doi: 10.1083/jcb.139.5.1337.
Epithelial cell adhesion molecule (Ep-CAM) modulates cell-cell interactions mediated by classic cadherins
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- PMID:9382878
- PMCID: PMC2140211
- DOI: 10.1083/jcb.139.5.1337
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Epithelial cell adhesion molecule (Ep-CAM) modulates cell-cell interactions mediated by classic cadherins
S V Litvinov et al. J Cell Biol..
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Abstract
The contribution of noncadherin-type, Ca2+-independent cell-cell adhesion molecules to the organization of epithelial tissues is, as yet, unclear. A homophilic, epithelial Ca2+-independent adhesion molecule (Ep-CAM) is expressed in most epithelia, benign or malignant proliferative lesions, or during embryogenesis. Here we demonstrate that ectopic Ep-CAM, when expressed in cells interconnected by classic cadherins (E- or N-cadherin), induces segregation of the transfectants from the parental cell type in coaggregation assays and in cultured mixed aggregates, respectively. In the latter assay, Ep-CAM-positive transfectants behave like cells with a decreased strength of cell-cell adhesion as compared to the parental cells. Using transfectants with an inducible Ep-CAM-cDNA construct, we demonstrate that increasing expression of Ep-CAM in cadherin-positive cells leads to the gradual abrogation of adherens junctions. Overexpression of Ep-CAM has no influence on the total amount of cellular cadherin, but affects the interaction of cadherins with the cytoskeleton since a substantial decrease in the detergent-insoluble fraction of cadherin molecules was observed. Similarly, the detergent-insoluble fractions of alpha- and beta-catenins decreased in cells overexpressing Ep-CAM. While the total beta-catenin content remains unchanged, a reduction in total cellular alpha-catenin is observed as Ep-CAM expression increases. As the cadherin-mediated cell-cell adhesions diminish, Ep-CAM-mediated intercellular connections become predominant. An adhesion-defective mutant of Ep-CAM lacking the cytoplasmic domain has no effect on the cadherin-mediated cell-cell adhesions. The ability of Ep-CAM to modulate the cadherin-mediated cell-cell interactions, as demonstrated in the present study, suggests a role for this molecule in development of the proliferative, and probably malignant, phenotype of epithelial cells, since an increase of Ep-CAM expression was observed in vivo in association with hyperplastic and malignant proliferation of epithelial cells.
Figures
Examples of Ep-CAM expression by some cells within the E-cadherin–positive cell population. (A) Heterogeneous expression of Ep-CAM in a basal cell carcinoma, as detected by immunofluorescent staining with mAb 323/A3 to Ep-CAM (green fluorescence); the red fluorescence indicates the expression of E-cadherin (mAb HECD-1). (B) The de novo expression of Ep-CAM in gastric mucosa in relation to the development of intestinal metaplasia; immunohistochemical staining with mAb 323/A3. Note the bordering Ep-CAM–positive and –negative cells. Bars, 30 μM.
Cell segregation directed by Ep-CAM. L cells were transfected with cDNA for E-cadherin (LEC) or Ep-CAM (LEp), and the E-cadherin transfectants were additionally supertransfected with either Ep-CAM cDNA (LEC-Ep) or the blank vector (LEC-C). (A) Pairs of transfected cells were tested in coaggregation assays: dispersed cells of two types (Type1 + Type2), each labeled with a different fluorescent dye, were mixed at equal concentrations. After 2 h of culturing in suspension, cell aggregates consisting of >10 cells were analyzed for the presence of cells of each type. The data is presented as percentage of aggregates (y-axis) containing the respective percentage of the Type 2 cells (x-axis). (B) Expression of E-cadherin and Ep-CAM in the transfectants, as determined by immunoblotting in total cell lysates using antibodies to E-cadherin (36) and to human Ep-CAM (323/A3), respectively.
Cell segregation directed by Ep-CAM. L cells were transfected with cDNA for E-cadherin (LEC) or Ep-CAM (LEp), and the E-cadherin transfectants were additionally supertransfected with either Ep-CAM cDNA (LEC-Ep) or the blank vector (LEC-C). (A) Pairs of transfected cells were tested in coaggregation assays: dispersed cells of two types (Type1 + Type2), each labeled with a different fluorescent dye, were mixed at equal concentrations. After 2 h of culturing in suspension, cell aggregates consisting of >10 cells were analyzed for the presence of cells of each type. The data is presented as percentage of aggregates (y-axis) containing the respective percentage of the Type 2 cells (x-axis). (B) Expression of E-cadherin and Ep-CAM in the transfectants, as determined by immunoblotting in total cell lysates using antibodies to E-cadherin (36) and to human Ep-CAM (323/A3), respectively.
Segregation of Ep-CAM–positive LEC cell transfectants from the parental cells in multicellular aggregates. LEC-C and LEC-Ep cells, labeled with fluorescent dyes PKH-26 and PKH-2, respectively, were mixed at a 1:1 ratio, sedimented, and allowed to form an aggregate. This aggregate, in which both cell types were represented in a random pattern, was mechanically dispersed, and the smaller aggregates obtained were further cultured in suspension for 24 h, fixed, and analyzed. The micrographs present optical cross sections at the equatorial area of the aggregates after 24 h, as seen with a confocal microscope. The artificial colors were assigned to the cells depending on the color of the fluorochrome and the cell type: LEC-C (red); LEC-Ep (white) cells. The figures show similar cell patterning in different size aggregates in the range of <100 to ∼1,000 cells. A dark area in the middle of some aggregates is an optically nontransparent zone.
Effect of increasing expression of Ep-CAM on cell–cell interactions in L cell E-cadherin transfectants (LEC). LEC cells were supertransfected with Ep-CAM cDNA under the control of the metallothionein promotor (clones LEC-MEp.2 andLEC-MEp.6), or with blank vector (LEC-MC). Induction of Ep-CAM expression with CdCl2 for 24 h resulted in an increased total Ep-CAM (A, immunoblot of total cell lysates) and in an increased presence of Ep-CAM molecules at the cell surface (B, flow cytometry with anti–Ep-CAM F(ab)-FLUOS conjugate). Cells detached with TC treatment were allowed to aggregate in suspension for 30 min in the presence of Ca2+, and the degree of cell aggregation was determined (C). The statistical significance of the observed differences in aggregation rates was determined using the Student'st-test (p). Where indicated, cells were cultured for 24 h before the assay in the presence of CdCl2 in culture medium. (D) The morphology of aggregates formed in 30 min by LEC-MC and LEC-MEp.6 cells (the latter noninduced and induced with 10 μM CdCl2).
Effect of increasing expression of Ep-CAM on cell–cell interactions in L cell E-cadherin transfectants (LEC). LEC cells were supertransfected with Ep-CAM cDNA under the control of the metallothionein promotor (clones LEC-MEp.2 andLEC-MEp.6), or with blank vector (LEC-MC). Induction of Ep-CAM expression with CdCl2 for 24 h resulted in an increased total Ep-CAM (A, immunoblot of total cell lysates) and in an increased presence of Ep-CAM molecules at the cell surface (B, flow cytometry with anti–Ep-CAM F(ab)-FLUOS conjugate). Cells detached with TC treatment were allowed to aggregate in suspension for 30 min in the presence of Ca2+, and the degree of cell aggregation was determined (C). The statistical significance of the observed differences in aggregation rates was determined using the Student'st-test (p). Where indicated, cells were cultured for 24 h before the assay in the presence of CdCl2 in culture medium. (D) The morphology of aggregates formed in 30 min by LEC-MC and LEC-MEp.6 cells (the latter noninduced and induced with 10 μM CdCl2).
Effect of increasing expression of Ep-CAM on cell–cell interactions in L cell E-cadherin transfectants (LEC). LEC cells were supertransfected with Ep-CAM cDNA under the control of the metallothionein promotor (clones LEC-MEp.2 andLEC-MEp.6), or with blank vector (LEC-MC). Induction of Ep-CAM expression with CdCl2 for 24 h resulted in an increased total Ep-CAM (A, immunoblot of total cell lysates) and in an increased presence of Ep-CAM molecules at the cell surface (B, flow cytometry with anti–Ep-CAM F(ab)-FLUOS conjugate). Cells detached with TC treatment were allowed to aggregate in suspension for 30 min in the presence of Ca2+, and the degree of cell aggregation was determined (C). The statistical significance of the observed differences in aggregation rates was determined using the Student'st-test (p). Where indicated, cells were cultured for 24 h before the assay in the presence of CdCl2 in culture medium. (D) The morphology of aggregates formed in 30 min by LEC-MC and LEC-MEp.6 cells (the latter noninduced and induced with 10 μM CdCl2).
Effect of increasing expression of Ep-CAM on cell–cell interactions in L cell E-cadherin transfectants (LEC). LEC cells were supertransfected with Ep-CAM cDNA under the control of the metallothionein promotor (clones LEC-MEp.2 andLEC-MEp.6), or with blank vector (LEC-MC). Induction of Ep-CAM expression with CdCl2 for 24 h resulted in an increased total Ep-CAM (A, immunoblot of total cell lysates) and in an increased presence of Ep-CAM molecules at the cell surface (B, flow cytometry with anti–Ep-CAM F(ab)-FLUOS conjugate). Cells detached with TC treatment were allowed to aggregate in suspension for 30 min in the presence of Ca2+, and the degree of cell aggregation was determined (C). The statistical significance of the observed differences in aggregation rates was determined using the Student'st-test (p). Where indicated, cells were cultured for 24 h before the assay in the presence of CdCl2 in culture medium. (D) The morphology of aggregates formed in 30 min by LEC-MC and LEC-MEp.6 cells (the latter noninduced and induced with 10 μM CdCl2).
The internal structure of the large aggregates formed by LEC-MC and LEC-MEp.6 cells in 24 h of suspension culture. The expression of Ep-CAM in LEC-MEp.6 cells was induced with 10 μM CdCl2 for 24 h before the aggregation assay (+). The aggregates were fixed with 2% paraformaldehyde/1.25% glutaraldehyde, embedded into Epon, and ultrathin sectioned. Sections were analyzed by reflection contrast microscopy. Bar, 25 μm.
Expression of E-cadherin in blank vector (−MC) and Ep-CAM transfectants (clones −MEp.2 and−MEp.6) of LEC cells. The cells were cultured for 24 h in the presence or absence of 10 μM CdCl2 in culture medium. Cells were either (A) detached from the substrate with EDTA and analyzed in flow cytometry after staining with mAb ECCD-2 to murine E-cadherin, or (B) lysed with 1% SDS, and E-cadherin was detected in total cell lysates (5 μg protein/sample) by immunoblotting with mAb 36.
Expression of E-cadherin in blank vector (−MC) and Ep-CAM transfectants (clones −MEp.2 and−MEp.6) of LEC cells. The cells were cultured for 24 h in the presence or absence of 10 μM CdCl2 in culture medium. Cells were either (A) detached from the substrate with EDTA and analyzed in flow cytometry after staining with mAb ECCD-2 to murine E-cadherin, or (B) lysed with 1% SDS, and E-cadherin was detected in total cell lysates (5 μg protein/sample) by immunoblotting with mAb 36.
The morphology of LEC-MC and LEC-MEp.6 cells (the latter shown after induction with 10 μM CdCl2, marked by +). Note the groups of the epithelial-like cells interconnected along their lateral domains in the culture of LEC-MC cells, which are absent in the LEC-MEp.6 cell culture.
Subcellular localization of E-cadherin in LEC-MC cells and in LEC-MEp.6 cells. Ep-CAM in LEC-MEp.6 was induced by 10 mM CdCl2 for 24 h before cell fixation. Immunofluorescent staining of methanol-fixed cells was performed as described in Materials and Methods using mAb ECCD-2 to murine E-cadherin. Bar, 10 μm.
E-cadherin and catenins in LEC cells and the derived Ep-CAM transfectants (MEp-6). (A) Cells were cultured in the absence (−) or presence (+) of CdCl2 for 24 h, and lysed in 1% SDS either directly (Total lysate), or after extraction with 0.5% Triton X-100 (Insoluble fraction). Samples of cell lysates, equalized by protein content, were analyzed by immunoblotting with antibodies to E-cadherin (mAb 36), β-catenin (mAb 14), or α-catenin (mAb 7A4). 5× more protein was taken for the extracted cell preparations. (B) LEC-MEp.6 cells cultured in the absence (−) or presence (+) of CdCl2 were lysed in buffer with 0.5% Triton X-100, and immunoprecipitations were performed from the soluble fraction. The immunoprecipitates were analyzed in Western blotting with antibodies to E-cadherin (E) or catenins (α- and β-, respectively). A rabbit polyclonal serum was used for α-catenin detection. The precipitations were performed from an excess of the lysate; therefore, the total amount of an antigen precipitated by an antibody remains equal.
Ultrastructure of the aggregates formed by HCA/C and HCA/CEp cells. Reflection contrast micrographs of cross sections through the aggregates formed in 90 min by each cell type reveals a tight organization of the HCA/C aggregates and loosely interconnected cells forming the HCA/CEp aggregates. (A) Electron microscopy on the preparations shows the abundant presence of the adherens junctions between HCA/C cells (arrows). In contrast, microvilli (B,arrow) were found at the intercellular space between the cells of internal layers of HCA/CEp cells. In HCA/C cells, microvilli were present almost exclusively at the apical membrane of the outer layer of cells in the aggregate and not on the surface of the cells from internal layers. Bars: (left) 10 μm; (right) 0.25 μm.
Changes in subcellular distribution of cadherins in HCA cells upon expression of Ep-CAM. HCA cells transfected with inducible pMEp4 vector, either blank (HMC) or containing the Ep-CAM cDNA (Wt), were induced with 50 μM CdCl2 24 h before fixation, fixed, and stained with anti-pan cadherin mAb CH-19 (reacts mainly with N-cadherin, the major cadherin of HCA cells). Note the redistribution of cadherins from adherens junctions to free domains of cell membrane in Wt Ep-CAM transfectants, and a general shift of the cell phenotype to a more scattered one. Bar, 5 μm.
The role of the cytoplasmic domain in the effect of Ep-CAM on cadherins. (A) The structural maps of the wild-type (wild-type) and mutant (Mu1) Ep-CAM molecules. The leader peptide (L), EGF-like domains (EGF-I,EGF-II) cysteine-poor region (CPR), the transmembrane domain (TM), and cytoplasmic domain (CYT) are marked. The cytoplasmic domain is deleted in the Mu1 molecule. (B) HCA cells were transfected with either blank vector (HMC), or the wild-type Ep-CAM (Wt), or Mu1, under the control of a metallothionein promotor. Where indicated (+Cd), the cells were cultured in the presence of 50 μm CdCl2 for 24h before lysis. Aliquots of total cell lysates equalized by protein were probed in immunoblotting using the Ep-CAM–specific mAB. Content of N-cadherin in whole cell lysates and after the extraction of cells with 0.5% Triton X-100 was analyzed with anti-pan cadherin mAb CH-19. Note that expression of Wt Ep-CAM, but not of an approximately equal level of Mu1, affects the detergent solubility of cadherins.
The role of the cytoplasmic domain in the effect of Ep-CAM on cadherins. (A) The structural maps of the wild-type (wild-type) and mutant (Mu1) Ep-CAM molecules. The leader peptide (L), EGF-like domains (EGF-I,EGF-II) cysteine-poor region (CPR), the transmembrane domain (TM), and cytoplasmic domain (CYT) are marked. The cytoplasmic domain is deleted in the Mu1 molecule. (B) HCA cells were transfected with either blank vector (HMC), or the wild-type Ep-CAM (Wt), or Mu1, under the control of a metallothionein promotor. Where indicated (+Cd), the cells were cultured in the presence of 50 μm CdCl2 for 24h before lysis. Aliquots of total cell lysates equalized by protein were probed in immunoblotting using the Ep-CAM–specific mAB. Content of N-cadherin in whole cell lysates and after the extraction of cells with 0.5% Triton X-100 was analyzed with anti-pan cadherin mAb CH-19. Note that expression of Wt Ep-CAM, but not of an approximately equal level of Mu1, affects the detergent solubility of cadherins.
Detergent extractability of catenins in HCA cells and Wt Ep-CAM transfectants. Immunoblotting was performed on total cell lysates and on lysates of cells preextracted with 0.5% Triton X-100. The + indicates induction of cells with 50 μM CdCl2 for 24 h before lysis.
References
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