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J. Lee, Y.-A. Lee, C.-K. Joo; Smad3 Regulate Rho Signal Pathway via Net1 in TGF-β Induced Human RPE. Invest. Ophthalmol. Vis. Sci. 2009;50(13):3421. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
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, Smad pathways have also been shown to mediate actin of TGF-β1. The purpose of this study were to examine what regulate Rho GTPase activity and to test whether Smad signaling cross-talks with Rho pathways during actin rearrangement induced by TGF-β1.
Serum-starved ARPE-19 cells were incubated with vehicle alone or 10ng/ml TGF-β1 and their morphological changes were examined by phase-contrast microscopy. Using siRNA targeting for NET1 and dominant negative Smad3 and active Smad3 DNA construct, we show that these proteins are critical to TGF-β1 induced cytoskeleton reorganization and RhoA activation. Actin reorganization was examined by immunochemistry and confocal microscopy. Protein expression was analyzed by Western blot analysis.
Using siRNA targeting for NET1, we show that NET1, the GEF of RhoA, is critical to TGF-β1 induced cytoskeleton reorganization and RhoA activation. In ARPE-19 cells that lack NET1, TGF-β1 induced stress fiber was not observed. Interestingly, dominant negative smad3 expressing cells, TGF-β1 failed to induce stress fiber formation.
We demonstrate that Smad3 regulate RhoA activation and cytoskeleton reorganization 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.
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