Figure 4.

LPA induces transcriptional activation of Cox-2 through C/EBP independently of AP-1 or NF-κB. A) Deletion and mutational analysis of the Cox-2 promoter. The unique AP-1-like, two NF-κB, and two C/EBP binding sites were deleted or point mutated as detailed in Materials and Methods. Caov-3 cells transfected with the wild-type or mutant constructs were treated with LPA for 6 h and assayed for luciferase activity. B) Requirement of Bcl10-dependent NF-κB activation for LPA-induced production of IL-8 but not that of Cox-2. The control and Bcl10 siRNA-treated Caov-3 cells were stimulated for 6 h with 10 μM LPA or vehicle. Cox-2 expression in cell lysates and IL-8 concentrations in culture supernatants were determined by immunoblotting and ELISA analysis, respectively. C) Inhibition of LPA-induced activation of the Cox-2 promoter and Cox-2 expression by the dominant-negative LIP. Caov-3 cells transfected with pGL2-Cox2–1kb-Luc along with pcDNA3 or pCDNA3-LIP were treated for 6 h with LPA (10 μM) and assayed for luciferase activity (left panel). Caov-3 cells were transfected with pcDNA3-LIP or pcDNA3 using Amaxa nucleofector, stimulated for 6 h with LPA (10 μM) and analyzed by immunoblotting for Cox-2 and C/EBPβ (right panel). The values beneath each lane represent relative intensities (%) quantified by densitometry with Cox-2 induced by LPA in pcDNA3-transfected cells defined as 100%. Data are representative of three independent experiments.