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.

先前我们已经表明，小的GTPases RhoA和RhoB在早期TGFβ诱导的肌动蛋白细胞骨架重组中起重要作用，并且RhoB被TGFβ及其信号传导因子Smad蛋白转录激活。但是，尚不知道TGFβ上调RhoB基因对细胞功能的长期影响。我们现在显示，RhoB的水平升高，而不是RhoA的水平升高，抑制了TGFβ/ Smad介导的细胞周期抑制剂p21 ^{WAF1 / Cip1的}转录诱导该基因以及一个通用的Smad反应启动子，提示RhoB可能是通过抑制对TGFβ的基因组应答而成为TGFβ信号转导自抑制环的一部分。我们显示，RhoB阻止了Smad3与I型TGFβ受体的相互作用，从而阻止了该受体的磷酸化和其向核的转运。使用体内GST下拉和免疫共沉淀试验，我们显示Smad3与RhoB物理相互作用，但不与RhoA相互作用。我们显示RhoB，而不是RhoA，通过以Smad依赖性方式诱导应激纤维形成，有效调节肌动蛋白细胞骨架重组。最后，我们显示Smad3下调Smad3中上皮黏附连接蛋白E-Cadherin的表达并上调纤连蛋白基因^-/-仅在存在RhoB的情况下JEG3细胞提示细胞质中的RhoB / Smad3复合物可能参与上皮到间充质的转变。总之，我们的数据提出了由小GTPase RhoB调节TGFβ/ Smad信号传导的新机制，并表明该TGFβ/ RhoB信号串扰以相反的方式影响对TGFβ的核和细胞质反应。