Analysis of the region responsible for the promoter activity of COX-2 gene in response to RANKL stimulation. (A) Putative consensus sequences in the 5′-flanking region of human COX-2 gene are illustrated in the upper left. Each deleted promoter fragment was inserted into a pGL3-basic luciferase (Luc) vector. Numbers indicate distance in base pairs from the start site of transcription. RAW264.7 cells were transiently transfected with these plasmids along with β-galactosidase plasmid. The transfected cells were stimulated with RANKL (50 ng/mL) for 6 hours, and then the activities of luciferase and β-galactosidase were measured. The normalized luciferase activities in the cells transfected with various reporter plasmids are presented as the fold induction calculated by dividing the relative luciferase value (RLV) of a given culture by the RLV of the pGL3-basic control. Data are expressed as mean (± SD) of triplicates. NFAT indicates nuclear factor of activated T cells; API, activator protein 1; ATF, activating transcription factor; CRE, cyclic adenosine monophosphate (cAMP)–responsive element; TATA, TATA box. (B) As described in panel A, except that RAW264.7 cells and RAW-RacN17 cells were transfected with the reporter plasmid P-1838 and stimulated with RANKL (50 ng/mL) and LPS (1μg/mL), respectively. PBS indicates phosphate-buffered saline used for a control. Data are expressed as mean (±SD) of triplicates.