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

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

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

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

)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

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-

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

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.

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

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

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

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

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

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