Dataset Information

β-Catenin/Smad3 Interaction Regulates Transforming Growth Factor-β-Induced Epithelial to Mesenchymal Transition in the Lens.

ABSTRACT: Cataracts are the leading cause of blindness worldwide. Although surgery is a successful method to restore vision loss due to cataracts, post-surgical complications can occur, such as secondary cataracts, also known as posterior capsular opacification (PCO). PCO arises when lens epithelial cells (LEC) are left behind in the capsular bag following surgery and are induced to undergo epithelial to mesenchymal transition (EMT). Following EMT, LEC morphology and phenotype are altered leading to a loss of transparency and vision. Transforming growth factor (TGF)-β-induced signaling through both canonical, TGF-β/Smad, and non-canonical, β-catenin/Wnt and Rho/ROCK/MRTF-A, pathways have been shown to be involved in lens EMT, and thus PCO. However, the interactions between these signaling pathways in the lens have not been thoroughly explored. In the current study we use rat LEC explants as an ex vivo model, to examine the interplay between three TGF-β-mediated pathways using α-smooth muscle actin (α-SMA) as a molecular marker for EMT. We show that Smad3 inhibition via SIS3 prevents nuclear translocation of β-catenin and MRTF-A, and α-SMA expression, suggesting a key role of Smad3 in regulation of MRTF-A and β-catenin nuclear transport in LECs. Further, we demonstrate that inhibition of β-catenin/CBP interaction by ICG-001 decreased the amount of phosphorylated Smad3 upon TGF-β stimulation in addition to significantly decreasing the expression levels of TGF-β receptors, TBRII and TBRI. Overall, our findings demonstrate interdependence between the canonical and non-canonical TGF-β-mediated signaling pathways controlling EMT in the lens.

SUBMITTER: Taiyab A

PROVIDER: S-EPMC6540099 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:PurposeTransforming growth factor-β (TGF-β) is considered to be essential to induce epithelial-to-mesenchymal transition (EMT) which plays central roles in wound healing in ocular fibrotic complication. The present study investigates whether small interference RNAs (siRNAs) targeting the type II receptor of TGF-β (TβRII) could be used to minimize the TGF-β action.MethodsTGF-β receptor type II (TβRII) specific siRNAs designed from the Nakamura human gene sequence were used to transfect the cultured lens epithelial cells (LECs). The optimal transfection of scramble siRNA-Cy3 labeled duplexes in cultured LECs were examined by laser scanning confocal microscope and flow cytometry. TβRII protein expression and transcript levels were analyzed by immunofluorescence, western blotting, and real time PCR, respectively. Western blotting was performed to examine protein expression of fibronectin and alpha-smooth muscle actin (α-SMA). Scratch assay was used to determine cell migration. Cell morphology was observed after transfection by inverted microscope.ResultsThe optimal transfection rate of scramble siRNA-Cy3 labeled duplexes was efficient in that nearly to 50% in cultured LECs. TβRII specific siRNAs significantly reduced the receptor transcript and protein expression in cultured LECs. The gene knockdown inhibited LECs transdifferentiation, as it abrogated the expression of fibronectin and α-SMA, and retarded cell migration on the scratch assay. In addition, after transfection with TβRII specific siRNA, the cultured LECs did not show fibroblast-like shape which was one of the feature signs of EMT. Wound scratch assays indicated that the number of cultured LECs migrated into the wounded area was significantly lower in TβRII specific siRNA treated group (12.8 ± 3.27/7.85 mm(2)), compared with normal (57.8 ± 3.06/7.85 mm(2)) and scrambled RNA transfected group (50.8 ± 3.64/7.85 mm(2); p<0.0001).ConclusionsOur results provided additional evidence to support that TGF-β pathway was involved in the development of EMT of human posterior capsule opacification, while how TβRII was involved should be further investigated.

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β-Catenin/Smad3 Interaction Regulates Transforming Growth Factor-β-Induced Epithelial to Mesenchymal Transition in the Lens.

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