Abstract
We previously demonstrated that RhoA-dependent signaling regulates transforming growth factor-beta1 (TGF-beta1)-induced cytoskeletal reorganization in the human retinal pigment epithelial cell line ARPE-19. Smad pathways have also been shown to mediate TGF-beta1 activity. Here, we examined what regulates Rho GTPase activity and tested whether Smad signaling cross-talks with Rho pathways during TGF-beta1-induced actin rearrangement. Using small interfering RNAs, we found that NET1, the guanine nucleotide exchange factor of RhoA, is critical for TGF-beta1-induced cytoskeletal reorganization, N-cadherin expression, and RhoA activation. In ARPE-19 cells lacking NET1, TGF-beta1-induced stress fibers and N-cadherin expression were not observed. Interestingly, in dominant-negative Smad3-expressing or constitutively active Smad7 cells, TGF-beta1 failed to induce NET1 mRNA and protein expression. Consistent with these results, both dominant-negative Smad3 and constitutively active Smad7 blocked the cytoplasmic localization of NET1 and inhibited interactions between NET1 and RhoA. Finally, we found that NET1 is a direct gene target of TGF-beta1 via Smad3. Taken together, our results demonstrate that Smad3 regulates RhoA activation and cytoskeletal reorganization by controlling NET1 in TGF-beta1-induced ARPE-19 cells. These data define a new role for Smad3 as a modulator of RhoA activation in the regulation of TGF-beta1-induced epithelial-mesenchymal transitions.
Highlights
Members of the transforming growth factor  (TGF-)[2] superfamily are multifunctional cytokines that regulate cellular processes, including cell-cycle arrest, differentiation, morphogenesis, and apoptosis (1–5)
Smad[3] Regulates RhoA via NET1 in transforming growth factor-1 (TGF-1)-induced Cells polymerization that may be responsible for the fibrotic response of retinal pigment epithelial (RPE) cells that can lead to proliferative vitreoretinopathy (PVR) in vivo (15)
In the spontaneously immortalized human RPE cell line ARPE-19, TGF-1 treatment led to dramatic morphological changes after 48 h (Fig. 1A)
Summary
Members of the transforming growth factor  (TGF-)[2] superfamily are multifunctional cytokines that regulate cellular processes, including cell-cycle arrest, differentiation, morphogenesis, and apoptosis (1–5). Our results demonstrate that Smad[3] regulates RhoA activation and cytoskeletal reorganization by controlling NET1 in TGF-1-induced ARPE-19 cells. Rho GTPases increase stress fiber formation, and we have previously proved that these small GTPases are key mediators of the TGF-1-induced changes in ARPE-19 cell cytoskeletal organization and F-actin expression (15).
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