Essential roles of the PI3 kinase/Akt pathway in regulating Nrf2-dependent antioxidant functions in the RPE.
OBJECTIVE
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 RNA approach 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.
Wang
Ling
L
Vanderbilt Eye Institute, Vanderbilt University Medical Center, Nashville, Tennessee 37232, USA.
Chen
Yan
Y
Sternberg
Paul
P
Cai
Jiyang
J
D000730:Androstadienes / Q000494:pharmacology; D000975:Antioxidants / Q000378:metabolism*; D018929:Cell Culture Techniques; D002867:Chromones / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D015536:Down-Regulation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005721:Glutamate-Cysteine Ligase / Q000235:genetics / Q000378:metabolism; D005978:Glutathione / Q000378:metabolism; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008928:Mitochondria / Q000378:metabolism; D009025:Morpholines / Q000494:pharmacology; D051267:NF-E2-Related Factor 2 / Q000378:metabolism*; D018384:Oxidative Stress; D019869:Phosphatidylinositol 3-Kinases / Q000502:physiology*; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010857:Pigment Epithelium of Eye / Q000378:metabolism*; D010957:Plasmids; D051057:Proto-Oncogene Proteins c-akt / Q000037:antagonists & inhibitors / Q000502:physiology*; D034741:RNA, Small Interfering / Q000494:pharmacology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction / Q000502:physiology*; D000077191:Wortmannin
D000730:Androstadienes; D000975:Antioxidants; D002867:Chromones; D004791:Enzyme Inhibitors; D005721:Glutamate-Cysteine Ligase; D005978:Glutathione; D005982:Glutathione Transferase; D009025:Morpholines; D051267:NF-E2-Related Factor 2; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D051057:Proto-Oncogene Proteins c-akt; D034741:RNA, Small Interfering; D000077191:Wortmannin; C495635:NFE2L2 protein, human; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one