Quercetin up-regulates expressions of peroxisome proliferator-activated receptor γ, liver X receptor α, and ATP binding cassette transporter A1 genes and increases cholesterol efflux in human macrophage cell line

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Quercetin up-regulates expressions of peroxisome proliferator-activated receptor γ, liver X receptor α, and ATP binding cassette transporter A1 genes and increases cholesterol efflux in human macrophage cell line Seung-Min Lee a , Jiyoung Moon a , Yoonsu Cho a , Ji Hyung Chung b , Min-Jeong Shin a,⁎ a b

Department of Food and Nutrition and Institute of Health Sciences, Korea University, Seoul, Republic of Korea Cardiovascular Research Institute, Yonsei University College of Medicine, Seoul, Republic of Korea

A R T I C LE I N FO

AB S T R A C T

Article history:

Cholesterol-laden macrophages trigger accumulation of foam cells and increase the risk of

Received 23 May 2012

developing atherosclerosis. We hypothesized that quercetin could lower the content of

Revised 1 November 2012

cholesterol in macrophages by regulating the expression of the ATP binding cassette

Accepted 14 November 2012

transporter A1 (ABCA1) gene in differentiated human acute monocyte leukemia cell line (THP-1) cells and thereby reducing the chance of forming foam cells. Quercetin, in

Keywords:

concentrations up to 30 μM, was not cytotoxic to differentiated THP-1 cells. Quercetin up-

Quercetin

regulated both ABCA1 messenger RNA and protein expression in differentiated THP-1 cells,

HDL

and its maximum effects were demonstrated at 0.3 μM for 4 to 8 hours in incubation. In

apoA1

addition, quercetin increased protein levels of peroxisome proliferator-activated receptor γ

ABCA1

(PPARγ) and liver X receptor α (LXRα) within 2 hours of treatment. Because PPARγ and LXRα

PPARγ

are important transcriptional factors for ABCA1, quercetin-induced up-regulation of ABCA1

LXRα

may be mediated by increased expression levels of the PPARγ and LXRα genes. Furthermore, quercetin-enhanced cholesterol efflux from differentiated THP-1 cells to both high-density lipoprotein (HDL) and apolipoprotein A1. Quercetin at the dose of 0.15 μM elevated the cholesterol efflux only for HDL. At the dose of 0.3 μM, quercetin demonstrated effects both on HDL and apolipoprotein A1. Our data demonstrated that quercetin increased the expressions of PPARγ, LXRα, and ABCA1 genes and cholesterol efflux from THP-1 macrophages. Quercetin-induced expression of PPARγ and LXRα might subsequently affect up-regulation of their target gene ABCA1. Taken together, ingestion of quercetin or quercetin-rich foods could be an effective way to improve cholesterol efflux from macrophages, which would contribute to lowering the risk of atherosclerosis. © 2013 Elsevier Inc. All rights reserved.

Abbreviations: ABCA1, ATP binding cassette transporter A1; apoA1, apolipoprotein A1; DMSO, dimethyl sulfoxide; HDL, high density lipoprotein; LDL, low-density lipoprotein; LXRα, liver X receptor α; mRNA, messenger RNA; MTT, 3-[4,5-dimethylthiazol-2-yl]-2,5diphenyltetrazolium bromide; NBD-cholesterol, 22-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-23,24-bisnor-5-cholen-3β-ol; PBS, phosphate-buffered saline; PCR, polymerase chain reaction; PMA, phorbol 12-myristate 13-acetate; PPARγ, peroxisome proliferator-activated receptor γ; SDS, sodium dodecyl sulfate; SR-A, scavenger receptor class A; THP-1, human acute monocyte leukemia cell line. ⁎ Corresponding author. Tel.: +82 2 940 2857; fax: +82 2 940 2850. E-mail address: [email protected] (M.-J. Shin). 0271-5317/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.nutres.2012.11.010

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1.

Introduction

Many chronic metabolic disorders, such as atherosclerosis, are not only related to dyslipidemia but also to aberrant lowgrade inflammation [1-3]. The presence of sustained activation of macrophages in the subendothelium in atherosclerotic lesions suggests that macrophage plays an essential role in atherosclerosis development [1,3]. During the initial phase of atherosclerosis, monocytes containing low-density lipoproteins (LDL) are recruited to the vascular lamina and differentiate into macrophages upon infiltration. These macrophages continue to receive oxidized LDL and to transform into foam cells [3]. This abnormal accumulation of foam cells is a hallmark of atherosclerosis [3]. In addition, the prolonged activation of macrophages produces inflammatory mediators and cytokines, further contributing to the inflammatory response [3]. In addition, the absence of inflammatory mediators or macrophage activation mitigates the development of atherosclerotic lesions in animals on genetically atherosclerotic background [4-6], implying that macrophages play a role in the development of atherosclerosis. Unlike other cell types, macrophages are relatively insensitive to feedback regulation in cholesterol metabolism. The expression of scavenger receptors is not negatively regulated by intracellular cholesterol levels, demonstrated in the case of LDL receptors. Macrophages incorporate modified LDL through the scavenger receptors, including scavenger receptor class A (SR-A) and CD36. A continual accumulation of oxidized LDL through these receptors in macrophages leads to the formation of foam cells [7]. Therefore, the reverse cholesterol transport mechanism is considered to be important in macrophages. Cholesterol efflux is mediated by cholesterol transporters, including SR-B1, ATP binding cassette transporter A1 (ABCA1), and ATP binding cassette transporter G1. Of these, ABCA1 is shown to be a major transporter that exports cholesterol out of the cells [8]. The ABCA1 incorporates cholesterol into apolipoprotein A1 (apoA1), forming pre-βhigh-density lipoprotein (HDL) [9]. This newly formed HDL is further loaded with cholesterol through the activity of the ATP binding cassette transporter G1, transforming into mature HDL [9]. Because ABCA1 plays a crucial role in cholesterol metabolism, substances that modulate the activity of ABCA1 can be beneficial in atherosclerosis. According to epidemiological and animal studies, high flavonoid intake is positively associated with a decreased incidence of disorders characterized by dyslipidemia, including atherosclerosis, coronary heart diseases, and diabetes [1013]. Quercetin, a substance rich in onions and apple [14], has been shown to possess antioxidant, anti-inflammatory, and antiatherogenic properties [15,16]. Its effects on atherosclerosis were demonstrated by a significant reduction of early stages of atherosclerotic lesion formation in humans [15] and animals that were either diet-induced or genetically engineered hypercholesterolemia [11,12,17,18]. The role of quercetin in macrophages has been recently studied in terms of the uptake of oxidized LDL. Quercetin prevented the activation of the protein kinase C and peroxisome proliferatoractivated receptor γ (PPARγ) signaling pathways, resulting in the reduction of scavenger receptor CD36 and SR-A gene expression. Through this mechanism, quercetin inhibited

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oxidized LDL uptake by macrophages [19]. Although quercetin has previously demonstrated the ability to significantly lower the risk of atherosclerosis, specific roles of quercetin on reverse cholesterol transport in macrophages and the possible mechanism of its action still remain elusive. Considering the antiatherogenic effect of quercetin through the macrophage, we hypothesized that quercetin could also play a protective role in accelerating reverse cholesterol transport from macrophages. To this end, we used the human macrophage human acute monocyte leukemia cell line (THP-1) cell line because these cell lines have been widely used to mimic many aspects of normal physiologic function of macrophages [20]. We also determined the effect of quercetin on the expression of the ABCA1 cholesterol efflux gene and its upstream transcriptional factors including PPARγ and liver X receptor α (LXRα) and examined cholesterol efflux in quercetin-treated cells.

2.

Methods and materials

2.1.

Reagent

Quercetin was purchased from Sigma-Aldrich (St Louis, MO, USA) and dissolved in sterile dimethyl sulfoxide (DMSO) as a 100-mM stock. RPMI-1640 was obtained from Lonza (Walkersville, MD, USA), and penicillin, streptomycin, and fetal bovine serum, from GIBCO BRL (Grand Island, NY, USA). Phorbol 12myristate 13-acetate (PMA) was purchased from SigmaAldrich, and 22-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)23,24-bisnor-5-cholen-3β-ol (NBD-cholesterol), from Invitrogen (Carlsbad, CA, USA). 3-[4,5-Dimethylthiazol-2-yl]-2,5diphenyltetrazolium bromide (MTT) was purchased from Amresco (Solon, OH, USA). The primary antibodies against ABCA1 and LXRα were purchased from Abcam (Cambridge, MA, USA), and PPARγ and β-actin, from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

2.2.

Cell culture

Human acute monocytic leukemia cell line (Korean Cell Line Bank, Seoul, Korea) was cultured in Roswell Park Memorial Institute (RPMI)-1640 supplemented with 10% fetal bovine serum, penicillin (100 U/mL), and streptomycin (100 μg/mL) at 37°C in 5% CO2 and 95% air. Cells (1 × 106) were seeded on each well of 6-well culture plates and differentiated by treatment of 200-nM PMA for 48 hours. The cells were serum-starved overnight before the addition of quercetin.

2.3.

Cell viability assay

Cells (1 × 106) were plated in 6-well plates and differentiated by treatment of 200-nM PMA for 48 hours; the cells were then serum-starved overnight before the addition of quercetin. The following day, they were treated with varying amounts of quercetin (0, 0.3, 1.5, 3, 15, and 30 μM) for 8 hours. Cell viability was measured by adding 1 mg/mL of MTT to each well and incubating at 37°C for 1 hour. After incubation, absorbance was measured at a wavelength of 570 nm with an Infinite M200 NanoQuant microplate reader (Tecan Group Ltd, Mannedorf, Switzerland). This assay was repeated 3 times.

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Total RNA was isolated from THP-1 cells using the RNeasy Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's protocol and stored at −80°C before use. One microgram of RNA was primed with oligo-dTs and reverse transcribed to generate complementary DNA using Superscript II Reverse Transcriptase (Invitrogen). Polymerase chain reaction (PCR) analysis was carried out using a thermal cycler (BIO-RAD Laboratories, Inc, Richmond, CA, USA). After an initial denaturation for 30 seconds at 97°C, the samples were amplified for each cycle, followed by 72°C for 1 minute. Each annealing condition was as follows: for ABCA1 (54°C, 22 cycles) and GAPDH (54°C, 22 cycles). Reactions were finished with 72°C with a 1-minute extension. Polymerase chain reaction primers for ABCA1 (forward primer 5’-CAAAGGGTCCTACCAGGTGA-3’ and reverse primer 5’AGTTCCAGGCTGGGGTACTT-3’) and GAPDH (forward primer 5’-TCCACCACCCTGTTGCTGTA-3’ and reverse primer 5’-ACCACAGTCCATGCCATCAC-3’). The PCR products were then separated by electrophoresis on a 1.5% agarose gel. The separated bands were visualized using an UV transilluminator (Fluorchem FC2; Alpha Innotech, San Leandro, CA, USA).

2.5.

Western blot analysis

THP-1–derived macrophage cells were treated with various concentrations of quercetin, harvested, and lysed in a radioimmunoprecipitation assay buffer (40 mM HEPES, pH 7.5, 120 mM NaCl, 1 mM EDTA, 1% Triton X-100) containing a protease inhibitor cocktail (Roche Diagnostics, Mannheim, Germany). Protein concentrations were determined by bicinchoninic acid method (Sigma-Aldrich). Equal amounts of protein lysates were mixed with 5× loading buffer (1 M Tris-HCl, pH 6.8, a trace amount of bromophenol blue, 50% glycerol, 10% sodium dodecyl sulfate [SDS], and distilled water) and lysis buffer and denatured at 95°C for 5 minutes. Samples were loaded onto 10% SDS–polyacrylamide gel. After electrophoresis, proteins were electrophoretically transferred from the gel onto polyvinylidene difluoride membrane in a buffer (2.5 mM Tris, 19.2 mM Glycine, pH 8.3) at 0.3 mA/cm2 for 1 hour 25 minutes at room temperature. Residual binding sites on the membrane were blocked by incubating the membrane in tris-buffered saline (pH 7.6) containing 0.1% Tween 20 and 5% nonfat dry milk for 1 hour at room temperature. The blots were washed in tris-buffered saline containing 0.1% Tween 20 and then incubated at 4°C with the appropriate antibody overnight. After washing, the membrane was incubated with anti-rabbit or mouse immunoglobulin G Ab conjugated with horseradish peroxidase, and bands were visualized using enhanced chemiluminescence (ECL; Young In Frontier, Seoul, Korea) and quantified by densitometery using an Alphaview software (Alpha Innotech).

2.6.

Cholesterol efflux analysis

Cholesterol efflux was measured according to the methods by Zhang et al [21]. In brief, THP-1 monocytes (1 × 105 cells) were seeded on each well of 96-well culture plates and differentiated by treatment of 200-nM PMA for 48 hours. To stop differentiation and recover cells, differentiated macrophages

were washed twice with 1× phosphate-buffered saline (PBS) and changed with the growth medium overnight. After 24 hours, the cells were incubated with 10-μM NBD-cholesterol for 24 hours and then serum starved overnight before the addition of 0.3-μM quercetin. The starved THP-1 cells were cultured in a serum-free medium with 0.3-μM concentrations of quercetin for 8 hours. At the end of the treatment time, media in all wells were carefully aspirated, and each well was washed twice with 1× PBS buffer and incubated in PBS containing 0.2% bovine serum albumin and 40 μg/mL apoA1 or 100 μg/mL HDL for 1 hour. Afterwards, the fluorescencelabeled cholesterol that was released from cells into the medium was collected and measured using a fluorescence microplate reader. Residual medium was suctioned, and cells in this plate were harvested in a lysis buffer (0.1% SDS and 5 mM Tris-HCl) by shaking the plate for 10 minutes. Fluorescent cholesterol from the medium and cell lysates was measured at wavelengths of 485 and 535 nm (Ex/Em). Cholesterol efflux was calculated by dividing the media-derived fluorescence by the total of the fluorescence in the medium and cells and expressed as a relative average to control (DMSO).

2.7.

Statistical analyses

Statistical analysis was performed using SPSS (SPSS, Inc, Chicago, IL, USA). The results are presented as means ± SD, and the differences among the experimental groups were analyzed using the Student t test, with P < .05 as the criterion of significance.

3.

Results

3.1.

Quercetin did not affect the viability of THP-1 cells

THP-1–derived macrophage cells were treated with various concentrations of quercetin ranging from 0.3 to 30 μM for 24 hours. The viability was not significantly reduced by the exposure to quercetin at all tested concentrations (Fig. 1). This result indicates that quercetin, at the 30-μM dose, does not inhibit cell viability. 1.2 1

Cell viability (fold of control)

2.4. RNA preparation and reverse transcription– polymerase chain reaction

0.8 0.6 0.4 0.2 0 QC 0

0.3

1.5

3

15

30µM

Fig. 1 – Quercetin effects on cell viability. The MTT assay was performed using cell lysates obtained from differentiated THP-1 cells that were treated with different concentrations of quercetin (QC) for 8 hours. Average value of 3 independent experiments was shown in graph.

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N U TR IT ION RE S E ARCH 3 3 ( 2 0 13 ) 13 6 –1 4 3

3.2. Treatment with quercetin leads to ABCA1 gene up-regulation

until 4 hours after the addition of quercetin but slowly decreased to its basal levels (Fig. 2C). Protein products of ABCA1 accumulated up to 8 hours and remained relatively higher than the control cells that were not treated with quercetin (Fig. 2D). Taken together, quercetin appears to up-regulate the transcription of ABCA1, increasing both the transcript and protein levels of the ABCA1 gene.

We investigated the expression of the ABCA1 gene in quercetintreated differentiated THP-1 cells. To identify the effective dose and time of quercetin in the modulation of ABCA1 expression, we varied the concentrations and incubation times of quercetin treatment. We found that 0.3 μM of quercetin induced the highest level of ABCA1 messenger RNA (mRNA) and protein expression (Fig. 2A and B). In these figures, the expression levels of ABCA1 mRNA and protein were measured at 4 and 8 hours, respectively, after incubation with different doses of quercetin in THP-1 cells. These conditions were determined by a timecourse experiment performed previously. A 24-hour treatment of quercetin in differentiated THP-1 cells showed similar effects (data not shown). We followed the changes in the expression levels of the ABCA1 gene after the addition of 0.3 μM of quercetin. The ABCA1 transcript levels gradually increased

2.5

When we observed an increase in ABCA1 transcript levels, we questioned whether quercetin would have an effect on transcription factors such as PPARγ and LXRα, which have been known to transcribe the ABCA1 gene. The protein levels of PPARγ and LXRα were measured in differentiated THP-1 cells at various time points over 24 hours after the treatment of 0.3-μM quercetin. We observed a sudden increase in the

B

*

ABCA1 protein expression (fold of control)

ABCA1 mRNA expression (fold of control)

A

3.3. Quercetin increased the expression of PPARγ and LXRα genes

2 1.5 1 0.5

*

2.5 2 1.5 1 0.5 0

0

QC (µM)

-

0.15

0.3

1.5

3

15 anti-ABCA1 anti- -actin

2.5

D

*

ABCA1 protein expression (fold of control)

ABCA1 mRNA expression (fold of control)

C 2 1.5 1 0.5 0

*

4 3.5 3 2.5 2 1.5 1 0.5 0

24

hr

24hr QC 0.3 µM 0

1

2

4

8

12

24hr anti-ABCA1 anti- -actin

Fig. 2 – Quercetin effects on expression of ABCA1 in THP-1–derived macrophage. A, Messenger RNA expression of ABCA1 in THP-1 cells was examined after 4-hour treatment of various amounts of quercetin, using reverse transcription–PCR. B, The ABCA1 protein levels in THP-1 cells were examined after 8-hour treatment of various amounts of quercetin, using immunoblotting. C, The ABCA1 mRNA was examined after treatment of the cells with 0.3-μM quercetin for 0 to 24 hours using reverse transcription–PCR. D, The ABCA1 protein expression after treatment of the cells with 0.3-μM quercetin for 0 to 24 hours using immunoblotting. The values from the independent experiments were quantified, normalized to GAPDH expression level, and expressed as fold changes (A and C). β-Actin was used as loading control. The representative image was shown (B and D). Significance of differences between groups was obtained by the Student t test. *P < .05.

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A

QC 0.3 µM 0

1

2

3

4

8

6

24hr anti-LXR-α anti-PPAR-γ anti-β-actin

Fig. 3 – Time course of LXRα and PPARγ with 0.3-μM quercetin for 0 to 24 hours using immunoblotting. β-Actin was used as loading control.

3.4. Quercetin increased reverse cholesterol transport in differentiated THP-1 cells Next, we examined if quercetin-mediated up-regulation of ABCA1 gene resulted in an increase in cholesterol efflux in differentiated THP-1 cells. After being loaded with cholesterol, the cells were treated with various concentrations of quercetin for 8 hours. Then, cholesterol transport to either apoA1 or HDL was measured for 1 hour after the addition of apoA1 or HDL. Quercetin at a dose higher than 0.3 μM resulted in the transport of cholesterol to apoA1 (Fig. 4A). However, quercetin at a dose as low as 0.15 μM was sufficient to induce significant amounts of cholesterol transported to HDL (Fig. 4B). The highest efflux of cholesterol to either of the cholesterol acceptors was achieved with 0.3-μM quercetin in cholesterol-loaded THP-1 cells (Fig. 4A and B). These findings demonstrated that quercetin promoted reverse cholesterol transport in differentiated macrophage cells.

was absent in THP-1 cells treated with 80 μM of quercetin [22]. A 0.3-μM concentration of quercetin in serum is in a physiologically achievable range according to a study by Egert et al [23]. The authors demonstrated that 2 weeks of quercetin intake at a dose of 150 mg/d was able to increase serum quercetin concentration to approximate 0.38 μM [23]. Because quercetin is abundant in various fruits and vegetables, including apples, onions, and various kind of herbs and spices [24], ingestion of food equivalent to 150 mg/d of quercetin through a dietary source could be feasible [25]. Overall, our data indicate that an optimal

A relative cholesterol efflux to apo-AI

protein levels of both PPARγ and LXRα genes within 2 hours of treatment (Fig. 3), which occurred before the induction of the ABCA1 gene (Fig. 2A-D). This suggests that the enhancement in the transcription of the ABCA1 gene by quercetin is likely to be mediated by increasing PPARγ and LXRα.

1.8

*

1.6 1.4 1.2 1 0.8 0.6 QC 0

Discussion

The current study supports the hypothesis that quercetin plays a crucial role in cholesterol reverse transport via up-regulating the expression of the cholesterol transport gene, ABCA1, as well as its transcription factors PPARγ and LXRα. This could lead to the suppression of foam cell formation as part of the antiatherogenic effects. The presence of sustained macrophage activation in the subendothelium of atherosclerotic lesions suggested that macrophages may play an essential role during the initiation and progression of atherosclerosis [1,3]. We noticed that concentration-dependent effects of quercetin were clear on ABCA1 expression but less clear in cholesterol efflux from THP-1–derived macrophages. However, it was clearly shown that the effective concentration of quercetin in THP-1 macrophage cells was around 0.3 μM without a further increase in effectiveness on up-regulation of ABCA1 expression and cholesterol efflux in doses higher than 0.3 μM. Sometimes, the treatment with quercetin in doses higher than 0.3 μM diminished its effects. However, this phenomenon is not likely to be due to its cytotoxic effects based on our cytotoxicity assay and a previous report [22], which demonstrated that apoptotic activity

relative cholesterol efflux to HDL

B 4.

*

0.15

1.8

0.3

1

3

15µM

1

3

15µM

* *

1.6

*

1.4 1.2 1 0.8 0.6 QC 0

0.15

0.3

Fig. 4 – Quercetin effects on cholesterol efflux in THP-1– derived macrophage. To load and label cholesterol, THP-1 cells were incubated with NBD-cholesterol (10 μM) for 24 hours. Efflux shown (A and B) was induced by addition of apoA1 (40 μg/mL) and HDL (100 μg/mL) after treatment of 0.3-μM quercetin for 8 hours. Values represent fold change relative to control (DMSO) given as means. Significance of differences between groups was obtained by the Student t test. *P < .05.

N U TR IT ION RE S E ARCH 3 3 ( 2 0 13 ) 13 6 –1 4 3

dosage range likely exists for quercetin to deliver its maximal protective effects against foam cell formation. The quercetin used in our study is aglycone; however, glycosides are more prevalent than algycones in nature [26]. The aglycone form of quercetin was shown to display faster kinetic behavior in absorption compared with the quercetin glycoside, rutin [26]. However, another report indicated that in the case of the 3-Oglucosylation, the glycosides resulted in higher plasma concentrations of quercetin after absorption than quercetin [27]. These results suggest that depending upon the types of glycodies, the bioeffectiveness of quercetin could differ. Therefore, to achieve effective concentrations of quercetin through diets, it is important to consider the forms of quercetin present in the diet. Quercetin-driven ABCA1 up-regulation appears to be mediated by an increase in the transcriptional activities of PPARγ and LXRα, as demonstrated by the elevated protein expression of these transcriptional factors with quercetin treatment. The quercetin-mediated induction of PPARγ and LXRα protein expression occurred earlier than that of ABCA1 protein expression. Therefore, it appears that quercetin upregulates the expression of PPARγ and LXRα genes first and then induces ABCA1 gene transcription. The rapid induction of PPARγ and LXRα genes indicate that quercetin may be directly involved in expression of these genes. This result using human macrophage is in accordance with a previous finding showing that the glycosylated quercetin, rutin, upregulated the expression of PPARγ in lung cells [28]. The effects of quercetin on PPARγ expression appear to be tissue specific because quercetin lowered PPARγ transcript levels in rat adipose tissue and exerted no effects on PPARγ transcriptional activity [29]. Although we have not examined whether quercetin acts as agonist or antagonist, it is possible that quercetin could have no measurable effects as PPARγ ligand as well. It is well established that ABCA1 gene expression is governed by LXRα via LXR-responsive elements residing in its promoter region [30,31]. Moreover, PPARγ regulates the transcription of the LXRα gene. Therefore, up-regulation of PPARγ may mediate ABCA1 gene expression through the regulation of LXRα expression, as indicated previously [32]. In addition, PPARγ has been shown to heterodimerize with LXRα [33], suggesting that these 2 factors may cooperatively affect ABCA1 transcription [34]. Taken all of these into consideration, quercetin may modulate ABCA1 gene expression at the transcriptional level by influencing the expression of the related transcription factors. The association of ABCA1 expression with cholesterol removal has been reported in many previous studies [30,32]. Ligand-activated PPARγ leads to the induction of LXRα and, subsequently, ABCA1 genes, thus resulting in the removal of cholesterol from macrophages [32]. However, a gene-targeted deletion of PPARγ caused a significant increase in atherosclerosis in low density lipoprotein receptor −/− mice [32]. A chemical LXR agonist also increased ABCA1 expression in LDL-loaded macrophages and lowered the occurrence of atherosclerotic lesions in mice models genetically engineered to be hyperlipidemic (low density lipoprotein receptor−/− and apolipoprotein E −/− mice) [30]. Therefore, the activities of PPARγ and LXRα in macrophages appear to be necessary to enhance the reverse cholesterol process. In line with these observations, we also demonstrated that quercetin promoted cholesterol efflux in

141

macrophages along with an up-regulation of PPARγ and LXRα. As a major regulator of reverse cholesterol transport, modulation of ABCA1 gene expression by quercetin may lower the deposition of cholesterol in macrophages. Considering the fact that cholesterol accumulation in foam cells is a critical feature in the initial stage of atherosclerosis, quercetin could ameliorate dysregulated cholesterol metabolism and prolonged inflammation, which are characterized in atherosclerosis [35]. Besides the quercetin effects that we observed, quercetin has been previously shown to provide health benefits by exerting its various antiatherogenic effects by down-regulating the expression of scavenger receptors such as SR-A and CD36 in macrophages and blocking free radical–mediated oxidative modification of LDL [1,11,12,36,37]. Furthermore, the detection of quercetin metabolites in the aorta with an atherosclerotic lesion and lipopolysaccharides-activated macrophages implied that quercetin could enter through the endothelium to interact with macrophages [37]. Therefore, this study corroborates these previous findings and provides a larger body of evidence to show favorable roles of quercetin in improving atherosclerosis. Several limitations in our studies include that, first, quercetindriven cholesterol efflux from macrophage was shown only in human macrophage cell lines and needs to be further demonstrated in animal models and human studies. Second, we did not distinguish whether quercetin effects on the expression of PPARγ, LXRα, and ABCA1 genes are mediated by only affecting PPARγ gene expression or affecting all 3 genes with different response times. However, overall, we propose quercetin as one of the flavonoids able to increase reverse cholesterol transport in macrophages as a strong regulator of the expression of PPARγ, LXRα, and ABCA1 genes. Our observation regarding the effects of quercetin on ABCA1 gene expression and cholesterol efflux from THP-1–derived macrophages is in accordance with recent reports describing the same quercetin effects in a murine macrophage cell line [38] as well as the effects of an ethanol extract of Brazilian red propolis to promote cholesterol efflux from THP-1 macrophages through up-regulation of ABCA1 expression [34]. Anthocyanin, another kind of flavonoid, was also reported to increase ABCA1 gene expression along with the stimulation of cholesterol efflux in peritoneal macrophages in an animal study [39]. The unique findings that we made in the current study were that quercetin was also able to increase the expression of PPARγ and LXRα genes. In all 3 cases, the identical mechanistic finding was the induction of the major cholesterol transport gene ABCA1 and cholesterol efflux in macrophages [34,39]. Our data suggest that quercetin or quercetin-rich foods can be used to lower the risk of atherosclerosis by promoting the expression of PPARγ, LXRα, andABCA1 as well as the cholesterol efflux from macrophages. This study provides a rationale to pursue antiatherogenic effects of quercetin in vivo. Further investigation using hyperlipidemic animal models and humans that are susceptible to develop dyslipidemia is warranted.

Acknowledgment This research was supported by the Basic Science Research Program through the National Research Foundation of Korea and funded by the Ministry of Education, Science and Technology (2012–0002119).

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