Transcriptional activation of the cyclooxygenase-2 gene in endotoxin-treated RAW 264.7 macrophages.
Cyclooxygenase-2 (COX-2), the enzyme primarily responsible for induced prostaglandin synthesis, is an immediate early gene induced by endotoxin in macrophages. We investigated the cis-acting elements of the COX-2 5'-flanking sequence, the transcription factors and signaling pathways responsible for transcriptional activation of the COX-2 gene in endotoxin-treated murine RAW 264.7 macrophages. Luciferase reporter constructs with alterations in presumptive cis-acting transcriptional regulatory elements demonstrate that the cyclic AMP-response element and two nuclear factor interleukin-6 (CCAAT/enhancer-binding protein (C/EBP)) sites of the COX-2 promoter are required for optimal endotoxin-dependent induction. In contrast, the E-box and NF-kappaB sites are not required for endotoxin-dependent induction. Inhibition of endotoxin-induced NF-kappaB activation by expression of an inhibitor-kappaB alpha mutant does not block endotoxin-dependent COX-2 reporter activity. Overexpression of c-Jun, C/EBPbeta, and C/EBPdelta enhances induction of the COX-2 reporter, while overexpression of cyclic AMP-response element-binding protein or "dominant negative" C/EBPbeta represses COX-2 induction. In addition, endotoxin rapidly and transiently elicits c-Jun phosphorylation in RAW 264.7 macrophages. Cotransfection of the COX-2 reporter with dominant negative expression vectors shows that endotoxin-induced COX-2 gene expression requires signaling through a Ras-independent pathway involving the adapter protein ECSIT and the signaling kinases MEKK1 and JNK. In contrast, endotoxin-induced COX-2 reporter activity is not blocked by overexpression of dominant-negative forms of Raf-1, ERK1, or ERK2.
Wadleigh
D J
DJ
Molecular Biology Institute, UCLA, Los Angeles, California 90095, USA.
Reddy
S T
ST
Kopp
E
E
Ghosh
S
S
Herschman
H R
HR
D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D022762:CCAAT-Enhancer-Binding Proteins; D000242:Cyclic AMP / Q000235:genetics; D051546:Cyclooxygenase 2; D004268:DNA-Binding Proteins / Q000235:genetics; D029721:Drosophila Proteins; D004731:Endotoxins / Q000494:pharmacology*; D017930:Genes, Reporter; D020672:I-kappa B Proteins / Q000235:genetics; D007527:Isoenzymes / Q000235:genetics*; D048031:JNK Mitogen-Activated Protein Kinases; D008070:Lipopolysaccharides / Q000494:pharmacology; D048728:MAP Kinase Kinase Kinase 1; D008264:Macrophages / Q000187:drug effects*; D051379:Mice; D020928:Mitogen-Activated Protein Kinases / Q000235:genetics; D009154:Mutation; D009687:Nuclear Proteins / Q000235:genetics; D010766:Phosphorylation; D011401:Promoter Regions, Genetic; D011451:Prostaglandin-Endoperoxide Synthases / Q000235:genetics*; D017346:Protein Serine-Threonine Kinases / Q000235:genetics; D011506:Proteins / Q000235:genetics; D016755:Proto-Oncogene Proteins c-jun / Q000378:metabolism; D015398:Signal Transduction; D015533:Transcriptional Activation
D048868:Adaptor Proteins, Signal Transducing; D022762:CCAAT-Enhancer-Binding Proteins; D004268:DNA-Binding Proteins; D029721:Drosophila Proteins; C121539:ECSIT protein, Drosophila; D004731:Endotoxins; D020672:I-kappa B Proteins; D007527:Isoenzymes; D008070:Lipopolysaccharides; D009687:Nuclear Proteins; D011506:Proteins; D016755:Proto-Oncogene Proteins c-jun; D000242:Cyclic AMP; D051546:Cyclooxygenase 2; D011451:Prostaglandin-Endoperoxide Synthases; D017346:Protein Serine-Threonine Kinases; D048031:JNK Mitogen-Activated Protein Kinases; D020928:Mitogen-Activated Protein Kinases; D048728:MAP Kinase Kinase Kinase 1; C482578:Map3k1 protein, mouse