Correspondence to: Arnoud Sonnenberg, Division of Cell Biology, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands. Tel:(31) 20 512 1942 Fax:(31) 20 512 1944 E-mail:asonn{at}nki.nl.
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Abstract |
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Adhesion receptors, which connect cells to each other and to the surrounding extracellular matrix (ECM), play a crucial role in the control of tissue structure and of morphogenesis. In this work, we have studied how intercellular adhesion molecules and ß1 integrins influence each other using two different ß1-null cell lines, epithelial GE11 and fibroblast-like GD25 cells. Expression of ß1A or the cytoplasmic splice variant ß1D, induced the disruption of intercellular adherens junctions and cell scattering in both GE11 and GD25 cells. In GE11 cells, the morphological change correlated with the redistribution of zonula occluden (ZO)-1 from tight junctions to adherens junctions at high cell confluency. In addition, the expression of ß1 integrins caused a dramatic reorganization of the actin cytoskeleton and of focal contacts. Interaction of ß1 integrins with their respective ligands was required for a complete morphological transition towards the spindle-shaped fibroblast-like phenotype. The expression of an interleukin-2 receptor (IL2R)-ß1A chimera and its incorporation into focal adhesions also induced the disruption of cadherin-based adhesions and the reorganization of ECMcell contacts, but failed to promote cell migration on fibronectin, in contrast to full-length ß1A. This indicates that the disruption of cellcell adhesion is not simply the consequence of the stimulated cell migration. Expression of ß1 integrins in GE11 cells resulted in a decrease in cadherin and -catenin protein levels accompanied by their redistribution from the cytoskeleton-associated fraction to the detergent-soluble fraction. Regulation of
-catenin protein levels by ß1 integrins is likely to play a role in the morphological transition, since overexpression of
-catenin in GE11 cells before ß1 prevented the disruption of intercellular adhesions and cell scattering. In addition, using biochemical activity assays for Rho-like GTPases, we show that the expression of ß1A, ß1D, or IL2R-ß1A in GE11 or GD25 cells triggers activation of both RhoA and Rac1, but not of Cdc42. Moreover, dominant negative Rac1 (N17Rac1) inhibited the disruption of cellcell adhesions when expressed before ß1. However, all three GTPases might be involved in the morphological transition, since expression of either N19RhoA, N17Rac1, or N17Cdc42 reversed cell scattering and partially restored cadherin-based adhesions in GE11-ß1A cells. Our results indicate that ß1 integrins regulate the polarity and motility of epithelial cells by the induction of intracellular molecular events involving a downregulation of
-catenin function and the activation of the Rho-like G proteins Rac1 and RhoA.
Key Words: ß1 integrins, cadherins, epithelial cells, Rho-like GTPases, migration
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Introduction |
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ADHESION molecules play an essential role in the organization of cells into tissues during embryonic development as well as in the adult. They not only maintain tissue structure and polarity but are also involved in the regulation of cell proliferation, migration, and differentiation. Intercellular adherens junctions, desmosomes, and tight junctions are the three major types of adhesive connections between cells (for review see
Cells also form adhesive contacts with proteins in the surrounding extracellular matrix (ECM) via different types of proteins, mostly of the integrin family. Integrins are heterodimeric transmembrane receptors, formed by the noncovalent association of an and a ß subunit. 18
and 8 ß subunits have been identified so far, giving rise to a family of >20 different dimers. Dimers containing the ß1 integrin subunit constitute the most abundantly expressed integrin subfamily. Antibody inhibition studies and disruption of the ß1 subunit gene by homologous recombination have demonstrated the critical role of ß1 integrins in development, cell differentiation, migration, and the assembly of the ECM proteins (for reviews see
Adhesive interactions between different cells or between a cell and the surrounding ECM can be either stable or dynamic. An example are the epithelium-mesenchyme transitions (EMT), which occur during specific stages of embryonic development but also under certain pathological conditions. EMT are morphogenetic events characterized by the loss of epithelial polarity, the disruption of intercellular adhesions, and the acquisition of a migratory, mesenchymal cell phenotype. Previous studies have indicated a role for integrins in the downregulation of cadherin activity during neural crest cell EMT (
Small GTPases of the Rho family are regulators of the actin cytoskeleton (6ß4 integrin stimulates Rac-dependent migration of colon carcinoma cells (
In this study, we have used two ß1-deficient cell lines, an epithelial cell line, GE11, which we isolated for this study, and the previously described fibroblast-like GD25 cell line (-catenin protein levels as well as their redistribution from the cytoskeleton-associated fraction to the soluble fraction. Overexpression of
-catenin inhibited the disruption of cellcell adhesions by ß1 in GE11 cells and prevented cells from scattering. We also found that expression of ß1 integrins in GE11 and GD25 cells resulted in the activation of both RhoA and Rac1. Experiments performed with dominant negative or active mutants showed that both RhoA and Rac1 were required but not sufficient for the phenotypic conversion induced by ß1 integrins.
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Materials and Methods |
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Antibodies and Adhesive Ligands
The mouse anti-3A and anti-
3B antibodies (29A3 and 54B3;
6 antibody (GoH3;
1,
2, and
v integrin subunits (
5 antibody (MBA5;
4 antibodies were purchased from PharMingen. The mouse mAb against vinculin (VIIF9;
) (TB30) was a gift of Dr. R. van Lier (Central Laboratory of the Netherlands Red Cross Blood Transfusion Service, Amsterdam, The Netherlands). The rabbit antipan-cadherin, the rabbit anti
-catenin, and the mouse antitalin (8D4) antibodies and rhodamine-labeled phalloidin were obtained from Sigma Chemical Co. The mouse antiß-catenin and mouse anti
-catenin antibodies were purchased from Transduction Laboratories, and the rabbit antiZO-1 and the rabbit anti-occludin antibodies were from Zymed. The monoclonal 9E10 antibody against the myc-epitope tag was purchased from Oncogene Research Products. Mouse anti-RhoA (26C4) and rabbit anti-Cdc42 antibodies were obtained from Santa Cruz Biotechnology, and mouse anti-Rac1 antibody from Transduction Laboratories. Secondary antibodies used were: FITC-conjugated goat antimouse antibody (Jackson ImmunoResearch Laboratories), Texas redconjugated goat antimouse and antirabbit antibodies (Molecular Probes), FITC-conjugated rabbit antihamster antibody (Nordic Immunological Laboratories), and FITC-conjugated goat antirabbit antibody (Zymed). Fibronectin was purchased from Sigma Chemical Co., and laminin-1 from Collaborative Biomedical Products.
GD 25 Cells and the Establishment of GE11 Cells
The previously described fibroblast-like GD25 cell line (
Generation of Retroviral Expression Constructs
A cDNA encoding full-length human ß1A (nucleotides -173096; -phage keratinocyte library with two ß1 oligonucleotide probes (nucleotides -825 and nucleotides 23572389) and subsequently cloned into pUC18. A Kozak consensus sequence was introduced by PCR, and the Kozak-containing human ß1A cDNA was then ligated into the retroviral LZRS-IRES-zeo expression vector, a modified LZRS retroviral vector conferring resistance to zeocin (
encoding sequence of the pCMV-IL2R vector (
-IRES-zeo vector.
Myc epitope-tagged dominant negative N17Cdc42, N17Rac1, N19RhoA, and dominant active V12Cdc42, V14RhoA, and V12Rac1 were cloned in the LZRS-IRES-zeo vector ((E)-catenin cDNA (
Retroviral Transduction and Cell Culture
Phoenix packaging cells (
Expression of the ß1 subunit or the IL2R-ß1 chimera was determined by FACS® analysis. Expression of the mutant forms of Rho-like GTPases was checked by immunoblotting as described previously (
Immunofluorescence and Flow Cytometry
Cells were grown on coverslips in DME, 10% FCS, fixed in 2% paraformaldehyde for 15 min, and permeabilized in PBS containing 0.2% Triton X-100 for 5 min. Cells were blocked in PBS, 2% BSA for 1 h, and incubated with primary antibodies for 1 h at room temperature. After washing in PBS, cells were incubated in the presence of FITC- or Texas redconjugated secondary antibodies or in the presence of rhodamine-labeled phalloidin for 1 h. Preparations were then washed in PBS, mounted in Vectashield (Vector Laboratories Inc.), and analyzed with a confocal Leica TCS NT microscope.
For flow cytometry and cell sorting, cultured cells were trypsinized, washed twice in PBS, 2% FCS, and incubated with primary antibodies for 45 min at 4°C. Cells were then washed in PBS, incubated with FITC-conjugated secondary antibodies for 45 min at 4°C, washed again, and analyzed in a FACScan® using Lysys II software (Becton Dickinson) for determination of integrin expression levels. Cells were sorted on a FACStar Plus® (Becton Dickinson).
Transwell Migration Assays and In Vitro Wound Healing
For the Transwell migration assay, 3 x 104 or 105 cells in DME, 0.5% BSA were seeded in the upper compartment of 8-µm Transwells (Costar) previously coated with 10 µg/ml fibronectin on the lower side on the filter, and allowed to migrate for 2 h at 37°C. Cells in the upper chamber were removed with a cotton swab and cells on the lower side of the filter were fixed in methanol and stained with crystal violet. The number of cells that had migrated was counted on photographs taken from the filters. For each filter, a total of three different 5-mm2 fields were photographed to obtain an average cell count.
For in vitro wound healing assay, cells were seeded for 2 h in DME, 10% FCS. After cell spreading, a cross was scratched in the cell monolayer to analyze wound closure and facilitate the localization of the same spot in time. Cells were photographed at the indicated time points (magnification 500x).
Detergent Solubility Assay
Subconfluent cell cultures were lysed for 10 min on ice in 1% Triton X-100, 50 mM Tris, pH 7.6, 150 mM NaCl, 2 mM EDTA in the presence of protease inhibitors and the lysates were centrifuged at 14,000 g for 15 min to obtain the soluble protein fraction. The pellet (the cytoskeletal, insoluble fraction) was resuspended in Laemmli sample buffer. For detection of total protein samples, cells were extracted with radio immunoprecipitation assay (RIPA) lysis buffer. Samples were adjusted to 50 µg of total proteins, separated by SDS-PAGE, and transferred to polyvinylidene difluoride membranes (Immobilon-P; Millipore). The membranes were incubated for 1 h with antipan-cadherin, anti-catenin, or antiß-catenin antibodies and then further incubated for 1 h with HRP-conjugated secondary antibodies. Immunoreactive proteins were visualized using enhanced chemiluminescence.
RhoA, Rac1, and Cdc42 Activity Assays
The biochemical activity assays were performed essentially as described previously (
For each measurement, two T75 flasks of subconfluent GE11 or GD25 cells were lysed for 5 min at 4°C in 1% NP-40, 50 mM Tris, pH 7.4, 10% glycerol, 100 mM NaCl, 2 mM MgCl2, in the presence of protease inhibitors. Lysates were clarified by centrifugation and the appropriate GST fusion protein was added for 30 min at 4°C, followed by three washes in lysis buffer. The beads were boiled in Laemmli sample buffer and protein samples were separated by SDS-PAGE and transferred to polyvinylidene difluoride membranes (Millipore). The blots were probed with anti-RhoA, anti-Rac1, or anti-Cdc42 antibodies and developed by enhanced chemiluminescence.
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Results |
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Expression of ß1A or ß1D in ß1-deficient Cells Induces the Disruption of Intercellular Adhesions and Cell Scattering
GE11 cells were isolated from a ß1 integrin subunit knockout chimeric embryo aged 10.5 d postcoitum and their ontogeny is unclear. They grow in epithelial colonies (Figure 1), contain keratin 8, no desmoplakin, and express the neural cell adhesion molecule NCAM-1. Electron microscopic studies showed that GE11 cells are polarized, have microvilli at their apical surface, and organized tight junctions, but do not assemble desmosomes (data not shown). Together, these observations suggest that GE11 cells are epithelial cells of neural origin, possibly the neuroepithelium. The previously described GD25 cells were obtained by in vitro differentiation and immortalization of ß1-knockout ES cells (
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We have expressed two cytoplamic splice variants of the ß1 integrin subunit, ß1A and ß1D, in GE11 and GD25 cells, by retroviral transduction. FACS® analysis revealed that 9095% of the cells express ß1 at their plasma membrane 24 h after the start of retroviral transduction (data not shown). Stable transfectants were selected with zeocin. Immunoprecipitation experiments showed that GE11 cells expressed the ß3 and ß5 subunits in association with v (data not shown). GE11-ß1A cells express several integrins of the ß1 family, including
3ß1,
5ß1,
6ß1, and
4ß1 at low levels, but not
1ß1 or
2ß1 (data not shown).
Infection of cells with the empty retroviral vector (GE11- and GD25-control) did not alter the epithelial phenotype of GE11 cells or the intercellular adhesions in GD25 colonies (Figure 1). In both cell types, the expression of the ß1A integrin subunit resulted in disruption of cellcell contacts and dissociation of cell colonies. Cells originally present in epithelial cell colonies separated from them and assumed a morphology resembling that of motile fibroblasts. These morphological changes were detected as early as 24 h after the start of retroviral infection (Figure 1). Similar effects, although somewhat less pronounced, were observed after the expression into GE11 and GD25 cells of the muscle-specific ß1D splice variant (Figure 1). The lower levels at which ß1D was expressed (50% of those of ß1A) probably account for this less pronounced phenotype.
Because the effect of ß1 expression was most dramatic on intercellular adhesions in GE11 cells, we concentrated our studies on stable GE11 cells expressing ß1A (GE11-ß1A). The distribution of various proteins associated with the actin cytoskeleton and with intercellular adhesions was analyzed in GE11 and GE11-ß1A cells (Figure 2). In GE11-control cells, actin filaments were organized in heavy peripheral bundles, which ran parallel to the outer membrane of cells at the periphery of the epithelial cell colonies. Actin filaments were also present in cortical bundles under the plasma membrane, along cellcell boundaries, and in stress fibers at the cell basis, where they were attached to the plasma membrane at sites of focal contacts (Figure 2). In GE11-ß1A cells, peripheral bundles of actin filaments were absent and stress fibers crossed the entire cell. Intercellular staining of cadherins, typical of epithelial cells, was observed in GE11-control cells, and no staining was found at the free cell border at the periphery of colonies (Figure 2). -, ß-, and
-catenins had a similar localization (data not shown). In contrast, cadherins and catenins were more diffusely distributed over the membrane of GE11-ß1A cells, and although there were some residual adherens junctions at high confluency, these proteins were also found in regions of the plasma membrane that were not in contact with other cells (Figure 2, medial plane). In GE11-control cells, vinculin was found in regions of cellcell contacts, where it was colocalized with cadherins (Figure 2, medial plane). In contrast, although GE11-ß1A cells developed cellcell contacts at high confluency, vinculin and cadherins were not colocalized in these cells (Figure 2, medial plane). Using interference reflection microscopy, we found that the number and size of focal contacts were different in GE11-control and GE11-ß1A cells (data not shown), and this was confirmed by the distribution of vinculin (Figure 2, basal plane) and talin (data not shown) at the basal surface of the cells. Typically, focal contacts were small and numerous in GE11-control cells, and distributed over the entire basal cell surface. However, at the periphery of the colonies they were more concentrated at the outer region of the cell, thus forming a characteristic interrupted ring-like structure. In GE11-ß1A cells, focal contacts were thick, and appeared to be arranged in long streaks frequently found at the end of actin stress fibers (Figure 2). In confluent GE11-ß1A cells, focal contacts were also found between two cells and sometimes on their apical surface, as seen by staining for talin and vinculin. Electron microscopic analysis revealed that this was likely to be due to the presence and assembly of secreted ECM proteins between cells and on their apical surface (data not shown). Another specialized membrane domain involved in intercellular adhesion of epithelial cells is the tight junction. A marker of tight junctions is ZO-1, but in cell types lacking these structures, such as fibroblasts or cardiac muscle cells, ZO-1 is colocalized with cadherins at adherens junctions (
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Finally, we have found that ß1 and the endogenous ß3 integrin subunits were both present in focal contacts formed by GE11-ß1A cells in the presence of FCS (data not shown).
In conclusion, these results show that the expression of the ß1 integrin subunit in GE11 cells induces a reorganization of the actin cytoskeleton and of focal contacts, accompanied by the disruption of both cadherin-based intercellular adhesions and tight junctions in epithelial cell colonies. However, adherens junctions of the type formed by fibroblasts (
Disruption of Intercellular Adhesions and Cell Scattering Is Dependent on ß1 IntegrinLigand Interactions
When GE11 cells were cultured on plastic in the presence of FCS, the mere expression of the ß1 subunit was sufficient for inducing the disruption of intercellular adhesions and the dissociation of cell colonies. Because GE11-ß1A cells express several integrins that can bind to fibronectin and vitronectin present in FCS, we investigated whether the change in morphology was due to the expression of ß1 or whether it was triggered by the interaction of ß1 integrins with their ligands. Although GE11-ß1A cells expressed the laminin receptor 6ß1, we have generated GE11 cells expressing
6ß1 at higher levels by coexpression of the human
6 integrin subunit in GE11-ß1A cells and by further selection by FACS®. The overexpression of
6 in GE11-
6ß1A cells resulted in a strong decrease of the percentage of cells expressing the fibronectin receptor
5ß1 as well as in a decrease of its average expression levels (Figure 3 A), probably because
6 associated with most of the available ß1 subunit. Although these cells could spread, they poorly scattered and developed strong cellcell adhesions when cultured on fibronectin (Figure 3 B), suggesting that the expression of all fibronectin-binding ß1 integrins (
5ß1 as well as
vß1) was reduced. However, scattering was induced when they were cultured on laminin-coated dishes (Figure 3 B). Together, these results indicate that the interaction of ß1 integrins with their ligand is required for the disruption of cadherin-based cellcell adhesion and cell scattering. They also show that several integrins of the ß1 family, which bind to various ECM proteins (
5ß1 or
vß1 to fibronectin, and
6ß1 to laminin), can trigger the described morphological transition.
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Expression of IL2R-ß1A Chimera Induces the Disruption of Cadherin-based CellCell Adhesions and Remodels Focal Contacts
The localization of a particular integrin in focal contacts is regulated by its subunit and requires the binding of the integrin to ligand (
-catenin, or ZO-1 (Figure 4, upper panels). Expression of the IL2R-ß1A chimera, on the contrary, induced the disruption of most intercellular adhesions. A few small epithelial-like colonies remained and cellcell adhesions had a tendency to reform, although they did not appear to be as stable as those between GE11-control cells. IL2R-ß1A expression induced an alteration of the peripheral bundles of actin filaments and changes in the localization of cadherins, catenins, and ZO-1 (Figure 4, lower panels), similar to the full-length ß1A subunit. In addition, the IL2R-ß1A chimera promoted cell spreading and induced a redistribution of vinculin: the ring of focal adhesions at the periphery of the colonies was no longer assembled in GE11-IL2R-ß1A cells and was replaced by thick and long streaks of vinculin at the base of cells. The arrows in Figure 4 indicate vinculin-positive rings in cells in which IL2R-ß1A was not expressed. In cells expressing IL2R-ß1A, the chimera was colocalized with vinculin (Figure 4) and the endogenous ß3 subunit (data not shown) in focal contacts. Together these results indicate that IL2R-ß1A induces both the disruption of intercellular adhesions and the reorganization of the cytoskeleton, thus mimicking the effects of full-length ß1A. Whether IL2R-ß1A is primarily incorporated into ß3-containing, preexisting focal adhesions and induces their remodeling, or whether it participates in the formation of new adhesion structures into which ß3 is eventually recruited will be discussed.
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The Expression of ß1, but Not That of IL2R-ß1, Enhances Cell Migration
To quantify potential changes in the motility of GE11-ß1A cells as compared with that of GE11-control cells, we have performed migration experiments using fibronectin-coated Transwells. As shown in Figure 5 A, although GE11-control cells are able to migrate to some extent on this substrate, the expression of ß1A strongly increased cell motility. The expression of IL2R-ß1A failed to enhance cell migration in any of the conditions tested.
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In addition, we tested random cell migration using an in vitro wound healing system. Figure 5 B shows that GE11-ß1A cells spread fast when grown under standard conditions on plastic, and that they migrate into the introduced wound. GE11-ß1D cells showed similar migration kinetics (data not shown). In contrast, GE11-control cells maintained stronger cellcell adhesions and spread and migrated more slowly.
Together, these results show that the motility of GE11-ß1 cells is increased. The fact that IL2R-ß1A is sufficient to trigger the disruption of intercellular adhesions, although it does not increase cell motility, indicates that cell migration is not simply the cause of the disruption of cellcell adhesions.
Expression of ß1 Integrins in GE11 Cells Induces a Decrease in Cadherin and -Catenin Protein Levels and Their Redistribution to the Detergent-soluble Fraction
Because the expression of ß1 integrins altered the integrity of intercellular adherens junctions, we compared the cadherin and - and ß-catenin protein levels as well as their detergent solubility in GE11-control, and GE11-ß1A and -ß1D cells. Immunoblotting of total cellular proteins revealed that GE11-ß1A cells contained smaller amounts of cadherin and
-catenin than their control counterparts (Figure 6). The amount of ß-catenin also appeared to be reduced in GE11-ß1A and GE11-ß1D cells, but to a lesser extent than the amounts of cadherin and
-catenin. Results of a detergent solubility assay, using 1% Triton X-100, indicated that the ratios between the insoluble and soluble fractions of cadherin and
-catenin were reduced in GE11-ß1A cells as compared with GE11-control cells. Thus, smaller amounts of these two proteins are associated with the detergent-insoluble cytoskeletal fraction in GE11-ß1A cells. Although the amount of ß-catenin is reduced in GE11-ß1A cells, its distribution in the cytoskeletal and soluble fractions is apparently not affected by ß1 expression. This might be due to the existence of a detergent-insoluble pool of ß-catenin in the nucleus of GE11-ß1A cells, although we could not confirm this hypothesis by immunofluorescence.
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These results indicate that ß1 integrins might cause the disruption of intercellular adhesions by inducing a downregulation of cadherin and/or catenin function. Moreover, immunofluorescence studies performed on cells that were first permeabilized in Triton X-100 and subsequently fixed in paraformaldehyde showed stainings for cadherins and catenins similar to those obtained with paraformaldehyde-fixed cells without prior solubilization (data not shown). This suggests that the redistribution of cadherins and -catenin from the Triton-insoluble to the -soluble fraction upon ß1 expression is due to a reduced number of cellcell adhesions rather than a decrease in their rigidity.
Overexpression of -Catenin in GE11 Cells Prevents the Morphological Change Induced by ß1 Integrins
By providing a link between cadherin complexes and the cytoskeleton, -catenin plays a major role in the establishment and maintenance of cadherin-based adhesions (
-catenin has been implicated in the formation of tight junctions (
-catenin were affected by ß1 integrins in GE11 cells, we have investigated the role of this protein in the ß1-induced phenotypic changes. First,
-catenin was retrovirally overexpressed in GE11 cells (GE11
-catenin cells). The effect of subsequent ß1 expression in GE11
-catenin cells was less pronounced than in GE11 cells or in cells previously transduced with the empty LZRS vector. Several clones were isolated in which the expression of ß1 and
-catenin was analyzed by FACS® and immunoblotting, respectively. Figure 7 shows the results obtained with three of them. The expression levels of
-catenin were higher in GE11-control cells and in cells from clones 3, 4, and 9, than in GE11-ß1A cells (Figure 7 A). In addition, ß-catenin and cadherin protein levels were greater in GE11-control cells and in the three GE11
-cateninß1A clones than in GE11-ß1A cells. Thus, when
-catenin is overexpressed in GE11 cells, protein levels of ß-catenin and cadherin are not decreased by the expression of ß1 integrins.
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Although the surface expression levels of ß1 integrins in clones 3 and 9 were similar to those in GE11-ß1A cells (Figure 7 B), cells from these clones remained clustered in epithelial cell colonies (Figure 7 C), indicating that overexpression of -catenin prevented the ß1-induced morphological change. In spite of ß1 expression, cells from clone 9 exhibited the peripheral bundles of actin filaments and the vinculin-positive ring found in GE11-control cells but not in GE11-ß1A cells. In addition, these cells presented well-developed intercellular adhesions containing both vinculin and cadherins (Figure 7 D). However, results obtained with clone 4 showed that a further increase of ß1 levels could overcome the inhibitory effect of
-catenin and allow cell scattering.
These results suggest that there is a tight balance between intercellular adhesion and ß1-mediated attachment to ECM proteins, which can be regulated at the level of -catenin.
Expression of ß1 Integrins Induces Activation of RhoA and Rac1
Small G proteins of the Rho family are involved in the regulation of the actin cytoskeleton, the turnover of focal adhesions, in cell migration, and in the assembly of intercellular adhesions (
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Dominant Negative Mutants of Small G Proteins of the Rho Family Inhibit and Reverse the ß1-Induced Phenotypic Changes and Migration
Next, we investigated whether the activation of RhoA and Rac1 is required for the phenotypic conversion induced by ß1 integrins. We first transduced dominant negative mutants of RhoA, Rac1, and Cdc42 in GE11 cells, and 2 d later, we introduced ß1A. Because the same retroviral vector was used for both proteins, the clones expressing ß1A could not be selected with antibiotics. Therefore, we isolated the cells expressing ß1 at high levels by FACS® analysis and sorting. In the second step, several individual clones were isolated from this ß1-positive cell population. The expression of both N17Rac1 (Figure 9 A) and ß1 (data not shown) was finally measured in these clones. We show that even when ß1 was strongly expressed at the cell surface, substoichiometric amounts of N17Rac1 inhibited the disruption of cellcell adhesion and cell scattering (Figure 9 B). It has been reported previously that amounts of RhoA or Rac1 mutants well below those of endogenous RhoA and Rac1 levels caused changes in the cellular organization (
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We have not been successful in isolating GE11 clones expressing detectable levels of dominant negative forms of RhoA and Cdc42, probably because these constructs have a toxic effect. This was most obvious for N19RhoA, of which expression induced cell rounding and detachment 2 d after the retroviral transduction. Therefore, we followed another approach in which the dominant negative mutants were expressed subsequently to the ß1 integrin subunit. All three RhoA, Rac1, and Cdc42 mutants inhibited cell scattering, but the morphology of the cells in the colonies was different from that of untransfected GE11 cells (Figure 10 A). In particular, the morphology of the cells in which N17Rac1 had been expressed after ß1 was clearly different from that of the cells that were first retrovirally transduced with N17Rac1 (compare with Figure 9 B). The expression of all three mutants N19RhoA, N17Rac1, and N17Cdc42 in GE11 cells subsequent to ß1 correlated with the localization of cadherins (data not shown), -catenin, and vinculin at cellcell junctions (see Figure 10 B for the GE11-ß1A-N17Rac). The inhibition of cell scattering was directly dependent on the amount of dominant negative GTPases: cells expressing N19RhoA, N17Rac1, or N17Cdc42 at high levels formed islands, whereas cells with low expression levels maintained a fibroblastic morphology and remained scattered (Figure 10 B). As a control, immunofluorescence staining for ß1 (Figure 10 B) and FACS® analysis (data not shown) demonstrated that the inhibition of cell scattering by dominant negative mutants of the Rho-like GTPases was not due to a decrease in the levels of surface expression of ß1 integrins. Finally, we have found that both N17Rac1 and N17Cdc42, but not N19RhoA, were enriched at cellcell contacts (Figure 10 A). This localization of N19RhoA (
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It is now well-established that RhoA plays a role in actin stress fiber and focal contact formation (
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To determine whether the activation of Rac1 or RhoA by ß1 integrins was sufficient for inducing the morphological change, dominant active mutants of RhoA (V14RhoA) or Rac1 (V12Rac1) were retrovirally expressed in GE11 cells. Although V14RhoA cells appeared to be more contracted and V12Rac1 cells displayed larger lamellipodia, neither of the constitutively active mutants separately nor when they were combined induced cell scattering (data not shown). Taken together, these results suggest that Rac, RhoA, and Cdc42 are required but not sufficient for the morphological changes induced by the expression of the ß1A integrin subunit.
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Discussion |
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In this study we have shown that the expression of either ß1A or ß1D integrin splice variants in two different ß1-deficient cell lines, GE11 and GD25 cells, induces the disruption of intercellular adhesions followed by cell scattering. This phenotypic conversion, which depends on the interaction of ß1 integrins with their respective ligands, is accompanied by the reorganization of the actin cytoskeleton and of focal contacts, and an increased ability of cells to migrate. However, loss of cellcell adhesions and the reorganization of the cytoskeleton does not require cell migration, since the expression of an IL2R-ß1A chimera had both these effects on cell morphology without stimulating cell motility.
Disruption of cellcell adhesions by ß1 integrins was correlated with a decrease in cadherin and -catenin protein levels and with their redistribution from the cytoskeleton-associated fraction to the soluble fraction. When the levels of
-catenin were increased in GE11 cells by retroviral transduction, the ß1-induced phenotypic changes did not occur, suggesting an important role for this catenin in the regulation of cadherin-based adhesions by ß1 integrins.
We have also found that the activity of RhoA and Rac1, but not that of Cdc42, was enhanced upon expression of ß1A, ß1D, or IL2R-ß1A in both GE11 and GD25 cells. These findings suggest that activation of these two Rho-like GTPases by ß1 integrins contributes to the loss of cellcell adhesions. Indeed, expression of either N17Rac1 or N19RhoA prevented the morphological transition induced by ß1 integrins. However, additional ß1-induced intracellular signals are required for the phenotypic change, since constitutively active mutants of either RhoA or Rac1 or both could not induce the disruption of cellcell contacts in GE11 cells.
Effects of the Interaction of ß1 Integrins with Their Respective ECM Ligands
The ß1-deficient GD25 cells, which have been described previously as fibroblast-like cells (6ß1 as the major ß1 integrin, scattered only when laminin-1 was provided as a substrate.
The IL2R-ß1A chimera is distributed to focal contacts, triggers phosphorylation signals independently of binding to the ligand, and functions as a constitutively active integrin when expressed at relatively low levels (
In many epithelial cells, and in particular those forming desmosomes, the mere expression of ß1 integrins is not sufficient to induce cell scattering. However, it has been shown previously that specific ECMintegrin interactions may lead to such changes in MDCK cells (
vß3-mediated cell migration by an unknown mechanism. In contrast, our results suggest a requirement for ß1-mediated effects for the regulation of cadherin activity, at least in some cell types.
vß3 has been shown previously to be involved in cell migration, and it is not clear why it does not efficiently support the motility of GE11 cells, given the strong homology between the cytoplasmic domains of ß1 and ß3 subunits. One possibility could be that the surface levels of
vß3 are too low to induce migration.
What Are the Molecular Mechanisms Responsible for the Disruption of Intercellular Adhesions Induced by ß1 Integrin Clustering?
Our results suggest that a decrease in cadherin and -catenin protein levels, together with their redistribution from the cytoskeleton-associated, Triton X-100insoluble to the -soluble fraction play a major role in the ß1-induced phenotypic changes. This was further supported by our finding that high levels of
-catenin expressed by retroviral transduction could inhibit cell scattering. Cadherin protein levels were increased in GE11
-cateninß1A cells as compared with those in GE11-ß1A cells, probably because overexpression of
-catenin stabilizes cadherin-based adhesions and prevents protein degradation. In addition, because
-catenin is also involved in the formation of tight junctions between epithelial cells (
Although our results clearly show that intercellular adhesions can be regulated by ß1 integrins via -catenin, the molecular mechanisms of this regulation remain to be elucidated. Integrin ligation triggers multiple intracellular events, among them the activation of various protein kinases (
-catenins and not on
-catenin, leading to their dissociation from the cytoskeleton. However, if the disruption of intercellular adhesions by ß1 integrins would be the result of ß- or
-catenin phosphorylation, we would not expect that
-catenin could compensate for such an effect, which suggests another type of regulation by ß1 integrins. It is also possible that ß1-induced phosphorylation of
-catenin-binding proteins other than ß- or
-catenin contributes to the disruption of intercellular adhesions.
Alternatively, overexpression of -catenin might stabilize adherens junctions by compensating for the redistribution of structural proteins. While many components of intercellular adherens junctions and focal contacts are specific to one or the other structure, other proteins such as vinculin and
-actinin, are found in both complexes. Previous studies have suggested that
-actinin and vinculin play an important role in the establishment and maintenance of intercellular adhesions. Notably, vinculin, which shares homology with
-catenin (
-catenin might provide alternative and possibly stronger links between cadherins and the actin cytoskeleton (
-catenin might compensate for this redistribution. However, this hypothesis implies that the amount of vinculin in the cell is a limiting factor, which does not seem to be the case, since it was found to reassociate with intercellular junctions in GE11
-cateninß1A cells.
Our data show that in addition to -catenin, Rho-like small G proteins also play a major role in the regulation of cell scattering by ß1 integrins. Recently, cell adhesion was found to regulate the activity of RhoA (
The finding that ß1 integrinmediated adhesion activates both RhoA and Rac1 is consistent with the changes in the organization of the cytoskeleton and in cell behavior that we have observed in GE11 cells. Upon ß1 expression, GE11 cells developed extensive lamellipodia and started to migrate. Whereas Rac1 stimulates the formation of lamellipodia and small focal complexes at the leading edge of migrating cells, RhoA stimulates the formation of new focal contacts and regulates the interaction of myosin-based motors with actin filaments to generate contractile forces required for cell motility. Although previous work suggested that cell adhesion induces the activation of the Cdc42 and Rac downstream effector PAK (
Immunofluorescence microscopy revealed that cadherins and catenins are redistributed to intercellular adherens junctions in GE11-ß1A cells upon expression of dominant negative mutants of RhoA, Rac1, and Cdc42. This indicates that in these cells, Rho-like GTPases are not strictly required for the assembly of cadherin-based adhesions. These observations are in contrast to previous studies that reported decreased staining intensity of cadherins and catenins induced by N17Rac1 or the RhoA inhibitor C3 in keratinocytes (
These discrepancies might reflect specific regulation mechanisms in different cell types, but also different methods used for the expression of dominant negative mutants, i.e., microinjection of recombinant proteins versus inducible expression systems. An explanation we favor is that the amounts of dominant negative mutants of Rho-like GTPases expressed by retroviral transduction are sufficient to prevent the migration of cells derived from the originally transduced single cell, which results in the formation of small epithelial-like colonies but still allows cellcell contacts to be formed.
When expressed before ß1, N17Rac1 was able to prevent GE11 cell scattering. Expression of N17Rac1 did not appear to affect the distribution of cadherins and catenins, and cells remained tightly attached to each other even in the presence of high levels of ß1. This suggests that Rac1 activation is required not only for cell migration but also for the disruption of cadherin-based adhesions in these cells. However, expression of either constitutively active RhoA, Rac1, or their simultaneous expression was not sufficient for dissociating the cells in GE11 colonies, suggesting that ß1 integrins trigger additional types of signals involved in the disruption of cellcell adhesions. Moreover, the phenotypic reversion induced by the dominant negative mutants of small GTPases was only partial, and the morphology of both cells and colonies was different from that of control GE11 cells. This suggests either that the prior expression of ß1 integrins induces irreversible morphogenetic events or that ß1 integrins continue to transduce GTPase-independent signals that modify the cell phenotype.
In conclusion, we have shown that the expression of ß1 integrins in two different ß1-deficient cell lines downregulates intercellular adhesions and stimulates cell scattering by inducing intracellular events involving both -catenin and Rho-like GTPases. Further studies are now in progress to elucidate the molecular mechanisms underlying this regulation.
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Footnotes |
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Clotilde Gimond and Arjan van der Flier contributed equally to this work.
1 . Abbreviations used in this paper: ECM, extracellular matrix; EMT, epithelium-mesenchyme transitions; ES, embryonic stem; GST, glutathione S-tranferase; IL2R, interleukin-2 receptor; PAK, p21-activated kinase; ZO, zonula occluden.
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Acknowledgements |
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We thank Frans Hogervorst for his contribution to the isolation and construction of the human ß1 cDNA, Jero Calafat and Hans Janssen for electron microscopic analysis, Lauran Oomen for his help with confocal laser scanning microscopy, Eric Noteboom and Anita Pfauth for cell sorting, Nico Ong for artwork, Frits Michiels and Rob van der Kammen for generating retroviral expression vectors for active and dominant negative Rho-like GTPases, and Anja Baathorn for technical assistance. We also thank our colleagues for their generous gifts of antibodies and constructs. We are grateful to Erik Danen, Lionel Fontao, Mirjam Nievers, Tim Reid, Eva Sander, and Ed Roos for helpful discussions and critical reading of the manuscript.
This work was supported by a grant from the Netherlands Heart Foundation (NHS 96.006) to C. Gimond, a Yamanouchi Research Studentship from Yamanouchi Research Institute, U.K. to A. van der Flier, a grant from the Dutch Cancer Society (96-1267) to S. van Delft, and a grant from the Swedish National Research Council to R. Fässler.
Submitted: 19 May 1999
Revised: 11 September 1999
Accepted: 2 November 1999
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