Tài liệu Báo cáo khoa học: Liver X receptor agonists inhibit tissue factor expression in macrophages docx

Liver X receptor agonists inhibit tissue factor expression
in macrophages
Naoki Terasaka
1
, Ayano Hiroshima
1
, Akiko Ariga
1
, Shoko Honzumi
1
, Tadashi Koieyama
1
,
Toshimori Inaba
2
and Toshihiko Fujiwara
1
1 Pharmacology and Molecular Biology Research Laboratories, Sankyo Co. Ltd, Tokyo, Japan
2 Pharmacodynamics Research Laboratories, Sankyo Co. Ltd, Tokyo, Japan
Tissue factor (TF) is the cell surface glycoprotein that
functions as the major cellular initiator of the coagula-
tion protease cascades [1–4]. It is a high-affinity recep-
tor for serine protease factors VII and VIIa. The
resulting TF–factor VIIa complex provides the first
catalytic event which is responsible for initiation of the
coagulation protease cascades. TF-initiated thrombosis
is associated with many diseases, including Gram-
negative sepsis, cancer, and atherosclerosis [5–8].
Atherosclerosis is a chronic inflammatory disease
as well as a disorder of lipid metabolism [9–11]. As
modulators of both lipid metabolism and immune

responses, macrophages play a central role in the ath-
erogenic process. The accumulation of cholesterol-
loaded macrophages in the arterial wall is the hallmark
of early atherosclerotic lesions. TF plays an important
role in the pathogenesis of thrombus formation at sites
of atherosclerotic plaque disruption resulting in acute
Keywords
atherosclerosis; genes; lipopolysaccharide;
liver X receptor; macrophage; tissue factor
Correspondence
N. Terasaka, Pharmacology and Molecular
Biology Research Laboratories, Sankyo Co.,
Ltd, 1-2-58 Hiromachi, Shinagawa-ku, Tokyo
140-8710, Japan
Fax: +81 3 5436 8566
Tel: +81 3 3492 3131
E-mail:
(Received 5 November 2004, revised 17
January 2005, accepted 4 February 2005)
doi:10.1111/j.1742-4658.2005.04599.x
Exposure of blood to tissue factor (TF) rapidly initiates the coagulation
serine protease cascades. TF is expressed by macrophages and other types
of cell within atherosclerotic lesions and plays an important role in throm-
bus formation after plaque rupture. Macrophage TF expression is induced
by pro-inflammatory stimuli including lipopolysaccharide (LPS), inter-
leukin-1b and tumor necrosis factor-a. Here we demonstrate that activation
of liver X receptors (LXRs) LXRa and LXRb suppresses TF expression.
Treatment of mouse peritoneal macrophages with synthetic LXR agonist
T0901317 or GW3965 reduced TF expression induced by pro-inflammatory
stimuli. LXR agonists also suppressed TF expression and its activity

in human monocytes. Human and mouse TF promoters contain binding
sites for the transcription factors AP-1, NFjB, Egr-1 and Sp1, but no
LXR-binding sites could be found. Cotransfection assays with LXR and
TF promoter constructs in RAW 264.7 cells revealed that LXR agonists
suppressed LPS-induced TF promoter activity. Analysis of TF promoter
also showed that inhibition of TF promoter activity by LXR was at least
in part through inhibition of the NFjB signaling pathway. In addition,
in vivo, LXR agonists reduced TF expression within aortic lesions in an
atherosclerosis mouse model as well as in kidney and lung in mice stimula-
ted with LPS. These findings indicate that activation of LXR results in
reduction of TF expression, which may influence atherothrombosis in
patients with vascular disease.
Abbreviations
ABC, ATP-binding cassette; apoE, apolipoprotein E; COX, cyclo-oxygenase; DMEM, Dulbecco’s modified Eagle’s medium; Egr-1, early
growth response-1; IL, interleukin; iNOS, inducible nitric oxide synthase; KLF, Kru
¨
ppel-like factor; LDLR, low-density lipoprotein receptor;
LPS, lipopolysaccharide; LPDS, lipoprotein protein-deficient serum; LXR, liver X receptor; MMP, matrix metalloproteinase; NFjB, nuclear
factor-jB; PPAR, peroxisome proliferator-activated receptor; RAR, retinoic acid receptor; RXR, retinoid X receptor; TBST, Tween 20 Tris
buffered saline; TF, tissue factor; TNFa, tumor necrosis factor-a.
1546 FEBS Journal 272 (2005) 1546–1556 ª 2005 FEBS
coronary events [5,12]. Physical disruption of the pla-
que promoted by macrophage-derived proteases such
as matrix metalloproteinases (MMPs) permits access of
blood coagulation proteins to TF in the lipid-rich core
[13,14].
Unlike other cofactors of the coagulation protease
cascades, which circulate as nonfunctional precursors,
TF is a potent initiator that is fully functional when
expressed on the cell surface. Therefore, transcriptional

regulation of TF would appear to be a crucial step in
the control of protease cascades. TF expression and
activation is increased by a variety of stimuli, such as
lipopolysaccharide (LPS), oxidized low-density lipo-
protein, shear stress, tumor necrosis factor-a (TNFa),
interleukin (IL)-1b, and CD40 ligand [14,15]. The tran-
scriptional regulation of the TF gene varies depending
on the cell type and stimulus. Functional analysis of
the TF promoter has identified putative AP-1, nuclear
factor-jB (NFjB), Sp1 and early growth response
(Egr)-1 binding sites [16].
Liver X receptors (LXRs), LXRa and LXRb, are
members of the nuclear receptor superfamily and are
involved in regulation of cholesterol and lipid metabo-
lism [17,18]. LXRs bind to DNA as obligate hetero-
dimers with retinoid X receptors (RXRs). Uptake of
oxidized low-density lipoprotein by macrophages leads
to increased cellular concentration of oxysterols, the
natural ligands for LXRs. LXRs directly regulate
the expression of ATP-binding cassette transporters
ABCA1 and ABCG1, and apolipoprotein E (apoE),
which mediate cellular cholesterol efflux in the pres-
ence of acceptors such as high-density lipoprotein
(HDL). In addition, recent studies suggest that LXRs
may also inhibit inflammatory responses [19,20]. LXR
agonists can suppress induction of inducible nitric
oxide synthase (iNOS), cyclo-oxygenase-2 (COX-2)
and MMP-9. In this study, we investigated whether
LXR activation inhibits inducible TF expression and
activity in macrophages.

Results
LXR agonists inhibit TF expression induced
by inflammatory stimuli in mouse peritoneal
macrophages
The effect of LXR activation on LPS-induced TF
mRNA concentrations in mouse peritoneal macro-
phages was determined by real-time quantitative PCR
assays. Macrophages were pretreated with 1 lm LXR
agonist T0901317 for 18 h and then stimulated with
LPS (100 ngÆmL
)1
). In a time course experiment,
macrophages stimulated with LPS exhibited a fivefold
induction of TF mRNA, which was maximal at 2–6 h
and reduced to low levels by 24 h (Fig. 1A). Preincu-
bation of macrophages with T0901317 resulted in a
reduction in TF mRNA concentrations (Fig. 1A).
Agonists for peroxisome proliferator-activated recep-
tor (PPAR)a (Wy14643), PPARc (rosiglitazone),
PPARoad (GW501516) and farnesoid X receptor
(GW4064) had minimal effects on TF expression at a
concentration of 1 lm (Fig. 1B). The inhibitory effect
of T0901317 on TF expression was dose-dependent
over the concentration range 0.01–1 lm (Fig. 1C).
LXR agonist GW3965, which has a different chemical
structure from T0901317, also inhibited TF expression
in a dose-dependent manner (Fig. 1C). We further
examined the effect of LXR agonists on induction
of TF mRNA by TNFa (20 ngÆmL
)1

) and IL-1b
(20 ngÆmL
)1
). Pretreatment with T0901317 also
reduced induction of TF mRNA by the stimuli in
macrophages (Fig. 1D).
LXR agonists inhibit LPS-induced expression of
TNFa, but not IL-1b or IL-6, in mouse peritoneal
macrophages
To investigate whether LXR agonists alter LPS-
induced inflammatory cytokine secretion in mouse
peritoneal macrophages, we examined the effects of
T0901317 and GW3965 on IL-1b, IL-6 and TNFa
protein secretion after LPS stimulation. Pretreatment
of macrophages for 18 h with T0901317 significantly
reduced LPS-induced TNFa protein secretion in a
dose-dependent manner (Fig. 2A), whereas neither
LPS-induced IL-1b nor IL-6 protein secretion was
affected (Fig. 2B,C). LPS-induced TNFa mRNA con-
centrations were also reduced by T0901317 (Fig. 2D).
LXR agonists inhibit LPS-induced TF expression
and activity in human monocytes
To determine whether LXR agonists had similar
effects on TF expression in human cells, human mono-
cytes were used for LPS stimulation experiments. The
data show that induction of TF mRNA was greater
in human monocytes than in mouse macrophages
(Fig. 3A). Pretreatment of human monocytes for 18 h
with T0901317 or GW3965 significantly reduced LPS-
induced TF activity in a dose-dependent manner in the

concentration range 0.01–1 lm (Fig. 3B). The effect on
inhibition of TF expression was greater than that
observed in mouse macrophages. LPS-induced TF
activity in human monocytes was also inhibited by pre-
treatment with T0901317 or GW3965 and correlated
with TF mRNA concentrations (Fig. 3B). In addition,
N. Terasaka etal. Repression of tissue factor in macrophages by LXR
FEBS Journal 272 (2005) 1546–1556 ª 2005 FEBS 1547
T0901317 reduced TF protein expression, as revealed
by western blot analysis (Fig. 3C).
LXR agonists inhibit LPS-induced TF promoter
activity
The results described above suggest that LXR agonists
inhibit transcription of the TF gene. However,
sequence analysis of the 5¢-flanking region of the TF
gene did not reveal the presence of potential LXR
response elements. The TF promoter contains binding
sites for AP-1, NFjB, Egr-1 and Sp1 [14]. These bind-
ing sites are highly conserved in human, porcine and
mouse TF genes. Then, we examined the possibility
that LXR agonists antagonize the signaling pathways
that induce TF expression. We investigated the effects
of LXRs on a luciferase reporter containing the human
TF gene promoter ()278 bp to 121 bp) (Fig. 4A). The
TF promoter was transiently transfected into RAW
264.7 macrophages along with expression plasmids for
LXRa and RXRa [21]. After transfection, cells were
treated with LPS and ⁄ or T0901317. TF promoter
activity was increased about 30-fold in response to
LPS (Fig. 4A). Pretreatment with T0901317 resulted in

a significant reduction in luciferase activity induced
by LPS when LXRa ⁄ RXRa expression plasmids were
cotransfected (Fig. 4A). Inhibition of TF promoter
activation induced by LPS was also observed when
Fig. 1. LXR agonists inhibit TF expression induced by inflammatory stimuli in mouse peritoneal macrophages. Thioglycolate-elicited peritoneal
macrophages were obtained from C57Bl ⁄ 6 J mice. For each condition, data are represented as mean ± SEM (n ¼ 4). (A) Mouse peritoneal
macrophages were pretreated with 1% Me
2
SO or 1 lM LXR agonist T0901317 for 18 h and then stimulated with LPS (100 ngÆmL
)1
) for 0.5,
1, 2, 4, 6, 8 and 24 h. TF mRNA concentrations were determined by real-time quantitative PCR assay. (B) Mouse peritoneal macrophages
were pretreated with 1% Me
2
SO or 1 lM T0901317 (LXR), Wy14643 (PPARa), rosiglitazone (PPARc), GW501516 (PPARoad) or GW-3965
(farnesoid X receptor) for 18 h and then stimulated with LPS (100 ngÆmL
)1
) for 6 h. TF mRNA concentrations were determined by real-time
quantitative PCR assay. (C) Mouse peritoneal macrophages were pretreated with 1% Me
2
SO or the indicated concentrations (lM)of
T0901317 or GW-3965 for 18 h and then stimulated with LPS (100 ngÆmL
)1
) for 6 h. TF mRNA concentrations were determined by real-time
quantitative PCR assay. (D) Mouse peritoneal macrophages were pretreated with 1% Me
2
SO or the indicated concentrations (lM)of
T0901317 for 18 h and then stimulated with TNFa (20 ngÆmL
)1
) or IL-1b (20 ngÆmL

)1
) for 6 h. TF mRNA concentrations were determined by
real-time quantitative PCR assay. *P<0.05, as compared with the vehicle control group using Dunnett’s multiple comparison test.
Repression of tissue factor in macrophages by LXR N. Terasaka etal.
1548 FEBS Journal 272 (2005) 1546–1556 ª 2005 FEBS
LXRb ⁄ RXRa expression plasmids were cotransfected
instead of LXRa ⁄ RXRa (Fig. 4A). The extent of inhi-
bition of TF promoter activity by LXRb ⁄ RXRa
cotransfection was equivalent to that by LXRa ⁄ RXRa.
Next, transient reporter assays were performed using
various TF gene promoter constructs (Fig. 4B). Serial
deletions revealed that the T0901317-mediated inhibi-
tion of luciferase activities induced by LPS was well
maintained when the constructs contained the reg-
ion )228 ⁄ )188, which contains AP-1-binding sites
(Fig. 4B). Deletion of the region )188 ⁄ )181, which
contains the NFjB-binding site, decreased T0901317-
mediated inhibition of luciferase activities (Fig. 4B).
To determine which binding site is important for
LXR-dependent repression of TF, transient reporter
assays were also performed using pGL3 ⁄ 3 · hTF-
dAP1-TK-Luc or pGL3 ⁄ 3 · hTFjB-TK-Luc (Fig. 4C).
Luciferase activity of these reporter plasmids was aug-
mented by LPS stimulation (Fig. 4C). Activation of
pGL3 ⁄ 3 · hTFjB-TK-Luc induced by LPS was sig-
nificantly inhibited by T0901317 when LXRa was
overexpressed. On the other hand, T0901317 did not
affect activation of pGL3 ⁄ 3 · hTFdAP1-TK-Luc by
LPS. These results indicate that the ability of LXRs to
inhibit the TF promoter requires the NFjB-binding

site.
To further investigate the mechanism of inhibition
of the NFjB pathway by LXR agonists, we examined
DNA-binding activity of NFjB. Mouse peritoneal
macrophages were preincubated with 1 lm T-0901317
Fig. 2. LXR agonists inhibit LPS-induced
expression of TNFa, but not IL-1b or IL-6, in
mouse peritoneal macrophages. Thioglyco-
late-elicited peritoneal macrophages were
obtained from C57Bl ⁄ 6J mice. For each
condition, data are represented as mean ±
SEM (n ¼ 3). Mouse peritoneal macropha-
ges were pretreated with 1% Me
2
SO or the
indicated concentrations (l
M) of LXR agon-
ists T0901317 or GW3965 for 18 h and then
stimulated with LPS (100 ngÆmL
)1
)for6h.
(A) IL-1a, (B) IL-6 and (C) TNFa protein con-
centrations in culture medium were deter-
mined as described in Experimental
procedures. (D) TNFa mRNA concentrations
were determined by real-time quantitative
PCR assay. *P<0.05, as compared with
the vehicle control group using Dunnett’s
multiple comparison test.
AB

C
Fig. 3. LXR agonists inhibit LPS-induced TF
expression and activity in human mono-
cytes. Human monocytes were pretreated
with 1% Me
2
SO or the indicated concentra-
tions (l
M) of T0901317 or GW-3965 for 18 h
and then stimulated with LPS (100 ngÆmL
)1
)
for 6 h. For each condition, data are repre-
sented as mean ± SEM. (n ¼ 3). (A) TF
mRNA concentrations were determined by
real-time quantitative PCR assay. (B) TF
activity was determined using a standard
chromogenic assay. (C) TF protein expres-
sion was analyzed by western blotting.
*P<0.05, as compared with the vehicle
control group using Dunnett’s multiple
comparison test.
N. Terasaka etal. Repression of tissue factor in macrophages by LXR
FEBS Journal 272 (2005) 1546–1556 ª 2005 FEBS 1549
or GW3965 and then activated with LPS (100
ngÆmL
)1
). After 6 h of LPS stimulation, nuclear
extracts were collected and then DNA-binding activity
of NFjB p50 and p65 was assessed. There were no sig-

nificant changes exhibited in the protein–DNA com-
plexes induced by LPS in the presence of LXR
agonists (Fig. 5).
LXR agonists inhibit TF expression induced
by LPS stimulation
To address whether the LXR pathway also functions
to modulate TF gene expression in vivo, we first inves-
tigated the effect of administration of LXR agonists
on induction of TF expression by LPS stimulation in
C57Bl ⁄ 6 mice. LPS at 4 mgÆkg
)1
induced TF mRNA
3.5-fold in kidney and 2.2-fold in lung 6 h after injec-
tion (Fig. 6). Administration of T0901317 at 3 mgÆkg
)1
or GW3965 at 30 mgÆkg
)1
significantly reduced induc-
tion of TF mRNA by LPS, but did not affect baseline
expression (Fig. 6).
LXR agonists inhibit TF expression in
atherosclerotic lesions
We previously observed that T0901317 augmented
ABCA1 expression in atherosclerotic lesions and resul-
ted in the prevention of lesion progression in LDLR
– ⁄ –
mice [22]. Next, we investigated the effect of T0901317
on expression of TF mRNA within the atherosclerotic
lesion in LDLR
– ⁄ –

mice. T0901317 significantly redu-
ced TF mRNA concentrations in the atherosclerotic
lesion in a dose-dependent manner (Fig. 7A). On the
other hand, ABCA1 mRNA concentrations in the
atherosclerotic lesion were increased by T0901317
(Fig. 7B), consistent with previous work [22], evaluated
by immunohistochemical analysis.
Discussion
LXRs are members of the nuclear receptor superfamily
and are highly expressed in macrophages. They play a
crucial role in the cholesterol efflux pathway through
Fig. 4. LXR agonists inhibit LPS-induced TF promoter activity. For each condition, data are represented as mean ± SEM. (n ¼ 4). (A) RAW
264.7 cells were transiently transfected with TF promoter construct (pGL3 ⁄ )278+121hTF-Luc) with or without pCMX, pCMX-LXRa, pCMX-
LXRb, pCMX-RXRa and pRL-CMV as described in Experimental procedures. After transfection, RAW 264.7 cells were incubated with 1%
Me
2
SO or 1 lM T-0901317 for 12 h in DMEM containing 10% LPDS. Cells were then stimulated with 100 ngÆmL
)1
LPS for 18 h. (B) RAW
264.7 cells were transiently transfected with TF promoter constructs (pGL3 ⁄ )228+121hTF-Luc, pGL3 ⁄ )211+121hTF-Luc, pGL3 ⁄
)188+121hTF-Luc and pGL3 ⁄ )181+121hTF-Luc) with or without pCMX-LXRa, pCMX-RXRa and pRL-CMV, respectively, as described in
Experimental procedures. (C) RAW 264.7 cells were transiently transfected with promoter reporter constructs (pGL3 ⁄ 3 · hTFdAP1-TK-Luc or
pGL3 ⁄ 3 · hTFjB-TK-Luc) with or without pCMX-LXRa, pCMX-RXRa and pRL-CMV, respectively, as described in Experimental procedures.
Luciferase activity was normalized to Renilla luciferase activities. *P<0.05, as compared with the vehicle control group using Dunnett’s
multiple comparison test.
Repression of tissue factor in macrophages by LXR N. Terasaka etal.
1550 FEBS Journal 272 (2005) 1546–1556 ª 2005 FEBS
the regulation of target genes, including ABCA1,
ABCG1 and apoE [23–26]. Recently we [22] and
Joseph et al. [27] demonstrated that synthetic LXR

agonists inhibit development of atherosclerotic lesion
area in LDLR
– ⁄ –
and apoE
– ⁄ –
mice without major
changes in plasma lipid concentrations. In addition,
Tangirala et al. [28] reported that LXRs in macro-
phages play a protective role in the development of
atherosclerosis in a bone marrow transplantation study
using LXR-deficient mice. In this study, we focused on
the molecular mechanism of the anti-atherosclerotic
effect of LXR agonists. We identified TF as a novel
LXR target gene in macrophages. We also demonstra-
ted that activation of LXRs in macrophages inhibits
expression of the TF gene in atherosclerotic lesions in
mice.
TF is abundantly expressed in macrophages and the
lipid-rich core in atherosclerotic plaques [5,29]. In
chronic atherosclerosis, macrophages appear to be the
major source of TF within the plaque. Macrophages
accumulate lipid, becoming foam cells, and finally
degenerating into a lipid core. Plaque rupture exposes
active TF in the lipid core to circulating blood, trigger-
ing thrombosis. As the initiator of coagulation, TF is
a potential target for inhibiting the thrombotic compli-
cations of atherosclerosis. Another study revealed that
deficiency of TFPI, an intrinsic TF inhibitor, reduced
atherosclerosis and thrombosis in apoE
– ⁄ –

mice [30]. In
addition, the absence of Egr-1, which is an important
regulator of TF expression, ameliorated progression of
atherosclerosis in apoE
– ⁄ –
mice [31]. These observa-
tions support the notion that TF has a crucial role in
the pathogenesis of atherosclerosis. However, the avail-
ability of synthetic TF inhibitors as therapeutic agents
has still not been reported. Our current data indicate
that LXR agonists would be useful as suppressants
of TF as well as activators of reverse cholesterol
transport.
Macrophages and foam cells secrete a number of
inflammatory mediators that increase inflammation in
the vessel wall and contribute to additional leukocyte
accumulation and smooth muscle cell proliferation.
Fig. 5. LXR agonists do not affect NFjB binding to DNA induced by
LPS. Thioglycolate-elicited peritoneal macrophages were obtained
from C57Bl ⁄ 6J mice. For each condition, data are represented as
mean ± SEM. (n ¼ 3). Mouse peritoneal macrophages were pre-
treated with 1% Me
2
SO or 1 lM T0901317 or GW-3965 for 18 h
and then stimulated with LPS (100 ngÆmL
)1
) for 6 h. Nuclear
extracts were prepared from peritoneal macrophages using a com-
mercial kit. DNA-binding activity of NFjB p50 (A) and p65 (B) was
assessed as described in Experimental procedures.

Fig. 6. LXR agonists inhibit TF expression induced by LPS stimulation. LXR agonist T-0901317 at 3 mgÆkg
)1
or GW-3965 at 30 mgÆkg
)1
was
orally administered daily to male C57Bl ⁄ 6J mice for 7 days (n ¼ 5 per group). The day after the last administration of LXR agonists, LPS at
4mgÆkg
)1
was intraperitoneally administered to C57Bl ⁄ 6 mice, and then mice were killed 6 h later. TF mRNA concentrations in kidney and
lung were determined by real-time quantitative PCR assay.
N. Terasaka etal. Repression of tissue factor in macrophages by LXR
FEBS Journal 272 (2005) 1546–1556 ª 2005 FEBS 1551
Thus, inflammation plays a central role in the incep-
tion and progression of atherosclerosis as well as accu-
mulation of lipid. Recently, LXR agonists have been
reported to repress expression of inflammatory genes
such as iNOS, COX-2 and MMP-9 in macrophages
[19,20]. In this study, we also found that LXR agonists
inhibit LPS-induced expression of TNFa in macro-
phages. Our observation also supports the notion that
LXRs play a role in the regulation of the macrophage
inflammatory response.
Activated NFjB can be detected in atherosclerotic
lesions, mainly within macrophages, whereas poor
NFjB activation is present in healthy vessels [32]. LXR
agonists appear to directly downregulate the enhanced
expression of inflammatory genes including TF during
the development of atherosclerosis induced by the acti-
vation of NFjB. The current data suggest that LXRs
allow binding of NFjB to the promoter, but prevent

functional activation of NFjB. Several models might
explain the inhibitory effect of LXR agonists on func-
tional NFjB activity. It is known that transcriptional
activation by nuclear receptors involves at least two
separate processes: derepression and activation [33].
Ligand binding triggers dissociation of corepressors
and recruitment of coactivators. Coactivators bridge
transcription factors including not only nuclear recep-
tors, but also CERB, STATs, bHLH factors, AP-1,
NFjB and the components of basal transcriptional
machinery. One possible explanation for the ligand-
dependent transcriptional repression is that LXRs com-
pete with NFjB for limited amounts of coactivators.
Indeed, LXRs and NFjB appear to recruit the same
coactivators, such as steroid receptor coactivator-1
(SRC-1) and activating signaling cointegrator-2 [34–39].
Another possible mechanism is that a direct protein–
protein interaction between LXR and NFjBor
between LXR and another transcription factor such as
Foxo1, a winged helix transcription factor, affects
coactivator recruitment to NFjB. Delerive et al. [40]
demonstrated that PPARa physically interacts with the
NFjB p65 subunit. PPARa agonists are also reported
to inhibit TF expression induced by LPS in macro-
phages [41,42]. Alternatively, a recent report by Dowell
et al. [43] revealed that PPARc interacts directly with
Foxo1 and inhibits each transcriptional activity in a
ligand-dependent manner. Although the mechanism of
PPARc-dependent repression remains to be elucidated,
PPARc agonists can also inhibit iNOS and COX-2

induction by LPS in macrophages as well as LXR
agonists [44]. Further studies focusing on coactivator
recruitment are needed to clarify the molecular basis
of inhibition of NFjB activity by LXR.
TF expression is also downregulated by all-trans-
retinoic acid, which is a ligand for a nuclear receptor
family known as the retinoic acid receptors (RARs)
[45–47]. In RAR activation, unlike LXR, the inhibitory
effect appears to be independent of the NFjB or AP-1
pathway. Whereas LXR and PPARa agonists inhibit
LPS-induced TNFa, no such inhibition by RAR agon-
ists was observed [45]. In addition, whereas LXR or
PPARa agonists do not affect basal TF expression,
RAR agonists inhibit both basal and LPS-induced TF
expression [45]. These data suggest that RAR activation
affects other nuclear factors in the transcription complex
required for TF expression. The zinc finger transcription
factor Egr-1 is also known to be a key player in
TF expression [16]. Shindo et al. [48] showed that a
RAR agonist reduces platelet-derived growth factor-A
Fig. 7. LXR agonists inhibit TF expression in atherosclerotic lesions.
Male LDLR
– ⁄ –
mice were fed an atherogenic diet (1.25% choles-
terol, 7.5% cocoa butter and 0.5% sodium cholate; Oriental Yeast,
Tokyo, Japan). T-0901317 at doses of 3 or 10 mgÆkg
)1
was orally
administered to LDLR
– ⁄ –

mice daily for 8 weeks (n ¼ 9 per group).
(A) TF and (B) ABCA1 mRNA concentrations in atherosclerotic
lesions were determined by real-time quantitative PCR assay.
*P<0.05, as compared with the vehicle control group using Dun-
nett’s multiple comparison test.
Repression of tissue factor in macrophages by LXR N. Terasaka etal.
1552 FEBS Journal 272 (2005) 1546–1556 ª 2005 FEBS
promoter activity via interaction with the transcription
factor Kru
¨
ppel-like factor 5 (KLF5), which is a target
gene of Egr-1. Furthermore, both KLF and Egr-1 tran-
scription factors appear to bind to the GC-rich binding
site of the platelet-derived growth factor-A promoter
[49]. Taken together, these findings suggest that suppres-
sion of TF expression by RAR is dominantly regulated
by the Egr-1 ⁄ KLF pathway, and not the NFjB or AP-1
pathway. These distinct mediations by nuclear receptors
provide clues to the elucidation of the cell-type-specific
manner of TF expression.
Finally, this study demonstrates that LXR agonists
antagonize inflammatory stimuli-induced TF expression
by inhibiting NFjB activity. Previous studies indicate
that LXR agonists strongly induce cholesterol efflux by
upregulation of ABCA1, ABCG1, and apoE expression
[22,27]. In addition, LXR agonists can suppress iNOS,
COX-2, MMP-9 and TNFa expression [19,20]. Taking
these observations together, the anti-atherosclerotic
effects of LXR agonists appear to be due to enhance-
ment of reverse cholesterol transport, anti-inflammation

and anti-thrombosis. Various mechanisms of action of
LXR agonists can be suggested from the function of
LXRs as positive, as well as negative, regulators of tar-
get genes. LXR agonists would be useful therapeutic
agents for the treatment of cardiovascular diseases.
Experimental procedures
Reagents
LXR agonists T0901317 and GW3965, farnesoid X recep-
tor agonist GW4064, peroxisome proliferator-activated
receptor-d (PPAR-d) agonist GW501516, and PPARc agon-
ist rosiglitazone were synthesized by Sankyo Co., Ltd.
PPARa agonist Wy14643 was purchased from Cayman
Chemical (Ann Arbor, MI, USA). 9-cis-Retinoic acid was
obtained from Sigma Chemical (St. Louis, MO, USA).
Agonists were dissolved in Me
2
SO before use in cell culture.
LPS from Salmonella typhimurium was purchased from
Sigma Chemical. Mouse TNFa and IL-1b were from Bio-
source International (Camarillo, CA, USA).
Plasmids
pCMX expression plasmids for LXRa, LXR b and RXRa
have been described previously [21]. The 5¢-flanking region
of the human TF gene ()278 ⁄ +121) was prepared by PCR
using human genomic DNA (Roche Applied Science, India-
napolis, IN, USA) as a template and a forward primer tailed
with a KpnI restriction site (5¢-AAGGTACCAACCCACCT
AAGCTGCACGT-3¢) and a reverse primer tailed with BglII
(5¢-GAAGATCTATGTCTACCAGTTGGCGGCGA-3¢). The
PCR product was digested with KpnI and BglII and sub-

cloned into the KpnI ⁄ BglII-digested luciferase reporter
plasmid pGL3-basic (Promega, Madison, WI, USA), gener-
ating pGL3 ⁄ )278+121hTF-Luc. For the construction
of pGL3 ⁄ )228+121hTF-Luc, pGL3 ⁄ )211+121hTF-Luc,
pGL3 ⁄ )188+121hTF-Luc and pGL3 ⁄ )181+121hTF-Luc,
the PCR products amplified by the reverse primer and a for-
ward primer tailed with a KpnI restriction site (5¢-AAGG
TACCGGTTGAATCACCTGGGGT-3¢)(5¢-AAGGTACC
TGAGTCATCCCTTGCAGGGT-3¢)(5¢-AAGGTACCGG
AGTTTCCTACCGGGAGGA-3¢) and (5¢-AAGGTACCT
ACCGGGAGGAGGCGG-3¢), respectively, was subcloned
into the pGL3-basic. For generation of pGL3 ⁄ 3 · hTF-
dAP1-TK-Luc and pGL3 ⁄ 3 · hTFjB-TK-Luc, oligonucleo-
tides encoding three copies of the downstream AP-1 site
(5¢-GGGTGAGTCATCC-3¢) and the NFjB site (5¢-CCC
GGAGTTTCCTA-3¢), respectively, on the 5¢-flanking
region of TF was subcloned into the HindIII and SalI sites
of TK-luc.
Cell culture and transfection
Peritoneal macrophages were obtained from thioglycolate-
injected C57Bl ⁄ 6J mice as described previously [22]. Cells
(4 · 10
5
) were plated on 24-well plates and cultured in Dul-
becco’s modified Eagle’s medium (DMEM) supplemented
with 10% lipoprotein protein-deficient serum (LPDS) (Sig-
ma Chemical). Human monocytes were isolated from lym-
phocyte preparations obtained from freshly drawn blood of
healthy volunteers. Lymphocyte preparations were diluted
with an equal volume of NaCl ⁄ P

i
. This mixture was under-
layered with Ficoll-Paque (Amersham Biosciences, Piscat-
away, NJ, USA) and centrifuged at 500 g for 10 min. The
lymphocytes were then harvested from the interface. The
lymphocytes were washed twice with NaCl ⁄ P
i
and resus-
pended in RPMI 1640 medium containing 10% LPDS
(1 · 10
6
cellsÆmL
)1
). Monocytes were isolated from lympho-
cytes by adherence (4 h at 37 °C). RAW 264.7 cells were
cultured in DMEM containing 10% fetal bovine serum. For
ligand treatment, cells were cultured in DMEM supplemen-
ted with 10% LPDS and receptor ligands for 18 h before
LPS or cytokine stimulation. Transient transfection of
RAW 264.7 cells was performed in triplicate in 48-well
plates. Cells (2 · 10
5
) were transfected for 6 h with reporter
plasmid (100 ng per well), receptor plasmids (50 ng per well)
and pRL-CMV (50 ng per well) as internal control using
OptiMEM medium and LipofectaminePlus reagent (Invitro-
gen, Carlsbad, CA, USA). After transfection, cells were
incubated in medium containing 10% LPDS and the indica-
ted ligands or vehicle for 12 h before stimulation with LPS
for another 18 h. Firefly and Renilla luciferase activities

were measured in a luminometer, Analyst
TM
HT (Molecular
Devices, Atlanta, GA, USA) using the Dual-Luciferase
Reporter Assay System (Promega).
N. Terasaka etal. Repression of tissue factor in macrophages by LXR
FEBS Journal 272 (2005) 1546–1556 ª 2005 FEBS 1553
Western blot analysis
Cell lysates (50 lg per lane) were separated by SDS ⁄ PAGE
using a 12% separating gel and transferred to an Immobi-
lon
TM
-P membrane (Millipore, Bedford, MA, USA) for
immunoblotting. The blot was blocked overnight at 4 °Cin
0.01% Tween 20 Tris buffered saline (TBST) containing
5% nonfat milk, incubated with goat antibody to human
TF (American Diagnostica, Greenwich, CT, USA) for 1 h
at room temperature, and washed with TBST. The blot was
then incubated with horseradish peroxidase-conjugated sec-
ondary antibody, washed in TBST, and proteins were
detected by ECL (enhanced chemoluminescence) (Amer-
sham Biosciences).
Assay of TF activity
TF activity was determined using a standard chromogenic
assay kit ActichromeÒ TF (American Diagnostica).
RNA analysis
Total RNA was extracted using RNeasyÒ mini kit (Qiagen,
Valencia, CA, USA). Real-time quantitative PCR (TaqMan)
assay was performed using an Applied Biosystems 7700
sequence detector as described [22]. The sequences of forward

primers, reverse primers and TaqMan probes, respectively,
were as follows: mouse TF: 5¢-GCTCTCAGGTGGGATG
CAG-3¢,5¢-GGCTCGTCCAGAATGACAAC-3¢,5¢-FAM-
CTTGGCCTTCGTGGGTGGATCC-TAMRA-3¢; human
TF: 5¢-CCCGTCAATCAAGTCTA CAC-3¢,5¢-GTCTGCT
TCACATCCTTCAC-3¢,5¢ -FAM-TACACAACAGACAC
AGAGTGTGACCTCACC-TAMRA-3¢; mouse TNFa:5¢-
CGGAGTCCGGGCAGGT-3¢,5¢-GCTGGGTAGAGAAT
GGATGAACA-3¢,5¢-FAM-ACTTTGGAGTCATTGCTCT
GTGAAGGG-TAMRA-3¢; cyclophilin: 5¢-CGATGACGAG
CCCTTGG-3¢,5¢-TCTGCTGTCTTTGGAACTTTGTC-3¢,
5¢-FAM-CGCGTCTCCTTTGAGCTGTTTGCA-TAMRA-
3¢. All assays were performed in duplicate, and cycle thresholds
of individual genes were normalized to that of cyclophilin.
Determination of cytokine concentrations
Concentrations of IL-1b, IL-6 and TNFa were determined
by the Bio-Plex Cytokine Assay (Bio-Rad Laboratories,
Hercules, CA, USA) according to the manufacturer’s
instructions.
Nuclear NFjB p50 and p65 activity
Nuclear extracts were prepared from peritoneal macro-
phages using a nuclear extraction kit (Transfactor Extraction
Kit; BD Biosciences Clontech Laboratories, Palo Alto, CA,
USA) following the manufacturer’s instructions. Briefly,
DNA-binding activity of NFjB p50 and p65 was assessed in
nuclear extracts using an ELISA-based format (BD Mercury
NFjB p50 and p65 Transfactor Kits; BD Biosciences Clon-
tech Laboratories) following the manufacturer’s instructions.
Animals
Male C57Bl ⁄ 6J mice were obtained from Charles River

Japan. Vehicle [propylene glycol ⁄ Tween 80 (4 : 1, v ⁄ v)] or
LXR agonists T-0901317 at 3 mgÆkg
)1
or GW-3965 at
30 mgÆkg
)1
was orally administered daily to the mice for
7 days (n ¼ 5 per group). The day after the last administra-
tion of LXR agonist, saline or LPS at 4 mgÆkg
)1
was intra-
peritoneally administered to C57Bl ⁄ 6 mice at 9 : 00 a.m.
The mice were anaesthetized with ethyl ether and killed at
3 : 00 p.m. (6 h after LPS administration). Blood was
obtained from the abdominal vein, and tissues were rapidly
removed and snap-frozen in liquid nitrogen for further ana-
lysis. Male low-density lipoprotein receptor (LDLR)
– ⁄ –
mice
were obtained from Charles River Japan. LDLR
– ⁄ –
mice
were fed an atherogenic diet (1.25% cholesterol, 7.5% cocoa
butter and 0.5% sodium cholate). Vehicle or T-0901317 at
doses of 3 or 10 mgÆkg
)1
was orally administered daily for
8 weeks (n ¼ 9 per group), and the tissues were obtained as
previously described in [22]. For gene expression analysis,
atherosclerotic lesions from aortic arch were visualized using

a stereo-microscope (Leica MZ12) and harvested using a
sterile scalpel to ensure the material collected contained only
atherosclerotic lesion and not underlying aortic tissue. All
animal care and experimental procedures complied with the
Sankyo Animal Care and Use Committee.
Statistical analysis
Significant difference from the vehicle control was assessed
using Dunnett’s multiple comparison test. A difference was
considered to be significant when the P value was less than
0.05.
Acknowledgements
We thank the Medicinal Chemistry Research Laborat-
ories at Sankyo for synthesizing ligands, Drs Richard
Heyman and Raju Mohan at X-Ceptor Therapeutics
for providing LXRs and RXR expression plasmids,
and Drs Hiroyuki Koike, Hiroaki Yanagisawa, Teii-
chiro Koga and Jun Ohsumi at Sankyo for critical
reading and helpful discussion.
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