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C Kathleen Dorey, Michael Ferguson, Matthew Gray, Kristin Knight; Zeaxanthin Increases Glutatione-S-Transferase (Pi isoform) Expression in ARPE-19 cells. Invest. Ophthalmol. Vis. Sci. 2019;60(9):1931. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
GSTP1 is a ubiquitously expressed phase II antioxidant enzyme that reverses membrane oxidation and detoxifies xenobiotics by conjugation with glutathione. Through glutathionylation of KEAP1 and release of Nrf2, GSTP1 also has the capacity to sustain production of protective phase II enzymes and thus limit oxidative damage. Zeaxanthin is accumulated in macular pigment by selective binding to GSTP1, which also increases the activity of GSTP1 several fold. These experiments tested the hypothesis that zeaxanthin delivered by human HDL induces expression of GSTP1 and/or scavenger receptor B1 (SRB1) in ARPE-19 cells.
ARPE-19 cells were cultured in DMEM/F12 media plus 10% FBS for 8-10 weeks postconfluence, then exposed to DMEM/F12 supplemented with 2% FBS and vehicle, human donor HDL plus vehicle, or HDL enriched with zeaxanthin by overnight swirling at 4o. Dose responses to HDL enriched with zeaxthin in DMSO or ethanol were compared, and further experiments were conducted in ethanol. After 48 hours exposure, cells were rinsed with PBS and harvested by scraping in cold RIPA buffer, run on 4-20% Western gels and transferred to a membrane, incubated in rabbit anti-GSTP1 (Invitrogen) or rabbit anti-SRB1 (AbCam), then HRP-tagged secondary antibody and HRP-tagged goat anti-actin. Chemiluminescence was imaged with Bio-Rad ChemiDot XRS. GSTP1, SRB1, and actin band areas were quantified using ImageJ software. Data were analyzed by student t-test.
Protein expression of GSTP1 was increased 3.5-fold (P=0.02) and that of SRB1 protein was increased more than 2.5-fold (P=0.04) in ARPE-19 cells exposed to HDL enriched with 200 nM zeaxanthin than in those exposed to native human donor HDL. Donor HDL alone tended to increase expression of GSTP1 but had no effect on SRB1 expression. These data suggest that zeaxanthin carried by donor HDL was sufficient to induce GSTP1 expression and that increasing the amount of zeaxanthin carried on HDL induces greater expression of GSTP1. Further experiments are necessary to evaluate whether increased SRB1 protein resulted in greater delivery of zeaxanthin.
Physiological levels of zeaxanthin induce GSTP1 expression in ARPE-19 cells. These data expand our understanding of the role of zeaxanthin in the RPE and retina and indicate that zeaxanthin’s protective role extends beyond its own antioxidant activity by inducing GSTP1, an important antioxidant enzyme.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
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