Am J Physiol Cell Physiol
279: C1472–C1482, 2000.
Differential expression of cell-cell adhesion proteins
and cyclin D in MEK1-transdifferentiated MDCK cells
INGRID MARSCHITZ,1 JUDITH LECHNER,1 IRENE MOSSER,1 MARTINA DANDER,1
ROBERTO MONTESANO,2 AND HERBERT SCHRAMEK1
1
Department of Physiology, University of Innsbruck, A-6010 Innsbruck, Austria; and
2
Department of Morphology, University Medical Center, CH-1211 Geneva 4, Switzerland
Received 22 February 2000; accepted in final form 22 May 2000
extracellular signal-regulated kinase; differentiation; proliferation; E-cadherin; ␤-catenin
CELL-CELL INTERACTIONS play an important role in development, tissue morphogenesis, and regulation of cell
migration and proliferation. The adherens junction,
representing one prominent cell-cell adhesive structure assembled by cells of epithelial origin, forms a
continuous belt and functions to hold neighboring cells
together through a family of Ca2⫹-dependent, trans-
Address for reprint requests and other correspondence: H.
Schramek, Dept. of Physiology, Fritz-Pregl-Strasse 3, A-6010 Innsbruck, Austria (E-mail: [email protected]).
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membrane cell-cell adhesion proteins called cadherins.
E-cadherin is one of several members of the cadherin
superfamily and is the predominant cadherin expressed in epithelial cells (5, 48). The role of this
cell-cell adhesion molecule is significant in the morphogenesis of epithelial tissues and the development of
polarized apical/basolateral membrane domains (33,
45). Downregulation of cadherin expression and/or cadherin function is associated with malignant transformation and invasiveness of tumor cells (45). In addition, evidence has accumulated indicating that
cadherins participate not only in cell-cell adhesion but
also in signaling events that affect cell differentiation,
proliferation, migration, and survival (18, 42).
Effective epithelial cell-cell adhesion depends on the
interaction of E-cadherin with the cytoskeleton
through proteins including ␣-, ␤-, and ␥-catenin (plakoglobin) (3, 17, 42). Of these, ␤-catenin represents a
multifunctional protein that is an integral component
of adherens junctions and intracellular signal transduction (7). At submembranal sites, ␤-catenin is anchored to the cytoplasmic tail of cadherin and, via
␣-catenin and vinculin, to the actin cytoskeleton. Moreover, a significant fraction of this protein is present in
the cytoplasm in a soluble form and is thus likely to
play an important role in signal transduction and gene
expression (41, 43). Binding to the adenomatous polyposis coli (APC) tumor suppressor protein can stimulate
the degradation of ␤-catenin, whereas signaling initiated by the extracellular Wnt-1 oncoprotein or selected
mutations in ␤-catenin itself result in the accumulation of higher levels of ␤-catenin in the cytoplasm (7).
Moreover, deregulated signal transduction involving
␤-catenin is a common event in tumor cells: the activation of ␤-catenin to an oncogenic state can result
from the inactivation of the tumor suppressor APC,
direct mutation in the ␤-catenin gene, or activation of
Wnt receptors (3, 28). Once activated, ␤-catenin may
promote tumor progression through its interaction
with one or more of its numerous targets (28).
We recently utilized Madin-Darby canine kidney
(MDCK) cell lines, which retain differentiated properThe costs of publication of this article were defrayed in part by the
payment of page charges. The article must therefore be hereby
marked ‘‘advertisement’’ in accordance with 18 U.S.C. Section 1734
solely to indicate this fact.
0363-6143/00 $5.00 Copyright © 2000 the American Physiological Society
http://www.ajpcell.org
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Marschitz, Ingrid, Judith Lechner, Irene Mosser,
Martina Dander, Roberto Montesano, and Herbert
Schramek. Differential expression of cell-cell adhesion proteins and cyclin D in MEK1-transdifferentiated MDCK cells.
Am J Physiol Cell Physiol 279: C1472–C1482, 2000.—
Overexpression of a constitutively active mutant of the mitogen-activated protein kinase kinase MEK1 (caMEK1) in
epithelial Madin-Darby canine kidney (MDCK)-C7 cells disrupts morphogenesis, induces an invasive phenotype, and is
associated with a reduced rate of cell proliferation. The role of
cell-cell adhesion molecules and cell cycle proteins in these
processes, however, has not been investigated. We now report loss of E-cadherin expression as well as a marked reduction of ␤- and ␣-catenin expression in transdifferentiated
MDCK-C7 cells stably expressing caMEK1 (C7caMEK1)
compared with epithelial mock-transfected MDCK-C7
(C7Mock1) cells. At least part of the remaining ␣-catenin was
coimmunoprecipitated with ␤-catenin, whereas no E-cadherin was detected in ␤-catenin immunoprecipitates. In both
cell types, the proteasome-specific protease inhibitors
N-acetyl-Leu-Leu-norleucinal (ALLN) and lactacystin led to
a time-dependent accumulation of ␤-catenin, including the
appearance of high-molecular-weight ␤-catenin species. Quiescent as well as serum-stimulated C7caMEK1 cells showed
a higher cyclin D expression than epithelial C7Mock1 cells.
The MEK inhibitor U-0126 inhibited extracellular signalregulated kinase phosphorylation and cyclin D expression in
C7caMEK1 cells and almost abolished their already reduced
cell proliferation rate. We conclude that the transdifferentiated and invasive phenotype of C7caMEK1 cells is associated
with a diminished expression of proteins involved in cell-cell
adhesion. Although ␤-catenin expression is reduced,
C7caMEK1 cells show a higher expression of U-0126-sensitive cyclin D protein.
MEK1-TRANSDIFFERENTIATED MDCK CELLS
mesenchymal-like cells (25). The invasive properties of
C7caMEK1 cells were associated with the expression of
activated type 2 matrix metalloproteinase (MMP-2) and
with elevated levels of membrane type 1 matrix metalloproteinase (MMP-1) (25). Moreover, addition of the
MEK inhibitor U-0126 induced a pronounced flattening of C7caMEK1 cells and markedly reduced the invasion of C7caMEK1 cells into the collagen matrix (25).
On the basis of these results, the present study was
performed to study the role of proteins involved in the
assembly of adherens junctions in epithelial mocktransfected MDCK-C7 (C7Mock1) cells compared with
transdifferentiated, invasive C7caMEK1 cells. To this
end, we investigated the expression of E-cadherin and
␤- and ␣-catenin in both cell lines. Inasmuch as ␤-catenin has recently been reported to regulate expression
of cyclin D1 (40, 47), we studied cyclin D expression
and its potential role for the alterations observed in
C7caMEK1 cell proliferation. Here we report loss of Ecadherin expression as well as marked reduction of ␤and ␣-catenin expression in dedifferentiated C7caMEK1
cells but not in epithelial C7Mock1 cells. Although ␤-catenin expression was reduced, C7caMEK1 cells showed a
higher expression of cyclin D protein. Inhibition of
MEK1 by administration of U-0126 substantially inhibited the increased cyclin D expression and the already reduced proliferation rate of C7caMEK1 cells. Collectively, these results suggest that the highly invasive
properties of fibroblastoid, mesenchymal-like C7caMEK1
cells are not only associated with elevated levels of
MMP-1 and with the expression of activated gelatinase
A (25), but also with an abolished/diminished expression of several proteins that are important for cell-cell
interactions between neighboring cells. Although ␤-catenin expression is reduced, C7caMEK1 cells show a higher
expression of U-0126-sensitive cyclin D protein.
MATERIALS AND METHODS
Reagents and antibodies. Leupeptin and pepstatin A were
purchased from Peninsula Laboratories (Belmont, CA), protein A-Sepharose from Amersham Pharmacia Biotech (Uppsala, Sweden), U-0126 from Alexis Biochemicals (San Diego,
CA), and cell culture media and all other reagents from
Sigma Chemical (St. Louis, MO).
The following antibodies were applied: mouse anti-E-cadherin (Transduction Laboratories, San Diego, CA), mouse
anti-␤-catenin (Transduction Laboratories), rabbit anti-␣catenin (Zymed, San Francisco, CA), rabbit anti-cyclin A
(Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anticyclin D1 (Santa Cruz Biotechnology), and rabbit anti-phospho-ERK1,2, which detects phosphorylated Tyr-204 of ERK1
and ERK2 (New England Biolabs, Beverly, MA).
Cell culture. Wild-type MDCK-C7 cells and their derivatives (see below) were grown in MEM with Earle’s salts,
nonessential amino acids, and L-glutamine at pH 7.4 (34).
MEM was supplemented with 10% FCS, 26 mM NaHCO3,
100 U/ml penicillin, and 100 ␮g/ml streptomycin. Cells were
grown at 37°C in 5% CO2-95% air, humidified atmosphere.
The MDCK-C7 cell line is a high-transepithelial resistance
subclone of the heterogenous MDCK strain from American
Type Culture Collection (CCL 34) (15, 50). Stable transfection of wild-type MDCK-C7 cells with caMEK1 has been
described previously (34). Briefly, MDCK-C7 cells were co-
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ties of renal tubular epithelium (15, 30, 33, 50), to
study the role of mitogen-activated protein kinases
(MAPK) in the regulation of renal epithelial cell differentiation, proliferation, and invasion (25, 29, 34, 35).
The best-studied MAPKs, the extracellular signal-regulated protein kinases 1 and 2 (ERK1 and ERK2), are
ubiquitous components of signal transduction pathways that transduce extracellular stimuli into diverse
biological responses. ERK1 and ERK2 are activated by
phosphorylation on threonine and tyrosine residues by
an upstream dual-specificity kinase called MAPK kinase (MAPKK or MKK) or MAPK/ERK kinase (MEK)
(14, 21, 31). In general, MEKs represent the fewest
members in the MAPK signaling module and have a
high specificity for their MAPK substrates. The MEKs
upstream of ERK1 and ERK2 constitute an evolutionary conserved family of protein kinases that includes at
least three highly homologous mammalian isoforms:
MEK1a, MEK1b, and MEK2 (36). Constitutively active
as well as dominant-negative MEK mutants have been
used by different laboratories to study the function of
the highly conserved MEK-ERK module in cell proliferation and differentiation. Expression of constitutively active MEK mutants, for example, has been
reported to transform fibroblasts and to induce tumor
formation in nude mice (6, 9, 23). Moreover, overexpression of an S222E mutant of MEK increased proliferation and altered morphology of NIH/3T3 fibroblasts
but failed to induce their growth in soft agar (37). In
contrast to these experiments performed in fibroblasts,
a constitutively active MEK1 mutant stimulated
PC-12 cell differentiation, whereas interfering mutants of MEK1 inhibited ligand-induced neurite outgrowth (9). Thus constitutive and sustained activation
of the only known ERK activator, MEK, leads to transformation of fibroblasts but differentiation of PC-12
cells, suggesting cell type-specific differences in the
function of the MEK-ERK signaling module.
In this context, we recently reported a differential
regulation of the MAPKs ERK1, ERK2, and c-Jun
NH2-terminal kinase 1 (JNK1) in alkali-dedifferentiated MDCK-C7Focus (MDCK-C7F) cells compared
with their parental epithelial MDCK-C7 cells (35). In
dedifferentiated MDCK-C7F cells, reduction of ERK1
protein expression, increases in ERK2 activity, and
slight decreases in JNK1 activity were accompanied by
an inhibition of serum-induced cell proliferation, suggesting that members of the MAPK family of protein
kinases could be involved in the regulation of the
epithelial MDCK cell phenotype and/or cell proliferation (35). These results were confirmed by expression of
a constitutively active mutant of MEK1 (caMEK1) in
epithelial MDCK-C7 cells, which resulted in pronounced phenotypic changes similar to those obtained
in MDCK-C7F cells, including the acquisition of a
fibroblastoid morphology, reduced cytokeratin and increased vimentin expression, and assembly of
␣-smooth muscle actin-containing stress fibers (29, 34).
In collagen gels, MDCK-C7 cells expressing caMEK1
(C7caMEK1 cells) were unable to generate cystlike
epithelial structures and behaved as highly invasive,
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MEK1-TRANSDIFFERENTIATED MDCK CELLS
the confocal image were adjusted for each antibody and
maintained for the analysis of C7Mock1 and C7caMEK1
cells.
Cell proliferation assay. The number of cells per square
centimeter of culture dish was evaluated by light microscopy
by utilizing an inverted microscope equipped with a camera
(model LDH 0670/00, Phillips) connected to a video graphic
printer (model UP-850, Sony) and a superimposed test frame,
as described previously (34, 35). For measurement of cell
number, quiescent MDCK-C7 cells, either mock-transfected
(C7Mock1) or transfected with constitutively active MEK1
(C7caMEK1), were stimulated with 10% FCS for 1 and 3 days
in the absence or presence of the synthetic, noncompetitive
MEK1 inhibitor U-0126 (10 ␮M). One measurement (n ⫽ 1)
represents the mean of cells per square centimeter determined from 10 video prints randomly taken from one petri
dish at each of days 0, 1, and 3.
RESULTS
Loss of E-cadherin expression in caMEK1-transfected
MDCK-C7 cells. Recently, evidence has accumulated
indicating that elements of the cadherin-associated
complex have roles in cell signaling, proliferation, and
differentiation (18). Increased and sustained activity of
the MEK1-ERK2 signaling module in epithelial
MDCK-C7 cells has been correlated with transdifferentiation of these cells, failure of morphogenesis, and
development of a highly invasive cell phenotype (25,
29, 34, 35). To assess the role of cell-cell adhesion
molecules in these morphoregulatory processes, we
first studied the expression of E-cadherin in MDCK-C7
cells stably transfected with a constitutively active
mutant of MEK1 (C7caMEK1 cells) compared with
mock-transfected MDCK-C7 cells (C7Mock1). In contrast to epithelial C7Mock1 cells, expression of E-cadherin has been abolished in soluble Triton X-100 lysates (Fig. 1), in soluble radioimmunoprecipitation
assay buffer lysates (data not shown), and in whole cell
lysates (data not shown) of transdifferentiated
C7caMEK1 cells. The amount of E-cadherin expression
in C7Mock1 cells and the alteration of E-cadherin
Fig. 1. Expression of E-cadherin and ␤- and ␣-catenin in soluble
Triton X-100 lysates of mock-transfected Madin-Darby canine kidney (MDCK)-C7 cells (C7Mock1) and MDCK-C7 cells stably expressing a constitutively active MEK1 mutant (C7caMEK1). After cells
were split and replated, they were grown in the presence of 10% FCS
for 24 h (d0), 48 h (d1), and 96 h (d3). Cells were then washed with
ice-cold PBS and harvested using Triton X-100 lysis buffer. Soluble
cell lysates were matched for protein content and analyzed by Western blotting with use of monoclonal E-cadherin and ␤-catenin antibodies or a polyclonal ␣-catenin antibody.
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transfected by the Lipofection technique with pREP4 expression vector together with the empty pECE vector (C7Mock1
cells) or pECE containing a hemagglutinin epitope-tagged
caMEK1 mutant (C7caMEK1 cells). caMEK1 was generated
by substitution of the Raf1-dependent regulatory phosphorylation sites (Ser-218 and Ser-222) with aspartic acid
(S218D/S222D mutant), as described previously (6). Experiments were performed with cells cultured in the presence of
10% FCS or with cells made quiescent by 24 h of incubation
in FCS-free medium.
Western blot analysis. Cells were washed three times with
ice-cold PBS and lysed in ice-cold Triton X-100 lysis buffer
(50 mM Tris 䡠 HCl, pH 7.5, 100 mM NaCl, 50 mM NaF, 5 mM
EDTA, 40 mM ␤-glycerophosphate, 200 ␮M sodium orthovanadate, 0.1 mM phenylmethylsulfonyl fluoride, 1 ␮g/ml
leupeptin, 1 ␮M pepstatin A, 1% Triton X-100) for 25 min at
4°C. Insoluble material was removed by centrifugation at
12,000 g for 15 min at 4°C. The protein content was determined using a micro-bicinchoninic acid assay (Pierce) with
BSA as the standard. Cell lysates were matched for protein,
separated on 12% SDS polyacrylamide gel, and transferred to
a polyvinylidene difluoride microporous membrane (Millipore). Subsequently, membranes were blotted with one of the
specific antibodies as listed above. The primary antibodies
were detected using horseradish peroxidase-conjugated goat
anti-rabbit IgG (for polyclonal antibodies except anti-phospho-ERK1,2) or horseradish peroxidase-conjugated rabbit
anti-mouse IgG (for monoclonal antibodies) visualized by
Amersham enhanced chemiluminescence system after intensive washing of the sheets or, when anti-phospho-ERK1,2
antibodies were applied, use of alkaline phosphatase-conjugated goat anti-rabbit IgG visualized by Phototope chemiluminescent Western detection system (New England Biolabs).
Immunoprecipitation. Cells were washed three times with
ice-cold PBS and lysed in ice-cold Triton X-100 lysis buffer for
25 min at 4°C. Insoluble material was removed by centrifugation at 12,000 g for 15 min at 4°C. The protein content was
determined using a micro-bicinchoninic acid assay (Pierce)
with BSA as the standard. Cell lysates were matched for
protein and precleared with preimmune serum preadsorbed
to 50 ␮l of protein A-Sepharose-coated beads for 2 h at 4°C.
The precleared supernatants were further incubated overnight with 4 ␮l of a monoclonal mouse anti-␤-catenin antibody preadsorbed to protein A-Sepharose.
Immunofluorescence microscopy. Cells were plated onto
Flexiperm slides (Heraeus, Hanau, Germany), rinsed three
times (in 3 steps from room temperature to 4°C) with PBS
containing Ca2⫹ and Mg2⫹, fixed for 10 min in 100% methanol at ⫺20°C, and then incubated for 4 min in the presence
of 100% acetone. After they were rinsed in PBS without Ca2⫹
and Mg2⫹ (in 3 steps from 4°C to room temperature) and
blocked with 5% milk, the cells were incubated with the
respective antibodies for 1 h at 37°C. Texas Red-X-conjugated secondary antibodies (Molecular Probes, Eugene, OR)
were diluted 1:50 in PBS. Indirect immunofluorescence microscopy was performed with a Zeiss Axiophot fluorescence
microscope or an MRC-600 Lasersharp system linked to a
Zeiss IM 35 inverted microscope for confocal microscopy.
With respect to the confocal immunofluorescence studies, a
series of confocal images was taken with 2 ␮m between single
pictures. After an initial focal plane was set by using phase
contrast, the confocal microscope was switched to the fluorescence imaging mode, and the focal plane was moved toward the apical or basal side of the cellular layer, respectively. The first and last images of the stack were defined by
the focal plane where the slight background staining of the
cytoplasm disappeared. Contrast and brightness settings on
MEK1-TRANSDIFFERENTIATED MDCK CELLS
change significantly over the period of time investigated (Fig. 1). In dedifferentiated C7caMEK1 cells,
however, ␤- and ␣-catenin protein expression was substantially reduced, although it was still clearly detectable (Fig. 1). Again, these results were confirmed by
conventional immunofluorescence analysis (Fig. 4). Although uniform, continuous ␤- and ␣-catenin staining
at apicolateral cell borders was detected in epithelial
C7Mock1 cells (Fig. 4, A and C), ␤- and ␣-catenin
expression was restricted to single patches along
C7caMEK1 cell borders (Fig. 4, B and D), suggesting
that in dedifferentiated C7caMEK1 cells the remaining
␤- and ␣-catenins might be colocalized at distinct regions close to the plasma membrane. To analyze ␣- and
␤-catenin localization in C7Mock1 and C7caMEK1
cells in more detail, we performed a confocal immunofluorescence analysis. Figures 5 and 6 show horizontal
cross sections starting from the apical plane toward the
basal plane for ␤- and ␣-catenin, respectively. In
C7Mock1 cells a continuous staining was detected for
␤- and ␣-catenin at the apicolateral cell membrane
marking the adherens junction. Staining became discontinuous and progressively more spotlike toward the
basal cell pole. In C7caMEK1 cells, overall staining
intensity was much reduced for ␤- and ␣-catenin compared with C7Mock1 cells. ␤- and ␣-catenin seemed to
be restricted to single patches at cell-cell borders, re-
Fig. 2. Conventional immunofluorescence analysis of E-cadherin expression in C7Mock1 and C7caMEK1 cells.
Cells were grown in the presence of 10% FCS, fixed, rinsed thoroughly, and finally stained with a monoclonal
E-cadherin antibody. Phase-contrast micrographs of confluent C7Mock1 (A) and C7caMEK1 (C) cells as well as
representative immunofluorescence pictures of C7Mock1 (B) and C7caMEK1 (D) cells are depicted. Photographs
were taken using a Zeiss ⫻20 objective.
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expression in C7caMEK1 cells remained unchanged for
ⱖ4 days after the cells were split and replated (Fig. 1).
These results were confirmed by conventional (Fig. 2)
and confocal (Fig. 3) immunofluorescence analysis. Although E-cadherin was prominent at cell-cell borders
in C7Mock1 cells (Fig. 2B), no specific staining was
detected in C7caMEK1 cells (Fig. 2D). A series of
confocal horizontal optical cross sections at successively lower focal planes from the apical side toward
the basal side revealed E-cadherin staining restricted
to the junctional belt at the apicolateral membrane in
C7Mock1 cells (Fig. 3). In C7caMEK1 cells, by contrast,
E-cadherin could not be detected by confocal immunofluorescence analysis (Fig. 3).
Attenuated expression of ␤- and ␣-catenin in transdifferentiated C7caMEK1 cells. The cytoplasmic domain of E-cadherin is known to interact with ␤- or
␥-catenin (plakoglobin), thereby binding to ␣-catenin,
which in turn is attached to the actin cytoskeleton.
Inasmuch as this protein complex has been discussed
to strengthen cell-cell adhesions and, in addition, to
participate in signaling events that affect cell differentiation, proliferation, migration, and cell survival, we
next studied the expression of ␤- and ␣-catenin in
C7Mock1 and C7caMEK1 cells. Western blot analysis
of epithelial C7Mock1 cell lysates revealed a strong
protein expression of ␤- and ␣-catenin, which did not
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MEK1-TRANSDIFFERENTIATED MDCK CELLS
sulting in a spotlike staining along lateral membranes.
Furthermore, we detected no increase in the nuclear
localization of one of the two catenins investigated
(Figs. 5 and 6, respectively). In addition, when ␤-catenin was immunoprecipitated from soluble lysates and
blotted with an antibody against ␣-catenin, we found
that, in C7caMEK1 cells, at least part of the remaining
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Fig. 3. Confocal immunofluorescence analysis of E-cadherin expression in C7Mock1 and C7caMEK1 cells. Cells were grown in the
presence of 10% FCS, fixed, rinsed thoroughly, and stained with a
monoclonal E-cadherin antibody. Fluorescence was observed by confocal laser scanning microscopy. A series of 5 horizontal optical cross
sections at successively lower focal levels (step sizes of 2 ␮m each) is
shown. A, image of the stack close to the apical cell surface; BL,
image taken near the basal side.
␤-catenin was still associated with ␣-catenin (Fig. 7,
top). In contrast, no E-cadherin was detected in ␤-catenin immunoprecipitates (Fig. 7, bottom).
Regulation of ␤-catenin expression by proteasomemediated proteolysis in C7caMEK1 and C7Mock1 cells.
Degradation of ␤-catenin is initiated by phosphorylation at specific serine and threonine residues in its NH2
terminus. This leads to ubiquitination of ␤-catenin,
which is a signal for proteasomal degradation (1). It is
well established that the peptide aldehyde ALLN (Nacetyl-Leu-Leu-norleucinal, LLnL, or calpain inhibitor
I) and the natural compound lactacystin inhibit proteasome-mediated proteolysis, leading to an accumulation of proteins that are usually metabolized by this
pathway (10). To study the posttranslational modifications of ␤-catenins in epithelial C7Mock1 cells compared with C7caMEK1 cells, we utilized these compounds in Western blot studies. ALLN (25 ␮M) led to a
time-dependent increase in ␤-catenin protein in
C7Mock1 and C7caMEK1 cells (Fig. 8A). This increase
in ␤-catenin protein expression started as early as 6 h
after ALLN application and was highest after 24 h of
incubation. In addition, ALLN led to the appearance of
a slower-migrating, hence larger, form of ␤-catenin
(Fig. 8A). Because ALLN also inhibits nonproteasomal
proteases, such as calpains and cathepsins, further
experiments were performed using lactacystin, which
represents a highly specific inhibitor of proteasomal
activity (13). Lactacystin (10 ␮M) led to a time-dependent increase in ␤-catenin protein and the appearance
of a slower-migrating ␤-catenin form, which, in contrast to ALLN, was highest after 12 h of incubation and
was decreased again after 24 h (Fig. 8B). ALLM (Nacetyl-Leu-Leu-methional, LLM, or calpain inhibitor
II; 25 ␮M), which is structurally related to ALLN and
a potent inhibitor of calpains and cathepsins but does
not inhibit the proteolytic activity of proteasomes (32),
did not affect ␤-catenin protein expression in C7Mock1
or C7caMEK1 cells (data not shown). In addition, another diffusible protease inhibitor, leupeptin A (10
␮M), as well as the phosphatase inhibitor vanadate (1
mM), had no effect (data not shown). From these results, it is concluded that accumulation of posttranslational modifications of ␤-catenin is induced by proteasome-specific protease inhibitors. It appears that these
posttranslational ␤-catenin modifications occur to a
comparable extent in both cell types. Thus it is unlikely
that the reduced ␤-catenin expression in C7caMEK1
cells is due to an increased proteasome-mediated proteolytic activity in these cells.
caMEK1-transfected MDCK-C7 cells show increased
cyclin D expression but decreased cell proliferation compared with C7Mock1 cells. It has been reported recently that HeLa cells, which express a mutant ␤-catenin lacking the phosphorylation sites necessary for
APC-mediated degradation, do express high levels of
cyclin D1 mRNA and protein (47). Furthermore, expression of a dominant-negative form of the transcription factor T cell factor (TCF) in colon cancer cells
strongly inhibited expression of cyclin D1 and caused
cells to arrest in the G1 phase of the cell cycle, suggest-
MEK1-TRANSDIFFERENTIATED MDCK CELLS
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ing that abnormal levels of ␤-catenin may contribute to
neoplastic transformation by causing accumulation of
cyclin D1 (40, 47). Thus, and because C7caMEK1 cells
show a reduced rate of cell proliferation (34), we next
investigated whether these cells, which express constitutively active MEK1 but substantially less ␤-catenin
than C7Mock1 cells, show alterations in cyclin D expression. As depicted in Fig. 9, quiescent C7caMEK1
cells showed a higher cyclin D expression than epithelial C7Mock1 cells, whereas cyclin A was equally expressed in both cell types. Stimulation of quiescent
C7Mock1 cells with 10% FCS for 1 and 3 days led to a
time-dependent increase in cyclin D1 but not cyclin A
expression (Fig. 9). In C7caMEK1 cells, cyclin D expression remained elevated after 1 and 3 days of serum
stimulation. To establish whether MEK1 is responsible
for the higher basal and/or the FCS-induced increase in
cyclin D expression in C7caMEK1 cells, additional experiments were performed utilizing the selective, noncompetitive MEK1 inhibitor U-0126 (12). U-0126 (10
␮M) inhibited the FCS-induced increase in ERK1 and
ERK2 phosphorylation in C7Mock1 cells and almost
completely blocked the constitutively increased ERK
phosphorylation in C7caMEK1 cells (Fig. 10, top).
These results suggest that U-0126 could inhibit MEK
directly by inhibiting the catalytic activity of the active
enzyme and, thus, is probably more effective against
constitutively active MEK1 than against intrinsic
MEK1 (12). In accordance with its effects on ERK
phosphorylation, 10 ␮M U-0126 inhibited the FCSinduced increase in cyclin D1 protein expression in
C7Mock1 cells and almost abolished the increased cyclin D expression in C7caMEK1 cells after 48 h (Fig.
10). These results provide evidence for an ERK-mediated increase in cyclin D expression in C7caMEK1
cells.
In this context, we recently reported that expression
of caMEK1 in MDCK-C7 cells is not only associated
with an epithelial-to-mesenchymal transdifferentiation of these cells but also with a decreased proliferation rate (34). If the MEK1-mediated increase in cyclin
D in C7caMEK1 cells is necessary for the cells to
maintain a certain level of cell proliferation, U-0126
should be able to further inhibit the already diminished cell proliferation rate of C7caMEK1 cells. Thus
we measured the number of FCS-stimulated C7Mock1
and C7caMEK1 cells per square centimeter in the
absence and presence of 10 ␮M U-0126. At day 0 there
were 2.57 ⫻ 104 ⫾ 0.11 and 2.59 ⫻ 104 ⫾ 0.08 C7Mock1
and C7caMEK1 cells/cm2 (n ⫽ 7), respectively. As we
previously reported (34), C7caMEK1 cells showed a
reduced rate of cell proliferation after 1 and 3 days
(4.98 ⫻ 104 ⫾ 0.16 and 11.36 ⫻ 104 ⫾ 1.10 cells/cm2,
respectively) compared with C7Mock cells (6.13 ⫻
104 ⫾ 0.38 and 18.36 ⫻ 104 ⫾ 0.64 cells/cm2, respectively). Indeed, after 3 days of incubation, U-0126 in-
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Fig. 4. Conventional immunofluorescence analysis of ␤-catenin (A and B) and ␣-catenin (C and D) expression in
C7Mock1 (A and C) and C7caMEK1 (B and D) cells. Cells were grown in the presence of 10% FCS, fixed, rinsed
thoroughly, and stained with a monoclonal ␤-cadherin antibody or a polyclonal ␣-catenin antibody. Photographs
were taken using a Zeiss ⫻20 objective.
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MEK1-TRANSDIFFERENTIATED MDCK CELLS
maintain a certain level of C7caMEK1 cell proliferation.
DISCUSSION
Three major findings can be derived from the present
study: 1) The transdifferentiated phenotype and the
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Fig. 5. Confocal immunofluorescence analysis of ␤-catenin expression in C7Mock1 and C7caMEK1 cells. Cells were grown in the
presence of 10% FCS, fixed, rinsed thoroughly, and stained with a
monoclonal ␤-catenin antibody. Fluorescence was observed by confocal laser scanning microscopy. A series of 5 horizontal optical cross
sections at successively lower focal levels (step sizes of 2 ␮m each) is
shown. A, image of the stack close to the apical cell surface; BL,
image taken near the basal side.
hibited the FCS-induced cell proliferation of C7Mock1
and C7caMEK1 cells by 43.8 and 61.1% (10.32 ⫻ 104 ⫾
0.61 and 4.42 ⫻ 104 ⫾ 0.55 cells/cm2, respectively, n ⫽
7) compared with untreated controls (Fig. 11). Together, these results suggest that the increased activity of the MEK1-ERK signaling module is necessary to
Fig. 6. Confocal immunofluorescence analysis of ␣-catenin expression in C7Mock1 and C7caMEK1 cells. Cells were grown in the
presence of 10% FCS, fixed, rinsed thoroughly, and stained with a
polyclonal ␣-catenin antibody. Fluorescence was observed by confocal laser scanning microscopy. A series of 5 horizontal optical cross
sections at successively lower focal levels (step sizes of 2 ␮m each) is
shown. A, image of the stack close to the apical cell surface; BL,
image taken near the basal side.
MEK1-TRANSDIFFERENTIATED MDCK CELLS
invasive properties of caMEK1-transfected MDCK-C7
cells are reflected by the downregulation of proteins
that are crucial for the assembly of actin-based adherens junctions in epithelial cells, such as E-cadherin
and ␤- and ␣-catenin. 2) Although C7caMEK1 cells do
not express E-cadherin, at least part of the remaining
Fig. 8. Proteasomal protease inhibitors lead to an increase in ␤-catenin protein expression and induce slower-migrating forms of ␤-catenin. Cells were treated with 25 ␮M N-acetyl-Leu-Leu-norleucinal (A)
or 10 ␮M lactacystin (B) for 1, 3, 6, 12, or 24 h. Soluble, proteinmatched Triton X-100 cell lysates were immunoblotted using a
monoclonal ␤-catenin antibody. One representative immunoblot of 3
separate experiments is depicted for each of the 2 conditions.
Fig. 9. Cyclin D and cyclin A protein expression in quiescent and
FCS-stimulated C7Mock1 and C7caMEK1 cells. MDCK-C7 cells
transfected with empty vectors (C7Mock1) or with caMEK1
(C7caMEK1) were made quiescent by 24 h of incubation in FCS-free
medium (d0). Thereafter, cells were stimulated with 10% FCS for
24 h (d1) or 72 h (d3), washed with ice-cold PBS, and finally harvested using Triton X-100 lysis buffer. Soluble cell lysates were
matched for protein content and analyzed by Western blotting with
use of a polyclonal cyclin D1 antibody or a polyclonal cyclin A
antibody. One representative immunoblot of 3 independent experiments is depicted.
␤- and ␣-catenin is localized at the cell membrane and
is restricted to single patches of C7caMEK1 cell-cell
contacts. 3) C7caMEK1 cells show an increased cyclin
D expression, despite their substantially reduced
␤-catenin expression.
With respect to alterations of cell-cell adhesion molecules in MDCK-C7 cells that stably express a constitutively active mutant of the ERK activator MEK1 (29,
34), our present results support recently published
findings in PC-12 cells (22): stable expression of an
ERK inhibitory mutant in PC-12 cells has been re-
Fig. 10. Extracellular signal-regulated kinase types 1 and 2 (ERK1
and ERK2) phosphorylation and cyclin D protein expression in the
absence and presence of the synthetic mitogen-activated protein
kinase/ERK (MEK) inhibitor U-0126. C7Mock1 and C7caMEK1 cells
were made quiescent by 24 h of incubation in FCS-free medium and
then stimulated for 24 h (top) or 48 h (bottom) with 10% FCS in the
absence or presence of 10 ␮M U-0126 (30 min of preincubation).
Thereafter, cells were washed with ice-cold PBS and harvested using
Triton X-100 lysis buffer. Soluble cell lysates were matched for
protein content and analyzed by Western blotting with use of a
polyclonal cyclin D1 antibody or a polyclonal anti-phospho-ERK1/2
antibody, which detects phosphorylated Tyr-204 of ERK1 (ERK1*)
and ERK2 (ERK2*). One representative immunoblot of 3 independent experiments is depicted.
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Fig. 7. Coimmunoprecipitation of ␤-catenin with ␣-catenin or Ecadherin in C7Mock1 and C7caMEK1 cells. Cells were washed with
ice-cold PBS and harvested using Triton X-100 lysis buffer. After
soluble cell lysates were matched for protein content, ␤-catenin was
immunoprecipitated and blotted with a polyclonal ␣-catenin antibody or a monoclonal E-cadherin antibody. One representative immunoblot of 3 separate experiments is depicted.
C1479
C1480
MEK1-TRANSDIFFERENTIATED MDCK CELLS
ported to lead to a reorganization of their actin cytoskeleton and to an increase in the expression of
several adherens junction proteins. Metabolic labeling
demonstrated an augmented synthesis of cadherin and
␤-catenin in these cells (22). Furthermore, the expression of adherens junction proteins increased after
treatment with the selective MEK inhibitor PD-098059
in nontransfected PC-12 cells and in a ras-transformed
MDCK cell line, and overexpression of ERK2 in PC-12
cells resulted in the reduced expression of cadherins
and catenins (22). We are now able to show that in
renal epithelial cells that express a constitutively active mutant of the ERK activator MEK1, but not in
mock-transfected MDCK-C7 cells, E-cadherin expression is abolished, whereas ␤- and ␣-catenin expression
is substantially reduced. Thus the highly invasive
properties of fibroblastoid, mesenchymal-like C7caMEK1
cells are not only associated with elevated levels of
MMP-1 and with the expression of activated gelatinase
A (25) but also with an abolished/diminished expression of several proteins, which are important for cellcell interactions between neighboring cells. Inhibitors
of proteasome-mediated proteolysis, such as ALLN and
lactacystin, lead to a ␤-catenin accumulation in epithelial C7Mock1 cells and transdifferentiated C7caMEK1
cells, providing evidence that ␤-catenin is regulated by
the ubiquitin-proteasome pathway. Inasmuch as these
posttranslational ␤-catenin modifications occur to a
comparable extent in both cell types, it is unlikely that
the reduced ␤-catenin expression in C7caMEK1 cells is
due to an increased proteasome-mediated proteolytic
activity.
The functional significance of adherens junctions is
not limited to the physical interactions among groups
of adhering cells. Rather, cadherin/catenin complexes
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Fig. 11. Effects of U-0126 on the rate of cell proliferation in C7Mock1
and C7caMEK1 cells. Twenty-four hours after cells were subcultured, medium was changed once (day 0, d0) and cells were kept in
culture for another day (d1) or for 3 days (d3) in the absence or
presence of the synthetic MEK inhibitor U-0126 (10 ␮M). One measurement (n ⫽ 1) represents the mean determined from 10 video
prints randomly taken from one petri dish at days 0, 1, and 3. Values
are means ⫾ SE of 7 experiments.
can participate in signaling events between and within
cells that affect differentiation, proliferation, and migration (18). Defective cadherin function, for example,
can render noninvasive cells invasive, and induced
expression of various cadherins in invasive cells, on the
other hand, can suppress their invasiveness in a cell
contact-dependent manner (8, 20, 51). Furthermore,
E-cadherin and ␣-catenin are frequently downregulated in human tumors (39), and several studies have
reported decreased expression of ␤-catenin in some
tumor types as well (26, 27, 38, 44). Thus, besides their
massive phenotypic alterations and their loss of morphogenetic competence associated with an increased
activity of the MEK1-ERK2 signaling module (29, 34),
C7caMEK1 cells show a substantial downregulation of
distinct cell-cell adhesion proteins, thereby fulfilling
additional criteria that are observed during progression of well-differentiated epithelial tumors to anaplastic and metastatic carcinomas (4). These results
support the idea that an increased activity of the
MEK1-ERK2 signaling module is associated with
downregulation of homotypic cell-cell interactions in
epithelial MDCK cells.
In addition to its role in cell-cell adhesion, ␤-catenin
is a signaling entity in the Wnt signaling pathway (3):
within this pathway, Wnt binding to its receptor results in inhibition of glycogen synthase kinase-3␤
(GSK-3␤) and, consequently, to the accumulation of
cytoplasmic ␤-catenin. The following unsuppressed accumulation of ␤-catenin leads to its translocation into
the nucleus, where it cooperates with lymphocyte enhancer binding factor-1 (LEF-1)/TCF in transcriptional
events. A recently discovered target of the ␤-catenin/
LEF-1 transactivation is the cyclin D1 gene (40, 47).
High levels of cyclin D1 in colon carcinoma cells are
dramatically reduced by overexpression of the N-cadherin cytoplasmic domain (40). Cells expressing mutant ␤-catenin produce high levels of cyclin D1 mRNA
and cyclin D1 protein constitutively, suggesting that
abnormal levels of ␤-catenin may contribute to neoplastic transformation by causing accumulation of cyclin D1 (47). Furthermore, expression of a dominantnegative form of TCF in colon cancer cells strongly
inhibits expression of cyclin D1 and causes cells to
arrest in the G1 phase of the cell cycle (47). Although
␤-catenin expression is markedly reduced in
C7caMEK1 cells and although immunofluorescence experiments suggest a predominant localization of the
remaining ␤-catenin close to the cell membrane, our
present studies revealed an increased cyclin D expression in quiescent and serum-stimulated C7caMEK1
cells. In this respect, several studies have provided
evidence for a role for sustained ERK activity in controlling G1 progression through positive regulation of
the continued expression of cyclin D1, which represents one of the earliest cell cycle-related events to
occur during G0/G1-to-S phase transition (2, 19, 46, 49).
Inhibition of the ERK1/ERK2 signaling by expression
of dominant-negative forms of MEK1 or ERK1 or by
expression of the MAPK phosphatase MKP-1 has been
reported to strongly inhibit expression of a reporter
MEK1-TRANSDIFFERENTIATED MDCK CELLS
We thank Dr. H. Oberleithner (University of Münster, Münster,
Germany) for providing MDCK-C7 cells and Dr. J. Pouysségur for
providing the MEK1 constructs. We gratefully acknowledge the
excellent technical assistance of Edna Nemati, Mario Hirsch, and
Markus Plank.
This work was supported by Austrian Science Foundation Grant
P13295-MED (to H. Schramek).
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