Abstract
Purpose: :
In previous studies we have demonstrated that RhoA dependent signaling regulate TGF-β1 induced cytoskeleton reorganization in human retinal pigment epithelium cell line, ARPE-19 cells. In addition to RhoA signaling, Rho GEF, NET1 have also been shown to mediate actin of TGF-β1. The purpose of this study were to examine what regulate Rho GEF activity and to test whether Smad signaling cross-talks with Rho pathways during actin rearrangement induced by TGF-β1.
Methods: :
Serum-starved ARPE-19 cells were incubated with vehicle alone or 10ng/ml TGF-β1. Using dominant negative Smad3 and active Smad3 DNA construct, we show that these proteins are critical to TGF-β1 induced cytoskeleton reorganization and NET1 expression. Actin reorganization was examined by immunochemistry and confocal microscopy. Protein expression was analyzed by Western blot analysis.
Results: :
Using DNA construct targeting for Smad, we show that Smad3 is critical to TGF-β1 induced cytoskeleton reorganization and NET1, the GEF of RhoA, expression. In ARPE-19 cells that lack Smad3, TGF-β1 induced stress fiber was not observed. Interestingly, dominant negative smad3 expressing cells, TGF-β1 failed to induce the NET1 mRNA and protein expression.
Conclusions: :
we demonstrate that Smad3 regulate RhoA activation and cytoskeleton reorganization via controlling NET1 in TGF-β1 induced ARPE-19 cells. These data define a new role for Smad as a modulator of RhoA activation while regulating TGF-β1 induced epithelial-mesenchymal transitions.
Keywords: EMT (epithelial mesenchymal transition) • retinal pigment epithelium • signal transduction