1 Laboratoire de Pharmacologie, Unite Mixte de Recherche Centre National de la Recherche Scientifique 7561, 54505 Vandoeuvre-lès-Nancy; and 2 Laboratoire de Biologie du Développement, Unite propre de l'enseignement superieur 2402, 54500 Vandoeuvre-lès-Nancy, France
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ABSTRACT |
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This work demonstrated the constitutive expression
of peroxisome proliferator-activated receptor (PPAR)- and PPAR-
in rat synovial fibroblasts at both mRNA and protein levels. A decrease in PPAR-
expression induced by 10 µg/ml lipopolysaccharide (LPS) was observed, whereas PPAR-
mRNA expression was not modified. 15-Deoxy-
12,14-prostaglandin J2
(15d-PGJ2) dose-dependently decreased LPS-induced cyclooxygenase (COX)-2 (
80%) and inducible nitric oxide synthase (iNOS) mRNA expression (
80%), whereas troglitazone (10 µM) only inhibited iNOS mRNA expression (
50%). 15d-PGJ2 decreased
LPS-induced interleukin (IL)-1
(
25%) and tumor necrosis factor
(TNF)-
(
40%) expression. Interestingly, troglitazone strongly
decreased TNF-
expression (
50%) but had no significant effect on
IL-1
expression. 15d-PGJ2 was able to inhibit
DNA-binding activity of both nuclear factor (NF)-
B and AP-1.
Troglitazone had no effect on NF-
B activation and was shown to
increase LPS-induced AP-1 activation. 15d-PGJ2 and
troglitazone modulated the expression of LPS-induced iNOS, COX-2, and
proinflammatory cytokines differently. Indeed, troglitazone seems to
specifically target TNF-
and iNOS pathways. These results offer new
insights in regard to the anti-inflammatory potential of the PPAR-
ligands and underline different mechanisms of action of
15d-PGJ2 and troglitazone in synovial fibroblasts.
peroxisome proliferator-activated receptor- ligands; rat
synovial fibroblasts; proinflammatory cytokines
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INTRODUCTION |
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RHEUMATOID ARTHRITIS
(RA) is a chronic inflammatory disease characterized by proliferative
and invasive synovitis. Proinflammatory cytokines such as interleukin
(IL)-1 and tumor necrosis factor (TNF)-
have been shown to play a
pivotal role in the pathogenesis of the synovitis in RA (1, 2, 7,
30). Therefore, counteracting the production of inflammatory
mediators by synoviocytes may be an important key in prevention of the
development of this articular pathology leading to cartilage degradation.
The peroxisome proliferator-activated receptors (PPARs) are members of
the nuclear receptor supergene family that function in ligand-activated
transcription (20, 21). PPARs bind to specific response
elements as heterodimers with the retinoid X receptor (14)
and activate transcription of target genes in response to a variety of
endogenous and exogenous ligands. PPARs consist in three isoforms,
which differ in their tissue distribution and ligand specificity.
PPAR- has been shown to play a major regulatory role in adipocyte
and macrophage differentiation and glucose homeostasis
(21). PPAR-
is mainly found in tissues that exhibit
high levels of lipid catabolism, such as the liver, whereas PPAR-
expression is ubiquitous and its physiological role is unclear.
It has been demonstrated that these nuclear receptors are involved in
inflammation control and, especially, in modulating proinflammatory
cytokine production. Indeed, PPAR- ligands inhibited production of
TNF-
, IL-1
, and IL-6 in monocytes (11) and
expression of inducible nitric oxide synthase (iNOS) and gelatinase B
in activated macrophages (5, 18) and reduced TNF-
expression in obese rat adipose tissue (15). Their
involvement in the inflammation process has been demonstrated in other
cell types including astrocytes, glial cells (16), and
mast cells (26) and in a mouse model of inflammatory bowel
disease (25). However, contradictory results exist,
because a recent work showed that thiazolidinediones were unable to
suppress the production of TNF-
and IL-6 in lipopolysaccharide (LPS)- or phorbol myristate acetate-stimulated monocytes or macrophages (29). The same observations were made with
thiazolidinedione-treated mice (29).
Concerning PPAR-, it was shown that inflammation due to either
arachidonic acid or leukotriene B4 was prolonged in
PPAR-
knockout mice compared with controls (6). Studies
performed in smooth muscle cells suggested that PPAR-
activators
could also modulate inflammation in the vascular wall by inhibiting the
IL-1-induced production of IL-6 and prostaglandins as well as
cyclooxygenase (COX)-2 expression (23).
The involvement of these nuclear receptors in inflammatory or
degenerative arthopathies is relatively unexplored, and we previously demonstrated (3) the presence of PPAR- and PPAR-
in
rat articular cartilage. We also showed (3) that
two PPAR-
ligands, 15-deoxy-
12,14-prostaglandin
J2 (15d-PGJ2) and, at a lesser level,
troglitazone, could counteract the decrease in proteoglycan synthesis
and the nitric oxide (NO) production induced by IL-1
. Recently, a
synovial tissue expression of PPAR-
was detected in patients with
RA, and this study showed that intraperitoneal administration of
PPAR-
ligands ameliorated adjuvant-induced arthritis with
suppression of pannus formation in rats (12).
In the present work, we demonstrated the presence of PPAR- and
PPAR-
in rat type B synovial cells at both mRNA and protein levels
and demonstrated a decrease in PPAR-
expression after treatment with
LPS. We further showed that 15d-PGJ2 and troglitazone modulate the expression of proinflammatory genes differently and that
15d-PGJ2 exerts its action at least partly by antagonizing the activities of transcription factors AP-1 and nuclear factor (NF)-
B. These results offer new insights in regard to the
implication of PPAR-
in the actions of 15d-PGJ2 and
troglitazone in LPS-stimulated B synoviocytes.
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MATERIALS AND METHODS |
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B synoviocyte isolation and culture. Synovial tissues were obtained from Wistar male rats (130-150 g; Charles River) killed under dissociative anesthesia [ketamine (Rhône-Mérieux) and acepromazine (Sanofi Santé Animale)]. Synoviocytes were obtained by sequential digestion with Pronase and collagenase B (Roche Molecular Biochemicals, Meylan, France). The cells were washed two times in phosphate-buffered saline (PBS) and cultured to confluence in 25-cm2 flasks at 37°C in a humidified atmosphere containing 5% CO2. The medium used was DMEM-Ham's F-12 supplemented with L-glutamine (2 mM), gentamicin (50 µg/ml), amphotericin B (0.5 µg/ml), and heat-inactivated fetal calf serum (10%; Life Technologies). The synovial cells were used were between passages 3 and 6, at which point they exhibited a fibroblastic morphology.
LPS treatment in presence of
PPAR- ligands.
Twelve hours before the treatments, complete medium was replaced by
serum-free medium. The ligands or the vehicle (0.1% Me2SO in final concentration) were then added to synovial cells 12 h before the addition of LPS. The ligands used were troglitazone (1 and
10 µM) and 15d-PGJ2 (1 and 10 µM; France-Biochem,
Meudon, France). Synoviocytes were then treated with LPS
(Escherichia coli 0111:B4, 10 µg/ml; Sigma) for 12 h.
Quantitative RT-PCR analysis.
Total RNA was extracted from cell cultures by a single-step guanidinium
thiocyanate-phenol chloroform method using Trizol reagent (GIBCO BRL,
Cergy-Pontoise, France). RNA was recovered in ultrapurified water and
quantified by spectrophotometry at 260 and 280 nm. PPAR-, PPAR-
,
iNOS, and COX-2 mRNA from rat synovial cells were assayed using a
quantitative multistandard RT-PCR method that takes advantage of both
gene of interest and L27 ribosomal protein sequence conservation
between rat and human (13). This protocol allowed us to
normalize the amounts of the gene mRNA to be measured with respect to
those of L27 mRNA. For each sample, RNA purified from rat synoviocytes
was mixed with a constant amount of total RNA prepared from human
chondrocyte cultures, which carried both competitive human L27 and gene
of interest sequences and thus acted as a multistandard source. The mixture was reverse-transcribed using hexamer random primers. PCRs for
each gene amplification were undertaken with oligonucleotide primers
that are able to hybridize with rat and human sequences with the same
efficiency. For each gene, preliminary kinetic experiments were
performed to determine the number of cycles allowed to be in the
exponential phase of amplification. For PPAR-
amplification [29
cycles, annealing temperature (AT) = 61°C], the direct primer extended from nucleotides 738 to 761 and the reverse primer from nucleotides 1084 to 1065, in accordance with the human sequence (GenBank accession no. NM005036). For PPAR-
amplification (30 cycles, AT = 64°C), the direct primer extended from nucleotides 841 to 866 and the reverse primer from nucleotides 1364 to 1339, in
accordance with the human sequence (GenBank accession no. U79012). For
iNOS amplification (27 cycles, AT = 62°C), the direct primer extended from nucleotides 1489 to 1512 and the reverse primer from
nucleotides 1921 to 1897, in accordance with the human sequence (GenBank accession no. L09210). For COX-2 amplification (28 cycles,
AT = 59°C), the direct primer extended from nucleotides 410 to
432 and the reverse primer from nucleotides 850 to 830, in accordance
with the human sequence (GenBank accession no. M90100). For L27
amplification (27 cycles, AT = 62°C), the direct primer extended
from nucleotides 63 to 81 and the reverse primer from nucleotides 287 to 268, in accordance with the human sequence (GenBank accession no.
L05094). Each amplification product was then distinguished by
restriction site polymorphism between the two species. Amplification
products were quantitated after electrophoresis and analysis of
ethidium bromide-stained gels, using the L27 from the same cDNA pools
as an internal control. Results are expressed as the ratio
(generat/genehuman) × (L27human/L27rat) in arbitrary units.
Western blot analysis.
Immunoblotting analysis of PPAR- and PPAR-
was performed on
synoviocytes treated or not with LPS for 12 h. Briefly, harvested cells were washed two times with ice-cold PBS and centrifuged at
170g for 10 min at 4°C. The cell pellet was resuspended in 200 µl of ice-cold buffer (in mM: 25 HEPES, 400 KCl, 1 EDTA, and 1.5 MgCl2) supplemented with a protease inhibitor mixture
(Roche Molecular Biochemicals), mixed vigorously, and incubated for 30 min on ice. The cell extract was centrifuged at 10,000 g for
10 min, and protein concentration was determined in the supernatant. Fifty micrograms of protein from each sample were mixed with gel loading buffer (50 mM Tris, 10% glycerol, 10% SDS, 10%
2-mercaptoethanol, and 2 mg bromphenol blue) in a volume ratio of 1:1,
boiled for 3 min, and electrophoresed on a discontinuous
SDS-polyacrylamide gel (4% stacking gel and 10% separative gel). The
proteins were transferred onto a polyvinylidene difluoride membrane
(Millipore), which was saturated by incubation for 1 h at room
temperature in Tris-buffered saline-Tween buffer containing 3% bovine
serum albumin. The membranes were then incubated with anti-rabbit
PPAR-
(1:1,000) or anti-rabbit PPAR-
(1:2,500) antibodies (Santa
Cruz Biotechnology) for 1 h at room temperature. They were further washed three times with Tris-buffered saline-Tween and then incubated with anti-rabbit or anti-goat immunoglobulins (IgGs) coupled to peroxidase (1:20,000; Interchim). The immunocomplexes were visualized by the enhanced chemiluminescence method (Interchim).
Immunocytochemical analysis.
Cell layers were fixed in 3% formaldehyde in PBS for 10 min and
permeabilized in methanol for 20 min at 4°C. They were then exposed
to the primary antibody (diluted 1:500 for anti-PPAR- and 1:2,000
for anti-PPAR-
-2 antibodies in PBS) for 30 min at 37°C. After two
washes in PBS, cells were incubated with fluorescein-conjugated goat
anti-rabbit IgG (diluted 1:100 in PBS) for 30 min at 37°C. Negative
controls were performed by replacing the primary antibody with
preimmune serum. Cells were then mounted in Vectashield medium and
photographed with a Polyvar microscope (Reichert-Jung, Vienna, Austria).
Nitrite and IL-1 assays.
NO production was determined spectrophotometrically by measuring the
accumulation of nitrite (NO
was
measured in the supernatants by an enzyme-linked immunosorbent assay
method according to the manufacturer's instructions (Biosource
International). Results were expressed in picograms per milliliter.
Nuclear extracts and electrophoretic mobility shift assay.
For electrophoretic mobility shift assay experiments, rat synovial
cells were stimulated or not with LPS for 1 h after a
preincubation of 12 h with the ligand or Me2SO
(0.1%). Cell monolayers were scrapped in a lysis buffer [in mM: 10 HEPES, pH 7.9, 10 KCl, and 1 mM dithiothreitol (DTT)] containing a
protease inhibitor cocktail and 0.5% Igepal. After a 15-min incubation
on ice, nuclei were collected by centrifugation at 1,500 g
for 5 min at 4°C. The nuclear pellets were resuspended in 50 µl of
the same buffer without Igepal and KCl but with 420 mM NaCl. After a
30-min incubation on ice, nuclear debris were removed by centrifugation
at 13,000 g for 10 min at 4°C and the supernatants were
collected and stored at 80°C before use.
Statistical analysis. After comparison of data by analysis of variance, different groups were compared with Fisher's t-test. Assays were made in triplicate (except where otherwise indicated), and P values (vs. control or LPS treatment) <0.05 were considered significant.
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RESULTS |
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Constitutive expression of PPAR- and
PPAR-
in rat B synovial cells:
modulation of expression by LPS at mRNA
and protein levels.
By the use of RT-PCR analysis, we first demonstrated the constitutive
expression of PPAR-
and PPAR-
mRNA in normal synovial B cell
cultures (Fig. 1). An immunocytochemical
analysis further allowed us to visualize the expression of these
receptors at the protein level as well as their intracellular
distribution (Fig. 2). Fluorescence
signals specific for PPAR-
and PPAR-
were mainly localized to the
nucleus with a lower presence in the cytoplasm (Fig. 2, A
and C). We thereafter studied the modulation of the nuclear
receptors' expression by LPS (10 µg/ml for 12 h) at both the
mRNA and protein levels. The use of a multistandard quantitative RT-PCR
analysis showed that PPAR-
mRNA expression was strongly decreased by
LPS treatment, whereas PPAR-
mRNA expression remained unmodified
(Fig. 1). The results of the Western blot analysis further showed that
changes in mRNA levels encoding PPAR-
and PPAR-
on treatment with
LPS could be related to the variations of the corresponding proteins
(Fig. 3).
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Ability of PPAR- ligands
15d-PGJ2 and troglitazone to modulate
LPS-induced iNOS and COX-2
mRNA expression.
To test the capability of specific agonists to modulate LPS-induced
iNOS and COX-2 mRNA expression, synoviocytes were preincubated for
12 h in the presence of PPAR-
ligands or vehicle (0.1%
Me2SO) alone before being stimulated with LPS (10 µg/ml)
for 12 h. The ligands used were 15d-PGJ2 (1 and 10 µM) and troglitazone (1 and 10 µM). It should be noted that both
agonists were tested at 100 µM, and at this concentration they were
found to be highly toxic to the cells, as shown by a strong increase in
extracellular lactate dehydrogenase activity (data not shown). LPS
treatment for 12 h induced strong COX-2 (Fig.
4A) and iNOS (Fig.
4B) mRNA expression in synovial cells. We observed that
15d-PGJ2 dose-dependently decreased both COX-2 and iNOS
mRNA expression (
80% at 10 µM). Conversely, at the two doses used,
troglitazone was less effective than 15d-PGJ2 in modulating
iNOS mRNA expression (
50% at 10 µM), whereas it was ineffective in
modulating the induction of COX-2 expression. Nitrite assay in
culture supernatants showed that 15d-PGJ2 and troglitazone,
at both concentrations tested, significantly decreased NO
production (Table 1).
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Ability of PPAR- ligands
15d-PGJ2 and troglitazone to modulate
LPS-induced IL-1
and
TNF-
expression.
As described above, synovial B cells were preincubated in the presence
of PPAR-
ligands for 12 h before being stimulated with LPS (10 µg/ml for 12 h). As expected, LPS induced strong IL-1
(Fig.
5A) and TNF-
(Fig.
5B) mRNA expression in synovial cells compared with
controls. Preincubation in the presence of the highest dose of
15d-PGJ2 led to the modulation of both IL-1
(
25%) and
TNF-
(
40%) mRNA expression. Interestingly, troglitazone at 10 µM had an important action in modulating the expression of TNF-
mRNA (
50%) but had no significant effect on the IL-1
mRNA
expression induced by LPS. The IL-1
protein assay showed that only
15d-PGJ2 at 10 µM was able to reduce IL-1
production (
50%; Table 1). Troglitazone had no effect, and this confirmed the
results at the mRNA level.
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Effect of PPAR- ligands
15d-PGJ2 and troglitazone on
NF-
B and AP-1 activation pathways in
LPS-stimulated synovial B cells.
The transcription factors NF-
B and AP-1 are well known to control,
at least in part, the induction of several genes involved in the
inflammatory process, such as IL-1
, TNF-
, COX-2, and iNOS. In
several cell lines, it was recently demonstrated (23, 25)
that the activation of PPAR-
or PPAR-
could lead to the inhibition of NF-
B and AP-1 activities. To observe the
potential antagonizing effect of PPAR-
ligands on NF-
B and AP-1
activation pathways in synovial fibroblast cultures, we preincubated
cells for 12 h in the presence of 10 µM 15d-PGJ2 or
10 µM troglitazone before stimulation with LPS (10 µg/ml) for
1 h. A gel-shift analysis was then performed on nuclear extracts
to evaluate the binding of NF-
B and AP-1 to specific radiolabeled
probes. We observed that 15d-PGJ2 induced a strong
inhibition of the DNA-binding activity of NF-
B (
48%; Fig.
6B) and AP-1 (
55%; Fig.
6A). In contrast, troglitazone had no significant effect on
NF-
B DNA-binding activity and, surprisingly, was shown to strongly
increase the DNA-binding activity of AP-1 (+160%). Incubation of
nuclear proteins with 100-fold concentrated respective unlabeled probe
was performed to indicate the specificity of binding of NF-
B or AP-1
to the DNA. Moreover, preincubation in the presence of specific
antibodies allowed identification of the components of the protein
complex as being p65-p50 heterodimer for NF-
B and c-jun-c-fos for
AP-1.
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DISCUSSION |
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Several cell types are present in joints, and each of them can
exert an action on the other, especially during the time course of RA.
Thus the synovium appears to be the principal tissue involved in the
pathogenesis of the disease by producing metalloproteinases, proinflammatory cytokines, and NO in quantities sufficient to cause cartilage damage (32). Indeed, in in vitro models,
human synovial fibroblasts, when cocultured with human macrophages, induced cartilage degradation that was mediated in part by IL-1 and
TNF-
(22). In a previous work (3), we
studied the potential role of PPAR-
ligands in IL-1
-stimulated
rat chondrocytes and showed that mainly 15d-PGJ2, but also
troglitazone, could counteract the deleterious effects of this
cytokine. In the present work, we focused on the pharmacological
effects of PPAR-
ligands on cells importantly involved in the
development of RA, synovial fibroblasts.
By RT-PCR, Western blot, and immunocytochemical analysis, we
demonstrated that PPAR- and PPAR-
are expressed in rat synovial B
cell cultures at both mRNA and protein levels. We then showed that the
expression of these nuclear receptors was modulated differently by LPS
stimulation. Indeed, only PPAR-
expression, at both mRNA and protein
levels, was decreased by the treatment. Thus PPAR-
seems not to be
involved in the cellular responses to LPS in synovial cells. Several
papers have reported that PPAR-
expression is affected by a number
of immune mediators. Among these, IL-4 (9), granulocyte/macrophage colony-stimulating factor, macrophage
colony-stimulating factor (17), glucocorticoids, and
9-cis-retinoic acid have been reported to upregulate
PPAR-
expression. On the other hand, TNF-
, IL-1, IL-6, leukemia
inhibitory factor (28), and leptin have been reported to
downregulate PPAR-
expression. It seems difficult to attribute
clinical relevance to these up- and downregulations of PPAR-
expression. However, the fact that only PPAR-
, and not PPAR-
, is
regulated by LPS may suggest that PPAR-
is involved in the LPS
pathway. The same modulation of PPAR-
expression was demonstrated
previously in rat chondrocytes stimulated with IL-1
(3).
Thus we further evaluated the ability of PPAR- ligands
(15d-PGJ2 and troglitazone) to modulate the effects of LPS
stimulation and, especially, their ability to counteract the production
of proinflammatory cytokines (IL-1
and TNF-
) after LPS treatment. Our results showed different abilities of 15d-PGJ2 and
troglitazone to counteract the effects of LPS. Indeed,
15d-PGJ2 dose-dependently decreased LPS-induced COX-2
(
80%) and iNOS (
80%) mRNA expression, whereas troglitazone (10 µM) only inhibited iNOS mRNA expression (
50%) and had no effect on
COX-2 mRNA expression. These effects were also observed at the level of
NO production. Concerning the induction of proinflammatory cytokines,
we observed that 15d-PGJ2 modulated LPS-induced IL-1
(
25%) and TNF-
(
40%) mRNA expression. However, it is
interesting that troglitazone only decreased LPS-induced TNF-
mRNA
expression (
50%) but had no significant effect on the induction of
IL-1
mRNA.
In the recent literature, it has been demonstrated that PPAR- (or
PPAR-
) is very specifically involved in the control of cell
activation, depending on both the cell type and the nature of the
stimulating agent. Indeed, 15d-PGJ2 suppressed the
LPS-induced expression of COX-2 in the macrophage-like differentiated
U937 cells but not in vascular endothelial cells (10). In
another work performed with smooth muscle cells, PPAR-
ligands, but
not PPAR-
ligands, were efficient inhibitors of IL-1
-induced
production of IL-6 and prostaglandins and expression of COX-2
(23). Interestingly, in neonatal rat cardiac myocytes
stimulated with LPS, both PPAR-
and PPAR-
ligands inhibited the
LPS-induced expression of TNF-
mRNA (27).
Concerning the nature of the stimulating agent, it was shown in
monocytes that cytokine synthesis induced by LPS was largely refractory
to the effects of 15d-PGJ2 and troglitazone, whereas phorbol ester and okadaic acid-induced cytokine synthesis is
susceptible to their action (11). Thus the cell type and
the nature of the stimulating agent are important factors necessary to
take into account in evaluating the effects of PPAR- ligands in the
control of cell activation. Moreover, controversy also exists
concerning the identification of intracellular targets involved in the
mechanism of action of 15d-PGJ2. In several recent papers,
some data suggested a main contribution of PPAR-
-independent
mechanisms on the action of this prostaglandin, mostly because of the
lack of effect of synthetic PPAR-
ligands such as
thiazolidinediones. For instance, in macrophage cultures and in a mouse
model of endotoxemia, 15d-PGJ2 inhibited the LPS-induced
production of TNF-
and IL-6, whereas other high-affinity PPAR-
ligands failed to affect cytokine production (29). The
same observations have been made in LPS-stimulated microglia
(16). In these different studies, and in others, new intracellular targets have been identified, especially in the NF-
B
signaling pathway (4, 19, 24, 31). Several lines of
evidence suggest that the involvement of PPAR-
in the effects of
15d-PGJ2 would depend on the expression level of the
nuclear receptor in the cell type under consideration. To our
knowledge, there are no data in the literature describing the level of
constitutive expression of PPAR-
protein and the effects of
activators in rat synovial fibroblasts. The results of the present work
demonstrate that 15d-PGJ2 is more efficient than
troglitazone in modulating the effects of LPS on B synovial cells. In
particular, it appeared that the prostaglandin exerted a broader action
compared with the drug. Indeed, troglitazone, at 10 µM, was found to
have an important and specific action on both iNOS and TNF-
.
Thiazolidinediones are synthetic compounds that are very specific
agonists for PPAR-
[dissociation constant
(Kd) = 30-700 nM], whereas
15d-PGJ2 has a much lower affinity for the receptor
(Kd = 2 µM). Several works showed
that the anti-inflammatory effects of 15d-PGJ2 occur in a
concentration range (1-10 µM) that is consistent with its
Kd for PPAR-
, whereas troglitazone or
BRL-49653 often required much higher concentrations (50 µM) to
accomplish the same effects. In the present study, we tested
troglitazone at 50 and 100 µM, but these concentrations were shown to
be highly cytotoxic to B cells, in contrast to other studies often
performed on much more resistant cell lines. Thus this would strongly
suggest that the mechanism of action of 15d-PGJ2 in
synovial B cells is, at least in large part, PPAR-
independent.
Moreover, the effects observed with 10 µM troglitazone would also
indicate that iNOS and TNF-
gene expression is much more sensitive
to regulation by PPAR-
than IL-1
and COX-2. This could be
explained by a particular organization of the promoter of these two genes.
Some of the anti-inflammatory effects obtained through the activation
of PPAR- have been shown to occur by antagonizing the activities of
the transcription factors AP-1 and NF-
B. These inhibitory effects
may be obtained through direct protein-protein or protein-DNA
interactions, but they also may be achieved by sequestration of
essential transcription coactivators such as cAMP binding protein-p300
or steroid receptor coactivator-1. Thus, in the present work, by a
gel-shift analysis, we investigated possible inhibiting interactions
between PPAR-
and NF-
B or AP-1 transcriptional pathways. We
observed that 15d-PGJ2, but not troglitazone, was very
potent to reduce DNA-binding activity of NF-
B and AP-1. Surprisingly, troglitazone was even shown to increase AP-1 binding activity. These observations demonstrate that troglitazone modulates LPS-induced iNOS and TNF-
mRNA expression without inhibiting the
DNA-binding activity of NF-
B and AP-1. Thus its inhibiting action
seems not to be the result of a physical interaction between proteins
or between protein and DNA but rather the consequence of the titration
of AP-1 and NF-
B coactivators by the PPAR system. However, at this
point, it is not possible to confirm this hypothesis. To
further evaluate the capability of troglitazone or 15d-PGJ2 to inhibit
specifically the LPS-induced gene expression, it would be necessary to
perform transient transfection experiments using adequate reporter
vectors. This would allow the demonstration of the inhibiting action of
a PPAR-
ligand on the promoter activity of a specific gene.
In summary, our study demonstrated that PPAR- is constitutively
expressed in rat synovial fibroblasts at both mRNA and protein levels.
We found that, by the use of PPAR-
activators, it is possible to
modulate the effects of LPS on cultured B cells. However, our results
also showed that 15d-PGJ2 and troglitazone modulated the
expression of proinflammatory genes differently, probably through
PPAR-
-independent and -dependent pathways, respectively. Thus the
use of PPAR-
agonists may offer new insights in regard to their
anti-inflammatory potential by the inhibition of TNF-
, and the
specific use of 15d-PGJ2 would be an interesting tool to
counteract the deleterious effects of IL-1
that are responsible for
the degradation of cartilage.
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ACKNOWLEDGEMENTS |
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This work was supported in part by European Contract No. QLK6-CT-1999-02072 and the Association de la Recherche contre la Polyarthrite.
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FOOTNOTES |
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Address for reprint requests and other correspondence: P. Netter, Laboratoire de Pharmacologie, UMR CNRS 7561, Faculté de Médecine de Nancy, BP 184, 54505 Vandoeuvre-lès-Nancy. (E-mail: pharmaco{at}facmed.u-nancy.fr).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 9 March 2001; accepted in final form 5 September 2001.
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