Elsevier

Biochemical Pharmacology

Volume 76, Issue 8, 15 October 2008, Pages 1006-1013
Biochemical Pharmacology

The RXR agonists PA024 and HX630 have different abilities to activate LXR/RXR and to induce ABCA1 expression in macrophage cell lines

https://doi.org/10.1016/j.bcp.2008.08.005Get rights and content

Abstract

Release of cellular cholesterol by ATP-binding cassette transporter (ABC)A1 and apolipoproteins is a major source of plasma high-density lipoprotein (HDL). Expression of ABC transporter A1 (ABCA1) is directly stimulated by liver X receptor (LXR)/retinoid X receptor (RXR) activation. We evaluated the abilities of two RXR agonists, PA024 and HX630, to increase ABCA1 expression. In differentiated THP-1 cells, the two agonists efficiently enhanced ABCA1 mRNA expression and apoA-I-dependent cellular cholesterol release. However, in RAW264 cells and undifferentiated THP-1 cells, PA024 was highly effective while HX630 was inactive in increasing ABCA1 mRNA. In parallel, the two agonists had different abilities to activate ABCA1 promoter in an LXR-responsive-element (LXRE)-dependent manner and to directly stimulate LXRα/RXR transactivation. The ability of HX630 to enhance ABCA1 expression was correlated closely with the cellular PPARγ mRNA level. Moreover, HX630 was able to activate PPARγ/RXR. Transfection of PPARγ in RAW264 cells induced HX630-mediated activation of LXRE-dependent transcription and ABCA1 promoter, suggesting the ability of HX630 to activate PPARγ-LXR-ABCA1 pathway. We conclude that RXR agonist PA024 and HX630 have different abilities to activate LXR/RXR, and that the cell-type-dependent effect of HX630 on ABCA1 expression and HDL generation is closely associated with this defect.

Introduction

ABC transporter A1 (ABCA1) mediates and rate-limits biogenesis of high-density lipoprotein (HDL) from helical apolipoprotein acceptors, such as apoA-I, and cellular cholesterol and phospholipids [1]. Mutations in the ABCA1 gene cause Tangier disease and other genetic HDL deficiencies [2], [3], [4]. Conversely, overexpression of ABCA1 in mice resulted in a mild elevation of HDL cholesterol [5], [6] and has been shown to protect animals from atherosclerosis [7], [8]. Plasma HDL levels are inversely related to the risk of atherosclerotic cardiovascular disease [9], presumably because HDL functions to remove excess cholesterol from peripheral tissues and transport it to the liver for conversion to bile acids [10]. Accordingly, therapies that increase ABCA1 expression are a promising strategy for preventing and treating atherogenesis.

Cellular expression of ABCA1 is highly regulated. Loading cholesterol into macrophages and fibroblasts resulted in enhanced transcription of the ABCA1 gene by the reaction mediated by the oxysterol-activated liver X receptor (LXR) [11], [12], [13], [14]. ABCA1 expression can also be increased by PPARα or PPARγ activators [15].

Retinoid X receptor (RXR) is a member of the nuclear receptor superfamily and forms heterodimers with a number of other receptors. RXR heterodimers, such as the peroxisome proliferator-activated receptor (PPAR)/RXR, LXR/RXR and the farnesoid X receptor (FXR)/RXR, can be activated by agonists for both RXR and the partner receptors and are classified as permissive heterodimers [16], [17], [18]. The thyroid hormone receptor/RXR or vitamin D receptor/RXR are not activated by RXR agonists and termed as non-permissive heterodimers [16], [17], [18]. A natural RXR agonist, 9-cis-retinoic acid, and synthetic RXR-selective ligands (rexinoids) have been shown to increase ABCA1 expression in macrophages [12], [19], [20].

In the present study, we evaluated the ability of two RXR agonists, PA024 and HX630, to induce ABCA1 expression in macrophage cell lines. Both PA024 and HX630 have been developed as RXR-selective agonists and are inactive alone in the HL-60 differentiation assay but strongly enhance the activity of low concentration of RAR-selective agonist AM80 [21]. However, their effects on the other RXR heterodimers are unknown. We found that PA024 potently induces ABCA1 expression in all cell models examined. However, HX630 failed to induce ABCA1 expression in RAW264 cells and undifferentiated THP-1 cells, and this defect was closely associated with the lack of ability to activate LXR/RXR. Instead, HX630 was able to activate PPARγ/RXR and induce ABCA1 expression in differentiated THP-1 cells. Our data also suggest the ability of HX630 to stimulate the PPARγ-LXR-ABCA1 pathway.

Section snippets

Materials

22(R)-hydroxycholesterol and phorbol 12-myristate 13-acetate (PMA) was obtained from Sigma; troglitazone from BIOMOL Research Laboratories Inc. (Plymouth Meeting, PA, USA) HX630, PA024, and Am80 were prepared as described previously [21], [22], [23].

Cell culture and real time quantitative RT-PCRs

RAW264 cells were obtained from the Riken Gene Bank (Tsukuba, Japan) and maintained in Dulbecco's modified Eagle's medium (DMEM)/F-12 (1:1) containing 10% fetal calf serum. Cells were incubated for 24 h in serum-free medium containing 0.1% BSA in the

RXR agonist PA024 and HX630 show different abilities to induce ABCA1 expression

We tested the ability of RXR agonist PA024 and HX630 to induce ABCA1 expression in murine macrophage-like cell line, RAW264. Treatment of RAW264 cells with PA024 markedly induced ABCA1 mRNA expression, which peaked at 100 nM, whereas HX630 up to 1 μM had no effect (Fig. 1A left). Expression of ABCA1 can be stimulated by oxysterol-activated LXR [11], [13], [14]. The addition of 22(R)-hydroxycholesterol to the medium increased ABCA1 expression and strongly enhanced the effect of PA024 (Fig. 1A

Discussion

In the present study, we found that RXR agonist PA024 efficiently enhanced ABCA1 mRNA expression in all cell lines tested and strongly promoted apoA-I-mediated cholesterol release (HDL generation) from PMA-differentiated THP-1 cells (Fig. 1B and C). However, HX630 was unable to raise the ABCA1 mRNA level in RAW264 cells (Fig. 1A) and undifferentiated THP-1 cells (Fig. 1B), but was active in differentiated THP-1 cells (Fig. 1B).

The different abilities of the two agonists to induce ABCA1 mRNA

Acknowledgments

This work was supported in part by a grant from the Japan Health Sciences Foundation, a grant (MF-16) from the Organization for Pharmaceutical Safety and Research, and Grant-in-Aid for Scientific Research 20590116 from Japan Society for the Promotion of Science.

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    Present address: Department of Molecular and System Pharmacology, Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka, Japan.

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