Treatment of mouse peritoneal macrophages with synthetic LXR agonist T0901317 or GW3965 reduced TF expression induced by pro-inflammatory stimuli.. Results LXR agonists inhibit TF express
Trang 1in macrophages
Naoki Terasaka1, Ayano Hiroshima1, Akiko Ariga1, Shoko Honzumi1, Tadashi Koieyama1,
Toshimori Inaba2and Toshihiko Fujiwara1
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 facrecep-tors 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: terasa@shina.sankyo.co.jp
(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.
Trang 2coronary 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,
Trang 3T0901317 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 l M 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% Me2SO or 1 l M 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 (l M ) 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 (l M ) 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.
Trang 4LXRb⁄ 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) for 6 h.
(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.
C
Fig 3 LXR agonists inhibit LPS-induced TF
expression and activity in human
mono-cytes Human monocytes were pretreated
with 1% Me2SO 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.
Trang 5or 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) Administrainjec-tion 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, LXRa, pCMX-LXRb, pCMX-RXRa and pRL-CMV as described in Experimental procedures After transfection, RAW 264.7 cells were incubated with 1%
Me2SO or 1 l M T-0901317 for 12 h in DMEM containing 10% LPDS Cells were then stimulated with 100 ngÆmL)1LPS 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.
Trang 6the 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% Me2SO or 1 l M 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)1or GW-3965 at 30 mgÆkg)1was 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
4 mgÆkg)1was 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.
Trang 7Thus, 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 transcripfunc-tional 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 facrecep-tors, 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 NFjB or 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)1was 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.
Trang 8promoter 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 expressuppres-sion 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 proliferarecep-tor-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 Me2SO 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, LXRb 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· 105) 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) Hu(Sig-man 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⁄ Pi 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⁄ Pi and resus-pended in RPMI 1640 medium containing 10% LPDS (1· 106cellsÆmL)1) Monocytes were isolated from lympho-cytes by adherence (4 h at 37C) 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· 105
) 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, AnalystTMHT (Molecular Devices, Atlanta, GA, USA) using the Dual-Luciferase Reporter Assay System (Promega)
Trang 9Western blot analysis
Cell lysates (50 lg per lane) were separated by SDS⁄ PAGE
using a 12% separating gel and transferred to an
Immobi-lonTM-P membrane (Millipore, Bedford, MA, USA) for
immunoblotting The blot was blocked overnight at 4C in
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 Actichrome TF (American Diagnostica)
RNA analysis
Total RNA was extracted using RNeasy 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¢-CCCGTCAATCAAGTCTACAC-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 (n¼ 5 per group) The day after the last administra-tion of LXR agonist, saline or LPS at 4 mgÆkg)1was 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)1was orally administered daily for
8 weeks (n¼ 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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