Cytokines regulate fibroblast-like synovial cell differentiation to adipocyte-like cells

S. Yamasaki, T. Nakashima1, A. Kawakami, T. Miyashita, F. Tanaka, H. Ida, K. Migita, T. Origuchi and K. Eguchi

First Department of Internal Medicine and 1Department of Oncology, Nagasaki University School of Medicine, 1-7-1 Sakamoto, Nagasaki 852-8501, Japan

Correspondence to: K. Eguchi. E-mail: eguchi{at}net.nagasaki-u.ac.jp


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Objectives. Our recent work showed that fibroblast-like synovial cells (FLS) could differentiate into adipocyte-like cells in vitro in response to stimulation with peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}) ligand. The aim of the present study was to determine the role of cytokines in the regulation of FLS differentiation to adipocyte-like cells.

Methods. FLS isolated from patients with rheumatoid arthritis (RA) and osteoarthritis (OA) and from normal synovial tissues were incubated with the synthetic PPAR{gamma} ligand troglitazone to induce adipocyte-like differentiation of the cells.

Results. Production of interleukin (IL)-6, IL-8 and matrix metalloproteinase-3 was reduced in adipocyte-like cells compared with FLS. DNA binding activity of nuclear factor {kappa}B (NF-{kappa}B) was clearly inhibited in adipocyte-like cells. Cultivation of FLS with interferon {gamma} (IFN-{gamma}), tumour necrosis factor-{alpha} (TNF-{alpha}) or IL-1ß inhibited the expression of PPAR{gamma} as well as CCAAT/enhancer binding protein (C/EBP) nuclear activity, and thus suppressed adipocyte-like cell differentiation in vitro.

Conclusion. Our results indicate the importance of PPAR{gamma} and C/EBP in adipocyte-like cell differentiation of FLS and that the process is influenced by inflammatory cytokines, and suggest that the proinflammatory character of FLS in patients with RA is diminished during adipocyte-like cell differentiation.

KEY WORDS: Adipogenesis, Cytokines, NF-{kappa}B, PPAR{gamma}, CCAAT/enhancer binding protein.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Hyperplasia of synovial tissues is the pathologic hallmark of rheumatoid arthritis (RA). Proliferating synovial cells produce a number of cytokines and proteases, leading to joint destruction [1, 2]. However, the mechanisms that alter the functions of synovial cells are not yet fully understood.

Our recent work has demonstrated the expression of peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}) in cultured fibroblast-like synovial cells (FLS) and revealed a notable characteristic of FLS: these cells can differentiate in vitro into adipocyte-like cells in response to stimulation with the PPAR{gamma} ligand troglitazone [3]. Since the gene expression pattern of FLS is similar to that of mesenchymal stem cells [4], FLS, acting as mesenchymal stem cells, might therefore exist as a progenitor cell population in the joints that can differentiate into multiple cell lineages during skeletal tissue regeneration or repair.

PPAR{gamma} is a nuclear transcriptional factor known to be essential for adipocyte differentiation from murine 3T3-L1 preadipocytes, in conjunction with another class of nuclear factor, CCAAT/enhancer binding protein (C/EBP) [58]. On the other hand, other investigators have demonstrated that PPAR{gamma} can inhibit cytokine synthesis [9] and that PPAR{gamma} can stimulate apoptotic cell death, including that of FLS [9, 10]. These results indicate that PPAR{gamma} is a multifunctional factor and that PPAR{gamma} stimulation could be beneficial for the treatment of inflammatory diseases, including RA. In fact, Kawahito et al. [10] have shown that administration of PPAR{gamma} ligand suppresses adjuvant arthritis in rats by induction of apoptosis.

The present study was designed to determine the importance of PPAR{gamma} and C/EBP in the differentiation of FLS and the role of inflammatory cytokines in this process. We first showed that adipocyte-like cell differentiation induced by PPAR{gamma} ligand was associated with suppression of nuclear factor {kappa}B (NF-{kappa}B) activity in FLS, with consequent reduction of production of interleukin (IL) 6, IL-8 and matrix metalloproteinase-3 (MMP-3). We next focused on cytokine regulation in PPAR{gamma}-mediated adipogenesis and showed that FLS differentiation into adipocyte-like cells in response to PPAR{gamma} ligand was suppressed in the presence of interferon-{gamma} (IFN-{gamma}), tumour necrosis factor-{alpha} (TNF-{alpha}) or IL-1ß, by inhibiting the expression of PPAR{gamma} and C/EBP nuclear activity in FLS. Our data demonstrated cross-talk between PPAR{gamma} and cytokine signalling pathways in FLS, and may flag a new therapeutic application of PPAR{gamma} signalling in RA.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Reagents
Troglitazone, a synthetic PPAR{gamma} ligand, was kindly provided by Sankyo (Tokyo, Japan). Rabbit polyclonal antibody against human PPAR{gamma} was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Recombinant human IFN-{gamma} was kindly provided by Shionogi (Osaka, Japan). Human recombinant TNF-{alpha} and IL-1ß were purchased from R & D Systems (Minneapolis, MN, USA).

Isolation of fibroblast-like synovial cells
We obtained synovial tissue specimens from patients with RA (n = 12) who met the American College of Rheumatology criteria for the disease [11], patients with osteoarthritis (OA, n = 5) and traumatic patients without arthritis (n = 4) at the time of orthopaedic surgery in the National Ureshino Hospital between April 2000 and November 2002. Informed consent was obtained from all participating subjects, and the study was conducted in accordance with the human experimental guidelines of our institution. Synovial cells were isolated from the synovial tissues by an enzymatic digestion as described previously [3]. Adherent synovial cells of at least four passages were examined for surface molecule expression with a flow cytometer (Epics XL; Beckman Coulter, Hialeah, FL, USA). Less than 1% of adherent synovial cells expressed CD2, CD3, CD20 and CD14, confirming that the adherent synovial cells used were FLS.

Adipocyte-like cell differentiation from FLS by troglitazone
Adipocyte-like cell differentiation from FLS was induced by stimulation with 10 µM troglitazone for 3 weeks, as described previously by our laboratory [3]. Briefly, FLS were cultured to confluence in Dulbecco's Modified Eagle Medium (DMEM) containing 10% fetal bovine serum (FBS). The cells (3 x 105 per well in 12-well culture plate; Figs 1 and 4) were cultured for another 3 weeks in the same media containing 10 µM troglitazone. The culture media were changed every 3 days for 3 weeks. After cultivation, adipogenesis was confirmed by intracellular lipid deposition by staining with Oil Red O, as described previously [3]. In some experiments, FLS were cultured in the presence of TNF-{alpha} (200 IU/ml), IL-1ß (20 IU/ml) or IFN-{gamma} (500 IU/ml) for 3 weeks with troglitazone, and adipocyte-like cell differentiation from FLS was also examined.



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FIG. 1. Adipocyte-like cell differentiation of FLS in response to troglitazone. Three weeks of cultivation with troglitazone (10 µM) induced adipocyte-like differentiation of FLS derived from normal synovial tissues (NoSC), OA (OASC) and RA (RASC). Intracellular lipid accumulation was observed by Oil Red O staining.

 


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FIG. 4. Inhibition of adipocyte-like cell differentiation from FLS by TNF-{alpha}, IL-1ß and IFN-{gamma}. FLS were cultured with troglitazone for 3 weeks in the presence or absence of TNF-{alpha} (200 IU/ml), IL-1ß (20 IU/ml) or IFN-{gamma} (500 IU/ml). After cultivation, adipocyte-like cell differentiation was examined by Oil Red O staining. (a) FLS cultured in the absence of troglitazone and cytokines. (b) FLS cultured with troglitazone. (c) FLS cultured with troglitazone in the presence of TNF-{alpha}. (d) FLS cultured with troglitazone in the presence of IL-1ß. (e) FLS cultured with troglitazone in the presence of IFN-{gamma}. Results shown are representative data from five RA patients. Magnification, x40.

 
Determination of IL-6, IL-8 and MMP-3 in culture supernatants from FLS and troglitazone-differentiated adipocyte-like cells
Production of IL-6, IL-8 and MMP-3 was determined in culture supernatants from FLS and troglitazone-differentiated adipocyte-like cells. Culture supernatants (5 x 106 in 100 mm dish; Fig. 2) were collected, and protein concentrations of IL-6, IL-8 and MMP-3 were examined by enzyme-linked immunosorbent assays (ELISA) (Fujirebio, Tokyo, Japan).



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FIG. 2. Low IL-6, IL-8 and MMP-3 production after adipocyte-like cell differentiation. (a) Protocol for sample collection for ELISA. Troglitazone was removed from the culture medium after 3 weeks of induction of adipocyte-like differentiation of FLS. Troglitazone-free medium was changed every 48 h (MC). Culture medium was collected twice (arrowheads; the first sampling was 24 h after the second medium change and the second sampling was 24 h after the fifth change). (b) Protein concentrations of IL-6, IL-8 and MMP-3 in undifferentiated FLS were calculated as 1.0 in each experiment. Data represent the mean ± S.D. of samples from seven RA patients. Note that the production of IL-6, IL-8 and MMP-3 was diminished after adipocyte-like cell differentiation but was restored 8 days after troglitazone withdrawal. *P < 0.01.

 
Expression of PPAR{gamma} in FLS
Expression of PPAR{gamma} was examined by Western blotting. Briefly, FLS (1.5 x 106 in 60 mm dish; Fig. 5) were cultured in the presence or absence of TNF-{alpha} (200 IU/ml), IL-1ß (20 IU/ml) or IFN-{gamma} (500 IU/ml) for the indicated times. After cultivation, the cells were washed three times with phosphate-buffered saline and lysed by addition of lysis buffer [50 mM Tris, pH 8.0, 150 mM NaCl, 0.1% sodium dodecyl sulphate (SDS), 1% NP-40 and 100 µg/ml phenylmethylsulfonyl fluoride (PMSF)]. Protein concentrations of cell extracts were determined using a protein assay kit (Bio-Rad, Melville, NY, USA). An identical amount of protein for each lysate (5 µg/well) was subjected to 12% SDS–polyacrylamide gel electrophoresis (PAGE). Proteins were transferred to a poly vinilidene difluoride (PVDF) filter, which was subsequently blocked for 1 h using 5% non-fat dried milk in TBS containing 0.5% Tween 20 (TBS-T). The filter was then washed with 1% non-fat dried milk in TBS-T, and incubated at room temperature for 1 h with anti-PPAR{gamma} antibody (0.4 µg/ml). The filter was washed with TBS-T and incubated with a 1:1000 dilution of sheep anti-rabbit IgG coupled with horseradish peroxidase. The enhanced chemiluminescence (ECL) system (Amersham, Amersham, UK) was used for detection.



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FIG. 5. Inhibition of PPAR{gamma} expression in FLS by IFN-{gamma}. FLS isolated from RA patients were cultured with IFN-{gamma} (500 IU/ml) for the indicated times and PPAR{gamma} expression was examined as described in the text. Results shown are representative data from four experiments. ß-Actin was assayed as an internal control protein.

 
Determination of nuclear NF-{kappa}B and C/EBP activities by electrophoretic mobility shift assay (EMSA)
NF-{kappa}B and C/EBP nuclear activities were examined by EMSA using the Gel Shift Assay System (Promega, Madison, WI, USA) as described previously [3, 12]. Briefly, nuclear proteins from the cells (5 x 106 in a 100 mm dish; Figs 3 and 6) extracted from the conditioned cells (7.5 µg protein from each cell lysate) were mixed with 32P-radiolabelled double-stranded oligonucleotide containing NF-{kappa}B binding sequence (5'-AGTTGAGGGGACTTTCCCAGGC-3') or C/EBP binding sequence (5'-TGCAGATTGCGCAATCTGCA-3') and mutant oligonucleotide of C/EBP (5'-TGCAGAGACTAGTCTCTGCA-3'), 0.25 mg/ml of poly(dI-dC) (Sigma, St Louis, MO, USA) in 10 mM Tris (pH 7.5), 50 mM NaCl, 0.5 mM EDTA, 1 mM MgCl2, 0.5 mM dithiothreitol and 4% glycerol. Reactions were incubated for 30 min at room temperature and analysed with 5% PAGE. Cold competition was performed by adding excess unlabelled oligonucleotide (data not shown).



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FIG. 3. Decrease in NF-{kappa}B DNA binding activity in FLS after adipocyte-like cell differentiation. Nuclear proteins were extracted from undifferentiated FLS and troglitazone-differentiated adipocyte-like cells, and NF-{kappa}B DNA binding activity was examined by EMSA. After 3 weeks of cultivation with troglitazone, adipocyte-like FLS were further incubated in the absence of troglitazone for 48 h to exclude a direct effect of troglitazone on NF-{kappa}B nuclear activity. Results shown are for three RA patients labelled as RA-1, RA-2 and RA-3. U, undifferentiated FLS, A, troglitazone-differentiated adipocyte-like cells.

 


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FIG. 6. Inhibition of C/EBP DNA binding activity in FLS by TNF-{alpha}, IL-1ß and IFN-{gamma}. FLS from RA patients were cultured in the presence or absence of TNF-{alpha} (200 IU/ml), IL-1ß (20 IU/ml) or IFN-{gamma} (500 IU/ml) for 12 h, nuclear proteins were extracted, and DNA binding activity of C/EBP was examined by EMSA. Mut, mutated oligo for negative control. Results shown are representative data from four experiments.

 
Statistical analysis
Data were expressed as mean ± S.D. Differences between groups were examined for statistical significance using the Student's t-test. A P value of less than 0.05 denoted the presence of a statistically significant difference.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Low production of IL-6, IL-8 and MMP-3 by adipocyte-like cells
Oil Red O staining demonstrated marked lipid deposition in all synovial cell cultures isolated from RA, OA and traumatic joints without arthritis in response following their culture with the synthetic PPAR{gamma} ligand troglitazone for 3 weeks (Fig. 1). The differentiation potential of FLS into adipocyte-like cells was similar among RA, OA and traumatic patients.

We next examined the functional changes in FLS during adipocyte-like cell differentiation based on our previous study that showed diminished IL-6 production by FLS during adipocyte-like differentiation [3]. Cytokines and proteases are crucial mediators for joint destruction in patients with RA [2, 13]. In addition to IL-6, protein concentrations of IL-8 and MMP-3 in culture supernatants of troglitazone-differentiated adipocyte-like cells were reduced compared with FLS. Interestingly, the production of IL-6, IL-8 and MMP-3 was restored 8 days after the withdrawal of troglitazone (Fig. 2). NF-{kappa}B is an indispensable transcription factor for IL-6, IL-8 and MMP-3 [14, 15], and therefore we studied NF-{kappa}B nuclear activity in FLS and troglitazone-differentiated adipocyte-like cells. NF-{kappa}B nuclear activity was not detected in either FLS or troglitazone-differentiated adipocyte-like cells (data not shown). Stimulation of FLS with TNF-{alpha} clearly induced NF-{kappa}B nuclear activity in FLS, which was diminished in troglitazone-differentiated adipocyte-like cells in response to TNF-{alpha} (Fig. 3).

Inhibition of troglitazone-induced adipocyte-like cell differentiation by cytokines
As shown in Fig. 1, FLS differentiation into adipocyte-like cells was similar in vitro in RA, OA and normal synovial tissues, indicating that the synovial microenvironment in vivo may be important for FLS differentiation. We focused on whether cytokines affect adipocyte-like cell differentiation from FLS in vitro. Confluent FLS were cultured with 10 µM troglitazone in the presence of TNF-{alpha}, IL-1ß or IFN-{gamma} for 3 weeks, and the results demonstrated that troglitazone-induced adipocyte-like cell differentiation from FLS was clearly inhibited by TNF-{alpha}, IL-1ß and IFN-{gamma} (Fig. 4). IFN-{gamma} exhibited the highest inhibitory effect on adipocyte-like cell differentiation (Fig. 4). Troglitazone-induced adipocyte-like cell differentiation was additively inhibited in the presence of TNF-{alpha} plus IL-1ß; however, the inhibition of which was less than that of IFN-{gamma} alone (data not shown).

Inhibition of adipogenic transcription factors by cytokines
Expressions of PPAR{gamma} and C/EBP are crucial for adipogenesis. As we described previously [3], PPAR{gamma} was expressed in FLS, but its expression was clearly suppressed by IFN-{gamma} (Fig. 5). We next examined C/EBP expression in FLS. Since constitutive expression of C/EBPß was found in FLS by western blotting (data not shown), C/EBP nuclear activity was studied by EMSA. As shown in Fig. 6, DNA binding activity of C/EBP was determined in untreated FLS. C/EBP DNA binding activity of FLS was suppressed by stimulation with TNF-{alpha}, IL-1ß or IFN-{gamma}.


    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Recent progress in research into the molecular basis of FLS has proved that FLS share some characteristics with malignant cells, including mutations in p53 protein and lack of expression of the novel tumour suppressor gene PTEN (phosphatase and tensin homologous on chromosome ten) [16, 17]. In addition, FLS can differentiate into mesenchymal lineage cells, such as osteoblasts, chondrocytes and adipocytes [4], suggesting that FLS possess characteristics of immature or undifferentiated cells. We have recently found that IL-6 production from troglitazone-differentiated adipocyte-like cells is suppressed compared with FLS [3]. These results implicate that effective differentiation of synovial cells into adipocytes could lead to reduced cytokine production in RA. Thus, we conducted the present study to examine the role of cytokines in adipogenesis from FLS.

Adipogenesis is a cell differentiation process dependent upon the coordinated expression of two classes of transcriptional factors, PPAR{gamma} and C/EBP [58]. C/EBP family members are bZIP transcription factors that possess a leucine zipper and a basic DNA binding domain. Following hormonal stimulation in 3T3-L1 preadipocytes, C/EBP proteins, including C/EBP{alpha}, ß and {delta}, are rapidly induced and function as indispensable nuclear factors for adipogenesis [7, 8]. PPAR{gamma} is expressed in FLS, and we showed here C/EBP nuclear activity in FLS, which may explain why FLS can differentiate into adipocyte-like cells. In addition to IL-6, endogenous production of IL-8 and MMP-3 in troglitazone-differentiated adipocyte-like cells was diminished. Furthermore, basal NF-{kappa}B nuclear activity was not detected in either FLS or troglitazone-differentiated adipocyte-like cells; however, TNF-{alpha}-induced NF-{kappa}B nuclear translocation was reduced in adipocyte-like cells compared with FLS. Since NF-{kappa}B is an indispensable transcription factor for IL-6, IL-8 and MMP-3 [14, 15], the small amount of active NF-{kappa}B that cannot be detected by EMSA might be suppressed during adipocyte-like cell differentiation. These transformations of FLS are very favourable for the regression of RA; however, the differentiation of the FLS into adipocyte-like cells may not be stable, because the productions of IL-6, IL-8 and MMP-3 were restored 8 days after the withdrawal of troglitazone.

We speculated that the inflammatory milieu in rheumatoid synovial tissues may contribute to the enrichment of immature FLS in the joints. Thus, we examined the effects of IFN-{gamma}, TNF-{alpha} and IL-1ß on troglitazone-mediated adipogenesis of FLS. Suzawa et al. [18] recently reported that troglitazone-induced adipocyte differentiation from mesenchymal stem cells can be inhibited by TNF-{alpha} and IL-1ß via NF-{kappa}B-inducing kinase (NIK)-mediated NF-{kappa}B activation. Similar to results for mesenchymal stem cells, our results showed that both TNF-{alpha} and IL-1ß suppressed troglitazone-mediated adipocyte-like cell differentiation from FLS. Interestingly, IFN-{gamma} also inhibited the process, and its inhibitory effect was more prominent than that of TNF-{alpha} and IL-1ß. These findings indicate that the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) cascade is another important inhibitory pathway for adipogenesis from FLS and our data are consistent with previous reports that stem cell self-renewal without differentiation is mediated through the JAK/STAT pathway in Drosophila spermatogenesis [19, 20]. Since TNF-{alpha}-induced NF-{kappa}B activation is inhibited in troglitazone-differentiated adipocyte-like cells, adipogenesis signals and cytokine signals apparently influence each other in FLS.

Pittenger et al. [21] showed that skin fibroblasts did not differentiate into mesenchymal lineage cells. However, FLS from RA, OA and normal synovial tissues can equally differentiate into adipocyte-like cells in vitro, suggesting that FLS are joint-specific pluripotent stem cells. Since the adipocyte-like cell differentiation potential of FLS did not differ among RA, OA and normal synovial tissues, the synovial microenvironment might be crucial in determining the mesenchymal lineage differentiation in vivo.

Our present data indicate the possibility that the inflammatory milieu affects the cell differentiation that results in cell proliferative disease in humans. Thus, we think it is possible to inhibit the activation of synovial cell in RA by artificial induction of adipogenesis of the cells.


    Acknowledgments
 
We thank Dr Itaru Furuichi and Dr Takahiko Aoyagi (Department of Orthopaedics, National Ureshino Hospital, Saga, Japan) for providing the synovial samples. We also thank Miss Uchiyama and Mrs Iwasaki for their excellent staining technique.


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Submitted 21 June 2003; Accepted 6 November 2003