Control of cellular cholesterol efflux by the nuclear oxysterol receptor LXR alpha

Abstract

LXR alpha is a nuclear receptor that has previously been shown to regulate the metabolic conversion of cholesterol to bile acids. Here we define a role for this transcription factor in the control of cellular cholesterol efflux. We demonstrate that retroviral expression of LXR alpha in NIH 3T3 fibroblasts or RAW264.7 macrophages and/or treatment of these cells with oxysterol ligands of LXR results in 7- to 30-fold induction of the mRNA encoding the putative cholesterol/phospholipid transporter ATP-binding cassette (ABC)A1. In contrast, induction of ABCA1 mRNA in response to oxysterols is attenuated in cells that constitutively express dominant-negative forms of LXR alpha or LXR beta that lack the AF2 transcriptional activation domain. We further demonstrate that expression of LXR alpha in NIH 3T3 fibroblasts and/or treatment of these cells with oxysterols is sufficient to stimulate cholesterol efflux to extracellular apolipoprotein AI. The ability of oxysterol ligands of LXR to stimulate efflux is dramatically reduced in Tangier fibroblasts, which carry a loss of function mutation in the ABCA1 gene. Taken together, these results indicate that cellular cholesterol efflux is controlled, at least in part, at the level of transcription by a nuclear receptor-signaling pathway. They suggest a model in which activation of LXRs by oxysterols in response to cellular sterol loading leads to induction of the ABCA1 transporter and the stimulation of lipid efflux to extracellular acceptors. These findings have important implications for our understanding of mammalian cholesterol homeostasis and suggest new opportunities for pharmacological regulation of cellular lipid metabolism.

Figure 1

Lipoproteins and oxysterols induce ABCA1 mRNA levels in human and murine macrophages. Total RNA (10 μg per lane) was separated on a 1% agarose/formaldehyde gel, transferred to nylon, and hybridized to radiolabeled cDNA probes. Membranes were probed with GAPDH cDNA to ensure equivalent loading and integrity of RNA. (A) Human macrophages were incubated for 48 h in medium A supplemented with 100 μM mevalonic acid. A lipid mixture (100 μg/ml Ac-LDL, 1 μg/ml 25-OHC, and 10 μg/ml cholesterol), 100 μg/ml Ac-LDL, Ox-LDL or LDL, or 4 μg/ml 25-OHC was added to the cells as indicated. (B) Human macrophages were incubated for 8 h in medium A in the absence or presence of the lipid mixture or cycloheximide (10 μg/ml) as indicated. (C) RAW264.7 macrophages were incubated for 48 h in medium A supplemented with 100 μM mevalonic acid in the absence or presence of lipid mixture or oxysterol (10 μM). (D) RAW264.7 cells were incubated for 8 h in medium A supplemented with 20(S)-OHC and/or actinomycin D (5 μg/ml), as indicated.

Figure 2

Retroviral expression of LXRα in RAW264.7 cells facilitates the induction of ABCA1 and ABCG1 mRNA in response to LXR and RXR ligands. (A) RAW264.7 cells were transduced with retroviral vector alone or LXRα expression vector as described in Materials and Methods. Cells were incubated for 24 h in medium A containing the indicated concentration of 20(S)-OHC and/or LG268 (50 nM). Northern analysis was performed as in Fig. 1. A cDNA probe for GAPDH was used as a control to ensure equivalent loading and integrity of RNA (not shown). (B) Quantitation of ABCA1 and ABCG1 mRNA levels shown in part A normalized for GAPDH expression. mRNA levels in untreated RAW-vector cells were arbitrarily assigned value of 1.0.

Figure 3

Retroviral expression of LXRα in NIH 3T3 cells promotes the induction of ABCA1 mRNA in response to LXR and RXR ligands. (A) NIH 3T3 cells were transduced with retroviral vector alone or LXRα expression vector as described in Materials and Methods. Cells were incubated for 24 h in medium A containing the indicated concentration of 20(S)-OHC, 22(R)-OHC, and/or LG268 (50 nM). Northern analysis was performed as in Fig. 1. A cDNA probe for GAPDH was used as a control to ensure equivalent loading and integrity of RNA (not shown). (B) Quantitation of ABCA1 mRNA levels shown in A normalized for GAPDH expression. mRNA levels in untreated NIH-vector cells were arbitrarily assigned value of 1.0.

Figure 4

Expression of dominant negative LXRα or LXRβ in NIH 3T3 cells blunts induction of ABCA1 mRNA in response to 20(S)-OHC. NIH-3T3 cells were transduced with retroviral vector alone or LXRαΔAF2 or LXRβΔAF2 expression vectors as described in Materials and Methods. Cells were incubated for 24 h in medium A containing the indicated concentration of 20(S)-OHC. Northern analysis was performed as in Fig. 1. ABCA1 mRNA levels were quantitated by PhosphorImager and normalized to GAPDH. For each experiment the level of ABCA1 mRNA in NIH-vector cells in the absence of oxysterol was assigned a value of 1.0. (A) Effect of a dominant-negative LXRα on ABCA1 expression. (B) Effect of a dominant-negative LXRβ on ABCA1 expression.

Figure 5

Expression and activation of LXRα stimulates cholesterol efflux to apoAI through the ABCA1 pathway. (A) Oxysterol ligands of LXR stimulate cholesterol efflux from NIH 3T3 cells. NIH-vector and NIH-LXRα cells were plated in 24-well plates and incubated for 24 h in medium A supplemented with [³H]cholesterol in the absence or presence of the indicated oxysterol (1 μg/ml) or LG268 (LG, 50 nM). ApoAI-dependent cholesterol efflux to the medium was determined as described in Materials and Methods. Data are presented as a percentage (±SE) of the total radioactivity in cells and medium; each point is the numerical average of triplicate experiments. (B) Stimulation of cholesterol efflux by LXR ligands is mediated by ABCA1. Normal or Tangier transformed human skin fibroblasts were plated in 24-well plates and incubated for 24 h in medium A supplemented with [³H]cholesterol in the absence or presence of the indicated oxysterol (1 μg/ml) or LG268 (50 nM). ApoAI-dependent cholesterol efflux to the medium was determined as described in Materials and Methods. Data are presented as a percentage (±SE) of the total radioactivity in cells and medium; each point is the numerical average of triplicate experiments. (Inset) Quantitation of Northern blot analysis of ABCA1 mRNA levels in normal and Tangier fibroblasts normalized for GAPDH expression.

Figure 6

Model for the regulation of ABCA1 expression and cellular cholesterol efflux by LXR in response to lipid loading. Binding of Ox-LDL to cell surface receptors (CD36, SR-A, SR-BI, etc.) leads to internalization and degradation in lysosomes (Lys). Oxysterols, either present in Ox-LDL or produced intracellularly from cholesterol, enter the nucleus and activate the LXR/RXR heterodimer on target genes such as ABCA1. Induction of ABCA1 protein expression results in increased efflux of cholesterol and/or phospholipids to apoAI.