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

J Natl Cancer Inst. 2003 Jan 1;95(1):53-66. doi: 10.1093/jnci/95.1.53.

Abstract

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: H4IIE 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 H4IIE 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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Anticarcinogenic Agents / pharmacology*
  • Bacterial Proteins*
  • Blotting, Northern
  • CCAAT-Enhancer-Binding Protein-beta / genetics
  • CCAAT-Enhancer-Binding Protein-beta / metabolism*
  • Carrier Proteins / drug effects*
  • Carrier Proteins / genetics
  • Cell Line
  • Electrophoretic Mobility Shift Assay
  • Enzyme Induction / drug effects
  • Gene Deletion
  • Gene Expression Regulation, Enzymologic / drug effects
  • Genes, Reporter / drug effects
  • Glutathione Transferase / biosynthesis*
  • Glutathione Transferase / metabolism
  • Hepatocytes
  • Immunoblotting
  • Immunohistochemistry
  • Intracellular Signaling Peptides and Proteins
  • Luciferases / drug effects
  • Luciferases / genetics
  • MAP Kinase Kinase 1
  • MAP Kinase Signaling System / drug effects
  • Mitogen-Activated Protein Kinase Kinases / metabolism
  • Phosphatidylinositol 3-Kinases / metabolism*
  • Phosphorylation / drug effects
  • Plasmids / metabolism
  • Protein-Serine-Threonine Kinases / metabolism
  • Pyrazines / pharmacology*
  • RNA, Messenger / drug effects
  • Rats
  • Rats, Inbred Strains
  • Thiones
  • Thiophenes
  • Transfection
  • Translocation, Genetic / drug effects
  • Up-Regulation / drug effects

Substances

  • Anticarcinogenic Agents
  • Bacterial Proteins
  • CCAAT-Enhancer-Binding Protein-beta
  • Carrier Proteins
  • GstA protein, bacteria
  • Intracellular Signaling Peptides and Proteins
  • Pyrazines
  • RNA, Messenger
  • Thiones
  • Thiophenes
  • oltipraz
  • Luciferases
  • Glutathione Transferase
  • Protein-Serine-Threonine Kinases
  • MAP Kinase Kinase 1
  • Mitogen-Activated Protein Kinase Kinases