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INTRODUCTION |
The acute phase response
(APR)1 is induced during
infection, inflammation, and injury and is associated with a wide
range of metabolic changes (1). Among these, changes in lipid
metabolism have received much attention due to the link between
infection/inflammatory diseases and atherosclerosis (2-6). The
characteristic changes in lipid metabolism during the APR include
hypertriglyceridemia (7), decreases in serum high density lipoprotein
cholesterol levels (8, 9), increased hepatic cholesterol synthesis, inhibition of bile acid synthesis (10), increased hepatic fatty acid
synthesis, and decreased hepatic fatty acid oxidation and ketogenesis
(11, 12). These changes are mediated by alterations in gene expression
caused by pro-inflammatory cytokines including TNF
, IL-1
, and
IL-6 (10). However, the underlying mechanism by which these cytokines
regulate gene transcription is not well understood, especially for the
negative acute phase proteins.
Nuclear hormone receptors are ligand-activated transcription factors
that are involved in various biological processes including development
and physiological homeostasis (13). Small lipophilic molecules such as
steroids, thyroid hormones, vitamin D, and retinoids bind to and
activate these receptors to exert their physiological effects by
regulating the transcription of specific genes (13-15). These
receptors share common structural features, including central, highly
conserved DNA binding domains and carboxyl-terminal ligand binding
domains (13-15). They can be divided into four major subgroups based
on their dimerization and DNA binding properties. Type II receptors are
characterized by their DNA binding as a heterodimer with the
9-cis-retinoic acid receptor (RXR) (13, 16). This group
includes the peroxisome proliferator-activated receptor (PPAR),
retinoic acid receptor, vitamin D receptor, liver X receptor (LXR), and
thyroid hormone receptors (TRs) (13, 17).
The farnesoid X receptor (FXR) was once an orphan receptor, and
recently, it was found that bile acids are the ligands for FXR
(18-20). FXR forms an obligate heterodimer with RXR and, thus, belongs
to the Type II nuclear receptor subgroup. FXR has been shown to bind to
FXR response elements (FXRE) composed of two inverted hexanucleotide
repeats (AGGTCA) spaced by one nucleotide (IR-1) (21). Chenodeoxycholic
acid (CDCA), the most potent ligand for FXR, induces the ileal bile
acid-binding protein (I-BABP) (18, 22), bile salt export pump (23),
phospholipid transfer protein (PLTP) (21, 24), apolipoprotein CII
(apoCII) (25), and SHP (26).
Activation of FXR down-regulates the expression of CYP7A1 via the
action of SHP protein (26). FXR-induced SHP binds to and inactivates
the liver receptor homolog 1(LRH), a transcription factor that is
required for the expression of cholesterol 7
-hydroxylase (CYP7A1)
(26, 27). CYP7A1 is a rate-limiting enzyme in the neutral pathway of
bile acid synthesis (28), and therefore, FXR activation inhibits bile
acid biosynthesis. Because bile acid is synthesized from cholesterol in
the liver and is the major route for the elimination of cholesterol
from the body, regulation of CYP7A1 transcription and activity
indicates a critical role of FXR in the regulation of cholesterol metabolism.
A number of genes involved in lipid metabolism whose hepatic expression
is decreased during the acute phase response are known to be regulated
by type II nuclear hormone receptors. Previously, we demonstrated that
in Syrian hamsters, LPS administration results in a decrease in hepatic
mRNA and/or protein levels of RXR
, -
, and -
, PPAR
and
-
, TR
and -
, and LXR
(29, 30). Furthermore, hepatic nuclear
extracts obtained from animals treated with LPS exhibited a reduced
binding activity to RXR-RXR, RXR-PPAR, RXR-TR, and RXR-LXR response
elements (29, 30). This suggests that reduced hepatic RXR levels alone
or in combination with decreases in PPARs and LXR could be a mechanism
for coordinately inhibiting the expression of multiple genes during the
acute phase response.
Expression of two proteins that are regulated by FXR, bile salt export
pump (BSEP) (31) and phospholipid transfer protein (PLTP) (32), are
decreased during the APR. Therefore, we hypothesized that the bile acid
receptor FXR is also suppressed during APR, affecting lipid/cholesterol
metabolism. In the present study, we demonstrate that LPS and
pro-inflammatory cytokines TNF
and IL-1
decrease the expression
of FXR, its DNA binding activity to FXR response element (IR1), and
transcription by FXR in studies of mice in vivo and the
Hep3B human hepatoma cells in vitro, which is accompanied by
decreased expression of FXR-regulated genes, SHP, and apoCII. The data
suggest that altered FXR activity may contribute to the changes in
lipid and cholesterol metabolism that occur during the APR.
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EXPERIMENTAL PROCEDURES |
Materials--
LPS (Escherichia coli 55:B5) was
obtained from Difco and freshly diluted to the desired concentration in
pyrogen-free 0.9% saline. Minimum essential medium (MEM) was purchased
from Fisher. Cytokines (human TNF
, human IL-1
, and human IL-6)
were from R&D Systems and were freshly diluted to desired
concentrations in serum-free MEM media containing 0.1% bovine serum
albumin (fatty acid-free). Tri-Reagent and fatty acid-free bovine serum
albumin were from Sigma. [
-32P]dCTP (3,000 Ci/mmol)
and [
-32P]dATP (3,000 Ci/mmol) were purchased from
PerkinElmer Life Sciences. Oligo(dT)-cellulose type 77F was from
Amersham Biosciences.
Animals--
Eight-week-old female C57BL/6 mice were purchased
from The Jackson Laboratory (Bar Harbor, ME). The animals were
maintained in a normal light-cycle room and were provided with rodent
chow and water ad libitum. Anesthesia was induced with
halothane. To determine the effect of APR on FXR and other mRNA
levels, mice were injected IP with 100 µg of LPS in saline or with
saline alone. Food was withdrawn at the time of injection, because LPS
induces anorexia in rodents (33). Livers were removed at the time
indicated in the figure legends (Figs. 1, 2, 4) after treatment.
The doses of LPS used in this study have significant effects on
triglyceride and cholesterol metabolism (7) but are not lethal because
the LD50 for LPS in rodents is ~5 mg/100 g of body weight.
Cell Culture--
Hep3B cells were maintained in MEM medium
supplemented with 10% fetal bovine serum in 75-cm2 flasks.
Cells were washed twice with phosphate-buffered saline (Ca2+- and Mg2+-free) and trypsinized before
seeding. For typical experiments, cells were seeded in 100-mm dishes at
a concentration of 2 × 106 cells/dish. After an
overnight incubation, cells were washed twice with phosphate-buffered
saline, and medium was replaced with fresh MEM (without serum) plus
0.1% bovine albumin and the appropriate cytokine concentration. For
transfection assays, 1.5 × 105 cells were used/well
in 6-well plates.
RNA Isolation and Northern Blot Analysis--
Total RNA from
mouse was isolated from 300-400 mg of snap-frozen whole liver and
~100 mg of kidney tissue using Tri-Reagent (Sigma).
Poly(A)+ RNA was subsequently purified using oligo(dT)
cellulose. RNA was quantified by measuring absorption at 260 nm. 10 µg of poly(A)+ were denatured and electrophoresed on a
1% agarose, formaldehyde gel. Total RNA from Hep3B was isolated from a
100-mm dish by the Tri-Reagent method and resuspended in diethyl
pyrocarbonate-treated water. 30 µg of total RNA was denatured and
electrophoresed as described above. The uniformity of sample loading
was checked by UV visualization of the ethidium bromide-stained gel
before electrotransfer to Nytran membrane (Schleicher & Schuell).
Prehybridization, hybridization, and washing procedures were performed
as described previously (34). Membranes were probed with
[
-32P]dCTP-labeled cDNAs using the random priming
technique (Amersham Biosciences). mRNA levels were detected by
exposure of the membrane to x-ray film and quantified by densitometry.
Glyceraldehyde-3-phosphate dehydrogenase was used as a control probe.
LRH-1 cDNA was kindly provided by Dr. Kristina Schoonjans (Institut
de Genetique et de Moleculaire et Cellulaire, Universite Louis Pasteur,
Paris, France). Mouse and human FXR, SHP, and apoCII probes were
prepared by PCR using the following primers: FXR 5'-CGT GAC TTG CGN CAA GTG ACC-3' (upper), 5'-CCA NGA CAT CAG CAT CTC AGC-3' (lower); SHP
5'-AGG GGT CTG CCC ATG CCA G-3' (upper), 5'-GGT CAC CTC AGC AAA AGC ATG
TC-3' (lower); apoCII 5'-GCC AAG GAG GTT GCC AAA G-3' (upper), 5'-GGT
CTG GTG ATG CGA GCA A-3' (lower).
Preparation of Nuclear Extracts--
Nuclear extracts were
prepared according to Neish et al. (35). Briefly, cells were
disrupted in a sucrose-HEPES buffer containing 0.5% Nonidet-P40 as a
detergent, protease inhibitors, and dithiothreitol. After disruption by
5 min of incubation on ice and centrifugation, nuclear proteins were
separated in a NaCl-HEPES buffer and re-suspended in a
glycerol-containing buffer. All the procedures were carried out on ice.
Protein quantification was determined by the Bradford assay (Bio-Rad),
and yields were similar in control and cytokine-treated groups.
Electromobility Shift Assay--
10 µg of crude nuclear
extract were incubated on ice for 30 min with 6 × 104
cpm of 32P-labeled oligonucleotides in 15 µl of binding
buffer consisting of 20% glycerol, 25 mM Tris-HCl, pH 7.5, 40 mM KCl, 0.5 mM MgCl2, 0.1 mM EDTA, 1 mM dithiothreitol, 2 µg of
poly(dI-dC), and 1 µg of salmon sperm DNA. Double-stranded
oligonucleotide probes were end-labeled with T4-polynucleotide kinase
(Amersham Biosciences) in the presence of 50 µCi of
[
-32P]dATP and purified on a Sephadex G-25 column
(Amersham Biosciences). DNA-protein complexes were separated by
electrophoresis (constant voltage of 200 V) on a 5% nondenaturing
polyacrylamide gel in 0.5× Tris-buffered EDTA at 4 °C. The gel was
dried, exposed to x-ray film, and quantified by densitometry. In the
competition assay, a 100-fold molar excess of the specific or mutated
unlabeled oligonucleotide was preincubated on ice for 1 h with 10 µg of nuclear extract from control cells. The following
oligonucleotides were used: IR1,
5'-GATCGGCCAGGGTGAATAACCTCGGGG-3'; mut-IR1,
5'-GATCGGCCAGGAAGAATATTCTCGGGG-3'.
Transfection Studies--
Hep3B cells were grown overnight in
35-mm plates and washed twice with serum-free medium. DNA-Lipofectin
complex containing 1.5 µg/ml FXRE-luciferase vector (a gift from Dr.
Mangelsdorf, University of Texas Southwestern Medical Center, Dallas,
TX), 0.5 µg/ml Rous sarcoma virus
-galactosidase vector (a gift
from Dr. Allan Pollock, Nephrology section at Veterans Affairs Medical Center, San Francisco, CA), and 5 µg/ml Lipofectin (Invitrogen) were
allowed to form at room temperature for 15 min. The cells were overlaid
with the DNA-Lipofectin complex and incubated at 37 °C for 4-6 h.
After washing the cells with serum-free medium, fresh growth medium
containing 10% fetal bovine serum was added.
Enzyme Assay--
Transfected cells were treated with lysis
buffer (Promega, Madison, WI), and aliquots of the lysates were assayed
for luciferase and
-galactosidase enzyme activity as described in
the manufacturer's instruction using Wallac
VICTOR2TM 1420 Multilabel Counter (PerkinElmer Life Sciences).
-Galactosidase enzyme activity was used to normalize for variability in the transfection efficiency.
Statistical Analysis--
Data are expressed as the mean ±S.E.
of experiments from 3-5 animals or plates for each groups or time
point. The difference between two experimental groups was analyzed
using the Student's t test. Differences among multiple
groups were analyzed using one way analysis of variance with the
Bonferroni's post hoc. A p value < 0.05 was considered significant.
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RESULTS |
LPS Decreases FXR mRNA Level--
We initially determined the
effect of LPS administration on the FXR mRNA levels in mouse liver
at various times up to 24 h. As shown in Fig.
1A, LPS (100 µg)
administration did not cause any significant change in the FXR mRNA
level at 2 h. However, FXR mRNA decreased significantly by
8 h to ~40% that of the control level, and the reduction
persisted for 24 h. Next, we determined if FXR mRNA decreases
in a dose-dependent manner in response to LPS
administration. As shown in Fig. 1B, the LPS-induced
decrease in FXR mRNA was dose-dependent in mouse liver,
with the half-maximal effect occurring at ~0.5 µg/100 g of body
weight. All doses tested in the experiment caused a significant
reduction in FXR mRNA levels. Thus, LPS decreases FXR mRNA
levels in the liver of mice at relatively low doses.

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Fig. 1.
Effect of LPS on FXR expression in
mouse. A, time course in liver. C57BL/6 mice were
injected IP with either saline or LPS (100 µg of LPS/mouse), and the
animals were sacrificed at the time indicated after LPS administration.
Poly(A)+ RNA was prepared from liver, and Northern blot
analysis was carried out as described under "Experimental
Procedures." B, dose response in liver. C57BL/6 mice were
injected IP with LPS at various concentrations as indicated, and
animals were sacrificed 16 h after LPS administration.
C, effect of LPS on FXR expression in the kidney. C57BL/6
mice were injected IP with LPS (100 µg of LPS/mouse), and animals
were sacrificed 16 h after LPS administration. Mouse kidney
poly(A)+ RNA was prepared, and Northern blot analysis was
carried out as described under "Experimental Procedures." Data
(means ± S.E., n = 4~5) are expressed as a
percentage of controls. *, p < 0.05 versus
control.
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Because hepatic expression of FXR is down-regulated by LPS
administration in mouse liver, next we examined if FXR expression is
altered in the intestine and the kidney, which also express FXR. As in
the liver, LPS administration for 16 h caused a significant decrease (~55%) in the level of FXR mRNA in the kidney (Fig.
1C). However, FXR mRNA was not reduced in the intestine
(~130%), suggesting that LPS causes tissue-specific responses in
terms of FXR expression. These results indicate that LPS-induced APR
reduces expression of FXR in mouse liver and kidney but not in the intestine.
LPS Administration Decreases the DNA Binding Activity of
FXR--
Nuclear hormone receptors exert their effect on
transcriptional regulation by binding to their cognitive response
element in the promoter region of target genes. To determine whether
the reduction of FXR mRNA caused by LPS administration affects the DNA binding activity of FXR, we isolated nuclei from mouse liver and
performed the electrophoretic gel mobility shift assay using a
32P-labeled DNA oligonucleotide containing FXR response
element IR-1. As shown in Fig.
2A, two major FXR·IR1
complexes were observed in the control samples. LPS
administration significantly decreased the binding of proteins in the
nuclear extract from mouse liver to IR1 when compared with the control.
LPS decreased FXR·IR1 binding by ~75 and ~50% for the upper and
the lower band, respectively (Fig. 2B). Competition with
100-fold molar excess of specific oligonucleotide (WT), but
not with mutated oligonucleotide (Mut), abolished the
complex formation of radiolabeled IR1 with FXR (Fig. 2A),
demonstrating the specificity of the two complexes. Furthermore, we
demonstrated that the complexes contain FXR and RXR. They were supershifted with anti-FXR (SS1, second lane) and anti-RXR
(SS2, third lane) antibody (Fig.
3). The migration of FXR·DNA complex was not affected by nonspecific IgG (fifth lane).

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Fig. 2.
Effect of LPS treatment on binding of hepatic
nuclear extracts to FXR response element IR1. C57BL/6 mice were
injected IP with either saline or LPS (100 µg of LPS/mouse). Sixteen
hours later, the animals were sacrificed, and hepatic nuclear extracts
(NE) were prepared as described under "Experimental
Procedures." Ten micrograms of nuclear extracts were incubated with
radiolabeled oligonucleotides IR1 representing binding site for FXR.
A, a representative electrophoretic gel mobility shift
assay. Unlabeled specific (100× WT) and nonspecific
(100× Mut) competing oligonucleotides were included at a
100-fold excess 1 h before the addition of the labeled probes.
Arrows represent specific IR1-bound complexes. B,
densitometric analysis of hepatic DNA-binding proteins. Data
(means ± S.E., n = 5) are expressed as a
percentage of controls. *, p < 0.05 versus
control.
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Fig. 3.
Protein IR1 complexes contains FXR and
RXR. Electrophoretic mobility shift assay was performed using a
nuclear extract from a control mouse in the presence of antibodies
raised against FXR (lane 2), RXR (lane 3), both
FXR and RXR (lane 4), and rabbit IgG (lane 5).
SS1 and SS2 represent the complexes supershifted by the FXR and RXR
antibodies, respectively.
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LPS Administration Reduces mRNA Levels of SHP and
ApoCII--
To determine whether the decreased binding of FXR to
DNA in hepatic nuclear extracts from LPS-treated mouse is associated with reduced transcription of FXR-regulated genes, we investigated the
effect of LPS administration on the mRNA levels of SHP and apoCII,
two of genes that are known to be regulated by FXR. SHP is a key
mediator of the FXR effect on the transcriptional regulation of CYP7A1
gene, and SHP is induced by FXR activation. As shown in Fig.
4, LPS administration decreases the
expression of SHP by ~70% and apoCII by 60% in mouse liver when
compared with control. These data demonstrate that FXR-regulated genes
are also repressed during LPS-induced APR in the mouse.

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Fig. 4.
Effect of LPS on the expression of FXR target
genes SHP and apoCII. Hepatic total RNA was prepared 16 h
after saline or LPS administration (100 µg of LPS/mouse) from mouse
liver. Northern blot analysis was performed as described under
"Experimental Procedures" using SHP and apoCII cDNAs. Data
(means ± S.E., n = 4~5) are expressed as a
percentage of controls. *, p < 0.05 versus
control.
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LRH Expression Decreases during APR--
It is well established
that FXR down-regulates CYP7A1 expression as a feedback mechanism to
maintain homeostasis of bile acid metabolism (26, 27). This is achieved
by FXR-induced stimulation of SHP expression (26). SHP binds to and
inactivates LRH, which is required for the transcriptional activation
of CYP7A1 (26). Therefore, based on the above results showing a
decrease in FXR and SHP, it is reasonable to expect an increase in the
level of CYP7A1 during the APR. However, our previous studies
demonstrated that the expression of Cyp7A1 is down-regulated during the
APR induced by LPS administration in vivo (36). Thus, we
determined the effect of LPS on the expression of LRH in mouse liver.
As shown in Fig. 5, LPS administration
for 16 h caused an ~65% reduction in the LRH mRNA levels in
mouse liver. This result suggests that the LPS-induced decrease in LRH
may play a major role in determining the transcriptional regulation of
CYP7A1.

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Fig. 5.
LPS administration decreases the expression
of LRH in mouse liver. Hepatic total RNA was prepared 16 h
after saline or LPS administration (100 µg of LPS/mouse) from mouse
liver. Northern blot analysis was performed as described under
"Experimental Procedures." Data (means ± S.E.,
n = 4~5) are expressed as a percentage of controls.
*, p < 0.05 versus control.
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TNF and IL-1, but Not IL-6, Decrease FXR mRNA in Hep3B Human
Hepatoma Cells--
It is well known that the physiological effect of
LPS is mediated by pro-inflammatory cytokines such as TNF, IL-1, and
IL-6. To determine whether these cytokines also decrease FXR mRNA
in vitro, as LPS does in mouse liver, human hepatoma cell
line Hep3B cells were treated with cytokines at 10 ng/ml for 24 h,
and RNA was isolated for Northern analysis. As shown in Fig.
6, TNF treatment decreased FXR mRNA
to less than 20% that of the control level. Also, IL-1 decreased FXR
mRNA by 60%. However, IL-6 did not affect FXR expression in Hep3B
cells (data not shown). These results suggest that the effect of LPS on
FXR expression is mediated by TNF and IL-1.

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Fig. 6.
Regulation of FXR expression by TNF and IL-1
in Hep3B human hepatoma cells. ~7 × 105 Hep3B
cells were plated in 100-mm Petri dishes in culture media containing
10% serum. After an overnight incubation, cells were washed twice with
phosphate-buffered saline, and medium was replaced with fresh MEM
(without serum) plus 0.1% bovine albumin and the appropriate cytokine
at 10 ng/ml. After 24 h of incubation, total RNA was isolated, and
Northern blot analysis was performed as described under "Experimental
Procedures." Data (means ± S.E., n = 3) are
expressed as a percentage of controls. *, p < 0.05 versus control.
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TNF Reduces the DNA Binding Activity of FXR in Hep3B
Cells--
Because the inflammatory cytokine TNF causes a
reduction in the FXR mRNA, we next determined if TNF also causes a
similar change in the DNA binding activity of nuclear extracts from
human hepatoma cells to an IR-1 FXR response element. For this
experiment, Hep3B cells were treated with TNF at 10 ng/ml. After
24 h the nuclei were isolated, and an electromobility shift assay
was conducted as described under "Experimental Procedures." As
shown in Fig. 7A, TNF
treatment at 10 ng/ml in Hep3B cells reduced the binding of FXR to IR1,
confirming the in vivo result. The reduction of DNA binding
activity was ~75 and ~55% that for the upper and the lower band,
respectively (Fig. 7B). Competition with 100-fold molar
excess of IR1 oligonucleotide (WT), but not of mutated
oligonucleotide (Mut), abolished the shift of DNA band (Fig.
7A), indicating the complex specificity. Furthermore, the
FXR·DNA complex showed supershifting after incubation of control
nuclear extract with anti-FXR antibodies (Fig.
8). These results indicate that the
decrease in FXR expression is associated with a decline in the DNA
binding activity of FXR during the acute phase caused by the
proinflammatory cytokine TNF in Hep3B cells.

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Fig. 7.
Effect of LPS treatment on binding of Hep3B
cell nuclear extracts to FXR response element IR1. Hep3B cells
were treated with either vehicle or TNF at 10 ng/ml for 24 h, and
hepatic nuclear extracts (NE) were prepared as described
under "Experimental Procedures." Ten micrograms of nuclear extracts
were incubated with radiolabeled oligonucleotides IR1 representing
binding site for FXR. A, a representative electrophoretic
gel mobility shift assay. Unlabeled specific (100× WT) and
nonspecific (100× Mut) competing oligonucleotides were
included at 100-fold excess 1 h before the addition of the labeled
probes. Arrows represent specific IR1-bound complexes.
B, densitometric analysis of DNA-binding proteins. Data
(means ± S.E., n = 4) are expressed as a
percentage of controls. *, p < 0.05 versus
control.
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Fig. 8.
Protein binding to IR1 contains FXR.
Electrophoretic mobility shift assay was performed as described under
"Experimental Procedures" using a nuclear extract from a
vehicle-treated Hep3B cells in the absence (lane 1) or
presence of antibody raised against FXR (lane 2).
Arrows indicate the complexes supershifted by the FXR
antibody.
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TNF and IL-1 Decrease the Expression of FXR-regulated Genes SHP and
ApoCII--
We next examined if the expression levels of two genes
that are known to be regulated by FXR are also affected by TNF and IL-1
in Hep3B human hepatoma cells. Treatment of Hep3B cells with TNF and
IL-1 at 10 ng/ml for 24 h decreased SHP mRNA level by ~60
and ~50%, respectively (Fig.
9A). Likewise, both TNF and
IL-1 significantly decrease apoCII mRNA by 65 and 57%,
respectively. Furthermore, TNF also inhibited CDCA-stimulated
expression of apoCII (Fig. 9B). These results
demonstrate that TNF and IL-1 decrease both the basal and stimulated
expression of FXR-regulated target genes in Hep3B human hepatoma
cells.

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Fig. 9.
TNF and IL-1 suppress the expression of FXR
target genes SHP and apoCII. A, TNF and IL-1 inhibit
the basal expression of SHP and apoCII in human hepatoma cells. Hep3B
cells were cultured for 24 h with vehicle or cytokine treatment
(10 ng/ml) as indicated in the figure. Total RNA was prepared, and
Northern blot analysis was performed as described under "Experimental
Procedures." Data (means ± S.E., n = 3) are
expressed as a percentage of controls. *, p < 0.05 versus control. B, inhibition of CDCA-stimulated
apoCII expression by TNF and IL-1. Hep3B cells were cultured in the
vehicle (Control) or 50 µM CDCA alone or with
TNF (CDCA + TNF), and apoCII mRNA was measured as described above.
Data (means ± S.E., n = 3) are expressed as a
percentage of controls. a, p < 0.05 versus control; b, p < 0.05 versus CDCA.
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Inhibition of FXRE-Luciferase Activity by TNF and IL-1--
To
further elucidate the effect of cytokine treatment on the expression of
genes regulated by FXR, we next carried out the transfection studies
using a FXRE construct linked to luciferase. The FXRE-Luc construct
contains five copies of FXR response element with basal promoter
activity. Luciferase activity was not observed in the absence of FXR
stimulation. Therefore, basal luciferase activity was stimulated with a
natural ligand of FXR, chenodeoxycholic acid. As shown in Fig.
10, both TNF and IL-1 almost completely abolished the activity of luciferase linked to FXRE when compared with
control. This result clearly demonstrates that TNF and IL-1 treatment
of Hep3B cells inhibits the expression of FXRE-regulated genes.

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Fig. 10.
Inhibition of FXRE-luciferase activity by
TNF and IL-1. Hep3B cells were plated in 6-well plates (1.5 × 105) the day before transfection in MEM media
supplemented with 10% fetal bovine serum. Transient transfections were
performed as described under "Experimental Procedures." The next
day cells were washed twice with serum-free media, and the basal
luciferase activity was stimulated by incubating cells in
CDCA-containing media supplemented with 0.1% bovine serum albumin.
After 30 min, cells were treated with 10 ng/ml TNF or IL-1 as indicated
in the figure using the serum-free media supplemented with 0.1% bovine
serum albumin and cultured for 24 h. At the end of the incubation,
cells were harvested in a lysis buffer, and luciferase and
galactosidase activity were determined (Promega). Data (means ± S.E., n = 3) are expressed as a percentage of controls.
*, p < 0.05 versus control.
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 |
DISCUSSION |
Infection, inflammation, and trauma induce a wide range of
metabolic changes in the liver as part of APR and result in the altered
concentration of plasma proteins that are collectively called acute
phase proteins (1, 37). During the APR, the levels of proteins such as
C-reactive protein and serum amyloid A increase (positive acute phase
proteins), whereas the levels of proteins such as albumin and
transferrin decrease (negative acute phase proteins) (1, 37, 38). It is
believed that positive acute phase proteins play a role in protecting
the host by neutralizing foreign agents to minimize tissue damage (39). Inflammatory cytokines such as TNF, IL-1, and IL-6 mediate the APR and
the transcriptional regulation of positive and negative acute phase
proteins. Activation of the TNF/IL-1 receptors stimulates membrane
sphingomyelinase to convert sphingomyelin to ceramide, which leads to
the activation and translocation of transcription factors activator
protein-1 and nuclear factor
B and induces class I acute phase
proteins (40, 41). On the other hand, activation of the IL-6 receptor
activates Janus tyrosine kinase and subsequently phosphorylates
tyrosine residue of signal transducer and activator of transcription
proteins, inducing transcription of class II proteins (41, 42).
The mechanisms regulating the negative acute phase proteins are not as
well understood. Recently, our studies together with others showed that
several nuclear hormone receptors including HNF-4, RXRs, retinoic acid
receptors, LXRs, PPARs, TR, constitutive androstane receptor, and
pregnane X receptor are down-regulated in the liver during the
APR (29, 30, 43, 44) and suggested that they might be involved in the
repression of several negative acute phase proteins. For example, a
decrease in PPAR
may contribute to the reduced expression of
acyl-CoA synthetase (29, 34) and carnitine palmitoyltransferase I (45)
during the APR. Similarly, a decrease in LXR, LRH, and SHP may
contribute to the decrease in cholesteryl ester transfer protein (46).
Because many of the proteins regulated by nuclear hormone receptors are
involved in the metabolism of triglyceride, cholesterol, and bile
acids, it is likely that repression of key nuclear receptors during the APR may be one of the mechanisms accounting for the changes in lipid metabolism.
In the present study, we show that the induction of the APR by LPS
administration decreases the level of FXR mRNA in mouse liver. The
decrease of FXR mRNA was significant as early as ~8 h after LPS
administration. Comparing this to our previous study, which showed that
RXR protein levels were significantly decreased as early as 2 h
(29), the decrease of FXR mRNA is preceded by the repression of
RXR. We also investigated the level of FXR mRNA in the kidney and
the intestine, two other organs involved in bile acid metabolism.
Interestingly, FXR mRNA expression was also down-regulated in the
kidney but not in the intestine, indicating that the response of FXR
mRNA to LPS is tissue-specific. In the liver, FXR mRNA is
decreased by low doses of LPS (the half-maximal dose for FXR mRNA
was ~0.5 µg/100g of body weight), indicating that the
reduction in FXR mRNA is a highly sensitive response to LPS.
Furthermore, in vitro experiments with Hep3B cells show that
TNF and IL-1, but not IL6, decreased the level of FXR mRNA, suggesting that the decrease of FXR mRNA by LPS in mouse liver is
mediated by the action of the inflammatory cytokines TNF and IL-1.
The decrease in FXR mRNA levels in the liver during the APR may
influence the transcription of its target genes. Our study shows that
FXR binding to FXR response element IR1 is decreased after LPS and
cytokine treatment in the nuclear extract from mouse liver and Hep3B
cells, respectively. In the electromobility shift assay, we were able
to observe two bands that were shifted from the unbound free probe, and
both bands were supershifted by the FXR antibody. The presence of two
bands could result from the presence of other proteins such as RXR,
coactivators, and/or corepressors. Also, the supershift experiment
confirms that RXR is a partner of FXR.
The decrease of FXR binding to the response element could result in the
reduced transcription of target genes such as I-BABP (ileal bile
acid-binding protein) (22), BSEP (23), PLTP (21, 24), and apoCII (25).
For example, the hepatic expression of BSEP, one of the FXR target
genes, has been reported to be down-regulated by LPS, IL-1, and TNF
administration (31). Also, Jiang and Bruce (32) report that LPS
administration decreased plasma activity of PLTP and its mRNA level
in the liver by ~66%. Transfection experiment in the present study
clearly demonstrates that TNF and IL-1 almost completely block
FXRE-linked luciferase activity, consistent with the decreased
expression of FXR target genes by TNF and IL-1. These results agree
with our finding that apoCII mRNA level is down-regulated by LPS in
mouse liver and by TNF and IL-1 in Hep3B human hepatoma cells. ApoCII
mRNA was reduced by ~60%, which is comparable with the changes
in PLTP and BSEP.
A decrease in the expression of FXR target genes may provide an
explanation for some of the changes in the lipid metabolism during the
APR. For example, APR-induced down-regulation of PLTP may disturb the
transfer of phospholipids and cholesterol from TG-rich lipoproteins to
high density lipoprotein and contribute to the increase of low density
lipoproteins. In the present study, we show that expression of apoCII
is decreased during the APR. ApoCII is a surface component of
chylomicrons, very low density lipoproteins, and high density
lipoproteins and plays an important role in plasma lipid metabolism as
an activator of lipoprotein lipase (47). Indeed, apoCII deficiency
has been linked to the increased plasma levels of triglycerides (48).
Therefore, lower apoCII expression during by the APR may contribute to
the increased levels of plasma triglycerides that occur during the
APR.
Altered expression of FXR target genes during the APR could also affect
bile acid synthesis and metabolism. When the high levels of bile acids
accumulate in the body, FXR is activated by its natural ligands and
negatively regulates the transcription of cholesterol 7
-hydroxylase
(CYP7A1) as a compensatory mechanism (27, 28). This negative regulation
by FXR is mediated by the induction of SHP, an orphan nuclear receptor
that inhibits LRH activation of CYP7A1 expression (26). Our present
study shows that FXR repression resulted in the reduced level of SHP
during the APR, which could theoretically lead to increased levels of CYP7A1 during the APR. However, our previous work demonstrated that
CYP7A1 is down-regulated during the LPS-induced APR in Syrian hamsters
(36). There are a number of possible explanations for the decrease in
CYP7A1. First, we showed that the level of LRH (mouse homologue of
CYP7A promoter-binding factor), a nuclear hormone receptor that
is required for the transcription of CYP7A1, is also decreased during
the APR; a decrease in LRH would decrease the transcription level of
CYP7A1 despite a reduction in SHP. Little is known about the
transcriptional regulation of LRH, and more studies need to be
conducted to further understand the implications of altered expression
of LRH during the APR. A second mechanism is that CYP7A1 is regulated
by other nuclear hormone receptors including HNF-4 (49, 50), chicken
ovalbumin upstream promoter-transcription factor II (50, 51), RXR (51),
and LXR
(52, 53). Transcription of CYP7A1 is induced by transfection
with HNF-4 alone in HepG2 cells and chicken ovalbumin upstream
promoter-transcription factor II cotransfection with HNF-4 had a
synergistic effect (50). RXRs bind to a DR1 motif in the promoter
region and transactivate gene transcription (51). In rats and mouse,
LXR is activated in response to oxysterols and induces CYP7A1
transcription to facilitate the excretion of excess cholesterol from
the body in the form of bile acids (52, 53). We and others demonstrate that RXR, LXR, and HNF-4 are decreased during the APR (29, 45), conferring limitations on the amount of the transcriptional activators for CYP7A1. A recent report on repression of CYP7A1 by TNF via the
HNF-4 site in the bile acid response element also supports the idea of
HNF-4 being another key player in the transcriptional regulation of
CYP7A1 (54). Finally, repression of CYP7A1 expression by high levels of
bile acids, but not by FXR ligands, in SHP-null mice suggests that
alternative mechanisms exist for the feedback regulation of CYP7A1
besides FXR/SHP pathway (55). Taken together with other studies (56,
57) that suggest redundant regulatory system for bile acid
biosynthesis, it is, thus, not surprising that decreased levels of SHP
would not necessarily result in the increased expression of CYP7A1
during the APR in the face of independent decreases in LRH, HNF-4,
LXRs, and RXRs.
The effects of LPS are mediated by its stimulation of various immune
cells to synthesize and secrete cytokines including TNF, IL-1, and IL-6
(1). TNF, IL-1, and IL-6 have all been shown to play important roles in
regulating the increased expression of positive APR proteins. However,
in our studies in which TNF and IL-1 inhibit the expression of nuclear
hormone receptors, IL-6 does not have any effect (58). Similarly in the
present study, TNF and IL-1 caused a decrease in the mRNA levels of
FXR and SHP, whereas IL-6 did not have any significant effect. The results suggest that TNF and IL-1 are major mediators for the negative
regulation of nuclear hormone receptors and that IL-6 is not involved
in this process.
In summary, the present study demonstrates that LPS causes a marked
decrease in FXR mRNA and activity in mouse liver. This was
associated with reduced expression of FXR target genes SHP and apoCII.
Furthermore, cytokines TNF and IL-1 caused a similar decrease in FXR
expression, DNA binding activity, transcription, and target genes in
Hep3B cells. Coupled with previous studies on the repression of RXRs,
retinoic acid receptors, PPARs, LXRs, TR, constitutive androstane
receptor, and pregnane X receptor, the results with FXR suggest that
decreases in type II nuclear hormone receptors are major contributors
to the negative APR. Considering the crucial physiological role of FXR
in the feedback regulation on bile acid synthetic pathway and in the
metabolism of cholesterol and lipoproteins, our study helps us to
understand the underlying mechanisms of the changes in lipid metabolism
during the APR.