PPAR-gamma ligands modulate effects of LPS in stimulated rat synovial fibroblasts

Marie-Agnès Simonin1, Karim Bordji1, Sandrine Boyault1, Arnaud Bianchi2, Elvire Gouze1, Philippe Bécuwe2, Michel Dauça2, Patrick Netter1, and Bernard Terlain1

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


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

This work demonstrated the constitutive expression of peroxisome proliferator-activated receptor (PPAR)-gamma and PPAR-alpha in rat synovial fibroblasts at both mRNA and protein levels. A decrease in PPAR-gamma expression induced by 10 µg/ml lipopolysaccharide (LPS) was observed, whereas PPAR-alpha mRNA expression was not modified. 15-Deoxy-Delta 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)-1beta (-25%) and tumor necrosis factor (TNF)-alpha (-40%) expression. Interestingly, troglitazone strongly decreased TNF-alpha expression (-50%) but had no significant effect on IL-1beta expression. 15d-PGJ2 was able to inhibit DNA-binding activity of both nuclear factor (NF)-kappa B and AP-1. Troglitazone had no effect on NF-kappa 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-alpha and iNOS pathways. These results offer new insights in regard to the anti-inflammatory potential of the PPAR-gamma ligands and underline different mechanisms of action of 15d-PGJ2 and troglitazone in synovial fibroblasts.

peroxisome proliferator-activated receptor-gamma ligands; rat synovial fibroblasts; proinflammatory cytokines


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

RHEUMATOID ARTHRITIS (RA) is a chronic inflammatory disease characterized by proliferative and invasive synovitis. Proinflammatory cytokines such as interleukin (IL)-1beta and tumor necrosis factor (TNF)-alpha 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-gamma has been shown to play a major regulatory role in adipocyte and macrophage differentiation and glucose homeostasis (21). PPAR-alpha is mainly found in tissues that exhibit high levels of lipid catabolism, such as the liver, whereas PPAR-delta 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-gamma ligands inhibited production of TNF-alpha , IL-1beta , 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-alpha 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-alpha 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-alpha , it was shown that inflammation due to either arachidonic acid or leukotriene B4 was prolonged in PPAR-alpha knockout mice compared with controls (6). Studies performed in smooth muscle cells suggested that PPAR-alpha 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-alpha and PPAR-gamma in rat articular cartilage. We also showed (3) that two PPAR-gamma ligands, 15-deoxy- Delta 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-1beta . Recently, a synovial tissue expression of PPAR-gamma was detected in patients with RA, and this study showed that intraperitoneal administration of PPAR-gamma ligands ameliorated adjuvant-induced arthritis with suppression of pannus formation in rats (12).

In the present work, we demonstrated the presence of PPAR-gamma and PPAR-alpha in rat type B synovial cells at both mRNA and protein levels and demonstrated a decrease in PPAR-gamma 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)-kappa B. These results offer new insights in regard to the implication of PPAR-gamma in the actions of 15d-PGJ2 and troglitazone in LPS-stimulated B synoviocytes.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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-gamma 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-gamma , PPAR-alpha , 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-alpha 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-gamma 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.

IL-1beta and TNF-alpha mRNA from rat synovial cell cultures were assayed using a semi-quantitative RT-PCR method. For IL-1beta amplification, the direct primer extended from nucleotides 501 to 522 and the reverse primer from nucleotides 843 to 867 (GenBank accession no. M98820). For TNF-alpha amplification, the direct primer extended from nucleotides 2 to 23 and the reverse primer from nucleotides 513 to 532 (GenBank accession no. X66539). 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.

Western blot analysis. Immunoblotting analysis of PPAR-gamma and PPAR-alpha 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-alpha (1:1,000) or anti-rabbit PPAR-gamma (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-alpha and 1:2,000 for anti-PPAR-gamma -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-1beta assays. NO production was determined spectrophotometrically by measuring the accumulation of nitrite (NO<UP><SUB>2</SUB><SUP>−</SUP></UP>) in culture supernatants by the Griess reaction (8). Briefly, 100 µl of culture supernatant were mixed with 100 µl of Griess reagent for 5 min at room temperature. The optical density was measured at 550 nm with a microplate reader, and nitrite concentrations were calculated with a standard curve of sodium nitrite ranging from 0 to 50 µM. IL-1beta 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.

The DNA sequences of the double-stranded oligonucleotide specific to NF-kappa B were 5'-GAT CCA GTT GAG GGG ACT TTC CCA GGC-3' and 5'-GAT CCG CCT GGG AAA GTC CCC TCA ACT G-3'. Those specific to AP-1 were 5'-GAT CCG CTT GAT GAC TCA GCC GGA AG-3' and 5'-GAT CCT TCC GGC TGA GTC ATC AAG CG-3'. Complementary strands were annealed, and double-stranded oligonucleotides were labeled with [32P]dCTP using the Klenow fragment of DNA polymerase (GIBCO BRL). Five micrograms of nuclear proteins were incubated for 10 min at 4°C in a binding buffer (20 mM Tris · HCl, pH 7.9, 5 mM MgCl2, 0.5 mM DTT, 0.5 mM EDTA, and 20% glycerol) in the presence of 2 µg of poly(dI-dC). The extracts were then incubated for 30 min at 4°C with 10,000 cpm of 32P-labeled NF-kappa B or AP-1 probes. The samples were loaded on a 5% native polyacrylamide gel and run in 0.5× Tris-borate-EDTA buffer. NF-kappa B- and AP-1-specific bands were confirmed by competition with a 100-fold excess of the respective unlabeled probe, which resulted in no shifted bands. For supershift experiments, after the addition of the labeled probe, the extracts were incubated for 30 min at 4°C in the presence of the specific antibody (anti-p65 or anti-p50 for NF-kappa B; anti-c-fos or anti-c-jun for AP-1).

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.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Constitutive expression of PPAR-alpha and PPAR-gamma 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-alpha and PPAR-gamma 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-alpha and PPAR-gamma 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-gamma mRNA expression was strongly decreased by LPS treatment, whereas PPAR-alpha mRNA expression remained unmodified (Fig. 1). The results of the Western blot analysis further showed that changes in mRNA levels encoding PPAR-alpha and PPAR-gamma on treatment with LPS could be related to the variations of the corresponding proteins (Fig. 3).


View larger version (49K):
[in this window]
[in a new window]
 
Fig. 1.   Expression at mRNA level of peroxisome proliferator-activated receptor (PPAR)-gamma (A) and PPAR-alpha (B) in lipopolysaccharide (LPS)-stimulated synovial cells (10 µg/ml for 12 h) and in control synovial cells. Left, electrophoretic profiles of RT-PCR products after specific amplification of each mRNA mixed with human mRNA. Rat PPAR-gamma product was digested in the presence of Alu I (A), and human PPAR-alpha product was digested by Stu I (B). Arrows indicate the bands corresponding to the rat product. Right, quantification by densitometric analysis with the L27 from the same cDNA pools as an internal control. Results are the means of 3 independent experiments in relative arbitrary units (RAU), and representative data are shown (n = 3; *P < 0.05 vs. control value).



View larger version (101K):
[in this window]
[in a new window]
 
Fig. 2.   Immunocytochemical analysis of PPAR-gamma (A and B) and PPAR-alpha (C and D) expression in LPS-stimulated rat synovial cells (10 µg/ml for 12 h; B and D) and in control cells (A and C).



View larger version (28K):
[in this window]
[in a new window]
 
Fig. 3.   Expression at the protein level of PPAR-gamma (A) and PPAR-alpha (B) in LPS-stimulated synovial cells (10 µg/ml for 12 h) and in control synovial cells. Top, Western blot analysis of nuclear receptor protein expression. Immunodetection was performed with polyclonal anti-PPAR-gamma or anti-PPAR-alpha antibodies and visualized with chemiluminescence. Bottom, densitometric analysis of each band. Results are representative of 3 independent experiments.

Ability of PPAR-gamma 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-gamma 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).


View larger version (47K):
[in this window]
[in a new window]
 
Fig. 4.   Effect of PPAR-gamma ligands [15-deoxy-Delta 12,14-prostaglandin J2 (15d-PGJ2), 1 and 10 µM; troglitazone, 1 and 10 µM] on LPS-induced cyclooxygenase (COX)-2 (A) and inducible nitric oxide synthase (iNOS; B) mRNA expression. Top, electrophoretic profiles of RT-PCR products for COX-2 (A) and iNOS (B) after specific amplification of mRNA with a quantitative multistandard RT-PCR method. A: rat but not human COX-2 product was digested by Ava I. Undigested product was loaded on lane 1; lane 2, control; lane 3, LPS induction; lane 4, LPS + 15d-PGJ2 1 µM; lane 5, LPS + 15d-PGJ2 10 µM; lane 6, LPS + troglitazone 1 µM; lane 7, LPS + troglitazone 10 µM; lane 8, molecular weight markers. B: human but not rat iNOS product was digested by Stu I. Lane 1, molecular weight markers. Undigested product was loaded on lane 2; lane 3, control; lane 4, LPS induction; lane 5, LPS + 15d-PGJ2 1 µM; lane 6, LPS + 15d-PGJ2 10 µM; lane 7, LPS + troglitazone 1 µM; lane 8, LPS + troglitazone 10 µM. Arrows indicate the bands corresponding to the rat product. Bottom, quantification by densitometric analysis with L27 from the same cDNA pools as an internal control. Results are means of 3 independent experiments, and representative data are shown (n = 3; *P < 0.05 vs. LPS treatment).


                              
View this table:
[in this window]
[in a new window]
 
Table 1.   Effect of PPAR-gamma ligands on LPS-induced IL-1beta and nitrite production

Ability of PPAR-gamma ligands 15d-PGJ2 and troglitazone to modulate LPS-induced IL-1beta and TNF-alpha expression. As described above, synovial B cells were preincubated in the presence of PPAR-gamma ligands for 12 h before being stimulated with LPS (10 µg/ml for 12 h). As expected, LPS induced strong IL-1beta (Fig. 5A) and TNF-alpha (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-1beta (-25%) and TNF-alpha (-40%) mRNA expression. Interestingly, troglitazone at 10 µM had an important action in modulating the expression of TNF-alpha mRNA (-50%) but had no significant effect on the IL-1beta mRNA expression induced by LPS. The IL-1beta protein assay showed that only 15d-PGJ2 at 10 µM was able to reduce IL-1beta production (-50%; Table 1). Troglitazone had no effect, and this confirmed the results at the mRNA level.


View larger version (49K):
[in this window]
[in a new window]
 
Fig. 5.   Effect of PPAR-gamma ligands (15d-PGJ2, 1 and 10 µM; troglitazone, 1 and 10 µM) on LPS-induced interleukin (IL)-1beta (A) and tumor necrosis factor (TNF)-alpha (B) mRNA expression. Top, electrophoretic profiles of RT-PCR products for IL-1beta (A) and TNF-alpha (B) after specific amplification of mRNA using a semiquantitative RT-PCR method: lane 2, control; lane 3, LPS induction; lane 4, LPS + 15d-PGJ2 1 µM; lane 5, LPS + 15d-PGJ2 10 µM; lane 6, LPS + troglitazone 1 µM; lane 7, LPS + troglitazone 10 µM; lane 1, molecular weight markers. Bottom, quantification by densitometric analysis using the L27 from the same cDNA pools as an internal control. Results are means of 3 independent experiments, and representative data are shown (n = 3; *P < 0.05 vs. LPS treatment).

Effect of PPAR-gamma ligands 15d-PGJ2 and troglitazone on NF-kappa B and AP-1 activation pathways in LPS-stimulated synovial B cells. The transcription factors NF-kappa 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-1beta , TNF-alpha , COX-2, and iNOS. In several cell lines, it was recently demonstrated (23, 25) that the activation of PPAR-alpha or PPAR-gamma could lead to the inhibition of NF-kappa B and AP-1 activities. To observe the potential antagonizing effect of PPAR-gamma ligands on NF-kappa 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-kappa B and AP-1 to specific radiolabeled probes. We observed that 15d-PGJ2 induced a strong inhibition of the DNA-binding activity of NF-kappa B (-48%; Fig. 6B) and AP-1 (-55%; Fig. 6A). In contrast, troglitazone had no significant effect on NF-kappa 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-kappa 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-kappa B and c-jun-c-fos for AP-1.


View larger version (63K):
[in this window]
[in a new window]
 
Fig. 6.   Electrophoretic mobility shift assay (EMSA). Synovial cells were cultured in 1% FCS-containing medium with 15d-PGJ2 (10 µM), troglitazone (10 µM), or vehicle (DMSO 0.1%) for 12 h and subsequently treated with LPS (10 µg/ml) for 1 h. Nuclear proteins were extracted, and 5 µg of each sample were subjected to EMSA using AP-1 (A) or nuclear factor (NF)-kappa B (B) consensus site radiolabeled probes. Complexes were visualized by autoradiography. Lane 1, probe alone; lane 2, control; lane 3, LPS 10 µg/ml; lane 4, LPS + 15d-PGJ2 10 µM; lane 5, LPS + troglitazone 10 µM; lane 6, LPS + 100-fold concentrated unlabeled probe. NS, nonspecific. Results are means of 3 independent experiments, and representative results are shown. C: EMSA "supershift" assays identifying the subunits components for NF-kappa B and AP-1 dimers.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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-1beta and TNF-alpha (22). In a previous work (3), we studied the potential role of PPAR-gamma ligands in IL-1beta -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-gamma ligands on cells importantly involved in the development of RA, synovial fibroblasts.

By RT-PCR, Western blot, and immunocytochemical analysis, we demonstrated that PPAR-gamma and PPAR-alpha 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-gamma expression, at both mRNA and protein levels, was decreased by the treatment. Thus PPAR-alpha seems not to be involved in the cellular responses to LPS in synovial cells. Several papers have reported that PPAR-gamma 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-gamma expression. On the other hand, TNF-alpha , IL-1, IL-6, leukemia inhibitory factor (28), and leptin have been reported to downregulate PPAR-gamma expression. It seems difficult to attribute clinical relevance to these up- and downregulations of PPAR-gamma expression. However, the fact that only PPAR-gamma , and not PPAR-alpha , is regulated by LPS may suggest that PPAR-gamma is involved in the LPS pathway. The same modulation of PPAR-gamma expression was demonstrated previously in rat chondrocytes stimulated with IL-1beta (3).

Thus we further evaluated the ability of PPAR-gamma ligands (15d-PGJ2 and troglitazone) to modulate the effects of LPS stimulation and, especially, their ability to counteract the production of proinflammatory cytokines (IL-1beta and TNF-alpha ) 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-1beta (-25%) and TNF-alpha (-40%) mRNA expression. However, it is interesting that troglitazone only decreased LPS-induced TNF-alpha mRNA expression (-50%) but had no significant effect on the induction of IL-1beta mRNA.

In the recent literature, it has been demonstrated that PPAR-gamma (or PPAR-alpha ) 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-alpha ligands, but not PPAR-gamma ligands, were efficient inhibitors of IL-1beta -induced production of IL-6 and prostaglandins and expression of COX-2 (23). Interestingly, in neonatal rat cardiac myocytes stimulated with LPS, both PPAR-gamma and PPAR-alpha ligands inhibited the LPS-induced expression of TNF-alpha 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-gamma 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-gamma -independent mechanisms on the action of this prostaglandin, mostly because of the lack of effect of synthetic PPAR-gamma 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-alpha and IL-6, whereas other high-affinity PPAR-gamma 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-kappa B signaling pathway (4, 19, 24, 31). Several lines of evidence suggest that the involvement of PPAR-gamma 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-gamma 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-alpha . Thiazolidinediones are synthetic compounds that are very specific agonists for PPAR-gamma [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-gamma , 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-gamma independent. Moreover, the effects observed with 10 µM troglitazone would also indicate that iNOS and TNF-alpha gene expression is much more sensitive to regulation by PPAR-gamma than IL-1beta 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-gamma have been shown to occur by antagonizing the activities of the transcription factors AP-1 and NF-kappa 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-gamma and NF-kappa B or AP-1 transcriptional pathways. We observed that 15d-PGJ2, but not troglitazone, was very potent to reduce DNA-binding activity of NF-kappa 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-alpha mRNA expression without inhibiting the DNA-binding activity of NF-kappa 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-kappa 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-gamma ligand on the promoter activity of a specific gene.

In summary, our study demonstrated that PPAR-gamma is constitutively expressed in rat synovial fibroblasts at both mRNA and protein levels. We found that, by the use of PPAR-gamma 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-gamma -independent and -dependent pathways, respectively. Thus the use of PPAR-gamma agonists may offer new insights in regard to their anti-inflammatory potential by the inhibition of TNF-alpha , and the specific use of 15d-PGJ2 would be an interesting tool to counteract the deleterious effects of IL-1beta that are responsible for the degradation of cartilage.


    ACKNOWLEDGEMENTS

This work was supported in part by European Contract No. QLK6-CT-1999-02072 and the Association de la Recherche contre la Polyarthrite.


    FOOTNOTES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Arend, WP, and Dayer JM. Inhibition of the production and effects of interleukin-1 and tumor necrosis factor alpha in rheumatoid arthritis. Arthritis Rheum 38: 151-160, 1995[ISI][Medline].

2.   Arend, WP, and Dayer JM. Cytokines and cytokine inhibitors or antagonists in rheumatoid arthritis. Arthritis Rheum 33: 305-315, 1999.

3.   Bordji, K, Grillasca JP, Gouze JN, Magdalou J, Schohn H, Keller JM, Bianchi A, Dauça M, Netter P, and Terlain B. Evidence for the presence of peroxisome proliferator-activated receptor (PPAR) alpha  and gamma  and retinoid Z receptor in cartilage. J Biol Chem 275: 12243-12250, 2000[Abstract/Free Full Text].

4.   Castrillo, A, Diaz-Guerra MJM, Hortelano S, Martin-Sanz P, and Bosca L. Inhibition of Ikappa B kinase and Ikappa B phosphorylation by 15-deoxy-Delta 12,14-prostaglandin J2 in activated murine macrophages. Mol Cell Biol 20: 1692-1698, 2000[Abstract/Free Full Text].

5.   Colville-Nash, PR, Qureshi SS, Willis D, and Willoughby DA. Inhibition of inducible nitric oxide synthase by peroxisome proliferator-activated receptor agonists: correlation with induction of heme oxygenase 1. J Immunol 161: 978-984, 1998[Abstract/Free Full Text].

6.   Devchand, PR, Keller H, Peters JM, Vasquez M, Gonzales FJ, and Wahli W. The PPAR alpha-leukotriene B4 pathway to inflammation control. Nature 384: 39-43, 1996[ISI][Medline].

7.   Fontana, A, Hengartner H, Weber E, Fehr K, Grob PJ, and Cohen G. Interleukin 1 activity in the synovial fluid of patients with rheumatoid arthritis. Rheumatol Int 2: 49-53, 1982[ISI][Medline].

8.   Green, LC, Wagner DA, Glogowski J, Skipper PL, Wishnok JS, and Tannenbaum SR. Analysis of nitrate, nitrite, and [15N]nitrate in biological fluids. Anal Biochem 126: 131-138, 1982[ISI][Medline].

9.   Huang, JT, Welch JS, Ricote M, Binder CJ, Willson TM, Kelly C, Witztum JL, Funk CD, Conrad D, and Glass CK. Interleukin-4-dependent production of PPARgamma ligands in macrophages by 12/15-lipoxygenase. Nature 400: 378-382, 1999[ISI][Medline].

10.   Inoue, H, Tanabe T, and Umesono K. Feedback control of cyclooxygenase-2 expression through PPARgamma . J Biol Chem 275: 28028-28032, 2000[Abstract/Free Full Text].

11.   Jiang, C, Ting AT, and Seed B. PPAR-gamma agonists inhibit production of monocyte inflammatory cytokines. Nature 391: 79-82, 1998[ISI][Medline].

12.   Kawahito, Y, Kondo M, Tsubouchi Y, Hashiramoto A, Bishop-Bailey D, Inoue K, Kohno M, Yamada R, Hla T, and Sano H. 15-Deoxy-Delta 12,14-PGJ2 induces synoviocyte apoptosis and suppresses adjuvant-induced arthritis in rats. J Clin Invest 106: 189-197, 2000[Abstract/Free Full Text].

13.   Khiri, H, Reynier P, Peyrol N, Lerique B, Torresani J, and Planells R. Quantitative multistandard RT-PCR assay using interspecies polymorphism. Mol Cell Probes 10: 201-211, 1996[ISI][Medline].

14.   Kliewer, SA, Ulesono K, Noonan DJ, Heyman RA, and Evans RM. Convergence of 9-cis retinoic acid and peroxisome proliferator signaling pathways through heterodimer formation of their receptors. Nature 358: 771-774, 1992[ISI][Medline].

15.   Peraldi, P, Xu M, and Spiegelman BM. Thiazolidinediones block tumor necrosis factor-alpha -induced inhibition of insulin signaling. J Clin Invest 100: 1863-1869, 1997[Abstract/Free Full Text].

16.   Petrova, TV, Akama KT, and Van Eldik LJ. Cyclopentenone prostaglandins suppress activation of microglia: down regulation of inducible nitric oxide synthase by 15d-PGJ2. Proc Natl Acad Sci USA 96: 4668-4673, 1999[Abstract/Free Full Text].

17.   Ricote, M, Huang J, Fajas L, Li A, Welch J, Najib J, Witztum JL, Auwerx J, Palinski W, and Glass CK. Expression of the peroxisome proliferator-activated receptor gamma (PPARgamma) in human atherosclerosis and regulation in macrophages by colony stimulating factors and oxidized low density lipoprotein. Proc Natl Acad Sci USA 95: 7614-7619, 1998[Abstract/Free Full Text].

18.   Ricote, M, Li AC, Willson TM, Kelly CJ, and Glass CK. The peroxisome proliferator-activated receptor gamma  is a negative regulator of macrophage activation. Nature 391: 79-82, 1998[ISI][Medline].

19.   Rossi, A, Kapahi P, Natoli G, Takahashi T, Chen Y, Karin M, and Santoro G. Anti-inflammatory cyclopentenone prostaglandins are direct inhibitors of Ikappa B kinase. Nature 403: 103-108, 2000[ISI][Medline].

20.   Schoonjans, K, Martin G, Dtaels B, and Auwerx J. Peroxisome proliferator-activated receptor, orphans with ligands and functions. Curr Opin Lipidol 8: 159-166, 1997[ISI][Medline].

21.   Schoonjans, K, Staels B, and Auwerx J. The peroxisome proliferator-activated receptors (PPARs) and their effects on lipid metabolism and adipocyte differentiation. Biochim Biophys Acta 1302: 93-109, 1996[ISI][Medline].

22.   Scott, BB, Waisbrot LM, Greenwood JD, Bogoch ER, Paige CH, and Keystone EC. Rheumatoid arthritis synovial fibroblast and U937 macrophage/monocyte cell line interaction in cartilage degradation. Arthritis Rheum 40: 490-498, 1997[ISI][Medline].

23.   Staels, B, Koenig W, Habib A, Merval R, Lebret M, Torra IP, Delerive P, Fadel A, Chinetti G, Fruchart JC, Najib J, Maclouf J, and Tedgui A. Activation of human aortic smooth-muscle cells is inhibited by PPARalpha but not by PPARgamma activators. Nature 393: 790-793, 1998[ISI][Medline].

24.   Straus, DS, Pascual G, Li M, Welch JS, Ricote M, Hsiang CH, Sengchanthalangsy LL, Gosh G, and Glass CK. 15-Deoxy-Delta 12,14-prostaglandin J2 inhibits multiple steps in the NF-kappa B signaling pathway. Proc Natl Acad Sci USA 97: 4844-4849, 2000[Abstract/Free Full Text].

25.   Su, CG, Wen X, Bailey ST, Jiang W, Rangwala SM, Keilbaugh SA, Flanigan A, Murthy S, Lazar MA, and Wu GD. A novel therapy for colitis utilizing PPARgamma ligands to inhibit the epithelial inflammatory response. J Clin Invest 104: 383-389, 1999[Abstract/Free Full Text].

26.   Sugiyama, H, Nonaka T, Kishimoto T, Komoriya K, Tsuji K, and Nakahata T. Peroxisome proliferator-activated receptors are expressed in mouse bone marrow-derived mast cells. FEBS Lett 467: 259-262, 2000[ISI][Medline].

27.   Takano, H, Nagai T, Asakawa M, Toyozaki T, Oka T, Komuro I, Saito T, and Masuda Y. Peroxisome proliferator-activated receptor activators inhibit lipopolysaccharide-induced tumor necrosis factor-alpha expression in neonatal rat cardiac myocytes. Circ Res 87: 596-602, 2000[Abstract/Free Full Text].

28.   Tanaka, T, Itoh H, Doi K, Fukunaga Y, Hosoda K, Shintani M, Yamashita J, Chun TH, Inoue M, Masatsugu K, Sawada N, Saito T, Inoue G, Nishimura H, Yoshimasa Y, and Nakao K. Down regulation of peroxisome proliferator-activated receptor gamma  expression by inflammatory cytokines and its reversal by thiazolidinediones. Diabetologia 42: 702-710, 1999[ISI][Medline].

29.   Thieringer, R, Fenyk-Melody JE, Le Grand CB, Shelton BA, Detmers PA, Somers EP, Carbin L, Moller DE, Wright SD, and Berger J. Activation of peroxisome proliferator-activated receptor gamma  does not inhibit IL-6 or TNF-alpha responses of macrophages to lipopolysaccharide in vitro or in vivo. J Immunol 164: 1046-1054, 2000[Abstract/Free Full Text].

30.   Ulfgren, AK, Andersson U, Engström M, Klareskog L, Maini RN, and Taylor PC. Systemic anti-tumor necrosis factor alpha  therapy in rheumatoid arthritis down-regulates synovial tumor necrosis factor alpha  synthesis. Arthritis Rheum 43: 2391-2396, 2000[ISI][Medline].

31.   Wang, C, Fu M, d'Amico M, Albanese C, Zhou JN, Brownlee M, Lisanti MP, Chatterjee VKK, Lazar MA, and Pestell RG. Inhibition of cellular proliferation through Ikappa B kinase-independent and peroxisome proliferator-activated receptor gamma -dependent repression of cyclin D1. Mol Cell Biol 21: 3057-3070, 2001[Abstract/Free Full Text].

32.   Zvaifler, NJ, and Firestein GS. Pannus and pannocytes: alternative models of joint destruction in rheumatoid arthritis. Arthritis Rheum 6: 783-789, 1994.


Am J Physiol Cell Physiol 282(1):C125-C133
0363-6143/02 $5.00 Copyright © 2002 the American Physiological Society