The Essential Role of Phosphatidylinositol 3-Kinase and of p38 Mitogen-Activated Protein Kinase Activation in the Antioxidant Response Element-Mediated rGSTA2 Induction by Decreased Glutathione in H4IIE Hepatoma Cells

Abstract

The protective adaptive response to electrophiles and reactive oxygen species is mediated by the enhanced expression of the phase II detoxifying genes through antioxidant response elements (AREs). The current study was designed to identify the signaling pathways responsible for the expression of rGSTA2 in response to cellular oxidative stress and to establish the molecular mechanistic basis. Deprivation of cystine and methionine caused oxidative stress in H4IIE hepatoma cells as evidenced by a marked decrease in the reduced glutathione (first order rate constant = 0.056 h(-1); t(1/2) = 12.6 h) and an increase in pro-oxidant production. Electrophoretic mobility shift assay revealed that the ARE complex, consisting of Nrf-1/2 and Maf proteins, was activated 12 to 48 h after sulfur amino acid deprivation (SAAD). The rGSTA2 mRNA level was elevated by SAAD beginning at 24 h, whereas the rGSTA2 subunit was maximally induced at 48 h. Nuclear ARE activation and rGSTA2 mRNA increase were both completely inhibited by wortmannin or LY294002, the phosphatidylinositol 3-kinase (PI3-kinase) inhibitors. The p38 mitogen-activated protein (MAP) kinase was activated at 0.5 to 3 h after SAAD, followed by sustained diminished activation up to 12 h. Inhibition of p38 MAP kinase by SB203580 prevented the ARE-mediated rGSTA2 induction. The activation of p38 MAP kinase, however, failed to be inhibited by wortmannin or LY294002, showing that PI3-kinase is not involved in the activation of p38 MAP kinase. Data showed that PI3-kinase plays an essential role in the ARE-mediated rGSTA2 induction by oxidative stress after SAAD, which activates the p38 MAP kinase and leads to rGSTA2 induction.

The Keap1 BTB/POZ Dimerization Function Is Required to Sequester Nrf2 in Cytoplasm

“…The current experiments demonstrate that Keap1 complex disruption in vivo occurs through an Erk-and p38-independent mechanism, thereby ruling out an Erk-or p38-dependent phosphorylation event as the proximate triggering signal (45). Other kinases such as protein kinase C (53) and phosphatidylinositol 3-kinase (54,55) have also been implicated in Nrf2 induction, leaving open the possibility that kinases activated by oxidant exposure provide the initiating signal for Keap1 complex disruption and Nrf2 release, although this remains to be demonstrated.…”

Transactivation of the PPAR-Responsive Enhancer Module in Chemopreventive Glutathione S -Transferase Gene by the Peroxisome Proliferator-Activated Receptor-γ and Retinoid X Receptor Heterodimer

“…The protein binding to the ARE consensus sequence involves the Nrf proteins, as well as Maf family members (6,7). Oxidative stress induces GSTA2 via ARE activation, which involves the Nrf proteins (8,9). Transcription factors of the CCAAT/enhancer binding protein (C/EBP) family play roles in cell differentiation and exert their function by regulating the expression of tissue-specific genes, as well as cell proliferation (10,11).…”

NRF2 and Age-Dependent RPE Degeneration

“…Results from the functional studies consistently showed that inhibition of the PI3K/Akt pathway decreased NRF2 activation induced by a variety of stimuli in different cell lines, while expression of a constitutive active mutant of Akt increased NRF2 activity, indicating that PI3K/Akt signalling is a positive regulator of NRF2 (Chen et al, 2009;Jain and Jaiswal, 2006;Kang et al, 2000;Lee et al, 2001;Wang et al, 2008). PI3K/Akt controls NRF2 via multiple indirect mechanisms.…”