Regulation of mouse sterol regulatory element-binding protein-1c gene (SREBP-1c) by oxysterol receptors, LXRalpha and LXRbeta

Abstract

The liver X receptors (LXRs) are members of the nuclear hormone receptor superfamily that are bound and activated by oxysterols. These receptors serve as sterol sensors to regulate the transcription of gene products that control intracellular cholesterol homeostasis through catabolism and transport. In this report, we describe a novel LXR target, the sterol regulatory element-binding protein-1c gene (SREBP-1c), which encodes a membrane-bound transcription factor of the basic helix-loop-helix-leucine zipper family. SREBP-1c expression was markedly increased in mouse tissues in an LXR-dependent manner by dietary cholesterol and synthetic agonists for both LXR and its heterodimer partner, the retinoid X receptor (RXR). Expression of the related gene products, SREBP-1a and SREBP-2, were not increased. Analysis of the mouse SREBP-1c gene promoter revealed an RXR/LXR DNA-binding site that is essential for this regulation. The transcriptional increase in SREBP-1c mRNA by RXR/LXR was accompanied by a similar increase in the level of the nuclear, active form of the SREBP-1c protein and an increase in fatty acid synthesis. Because this active form of SREBP-1c controls the transcription of genes involved in fatty acid biosynthesis, our results reveal a unique regulatory interplay between cholesterol and fatty acid metabolism.

Figure 1

Effect of dietary cholesterol on the liver expression of SREBPs and lipogenic enzymes in wild-type and Lxr-knockout mice. Mixed strain (A129/C57Bl6) male mice of various Lxr genotypes were fed basal (0.02% cholesterol) or high cholesterol (2% cholesterol) diets for seven days. Liver mRNA was pooled from six animals per treatment group and Northern analysis performed using probes for SREBP-1 and -2, SCD-1, FAS, and ACC (Shimano et al. 1996). All RNA levels are expressed relative to wild-type mice on basal diet. Numbers above bars refer to relative fold changes in mRNA levels between animals of the same genotype after high cholesterol feeding. Genotypes denoted are: wild-type (WT); Lxrα−/− (α−/−); Lxrβ−/− (β−/−); and Lxrα/β−/− (α/β−/−).

Figure 2

Effect of nuclear receptor agonists on SREBP-1 mRNA expression. Male mice were fed diets containing 0.2% cholesterol plus vehicle or the following agonists for 12 h: 30 mpk LG268 (RXR), 1000 mpk fenofibrate (PPARα), 150 mpk troglitazone (PPARγ), 100 mpk pregnenolone α-carbonitrile (PXR), 1000 mpk chenodeoxycholic acid (FXR), or 50 mpk T0901317 (LXR). Northern analysis was performed on mRNA from liver or duodenal mucosa (n = 4 animals/group) using a SREBP-1 probe.

Figure 3

Regulation of SREBP-1 mRNA by RXR and LXR agonists in liver (A), white adipose tissue (B), and small intestine (C). Male wild-type and Lxrα/β−/− mice were fed diets containing 0.2% cholesterol plus vehicle (open bars), 30 mpk LG268 (hatched bars), or 50 mpk T0901317 (black bars) for 12 h. Northern analysis was performed on mRNA individually (A) or as a pooled sample consisting of three to four animals per group (B,C). Relative mRNA levels in liver (A) are expressed as the mean ± SEM, and a representative Northern blot is shown above. Small intestine mRNA was isolated from the mucosa of the duodenum (D), jejunum (J), and ileum (I).

Figure 4

Expression of SREBP-1c mRNA is regulated by rexinoid. (A) Three mice per group were fed diets containing 0.2% cholesterol plus vehicle (−) or 30 mpk LG268 (+) for 16–18 h. Pooled RNA was isolated from liver, intestine, and epididymal white adipose depots from each group. Aliquots of the pooled RNA (20μg) were subjected to RNase protection assays using a ³²P-labeled cRNA probe that can distinguish between 1a and 1c isoforms of SREBP-1 (BP1). RNA samples from two independent studies were examined in the same blot. Lanes 1 and 3 contain the samples from one experiment; lanes 2 and 4 are from an independent experiment. (B) Data in A were quantified as described in Materials and Methods and normalized relative to the β-actin signal. The data for SREBP-1a and SREBP-1c mRNA are plotted as the fold change relative to the SREBP1a mRNA level in the liver of the first vehicle group. Autoradiography was performed for 8 h at −80°C on Kodak X-Omat (XB-1) film with an intensifying screen.

Figure 5

Regulation of SREBP-1 protein expression by rexinoid. Immunoblot analysis of SREBP-1 and SREBP-2 was performed on fractionated cell extracts from liver and intestine of wild-type male mice fed diets containing 0.2% cholesterol plus vehicle (−) or 30 mpk LG268 (+) for 18 h. P and N denote the precursor and cleaved nuclear forms of SREBP, respectively, and the asterisk indicates a nonspecific band. The filters for liver and intestine were exposed to film at room temperature for 15 sec and 45 sec, respectively.

Figure 6

RXR and LXR agonists activate mouse SREBP-1c promoter. (A) The sequence of the mouse SREBP-1c 5′-flanking region (∼1.3 kb) is shown with the DR4 LXRE motif denoted by arrows. The putative transcription start site at −63 is boxed. (B) RXR and LXR agonists (LG268 and T0901317) synergistically activate a luciferase reporter driven by the SREBP-1c promoter. A 6.5-kb fragment of the SREBP-1c promoter was linked to pGL3 basic luciferase reporter. The resulting plasmid pBP1c(6500)-Luc was cotransfected into HEK-293 cells with a control reporter plasmid pCMV-βGal as described in Materials and Methods. Twenty hours after transfection, cells were treated with vehicle (DMSO and/or ethanol), LG268 (1 μM), T0901317 (10 μM), or LG268 (1 μM) plus T0901317 (10 μM). Normalized luciferase activity of cells treated with vehicle is arbitrarily defined as 1, and relative luciferase activities from different treatments are shown as “fold increase”. Each value represents data from four independent transfection experiments (each in duplicate). (C) Deletions of the ∼6500 kb mouse SREBP-1c promoter were assayed as described in B. Data shown are the average of three independent transfection experiments (each in duplicate) in the upper panel and two independent experiments in the lower panel. The 100% value for basal activity corresponds to the normalized luciferase activity obtained with the pBP1c(6500)-Luc promoter construct in the absence of LG268 and T0901317. Individual values for fold-activations were: upper panel 8.1 (4.9, 7.4, 12), 4.5 (3.8, 4.8, 5.0), 5.0 (2.4, 5.5, 7.0), 0.73 (0.4, 0.8, 1.0); lower panel 7.8 (5.1, 10.5), 0.85 (0.6, 1.1), 4.4 (4, 4.8), 0.98 (0.95, 1.0).

Figure 7

The LXRE in mouse SREBP-1c promoter is a high affinity binding site for RXR/LXR heterodimer. (A) Sequences of the LXRE derived from the mouse mammary tumor virus LTR (ΔMTV-LXRE), wild-type mouse SREBP-1c promoter region (1c-WT), and mutant mouse SREBP-1c promoter region (1c-MUT). For comparison, the LXRE sequences of the bottom strands of 1c-WT and 1c-MUT are aligned with the top strand sequence of ΔMTV. (B) Electrophoretic-mobility shift assays were performed as described in Materials and Methods. The ³²P-labeled 1c-WT LXRE probe was incubated with in vitro synthesized human LXRα and RXRα proteins as indicated. For antibody supershift experiments, anti-RXRα antibody (αRXR) or a non-specific antibody (ns) were used. Free probe, RXR/LXR complex, and antibody-supershifted RXR/LXR complex are denoted by arrows. Nonspecific bands are indicated by asterisks. (C) Competition gel mobility-shift assays were performed as described in Materials and Methods using ³²P-labeled 1c-WT LXRE as input probe and unlabeled oligonucleotides as competitors at 10- and 50-fold molar excesses. Autoradiography was performed for 16 h at −80°C with an intensifying screen.

Figure 8

Mutation of the LXRE sequence abolishes activation of mouse SREBP-1c promoter by RXR/LXR. (A) Schematic diagrams of pBP1c(6500)-Luc wild-type and mutant reporter plasmids. Differences between wild-type and mutant plasmids (nucleotides −300 to −280) are shown for comparison. (B) Activation of wild-type, but not mutant, LXRE reporter plasmids. Wild-type and mutant pBP1c(6500)-Luc were cotransfected into HEK-293 cells. For each 60 mm dish 0.5 μg pBP1c-Luc, 25 ng, CMV-βgal and CMX-LXRα or pCDNA3 were used. Three hours after transfection, cells were switched to medium A containing vehicle (DMSO) or 10 μM T0901317, and assayed as described in Figure 6. Data represent three independent transfection experiments (each in duplicate).

Figure 9

Model depicting LXR-activated target genes that regulate cellular cholesterol metabolism. Cholesterol-derived oxysterols function as LXR ligands to regulate the expression of RXR/LXR target genes (shown in black boxes), which, in turn, modulate intracellular free cholesterol levels through increased elimination, efflux, and storage. The identification of SREBP-1c as an RXR/LXR-regulated gene links cholesterol and fatty acid metabolism, perhaps as a means for the cell to achieve the appropriate ratio of cholesterol to other lipids and thereby maintain cellular membrane integrity. LXR-regulated genes include cholesterol 7α-hydroxylase (CYP7A1; Peet et al. 1998), the ATP-binding cassette transporter-1 (ABC1; Repa et al. 2000), and sterol regulatory element binding protein 1c (SREBP-1c), which in turn governs the expression of stearoyl CoA desaturase (SCD-1).