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Eri Kubo, Hiromi Osada, Naoko Shibata, Etsuko Kiyokawa, Hiroshi Sasaki, Dhirendra P Singh; Tropomyosin Regulation of Stress Fiber Formation and EMT in Lens Epithelial Cells. Invest. Ophthalmol. Vis. Sci. 2014;55(13):1222.
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The epithelial-mesenchymal transition (EMT) of lens epithelial cells (LECs) is a leading cause of posterior capsule opacification (PCO) after cataract surgery. Transforming growth factor (TGF)-β2 and fibroblast growth factor (FGF)-2 are frequently and abundantly expressed in LECs and are considered key mediators of EMT by regulating the expression of extracellular matrix genes, including the tropomyosin (Tpm) family of proteins. We previously showed that the elevated expression of Tpm1α/2β was related to the progression of rat PCO and of human cataracts with anterior subcapsular fibrosis. Using mouse and human LECs, we analyzed the effects of FGF2 and/or TGFβ2 on the expression and role of Tpm1α/2β and α-smooth muscle actin (αSMA) in stress fiber formation, as well as assessing EMT related gene expression.
Primary cultured mouse and human LECs showing reduced expression of Tpm1α/2β were treated with 0 to 10 ng/ml of FGF-2 and/or TGFβ-2. Tpm1α and 2β were selectively knocked down by transfecting their corresponding siRNAs using lipofectin. Mouse and human Tpm1α/2β were overexpressed by transfecting GFP-Tpm1α and/or GFP-Tpm2β. The expression of proteins related to EMT was assayed by western blotting using specific antibodies. F-actin distribution in LECs was visualized using fluorescent phalloidin.
Stress fiber formation was induced by the addition of TGFβ2 and inhibited by the addition of FGF2. Silencing of Tpm1α and/or -2β expression significantly inhibited TGF-β2-mediated stress fiber formation and αSMA expression. In contrast, overexpression of Tpm-1α and/or -2β induced stress fiber formation and αSMA expression. LECs overexpressing Tpm1α and or 2β displayed overmodulation of genes related to EMT.
Tpm expression mediates the regulation and reprogramming of the actin cytoskeleton during the process of EMT and fiber regeneration in PCO, and may lead to innovative avenues of investigation and treatment of PCO.
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