Essential role of phosphatidylinositol 3-kinase-dependent CCAAT/enhancer binding protein β activation in the induction of glutathione S-transferase by oltipraz

Background: Cancer chemopreventive agents transcriptionally induce genes whose protein products can protect cells from chemical-induced carcinogenesis. Oltipraz, a dithiolthione, exerts chemopreventive responses through glutathione S-transferase (GST) induction. We investigated the role of the CCAAT/enhancer binding protein (C/EBP) in the induction of the GSTA2 gene (alpha class) by oltipraz and identified the enhancer element(s) responsible for GSTA2 gene expression. Methods: H411E rat hepatocyte-derived cells were treated with oltipraz, and GSTA2 expression was determined by northern and immunoblot analyses. The activation of C/EBPbeta and alpha forms and NF-E2-related factor 2 (Nrf2) was assessed by immunochemical assays. C/EBPbeta-DNA binding activity was determined by subcellular fractionation and electrophoretic mobility shift assays. The role of the C/EBP binding site in the induction of the GSTA2 gene was assessed by luciferase reporter-gene activity. The role of phosphatidylinositol 3-kinase (PI3-kinase) and mitogen-activated protein (MAP) kinase signaling pathways in C/EBP-mediated GSTA2 induction was studied by using chemical inhibitors, overexpression vectors, and dominant-negative mutants. All statistical tests were two-sided. Results: Oltipraz induced GSTA2 mRNA and protein expression. In oltipraz-treated cells, C/EBPbeta translocated to the nucleus and bound to the consensus sequence of C/EBP (TTGCGCAA). Oltipraz treatment increased luciferase reporter-gene activity in H411E cells transfected with the C/EBP-containing regulatory region of the GSTA2 gene. Deletion of the C/EBP binding site or overexpression of a dominant-negative mutant form of C/EBP (AC/EBP) abolished the reporter gene activity. PI3-kinase, but not MAP kinases, was required for C/EBPbeta-dependent induction of GSTA2 by oltipraz. Conclusions: 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.