Essential Roles of the PI3 Kinase/Akt Pathway in Regulating Nrf2-Dependent Antioxidant Functions in the RPE

Cumulative oxidative injury is an important environmental factor contributing to the development and progression of age-related macular degeneration (AMD).1-3 In animal models of chronic oxidative stress, the retina and retinal pigment epithelium (RPE) develop pathologic lesions that are characteristic of early AMD.4-8 In the Age-Related Eye Disease Study (AREDS), supplementation with antioxidant vitamins, zinc, or both was shown to significantly reduce the risk for progression of AMD.9 Characterizing the antioxidant defense system and its regulatory mechanisms will be essential in defining the vulnerability of the retina to oxidative injury and in developing new treatment strategies for AMD. Nuclear factor erythroid 2-related factor 2 (Nrf2) is commonly involved in the transcriptional regulation of genes encoding antioxidant proteins under stress conditions.10,11 It heterodimerizes with members of the small Maf family of transcription factors and binds to the cis-acting antioxidant response element (ARE) sequence in the promoter regions of various phase 2 detoxification genes. The latter encode for a group of enzymes, such as glutamate-cysteine ligase (GCL) and glutathione S-transferase (GST), essential in the detoxification of xenobiotics and endogenous reactive electrophilic compounds.12,13 Agents that activate Nrf2 can protect RPE cells from oxidative injury.14-17 The phosphatidylinositol 3-kinase (PI3K)/Akt pathway plays a major role in cell survival signaling and is implicated in a plethora of biological responses such as apoptosis, cell growth, differentiation, calcium signaling, and insulin signaling.18,19 The PI3K consists of an 85-kDa and a 110-kDa subunit; once activated, it phosphorylates phosphatidylinositol at the D-3 position of the inositol ring.20 LY294002 and wortmannin are two highly specific inhibitors that block PI3K activity through direct interaction with the p85 subunit.21,22 Oxidative stress has been shown to regulate the PI3K/Akt and, consequently, to alter the downstream signaling events in cultured RPE cells.23-25 The interactions between the Nrf2-dependent antioxidant system and other cellular signaling pathways have not been well characterized in the retina. Previous studies using several cancer cell lines reported that PI3K and Akt functions are required for Nrf2 activation by various inducers such as tert-butyl hydroquinone (tBH), hemin, and peroxynitrite.26-28 In the present study, we investigated the functional interactions between the PI3K/Akt pathway and Nrf2 in cultured ARPE-19 cells. Our results indicated that exposure of the ARPE cells to PI3K inhibitors caused decreased Nrf2 activity, downregulation of GCL and GST, and the consequent reduction of cellular and mitochondrial GSH contents. On the other hand, cells overexpressing constitutively active Akt showed enhanced Nrf2 activity and increased response to sulforaphane. The data suggest that the PI3K/Akt pathway is essential in regulating the Nrf2-ARE-dependent protection against oxidative stress in the RPE.