Suppression of KLF5 expression is mediated by PPAR-γ activation.
Cells were pretreated with or without PPAR-γ antagonist GW9662 (3 mM) or BADGE (1 μM) for 30 min or PPAR-γ specific siRNA for 48 hrs prior before the addition of PPAR-γ activator rosiglitazone (Ros) (5μM), 15-d-PGJ2 (15D) (5μM) or pioglitazone (Pio) (50 μM) for 1 hr, and subsequently stimulated with Ang II (0.1 μM) for 24 hrs. (A) Representative immunoblots for PPAR-γ and β-actin from 3 separate experiments. PPAR-γ protein expression is shown as fold increase over control group. Results are mean ± S.E.M. β-actin was used as an internal control. (B) PPAR-γ activation was analyzed by DNA-binding assay. NSB: non-specific binding, C1: competitor binding, PC: positive control binding. Results are mean ± S.E.M. of 3 independent experiments, expressed as OD 450. (C) Western blot analysis of KLF5 protein expression in VSMCs. Representative western blot (upper panel), and data are mean ± S.E.M. (bottom panel) of 3 independent experiments. Results are expressed as fold increase over control group. β-actin served as an internal control. (D) Real-time RT-PCR analysis of KLF5 mRNA expression in response to different treatment in VSMCs. Results are fold increase over control, and data are mean ± S.E.M. of 3 independent experiments. GAPDH served as an internal control. (*P<0.05 vs. control; # P<0.05 vs. Ang II; †P<0.05 vs. Ang II + Ros).