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


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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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 beta - and alpha -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 alpha -catenin was coimmunoprecipitated with beta -catenin, whereas no E-cadherin was detected in beta -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 beta -catenin, including the appearance of high-molecular-weight beta -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 beta -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; beta -catenin


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ABSTRACT
INTRODUCTION
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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 alpha -, beta -, and gamma -catenin (plakoglobin) (3, 17, 42). Of these, beta -catenin represents a multifunctional protein that is an integral component of adherens junctions and intracellular signal transduction (7). At submembranal sites, beta -catenin is anchored to the cytoplasmic tail of cadherin and, via alpha -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 beta -catenin, whereas signaling initiated by the extracellular Wnt-1 oncoprotein or selected mutations in beta -catenin itself result in the accumulation of higher levels of beta -catenin in the cytoplasm (7). Moreover, deregulated signal transduction involving beta -catenin is a common event in tumor cells: the activation of beta -catenin to an oncogenic state can result from the inactivation of the tumor suppressor APC, direct mutation in the beta -catenin gene, or activation of Wnt receptors (3, 28). Once activated, beta -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 alpha -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 beta - and alpha -catenin in both cell lines. Inasmuch as beta -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 beta - and alpha -catenin expression in dedifferentiated C7caMEK1 cells but not in epithelial C7Mock1 cells. Although beta -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 beta -catenin expression is reduced, C7caMEK1 cells show a higher expression of U-0126-sensitive cyclin D protein.


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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-beta -catenin (Transduction Laboratories), rabbit anti-alpha -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 beta -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-beta -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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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 beta - and alpha -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 beta -catenin antibodies or a polyclonal alpha -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.

Attenuated expression of beta - and alpha -catenin in transdifferentiated C7caMEK1 cells. The cytoplasmic domain of E-cadherin is known to interact with beta - or gamma -catenin (plakoglobin), thereby binding to alpha -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 beta - and alpha -catenin in C7Mock1 and C7caMEK1 cells. Western blot analysis of epithelial C7Mock1 cell lysates revealed a strong protein expression of beta - and alpha -catenin, which did not change significantly over the period of time investigated (Fig. 1). In dedifferentiated C7caMEK1 cells, however, beta - and alpha -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 beta - and alpha -catenin staining at apicolateral cell borders was detected in epithelial C7Mock1 cells (Fig. 4, A and C), beta - and alpha -catenin expression was restricted to single patches along C7caMEK1 cell borders (Fig. 4, B and D), suggesting that in dedifferentiated C7caMEK1 cells the remaining beta - and alpha -catenins might be colocalized at distinct regions close to the plasma membrane. To analyze alpha - and beta -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 beta - and alpha -catenin, respectively. In C7Mock1 cells a continuous staining was detected for beta - and alpha -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 beta - and alpha -catenin compared with C7Mock1 cells. beta - and alpha -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 beta -catenin was immunoprecipitated from soluble lysates and blotted with an antibody against alpha -catenin, we found that, in C7caMEK1 cells, at least part of the remaining beta -catenin was still associated with alpha -catenin (Fig. 7, top). In contrast, no E-cadherin was detected in beta -catenin immunoprecipitates (Fig. 7, bottom).


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Fig. 4.   Conventional immunofluorescence analysis of beta -catenin (A and B) and alpha -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 beta -cadherin antibody or a polyclonal alpha -catenin antibody. Photographs were taken using a Zeiss ×20 objective.



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Fig. 5.   Confocal immunofluorescence analysis of beta -catenin expression in C7Mock1 and C7caMEK1 cells. Cells were grown in the presence of 10% FCS, fixed, rinsed thoroughly, and stained with a monoclonal beta -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 alpha -catenin expression in C7Mock1 and C7caMEK1 cells. Cells were grown in the presence of 10% FCS, fixed, rinsed thoroughly, and stained with a polyclonal alpha -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 beta -catenin with alpha -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, beta -catenin was immunoprecipitated and blotted with a polyclonal alpha -catenin antibody or a monoclonal E-cadherin antibody. One representative immunoblot of 3 separate experiments is depicted.

Regulation of beta -catenin expression by proteasome-mediated proteolysis in C7caMEK1 and C7Mock1 cells. Degradation of beta -catenin is initiated by phosphorylation at specific serine and threonine residues in its NH2 terminus. This leads to ubiquitination of beta -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 beta -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 beta -catenin protein in C7Mock1 and C7caMEK1 cells (Fig. 8A). This increase in beta -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 beta -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 beta -catenin protein and the appearance of a slower-migrating beta -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 beta -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 beta -catenin is induced by proteasome-specific protease inhibitors. It appears that these posttranslational beta -catenin modifications occur to a comparable extent in both cell types. Thus it is unlikely that the reduced beta -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 beta -catenin protein expression and induce slower-migrating forms of beta -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 beta -catenin antibody. One representative immunoblot of 3 separate experiments is depicted for each of the 2 conditions.

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 beta -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 beta -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 beta -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
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 beta - and alpha -catenin. 2) Although C7caMEK1 cells do not express E-cadherin, at least part of the remaining beta - and alpha -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 beta -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 beta -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 beta - and alpha -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 beta -catenin accumulation in epithelial C7Mock1 cells and transdifferentiated C7caMEK1 cells, providing evidence that beta -catenin is regulated by the ubiquitin-proteasome pathway. Inasmuch as these posttranslational beta -catenin modifications occur to a comparable extent in both cell types, it is unlikely that the reduced beta -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 alpha -catenin are frequently downregulated in human tumors (39), and several studies have reported decreased expression of beta -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, beta -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-3beta (GSK-3beta ) and, consequently, to the accumulation of cytoplasmic beta -catenin. The following unsuppressed accumulation of beta -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 beta -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 beta -catenin produce high levels of cyclin D1 mRNA and cyclin D1 protein constitutively, suggesting that abnormal levels of beta -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 beta -catenin expression is markedly reduced in C7caMEK1 cells and although immunofluorescence experiments suggest a predominant localization of the remaining beta -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-3beta 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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