Chenodeoxycholic acid (CDCA), a primary bile acid, has recently been shown to activate the farnesoid X receptor (FXR), a member of the nuclear hormone receptor superfamily. Recent data suggest that activated FXR controls both bile acid biosynthesis and plasma lipid levels. As such, FXR may affect the development of gallstones and/or atherosclerosis. However at the current time, very little is known about the target genes and metabolic pathways that are affected by activated FXR. In the first specific aim, we propose to use Suppression Subtractive Hybridization and DNA microarrays to identify genes that are regulated by CDCA-activated FXR. These studies will utilize HepG2 and Caco2 cells that stably overexpress high levels of FXR in order to more easily identify FXR-target genes. We will use normal, FXR-/- or VP16-FXR transgenic mice, treated with FXR ligands, to demonstrate (i) that these same genes are induced in vivo and (ii) that activation of FXR results in a decrease in plasma lipids. In the second specific aim, we will identify FXREs and other critical cis elements in the promoters of a few selected genes that have been identified in aim 1, so as to confirm that these genes are direct targets of FXR/CDCA. In the third aim, we will generate mice that overexpress rat VP16-FXR in their livers (see aim 1). Finally, in specific aim 4, we will isolate cell lines derived from HepG2 and Caco2 cells that express either FXR1, FXR2, or FXR3. The induction of target genes, identified in aim 1, by each FXR isoform will be determined in order to test the hypothesis that specific genes/metabolic pathways are activated by each FXR isoforms.
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