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Keijiro Ishikawa, Ram Kannan, Christine Spee, Parameswaran G Sreekumar, David R Hinton; Inhibition of αB Crystallin Induces Mesenchymal to Epithelial Transition in RPE cells Through Down-regulation of Snail and Slug. Invest. Ophthalmol. Vis. Sci. 2014;55(13):363.
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Epithelial-to-mesenchymal transition (EMT) of the retinal pigment epithelium (RPE) is a hallmark of proliferative vitreoretinopathy and age-related macular degeneration. The role of small heat shock proteins (sHSPs) in EMT has not been studied. The aim of this study was to investigate the role of αB crystallin, a prominent member of sHSP family, in EMT of RPE cells.
All studies were conducted in cultured human primary fetal RPE cells at two to four passages. We studied the expression changes of αB crystallin after stimulation with TGFβ2 (5ng/ml) by Western blotting and realtime RT-PCR analysis. To examine whether modulation of αB crystallin expression can alter EMT markers, we investigated the effects of αB crystallin silencing on the expression of E-cadherin, α-SMA and the transcriptional factors such as Snail and Slug with or without TGFβ2 by Western blotting and realtime RT-PCR. To examine whether αB crystallin is involved in TGFβ/SMAD signaling pathway, we also studied the effects of αB crystallin knockdown on phosphorylation of SMAD2/3 induced by TGFβ2 stimulation.
The levels of mRNA and protein of αB crystallin were not significantly altered in RPE cells after TGFβ2 stimulation. Suppression of αB crystallin by siRNA induced significant up-regulation of E-cadherin and down-regulation of α-SMA, Snail and Slug in mRNA and protein expressions (p<0.05 vs controls). Knockdown of αB crystallin did not alter TGFβ2-induced phosphorylation of SMAD2/3.
Our results show that αB crystallin plays a critical role through modulation of Snail and Slug in EMT but not through TGFβ/SMAD signaling pathway in this process.
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