The multidrug resistance-associated protein 2 (MRP2, ABCC2), mediates the efflux of several conjugated compounds across the apical membrane of the hepatocyte into the bile canaliculi. We identified MRP2 in a screen designed to isolate genes that are regulated by the farnesoid X-activated receptor (FXR, NR1H4). MRP2 mRNA levels were induced following treatment of human or rat hepatocytes with either naturally occurring (chenodeoxycholic acid) or synthetic (GW4064) FXR ligands. In addition, we have shown that MRP2 expression is regulated by the pregnane X receptor (PXR, NR1I2) and constitutive androstane receptor (CAR, NR1I3). Thus, treatment of rodent hepatocytes with PXR or CAR agonists results in a robust induction of MRP2 mRNA levels. The dexamethasone-and pregnenolone 16␣-carbonitrile-dependent induction of MRP2 expression was not evident in hepatocytes derived from PXR null mice. In contrast, induction of MRP2 by phenobarbital, an activator of CAR, was comparable in wild-type and PXR null mice. An unusual 26-bp sequence was identified 440 bp upstream of the MRP2 transcription initiation site that contains an everted repeat of the AGTTCA hexad separated by 8 nucleotides (ER-8). PXR, CAR, and FXR bound with high affinity to this element as heterodimers with the retinoid X receptor ␣ (RXR␣, NR2B1). Luciferase reporter gene constructs containing 1 kb of the rat MRP2 promoter were prepared and transiently transfected into HepG2 cells. Luciferase activity was induced in a PXR-, CAR-, or FXR-dependent manner. Furthermore, the isolated ER-8 element was capable of conferring PXR, CAR, and FXR responsiveness on a heterologous thymidine kinase promoter. Mutation of the ER-8 element abolished the nuclear receptor response. These studies demonstrate that MRP2 is regulated by three distinct nuclear receptor signaling pathways that converge on a common response element in the 5-flanking region of this gene.Members of the nuclear receptor superfamily of ligand-activated transcription factors have critical roles in many aspects of development and adult physiology, including cholesterol homeostasis, bile acid biosynthesis and transport, and xenobiotic metabolism. Recently, two orphan nuclear receptors, the farnesoid X-activated receptor (FXR, 1 NRIH4) and the pregnane X receptor (PXR, NR1I2) were shown to be activated by an overlapping spectrum of bile acids (1-5). These results indicate that bile acids function as hormonal ligands, in addition to their well established roles in the solubilization and absorption of lipids and fat-soluble vitamins from the intestinal lumen.

Apolipoprotein E (apoE) secreted by macrophages in the artery wall exerts an important protective effect against the development of atherosclerosis, presumably through its ability to promote lipid efflux. Previous studies have shown that increases in cellular free cholesterol levels stimulate apoE transcription in macrophages and adipocytes; however, the molecular basis for this regulation is unknown. Recently, Taylor and colleagues [Shih, S. J., Allan, C., Grehan, S., Tse, E., Moran, C. & Taylor, J. M. (2000) J. Biol. Chem. 275, 31567-31572] identified two enhancers from the human apoE gene, termed multienhancer 1 (ME.1) and multienhancer 2 (ME.2), that direct macrophage- and adipose-specific expression in transgenic mice. We demonstrate here that the nuclear receptors LXRalpha and LXRbeta and their oxysterol ligands are key regulators of apoE expression in both macrophages and adipose tissue. We show that LXR/RXR heterodimers regulate apoE transcription directly, through interaction with a conserved LXR response element present in both ME.1 and ME.2. Moreover, we demonstrate that the ability of oxysterols and synthetic ligands to regulate apoE expression in adipose tissue and peritoneal macrophages is reduced in Lxralpha-/- or Lxrbeta-/- mice and abolished in double knockouts. Basal expression of apoE is not compromised in Lxr null mice, however, indicating that LXRs mediate lipid-inducible rather than tissue-specific expression of this gene. Together with our previous work, these findings support a central role for LXR signaling pathways in the control of macrophage cholesterol efflux through the coordinate regulation of apoE, ABCA1, and ABCG1 expression.

The farnesoid X-activated receptor (FXR; NR1H4) is a member of the nuclear hormone receptor superfamily and functions as a heterodimer with the 9-cis-retinoic acid receptor (RXR) .

The farnesoid X-activated receptor (FXR; NR1H4), a member of the nuclear hormone receptor superfamily, induces gene expression in response to several bile acids, including chenodeoxycholic acid. Here we used suppression subtractive hybridization to identify apolipoprotein C-II (apoC-II) as an FXR target gene. Retroviral expression of FXR in HepG2 cells results in induction of the mRNA encoding apoC-II in response to several FXR ligands. EMSAs demonstrate that recombinant FXR and RXR bind to two FXR response elements that are contained within two important distal enhancer elements (hepatic control regions) that lie 11 kb and 22 kb upstream of the transcription start site of the apoC-II gene. A luciferase reporter gene containing the hepatic control region or two copies of the wild-type FXR response element was activated when FXR-containing cells were treated with FXR ligands. In addition, we report that hepatic expression of both apoC-II and phospholipid transfer protein mRNAs increases when mice are fed diets supplemented with cholic acid, an FXR ligand, and this induction is attenuated in FXR null mice. Finally, we observed decreased plasma triglyceride levels in mice fed cholic acid- containing diets. These results identify a mechanism whereby FXR and its ligands lower plasma triglyceride levels. These findings may have important implications in the clinical management of hyperlipidemias.

During the last three years there have been a plethora of publications on the liver X-activated receptors (LXR ␣ , NR1H3, and LXR ␤ , NR1H2), the farnesoid X-activated receptor (FXR, NR1H4), and the pregnane X receptor (PXR, NR1I2) and the role these nuclear receptors play in controlling cholesterol, bile acid, lipoprotein and drug metabolism. The current interest in these nuclear receptors is high, in part, because they appear to be promising therapeutic targets for new drugs that have the potential to control lipid homeostasis. In this review we emphasize i ) the role of LXR in controlling many aspects of cholesterol and fatty acid metabolism, ii ) the expanded role of FXR in regulating genes that control not only bile acid metabolism but also lipoprotein metabolism, and iii ) the regulation of bile acid transport/metabolism in response to bile acid-activated

The CTP:phosphocholine cytidylyltransferase (CT) gene encodes the rate-controlling enzyme in the phosphatidylcholine biosynthesis pathway. CT ␣ mRNA levels, like farnesyl diphosphate synthase and the LDL receptor, are repressed when human or rodent cells are incubated with exogenous sterols and induced when cells are incubated in lipid-depleted medium. A putative sterol response element (SRE) was identified 156 bp upstream of the transcription start site of the CT ␣ gene. Electrophoretic mobility shift assays demonstrate that recombinant SREBP-1a binds to the wild-type SRE identified in the CT ␣ promoter but not to oligonucleotides containing two mutations in the SRE. In other studies, a luciferase reporter construct under the control of the murine CT ␣ proximal promoter was transiently transfected into cells. The activity of the reporter was repressed after addition of sterols to the medium and induced when the cells were incubated in lipid-depleted medium. The activity of the CT ␣ -luciferase reporter was also induced when cells were cotransfected with plasmids encoding either SREBP-1a or SREBP-2. In contrast, no induction was observed under the same conditions when the CT ␣ promoterreporter gene contained two mutations in the SRE. In addition, the induction of the wild-type CT ␣ promoter-reporter gene that occurs in cells incubated in lipid-depleted medium is attenuated when dominant-negative SREBP is cotransfected into the cells. These studies demonstrate that transcription of the CT ␣ gene is inhibited by sterols and activated by mature forms of SREBP.We conclude that SREBP-regulated genes are involved not only in the synthesis of cholesterol, fatty acids, triglycerides, and NADPH, but also, as shown here, in the synthesis of phospholipids.