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. 2004 Jan 15;18(2):157–169. doi: 10.1101/gad.1138104

Figure 2.

Figure 2.

PGC-1α coactivates PPARγ and HNF4α to enhance FXR gene transcription. (A) PPARγ ligand induces FXR expression. Mouse primary hepatocytes were treated for 24 h with either vehicle (DMSO) or ligands for FXR (GW4064, 1 μM), PXR (PCN, 10 μM), LXR (T0901317, 1 μM), or PPARγ (GW7845, 1 μM). Total RNA was used for Northern blot assay. (B) Schematic representation of the 5′-end of the FXR gene showing the two FXR promoters and the nucleotides corresponding to the two DR-1 elements. FXRα1/α2 and FXRα3/α4 mRNAs are the products of transcription initiated from exon 1 or exon 3, respectively. Exon 1 is 26.6 kb upstream of exon 3. The sequences of DR-1 elements, and the incorporated mutations, are shown. The corresponding sequences of DR-1 elements in the promoters of the human FXR gene are also shown. (C,D) Activation of FXR promoters by PPARs. CV-1 cells were transiently transfected with either the FXR promoter-reporter constructs pGL3-FXRα1/α2-luc or pGL3-FXRα3/α4-luc together with PPARα, PPARδ, or PPARγ. The cells were treated with either vehicle or corresponding ligands (PPARα, 1 μM GW7847; PPARδ, 1 μM GW2433; PPARγ, 1 μM GW7845) for 36 h. Luciferase activity was analyzed and normalized with β-galactosidase activity. The values represent three independent experiments. (E,F) PPARγ/RXR binds to the DR-1 elements in both FXR promoters. Electrophoretic mobility-shift assays were performed using in vitro transcribed/translated receptors and radiolabeled FXRα1/α2 (E) or FXRα3/α4 (F) probes. For competition experiments, excess unlabeled competitor DNA, containing wild-type or mutant DR-1 sequences (B), was used at 20×, 100×, and 500×, respectively. (G,H) PGC-1α coactivates PPARγ to enhance FXR transcription via a DR-1 element. CV-1 cells were transfected with plasmids encoding PPARα, PPARγ, or PGC-1α together with either pGL3-FXRα1/α2-luc and pGL3-FXRα1/α2mut-luc (G) or pGL3-FXRα3/α4-luc and pGL3-FXRα3/α4mut-luc (H). Cells were incubated for 36 h in the presence or absence of specific PPAR ligands prior to determination of the luciferase activity. Values, normalized to β-galactosidase activity, are the means (±SE) of three experiments. (I,J) PGC-1α coactivates HNF4α to enhance FXR transcription via a DR-1 element. CV-1 cells were transfected with plasmids encoding PGC-1α or HNF4α together with FXR promoters as described in G and H. These values represent three independent experiments.