The purpose of the present study was to investigate the mechanism of transcriptional induction of the small GTPase RhoB gene by the transforming growth factor beta (TGFbeta) signaling pathway and the role of this regulation in TGFbeta-induced cell migration. To achieve our goals, we utilized a combination of siRNA-mediated gene silencing, adenovirus-mediated gene transfer receptor and MAPK inhibition, transactivation assays, and DNA-protein interaction assays in human HaCaT keratinocytes. We found that the RhoB gene is a direct transcriptional target of TGFbeta. We show that TGFbeta activates an early MEK/ERK pathway and that this activation is required for the recruitment of Smad3 to a novel, nonclassical, Smad binding element in the proximal RhoB promoter, in a p53-dependent manner. This element is overlapping with a CCAAT box that constitutively binds nuclear factor Y. Mutagenesis of this site abolished the Smad-mediated transactivation of the RhoB promoter. Finally, silencing of RhoB gene expression via siRNA or utilization of a dominant negative form of RhoB significantly inhibited TGFbeta-induced migration of HaCaT keratinocytes and DU145 prostate cancer cells. Our findings establish RhoB as a direct transcriptional target of TGFbeta in human keratinocytes and identify an important role of RhoB in TGFbeta-induced cell migration.-Vasilaki, E., Papadimitriou, E., Tajadura, V., Ridley, A. J., Stournaras, C., Kardassis, D. Transcriptional regulation of the small GTPase RhoB gene by TGFbeta-induced signaling pathways.