Integrin-specific activation of Rho GTPases, their roles in mechanosignalling and cancer.

COLÓ, G.P.; HAGA, R.B.; FERNÁNDEZ CHÁVEZ, L.; ALONSO, E.N; GANDINI, N.A.; FERMENTO, M.E.; GUEVARA, J.A.; MASCARÓ, M.; FERRONATO, M.J.; GRIOLI, S.M.; FACCHINETTI, M.M.; CURINO, A.C.; FÄSSLER, R.

Congreso; LXIII Reunión Científica Anual de la Sociedad Argentina de Investigación Clínica (SAIC); 2018

Mechanotransduction is mediated by the integrin family of cell adhesion receptors. Integrins bind cell extracellular matrix proteins and connect to the F-actin cytoskeleton and non-muscle-myosin inside the cells. Using genetically engineered cells, biochemical assays, in combination with mass spectroscopy (MS), traction force microscopy and micropatterns, we observed that α5β1-integrin expressing cells promote the formation of small nascent adhesions, low RhoA activation and high force, while αVβ3-integrin expressing cells showed large focal adhesions connected to contractile stress fibers (SFs), resulting in high RhoA but low force. To further analyze pKO-cells phenotypes, we looked for specific RhoA activators (GEFs). We performed a MS-proteomic analysis and amongst the interesting hits was GEF-H1, together with biochemical assays we observed that GEF-H1 activation is dependent on a specific integrin-class suggesting that integrins may activate specific GEFs during adhesion, migration and invasion. Recent studies have also shown that an increase in GEF-H1 expression correlates with an increase in tumor progression and metástasis. In addition, GEF-H1 is involved in the cross-talk between microtubules and the actin cytoskeleton. Our data shows that GEF-H1 is localized in the cytoplasm and more active in αVβ3-cells when compared to α5β1-cells, where GEF-H1 is in an inactive state bounded to the microtubules. Similar results we observed in breast cancer cells depending their invasiveness and the integrin-class-expression. These results could explain the increase in SFs formation in αVβ3-cells and RhoA activation. GEF-H1 can be released to the cytoplasm either by microtubule depolymerization or by protein phosphorylation. A phosphoenrichment-label-free MS analysis revealed that GEF-H1 is highly phosphorylated in αVβ3-cells. Furthermore, using integrin-tail pull-down and MS assay, we observed that GEF-H1 binds to β3-integrin tail. These results show for the first time that GEF-H1-RhoA activation is αVβ3-integrin dependent and it can mediate the signaling involved in controlling cell structure, force generation, migration and invasion.