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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 91 No. 8 (April 15), 1998:
pp. 2857-2865
Retinoic Acid Selectively Inhibits Lipopolysaccharide Induction of
Tissue Factor Gene Expression in Human Monocytes
By
Paul Oeth,
Jin Yao,
Sao-Tah Fan, and
Nigel Mackman
From the Departments of Immunology and Vascular Biology, The Scripps
Research Institute, La Jolla, CA.
 |
ABSTRACT |
Expression of tissue factor (TF) by activated monocytes in several
diseases leads to disseminated intravascular coagulation. Lipopolysaccharide (LPS)-induced monocyte TF expression is
downregulated by the nuclear hormone all-trans retinoic acid
(ATRA). In this study, we examined the mechanism by which ATRA inhibits
monocyte TF expression. We show that ATRA selectively inhibited LPS
induction of TF expression in human monocytes and monocytic THP-1 cells without affecting LPS induction of tumor necrosis factor- (TNF-) and interleukin-8 (IL-8). Inhibition of TF expression occurred at the
level of transcription as determined by nuclear run-on. ATRA did not
significantly alter the binding or functional activity of the
transcription factors c-Fos/c-Jun and
c-Rel/p65, which are required for LPS induction of the TF
promoter in monocytic cells. In contrast to the ATRA inhibition of the
endogenous TF gene, LPS induction of the cloned TF promoter was not
inhibited by ATRA in transiently transfected THP-1 cells. Our results
demonstrate that ATRA selectively inhibited LPS-induced TF gene
transcription in human monocytic cells by a mechanism that does not
involve repression of AP-1- or
NF- B-mediated transcription.
| |
INTRODUCTION |
TISSUE FACTOR (TF) is the primary
cellular initiator of the coagulation protease cascades.1,2
TF-initiated thrombosis is associated with many disease states,
including gram-negative sepsis, atherosclerosis, and
cancer.3-6 TF expression by leukemic cells in patients with
acute promyelocytic leukemia (APL) may initiate disseminated
intravascular coagulation and lead to a consumptive
coagulopathy.7-9 All-trans-retinoic acid (ATRA)
therapy downregulates TF expression in these patients.8-10
In addition, several studies have established that ATRA also inhibits
TF expression in bacterial lipopolysaccharide (LPS)-stimulated
peripheral blood monocytes, as well as in tumor necrosis factor-
(TNF-)-stimulated human endothelial cells.7,11,12
ATRA is a member of the retinoid family of hormones, which are ligands
for a nuclear hormone receptor family known as the retinoic acid
receptors (RARs).13 Recent studies have shown that RAR
and RAR are required for retinoid-induced downregulation of monocyte
TF expression.14 Retinoids are lipophilic molecules that
can pass through plasma membranes and effectively enter the nucleus of
cells to bind RARs. Binding of retinoids to RARs induces allosteric
changes that allow RARs to bind to specific DNA recognition sites and
regulate gene transcription.13,15 Liganded RAR has been
shown to modulate gene transcription by directly interacting with other
proteins, such as c-Jun.16
LPS stimulates monocytes to rapidly and transiently express several
proinflammatory mediators, including TF, TNF-, and interleukin-8 (IL-8).17 LPS induces the transcriptional activation of
these genes, which require cooperative interaction between several
transcription factors, including those belonging to the NF-B/Rel and
AP-1 families.18-21 We have established that binding of
proteins to a 56-bp ( 227 bp to 172 bp) region located in the
human TF promoter mediates LPS induction of the TF gene in
monocytes.22 This region contains two AP-1-binding sites
and a nonconsensus B-binding site, which bind
c-Fos/c-Jun and c-Rel/p65 heterodimers,
respectively.23,24 Functional interaction between these
transcription factors is required for maximal LPS induction of TF gene
transcription in monocytic cells.22,23
Our laboratory and others have shown that agents that block the nuclear
translocation of c-Rel/p65 heterodimers by preventing IB
phosphorylation or proteolytic degradation inhibit LPS-induced TF gene
transcription in monocytic cells. These agents include protease
inhibitors and salicylates.25-27 Additionally, agents that
elevate intracellular cyclic adenosine monophosphate (cAMP) levels can
inhibit LPS-induced TF gene transcription in monocytic cells and
endothelial cells by affecting nuclear NF-B activity.28 Our recent studies indicate that cAMP inhibits NF-B activity by
activating the PKA signaling pathway, which leads to competition between p65 and CREB for limiting amounts of the coactivator
CBP.29 Importantly, all of these NF-B inhibitors also
inhibit LPS induction of TNF- gene transcription in human monocytic
cells.25-28,30
Previous studies report that ATRA can inhibit both AP-1- and
NF-B-dependent transcription.31-32 RAR liganded with
ATRA is a negative regulator of AP-1-dependent genes, such as the
stromolysin and collagenase genes, which is proposed to be the result
of direct interactions between RARs and c-Jun or binding of
RARs to c-Jun sites.31,32 In addition, ATRA was
recently shown to selectively inhibit TNF--induced vascular
cell-adhesion molecule-1 (VCAM-1) expression in cultured dermal
microvascular endothelial cells without affecting other
NF-B-regulated genes, such as E-selectin and intracellular adhesion
molecule 1.33 The mechanism of this ATRA inhibition was
proposed to be due to a specific reduction of NF-B binding to B
sites in the VCAM-1 promoter without affecting binding to other B
sites.
In this study, we examined the mechanism by which ATRA inhibits
LPS-induced TF gene expression in human monocytic cells.
| |
MATERIALS AND METHODS |
Materials.
LPS (Escherichia coli serotype O111:B4) was purchased from
CalBiochem (San Diego, CA). ATRA was obtained from Sigma (St Louis, MO)
and was dissolved in ethanol.
Monocyte isolation and cell culture.
Human peripheral blood mononuclear cells (PBMCs) were isolated by
centrifugation from heparinized blood drawn from volunteers. Cells were
diluted 1:1 (vol:vol) in endotoxin-free RPMI 1640 medium (BioWhittaker,
Walkersville, MD) and were overlaid onto Ficoll-Hypaque (Pharmacia,
Uppsala, Sweden). After centrifugation at 400g, the PBMCs were
removed and washed twice with medium and resuspended in RPMI 1640 media
containing 2% fetal calf serum (Gemini Bioproducts, Calabasas, CA).
Monocytes were isolated from PBMCs using Sepracell-MN (Sepratech,
Oklahoma City, OK). Monocytes were cultured in RPMI 1640 medium
supplemented with 10% fetal calf serum. The purity of the monocyte
preparations was typically 80% to 90% as determined by nonspecific
esterase staining.34 Human monocytic leukemia THP-1 cells
were obtained from the American Type Culture Collection (Rockville, MD)
and were cultivated (nonsynchronized) at a density of 2 to 5 × 106 cells/mL as described.35 All reagents were
tested by Limulus amebocyte lysate assay (BioWhittaker) and contained
less than 0.005 ng/mL of endotoxin.
TF activity.
THP-1 cell pellets were solubilized at 37°C for 15 minutes using 15 mmol/L octyl -D-glucopyranoside. TF activity in cell lysates was assayed using a one-stage clotting assay as
described.36 Clotting times were converted to milliunits of
TF activity by comparison with a standard curve established with
purified human brain TF.36 For reference, a clotting time
of 50 seconds corresponds to 1,000 mU of TF activity.
TF, TNF-, and IL-8 enzyme-linked immunosorbent
assays.
Monocytes (1 × 106 cells) or THP-1 cells
(1 × 106 cells) were pretreated with various doses of
ATRA for 30 minutes before LPS stimulation. Monocytes were stimulated
with 100 ng/mL of LPS and THP-1 cells were stimulated with 10 µg/mL
of LPS. Cells were incubated for 5 hours before measuring TF antigen
levels in cell lysates and 24 hours before measuring TNF- and IL-8
antigen levels in culture supernatants. The TF enzyme-linked
immunosorbent assay (ELISA) kit was purchased from American Diagnostica
(Greenwich, CT) and the TNF- and IL-8 ELISA kits were purchased from
R & D Systems (Minneapolis, MN). TF, TNF-, and IL-8 protein
concentrations from the ELISAs were determined using standard curves
for each assay.
Analysis of TF, TNF-, and IL-8 mRNA.
Total cellular RNA, purified using TRIZOL reagent (GIBCO-BRL,
Gaithersburg, MD), was analyzed by Northern blotting as
described.37 THP-1 cells were pretreated for 30 minutes
with ATRA followed by a 2-hour induction with LPS (10 µg/mL). A
641-bp human TF cDNA fragment,38 an 800-bp human TNF-
cDNA fragment,39 and a 400-bp human IL-8 cDNA fragment
(kindly provided by Dr R. Terkeltaub) were used as probes. cDNA
fragments were labeled with [-32P]deoxycytidine
triphosphate (ICN, Costa Mesa, CA) using the Prime-It Kit (Stratagene,
La Jolla, CA). Blots were rehybridized with the housekeeping gene
glyceraldehyde 3-phosphate dehydrogenase (G3PDH; Clontech Laboratories,
Palo Alto, CA). Autoradiography was performed at 70°C using Kodak
Biomax film (Eastman-Kodak, Rochester, NY). Band intensities were
quantified using a Personal Densitometer and ImageQuant software
(Molecular Dynamics, Sunnyvale, CA).
Nuclear run-on.
Nuclear run-on assays were performed as described
previously.40 Cells were pretreated for 30 minutes with
ATRA (1 × 10 5 mol/L) before a 1-hour stimulation
with LPS. Cells (5 × 107) were lysed in a homogenizer
and nuclei collected by centrifugation using a 2-mmol/L sucrose
cushion. Nuclear RNA was labeled using [-32P]uridine
triphosphate (ICN). Samples were treated sequentially with DNase I
(final concentration, 1 U/µL) for 10 minutes at 37°C and with
proteinase K (final concentration, 200 µg/mL) for 45 minutes at
37°C. Nuclear RNA was extracted with phenol/chloroform, ethanol-precipitated, and purified using a G-50 sephadex column. TF,
G3PDH, and TNF- cDNAs present in the vectors pGEM3Z and pSP73 (Promega, Madison, WI) were used as targets in the nuclear
run-on assays. Prehybridization and washing of the filters was
performed as described.41 Radioactivity was quantified
using a Phosphoimager and ImageQuant software (Molecular Dynamics).
Plasmids and transfections.
p(AP-1)4LUC and p(B)4LUC contain four copies
of the proximal TF AP-1 site and TF B site, respectively, cloned
upstream of the minimal simian virus 40 (SV40) promoter expressing the
firefly luciferase (LUC) reporter gene in pGL2-Promoter
(Promega).24 pTF(2106)LUC has previously been
described.22 Cells were transfected using diethyl
aminoethyl (DEAE)-dextran22 and were cultivated for 46 hours before a 5-hour stimulation with LPS (10 µg/mL). Luciferase
activity was determined using the Luciferase Assay System (Promega) and
Monolight 2010 luminometer (Analytical Luminescence, San Diego, CA).
Electrophoretic mobility shift assay.
Nuclear extracts were prepared from 5 × 106 cells as
described.42 Protein concentrations were 1 to 5 mg/mL, as
determined by a bicinchoninic acid (BCA) protein assay (Price,
Rockford, IL). An oligonucleotide (Operon Technologies, Alameda, CA)
containing the TF B site (underlined),
5 -GTCCCGGAGTTTCCTACC-3, was annealed with a complementary
primer and was radiolabeled using [-32P]deoxycytidine
triphosphate (ICN) as described.24 Similarly, binding of
AP-1 was performed using a radiolabeled oligonucleotide containing
the proximal TF AP-1 site (underlined),
5-CTGGGGTGAGTCATCCCTT-3.23 An oligonucleotide
containing an Sp1 site (underlined),
5-ATTCGATCGGGGCGGGGCGAGC-3, was purchased from Promega.
Protein-DNA complexes were separated from free DNA probe by
electrophoresis through 6% nondenaturing acrylamide gels (NOVEX, San
Diego, CA) in 0.5× Tris borate-EDTA and bands visualized by
autoradiography. Band intensities were quantified by densitometric
analysis using a Personal Densitometer and ImageQuant software. The
following antibodies from Santa Cruz Biotechnology were used for
supershift studies: anti-p50 sc-114; anti-p65 sc-109;
anti-c-Rel sc-070; anti-Sp1 (PEP2).
Statistical analysis.
Statistical significance was calculated using a paired Student's
t test and was considered significant at P values
 .05.
| |
RESULTS |
ATRA selectively inhibits LPS induction of TF expression in human
monocytes and monocytic cells. ATRA has previously been shown to
downregulate TF procoagulant activity in LPS-stimulated human
monocytes.7,11 To determine if ATRA was a general inhibitor of LPS-induced genes in human monocytes, we analyzed the effect of ATRA
on LPS induction of TF, TNF-, and IL-8. Monocytes were incubated for
30 minutes with various doses of ATRA (1 × 109 mol/L
to 1 × 105 mol/L) before LPS stimulation. ATRA
inhibited LPS induction of TF expression in a dose-dependent manner
(Fig 1A). Statistically significant inhibition (P = .005) was
observed using 109 mol/L ATRA. In contrast, ATRA had no
effect on LPS induction of TNF- expression (Fig
1B) or IL-8 expression (Fig 1C). These results indicate that ATRA selectively inhibits the LPS induction of TF
expression in human monocytes without affecting induction of TNF-
and IL-8 expression.
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| Fig 1.
Effect of ATRA on TF, TNF-, and IL-8 protein
expression in LPS-stimulated human monocytes. Freshly isolated human
monocytes (1 × 106 cells) were incubated with LPS
alone, ATRA alone (105 mol/L) or with various doses of
ATRA (1 × 109 to 1 × 105 mol/L)
for 30 minutes before LPS stimulation (100 ng/mL). (A) Monocytes were
incubated for 5 hours and TF antigen levels were measured by ELISA. (B)
Monocytes were incubated for 24 hours and TNF- antigen level in
culture supernatants were measured by ELISA. (C) Monocytes were
incubated for 24 hours and IL-8 antigen levels in culture supernatants
were measured by ELISA. Results from three independent experiments
(mean ± SE) are shown.
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We next determined if ATRA selectively inhibited LPS induction of TF
expression in human monocytic THP-1 cells, which we use as a model of
human monocytes. THP-1 cells were incubated for 30 minutes with ATRA at
various doses (1 × 109 to 1 × 105
mol/L) before LPS stimulation. LPS induction of TF activity in THP-1
cells was inhibited by ATRA in a dose-dependent manner (Fig 2A). Statistically significant inhibition
(P = .01) was observed using 109 mol/L ATRA.
Similar to the results observed with human monocytes, ATRA did not
inhibit the LPS induction of TNF- (Fig 2B) or IL-8 (Fig 2C). These
results indicated that THP-1 cells were a valid model to examine the
molecular mechanism by which ATRA selectively inhibits LPS-induced TF
expression in human monocytic cells.
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| Fig 2.
Effect of ATRA on TF, TNF-, and IL-8 expression
LPS-stimulated THP-1 cells. THP-1 cells (1 × 106 cells)
were incubated LPS alone, ATRA (105 mol/L), or various
doses of ATRA (1 × 109 to 1 × 105
mol/L) before LPS stimulation (10 µg/mL). (A) THP-1 cells were exposed to LPS for 5 hours and TF activity was determined using a
one-stage clotting assay. (B) THP-1 cells were exposed to LPS for 24 hours and TNF- antigen levels in culture supernatants measured by
ELISA. (C) THP-1 cells were exposed to LPS for 24 hours and IL-8
antigen levels in culture supernatants measured by ELISA. Results from
three independent experiments (mean ± SE) are shown.
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ATRA selectively inhibits LPS induction of TF mRNA expression.
To determine if ATRA inhibited LPS-induced TF mRNA expression, we
analyzed TF mRNA levels by Northern blot analysis in LPS-stimulated THP-1 cells with or without ATRA. THP-1 cells were incubated for 30 minutes with ATRA (105 mol/L) before LPS stimulation for
2 hours. The basal levels of TF mRNA observed in unstimulated cells
were strongly induced by LPS (Fig 3A). ATRA
reduced the basal levels of TF mRNA in unstimulated cells and abolished
the LPS induction of TF mRNA (Fig 3A). ATRA did not inhibit LPS
induction of TNF- mRNA (Fig 3B) or IL-8 mRNA (Fig 3C). We also
performed dose-titration experiments. ATRA inhibited LPS induction of
TF mRNA expression in a dose-dependent manner (Fig
4A), but did not inhibit LPS induction of
TNF- mRNA expression at any concentration (Fig 4B). These results
indicate that ATRA selectively inhibits LPS induction of TF mRNA
expression in human monocytic cells.
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| Fig 3.
Effect of ATRA on TF, TNF-, and IL-8 mRNA expression
in LPS-stimulated THP-1 cells. Total RNA was extracted from THP-1 cells exposed to LPS (10 µg/mL) for 2 hours with or without a 30-minute pretreatment with ATRA (1 × 105 mol/L). Control
samples were incubated with ATRA alone or without LPS or ATRA. Various
mRNA levels were determined by Northern blot analysis using
radiolabeled human cDNA probes. Membranes were reprobed with a G3PDH
cDNA probe to assess RNA loading. The positions of TF (A), TNF- (B),
and IL-8 (C) mRNAs are indicated. Similar results were observed in one
or more independent experiments.
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| Fig 4.
Dose-dependent ATRA inhibition of TF mRNA expression in
LPS-stimulated THP-1 cells. Total RNA was extracted from THP-1 cells exposed to LPS (10 µg/mL) for 2 hours with or without a 30-minute pretreatment with ATRA at doses ranging from 1 × 1010
to 1 × 105 mol/L. The control sample was incubated
without LPS or ATRA. TF and TNF- mRNA levels were determined by
Northern blot analysis using radiolabeled human cDNA probes. Blots were
reprobed with a G3PDH cDNA probe to assess RNA loading. The positions
of TF (A) and TNF- (B) mRNAs are indicated.
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ATRA selectively inhibits LPS induction of TF gene transcription.
ATRA inhibition of TF mRNA expression could be due to changes in TF
mRNA stability and/or an inhibition of TF gene transcription. To determine if ATRA inhibited LPS induction of TF gene transcription, nuclear run-on experiments were performed. THP-1 cells were incubated with ATRA (105 mol/L) for 30 minutes before a 1-hour
stimulation with LPS. LPS stimulation of THP-1 cells increased the rate
of TF gene transcription, which was abolished by ATRA (Fig 5). In
contrast, ATRA did not affect the LPS induction of TNF- gene
transcription (Fig 5). The band intensity
representing the rate of transcription of the housekeeping gene, G3PDH,
was slightly reduced in cells incubated with ATRA and LPS in this
experiment (Fig 5). However, this decrease was not observed in an
independent experiment and is likely to be due to experimental
variations in the assay. Importantly, ATRA and LPS did not decrease the
levels of G3PDH mRNA in cells (Figs 3 and 4). These results demonstrate
that ATRA selectively inhibits TF gene transcription in human monocytic
cells.
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| Fig 5.
Effect of ATRA on TF and TNF- gene transcription in
LPS stimulated THP-1 cells. Nuclei were isolated from unstimulated
cells, cells stimulated with LPS (10 µg/mL) for 1 hour, and cells
treated with ATRA (105 mol/L) for 30 minutes before LPS
stimulation for 1 hour. The rate of transcription of the TF, TNF-,
and G3PDH gene was determined by nuclear run-on. Vector controls used
in the experiment were pSP73 and pGEM3Z. The autoradiogram was exposed
for 7 days at 80°C with intensifier screens. Similar results were
observed in an independent experiment.
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ATRA does not affect DNA binding of c-Fos/c-Jun, c-Rel/p65, or Sp1
transcription factors.
LPS induction of TF gene transcription in monocytic cells is primarily
regulated by the cooperative interaction of c-Fos/c-Jun and c-Rel/p65 heterodimers bound to a 56-bp LPS response
element in the TF promoter.22,23 Previous electrophoretic
mobility shift assays (EMSAs) and antibody supershift analysis of
complexes have established that c-Fos/c-Jun and
c-Rel/p65 heterodimers bind to the TF promoter. To examine if
ATRA inhibited TF gene transcription by preventing binding of either
c-Fos/c-Jun or c-Rel/p65 heterodimers, we
performed EMSAs using oligonucleotides containing either the proximal
AP-1 site or the B site from the human TF promoter. THP-1 cells were
incubated with ATRA for 30 minutes before a 2-hour stimulation with
LPS. The binding of c-Fos/c-Jun heterodimers and Sp1 in
unstimulated or LPS-stimulated THP-1 cells was not affected by ATRA
(Fig 6A). We have shown that LPS
stimulation of THP-1 cells induces nuclear translocation of
c-Rel/p65 heterodimers and binding to the TF B
site.24 LPS-induced nuclear translocation and DNA binding
of c-Rel/p65 heterodimers was also not inhibited by ATRA (Fig
6A). Similar results were observed using a B site that binds p50/p65
heterodimers (data not shown), which regulate TNF- gene
transcription.43 To exclude the possibility that ATRA
induced binding of an inhibitory complex to the TF B site, we
performed supershift experiments using antibodies raised against NF-B proteins. These studies demonstrated that ATRA did not change the composition of the c-Rel/p65 complex (Fig 6B). These
results indicated that ATRA does not inhibit TF gene transcription by preventing the binding of Sp1, c-Fos/c-Jun, or
c-Rel/p65 to the TF promoter.
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| Fig 6.
The effect of ATRA on the binding of transcription
factors to the TF promoter. (A) Effect of ATRA on the binding of
c-Fos/c-Jun, Rel/p65, and Sp1. Nuclear extracts were
isolated from THP-1 cells treated with or without ATRA
(1 × 105 mol/L) for 30 minutes before the addition
of LPS (10 µg/mL) for 2 hours. EMSAs were performed using AP-1, B,
and Sp1 sites. The position of c-Fos/c-Jun,
c-Rel/p65, and Sp1 is indicated. Similar results were observed
in an independent experiment. (B) Antibody supershift analysis of the
protein-DNA complex formed using nuclear extracts from ATRA- and
LPS-treated cells. Antibodies to various NF-B proteins and Sp1 were
incubated with nuclear extracts before the addition of a radiolabeled
TF B site.
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ATRA does not inhibit LPS-induced AP-1- or
NF-B/Rel-dependent transcription in monocytic cells.
EMSAs analyze DNA binding of transcription factors, but do not measure
the transcriptional activity of these proteins. For instance,
phosphorylation of c-Jun increases the transcription activity
of AP-1 without affecting DNA binding.44 Therefore, we
performed functional studies to analyze the effect of ATRA on AP-1-
and NF-B/Rel-dependent transcription in THP-1 cells. Cells
were transfected with either p(AP-1)4LUC,
p(B)4LUC, or the control plasmid pGL2-Promoter. LPS
induced a twofold increase in luciferase activity expressed by
p(AP-1)4LUC (Fig 7A). ATRA (105 mol/L) did not significantly reduce LPS induction
of AP-1-dependent transcription (Fig 7A). LPS induced a 21-fold
increase in luciferase activity expressed by p(B)4LUC,
which was slightly reduced (17%) by ATRA. LPS did not induce
luciferase activity expressed by pGL2-Promoter, which contains only the
minimal SV40 promoter (data not shown). These results indicated that
ATRA did not significantly inhibit LPS-induced AP-1- or
NF-B-dependent transcription in THP-1 cells.
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| Fig 7.
Effect of ATRA on LPS-induced AP-1- and
NF-B-dependent transcription in THP-1 cells. THP-1 cells were
transfected with p(AP-1)4LUC or p(B)4LUC (6 µg) using DEAE-dextran. After 46 hours, cells were divided into four
equal portions and incubated with or without LPS (10 µg/mL) for 5 hours in the presence or absence of a 30-minute pretreatment with ATRA
(1 × 105 mol/L). AP-1-mediated transcription (A) and
c-Rel/p65-mediated transcription (B) are shown. Luciferase
activity (light units) expressed by p(AP-1)4LUC and
p(B)4LUC was used to determine fold inductions
(mean ± SE) in three independent experiments.
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ATRA does not inhibit LPS induction of the cloned human TF promoter.
To determine if ATRA inhibited LPS induction of TF gene transcription
by assembling an inhibitory complex at putative retinoic acid response
elements at 1050 and 615 in the human TF promoter, we assessed
the effect of ATRA on LPS induction of the cloned TF promoter in
transiently transfected THP-1 cells. pTF(2106)LUC contains the
wild-type TF promoter (2106 to +121 bp, relative to the start site
of transcription) and is strongly induced by LPS.22,23 LPS
induced a fivefold increase in luciferase activity expressed by
pTF(2106)LUC, which was not affected by ATRA (Fig 8). These results indicated that ATRA does
not inhibit the cloned TF promoter in transiently transfected THP-1
cells.
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| Fig 8.
Effect of ATRA on LPS-induced TF promoter activity in
THP-1 cells. pTF(2106)LUC (6 µg) was transfected into THP-1 cells
using DEAE-dextran. After 46 hours, cells were divided into four equal portions and were treated with LPS (10 µg/mL) for 5 hours with or
without a 30-minute pretreatment of ATRA (1 × 105
mol/L). The control sample was incubated without LPS or ATRA. Luciferase activity (light units) expressed by pTF(2106)LUC was used
to determine fold inductions (mean ± SE) in three independent experiments.
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| |
DISCUSSION |
This study showed that ATRA inhibited LPS-induced TF expression in
human monocytic THP-1 cells by preventing induction of TF gene
transcription. ATRA inhibited monocyte TF expression at clinically
relevant doses and required only a short preincubation period. A
previous study showed that the inhibitory effect of ATRA was also
observed when monocytes were exposed to retinoids for 10 minutes and
washed before challenge with LPS, indicating that the inhibitory effect
was rapid and irreversible.11 Short-term incubation with
ATRA was not a general inhibitor of LPS signaling pathways in
monocytes, because it had no effect on the LPS induction of TNF- and
IL-8. In contrast, long-term incubation of human monocytes (>72
hours) with ATRA inhibits LPS induction of cytokine expression,
including IL-8,45 suggesting a different inhibitory mechanism.
The mechanism by which ATRA inhibits monocyte TF gene transcription is
not known. Here, we showed that inhibition was not due to reduced DNA
binding or reduced functional activity of c-Fos/c-Jun or c-Rel/p65 heterodimers in monocytic cells. These results are consistent with the failure of ATRA to inhibit LPS induction of the
TNF- and IL-8 expression, both of which are regulated by AP-1 and
NF-B proteins.18,21,43,46 The small reduction in AP-1-
and NF-B-dependent transcription observed in transfected cells in
the presence of ATRA may be due to competition for limiting amounts of
the coactivator CBP. Recent studies have shown that CBP functions as a
coactivator in transcription mediated by AP-1, NF-B, and nuclear
hormone receptors, such as RAR.29,47-50
Low-level (basal) expression of TF activity in unstimulated PBMCs is
abolished by NF-B inhibitors.25 Similarly, basal
expression of TF mRNA expression in unstimulated THP-1 cells is
abolished by NF-B inhibitors (N. Mackman, unpublished data, July
1994), suggesting that it is mediated by c-Rel/p65. Thus, the
same transcription factor complex appears to regulate both basal and
LPS-induced TF expression in monocytic cells. We observed that ATRA
inhibited both basal and LPS-induced TF mRNA expression in THP-1 cells
(Fig 3), suggesting that it affected a common factor in the
transcription complex that is required for monocyte TF expression. ATRA
did not fully inhibit LPS-induced TF mRNA expression in all
experiments, which is probably due to slight variations in the LPS and
ATRA response of different cultures of THP-1 cells. Clinically, ATRA is
used to treat APL patients suffering from coagulation disorders that
often result in disseminated intravascular coagulation.10 TF expression by the leukemic cells themselves, as well as monocytes, is thought to significantly contribute to this condition.9 Our studies and others have shown that ATRA inhibits TF expression in
both APL cells and monocytes.8,10,11 At present, there are
no studies on how the TF gene is regulated in APL cells. However, the
ability of ATRA to inhibit both constitutive TF expression in APL cells
and LPS-induced TF expression in monocytes suggest that a similar
regulatory mechanism may be employed in both cell types. This is
currently being investigated.
ATRA inhibited LPS induction of the endogenous TF gene, but not the
cloned TF promoter (2106 to +121 bp) in transiently transfected THP-1 cells. The failure to inhibit the cloned TF promoter was not due
to limiting amounts of RAR, because similar results were observed in
cells cotransfected with a plasmid expressing RAR (data not shown).
These results suggest that liganded RAR may assemble a repressor
complex at a retinoic acid response element outside the cloned TF
promoter region examined in this study. RAR has been shown to bind a
transcriptional repressor called N-CoR.51,52 Recent studies
have shown that unliganded RXR/TR heterodimers assemble a repressor
complex that includes N-CoR.53,54 Importantly, in
the presence of thyroid hormone this repressor complex is disrupted and
the liganded thyroid receptor now recruits an activator complex. Our
results suggest a novel inhibitory mechanism for ATRA because
inhibition requires liganded RAR. Alternatively, inhibition may
require assembly of the DNA into chromatin for recognition by ATRA. A
recent study indicated that chromatin structure played a role in the
regulation of gene expression by steroid hormone
receptors.55 Finally, liganded RAR may compete with an
as yet unidentified activator that induces the endogenous TF gene, but
does not contribute to the regulation of the cloned TF promoter. ATRA
inhibition of vitamin D3-stimulated transcription of the avian
3-integrin gene is mediated by competition for binding of VDR/RXR
heterodimers and RAR/RXR heterodimers to a unique response element.56 Further studies are required to elucidate the
molecular mechanism by which ATRA selectively inhibits TF gene
expression in monocytes. This may require cloning of additional 5 and
3 flanking regions of the TF gene and/or the generation of
stable THP-1 cell lines containing TF promoter-luciferase reporter
genes.
| |
FOOTNOTES |
Submitted July 7, 1997;
accepted December 10, 1997.
Supported by Grant No. HL-48872 (N.M.) from the National Institutes of
Health, and performed during the tenure of an Established Investigatorship from the American Heart Association (N.M.).
Address reprint requests to Nigel Mackman, PhD, The Scripps Research
Institute, 10660 N Torrey Pines Rd, IMM-17, La Jolla, CA 92037.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. section 1734 solely to indicate this fact.
| |
ACKNOWLEDGMENT |
We acknowledge H. McClary for technical assistance, G. Parry for
helpful discussion, J. Erlich and M. O'Connell for critical reading of
the manuscript, C. Glass for providing pRAR, and J. Robertson for
preparing the manuscript.
| |
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Abstract
Introduction
Abstract