Abstract
Purpose:
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.
Methods:
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.
Results:
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.
Conclusions:
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.
Keywords: 512 EMT (epithelial mesenchymal transition) •
701 retinal pigment epithelium •
655 proliferative vitreoretinopathy