Oltipraz-induced GSTA2 gene expression is dependent upon PI3-kinase-mediated nuclear translocation and binding of C/EBPbeta to the C/EBP response element in the GSTA2 gene promoter.

Arachidonic acid (AA, a proinflammatory fatty acid) in combination with iron promotes excess reactive oxygen species (ROS) production and exerts a deleterious effect on mitochondria. We have shown previously that activation of AMP-activated protein kinase (AMPK) protects hepatocytes from AA ϩ iron-induced apoptosis. Resveratrol, a polyphenol in grapes, has beneficial effects mediated through SIRT1, LKB1, and AMPK. This study investigated the potential of resveratrol to protect against the mitochondrial impairment induced by AA ϩ iron and the underlying mechanism for this cytoprotection. Resveratrol treatment inhibited apoptosis, ROS production, and glutathione depletion elicited by AA ϩ iron in HepG2 cells. In addition, resveratrol attenuated superoxide generation in mitochondria and inhibited mitochondrial dysfunction induced by AA ϩ iron. Overall, AMPK activation by resveratrol contributed to cell survival, as supported by the reversal of its restoration of mitochondrial membrane potential by either overexpression of a dominant-negative mutant of AMPK␣ or compound C treatment. Resveratrol increased inhibitory phosphorylation of glycogen synthase kinase-3␤ (GSK3␤) downstream of AMPK, which contributed to mitochondrial protection and cell survival. Likewise, small interfering RNA knockdown of LKB1, an upstream kinase of AMPK, reduced the ability of resveratrol to protect cells from mitochondrial dysfunction. Furthermore, this LKB1-dependent mitochondrial protection resulted from resveratrol's poly(ADP-ribose)polymerase activation, but not SIRT1 activation, as supported by the experiment using 3-aminobenzamide, a poly(ADP-ribose)polymerase inhibitor. Other polyphenols, such as apigenin, genistein, and daidzein, did not activate AMPK or protect mitochondria against AA ϩ iron. Thus, resveratrol protects cells from AA ϩ iron-induced ROS production and mitochondrial dysfunction through AMPKmediated inhibitory phosphorylation of GSK3␤ downstream of poly(ADP-ribose)polymerase-LKB1 pathway.

No abstract

ABSTRACT

The expression of the glutathione S-transferase gene (GST), whose induction accounts for cancer chemoprevention, is regulated by activation of CCAAT/enhancer binding protein ␤ (C/EBP␤) and NF-E2-related factor 2 (Nrf2). The present study investigated the repressing effects of activating glucocorticoid receptor (GR) on C/EBP␤-and Nrf2-mediated GSTA2 gene induction and the mechanism. Dexamethasone that activates GR inhibited constitutive and oltipraz-or tert-butylhydroquinone (t-BHQ)-inducible GSTA2 expression in H4IIE cells. Also, dexamethasone repressed GSTA2 promoter-luciferase gene activity. Dexamethasone-GR activation did not inhibit nuclear translocation of C/EBP␤ or Nrf2 nor their DNA binding activities induced by oltipraz or t-BHQ. Deletion of the glucocorticoid response element (GRE) in the GSTA2 promoter abolished dexamethasone inhibition of the gene induction. Immunoprecipitation-immunoblotting, chromatin immunoprecipitation, and GST pull-down assays revealed that silencing mediator for retinoid and thyroid hormone receptors (SMRT), a corepressor recruited to steroid-GR complex for histone deacetylation, bound to TAD domain of C/EBP␤ and Neh4/5 domain of Nrf2. The GSTA2 promoter-luciferase activities were decreased by SMRT but not by truncated SMRTs. The small interference RNA (siRNA) against SMRT abolished SMRT repression of the gene induction by C/EBP␤ or Nrf2. The plasmid transfection and siRNA experiments directly evidenced the functional role of SMRT in GSTA2 repression. In conclusion, dexamethasone antagonizes C/EBP␤-and Nrf2-mediated GSTA2 gene induction via ligand-GR binding to the GRE, and steroid-mediated GSTA2 repression involves inactivation of C/EBP␤ and Nrf2 by SMRT recruited to steroid-GR complex.