TGFβ1-induced aortic endothelial morphogenesis requires signaling by small GTPases Rac1 and RhoA

TGFβ is a potent regulator of cell differentiation in many cell types. On aortic endothelial cells, TGFβ1 displays angiogenic properties in inducing capillary-like tube formation in collagen I gels, in vitro. We investigated cytoskeletal changes that precede tube formation and related these alterations to the effects of TGFβ1 on the activation state of members of the RhoGTPase family. TGFβ1 promotes cell elongation and stress fiber formation in aortic endothelial cells. Using cell lines with inducible expression of Rac1 mutants, we show that these events are mimicked by expression of dominant-negative Rac1 whereas the constitutively active mutant prevents the TGFβ1-mediated change of phenotype. Although TGFβ1 induces an initial rise in the Rac1-GTP content, this phase is followed by a prolonged loss of the active form. In contrast, RhoA activity increases progressively and reaches a plateau when Rac1-GTP is no longer detectable. Prolonged inhibition of Rac1 appears necessary and sufficient for the increase in RhoA-GTP. In situ examination of Rho activity in TGFβ1-treated cells provides evidence that active RhoA relocalizes to the tips of elongated cells. Inhibiting the Rho effector ROCK abrogates tube formation. Thus, Rac1 and RhoA are regulated by TGFβ1 in the process of endothelial tube formation in collagen I gels.