ORIGINAL PAPER

The Effect of PPARa and PPARc Ligands on Inflammation andABCA1 Expression in Cultured Gallbladder Epithelial Cells

Jin Lee Æ Eun Mi Hong Æ Hyun Woo Byun Æ Min Ho Choi Æ Hyun Joo Jang ÆChang Soo Eun Æ Sea Hyub Kae Æ Ho Soon Choi

Received: 8 March 2007 / Accepted: 19 September 2007 / Published online: 12 October 2007

� Springer Science+Business Media, LLC 2007

Abstract The preservation of gallbladder function by

control of inflammation and elimination of cholesterol

accumulation in gallbladder epithelial cells (GBEC) could

contribute to the prevention of gallstone formation and

cholecystitis. Peroxisome proliferator-activated receptors

(PPARs) modulate inflammation and lipid metabolism in

various cells and GBEC efflux of excessive amounts of

absorbed cholesterol through the ATP-binding cassette

transporter A1 (ABCA1)-mediated pathway. The aim of

this study was to determine whether ligands of PPARa and

PPARc modulate inflammation and have an effect on

ABCA1 expression in GBEC. Canine GBEC were cultured

on dishes coated with collagen matrix. We performed

Western blot analysis for the expression of specific protein

and/or RT-PCR for the expression of specific mRNA.

PPARa and PPARc expression was observed and increased

in GBEC treated with WY-14643 (PPARa ligand), trog-

litazone (PPARc ligand), and lipopolysaccharide (LPS)

compared to the no-treatment control and PPARa antago-

nist (GW-9662) treatment group. WY-14643, troglitazone,

and LPS also induced an increase in the expression of

ABCA1 protein and mRNA in cultured GBEC. LPS-

induced TNFa mRNA expression was suppressed by pre-

treatment with WY-14643 and troglitazone preceding LPS

treatment in GBEC. PPAR ligands, especially PPARc, may

preserve gallbladder function by suppression of inflam-

matory reaction and prevention of cholesterol

accumulation in GBEC, contributing to the prevention of

gallstone formation and progression to cholecystitis.

Keywords Gallbladder � Gallstone �Peroxisome proliferator-activated receptor (PPAR) �ATP-binding cassette transporter A1 (ABCA1)

Introduction

Cholesterol supersaturation in bile, the loss of balance

between the nucleation promoter and inhibitor, and gall-

bladder (GB)-associated factors are still regarded as major

elements in the pathogenesis of cholesterol gallstones [1,

2]. Among these elements, GB-associated factors could be

the most important target in the treatment or prevention of

gallstones in view of the possibility of control. The known

GB-associated factors are chronic inflammation of GB

epithelium, excessive accumulation of cholesterol in epi-

thelium and subcutaneous tissue, hypersecretion of mucin,

GB hypomotility, and increased prostaglandin E, that have

an effect on one another [1–6]. If we could control the

inflammation and excessive accumulation of cholesterol in

GB, the other GB-associated factors described above could

be controlled, resulting in a contribution to the treatment or

prevention of gallstones.

ABCA1 (ATP-binding cassette transporter A1) is a

membrane-bound ABC transporter that is mutated in

patients with Tangier disease. These patients have a defect

in reverse cholesterol transport, whereby cholesterol cannot

be mobilized from peripheral tissues. The patients have

J. Lee (&) � E. M. Hong � H. W. Byun �M. H. Choi � H. J. Jang � C. S. Eun � S. H. Kae

Division of Gastroenterology, Department of Internal Medicine,

Hallym University Hangang Sacred Heart Hospital, 94-200,

Youngdungpo-Dong, Youngdungpo-Gu, Seoul 150-030, South

Korea

e-mail: jinlee@medimail.co.kr

H. S. Choi

Division of Gastroenterology, Department of Internal Medicine,

Hanyang University College of Medicine, 17, Haengdang-Dong,

Seongdong-Gu, Seoul 133-792, South Korea

123

Dig Dis Sci (2008) 53:1707–1715

DOI 10.1007/s10620-007-0029-5

very low levels of high-density lipoprotein (HDL) choles-

terol, which manifests itself as excessive peripheral deposits

of cholesterol ester, and die prematurely from atheroscle-

rosis [7–9]. Earlier we showed evidence for an ABCA1-

mediated reverse cholesterol efflux in cultured gallbladder

epithelial cells (GBEC), and the pathway is also regulated

by liver X receptor-a (LXRa)/retinoid X receptor (RXR)-

heterodimer, which are upper-level nuclear hormone regu-

lators. It is suggested that ABCA1 have a key role in

eliminating excessive cholesterol in GBEC, preventing

hypomotility of GB as a defense mechanism [10, 11].

Peroxisome proliferator-activated receptors (PPARs)

belong to the nuclear receptors that, upon heterodimeriza-

tion with RXR, function as transcriptional regulators of

glucose and lipid metabolism [12, 13]. So far, three PPAR

isoforms have been identified and cloned: PPARa, PPARb/

d, and PPARc. PPARa is highly expressed in the liver,

heart, muscle, and kidney, and in cells of the artery. Fatty

acid, fibrates, and eicosanoids are ligands of PPARa.

PPARc is the molecular target of thiazolidinedione anti-

diabetic agents and is mainly expressed in adipose tissue

[14, 15]. It has recently been discovered that PPARs are

also strongly linked to inflammatory reaction. Inflamma-

tion inducers such as lipopolysaccharide (LPS) and tumor

necrosis factor (TNFa) evoke activation of nuclear factor-

jB (NF-jB), a major transcription factor in the inflam-

matory process, and promote the secretion of a series of

inflammatory cytokines in various cells. PPARa and

PPARc ligands can block the NF-jB pathway, modulating

inflammatory reaction [16, 17]. It is also known that acti-

vated PPARs induce LXRa activation, resulting in ABCA1

activation in macrophages [18–21]. There are no reports of

direct evidence of PPAR expression in GBEC except one

paper which showed the possibility that PPARc ligand

could suppress the inflammation of human GBEC [22].

It could be expected that ABCA1 activation preserves

the motility of GB by the elimination of excessive cho-

lesterol and suppression of inflammation, which prevents

excessive mucin secretion and prostaglandin production,

preventing gallstone formation and the progression of

cholecystitis [10, 11, 22]. However, it is impossible to put

known ABCA1 ligands or activators such as retinoic acid

(RXR-agonist), hydroxycholestrol (LXRa agonist), or

synthetic LXRa agonists to practical use because of strong

adverse effects or lack of clinical evidence [23]. There also

are clinical problems in using non-steroidal anti-inflam-

matory drugs (NSAIDs) for control of inflammation,

because of side-effects, even though some NSAIDs are

known to have anti-inflammatory and anti-lithogenic

effects on the GB [24].

We herein demonstrate that in GBEC: PPARa and

PPARc exist, PPARs expression is increased after inflam-

matory stimulation with LPS, the ligands for PPARs

activate ABCA1 and suppress pro-inflammatory cytokines,

and the mechanism of ABCA1 activation by PPAR ligands

is the LXRa-mediated transcriptional pathway.

Materials and methods

Materials

Eagle’s minimum essential medium (EMEM), fetal bovine

serum (FBS), and penicillin/streptomycin were from Hy-

clone (South Logan, UT, USA). Trypsin/EDTA, non-

essential amino acid solution, vitamin solution, LPS, dime-

thyl sulfoxide (DMSO), and 3-[4,5-dimethylthiazol-2-

yl]diphenyl tetrazolium bromide (MTT) were from Sigma

Chemicals (St Louis, MO, USA). Vitrogen was purchased

from Inamed (Fremont, CA, USA). WY-14643, troglitaz-

one, and GW-9662 were purchased from Cayman Chemicals

(Ann Arbor, MI, USA). The rabbit polyclonal anti-human

PPARa antibody, the rabbit polyclonal anti-human PPARcantibody, and the mouse monoclonal anti-human ABCA1

antibody were from Abcam (Cambridge, UK), The peroxi-

dase-conjugated anti-rabbit IgG antibody was from

Amersham Biosciences (Buckinghamshire, UK), The per-

oxidase-conjugated anti-mouse IgG antibody was from

Pierce (Rockford, IL, USA). The Western blot detection kit

(Visualizer) was from Upstate (Lake Placid, NY, USA).

Cell culture

Epithelial cells were isolated from canine GB as described

previously [25]. Stock cultures were grown on 100 mm

dishes with 2 mL Vitrogen gel coating (1:1 mixture of

Vitrogen and medium) in EMEM supplemented with 10%

FBS, 2 mmol L–1 L-glutamine, 20 mmol L–1 Hepes,

100 IU mL–1 penicillin, and 100 lg mL–1 streptomycin.

Medium was changed twice a week, and the cells were

maintained at 37�C in an incubator with 5% CO2. Cells

were passaged when confluent (every 7–10 days) using

trypsin (2.5 g L–1) and EDTA (1.0 g L–1). For experi-

ments, the cells were grown on 60-mm dishes without

Vitrogen coating, and the medium was changed to serum-

free medium (SFM) containing 0.2% bovine serum albu-

min (BSA) (Sigma).

MTT (3-(4,5-dimethylthiazol-2-yl)-diphenyl

tetrazolium bromide) assay

GBEC were plated at a density of 5 · 104 cells mL–1 in 24-

well plates. Cells were cultured to 60% confluency in

serum-containing regular medium and then incubated with

1708 Dig Dis Sci (2008) 53:1707–1715

123

or without various concentrations of reagents (DMSO,

LPS, WY-14643, GW-9662, and troglitazone) in SFM for

24 h. MTT (0.5 mg mL–1) was then added to each well and

incubated for further 4 h at 37�C. After decanting of the

medium, 500 lL DMSO was added to each well. After

10 min of constant and gentle shaking, the color intensity

(proportional to the number of live cells) was assessed with

the ELX800 (Biotek, Winooski, VT, USA) at 570 nm

wavelength.

RNA extraction and reverse transcription-PCR analysis

GBEC were cultured to confluency on 60-mm dishes and

then treated as outlined below. Treatment groups for TNFamRNA were:

1 No-treatment: the cells were incubated in SFM con-

taining 0.2% BSA;

2 Treatment with LPS (200 lg mL–1) alone for 1 h in

SFM containing 0.2% BSA;

3 LPS (200 lg mL–1) treatment for 1 h following pre-

treatment with WY-14643 (100 lmol L–1) for 18 h in

SFM containing 0.2% BSA;

4 LPS (200 lg mL–1) treatment for 1 h following pre-

treatment with troglitazone (10 lmol L–1) for 18 h in

SFM containing 0.2% BSA;

5 Simultaneous treatment with LPS (200 lg mL–1) and

WY-14643 (100 lmol L–1) for 1 h in SFM containing

0.2% BSA; and

6 Simultaneous treatment with LPS (200 lg mL–1) and

troglitazone (10 lmol L–1) for 1 h in SFM containing

0.2% BSA.

For ABCA1 mRNA, the cells were incubated in SFM

containing 0.2% BSA with LPS (200 lg mL–1) or

WY14643 (100 lmol L–1), or troglitazone (10 lmol L–1)

for 24 h. The cells were harvested, and RNA was extracted

using Trizol reagent (Invitrogen, Carlsbad, CA, USA). RT-

PCR was performed with the RT-PCR system according to

the manufacturer’s instruction (Promega, Madison, WI,

USA). The sequences of primers were: canine ABCA1,

forward, 50-CAGCTTCGTTGTGTTCCTGA-30, reverse,

50-GAGCTAGGACAGGCAGGTTG-30; canine GAPDH,

forward, 50-ATCACTGCCACCCAGAAGAC-30, reverse,

50-GCCAGGTCAGATCCACAACT-30; canine LXRa,

forward, 50-TCAACCCCATCTTCGAGTTC-30, reverse,

50-TTGCTCTGAATGGACACTGC-30; canine TNFa pri-

mer set (the manufacturer did not disclose the sequence)

(Endogen, Rockford, IL, USA). The total reaction was

performed in 50-lL mixtures containing total RNA (1 lg

of each), 10 pmol of each primer, 5 U of AMV transcrip-

tase, 1.25 U of Taq DNA polymerase, 10 mmol L–1 dNTP,

PCR buffer (10X), and 2 mmol L–1 MgCl2. The RT

reaction was set at 45�C for 45 min and then at 95�C for

2 min. The PCR conditions were: denaturation at 95�C for

30 s, annealing at 55�C for 30 s for TNFa, and extension at

72�C for 1 min. The annealing temperature for ABCA1

and LXRa was adjusted to 54�C. The reaction was ended

with an additional extension at 72�C for 5 min, then chilled

to 4�C. TNFa mRNA was amplified during 35 cycles, and

ABCA1 mRNA was amplified during 30 cycles. The size

of the PCR products was 206 bp (ABCA1), 308 bp

(GAPDH), 266 bp (LXRa), and 597 bp (TNFa). The PCR

products were fractionated by electrophoresis on 2% aga-

rose gels containing ethidium bromide.

Western blot analysis

GBEC were cultured to confluency on 60 mm dishes with

regular media. Cells were treated with LPS (50, 100, 200,

400 lg mL–1) or WY-14643 (25, 100 lmol L–1) or trog-

litazone (5, 10 lmol L–1), or GW-9662 (10 lmol L–1) in

SFM containing 0.2% BSA for 24 h, as indicated. They

were then washed with PBS and harvested with lysis buffer

(50 mmol L–1 Tris pH 7.5, 150 mmol L–1 NaCl, 1 mmol

L–1 EDTA, 1% Tripton X-100, 1% sodium deoxycholate,

0.1% SDS, 1 lmol L–1 phenylmethylsulfonyl fluoride

(PMSF), 5 lg mL–1 aprotinin, 5 lg mL–1 leupeptin). Pro-

tein contents were analyzed by the Bradford assay (Sigma).

SDS-PAGE was performed with a 4% stacking gel and a

6% (for ABCA1) or 10% (for PPARa and PPARc)

resolving gel, followed by transfer to nitrocellulose mem-

brane (Bio-Rad, Hercules, CA, USA). The membranes

were blocked overnight at 4�C in blocking solution (5%

skim milk in Tris-buffer with Tween-20 [TBS-T]:

200 mmol L–1 Tris, 500 mmol L–1 NaCl, pH 7.5, 0.05% v/

v Tween-20), and then incubated with the mouse mono-

clonal anti-ABCA1 antibody or rabbit polyclonal anti-

PPARa antibody or rabbit polyclonal anti-PPARc antibody

for 1 h at room temperature. The membranes were washed

with TBS-T and incubated with the peroxidase conjugated

anti-mouse IgG or anti-rabbit IgG for 1 h at room tem-

perature. The membrane was washed and incubated with

Visualizer western blot detection kit for 5 min, and auto-

radiography was performed. The signal intensities for

specific bands on the Western blots were quantified using

NIH Image J density analysis software (Version 1.20).

Statistical analysis

Results from each experiment are expressed as the

means ± SD of duplicate cultures, and all results described

are representative of at least three separate experiments.

One-way analysis of variance (ANOVA) for three or more

Dig Dis Sci (2008) 53:1707–1715 1709

123

unpaired groups or Student’s t test for two unpaired groups

was used, and P \ 0.05 was considered significant. The

results are expressed as means ± SE, where the results of

multiple experiments are pooled.

Results

PPARa and PPARc exist in GBEC

Canine GBEC were cultured to confluency on 60-mm

dishes. After changing to SFM, the PPARa ligand (WY-

14643, 50 lmol L–1 or 100 lmol L–1) or the PPARc ligand

(troglitazone, 5 lmol L–1 or 10 lmol L–1) or the PPARaantagonist (GW-9662, 10 lmol L–1) were added to the

media for 24 h as indicated, and then Western blotting was

performed to observe the expressional change of PPARaand PPARc in GBEC in proportion to loading dose of

ligands compared to no-treatment control as described in

Materials and methods. Expression of both PPARa and

PPARc increased significantly in accordance with the

increase in the concentration of each loading ligand

(P \ 0.01 versus no-treatment control; P \ 0.05, versus

no-treatment control and the lower concentrations of

treatment with PPARa; Fig. 1a) (P \ 0.001 versus no-

treatment control; P \ 0.01, versus no-treatment control

and the lower concentrations of treatment with PPARc;

Fig. 1b). The basic expression of PPARa was relatively

more abundant than that of PPARc, however, the response

after stimulation with a ligand was much more prominent

for PPARc than for PPARa. These findings suggest that

PPARc is more inducible and may be a target of manipu-

lation in GBEC.

Another Western blotting was performed to see whether

the PPARa ligand and the PPARa antagonist have an effect

on PPARc expression in GBEC. The PPARa ligand also

significantly increased PPARc expression, though weaker

than the PPARc ligand, and the PPARa antagonist signif-

icantly suppressed PPARc expression (P \ 0.01 or

P \ 0.05 versus no-treatment control, P \ 0.01 versus no-

treatment control, GW-9662 and WY-14643 treatment

groups; Fig. 2). These findings could be explained by two

possibilities, that PPARa could share the properties and

mechanism of action of PPARc or that the ligands (WY-

14643 and GW-9662) are not very specific to the receptors,

though it is known that WY-14643 is the most specific

PPARa ligand. However, this remains to be clarified.

LPS induces PPARa and PPARc expression in GBEC

Canine GBEC were cultured to confluency on 60-mm

dishes. After changing to SFM, various concentrations of

LPS was added to the media for 24 h, as indicated, and

then Western blotting was performed to observe the

expressional change of PPARa and PPARc in GBEC in

RAPP

0

1

2

3

001YW05YWtnoC

Exp

ress

ion

RAPP

0

5

01

01RT5RTtnoC

Exp

ress

ion

(B)

(A)

*

†

**

††

Fig. 1 PPARa and PPARc exist in GBEC. (a) Western blot analysis

of PPARa protein in GBEC following treatment with PPARa ligand

(WY-14643, 50 lmol L–1 (WY 50) or 100 lmol L–1 (WY 100)) for

24 h. (b) Western blot analysis of PPARc protein in GBEC following

treatment with PPARc ligand (troglitazone, 5 lmol L–1 (TR 5) or

10 lmol L–1 (TR 10)) for 24 h. *P \ 0.01 versus no-treatment

control (Cont), **P \ 0.05 versus no-treatment control and 50 lmol

L–1 WY-14643 treatment. �P \ 0.001 versus no-treatment control, ��P\ 0.01 versus no-treatment control and 5 lmol L–1 troglitazone

treatment groups

RAPP

0

1

2

3

4

5

6

RTYWWGtnoC

Exp

ress

ion

**

*

†

Fig. 2 The PPARa ligand and antagonist have an effect on PPARcexpression in GBEC. Western blot analysis of PPARc protein in

GBEC was performed following treatment with 10 lmol L–1 GW-

9662 (PPARa antagonist, GW) or 100 lmol L–1 WY-14643 (WY), or

10 lmol L–1 troglitazone (TR) for 24 h. *P \ 0.01 or �P \ 0.05

versus no-treatment control, **P \ 0.01 versus no-treatment control

(Cont), GW-9662 and WY-14643 treatment groups

1710 Dig Dis Sci (2008) 53:1707–1715

123

proportion to the concentrations of LPS compared to no-

treatment control. Expression of both PPARa and PPARcwere significantly increased in accordance with the

increase in the concentrations of LPS (P \ 0.05 versus no-

treatment control, P \ 0.05 versus no-treatment control

and the lower concentrations of LPS-treatment groups;

Fig. 3a) (P \ 0.001 versus no-treatment control, P \ 0.01

versus no-treatment control and the lower concentrations of

LPS-treatment groups; Fig. 3b). In particular, PPARcexpression was much more remarkable than that of PPARa,

which suggests that PPARc is more strongly associated

with the modulation of inflammatory response.

PPARa and PPARc ligands suppress TNFa production

induced by LPS in GBEC

In order to evaluate the attenuation effect of PPARa and

PPARc ligands on inflammation, RT-PCR for TNFa, a

representative pro-inflammatory cytokine, was performed

and compared to no-treatment control (lane 1) as described

in Materials and methods. Before RT-PCR, Some cells

were treated with LPS alone for 1 h (lane 2), or LPS and

the PPARa ligand simultaneously for 1 h (lane 5), or LPS

and the PPARc ligand simultaneously for 1 h (lane 6), and

the other cells were pre-treated with PPARa (lane 3) or

PPARc ligands (lane 4) for 18 h followed by LPS loading

for 1 h. We observed that LPS-induced TNFa mRNA

production was almost completely repressed in the cells

pre-treated with PPARa and PPARc ligands preceding LPS

treatment. However, this suppressive action against TNFawas not prominent in the cells simultaneously treated with

LPS and PPARa or PPARc ligands (Fig. 4). We made the

conclusion that both the PPARa and PPARc ligands have a

powerful preventive effect on the inflammatory process.

PPARa and PPARc ligands activate ABCA1 by the

LXRa-mediated pathway in GBEC

RT-PCR and Western blotting were preformed to test the

capability of specific ligands and LPS to activate ABCA1

protein and mRNA as described in Materials and methods.

GBEC were pre-incubated for 24 h in the presence of LPS

(200 lg mL–1) or WY-14643 (100 lmol L–1), or troglit-

azone (10 lmol L–1) in SFM containing 0.2% BSA. We

found that LPS treatment and PPARa and PPARc ligands

significantly increased expression of ABCA1 protein and

mRNA as a similar pattern in both the RT-PCR (Fig. 5a)

and Western blotting (P \ 0.001 versus no-treatment

control, P \ 0.01 versus no-treatment control and WY-

14643 treatment; Fig. 5), and PPARc ligand had an more

powerful effect than PPARa ligand on ABCA1 induction.

Another RT-PCR for LXRa was performed to demon-

strate the transcriptional mechanism of activation of

0123

004SPL001SPLtnoC

Exp

ress

ion

RAPP

RAPP

0

2

4

6

8

01

002SPL05SPLtnoC

Exp

ress

ion

(B)

(A)

* **

†

††

Fig. 3 LPS induces PPARa and PPARc expression in GBEC.

Western blot analysis of PPARa (a) and PPARc (b) protein in GBEC

were performed following treatment with various concentrations of

LPS for 24 h. *P \ 0.05 versus no-treatment control (Cont),**P \ 0.05 versus no-treatment control and 100 lg mL–1 LPS (LPS100) treatment groups. �P \ 0.001 versus no-treatment control, ��

P \ 0.01 versus no-treatment control and 50 lg mL–1 LPS treatment

groups

FNT

HDPAG

21CM 6543

Fig. 4 PPARa and PPARc ligands suppress TNFa production

induced by LPS in GBEC. RT-PCR of TNFa mRNA was performed

as follows: M, DNA Marker; C, positive control provided by

manufacturer; 1, no-treatment control; 2, LPS (200 lg mL–1) alone

treatment for 1 h; 3, LPS (200 lg mL–1) treatment for 1 h following

pre-treatment with WY-14643 (100 lmol L–1) for 18 h; 4, LPS

(200 lg mL–1) treatment for 1 h following pre-treatment with

troglitazone (10 lmol L–1) for 18 h; 5, treatment with LPS (200 lg

mL–1) and WY-14643 (100 lmol L–1) simultaneously for 1 h; 6,

treatment with LPS (200 lg mL–1) and troglitazone (10 lmol L–1)

simultaneously for 1 h

Dig Dis Sci (2008) 53:1707–1715 1711

123

ABCA1 in the presence of LPS, PPARa, and PPARcligands in GBEC. LXRa mRNA was increased following

treatment with LPS (200 lg mL–1) or WY-14643

(100 lmol L–1), or troglitazone (10 lmol L–1), or 22-(R)-

hydroxycholesterol (positive control) in SFM containing

0.2% BSA compared to vehicle alone (Fig. 5c). We dem-

onstrated in GBEC that activated PPARs induced LXRaactivation, resulting in ABCA1 activation as observed in

macrophages.

Discussion

The GB epithelium absorbs cholesterol through both pas-

sive and active mechanisms [26]. Studies in vitro have

shown that absorption by the GB epithelium alters cho-

lesterol solubility in bile [6]. However, the fate of the

cholesterol absorbed by GBEC was not clear. Recently, we

have documented that cholesterol absorbed by GBEC is

eliminated by the cAMP-dependent ABCA1-LXRa/RXR-

mediated basolateral reverse cholesterol efflux system, and

GBEC synthesize apolipoprotein A-I and E as acceptors for

basolaterally effluxed cholesterol. We concluded that this

reverse cholesterol system in GBEC may alter cholesterol

concentrations in bile and allows the GB to unload

excessive amounts of absorbed cholesterol [10, 11]. Con-

sidering the fact that prolonged deposition of cholesterol

ester in vascular endothelium causes chronic inflammation

and loss of elasticity, which consequently induces athero-

sclerosis [27], the ABCA1-LXRa/RXR-mediated

cholesterol efflux system in GBEC could be a key defense

mechanism to prevent chronic inflammation and loss of

motility in the GB. This theory was supported by another

study of ours in which ABCA1 expression in GB epithe-

lium is increased in patients with cholesterol polyp and

cholesterol stones compared to normal GB [28]. These

findings in vivo showed the possibility that ABCA1 in

human GBEC also play a role in the control of excessively

loaded cholesterol.

Although many researchers have made an effort to find

effective and safe drugs that activate ABCA1 or LXR,

because ABCA1 is a target of new therapeutic drug for

atherosclerosis or cholesterol control [29, 30], the results

were not satisfactory. Most recently, the good news that

PPARs activate ABCA1 mediated by their stimulatory

action on LXRa expression and activity in macrophages

was reported, and was also proved in various other cells. It

is known that ABCA1 mRNA and promoter transcription

are induced by oxysterols acting via LXRa [31, 32]. In

addition, unsaturated fatty acid and synthetic PPARaligands such as WY-14643 induce LXRa in cultured

hepatocytes and in vivo in liver [31, 32]. PPARa ligands

such as fenofibrate and clofibrate are being prescribed as

therapeutic agents for hypertriglyceridemia [33], and

PPARc ligands such as rosiglitazone and pioglitazone are

being prescribed as antidiabetic agents with clinical safety

[34]. Therefore, we think that PPAR ligands can be used as

ABCA1 activators in various clinical fields. We demon-

strated that PPARa and PPARc ligands also induced the

expression of ABCA1 gene and protein in GBEC, which

was regulated by LXRa, and that PPARc ligands had a

more powerful effect on ABCA1 induction. These results

mean that PPAR ligands, especially PPARc, can contribute

to prevention of excessive cholesterol accumulation in

GBEC, keeping the GBEC healthy.

In this study we found that LPS-induced inflammation

increased expression of ABCA1 in GBEC, which was

contrary to results that LPS suppressed ABCA1 expression

in macrophages or renal tubular cells [35–37]. Those

results suggested that LPS could block the transcriptional

pathway of the ABCA1 gene. However, other studies have

demonstrated that LPS induced expression of ABCA1 in

macrophages and monocytes, and suggested that ABCA1

activation by LPS contributed to attenuation of the

inflammatory process [38, 39]. Possible mechanisms of

LPS induction of ABCA1 activation in GBEC are:

1ACBA

HDPAG

SPLtnoC RTYW

1ACBA

0246801

RTYWSPLtnoC

Exp

ress

sion

YWSPLtnoC C-HORT

HDPAG

RXL(C)

(B)

(A)

**

*, **

Fig. 5 LPS, PPARa, and PPARc ligands activate ABCA1 by the

LXRa-mediated pathway in GBEC. Western blot analysis of ABCA1

protein (a) and RT-PCR of ABCA1 mRNA (b) were performed

following treatment with LPS (200 lg mL–1) or WY-14643

(100 lmol L–1, WY), or troglitazone (10 lmol L–1, TR) for 24 h. (c)

Another RT-PCR for LXRa was performed to demonstrate the

transcriptional mechanism of activation of ABCA1 in the presence of

LPS (200 lg mL–1) or WY-14643 (100 lmol L–1) or troglitazone

(10 lmol L–1), or 22-(R)-hydroxycholesterol (LXRa ligand, 10 lmol

L–1, OH-C) for 24 h. *P \ 0.001 versus no-treatment control (Cont),**P \ 0.01 versus no-treatment control and WY-14643 treatment

1712 Dig Dis Sci (2008) 53:1707–1715

123

1 LPS activates PPARs that block the inflammatory

process, consequently inducing expression of LXRawhich is a regulator of ABCA1 [16–18];

2 LPS-induced inflammation can oxidize cholesterol in

GBEC to form oxysterol which is a potent LXRaligand, resulting in activation of LXRa [40]; and

3 GBEC are naturally sufficiently tolerant to maintain

transcriptional activity in and inflammatory

environment.

According to our MTT results for LPS, more than 80% of

GBEC survived the very high concentration of LPS

(800 lg mL–1 for 24 h). The reason why ABCA1 in GBEC

is activated by LPS may be another protective mechanism to

remove oxidized cholesterol which can be toxic to cells [40].

It has recently been shown that PPARs have an impor-

tant role in the control of various types of inflammatory

response. These functions are mediated largely through the

abilities of the PPARa and PPARc isoforms to repress the

activities of many activated transcription factors including

nuclear factor-kB (NF-kB), signal transducers and activa-

tors of transcription (STATs), activator protein 1 (AP1),

and nuclear factor of activated T-cells (NFAT), causing

suppression of the production of various pro-inflammatory

cytokines such as TNFa, IL-1b, and IL-6 [16, 17, 41, 42].

Furthermore, PPARs also suppress the activation of COX-

2, prostaglandin, and iNOS, which are strongly associated

with inflammatory reaction and cell proliferation. These

actions of PPARs are regarded a beneficial in the preven-

tion of inflammation and the progression of atherosclerosis

by maintaining vascular endothelial elasticity [15]. In

GBEC, only one study has demonstrated indirect evidence

that PPARc ligand suppresses the activity of IL-6 under the

conditions of IL-1b treatment compared to the control

group [22]. In the current study, LPS treatment on GBEC

evoked PPARa and PPARc expression, which was perhaps

intended to modulate the inflammatory process. This

response was observed more prominently in PPARc, which

means PPARc has a more important role in modulating

inflammation than PPARa. In practice, TNFa mRNA

production was totally blocked in the GBEC pre-treated

with PPARa and PPARc ligands before LPS loading.

However, this phenomenon was not observed in cells

treated simultaneously with LPS and PPARc or PPARaligands. We think that once the inflammatory reaction

begins to progress, PPAR ligands are no longer effective in

controlling inflammation, which suggests that prevention is

more important in preserving GB function in patients at

high risk from GB stones, such as those with diabetes,

severe obesity, hypercholesterolemia, etc [43].

Contrary to our expectation, long-term practical use of

fibrates, PPAR-alpha ligands, have been reported to

increase the risk of cholesterol gallstone formation [44]. In

hepatocytes, fibrates paradoxically inhibit 7-alpha-

hydroxylase (Cyp7A1), a key enzyme in the synthesis of

primary bile acid, in spite of LXRa activation [45, 46], and

suppress acyl-CoA:cholesterol acyltransferase (ACAT)

that involves the esterification of free cholesterol [47], and

increase LXR-mediated ABCG5 and ABCG8 activity that

facilitates cholesterol efflux into bile [45, 48]. A series of

these phenomena could involve supersaturation of bile,

contributing to gallstone formation although fibrates have

evidently affirmative actions to preserve GB function. This

fact means that fibrates have some limitations in practical

application to prevention of gallstone formation. Fortu-

nately, there have been no reports of risk of gallstone

formation associated with rosiglitazone, a representative

PPARc agonist. On the contrary, it has been reported that

rosiglitazone blocks repression of CyP7A1 in HepG2 cells;

failure of the generation of endogenous PPARc agonists

leads to cholesterol supersaturation and gallstone forma-

tion, and decreased hepatic expression of PPARccoactivator-1 increases the cholesterol gallstone formation

[49, 50]. These facts, along with our data that PPARc is

more strongly associated with ABCA1 expression and

inflammatory reaction, suggest that PPARc ligands can be

more effective and safer drugs than PPARa ligands in the

prevention of gallstone formation and progression to

cholecystitis.

In this study, we demonstrated that PPAR ligands,

especially PPARc, can preserve GB function by suppres-

sion of inflammatory reaction and the prevention of

cholesterol accumulation in GBEC, by LXRa-mediated

ABCA1 activation, resulting in a contribution to the pre-

vention of gallstone formation and progression to

cholecystitis. Our studies were performed on cultured

canine GBEC, a model system that has provided insights

into several areas of GB cell physiology [25–27]. Future

investigations with an animal model will no doubt reveal

the actual effects of PPAR ligands in vivo on the preven-

tion of gallstone formation and attenuation of inflammation

in the GB.

Acknowledgment This study was supported by the 2006 Clinical

Research Fund of Hallym Medical Center, Seoul. Korea. We thank

Rahul Kuver and Sum P. Lee (Division of Gastroenterology, Uni-

versity of Washington School of Medicine, Seattle, USA) for the kind

gift of the GBEC.

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