| The identification of bile acids as ligands for the nuclear receptor FXR, and the identification of a small set of FXR target genes, has provided the first evidence that the end products of cholesterol catabolism play important roles in this lipid metabolism. As such, FXR governs expression of key genes involved in hepatic cholesterol homeostasis and serum lipoprotein metabolism.; In an effort to attain a more complete understanding of FXR signaling pathways, and the effects of bile acids on gene expression, we engineered a stable hepatoma cell line which overexpresses FXR (HepG2-FXR) as a tool to search for novel FXR target genes. To this end, we treated HepG2-FXR tools with FXR ligands or vehicle, and used several methods to screen for genes for which mRNA levels were enriched in ligand versus vehicle treated cells.; Our initial screen, using a suppression subtractive hybridization (SSH) technique led to the identification of the multi-drug resistance protein 2 (MRP2) as an FXR target. MRP2 mediates the efflux of conjugated organic anions across the apical membrane of the hepatocyte into the bile canaliculi, thereby ridding the hepatocyte of toxic bile acids and xenobiotics.; To achieve a more global understanding of FXR dependent changes in gene expression, we used mRNAs from HepG2-FXR cells treated with FXR ligands or vehicle to synthesize probes for Affymetrix Gene array chips. This technique led to the identification of several candidate targets. Of these candidates, we chose to explore the expression induction of Syndecan-1 (SDC1) because of its reported role in lipoprotein metabolism. Treatment of human hepatocytes with either naturally occurring or synthetic FXR ligands resulted in both (i) induction of SDC1 mRNA and (ii) enhanced binding, internalization, and degradation of a methylated low density lipoprotein particle. The identification of SDC1 as a target, coupled with the identification of the phospholipid transfer protein (PLTP) and apolipoprotein C-II (apoC-II) as FXR targets, suggests an important role for FXR and bile acids in modulating lipoprotein metabolism, and begins to provide a molecular framework to explain the observation that treatment of patients with gallstones, or feeding of rodents with FXR ligands leads to a dramatic reduction in serum triglyceride levels.; Finally, preliminary analysis of other FXR targets gleaned from the microarray data implicate a role for FXR in blood coagulation through the induction of three genes: Fibrinogen α, β, and γ (FBGα, β, γ). mRNAs for all three genes are induced following incubation with natural or synthetic FXR ligands in multiple human hepatoma cell types. The identification of the genes encoding all of the components necessary for the formation of the mature fibrinogen protein as FXR targets suggests a novel bile acid mediated signaling pathway, which may serve to trigger components of the coagulation system as a protection mechanism in response to increases in bile acid concentration. |
