Kanaya Eiko, Shiraki Takuma, Jingami Hisato
Department of Molecular Biology, Biomolecular Engineering Research Institute (BERI), 6-2-3 Furuedai, Suita-City, Osaka 565-0874, Japan.
Biochem J. 2004 Sep 15;382(Pt 3):913-21. doi: 10.1042/BJ20040432.
The nuclear bile acid receptor FXR (farnesoid X receptor) is one of the key factors that suppress bile acid biosynthesis in the liver. PGC-1alpha [PPARgamma (peroxisome-proliferator-activated receptor gamma) co-activator-1alpha] is known to control energy homoeostasis in adipose tissue, skeletal muscle and liver. We performed cell-based reporter assays using the expression system of a GAL4-FXR chimaera, the ligand-binding domain of FXR fused to the DNA-binding domain of yeast GAL4, to find the co-activators for FXR. We found that the transcriptional activation of a reporter plasmid by a GAL4-FXR chimaera was strongly enhanced by PGC-1alpha, in a ligand-dependent manner. Transcriptional activation of the SHP (small heterodimer partner) gene by the FXR-RXRalpha (retinoid X receptor alpha) heterodimer was also enhanced by PGC-1alpha in the presence of CDCA (chenodeoxycholic acid). Co-immunoprecipitation and pull-down studies using glutathione S-transferase-PGC-1alpha fusion proteins revealed that the ligand-binding domain of FXR binds PGC-1alpha in a ligand-influenced manner both in vivo and in vitro. Furthermore, our studies revealed that SHP represses its own transcription, and the addition of excess amounts of PGC-1alpha can overcome the inhibitory effect of SHP. These observations indicate that PGC-1alpha mediates the ligand-dependent activation of FXR and transcription of SHP gene.
核胆汁酸受体FXR(法尼酯X受体)是抑制肝脏中胆汁酸生物合成的关键因素之一。已知PGC-1α[PPARγ(过氧化物酶体增殖物激活受体γ)共激活因子-1α]可控制脂肪组织、骨骼肌和肝脏中的能量稳态。我们使用GAL4-FXR嵌合体的表达系统(FXR的配体结合结构域与酵母GAL4的DNA结合结构域融合)进行基于细胞的报告基因检测,以寻找FXR的共激活因子。我们发现,PGC-1α以配体依赖的方式强烈增强了GAL4-FXR嵌合体对报告质粒的转录激活。在鹅去氧胆酸(CDCA)存在的情况下,PGC-1α也增强了FXR-RXRα(视黄酸X受体α)异二聚体对小异二聚体伴侣(SHP)基因的转录激活。使用谷胱甘肽S-转移酶-PGC-1α融合蛋白进行的免疫共沉淀和下拉实验表明,FXR的配体结合结构域在体内和体外均以配体影响的方式与PGC-1α结合。此外,我们的研究表明,SHP可抑制其自身转录,添加过量的PGC-1α可克服SHP的抑制作用。这些观察结果表明,PGC-1α介导了FXR的配体依赖性激活以及SHP基因的转录。