Essential roles of the PI3 kinase/Akt pathway in regulating Nrf2-dependent antioxidant functions in the RPE

Ling Wang, Yan Chen, Paul Sternberg, Jiyang Cai

Research output: Contribution to journalArticle

117 Citations (Scopus)

Abstract

Purpose. To investigate functional interactions between the phosphatidylinositol 3-kinase (PI3K)/Akt pathway and the nuclear factor erythroid 2-related factor 2 (Nrf2)-dependent antioxidant system in cultured human retinal pigment epithelium (RPE) cells. Methods. Cultured ARPE-19 cells were treated with different concentrations of PI3K inhibitors followed by exposure to sulforaphane, an Nrf2 inducer. Akt phosphorylation was detected by Western blot analysis. Intracellular glutathione (GSH) content was measured by HPLC. Expression of genes downstream of Nrf2 including glutamate-cysteine ligase (GCL) and glutathione S-transferase was measured by quantitative RT- PCR. Nrf2 activity was measured by a dual luciferase assay after transfection of a reporter plasmid containing the antioxidant response element (ARE). The small interference RNAapproach was used to knock down Nrf2 in the RPE. Nrf2 localization was determined by subcellular fractionation and Western blot analyses. Results. PI3K inhibitors wortmannin and LY294002 caused dose-dependent cellular and mitochondrial GSH depletion and downregulation of the modulatory subunit of GCL in cultured RPE cells. Both the basal and the induced Nrf2 activities were inhibited by wortmannin and LY294002. Overexpression of a constitutively active form of Akt potentiated Nrf2 activation and the effect of Akt was blocked by siRNA that knocked down Nrf2. LY294002 also inhibited sulforaphane-induced Nrf2 nuclear translocation. Conclusions. The PI3K/Akt pathway plays key roles in regulating Nrf2-ARE-dependent protection against oxidative stress in the RPE.

Original languageEnglish (US)
Pages (from-to)1671-1678
Number of pages8
JournalInvestigative Ophthalmology and Visual Science
Volume49
Issue number4
DOIs
StatePublished - Apr 2008
Externally publishedYes

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Phosphatidylinositol 3-Kinase
Retinal Pigment Epithelium
Phosphatidylinositol 3-Kinases
2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one
Antioxidants
Antioxidant Response Elements
Glutamate-Cysteine Ligase
Western Blotting
Glutathione Transferase
Luciferases
Small Interfering RNA
Transfection
Glutathione
Oxidative Stress
Plasmids
Down-Regulation
High Pressure Liquid Chromatography
Phosphorylation
Gene Expression
Polymerase Chain Reaction

ASJC Scopus subject areas

  • Ophthalmology
  • Sensory Systems
  • Cellular and Molecular Neuroscience

Cite this

Essential roles of the PI3 kinase/Akt pathway in regulating Nrf2-dependent antioxidant functions in the RPE. / Wang, Ling; Chen, Yan; Sternberg, Paul; Cai, Jiyang.

In: Investigative Ophthalmology and Visual Science, Vol. 49, No. 4, 04.2008, p. 1671-1678.

Research output: Contribution to journalArticle

Wang, Ling ; Chen, Yan ; Sternberg, Paul ; Cai, Jiyang. / Essential roles of the PI3 kinase/Akt pathway in regulating Nrf2-dependent antioxidant functions in the RPE. In: Investigative Ophthalmology and Visual Science. 2008 ; Vol. 49, No. 4. pp. 1671-1678.
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