Modulation of TGFβ/Smad signaling by the small GTPase RhoB

Abstract

We have shown previously that the small GTPases RhoA and RhoB play important roles in early TGFβ-induced actin cytoskeleton reorganization and that RhoB is transcriptionally activated by TGFβ and its signaling effectors, the Smad proteins. However, this long-term impact of RhoB gene upregulation by TGFβ on cellular functions is not known. We now show that increased levels of RhoB, but not of RhoA, inhibit the TGFβ/Smad-mediated transcriptional induction of the cell cycle inhibitor p21^WAF1/Cip1 gene as well as of a generic Smad-responsive promoter suggesting that RhoB could be part of an auto-inhibitory loop in TGFβ signaling by inhibiting the genomic responses to TGFβ. We show that RhoB blocks the interaction of Smad3 with the type I TGFβ receptor which prohibits its phosphorylation by this receptor and its translocation to the nucleus. Using in vivo GST pull-down and co-immunoprecipitation assays we show that Smad3 physically interacts with RhoB but not with RhoA. We show that RhoB, but not RhoA, potently regulates actin cytoskeleton reorganization by inducing stress fiber formation in a Smad-dependent manner. Finally we show that Smad3 downregulates the expression of the epithelial adherens junctions protein E-Cadherin and upregulates the fibronectin gene in Smad3^-/- JEG3 cells only in the presence of RhoB suggesting that RhoB/Smad3 complexes in the cytoplasm may be involved in epithelial to mesenchymal transitions. In summary, our data propose a novel mechanism of TGFβ/Smad signaling modulation by the small GTPase RhoB and show that this TGFβ/RhoB signaling cross talk affects the nuclear and cytoplasmic responses to TGFβ in opposite ways.

Keywords: Actin cytoskeleton; EMT; Rho GTPases; Smad3; TGFβ.