LXRs control lipid-inducible expression of the apolipoprotein E gene in macrophages and adipocytes

Abstract

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.

Figure 1

Coordinate induction of apoE, ABCA1, and ABCG1 expression in THP-1 macrophages by modified LDL. Differentiated THP-1 macrophages were incubated for 48 h in RPMI medium 1640 containing 10% (vol/vol) LPDS with mevinolin and mevalonic acid, and 100 μg/ml (protein) LDL, highly oxidized LDL, or acetylated LDL, as indicated. Total RNA (10 μg per lane) was electrophoresed through formaldehyde-containing gels, transferred to Nylon, and hybridized to ³²P-labeled cDNA probes. 36B4 was used as a control (ctrl) for loading and integrity of the RNA.

Figure 2

Coordinate regulation of apoE, ABCG1, and ABCA1 expression in THP-1 macrophages by LXR ligands. Differentiated THP-1 macrophages were incubated for 48 h in RPMI medium 1640 containing 10% (vol/vol) LPDS with 50 nM LG268 (LG), 2.0 μg/ml 22(S)-hydroxycholesterol [22(S)HC], 2.0 μg/ml 20(S)HC, 2.0 μg/ml 22(R)HC, or vehicle control (ctrl), as indicated. Northern analysis was performed as described above. Blots were quantified by PhosphorImaging and normalized to 36B4.

Figure 3

Macrophage- and adipocyte-specific induction of ApoE expression by LXR and LXR ligands. (A) Differentiation-dependent induction of apoE expression by LXR ligands. Undifferentiated (monocyte) or differentiated (macrophage) THP-1 cells were incubated in RPMI medium 1640 containing 10% (vol/vol) LPDS and 2.0 μg/ml 22(S)HC, 2.0 μg/ml 20(S)HC, 2.0 μg/ml 22(R)HC, or vehicle control (ctrl) for 48 h. (B and C) Retroviral expression and activation of LXRα induces apoE expression in preadipocytes but not in fibroblasts. NIH 3T3 fibroblasts and 3T3-F442A preadipocytes were transduced with a retroviral vector encoding LXRα (NIH-LXRα, 442A-LXRα) or the empty vector alone (NIH-vector, 442A-vector). Stable cell lines were cultured for 48 h in DMEM containing 10% (vol/vol) LPDS in the presence of 50 nM LG268 (LG), 2.0 μg/ml 22(R)HC, or vehicle control (ctrl). Northern analysis was performed as described above.

Figure 4

The LXR/RXR heterodimer activates the apoE ME.1 and ME.2 enhancers. (A) LXRα/RXRα activates the −890-bp apoE proximal promoter fused to either ME.1 or ME.2. HepG2 cells were transfected with pGL-890, pGL-890-ME.1, or pGL-890-ME.2 with or without CMX-mLXRα and CMX-RXRα and CMV-β-galactosidase. After transfection, cells were incubated for 24 h in MEM supplemented with 10% (vol/vol) LPDS and 22(R)HC (5.0 μg/ml), LG268 (50 nM), or vehicle control. Luciferase activity was normalized for transfection efficiency with the use of β-galactosidase activity. The data are expressed as fold activation in the presence of the indicated ligand versus in the absence of ligand and represent the average of triplicate experiments. (B) VP16-LXRα activates the apoE proximal promoter fused to either ME.1 or ME.2. Transient transfections were performed by using CMX-VP16-LXRα and CMX-RXRα as indicated. The results are shown as normalized luciferase units. (C) ME.1 and ME.2 function as LXRα/RXR-responsive enhancers when fused to a heterologous promoter. Transfections were performed with the use of pTK-Luc, pTK-ME.1-Luc, or pTK-ME.2-Luc reporters. (D) ME.1 and ME.2 are activated by VP16-LXRα. Transfections were performed with the use of pTK-Luc, pTK-ME.1-Luc, or pTK-ME.2-Luc along with CMX-VP16-LXRα and CMX-RXRα.

Figure 5

The apoE ME.1 and ME.2 enhancers are direct targets for binding LXR/RXR heterodimers. (A) Genomic structure of the apoE locus and location of potential LXREs. ApoE LXRES are aligned with a known LXRE from the cholesterol-7-α-hydroxylase gene (CYP7α LXRE) and an idealized LXRE (β-DR-4). Also shown is the sequence of a mutant ME LXRE (ME MUT LXRE) used in C and D. (B) Direct binding of LXRα/RXRα and LXRβ/RXRα to a low-affinity LXRE present in the proximal promoter and a high-affinity site conserved in both ME.1 and ME.2. Gel mobility-shift assays were performed with the use of in vitro translated receptors as described in Materials and Methods. (C and D) Sequence-specific competition for LXRα/RXRα (C) and LXRβ/RXRα (D) binding to the ME LXRE.

Figure 6

Tissue-specific induction of apoE expression in vivo by LXR ligands. (A) Sterol-induced expression of apoE in macrophages requires LXR. Peritoneal macrophages were isolated as described in Materials and Methods and cultured for 42 h in the presence of 100 μM mevalonic acid (control) or mevalonic acid plus 5 μM compactin (unloaded), 10 μM 22(R)-hydroxycholesterol [22(R)HC], or 10 μM T0901317. (B and C) LXR and RXR ligands induce apoE expression in an LXR-dependent manner in adipose tissue (B) but not in liver (C). Wild-type or Lxrα/β−/− mice were fed ad libitum diets containing 0.2% cholesterol and vehicle (Veh), LXR agonist (T0901317, 50 mg/kg body weight) or RXR-specific agonist (LG268, 30 mg/kg body weight) for 10 days. Northern analysis was performed as above. Blots were quantitated by PhosphorImager, standardized against actin or cyclophilin (cyclo), and mathematically adjusted to establish a unit of 1 for the wild-type group receiving vehicle.

Figure 7

Coordinate regulation of genes involved in macrophage sterol efflux by LXRs. Peritoneal macrophages were isolated from wild-type or Lxrα/β−/− mice and incubated for 42 h with 100 μM mevalonic acid (control) or mevalonic acid plus 5 μM compactin (unloaded), 10 μM 20(S)-hydroxycholesterol [20(S)HC], 10 μM 22(R)-hydroxycholesterol [22(R)HC], or 10 μM lanosterol. Northern analysis was performed as described above.