Functional role of PPAR-γ on the proliferation and migration of fibroblast-like synoviocytes in rheumatoid arthritis

Peroxisome proliferator-activated receptor (PPAR)-γ is involved in both normal physiological processes and pathology of various diseases. The purpose of this study was to explore the function and underlying mechanisms of PPAR-γ in rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLSs) proliferation and migration. In the present study, we found PPAR-γ expression was remarkably reduced in RA synovium patient compare with OA and normal, as well as it was low-expression in Adjuvant-induced arthritis (AA). Moreover, inhibition PPAR-γ expression by T0070907 (12.5 μM) or PPAR-γ siRNA could promote FLSs proliferation and expressions of c-Myc, Cyclin D1, MMP-1, and MMP-9 in AA FLSs, except for TIPM-1. These date indicate that up-regulation of PPAR-γ may play a critical role in RA FLSs. Interestingly, co-incubation FLSs with Pioditazone (25 μM) and over expression vector with pEGFP-N1-PPAR-γ reduced proliferation and expressions of c-Myc, Cyclin D1, MMP-1, and MMP-9 in AA FLSs, besides TIMP-1. Further study indicates that PPAR-γ may induce activation Wnt/β-catenin signaling. In short, these results indicate that PPAR-γ may play a pivotal role during FLSs activation and activation of Wnt/β-catenin signaling pathway.


Results
The expression of PPAR-γ was down-regulated in RA FLSs. To affirm the role of PPAR-γ in RA, model of AA was established by injection with the complete Freund's adjuvant. Histopathological analysis (Fig. 1a) confirmed the model of AA was established successfully, increased remarkable inflammatory cells infiltrations. We performed immunohistochemical and western blot analysis the expression of PPAR-γ was down-regulated observably in RA FLSs compare with normal, as show Fig. 1b,c and d. The expression of Vimentin studies (Fig. 1e) indicated that the cells were derived from synovial tissues was FLSs. Similarly, western blot and Q-PCR analysis showed PPAR-γ mRNA and protein levels (Fig. 1f,g) were down-regulated significantly in FLSs isolated from AA rats' synovium. Moreover, we also have measured PPAR-γ protein expression by immunofluorescence staining (Fig. 1e) in AA FLSs was lower than normal FLSs. Thus, these results suggesting that the expression of PPAR-γ was observably reduced in RA FLSs.

Effect of PPAR-γ inhibitor increases FLSs proliferation and migration.
To identify the significance of PPAR-γ in the RA FLSs, we have measured the effect of PPAR-γ on the proliferation and migration FLSs by treated with PPAR-γ inhibitor T0070907 in AA and normal rats. First, we investigated the effect of T0070907 on the expression of PPAR-γ (Fig. 2a), addition of 12.5 μM suppressed obviously the expression of PPAR-γ mRNA with Q-PCR assays in normal FLSs as show Fig. 2b and c. The results of western blot and Q-PCR showed the expression of PPAR-γ was down-regulated markedly in varying degrees with 12.5 μM T0070907 in normal and AA FLSs as show Fig. 2b and c. More significantly, western blot and Q-PCR has verified expression of protooncogene c-Myc and cyclin protein Cyclin D1 were up-regulated markedly by PPAR-γ inhibitor 12.5 μM T0070907 in normal and AA FLSs (Fig. 2b and c). In addition, MMP-3 and MMP-9 mRNA and protein expression also were up-regulated observably by T0070907 in normal and AA FLSs, whereas TIPM-1 was down-regulated ( Fig. 2d and e). Similarly, cell cycle analysis (Fig. 2f) also suggested that treatment of FLSs with T0070907 resulted in increased varying degrees in S phase and G2/M phase. Remarkably, evidences were collected by CFDA SE cell proliferation assay and BrdU cell proliferation ELISA, as show Fig. 2b and c, that the normal and AA FLS were promoted observably proliferation with T0070907. What's more, wound-healing assay and cell invasion assay have proved that the normal and AA FLS were promoted observably migration with T0070907 ( Fig. 2i and j). Thus, these researches had proved that effect of PPAR-γ inhibitor T0070907 increased markedly FLSs proliferation and migration in normal and AA rats.
Effect of PPAR-γ siRNA increases FLSs proliferation and migration. In order to provide additional evidence that PPAR-γ is involved in the proliferation and migration of RA FLSs, siRNA specific for rat PPAR-γ was used to knockdown gene expression in AA FLSs. Normal and AA FLSs treated with control siRNA or PPAR-γ siRNA was exposed to 48 h following the transfection with 100 nM PPAR-γ siRNA, the level of PPAR-γ mRNA and protein were reduced remarkably compared with the cells transfected with control siRNA (Fig. 3a). As same as expected, the expression of c-Myc and Cyclin D1 mRNA and protein were up-regulated by PPAR-γ siRNA in normal and AA FLSs as show Fig. 3a and b. Furthermore, MMP-3 and MMP-9 mRNA and protein expression also were up-regulated observably by PPAR-γ siRNA, however, TIPM-1 were down-regulated markedly in normal and AA FLSs as show Fig. 3c and d. Similarly, cell cycle analysis also suggested that treatment of FLSs with PPAR-γ siRNA resulted in increased varying degrees in S phase and G2/M phase in normal and AA rats (Fig. 3e). Remarkably, evidence was collected by CFDA SE cell proliferation assay and BrdU cell proliferation ELISA, as show Fig. 3f and g, that the normal and AA FLS were promoted observably proliferation with PPAR-γ siRNA. What's more, wound-healing assay and cell invasion assay ( Fig. 3h and i) have proved that the normal and AA FLS were promoted observably migration with PPAR-γ siRNA. Taken together, our findings suggested that inhibited PPAR-γ expression could increase FLSs proliferation and migration in normal and AA rats and PPAR-γ may contribute to the progression of RA.

Effect of PPAR-γ agonist inhibits FLSs proliferation and migration. This is so clear that inhibited
PPAR-γ expression could increase FLSs proliferation and migration in normal and AA rats. Based on the above research, we proposed over-expression of PPAR-γ may inhibit FLSs proliferation and migration. To further determine the underlying mechanism of PPAR-γ during FLSs proliferation and migration, PPAR-γ agonist Pioditazone hydrochloride was used to over-expression of PPAR-γ. Q-PCR assays showed 25 μM Pioditazone could increase obviously the expression of PPAR-γ mRNA in AA FLSs (Fig. 4a). In addition, with 25 μM Pioditazone in FLSs, the expression of PPAR-γ was up-regulated significantly in normal and AA FLSs ( Fig. 4b and c). Conversely, after treating with 25 μM Pioditazone, expressions of c-Myc and Cyclin D1 mRNA and protein were down-regulated remarkably in normal and AA FLSs, as show Fig. 4b and c. Meanwhile, MMP-3 and MMP-9 mRNA and  . All values were expressed as mean ± SEM. # P < 0.05, ## P < 0.01 vs normal group. *P < 0.05, **P < 0.01 vs model group. . All values were expressed as mean ± SEM. # P < 0.05, ## P < 0.01 vs normal group. *P < 0.05, **P < 0.01 vs model group. . All values were expressed as mean ± SEM. # P < 0.05, ## P < 0.01 vs normal group. *P < 0.05, **P < 0.01 vs model group.
protein expression (Fig. 4d and e) also were down-regulated observably by Pioditazone, whereas TIPM-1 were up-regulated markedly in normal and AA FLSs. Consistent with the cell proliferation results, as same as expected flow cytometry analysis showed over-expression of PPAR-γ by 25 μM Pioditazone for 48 h resulted in decreased in S phase and G2/M phase significantly in normal and AA rats, as show Fig. 4f. Remarkably, evidence was collected by CFDA SE cell proliferation assay and BrdU cell proliferation ELISA ( Fig. 4g and h) that the normal and AA FLS were suppressed observably proliferation with Pioditazone. What's more, wound-healing assay and cell invasion assay have proved that the normal and AA FLS were suppressed observably proliferation with Pioditazone, as show Fig. 4i and j. These results suggested that treatment of FLSs with Pio 25 μM had a profound inhibitory effect on proliferation and migration of FLSs.

Effect of over expression vector of PPAR-γ inhibits FLSs proliferation and migration.
In order to provide additional evidence that PPAR-γ is involved in the proliferation and migration of RA FLSs, over expression vector with pEGFP-N1-PPAR-γ for rat was used to over expression of PPAR-γ in AA FLSs. Western blot and Q-PCR ( Fig. 5a and b) showed the expression of PPAR-γ was up-regulated observably with pEGFP-N1-PPAR-γ in normal and AA FLSs. In addition, after treating with pEGFP-N1-PPAR-γ, expressions of c-Myc and Cyclin D1 mRNA and protein were down-regulated remarkably in normal and AA FLSs ( Fig. 5a and b). Meanwhile, MMP-3 and MMP-9 mRNA and protein expression also were down-regulated observably by pEGFP-N1-PPAR-γ, conversely, TIPM-1 were up-regulated markedly, as show Fig. 5c and d. As same as Pioditazone, over-expression of PPAR-γ by pEGFP-N1-PPAR-γ resulted in decreased in S phase and G2/M phase significantly by flow cytometry analysis in normal and AA rats (Fig. 5e). Consistent with the cell proliferation results, CFDA SE cell proliferation assay and BrdU cell proliferation ELISA ( Fig. 5f and g) has proved that the normal and AA FLS were suppressed observably proliferation with pEGFP-N1-PPAR-γ. What's more, wound-healing assay and cell invasion assay have proved that the normal and AA FLS were suppressed observably proliferation with pEGFP-N1-PPAR-γ ( Fig. 5h and i). These evidences proved that treatment of FLSs with pEGFP-N1-PPAR-γ had a profound inhibitory effect on proliferation and migration of FLSs. It may be involved in the proliferation of FLSs, and play a pivotal role in the pathogenesis of RA.

PPAR-γ may modulate FLSs proliferation and migration and be closely associated with
Wnt/β-catenin signaling pathway. It has proved Wnt/β-catenin signaling plays a fundamental role in cell differentiation and proliferation. To investigate the effect of PPAR-γ on Wnt/β-catenin signaling in FLSs proliferation and migration, we firstly examined the expression profiles of β-catenin, a major component of Wnt/β-catenin pathway. Western blot analysis performed that β-catenin was up-regulated obviously in AA FLSs compare with normal. Moreover, inhibition PPAR-γ by T0070907 (12.5 μM) or PPAR-γ siRNA ( Fig. 6a and b), β-catenin expression was inhanced significantly in normal and AA FLSs. In particular, Pioditazone (25 μM) or pEGFP-N1-PPAR-γ ( Fig. 6c and d) supressed Wnt/β-catenin signaling, the expression of β-catenin was decreased substantially in normal and AA FLSs. In addition, Fig. 6e showed that after treating with PPAR-γ siRNA and XAV-939, expressions of c-Myc, Cyclin D1, MMP-3 and MMP-9 proteins were down-regulated remarkably, however, they were unchanged between with XAV-939 in AA FLSs. Taken together, all the above results indicated that PPAR-γ could modulate FLSs proliferation and migration and be closely associated with Wnt/β-catenin signaling pathway.

Discussion
The results of this study show that: 1) the expression of PPAR-γ was down-regulated substantially in RA FLSs; 2) PPAR-γ inhibitor T0070907 or PPAR-γ siRNA could increase observably FLSs proliferation and migration in normal and AA; 3) PPAR-γ agonist Pioditazone or pEGFP-N1-PPAR-γ could suppress substantially FLSs proliferation and migration in normal and AA; 4) PPAR-γ could modulate FLSs proliferation and migration and be closely associated with Wnt/β-catenin signaling pathway.
Adjuvant arthritis (AA) is a model of experimental RA that is induced by injection complete Freund's adjuvant (CFA) 25,26 . The AA in rats has similar characteristics to RA in aspects of histology and immunology and is a useful test system for evaluating therapies for RA. Therefore, we chose AA rats to affirm the role of PPAR-γ rather than RA. Recent progress in research has substantiated FLSs are key effector cells in inflammatory arthritic diseases 1,5 . In addition, the hyperplastic FLSs population potentially promotes lymphocyte and macrophage infiltration 27 , recruitment and retention by produces cytokines 28 , chemokines 29 , extracellular matrix proteins 27 and cell adhesion molecules 30 . Migration and invasion of activated FLSs into cartilage and bone are critical events during invasive pannus formation in RA synovium 31 . However, there are no approved drugs that are known to target the FLSs in RA, and the underlying mechanisms driving FLSs activation remain unresolved. Hence, targeted suppress proliferation and migration of FLSs may potentially complement the current therapeutics without major deleterious effects on adaptive immune responses.
PPAR-γ is the most ample PPAR subtype in white adipose tissue, and its main function is administer of insulin sensitivity, fatty acid uptake and glucose homeostasis 32 . First, in this paper, we agree with Marder W and his colleagues 18 that PPAR-γ is observably reduced by immunohistochemical and western blot in RA and AA FLSs compare with normal. Moreover, we also have measured PPAR-γ expression by immunofluorescence staining in AA FLSs was lower than normal. What's more, inhibition of PPAR-γ expression with T0070907 12.5 μM or PPAR-γ siRNA could active protooncogene c-Myc, cyclin protein Cyclin D1, matrix metalloproteinases (MMPs) proteins MMP-3 and MMP-9 over-expression, whereas TIPM-1 was down-regulated. It is known that Cyclin D1 and c-Myc are common downstream molecules of this pathway, which are proved to be related with cell proliferation 33 . In addition, numerous experimental and clinical studies indicating the expression of MMP-3 and MMP-9 a family of zinc-dependent endopeptidases, provide condition for the cell migration and invasion 34 . On  the other hand, the action of all of MMPs is regulated by a group of endogenous tissue inhibitors of metalloproteinases (TIMPs). More significantly, cell cycle analysis and CFDA SE cell proliferation assay, BrdU cell proliferation ELISA, wound-healing assay and cell invasion assay also suggested that inhibition of PPAR-γ expression increased markedly FLSs proliferation and migration in normal and AA rats.
It is necessary to consider whether over-expression of PPAR-γ could improve or regulate FLSs proliferation and migration. Indeed, PPAR-γ has been regarded as the receptor of the thiazolidinedione, a resultant chemical among anti-diabetic drugs, and in consequence, it could be targeted by many drug candidates 35 . Although not explicitly demonstrated, it is very likely that the side effects result from high doses of full agonists, such as Pioglitazone 36 , Bezafibrate 37 and metaglidasen 38 . As a result, with treatment of FLSs with Pioglitazone 25 μM or over expression vector with pEGFP-N1-PPAR-γ had a profound inhibitory effect on proliferation and migration of FLSs in normal and AA rats. More importantly, PPAR-γ has been known to have remarkably anti-inflammatory activities 16,18 . In this study, our results showed that over-expression PPAR-γ could down-regulation expression of c-Myc, Cyclin D1, MMP-3 and MMP-9, and up-regulation expression of TIPM-1. To summarize, over-expression of PPAR-γ expression reduced significantly proliferation and migration of FLSs in AA. Regarding this last comment, of course, we suppose that PPAR-γ inhibits FLSs proliferation and migration through reduction insulin resistance and lipid metabolism, and further studies are required to comprehensively explore the role of PPAR-γ in RA.
This is so clear that activation of Wnt signaling disrupts this destruction complex, which leads to accumulation of β-catenin in the cytoplasm and finally translocation to nucleus. Our results also demonstrate this viewpoint that inhibition PPAR-γ by T0070907 12.5 μM or PPAR-γ siRNA, β-catenin expression was inhanced significantly in normal and AA FLSs. In particular, Pioglitazone 25 μM or pEGFP-N1-PPAR-γ supressed Wnt/β-catenin signaling, the expression of β-catenin was substantially decreased in normal and AA FLSs. Take together, these dates suggested that PPAR-γ mediated FLSs activation, proliferation and migration and was closely associated with activation of Wnt/β-catenin signaling pathway in RA.
In summary, our findings in the present study suggested that PPAR-γ might play a pivotal role during FLS activation and be closely associated with activation of Wnt/β-catenin signaling pathway. It is one more competent and qualified opinion that the over-expression of PPAR-γ suppresses AA FLSs proliferation and migration, indicating the potential of PPAR-γ as a therapeutic target for RA. Further research is needed to clarify if PPAR-γ could be used as a diagnostic marker and prognostic indicators of RA.

Materials and Methods
Human synovial tissue collection. Synovial tissue was obtained from patients with RA (n = 5) and osteoarthritis (OA, n = 15) according to the American College of Rheumatology 1987 revised criteria during joint synovectomies and 4 trauma patients with no history of acute or chronic arthritis served as controls. All patients signed informed consent to take part in the study. The study protocol was approved by the ethics boards of Anhui Medical University, and tissue specimen acquisition was performed in accordance with the institutional guidelines. The written informed consent was obtained from all subjects. Histopathology. The synovium specimens were fixed with 4% paraformaldehyde for 24 h and embedded in paraffin. Hematoxylin and eosin (H&E) staining and immunohistochemistry were performed according to a standard procedure. The pathological changes were assessed and photographed under an Olympus BX-51 microscope.

Materials and reagents. T0070907 and Cell
Cell culture. FLSs were derived from the synovial tissues of AA and control rats. The cells were cultured in cell culture flasks in high-glucose DMEM medium (Hyclone, USA) supplemented with 20% (v/v) heat-inactivated fetal bovine serum (FBS) (Millipore, USA), 100 U/ml of penicillin, and 100 mg/ml of streptomycin (both from Beyotime, China). Cell cultures were maintained at 37 °C at an atmosphere of 5% CO 2 .
Transwell experiments. For the migration assays, after with T0070907, PPAR-γ-siRNA and Pioditazone, pEGFP-N1-PPAR-γ, 1 × 10 5 cells in 1% FBS media were placed into the upper chamber of an insert (8-μm pore size; Millipore, USA). And medium containing 20% FBS was added to the lower chamber. After incubation for 48 h, Cells that had migrated or invaded through the membrane were stained with methanol and 0.1% crystal violet stain, and viewed under an Olympus BX-51 microscope.

Statistical analysis.
Data are presented as means ± SD and were analyzed using SPSS16.0 software. Statistical significances were determined by one-way ANOVA with the post-hoc Dunnett's test. In all cases, values of P < 0.05 were considered to be statistically significant.