| Coordination of cell-cell and cell-matrix adhesions is crucial for proper establishment and maintenance of epithelial tissue structure. Breakdown of this coordination can cause disruption of epithelial architecture, leading to the development of pathologies including tumor growth and metastasis. Currently, the pathways by which cell-cell and cell-matrix adhesions interact are poorly defined.;Activation of R-Ras, a member of the Ras family of small GTPases, disrupts ductal morphogenesis of mammary epithelial cells (MECs) in 3D collagen gels and promotes cell migration, causing loss of epithelial characteristics and acquisition of a migratory phenotype. I show in this thesis that constitutive activation of R-Ras in cells plated on collagen causes a decrease in adherens junction (AJ) formation and a corresponding increase in focal adhesions (FAs), i.e. a decrease in cell-cell adhesions and an increase in cell-matrix adhesions. This inverse relationship between AJs and FAs can be controlled by varying the concentration of collagen substrate on which the cells are cultured. It has been shown previously that R-Ras regulates the affinity and avidity of the alpha2beta1 integrin, a collagen and laminin receptor that mediates MEC polarization in 3D collagen matrices. Inhibition of the alpha2beta1 integrin through siRNA or addition of a blocking antibody, P1E6, causes a partial rescue of AJ formation on collagen in cells expressing activated R-Ras (38V), indicating that R-Ras enhancement of cell-matrix adhesions (FAs) down-regulates cell-cell adhesions (AJs). I show that R-Ras increases association of a FA component (alpha2 integrin) with the cytoskeleton and decreases association of AJ components with the cytoskeleton, suggesting that R-Ras regulates AJs by regulating the cytoskeleton. Consistent with this, disruption of R-Ras-enhanced actin polymerization with jasplakinolide rescues AJ formation and AJ component association with the cytoskeleton, indicating that R-Ras enhancement of alpha2beta1-based cell-matrix contacts affects cell-cell contacts through regulation of the actin cytoskeleton.;In addition, I have shown that R-Ras regulation of cell-cell vs. cell-matrix adhesions may also be present on fibronectin, an alpha5beta1 ligand. These data indicate that R-Ras may be a general regulator of cell-cell/cell-matrix balance in mammary epithelial cells. |
