Differential expression of cell-cell adhesion proteins and
cyclin D in MEK1-transdifferentiated MDCK cells
Ingrid
Marschitz1,
Judith
Lechner1,
Irene
Mosser1,
Martina
Dander1,
Roberto
Montesano2, 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
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ABSTRACT |
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 signal-regulated 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.
extracellular signal-regulated kinase; differentiation; proliferation; E-cadherin;
-catenin
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INTRODUCTION |
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, transmembrane 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 properties 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,
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 mock-transfected 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 E-cadherin
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.
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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 anti-cyclin 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 µg/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 cotransfected 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 · HCl, pH 7.5, 100 mM
NaCl, 50 mM NaF, 5 mM EDTA, 40 mM
-glycerophosphate, 200 µM sodium
orthovanadate, 0.1 mM phenylmethylsulfonyl fluoride, 1 µg/ml
leupeptin, 1 µM 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 µl of
protein A-Sepharose-coated beads for 2 h at 4°C. The precleared
supernatants were further incubated overnight with 4 µl 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 µm 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 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 µM). 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.
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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
expression in C7caMEK1 cells remained unchanged for
4 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).

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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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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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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 µm each)
is shown. A, image of the stack close to the apical cell surface; BL,
image taken near the basal side.
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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 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, resulting 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
-catenin was still associated with
-catenin (Fig. 7, top). In
contrast, no E-cadherin was detected in
-catenin immunoprecipitates
(Fig. 7, bottom).

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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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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 µm each)
is shown. A, image of the stack close to the apical cell surface; BL,
image taken near the basal side.
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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 µm each)
is shown. A, image of the stack close to the apical cell surface; BL,
image taken near the basal side.
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Fig. 7.
Coimmunoprecipitation of -catenin with -catenin or
E-cadherin 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.
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Regulation of
-catenin expression by proteasome-mediated
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 (N-acetyl-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 µM) 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 µM) 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
(N-acetyl-Leu-Leu-methional, LLM, or calpain inhibitor II;
25 µM), 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 µM),
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.

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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 µM
N-acetyl-Leu-Leu-norleucinal (A) or 10 µM
lactacystin (B) for 1, 3, 6, 12, or 24 h. Soluble,
protein-matched 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.
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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,
suggesting 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 µM) 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 µM U-0126 inhibited the FCS-induced 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.

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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.
|
|

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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 µM
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.
|
|
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 µM 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 inhibited 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 maintain a certain level of C7caMEK1 cell proliferation.

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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 µM).
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.
|
|
 |
DISCUSSION |
Three major findings can be derived from the present study:
1) The transdifferentiated phenotype and the 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
- 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 reported
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 cell-cell 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 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 dominant-negative 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 gene driven by the human cyclin D1 promoter as well as the
endogenous cyclin D1 protein (19). Furthermore, it has
been shown in Chinese hamster embryo fibroblasts (IIC9 cells) that
application of the synthetic MEK inhibitor PD-098059 or expression of
dominant-negative ERK1, but not dominant-negative JNK, results in a
dramatic decrease in cyclin D1 protein and mRNA levels
(49). Conversely, activation of the MEK1/2-ERK1/2 pathway
by expression of a constitutively active MEK1 mutant in CCL39 cells
increases cyclin D1 promoter activity and cyclin D1 protein expression
(19). In our present experiments utilizing C7caMEK1 cells,
we found U-0126-sensitive, increased basal and FCS-inducible cyclin D
protein levels, suggesting that the high cyclin D expression in these
cells is at least partially due to the constitutively active
MEK1-ERK1/2 signaling pathway. In this context, it is important to note
that, in cells where cyclin D1 levels are high in the absence of growth
factors, phosphatidylinositol 3-kinase (PI3K)-dependent regulation of
cyclin D1 protein stability and translation may also contribute to cell
cycle progression (24). Moreover, PI3K is able to regulate
cyclin D1 at a transcriptional level as well as posttranslationally.
Thus inactivation of GSK-3
through phosphorylation by protein kinase
B could provide an additional PI3K-dependent route to the stabilization
of cyclin D1 (11). In addition to MEK1-ERK1/2 and PI3K,
multiple small GTPase signaling pathways, such as signals activated by
Ral, Rac, and Cdc42, are likely to regulate cyclin D1 expression as
well (24). We obtained a reduced rate of cell
proliferation in two different and independent MDCK cell lines, both of
which show a fibroblastoid phenotype and express highly active ERK2
(34, 35). In the present study we report that, in MDCK-C7
cells stably expressing constitutively active MEK1, administration
of the MEK inhibitor U-0126 further reduces the already diminished
serum-induced proliferation rate. Thus it is tempting to speculate that
the augmented cyclin D expression in C7caMEK1 cells might be necessary
to overcome a negative effect of loss of cell-cell contact,
cell-substrate interaction, and/or loss of cell polarity on cell
proliferation in these cells.
 |
ACKNOWLEDGEMENTS |
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.
 |
FOOTNOTES |
This work was supported by Austrian Science Foundation Grant P13295-MED
(to H. Schramek).
Address for reprint requests and other correspondence: H. Schramek, Dept. of Physiology, Fritz-Pregl-Strasse 3, A-6010 Innsbruck, Austria (E-mail: herbert.schramek{at}uibk.ac.at).
The 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.
Received 22 February 2000; accepted in final form 22 May 2000.
 |
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