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Epithelial cell adhesion molecule (Ep-CAM) modulates cell-cell interactions mediated by classic cadherins.

Litvinov SV, Balzar M, Winter MJ, Bakker HA, Briaire-de Bruijn IH, Prins F, Fleuren GJ, Warnaar SO - J. Cell Biol. (1997)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Leiden University, Leiden 2300 RC, The Netherlands. slitvinov@pathology.medfac.leidenuniv.nl

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.

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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.
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Figure 13: 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.

Mentions: Induction of Ep-CAM in cells did not affect either the total level of cell cadherins (as was detected with immunoblotting with pancadherin antibody, (see Fig. 13), or the surface expression of N-cadherin (as was detected with flow cytometry, not shown). The surface expression of E- and P-cadherins in HCA cells was too low to be measured by flow cytometry. However, upon induction of Ep-CAM, the subcellular distribution of cadherins was changed: the typical junctional structures, including cadherins that were present in HCA cells (or control transfectants), were absent in induced (Fig. 11) but not in noninduced Ep-CAM transfectants. The disturbance of cadherin's subcellular localization suggested that the effect of Ep-CAM was similar to that observed for LEC cells. Indeed, the detergent solubility of the cadherins in HCA cells was increased after 24 h of Ep-CAM (see Fig. 13). Induction of Ep-CAM had no effect on total level of cellular β-catenin, but it negatively affected the level of total α-catenin (Fig. 12); the detergent-insoluble fraction for both molecules was reduced in relation to the expression of Ep-CAM (Fig. 12). All observations are based on 24-h induction of Ep-CAM.


Epithelial cell adhesion molecule (Ep-CAM) modulates cell-cell interactions mediated by classic cadherins.

Litvinov SV, Balzar M, Winter MJ, Bakker HA, Briaire-de Bruijn IH, Prins F, Fleuren GJ, Warnaar SO - J. Cell Biol. (1997)

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.
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Related In: Results  -  Collection

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Figure 13: 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.
Mentions: Induction of Ep-CAM in cells did not affect either the total level of cell cadherins (as was detected with immunoblotting with pancadherin antibody, (see Fig. 13), or the surface expression of N-cadherin (as was detected with flow cytometry, not shown). The surface expression of E- and P-cadherins in HCA cells was too low to be measured by flow cytometry. However, upon induction of Ep-CAM, the subcellular distribution of cadherins was changed: the typical junctional structures, including cadherins that were present in HCA cells (or control transfectants), were absent in induced (Fig. 11) but not in noninduced Ep-CAM transfectants. The disturbance of cadherin's subcellular localization suggested that the effect of Ep-CAM was similar to that observed for LEC cells. Indeed, the detergent solubility of the cadherins in HCA cells was increased after 24 h of Ep-CAM (see Fig. 13). Induction of Ep-CAM had no effect on total level of cellular β-catenin, but it negatively affected the level of total α-catenin (Fig. 12); the detergent-insoluble fraction for both molecules was reduced in relation to the expression of Ep-CAM (Fig. 12). All observations are based on 24-h induction of Ep-CAM.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Leiden University, Leiden 2300 RC, The Netherlands. slitvinov@pathology.medfac.leidenuniv.nl

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.

Show MeSH
Related in: MedlinePlus