Direct and Indirect Suppression of Interleukin-6 Gene Expression in Murine Macrophages by Nuclear Orphan Receptor REV-ERBα

Figure 4

Effect of RORα on il6 promoter activity. (a) RAWror and RAWvecA cells were transiently transfected with luciferase reporter construct containing either a distal or a proximal construct of il6 promoter. After treatment with or without 1 μg/mL LPS for 24 h, luciferase activities were determined. ROREd, distal RORE; ROREp, proximal RORE. (b) The AGGTCA half-site in the proximal RORE was changed to AGGCCT by site-directed mutagenesis of nucleotides −518 (A to T) and −520 (T to C), and luciferase activities of each cell either untreated or treated with LPS for 24 h were determined. ROREpm, proximal RORE mutant. (c) The GGGATTTTCC half-site in the NFκBRE was changed to GGGCCCTTCC by site-directed mutagenesis of nucleotides −86 (T to C), −87 (T to C), and −88 (A to C), and luciferase activities of each cell either untreated or treated with LPS for 24 h were determined. NFκBREm, NFκBRE mutant. Luciferase values were normalized using Renilla luciferase. The data are presented as the means ± S.E. from sextuplicate cultures. versus vector control.

(a)

(b)

(c)