Increased glutathione synthesis through an ARE-Nrf2-dependent pathway by zinc in the RPE

Implication for protection against oxidative stress

Khoi Nguyen Ha, Yan Chen, Jiyang Cai, Paul Sternberg

Research output: Contribution to journalArticle

88 Citations (Scopus)

Abstract

PURPOSE. To determine the molecular mechanisms underlying the protective effects of zinc against oxidative stress in cultured retinal pigment epithelial (RPE) cells. METHODS. Cultured ARPE-19 cells were treated with different concentrations of zinc for various times. Cellular glutathione (GSH) and glutathione disulfide (GSSG) levels were measured by high-performance liquid chromatography (HPLC). Glutamate-cysteine ligase (GCL) expression was measured by quantitative reverse transcription-PCR (RT-PCR). Nuclear factor erythroid2-related factor (Nrf2) activity was measured in a dual luciferase assay after transfection of reporter plasmids containing the antioxidant response element (ARE). The small interference (si)RNA approach was used to knock down the expression of Nrf2. RESULTS. Zinc significantly increased GSH levels in ARPE-19 cells through induction of the de novo synthesis pathway. At 150 μM, zinc increased the GSH level by 70%. At similar concentrations, zinc upregulated the mRNA level of GCL and activated the ARE-Nrf2 pathway. The effects of zinc on ARE activation and GSH synthesis were inhibited by knockdown of Nrf2 expression using the siRNA approach. CONCLUSIONS. Induction of the ARE-Nrf2 pathway by zinc provides powerful and prolonged antioxidation and detoxification that may explain the beneficial effects of zinc observed in the treatment of age-related macular degeneration (AMD).

Original languageEnglish (US)
Pages (from-to)2709-2715
Number of pages7
JournalInvestigative Ophthalmology and Visual Science
Volume47
Issue number6
DOIs
StatePublished - Jun 2006
Externally publishedYes

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Antioxidant Response Elements
Retinal Pigments
Glutathione
Zinc
Oxidative Stress
Glutamate-Cysteine Ligase
Glutathione Disulfide
Macular Degeneration
RNA Interference
Luciferases
Small Interfering RNA
Reverse Transcription
Transfection
Plasmids
Epithelial Cells
High Pressure Liquid Chromatography
Polymerase Chain Reaction
Messenger RNA

ASJC Scopus subject areas

  • Ophthalmology

Cite this

Increased glutathione synthesis through an ARE-Nrf2-dependent pathway by zinc in the RPE : Implication for protection against oxidative stress. / Ha, Khoi Nguyen; Chen, Yan; Cai, Jiyang; Sternberg, Paul.

In: Investigative Ophthalmology and Visual Science, Vol. 47, No. 6, 06.2006, p. 2709-2715.

Research output: Contribution to journalArticle

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abstract = "PURPOSE. To determine the molecular mechanisms underlying the protective effects of zinc against oxidative stress in cultured retinal pigment epithelial (RPE) cells. METHODS. Cultured ARPE-19 cells were treated with different concentrations of zinc for various times. Cellular glutathione (GSH) and glutathione disulfide (GSSG) levels were measured by high-performance liquid chromatography (HPLC). Glutamate-cysteine ligase (GCL) expression was measured by quantitative reverse transcription-PCR (RT-PCR). Nuclear factor erythroid2-related factor (Nrf2) activity was measured in a dual luciferase assay after transfection of reporter plasmids containing the antioxidant response element (ARE). The small interference (si)RNA approach was used to knock down the expression of Nrf2. RESULTS. Zinc significantly increased GSH levels in ARPE-19 cells through induction of the de novo synthesis pathway. At 150 μM, zinc increased the GSH level by 70{\%}. At similar concentrations, zinc upregulated the mRNA level of GCL and activated the ARE-Nrf2 pathway. The effects of zinc on ARE activation and GSH synthesis were inhibited by knockdown of Nrf2 expression using the siRNA approach. CONCLUSIONS. Induction of the ARE-Nrf2 pathway by zinc provides powerful and prolonged antioxidation and detoxification that may explain the beneficial effects of zinc observed in the treatment of age-related macular degeneration (AMD).",
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T1 - Increased glutathione synthesis through an ARE-Nrf2-dependent pathway by zinc in the RPE

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AU - Sternberg, Paul

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N2 - PURPOSE. To determine the molecular mechanisms underlying the protective effects of zinc against oxidative stress in cultured retinal pigment epithelial (RPE) cells. METHODS. Cultured ARPE-19 cells were treated with different concentrations of zinc for various times. Cellular glutathione (GSH) and glutathione disulfide (GSSG) levels were measured by high-performance liquid chromatography (HPLC). Glutamate-cysteine ligase (GCL) expression was measured by quantitative reverse transcription-PCR (RT-PCR). Nuclear factor erythroid2-related factor (Nrf2) activity was measured in a dual luciferase assay after transfection of reporter plasmids containing the antioxidant response element (ARE). The small interference (si)RNA approach was used to knock down the expression of Nrf2. RESULTS. Zinc significantly increased GSH levels in ARPE-19 cells through induction of the de novo synthesis pathway. At 150 μM, zinc increased the GSH level by 70%. At similar concentrations, zinc upregulated the mRNA level of GCL and activated the ARE-Nrf2 pathway. The effects of zinc on ARE activation and GSH synthesis were inhibited by knockdown of Nrf2 expression using the siRNA approach. CONCLUSIONS. Induction of the ARE-Nrf2 pathway by zinc provides powerful and prolonged antioxidation and detoxification that may explain the beneficial effects of zinc observed in the treatment of age-related macular degeneration (AMD).

AB - PURPOSE. To determine the molecular mechanisms underlying the protective effects of zinc against oxidative stress in cultured retinal pigment epithelial (RPE) cells. METHODS. Cultured ARPE-19 cells were treated with different concentrations of zinc for various times. Cellular glutathione (GSH) and glutathione disulfide (GSSG) levels were measured by high-performance liquid chromatography (HPLC). Glutamate-cysteine ligase (GCL) expression was measured by quantitative reverse transcription-PCR (RT-PCR). Nuclear factor erythroid2-related factor (Nrf2) activity was measured in a dual luciferase assay after transfection of reporter plasmids containing the antioxidant response element (ARE). The small interference (si)RNA approach was used to knock down the expression of Nrf2. RESULTS. Zinc significantly increased GSH levels in ARPE-19 cells through induction of the de novo synthesis pathway. At 150 μM, zinc increased the GSH level by 70%. At similar concentrations, zinc upregulated the mRNA level of GCL and activated the ARE-Nrf2 pathway. The effects of zinc on ARE activation and GSH synthesis were inhibited by knockdown of Nrf2 expression using the siRNA approach. CONCLUSIONS. Induction of the ARE-Nrf2 pathway by zinc provides powerful and prolonged antioxidation and detoxification that may explain the beneficial effects of zinc observed in the treatment of age-related macular degeneration (AMD).

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