Figure 3.

Atorvastatin and its metabolites neither induce the assembly of CAR-LBD nor the in vitro interaction with co-activator SRC-1. (A) CAR assembly assay in COS1 cells, which were co-transfected with expression plasmids encoding GAL4-DBD/hCAR-LBD (105–150) and VP16-AD/hCAR-LBD (151–348) fusion proteins (open columns) or empty expression vector pVP16-AD (filled columns). Cells were treated for 40 h with 1 µM CITCO, 30 µM of atorvastatin or metabolites or 0.1% DMSO only. Mean fold activation (±SD) of the normalized activity of co-transfected reporter plasmid pGL3-G5 by treatment with the indicated compounds is shown. The respective activity of cells transfected with GAL4-DBD/hCAR-LBD(105–150) expression plasmid and pVP16-AD, treated with DMSO only, was designated as 1. Data were analysed by one-way anova with the Tukey-Kramer post-test. ***P < 0.001, significant effect of CITCO. (B) Biacore analysis of ligand-induced interaction of co-activator SRC-1 with CAR in vitro. CAR-LBD protein, pre-incubated with 10 µM CITCO or 100 µM of the indicated atorvastatin metabolites or 1% DMSO only, was injected onto immobilized SRC-1-RID protein (+CAR). As a control, compounds were also injected alone (-CAR). Data are shown as means ± SD of four to six independent experiments. Open columns show increase in binding by pre-incubation of CAR with compounds. Binding of CAR to SRC-1 in the presence of DMSO only, was designated as 1. Data were analysed by one sample t-test and P-values corrected for multiple testing by the method of Bonferroni, *P < 0.05, significant effect of CITCO. The inset shows representative individual sensorgrams of CAR pre-incubated with ATV, ATV-L or vehicle DMSO only. ATV, atorvastatin; ATV-L, atorvastatin lactone, o-OH-ATV, ortho-hydroxy atorvastatin; p-OH-ATV, para-hydroxy atorvastatin.