Fig. 4.

RARα, not RXR, appears to suppress ARE function. (A) Effect of RAR and RXR agonists on ARE inhibition. AREc32 cells were seeded in 96-well plates. After 24 h, tBHQ (10 μM) and the retinoids ATRA, TTNPB, AM580, or MA were added concomitantly to the medium. After a further 24 h, samples were assayed for luciferase activity. The value of luciferase activity of cells treated with 10 μM tBHQ alone (control) was taken as 100%. Values shown are mean ± SD. (B) RARα antagonist RO-41-5253 reversed RA-mediated ARE inhibition. AREc32 cells were incubated with 2 μM or 20 μM RO-41-5253 for 1 h. After pretreatment with RO-41-5253, the culture medium was replaced with fresh DMEM containing tBHQ (10 μM) in the presence or absence of ATRA (1 μM). The cells were then incubated for a further 24 h and assayed for luciferase activity, expressed relative to DMSO control. (C) Reduction of RARα expression increased induction of AKR1C1 and AKR1C2 mRNA by tBHQ and, in part, blocked the inhibitory effect of ATRA. AREc32 cells were seeded at 4 × 10⁵ cells per well in six-well plates, containing diluted RNAi (200 pmol per well) and Lipofectamine 2000 (10 μl per well). After 24 h, the culture medium was replaced with fresh DMEM containing tBHQ (10 μM), ATRA (1 μM), or tBHQ (10 μM) plus ATRA (1 μM), and the cells were incubated for a further 24 h. The mRNA levels of AKR1C1 (Left) and AKR1C2 (Right) were measured by TaqMan. Values are expressed as the fold change relative to cells transfected with mock RNAi and treated with DMSO. The level of 18S rRNA was used as an internal standard. *, P < 0.05; **, P < 0.005. (D) V5-mNrf2 associates with endogenous RARα. AREc32 cells were seeded in a 150-mm dish at 5 × 10⁶ cells per dish in the growth medium. After 24 h, 48 μg per dish of V5-mNrf2 plasmid DNA was transiently transfected by using Lipofectamine 2000. The culture medium was then replaced after 5 h with fresh DMEM containing 10 μM tBHQ with or without 1 μM ATRA. After 24 h Nrf2 was immunoprecipitated from the lysate by using anti-V5 antibody. The immunoprecipitated complexes were then fractionated by SDS/PAGE and immunoblotted with anti-RARα antibody. Data are representative of two independent experiments. (E) Endogenous RARα associates with Nrf2 protein. AREc32 cells were incubated with DMEM containing 10 μM tBHQ with or without 1 μM ATRA for 24 h. RARα was then immunoprecipitated from the lysate by using anti-RARα antibody. The immunoprecipitated complexes were then fractionated by SDS/PAGE and immunoblotted with anti-Nrf2 antibody. Data shown are representative of two independent experiments.