We have shown previously that
We have shown previously that activation of the TGFβ/Smad signaling pathway in keratinocytes regulates BMS-911543 cytoskeleton organization via non-genomic and genomic mechanisms and that RhoB gene is a target of Smad proteins [12,13]. Although the role of RhoB in early non-genomic TGFβ-induced cell responses is well established, the role of the the transcriptional upregulation of the RhoB gene by TGFβ and Smads is still unknown. In the present study we propose a novel mechanism of TGFβ/Smad signaling modulation involving RhoB and show that this TGFβ/Rho signaling cross talk differentially affects the nuclear and cytoplasmic responses to TGFβ.
Materials and methods
Discussion We have shown previously that activation of the TGFβ/Smad signaling pathway in different cell types regulates actin cytoskeleton organization via non-genomic and genomic mechanisms [8,12,13,24]. The rapid non-genomic cellular responses to TGFβ involve both Smad-dependent and Smad-independent pathways such as the Ras-ERK1/2 pathway and the RhoA/B/ROCK/LIMK2/cofilin pathway depending on the particular cellular context which in the case of Rho GTPases could be the levels of cell-specific Rho modulatory proteins such as guanosine nucleotide exchange factors (GEFs), GTPase-activating proteins (GAPs) and guanosine nucleotide dissociation inhibitors (GDIs) . In support of this hypothesis, we have shown that TGFβ activates the RhoA gene via Src and the GEF Vav2 in JEG3 choriocarcinoma cells  whereas in keratinocytes TGFβ activates RhoA via the selective upregulation of the GEF Net1 . Thus, it appears that in certain cell types Smad and Rho proteins cooperate in TGFβ signal transduction but the precise mechanism for this cooperation is still unknown. In the present study we show for the first time that under conditions of RhoB overexpression, RhoB physically interacts with Smad3 and this interaction inhibits the translocation of Smad3 from the cytoplasm to the nucleus causing the accumulation of cytoplasmic Smad3/RhoB protein complexes. This interaction has two major consequences: a) it inhibits the transcriptional activation of genes by the TGFβ/Smad pathway; and b) it enhances responses to TGFβ such as actin cytoskeleton reorganization and the reduction in the expression of E-Cadherin which is a critical step in EMT. The inhibition of TGFβ/Smad signaling to the nucleus by RhoB was demonstrated by several complementary approaches. First, we examined the mRNA levels and promoter activity of the gene encoding the cell cycle inhibitor p21WAF1/Cip1 gene, which is an established TGFβ target and is considered a critical protein for the tumor suppressor program of TGFβ . We have shown previously that the p21WAF1/Cip1 promoter contains several binding sites for Sp1 in the proximal region which are required for the transactivation of the p21WAF1/Cip1 promoter by Smad proteins via complexes with Sp1 [17,18]. We have also shown that blocking the activity of Smad proteins by dominant negative Smad forms or by the overexpression of Smad7 inhibited the induction of the p21WAF1/Cip1 promoter by TGFβ in HaCaT keratinocytes which is in line with the findings of the present study . Second, we showed that the inhibitory role of RhoB in TGFβ signaling may not be limited to the regulation of p21WAF1/Cip1 gene but may have broader consequences for the genomic effects of TGFβ. This hypothesis is supported by the observation that overexpression of RhoB inhibits the induction of the generic TGFβ-responsive promoter (CAGA)12 by TGFβ stimulation or Smad3 overexpression. This finding indicated that RhoB may regulate negatively a large number of genes that depend strictly on Smads. In favor of this hypothesis we have shown that RhoB inhibits the expression of the two additional Smad-responsive genes: the gene encoding the plasminogen activator inhibitor 1 (PAI 1) and the gene encoding RhoB itself (data not shown). These data suggested that RhoB may be implicated in a feedback inhibitory loop. This is in agreement with a recent study which showed that RhoB inhibits its own expression in stem cells .