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Prepublished online as a Blood First Edition Paper on August 29, 2002; DOI 10.1182/blood-2002-06-1762.
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Blood, 15 January 2003, Vol. 101, No. 2, pp. 545-551
HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
Fibrates down-regulate IL-1-stimulated C-reactive protein gene
expression in hepatocytes by reducing nuclear p50-NF B-C/EBP-
complex formation
Robert Kleemann,
Philippe P. Gervois,
Lars Verschuren,
Bart Staels,
Hans M. G. Princen, and
Teake Kooistra
From the Gaubius Laboratory Nederlandse Organisatie
voor toegepast natuurwetenschappelijk onderzoek (TNO)
Prevention and Health, Leiden, The Netherlands; Département
d'Athérosclérose, U.545 INSERM, Institut Pasteur de Lille;
and Faculté de Pharmacie, Université de Lille II,
France.
 |
Abstract |
C-reactive protein (CRP) is a major acute-phase protein in humans.
Elevated plasma CRP levels are a risk factor for cardiovascular disease. CRP is predominantly expressed in hepatocytes and is induced
by interleukin-1 (IL-1) and IL-6 under inflammatory situations, such as
the acute phase. Fibrates are hypolipidemic drugs that act through the
nuclear receptor peroxisome proliferator-activated receptor-
(PPAR-). Fibrates have been shown to reduce elevated CRP levels in
humans, but the molecular mechanism is unknown. In this study, we
demonstrate that different PPAR- activators suppress IL-1-induced,
but not IL-6-induced, expression of CRP in primary human hepatocytes
and HuH7 hepatoma cells. Induction of CRP expression by IL-1 occurs at
the transcriptional level. Site-directed mutagenesis experiments show
that IL-1 induces CRP expression through 2 overlapping response
elements, the binding sites for CCAAT-box/enhancer-binding protein-
(C/EBP-) and p50-nuclear factor- B (p50-NFB). Cotransfection of
C/EBP- and p50-NFB enhances CRP promoter activity, and
coimmunoprecipitation experiments indicate that the increase in CRP
promoter activity by IL-1 is related to the generation and nuclear
accumulation of C/EBP--p50-NFB complexes. Interestingly,
PPAR- activators reduce the formation of nuclear
C/EBP--p50-NFB complexes, and thereby CRP promoter activity, by
2 mechanisms. First, PPAR- increases IB- expression and thus
prevents p50-NFB translocation to the nucleus. Second, fibrates
decrease hepatic C/EBP- and p50-NFB protein levels in mice in a
PPAR--dependent way. Our findings identify C/EBP- and p50-NFB
as novel targets for PPAR- and provide a molecular explanation for
the reduction of plasma CRP levels by fibrates.
(Blood. 2003;101:545-551)
© 2003 by The American Society of Hematology.
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Introduction |
Among the liver-specific or liver-enriched genes
whose expression is strongly modulated during the acute phase of
inflammation is C-reactive protein (CRP). CRP is a major acute-phase
protein in humans, its plasma concentration increasing more than
1000-fold in severe inflammatory states.1,2 Several
studies have reported a predictive association between elevated plasma
CRP and coronary artery disease.3,4 There is increasing
evidence that CRP is not merely an important and unique risk marker but
that it also has a role in the pathogenesis of inflammation and
atherosclerosis.5-8 CRP is synthesized predominantly in
human liver, and the stimulation of CRP biosynthesis in response to
trauma and inflammation is mainly mediated by interleukin-1 (IL-1)
and IL-6.
Treatment of hyperlipidemia with fibrates reduces plasma CRP
concentrations.9,10 Fibrates are clinically used
hypolipidemic drugs that lower plasma levels of triglycerides and
cholesterol, both of which are established risk factors for
cardiovascular disease. Fibrates exert these beneficial activities on
lipid and lipoprotein metabolism through the activation of the nuclear
receptor, peroxisome proliferator-activated receptor-
(PPAR-).11 It is unclear whether PPAR- is also
involved in the down-regulation of CRP by fibrates.
PPAR- belongs to the superfamily of nuclear receptors that activate
gene expression on ligand binding and dimerization with the retinoid X
receptor (RXR). PPAR- RXR heterodimers bind to specific sequences
localized in the promoter region of target genes, termed peroxisome
proliferator response elements. In addition to its role in mediating
the hypolipidemic effects of fibrates, PPAR- has been shown to act
as a negative regulator of inflammatory processes by antagonizing the
activity of the transcription factor pathways, such as NF-B and
AP-1.9,12
Recently, we reported another anti-inflammatory mode of action of
fibrate-activated PPAR-, namely through the binding of GRIP1/TIF2
(glucocorticoid receptor-interacting protein 1/transcription intermediary factor 2), a coactivator of the CCAAT box/enhancer-binding protein- (C/EBP-).13 This mechanism appeared
relevant for quenching the expression of several
C/EBP-/GRIP1-regulated acute-phase response genes by fibrates,
including the down-regulation of the expression of fibrinogen-,
fibrinogen-, and serum amyloid A (SAA) genes. In
accordance with these findings, the effect of fibrates on the
fibrinogen gene expression was absent in PPAR- knock-out
mice.14 Because CRP is not an acute-phase protein in
mice,2 knowledge of the role of PPAR- in the regulation of CRP remained elusive.
Several recent studies indicate that the induction of CRP by IL-1
and IL-6 is at the transcriptional level, and it has been narrowed down
to a 300-bp promoter fragment that harbors binding sites for the
transcription factors signal transducer and activator of transcription
3 (STAT3),15 C/EBP-, and p50-nuclear factor-B (p50-NFB).16,17 Because the classical binding partner
of p50-NFB p65-NFBis not involved in the transcriptional
activation of CRP and p50/p65 heterodimers are not capable of binding
to the promoter, p50-NFB is thought to enhance CRP transcription by
facilitating the binding of transcriptionally active
C/EBP.16 It is possible that transcription factors that
transduce the effects of IL-1 and IL-6 on CRP expression are targets
for fibrate-activated PPAR-.
In the present paper we have investigated in detail the induction of
CRP by IL-1 and the mechanism of CRP promoter inhibition by fibrates.
It is shown that IL-1 and IL-6 strongly induce CRP expression in
primary cultures of human hepatocytes, but only the IL-1 effect could
be suppressed by fibrates and by a specific PPAR- activator, Wy
14643. Evidence is provided that the induction of CRP gene expression
by IL-1 requires the integrity of the overlapping response elements
(REs) for p50-NFB and C/EBP- and correlates with the accumulation
of IL-1-inducible p50-NFB-C/EBP- complexes in the nucleus. We
demonstrated that fibrates and Wy 14643 reduce the amount of
p50-NFB-C/EBP- complexes in the nucleus, and thereby CRP gene
expression, by preventing the translocation of p50-NFB from the
cytosol into the nucleus. It is shown that fibrates and Wy 14643 induce
a cytoplasmic inhibitor of NF-BIB- thus trapping p50-NFB in the cytosol. A second effect of fibrates on
p50-NFB-C/EBP- was observed in vivo when fenofibrate treatment
almost completely blocked the basal generation of p50-NFB and
C/EBP- in the livers of wild-type, but not PPAR- knock-out, mice.
Our findings provide a specific molecular mechanism for the
fibrate-induced down-regulation of CRP not shared with other
acute-phase response genes repressed by these drugs, and they explain
the uniqueness of CRP as a marker for inflammatory processes.
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Materials and methods |
Reagents
Ciprofibrate and bezafibrate were from Sanofi-Synthelabo
(Aramon, France) and Roche Molecular Biochemicals (Almere, The
Netherlands), respectively. Fenofibric acid was a kind gift of Dr A. Edgar (Laboratoires Fournier, Daix, France). Wy 14643 was from Chemsyn
(Lenexa, KS), and simvastatin was from Merck (Amsterdam, The
Netherlands). Human recombinant IL-1 and IL-6 were purchased from
Sanvertech (Heerhugowaard, The Netherlands). All antibodies used in
this study were from Santa Cruz Biotechnology (Heerhugowaard, The
Netherlands). Molecular biology reagents were obtained from Life
Technologies (Breda, The Netherlands). All other chemicals were
specified in the references cited or were purchased from Sigma-Aldrich
Chemicals (Zwijndrecht, The Netherlands).
Cell culture
Primary hepatocytes were isolated from human donor livers as
described.18 Experiments with primary hepatocytes were
approved by the institutional committees of the Leiden University
Medical Center and TNO Prevention and Health. Cellular viability was
greater than 85%, as determined by trypan blue exclusion. Cell culture conditions and experimental conditions for primary hepatocytes were as
reported.19
The human hepatoma cell line HuH7, a cell line with endogenous
CRP expression and responsiveness to IL-1, was a kind gift of Dr J. Rijntjes (Organon Teknika, Boxtel, The Netherlands). Hepatoma cells
were cultured in Dulbecco modified Eagle medium (DMEM) (Life
Technologies, Breda, The Netherlands) supplemented with 10% (vol/vol)
fetal calf serum, 100 IU penicillin, and 100 µg/mL streptomycin.
Animal studies
Animal studies were performed in compliance with European
Community specifications regarding the use of laboratory animals. Experimental conditions have been described previously.14
Briefly, male Sv/129 homozygous wild-type (+/+) and PPAR- knock-out
( /) mice (10 to 12 weeks of age) were fed for 17 days with either a
standard mouse chow or one containing 0.2% (wt/wt) fenofibrate. Animals were killed by exsanguination under ether anesthesia. Livers
were removed immediately, weighed, rinsed with 0.9% (wt/vol) NaCl,
frozen in liquid nitrogen, and stored at 80°C until use. For
immunoblotting, livers were homogenized in phosphate-buffered saline
(PBS) containing proteinase inhibitor (PI) (Roche Diagnostics) and
centrifuged at 10 000g and 4°C for 5 minutes, and soluble proteins were immediately boiled in Laemmli electrophoresis buffer for
immunoblot analysis.
Enzyme-linked immunosorbent assay measurements
Fibrinogen concentrations were measured by an enzyme-linked
immunosorbent assay (ELISA) procedure as previously
described.14 ELISA for CRP was performed according to an
established protocol20 using antibodies from DAKO
Diagnostics B.V. (Glostrup, Denmark).
Plasmids and luciferase assay
A genomic fragment corresponding to nucleotides 300 to 1 of
the human CRP promoter21 was amplified using the primers
5' CCT AGA TCT AGA GCT ACC TCC TCC TGC CTG G 3' and 5' CCG ACG CGT ACC
CAG ATG GCC ACT CGT TTA ATA TGT TAC C 3'. The primers were designed to
contain the BglII and MluI restriction sites,
respectively. PCR products were then cloned into the luciferase
reporter vector pGL3 (Promega, Leiden, The Netherlands). DNA sequences
were confirmed by bidirectional sequencing of the clones. Constructs
containing mutated binding sites for C/EBP- and p50-NFB were
generated following a published procedure17 using the
Quick Change Mutagenesis kit (Stratagene, Amsterdam, The Netherlands)
and the primers 5' GGA AAA TTA TTT ACA TAG TGT AGC TTA CTC CCT TAC TGC
TTT GG 3' and 5' CCA AAG CAG TAA GGG AGT AAG CTA CAC TAT GTA AAT AAT
TTT CC 3' for mutation of the C/EBP- binding site and 5' CAT AGT GGC
GCA AAC GAT ATT ACT GCT TTG GAT A 3' and 5' TAT CCA AAG CAG TAA TAT CGT
TTG CGC CAC TAT G 3' for mutation of the p50-NFB-binding site.
Mutant plasmids were bidirectionally sequenced to confirm sequence
identity. Generation of the human fibrinogen- promoter construct was
described previously.13
The human hepatoma cell line HuH7 was used for all reporter gene
assays. Applied fibrate concentrations did not affect cell viability,
as determined by the MTT (3-(4,5-cimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) viability assay.22 Reporter gene
assays were performed as described13 with the following
modifications: the FUGENE6 (Roche Diagnostics) reagent was used, and
100 ng luciferase reporter plasmid was transiently transfected in
1.2 × 105 cells. After 20 hours, luciferase activity was
quantified with the dual-luciferase reporter assay system (Promega)
according to the manufacturer's protocol.
Cell extracts and coimmunoprecipitations
Total and nuclear cell extracts were prepared in the presence of
PI (Roche Diagnostics), and all steps were performed at
4°C.23,24 Briefly, cells were washed twice with cold
PBS, scraped off in 1.3 mL cold PBS containing PI, and collected by
centrifugation at 800g for 5 minutes. Cell pellets were
resuspended in cold PBS-protease inibitor, and equal amounts were used
for the preparation of total and nuclear extracts. For the preparation
of total cell extracts, resuspended cells were boiled in Laemmli
electrophoresis buffer and stored at 80°C until use. For
preparation of nuclear extracts, cells were pelleted again and
resuspended in 1 mL cold hypotonic buffer containing 10 mM HEPES
(N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid), pH 7.9, 10 mM KCl, 0.1 mM EDTA (ethylenediaminetetraacetic acid), 0.1 mM EGTA (ethyleneglycoltetraacetic acid), 1 mM
dithiothreitol (DTT), and PI. Cells were allowed to swell for 15 minutes on ice. Then 62.5µL 10% (vol/vol) Nonidet P-40 was added,
and cells were lysed within 2 minutes under shaking. After
centrifugation at 1000g for 10 minutes, the supernatant was
removed and the pellet containing the nuclei was washed with cold
PBS/PI. Nuclei were incubated for 30 minutes in 20 mM HEPES, pH 7.9, 0.4 M KCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, and PI and were centrifuged
for 5 minutes at 10 000g, and the supernatant corresponding
to the nuclear extract was collected, boiled in Laemmli electrophoresis buffer, and stored at 80°C until use. Protein concentration was determined by the method of Bradford using a kit from Bio-Rad Laboratories (Veenendaal, The Netherlands).
Coimmunoprecipitation was performed as described
previously.13 Briefly, lysed nuclei were centrifuged, and
soluble nuclear proteins were incubated in 1.3 mL PBS/PI with
anti-p50-NFB antibody (1.5 µg/mL) or anti-MMP-8 (anti-matrix
metalloproteinase 8) control antibody (1.5 µg/mL) for 16 hours at
4°C. Complexes were immunoprecipitated with protein A-Sepharose
(Amersham Pharmacia Biotech, Roosendaal, The Netherlands) and washed 3 times in PBS/PI and 3 times in 50 mM Tris-HCl, pH 8.0, 170 mM NaCl,
0.5% (vol/vol) Nonidet P-40, and 50 mM NaF in the presence of PI.
Washed complexes were immediately boiled in Laemmli electrophoresis
buffer and analyzed by immunoblotting.
Western blotting
For sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE), samples were electrophoresed as described.13
Proteins were blotted onto Immobilon-P polyvinylidene fluoride transfer membranes (Millipore, Bedford, MA). Blots were blocked with 5% (wt/vol) skim milk powder (Merck, Amsterdam, The Netherlands) diluted
in 20 mM Tris (pH 7.4), 55 mM NaCl, and 0.1% (vol/vol) Tween-20. Blots
were developed with a goat anti-p50-NFB primary antibody, a rabbit
anti-C/EBP- primary antibody, or a rabbit anti-IB- primary
antibody and horseradish peroxidase-conjugated secondary
immunoglobulin, respectively. Antihistone H1 and anti--actin antibodies were used for control. All antibodies were diluted in 20 mM
Tris (pH 7.4), 55 mM NaCl, 0.1% (vol/vol) Tween-20, and 5% (wt/wt)
bovine serum and were used at a final concentration of 0.2 µg/mL. The
Super Signal West Dura Extended Duration Substrate (Pierce, St
Augustin, Germany) and the luminescent image workstation (Roche
Diagnostics) were used for visualization.
Statistical analysis
All data are presented as mean ± SD. Statistical analysis
was performed with the Student t test, and
P < .05 was considered statistically significant.
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Results |
Effects of ciprofibrate and Wy 14643 on CRP and fibrinogen
expression in primary human hepatocytes
Figure 1 shows the effects of
ciprofibrate and the PPAR- activator Wy 14643 on the synthesis of
CRP and, as a control, fibrinogen in primary human hepatocytes under
basal and IL-1- or IL-6-induced conditions during a 24-hour
incubation period. CRP concentrations were increased 23 times and 68 times by IL-1 and IL-6, respectively (Figure 1A). Ciprofibrate and Wy
14643 had no significant effect on basal CRP expression but strongly
reduced the IL-1 induction of CRP. In contrast, IL-6-induced CRP
expression was not or was only slightly reduced in the presence of
ciprofibrate and Wy 14643, respectively. Fibrinogen concentrations were
moderately but significantly increased 1.3 times with IL-1 and 2.1 times with IL-6 (P < .05). Ciprofibrate and Wy 14643 strongly decreased IL-6-induced fibrinogen production but showed no
(ciprofibrate) or only moderate (Wy 14643) suppressive effect on basal
and IL-1-increased fibrinogen synthesis (Figure 1B). These
differential effects of ciprofibrate and Wy 14643 on IL-1- and
IL-6-induced CRP and fibrinogen expression point to differences in CRP
and fibrinogen gene regulation mechanisms.
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| Figure 1.
Effect of ciprofibrate and Wy 14643 on CRP and
fibrinogen expression in human hepatocytes stimulated by IL-1 and IL-6.
CRP (A) and fibrinogen (B) concentrations were measured in culture
medium by ELISA and were expressed as means ± SDs. Human
hepatocytes were isolated and treated for 16 hours with 250 µM
ciprofibrate (CF), 250 µM Wy 14643 (WY), or vehicle (dimethyl
sulfoxide [DMSO]; C) and subsequently were stimulated with 25 ng/mL
IL-1 or IL-6 for an additional 24 hours. Results of 1 representative
experiment of 3 experiments with different donors are shown.
*P < .05 compared with control.
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CRP repression by fibrates is at the transcriptional level and
is specific for activators of PPAR-
We next investigated whether the negative interference of fibrates
with the IL-1-induced expression of CRP is at the transcriptional level. A 300-bp promoter fragment of the human CRP
gene21,25 containing the essential regulatory elements for
IL-1-mediated promoter activity26,27 was cloned in front
of a luciferase reporter gene, giving rise to pCRP-luc. The human
hepatoma cell line HuH7 was transiently transfected with pCRP-luc, and
cells were incubated with IL-1. Induction was optimal at 10 ng/mL IL-1 and resulted in 5- to 6-fold increased luciferase activity, as shown in
Figure 2. Treatment of HuH7 cells with
increasing concentrations (10-250 µM) of the structurally different
fibrates, gemfibrozil, ciprofibrate, or bezafibrate or the PPAR-
activator Wy 14643 resulted in a concentration-dependent inhibition of
IL-1-stimulated CRP promoter activity (Figure 2), with the strongest
inhibitory effect observed with the specific PPAR- activator Wy
14643. By contrast, BRL 49653 (10 µM), a specific activator of
PPAR- ,28 did not display an effect (Figure 2),
supporting the notion that specific activation of PPAR- is necessary
to inhibit CRP transcription.
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| Figure 2.
Inhibition of IL-1-induced human CRP promoter activity
by PPAR- activators in HuH7 cells.
HuH7 cells were transiently transfected with the human CRP promoter
(pCRP-luc) linked to a luciferase reporter and incubated with
increasing concentrations (10, 30, 50, 125, 250 µM) of gemfibrozil
(GF), ciprofibrate (CF), bezafibrate (BF), and Wy 14643 (WY) or
BRL49653 (BRL) (10 µM) for 5 hours and subsequently stimulated with
IL-1 (10 ng/mL) for 18 hours. Luciferase activities are expressed as
means ± SDs of several (3 or more) transfection experiments
performed in triplicate.
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IL-1-induced CRP expression is mediated by p50-NFB and
C/EBP-
Transcription factors that transduce the IL-1 effect on CRP
expression have not been investigated. To that end we analyzed the CRP
promoter fragment cloned in front of the luciferase reporter gene. This
fragment contains a 17-bp stretch with 2 distinct response elements.
One was a binding site for C/EBP- and the other a binding site for
p50-NFB (Figure 3A).16 To
delineate whether either of these sites was involved in
IL-1-stimulated CRP promoter activity, binding sites for C/EBP- and
p50-NFB within the pCRP-luc construct were mutated (Figure 3B).
Mutation of the p50-NFB binding site did not affect basal pCRP-luc
activity, but it completely repressed induction by IL-1 (Figure 3B).
Mutation of the C/EBP- response element core site in pCRP-luc did
not affect basal transcriptional activity either, but it significantly
reduced the induction of CRP promoter activity by IL-1. These results
point to a crucial role of p50-NFB and C/EBP- binding sites in
IL-1-induced CRP promoter activity.
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| Figure 3.
C/EBP- and p50-NFB mediate the induction of the CRP promoter by
IL-1.
(A) The fragment of the human CRP core promoter containing the
overlapping REs for C/EBP- and p50-NFB. (B) HuH7 cells were
transfected with the wild-type human CRP promoter construct pCRP-luc
and the mutated constructs pCRP p50NF-B-luc and
pCRPC/EBP--luc, as indicated, and subsequently were stimulated
with IL-1. (C) Dose-dependent 18-hour stimulation of HuH7 cells with
IL-1, as indicated, and Western blot analysis of p50-NFB and
C/EBP- in nuclear extracts. Levels of histone H1 are shown for
confirmation of equal loading. (D) HuH7 cells were transfected
with pCRP-luc in the presence of p50-NFB and C/EBP- expression
vectors or control vector (Con) and were stimulated with 10 ng/mL IL-1
for 18 hours. Luciferase activities represent means ± SDs of
several (3 or more) transfection experiments.
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In accordance with this, stimulation with IL-1 increased the nuclear
amount of p50-NFB and C/EBP- (Figure 3C), and overexpression of
p50-NFB or C/EBP- by cotransfection resulted in a 2-fold and a
10-fold increase in basal CRP promoter activity, respectively (Figure
3D). These increases in luciferase activity were further enhanced in
the presence of IL-1, which can be explained by the increased nuclear
translocation of p50-NFB (data not shown).
The absence of an IL-1 effect on mutation of the p50-NFB binding
site and the proximity of the binding sites for C/EBP- and
p50-NFB on the CRP promoter suggested to us that the 2 transcription factors act as a complex. To determine whether C/EBP- and p50-NFB are associated in regular human hepatocytes and HuH7 cells,
coimmunoprecipitation experiments were performed with nuclear protein
extracts. Specific coimmunoprecipitation of C/EBP--p50-NFB was
detected by anti-C/EBP- Western blot when anti-p50-NFB, but not
control antibody, was used for precipitation (Figure
4). Treatment of HuH7 cells and primary
human hepatocytes with IL-1 strongly increased nuclear C/EBP-p50-NFB levels, thus providing a molecular mechanism for the
IL-1-stimulated CRP transcription rate.
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| Figure 4.
Effect of PPAR- activators on the IL-1-induced
formation of C/EBP--p50-NFB complexes.
Coimmunoprecipitation was performed on nuclear extracts prepared from
HuH7 cells (A) and freshly isolated primary human hepatocytes (B),
preincubated for 6 hours with Wy 14643 (WY), fenofibric acid (FFA), or
vehicle, and subsequently stimulated with 10 ng/mL IL-1 (HuH7 cells) or
25 ng/mL IL-1 (primary hepatocytes) for 17 hours. PPAR- activators
were used at a final concentration of 50 µM and 250 µM in HuH7
cells and primary hepatocytes, respectively. Anti-p50-NFB or
anti-MMP-8 control antibody (Con) were used for immunoprecipitation.
C/EBP- bound to precipitated p50-NFB was detected by Western
blot analysis.
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Fibrates and Wy 14643 prevent nuclear C/EBP--p50-NFB
complex formation
To determine whether the suppressors of IL-1-induced CRP promoter
activitythat is, fibrates and Wy 14643interfere with nuclear C/EBP--p50-NFB complex formation, we performed
coimmunoprecipitation on nuclear extracts from IL-1-treated HuH7 cells
and primary human hepatocytes preincubated with activators of PPAR-.
As shown in Figure 4A-B, Wy 14643 and fenofibric acid markedly reduced
nuclear C/EBP-p50-NFB complex accumulation in IL-1-treated
hepatocytes, thus explaining their suppressive effects on
IL-1-stimulated CRP expression.
To evaluate at which level the various compounds interfere with
IL-1-induced nuclear C/EBP--p50-NFB accumulation, we analyzed total cellular and nuclear levels of C/EBP- and p50-NFB in HuH7 cells stimulated with IL-1 in the presence or absence of Wy 14643 or
ciprofibrate by Western blotting (Figure
5A). None of the compounds affected
IL-1-induced total and nuclear C/EBP- levels. However, though Wy
14643 and ciprofibrate were without effect on IL-1-induced total
p50-NFB levels, they strongly reduced IL-1-induced nuclear p50-NFB accumulation. These findings indicate that the inhibition of
p50-NFB translocation is the prime cause for the suppression of the
IL-1-induced C/EBP-p50-NFB accumulation by PPAR- activators in cultured human hepatocytes.
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| Figure 5.
Effect of PPAR- activators on the expression of
C/EBP-, p50-NFB, and IB- in HuH7 cells.
(A) HuH7 cells were preincubated with Wy 14643 (WY; 50 µM),
ciprofibrate (CF; 125 µM), or DMSO (Con) for 5 hours and subsequently
were stimulated with 10 ng/mL IL-1 for 17 hours. Total cellular (upper
panel) and nuclear (lower panel) extracts were analyzed for p50-NFB
and C/EBP- expression. (B) HuH7 cells were incubated with PPAR-
activators for 24 hours, and total cellular extracts were analyzed for
IB- by Western blotting. Equal loading was ensured by
demonstration of uniform -actin and histone H1 expression for total
and nuclear extracts, respectively.
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A possible explanation for impaired p50-NFB translocation from the
cytosol to the nucleus would be trapping of p50-NFB by a cytoplasmic
inhibitor of NFB, termed IB-. Indeed, Wy 14643 and
ciprofibrate all up-regulated IB- protein concentrations (Figure
5B). Because IB- effectively binds p50-NFB in the cytosol, it
prevents nuclear C/EBP--p50-NFB complex formation.
PPAR--dependent down-regulation of basal C/EBP- and
p50-NFB expression in vivo
Previously, it had been shown that PPAR- activation also
induces IB- expression in mice.29 Evaluation of the
hepatic expression of C/EBP- and p50-NFB in fenofibrate-treated
mice revealed novel targets for the activity of PPAR-. Treatment of mice with fenofibrate for 17 days resulted in strongly reduced C/EBP- and p50-NFB protein levels as analyzed by Western blotting (Figure 6). This reduction of C/EBP-
and p50-NFB was dependent on PPAR- because the effect of
fenofibrate was absent in PPAR- (/) mice. These effects may
further contribute to the observed reduction of plasma CRP levels in
patients treated with fibrates. The results also suggest that under
chronic conditions of PPAR- activation, a second
mechanismsuppression of C/EBP- and p50-NFB levelsis
operative.
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| Figure 6.
Down-regulation of basal p50-NFB and C/EBP-
expression on activation of PPAR- in vivo.
Wild-type (+/+) and PPAR- (/) mice were fed
normal chow or 0.2% (w/w) fenofibrate (FF)-containing chow
for 17 days, and total liver extracts were analyzed for p50-NFB and
C/EBP- by Western blotting. Levels of -actin are shown for
confirmation of equal loading.
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| |
Discussion |
Fibrates reportedly lower plasma CRP levels in
humans,9 but the regulatory mechanism of this effect
remains to be clarified. In this report, we demonstrate that fibrates
strongly inhibit IL-1-induced, but not IL-6-induced, CRP expression
in human hepatocytes. We show that the induction of CRP expression by
IL-1 is at the transcriptional level, requires the integrity of the
overlapping REs for p50-NFB and C/EBP-, and correlates with
increasing nuclear concentrations of C/EBP-p50-NFB complexes. We
found that fibrates and the PPAR- activator Wy 14643 inhibit CRP
transcription by reducing the formation of nuclear
C/EBP--p50-NFB complexes in 2 ways. First, we demonstrated that
PPAR- activators up-regulate IB- expression in vitro (current
study) and in vivo,29 thereby preventing p50-NFB
translocation into the nucleus. Second, we showed a PPAR--dependent
strong reduction of basal C/EBP- and p50-NFB expression levels in
the livers of mice treated with fibrate for several days. More
important, the results presented here give detailed insight into how
fibrates inhibit CRP expression in humans.
Our findings show that IL-1 and IL-6 strongly induce the expression of
CRP in human hepatocytes. This is in line with previous reports in
which dual control of CRP gene expression by IL-1 and IL-6 was
demonstrated in hepatoma cells,27 primary human
hepatocytes,30 and mice carrying the human CRP transgene
(hCRP).31 Interestingly, we found that fibrates and the
PPAR- activator Wy 14643 strongly suppressed the induction of CRP by
IL-1 but did not affect or only slightly affected the induction by IL-6
in primary human hepatocytes, suggesting different regulatory
mechanisms. Induction of CRP by IL-6 has been reported to involve the
IL-6-inducible transcription factors STAT3,15 C/EBP-,
and C/EBP- ,21,32 but the factors that participate in
transducing the effects of IL-1 on CRP expression, such as those
observed in IL-6(/) hCRP transgenic mice,31 remained
unclear. Our study provides the first molecular explanation for the
induction of CRP by IL-1. This IL-1 induction pathway in HuH7 cells is
direct and independent of IL-6, because an IL-6-inducible,
IL-6-RE-containing promoter fragment was not activated by IL-1 (R.K.,
unpublished data, December 2001). Functional analysis of the
promoter of the human CRP gene in HuH7 human hepatoma cells
revealed the existence of 2 overlapping response elements that are
crucial for full induction by IL-1the binding sites for C/EBP- and
p50-NFB. Because IL-1 induces the expression and nuclear
translocation of C/EBP- and p50-NFB and the cotransfection of
C/EBP- and p50-NFB was found to enhance CRP promoter activity, we
conclude that the induction of CRP by IL-1 involves the binding of the
transcription factors C/EBP- and p50-NFB to their respective CRP
promoter-binding sites. These findings also explain the unique position
of CRP among the acute-phase proteins. Although C/EBP- and STAT3
also mediate the expression of other IL-6-inducible acute-phase
proteins, such as haptoglobin and fibrinogen,13,32,33 the
stimulation of promoter activity by p50-NFB and C/EBP- is
specific for CRP. Indeed, a computer search for sequences similar to
the CRP promoter region harboring the (overlapping) REs for C/EBP-
and p50-NFB was without results. These differences in gene
regulation also provide a rationale for the use of CRP and fibrinogen
as independent risk markers34 in clinical and
epidemiologic studies to predict future cardiovascular events and to
underscore their additive value.
Our observation that deletion of the p50-NFB binding site completely
abolishes CRP transcription by IL-1 supports the idea of concerted
action of p50-NFB and C/EBP- and suggests interaction between the
2 transcription factors.17 We demonstrate for the first
time that C/EBP- and p50-NFB physically interact in the nucleus
under physiologically relevant expression levels, thereby providing a
physiologic basis and a functional role for earlier in vitro
overexpression studies.35,36 Because p50-NFB lacks a
transactivation domain and p65-NFB abolishes CRP
transcription,17 the mechanism by which p50-NFB
transactivates CRP is uncertain. Interaction with C/EBP- under
physiologic conditions gives strength to the hypothesis put forward by
Cha-Molstad et al17 that p50-NFB binds to the promoter
region followed by C/EBP-, which carries the required
transactivation domain.
The finding that ciprofibrate and Wy 14643 strongly suppress IL-1- but
not IL-6-induced CRP expression, together with the finding that these
2 cytokines differ in the induction of p50-NFB, suggested to us that
PPAR- activation might interfere with the induction or the nuclear
translocation of p50-NFB. Indeed, activators of PPAR- reduced the
formation of IL-1-inducible nuclear p50-NFB-C/EBP- complexes by
inhibition of the p50-NFB translocation. Cytosolic retention of
p50-NFB extends the anti-inflammatory properties of PPAR- that
have been mainly characterized by direct binding and inactivation of
transcription factors, such as p65-NFB and c-jun.9,37
Induction of IB- may also explain the broad inhibitory effect of
PPAR- activators on NF-B-regulated genes, including IL-6,
VCAM-1, and SAA.38,39
Among the nonlipid blood markers, CRP and fibrinogen are widely used in
clinical and epidemiologic studies as independent risk
markers34 to predict future cardiovascular events, but it
has remained unclear why they provide additive predictive value. We
showed that fibrates and activators of PPAR- markedly inhibited the
expression of fibrinogen, which is in accordance with previous observations.13,14 In contrast to fibrinogen,
IL-6-induced expression of CRP was not, or was only slightly,
inhibited by fibrates and Wy 14643, pointing to differences in CRP and
fibrinogen gene regulation mechanisms and their additive predictive
value as inflammation markers. This observation is of clinical
relevance because CRP levels provide additional information on the
activities of a given drug caused by its unique molecular mechanism of regulation.
Elevated plasma CRP levels are associated with an increased
inflammatory state and a higher risk for atherosclerosis and coronary heart disease.40 It is becoming increasingly clear that
CRP is not only a risk marker but also a risk factor, playing an active role in inducing adhesion molecule and monocyte chemoattractant protein-1 (MCP-1) expression.5,6 Given that direct CRP
effects are considered to worsen the patient's situation, the
down-regulation of CRP expression, as demonstrated here by the
activation of PPAR-, may contribute to the prevention and treatment
of cardiovascular diseases beyond merely risk factor correction.
The results of the present study demonstrate that IL-1 stimulates
hepatic CRP gene expression through C/EBP- and p50-NFB. Fibrates
and PPAR- activators reduce the formation of nuclear p50-NFB-C/EBP- complexes, and thereby CRP promoter activity, by
2 novel mechanisms, the inhibition of p50-NFB translocation and the
down-regulation of basal p50-NFB and C/EBP- expression in a
PPAR- dependent fashion, providing a first molecular explanation for
the reduced plasma CRP levels of humans treated with
fibrates9 and possibly contributing to a general
PPAR--dependent reduction of other acute-phase genes. These
observations extend our knowledge of the mechanisms of
anti-inflammatory activities of PPAR- effects, which are undoubtedly
complimentary to the beneficial effects of fibrates on lipid metabolism
and provide an additional rationale for the use of these drugs in
atherosclerosis therapy.
| |
Acknowledgments |
We thank Dr Alt Zantema for helpful discussions and Annette de Jong
for assistance with the experiments.
| |
Footnotes |
Submitted June 13, 2002; accepted August 14, 2002.
Prepublished online
as Blood First Edition Paper, August 29, 2002; DOI
10.1182/blood-2002-06-1762.
Supported by Netherlands Heart Foundation grants 99.104 (L.V.)
and 99.110 (R.K.) and by European Community Marie Curie Fellowship QLK-1-CT-1999-51206 (P.P.G.).
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
section 1734.
Reprints: R. Kleemann, Gaubius Laboratory, TNO
Prevention and Health, PO Box 2215, 2301 CE Leiden, The Netherlands;
e-mail: r.kleemann{at}pg.tno.nl.
| |
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Modulation of C-rea |
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Abstract
Introduction