Paraoxonase 1

1. Free Radical Biology & Medicine 52 (2012) 1372–1381
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Free Radical Biology & Medicine
journal homepage: www.elsevier.com/locate/freeradbiomed
Original Contribution
Purified human paraoxonase-1 interacts with plasma membrane lipid rafts and
mediates cholesterol efflux from macrophages
Hicham Berrougui a,b, Soumaya Loued a,b,c, Abdelouahed Khalil a,b,c,⁎
a Research Center on Aging, Faculty of Medicine, University of Sherbrooke, Sherbrooke, Canada QC J1H 4C4
b Department of Medicine, Geriatrics Service, Faculty of Medicine, University of Sherbrooke, Sherbrooke, Canada QC J1H 4C4
c Programs of Physiology, Faculty of Medicine, University of Sherbrooke, Sherbrooke, QC Canada J1H 4C4
a r t i c l e i n f o a b s t r a c t
Article history:
Received 12 September 2011
Revised 17 January 2012
Accepted 23 January 2012
Available online 1 February 2012
Keywords:
Paraoxonase-1
HDL
Cholesterol efflux
Lipid raft
Free radicals
Macrophages
Paraoxonase-1 (PON1) is a high-density lipoprotein (HDL)-associated serum enzyme thought to make a
major contribution to the antioxidant and anti-inflammatory capacities of HDLs. However, the role of PON1
in the modulation of cholesterol efflux is poorly understood. The aim of our study was to investigate the in-volvement
of PON1 in the regulation of cholesterol efflux, especially the mechanism by which it modulates
HDL-mediated cholesterol transport. The enrichment of HDL3 with human PON1 enhanced, in a dose-dependent
manner, cholesterol efflux from THP-1 macrophage-like cells and ABCA1-enriched J774 macro-phages.
Moreover, an additive effect was observed when ABCA1-enriched J774 macrophages were incubated
with both PON1 and apo-AI. Interestingly, PON1 alone was able to mediate cholesterol efflux from J774 mac-rophages
and to upregulate ABCA1 expression on J774 macrophages. Immunofluorescence measurement
showed an increase in PON1 levels in the cytoplasm of J774 macrophages overexpressing ABCA1. PON1
used an apo-AI-like mechanism to modulate cholesterol efflux from rapid and slow efflux pools derived
from the lipid raft and nonraft domains of the plasma membrane, respectively. This was supported by the
fact that ABCA1 protein was incrementally expressed by J774 macrophages within the first few hours of in-cubation
with cholesterol-loaded J774 macrophages and that cyclodextrin significantly inhibited the capacity
of PON1 to modulate cholesterol efflux from macrophages. This finding suggested that PON1 plays an impor-tant
role in the antiatherogenic properties of HDLs and may exert its protective function outside the lipopro-tein
environment.
© 2012 Elsevier Inc. All rights reserved.
It has long been recognized that the concentration of plasma high-density
lipoproteins (HDLs) is inversely related to the risk of cardio-vascular
disease. The atheroprotective effect of HDLs is largely attrib-uted
to its key role in reverse cholesterol transport (RCT) whereby
excess cholesterol from peripheral cells is transported back to the
liver for excretion [1]. Macrophages possess a number of mechanisms
to regulate the equilibrium between cholesterol uptake/synthesis and
export, including transport mechanisms that promote the efflux of
excess cholesterol to extracellular acceptors [1]. Although cholesterol
efflux from macrophages makes up only a small fraction of overall
flux through the RCT pathway, it is considered a relevant component
of atheroprotection [1]. HDL-mediated cholesterol efflux is the natu-ral
rate-limiting step of RCT [2] and occurs via three pathways. The
first pathway is aqueous diffusion by which free cholesterol mole-cules
spontaneously desorb from the plasma membrane, diffuse
through the aqueous phase, and become adsorbed on acceptor
particles by collision [3]. The second pathway involves scavenger re-ceptor
class B type I (SR-BI)-mediated bidirectional free cholesterol
exchanges depending on the cholesterol gradient, this pathway medi-ates
cholesterol efflux to a wide variety of cholesterol acceptors [4].
The third pathway involves the ATP-binding cassette receptors
ABCA1 and ABCG1, which mediate cholesterol efflux in a unidirec-tional
manner to lipid-poor apolipoprotein-AI (apo-AI) and to other
subfamily members of HDL, respectively [5–7].
The rate of cholesterol efflux, however, depends also on the capac-ity
of cells to release excess cholesterol and of plasma acceptors to
transport the released cholesterol, which is determined by the con-centration
and physical and functional states of HDLs and apo-AI [2].
Apo-AI is a major protein component of HDL and is a critical ele-ment
in cholesterol metabolism. Apo-AI reacts specifically with
ABCA1 to generate nascent HDLs, which are then enriched with ester-ified
cholesterol by LCAT (activated by apo-AI) to form mature spher-ical
HDLs [8]. A number of other proteins confer additional properties
on HDLs. Vaisar et al. identified 48 proteins in HDLs associated with
clinically significant cardiovascular disease [9]. Paraoxonase 1
(PON1) is one of the 8 most abundant HDL-associated proteins and
may have atheroprotective properties [9]. PON1 has been extensively
⁎ Corresponding author at: Research Center on Aging, 1036 rue Belvédère Sud, Sher-brooke,
QC, Canada J1H 4C4. Fax: +1 819 829 7141.
E-mail address: Abdelouahed.Khalil@USherbrooke.ca (A. Khalil).
0891-5849/$ – see front matter © 2012 Elsevier Inc. All rights reserved.
doi:10.1016/j.freeradbiomed.2012.01.019

2. H. Berrougui et al. / Free Radical Biology & Medicine 52 (2012) 1372–1381 1373
studied with respect to its association with cardiovascular risk, oxida-tive
stress, and inflammation [10].
PON1 is mainly synthesized in the liver and 95% is transferred to
HDLs via an SR-BI-mediated mechanism. Small amounts of plasma
PON1 (5%) are associated with chylomicrons and very low density li-poproteins
[11–13]. In vitro studies have shown that PON1 inhibits
low-density lipoprotein (LDL) and HDL lipid peroxidation and may
be a primary determinant of the anti-inflammatory capacity of HDLs
[14,15]. PON1 also attenuates oxidized LDL uptake via scavenger re-ceptor
CD-36 and directly reduces oxidative stress in macrophages
[16,17]. It also inhibits oxidized LDL-induced MCP-1 production via
a mechanism that is probably related to its capacity to hydrolyze
some activated phospholipids as well as lipid peroxide [18]. Cys284
of the free sulfhydryl of PON1 is required for its atheroprotective ef-fect
and is thought to be the active site for its antioxidant activity
[19]. We previously showed that exposure of purified human PON1
to oxygen free radicals induced a significant decrease in PON1 para-oxonase
and antioxidant activities, which was accompanied by a de-crease
in free sulfhydryl groups [19].
PON1 expression is inversely correlated with atherosclerotic de-velopment
in animal models [20,21]. PON1 accumulates in arterial
walls as human plaque progresses from fatty streaks into advanced
lesions. This process is associated with macrophages and seems to
protect against the increasing oxidation associated with plaque pro-gression
[22]. Treating human carotid lesion extracts with recombi-nant
PON1 significantly decreases the oxidative potential of the
extracts [23]. These properties may be the main explanation for the
beneficial effect of PON1 on the atherosclerotic process [24].
Recent research has focused on investigating the potential anti-atherogenic
role played by PON1 in HDL-mediated cholesterol efflux
from macrophages [25]. Rosenblat et al. suggested that PON1 in-creases
lysophosphatidylcholine (lyso-PC) formation, which in turn
stimulates HDL binding and HDL-mediated cholesterol efflux [26].
The H115Q and H134Q histidine residues of PON1 seem to be re-quired
for this process [25].
Lipid rafts are cholesterol- and sphingolipid-rich lateral domains
containing approximately double the molar percentages of these
lipids and much lower proportions of protein than other areas of
the plasma membrane [27]. Research on the role of lipid rafts in cho-lesterol
transport and homeostasis has concentrated mainly on
caveolin-1, a structural protein in a subset of lipid rafts (caveolae)
that directly interacts with cholesterol [28]. Although caveolin-1 is a
useful marker for isolating rafts and seems to be involved in intracel-lular
cholesterol transport, there is some controversy concerning the
role of caveolae cholesterol in cholesterol efflux [29], given that
caveolin-1 is not expressed in primary human macrophages or J774
macrophages [30] and that cholesterol efflux in these cells normally
occurs via apo-AI [31]. Gaus et al. recently demonstrated that choles-terol
exported to apo-AI from the major slow efflux pool comes from
nonraft regions of the plasma membrane and that the interaction of
apo-AI with lipid rafts is required to stimulate this efflux [28].
We investigated the role of purified human PON1 on HDL-mediated
and non-HDL-mediated cholesterol efflux and propose an
explanation of the mechanism by which PON1 enhances cholesterol
efflux, especially via its interaction with lipid and nonlipid raft
domains. We provide further evidence that the PON1–lipid raft inter-action
is an essential event in PON1-mediated cholesterol efflux.
Material and methods
Acetic acid, sulfuric acid, sodium phosphate, thiobarbituric acid,
n-butanol, methanol, ethanol, n-isopropanol, hexane, ammonium
hydroxide, chloroform, and methanol were purchased from Fisher
Scientific (Montreal, QC, Canada). N-ethylmaleimide (NEM), 1,1,3,3,-
tetraethoxypropane, ethylenediaminetetraacetic acid (EDTA), 1,6-
diphenyl-1,3,5-hexatriene, phosphatidylcholine, sphingomyelin, 8-
(4-chlorophenylthio)adenosine 3′:5′-cyclic monophosphate (cAMP),
methyl-β-cyclodextrin (MβCD), and [3H]cholesterol were from
Sigma (St. Louis,MO, USA). THP-1 and J774 cellswere fromtheAmerican
Type Culture Collection (Manassas, VA, USA). RPMI 1640 and Dulbecco's
modified Eagle's medium (DMEM) were from Invitrogen Canada
(Burlington, ON, Canada). Fetal bovine serum(FBS) was fromWisent
(St-Bruno, QC, Canada).
Subjects
Plasma samples were obtained from young healthy volunteers 20
to 30 years of age. They were all healthy, normolipidemic non-smokers
and nonobese. None had clinical or laboratory signs of hy-pertension,
inflammation, or diabetes, and all had normal thyroid
function test results. None were taking medications or oral antioxi-dant
supplements. The ethics committee of the Sherbrooke Geriatric
University Institute approved the study. All the volunteers provided
written informed consent.
Blood collection
After overnight fasting, 80-ml blood samples were collected in
EDTA or citrate vacuum tubes. The plasma was separated by low-speed
centrifugation (1000 g), and 15 ml was used immediately to
isolate lipoproteins. The remaining plasma was stored at −80 °C
until used for the PON1 purification procedure.
Lipoprotein isolation
Human plasma was collected and HDL and HDL3 were isolated by
sequential ultracentrifugation according to the method of Sattler et al.
[32]. In brief, whole HDL (1.063bdb1.19 g/ml) and HDL3 (1.125b
db1.21 g/ml) were isolated by respectively 2 and 4 h of ultracentrifu-gation
in a Beckman TLA 100.4 rotor (100,000 rpm at 15 °C). Isolated
lipoproteins were dialyzed overnight at 4 °C in 10 mM sodium
phosphate buffer (pH 7). Protein concentrations in the samples
were measured using a commercial assay (Bio-Rad, Mississauga, ON,
Canada).
Cell cultures
Human THP-1 monocytes and J774 macrophages were grown in
RPMI 1640, and Fu5AH hepatoma cells were grown in DMEM. The
media were supplemented with 10% heat-inactivated FBS, 50 nM
2-mercaptoethanol (only for THP-1 cells), 2 mM L-glutamine, 100 U/
ml penicillin, and 1.5 mg/ml glucose in a humidified atmosphere
(5% CO2 and 95% air) at 37 °C. The THP-1 monocytes (105 cells/cm2
in six-well plates) were incubated in RPMI–FBS containing 10 ng/ml
phorbol 12-myristate 13-acetate for 96 h to induce differentiation
into adherent macrophage-like cells.
Paraoxonase purification
PON1 was purified using blue agarose and DEAE chromatography
as described by Gan et al. [33], with some modifications. Briefly, plas-ma
was mixed with blue agarose (Cibadron Blue 3GA; Sigma–Aldrich,
Oakville, ON, Canada) in a solution containing 3 M NaCl, 50 mM Tris–
HCl buffer (pH 8), 1 mM CaCl2, and 5 μM EDTA. The PON1 was eluted
with 0.1% deoxycholate. The blue agarose-eluted PON1 was further
purified by anion-exchange chromatography (DEAE-Biogel; Sigma)
using a NaCl linear gradient. The paraoxonase and arylesterase activities
of the purified PON1 were measured as described previously [33,34].
The purity of PON1 was verified by SDS–PAGE.

3. 1374 H. Berrougui et al. / Free Radical Biology & Medicine 52 (2012) 1372–1381
Cholesterol efflux measurements
THP-1 macrophage-like cells, J774 macrophages, and Fu5AH
cells were incubated in fresh medium containing 2 μCi/ml [3H]cho-lesterol
for 24 h. Labeled cells were washed and equilibrated in
serum-free medium containing 1% bovine serum albumin (BSA)
for an additional 12 h. To evaluate the interaction between PON1
and the ABCA1 receptor, [3H]cholesterol-loaded J774 macrophages
were washed three times and incubated with 1% BSA in DMEM
alone (control) or with DMEM containing 0.3 mM cAMP for 12 h
to yield ABCA1-enriched cells (cAMP stimulates ABCA1 gene tran-scription
and surface protein expression) [35]. After the equilibra-tion
period, the THP-1 macrophage-like cells and J774
macrophages were washed three times before being incubated
with various cholesterol acceptors (native or oxidized PON1, HDL3,
apo-AI) depending on the experimental design. The cells were sedi-mented
by centrifugation (350 g for 10 min) and were lysed in
0.1 M NaOH. The counts per minute (cpm) in the supernatant and
cell lysates were determined using a liquid scintillation counter.
Cholesterol efflux (radiolabeled cholesterol released from cells)
was calculated using the following formula: (radioactivity (cpm)
in supernatant/radioactivity (cpm) in cells+medium)×100.
Inactivation of PON1
PON1 was inactivated by exposure to oxygen free radicals pro-duced
by γ-radiolysis of water. Exposure of PON1 to oxygen free rad-icals
has been shown to oxidize PON1 and inhibits its enzymatic
activity without causing protein fragmentation [19]. PON1 was also
inactivated by heat (1 h at 70 °C) or with 10 μM NEM, which scav-enges
the free sulfhydryl group on PON1 (Cys284) [19]. The inactiva-tion
of PON1 was confirmed by measuring PON1 activity.
Lipid raft extraction
Lipid rafts were extracted as previously described [36]. Briefly,
cells were resuspended in 300 μl of ice-cold lipid raft lysis buffer
and mixed with 300 μl of 85% sucrose in Hepes-buffered saline solu-tion
(pH 6.9). The samples were overlaid with 1 ml of 35% sucrose
and then 300 μl of 5% sucrose. The preparations were centrifuged at
200,000 g at 4 °C for 16 h using a Beckman TLA-100.4 rotor (Beckman
Instruments, Montreal, QC, Canada). Nine 200-μl fractions were col-lected
from the top of the gradient. They were separated on 10%
SDS–PAGE gels and were transferred to nitrocellulose membranes,
which were blotted and incubated with anti-flotillin antibody.
Immunofluorescence microscopy
J774 macrophages were seeded in eight-well glass culture slides
(BD Falcon, Bedford, MA, USA) at a concentration of 2.5×105 cells
per well and were incubated with 0.3 mM cAMP for 12 h to generate
ABCA1-enriched cells. The enriched cells were incubated with 20 U/
ml PON1 for 4 h, washed twice with PBS, fixed with 4% formaldehyde
for 10 min, and incubated with 0.1% Triton X-100 in PBS for 2 min.
After being washed with PBS, the cells were incubated with blocking
solution (5% goat serum, 5% BSA, and 0.01% Tween 20) and then with
primary antibody in blocking solution for 16 h at 4 °C. Alexa 488-
labeled goat anti-mouse IgG was used as the secondary antibody.
The slides were washed, stained with DAPI, and mounted in Vecta-shield
(Vector Laboratories, Burlington, ON, Canada) to visualize the
shapes of the nuclei.
Western blotting
Cell proteins were solubilized in RIPA buffer. Identical amounts of
cell lysate protein were separated on 10% SDS–PAGE gels and were
transferred to nitrocellulose membranes. The membranes were
blocked with 5% fat-free powdered milk in TBST (10 mM Tris–HCl,
pH 7.4, 150 mM NaCl, 0.1% Tween 20) and then incubated with
ABCA1, ABCG1, or SR-BI primary antibodies (Abcam, Cambridge,
MA, USA). After three washes with TBST, the membranes were incu-bated
with horseradish peroxidase-conjugated goat anti-rabbit, goat
anti-mouse IgG, or rabbit anti-goat IgG (Sigma), and the signals
were visualized using the ECL Western blotting system (GE Health-care,
Piscataway, NJ, USA).
Coimmunoprecipitation assays
Coimmunoprecipitation was used to confirm the formation of
ABCA1–PON1 complex in lysates from J774 macrophages. cAMP
(0.3 mM) was used to stimulate ABCA1 overexpression on J774 mac-rophages.
PON1 (20 U/ml) was added to ABCA1-enriched or none-nriched
J774 macrophages for 3 h and then cell lysates were
prepared with RIPA buffer and protease inhibitor cocktail. Immuno-precipitations
were carried out using Dynabeads protein G (Invitro-gen)
according to the manufacturer's protocol. Briefly, 1.5 mg of
Dynabeads was incubated with 5 μg of ABCA1 monoclonal antibody
(mAb) for 60 min. After overnight incubation at 4 °C of Dynabeads–
mAb complex with cell lysates, beads were washed three times
with PBS and then 20 μl of 2× sample buffer (100 mM Tris, pH 6.8,
40 g/L SDS, 200 ml/L glycerol, 20 mg/ml bromophenol blue, 0.05% 2-
mercaptoethanol) was added to the beads. The sample/supernatant
was separated from the beads by placing the tube on a magnet and
then loaded on the SDS–PAGE (10%) gel. After being transferred to a
polyvinylidene difluoride membrane, PON1 was detected using an
anti-PON1 mouse monoclonal antibody (Abcam).
Quantitative RT-PCR
Total RNA from J774 macrophages treated with 20 U/ml PON1 for
0, 1, 3, or 16 h was extracted using RNeasy extraction mini kits (Invi-trogen)
and treated with DNase I (Qiagen, Mississauga, ON, Canada)
according to the manufacturer's protocol. Two micrograms of RNA
was transcribed using Reverse Transcriptase Superscript II (Invitro-gen).
The expression of the ABCA1 gene was normalized to the corre-sponding
amount of β-actin. Amplifications were performed using
the following primers (sense and antisense): ABCA1, 5′-TCATCTT-CATCTGCTTCCAGC-
3′ and 5′-GTGCTGGGGATCTTGAACAC-3′; β-actin,
5′-GAACGGTGAAGGTGACA-3′ and 5′-TAGAGAGAAGTGGGGTGG-3′.
The quantitative PCR assays were performed using the Stratagene
MX3005P system (Agilent Technologies, Mississauga, ON, Canada)
and Brilliant II SYBR Green QPCR Master Mix (Agilent). The qPCR as-says
were performed using 25 ng of template cDNA. Samples were in-cubated
at 95 °C for 10 min followed by 45 cycles using the following
conditions: 95 °C for 40 s, 56 °C for 40 s, and 72 °C for 40 s. All reac-tions
were run in triplicate for each replicate, and the average values
were used for quantification purposes. The relative quantities of tar-get
genes were determined using the ΔΔCt method. Briefly, the Ct
(threshold cycle) values of target genes were normalized to an en-dogenous
control gene, β-actin (ΔCt=Ct target−Ct β-actin), and were
compared with a calibrator (ΔΔCt=ΔCt sample−ΔCt calibrator). Relative
expression (RQ) was calculated using a sequence detection system
(MxPro and QPCR software; Agilent) and the formula RQ=2−ΔΔCt.
Statistical analysis
Values are expressed as means ± SEM. A one-way analysis of var-iance
was used for multiple comparisons. A linear regression analysis
was used to assess the association between two continuous variables.
Statistical analyses were performed using Prism 5.0 version software.
A p value of b0.05 was considered statistically significant.

4. Results
PON1 has an effect on HDL-mediated cholesterol efflux
HDL-associated proteins other than apo-AI can stimulate HDL-mediated
cholesterol efflux [37,38]. The aim of this study was to in-vestigate
the role of PON1 in cholesterol efflux. Loading THP1
macrophage-like cells with [3H]cholesterol and purified human plas-ma
PON1 5, 10, and 20 U/ml (corresponding to 12.5, 25, and 50 μg/ml
of protein) increased HDL3-mediated cholesterol efflux from THP-1
macrophage-like cells by 20.8, 24, and 63% (pb0.05), respectively
(Fig. 1A). The increase in cholesterol efflux from THP1 macrophage-like
cells was dependent on the concentration of PON1 (r2=0.91,
pb0.05, data not shown). It is noteworthy that the physiological con-centration
of human plasma PON1 is between 50 and 100 μg/ml pro-tein
[39,40].
Although PON1 significantly increased cholesterol efflux to J774
macrophages (Fig. 1B) in a concentration-dependent manner
(r2=0.94, pb0.05, data not shown), stimulating J774 macrophages
with cAMP to induce ABCA1 overexpression significantly potentiated
the effect (r2=0.97, pb0.05). A comparison of the slopes of the two
correlations shows that cholesterol efflux increased 1.82-fold when
PON1 was added to cAMP-stimulated J774 macrophages compared
to unstimulated cells (1.03±0.11 vs 0.55±0.1, respectively,
pb0.05). This suggests that PON1 enhances HDL3-mediated choles-terol
efflux and that the ABCA1 transporter pathway mediates this
effect.
PON1 is a novel independent transporter involved in cholesterol efflux
We performed experiments in more than one cell type to deter-mine
which of the three cholesterol efflux pathways might be stimu-lated
by PON1. PON1-mediated cholesterol efflux was first studied in
THP-1 macrophage-like cells, which equally express ABCA1/ABCG1
transporters and the SR-BI receptor. Our results show that incubation
of cholesterol-loaded THP-1 with 0, 5, 10, or 20 U/ml PON1 alone re-sults
in a significant increment in cholesterol efflux by 2.5-, 3-, and
4.6-fold compared to the control (Fig. 2A).
To better understand the mechanism by which PON1 mediates
cholesterol efflux, we investigated the role of the ABCA1 pathway in
this process. For that purpose we used cAMP-pretreated J774 macro-phages,
which express high levels of ABCA1 [4]. ABCA1-enriched and
nonenriched J774 macrophages (control) were incubated with in-creasing
concentrations of PON1. The results show that PON1 signifi-cantly
promoted cholesterol efflux in ABCA1-enriched macrophages
in a concentration-dependent manner, whereas no significant in-crease
was observed in the control (Fig. 2B).
Taking into account that PON1 and apo-AI are both associated
with HDL, we investigated a possible synergistic effect between
apo-AI and PON1 to mediate cholesterol efflux. For that purpose, we
incubated [3H]cholesterol-loaded ABCA1-enriched J774 cells with in-creasing
concentrations of PON1 (0, 5, 10, and 20 U/ml) in the pres-ence
or not of apo-AI (25 μg/ml) for 4 h. Results in Fig. 2C, shows
that apo-AI-mediated cholesterol efflux was significantly potentiated,
and in a dose-dependent manner, in the presence of PON1 by an av-erage
of 37.24±5.35% (pb0.01).
The incubation of cholesterol-loaded ABCA1-enriched J774 with
increasing concentrations of BSA (negative control) had no effect,
whereas 25 μg/ml apo-AI (positive control) significantly enhanced
cholesterol efflux (data not shown). This confirms that PON1 en-hances
ABCA1-dependent cholesterol efflux from J774 macrophages.
To assess SR-BI-mediated cholesterol efflux, we used Fu5AH rat
hepatoma cells, which express high levels of SR-BI and lack a func-tional
ABCA1 [41]. Interestingly, incubating PON1 (0, 5, 10, or 20 U/
ml) with SR-BI-overexpressing Fu5AH cells did not cause a significant
change in cholesterol efflux (Fig. 2D). This effect was nonsignificant
even when PON1 was incubated in the presence of HDL (50 μg/ml),
which leads to the suggestion that the SR-BI receptor was not or
was less implicated in the PON1-mediated cholesterol efflux.
ABCA1 overexpression increases PON1 internalization
cAMP-treated J774 macrophages exhibited more intense immuno-fluorescence
than the control cells (Figs. 3A and B), indicating that
ABCA1 overexpression increases the internalization of PON1, which
leads to its localization in the cell's cytoplasm (Fig. 3C, 100× original
magnification). This is in accordance with the results reported by
Efrat and Aviram [42], who used FITC fluorescence and confocal mi-croscopy
to demonstrate an internalization of PON1 and its localiza-tion
in the cell's cytoplasm compartment.
ABCA1-enriched and nonenriched J774 macrophages were incu-bated
with PON1 for 3 h. PON1 was detected in the ABCA1
A
40
30
20
10
0
* *
**
+ + + + HDL3 [50μg/ml]
0 5 10 20 PON1 [U/ml]
Cholesterol efflux (%)
30
20
10
0
ns
*
**
**
+ + + + + + + + HDL3 [50 μg/ml]
- + - + - + - + cAMP [0.3 mM]
0 5 10 20 PON1 [U/ml]
B
Cholesterol efflux (%)
Fig. 1. HDL3-associated PON1 mediates cholesterol efflux from THP-1 and J774 macro-phages.
(A) THP-1 macrophages were loaded with [3H]cholesterol (2 μCi/ml) for 24 h.
The cells were then washed and further incubated for 24 h with 50 μg/ml HDL3 alone
(control) or in the presence of increasing concentrations of PON1 (0–20 U/ml;
0–50 μg/ml PON1 protein concentration). (B) [3H]Cholesterol-loaded J774 macro-phages
were incubated overnight at 37 °C without (control) or with 0.3 mM cAMP
(ABCA1-enriched cells). After being washed, the cells were incubated for 24 h with
HDL3 alone or with HDL3 supplemented with increasing concentrations of PON1
(0–20 U/ml; 0–50 μg/ml). Results are expressed as the means ± SEM of three indepen-dent
experiments. *pb0.05, **pb0.01.
H. Berrougui et al. / Free Radical Biology & Medicine 52 (2012) 1372–1381 1375

5. 1376 H. Berrougui et al. / Free Radical Biology & Medicine 52 (2012) 1372–1381
0 5 10 20 PON1 [U/ml] 5 10 20 5 10 20
0 5 10 15 20 25 0 5 10 20 20
+
20
15
10
5
0
**
THP1
Cholesterol efflux (%)
15
10
5
PON1
cAMP -
+
+
0
0 0 0 0 0
- - - + + +
+
+
Magnification (x 100)
**
***
10
8
6
4
2
0
Cholesterol efflux (%)
apo-AI 25μg/ml
15
10
5
A B C
Fig. 3. ABCA1 overexpression increases PON1 internalization. (A) Control and (B and C) ABCA1-enriched J774 cells (original magnification 40× and 100×, respectively) were incu-bated
with PON1 (20 U/ml; 50 μg/ml) before their fixation with 4% paraformaldehyde and their immunostaining with primary antibody for PON1 (mouse). The cells were then
incubated with Alexa 488-labeled goat anti-mouse IgG secondary antibody and processed for immunofluorescence microscopy.
PON1 [U/ml]
**
***
***
J774
HDL
cAMP [0.3 mM]
0
apo-AI-free
PON1 [U/ml]
Cholesterol efflux (%)
0
ns Fu5AH
ns
PON1 [U/ml]
HDL [μg/ml]
0
50 50
0 0 0 0
Cholesterol efflux (%)
ns
A B
C D
Fig. 2. Effects of purified PON1 on the cholesterol efflux. (A) [3H]Cholesterol-loaded THP-1 cells were incubated with PON1 (0–20 U/ml; 0–50 μg/ml) for 24 h. (B) J774 macrophages
were loaded with [3H]cholesterol and then incubated for 12 h in the presence of cAMP to yield ABCA1 expression. Cholesterol efflux from ABCA1-enriched and nonenriched J774
was then initiated by the addition of PON1 (0–20 U/ml; 0–50 μg/ml) for 4 h. (C) [3H]Cholesterol-loaded and ABCA1-enriched J774 cells were incubated for 4 h with increasing con-centrations
of PON1 (0–50 μg/ml) in presence or not of apo-AI. (D) [3H]Cholesterol-loaded Fu5AH cells were incubated with PON1 (0–20 U/ml; 0–50 μg/ml) and/or HDL3 (50 μg/ml,
positive control) for 24 h. To investigate if PON1 require HDL for its interaction with SR-BI receptors, [3H]cholesterol-loaded Fu5AH cells were incubated with PON1 (20 U/ml; 50 μg/
ml) in the presence of HDL3 (50 μg/ml) for 24 h. Results are expressed as the means ± SEM of more than three independent experiments. **pb0.01, ***pb0.001; ns, nonsignificant.

6. PON1 (20 U/ml)
1 2
+ +
immunoprecipitate, with the highest amount in the precipitate from
ABCA1-enriched J774 (Fig. 4), suggesting that PON1 binds with
ABCA1 to form a protein complex.
We previously showed that the oxidation of purified human PON1
causes a decrease in its antioxidant and paraoxonase activities [19].
This effect was attributed to the loss of the free sulfhydryl group
[19]. In this study, we show that exposure of PON1 to oxygen free rad-icals
produced by γ-radiolysis affects significantly its ability to pro-mote
HDL3-mediated cholesterol efflux from THP-1 macrophage-like
cells (−29.04%, pb0.01, Fig. 5A). In addition, heat (70 °C for
1 h) and NEM treatment, which irreversibly blocks SH groups by
covalent binding [43], significantly decreased (pb0.01) paraoxonase
activity as well as its ability to promote cholesterol efflux, especially
from ABCA1-enriched macrophages (Fig. 5B).
PON1 interacts with lipid raft domains to initiate cholesterol efflux from
macrophages
In the second part of our study, we investigated the capacity of
PON1 to interact with lipid rafts and to mediate cholesterol efflux. Su-crose
gradients separated the plasma membranes into two fractions:
40
30
20
10
0
*
A
Cholesterol efflux (%)
10
8
6
4
2
0
HDL3-nPON1 [20U/ml] HDL3-oxPON1 [20U/ml]
** **
nPON1 Heat-inactivated
PON1
NEM-inactivated
PON1
B
Cholesterol efflux (%)
Fig. 5. PON1 oxidation impairs its capacity to stimulate HDL-mediated cholesterol
efflux. (A) [3H]Cholesterol-loaded THP-1 macrophages were incubated with HDL3 in
the presence of native (nPON1) or oxidized PON1 (oxPON1) for 24 h. Oxidation of
PON1 was induced by its exposure to oxygen free radicals produced by γ-radiolysis
of water. (B) Denaturing PON1 reduces its capacity to interact with ABCA1 receptors
and to promote cholesterol efflux. 20 U/ml (50 μg/ml) PON1 was inactivated by heat
(70 °C/1 h) or by NEM treatment (1 h) before starting the experiments. J774 macro-phages
were loaded with [3H]cholesterol and treated with cAMP to upregulate
ABCA1 expression. Treated and control cells were incubated with native PON1, heat-inactivated
PON1, or NEM-treated PON1 for 4 h. Data are given as means ± SEM of
more than three independent experiments. *pb0.05, **pb0.01.
8×10 04
6×10 04
4×10 04
2×10 04
2.0×1004
1.5×1004
1.0×1004
5.0×1003
Control
*
apoA1
PON1
Control
apoA1
PON1
0.0
Raft fractions
**
*
Cholesterol contents
(cpm)
12 **
9
6
3
Control-CD free
apoA1-CD
PON1-CD free
Control-CD
apoA1-CD free
PON1-CD
0
*
Cholesterol efflux (%)
A
B
Non-Raft fractions
Fig. 6. PON1 and apo-AI start mediating cholesterol efflux from lipid raft domains. THP-
1 macrophages were loaded with [3H]cholesterol and then incubated for 4 h with apo-
AI or PON1. Plasma membranes were isolated on a sucrose gradient as described under
Material and methods. (A) Cholesterol contents (cpm) in associated lipid raft and non-raft
fractions. (B) [3H]Cholesterol-loaded and ABCA1-enriched J774 macrophages were
pretreated for 1 h at 37 °C with medium containing BSA alone or plus methyl-β-
cyclodextrin (CD; 2 mg/ml). After the above preincubations, the media were removed
and efflux to fresh medium containing or not apo-AI or PON1 over 4 h was measured.
Results are expressed as the means ± SEM of more than three independent experi-ments.
*pb0.05, **pb0.01 vs control.
PON1 45Kda
cAMP
+
Fig. 4. Coimmunoprecipitation of ABCA1 and PON1 in lysates from J774 macrophages
shows that PON1 binds to ABCA1. PON1 (20 U/ml) was added to ABCA1-enriched
(lane 1) or nonenriched (lane 2) J774 for 3 h and then ABCA1 immunoprecipitation
(IP) from cell lysates was performed using Dynabeads protein-G and ABCA1 monoclo-nal
antibody. ABCA1 IP was followed by PON1 Western blot (10% SDS–PAGE) using
mouse anti-human PON1 monoclonal antibody.
H. Berrougui et al. / Free Radical Biology & Medicine 52 (2012) 1372–1381 1377

7. 1378 H. Berrougui et al. / Free Radical Biology & Medicine 52 (2012) 1372–1381
light membranes (rafts) and heavy membranes (nonrafts). Western
blotting using fotilin-1, which migrated with the rafts, confirmed
the separation (data not shown).
Incubating [3H]cholesterol-loaded THP-1 macrophage-like cells
with PON1 and apo-AI resulted in a significant decrease in the choles-terol
content of the lipid raft fractions, by approximately 38.0
(pb0.01) and 20.5% (pb0.05), respectively. However, in the nonraft
fractions, cholesterol decreased by only 23.1 and 12.3% (pb0.05) in
the presence of PON1 and apo-AI, respectively (Fig. 6A). This sug-gested
that PON1 interacts with lipid rafts to initiate cholesterol ef-flux
from macrophages.
To gain more insight into the mechanismby which the PON1–lipid
raft interaction modulates cholesterol efflux, we studied the effect of
a short MβCD pretreatment on the increase in cholesterol efflux from
cholesterol-loaded macrophages by PON1 and apo-AI (control). The
MβCD treatment caused a selective removal of cholesterol from
lipid raft domains and induced the dispersal of their lipid and protein
components [28]. Interestingly, the exposure of ABCA1-enriched J774
macrophages to MβCD for 1 h resulted in a significant impairment of
the cholesterol efflux mediated by PON1 (57.21%, pb0.05) and by
apo-AI (63.89%, pb0.01) (Fig. 6B). This suggested that the presence
of intact lipid rafts seems to be essential for initiating PON1 to stimu-late
the cholesterol efflux process.
Modeling the kinetics of cholesterol export from macrophages to
apo-AI predicted a rapid initial efflux from the small pool, followed
by a slower but substantial and sustained efflux from the separate
larger pool, which contributed to most of the exported cholesterol
[44].We examined the kinetics model of cholesterol efflux from mac-rophages
to PON1 compared to apo-AI. Fig. 7 shows a typical choles-terol
efflux experiment (0 to 16 h) from cholesterol-loaded J774
macrophages treated with cAMP. Cholesterol efflux to DMEM alone
containing 1% BSA was minimal compared to apo-AI- and PON1-
mediated cholesterol efflux.
Like apo-AI, PON1-induced cholesterol efflux can be qualitatively
split into two phases, an initial high cholesterol efflux rate phase
that lasts from 0 to 3 h followed by a slower cholesterol efflux rate
phase.
PON1 has an effect on ABCA1 protein and gene expression
To investigate the effects of PON1 on the ABCA1 level during the
distinct phases of the cholesterol efflux process, ABCA1 protein and
gene expression was analyzed by Western blotting and qPCR,
10000
8000
6000
4000
2000
15
10
5
1 3 16 1 3 16
0 1 3 16 1 3 16
*** ***
A
respectively, in J774 macrophages incubated in the presence or
absence of PON1. Interestingly, a short incubation of the macrophages
with PON1 increased ABCA1 protein levels after 1 and 3 h, but the
effect disappeared after a longer incubation period (16 h; Fig. 8A).
More importantly, the upregulation by PON1 was accompanied by
an increase in ABCA1 mRNA levels (Fig. 8B). The same effect on
ABCA1 protein expression was observed when apo-AI was used as a
control. These results demonstrate that PON1 modulates ABCA1
expression on macrophages.
Discussion
Cholesterol efflux is thought to be one of the most important
atheroprotective properties of HDLs. This process is mediated by the
interaction of lipid-free apo-AI and HDLs with ABCA1, ABCG1, and
SR-B1 membrane proteins as well as via other mechanisms, including
passive diffusion. Apo-AI is a key factor in the enhancement of HDL-mediated
cholesterol efflux. However, plasma HDL particles contain
0 4 8 12 16
25
20
15
10
5
0
Apo-AI [25 μg/ml]
PON1 [20 U/ml: 50 μg/ml]
Control [BSA: 50 μg/ml]
Time [hours]
Cholesterol efflux (%)
Fig. 7. PON1 enhances cholesterol efflux from the ABCA1-dependent pathway in rapid-low
and slow-high pools. Kinetics of cholesterol efflux to apo-AI (25 μg/ml) and PON1
(20 U/ml; 50 μg/ml) over the first 16 h in J774 [3H]cholesterol-loaded macrophages
enriched in ABCA1 is shown. BSA (50 μg/ml) was used as control. Results are expressed
as the means ± SEM of more than three independent experiments.
0
**
***
PON1
expression
gene ABCA1 Relative 0 1 3 16
PON1 Incubation time [hours] 0
*** ***
*** ***
Incubation time [Hours]
PON1
apo-AI
Hours
ABCA1
0
apo-AI PON1
Densitometry Analysis
B
Fig. 8. Effects of short and long pretreatment of J774 macrophages with apo-AI or PON1
on ABCA1 protein and RNA expression. Kinetics analysis of ABCA1 expression (0, 3, and
16 h) upon incubation with apo-AI (25 μg/ml) or PON1 (20 U/ml; 50 μg/ml) was car-ried
out in non-ABCA1-enriched J774 cells. Protein levels and RNA expression of
ABCA1 were respectively determined by (A) Western blot and (B) RT-PCR analyses.
Quantitative analysis was determined using densitometry from three independent
experiments. Results are expressed as means ± SEM. **pb0.01, ***pb0.001.

8. H. Berrougui et al. / Free Radical Biology & Medicine 52 (2012) 1372–1381 1379
a variety of proteins with antiatherogenic potential, including PON1,
that may also contribute significantly to the cholesterol efflux pro-cess.
PON1 is a protein that is mainly complexed to HDLs and has
been associated, in part, with the antioxidant and anti-inflammatory
properties of HDLs. The goal of this study was to investigate the in-volvement
of PON1 in the regulation of the cholesterol efflux process,
especially the mechanism by which PON1 modulates HDL-mediated
cholesterol transport.
Our results showed that PON1 enhances HDL-mediated cholester-ol
efflux in THP-1 macrophage-like cells and in J774 macrophages.
The effect was more pronounced with ABCA1-enriched J774 macro-phages,
suggesting that an interaction between PON1 and ABCA1 re-ceptors
promotes cholesterol efflux. Our results showed that purified
PON1 is highly effective in stimulating cholesterol efflux via the
ABCA1 pathway, whereas no effect was observed with respect to
SR-BI receptors. This finding is in agreement with that of Rosenblat
et al., who used purified human PON1 and HDLs from PON1 knockout
and PON1Tg mice [26]. Moreover, Fuhrman et al. recently reported
that PON1 may protect macrophages from apoptosis by upregulating
macrophage SR-BI-mediated HDL binding to the cells. However, the
involvement of SR-BI in the interaction of HDLs with macrophages
does not affect cellular cholesterol efflux [45].
Although apo-AI is the principal HDL-associated protein that in-teracts
with membrane proteins to promote cholesterol efflux, other
HDL-associated proteins may play this role. A recent study by De
Beer et al. [46] showed that lipid-free serum amyloid A (SAA) acts
like apo-AI by mediating sequential cholesterol efflux via ABCA1
and ABCG1 [46]. Remaley et al. showed that ABCA1-mediated lipid ef-flux
is not specific to apo-AI but can also occur with other apolipopro-teins
that contain multiple amphipathic helical domains [47]. A study
by Xie et al. [49] showed that an apo-AI mimetic peptide (D-4F),
which has no sequence homology with apo-AI but possesses the
class-A amphipathic helical motif, promotes cholesterol efflux
through the ABCA1 pathway [48,49]. It is thus not surprising that
PON1 modulates cholesterol efflux from macrophages principally
via the ABCA1 pathway. Indeed, like apo-AI and SAA, the secondary
structure of PON1 contains amphipathic α-helices with approximate-ly
22 amino acids [50] that enable HDL particles to tightly bind, stabi-lize,
and stimulate PON1 [51].
Our results demonstrate that PON1 enhances cholesterol efflux
and induces an upregulation of ABCA1. Although we did not investi-gate
the mechanism of PON1-induced upregulation of ABCA1, it has
been suggested that this effect could be attributed to the capacity of
PON1 to hydrolyze oxidized phospholipids to form lyso-PC, which
in turn induces ABCA1 expression [26]. In accordance, Hou et al. dem-onstrated
that lyso-PC increases the mRNA and protein expression of
PPARγ, LXRα, and ABCA1 of macrophage foamcells in a concentration-dependent
manner [52]. The upregulation of ABCA1, in this study,
was confirmed by the measurement of ABCA1 protein and mRNA
expression and also evidenced by the internalization of PON1 in mac-rophages.
Our results from the immunofluorescence and cholesterol
efflux assays showed that the internalization of PON1 in macrophages
is ABCA1 dependent. A reduction in ABCA1 content or an alteration in
the structure of PON1 significantly affected PON1-mediated cholesterol
efflux.
Sorenson et al. showed that purified human and rabbit PON1 both
have a single free sulfhydryl group (Cys284) and suggested that this
cysteine is part of the active site of PON1 required for its antioxidant
activity [53]. Previous studies, including ours, showed that PON1 mu-tants
display significantly less antioxidant activity and that blocking
the free sulfhydryl groups with NEM or p-hydroxymercuribenzoate
significantly reduces the antioxidant activity of PON1 [19,54]. In this
study, we showed that exposure of PON1 to free radicals or its inacti-vation
by heat or NEM treatment induced a net reduction in its ability
to promote cholesterol efflux from THP-1 macrophage-like cells and
ABCA1-enriched J774 macrophages. These results confirm that the
capacity of PON1 to stimulate cholesterol efflux is dependent on its
structural integrity.
The mechanism by which apo-AI removes excess cholesterol and
phospholipids from macrophages has been extensively investigated.
Gaus et al. [28,44] studied the interaction between apo-AI and lipid
raft microdomains and concluded that cholesterol efflux to apo-AI oc-curs
sequentially from two membrane pools with different kinetics,
that is, rapid release from a small rapid efflux pool within the first
few hours followed by progressive release from a major slow efflux
pool over several hours. They proposed that cholesterol exported to
apo-AI from this major slow efflux pool comes from nonraft regions
of the plasma membrane. However, the interaction of apo-AI with
lipid rafts is required to stimulate this efflux. Our results showed
that PON1-mediated cholesterol efflux also occurs via two steps:
rapid cholesterol efflux during the first 3 h followed by slow choles-terol
efflux thereafter. The level of cholesterol efflux mediated by
PON1 was comparable to that mediated by apo-AI, at least during
the rapid cholesterol efflux step. This suggested that PON1 acts via
an apo-AI-like mechanism to mediate cholesterol efflux. This mecha-nistic
assumption was supported by the results of ABCA1 expression
measurements and experiments involving a pretreatment with cyclo-dextrin,
which selectively depletes cholesterol from lipid raft do-mains
(~50%) but not from nonraft domains [28]. First, the ABCA1
transporter was overexpressed within the first 3 h of incubation of
PON1 with cholesterol-loaded J774 macrophages, which covers the
period during which cholesterol is released from the small rapid ef-flux
pool. After 16 h, ABCA1 expression was downregulated, which
was probably related to the involvement of the ABCG1 transporter
or SR-BI receptor. These two proteins do not interact with lipid-free
apo-AI [55,56]. Second, cyclodextrin significantly affected the ability
of PON1 and apo-AI to mediate cholesterol efflux from macrophages
(Fig. 6B).
Studies investigating the relationship between membrane plasma
microdomains and apo-AI with respect to the cholesterol efflux ca-pacity
of apo-AI have given rise to conflicting results. The distribution
of ABCA1 between lipid raft and nonraft domains depends on the
macrophage cell line and the method used to prepare the rafts.
ABCA1 is not present in Triton-resistant membrane domains, whereas
a significant proportion can be detected in Lubrol-resistant domains
[57,58]. Mendez et al. reported that membrane raft lipids are not di-rectly
available for lipid efflux by apolipoprotein-dependent mecha-nisms
[58]. In contrast, cholesterol efflux from cells to HDL particles
or to plasma can be partially explained by the depletion of cholesterol
from membrane rafts. This suggests that apo-AI– and PON1–ABCA1-
mediated cellular cholesterol efflux occurs by mechanisms distinct
from those mediated by lipoprotein particles containing lipids as
their major constituent. More interestingly, this study showed that
PON1 plays an important role in the antiatherogenic properties of
HDLs and can exert its protective function outside the lipoprotein en-vironment.
This is supported by a recent study by Deakin et al. show-ing
that PON1 is not a fixed component of HDLs because it can be
transferred from HDLs to the external face of the plasma membrane
of cells in an enzymatically active form [59].
These findings open a new window on the association between
PON1 and HDLs with respect to their antiatherogenic function as
well as on the mechanisms by which PON1 may exert its atheropro-tective
effect, especially by regulating cholesterol efflux from macro-phages
and maintaining cholesterol homeostasis. However, further
studies are needed to elucidate the nature of the pathways involved
in the promotion of cholesterol efflux by PON1 and to determine
the activity of free, non-HDL-associated PON1.
Acknowledgment
This work was supported by a grant from the Canadian Institutes
of Health Research (MOP-89912).

9. 1380 H. Berrougui et al. / Free Radical Biology & Medicine 52 (2012) 1372–1381
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