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Keijiro Ishikawa, Parameswaran G Sreekumar, Christine Spee, Ram Kannan, David R Hinton; Inhibition of αB-Crystallin Suppresses Nuclear translocation of SMAD4 Resulting in Mesenchymal to Epithelial Transition in RPE cells.. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):5586.
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© ARVO (1962-2015); The Authors (2016-present)
We previously reported that αB-crystallin plays a critical role through modulation of Snail in epithelial-to-mesenchymal transition (EMT) of the retinal pigment epithelium (RPE). The aim of this study was to investigate the possible role of αB-crystallin as a chaperone for SMAD4, an important effector of EMT in RPE cells.
All studies were conducted in cultured human primary fetal RPE cells at two to four passages. We studied the expression changes of SMAD4 in the cytosolic and nuclear fraction after silencing αB-crystallin with or without TGFβ-2 treatment by Western blotting. To assess possible translocation of SMAD4, we performed immunofluorescent staining in cells after silencing αB-crystallin with or without TGFβ-2. To test if αB-crystallin suppression enhances mono-tetra ubiquitination that can impair nuclear translocation of SMAD4, we isolated ubiquitinated proteins from the cells treated with siRNA and analyzed SMAD4 expression.
Western blotting demonstrated that silencing of αB-crystallin resulted in a decrease in SMAD4 expression in nuclear fraction and an increase in cytosolic fraction of RPE cells. TGFβ-2 (10ng/ml) caused an increase in nuclear expression of SMAD4 with a concomitant decrease in cytosol. Silencing of αB-crystallin inhibited the TGFβ-2-induced expression change of SMAD4 in cytosolic and nuclear fractions. Immunofluorescent staining confirmed the Western blot findings: SMAD4 in both cytosol and nucleus in control and TGFβ-2 induced accumulation of SMAD4 in the nucleus. SMAD4 expression was not seen in the nucleus after silencing αB-crystallin with or without TGF-β2 treatment. In cells transfected with αB-crystallin siRNA, similar levels of expression of polyubiquitinated SMAD4 were found; however, mono-tetra ubiquitinated SMAD4 increased as compared to control siRNA.
Our results suggest that suppression of αB-crystallin could inhibit SMAD4 nuclear translocation which could be mediated by mono- tetra ubiquitination in RPE cells.
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