anti-a- or anti-b-catenin (Fig. 7; SCC9; N-cad -.
a-catenin; N-cad -. b-catenin), indicating that N-cadherin
formed complexes with both catenins. In these cells, no
protein bands were detected in sequential immunoprecipitation of N-cadherin, followed by Pg (Fig.7; SCC9;
N-cad -. Pg).
In SCC9P cell extracts, the cadherin antibodies
brought down 4 bands corresponding to N-cadherin,
a-catenin, b-catenin, and Pg (Fig.7; SCC9P). The subse-
Fig. 7. Intracellular interactions of newly synthesized adhesion molecules in SCC9 and SCC9P cells. Cultures of SCC9 and SCC9P cells
were grown to confluency and 35S-labeled for a total of 4 h, as
described under Materials and Methods. After cell fractionation, equal
aliquots of the soluble fractions were processed for immunoprecipitation with the Pan-cadherin antibodies, which detect N-cadherin (Ncad). In duplicate samples, immune complexes were eluted and
reprecipitated with a-catenin (N-cad -. a-cat), b-catenin (N-cad -.
b-cat), or Pg (N-cad -. Pg).
quent second immunoprecipitations with the anticatenins and anti-Pg antibodies, verified the interactions
between these proteins and N-cadherin. Taken together,
exogenous expression of Pg did not seem to alter the
interactions significantly between the catenins and
N-cadherin.
DISCUSSION
Pg is a member of the Arm family of proteins,
which also includes b-catenin and the product of the
Drosophila segment polarity gene Armadillo [Peifer and
Wieschaus, 1990; Peifer et al., 1992, 1994].These proteins are involved in pathways regulating cell–cell adhesion, fate determination, and cell proliferation. The
adhesive function of these proteins is mediated by
Plakoglobin Induces Fibroblast to Epidermoid Transition
interactions with cadherins. Whereas b-catenin interacts
only with the classic cadherins (e.g., E-, N-, P-, M-), Pg
associates with both classic and desmosomal cadherins
[Knudsen and Wheelock, 1992; Cowin and Burke, 1995;
Marrs and Nelson, 1996; Gumbiner, 1996]. The necessity
of Pg–desmosomal cadherin interactions for the assembly
of desmosomes is well documented and supported by the
presence of the insoluble (cytoskeleton-associated) Pg–
desmosomal cadherin complexes [Mathur et al., 1994;
Troyanovsky et al., 1994; Kowalczyk et al., 1994; Pasdar
et al., 1995a; Bornslaeger et al., 1996]. However, the
precise role of Pg in complexes involving the classic
cadherins is unclear. Two lines of evidence suggest that
this Pg–cadherin complex may have regulatory functions:
Pg-containing classic cadherin complexes are primarily
present in soluble forms, indicating the lack of strong
interactions with the cytoskeleton; and Pg and b-catenin
interact with the classic cadherins in a mutually exclusive
manner [Hinck et al., 1994]. The latter finding also
suggests that b-catenin and Pg binding sequences are
identical or overlapping.
Arm is a component of the Drosophila Wg (wingless) signaling pathway, which regulates developmental
patterning. Pg and b-catenin are components of the Wnt
(mammalian homologue of Wg) pathway [for a recent
review, see Peifer, 1996]. Within this pathway, Pg and
b-catenin interact with the tumor suppressor protein APC
and transcription factors Lef-1/Tcf-1 [Behrens et al.,
1996; Huber et al., 1996b]. Pg and b-catenin interact with
cadherins and signaling proteins in a mutually exclusive
manner, thus regulating the balance between cell adhesion and cell proliferation. Several recent studies have
characterized the regulation of b-catenin function through
its interactions with APC and transcription factors [Peifer,
1996; Korinek et al., 1997; Rubinfeld et al., 1997; Morin
et al., 1997], but the role of Pg in regulating cell growth
and proliferation is not as clear.
SCC9 cells are carcinoma cells with a transformed
phenotype that lack Pg but express b-catenin and can
therefore be a useful model to study the adhesive and
signaling functions of Pg. These cells also lack E-cadherin
but express N-cadherin, which is the primary cadherin of
nonepithelial cells. We previously showed that the exogenous expression of L-CAM or E-cadherin in the absence
of Pg could induce epidermoid phenotype in SCC9 cells;
this phenotypic transition coincided with decreased synthesis and stability of the endogenous N-cadherin and
increased synthesis and stability of both a- and bcatenins [Li et al., in press]. These results were consistent
with the necessity of E-cadherin expression for the
epidermoid phenotype.
This study shows that, in the absence of E-cadherin,
Pg expression enabled SCC9 cells to form desmosomes
and induced the same phenotypic transition as that
97
induced by E-cadherin expression. Interestingly, unlike
E-cadherin, Pg expression coincided with increased synthesis and stability of N-cadherin and decreased levels
and stability of b-catenin. The Pg-induced epidermoid
phenotype was associated with increased adhesiveness
and decreased growth rate at confluency. In fact, the
initial adhesion ability was significantly more (&mt;50%)
in Pg-expressing cells than those expressing L-CAM or
E-cadherin [Li et. al., in press] (Fig. 2C). Unlike L-CAMexpressing SCC9 cells, Pg-expressing cells are capable of
forming desmosomes and therefore, the initial cell–cell
adhesion may be more in these cells due to the presence
of both desmosomes and adherens junctions. E-Cadherin
expression and Pg-E–cadherin interaction have been
known to be necessary for, and to precede, the formation
of desmosomes [Gumbiner et al., 1988; Lewis et al.,
1994, 1997; Jensen et al., 1997]. Our results suggest that
E-cadherin can be replaced by N-cadherin and that
N-cadherin–Pg complex is, presumably, as effective in
initiating desmosome formation as Pg-E–cadherin complex. A similar fibroblast-to-epidermoid transition concurrent with desmosome assembly and upregulation of
N-cadherin expression has been reported for HeLa cells
transfected with cDNAs encoding P0 cell adhesion
protein [Doyle et al., 1995].
The induction of epidermoid phenotype and decreased proliferation rate of SCC9P cells are consistent
with the suggested tumor suppressor activity of Pg
[Simcha et al., 1996]. The exogenous expression of Pg
(with or without cadherin) in an SV40-transformed 3T3
fibroblast cell line lacking both Pg and cadherins led to
suppression of tumorgenicity. When expressed alone, Pg
did not modify the morphology of the transfected cells
but decreased their tumorgenicity. By contrast, transfection with N-cadherin induced a fibroblast to epidermoid
transition in the morphology but had no effect on their
tumorgenicity. When expressed together with N-cadherin, Pg was much more effective in decreasing tumorigenicity. Simcha et al. [1996] suggested two possible
mechanisms for the effects of Pg on the ability of cells to
form tumors. Either the Pg interacts with components of
signal pathways that influence cell proliferation, morphology, and other cellular processes; or Pg enhances cellular
adhesion, thereby inhibiting tumor-forming ability. We
have not tested the tumorigenicity of SCC9 cells after
expression of Pg, but the phenotypic transformation from
fibroblast to epidermoid and decreased growth rate are
consistent with the increased adhesivity and possible
tumor suppressor activity of Pg. Furthermore, the upregulation of N-cadherin and its increased stability are
consistent with the possibility that the tumor suppressor
activity of Pg may be mediated by its involvement in
adhesion complexes (see below).
98
Parker et al.
The expression of N-cadherin by SCC9 cells was an
unexpected finding, as this is a squamous cell carcinoma
derived line and epidermoid cells generally express Eand P-cadherin [Nicholson et al., 1991; Johnson et al.,
1993]. A recent study by Islam et al. [1996] also reported
N-cadherin expression in two other transformed squamous carcinoma cell lines expressing low levels of E- and
P-cadherins. The authors suggested that the inappropriate
N-cadherin expression contributed directly to the invasive phenotype because transfection of these carcinoma
cells with N-cadherin anti-sense RNAs downregulated
N-cadherin and induced a fibroblast–epidermoid phenotypic transition, coincident with the upregulation of Eand P-cadherin expression. We have not detected any
E-cadherin in SCC9 cells, and the Pg-induced epidermoid
morphology coincided with increased amounts of
N-cadherin. Our data suggest that, at least in SCC9 cells,
N-cadherin expression per se is not responsible for the
transformed phenotype. In addition our results suggested
that Pg could form an apparently functional complex with
N-cadherin to induce desmosome assembly and epidermoid morphology. These observations underscore the
pivotal role of Pg in regulating cell adhesive properties
and epithelial morphology.
What are the mechanisms by which Pg induces
epidermoid phenotype? We suggest that the transformed
phenotype of SCC9 cells could result from either decreased adhesive properties due to the absence of Pg, or
excess b-catenin, or both. Several processes could result
in excess b-catenin. For example, inefficient interactions
between N-cadherin and b-catenin, due to alterations in
b-catenin binding domain of N-cadherin, deficiency in
APC, or abnormalities in APC–b–catenin interactions. It
is well documented that, if not sequestered by APC,
excess b-catenin could interact with Lef-1 transcription
factors and translocate into the nucleus. Once in the
nucleus, Lef-1–b-catenin complex can activate the target
genes involved in cell proliferation and mesenchymal
phenotype [Korinek et al., 1997; Huber et al., 1996b].This
complex also has been shown to bind to the E-cadherin
promoter in vitro, resulting in decreased E-cadherin
expression [Huber et al., 1996a,b].
It has been suggested that Pg and b-catenin may
have overlapping binding domains in classic cadherins
[Hinck et al., 1994]. One could imagine that a small
alteration in the nonoverlapping area of binding for one of
the two proteins could result in inefficient binding of that
molecule without affecting the other. Therefore, if the
SCC9 N-cadherin is defective in the b-catenin binding
region, this could affect the stability of the cadherin–
catenin complex. This deficiency could lead to excess
b-catenin and can be corrected by introducing either a
‘‘normal’’ cadherin molecule, to interact with the excess
b-catenin or by expressing Pg to substitute for b-catenin
and forming stable complexes. An exogenously expressed functional cadherin such as L-CAM or E-cadherin
would remove the excess b-catenin by forming efficient
and functional cadherin–catenin complexes. This effect is
similar to the suppression of dorsal signaling activity of
the soluble b-catenin by overexpression of cadherins in
Xenopus embryo [Fagotto et al., 1996; Miller and Moon,
1997]. Similar results would be expected by the exogenous expression of Pg. In that case, Pg could potentially
function in two capacities. It could replace b-catenin and
form stable N-cadherin–Pg complexes that are necessary
for, and that initiate, desmosome formation, leading to
increased adhesiveness and epidermoid morphology. Alternatively or in addition, the exogenously expressed Pg
could directly decrease b-catenin levels, consistent with
decreased synthesis and faster degradation of b-catenin in
Pg-expressing SCC9P cells (Fig.5) and the recent study
by Salomon et al. [1997] suggesting co-regulation of Pg
and b-catenin. Finally, Pg could bind and sequester the
transcription factors (Lef-1) in the cytoplasm inhibiting
its interaction with b-catenin and the subsequent translocation of the complex into the nucleus, as has been
suggested previously [Merriam et al., 1997].
In conclusion, this study shows that, in the absence
of E-cadherin, Pg can induce desmosome formation and a
transition from transformed to epidermoid morphology
by interacting with the available cadherin, N-cadherin.
This observation would suggest that the regulatory role of
the Pg–classic cadherins complex may be specified by Pg.
Furthermore, the data suggest that Pg may also function
as one of a regulators of b-catenin levels in cells.
ACKNOWLEDGMENTS
We are grateful to Drs. A. Klein-Santoz, John
Hemperly, and Lionel Larue for their gifts of cells and
cDNAs, to Honey Chan for her valuable assistance in
electron microscopy and to Drs. W.J. Gallin and E.K.
Shibuya for their critical review of the manuscript . This
research is supported by a grant from Medical Research
Council of Canada (to M.P.). We also acknowledge
financial assistance from Leo Laboratories Canada Ltd.,
as part of the Dermatology Foundation grant. The University of Alberta Faculty of Medicine Confocal Laser
Scanning Microscopy Facility is supported in part by
funds from the Medical Research Council and the Alberta
Heritage Foundation for Medical Research (AHFMR).
M. Pasdar is an AHFMR scholar.
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