Biochemical and Biophysical Research Communications
Mycophenolic acid induces ATP-binding cassette transporter A1 (ABCA1) expression through the PPARγ–LXRα–ABCA1 pathway
Highlights
► Using an ABCA1p-LUC HepG2 cell line, we found that MPA upregulated ABCA1 expression. ► MPA induced ABCA1 and LXRα protein expression in HepG2 cells. ► PPARγ antagonist GW9662 markedly inhibited MPA-induced ABCA1 and LXRα protein expression. ► The effect of MPA upregulating ABCA1 was due mainly to activation of the PPARγ-LXRα-ABCA1 pathway.
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.
Section snippets
Cell culture
Human hepatoma HepG2 cells were grown in RPMI 1640 medium (Hyclone, Logan, UT, USA) with Earle’s balanced salt and 2 mM l-glutamine solution (medium A) supplemented with 10% heat-inactivated fetal bovine serum (medium B). A stably transformed ABCA1p-LUC HepG2 cell line was maintained in medium C (medium B supplemented with 500 μg/ml G418). Rat peritoneal RAW264.7 macrophages were passaged in Dulbecco’s modified Eagle’s medium (Hyclone) containing 10% fetal bovine serum (medium D). All cells were
MPA induced ABCA1 expression in ABCA1p-LUC HepG2 cells
MPA was isolated from strain I07F-02378, which tested positive for potential ABCA1 upregulators. The effect of MPA on the transcriptional activity of the ABCA1 gene promoter was detected in ABCA1p-LUC HepG2 cells. MPA induced ABCA1 transcription in a dose-dependent manner, with an EC50 of 0.09 μM. MPA upregulated ABCA1 transcriptional activity to a maximal value of 360% (Fig. 1B).
MPA induced ABCA1 expression in HepG2 cells
To confirm ABCA1 upregulation by MPA in the luciferase reporter assay, we examined the effect of MPA on ABCA1 mRNA
Discussion
The efficacy of MPA, which inhibits inosine monophosphate dehydrogenase, in preventing allograft rejection and the treatment of rejection is now firmly established. Recent studies have shown that MMF significantly reduces plaque development in a hypercholesterolemic model of atherosclerosis [14], [15]. This effect is associated with a reduction in macrophage and foam cell infiltration, an inhibition of smooth muscle cell proliferation and infiltration, and a decrease in the recruitment of
Acknowledgments
This work was supported by the Key New Drug Creation and Manufacturing Program (2009ZX09302-004), the National Natural Science Foundation of China (81102443) and National Natural Science Foundation of China-Guangdong Provincial People’s Government of the Joint Natural Science Fund Projects (U1032007).
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These authors contributed equally to this study.