Selenomethionine (Se-Met) Induces the Cystine/Glutamate Exchanger SLC7A11 in Cultured Human Retinal Pigment Epithelial (RPE) Cells: Implications for Antioxidant Therapy in Aging Retina

Abstract

Oxidative damage has been identified as a major causative factor in degenerative diseases of the retina; retinal pigment epithelial (RPE) cells are at high risk. Hence, identifying novel strategies for increasing the antioxidant capacity of RPE cells, the purpose of this study, is important. Specifically, we evaluated the influence of selenium in the form of selenomethionine (Se-Met) in cultured RPE cells on system xc- expression and functional activity and on cellular levels of glutathione, a major cellular antioxidant. ARPE-19 and mouse RPE cells were cultured with and without selenomethionine (Se-Met), the principal form of selenium in the diet. Promoter activity assay, uptake assay, RT-PCR, northern and western blots, and immunofluorescence were used to analyze the expression of xc-, Nrf2, and its target genes. Se-Met activated Nrf2 and induced the expression and function of xc- in RPE. Other target genes of Nrf2 were also induced. System xc- consists of two subunits, and Se-Met induced the subunit responsible for transport activity (SLC7A11). Selenocysteine also induced xc- but with less potency. The effect of Se-met on xc- was associated with an increase in maximal velocity and an increase in substrate affinity. Se-Met increased the cellular levels of glutathione in the control, an oxidatively stressed RPE. The Se-Met effect was selective; under identical conditions, taurine transport was not affected and Na+-coupled glutamate transport was inhibited. This study demonstrates that Se-Met enhances the antioxidant capacity of RPE by inducing the transporter xc- with a consequent increase in glutathione.

Keywords: SLC7A11; age-related macular degeneration (AMD), antioxidants; glutathione; oxidative stress; retina; retinal pigment epithelium (RPE); selenium; selenomethionine (Se-Met); system xc; xCT.

Figure 1

Selenomethionine (Se-Met)-induced upregulation of Nrf2 in ARPE-19 cells: (A) immunocytochemical analysis of Nrf2 protein in ARPE-19 cells cultured in the presence (+Se-Met) or absence (−Se-Met) of selenomethionine (1 mM, 16 h), where cells in which PBS was substituted for the primary antibody served as the negative control (Neg. Con); (B) western blot analysis of Nrf2 in control and Se-Met-treated ARPE-19 cells; (C) RT-PCR analysis of mRNA transcripts specific for xCT (SLC7A11) and 4F2hc in control (−Se-Met) and Se-Met-treated ARPE-19 cells; and (D) RT-PCR analysis of Nrf2 target genes: glutamate-cysteine ligase catalytic subunit (GCLC), glutamate-cysteine ligase regulatory subunit (GCLM), heme oxygenase-1 (HO-1), glutathione S-transferase A1 (GSTA1), glutathione S-transferase A2 (GSTA2), thioredoxin reductase (TXNRD), glutathione peroxidase (GPX), and NAD(P)H oxidase: quinone 1 (NQO1), in ARPE-19 cells cultured in the presence (+Se-Met) or absence (−Se-Met) of selenomethionine (1 mM, 16 h).

Figure 2

Se-Met-induced expression of xCT (SLC7A11): (A) luciferase reporter assay of Se-Met (1 mM)-induced SLC7A11 (xCT) promoter activation in in ARPE-19 cells transfected with luciferase reporter constructs containing either the short or long form of SLC7A11 promoter (* p < 0.001 compared to the control (−Se-Met) SLC7A11 promoter-long cells); (B) northern blot analysis of xCT and 4F2hc expression in control and Se-Met-treated ARPE-19 cells; (C) immunocytochemical analysis of xCT protein in ARPE-19 cells cultured in the presence (+Se-Met) or absence (−Se-Met) of selenomethionine (1 mM, 16 h), where cells in which PBS was substituted for primary antibody served as the negative control (Neg. Con); and (D) western blot analysis of xCT protein in plasma membrane versus cytosolic protein fractions isolated from ARPE-19 cells cultured in the presence or absence of Se-Met (1 mM, 16 h).

Figure 3

Selenomethionine (Se-Met)-induced stimulation of xc- transport activity is time- and dose-dependent. (A) Uptake of [3H]-glutamate (2.5 μM) was measured for 15 min in ARPE-19 cells cultured in the presence or absence of Se-Met (1 mM) for 0–16 h. Uptake was monitored at 37 °C in the absence of Na+. (B) Na+-independent [3H]-glutamate (2.5 μM) uptake was measured for 15 min in ARPE-19 cells cultured in the presence or absence of varying concentrations (0–2500 μM) of selenomethionine (Se-Met) or methionine (Met) for 16 h. (* p < 0.05; ** p < 0.01; compared to corresponding concentration of Met). (C) Cell viability was determined by trypan blue exclusion assay.

Figure 4

Substrate specificity of system xc- in control and selenomethionine (Se-Met)-treated ARPE-19 cells: uptake of [3H]-glutamate (2.5 μM) was measured in control and Se-Met-treated ARPE-19 cells in the absence of Na+ for 15 min at 37 °C and in the presence or absence of excess unlabeled amino acids, each at a concentration of 5 mM (* p < 0.01; ** p < 0.001).

Figure 5

Differential influence of selenoamino acids (Se-Met and Se-Cys) and sulfur amino acids (Met and Cys) on system xc- transport activity: ARPE-19 cells were cultured in the presence or absence of selenomethionine (Se-Met), selenocysteine (Se-Cys), methionine (Met), and cysteine (Cys), each at 1 mM, for 16 h. Uptake of [3H]-glutamate (2.5 μM) was measured for 15 min at 37 °C in the presence and absence of Na+: (A) Na+-independent, xc- -specific glutamate uptake; (B) total glutamate uptake; and (C) Na+ -dependent, excitatory amino acid transporter (EAAT)-specific glutamate uptake. (* p < 0.05, ** p < 0.01 compared to control).

Figure 6

Kinetic analysis of system xc- activity in the presence or absence of Se-Met in ARPE-19 cells: saturation kinetics of glutamate uptake in ARPE-19 cells cultured for 16 h in the presence or absence of 1 mM selenomethionine (Se-Met) was evaluated by monitoring the uptake of [3H]-glutamate in Na+-free medium with various concentrations of unlabeled glutamate (0–2000 μM). Inset: Eadie–Hofstee plot.

Figure 7

Se-Met raises intracellular levels of glutathione (GSH) protecting ARPE-19 cells against H₂O₂-induced GSH depletion. Oxidative stress was induced in ARPE-19 cells using hydrogen peroxide (H₂O₂, 500 μM) in the presence or absence of Se-Met (1 mM). Intracellular levels of glutathione (GSH) were then measured using the GSH-Glo Assay kit (Promega). (* p < 0.01; ** p < 0.001).

Figure 8

Induction of xc- by selenomethionine (Se-Met) in mouse primary RPE cells: Primary mouse RPE cells were cultured in the presence or absence of selenomethionine (Se-Met, 1 mM) for 16 h followed by measurement of [3H]-glutamate (2.5 μM) or [3H]-taurine (25 nM) uptake (15 min, 37 °C) in the presence and absence of Na+. (A) Na+-independent, (xc-)-specific glutamate uptake; (B) Na+-dependent, excitatory amino acid transporter (EAAT)-specific glutamate uptake; and (C) Na+-dependent taurine (TAUT) uptake. (* p < 0.01).