Ceramide, an apoptotic rheostat, inhibits CCAAT/enhancer binding protein-beta and NF-E2-related factor-2 activation: the role in glutathione S-transferase A2 gene repression.
Ceramide is a sphingolipid that acts as a second messenger in signaling systems. Sphingomyelinase generates ceramide in response to cytotoxic stimuli. CCAAT/enhancer binding protein-beta (C/EBPbeta) and NF-E2-related factor-2 (Nrf2) are both involved in the regulation of the genes encoding phase II detoxification enzymes including glutathione S-transferase (GST). In the present study, we examined the effects of ceramide on C/EBPbeta or Nrf2 activation and on the inducible GSTA2 gene transactivation. C2-ceramide (C2), a cell-permeable analog, inhibited GSTA2 induction by oltipraz or tert-butylhydroquinone (t-BHQ) in H4IIE cells, whereas dihydro-C2-ceramide (dihydro-C2), an inactive analog, had no effect. Immunoblot analysis revealed that C2 prevented increase in the level of nuclear C/EBPbeta by oltipraz, whereas the level of C/EBPbeta in total cell lysates was not changed. Increase in nuclear Nrf2 by t-BHQ was also prevented by C2 treatment. Decreases in nuclear C/EBPbeta and Nrf2 by C2 were reversed by treatment of cells with N-benzoyloxycarbonyl (Z)-Leu-Leu-leucinal (MG132), a proteasome inhibitor, verifying the previous observations that the transcription factors were degraded by the proteasome system. In another study, we found that ceramide decreased nuclear hepatic nuclear factor-1 (HNF1), whose binding to the HNF1-response element in the GSTA2 gene was responsible for the constitutive and inducible gene expression. To define the role of C/EBPbeta or Nrf2 repression in GST expression under the condition excluding the negative regulation by C2-mediated HNF1 suppression, luciferase activity was determined in the cells transfected with DeltaHNF-pGL-1651 plasmid lacking the HNF1-response element. In the cells transfected with DeltaHNF-pGL-1651, C2 decreased the luciferase induction by oltipraz or t-BHQ. Thus, ceramide inhibits C/EBPbeta or Nrf2 activation, which contributes to repression of GSTA2 gene transactivation.
National Research Laboratory, College of Pharmacy and Research Institute of Pharmaceutical Sciences, Seoul National University, South Korea.
I-Na
IN
Park
Il Je
IJ
Cho
Sang Geon
SG
Kim
D000818:Animals; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology; D022782:CCAAT-Enhancer-Binding Protein-beta / Q000037:antagonists & inhibitors* / Q000187:drug effects / Q000378:metabolism; D002460:Cell Line; D002518:Ceramides / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D004268:DNA-Binding Proteins / Q000037:antagonists & inhibitors* / Q000187:drug effects / Q000378:metabolism; D020868:Gene Silencing / Q000187:drug effects* / Q000502:physiology; D005982:Glutathione Transferase / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism*; D022781:Hepatocytes / Q000166:cytology; D006873:Hydroquinones / Q000037:antagonists & inhibitors / Q000494:pharmacology; D007527:Isoenzymes / Q000235:genetics*; D051267:NF-E2-Related Factor 2; D011719:Pyrazines / Q000037:antagonists & inhibitors / Q000494:pharmacology; D051381:Rats; D013107:Sphingolipids / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D013871:Thiones; D013876:Thiophenes; D015534:Trans-Activators / Q000037:antagonists & inhibitors* / Q000187:drug effects / Q000378:metabolism
D022782:CCAAT-Enhancer-Binding Protein-beta; D002518:Ceramides; D004268:DNA-Binding Proteins; D006873:Hydroquinones; D007527:Isoenzymes; D051267:NF-E2-Related Factor 2; C495637:Nfe2l2 protein, rat; D011719:Pyrazines; D013107:Sphingolipids; D013871:Thiones; D013876:Thiophenes; D015534:Trans-Activators; C026209:oltipraz; C018855:2-tert-butylhydroquinone; D005982:Glutathione Transferase; C119995:glutathione S-transferase alpha