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Anna Korol, AFTAB TAIYAB, Judith A West-Mays; Transforming Growth Factor β (TGFβ) regulates stress fiber formation in lens epithelial cells through Fascin. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):2647.
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© ARVO (1962-2015); The Authors (2016-present)
Posterior Capsular Opacification (PCO) or secondary cataract is a common postoperative complication after cataract surgery. Numerous studies have shown TGFβ-induced epithelial to mesenchymal transition (EMT) is a main contributor to PCO. However, the signaling events that lead to the cytoskeletal changes observed during TGFβ-induced EMT and PCO remains poorly understood. Here, we have examined the cellular distribution and expression of fascin, an actin bundling protein, in rat lens epithelial explants following TGFβ stimulation. We further examine the relationship of fascin with Matrix Metalloproteinases (MMP) -2 and -9 and β-catenin, two previously identified regulators of EMT in the lens.
Wistar rats (P17 and P19) were sacrificed by asphyxiation and their eyes were removed. Lens epithelial explants were isolated and cultured in medium M199 in the presence and absence of TGFβ2 (6 ng/ml), and TGFβ2 in combination with MMP2/9 inhibitor II or β-catenin inhibitor (PNU-74654; 20 µM) for 48 hrs (n=6 per treatment). Western blot and Immunofluorescence analyses were carried out using mouse anti-fascin antibody, mouse anti-GAPDH antibody, and a Rhodamine Phalloidin stain for F-actin.
An increase in the expression of fascin was observed in rat lens epithelial explants upon stimulation with TGFβ as determined by Western blot and Immunofluorescence analysis. Further, fascin was observed to be organized in filaments which co-localized with F-actin. Inhibition of MMP2/9 activity, as well as abrogation of β-catenin transcriptional activity led to a decrease in the expression of fascin. Additionally, inhibition of MMP2/9 also led to a decrease in the formation of stress fibers as determined by F-actin staining.
The induced expression of fascin by TGFβ suggests the possible role of fascin in formation of actin bundles in stress fiber formation during EMT in the lens. The decrease in TGFβ-induced expression of fascin along with a decrease in the stress fiber formation upon inhibition of MMP2/9, as well as β-catenin activity, demonstrates that fascin is downstream of these regulators. Further experiments will elucidate the signal integration between MMP2/9 and β-catenin in the regulation of fascin during TGFβ-induced EMT using rat lens epithelial explants.
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