Rheumatoid arthritis (RA) is a common chronic inflammatory disease characterized by infiltrations of macrophages and T cells into the joints, as well as synovial hyperplasia. Inflammatory cytokines have been recognized as a significant factor in the pathogenesis of RA (1). The success of anticytokine therapies in RA, particularly anti–tumor necrosis factor α (anti-TNFα) and anti–interleukin-1 (anti–IL-1), has revealed the critical pathogenetic importance of cytokines (2). However, anticytokine therapies have generally involved biologic agents that are selective for a single factor. Thus, the focus has begun to shift to the development of biologic agents that target specific signal transduction pathways, which can regulate the expression of an array of cytokines.
NF-κB seems to have a central role in mediating a variety of immune and inflammatory responses. Indeed, NF-κB has been shown to be involved in regulating the expression of genes that encode inflammatory cytokines, immune growth factors, immunoreceptors, cell adhesion molecules, and acute-phase proteins (3). In resting cells, NF-κB is sequestered in the cytoplasm and is therefore inhibited by members of the IκB family, including IκBα and IκBβ. Once activated, IκB proteins are phosphorylated by a complex of IκB kinases and then ubiquitinated and rapidly degraded by the proteasome, allowing NF-κB to be released from IκB and to translocate to the nucleus and initiate transcription by binding to numerous specific gene promoter elements (4, 5).
There is increasing evidence to suggest that NF-κB activation participates in the pathogenesis of RA. For instance, it has been demonstrated that NF-κB activation is significantly higher in RA synovium than in osteoarthritis synovium (6, 7). Furthermore, immunohistochemical analysis has demonstrated nuclear translocation of the p50 and p65 NF-κB proteins in the synovial intimal lining (7). NF-κB in cultured fibroblast-like synoviocytes is rapidly activated after stimulation by TNFα and induces production of many cytokines, such as IL-6 and IL-1β (8, 9). Several animal models of inflammatory arthritis have also shown that inhibition of NF-κB in vivo can suppress joint inflammation (10–12). These findings suggest that NF-κB may be an attractive therapeutic target for RA (13).
The Rho family of small GTPases, consisting of Rho, Rac, and Cdc42, are 20- to 40-kd monomeric G proteins that can cycle between 2 interconvertible forms: the GDP-bound (inactive) state and GTP-bound (active) state (14, 15). Activated Rho binds to specific downstream effectors, resulting in several cellular biologic functions, including formation of actin stress fibers, focal adhesion, cell motility, aggregation of cells, proliferation, and transcriptional regulation (15, 16). There is increasing evidence to support the notion that small Rho GTPases and their exchange factors are important components of the signaling pathways used by antigen, costimulatory, cytokine, and chemokine receptors to regulate the immune response (17–20).
Rho GTPases, for example, have been implicated in the regulation of NF-κB activation and proliferation in T cells (17, 20). It has been reported that signaling mediated by the Rho small GTP-binding protein promotes proliferation of rheumatoid synovial fibroblasts (21), and that the Rho pathway also mediates activation of NF-κB and expression of cytokines in TNFα-induced peripheral blood mononuclear cells (PBMCs) from patients with Crohn's disease, a chronic inflammatory disease (22). These studies indicate that the Rho pathway may play an important role in a few chronic inflammatory diseases; however, it remains unknown whether Rho GTPases mediate the inflammatory responses in RA.
The 3-hydroxy-3-methylglutaryl-CoA reductase inhibitors, or statins, are potent inhibitors of cholesterol biosynthesis that are used extensively in the treatment of patients with hypercholesterolemia (23, 24). Recent studies indicate that statins may exert antiinflammatory effects and immunomodulatory activities (25). For instance, it has been reported that statins can abrogate the Th1 immune response, promote the release of Th2 cytokines, prevent the production of chemokines, and inhibit the proliferation of T cells and endothelial cells (26–29). Moreover, simvastatin (SMV) can markedly inhibit murine collagen-induced arthritis, a surrogate model for human RA, via specific suppression of the pathogenic Th1 and proinflammatory responses (30). However, the effects of statins on intracellular signaling in RA remain unknown.
It is usually assumed that the beneficial effects of statins result from the competitive inhibition of cholesterol synthesis. However, statins may exert additional beneficial effects beyond their cholesterol-lowering properties by preventing the synthesis of various isoprenoid intermediates, such as farnesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate (GGPP), which serve as lipid attachments for a variety of intracellular signaling molecules, including small GTP-binding proteins (31–33). We have also recently shown that some of the beneficial effects of statins may be mediated via modulation of the activity of the Rho GTPase (34, 35).
In the present study, we examined the hypothesis that TNFα may contribute to the activation of NF-κB via a RhoA-mediated pathway in rheumatoid synoviocytes, and also postulated that statins, such as SMV, may be beneficial in RA by modulating the TNFα-induced, RhoA-mediated NF-κB signaling pathway and by preventing the enhanced secretion of cytokines induced by TNFα in rheumatoid synoviocytes.
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The present study in cultured rheumatoid synoviocytes shows that TNFα-induced NF-κB activation is dependent on the activity of RhoA. Our results also provide evidence that SMV inhibits TNFα-induced activation of NF-κB and secretion of IL-1β and IL-6 in rheumatoid synoviocytes by preventing signaling in the RhoA pathway.
A great deal of evidence indicates that in activated rheumatoid synoviocytes, many pathologic processes, including production of inflammatory cytokines, are regulated by intracellular signaling. The Rho family of small GTPases contains important regulators that are involved in a number of intracellular signaling pathways, such as actin stress fiber formation, cell proliferation, and transcriptional regulation (15, 16, 39). Although it has been reported that thrombin can induce proliferation, progression of the cell cycle to the S phase, and IL-6 secretion by RA synovial fibroblasts through Rho and one of its guanine nucleotide exchange factors (GEFs), p115RhoGEF (21), the role of Rho-mediated signaling in inflammatory processes in RA is still unknown.
Therefore, in the present study using cultured human rheumatoid fibroblast-like synoviocytes, we elucidated the pivotal role of RhoA in TNFα-induced activation of the NF-κB pathway, a critical signaling pathway for regulating the inflammatory response. We demonstrated that in rheumatoid synoviocytes, TNFα stimulation induced a dose-dependent increase in RhoA activity, suggesting that RhoA may play a role downstream in TNFα-stimulated signaling. We also showed that TNFα induced nuclear NF-κB translocation, DNA binding, and gene transcription, and further demonstrated that these effects were mediated by RhoA, since rheumatoid synoviocytes transfected with dominant-negative RhoA mutant failed to exhibit increased nuclear NF-κB translocation, DNA binding activity, and gene transcription in response to TNFα. This finding was consistent with the observed inhibition of degradation of IκB, a protein kinase that inhibits NF-κB activity, in the cells infected with dominant-negative RhoA plasmid. These results are identical to the findings in recent studies in which inactivation of the Rho protein could reduce TNFα-stimulated NF-κB activity in other cell lines (22, 38, 40, 41).
IL-1β and IL-6 are critical cytokines in the pathogenesis of RA. These cytokines exhibit abundant production in RA synovium and high concentrations in the synovia and serum of patients with RA. Previous studies indicate that IL-1β can be induced via the RhoA-mediated NF-κB pathway in PBMCs from patients with Crohn's disease (22). Moreover, thrombin-induced IL-6 secretion is mediated by the RhoA pathway in rheumatoid synoviocytes (21). In this study, the obtained results showed that the TNFα-induced secretion of IL-1β and IL-6 was inhibited in rheumatoid synoviocytes expressing the dominant-negative mutant of RhoA, suggesting that RhoA plays a role in mediating the secretion of IL-1β and IL-6 by rheumatoid synoviocytes. We also found that PDTC, a specific inhibitor of NF-κB, markedly reduced supernatant levels of IL-1β and IL-6 following stimulation with TNFα. Taken together, these data indicate that RhoA plays a key role in synovial NF-κB activation and cytokine secretion in the TNFα-stimulated process of synovial inflammation.
Because the role of NF-κB activation in RA is well documented, inhibition of cytoplasmic components of NF-κB, such as RhoA, may be an effective strategy for blocking the inflammatory process. Our study findings suggest that specific inhibition of RhoA activation may be considered a promising antiinflammatory approach with therapeutic potential in RA.
Statins are drugs that are commonly prescribed for the treatment of patients with hypercholesterolemia and have been suggested to exert an antiinflammatory role by lipid-lowering–independent functions. Although it has been reported that SMV is beneficial for inflammatory arthritis (30), the exact mechanisms by which statins modulate the RA inflammatory response are still unknown in detail. Recent reports indicate that statins can influence the signaling pathways implicated in the modulation of inflammatory processes in several cell lines (25). For instance, Hernändez-Presa et al reported that SMV prevented NF-κB activation in PBMCs (42). Meroni et al also demonstrated that statins could inhibit antiphospholipid antibody– and TNFα-induced activation of NF-κB and cytokine expression in endothelial cells (43). In the present study, we demonstrated that SMV inhibited TNFα-induced nuclear NF-κB translocation, DNA binding, and luciferase reporter gene expression in cultured rheumatoid synoviocytes, and further showed that SMV prevented degradation of IκBα, suggesting that SMV can modulate NF-κB activity and its related gene transcription in rheumatoid synoviocytes.
Activation of the NF-κB transcription factor family plays a central role in inflammatory responses through the ability of NF-κB to regulate proinflammatory gene transcription. Moreover, excessive NF-κB activation has been implicated in diverse chronic diseases, including RA (12, 44). Inhibition of NF-κB activity has been shown to have antiinflammatory effects, both in cultured cells and in animal models of inflammatory arthritis (6–12). Thus, SMV may be effective by inducing an antiinflammatory response in RA through the modulation of inflammatory gene activation, via inhibition of NF-κB activity.
Increasing evidence suggests that statins exhibit significant pleiotropic effects on cell signaling pathways, largely by preventing posttranslational lipid modification (isoprenylation) of small GTPase proteins, a process essential for the translocation of Rho GTPases from the cytosol to the membrane, where activation of these proteins takes place, and which influences numerous inflammatory signaling pathways (45–49). Previous studies from our laboratory and other investigators have indicated that statins modulate several cellular processes by preventing prenylation of small Rho GTPases such as Rho and Rac1 GTPases (34, 35).
In the present study, we found that cotreatment of rheumatoid synoviocytes with SMV prevented the activation of RhoA induced by TNFα, and at the same concentrations as those that were found to inhibit NF-κB activation, luciferase reporter gene expression, and cytokine secretion. Therefore, our results suggest that SMV plays a beneficial role in inflammatory arthritis by, at least in part, preventing RhoA-mediated NF-κB signaling and inhibition of proinflammatory cytokine secretion. Furthermore, MEV and GGPP not only reversed the inhibitory effect of SMV on RhoA activation, but also reversed the SMV inhibition of NF-κB activation and proinflammatory cytokine secretion by rheumatoid synoviocytes. These data suggest that SMV interfered with TNFα-induced NF-κB activation in rheumatoid synoviocytes via inhibition of geranylgeranylation of Rho.
In conclusion, on the basis of our findings, we propose that RhoA is involved in TNFα-induced NF-κB activation and cytokine secretion, suggesting a key role of the Rho GTPase in inflammatory responses and arthritic inflammation. Our study also demonstrates, for the first time, that SMV, by preventing RhoA activity, modulates TNFα-induced NF-κB activation and proinflammatory cytokine secretion in RA.