Rev-erbα Antagonizes LXR-Mediated TLR-4 Induction
A, C, and E, Primary human macrophages were infected with a Rev-erbα expressing adenovirus (ad-Rev-erbα) or a GFP (ad-GFP) expressing adenovirus and subsequently treated with T0901317 (1 μm) or vehicle (Control) for 24 h. B, D, and F, Primary human macrophages transfected with siRNA against the hRev-erbα sequence or with the scrambled control. Nuclear protein extracts were prepared and Rev-erbα and β-actin protein levels were measured by western blot analysis. Scr, Scrambled; Rα, Rev-erbα (B). Cells were treated with T0901317 (1 μm) or vehicle (Control) for 24 h and total RNA was extracted and treated with deoxyribonuclease I. mRNA levels of Rev-erbα (A and B), ABCA1 (C and D) and TLR-4 (E and F) were quantified by Q-PCR and normalized to cyclophilin mRNA levels. Results are representative of those obtained from three independent macrophage preparations and are expressed relative to the levels in untreated cells set as 1. Each bar is the mean value ± sd of triplicate determinations. Statistically significant differences are indicated (ANOVA/t test; adGFP vs. ad-Rev-erbα or scramble vs. siRNA-Rev-erbα: §, P < 0.05; §§, P < 0.01; §§§, P < 0.001 and control vs. T0901317: *, P < 0.05; **, P < 0.01; ***, P < 0.001). Based on two-way ANOVA analysis, Rev-erbα expression significantly affected TLR-4 mRNA induction by T0901317 treatment.