Essential role of phosphatidylinositol 3-kinase-dependent CCAAT/enhancer binding protein beta 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
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.
Kang
Keon Wook
KW
National Research Laboratory, College of Pharmacy and Research Institute of Pharmaceutical Sciences, Seoul National University, Korea.
Cho
Il Je
IJ
Lee
Chang Ho
CH
Kim
Sang Geon
SG
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
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