Transcriptional activation of the cyclooxygenase-2 gene in endotoxin-treated RAW 264.7 macrophages

Cyclooxygenase-2 (COX-2), the enzyme primarily responsible for induced pros taglandin synthesis, is an immediate early gene induced by endotoxin in mac rophages, 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 RA W 264.7 macrophages, Luciferase reporter constructs with alterations in pre sumptive cis-acting transcriptional regulatory elements demonstrate that th e cyclic AMP-response element and two nuclear factor interleukin-6 (CCAAT/e nhancer-binding protein (C/EBP)) sites of the COX-2 promoter are required f or optimal endotoxin-dependent induction. In contrast, the E-box and NF-kap pa B sites are not required for endotoxin-dependent induction. Inhibition o f endotoxin-induced NF-kappa B activation by expression of an inhibitor-kap pa B a mutant does not block endotoxin-dependent COX-2 reporter activity, O verexpression of c-Jun, C/EBP beta, and C/EBP delta enhances induction of t he COX-2 reporter, while overexpression of cyclic AMP-response element-bind ing protein or "dominant negative" C/EBP beta 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 dominan t negative expression vectors shows that endotoxin-induced COX-2 gene expre ssion requires signaling through a Ras-independent pathway involving the ad apter protein ECSIT and the signaling kinases MEKK1 and JNK. In contrast, e ndotoxin-induced COX-2 reporter activity is not blocked by overexpression o f dominant-negative forms of Raf-1, ERK1, or ERR2.