title: Smad3 regulates Rho signaling via NET1 in the transforming growth factor-beta-induced epithelial-mesenchymal transition of human retinal pigment epithelial cells.
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.
authors.affiliation: Laboratory of Ophthalmology and Visual Science, Catholic Research Institutes of Medical Sciences, Korean Eye Tissue and Gene Bank Related to Blindness, College of Medicine, The Catholic University of Korea, Seoul, Korea. leeje@catholic.ac.kr
authors.forename: Jungeun
authors.initials: J
authors.lastname: Lee
authors.forename: Hyun-Jeong
authors.initials: HJ
authors.lastname: Moon
authors.forename: Jong-Min
authors.initials: JM
authors.lastname: Lee
authors.forename: Choun-Ki
authors.initials: CK
authors.lastname: Joo
mesh_terms: D002460:Cell Line; D003599:Cytoskeleton / Q000378:metabolism / Q000648:ultrastructure; D004847:Epithelial Cells / Q000166:cytology; D006801:Humans; D059630:Mesenchymal Stem Cells / Q000166:cytology; D015513:Oncogene Proteins / Q000378:metabolism*; D055213:Retinal Pigment Epithelium / Q000166:cytology*; D015398:Signal Transduction; D051900:Smad3 Protein / Q000502:physiology*; D053773:Transforming Growth Factor beta1 / Q000494:pharmacology*; D020742:rhoA GTP-Binding Protein / Q000378:metabolism*
chemical_list: C099763:NET1 protein, human; D015513:Oncogene Proteins; D051900:Smad3 Protein; D053773:Transforming Growth Factor beta1; D020742:rhoA GTP-Binding Protein