Redundancy in the signaling pathways and promoter elements regulating cyclooxygenase-8 gene expression in endotoxin-treated macrophage/monocytic cells

Macrophage expression of cyclooxygenase-2 (COX-2), the inducible isoform of COX, is up-regulated by proinflammatory stimuli both in vivo and in vitro. Here we investigated the mechanisms regulating COX-2 gene expression in macrophage/monocytic cells. Lipopolysaccharide (LPS) is known to induce de novo COX-2 mRNA expression in these cells. Transient cotransfections with a COX-2 promoter-luciferase construct and different expression vectors showed that LPS up-regulates COX-2 transcription through both mitogen-activated protein kinase (MAPK) and protein kinase C (PKC) pathways. Cotransfections with expression vectors for dominant negative mutants of MAPK and PKC isoforms did not suppress the effects of LPS on COX-2. Electrophoretic mobility shift assays and transient transfection experiments with deleted and mutated variants of a COX-2 promoter-luciferase construct showed that NFKB, NF-IL6, and CRE promoter sites mediate gene transcription independently in response to LPS treatment. In these experiments, isolated NFKB, NF-IL6, and CRE promoter sites were less effective than the intact promoter in mediating COX-2 transcription. Cotransfections with mutated COX-2 promoter-luciferase constructs and expression vectors showed that each one of these promoter elements can be activated by LPS through both MAPK and PKC pathways to induce gene expression. In summary, there is redundancy in the signaling pathways and promoter elements regulating COX-2 transcription in endotoxin-treated cells of macrophage/monocytic lineage.