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Induction of cell scattering by expression of beta1 integrins in beta1-deficient epithelial cells requires activation of members of the rho family of GTPases and downregulation of cadherin and catenin function.

Gimond C, van Der Flier A, van Delft S, Brakebusch C, Kuikman I, Collard JG, Fässler R, Sonnenberg A - J. Cell Biol. (1999)

Bottom Line: Expression of beta1 integrins in GE11 cells resulted in a decrease in cadherin and alpha-catenin protein levels accompanied by their redistribution from the cytoskeleton-associated fraction to the detergent-soluble fraction.In addition, using biochemical activity assays for Rho-like GTPases, we show that the expression of beta1A, beta1D, or IL2R-beta1A in GE11 or GD25 cells triggers activation of both RhoA and Rac1, but not of Cdc42.Our results indicate that beta1 integrins regulate the polarity and motility of epithelial cells by the induction of intracellular molecular events involving a downregulation of alpha-catenin function and the activation of the Rho-like G proteins Rac1 and RhoA.

View Article: PubMed Central - PubMed

Affiliation: Division of Cell Biology, The Netherlands Cancer Institute, 1066 CX Amsterdam,The Netherlands.

ABSTRACT
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 beta1 integrins influence each other using two different beta1- cell lines, epithelial GE11 and fibroblast-like GD25 cells. Expression of beta1A or the cytoplasmic splice variant beta1D, 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 beta1 integrins caused a dramatic reorganization of the actin cytoskeleton and of focal contacts. Interaction of beta1 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)-beta1A chimera and its incorporation into focal adhesions also induced the disruption of cadherin-based adhesions and the reorganization of ECM-cell contacts, but failed to promote cell migration on fibronectin, in contrast to full-length beta1A. This indicates that the disruption of cell-cell adhesion is not simply the consequence of the stimulated cell migration. Expression of beta1 integrins in GE11 cells resulted in a decrease in cadherin and alpha-catenin protein levels accompanied by their redistribution from the cytoskeleton-associated fraction to the detergent-soluble fraction. Regulation of alpha-catenin protein levels by beta1 integrins is likely to play a role in the morphological transition, since overexpression of alpha-catenin in GE11 cells before beta1 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 beta1A, beta1D, or IL2R-beta1A 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 cell-cell adhesions when expressed before beta1. 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-beta1A cells. Our results indicate that beta1 integrins regulate the polarity and motility of epithelial cells by the induction of intracellular molecular events involving a downregulation of alpha-catenin function and the activation of the Rho-like G proteins Rac1 and RhoA.

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Scattering of GE11 cells is dependent on β1 integrin-ligand interaction. (A) Expression of β1, α5, and α6 integrin subunits in GE11-β1 and GE11-α6β1 cells overexpressing the α6β1 integrin, as determined by FACS® analysis. Both the percentage of cells expressing the different subunits and their expression levels in positive cells in mean fluorescence (arbitrary units) are indicated. (B) Phase-contrast microscopy of GE11-α6β1 grown on plastic in the presence of FCS (FN/VN) or on laminin-1 substratum (LN-1).
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Figure 3: Scattering of GE11 cells is dependent on β1 integrin-ligand interaction. (A) Expression of β1, α5, and α6 integrin subunits in GE11-β1 and GE11-α6β1 cells overexpressing the α6β1 integrin, as determined by FACS® analysis. Both the percentage of cells expressing the different subunits and their expression levels in positive cells in mean fluorescence (arbitrary units) are indicated. (B) Phase-contrast microscopy of GE11-α6β1 grown on plastic in the presence of FCS (FN/VN) or on laminin-1 substratum (LN-1).

Mentions: When GE11 cells were cultured on plastic in the presence of FCS, the mere expression of the β1 subunit was sufficient for inducing the disruption of intercellular adhesions and the dissociation of cell colonies. Because GE11-β1A 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 β1 or whether it was triggered by the interaction of β1 integrins with their ligands. Although GE11-β1A cells expressed the laminin receptor α6β1, we have generated GE11 cells expressing α6β1 at higher levels by coexpression of the human α6 integrin subunit in GE11-β1A cells and by further selection by FACS®. The overexpression of α6 in GE11-α6β1A cells resulted in a strong decrease of the percentage of cells expressing the fibronectin receptor α5β1 as well as in a decrease of its average expression levels (Fig. 3 A), probably because α6 associated with most of the available β1 subunit. Although these cells could spread, they poorly scattered and developed strong cell–cell adhesions when cultured on fibronectin (Fig. 3 B), suggesting that the expression of all fibronectin-binding β1 integrins (α5β1 as well as αvβ1) was reduced. However, scattering was induced when they were cultured on laminin-coated dishes (Fig. 3 B). Together, these results indicate that the interaction of β1 integrins with their ligand is required for the disruption of cadherin-based cell–cell adhesion and cell scattering. They also show that several integrins of the β1 family, which bind to various ECM proteins (α5β1 or αvβ1 to fibronectin, and α6β1 to laminin), can trigger the described morphological transition.


Induction of cell scattering by expression of beta1 integrins in beta1-deficient epithelial cells requires activation of members of the rho family of GTPases and downregulation of cadherin and catenin function.

Gimond C, van Der Flier A, van Delft S, Brakebusch C, Kuikman I, Collard JG, Fässler R, Sonnenberg A - J. Cell Biol. (1999)

Scattering of GE11 cells is dependent on β1 integrin-ligand interaction. (A) Expression of β1, α5, and α6 integrin subunits in GE11-β1 and GE11-α6β1 cells overexpressing the α6β1 integrin, as determined by FACS® analysis. Both the percentage of cells expressing the different subunits and their expression levels in positive cells in mean fluorescence (arbitrary units) are indicated. (B) Phase-contrast microscopy of GE11-α6β1 grown on plastic in the presence of FCS (FN/VN) or on laminin-1 substratum (LN-1).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2168093&req=5

Figure 3: Scattering of GE11 cells is dependent on β1 integrin-ligand interaction. (A) Expression of β1, α5, and α6 integrin subunits in GE11-β1 and GE11-α6β1 cells overexpressing the α6β1 integrin, as determined by FACS® analysis. Both the percentage of cells expressing the different subunits and their expression levels in positive cells in mean fluorescence (arbitrary units) are indicated. (B) Phase-contrast microscopy of GE11-α6β1 grown on plastic in the presence of FCS (FN/VN) or on laminin-1 substratum (LN-1).
Mentions: When GE11 cells were cultured on plastic in the presence of FCS, the mere expression of the β1 subunit was sufficient for inducing the disruption of intercellular adhesions and the dissociation of cell colonies. Because GE11-β1A 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 β1 or whether it was triggered by the interaction of β1 integrins with their ligands. Although GE11-β1A cells expressed the laminin receptor α6β1, we have generated GE11 cells expressing α6β1 at higher levels by coexpression of the human α6 integrin subunit in GE11-β1A cells and by further selection by FACS®. The overexpression of α6 in GE11-α6β1A cells resulted in a strong decrease of the percentage of cells expressing the fibronectin receptor α5β1 as well as in a decrease of its average expression levels (Fig. 3 A), probably because α6 associated with most of the available β1 subunit. Although these cells could spread, they poorly scattered and developed strong cell–cell adhesions when cultured on fibronectin (Fig. 3 B), suggesting that the expression of all fibronectin-binding β1 integrins (α5β1 as well as αvβ1) was reduced. However, scattering was induced when they were cultured on laminin-coated dishes (Fig. 3 B). Together, these results indicate that the interaction of β1 integrins with their ligand is required for the disruption of cadherin-based cell–cell adhesion and cell scattering. They also show that several integrins of the β1 family, which bind to various ECM proteins (α5β1 or αvβ1 to fibronectin, and α6β1 to laminin), can trigger the described morphological transition.

Bottom Line: Expression of beta1 integrins in GE11 cells resulted in a decrease in cadherin and alpha-catenin protein levels accompanied by their redistribution from the cytoskeleton-associated fraction to the detergent-soluble fraction.In addition, using biochemical activity assays for Rho-like GTPases, we show that the expression of beta1A, beta1D, or IL2R-beta1A in GE11 or GD25 cells triggers activation of both RhoA and Rac1, but not of Cdc42.Our results indicate that beta1 integrins regulate the polarity and motility of epithelial cells by the induction of intracellular molecular events involving a downregulation of alpha-catenin function and the activation of the Rho-like G proteins Rac1 and RhoA.

View Article: PubMed Central - PubMed

Affiliation: Division of Cell Biology, The Netherlands Cancer Institute, 1066 CX Amsterdam,The Netherlands.

ABSTRACT
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 beta1 integrins influence each other using two different beta1- cell lines, epithelial GE11 and fibroblast-like GD25 cells. Expression of beta1A or the cytoplasmic splice variant beta1D, 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 beta1 integrins caused a dramatic reorganization of the actin cytoskeleton and of focal contacts. Interaction of beta1 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)-beta1A chimera and its incorporation into focal adhesions also induced the disruption of cadherin-based adhesions and the reorganization of ECM-cell contacts, but failed to promote cell migration on fibronectin, in contrast to full-length beta1A. This indicates that the disruption of cell-cell adhesion is not simply the consequence of the stimulated cell migration. Expression of beta1 integrins in GE11 cells resulted in a decrease in cadherin and alpha-catenin protein levels accompanied by their redistribution from the cytoskeleton-associated fraction to the detergent-soluble fraction. Regulation of alpha-catenin protein levels by beta1 integrins is likely to play a role in the morphological transition, since overexpression of alpha-catenin in GE11 cells before beta1 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 beta1A, beta1D, or IL2R-beta1A 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 cell-cell adhesions when expressed before beta1. 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-beta1A cells. Our results indicate that beta1 integrins regulate the polarity and motility of epithelial cells by the induction of intracellular molecular events involving a downregulation of alpha-catenin function and the activation of the Rho-like G proteins Rac1 and RhoA.

Show MeSH
Related in: MedlinePlus