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Hiroto Terasaki, Parameswaran G Sreekumar, Shozo Sonoda, Christine Spee, Taiji Sakamoto, David R Hinton, Ram Kannan; Increased expression and apical distribution of betaA3/A1-Crystallin in polarized RPE cells. Invest. Ophthalmol. Vis. Sci. 2016;57(12):263.
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© ARVO (1962-2015); The Authors (2016-present)
While recent studies have shown that alpha-crystallins play an important role in retinal function, not much is known about expression and function of beta-crystallins in the retinal pigment epithelium (RPE). The purpose of this study was to investigate the expression of betaA3/A1-crystallin in human RPE cells and the effect of polarity and oxidative stress on its expression.
Polarized human fetal RPE cells with a mean TER of 385 ± 13.1 Ω･cm2 were cultured following an established protocol in our laboratory (Sonoda et al. Nature protocols 2009). Initial microarray analysis of polarized and non-polarized H-RPE cells (Lonza, Walkersville, MD) was carried out using a commercial array service (Agilent Expression Array; Takara Bio, Yokkaichi, Japan). Expression of betaA3/A1-crystallin in polarized and non-polarized human RPE cells was assessed by western blot and real-time PCR. Apical/basolateral localization of beta A3/A1 crystallin was determined by immunostaining. To study the effect of oxidative stress on expression of betaA3/A1 crystallin, polarized RPE cell were exposed to 500uM of H2O2 for 24 h and expression was determined by western blot and real-time PCR analysis.
Microarray analysis revealed that, among crystallins, betaA3/A1 crystallin was highly expressed in polarized RPE cells (about 6.5 times higher compared to non-polarized RPE cells). This finding was confirmed by western blot, confocal microscopy and real-time PCR which showed higher expression of betaA3/A1 crystallin in polarized RPE cells while non-polarized RPE cells had negligible expression. Oxidative stress caused a decrease in gene and protein expression of betaA3/A1 crystallin in polarized RPE cells.
BetaA3/A1 crystallin was highly expressed in polarized RPE cells and its expression decreased with oxidative stress. The increased expression in the apical domain suggests a possible role for betaA3/A1 crystallin to protect the neural retina.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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