In the present study we analyzed the regulation of the two isoforms of the RhoA-specific guanine nucleotide exchange factor Net1 by transforming growth factor-b (TGF-b) in keratinocytes. We report that short-term TGF-b treatment selectively induced Net1 isoform2 (Net1A) but not Net1 isoform1. This led to upregulation of cytoplasmic Net1A protein levels that were necessary for TGF-b-mediated RhoA activation. Smad signaling and the MAPK/ERK kinase (MEK)/extracellular signal-regulated kinase (ERK) pathway were involved in Net1A upregulation by TGF-b. Interestingly, long-term TGF-b treatment resulted in Net1 mRNA downregulation and Net1A protein degradation by the proteasome. Furthermore, we identified the microRNA miR-24 as a novel post-transcriptional regulator of Net1A expression. Silencing of Net1A resulted in disruption of E-cadherin-and zonula occludens-1 (ZO-1)-mediated junctions, as well as expression of the transcriptional repressor of E-cadherin, Slug and the mesenchymal markers N-cadherin, plasminogen activator inhibitor-1 (PAI-1) and fibronectin, indicating that late TGFb-induced downregulation of Net1A is involved in epithelial-to-mesenchymal transition (EMT). Finally, miR-24 was found to be implicated in the regulation of the EMT program in response to TGF-b and was shown to be directly involved in the TGF-b-induced breast cancer cell invasiveness through Net1A regulation. Our results emphasize the importance of Net1 isoform2 in the short-and long-term TGF-b-mediated regulation of EMT.

In previous studies, we have demonstrated that RhoA/B‐dependent signaling regulates TGFβ‐induced rapid actin reorganization in Swiss 3T3 fibroblasts. Here we report that TGFβ regulates long‐term actin remodeling by increasing the steady‐state mRNA levels of the RhoB gene in mouse Swiss 3T3 fibroblasts and human hepatoma HepG2 cells. We show that this regulation is specific for the RhoB gene and is facilitated by enhanced activity of the RhoB promoter. Adenovirus‐mediated gene transfer of Smad2 and Smad3 in Swiss 3T3 fibroblasts induced transcription of the endogenous RhoB gene but not the RhoA gene. Interestingly, in JEG‐3 choriocarcinoma cells that lack endogenous Smad3, TGFβ‐induced transcriptional up‐regulation of the RhoB gene was not observed, but it was restored by adenoviral Smad3 overexpression. In addition, Smad2 and Smad3 triggered activation of RhoA and RhoB GTPases and long‐term actin reorganization in Swiss 3T3 fibroblasts. Finally, Smad3, and to a lesser extent Smad2, induced transcription of the α‐smooth muscle actin (α‐SMA) gene, and enhanced the incorporation of α‐SMA into microfilaments in Swiss 3T3 fibroblasts. These data reveal a novel mechanism of cross‐talk between the classical TGFβ/Smad pathway and Rho GTPases, regulating the rapid and the long‐term actin reorganization that may control the fibroblast–myofibroblast differentiation program.

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.

TGFβ has been shown to induce short- and long-term actin reorganization controlled by Rho-GTPase signaling. A number of direct Smad target genes, rapidly activated by TGFβ, have been previously reported to control the long-term Rho activation and actin reorganization. However, the molecular mechanisms that regulate the prompt stimulation of Rho GTPases by TGFβ remain unknown. In the present study we report that TGFβ rapidly stimulated RhoA and RhoB activation in JEG3 choriocarcinoma cells that lack endogenous Smad3. Inhibition of Smad2 expression via siRNA-mediated silencing or by blocking its phosphorylation using the TβRI inhibitor SB431542 did not prevent the early RhoA/B activation by TGFβ indicating that this effect is Smad2/3-independent. Pre-treatment of the cells with the general tyrosine kinase inhibitor Genistein blocked the TGFβ-induced early RhoA activation. In line with this finding, TGFβ-stimulation resulted in a quick activation of the non-receptor tyrosine kinase Src, followed by activation of the guanine nucleotide exchange factor (GEF) Vav2. Inhibition of Src kinase by the selective inhibitor of the Src family tyrosine kinases PP2 totally blocked the early TGFβ-induced RhoA activation. Similarly, Vav2 silencing via siRNA reduced the TGFβ-induced RhoA activation implying that the rapid Src/Vav2 stimulation was effective in regulating RhoA activation. Our present findings provide for the first time a clear evidence for the role of Src and Vav2-GEF in the early Smad2/3-independent Rho activation by TGFβ.

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