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Protein kinase C activation upregulates intercellular adhesion of alpha-catenin-negative human colon cancer cell variants via induction of desmosomes.

van Hengel J, Gohon L, Bruyneel E, Vermeulen S, Cornelissen M, Mareel M, von Roy F - J. Cell Biol. (1997)

Bottom Line: Interestingly, this normalizing TPA effect was not associated with alpha-catenin induction.Addition of an antibody against the extracellular part of desmoglein-2 blocked the TPA effect, too.Remarkably, the combination of anti-E-cadherin and anti-desmoglein antibodies synergistically inhibited the TPA effect.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology, Laboratory of Molecular Cell Biology, University of Ghent and Flanders Interuniversity Institute of Biotechnology (V.I.B.), B-9000 Ghent, Belgium.

ABSTRACT
The alpha-catenin molecule links E-cadherin/ beta-catenin or E-cadherin/plakoglobin complexes to the actin cytoskeleton. We studied several invasive human colon carcinoma cell lines lacking alpha-catenin. They showed a solitary and rounded morphotype that correlated with increased invasiveness. These round cell variants acquired a more normal epithelial phenotype upon transfection with an alpha-catenin expression plasmid, but also upon treatment with the protein kinase C (PKC) activator 12-O-tetradecanoyl-phorbol-13-acetate (TPA). Video registrations showed that the cells started to establish elaborated intercellular junctions within 30 min after addition of TPA. Interestingly, this normalizing TPA effect was not associated with alpha-catenin induction. Classical and confocal immunofluorescence showed only minor TPA-induced changes in E-cadherin staining. In contrast, desmosomal and tight junctional proteins were dramatically rearranged, with a conversion from cytoplasmic clusters to obvious concentration at cell-cell contacts and exposition at the exterior cell surface. Electron microscopical observations revealed the TPA-induced appearance of typical desmosomal plaques. TPA-restored cell-cell adhesion was E-cadherin dependent as demonstrated by a blocking antibody in a cell aggregation assay. Addition of an antibody against the extracellular part of desmoglein-2 blocked the TPA effect, too. Remarkably, the combination of anti-E-cadherin and anti-desmoglein antibodies synergistically inhibited the TPA effect. Our studies show that it is possible to bypass the need for normal alpha-catenin expression to establish tight intercellular adhesion by epithelial cells. Apparently, the underlying mechanism comprises upregulation of desmosomes and tight junctions by activation of the PKC signaling pathway, whereas E-cadherin remains essential for basic cell-cell adhesion, even in the absence of alpha-catenin.

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Upon TPA treatment, α-catenin–negative  cells start to exhibit various  cell junctions as demonstrated by electron microscopy. Untreated HCT-8/E8  cells (a) show tight-junctionlike structures (small arrows),  desmosomes (large arrows),  and membrane interdigitations (arrowhead) at lateral  cell–cell contacts. All these  features are consistently  lacking in untreated HCT-8/ R1 cells (b and d). In contrast, TPA-treated HCT-8/ R1 cells (c and e) form morphologically typical tight  junctions (small arrows) and  desmosomes (large arrows).  The latter are clearly linked  to 10-nm intermediate filaments (e). A, apical side of  the cells. Bars: (a–c) 280 nm;  (d and e) 120 nm.
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Figure 8: Upon TPA treatment, α-catenin–negative cells start to exhibit various cell junctions as demonstrated by electron microscopy. Untreated HCT-8/E8 cells (a) show tight-junctionlike structures (small arrows), desmosomes (large arrows), and membrane interdigitations (arrowhead) at lateral cell–cell contacts. All these features are consistently lacking in untreated HCT-8/ R1 cells (b and d). In contrast, TPA-treated HCT-8/ R1 cells (c and e) form morphologically typical tight junctions (small arrows) and desmosomes (large arrows). The latter are clearly linked to 10-nm intermediate filaments (e). A, apical side of the cells. Bars: (a–c) 280 nm; (d and e) 120 nm.

Mentions: We performed an ultrastructural analysis to determine whether our immunofluorescence data represented the TPA-induced assembly of genuine cell junctions in R-variants. E-variants exhibited many well developed intercellular junctions and showed interdigitation of juxtaposed plasma membranes (Fig. 8 a). Moreover, these cells possessed short microvilli (not shown). In contrast, untreated R-variants revealed no detectable adhesion specializations along the whole length of their non-interdigitated cell–cell contact surfaces (Fig. 8, b and d). The TPA-treated R-variants, however, demonstrated an evident array of junctions, including tight junction–like structures and classical desmosomes with attached intermediate filaments (Fig. 8, c and e).


Protein kinase C activation upregulates intercellular adhesion of alpha-catenin-negative human colon cancer cell variants via induction of desmosomes.

van Hengel J, Gohon L, Bruyneel E, Vermeulen S, Cornelissen M, Mareel M, von Roy F - J. Cell Biol. (1997)

Upon TPA treatment, α-catenin–negative  cells start to exhibit various  cell junctions as demonstrated by electron microscopy. Untreated HCT-8/E8  cells (a) show tight-junctionlike structures (small arrows),  desmosomes (large arrows),  and membrane interdigitations (arrowhead) at lateral  cell–cell contacts. All these  features are consistently  lacking in untreated HCT-8/ R1 cells (b and d). In contrast, TPA-treated HCT-8/ R1 cells (c and e) form morphologically typical tight  junctions (small arrows) and  desmosomes (large arrows).  The latter are clearly linked  to 10-nm intermediate filaments (e). A, apical side of  the cells. Bars: (a–c) 280 nm;  (d and e) 120 nm.
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Related In: Results  -  Collection

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Figure 8: Upon TPA treatment, α-catenin–negative cells start to exhibit various cell junctions as demonstrated by electron microscopy. Untreated HCT-8/E8 cells (a) show tight-junctionlike structures (small arrows), desmosomes (large arrows), and membrane interdigitations (arrowhead) at lateral cell–cell contacts. All these features are consistently lacking in untreated HCT-8/ R1 cells (b and d). In contrast, TPA-treated HCT-8/ R1 cells (c and e) form morphologically typical tight junctions (small arrows) and desmosomes (large arrows). The latter are clearly linked to 10-nm intermediate filaments (e). A, apical side of the cells. Bars: (a–c) 280 nm; (d and e) 120 nm.
Mentions: We performed an ultrastructural analysis to determine whether our immunofluorescence data represented the TPA-induced assembly of genuine cell junctions in R-variants. E-variants exhibited many well developed intercellular junctions and showed interdigitation of juxtaposed plasma membranes (Fig. 8 a). Moreover, these cells possessed short microvilli (not shown). In contrast, untreated R-variants revealed no detectable adhesion specializations along the whole length of their non-interdigitated cell–cell contact surfaces (Fig. 8, b and d). The TPA-treated R-variants, however, demonstrated an evident array of junctions, including tight junction–like structures and classical desmosomes with attached intermediate filaments (Fig. 8, c and e).

Bottom Line: Interestingly, this normalizing TPA effect was not associated with alpha-catenin induction.Addition of an antibody against the extracellular part of desmoglein-2 blocked the TPA effect, too.Remarkably, the combination of anti-E-cadherin and anti-desmoglein antibodies synergistically inhibited the TPA effect.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology, Laboratory of Molecular Cell Biology, University of Ghent and Flanders Interuniversity Institute of Biotechnology (V.I.B.), B-9000 Ghent, Belgium.

ABSTRACT
The alpha-catenin molecule links E-cadherin/ beta-catenin or E-cadherin/plakoglobin complexes to the actin cytoskeleton. We studied several invasive human colon carcinoma cell lines lacking alpha-catenin. They showed a solitary and rounded morphotype that correlated with increased invasiveness. These round cell variants acquired a more normal epithelial phenotype upon transfection with an alpha-catenin expression plasmid, but also upon treatment with the protein kinase C (PKC) activator 12-O-tetradecanoyl-phorbol-13-acetate (TPA). Video registrations showed that the cells started to establish elaborated intercellular junctions within 30 min after addition of TPA. Interestingly, this normalizing TPA effect was not associated with alpha-catenin induction. Classical and confocal immunofluorescence showed only minor TPA-induced changes in E-cadherin staining. In contrast, desmosomal and tight junctional proteins were dramatically rearranged, with a conversion from cytoplasmic clusters to obvious concentration at cell-cell contacts and exposition at the exterior cell surface. Electron microscopical observations revealed the TPA-induced appearance of typical desmosomal plaques. TPA-restored cell-cell adhesion was E-cadherin dependent as demonstrated by a blocking antibody in a cell aggregation assay. Addition of an antibody against the extracellular part of desmoglein-2 blocked the TPA effect, too. Remarkably, the combination of anti-E-cadherin and anti-desmoglein antibodies synergistically inhibited the TPA effect. Our studies show that it is possible to bypass the need for normal alpha-catenin expression to establish tight intercellular adhesion by epithelial cells. Apparently, the underlying mechanism comprises upregulation of desmosomes and tight junctions by activation of the PKC signaling pathway, whereas E-cadherin remains essential for basic cell-cell adhesion, even in the absence of alpha-catenin.

Show MeSH
Related in: MedlinePlus