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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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Expression of β1 integrins in GE11 and GD25 cells activates RhoA and Rac1 but not Cdc42. Lysates of GE11 and GD25 cells expressing either the control vector alone, β1A, β1D, IL2R, or IL2R-β1A were incubated with GST-rhotekin fusion protein for the RhoA assay, or with GST-PAK fusion protein for the Rac1 and Cdc42 assays. The presence of active, GTP-bound RhoA, Rac1, or Cdc42 was analyzed by immunoblotting. Total amounts of RhoA, Rac1, and Cdc42 were determined in total cell lysates.
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Figure 8: Expression of β1 integrins in GE11 and GD25 cells activates RhoA and Rac1 but not Cdc42. Lysates of GE11 and GD25 cells expressing either the control vector alone, β1A, β1D, IL2R, or IL2R-β1A were incubated with GST-rhotekin fusion protein for the RhoA assay, or with GST-PAK fusion protein for the Rac1 and Cdc42 assays. The presence of active, GTP-bound RhoA, Rac1, or Cdc42 was analyzed by immunoblotting. Total amounts of RhoA, Rac1, and Cdc42 were determined in total cell lysates.

Mentions: Small G proteins of the Rho family are involved in the regulation of the actin cytoskeleton, the turnover of focal adhesions, in cell migration, and in the assembly of intercellular adhesions (Mackay and Hall 1998). Therefore, we have studied their role in the cell scattering induced by β1 integrins. GST-rhotekin and GST-PAK fusion proteins were used to precipitate the active form (GTP-bound) of RhoA, and of Rac1 and Cdc42, respectively, from cell lysates of stably transfected GE11 cells expressing either β1A, β1D, IL2R, or IL2R-β1A. The total amounts of GTPases (guanosine diphosphate (GDP)- and GTP-bound) were measured in parallel in the same lysates. Increased amounts of GTP-RhoA were precipitated from the lysates of cells expressing either β1A or β1D, when compared with vector controls (Fig. 8). Experiments performed with transduced GD25 cells confirmed the activation of RhoA by β1A, and to a lesser extent, by the β1D integrin splice variant. Similarly, the expression of either splice variant of the β1 integrin subunit induced the activation of Rac1 in both GE11 and GD25 cells. Fig. 8 also shows that the activation of RhoA and Rac1 mediated by IL2R-β1A was less efficient than that observed with full-length β1A. Finally, in contrast to RhoA and Rac1, the activity of Cdc42 was not significantly increased upon expression of β1 integrins. These data demonstrate that expression of either the β1A or the β1D integrin splice variant activates RhoA and Rac1 in both GE11 and GD25 cells.


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)

Expression of β1 integrins in GE11 and GD25 cells activates RhoA and Rac1 but not Cdc42. Lysates of GE11 and GD25 cells expressing either the control vector alone, β1A, β1D, IL2R, or IL2R-β1A were incubated with GST-rhotekin fusion protein for the RhoA assay, or with GST-PAK fusion protein for the Rac1 and Cdc42 assays. The presence of active, GTP-bound RhoA, Rac1, or Cdc42 was analyzed by immunoblotting. Total amounts of RhoA, Rac1, and Cdc42 were determined in total cell lysates.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2168093&req=5

Figure 8: Expression of β1 integrins in GE11 and GD25 cells activates RhoA and Rac1 but not Cdc42. Lysates of GE11 and GD25 cells expressing either the control vector alone, β1A, β1D, IL2R, or IL2R-β1A were incubated with GST-rhotekin fusion protein for the RhoA assay, or with GST-PAK fusion protein for the Rac1 and Cdc42 assays. The presence of active, GTP-bound RhoA, Rac1, or Cdc42 was analyzed by immunoblotting. Total amounts of RhoA, Rac1, and Cdc42 were determined in total cell lysates.
Mentions: Small G proteins of the Rho family are involved in the regulation of the actin cytoskeleton, the turnover of focal adhesions, in cell migration, and in the assembly of intercellular adhesions (Mackay and Hall 1998). Therefore, we have studied their role in the cell scattering induced by β1 integrins. GST-rhotekin and GST-PAK fusion proteins were used to precipitate the active form (GTP-bound) of RhoA, and of Rac1 and Cdc42, respectively, from cell lysates of stably transfected GE11 cells expressing either β1A, β1D, IL2R, or IL2R-β1A. The total amounts of GTPases (guanosine diphosphate (GDP)- and GTP-bound) were measured in parallel in the same lysates. Increased amounts of GTP-RhoA were precipitated from the lysates of cells expressing either β1A or β1D, when compared with vector controls (Fig. 8). Experiments performed with transduced GD25 cells confirmed the activation of RhoA by β1A, and to a lesser extent, by the β1D integrin splice variant. Similarly, the expression of either splice variant of the β1 integrin subunit induced the activation of Rac1 in both GE11 and GD25 cells. Fig. 8 also shows that the activation of RhoA and Rac1 mediated by IL2R-β1A was less efficient than that observed with full-length β1A. Finally, in contrast to RhoA and Rac1, the activity of Cdc42 was not significantly increased upon expression of β1 integrins. These data demonstrate that expression of either the β1A or the β1D integrin splice variant activates RhoA and Rac1 in both GE11 and GD25 cells.

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