title: Essential role of phosphatidylinositol 3-kinase-dependent CCAAT/enhancer binding protein beta activation in the induction of glutathione S-transferase by oltipraz.
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.
authors.affiliation: National Research Laboratory, College of Pharmacy and Research Institute of Pharmaceutical Sciences, Seoul National University, Korea.
authors.forename: Keon Wook
authors.initials: KW
authors.lastname: Kang
authors.forename: Il Je
authors.initials: IJ
authors.lastname: Cho
authors.forename: Chang Ho
authors.initials: CH
authors.lastname: Lee
authors.forename: Sang Geon
authors.initials: SG
authors.lastname: Kim
mesh_terms: D000818:Animals; D016588:Anticarcinogenic Agents / Q000494:pharmacology*; D001426:Bacterial Proteins; D015152:Blotting, Northern; D022782:CCAAT-Enhancer-Binding Protein-beta / Q000235:genetics / Q000378:metabolism*; D002352:Carrier Proteins / Q000187:drug effects* / Q000235:genetics; D002460:Cell Line; D024202:Electrophoretic Mobility Shift Assay; D004790:Enzyme Induction / Q000187:drug effects; D017353:Gene Deletion; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D017930:Genes, Reporter / Q000187:drug effects; D005982:Glutathione Transferase / Q000096:biosynthesis* / Q000378:metabolism; D022781:Hepatocytes; D015151:Immunoblotting; D007150:Immunohistochemistry; D047908:Intracellular Signaling Peptides and Proteins; D008156:Luciferases / Q000187:drug effects / Q000235:genetics; D048369:MAP Kinase Kinase 1; D020935:MAP Kinase Signaling System / Q000187:drug effects; D020929:Mitogen-Activated Protein Kinase Kinases / Q000378:metabolism; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D010957:Plasmids / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D011719:Pyrazines / Q000494:pharmacology*; D012333:RNA, Messenger / Q000187:drug effects; D051381:Rats; D011919:Rats, Inbred Strains; D013871:Thiones; D013876:Thiophenes; D014162:Transfection; D014178:Translocation, Genetic / Q000187:drug effects; D015854:Up-Regulation / Q000187:drug effects
chemical_list: D016588:Anticarcinogenic Agents; D001426:Bacterial Proteins; D022782:CCAAT-Enhancer-Binding Protein-beta; D002352:Carrier Proteins; D005982:Glutathione Transferase; D047908:Intracellular Signaling Peptides and Proteins; D008156:Luciferases; D048369:MAP Kinase Kinase 1; D020929:Mitogen-Activated Protein Kinase Kinases; D019869:Phosphatidylinositol 3-Kinases; D017346:Protein Serine-Threonine Kinases; D011719:Pyrazines; D012333:RNA, Messenger; D013871:Thiones; D013876:Thiophenes; C105760:GstA protein, bacteria; C026209:oltipraz