Autoregulation of the human liver X receptor alpha promoter - PubMed (original) (raw)

Autoregulation of the human liver X receptor alpha promoter

B A Laffitte et al. Mol Cell Biol. 2001 Nov.

Abstract

Previous work has implicated the nuclear receptors liver X receptor alpha (LXR alpha) and LXR beta in the regulation of macrophage gene expression in response to oxidized lipids. Macrophage lipid loading leads to ligand activation of LXRs and to induction of a pathway for cholesterol efflux involving the LXR target genes ABCA1 and apoE. We demonstrate here that autoregulation of the LXR alpha gene is an important component of this lipid-inducible efflux pathway in human macrophages. Oxidized low-density lipoprotein, oxysterols, and synthetic LXR ligands induce expression of LXR alpha mRNA in human monocyte-derived macrophages and human macrophage cell lines but not in murine peritoneal macrophages or cell lines. This is in contrast to peroxisome proliferator-activated receptor gamma (PPAR gamma)-specific ligands, which stimulate LXR alpha expression in both human and murine macrophages. We further demonstrate that LXR and PPAR gamma ligands cooperate to induce LXR alpha expression in human but not murine macrophages. Analysis of the human LXR alpha promoter led to the identification of multiple LXR response elements. Interestingly, the previously identified PPAR response element (PPRE) in the murine LXR alpha gene is not conserved in humans; however, a different PPRE is present in the human LXR 5'-flanking region. These results have implications for cholesterol metabolism in human macrophages and its potential to be regulated by synthetic LXR and/or PPAR gamma ligands. The ability of LXR alpha to regulate its own promoter is likely to be an integral part of the macrophage physiologic response to lipid loading.

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Figures

FIG. 1

Induction of LXRα expression in human macrophages in response to modified LDL loading. Differentiated THP-1 macrophages, primary human monocyte-derived macrophages (Mφ), or thioglycolate-elicited mouse peritoneal macrophages were incubated for 48 h in RPMI medium containing 10% LPDS, 5 μM simvastatin, and 100 μM mevalonic acid. Cells were treated with vehicle control or 100 μg (protein) of either LDL, oxLDL, or acLDL per ml as indicated. Total RNA (10 μg/lane) was electrophoresed through formaldehyde-containing gels, transferred to nylon, and hybridized to 32P-labeled cDNA probes. 36B4 was used as a control for loading and integrity of the RNA.

FIG. 2

Oxysterols and synthetic LXR ligands stimulate LXRα expression in THP-1 macrophages. Differentiated THP-1 macrophages (A and B) or thioglycolate-elicited mouse peritoneal macrophages (Mφ) (C) were incubated for 48 h in RPMI medium plus 10% LPDS or RPMI medium plus 10% LPDS, 5 μM simvastatin, and 100 μM mevalonic acid (unloaded). Oxysterols [20(S)HC, 22(R)HC, or 22(S)HC, 2.0 μg/ml], synthetic LXR ligand (GW3965 or T1317, 0.1 to 10.0 μM), or RXR ligand (LG268, 50 nM) was included as indicated. Northern analysis was performed as described in the legend to Fig. 1.

FIG. 3

Differential induction of the LXR target gene apoE by LXR ligands in human and murine macrophages. Differentiated THP-1 macrophages or thioglycolate-elicited mouse peritoneal macrophages were incubated for 48 h in RPMI medium plus 10% LPDS, 5 μM simvastatin, and 100 μM mevalonic acid. Cells were treated with the indicated concentrations of either T1317 or GW3965. The expression of apoE mRNA was monitored by real-time quantitative PCR (Taqman) assays (see Materials and Methods).

FIG. 4

Ligands for LXR and PPARγ additively induce LXRα expression in human macrophages. Differentiated THP-1 macrophages, MonoMac-6 cells, human monocyte-derived macrophages (Mφ), or thioglycolate-elicited mouse peritoneal macrophages were incubated for 48 h in RPMI medium plus 10% LPDS. Oxysterols {20(S)HC [20 (S)] or 22(R)HC [22 (R)], 2.0 μg/ml}, synthetic LXR ligands (GW3965 or T1317, 5 μM), and/or PPARγ ligands (rosiglitazone [Rosi] or GW7845, 5 μM) were included as indicated. Northern blots (A) were quantitated by phosphorimager analysis and normalized to 36B4. The level of expression relative to LPDS control (fold induction) is indicated; real-time quantitative PCR assays (B) were performed in duplicate as described in Materials and Methods and normalized to 36B4 or β-actin. The level of mRNA expression relative to control (fold induction) is indicated.

FIG. 5

Autoregulation of the hLXRα gene in preadipocytes and HepG2 cells. Primary human preadipocytes (Pre Ad), HepG2 cells, or 3T3-F442A murine preadipocytes were cultured for 48 h in media containing 10% LPDS and one or more of the following nuclear receptor ligands as indicated: GW3965 (5 μM), T1317 (5 μM), GW7845 (5 μM), and Wy14643 (50 μM).

FIG. 6

Sequence comparison of the hLXRα and mLXRα transcriptional start sites. The putative transcriptional start sites are marked by arrows. The initiator element is indicated in bold type. The previously published mouse transcriptional start site is designated as position +1 (1).

FIG. 7

Comparison of the hLXRα and mLXRα promoter regions. (A) Schematic representation of the hLXRα and mLXRα genes. Transcription start sites, genomic structure, sequence identity, and nuclear receptor binding sites are indicated. The asterisk denotes the major start site in macrophages. The arrows indicate hormone response element half sites. (B) Sequences of the LXREs and PPREs from the hLXRα and mLXRα promoters.

FIG. 8

PPARγ and LXRα bind to response elements in the hLXRα promoter. Gel mobility shift assays were performed using in vitro-translated receptors and end-labeled oligonucleotide probes as described in Materials and Methods. (A) Direct binding of PPARγ/RXR heterodimers to a putative PPRE from the hLXRα promoter. (B) Direct binding of LXRα/RXR heterodimers to the LXRE-A, LXRE-B, and LXRE-C sites from the hLXRα promoter. (C) Competition for LXRα/RXR binding to LXRE-C. Unlabeled oligonucleotide was included in the binding reaction at the indicated molar excess.

FIG. 9

LXRα, LXRβ, and PPARγ activate the hLXRα promoter. (A) LXRα/RXRα and LXRβ/RXR heterodimers activate the −2625-bp LXRα proximal promoter. HepG2 cells were transfected with either control pGL3-luc or -2625 LXRα-luc reporters with or without CMX-mLXRα/CMX-RXRα or CMX-mLXRβ/CMX-RXRα and CMV–β-galactosidase. Following transfection, cells were incubated for 24 h in MEM supplemented with 10% LPDS and 1 μM GW3965 or vehicle control. Luciferase activity was normalized for transfection efficiency using β-galactosidase activity. (B) Ligand activation of PPARγ and LXRα has an additive effect on the −2625-bp LXRα promoter. HepG2 cells were transfected as in panel A except that CMX-mPPARγ1 and GW7845 (1 μM) were included as indicated.

FIG. 10

Deletion and mutation analysis of the hLXRα proximal promoter. (A) Luciferase reporters containing bp −2625 to +345, −2210 to +345, −1310 to + 345, or 560 to +345 of the hLXRα promoter were transfected into HepG2 cells in the presence or absence of CMX-hLXRα, CMX-RXRα, and 5 μM T1317. Luciferase activity was normalized for transfection efficiency using β-galactosidase activity. The data are expressed as fold activations in the presence of the indicated ligand versus in the absence of ligand and represent the average of triplicate experiments. (B) hLXRα promoter constructs were cotransfected into HepG2 cells along with CMX vector or CMX-VP16-LXRα in the presence of 5 μM T1317. Data are expressed as relative luciferase activities normalized to β-galactosidase activities and represent the average of triplicate experiments. (C) Mutations were introduced into individual LXREs in the bp −2625 to +345 hLXRα promoter construct by site-directed mutagenesis. Wild-type (WT) and mutant (Mut) reporters were transfected into HepG2 cells along with CMX-hLXRα and CMX-RXRα in the presence or absence of 1 μM GW3965. Data are expressed as luciferase activities normalized to β-galactosidase activities and represent the averages of triplicate experiments.

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Autoregulation of the human liver X receptor alpha promoter - PubMed (original) (raw)