Potentiation of lipopolysaccharide-inducible cyclooxygenase 2 expression by C2-ceramide via c-Jun N-terminal kinase-mediated activation of CCAAT/enhancer binding protein beta in macrophages.
Ceramide, formed by sphingomyelinase, is involved in the expression of cyclooxygenase-2 (COX-2). This study examines the effect of C2-ceramide (C2), a cell-permeable ceramide analog, on the lipopolysaccharide (LPS)-inducible COX-2 expression and signaling pathways. C2 did not induce COX-2 but potentiated LPS-inducible COX-2 expression in Raw264.7 cells, whereas dihydro-C2 was inactive. Treatment of cells with C2 notably increased LPS-inducible CCAAT/enhancer binding protein (C/EBP) DNA binding. Antibody supershift experiments revealed that LPS-induced C/EBP DNA binding activity depended on C/EBP beta and C/EBP delta but not C/EBP alpha, C/EBP epsilon or CBP/p300. C/EBP beta contributed to C2-enhanced DNA binding activity. 4-(4-Fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl) 1H-imidazole (SB203580), a p38 kinase inhibitor, completely inhibited LPS-inducible and C2-potentiated LPS-inducible COX-2 expression. Enhancement of LPS-inducible COX-2 expression and C/EBP DNA binding by C2 was abrogated in dominant-negative mutant of JNK1 [JNK1(-)] cells. 2'-Amino-3'-methoxyflavone (PD98059) or stable transfection with dominant-negative mutant of MKK1 decreased COX-2 induction by LPS but failed to inhibit C2-enhanced LPS induction of COX-2. Transfection with dominant-negative mutant of C/EBP inhibited the ability of C2 to potentiate the induction of COX-2 by LPS. In LPS-treated cells, C2 enhanced both the nuclear translocation and the expression of LPS-inducible C/EBP beta with an increase in AP-1 DNA binding activity. These enhancements were abolished by JNK1(-) transfection. AP-1 decoy oligonucleotide suppressed C2-potentiated C/EBP beta expression, indicating that AP-1 was responsible for C2-mediated C/EBP beta expression. These results demonstrate that C2 increases C/EBP beta-mediated COX-2 induction by LPS and that the pathway of JNK1 but not ERK1/2 is responsible for C/EBP beta activation involving activator protein-1-mediated enhanced C/EBP beta expression.
Cho
Yang Hee
YH
National Research Laboratory (MDT), College of Pharmacy, Seoul National University, Seoul, Korea.
Lee
Chang Ho
CH
Kim
Sang Geon
SG
D000818:Animals; D022782:CCAAT-Enhancer-Binding Protein-beta / Q000378:metabolism*; D051743:CCAAT-Enhancer-Binding Protein-delta; D022762:CCAAT-Enhancer-Binding Proteins / Q000378:metabolism; D002467:Cell Nucleus / Q000187:drug effects / Q000378:metabolism; D002478:Cells, Cultured; D024042:Collagen Type I / Q000494:pharmacology; D017362:Cyclic AMP Response Element-Binding Protein / Q000378:metabolism; D051546:Cyclooxygenase 2; D004247:DNA / Q000187:drug effects / Q000378:metabolism; D004357:Drug Synergism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D007527:Isoenzymes / Q000096:biosynthesis*; D048031:JNK Mitogen-Activated Protein Kinases; D008070:Lipopolysaccharides / Q000494:pharmacology*; D008264:Macrophages / Q000187:drug effects* / Q000201:enzymology / Q000378:metabolism; D051379:Mice; D020928:Mitogen-Activated Protein Kinases / Q000235:genetics / Q000378:metabolism*; D016328:NF-kappa B / Q000378:metabolism; D011451:Prostaglandin-Endoperoxide Synthases / Q000096:biosynthesis*; D015398:Signal Transduction; D013110:Sphingosine / Q000031:analogs & derivatives* / Q000494:pharmacology*; D018808:Transcription Factor AP-1 / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
D022782:CCAAT-Enhancer-Binding Protein-beta; D022762:CCAAT-Enhancer-Binding Proteins; C497253:Cebpd protein, mouse; D024042:Collagen Type I; D017362:Cyclic AMP Response Element-Binding Protein; D004791:Enzyme Inhibitors; D007527:Isoenzymes; D008070:Lipopolysaccharides; C064769:N-acetylsphingosine; D016328:NF-kappa B; D018808:Transcription Factor AP-1; D014157:Transcription Factors; D014409:Tumor Necrosis Factor-alpha; D051743:CCAAT-Enhancer-Binding Protein-delta; D004247:DNA; D051546:Cyclooxygenase 2; D011451:Prostaglandin-Endoperoxide Synthases; D048031:JNK Mitogen-Activated Protein Kinases; D020928:Mitogen-Activated Protein Kinases; D013110:Sphingosine