September 2016
Volume 57, Issue 12
Open Access
ARVO Annual Meeting Abstract  |   September 2016
Increased expression and apical distribution of betaA3/A1-Crystallin in polarized RPE cells
Author Affiliations & Notes
  • Hiroto Terasaki
    Arnold and Mabel Beckman Macular Research Center, Doheny Eye Institute, Los Angeles, California, United States
    Ophthalmology, Kagoshima University Graduate School of Medical and Dental Sciences, Kagoshima, Japan
  • Parameswaran G Sreekumar
    Arnold and Mabel Beckman Macular Research Center, Doheny Eye Institute, Los Angeles, California, United States
  • Shozo Sonoda
    Ophthalmology, Kagoshima University Graduate School of Medical and Dental Sciences, Kagoshima, Japan
  • Christine Spee
    Ophthalmology, Keck School of Medicine of the University of Southern California, Los Angeles, California, United States
  • Taiji Sakamoto
    Ophthalmology, Kagoshima University Graduate School of Medical and Dental Sciences, Kagoshima, Japan
  • David R Hinton
    Ophthalmology, Keck School of Medicine of the University of Southern California, Los Angeles, California, United States
    Pathology, Keck School of Medicine of the University of Southern California, Los Angeles, California, United States
  • Ram Kannan
    Arnold and Mabel Beckman Macular Research Center, Doheny Eye Institute, Los Angeles, California, United States
  • Footnotes
    Commercial Relationships   Hiroto Terasaki, None; Parameswaran Sreekumar, None; Shozo Sonoda, None; Christine Spee, None; Taiji Sakamoto, None; David Hinton, None; Ram Kannan, None
  • Footnotes
    Support  EY01545 and a grant from the Arnold and Mabel Beckman Foundation
Investigative Ophthalmology & Visual Science September 2016, Vol.57, 263. doi:
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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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      © 2017 Association for Research in Vision and Ophthalmology.

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Abstract

Purpose : 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.

Methods : 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.

Results : 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.

Conclusions : 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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