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The role of the synovial fibroblast in rheumatoid arthritis: cartilage destruction and the regulation of matrix metalloproteinases.

Abstract

Rheumatoid arthritis (RA) is a complex multisystem disease, the hallmark of which is pannus, the abnormal proliferative synovial tissue that serves as both propagator of the immune response and as the engine of tissue damage. Conceptually, pannus may be divided into two compartments (Fig. 1). The first, comprised by T cells, B cells, macrophages, and dendritic cells, is an immune compartment that exists in what was formerly the subintimal layer of normal synovium. These immune cells partake in antigen presentation, immunoglobulin production (including rheumatoid factor and anti-cyclic citrullinated protein [CCP] antibodies) and cytokine generation; the T cell is thought by many investigators to be the driving force coordinating these various activities.

[FIGURE 1 OMITTED]

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The other compartment of the rheumatoid pannus, the erosive compartment (Fig. 1), consists of cells adjacent to bone and cartilage that contribute directly to the erosion and destruction of those tissues. Although so-called marginal erosions of joints (the radiographic hallmark of RA) appear as negative spaces in the juxta-articular bone, they actually represent the presence of radiolucent pannus directly invading into bone. While several RA cell types contribute to cartilage and bone destruction, including neutrophils (in the joint space itself) and chondrocytes (sequestered deep within the cartilage), the primary cells that propagate articular damage at the pannus's leading edge are osteoclasts and synovial fibroblasts (SF).

It is increasingly appreciated that the osteoclast is a key player in RA bone destruction, and ongoing trials are investigating whether antiresorptive agents such as bisphosphonates, which act at the level of the osteoclast, can reduce bone damage. Additionally, SF (also referred to in the literature as synoviocytes or fibroblast-like synovial cells) are understood to play both direct and indirect roles in the erosion of bone and, more prominently, of cartilage. Moreover, SF regulate multiple other processes in the RA joint, including propagation of inflammation, maintenance of pannus architecture, and regulation of other cell types. In this discussion, we focus on the SF and its role in RA.

The RA Synovial Fibroblast: A Cell of Many Functions

Synovial fibroblasts play critical roles in normal embryogenesis and mature joint functioning. During development, it is the SF that forms the main element of the nascent joint tissue and that, in response to hyaluronic acid and other signals, begins to define the joint space and capsule. In the healthy adult joint, the SF performs several functions. As the primary stromal cell of the joint, the SF appears responsible for the production of collagen and other connective tissue molecules that form and maintain the joint capsule. The SF is also the major secretor of hyaluronic acid and other molecules into the joint space itself, providing lubrication to the joint surface as well as signaling functions to the joint tissues. Healthy SF are also likely to secrete controlled amounts of enzymes, such as matrix metalloproteinases (MMP, see below), that have the capacity to digest connective tissue and presumably maintain the structure and pliability of the joint capsule through remodeling. (1)

In RA, however, SF take on both a different character and a different set of roles. Most prominently, one could describe the RA SF as tumor-like, in that the tissue it constitutes becomes hyperplastic. The RA SF also resembles tumor in that it becomes invasive, advancing past its normal barriers to invade bone and cartilage. In contrast to many tumors, however, the RA SF is not autonomous, instead responding to a wide range of inflammatory signals, including proinflammatory cytokines. Nonetheless, RA SF has been shown to accumulate a number of mutations similar to those seen in malignancy, including mutations in tumor suppressor genes. (2) These mutations appear to be a consequence of inflammatory environment, rather than a primary cause of RA, but once established, they may facilitate additional RA SF proliferation.

The RA SF as a Producer of Proinflammatory Mediators

Another important feature of RA SF is their capacity to produce and secrete a wide range of proinflammatory mediators, including cytokines, growth factors, and lipid mediators of inflammation. These mediators impact a number of cell types (including lymphocytes, neutrophils, endothelial cells, and osteoclasts) and a range of processes in the rheumatoid joint, including angiogenesis, inflammation, chemoattraction, and bone erosion (Table 1). For example, the production of a wide range of growth factors by RA SF, including platelet-derived growth factor, fibroblast growth factor, vascular endothelial growth factor, epidermal growth factor, GM-CSF, G-CSF, and TGF-[beta], all contribute to neoangiogenesis. (1) A number of products elaborated by RA SF, including IL-8, IL-16, MCP-1, MIP-1a, and RANK ligand serve as potent chemoattractants, drawing various inflammatory cells from the bloodstream into the joint. These chemoattractants act in concert with other proinflammatory cytokines, such as IL-1[beta], IL-6, TNF-[alpha], IL-11, IL-15, RANK ligand, and MIF, as well as [PGE.sub.2], to render the vasculature more permeable, as well as adherent, to inflammatory cells. (1) One of the more important of these mediators vis-a-vis joint destruction is RANK ligand, which has the ability to both recruit monocytes and drive their differentiation into active osteoclasts, which can then interact with and erode bone.

RA SF as Mediators of Cartilage Erosion

One of the most significant features of RA SF is their ability to destroy cartilage, and indeed, RA SF are considered the primary cells responsible for marginal cartilage destruction. A large body of evidence supports this view, including a series of elegant studies by Muller-Ladner and Gay, who harvested human cartilage as well as pannus from RA patients, isolated the individual cell types from the pannus, and co-implanted cartilage disks and RA synovial fibroblasts under the renal capsules of immunodeficient mice. (3) Sometime later, they harvested the cartilage and examined it for damage. Erosion was prevalent primarily in cartilage implanted in the presence of RA SF, confirming that these cells do not need the presence of other cell types to damage cartilage.

The primary mechanism by which RA SF erode cartilage appears to be via the synthesis and secretion of matrix metalloproteinases (MMPs). MMPs are a family of more than 20 enzymes, each of which has the potential to digest a nonidentical but overlapping group of connective tissue components. MMPs produced by RA SF include MMP-1, 3, 9, 10 and 13. Of these, MMP-1 (also known as collagenase) may be particularly important in RA, both because it is copiously produced in response to cytokine stimulation, and because it is the only MMP secreted by RA SF that has the capacity to digest type II collagen (the major collagen in cartilage) in the absence of prior collagen degradation. The mechanism through which MMP-1 digests collagen has recently been elucidated by Saffarian et al, who demonstrated that MMP-1 sets down on one of the strands of collagen and has the ability to slide bidirectionally along the strand, driven by Brownian motion. (4) MMP-1 also has the capacity to cleave the strand either to the right or left of the enzyme, effectively blocking its own migration in the direction of cleavage. Thus, MMP-1 moves by what is effectively a ratchet mechanism. However, MMP-1 also has the capacity to jump from one strand to another and to recapitulate the process on the new strand. Thus, a single MMP-1 molecule has the capacity to digest multiple collagen strands in multiple places.

Given the centrality of MMPs to cartilage erosion in RA, the mechanisms by which MMP secretion is regulated from RA SF represent potential targets for RA therapy. Our laboratory has been studying the regulation of MMP secretion, with particular regard to the regulation of MMP-1. In the following sections, we discuss some of our findings, as well as those from other investigators.

Regulation of MMP-1 Secretion by Mitogen-activated Protein Kinases

The mitogen-activated protein kinases (MAPKs) are serine-threonine kinases that share common structural features as well as homologous pathways of activation. MAPKs are unusual in that they require dual threonine/ tyrosine activation for maximal activation; the enzymes responsible for this activation themselves share common features and are known as MAPK kinases (MAPKK). In turn, a series of MAPKK kinases (MAPKKK) stand at the head of these similar signaling cascades. Three families of MAPK have been identified: the Jnk family (Jun kinases), p38 family, and Erk (extracellular signal-regulated kinase) family.

Both the p38 and Jnk families are activated primarily in inflammatory states, suggesting their role in the propagation of such states, and indeed, inhibition of both p38 and Jnk have been proposed as therapeutic strategies in RA. Work by Firestein et al has demonstrated that Jnk regulates MMP-1 secretion from RA SF. (5) In contrast, most evidence, including studies from our own laboratory, suggests that p38 does not regulate MMP-1 secretion, though it undoubtedly regulates other RA SF processes.

In contrast to p38 and Jnk, Erk was identified first as a signal for cell growth and division. Subsequent studies, however, demonstrated that Erk mediates a number of inflammatory responses. For example, we observed that Erk has the ability to regulate neutrophil adhesion, a critical early stage in acute inflammation. (6,7) We then sought to determine the role of Erk in RA SF MMP-1 secretion. Incubation of RA SF with PD98059 or UO126, selective inhibitors of the MAPKK Mek1 (proximal activator of Erk), dramatically inhibited MMP-1 secretion in response to IL-1[beta] or TNF-[alpha]. (8) MMP-3 and 9, but not MMP-13, secretion were also inhibited. Hence, Erk regulates the secretion of some, but not all, MMPs from RA SF (Fig. 2).

[FIGURE 2 OMITTED]

Role of [PGE.sub.2] in the Regulation of Erk Activation and MMP-1 Secretion

[PGE.sub.2] mediates vasodilatation, vascular permeability, and pain. Accordingly, inhibition of [PGE.sub.2] production through the use of NSAIDs and/or selective COX-2 inhibitors has been a standard approach to reducing the signs and symptoms of inflammation. Nonetheless, a number of in vitro and animal studies suggest that [PGE.sub.2] also has anti-inflammatory properties. For example, Zurier's group has demonstrated the capacity of PGE to ameliorate an animal model of arthritis, (9) and our own laboratory has documented that [PGE.sub.2] inhibits both Erk activation and activation of adhesion molecules in neutrophils. (10) Given our prior findings linking Erk to MMP-1 production, we were therefore interested in examining the effects of [PGE.sub.2] on MMP-1 secretion.

Exposure of RA SF to [PGE.sub.2] inhibited MMP-1 secretion, and at the same time, inhibited Erk activation, indicating that the effect of [PGE.sub.2] was due at least in part to Erk inhibition (Fig. 2). [PGF.sub.2[alpha]], prostaglandin that does not interact with [PGE.sub.2] receptors, did not inhibit either Erk or MMP-1, confirming the specificity of the [PGE.sub.2] effect. Moreover, administration of NSAIDs for times sufficient to deplete cellular [PGE.sub.2] resulted in spontaneous Erk activation and enhanced MMP-1 secretion; both of these effects could be overcome by the introduction of exogenous [PGE.sub.2]. These data confirm that [PGE.sub.2] has complex, and in some respects, anti-inflammatory/anti-erosive properties in the rheumatoid joint. Moreover, these findings may explain why NSAIDs do not reduce cartilage erosion in the RA joint. Of interest, the effects of NSAIDs on Erk activation and MMP-1 secretion could be reproduced by selective COX-2 inhibitors, but not by a selective inhibitor of COX-1 (SC-560), indicating that the effect of [PGE.sub.2] on Erk and MMP-1 secretion is largely mediated by [PGE.sub.2] derived from a COX-2 source.

Regulation of NF-[kappa]B by Erk and [PGE.sub.2]

Nuclear factor-[kappa]B (NF-[kappa]B) is a transcriptional factor critical to the regulation of inflammatory responses; it regulates the synthesis of more than 40 inflammatory mediators, including cytokines, COX-2, and MMP-1. NF-[kappa]B is actually a family of molecules that assemble in various combinations to form transcription factors. Most prominent among these is the p50/p65 heterodimer, which contains a nuclear localization sequence but which is sequestered in the cytoplasm owing to an interaction with a regulatory subunit, inhibitor of NF-[kappa]B (I[kappa]B). Various inflammatory stimuli activate a kinase (IKK, or I[kappa]B kinase) that phosphorylates I[kappa]B, leading to its degradation. Once released from I[kappa]B, p50/p65 is free to translocate to the nucleus, where it carries out its proinflammatory effects. (11) It should be noted that other dimers in the NF-[kappa]B family may have anti-inflammatory effects. For example, p50/p50 homodimers can translocate to the nucleus but do not induce transcription of inflammatory genes. Rather, they may block the access of p50/p65 to sites of DNA binding and transcriptional activity.

In our laboratory, we have confirmed that inflammatory cytokines such as IL-1[beta] and TNF-[alpha] are capable of activating NF-[kappa]B in RA SF, measured as both p50/p65 translocation to the nucleus, and as p65 binding to nuclear DNA. (12) We have further observed that NF-[kappa]B activation is Erk-dependent, since pharmacologic Erk inhibitors blocked nuclear translocation of p65. (12) Interestingly, Erk inhibitors actually enhanced the translocation of p50, suggesting that Erk inhibition enhances the binding of p50/p50 homodimers to nuclear DNA. We next examined the role of [PGE.sub.2] in the regulation of NF-[kappa]B. Strikingly, [PGE.sub.2] inhibited p65 translocation, but enhanced p50 translocation, consistent with its ability to inhibit Erk. [PGE.sub.2] also appeared to inhibit NF-[kappa]B by an additional mechanism by enhancing the expression of I[kappa]B. In keeping with the effect of [PGE.sub.2], agents that inhibit [PGE.sub.2] production (eg, NSAIDs, selective COX-2 inhibitors) actually enhanced NF-[kappa]B activation. These data indicate that the effects of [PGE.sub.2] (and agents that inhibit [PGE.sub.2] synthesis) are complex, and that [PGE.sub.2] may in some instances actually reduce cellular inflammatory responses.

Regulation of MMP-1 by Cholesterol Pathway Intermediates and Prenyltransferase Inhibitors

Given the accumulating evidence that statins may be useful as therapeutic agents in inflammatory diseases including RA, we investigated the role of cholesterol pathway intermediates in MMP-1 secretion from RA SF. Statins are widely-used cholesterol-lowering agents that inhibit the rate-limiting step of cholesterol synthesis (the conversion of HMG-COA to mevalonic acid). They also have a number of anti-inflammatory effects independent of their cholesterol-lowering effect. In large part, the anti-inflammatory actions of statins result from the depletion of two biologically important cholesterol pathway intermediates, farnesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate (GGPP), which are required for posttranslational modification of small GTP-binding proteins (small GTPases) such as Ras (FPP) or Rac and Rho (GGPP) (Figure 3). (13) Farnesylation or geranylgeranylation of these proteins directs their cellular localization and function. In turn, small GTPases are important regulators of inflammatory pathways including, but not limited to, NF-[kappa]B and the MAPKs Jnk and Erk. By depleting both FPP and GGPP, statins indirectly decrease the intracellular level of properly functioning small GTPases, and consequently dampen the inflammatory pathways through which these proteins signal. (13)

[FIGURE 3 OMITTED]

While statins deplete both FPP and GGPP, agents that specifically target these intermediates are also available. Farnesyltransferase inhibitors (FTI), prevent FPP from attaching to its protein substrates and are currently being tested in several oncology trials. Geranylgeranyl transferase inhibitors (GGTI) prevent GGPP from modifying its protein targets and are also commercially available, although they are not yet being evaluated clinically.

In our studies, statins (lovastatin and simvastatin) did not inhibit MMP-1 secretion in cytokine-stimulated RA SF in a time- or concentration-dependent manner. Because statins have complex downstream effects through different isoprenoid intermediates, we further investigated whether inhibition of the attachment of one or more of these intermediates to their target proteins would independently regulate MMP-1 secretion. We first investigated FTI, as farnesyltransferase inhibition is known to inhibit both Erk and NF-[kappa]B. In fact, pretreating RA SF with an FTI (FTI-276) prior to cytokine stimulation led to dramatic inhibition of MMP-1 secretion. To confirm that this effect was cholesterol-independent, we tested the effects of squalestatin, a cholesterol synthesis inhibitor that acts downstream (ie, independently of) FPP and GGPP; squalestatin did not inhibit MMP-1 secretion. Moreover, increasing cellular protein farnesylation by supplying the cell with exogenous FPP produced an effect opposite to farnesyltransferase inhibition and significantly upregulated MMP-1 secretion. These data led to the conclusion that farnesylation of one or more proteins regulates MMP-1 secretion in RA SF. (14)

FTI-276 appears to mediate its effect on MMP-1 secretion at least in part via effects on NF-[kappa]B and Jnk. In further experiments, FTI-276 strongly inhibited both Jnk phosphorylation (a measure of Jnk activation) and NF-[kappa]B activation (as measured by nuclear translocation of p65). We were surprised, however, to find that FTI-276 did not inhibit Erk activation, indicating that Erk activation in response to cytokines is a farnesylation-independent process. (14)

We also examined whether altering the remaining process affected by statins, geranylgeranylation, could effect a change in MMP-1 secretion, by pretreating RA SF with a geranylgeranyl transferase inhibitor (GGTI-298) prior to stimulation. In contrast to FTI-276, GGTI-298 dramatically enhanced MMP-1 secretion in stimulated fibroblasts, whereas increasing cellular protein geranylgeranylation (by supplying SF with exogenous GGPP) decreased MMP-1 by about 50%. These data suggest that geranylgeranylation of one or more proteins is required to suppress MMP-1 secretion in RA SF. (14)

In summary, we demonstrated that protein farnesylation is necessary for cytokine-stimulated MMP-1 secretion from RA SF, in part via mediation of Jnk and NF-[kappa]B activation. In contrast, geranylgeranylation of one or more proteins provides an inhibitory signal for MMP-1 secretion. Although statins have anti-inflammatory effects, their ability to simultaneously inhibit the opposing effects of farnesylation and geranylgeranylation leads to no net effect on MMP-1 secretion in the rheumatoid joint (Fig. 3). FTIs, however, have already been shown to be effective in one animal model of RA and may hold promise for controlling RA joint destruction in the future.

Conclusion

The SF is a key player in propagating both inflammation and joint destruction in RA. In contrast to the SF in a normal joint, the RA SF is hyperplastic and invasive, and helps to perpetuate the RA joint's inflammatory microenvironment by both elaborating inflammatory mediators and by stimulating other localized inflammatory cells. RA SF are largely responsible for cartilaginous destruction in the RA joint through production and secretion of MMP-1. MMP-1 production by these cells depends on the interplay of a number of extracellular (including TNF-[alpha] and IL-1[beta]) and intracellular (including Erk, Jnk, and NF-[kappa]B) signals. We and other groups have demonstrated that interrupting these signaling pathways in RA SF dramatically diminishes MMP-1 secretion, which in turn should prevent the destruction of joint cartilage. Currently available medications for RA indirectly target RA SF, but directly targeting these cells would potentially be both safe (given their particular distribution) and effective. As yet, there is no known cell surface molecule (such as CD20 on B cells) to unequivocally distinguish SF from other cells, and therefore we cannot yet engineer a biological "smart bomb" to take aim at SF. Nevertheless, our expanding knowledge of RA SF biology and pathophysiology hold promise for novel approaches and future therapies, for the problem that is RA.

References

(1.) Mor A, Abramson SB, Pillinger MH. The fibroblast-like synovial cell in rheumatoid arthritis: a key player in inflammation and joint destruction. Clin Immunol. 2005;115:118-28.

(2.) Reme T, Travaglio A, Gueydon E, Adla L, Jorgensen C, Sany J. Mutations of the p53 tumour suppressor gene in erosive rheumatoid synovial tissue. Clin Exp Immunol. 1998;111:353-8.

(3.) Muller-Ladner U, Kriegsmann J, Franklin BN, et al. Synovial fibroblasts of patients with rheumatoid arthritis attach to and invade normal human cartilage when engrafted into SCID mice. Am J Pathol. 1996;149:1607-15.

(4.) Saffarian S, Collier IE, Marmer BL, Elson EL, Goldberg G. Interstitial collagenase is a Brownian ratchet driven by proteolysis of collagen. Science. 2004;306:108-11.

(5.) Han Z, Boyle DL, Chang L, et al. c-Jun N-terminal kinase is required for metalloproteinase expression and joint destruction in inflammatory arthritis. J Clin Invest. 2001;108:73-81.

(6.) Pillinger MH, Feoktistov AS, Capodici C, et al. Mitogen-activated protein kinase in neutrophils and enucleate neutrophil cytoplasts: evidence for regulation of cell-cell adhesion. J Biol Chem. 17 1996;271:12049-56.

(7.) Capodici C, Pillinger MH, Han G, Philips MR, Weissmann G. Integrin-dependent homotypic adhesion of neutrophils. Arachidonic acid activates Raf-1/Mek/Erk via a 5-lipoxygenase-dependent pathway. J Clin Invest. 1998;102:165-75.

(8.) Pillinger MH, Rosenthal PB, Tolani SN, et al. Cyclooxygenase-2-derived E prostaglandins down-regulate matrix metalloproteinase-1 expression in fibroblast-like synoviocytes via inhibition of extracellular signal-regulated kinase activation. J Immunol. 1 2003;171:6080-9.

(9.) Kunkel SL, Ogawa H, Conran PB, Ward PA, Zurier RB. Suppression of acute and chronic inflammation by orally administered prostaglandins. Arthritis Rheum. 1981;24:1151-8.

(10.) Pillinger MH, Philips MR, Feoktistov A, Weissmann G. Cross-talk in signal transduction via EP receptors: prostaglandin E1 inhibits chemoattractant-induced mitogen-activated protein kinase activity in human neutrophils. Adv Prostaglandin Thromboxane Leukot Res. 1995;23:311-6.

(11.) Ghosh S, May MJ, Kopp EB. NF-kappa B and Rel proteins: evolutionarily conserved mediators of immune responses. Annu Rev Immunol. 1998;16:225-60.

(12.) Gomez PF, Pillinger MH, Attur M, et al. Resolution of inflammation: prostaglandin E2 dissociates nuclear trafficking of individual NF-kappaB subunits (p65, p50) in stimulated rheumatoid synovial fibroblasts. J Immunol. 2005;175:6924-30.

(13.) Abeles AM, Pillinger MH. Statins as antiinflammatory and immunomodulatory agents: a future in rheumatologic therapy? Arthritis Rheum. 2006;54:393-407.

(14.) Abeles AM, Marjanovic N, Al-Mussawir H, Abramson SB, Pillinger MH. Farnesyltransferase inhibitors, but not statins, inhibit matrix metalloproteinase-1 secretion from rheumatoid arthritis synovial fibroblasts. [Abstract]. Arthritis Rheum. 2005;52:S453.

Aryeh M. Abeles, M.D., and Michael H. Pillinger, M.D., are from The Department of Medicine, Division of Rheumatology, New York University Hospital for Joint Diseases; and The Department of Medicine, New York Harbor Healthcare System New York Campus of the Veterans Administration, New York, New York.

Correspondence: Michael Pillinger, M.D., Division of Rheumatology, NYU Hospital for Joint Diseases, 301 East 17th Street, New York, New York 10003.
Table 1 Synovial Fibroblasts Affect the Behavior of
Other Cells in Rheumatoid Synovium

Target Cell Effect

Monocyte Recruitment, activation
 Differentiation into osteoclasts
T cell Recruitment
 Prevention of T-cell apoptosis
B cell Prevention of B-cell apoptosis
Neutrophil Recruitment, activation
 Passage into joint space
Endothelium Angiogenesis
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Author:Abeles, Aryeh M.; Pillinger, Michael H.
Publication:Bulletin of the NYU Hospital for Joint Diseases
Geographic Code:1USA
Date:Jun 22, 2006
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Previous Article:Pharmacogenetics in the rheumatic diseases.
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