Rheumatoid arthritis (RA) is a chronic autoimmune inflammatory disease that affects the synovial joints. Development of RA is characterized by hyperplasia of the synovial lining, in which synovial fibroblasts and other immune cells interact with one another to create a pathologic tissue that provokes the destruction of cartilage and bone. Activated synovial fibroblasts play a key role in RA pathogenesis by producing proteases, cytokines, and chemokines that contribute to joint destruction and perpetuate the inflammatory process (1). Although previous studies showed that a network of cytokines, including tumor necrosis factor α (TNFα) and interleukin-1β (IL-1β), contribute to the activation of RA synovial fibroblasts (RASFs), the exact signaling mechanisms that participate in the inflammatory responses of RASFs are still not fully understood (1).
Cysteine-rich protein 61 (CYR-61; also known as CCN1) belongs to the CCN family of matricellular signaling molecules (2). Functionally, CYR-61 has been shown to regulate angiogenesis and the proliferation, adhesion, migration, and differentiation of cells (2) and is important for wound healing (3) and embryo development (4). Previous studies have implicated CYR-61 in the pathogenesis of inflammatory diseases, including RA. A complementary DNA (cDNA) microarray analysis of B cells from monozygotic twins revealed significantly higher expression of CYR-61 in the twin with RA compared with the healthy twin (5).
In a recent study, we found that CYR-61 stimulated CCL2 expression in human osteoblastic cells, and the levels of CYR-61 in osteoblasts correlated with disease progression in an animal model of inflammatory arthritis (6). Zhang et al demonstrated that CYR-61 was overexpressed in RA synovial tissue and that it played a critical role in IL-17–mediated proliferation of RASFs (7). Those authors further reported that CYR-61 activated the production of IL-6 in RASFs via the αvβ5/Akt/NF-κB signaling pathway, and stimulation of the differentiation of IL-17–producing Th17 cells by RASFs was dependent on CYR-61 (8).
Since RASFs also produce a large amount of CCL20, a chemokine required for the recruitment of arthritogenic Th17 cells (9), it would be of interest to investigate whether CYR-61 is also involved in the regulation of CCL20 production by RASFs. More importantly, the regulatory mechanism of CYR-61 expression in RASFs needs to be clarified.
Recently, Lee et al (10) found that the promoter of the Cyr61 gene contains the forkhead factor binding motif, and FoxO3a is a novel negative regulator of the Cyr61 gene in vascular smooth muscle cells. The FoxO subfamily of forkhead transcription factors plays several key roles in cellular survival and metabolism, and these factors are crucial in immune regulation (11). A growing body of evidence indicates that FoxO3a and other FoxO proteins are important for maintaining immune cell homeostasis in RA (12–15). However, the role of FoxO proteins in the effector mechanisms of RASFs has not been studied.
The activity of FoxO proteins is tightly regulated by posttranslational modifications, including phosphorylation, acetylation, and ubiquitination (16, 17). In general, the phosphorylation status governs the nucleus–cytoplasm shuttling, and acetylation/deacetylation can alter the transcriptional program of FoxO proteins (16, 17).
The sirtuins (SIRT-1–SIRT-7) are a family of NAD+-dependent protein deacetylases that modulate the function of many cellular proteins, including FoxO transcription factors (18). Brunet et al (19) reported that SIRT-1, the best characterized member of the family, has a dual effect on FoxO3a function. SIRT-1 enhances the ability of FoxO3a to confer cell cycle arrest and resistance to oxidative stress, but also inhibits the ability of FoxO3a to induce cell death. It is therefore interesting to examine the regulatory role of SIRT-1/FoxO3a in CYR-61 expression in RASFs.
Although hydroxymethylglutaryl-coenzyme A reductase inhibitors (statins) are not routine treatment agents for RA patients, mounting evidence indicates that they have beneficial effects against RA progression (20, 21). However, previous studies using collagen-induced arthritis (CIA) in rodent models to examine the therapeutic effects of systemically delivered statins have yielded conflicting results (22–25). The disparity may be due to the liver-specific pharmacokinetics and poor distribution of statins to the bone and joints when given systemically. In an earlier study using intraarticular drug administration in a rat CIA model, we confirmed the beneficial effect of simvastatin on inflammatory arthritis (6), which suggests that intraarticular injection of statins may be a useful therapy for RA. Although previous studies showed that the protective effects of statins on RA may derive from multiple mechanisms (21, 25, 26), their action on SIRT-1 signaling in RASFs has not been investigated.
The aims of this study were to examine the role of SIRT-1/FoxO3a in the expression of CYR-61 in RASFs and determine the influence of simvastatin on this pathway. In addition, with the use of a rat model of CIA, the relationship between disease progression and FoxO3a/CYR-61 signaling in synovial fibroblasts was assessed in vivo.
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The present study demonstrated that CYR-61 is an important effector molecule in RA pathogenesis. In cultured RASFs, TNFα enhanced CYR-61 synthesis, and the two molecules additively induced CCL20. Our experiments further showed that simvastatin has a therapeutic effect by reducing the expression of CYR-61 and CCL20 in RASFs. The transcription factor FoxO3a plays a major role in the inhibitory action of simvastatin on CYR-61 induction by TNFα. Based on our findings in a rat CIA model, the critical role of FoxO3a/CYR-61 signaling in the therapeutic effect of simvastatin was confirmed.
Previous studies have shown that CYR-61 contributes to the pathogenesis of RA by mediating IL-17–induced proliferation of RASFs (7) and stimulating the differentiation of IL-17–producing Th17 cells (8). Th17 cells are now believed to play a crucial role in the pathogenesis of RA (30, 31), and CCL20 is required for the migration of Th17 cells to initiate self-destructive immune reactions in RA (9). Once synovial inflammation occurs, RASFs may further recruit Th17 cells through CCL20 production (9). In our study, we demonstrated, for the first time, that CYR-61 up-regulated CCL20 secretion by RASFs, and an additive effect was observed when cells were exposed to both TNFα and CYR-61. Since CYR-61 plays pivotal roles in both cell proliferation and chemokine secretion by RASFs, it may serve as a therapeutic target in the treatment of RA.
Jonsson et al showed that FoxO3a is required for the survival of neutrophils in inflammatory arthritis (12), but its function in RASFs has not been studied. In our experiments, we found that FoxO3a is a negative regulator of CYR-61 expression in RASFs. TNFα treatment abrogated the function of FoxO3a by inducing its nuclear export. Forced expression of FoxO3a inhibited TNFα-enhanced synthesis of CYR-61 and suppressed the promoter activity of the Cyr61 gene.
The activity of FoxO proteins is tightly regulated by phosphorylation and acetylation (16, 17). Akt-dependent phosphorylation of the transcription factors inhibits their DNA-binding activity and results in nuclear export (16, 17). TNFα may inhibit the function of FoxO3a in RASFs through the activation of Akt, since previous studies (32, 33) and our experiments (results not shown) revealed that TNFα is an activator of Akt in RASFs. In support of this notion, we found that TNFα induced phosphorylation of FoxO3a at the Akt-specific site Ser253 (34).
Besides Akt-dependent phosphorylation, other mechanisms may also be involved in TNFα-induced suppression of FoxO3a in RASFs, since we observed that TNFα enhanced acetylation of the transcription factor. Acetylation by CREB binding protein/p300 has been identified as an important regulatory pathway for the function of FoxO proteins (35). Although the exact consequences of acetylation remain unclear, one study demonstrated that acetylation of FoxO proteins attenuates their transcriptional activity (35). In our study, we found that FoxO3a acetylation was associated with a decrease in its repressive action on Cyr61 transcription. TNFα has been shown to enhance the binding between p300 and other transcription factors and promote their acetylation (36, 37). Whether this pathway is involved in TNFα-induced acetylation of FoxO3a remains to be determined.
Recently, the therapeutic effects of statins against RA progression have drawn the attention of many researchers. In our study, we confirmed that simvastatin is beneficial to RA by its ability to maintain the repressive action of FoxO3a on Cyr61 transcription in RASFs. In TNFα-treated RASFs, simvastatin kept FoxO3a in the nucleus and retained its binding to the Cyr61 gene promoter. The effect of simvastatin on FoxO3a may derive from its inhibitory action of this treatment on Akt and the enhancement of SIRT-1 activity. In RASFs, it was reported that statins suppressed the TNFα-induced activation of Akt (38), and we observed a similar effect on RASFs after treatment with simvastatin (results not shown). We also showed that simvastatin treatment resulted in a decrease in Akt-specific Ser253-phosphorylation of FoxO3a. On the other hand, the other pathway through which simvastatin influences FoxO3a activity is via deacetylation of the protein. For the first time, we demonstrated that simvastatin decreased the acetylation of FoxO3a in RASFs and maintained its repressive action on gene expression. It is noteworthy that acetylation/deacetylation of FoxO proteins may also influence the sensitivity for phosphorylation. It was shown that, in addition to an inhibitory effect on DNA binding, acetylation of FoxO1 caused an increase in Akt-mediated phosphorylation, leading to the subsequent nuclear export of the transcription factor (39).
The mechanism by which simvastatin promotes deacetylation of FoxO3a appears to involve SIRT-1. SIRT-1 is a key regulator of the transcriptional activity of FoxO3a (19), and therefore its function in the modulating effect of FoxO3a on CYR-61 synthesis and its relationship to the action of simvastatin were examined. In our study, we documented that SIRT-1 suppressed TNFα-induced CYR-61 expression in RASFs, possibly through deacetylation of FoxO3a. It is obvious that the deacetylase activity of SIRT-1 is pivotal to its action, since forced expression of a catalytically inactive SIRT-1 mutant instead of the wild-type protein had little effect on the promoter activity of the Cyr61 gene. More importantly, we further observed that simvastatin enhanced the level of SIRT-1 in RASFs and promoted SIRT-1/FoxO3a binding. Our results indicate that SIRT-1/FoxO3a signaling plays a critical role in the inhibitory effect of simvastatin on CYR-61 expression in RASFs.
Sirtuins have attracted much attention recently because of their role in lifespan regulation in lower organisms and their capacity to regulate mammalian cell growth and survival in response to stress. The identification of several transcription factors known to play a role in the immune system as substrates of sirtuins has suggested that they may also participate in the regulation of inflammation, but a conclusive role for sirtuins as pro- or antiinflammatory regulators is still a matter of debate (40). In the case of arthritis, Niederer et al (41) found that persistent overexpression of SIRT-1 inhibited apoptosis and promoted proinflammatory cytokine production in RASFs. In contrast, Nakayama et al (42) demonstrated that resveratrol-induced apoptosis of synovial cells was sirtuin dependent. Moreover, SIRT-1 was shown to have the ability to inhibit osteoclastogenesis (43) and enhance the survival of osteoarthritic chondrocytes (44). In the present study, we also found that SIRT-1 is antiinflammatory, through its suppression of CYR-61 production in RASFs. Obviously, careful investigation of the cell- and disease-specific effects of sirtuins is needed before clinically relevant applications of agents targeting these molecules could be envisioned.
Our experiments with the rat CIA model confirmed the importance of CYR-61 expression and FoxO3a signaling in the pathogenic mechanism of RASFs. Overexpression of CYR-61 has been demonstrated in synovial fibroblasts from RA patients (7), but a relationship between expression levels of CYR-61 and the extent of disease activity in arthritis was not reported. In our study, we showed that alleviation of arthritis in a rat CIA model by intraarticular delivery of simvastatin was accompanied by a reduction in CYR-61 expression in synovial fibroblasts.
With regard to the relationship between FoxO3a expression and disease activity in inflammatory arthritis, previous studies demonstrated that FoxO3a may favor the survival of neutrophils in the blood (12, 15) and T cells in the synovium from RA patients (13, 15), and high phosphorylation levels of FoxO3a were found in synovial T cells from patients displaying low disease activity (13). In contrast, we observed increased accumulation of inactive phospho-FoxO3a in the synovial fibroblasts from rat ankle joints with a higher arthritis inflammation index. It is obvious that the exact mechanisms by which FoxO3a is involved in RA are complex and the transcription factor may exert disparate functions in different cell types important for the pathogenesis of RA.
In conclusion, we demonstrated that CYR-61 is important in the effector mechanisms of RASFs and confirmed the beneficial effect of simvastatin on inflammatory arthritis. Our study is the first to reveal that SIRT-1/FoxO3a signaling is crucial to the induction of CYR-61 in RASFs and that simvastatin has a modulating effect on this pathway. Continued study of the pathways linking sirtuins, FoxO proteins, and inflammatory responses of RASFs may provide new insights into the pathophysiology of RA, with possible therapeutic applications.
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All authors were involved in drafting the article or revising it critically for important intellectual content, and all authors approved the final version to be published. Dr. S.-K. Lin had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.
Study conception and design. Kok, L.-D. Lin, Hou, Hong, Chang, Hsiao, S.-K. Lin.
Acquisition of data. Kok, L.-D. Lin, Chang, Wang, Lai, S.-K. Lin.
Analysis and interpretation of data. Kok, L.-D. Lin, Hou, Hong, Hsiao, Wang, Lai, S.-K. Lin.