1 University of Nebraska Medical Center, College of Dentistry, 769605 Nebraska Medical Center, Omaha, NE 68583, USA
2 Lankenau Institute for Medical Research, 100 Lancaster Avenue, Wynnewood, PA 19096, USA
3 Biology Department, Saint Joseph's University, 5600 City Avenue, Philadelphia, PA 19131, USA
Author for correspondence (e-mail: knudsenk{at}mlhs.org)
Accepted 23 May 2005
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Summary |
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Key words: Cell-cell adhesion, Junctions, Cadherin, Catenins, Actin, Vimentin
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Introduction |
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The strength of cadherin-mediated cell-cell adhesion is regulated in a dynamic manner to accommodate such processes as cell migration during embryogenesis, cell renewal in epithelia and wound healing. In addition, abnormal changes in cadherin adhesion contribute to pathological processes, including cancer and its metastasis (Conacci-Sorrell et al., 2002; Hajra and Fearon, 2002
; Christofori, 2003
; Cavallaro and Christofori, 2004
). Multiple mechanisms modulate the strength of adhesion, including the nature and level of the cadherin(s) expressed, cadherin clustering within the plasma membrane, presence of growth factors (e.g. EGF and scatter factor/HGF), phosphorylation state of catenins and anchorage to the cytoskeleton (Gumbiner, 2000
). Additionally, the formation of multiple junction types in cells affects the strength of their interactions. In particular, desmosomes play an important role in stabilizing cell-cell interactions in tissues that are subject to mechanical stress, such as the skin and myocardium.
Studying the dynamic role of junctions within cells is complicated by the fact that under most physiological conditions, cells are in constant contact. To overcome this obstacle, several model systems have been established. One system involves exposing cadherin-expressing cells to a substrate comprised of the corresponding cadherin extracellular domain or to beads bearing immobilized anti-cadherin antibodies (Kovacs et al., 2002; Betson et al., 2002
). This allows one to investigate initial cell-cell contacts in a system analogous to those used to study cell-extracellular matrix interactions. A second model involves a calcium switch. As cadherins require calcium for their activity, cell-cell contact can be initiated by elevating the concentration of extracellular calcium for cells grown under low-calcium conditions. This system has been used by a number of groups studying cadherin function (Nelson et al., 1990
; Wheelock and Jensen, 1992
). A third model involves plating cells in sparse cultures and observing them as they proliferate and finally make contact with one another (Ehrlich et al., 2002
). Each model system has its limitations, and one goal of this study was to develop a new system in which the cadherin could be rapidly activated in order to further understand its function.
It has been established that fusion of the hormone-binding domain of the estrogen receptor to a heterologous protein can result in a protein whose function is hormone-regulated. Normally the estrogen receptor exists in cells as an inactive protein tightly associated with the HSP90 protein chaperone. When estrogen enters the cell and binds to its receptor, HSP90 is displaced, allowing the receptor to interact with DNA and initiate transcription of estrogen-responsive genes (Beato and Klug, 2000). One group has made use of this HSP90/estrogen receptor interaction to develop a chimeric system whereby heterologous proteins can be inactive or active, depending on the absence or presence of hormone (Picard et al., 1988
). In the ideal case, the chimeric protein is expressed, but is inactive until hormone is added. Upon addition of hormone, HSP90 is displaced, and the hormone-binding domain undergoes a conformational change that relieves the inhibition of the heterologous protein and it becomes fully active. This system has mainly been used to create transcription factors and kinases that can be readily activated (Picard, 2000
); however it has also been used to create a hormone-responsive form of two transmembrane proteins, Fas and the epidermal growth factor receptor (Picard, 2000
), which suggested that we could use the system with a classical cadherin. Thus, we made a chimeric cDNA construct comprised of a modified estrogen receptor ligand binding domain (ER-LBD) fused to the 3' end of the full-length N-cadherin cDNA (i.e. C-terminus of the protein). Our aim was to express the mutant N-cadherin (NcadER) in cells lacking an endogenous cadherin and to compare cell behavior in the absence and presence of 4-hydroxytamoxifen (4OHT) to activate the NcadER.
We expressed the mutant N-cadherin in mouse L fibroblasts, which lack an endogenous cell-cell adhesion system. Using this model system we discovered strong evidence for a novel vimentin-based classical cadherin adhesion system. We propose that the vimentin-based classical cadherin adhesion system acts in concert with the traditional actin-based system to strengthen cell-cell adhesion in fibroblasts, and perhaps other cell types.
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Materials and Methods |
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Immunofluorescent light microscopy
Cells were grown on glass coverslips for 3-5 days. Fixation for vimentin immunofluorescence was done with ice-cold methanol for 5 minutes. Fixation for actin and tubulin immunofluorescence was done with 10% formaldehyde for 15 minutes followed by permeabilization with 0.2% Triton-X-100 for 15 minutes. Cells were fixed for N-cadherin and catenin immunofluorescence with ice-cold methanol for 10 minutes or Histochoice (Amresco, Solon, OH) for 15 minutes. Primary antibodies were diluted in 10% goat serum in phosphate-buffered saline (PBS) and applied to the cells at room temperature for 60 minutes. The cells were washed three times with PBS and exposed to Cy3- or FITC-conjugated, species-specific secondary antibodies for 60 minutes at room temperature. When indicated, live cells were exposed to 0.05% NP40 detergent for 1 minute at room temperature prior to being fixed and exposed to primary and secondary antibodies for antigen detection. Primary antibodies to N-cadherin included 13A9 (Knudsen et al., 1995) and 3B9 (Zymed Laboratories, South San Francisco, CA) developed by our laboratories. Other antibodies included: anti-ß-catenin (15B8 or 5H10, BD PharMingen Transduction Laboratories, San Diego, CA), anti-plakoglobin (PG-11E4; Zymed Laboratories), anti-
-catenin (
CAT-7A4, Zymed Laboratories), monoclonal anti-p120 catenin (15D2, Zymed), polyclonal anti-p120 catenin (kind gift of Albert Reynolds, Vanderbilt University), anti-desmoplakin (multi-epitope cocktail to desmoplakin 1 and 2; Research Diagnostics, Flanders NJ), anti-vimentin (Vim 13.2; Sigma, St Louis, MO) and anti-actin (AC-15, Sigma). Rhodamine phalloidin used to detect filamentous actin (F-actin) was from Molecular Probes (Eugene, Oregon). Light microscopic images were acquired on a Zeiss Axiovert 200M microscope (Carl Zeiss, Thornwood, NY) equipped with an ORCA-ER digital camera (Hamamatsu, Houston, TX). Images were collected and processed using OpenLab software from Improvision (Boston, MA). Confocal images were acquired on an LSM 5 Pascal confocal microscope (Carl Zeiss, Thornwood, NY) by using a 63x 1.4 numerical aperture with appropriate filters.
Immunoblot analysis
Immunoblotting was performed essentially as described (Johnson et al., 1993). Briefly, cells were washed with phosphate-buffered saline, scraped from the culture dish, pelleted by centrifugation and extracted on ice in 10 mM Tris-acetate, pH 8.0, containing 0.5% Nonidet P-40 (NP40), 1 mM EDTA, and 2 mM phenylmethylsulfonyl fluoride (PMSF; Sigma). Cells were triturated vigorously and agitated for 20 minutes at 4°C. Non-solubilized material was removed by centrifugation at 14,000 g for 20 minutes at 4°C. Proteins in the supernatant fraction were separated by SDS-PAGE under reducing conditions, and for Western immunoblot analysis transblotted electrophoretically to nitrocellulose, which was then blocked with either 3% bovine serum albumin or 5% non-fat dry milk. Proteins were detected by the primary antibodies listed above, followed by alkaline phosphatase- or horseradish peroxidase-conjugated species-specific secondary antibodies (Southern Biotechnology Associates, Birmingham, AL) and substrates 5-bromo-4-chloro-3-indolyl phosphate (BCIP) and nitro blue tetrazolium (NBT; Sigma) or Pierce supersignal substrate (Rockford, IL) as described (Radice et al., 2003
; Maeda et al., 2005
).
Immunoprecipitation and immunoblot analysis
Unlabeled or metabolically radiolabeled cells were extracted as described above for immunoblot analysis. Proteins were immunoprecipitated with specific antibodies as described (Wheelock et al., 1987; Knudsen et al., 1995
; Wahl et al., 2003
). Immunoprecipitates were resolved by SDS-PAGE. Radiolabeled bands were detected using Biomax MR1 film (Kodak). Non-radiolabeled immunoprecipitates were transferred to nitrocellulose and immunoblotted as described above for western immunoblot analysis. Primary and secondary antibodies were as listed above. When mouse monoclonal antibodies were used for immunoprecipitation and the immunoprecipitate subsequently immunoblotted using mouse antibodies, the heavy chain of the precipitating mouse antibody was routinely detected by the secondary anti-mouse IgG antibody. When immunoprecipitating vimentin, the cell extract was first centrifuged at 100,000 g for 60 minutes at 4°C then pre-cleared by incubating the cell extract with anti-mouse IgG beads (MP Biomedicals, Aurora, OH) for 60 minutes followed by a second incubation with anti-mouse IgG beads coated with anti-myc antibody (9E10; kindly provided by Kathleen Green, Northwestern University). The cleared cell extract was used for co-immunoprecipitation experiments. This was done to avoid the possibility that polymerized vimentin might contaminate the immunoprecipitates non-specifically during the immunoprecipitation procedure, which includes centrifugation steps.
Aggregation assays
Two types of aggregation assay were performed to evaluate cell-cell adhesion activity. In one case monolayer cultures were trypsinized, triturated into single cell suspensions, placed in suspension and mixed for up to 24 hours as described (Knudsen and Horwitz, 1978). In the second assay, trypsinized single cells were placed in hanging drop cultures for up to 24 hours as described (Redfield et al., 1997
). At the end of the assay, cells and aggregates were collected by pipette, triturated to disperse loosely associated cells, visualized live by phase-contrast light microscopy and photographed.
siRNA targeting of mouse vimentin
Synthetic siRNAs were purchased from Dharmacon (Chicago, IL, M-061596-00-0005, mouse VIM, NM_011701). 7x104 LNER cells were transfected with 90 nM siRNA using siPort Amine (Ambion, Austin, TX) according to the manufacturer's instructions. Briefly, 90 nM mouse vimentin siRNA was mixed with siPort Amine/OPTI-MEM I (Gibco, Grand Island, NY) and incubated at room temperature for 10 minutes. The siRNA mixture was then added to a 12-well cell culture plate (90 nM of siRNA per well) followed by addition of 7x104 LNER cells. After overnight incubation at 37°C the transfection medium was replaced with HamsF12/DMEM (1:1) containing 10% fetal bovine serum. Target gene expression was assayed 96 hours after transfection with 90 nM RISC-Free siRNA (Dharmacon) as a control.
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Results |
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Immunoblot analysis confirmed that the parent L cells lack N-cadherin (Fig. 1B). The L cells also lack - and ß-catenin, which are degraded in the absence of a cadherin (Nagafuchi et al., 1987
). In contrast, the p120 catenin (p120ctn) is stable in the absence of a cadherin (Thoreson et al., 2000
) and was detected in the L cells (Fig. 1B). In the LN cells, which express wild-type N-cadherin, all three catenins,
-catenin, ß-catenin and p120ctn, were detected (Fig. 1B). The NcadER fusion protein was expressed by the LNER cells, and its level was not affected by 4OHT. In addition, all three catenins,
-catenin, ß-catenin and p120ctn, were detected in the LNER cells, and no significant difference was seen in the levels or molecular weights of these catenins when comparing the LNER cells treated or not with 4OHT. Plakoglobin was not detected (data not shown), consistent with its reported absence in L cells (Kowalczyk et al., 1996
). Together, our data indicated that the cellular level of NcadER and catenins in LNER cells was not altered by 4OHT.
We examined localization of the NcadER in LNER cells with or without 4OHT treatment using immunofluorescent light microscopy (Fig. 1C). This revealed an interesting difference. In the absence of 4OHT, the NcadER signal was largely diffuse, whereas in its presence the NcadER staining was more organized and prominent at cell-cell borders. The pattern of NcadER in LNER cells treated with 4OHT closely resembled that in LN cells (Fig. 1D). As a control, the parent L cells lacked N-cadherin staining (Fig. 1D). The data suggested that 4OHT induces a change in the localization of the NcadER. We considered the possibility that in the absence of 4OHT the mutant cadherin might not be on the cell surface where it could promote cell-cell adhesion. To test this possibility we performed aggregation assays with LNER cells exposed or not to 4OHT, and compared their aggregate formation to that of parent L cells and LN cells expressing wild-type N-cadherin.
As expected, the parent L cells exhibited little or no propensity to adhere to one another (Fig. 2A). In contrast, the LN cells expressing wild-type N-cadherin formed tightly compacted cell aggregates (Fig. 2A). The LNER cells aggregated in the absence of 4OHT, indicating that even in the non-activated state the NcadER was present on the cell surface and was able to promote cell-cell adhesion (Fig. 2A). However, we noted that LNER cells without 4OHT treatment failed to form the tightly compacted aggregates formed by the LN cells. In contrast, the LNER cells treated with 4OHT, like the LN cells, formed tightly compacted aggregates (Fig. 2A). Compaction of cell aggregates was identified by two properties detected by phase-contrast light microscopy of live cells: (1) difficulty in discerning individual cells in the aggregate and (2) the presence of a phase-dense line at the periphery of the aggregate (arrows Fig. 2A). Compaction followed aggregation in time, with aggregation of LNER cells detectable within 30 minutes and compaction barely detectable by
2 hours, obvious by 6 hours and maximal by 12-24 hours. The difference in the behavior of the LNER cells with or without 4OHT treatment suggested that cadherin-mediated adhesion could be separated functionally into two processes, aggregation followed by a strengthening of adhesion detected as compaction.
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A similar difference in the strength of LNER cell interaction depending on 4OHT treatment was observed in monolayer culture. Cells with or without 4OHT were plated at high density (50,000 cells/100 µl medium) in a small circle in the center of the well of a six-well culture plate. After the cells attached to the plastic, the wells were gently flooded with 2 ml medium, and the cells were observed for 3 days. In the absence of 4OHT, the LNER cells piled up on one another, and although most cells remained attached to one another in the original area of plating, many were observed outside the area of plating. On the other hand, the LNER cells treated with 4OHT remained predominantly as a dense monolayer within the area of plating (Fig. 2B). The cells did not pile up on one another and few cells were found outside the original plating area.
Analysis of proteins interacting with NcadER in LNER cells with or without 4OHT
Our data suggested that 4OHT induced a strengthening of LNER cell-cell adhesion mediated by NcadER. To begin to understand the mechanism involved we conducted a series of experiments. We initially considered the possibility that the ER-LBD, being located at the C-terminus of the N-cadherin, altered ß- and or -catenin binding. Reduced
- and ß-catenin binding to NcadER would be predicted to affect its linkage to actin, thereby altering the strength of adhesion. One argument against this idea was that
- and ß-catenin were stabilized at similar levels in LNER cells, with or without 4OHT (Fig. 1B). Nevertheless, to test the interaction of the mutant N-cadherin with ß- and
-catenin we immunoprecipitated the NcadER from LNER cells treated with or without 4OHT, using anti-N-cadherin antibodies. We then immunoblotted the immunoprecipitate with antibodies to
-catenin, ß-catenin and actin, as well as p120ctn, which is known to regulate cadherin adhesive strength (Yap et al., 1998
; Ohkubo and Awawa, 1999
; Thoreson et al., 2000
). As a control, we immunoprecipitated wild-type N-cadherin from the LN cells (Fig. 3A). Using wild-type or mutant N-cadherin as a reference point, similar levels of
- and ß-catenin and actin, were detected in the immunoprecipitates, comparing LN cells and LNER with or without 4OHT treatment (Fig. 3A,B). Interestingly, the level of p120ctn in immunoprecipitates of LNER cells treated with 4OHT was approximately twice that in immunoprecipitates of LNER cells without 4OHT (Fig. 3A), although it was also higher than for the LN cells. Thus, the level of p120ctn in the immunoprecipitate relative to N-cadherin did not strictly correlate with the compaction phenotype.
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As another approach to examining a possible role for actin in the 4OHT-induced strengthening of cell-cell adhesion, we treated LNER cells, plus or minus 4OHT, with cytochalasin B to disrupt actin filaments. Fluorescence microscopy confirmed that cytochalasin B treatment disrupted the actin cytoskeleton as detected by phalloidin staining (Fig. 5). In the absence of 4OHT treatment, the cytochalasin B had a striking negative impact on the aggregation of LNER cells, indicating that this aspect of cell-cell adhesion requires an intact actin cytoskeleton. However, the effect of cytochalasin B on LNER cells treated with 4OHT was tempered. Aggregation of these cells (i.e. +4OHT, +cytoB) was greater than that of cytochalasin B-treated LNER cells without 4OHT, although it was less than that of 4OHT-treated cells without cytochalasin B (Fig. 5). Moreover, the aggregates of 4OHT+cytochalasin B-treated LNER cells that did form showed evidence of compaction, as detected by the presence of the characteristic phase-dense lines at the periphery of aggregates (arrows).
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We considered the possibility that, although actin was involved in aggregation, another cytoskeletal system was involved in compaction. To begin to explore this possibility we performed immunofluorescent light microscopy on monolayer cultures of LNER cells treated or not with 4OHT. We reasoned that we might detect a change in the organization of a particular cytoskeletal system if it interacted differently with the NcadER activated by 4OHT compared to the non-activated cadherin. We looked at F-actin, tubulin, and vimentin as markers of the actin, microtubule, and intermediate filament systems, respectively. No change was detected in the F-actin pattern of LNER cells upon 4OHT treatment as detected under lower magnification using conventional fluorescence microscopy (Fig. 7A) or higher magnification using confocal microscopy (Fig. 7B). This was predicted, as our data did not support a role for actin in 4OHT-induced compaction. Nor was there any noticeable difference in the tubulin pattern at low or high magnification, suggesting the microtubule system was not involved in compaction. On the other hand, the vimentin pattern was noticeably different comparing LNER cells with or without 4OHT treatment. In the absence of 4OHT much of the vimentin signal was perinuclear in the LNER cells, although vimentin extensions in the cell body were observed in some cells (Fig. 7A,B). In the presence of 4OHT the vimentin appeared more filamentous and more extended to the cell periphery (Fig. 7A,B). These data suggested to us that perhaps vimentin intermediate filaments were associating with the cadherin/catenin complex at the cell surface and were involved in strengthening the cadherin-mediated adhesion.
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Evidence for a role for vimentin in strengthening cell-cell adhesion
Our data suggested that upon 4OHT activation NcadER might interact with vimentin intermediate filaments, thereby strengthening cell-cell adhesion. To test this idea we determined if vimentin co-immunoprecipitated with the mutant N-cadherin isolated from LNER cells treated or not with 4OHT. This was accomplished by immunoprecipitating NcadER or vimentin and subsequently immunoblotting for both proteins. When anti-N-cadherin was used to isolate the cadherin/catenin complex from LNER cells, vimentin was detected by immunoblotting (Fig. 8C). Conversely, NcadER was detected in vimentin immunoprecipitates, suggesting that vimentin interacts with the cadherin/catenin complex. The data were not entirely clear, however, because we expected to see more NcadER co-immunoprecipitating with vimentin in the case of the LNER cells treated with 4OHT, compared to cells without 4OHT. These co-immunoprecipitations did not consistently bring down equivalent amounts of the co-immunoprecipitating protein and we think this is because we could only immunoprecipitate the vimentin that was in the NP40-soluble fraction, whereas the interactions we are most interested in might reside in the NP40-insoluble fraction.
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Discussion |
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Here we present strong evidence that a novel classical cadherin adhesion system strengthens the interaction of fibroblasts. Expression of a mutant, hormone-activated N-cadherin in mouse fibroblasts has revealed a vimentin-based cadherin adhesion complex that cooperates with the traditional actin-based complex to promote strong cell-cell adhesion. N-cadherin fused at the C-terminus to a mutant estrogen receptor ligand-binding domain (NcadER) that binds 4-hydroxytamoxifen (4OHT) was stably expressed in mouse L cells, which lack an endogenous cadherin. L cells expressing the NcadER (LNER cells) formed aggregates in suspension with or without 4OHT treatment. However, addition of 4OHT strengthened the adhesion of the LNER cells, causing the aggregates to compact, similar to L cells expressing wild-type N-cadherin (LN cells).
Compaction of 4OHT-treated LNER cells followed aggregation in time and did not correlate with a change in the cellular level of cadherin or catenins, including -catenin, ß-catenin and p120ctn. Moreover, typical cadherin/catenin complexes formed in LNER cells, with or without 4OHT treatment, although when normalized to the NcadER, more p120ctn was present in immunoprecipitates from 4OHT-treated cells, compared to non-treated cells. In addition, 4OHT-induced compaction of LNER cells correlated with enhanced interactions between the mutant N-cadherin and the cytoskeleton. Aggregation of LNER cells required an intact actin cytoskeleton, consistent with the known interaction of classical cadherins with the actin cytoskeleton, and its role in promoting cell-cell adhesion. In contrast, compaction clearly involved more than the actin cytoskeleton, which led us to consider other cytoskeletal systems. We noted that vimentin became more organized when the LNER cells were treated with 4OHT, compared to non-treated cells. Moreover, better-organized vimentin correlated with the compaction phenotype exhibited by both 4OHT-treated LNER cells and LN cells expressing wild-type N-cadherin. These data suggested that the vimentin intermediate filament system might interact with the cadherin to bring about compaction.
A physical interaction between vimentin and NcadER was supported by their co-immunoprecipitation from LNER cells. Moreover, a vital role for vimentin in compaction was shown by using siRNAs to knockdown vimentin. Decreasing vimentin by approximately 50% inhibited compaction, while having no effect on aggregation, which is actin-dependent. These data provide strong evidence that vimentin is involved in the compaction process. The mechanism by which vimentin interacts with cadherin appears to involve p120ctn, as vimentin and p120ctn co-immunoprecipitate even when a cadherin and - and ß-catenin are absent. Whether the interaction of vimentin and p120ctn is direct or involves some linker protein is a topic for future study, as is regulation of the interaction by phosphorylation. We do know that plakoglobin is not involved in linking the cadherin to vimentin, as it is in endothelial cells (Kowalczyk et al., 1998
), since the L cells do not express plakoglobin.
In summary, we propose that classical cadherins can form two types of adhesion complex in fibroblasts. One complex is comprised of the cadherin linked through ß- and -catenin to the actin cytoskeleton. This actin-based cadherin complex promotes the aggregation of cells. A second adhesion complex is comprised of the cadherin linked through p120ctn to vimentin intermediate filaments. This adhesion system promotes compaction of fibroblast aggregates and may be functionally analogous to desmosomes in epithelial cells. Together, the actin- and vimentin-based classical cadherin adhesion systems initiate and strengthen cell-cell adhesion in fibroblasts, and perhaps other cell types.
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Acknowledgments |
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Footnotes |
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