Curcumin enhances LXRα in an AMP-activated protein kinase dependent manner in human macrophages

Javier Sa´enz, Gonzalo Alba, Mar´ıa E. Reyes-Quiroz, Isabel Geniz, Juan Jime´nez, Francisco Sobrino, Consuelo Santa-Mar´ıa
PII: S0955-2863(17)30369-8
DOI: doi: 10.1016/j.jnutbio.2017.11.006
Reference: JNB 7885

To appear in: The Journal of Nutritional Biochemistry

Received date: 26 April 2017
Revised date: 11 September 2017
Accepted date: 11 November 2017

Please cite this article as: S´aenz Javier, Alba Gonzalo, Reyes-Quiroz Mar´ıa E., Geniz Isabel, Jim´enez Juan, Sobrino Francisco, Santa-Mar´ıa Consuelo, Curcumin enhances LXRα in an AMP-activated protein kinase dependent manner in human macrophages, The Journal of Nutritional Biochemistry (2017), doi: 10.1016/j.jnutbio.2017.11.006

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Curcumin enhances LXRα in an AMP-activated protein kinase dependent manner in human macrophages

Javier Sáenza,1, Gonzalo Albaa,1, María E. Reyes-Quiroza, Isabel Genizc, Juan Jiméneza,

Francisco Sobrinoa, Consuelo Santa-Maríab,*

aDepartamento de Bioquímica Médica y Biología Molecular, Universidad de Sevilla, Sevilla, Spain, bDepartamento de Bioquímica y Biología Molecular, Universidad de Sevilla, Sevilla, Spain, cHospital Nuestra Señora de Valme, Servicio Andaluz de Salud, Sevilla, Spain

*Corresponding author: Consuelo Santa-María. Departamento de Bioquímica y Biología Molecular, Facultad de Farmacia, Universidad de Sevilla, C/ Profesor García González 2, 41012 Sevilla, Spain
E-mail: [email protected], Fax: +34954907048

Keywords: ABCA1; AMPK; Curcumin; LXRa; Reverse Cholesterol Transport

Abbreviations: LXR, liver X receptor; ABCA1, ATP-binding cassette transporter A1; ABCG1, ATP-binding cassette transporter G1; SREBP1c, sterol response element binding protein 1c; AMPK, AMP-activated protein kinase; PMA, phorbol myristate acetate; fMLP, formyl-Met-Leu- Phe; siRNA, small interference RNA; ROS, reactive oxygen species; GAPDH, glyceraldehyde 3- phosphate dehydrogenase; MAPK, mitogen activated protein kinase; apo A-I, apolipoprotein A- I; AA actimicyn A; 2DG, deoxyglucose; COX-2, cyclooxygenase-2; MMP-9, matrix metalloproteinase-9; HDL, high density lipoprotein; oxLDL, oxidized low density lipoprotein; NF- κB, nuclear factor kappa B; Nrf2, nuclear factor E2-related factor 2

Summary

Liver X receptor alpha (LXRα) is a nuclear receptor involved in cholesterol homeostasis. Curcumin, a traditional Chinese derivative from the rhizomes of Curcuma longa and a well known AMP-activated protein kinase (AMPK) activator, possess hypocholesterolemic activity, however, the possible link between AMPK and cholesterol is unknown. In this study, we have investigated whether curcumin regulates metabolic changes in cholesterol metabolism via LXRα in THP-1 human macrophages, the cells implicated in atheroma plaques formation. Results showed that curcumin induced AMPK phosphorylation, increased LXRα mRNA and protein expression. Curcumin up-regulated mRNA expression of genes involved in cholesterol transport and metabolism as ATP-binding cassette (ABC) transporters ABCA1 and ABCG1, and the sterol response element binding protein 1c (SREBP1c). On the other hand, this increased LXRα mRNA and protein expression was reverted when AMPK was inhibited by its chemical inhibitor, compound C. Transfection with AMPK α1 and α2 siRNA decreased the LXRα mRNA expression and its target genes. Curcumin treatment inhibited cell migration and was also able to promote reverse cholesterol transport in THP-1 cells. This enhanced reverse cholesterol transport might be related to the upregulating of ABCA1 and ABCG1 mRNA expression by activating AMPK-LXRα signaling in THP-1 cells. This study describes a possible mechanism for understanding the hypocholesterolemic effects of curcumin and expand knowledge about the LXRα regulation by AMPK.

1. Introduction

Liver X receptors (LXRs) are ligand-activated transcription factors that belong to the nuclear receptor superfamily. LXRs act as key sensors of intracellular sterol levels that trigger a series of adaptive mechanisms in response to cholesterol overload [1]. They control the expression of genes important for cholesterol uptake, efflux, transport, and excretion, such as those encoding the ATP-binding cassette (ABC) transporters ABCA1 and ABCG1 responsible for the reverse cholesterol transport. Moreover, they are involved in other metabolic functions, such as fatty acid synthesis (regulating the expression of sterol response element binding protein 1c, SREBP1c), glucose homeostasis, steroidogenesis and neuronal homeostasis [2].
LXRs also attenuate the transcription of many genes associated with inflammation [3]. The ability of LXRs to coordinate metabolic and immune responses constitutes an attractive therapeutic target for the treatment of chronic inflammatory disorders such as atherosclerosis. There are two LXR isoforms; α, which is expressed in a restricted set of tissue/cell types, including macrophages, and β, which displays a ubiquitous expression pattern.
The presence of LXRα in macrophages is crucial because of its main role in the reverse cholesterol transport pathway that contributes significantly to prevent foam cell formation and atherosclerosis development [4].
It has been described that LXRs, like other nuclear receptors, may be modulated by phosphorylation [5–10]. LXRs phosphorylation modulates its transcriptional activity in a gene- specific manner [6]. However, the signaling pathways on LXRs action are poorly understood. Some kinases, such as casein kinase-2 (CK2) [6], mitogen-activated protein kinases (MAPKs) [7], protein kinase A (PKA) [8], protein kinase C (PKC) [9] or adenosine monophosphate activated protein kinase (AMPK) [10] have been suggested for LXRs phosphorylation in serine and/or threonine residues.
AMPK is a member of a metabolite-sensing protein kinase family and operates as an energy status sensor that maintains cellular energy homeostasis [11]. The activation of AMPK attenuates anabolic processes, such as the synthesis of proteins, fatty acids, and cholesterol, and stimulates catabolic pathways such as glycolysis and β-oxidation [11]. Activation of AMPK inhibits lipid synthesis by suppressing the SREBP-1c in post-transductional regulation [12]. However, the relationship between AMPK and LXRs is still little studied.

It has been described that the polyphenol curcumin may activate AMPK [13,14]. Curcumin is a lipophilic phenolic substance with a characteristic yellow colour derived from the rhizome of the plant turmeric (Curcuma longa) and is commonly used as an additive (E100) for food coloring and flavoring by the food industry and in private homes, especially in oriental diet and also in medicine [15]. The mean daily intake of curcumin was estimated to be 0.4–1.5 mg/kg bodyweight in India and 0.48 mg/kg bodyweight in France, generally in turmeric form [16]. Curcumin is save and well tolerated even at high doses [17].

Curcumine possesses hypocholesterolemic [18], antioxidant [19], anti-inflammatory [20], antineoplasic [21] and other beneficial properties [22]. The molecular mechanisms of this pleiotropic activity seem to be mediated by inhibition of several cellular signaling pathways at many levels [22]. The common molecular targets of curcumin include transcription factors [23], inflammatory mediators [20] and protein kinases [24]. Curcumin suppresses numerous cell signaling pathways including PI3K [23], NF-κB [24], Nrf2 [25], ROS [25] and COX-2 [26]. It has also been reported that curcumin inhibits cancer progression through regulating expression of microRNAs [27].
The hipocholesterolemic and/or antiatherosclerosis curcumin effect has been described by many authors. Curcumin reduced the infiltration of immune cells into the vascular wall and prevented atherosclerosis [28]. A curcuminoid extract reduces the atherogenic risk in patients with type 2 diabetes [29]. Curcumin inhibites atherogenesis by down-regulating lipocalin- 2 expression [30]. Curcumin enhances cell-surface LDLR level and promotes LDL uptake through downregulation of PCSK9 gene expression in HepG2 cells [18].
The present paper examines, in THP1 human macrophages, the effect of curcumin on LXRα and its target genes expression and analyses the implication of AMPK in this process. Besides the cholesterol efflux, cell migration and ROS (reactive oxygen species) production exerted by curcumin in these cells has also been studied.

2. Materials and methods

2.1. Materials

THP-1 human cell line was from the American Type Culture Collection (ATCC, Virginia, USA). Phorbol myristate acetate (PMA), formyl-Met-Leu-Phe (fMLP), 2,7-diclorodihydro- fluorescein diacetate (DCF-DA), 2-β-mercaptoetanol, isopropanol, 2-deoxy-D-glucose (2DG), actimycin A (AA), compound C, apolipoprotein A1 (Apo A1), staurosporine, Tween-20, casein, phosphate buffer saline (PBS), paraformaldehide and curcumin were purchased from Sigma- Aldrich (Madrid, Spain). Trypan blue (Gibco) was from Thermo Fisher. Scientific fetal bovine serum (FBS), bovine serum albumin (BSA), L-glutamine, streptomycin, penicillin and amphotericin B were obtained from Bio-Whittaker (Basel, Switzerland). Polyvinylidene difluoride (PVDF) membranes were from Pall (Madrid, Spain). Lipofectamin 2000 reagent, Opti-MEM (Invitrogen), and RPMI-1640 (Gibco) were obtained from Thermo Fisher Scientific (Waltham, MA, USA). TO901317 was purchased from Cayman Chemical (Ann Arbor, MI). Rabbit monoclonal antibody against phosphorylated (Thr172) AMPK, from Cell Signaling (Danvers, MA, USA) and mouse monoclonal anti-human LXRα were obtained from Perseus Proteomics (Tokyo, Japan). siRNA AMPKα1/α2 came from Santa Cruz Biotechnology (Dallas, TX, USA). Mouse monoclonal anti-GAPDH was purchased from Chemicon International (Madrid, Spain). Horseradish peroxidase (HRP)-conjugated goat anti-rabbit and anti-mouse IgGs were from Promega (Madison, WI, USA). Liquid scintillation and [3H]-cholesterol were from Perkin Elmer (Madrid, Spain). Transwell migration chambers from Corning Inc (New York, USA). Fluorescein isothiocyanate (FITC)-conjugated monoclonal antibody against CD16 from Immunotech, Marseille, France.

2.2. Cell culture and curcumine treatment

THP-1 cells were maintained in RPMI-1640 supplemented with 10% FBS (v/v), 2 mM L- glutamine, 100 U/mL penicillin, 100 µg/mL streptomycin and 2.5 µg/mL amphotericin B at 37ºC and 5% CO2. These cells were sub-cultured every 72 h. Cells were differentiated into macrophages by incubation with PMA 80 nM for 72 h at 37ºC, once cells have been passagedapproximately 10 times. For cell number quantification and cell viability analysis, cells obtained were analyzed by an exclusion test with trypan bluestain.

For curcumine treatment, cells were treated with this molecule dissolved in DMSO at a final concentration of 5 µM. This concentration was determined after the realization of a dose response curve.

2.3. ROS detection with 2′,7′-dichlorodihydrofluorescein staining

The oxidation of 2′,7′-dichlorodihydrofluorescein diacetate (DCF-DA) to fluorescent 2′,7′- dichlorodihydrofluorescein (DCF) was measured to determine ROS levels. Cells were treated with or without TO901317 and curcumin as indicated in each experiment. Then, cells were incubated for 1 h at 37ºC in darkness with 10 µmol/L DCF-DA as described [31]. Finally, a positive control was performed with PMA. Emission of trapped, oxidized DCF was assayed by flow cytometry using a flow cytometer Cytomics FC 500 Beckman Coulter (Brea, CA, USA). Data were analyzed with CXP Software.

2.4. Real time RT-PCR analyses of mRNA levels

For real time RT-PCR analysis, 2 µg of total RNA was reverse-transcribed into cDNA as described [32]. Real time RT-PCR was carried out in an ABI Prism 7300 Sequence Detection System from Applied Biosystems (Foster City, CA, USA) using the specific thermocycler conditions recommended by the manufacturer. The PCR reactions were performed in triplicate and contained 2 mL of cDNA and SYBR Green PCR Master Mix (Applied Biosystems) in a total volume of 25 µL. Each sample was also analyzed for β-actin transcript levels to normalize for RNA input amounts [32]. The following primers were used: LXRα: forward, 5’- AAGCCCTGCATGCCTACGT-3’, reverse, 5’-TGCAGACGCAGTGCAAACA-3’; ABCA1: forward, 5’-CCCTGTGGAATGTACCTATGTG-3’, reverse, 5’-GAGGTGTCCCAAAGATGCAA-3’; ABCG1: forward,5’-CAGTCGCTCCTTAGCACCA-3’, reverse, 5’-TCCATGCTCGGACTCTCTG-3’; SREBP1c: forward, 5’-TCAGCGAGGCGGCTTTGGAGCAG-3’, reverse, 5’- CATGTCTTCGATGTCGGTCAG-3’; AMPKα1: forward, 5’-TGTAAGAATGGAAGGCTGGATGA- 3’, reverse, 5’-GGACCACCATATGCCTGTGA-3’, AMPKα2: forward, 5’-
GGTGATCAGCACTCCAACAGA-3’, reverse, 5’-TCTCTTCAACCCGTCCATGC-3’ and β-actin: forward, 5’-CCAGCTCACCATGGATGATG-3’, reverse, 5’-ATGCCGGAGCCGTTGTC-3’.

2.5. Western blotting analysis of LXRα and AMPK

Western blotting analysis of phosphorylated AMPK and LXRα protein levels was performed on total cell lysates with modifications as described [7]. Blots were probed with rabbit monoclonal anti-phospho AMPK (Thr172), at a 1:1000 dilution or mouse monoclonal anti-LXRα, at a 1:2000 dilution, in PBS plus 0.5% BSA and 0.02% Tween-20. Then the membranes were incubated with horseradish peroxidase (HRP)-conjugated to anti-rabbit or anti-mouse IgG used at a 1:5000 dilution in PBS plus 0.5% casein, and detection was carried out by enhanced chemiluminescence [33]. To verify even protein loading, the blots were subsequently stripped and re-probed with polyclonal antibodies against GAPDH at a 1:5000 dilution. Band intensities were quantitated using the ImageQuant TL software from GE Healthcare Life Sciences (Uppsala, Sweden) and corrected for differences in GAPDH levels.

2.6. Chemotaxis assay

Migration of THP-1 human cells was assessed in Trans-well migration chambers (diameter, 6.5 mm; pore size, 5 µm; Costar plates type 3421) [7] . Cells were preincubated with or without TO901317 for 18 h at 37 °C. The chemoattractant peptide, fMLP, was deposited in the lower compartment in a final volume of 0.6 mL of RPMI-1640, and 106 cells/mL were deposited on each detachable insert and incubated for 2 h at 37°C in a humidified atmosphere of 5% CO2 in air. In separate wells, THP-1 human cells were added to the lower compartment and used as controls, representing 100% migration. At the end of the incubation period, the cells that had migrated into the bottom chambers were collected and centrifuged at 400 g for 5 min. After being stained with fluorescein isothiocyanate (FITC)-conjugated monoclonal antibody against CD16, the cells were fixed with 1% paraformaldehyde and finally counted on a flow cytometer using a flow cytometer Cytomics FC 500 Beckman Coulter (Brea, CA, USA). The results are presented as the mean ± standard error media (SEM) from four separate experiments, and are expressed as the percentage of total THP-1 human cells initially added to each chamber.

2.7. Reverse cholesterol transport

Reverse cholesterol transport assays were performed as described [34], with modifications. THP-1 human cells (5×106 cells/mL) were incubated for 72 h in RPMI-1640 supplemented with 2% FBS, 80 nM PMA and 2.0 µCi/mL [3H]-cholesterol. To equilibrate cholesterol pools, cells were washed twice with PBS and incubated for 18 h in RPMI-1640 containing 0.2% BSA plus the indicated ligands TO901317 or curcumin. Then we added 15 mg/mL Apo A1 for 12 h. Aliquots of the medium were removed, scintillation liquid was added, and centrifuged at 14,000 x g for 2 min. Total cell-associated radioactivity was determined by dissolving the cells in 0.2 M NaOH and then in scintillation liquid. The cholesterol efflux was calculated as the percent of [3H]-cholesterol released into the medium after subtraction of values obtained in the absence of Apo A1. The radioactivity was determined by liquid scintillation counting Wallac 1450 MicroBeta Triux from Perkin Elmer (Madrid, Spain).

2.8. Detection of cellular apoptosis

Apoptosis, measured as DNA fragmentation, was tested using the Cell Death Detection ELISA plus kit from Roche Applied Science (Barcelona, Spain), according to the manufacturer’s instructions. No evidence of apoptotic death was found in our cells, even after 18 h of incubation in our experimental conditions. Cells treated with 100 nM staurosporine were used as positive control.

2.9. Cell culture and siRNA transfection

THP1 were transfected with a transfection reagent according to manufacturer’s instructions. Briefly, THP-1 human cells (1×105 cells/mL) were seeded in a 6-well plate and differentiated into macrophages as described above. For siRNA transient transfections experiments, THP-1 differentiated cells were transfected with siRNAs (40 nM) directed against AMPKα1 and α2 or control siRNAs by using lipofectamine 2000 for 12 h. Then, cells were treated with different reagents for 18 h. Cells were used for experiments and target gene knockdown was validated by real time RT-PCR. A β-actin expression vector was used as an internal control.

2.10. Statistical analyses

All data was recorded as mean ± standard error of the mean (SEM). mRNA levels quantitated by RT-PCR are expressed as fold induction relative to untreated cells. Protein levels quantitated from Western blots are expressed in arbitrary units. The results were statistically analyzed using the SigmaPlot 12 software from Systat Software GmbH (Erkrath, Germany) by means of ANOVA and the Student’s paired t-test.

3. Results

3.1. Curcumin treatment increased LXRα mRNA and its protein expression in THP-1 macrophages
The human THP-1 cell line is a widely used model system for studies on macrophage gene expression, including the actions of nuclear receptors [35]. In our studies, we have used the LXRα isoform present in phagocytic cells [4]. Initial experiments were designed to analyze the curcumin effects on TO901317-induced mRNA expression of LXRα and its target genes in human macrophages. Relative quantification of LXRα and its target genes mRNA levels in human THP-1 macrophages treated with TO901317 (a synthetic LXRα agonist) was analyzed by means of real time RT-PCR. As shown in Fig 1A, THP-1 macrophages increased the mRNA expression of LXRα after treatment with TO901317 for 18 h. Treatment of macrophages with curcumin (5 µM) raised the LXRα mRNA expression two fold. This effect was lesser than that observed with the synthetic agonist, although it was statistically significant (Fig 1A). Interestingly, when cells were incubated with both molecules, curcumin and TO901317, a synergic effect was produced, observing an increase of 100% (Fig 1A). We next performed assays of curcumin and TO901317 effect on LXRα protein expression. These studies corroborated the results found over LXRα mRNA expression after treatment with TO901317, curcumin, or both compounds together, although the synergistic effect was less pronounced (Fig 1B).
To assess whether the TO901317 and curcumin effect on LXRα mRNA expression was extensible to its target genes, the expression of genes involved in cholesterol transport and metabolism such as ATP-binding cassette (ABC) transporters ABCA1 and ABCG1, and the sterol response element binding protein 1c (SREBP1c) was evaluated. Figure 1C shows that curcumin up-regulated mRNA of all these genes similarly to LXRα. Furthermore, a synergic effect was also observed in treatments with TO901317 and curcumin, being the most pronounced in the cholesterol transporter ABCA1 gene (Fig 1C).

3.2. Compound C decreased LXRα mRNA expression produced by curcumin in THP-1 macrophages

It has been described by our group and others that LXRs may be regulated by phosphorylation by different kinases, such as mitogen-activated protein kinases (MAPKs) [7] or adenosine monophosphate activated protein kinase (AMPK) [10]. Curcumin is a well-known AMPK activator [13,14]. Thus, we tried to test whether the AMPK curcumin stimulation has any effect on LXRα activation. First, we explored whether treatment with curcumin or TO901317 increased the AMPK phosphorylated levels in THP-1 macrophages. As shown in Fig 2A, a clear activation of AMPK by curcumin treatment was found. Interestingly, a TO901317-induced AMPK phosphorylated stimulation was also found. Depletion of ATP with treatment with actimicyn A (AA) and deoxyglucose (2DG) was used as a positive control (Fig 2A). A comparative induction of LXRα mRNA levels after treatment with TO901317, curcumin or AA/2DG is represented in Figure 2B. All AMPK stimulators used induce a clear and significant LXRα mRNA expression increment. To explore the possibility that AMPK could participate in the LXRα activation exerted by curcumin or TO901317, the specific AMPK inhibitor, compound C was used.
The TO901317 effect on LXRα mRNA expression was inhibited by compound C about 46% and the same compound C treatment totally reverted the curcumin effect (Fig 2C).
In complementary experiments, the levels of LXRα protein expression were also analyzed (Fig 2D). As expected similar results were observed in curcumin or TO901317- induced LXRα protein levels in THP-1 cells incubated with compound C (Fig 2D).

3.3. Transfection with specific AMPKα1/α2 siRNA decreased curcumin-stimulated LXRα mRNA expression and its target genes in THP-1 macrophages
The alternative trial methodology to inhibit AMPK was the use of small interference RNA (siRNA) against both α1 and α2 isoforms of the catalytic subunits of the enzyme. First, differentiated THP-1 human cells treated with siRNA showed lower AMPKα1 and α2 mRNA expression levels than those treated with RNA silencing control (Fig 3A) when tested. As shown in Fig 3B, transfection with AMPK α1 and α2 siRNA fully reverted the LXRα mRNA expression in cells treated with curcumin or TO901317 compared to LXRα levels in untreated cells. Similar results were found in LXRα target genes compared with control siRNA transfected cells (Fig 3B- E). In treatment with both molecules, curcumin and TO901317, the inhibition by siRNA was

lesser than in the above cases, although this inhibition always exceeded 50% in every gene tested (Fig 3B-E).

3.4. Curcumin treatments decreased reactive oxygen species production in THP-1 macrophages
An essential function in phagocytic cells is the production of reactive oxygen species (ROS) by different systems such as the NADPH oxidase enzyme [36]. Our group has documented that TO901317 stimulated LXRα decreased ROS production in human neutrophils [7,37]. Thus, the following experiments were designed to analyse whether TO901317 and curcumin affected ROS production in THP-1 macrophages. A sensitive system, flow cytometry, was used to analyze ROS production in oxidant condition incubating cells with PMA, a well- known prooxidant agent [38]. Figure 4A shows that PMA managed to induce ROS production in THP-1 macrophages. When THP-1 human cells were preincubated with 1 mM TO901317 or 5 µM curcumin at 37ºC for 18 h and finally treated with 100 nM PMA for 1 h, reverted ROS levels were observed when compared to PMA-stimulated cells alone (Fig 4B and C). Similar results were obtained with other prooxidant agents such as hydrogen peroxide or LPS/TNFα (data not shown).

3.5. Curcumin treatments inhibited cell migration and increased reverse cholesterol transport in THP-1 macrophages
We also investigated whether curcumin treatment altered particular inflammatory responses by THP-1 macrophages. Migratory capacity is an important phagocytic cells property. This property was tested following TO901317 or curcumin treatment in THP-1 macrophages. Cells treated for 18 h with TO901317 or curcumin migrated, to a lesser extent, than untreated cells. When the cell migration was stimulated with the chemotactic peptide fMLP, the LXRα agonist or curcumin decreased the migration capacity to around 68% and 57% respectively (Fig 5A).
The most significant function of LXRα activation is the reverse cholesterol transport stimulation. Reverse cholesterol transport is a multi-step process resulting in the net movement of cholesterol from peripheral tissues back to the liver. Cholesterol from non-hepatic peripheral

tissues is transferred to HDL by the ABCA1 (ATP-binding cassette transporter). Apolipoprotein A1 (Apo A1), the major protein component of HDL, acts as an acceptor [39]. The next question was whether the increase in LXRα, ABCA1 and ABCG1 mRNA levels was accompanied by a greater reverse cholesterol transport in THP-1 cells. Therefore, the reverse cholesterol transport mediated by Apo A1 in differentiated THP-1 cells was measured using radiolabelled cholesterol. As shown in Figure 5B the treatment with TO901317 (positive control), induced reverse cholesterol transport in differentiated THP-1 cells when compared to untreated cells. Curcumin had higher reverse cholesterol transport compared to untreated cells, but this increase was lower than those observed with TO901317 treatment. Treatment with the synthetic LXRα agonist induced reverse cholesterol transport over 43.5% and curcumin over 20.3%. Control refers to reverse cholesterol transport constitutively due to the presence of Apo A1 in culture medium.

4. Discussion

It has been suggested that LXRα is regulated post-transcriptionally by phosphorylation [5]. The AMPK enzyme is one of the kinases proposed for this modification [10]. LXRα and AMPK are related to the energy cell status, however, the relationship between these enzymes is still not well established in phagocytic cells. Both molecules, LXRα and AMPK, participate in cholesterol homeostasis [1,11]. The regulation of cholesterol metabolism in macrophages is crucial to prevent them from turning into foam cells.
Curcumin, a polyphenol derived from Curcuma longa, has hypocholesterolemic properties and also activates the AMPK enzyme [13,14]. Several mechanisms have been proposed to explain the hypolipidemic effects of curcumin; although, the molecular mechanisms are not yet completely clear. Suggested curcumin actions are a decreased expression of CD36 (a fatty acid transporter located on the adipocyte membrane), fatty acid synthase and hydroxymethylglutaryl-CoA reductase. Moreover, curcumin enhances fatty acid β-oxidation increasing the expression of carnitine palmitoyltransferase-1 [40].

This present work analyzes the effect of this molecule on LXRa gene expression in THP- 1 human cells and the possible AMPK implication in this process. First, the LXRa mRNA and its protein expression in THP-1 macrophages after treatment with a LXRa synthetic ligand (TO901317) were tested. Cell incubation with this molecule increased both LXRα mRNA and protein levels. These results are consistent with those described by our group and others in different cell types [7,37,41]. Curcumin also elevated the LXRα mRNA and protein expression although in to a lesser extent than those observed with the LXRα synthetic agonist (TO901317). It is worth noting the synergic effect detected when cells were incubated with both molecules curcumin and TO901317. Curcumin has also been shown to be synergistic with other compounds such as resveratrol, piperine, catechins, quercetin and genistein [22].
Next, the study was extended to some LXRα target genes. The influence of TO901317 and curcumin on mRNA levels of the genes involved in cholesterol transport and metabolism as ABCA1 and ABCG1, and the sterol response element binding protein 1c (SREBP1c) was assessed. Both TO901317 and curcumin activated mRNA of all these genes in a similar manner to LXRα. The synergic effect mentioned above was also detected in treatments with TO901317 and curcumin being the most prominent in the cholesterol transporter ABCA1 gene.

Few, andeven contradictory, reports about curcumin effects on LXRα have previously been documented. Curcumin increased LXRα and ABCA1 mRNA expression in rabbit subcutaneous adipocytes [42], in rat brains [43] and in the human hepatoma cell line HepG2 [44]. An increase in the mRNA expression of LXRα and ABCA1 [45] or a decrease in the LXRα, ABCA1 and ABCG1 mRNA expression have been reported [46] in both mice and human macrophages.
Afterwards, the potential AMPK repercussion in the LXRα mRNA up-expression produced by curcumin or TO901317 treatment was tested. Current work confirms that AMPK phosphorylation is induced by curcumin in THP-1 human cells, as other authors have also described in other cells [13,14]. The opposite effect [47] or even inconclusive results [48] have been also reported in some cellular types. Subsequently, a specific AMPK inhibitor compound C was used [49] and LXRα mRNA and protein expression after curcumin or TO901317 treatment was measured. The curcumin effect on LXRα mRNA and protein expression was reverted by compound C. The effect of the synthetic agonist on LXRα activation was also repressed although not totally (at mRNA and protein level). Compound C is also able to inhibit the up- regulation LXRα produced by TO901317 on endothelial cells [50] or mouse hepatocytes [51].
Another experimental approach to inhibit AMPK was by silencing RNA (siRNA) both α1 and α2 isoforms of the catalytic subunit of the enzyme. By inhibiting AMPK activity, LXRα expression and its target genes decreased in cells stimulated with TO901317 or curcumin. Recently, it has been published in macrophages from buffy-coat, that the other isoform of LXR, LXRβ, decreases drastically when a RNA interference specific to AMPK α1 subunit was used [52]. Curcumin decreases renal triglyceride accumulation through AMPK-SREBP signaling pathway in streptozotocin-induced type 1 diabetic rats [53]. A suppression of colon cancer cell invasion via AMPK-induced inhibition of NF-κB, and MMP-9 [54] and a protective effect of curcumin on LPS-induced acute lung injury have been also associated with AMPK activation
[55] that has been also reported recently.

In terms of cellular function modifications exerted by TO901317 and curcumin in THP-1 cells, our group has previously reported that LXRα activation by TO901317 decreases ROS production in human macrophages and neutrophils [7,37]. Herein, we have described that treatments with the LXRα synthetic ligand or curcumin decreased ROS production in THP-1 cells. These results are consistent with those described by Barzegar and Moosavi-Movahedishowing that curcumin decreases the hydroperoxide-stimulated ROS production in rat skeletal muscle cells [56]. Curcumin is a potent inhibitor of enzymes such as lipoxygenase/cyclooxygenase, xanthine oxidase, inducible nitric oxide synthase or NADPH oxidase [57], responsible for the ROS generation. Besides, it has been indicated that curcumin can act as a bifunctional antioxidant first, because of its ability to react with ROS directly, and remove it rapidly and second, by inducing various regulatory cytoprotective and antioxidant proteins [58]. Thus, antioxidant properties have been attributed to curcumin, although, it has been also been described that, depending on cell types and doses, they may act as pro-oxidant [59].

Migratory capacity in response to chemo-attractants is a specific function of phagocytic cells. We observed that pre-incubation with TO901317 decreased the fMLP-induced THP-1 cells migration. Curcumin also reduced the migration induced by fMLP in THP-1 cells. The inhibitory effect of curcumin on chemotaxis may contribute to its anti-inflammatory activity. Likewise, curcumin has been described to inhibit migration in Raw 264.7 (murine macrophage cell line) [60] and circulating fibrocytes [61]. It has also been shown that curcumin may inhibit migration of cancer cells in breast cancer stem cells [62], human lung cancer cells [63] and human thyroid cancer [64].

Curcumin has been shown to improve lipoprotein metabolism by reducing LDL and triglycerides, and increasing HDL [65]. The main function of HDL refers to its role in reverse cholesterol transport, in which excess cholesterol from peripheral cells is taken up by HDL and delivered to the liver for catabolism and excretion in bile. The is one of the most significant function of LXRα. THP-1 cells treated with TO901317 showed an increase in reverse cholesterol transport mediated by Apo A1. Synthetic LXRα agonist TO901317 has previously been reported to increase the reverse cholesterol transport and ABCA1 expression in macrophages or monocytes [41,66]. Curcumin treatment also produced an increased reverse cholesterol transport in THP-1 cells, although less pronounced than that caused by the synthetic agonist, similarly to data described for LXRα expression. This rise may reflect the increased ABCA1 y ABCG1 mRNA expression that was found after curcumin treatment. In other cells, such as subcutaneous adipocytes isolated from rabbits [42] and cerebral cells [43], curcumin also increased the inverse cholesterol transport by activating the LXRα expression.

The hypocholesterolaemic effects by curcumine in humans have been described in several randomized controlled trials. In healthy volunteers 0.5–6 g/day for 7 days decreased lipid levels [67]. In patients with acute coronary syndrome 15–60 mg/day for 2 years reduced total and LDL cholesterol [68]. In patients with metabolic syndrome, approximately 1890 mg/ for 12 weeks lowered lipid level [69].
In spite of the multiple beneficial properties described for curcumin, the handicaps for this use is its low bioavailability due to its limited intestinal uptake and rapid metabolism [16]. Maximum plasma curcumin concentrations in humans, even upon intake of doses as high as 10 or 12 g curcumin, remain in the low nanomolar range (<160 nmol/L) [70]. Although, levels in the microgram range have been necessary to show beneficial effects in in vitro studies [71]. However, it is well stablished that concomitant administration of piperine 20 mg produced a much higher plasma concentration increasing the bioavailability by about 2000% [72]. Besides, the bioavailability can be increased by heating while remaining biologically active [73]. On the other hand, we propose the possibility that curcumin would also be administered as nutritional supplements or functional foods. Although further studies are needed.

In summary, we conclude that AMPK activation, LXRα stimulation and higher mRNA expression of its target genes exerted by curcumin in THP-1 human cells might contribute to the beneficial role of curcumin in the prevention of atherosclerosis.

Disclosures

None declared

Funding

M.E.R-Q was supported by a fellowship from the Asociación Virgen Macarena, Hospital Universitario Virgen Macarena, Sevilla. G.A. was supported by fellowships from the Ministerio de Educación y Ciencia (BFU2006-13802) and the Consejería de Innovación, Ciencia y Empresa, Junta de Andalucía (P08-CVI-03550). This work was funded by grants from the latter (P06-CTS-01936 and P08-CVI-03550) to F.S., and from the Consejería de Salud, Junta de Andalucía (CS 0116/2007) to E. P.
F.S., G.A. and C.S-M. conceived and designed research project; J.S, G.A. and M.E.R-Q. performed the experiments; I.G. and J.J. performed statistical analysis and revised the manuscript; G.A. and C.S-M. wrote the manuscript.

Acknowledgements

The authors would like to thank Margarita Rodriguez Borrego for technical assistance. We are also grateful to Modesto Carballo of the Servicio de Biología, CITIUS, Universidad de Sevilla, for helping with experiments performed at the facility.

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