Phosphatidylinositol 3 kinase pathway and 4-hydroxy-2-nonenal-induced oxidative injury in the RPE.
OBJECTIVE
4-Hydroxy-2-nonenal (4-HNE) is a major lipid peroxidation product in the retina and the retinal pigment epithelium. The purpose of the present study was to investigate how NF-E2-related factor-2 (Nrf2) and phosphatidylinositol 3 (PI3K) pathways affect the responses of cultured human retinal pigment epithelial (RPE) cells to 4-HNE.


METHODS
Cultured ARPE-19 cells were treated with different concentrations of 4-HNE and a PI3K inhibitor, LY294002. Intracellular glutathione (GSH) was measured by high-performance liquid chromatography (HPLC). The transcriptional activity of Nrf2 was measured by dual luciferase assay after transient transfection with reporter plasmids. The mRNA level of glutamate cysteine ligase (GCL) was quantified by real-time RT-PCR. Formation of HNE adduct on heat shock cognate protein 70 (Hsc70) was measured by immunoprecipitation and Western blot analyses.


RESULTS
Treatment with 4-HNE increased Nrf2 activity and GSH synthesis in a dose-dependent manner in cultured RPE cells. The modulatory subunit of GCL was upregulated by 4-HNE. Antioxidant responses were largely abolished by pretreatment with LY294002. The modification of Hsc70 by 4-HNE was increased when PI3K was inhibited.


CONCLUSIONS
The Nrf2-dependent antioxidant response protects against 4-HNE toxicity, and this protective mechanism is dependent on the functions of the PI3K pathway.
Chen
Jianbin
J
Vanderbilt Eye Institute, Vanderbilt University Medical Center, Nashville, Tennessee 37232, USA.
Wang
Ling
L
Chen
Yan
Y
Sternberg
Paul
P
Cai
Jiyang
J
D000447:Aldehydes / Q000494:pharmacology*; D015153:Blotting, Western; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid; D002867:Chromones / Q000494:pharmacology; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005434:Flow Cytometry; D005721:Glutamate-Cysteine Ligase / Q000235:genetics; D005978:Glutathione / Q000378:metabolism; D050883:HSC70 Heat-Shock Proteins / Q000378:metabolism; D006801:Humans; D009025:Morpholines / Q000494:pharmacology; D051267:NF-E2-Related Factor 2 / Q000378:metabolism; D018384:Oxidative Stress / Q000187:drug effects; D019869:Phosphatidylinositol 3-Kinases / Q000502:physiology*; D000081082:Phosphoinositide-3 Kinase Inhibitors; D012333:RNA, Messenger / Q000378:metabolism; D055213:Retinal Pigment Epithelium / Q000187:drug effects* / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction / Q000502:physiology; D015854:Up-Regulation
D000447:Aldehydes; D002867:Chromones; D015853:Cysteine Proteinase Inhibitors; D004791:Enzyme Inhibitors; D005721:Glutamate-Cysteine Ligase; D005978:Glutathione; D050883:HSC70 Heat-Shock Proteins; D009025:Morpholines; D051267:NF-E2-Related Factor 2; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D012333:RNA, Messenger; C495635:NFE2L2 protein, human; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; C027576:4-hydroxy-2-nonenal