The Peroxisomal 3-keto-acyl-CoA thiolase B Gene Expression Is under the Dual Control of PPARα and HNF4α in the Liver
Figure 1
Wy induces hepatic Thb gene expression in a PPARα-dependent manner through a novel PPRE. (a) Northern blot was performed with liver RNA from wild-type () and PPARα −/− mice () fed Wy (30 mg kg−1·day−1) for 8 days. Autoradiographs were quantified and average fold changes indicated below the different blots. The signal for WT mice NOT fed Wy was arbitrarily set to 1. 36B4 mRNA levels were evaluated and used as internal control of loading. Western blot experiment was conducted with liver protein samples of the same animals using an antibody that recognizes the two peroxisomal 3-ketoacyl-CoA thiolases (PTL) identified in rodents [35]. (b) Rat hepatoma H4IIEC3 cells were treated with Wy (10 or 50 μM, as indicated) for 48 h. Expression of Thiolase b (Thb), AcylCoA oxidase-1 (Acox-1), and Multifunctional protein-1 (Mfp1) was determined by quantitative RT-PCR analysis. Values are mean of three independent experiments ±SEM. Wy: Wy14,643. (c) The Thb gene contains a composite PPRE in intron 3. The nucleotide sequence of the different PPREs (TB PPRE3, ACOX-I PPRE, and MFP-1 PPRE) used in this study is shown. The nucleotide positions are given taking as +1 the transcription initiation site. The different arrows indicate half-site of a Direct Repeat (DR). (d) A double-strand oligonucleotide containing TB PPRE3 or MFP1-PPRE was incubated with increasing amounts of in vitro translated PPARα and RXRα proteins. Binding complexes were separated by electrophoresis. (e) Transactivation assays were performed in COS-7 cells with a Luc reporter vector containing either a single copy of TB PPRE3 or a copy of ACOX-I PPRE or MFP-1 PPRE. These constructs were transfected together with an expression vector for mouse PPARα (pSG5 mPPARα) in presence or absence of Wy (10 μM). DMSO was used as vehicle. Values are mean of four independent experiments ±SEM. ***Effect statistically significant compared to control (no PPARα transfected, DMSO) with *** with one-way Anova test. Errors bars represent SEM.
