The Foreign Investigator (Fl) of this project has recently discovered that alphavbeta3 integrin in astrocytes is a receptor for Thy-1 and shown that treatment of astrocytes with Thy- 1 induces signaling linked to focal adhesions (FA) and stress fiber formation. FAs anchor stress fibers to the plasma membrane, contain multiple structural, signaling and adaptor molecules and are implicated in cell migration, growth and differentiation. Astrocytes change from stellate to fibroblast-like morphology upon brain damage and the GTPase RhoA has been implicated in these changes. Our preliminary data reveal that RhoA is implicated in Thy-1-induced focal adhesion formation in astrocytes. This collaborative grant is aimed at understanding how cell-cell interaction mediated by Thy-1 binding to integrin alphavBeta3 activates RhoA, whether integrin signaling triggered by Thy-1 binding stimulates cell migration and what are the signaling events occurring downstream from FAs. We will explore the possibility that Thy-1-astocyte interaction is mediated by two different domains on Thy-1, one binding integrins and one binding syndecans. We will look for possible guanine nucleotide exchange factors (GEFs) that may activate RhoA using dominant negative constructs of RhoA, which have high affinity for Rho GEFs. To study the effect of Thy-1- alphavBeta3 integrin interaction on cell migration we will use different wound healing assays currently in use in the PI's lab. We will also test whether changes in motility correlate with modulation of Rac 1 or Cdc42 activity, measured with pulldown assays. We will test the hypothesis that Thy-1 activated integrins trigger signaling events in astrocytes that are similar to those described in fibroblasts, with particular interest on the involvement of PI 3-kinase and MAP kinase pathways, given their known participation in cell migration and proliferation, respectively. Thus, in this proposal we will study the molecular response of astrocytes to Thy-1- alphavBeta3 integrin interaction in vitro. Learning about such changes should lead to a better understanding of """"""""astrogliosis,"""""""" a migratory and proliferative reaction of astrocytes to wounding, that creates an environment antagonistic to neuronal regeneration following brain injury.
