Enzymatic reduction of oxysterols impairs LXR signaling in cultured cells and the livers of mice

Abstract

Liver X receptors (LXRs) are nuclear receptors that play crucial roles in lipid metabolism in vivo and are activated by oxysterol ligands in vitro. The identity of the ligand that activates LXRs in vivo is uncertain. Here we provide two lines of evidence that oxysterols are LXR ligands in vitro and in vivo. First, overexpression of an oxysterol catabolic enzyme, cholesterol sulfotransferase, inactivates LXR signaling in several cultured mammalian cell lines but does not alter receptor response to the nonsterol agonist T0901317. Adenovirus-mediated expression of the enzyme in mice prevents dietary induction of hepatic LXR target genes by cholesterol but not by T0901317. Second, triple-knockout mice deficient in the biosynthesis of three oxysterol ligands of LXRs, 24S-hydroxycholesterol, 25-hydroxycholesterol, and 27-hydroxycholesterol, respond to dietary T0901317 by inducing LXR target genes in liver but show impaired responses to dietary cholesterol. We conclude that oxysterols are in vivo ligands for LXR.

Figure 1. Inactivation of Oxysterol, but Not Nonsterol, Ligands by Sulfotransferase

(A) HEK293 cells were transfected with plasmids of the GAL4-LXRα/GAL4-luciferase receptor-reporter system for 4 hr. Thereafter, the indicated ligands were added to the medium at either 1.25 µM (sterols) or 0.1 µM (T0901317) concentration, and the incubation was continued for 16 hr. Cells were harvested and luciferase activity was determined. (B) HEK293 cells were transfected with plasmids of the full-length FXR/FXRE-luciferase receptor-reporter system as above. The ligands chenodeoxycholic acid (CDCA) and GW4064 were added at concentrations of 25 and 0.5 µM, respectively. (C) HEK293 cells were transfected with plasmids of the full-length LXRα/RXRα/LXRE-luciferase receptor-reporter system. Ligands added were 24,25-epoxycholesterol (24,25-EC, 0.625 µM), T1091317 (0.1 µM), and 9-cis-retinoic acid at the indicated concentrations.

Figure 2. Sulfotransferase Blocks the Ability of Insulin to Activate LXR in Primary Hepatocytes

(A) Cells were prepared from rat liver and plated on collagen-coated dishes for 3–4 hr. The plating medium was replaced with fresh medium containing either no adenovirus (−) or adenovirus expressing the mouse cholesterol sulfotransferase cDNA at the designated multiplicities of infection (MOI). After an additional 14–16 hr incubation, cells were washed, and medium containing vehicle or the indicated concentrations of insulin or T0901317 was added. Nine hours later, total RNA was extracted from the cells, and levels of SREBP-1c mRNA were determined by real-time RT-PCR. (B) Primary hepatocytes were prepared, plated, and infected with adenovirus expressing either the E. coli β-galactosidase gene (β-Gal) or the mouse cholesterol transferase gene (ST) and then treated with vehicle or insulin as described in (A). The experiments in (A) and (B) were performed on different days.

Figure 3. Expression of Sulfotransferase in Mice Disrupts LXR-Mediated Gene Activation

Male C57BL/6 mice (n = 3 per group) were infected with the indicated adenovirus (7.8 × 10⁸ pfu/mouse) on day 0 of the experiment and then maintained on diets containing 0.02% cholesterol (−), 1% cholesterol (C), or 0.025% T0901317 (T). On day 4, animals were killed, and total RNA was prepared from individual livers. Equal amounts of RNA from each animal in an experimental group were pooled, and levels of four LXR target-gene mRNAs were determined by real-time RT-PCR. The results are representative of two separate infection/feeding experiments using the same number of animals of each genotype.

Figure 4. Knockout of Oxysterol Biosynthetic Genes Attenuates LXR-Mediated Gene Transcription in Liver

Male wild-type (+/+) or Cyp46a1^−/− Ch25h^−/− Cyp27a1^−/− (3KO) C57BL/6;129S6/SvEv mice (n = 6 per group) were fed chow with 0.025% cholic acid and 0.02% cholesterol (−), 1% cholesterol (C), or 0.025% T0901317 (T) for 7 days. Animals were killed, total RNA was prepared from individual livers, and equal amounts of RNA from each animal were pooled. The levels of five LXR target-gene mRNAs were determined by real-time RT-PCR. The results are representative of two separate feeding experiments using the same number of animals for each genotype.