Redundancy in the Signaling Pathways and Promoter Elements Regulating Cyclooxygenase-2 Gene Expression in Endotoxin-treated Macrophage/Monocytic Cells

Abstract

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 NFB, NF-IL6, and CRE promoter sites mediate gene transcription independently in response to LPS treatment. In these experiments, isolated NFB, 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.

Overlapping CRE and E‐box promoter elements can independently regulate COX‐2 gene transcription in macrophages

“…The single mutants designated CRM and EBM and the triple mutants designated E-box and CRE were created using site-directed mutagenesis on WT or CRE/E-box templates, respectively. Brie£y, primers that incorporated mutations (lower-case letters) for CRE (sense: 5P-CAGTCATTTgaT-CACATGGGCTTGG-3P) or E-box (sense: 5P-CAGTCATTTCGTCACActGGCTTGG-3P) were used to amplify the template constructs as previously described [6]. Incorporation of the desired mutations was con¢rmed by DNA sequencing (see Fig.…”

“…Cells were then treated with fresh 3% FBS RPMI with or without LPS (50 ng/ml). Nuclear extracts were obtained by high-salt extraction (0.5 M NaCl) 30 min later as described previously [6]. In some experiments, nuclear extracts were incubated with rabbit IgG, anti-c-Jun, anti-CREB or anti-USF-1 for 2 h, followed by incubation with protein A-agarose beads for 2 h. After centrifugation, transcription factor-depleted supernatants were used in EMSA.…”

“…Cold chase was carried out with a 50Umolar excess of the same, unlabeled probe or with a probe that contained mutated binding sequences (lower-case letters) for CRE (TTgaTCA-CATG) or E-box (TTCGTCACAct). Nuclear extract^DNA complexes were resolved in 4% polyacrylamide gels using 0.5UTBE at 150 V and the gels were then dried and autoradiographed [6].…”

“…In previous work [6], we had made the observation that maximal endotoxin-mediated induction of COX-2 transcription in macrophages necessitated either an NF-UB or an NF-IL6 promoter element and an intact CRE/E-box consensus. In order to assess the relative contribution of the CRE and Ebox elements, we constructed COX-2 promoter-luciferase reporter elements in which the CRE, E-box or both elements had been selectively mutated.…”

“…We recently reported that in the COX-2 gene (Fig. 1), promoter elements for nuclear factor UB (NF-UB, 3223/3214), nuclear factor interleukin 6 (NF-IL6, 3132/3124) and a cAMP responsive element overlapping a non-canonical E-box (CRE/E-box, 359/349) regulate transcription in macrophages exposed to endotoxin [6]. Although not su¤cient by itself to confer maximal COX-2 transcription, the overlapping CRE/E-box appeared to be the most active promoter element in response to endotoxin.…”