Phosphatidylinositol 3 kinase pathway and 4-hydroxy-2-nonenal-induced oxidative injury in the RPE

Jianbin Chen, Ling Wang, Yan Chen, Paul Sternberg, Jiyang Cai

Research output: Contribution to journalArticle

21 Citations (Scopus)

Abstract

PURPOSE. 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.

Original languageEnglish (US)
Pages (from-to)936-942
Number of pages7
JournalInvestigative Ophthalmology and Visual Science
Volume50
Issue number2
DOIs
StatePublished - Feb 2009
Externally publishedYes

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Phosphatidylinositol 3-Kinase
Retinal Pigments
NF-E2-Related Factor 2
Phosphatidylinositol 3-Kinases
Wounds and Injuries
HSC70 Heat-Shock Proteins
Glutamate-Cysteine Ligase
2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one
Antioxidants
Epithelial Cells
Retinal Pigment Epithelium
Phosphatidylinositols
4-hydroxy-2-nonenal
Luciferases
Immunoprecipitation
Lipid Peroxidation
Transfection
Glutathione
Retina
Real-Time Polymerase Chain Reaction

ASJC Scopus subject areas

  • Ophthalmology
  • Sensory Systems
  • Cellular and Molecular Neuroscience
  • Medicine(all)

Cite this

Phosphatidylinositol 3 kinase pathway and 4-hydroxy-2-nonenal-induced oxidative injury in the RPE. / Chen, Jianbin; Wang, Ling; Chen, Yan; Sternberg, Paul; Cai, Jiyang.

In: Investigative Ophthalmology and Visual Science, Vol. 50, No. 2, 02.2009, p. 936-942.

Research output: Contribution to journalArticle

Chen, Jianbin ; Wang, Ling ; Chen, Yan ; Sternberg, Paul ; Cai, Jiyang. / Phosphatidylinositol 3 kinase pathway and 4-hydroxy-2-nonenal-induced oxidative injury in the RPE. In: Investigative Ophthalmology and Visual Science. 2009 ; Vol. 50, No. 2. pp. 936-942.
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N2 - PURPOSE. 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.

AB - PURPOSE. 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.

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