Mycophenolic acid induces ATP-binding cassette transporter A1 (ABCA1) expression through the PPARγ–LXRα–ABCA1 pathway

Abstract

ATP-binding cassette transporter A1 (ABCA1) promotes cholesterol and phospholipid efflux from cells to lipid-poor apolipoprotein A-I and plays an important role in atherosclerosis. In a previous study, we developed a high-throughput screening method using an ABCA1p-LUC HepG2 cell line to find upregulators of ABCA1. Using this method in the present study, we found that mycophenolic acid (MPA) upregulated ABCA1 expression (EC50 = 0.09 μM). MPA upregulation of ABCA1 expression was confirmed by real-time quantitative reverse transcription-PCR and Western blot analysis in HepG2 cells. Previous work has indicated that MPA is a potent agonist of peroxisome proliferator-activated receptor gamma (PPARγ; EC50 = 5.2–9.3 μM). Liver X receptor α (LXRα) is a target gene of PPARγ and may directly regulate ABCA1 expression. Western blot analysis showed that MPA induced LXRα protein expression in HepG2 cells. Addition of PPARγ antagonist GW9662 markedly inhibited MPA-induced ABCA1 and LXRα protein expression. These data suggest that MPA increased ABCA1 expression mainly through activation of PPARγ. Thus, the effects of MPA on upregulation of ABCA1 expression were due mainly to activation of the PPARγ–LXRα–ABCA1 signaling pathway. This is the first report that the antiatherosclerosis activity of MPA is due to this mechanism.

Introduction

Atherosclerotic cardiovascular disease is a major cause of morbidity and mortality worldwide. The pathological hallmark of atherosclerosis is excessive accumulation of cholesterol by macrophages and their subsequent transformation into foam cells [1]. Removal of excess free cholesterol from arterial cells is widely accepted as important for maintaining cellular cholesterol homeostasis, decreasing the size of atherosclerotic plaques, and protecting against atherosclerosis.

ATP-binding cassette transporter A1 (ABCA1) is a member of the ABC superfamily and is highly expressed in numerous human organs, including placenta, liver, lungs, adrenal glands, and fetal tissues [2]. ABCA1 mediates cellular cholesterol and phospholipid efflux to lipid-poor apolipoprotein A-I (apoA-I) and plays a key role in high-density lipoprotein (HDL) synthesis and reverse cholesterol transport [3], [4]. Loss-of-function mutations in the ABCA1 gene result in Tangier disease, which is characterized by cholesterol accumulation in tissue macrophages, decreased plasma HDL cholesterol, and increased incidence of cardiovascular disease [5]. Transgenic mice that strongly express ABCA1 elevate levels of plasma HDL and apoA-I, increase cholesterol efflux from macrophages and result in significantly less aortic atherosclerosis [6]. ABCA1 is thought to be an attractive target for the development of drugs for preventing atherosclerosis.

Expression of the ABCA1 gene is transcriptionally regulated [7]. Recent studies indicate that peroxisome proliferator-activated receptor gamma (PPARγ), a member of the nuclear receptor superfamily, enhances cholesterol efflux by inducing transcription of the liver X receptor (LXRα) gene and thus ABCA1 [8]. LXRα forms obligate heterodimers with retinoid X receptor, and recognizes specific DNA response elements in the ABCA1 promoter and stimulates the transcription of the ABCA1 gene in this configuration. This stimulation increases ABCA1-dependent cholesterol efflux to apoA-I [9]. The PPARγ–LXRα–ABCA1 pathway represents a powerful means of stimulating cholesterol efflux from macrophages and therefore could strongly influence the progression of atherosclerotic plaques [8].

In our previous study, a high-throughput screening (HTS) method for ABCA1 up-regulators was developed. Strain I07F-02378 was a positive hit in the HTS assay. In this study, we found that mycophenolic acid (MPA; Fig. 1A) produced by strain I07F-02378 upregulated ABCA1 expression. MPA has been used as an immunosuppressant to prevent rejection in organ transplantation. Mycophenolate mofetil (MMF) is the prodrug of MPA, which is a potent and noncompetitive inhibitor of inosine monophosphate dehydrogenase (MPDH). MMF is also efficacious in several experimental animal models for chronic rejection, such as kidney diseases, autoimmune uveoretinitis, experimental autoimmune encephalomyelitis, and adjuvant arthritis. Recent evidence indicates that MPA is active against tumors, Pneumocystis carinii, hepatitis C virus, and human immunodeficiency virus. Previous studies showed that the effects of MPA on adipocyte-like terminal differentiation of breast cancer cells are partially due to PPARγ activation [10], [11].

The current study was designed to investigate the effect of MPA on ABCA1 expression and to explore the possible mechanism of the effects of MPA on ABCA1 regulation.