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Cell Growth & Differentiation Vol. 10, 317-332, May 1999
© 1999 American Association for Cancer Research

Constitutively Active Mitogen-activated Protein Kinase Kinase MEK1 Disrupts Morphogenesis and Induces an Invasive Phenotype in Madin-Darby Canine Kidney Epithelial Cells1

Roberto Montesano2, Jesus V. Soriano, Ghamartaj Hosseini, Michael S. Pepper and Herbert Schramek

Department of Morphology, University Medical Center, CH-1211 Geneva 4, Switzerland [R. M., J. V. S., G. H., M. S. P.], and Department of Physiology, University of Innsbruck, A-6020 Innsbruck, Austria [H. S.]


    Abstract
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
During certain developmental processes, as well as during tumor progression, polarized epithelial cells integrated within multicellular structures convert into scattered, freely migrating fibroblast-like cells. Despite the biological and clinical importance of this phenomenon, the intracellular biochemical cascades that control the switch between the epithelial and mesenchymal phenotypes have not been elucidated. Using Madin-Darby canine kidney (MDCK) cells (clone C7) as a model system, we have assessed the potential role of the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) cascade in the modulation of epithelial plasticity. When grown in three-dimensional collagen gels, MDCK-C7 cells form spherical cysts composed of polarized epithelial cells circumscribing a central lumen. This morphogenetic behavior is profoundly subverted in MDCK-C7 cells expressing a constitutively active MAPK/ERK kinase 1 (caMEK1) mutant (C7-caMEK1 cells). When suspended in collagen gels, C7-caMEK1 cells assume an elongated fibroblastoid shape and are unable to generate multicellular cysts. In addition, when seeded onto the surface of a collagen gel, C7-caMEK1 cells penetrate extensively into the underlying matrix, unlike wild-type and mock-transfected MDCK-C7 cells, which remain confined to the surface of the gel. Similar changes in morphogenetic and invasive properties are observed in MDCK-C7F cells, a nontransfected, stably dedifferentiated derivative of MDCK-C7 cells that expresses substantially increased ERK2 activity. Both C7-caMEK1 and MDCK-C7F cells but not wild-type or mock-transfected MDCK-C7 cells express activated Mr 72,000 gelatinase A [matrix metalloproteinase (MMP)-2] as well as elevated levels of membrane type-1 MMP. Synthetic MMP inhibitors as well as recombinant tissue inhibitor of metalloproteinases 2 and 3 suppress the invasion of collagen gels and restore the capacity of C7-caMEK1 cells to form cysts, thereby implicating the membrane type-1 MMP/MMP-2 proteolytic system in epithelial cell invasiveness and loss of multicellular organization. Taken together, our data demonstrate that increased activity of the MEK1-ERK2 signaling module in MDCK-C7 cells is associated with failure of morphogenesis and expression of a highly invasive phenotype. Sustained activation of the MAPK cascade therefore results in the destabilization of the three-dimensional architecture and the conversion of polarized epithelial cells into migrating mesenchymal-like cells.


    Introduction
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
A major challenge in developmental biology is to elucidate the mechanisms underlying the biogenesis, maintenance, and interconversion of different tissue phenotypes. The primary tissues involved in the formation of parenchymal organs during embryonic and postnatal life are epithelium and mesenchyme. Epithelial cells form coherent layers that line body surfaces and fold into complex glandular structures such as follicles, alveoli, and branching tubules. They are characterized by a well-defined apical-basal polarity, are tightly connected to each other by different types of intercellular junctions, interact with the extracellular matrix (basement membrane) via their basal surface (1, 2, 3) , and lack invasive properties when grown on three-dimensional collagen matrices (4 , 5) . In contrast, mesenchymal cells usually reside in connective tissues as individual elongated cells that are entirely surrounded by the extracellular matrix and have a pronounced ability to invade collagen lattices in vitro (1 , 4 , 5) .

Once they are established as epithelial cells, cells are not irreversibly maintained in this phenotype. Under specific circumstances, epithelial cells can modify their differentiation program and convert into mesenchymal-like cells capable of freely migrating through the extracellular matrix (reviewed in Refs. 6, 7, 8, 9 ). This phenotypic switch, designated EMT,3 plays a fundamental role in morphogenetic tissue remodeling during embryogenesis (1) . A temporally and spatially restricted loss of epithelial characteristics and the acquisition of mesenchymal features are indeed necessary for a number of developmental processes that require cell translocation and matrix invasion, such as mesoderm formation (10) , the migration of neural crest cells out of the neural tube (11) , the formation of cardiac cushion mesenchyme (12) , the fusion of palatal medial edges (13) , and Müllerian duct regression (14) .

In postnatal life, the genetic program responsible for EMT is apparently repressed or switched off, at least under physiological conditions. The EMT program, however, can be inappropriately reactivated during pathological processes, most notably during the transition from benign adenomas to metastatic carcinomas. In fact, the disruption of polarized tissue architecture, loss of cell-cell adhesion, and acquisition of an invasive phenotype are hallmarks of malignancy (reviewed in Refs. 15, 16, 17, 18 ). Elucidating the molecular mechanisms that govern EMT is therefore essential for understanding the process of tumor progression.

A variety of molecular cues have been proposed to play a role in EMT. These include growth factors (19, 20, 21, 22) , pleiotropic cytokines such as transforming growth factor ß (23, 24, 25, 26, 27, 28, 29, 30, 31) , extracellular matrix components (32, 33, 34) , cell adhesion molecules (35, 36, 37) , oncogenes (36 , 38, 39, 40, 41) , matrix-degrading proteases (42 , 43) , cell surface proteoglycans (44 , 45) , transcription factors (46 , 47) , and other intracellular proteins (48, 49, 50, 51) . The finding that a number of different growth factors, matrix components, and adhesion receptors trigger a similar EMT program raises the possibility that the initial biochemical events elicited by these molecules converge upon a common intracellular signal transduction pathway, eventually leading to changes in gene expression. To date, however, very little information is available concerning the biochemical cascades that control EMT (52, 53, 54, 55) .

A potential signaling pathway that might be involved in the control of EMT is the MAPK cascade, which is known to play a central role in the transduction of extracellular stimuli into diverse biological responses. The MAPK cascade, also known as the ERK cascade, is triggered by the binding of a variety of ligands to their cognate cell surface receptors, resulting in the activation of a Ras GTPase. Ras then activates the Raf-1 serine/threonine kinase, which in turn phosphorylates and thereby activates MAPK kinases, exemplified by MEK1 and MEK2. MEK1 and MEK2 are dual-specificity kinases that phosphorylate their downstream targets ERK1 and ERK2 on both threonine and tyrosine residues. Upon activation, ERK1 and ERK2 phosphorylate cytoplasmic targets and translocate into the nucleus, where they stimulate the activity of a number of transcription factors, thereby altering the expression of genes involved in the regulation of cell growth and differentiation (reviewed in Refs. 56, 57, 58, 59, 60, 61 ).

We have used MDCK cells, a cell line that retains differentiated properties of renal tubular epithelium (62 , 63) , to study the role of the MAPK pathway in the modulation of the epithelial phenotype. In previous reports, we showed that transfection of a constitutively active mutant of the upstream ERK activator MEK1 into MDCK-C7 cells results in pronounced phenotypic changes, including the acquisition of a fibroblastoid morphology, reduced cytokeratin and increased vimentin expression (64) , and the assembly of {alpha}-smooth muscle actin-containing stress fibers (65) .

A remarkable property of MDCK cells is their ability to arrange themselves into precisely organized multicellular structures when grown in a three-dimensional extracellular matrix environment. Thus, when embedded in collagen gels, MDCK cells form spherical cysts composed of a monolayer of polarized epithelial cells surrounding a central lumen (66, 67, 68) . In this study, we have taken advantage of the morphogenetic competence of MDCK cells to investigate the potential involvement of the MAPK pathway in the regulation of epithelial architecture. We show that MDCK-C7 cells expressing caMEK1 are unable to generate cyst-like epithelial structures in collagen gels and behave like highly invasive, mesenchymal-like cells. Similar findings were obtained with MDCK-C7F cells, a nontransfected, dedifferentiated derivative of MDCK-C7 cells that harbor substantially increased ERK2 activity (69) . Collectively, these results strongly support the notion that sustained activation of the MAPK pathway results in the destabilization of the epithelial architecture and the induction of EMT.


    Results
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
caMEK1 Disrupts the Morphogenetic Properties of MDCK-C7 Cells.
To assess the role of the MAPK pathway in the regulation of epithelial morphogenesis, MDCK-C7 cells transfected with caMEK1 (C7-caMEK1) cells (64) were grown in three-dimensional collagen gels. Under these conditions, C7-caMEK1 cells became elongated and did not form organized multicellular structures, unlike wild-type and mock-transfected MDCK-C7 cells that generated spheroidal cysts. However, in these initial experiments, the poor viability of C7-caMEK1 cells in collagen gels resulted in progressive cell loss during the course of the assay (4–7 days). In an attempt to overcome this drawback, we tested a number of growth factors for their potential effect on cell survival and found that bFGF efficiently prevented C7-caMEK1 cell death in collagen gels, whereas epidermal growth factor, hepatocyte growth factor, and insulin-like growth factor I and II had no obvious effect (data not shown). Because bFGF did not influence the three-dimensional organization of control or transfected MDCK-C7 cells, it was added to all gel cultures in subsequent experiments to maintain adequate cell viability.

The dramatically altered behavior of MDCK-C7 cells expressing caMEK1 is illustrated in Fig. 1Citation . Whereas both wild-type (Fig. 1A)Citation and mock-transfected (not shown) MDCK-C7 cells formed spherical cysts when suspended in collagen gels, two different clones of caMEK1-transfected cells, i.e., C7-caMEK1-1 (not shown) and C7-caMEK1-4 (Fig. 1B)Citation assumed a spindle-shaped, fibroblastoid morphology and failed to organize into multicellular structures. Similar results were obtained by sandwiching MDCK-C7 cells between two collagen layers, as described in "Materials and Methods." Under these conditions, both wild-type (Fig. 1C)Citation and mock-transfected (not shown) MDCK-C7 cells formed irregularly shaped flattened cysts enclosing a central lumen, whereas C7-caMEK1-1 cells (not shown) and C7-caMEK1-4 cells (Fig. 1D)Citation acquired a very elongated shape and did not associate with each other to form lumen-containing structures.



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Fig. 1. Sustained expression of caMEK1 in MDCK-C7 cells is associated with the failure of cystogenesis. When suspended in a three-dimensional collagen gel (A) or sandwiched between two collagen layers (C), wild-type MDCK-C7 cells form spherical cysts or irregularly shaped lumen-containing structures, respectively. In contrast, when suspended in collagen gels (B) or sandwiched between two collagen layers (D), caMEK1-4 cells acquire an elongated fibroblastoid morphology and do not form organized multicellular structures. A and B, bright-field microscopy; C and D, phase-contrast microscopy. Bar, 200 µm.

 
Thin sections of the cysts formed in collagen gels by mock-transfected MDCK-C7 cells showed that the lumen was delimited by correctly polarized epithelial cells provided with apical microvilli and lateral junctional complexes (Fig. 2A)Citation . In contrast, thin sections of collagen gel cultures of C7-caMEK1-1 cells confirmed the absence of organized lumen-containing structures and showed that the gels were populated by elongated cells devoid of morphologically detectable apical-basal polarity (Fig. 2B)Citation .



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Fig. 2. Thin sections of collagen gel cultures of mock- or caMEK1-transfected cells. A, mock-transfected MDCK-C7 cells grown for 4 days in a collagen gel form spherical cysts consisting of well-polarized cuboidal epithelial cells with a basal surface in contact with the collagen matrix and a microvilli-rich apical surface facing the lumen of the cyst. B, caMEK1-expressing MDCK-C7 cells (C7-caMEK1-1 cells) fail to form lumen-containing multicellular structures and lack obvious structural polarity. Bars, 10 µm.

 
Proliferation assays in collagen gels demonstrated that despite the addition of bFGF, the number of C7-caMEK1-1 cells did not increase over a 6-day period, whereas mock-transfected cells underwent a 23-fold increase in cell number (data not shown). It could therefore be argued that the inability of C7-caMEK1-1 cells to form cystic structures is due to their impaired growth in collagen gels. To clarify this issue, we examined whether three-dimensional matrices other than collagen gels might allow the proliferation of C7-caMEK1-1 cells. We found that fibrin gels were permissive for sustained growth (a 6-fold increase in cell number after 7 days of culture) of C7-caMEK1-1 cells, resulting in the formation of solid (i.e., lumen-less) multicellular cords (Fig. 3B)Citation . Similar results were obtained with a different clone of caMEK1-transfected cells (C7-caMEK1-4 cells; data not shown). Remarkably, under the same conditions, mock-transfected cells exclusively formed spherical cysts (Fig. 3A)Citation , similar to those observed in collagen gels. The fibrin gel experiments thus demonstrated that the inability of C7-caMEK1 cells to form cystic structures is not the consequence of constitutively impaired proliferation but results from a profound subversion of the normal morphogenetic properties of MDCK-C7 cells.



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Fig. 3. Behavior of mock- and caMEK1-transfected cells in fibrin gels. When grown in three-dimensional fibrin gels for 4 days, mock-transfected MDCK-C7 cells generate spherical cysts (A) similar to those formed in collagen gels (inset, semithin section of a cyst). Under the same experimental conditions, C7-caMEK1-1 cells form branching cords devoid of lumen (B; inset, semithin section of a cord). Bars, 200 µm. Inset bars, 20 µm.

 
caMEK1 Confers an Invasive Phenotype to MDCK-C7 Cells.
Taken together with our previous findings in conventional monolayer culture (64) , the results described above indicated that MDCK-C7 cells expressing caMEK1 had lost many of the features associated with the epithelial phenotype and had acquired morphological and functional traits characteristic of mesenchymal cells. Because one of the hallmarks of the mesenchymal phenotype is the ability to actively migrate through the extracellular matrix, we asked whether C7-caMEK1 cells might be endowed with invasive properties. To address this issue, wild-type and caMEK1-transfected MDCK-C7 cells were seeded onto three-dimensional collagen gels, and their ability to invade the underlying matrix was evaluated by phase-contrast microscopy by focusing at different levels below the surface of the gel.

As expected (36 , 68) , both wild-type and mock-transfected MDCK-C7 cells formed a cobblestone-like monolayer and remained strictly confined to the surface of the gel (data not shown). In contrast, C7-caMEK1–1 (not shown) and C7-caMEK1–4 cells (Fig. 4)Citation did not form a confluent monolayer; instead, they invaded the underlying collagen matrix, either as individual spindle-shaped cells or as thin cell cords. Invasion of the gels was already extensive 48 h after seeding (Fig. 4)Citation , and after an additional 4 days, some cells had migrated to a depth as great as 450 µm below the surface of the gel, as determined using the calibrated micrometer of the inverted microscope. Semithin sections perpendicular to the culture plane confirmed that wild-type MDCK-C7 cells form a sheet of tightly interconnected cells on the surface of the collagen gel (Fig. 5A)Citation , whereas C7-caMEK1-4 cells form a discontinuous layer of loosely associated cells on the gel surface and penetrate deeply into the collagen matrix (Fig. 5B)Citation . C7-caMEK1 cells also exhibited invasive properties when grown on fibrin gels. However, under this experimental condition, the cells penetrated the underlying substrate mostly as multicellular cords (data not shown).



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Fig. 4. caMEK1-transfected MDCK-C7 cells exhibit a highly invasive behavior on collagen gels. C7-caMEK1-4 cells were plated onto a three-dimensional collagen gel. The same field was photographed 48 h later at the gel surface (A) and at 20 (B), 50 (C), and 90 µm (D) below the surface. A, subconfluent monolayer at the surface of the gel; B-D, focusing progressively deeper into the collagen gel reveals elongated cells that have migrated to various depths from the surface of the gel into the underlying matrix. Bar, 100 µm.

 


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Fig. 5. Semithin sections of collagen gel cultures of wild-type and caMEK1-transfected MDCK-C7 cells. A, wild-type MDCK-C7 cells grown on a collagen gel form a confluent monolayer of tightly apposed cuboidal cells and remain strictly confined to the gel surface. B, in marked contrast, C7-caMEK1-4 cells do not form a confluent monolayer but invade deeply into the underlying collagen matrix (arrows). Bar, 50 µm.

 
To establish whether activated MEK1 was responsible for the invasive properties of C7-caMEK1 cells, collagen gel invasion assays were performed in the presence of U0126, a selective, noncompetitive MEK1 inhibitor (70) . The addition of 10 µM U0126 to the cultures induced a pronounced flattening of C7-caMEK1-4 cells on the gel surface (compare Fig. 6A, and BCitation ) and markedly reduced their invasion into the collagen matrix (Fig. 6C)Citation . These findings demonstrate that inhibition of MEK1 activation can partially reverse the invasive phenotype of C7-caMEK1 cells.



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Fig. 6. The MEK1 inhibitor U0126 reduces the invasion of C7-caMEK1 cells into collagen gels. A, C7-caMEK1-4 cells grown on a collagen gel for 48 h extensively invade the underlying matrix (focus is slightly beneath the gel surface). Bar, 100 µm. B, C7-caMEK1-4 cells grown on a collagen gel for 48 h in the presence of 10 µM U0126 are more flattened. Focusing below the gel surface reveals that the cells exclusively invade the more superficial layers of the collagen matrix (data not shown). Bar, 100 µm. C, C7-caMEK1-4 cells were plated onto a three-dimensional collagen gel and incubated in the presence or absence of 10 µM U0126. Invasion was quantified 48 h later as described in "Materials and Methods" by determining the mean total additive length ± SEM (in µm) of all cellular structures (i.e., single cells or cell cords) present in a photographic field 60 µm beneath the surface monolayer. Three randomly selected fields per condition were analyzed in each of three separate experiments. Values for U0126 are significantly (P < 0.001) different from control values.

 
A Derivative of MDCK-C7 Cells Expressing Increased ERK2 Activity Exhibits Properties Similar to Those of caMEK1-transfected Cells.
Having established that MDCK-C7 cells transfected with a caMEK1 have lost their normal morphogenetic potential and acquired an invasive phenotype, we next wished to examine the behavior of epithelial cells spontaneously expressing elevated levels of MAPK/ERK activity. For this purpose, we took advantage of MDCK-C7F cells, a stably dedifferentiated cell line generated by transient culture of MDCK-C7 cells in alkaline medium (71) . Unlike the parental cell line, C7F cells display a spindle-shaped morphology (71) , have a reduced cytokeratin content (65) , and express the {alpha}-smooth muscle actin isoform (65) . In addition, we have previously demonstrated that MDCK-C7F cells exhibit substantially increased ERK2 activity as compared with parental MDCK-C7 cells (69) . This cell line therefore provides a complementary model system to study the modulation of the epithelial phenotype by the MAPK pathway.

When embedded in collagen gels, MDCK-C7F cells assumed an elongated fibroblastoid shape and did not form cystic structures (Fig. 7A)Citation , in contrast to the results observed with parental MDCK-C7 cells (compare Fig. 1ACitation ). Thus, the behavior of MDCK-C7F cells in collagen gels was remarkably similar to that of caMEK1-transfected MDCK-C7 cells (compare Fig. 1BCitation ). Likewise, when grown in fibrin gels, MDCK-C7F cells formed thin branching cords instead of cysts (Fig. 7B)Citation , similar to the results observed with C7-caMEK1 cells (compare Fig. 3BCitation ). In addition, when seeded onto the surface of three-dimensional collagen or fibrin gels, MDCK-C7F cells invaded the underlying matrix as spindle-shaped cells (Fig. 7C)Citation or as thin cord-like structures (Fig. 7D)Citation , respectively. This invasive behavior contrasted sharply with that of parental MDCK-C7 cells, which remained confined to the surface of the gel (data not shown; compare Fig. 5ACitation ).



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Fig. 7. MDCK-C7F cells exhibit properties similar to those of caMEK1-transfected MDCK-C7 cells. MDCK-C7F cells are a nontransfected derivative of wild-type MDCK-C7 cells that harbor substantially increased ERK2 activity. When suspended in collagen gels, they acquire an elongated shape and remain mostly as single cells (A), whereas in fibrin gels, they tend to form thin cord-like structures (B). Cyst formation is not observed under either condition. Likewise, when seeded on collagen (C) or fibrin (D) gels, MDCK-C7F cells invade the underlying matrix mostly as single elongated cells (C) or thin branching cords (D), respectively. The behavior of MDCK-C7F cells is therefore strikingly similar to that of caMEK1-transfected MDCK-C7 cells. Bars, 100 µm.

 
Thus, both when grown in suspension in collagen or fibrin gels (morphogenesis assay) or when seeded onto the gel surface (invasion assay), the behavior of MDCK-C7F cells correlated closely with that of C7-caMEK1 cells. These results indicate that sustained activation of the MAPK pathway resulting from either constitutively increased ERK2 activity or transfection with caMEK1 induces similar phenotypic changes in MDCK cells, namely, the loss of morphogenic competence and gain of invasive properties.

C7-caMEK1 and C7F Cells Express Activated Gelatinase A and MT1-MMP.
The migration of cells through collagenous matrices requires the activity of MMPs (72) . Therefore, we used gelatin zymography to determine whether C7-caMEK1 and C7F cells secreted gelatin-degrading MMP. The Mr 72,000 MMP-2 proenzyme (progelatinase A) was revealed in the conditioned media of wild-type or mock-transfected MDCK-C7 cells (Fig. 8A)Citation . However, Mr 64,000 and Mr 62,000 activated MMP-2 species were only found in the conditioned media of C7-caMEK1-4 and C7F cells. MMP-9 (gelatinase B) activity was expressed at similar levels in all cell lines (Fig. 8A)Citation .



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Fig. 8. C7-caMEK1 and C7F cells show increased activation of MMP-2 and express high levels of MT1-MMP. A, gelatin zymography of conditioned medium from mock-transfected MDCK-C7 cells (C7-mock), C7-caMEK1-4 cells (C7-caMEK), MDCK-C7 cells (C7), and MDCK-C7F cells (C7F). Cells were seeded onto the surface of collagen gels and incubated in serum-free medium supplemented with 10 ng/ml bFGF for 24 h, after which conditioned media were harvested and loaded onto SDS-PAGE/gelatin gels under nonreducing conditions, as described in "Materials and Methods." B, Northern blot analysis of total cellular RNA. Cells were seeded into tissue culture dishes and incubated in complete medium supplemented with 10 ng/ml bFGF for 24 h, after which RNA samples (5 µg/lane) were prepared and hybridized with either MT1-MMP, TIMP-2, or P0 cRNA probes, as described in "Materials and Methods."

 
To exert its enzymatic activity, MMP-2 requires cleavage and activation at the cell surface by MT1-MMP or MMP-14 (73) . To assess whether C7-caMEK1 and C7F cells produce MT1-MMP, total cellular RNA was analyzed by Northern blot hybridization. MT1-MMP mRNA was barely detectable in mock-transfected or wild-type MDCK-C7 cells, but very high levels of expression were observed in both the C7-caMEK1-1 and C7-caMEK1-4 clones and in C7F cells (Fig. 8B)Citation . MT1-MMP mRNA expression by C7-caMEK1-4 cells was reduced by approximately 20% after an 18-h incubation with the MEK inhibitor U0126 (10 µM; data not shown).

MT1-MMP-induced activation of MMP-2 requires the binding of TIMP-2 to MT1-MMP. This results in the generation of a heteromolecular receptor complex, which in turn allows the binding and subsequent activation of the MMP-2 proenzyme (74) . By Northern blot analysis, both the 1-kb and the 3.5-kb TIMP-2 transcripts were expressed at equivalent levels in all cell lines examined (Fig. 8B)Citation .

MMP Inhibitors Suppress Collagen Gel Invasion and Restore Cyst Morphogenesis.
To determine whether MMP activity is required for the invasion of C7-caMEK1 cells into collagen gels, we examined the behavior of C7-caMEK1-4 cells grown on collagen gels in the presence or absence of MMP inhibitors (Fig. 9)Citation . A quantitative analysis demonstrated that collagen gel invasion was suppressed in a dose-dependent manner by the synthetic MMP inhibitor BB94 (92% inhibition at 10 µM) but was only marginally (nonsignificantly) reduced by the inactive isomer BB1268 (Fig. 9C)Citation . Invasion was also suppressed in a dose-dependent manner by the synthetic MMP inhibitor CT1847 (data not shown). The role of MMP in collagen gel invasion was further investigated using recombinant TIMPs. Invasion was only slightly decreased by TIMP-1 (a significant 30% inhibition at 10 µg/ml) but was virtually completely suppressed by TIMP-2 and TIMP-3 (95% and 100% inhibition, respectively, at 10 µg/ml; Fig. 9, B and DCitation ).



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Fig. 9. MMP inhibitors abrogate the invasion of C7-caMEK1 cells into collagen gels. A, C7-caMEK1-4 cells grown on a collagen gel for 48 h extensively invade the underlying matrix (focus is slightly beneath the gel surface). B, C7-caMEK1-4 cells incubated for 48 h with 10 µg/ml TIMP-2 form a continuous monolayer on the surface of the gel and do not invade the underlying matrix. C, dose-dependent inhibition of invasion by the synthetic MMP inhibitor BB94. Invasion was quantified as described in "Materials and Methods" by determining the mean total additive length ± SEM (in µm) of all cellular structures (i.e., single cells or cell cords) present in a photographic field 20 µm beneath the surface monolayer. Values for BB94 are significantly (P = 0.001 for 0.1 µM; P < 0.001 for 1 µM and 10 µM) different from control values (in contrast, invasion is not significantly inhibited by the inactive isomer BB1268). D, dose-dependent inhibition of invasion by TIMPs. TIMP-1, which is not an effective inhibitor of MT1-MMP, only reduces invasion by 30% at 10 µg/ml. In contrast, at the same concentrations, TIMP-2 and TIMP-3 virtually completely abrogate the invasion of collagen gels (95% and 100% inhibition, respectively). Bars in A and B, 200 µm.

 
We next investigated the potential effect of MMP inhibitors in the morphogenesis assay, i.e., on C7-caMEK1 cells grown in suspension in collagen gels. We found that BB94, CT1847, and TIMP-2 did not prevent the elongation of most C7-caMEK1-4 cells in collagen gels. Interestingly, however, the addition of these MMP inhibitors (but not the addition of either the inactive compound BB1268 or TIMP-1) resulted in the formation of tiny cysts interspersed among the elongated cells. The cysts were composed of a single layer of plump epithelial cells circumscribing a clearly defined lumen (Fig. 10)Citation . Cystic structures were never observed in cultures incubated in the absence of MMP inhibitors. In light of the recent report that MT1-MMP mediates the cell invasion of fibrin matrices (75) , we also examined the effect of MMP inhibitors on the behavior of C7-caMEK1-4 cells suspended in fibrin gels. We found that the addition of either BB94 or CT1847 abrogated the formation of branching cords normally seen under control conditions (compare Fig. 3BCitation ), resulting instead in the formation of spherical cysts similar to those observed in fibrin gel cultures of mock-transfected MDCK-C7 (compare Fig. 3ACitation ; data not shown). Taken together, these findings demonstrate that the reduction of elevated MMP activity is sufficient to restore normal morphogenetic properties to C7-caMEK1 cells.



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Fig. 10. MMP inhibitors restore the capacity of C7-caMEK1 cells to form cysts. The addition of the synthetic MMP inhibitor CT1847 (100 nM) to C7-caMEK1-4 cells suspended in collagen gels (morphogenesis assay) results in the formation of small cystic structures (arrows) interspersed among a population of elongated cells (5-day-old culture). In the inset, the cyst in the lower left portion of the image was photographed with a high power (x40) objective with a narrow depth of field (focus is approximately on the equatorial plane of the cyst). The cells lining the floor of the cavity appear as a blurred image in the center of the cyst. Serial micrographs taken at different focal planes (data not shown) revealed that the wall of the cyst consisted of a single layer of plump epithelial cells. Bar, 200 µm. Inset bar, 20 µm.

 

    Discussion
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Although the conversion of polarized epithelial cells into migrating mesenchymal-like cells plays a crucial role in embryonic development as well as in the malignant progression of epithelial tumors, the intracellular signaling pathways that trigger this phenotypic switch are still largely unknown. To assess the potential involvement of the MAPK cascade in the process of EMT, we have used MDCK-C7 cells, a clonal kidney-derived cell line that exhibits differentiated properties of collecting duct epithelium. Using two independent cellular models, we found that constitutive activation of the MAPK pathway in MDCK-C7 cells disrupts their ability to generate complex three-dimensional structures and confers an invasive phenotype upon them, which correlates with the expression of activated gelatinase A. Sustained activation of the MAPK cascade therefore appears to result in destabilization of the epithelial architecture and the induction of EMT.

The MAPK signaling pathway is a multistep phosphorylation cascade that transmits a variety of stimuli from cell surface receptors to cytosolic and nuclear targets (reviewed in Refs. 56, 57, 58, 59, 60 ). Whereas it was originally thought to be primarily involved in the control of cell growth, the MAPK pathway has now emerged as a central regulator of a wide range of biological responses (61) . Thus, the expression of constitutively active MEK mutants has been reported to induce proliferation and/or morphological transformation in fibroblasts (76, 77, 78, 79) and differentiation in PC12 cells (77) . Interestingly, recent studies have shown that the MAPK pathway can either promote (80, 81, 82, 83) or inhibit (84, 85, 86, 87) cell differentiation, depending on the biological assay and target cell type. However, despite the increasingly recognized involvement of the MAPK cascade in a broad spectrum of biological processes, virtually nothing is known concerning its role in the regulation of the differentiated epithelial phenotype. To address this question, we chose to assess the potential involvement of the MAPK pathway in the modulation of three-dimensional epithelial architecture.

The ability to arrange into orderly, highly organized multicellular structures is a fundamental property of glandular epithelial cells. This morphogenetic process can be recapitulated in vitro by growing epithelial cells within reconstituted extracellular matrices. Thus, when suspended in three-dimensional collagen gels, MDCK cells proliferate clonally to form spherical cysts composed of a closed monolayer of epithelial cells whose apical pole faces the lumen of the cyst, whereas the basal pole is in contact with the collagen matrix (66, 67, 68 , 88 , 89) . Cyst formation by MDCK cells therefore represents a useful model system to decipher the molecular signals that either elicit additional tissue-specific morphogenic events (90) or induce architectural destructuring (67 , 91) .

We have recently reported that stable transfection of MDCK-C7 cells with caMEK1 results in increased enzymatic activity of ERK2 as well as in phenotypic changes consistent with epithelial dedifferentiation and EMT, including acquisition of a fibroblast-like shape, reduced cytokeratin and increased vimentin expression (64) , and assembly of {alpha}-smooth muscle actin-containing stress fibers (65) . These findings prompted us to determine whether caMEK1-transfected MDCK-C7 cells still retained the morphogenetic competence to form multicellular cysts in collagen gels. We found that, unlike wild-type and mock-transfected cells, caMEK1-transfected MDCK-C7 cells (C7-caMEK1 cells) are unable to form multicellular cysts in collagen gels; instead, they assume the morphology of elongated, mesenchymal-like cells. Although C7-caMEK1 cells do not proliferate in collagen gels, the failure of cystogenesis cannot be attributed to a growth defect. Indeed, when grown in fibrin instead of collagen gels, C7-caMEK1 cells actively proliferate, yet they form thin branching cords rather than spherical cysts.

In postnatal life, most epithelial cell types adopt a sedentary phenotype and do not invade the connective tissues matrix under physiological conditions. This behavior is usually maintained when epithelial cells are grown in vitro on three-dimensional extracellular matrix substrata (4 , 5) . Thus, MDCK cells form confluent monolayers of tightly connected polygonal cells on the surface of collagen gels and do not invade the underlying matrix (36 , 68) . Similar findings were obtained in this study with both wild-type and mock-transfected MDCK-C7 cells. In striking contrast, we found that when seeded on the surface of collagen gels, caMEK1-transfected MDCK-C7 cells infiltrate into the underlying matrix either as single spindle-shaped cells or as thin cell cords. In this respect, the behavior of C7-caMEK1 cells in the collagen gel invasion assay is similar to that of MDCK cells that have been transformed by viral oncogenes (36 , 37 , 39 , 40 , 92) . The finding that the selective MEK1 inhibitor U0126 markedly reduces cell invasion into collagen gels demonstrates that activated MEK1 is responsible for the invasive properties of C7-caMEK1 cells. Interestingly, Kss1, a MAPK homologue in yeast, has recently been shown to be a potent negative regulator of invasive growth in its inactive form. In this system, the MAPK kinase homologue Ste7 acts to relieve this negative regulation by switching Kss1 from an inhibitor to an activator (93) . Our data suggest that the MEK1-ERK2 module plays a similar role in renal epithelial cells.

Collectively, the results obtained through the analysis of transfected MDCK-C7 cells indicate that expression of a caMEK1 mutant is associated with the loss of morphogenetic competence and the acquisition of invasive properties. To further assess the potential effects of MAPK activation in an independent cellular system, we used MDCK-C7F cells, a stably dedifferentiated derivative of parental MDCK-C7 cells (71) that harbor substantially increased ERK2 activity (69) . MDCK-C7F cells have a fibroblastoid morphology (71) , exhibit a decreased cytokeratin content (65) , and express the {alpha}-smooth muscle actin isoform (65) . We found that MDCK-C7F cells, like caMEK1-transfected MDCK-C7 cells, are unable to form multicellular cysts and exhibit an invasive behavior in both collagen and fibrin gels. The results obtained with caMEK1-transfected MDCK-C7 cells and nontransfected MDCK-C7F cells therefore mutually corroborate and point to an important role of the MAPK pathway in the destabilization of epithelial architecture and the induction of cell invasiveness. This conclusion is in accord with the recent observation that expression of activated Raf induces disorganized growth of MDCK cells in collagen gels (94) . However, although no MEK1 substrate other than ERK1 and ERK2 has been identified to date, recent findings in fibroblasts suggest that activated MEK1 could elicit certain cellular responses via both ERK-dependent and ERK-independent pathways (95, 96, 97) . Thus, it cannot be excluded that as yet undefined downstream effectors of MEK1 may act in concert with ERK-mediated events in epithelial MDCK-C7 cells, leading to the observed mesenchymal transition and/or to the induction of cell invasiveness.

Extracellular matrix remodeling by MMP is a key event in the regulation of cell behavior in both physiological and pathological processes. MMP expression is usually coordinated with the expression of physiological MMP inhibitors (TIMPs). MMP expression is regulated at both transcriptional and posttranscriptional levels (reviewed in Refs. 98 and 99 ). With regard to the latter, proteolytic activation of latent MMP is of particular importance, the most striking and extensively studied example of which is the activation of progelatinase A (latent MMP-2) by MT1-MMP. MT1-MMP is expressed at the cell surface and, through its catalytic domain, binds to the NH2-terminal domain of TIMP-2. TIMP-2 also binds to progelatinase A via COOH-terminal domain interactions. TIMP-2 thus serves as a bridge between MT1-MMP and progelatinase A, thereby localizing the latter to the cell surface. In the presence of an excess of MT1-MMP, free MT1-MMP (which is not bound and inactivated by TIMP-2) activates progelatinase A (reviewed in Refs. 100 and 101 ). Zymographic analysis of conditioned media demonstrated that whereas wild-type and mock-transfected MDCK-C7 cells express progelatinase A, both C7-caMEK1 and C7F cells express activated gelatinase A species. The Mr 92,000 gelatinase B (MMP-9) was expressed at comparable levels in all cell lines and is therefore unlikely to be regulated by the MAPK cascade. The latter findings differ from those of Gum et al. (102) , who reported up-regulation of MMP-9 by the MAPK pathway in a squamous carcinoma cell line. Wild-type and mock-transfected MDCK-C7 cells expressed extremely low levels of MT1-MMP mRNA, which is in agreement with the results of Kadono et al. (92) . In contrast, both clones of C7-caMEK1 cells, as well as C7F cells, expressed very high levels of MT1-MMP mRNA. Kadono et al. (92) recently reported an elevated expression of MT1-MMP in v-src-transformed MDCK cells, but they did not investigate whether the effect of v-src was mediated via the MAPK pathway.

The functional relevance of the MT1-MMP/MMP-2 system was demonstrated by the finding that collagen gel invasion of C7-caMEK1-4 cells was almost totally suppressed by the addition of synthetic MMP inhibitors as well as by the addition of TIMP-2 and TIMP-3, but not by the addition of TIMP-1, which is a poor inhibitor of MT1-MMP (103) . Significantly, our demonstration of activated MMP-2 and high levels of MT1-MMP in caMEK1-transfected cells complements previous studies showing that the MAPK pathway regulates the expression of another class of matrix-degrading proteinases, namely plasminogen activators (104) . Collagen gels consist largely of fibrillar type I collagen, which is generally assumed to be resistant to gelatinase A activity. However, by acting sequentially after the initial cleavage of the triple helix by interstitial collagenase (MMP-1), gelatinase A may act as a key and possibly rate-limiting step in the degradation of fibrillar collagens (105) . In addition, it has been reported that gelatinase A does have the ability to at least partially degrade native type I collagen (106) . Finally, MT1-MMP itself has recently been shown to directly cleave type I collagen in addition to activating progelatinase A (107) . Therefore, the elevated expression of both MT1-MMP and active MMP-2 by C7-caMEK1 and C7F cells is likely to synergistically promote collagen degradation at the cell-matrix interface, thereby allowing cell invasion.

Interestingly, MMP inhibitors were able to restore cyst formation when added to C7-caMEK1 cells suspended in three-dimensional collagen or fibrin gels. This suggests that elevated expression of MT1-MMP and activated gelatinase A contributes to the disruption of cyst morphogenesis, presumably by acting in concert with additional MAPK-mediated events, such as the modulation of cell-cell and cell-matrix interactions (108, 109, 110) , cytoskeletal remodeling (111) , and stimulation of motility (112 , 113) . This hypothesis is in accord with the notion (114) that unbalanced extracellular proteolysis is incompatible with normal morphogenesis.

The MAPK cascade has been shown to phosphorylate both cytosolic targets (e.g., phospholipase A2) and nuclear transcription factors, including Elk-1, which in turn controls the expression of c-fos (61) . Although the MAPK targets responsible for the observed changes in MDCK cell phenotype are not known, it is noteworthy that prolonged activation of the c-fos oncoprotein has been reported to induce EMT and up-regulation of extracellular proteinases in mammary epithelial cells (38) . It is therefore conceivable that the profound phenotypic changes induced in MDCK-C7 cells by constitutive MAPK activation are mediated in part through an increased transcription of c-fos.

What is the significance of our findings? The dramatic alterations we have observed in the morphogenetic, invasive, and proteolytic properties of MDCK-C7 cells are associated with constitutive, long-lasting activation of the MAPK cascade. These changes may therefore be viewed as an exaggeration of cellular responses that occur physiologically under conditions of transient, reversible MAPK activation. Temporal parameters of MAPK activation are known to play a critical role in determining the nature of the ensuing cellular response (115, 116, 117) . In accord with this notion, we suggest that a function of the MAPK pathway is to promote graded changes in epithelial plasticity, depending on the duration of signaling. Thus, short-lived activation of the kinase cascade by extracellular stimuli may lead to transient loosening of intercellular adhesion and increased cell motility. This would in turn result in ordered cell repositioning, as observed in vitro in response to morphogenetic growth factors (90) and in vivo during organogenesis and tissue regeneration. On the other hand, sustained MAPK signaling may lead to a more pronounced and irreversible phenotypic conversion characterized by disruption of the orderly three-dimensional architecture, loss of apical-basal polarity, and the acquisition of invasive properties, as observed during the progression of well-differentiated epithelial tumors to anaplastic and metastatic carcinomas (17) . In accord with this hypothesis, it has recently been reported that MAPK is hyperexpressed in human breast malignancies (118) . Components of the MAPK cascade therefore represent potential targets for therapeutic interventions aimed at reducing epithelial cell invasion during tumor progression.


    Materials and Methods
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Cells and Reagents.
Wild-type MDCK-C7 cells and their derivatives (see below) were grown in MEM with Earl’s salts and L-glutamine (Life Technologies, Inc., Basel, Switzerland) supplemented with 1% nonessential amino acids (Life Technologies, Inc.) and 10% FCS (Life Technologies, Inc.). The MDCK-C7 cell line is a high transepithelial resistance subclone (71 , 119) of the heterogenous MDCK strain from the American Type Culture Collection (CCL 34). MDCK-C7F cells are a stably dedifferentiated population derived from parental MDCK-C7 cells via transient exposure to alkaline stress (71) . Stable transfection of wild-type MDCK-C7 cells with caMEK1 has been described previously in detail (64) . Briefly, MDCK-C7 cells were cotransfected by the lipofection technique with pREP4 expression vector together with either the empty pECE vector or pECE containing a hemagglutinin epitope-tagged caMEK1 mutant (C7-caMEK1 cells). caMEK1 was generated by substitution of the Raf1-dependent regulatory phosphorylation sites (serine 218 and serine 222) with aspartic acid (S218D/S222D mutant), as described previously (76) . Two stable clones obtained after hygromycin B selection, designated C7-caMEK1-1 and C7-caMEK1-4 (64) , were used in this study.

The synthetic MMP inhibitor BB94 and the inactive isomer BB1268 were a gift of Dr. P. Brown (British Biotech Pharmaceuticals Ltd., Oxford, United Kingdom). The MMP inhibitor CT1847 was kindly provided by Dr. A. J. P. Docherty (Celltech Ltd., Slough, United Kingdom). Recombinant TIMP-1, TIMP-2, and TIMP-3 were generously provided by Dr. G. Murphy (University of Norwich, Norwich, United Kingdom). The MEK inhibitor U0126 (70) was generously provided by Dr. J. M. Trzaskos (DuPont Pharmaceuticals Co., Wilmington, DE). Recombinant human bFGF was a gift of Dr. P. Sarmientos (Farmitalia Carlo Erba, Milan, Italy).

Assays of Morphogenesis in Collagen and Fibrin Gels.
For embedding in collagen gels, wild-type MDCK-C7 cells and their derivatives were harvested using trypsin-EDTA, centrifuged, and resuspended on ice in a collagen solution prepared as described previously (120) . In brief, 8 volumes of rat tail tendon collagen solution (approximately 1.5 mg/ml) were mixed with 1 volume of 10x MEM and 1 volume of sodium bicarbonate (11.76 mg/ml) in a sterile flask kept on ice to prevent premature collagen gelation. Cells were suspended in the cold mixture at concentrations ranging from 2 x 104 to 2 x 105 cells/ml collagen, and either 0.4- or 2-ml aliquots were dispensed into 16-mm wells (Nunc, Kampstrup, Roskilde, Denmark) or 35-mm dishes (Nunc), respectively. After incubation at 37°C for at least 10 min to allow collagen gelation, complete medium supplemented with 10 ng/ml bFGF was added and changed every 2–3 days.

The potential ability of wild-type and transfected MDCK-C7 cells to form lumina was more specifically assessed by sandwiching the cells between two collagen layers (121) . To this end, cells were seeded at a concentration of 7.5 x 103 to 1.5 x 104 cells/dish onto the surface of a collagen gel (1 ml) cast in a 35-mm dish and allowed to attach for approximately 1 h. The cells were subsequently overlaid with a second collagen gel (1 ml) as described previously (122) and incubated at 37°C in 2 ml of complete medium.

For incorporation into fibrin gels, cells were suspended in a polymerizing fibrinogen solution prepared essentially as described previously (114) . Briefly, bovine fibrinogen (catalogue number F-4753; Sigma, St. Louis, MO) was dissolved at 37°C in calcium-free MEM (Life Technologies, Inc.) to obtain a final protein concentration of 2.5 mg/ml. MDCK-C7 cells were suspended in the fibrinogen solution at a concentration of 5 x 104 to 1 x 105 cells/ml, and clotting was initiated by adding 1:10 v/v of CaCl2 (2 mg/ml) and thrombin (25 units/ml; Sigma; catalogue number T4684). The mixture was immediately transferred into either 35-mm dishes (2 ml) or 16-mm wells (400 µl) and allowed to gel for at least 2 min at room temperature before adding complete culture medium supplemented with 10 ng/ml bFGF. Trasylol (Bayer Pharma, Zurich, Switzerland) was added to the culture medium at a concentration of 500 Kallicrein inhibitory units (KIU)/ml to prevent lysis of the fibrin substrate (123) .

Invasion Assay.
Wild-type MDCK-C7 cells and their derivatives were harvested by trypsinization and seeded onto the surface of collagen or fibrin gels, which were prepared as described above in either 35-mm dishes (1500 µl) or 16-mm wells (400 µl). After a 2–4-day incubation in complete medium supplemented with 10 ng/ml bFGF, invading cells were identified by focusing at different levels below the surface of the gel, using the x20 or x40 phase-contrast objective of a Nikon Diaphot TMD inverted photomicroscope.

To quantitate the effect of either MMP or MEK inhibitors on invasion, C7-caMEK1-4 cells were seeded onto collagen gels at saturation density (5 x 104 cells/well) in 400 µl of complete medium with 10 ng/ml bFGF and the indicated concentration of inhibitor. After 48 h, the cultures were fixed (see below), and three randomly selected fields measuring 0.5 x 0.7 mm were photographed in each well with a x20 phase-contrast objective at a focal level 20 µm (for MMP inhibitors) or 60 µm (for the MEK inhibitor U0126) beneath the surface monolayer. Invasion was quantified as described previously (124) by determining the total additive length of all cellular structures that had penetrated beneath the surface monolayer either as single cells or in the form of thin cords. Data are expressed as mean additive cell/cord length ± SEM (in µm) of three photographic fields per experiment for each of three experiments per condition. Statistical significance was determined using Student’s unpaired t test.

Processing for Light and Electron Microscopy.
Collagen or fibrin gel cultures were fixed in situ overnight with 2.5% glutaraldehyde in 100 mM sodium cacodylate buffer (pH 7.4). After extensive rinsing in the same buffer, the gels were gently removed from the dishes or wells and cut into 2 x 2-mm fragments. These were postfixed in 1% osmium tetroxide in Veronal acetate buffer for 45 min, stained en bloc with 2.5% uranyl acetate in 50% ethanol, dehydrated in graded ethanols, and embedded in Epon 812 in flat moulds. Semithin (1-µm-thick) and thin sections were cut with an LKB ultramicrotome (LKB Instruments, Gaithersburg, MD). Semithin sections were stained with 1% methylene blue and photographed under transmitted light using a Zeiss photomicroscope (Carl Zeiss, Orberkochen, Germany). Thin sections were stained with uranyl acetate and lead citrate and examined in a Philips CM 10 electron microscope (Philips, Eindhoven, the Netherlands).

Gelatin Zymography.
Wild-type MDCK-C7 cells and their derivatives were seeded onto the surface of collagen gels in 22-mm wells (1.6 x 105 cells/well in 1600 µl of complete medium) and allowed to attach at 37°C for 1 h, at which time the medium was aspirated and replaced with fresh serum-free DMEM/F-12 supplemented with 10 ng/ml bFGF. After an additional 24-h incubation, conditioned media were collected, supplemented with 0.5 mM phenylmethylsulfonyl fluoride and 15 mM HEPES, and centrifuged at 1500 rpm for 5 min, and the resulting supernatants were stored at -80°C until use. Cells from each experimental condition were harvested by trypsinization and counted with a hemocytometer, and the sample volume was normalized to the cell number. Samples were mixed with SDS-PAGE buffer without denaturing agent and subjected to electrophoresis in 10% SDS-PAGE copolymerized with 1 mg/ml gelatin. After soaking in 2.5% Triton X-100 for 30 min to remove SDS, the gels were incubated in reaction buffer [50 mM Tris-HCl (pH 8) containing 150 mM NaCl, 10 mM CaCl2, and 0.02% NaN3] at 37°C for 16 h and stained with ethanol:acetic acid:water (30:10:60) containing 0.25% Coomassie Blue R250 for 4 h. The location of gelatinolytic activity was detected as clear bands on the background of uniform staining.

Plasmid Construction and in Vitro Transcription.
pPSMT1-MMP3` was constructed by subcloning a 630-bp fragment of human MT1-MMP (73) into the XhoI-BamHI sites of pBluescript KS. pGEM-hTIMP2 was constructed by subcloning a 791-bp fragment of human TIMP-2 cDNA (125) into the EcoRI-XbaI sites of pGEM-4. pBS-hP0 contains a 200-bp fragment of human ribosomal P0 phosphoprotein (126) amplified by reverse transcription-PCR from human umbilical vein endothelial cell total RNA and cloned into the EcoRI-BamHI sites of Bluescript KS. In vitro transcription of 32P-labeled cRNA probes was performed as described previously (127) .

RNA Extraction and Northern Blot Hybridization.
Wild-type MDCK-C7 cells and their derivatives were seeded into tissue culture dishes at 1 x 105 cells/ml in complete medium supplemented with 10 ng/ml bFGF and incubated at 37°C for 24 h, and then total RNA was extracted with Trizol (Life Technologies, Inc., Paisley, Scotland) according to manufacturer’s instructions. RNA was denatured with glyoxal, electrophoresed in a 1% agarose gel (5 µg RNA/lane), and transferred overnight onto nylon membranes (Hybond-N; Amersham, Buckinghamshire, United Kingdom). RNAs were cross-linked by the incubation of filters at 80°C for 2 h and stained with methylene blue to assess the integrity of 18S and 28S rRNA. Filters were prehybridized for 4 h at 65°C with 1.5 x 106 cpm/ml of 32P-labeled human MT1-MMP, or TIMP-2 cRNA probes. As an internal control for the determination of the amount of RNA loaded, the filters were simultaneously hybridized with 2 x 105 cpm/ml of 32P-labeled P0 cRNA probe. Posthybridization washes were performed as described previously (127) . Filters were exposed to Kodak XAR-5 films at -80°C between intensifying screens.


    Acknowledgments
 
We thank Dr. H. Oberleithner (University of Münster, Münster, Germany) for providing MDCK-C7 and -C7F cells; Dr. J. Pouysségur (Nice, France) for providing the MEK1 constructs; Dr. G. Murphy (University of Norwich, Norwich, United Kingdom) for providing recombinant TIMP-1, TIMP-2, and TIMP-3; Drs. P. Brown (British Biotech Pharmaceuticals Ltd., Oxford, United Kingdom) and A. J. P. Docherty (Celltech Ltd., Slough, United Kingdom) for providing the synthetic MMP inhibitors; A. Christin Tabaka and Jia-Sheng Yan (DuPont Pharmaceuticals Co., Wilmington, DE) for synthesis of U0126; Dr. M. Seiki (University of Tokyo, Tokyo, Japan) for providing the MT1-MMP cRNA probe; and Dr. S. Mandriota (University of Geneva, Geneva, Switzerland) for the P0 cDNA. We also thank Dr. L. Orci for continuing support, J. Rial-Robert and F. Doffey-Lazeyras for excellent technical assistance, J.-P. Gerber for photographic work, and F. Hellal and S. Kang for assistance in manuscript preparation.


    Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported by Swiss National Science Foundation Grant 31-43364.95 (to R. M.) and Austrian Science Foundation Grant P13295-MED (to H. S.). Back

2 To whom requests for reprints should be addressed, at Department of Morphology, University of Geneva Medical Center, rue Michel-Servet 1, CH-1211 Geneva 4, Switzerland. Phone: 4122-702-52-73; Fax: 4122-347-33-34; E-mail: Roberto.Montesano{at}medecine.unige.ch Back

3 The abbreviations used are: EMT, epithelial-mesenchymal transition; bFGF, basic fibroblast growth factor; caMEK1, constitutively active MEK1; ERK, extracellular signal-regulated kinase; MAPK, mitogen-activated protein kinase; MDCK, Madin-Darby canine kidney; MEK, MAPK/ERK kinase; MMP, matrix metalloproteinase; MT1-MMP, membrane type-1 MMP; TIMP, tissue inhibitor of metalloproteinases. Back

Received for publication 12/16/98. Revision received 3/ 8/99. Accepted for publication 3/17/99.


    References
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 

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MAP Kinases: From Intracellular Signals to Physiology and Disease
Physiology, April 1, 2002; 17(2): 62 - 67.
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Genes Dev.Home page
A. Schulze, K. Lehmann, H. B.J. Jefferies, M. McMahon, and J. Downward
Analysis of the transcriptional program induced by Raf in epithelial cells
Genes & Dev., April 15, 2001; 15(8): 981 - 994.
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IOVSHome page
A. Zubilewicz, C. Hecquet, J.-C. Jeanny, G. Soubrane, Y. Courtois, and F. Mascarelli
Proliferation of CECs Requires Dual Signaling through Both MAPK/ERK and PI 3-K/Akt Pathways
, February 1, 2001; 42(2): 488 - 496.
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JCBHome page
H. Reuveni, T. Geiger, B. Geiger, and A. Levitzki
Reversal of the Ras-Induced Transformed Phenotype by Hr12, a Novel Ras Farnesylation Inhibitor, Is Mediated by the Mek/ERK Pathway
J. Cell Biol., December 11, 2000; 151(6): 1179 - 1192.
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Am. J. Physiol. Cell Physiol.Home page
I. Marschitz, J. Lechner, I. Mosser, M. Dander, R. Montesano, and H. Schramek
Differential expression of cell-cell adhesion proteins and cyclin D in MEK1-transdifferentiated MDCK cells
Am J Physiol Cell Physiol, November 1, 2000; 279(5): C1472 - C1482.
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JCBHome page
K. Hotary, E. Allen, A. Punturieri, I. Yana, and S. J. Weiss
Regulation of Cell Invasion and Morphogenesis in a Three-Dimensional Type I Collagen Matrix by Membrane-Type Matrix Metalloproteinases 1, 2, and 3
J. Cell Biol., June 12, 2000; 149(6): 1309 - 1323.
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J. Pharmacol. Exp. Ther.Home page
M. Gekle, G. Schwerdt, R. Freudinger, S. Mildenberger, D. Wilflingseder, V. Pollack, M. Dander, and H. Schramek
Ochratoxin A Induces JNK Activation and Apoptosis in MDCK-C7 Cells at Nanomolar Concentrations
J. Pharmacol. Exp. Ther., June 1, 2000; 293(3): 837 - 844.
[Abstract] [Full Text]


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