May 2008
Volume 49, Issue 13
Free
ARVO Annual Meeting Abstract  |   May 2008
Functional Interaction Between the PI3K/Akt-Mediated Cell Survival Pathway and Nrf2-Dependent Antioxidant Responses in the RPE
Author Affiliations & Notes
  • L. Wang
    Ophthalmology, Vanderbilt University, Nashville, Tennessee
  • Y. Chen
    Ophthalmology, Vanderbilt University, Nashville, Tennessee
  • P. Sternberg
    Ophthalmology, Vanderbilt University, Nashville, Tennessee
  • J. Cai
    Ophthalmology, Vanderbilt University, Nashville, Tennessee
  • Footnotes
    Commercial Relationships  L. Wang, None; Y. Chen, None; P. Sternberg, None; J. Cai, None.
  • Footnotes
    Support  International Retinal Research Foundation, NIH Grants EY07892 and EY08126 and Research to Prevent Blindness, Inc.
Investigative Ophthalmology & Visual Science May 2008, Vol.49, 226. doi:https://doi.org/
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      L. Wang, Y. Chen, P. Sternberg, J. Cai; Functional Interaction Between the PI3K/Akt-Mediated Cell Survival Pathway and Nrf2-Dependent Antioxidant Responses in the RPE. Invest. Ophthalmol. Vis. Sci. 2008;49(13):226. doi: https://doi.org/.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose: : To investigate the 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 (GST), 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 RNA (siRNA) approach was used to knock down Nrf2 in the RPE. Nrf2 localization was determined by subcellular fractionation and western blot analyses.

Results: : PI3K inhibitors wortmaninn 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 induced Nrf2 activities were inhibited by wortmaninn 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 the Nrf2-ARE-dependent protection against oxidative stress in the RPE.

Keywords: age-related macular degeneration • retinal pigment epithelium • signal transduction 
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