LXRα regulates human CETP expression in vitro and in transgenic mice

doi:10.1016/j.atherosclerosis.2010.04.025

Atherosclerosis

Volume 212, Issue 1, September 2010, Pages 139-145

https://doi.org/10.1016/j.atherosclerosis.2010.04.025 Get rights and content

Abstract

Liver X receptors (LXRs), LXRα and LXRβ, are members of the nuclear receptor superfamily and regulate the expression of genes involved in the regulation of cholesterol and fatty acid metabolism. Human plasma, unlike mouse plasma, contains cholesteryl ester transfer protein (CETP), which plays an important role in reverse cholesterol transport (RCT). LXRs induce CETP transcription via a direct repeat 4 element in the CETP promoter. However, the specific roles of the individual LXR subtypes in CETP expression and their consequences on plasma lipoprotein metabolism are still unclear. Here we showed that synthetic LXR agonist enhanced plasma CETP activity and resulted in non-high density lipoprotein (non-HDL) increase and HDL decrease in cynomolgus monkeys and human CETP transgenic mice. To address the relative importance of the two LXR subtypes, we investigated the effect of the suppression of both LXR subtypes on CETP expression in HepG2 cells. CETP expression induced by the LXR agonist was significantly reduced by LXRα knock-down, but not by LXRβ. Consistent with these data, CETP promoter activity was enhanced by LXRα activation, whereas LXRβ activation had only a minor effect. Furthermore, we investigated the effect of genetic deficiency of both LXR subtypes in human CETP transgenic mice. LXRα deficiency abolished the augmentation of plasma CETP activity and hepatic CETP expression induced by the synthetic LXR agonist, consequently increasing HDL and decreasing non-HDL, whereas LXRβ deficiency did not affect CETP activation. These findings indicate that LXRα has an essential role in the regulation of CETP expression and maintaining RCT.

Introduction

Liver X receptors (LXRs), LXRα and LXRβ, are members of the nuclear receptor superfamily of transcription factors. The LXR transcription factors form heterodimers with the retinoid X receptor (RXR) and regulate target gene expression through interaction with a direct repeat 4 (DR-4) element, aka., LXR response element (LXRE) [1], [2]. LXRα is predominantly expressed in tissues which play important roles in lipid homeostasis, such as the liver, small intestine, adipocytes and macrophages, while LXRβ is ubiquitously expressed [3], [4], [5], [6]. The natural agonists for both LXR isoforms include oxidized derivatives of cholesterol, such as 22(R)-hydroxycholesterol and 27-hydroxycholesterol [7]. Both LXRs function as intracellular sensors of cholesterol levels. The activation of LXRs by synthetic agonists has been reported to decrease intestinal cholesterol absorption [8], [9]. It was also documented that the activation of both LXRs increases the expression of the gene encoding ATP binding cassette protein A1 (Abca1) and G1 (Abcg1) [9], [10], [11], [12], which induces cholesterol efflux in macrophages. In addition, synthetic LXR agonists have been shown to protect the progression of atherosclerotic lesions in mouse models [13], [14]. Therefore, LXRs are believed to be a therapeutic target for atherosclerosis.

The expression of cholesteryl ester transport protein (CETP), which mediates the transfer of cholesteryl ester (CE) from high density lipoprotein (HDL) to triglyceride (TG) rich lipoproteins, was also documented to be regulated by LXRs [12], [15]. CETP is synthesized in the liver and secreted into plasma. Plasma CETP activity influences the atherogenicity of the lipoprotein profile and cholesterol efflux in macrophages [12]. In humans, CETP deficiency characteristically exhibits high HDL and low low-density lipoprotein (LDL) [16], [17]. Most of the previous studies addressing the role of LXRs in lipoprotein metabolism were conducted in mice [18], [19], but they do not have plasma CETP activity. The first aim of the present study was to address the impact of synthetic LXR agonist on plasma CETP activity and lipoprotein profiles in animals exhibiting CETP, such as cynomolgus monkeys and mice expressing human CETP under the control of its natural flanking regions containing DR-4 [20].

The second aim of the present study was to determine the relative role of LXR subtypes on CETP regulation. Abca1 and Abcg1 have been reported to be regulated by both LXRs in macrophages [9], [10], [11], [21]. Thus, there is a functional redundancy with respect to the cholesterol efflux. In contrast, TG metabolism in the liver appears to be predominantly regulated by LXRα [22], [23]. Here we demonstrate that LXRα isotype plays a critical role in the regulation of CETP expression by two different techniques, small interfering RNA (siRNA) knock-down in culture cells and genetic deficiency in human CETP transgenic (Tg) mice.

Section snippets

Reagent

LXR agonist T0901317 [24] was synthesized at Daiichi Sankyo Co., Ltd.

Animals

Male C57BL/6J mice were obtained from Charles River Japan and male CETP-Tg mice [20] were obtained from The Jackson Laboratory. CETP-Tg mice were crossbred with LXRα^−/− mice or LXRβ^−/− mice [25], [26]. The vehicle [propylene glycol (PG)/Tween 80 (4:1, v/v)] or LXR agonist T0901317 at 1 mg/kg was administered daily to the mice for 7 days. At 9:00 on the day after the last administration, plasma samples were obtained from the

Effect of LXR agonist on plasma lipids, lipoproteins and CETP activity in cynomolgus monkeys

The vehicle or T0901317 (3 or 10 mg/kg) was orally administered to cynomolgus monkeys once a day for 7 days. T0901317 at 10 mg/kg significantly increased non-HDL cholesterol (Fig. 1A) and decreased HDL cholesterol (Fig. 1B). There was no significant difference in the TC levels (Fig. 1C). T0901317 significantly elevated the plasma TG levels (Fig. 1D). We also determined plasma ANGPTL3 levels, since ANGPTL3, the endogenous LPL inhibitor, which is transcriptionally regulated by LXR, is important in

Discussion

Pharmacological activation of the nuclear receptor LXRs dramatically influences cholesterol homeostasis [1], [31], [32]. The administration of a potent synthetic LXR agonist T0901317 in mice increased plasma HDL and TG (Fig. 4A and Table 1) [24], [33], [34] and ameliorated atherosclerosis [13]. Most of these studies have been conducted in mice, a mammal species with no detectable plasma CETP activity. Humans exhibit CETP gene, which is considered to have an influence on reverse cholesterol

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The findings with BMS-852927 are consistent with the differences between rodent and human lipid physiology that will need to be addressed in future LXR-based drug development efforts. Specifically, rodents lack the plasma cholesterol ester transfer protein (CETP) that humans express, and LXR produces a distinct response on inducible degrader of LDL (IDOL) protein in rodents versus humans342,344,345. Approaches to modulate LXR should be informed by the clinically successful modulation of other NHRs, notably ER.
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A Novel Role for CETP as Immunological Gatekeeper: Raising HDL to Cure Sepsis?
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Most of the cholesterol in plasma is in an esterified form that is generated in potentially cardioprotective HDLs. Cholesteryl ester transfer protein (CETP) mediates bidirectional transfers of cholesteryl esters (CEs) and triglycerides (TGs) between plasma lipoproteins. Because CE originates in HDLs and TG enters the plasma as a component of VLDLs, activity of CETP results in a net mass transfer of CE from HDLs to VLDLs and LDLs, and of TG from VLDLs to LDLs and HDLs. As inhibition of CETP activity increases the concentration of HDL-cholesterol and decreases the concentration of VLDL- and LDL-cholesterol, it has the potential to reduce atherosclerotic CVD. This has led to the development of anti-CETP neutralizing monoclonal antibodies, vaccines, and antisense oligonucleotides. Small molecule inhibitors of CETP have also been developed and four of them have been studied in large scale cardiovascular clinical outcome trials. This review describes the structure of CETP and its mechanism of action. Details of its regulation and nonlipid transporting functions are discussed, and the results of the large scale clinical outcome trials of small molecule CETP inhibitors are summarized.

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¹: These authors contributed equally to this work.

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LXRα regulates human CETP expression in vitro and in transgenic mice

Abstract

Introduction

Section snippets

Reagent

Animals

Effect of LXR agonist on plasma lipids, lipoproteins and CETP activity in cynomolgus monkeys

Discussion

J Biol Chem

Gene

J Biol Chem

J Biol Chem

J Biol Chem

Cell Metab

FEBS Lett

J Biol Chem

Biochem Pharmacol

Cell

Atherosclerosis

J Biol Chem

J Lipid Res

Curr Opin Pharmacol

J Biol Chem

J Biol Chem

J Lipid Res

Curr Opin Genet Dev

J Biol Chem

Biochem Biophys Res Commun

J Lipid Res

Nature

Mol Endocrinol