Research article
Unsaturated fatty acids repress expression of ATP binding cassette transporter A1 and G1 in RAW 264.7 macrophages

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Abstract

Reverse cholesterol transport (RCT), a process to deliver excess cholesterol from the periphery to the liver for excretion from body, is a major atheroprotective property of high-density lipoproteins. As major transporters for cholesterol efflux in macrophages, ATP-binding cassette transporter A1 (ABCA1) and G1 (ABCG1) are critical for RCT. We investigated mechanisms for the regulation of ABCA1 and ABCG1 expression by fatty acids (FA) in RAW264.7 macrophages. Cells were incubated with 100 μmol/L of palmitic, oleic, linoleic, linolenic or eicosapentaenoic acids in the absence or presence of T0901317, a liver X receptor (LXR) agonist. Unsaturated FA, but not saturated FA, significantly reduced ABCA1 and ABCG1 mRNA without the agonist. Trichostatin A (TSA), a histone deacetylase inhibitor, not only increased basal ABC transporter expression but abrogated the transcriptional repression by unsaturated FA. The increased basal ABCA1 and ABCG1 mRNA by TSA paralleled the increased peroxisome proliferator-activated receptor γ (PPARγ) and PPARγ coactivator 1α expression, whereas LXRα and PGC-1β expression was significantly lowered. Although the repressive effect of ABCA1 and ABCG1 mRNA by unsaturated FA was abolished by T0901317, protein levels remained diminished. Chemical and genetic deficiency of protein kinase C δ did not abolish the repressive effect of linoleic acid on ABCA1 and ABCG1. In conclusion, unsaturated FA repressed ABCA1 and ABCG1 expression by two distinct mechanisms in RAW 264.7 macrophages: LXR-dependent transcriptional repression possibly by modulating histone acetylation state and LXR-independent posttranslational inhibition.

Introduction

Since the hypothesis that high levels of plasma high-density lipoprotein (HDL) cholesterol are protective against coronary heart disease (CHD) was initially proposed in the early 1950s [1], epidemiological studies have consistently shown a strong inverse relationship between plasma HDL cholesterol levels and the incident of CHD [2], [3], [4], [5]. Dyslipidemia, an underlying pathological condition for CHD and type 2 diabetes, is characterized by high plasma concentrations of total cholesterol, low-density lipoprotein (LDL) cholesterol and triglycerides as well as low plasma HDL cholesterol levels [6]. The antiatherogenic effect of HDL is due in part to the ability of HDL to promote cholesterol efflux from cells and to participate in reverse cholesterol transport (RCT) [7], [8], [9]. In the RCT, excess cholesterol is transported from the periphery to the liver for ultimate excretion from body, and HDL functions as the primary acceptor of cellular free cholesterol [10].

Removal of cholesterol from macrophages in arterial wall via the RCT is of significant importance in the prevention of atherosclerosis development. ATP binding cassette transporter A1 (ABCA1) and G1 (ABCG1) play a pivotal role in this process. ABCA1 facilitates the efflux of cellular cholesterol to extracellular acceptors, namely, lipid-free or lipid-poor apolipoprotein A-I [11], [12], [13], [14]. In contrast, ABCG1 is highly expressed in macrophages and mediates the efflux of cholesterol to HDL2 [15], [16]. Deletion of macrophage Abcg1 led to the deposition of free cholesterol and cholesteryl esters in various tissues, implicating its role in maintaining cellular cholesterol level [17], [18]. Studies have shown that dysfunction of ABCA1 and ABCG1 in macrophages induces cholesterol accumulation and accelerates atherosclerosis, although some contradictory observations exist as to the role of ABCG1 in atherogenesis [19], [20], [21], [22], [23], [24].

Transcription of ABCA1 and ABCG1 is primarily under the control of liver X receptors (LXRs) in response to cellular cholesterol levels [25]. Studies have shown that fatty acids can also alter ABCA1 expression at the transcriptional and posttranscriptional levels. Posttranscriptional repression of ABCA1 expression by unsaturated fatty acids in macrophages has been demonstrated by a series of studies conducted by Wang et al. [26] and Wang and Oram [27], [28], [29].In their studies, unsaturated fatty acids increase ABCA1 protein degradation in macrophages by activating phospholipase D2 (PLD2) and subsequently protein kinase C δ (PKCδ). This, in turn, phosphorylates ABCA1 serine residues for protein degradation. Other studies exist to demonstrate that fatty acids could alter transcription of ABCA1 and ABCG1. In human monocyte-derived macrophages, basal ABCA1 and ABCG1 mRNA levels were lowered by linoleic acid (18:2) compared with palmitic acid (16:0) [6]. Basal ABCA1 mRNA abundance in HepG2 and RAW 264.7 macrophages was reduced by eicosapentaenoic acid in a dose-dependent manner [30]. Unsaturated fatty acids, including oleic acid, arachidonic acid and eicosapentaenoic acid, also significantly lowered ABCA1 expression when an LXR agonist was present by decreasing ABCA1 promoter activity [30]. In RAW 264.7 macrophages, unsaturated fatty acids repressed ABCA1 and ABCG1 mRNA levels in the absence or presence of an LXR agonist, and mutations or deletion of direct repeat 4, a cis-acting element for LXR binding, in the promoters of ABCA1 and ABCG1 abolished the suppressive effects [31]. These studies suggest that unsaturated fatty acids repress the basal as well as LXR-activated ABC transporter expression.

Studies have demonstrated that unsaturated fatty acids, but not saturated fatty acids, reduce ABCA1 and ABCG1 expression. However, limited and contradictory observations exist as to the molecular mechanisms underlying the repressive effect of unsaturated fatty acids. This study shows that fatty acids commonly present in diet can alter gene expression via a modulation of histone deacetylation. In addition, an LXR-independent posttranscriptional inhibition of the ABC transporter expression exists in macrophages.

Section snippets

Cell culture and fatty acid preparation

RAW 264.7 macrophages were purchased from ATCC (Manassas, VA, USA) and maintained in RPMI medium containing 10% fetal bovine serum (FBS), 100 kU/L penicillin,100 mg/L streptomycin, 1× vitamins and 2 mmol/L l-glutamine in a humidified incubator at 37°C with 5% CO2. All cell culture supplies were purchased from Mediatech (Manassas, VA, USA).

Sodium salts of fatty acids (Nu-Chek, Elysian, MN, USA) were dissolved in the fatty-acid-poor and endotoxin-free bovine serum albumin (BSA) stock solution (2

Macrophage ABCA1 and ABCG1 expression was repressed by unsaturated fatty acids

RAW264.7 macrophages were treated with various fatty acids including palmitic acid (16:0), palmitoleic acid (16:1), oleic acid (18:1), 18:2, linolenic acid (18:3) and eicosapentaenoic acid (20:5) in the absence or presence of T091317, an LXR agonist. In the absence of the LXR agonist, both ABCA1 and ABCG1 mRNA levels were significantly repressed by all the unsaturated fatty acids tested compared with the control (Fig. 1A). However, when T0901317 was present, the repressive effect was completely

Discussion

As major transporters for cholesterol efflux in macrophages, ABCA1 and ABCG1 play a pivotal role in maintaining cellular cholesterol homeostasis. Disturbance of the ABC transporter functions in macrophages, particularly in the arterial wall, could facilitate foam cell formation and consequently atherogenesis. The goal of this study was to understand molecular mechanisms for the regulation of macrophage ABCA1 and ABCG1 by fatty acids, whose plasma concentrations are commonly elevated in

Acknowledgments

C. S. Ku, Y. Park, and S. L. Coleman conducted experiments; J. Lee designed the experiments and wrote the manuscript. All authors read and approved the final manuscript.

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    Funding sources: This work was supported by National Science Foundation-EPSCoR grant EPS-0346476 and University of Nebraska Foundation Layman Award to J. Lee.

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