ARTHRITIS & RHEUMATISM Vol. 58, No. 12, December 2008, pp 3644–3656 DOI 10.1002/art.24046 © 2008, American College of Rheumatology REVIEW Emerging Roles of Serine Proteinases in Tissue Turnover in Arthritis J. M. Milner, A. Patel, and A. D. Rowan subdivided into 13 clans on the basis of sequence similarity, tertiary structure, and the sequential order of catalytic residues (5). A vast array of targets includes extracellular matrix (ECM), zymogens, prohormones, cytokines, growth factors, and chemokines. Some of the best-known serine proteinases have roles in the digestion of food proteins (chymotrypsin, trypsin, and elastase), blood clot formation (coagulation factors and thrombin), and fibrinolysis (plasmin). Furthermore, serine proteinases have been shown to be major players in initiating the immune responses that are thought to drive inflammation in arthritis (6,7). In recent years, it has emerged that serine proteinases can also activate signaling cascades by such mechanisms as cleaving proteinase-activated receptors (PARs), which are G protein–coupled receptors (GPCRs). Thus, serine proteinases should no longer be viewed simply as ECM-degrading enzymes. Other novel functions such as the regulation of cell signaling and modulating the biologic activity of growth factors can also significantly alter an inflammatory response. In this review, we will discuss the traditional roles of serine proteinases (coagulation, complement, and fibrinolysis) and the new emerging roles of these and novel serine proteinases in relation to the tissue turnover associated with joint destruction in RA and OA. Introduction The end stage of arthritides such as osteoarthritis (OA) and rheumatoid arthritis (RA) is characterized by the essentially irreversible loss of articular cartilage. Although different etiologies perpetuate these diseases, inflammation (to a greater extent in RA and a lesser extent in OA) contributes to this proteolysis, which is widely believed to be metalloproteinase mediated. The matrix metalloproteinases (MMPs) have received most attention, not least as potential therapeutic targets (1). Indeed, among ⱖ569 proteinases in the human degradome, ⬃34% are metalloproteinases, but to date no MMP inhibition–based therapeutic approach has demonstrated suitable clinical efficacy (1). This failure has been attributed to a lack of selectivity of MMP inhibitors as a consequence of the high degree of similarity between MMPs. Another major class of extracellular enzymes is the serine proteinases, which constitute an additional 31% of the degradome (2). Historically, the serinedependent enzymes have been known about longer than have the MMPs, which rapidly became in vogue following their discovery. Indeed, arthritis was the first disease to be associated with an MMP, when a collagenase was detected in diseased synovium (3). The serine proteinases are perhaps better known as enzymes used in medicine (for review, see ref. 4) and are considered to be important in many fundamental processes. Recent genome sequencing has led to a considerable expansion in the number of human serine proteinases that can be Coagulation cascade The coagulation cascade involves a series of proteolytic events whereby coagulation factors circulating as zymogens are cleaved into active forms, culminating in crosslinked fibrin formation. The main initiator of this cascade is tissue factor (8), an integral membrane glycoprotein that forms a ternary complex with the serine proteinase factor VIIa and zymogen factor X. Factor Xa is generated, cleaving prothrombin into thrombin, which then converts soluble fibrinogen into insoluble fibrin monomers (Figure 1). Increased expression of coagulation factors occurs in arthritic joints and Supported by grant 17165 from the Arthritis Research Campaign and by the Medical Research Council. J. M. Milner, BSc, MSc, PhD, A. Patel, BSc, A. D. Rowan, BSc, PhD: Institute of Cellular Medicine, Newcastle University, Newcastle upon Tyne, UK. Address correspondence and reprint requests to J. M. Milner, BSc, MSc, PhD, Musculoskeletal Research Group, Institute of Cellular Medicine, 4th Floor Cookson Building, Newcastle University, Newcastle upon Tyne NE2 4HH, UK. E-mail: firstname.lastname@example.org. Submitted for publication May 1, 2008; accepted in revised form August 15, 2008. 3644 SERINE PROTEINASES AND TISSUE TURNOVER IN RA AND OA 3645 and cartilage, leading to hypoxia and acidosis. In addition, fibrin can enhance inflammation and provides a matrix for cell adhesion and migration (9). A role for fibrin in arthritis development via ␣M␤2-dependent leukocyte activation and changes in the inflammation status is supported by the reduction in collagen-induced arthritis (CIA) disease severity in mice lacking either fibrinogen or the leukocyte receptor integrin ␣M␤2– binding motif for fibrinogen (14). Coagulation enzymes have additional functions such as activating signaling cascades via PARs (8,15). For example, the tissue factor–factor VIIa complex and factor Xa activate PAR-2 (16), while thrombin activates PAR-1, PAR-3, and PAR-4 (for review, see ref. 17). Thus, some coagulation cascade serine proteinases have noncoagulant functions in the joint that can modulate various cellular processes such as cell proliferation and survival, gene transcription, and protein translation (18). Complement cascade Figure 1. Schematic representation of key interactions of serine proteinase cascades leading to tissue turnover in the arthritic joint. Activation of the coagulation cascade culminates in the release of thrombin and fibrin deposition. Thrombin and several coagulation factors cleave proteinase-activated receptors (PARs), leading to activation of signaling cascades. Activated protein C (APC) can inhibit the coagulation cascade, activate pro–matrix metalloproteinase 2 (proMMP-2), and down-regulate proMMP-9 synthesis (not shown). The plasminogen activator (PA)/plasmin cascade generates plasmin, which can degrade fibrin and activate growth factors, PA receptors (PARs), and proMMPs. Binding of urokinase PA (uPA) to its receptor (uPAR) can activate signaling cascades. Complement activation promotes inflammation via recruitment of immune cells and inflammatory mediators (not shown). Immune cells release serine proteinases that promote the inflammatory response via activation of PARs, generation of mediators of inflammation (not shown), and matrix degradation. Furin can activate pro-metalloproteinases (proMPs). Fibroblast activation protein ␣ (FAP␣) and dipeptidylpeptidase 4 (DPP4) may activate signaling cascades, modulate chemokine activity (not shown), and degrade matrix. High-temperature requirement proteinase A1 (HtrA1) and HtrA3 may contribute by inhibiting transforming growth factor ␤ signaling (not shown) and degrading matrix. tPA ⫽ tissue PA; FDP ⫽ fibrin degradation products; PR3 ⫽ proteinase 3; CTG ⫽ cathepsin G; NE ⫽ neutrophil elastase; ECM ⫽ extracellular matrix. experimental arthritis (for review, see ref. 9). Furthermore, blockade of this cascade using the thrombin inhibitor hirudin or inhibition of tissue factor reduces the severity of murine arthritis (10–12). Fibrin accumulation in the joint, a characteristic of arthritis (especially RA), is a consequence of this cascade (9,13) and may be detrimental due to impaired nutrition for the synovium The complement cascade is important in innate immunity and autoimmunity; during disease, however, inappropriate or excessive complement activation can occur, resulting in tissue injury. The complement cascade is implicated in many acute and chronic inflammatory disease processes, including the pathogenesis of RA (for review, see ref. 7). Deposited autoantibodies, immune complexes, apoptotic cells, and necrotic cells can activate this cascade, leading to the release of proinflammatory activators, recruitment of inflammatory cells (Figure 1), and formation of membrane attack complexes. The levels of complement activation components are elevated in the synovial fluid, synovium, and cartilage of patients with arthritis (19–23), and targeted deletion/inhibition reduces disease severity in murine arthritis models (7,24,25). These previous studies have led to the development of therapeutic approaches targeting complement components for the treatment of RA (for review, see ref. 7). Complement activation also occurs at the cartilage surface; both intact and degraded fibronectin can activate the complement cascade, and cartilage degradation and fibronectin release may be an important mechanism in promoting joint inflammation (7,26). Serine proteinase C1s can also degrade insulinlike growth factor binding protein 5 (IGFBP-5) to release active IGF-1 (27), a growth factor integral to controlling cartilage damage. Conversely, C1s can also degrade type I collagen, type II collagen, and gelatin (28), although the typical three-quarter– and one- 3646 quarter–size fragments effected by collagenasemediated collagenolysis are not observed. Plasminogen activator (PA)/plasmin cascade Plasmin is a broad-spectrum serine proteinase generated from its inactive precursor plasminogen by PAs (Figure 1). There are 2 known PAs, urokinase PA (uPA) and tissue PA (tPA), both of which are serine proteinases with limited substrate specificities. A specific cell surface receptor for uPA (uPAR) localizes uPA activity to the pericellular environment (29). Most cells within the arthritic joint express PAs and uPAR, and the levels of these are elevated in the tissue and synovial fluid of patients with arthritis (for review, see ref. 9). Plasminogen is also present in synovial fluid, and plasmin activity has been detected in the cartilage of patients with arthritis (30). Plasmin has many potential targets in the arthritic joint, the most important of which may be fibrin. As discussed previously, fibrin accumulation is detrimental and contributes to arthritis, and deposition of fibrin in the joint correlates with disease severity in murine arthritis models (31). Targeted deletion of components of the PA/ plasmin cascade has produced conflicting results in murine models (32). In some models, deletion of uPA, plasminogen, or tPA exacerbated arthritis, with increased fibrin accumulation in the joint (31,33), while uPA or plasminogen deletion in other models resulted in only mild disease (31,34,35). The reasons for such conflicting observations are unknown, but the differences may be partly attributable to the different pathologies reported in the models used (32). Furthermore, plasmin has contrasting roles in the context of arthritis: plasmin-mediated fibrinolysis is important in maintaining a healthy joint, while plasmin can also contribute directly to ECM proteolysis by cleaving matrix components (glycoproteins, fibronectin, and proteoglycans) or indirectly by proMMP activation and subsequent ECM degradation (36–39). Other arthritis-relevant roles for plasmin include intracellular signaling via activation of PAR-1 in association with integrin ␣9␤1 (40), activation of growth factors such as transforming growth factor ␤ (TGF␤) (41,42), release of IGF-1 from IGFBPs (43), and activation of the complement cascade (35). Independent of plasmin generation, uPAR also has roles in cellular adhesion, differentiation, proliferation, and migration (44), and although uPAR lacks a transmembrane domain, it is capable of mediating cell signaling. This occurs via association with ligands such as integrins, MILNER ET AL vitronectin, GPCRs, and uPAR-associated protein, interactions that appear to be dependent on uPA being bound to its receptor (45). Activated protein C (APC) APC is a serine proteinase generated from its precursor, protein C, by the action of thrombin bound to thrombomodulin. APC is best known for its ability to prevent blood clot formation via the proteolysis of active coagulation factors factor Va and factor VIIIa. However, APC also has antiinflammatory and antiapoptotic properties (for review, see ref. 46). APC directly activates proMMP-2 (47), and the level of APC is elevated in RA synovial joints, where it colocalizes with MMP-2 (48). In RA fibroblasts and monocytes, APC downregulates proMMP-9 synthesis, reduces tumor necrosis factor ␣ (TNF␣) production, and inhibits NF-B– and p38 mitogen–activated protein kinase activity (49). These observations are dependent on APC binding to its specific receptor, endothelial protein C receptor (EPCR), although this receptor does not signal. The proposed mechanism involves APC binding to EPCR, activating PAR-1, and transactivating the epidermal growth factor receptor (46,50,51). Cleavage of PAR-1 by APC bound to EPCR within a lipid raft activates Gi and subsequent antiinflammatory signals. When PAR-1 is activated outside the lipid raft by other serine proteinases, signaling via Gq and/or G12/13 occurs, leading to proinflammatory signals (50). Immune cell–derived serine proteinases The serine proteinases cathepsin G (CTG), neutrophil elastase (NE), and proteinase 3 (PR3) are stored in azurophil granules and released following neutrophil exposure to inflammatory stimuli (Figure 1). RA is characterized by neutrophil infiltration into the joint, and these proteinases are important in arthritis development (52). Dipeptidylpeptidase 1 (DPP-1 or cathepsin C), a lysosomal cysteine proteinase, is an important activator of neutrophil serine proteinases. In DPP-1⫺/⫺ or NE⫺/⫺:CTG⫺/⫺ mice, there is reduced severity of experimental arthritis (52,53), with reduced cytokine levels and inflammatory cell recruitment. These studies reveal that neutrophil proteinases are important in promoting the inflammatory process whereby NE and CTG help establish chemotactic gradients that recruit immune cells and enhance inflammation (52–54). The mechanism(s) by which neutrophil serine proteinases participate in inflammation probably involves proteolysis of chemokines (CXCL8, CXCL2) or cytokines (pro- SERINE PROTEINASES AND TISSUE TURNOVER IN RA AND OA TNF␣, pro–interleukin-1␤ [proIL-1␤], IL-6), or modulating integrin clustering and activation of Toll-like receptor 4 and PARs (for review, see ref. 6). Such diverse biologic mechanisms further highlight the non– ECM-degrading functions of serine proteinases that have direct relevance to arthritis. Granzyme A and granzyme B are serine proteinases stored in the granules of activated cytotoxic T cells and natural killer cells and are involved in the cytotoxic immune response by inducing apoptosis in target cells. T cells from the synovial fluid and synovium of patients with RA, and to a lesser extent from patients with OA, contain these enzymes (55–59), and raised levels of granzyme B correlate with joint damage in RA (60,61). Extracellular granzyme A and granzyme B can also be found in the synovial fluid of patients with RA (62), but their role here is less well characterized than the intracellular cytotoxic function. However, granzyme A has been reported to stimulate production of TNF␣, IL-6, and IL-8 by monocytes (63) and IL-6 and IL-8 by fibroblasts (64), although the mechanism is unclear. Therefore, soluble granzyme A in the arthritic joint could promote synovial inflammation due to its effects on cytokine production. Granzyme B–positive cells are detectable at the cartilage–pannus junction (65,66), and granzyme B is expressed by chondrocytes (67). Thus, granzyme B may contribute directly to ECM degradation, because it can degrade aggrecan, vitronectin, fibronectin, and laminin (66,68,69) (Figure 1). Increased numbers of mast cells are found in the synovium and synovial fluids of patients with arthritis (for review, see ref. 70). Tryptase and chymase are the major serine proteinases stored and secreted by mast cells and are present in synovium and the cartilage– pannus junction in arthritis (71,72). Both enzymes promote inflammation, ECM destruction, and remodeling by several mechanisms (for review, see ref. 73) (Figure 1). Tryptase can process prothrombin (74), generate C3a from complement C3 (75), and degrade ECM components including fibrinogen (76,77), denatured type I collagen (78), and fibronectin (79). Tryptase and chymase can further promote ECM degradation by activating proMMPs (80–84) and pro-uPA (85). Mast cell tryptase is important in activating PAR-2 on synovial fibroblasts and inducing proinflammatory cytokine release (86,87). Tryptase can also enhance the release of vascular endothelial growth factor from chondrocytes, although this appears to be a PAR-independent event (88). High-temperature requirement proteinases The high-temperature requirement A (HtrA) family of serine proteinases has 4 members, all of which 3647 have at least 1 C-terminal PDZ domain that is thought to be involved in protein–protein interactions and regulating proteinase activity (89). HtrA1, HtrA3, and HtrA4 are secreted and all consist of a highly conserved trypsinlike serine proteinase domain, an IGFBP domain, and a Kazal-type serine proteinase inhibitor motif at the N-terminus. HtrA2 (OMI; PRSS25) is quite distinct, and instead of the IGFBP and Kazal-type domains, it localizes to the intermembrane space of mitochondria and is thought to be involved in programmed cell death and handling misfolded mitochondrial proteins (90). The expression of HtrA1 and HtrA3 is decreased in several cancers, while overexpression of HtrA1 in human cancers inhibits cell growth and proliferation (91–94). These results suggest a tumor suppressor function for HtrA proteinases. In contrast, HtrA1 is upregulated in the skeletal muscle of patients with Duchenne’s muscular dystrophy (95) or Alzheimer’s disease (96) and in OA cartilage (97,98). HtrA1 levels are also increased in both OA and RA synovial fluids, and synovial fibroblasts secrete HtrA1 (99). Furthermore, levels of both HtrA1 and HtrA3 are elevated in experimental arthritis (100,101). The precise functions of HtrA1, HtrA3, and HtrA4 are unclear. HtrA1 and HtrA3 are characteristically expressed in embryonic tissues, where TGF␤ family proteins are developmentally important (100), and both enzymes bind to and inhibit the signaling of TGF␤ proteins in a proteinase activity–dependent manner (100,102). HtrA1 is expressed by hypertrophic chondrocytes in both normal and pathologic situations. During bone formation, HtrA1 is expressed by hypertrophic chondrocytes, and this may be important in inhibiting local TGF␤ signaling and allowing chondrocyte differentiation (101). HtrA1 is also increased in the setting of CIA, where resting chondrocytes proceed to terminal hypertrophy (101). In the normal adult joint, TGF␤ proteins are important in maintaining a layer of articular cartilage by preventing chondrocyte differentiation and hypertrophy and stimulating the synthesis of proteoglycans and collagens (103,104). Overexpression of HtrA1 may promote arthritis by inhibiting TGF␤ and accelerating chondrocyte hypertrophic differentiation (101). Furthermore, HtrA1 can digest biglycan, decorin, aggrecan, fibromodulin, fibronectin, and matrix Gla protein and thus promote cartilage ECM degradation (99– 101,105). HtrA3 can also degrade biglycan and decorin (100). HtrA1-generated fibronectin fragments have been shown to stimulate synovial fibroblasts to secrete MMP-1 and MMP-3 and thus further enhance cartilage breakdown (99). Therefore, there are several mechanisms by 3648 which HtrA proteinases may contribute to arthritis pathogenesis. HtrA1 is also expressed by osteoblasts and inhibits matrix mineralization, which may occur via modulating gene expression, inhibiting TGF␤ family protein signaling, and/or degradation of ECM proteins (105). DPPs The proline-specific DPP family of serine proteinases includes DPP-2 (DPP-7 or quiescent cell proline dipeptidase), DPP-4 (CD26), and fibroblast activation protein ␣ (FAP␣ or seprase). DPP-2 is lysosomal (106), whereas DPP-4 and FAP␣ are both type II transmembrane proteins with a large C-terminal extracellular domain, a transmembrane domain, and a short cytoplasmic tail (107,108). These enzymes have the unique proteolytic ability to cleave N-terminal dipeptides from proteins with proline or alanine in the penultimate position. Processing of such N-terminal dipeptides is an important control mechanism and can result in the activation or inactivation of a substrate, modification of receptor binding, and alterations in downstream signaling. The DPPs have numerous targets, including cytokines, chemokines, neuropeptides, and peptide hormones, and are therefore important regulators of biologic processes. They have been linked to several diseases, including some cancers (107), liver disorders (109), type 2 diabetes mellitus (110,111), skin diseases (112), and arthritis (113). DPP-4 is the most extensively studied DPP family member. Enzyme activity depends on homodimerization of its 110-kd subunits. A soluble form is cleaved from the cell membrane by an unknown proteinase. Its designation as CD26 (114) serves to exemplify its expression on the surface of T cells, B cells, and macrophages, although it is also present on other joint cells such as fibroblasts and chondrocytes (113,115,116). DPP-4 activity is significantly higher in OA synovial fluid compared with RA synovial fluid (117), whereas synovial membrane activity has been reported to be either similar in patients with RA and those with OA (118) or higher in patients with RA (119). In RA animal models (adjuvant-induced arthritis, alkyldiamine-induced arthritis, or CIA), DPP-4 inhibition suppressed arthritis development (120,121), although the severity of antigeninduced arthritis increased in DPP-4⫺/⫺ mice (122). DPP-4 has a multitude of known biologic substrates, yet the exact role(s) of DPP-4 in arthritis are unknown. A potentially important role is its ability to modulate the bioactivity of chemokines such as stromal MILNER ET AL cell–derived factor 1 (SDF-1 or CXCL12) and RANTES. SDF-1 interacts with its unique receptor, CXCR4, and stimulates angiogenesis and mononuclear cell trafficking into the joint as well as MMP-3, MMP-9, and MMP-13 release from chondrocytes (123–125). DPP-4–mediated removal of the N-terminal dipeptide of SDF-1 reduces leukocyte chemotaxis (122), and this cleavage significantly alters the functionality of the SDF-1:CXCR4 axis (126). For example, SDF-1 can induce chondrocyte cell death (127), and DPP-4 could protect against chondrocyte cell death. Similarly, fulllength RANTES promotes monocyte chemotaxis, while DPP-4–cleaved RANTES does not (128). Thus, within inflamed joints, DPP-4 activity could serve to regulate both the magnitude and longevity of an inflammatory response. Other biologic functions unrelated to its proteolytic abilities have been described: DPP-4 is a cell surface plasminogen receptor on human RA synovial fibroblasts (129). DPP-4 also associates with adenosine deaminase, CD45, and IGF receptor II and binds to the ECM proteins collagen and fibronectin. These interactions have been shown to be significant to the progression and suppression of cancers (for review, see ref. 130) and in T cell regulation (114), such that targeting DPP-4 may be useful for suppressing the immune response in RA and other autoimmune diseases (for review, see ref. 114). FAP␣ is structurally very similar to DPP-4. The active enzyme is a homodimer of two 97-kd subunits, and a soluble form of FAP␣ has also been reported (131,132). In contrast to DPP-4, FAP␣ typically is not expressed in normal tissues. FAP␣ is strongly expressed by reactive stromal fibroblasts within the stroma of the majority of human epithelial tumors but not in carcinoma cells (133). It is also expressed on reactive fibroblasts in granulation tissue of healing wounds, on stellate cells at the tissue remodeling interface in cirrhosis (134), and in lung tissue in idiopathic pulmonary fibrosis (135). Due to this rather “disease-specific” expression, FAP␣ is an attractive therapeutic target. In patients with advanced or metastatic FAP␣positive cancers, a humanized antibody directed against FAP␣ (sibrotuzumab) rapidly and selectively localized to tumors (136). Interestingly, sibrotuzumab also localized to the knees and shoulders in 3 patients in that study, and, although no obvious clinical symptoms of arthritis were reported, it is intriguing to speculate that arthritis may develop in these patients. FAP␣ expression is associated with arthritis, and we have shown active FAP␣ on the surface of chondrocytes and elevated expression in OA cartilage compared with control carti- SERINE PROTEINASES AND TISSUE TURNOVER IN RA AND OA lage (137). FAP␣ is also present in OA and RA synovial tissue (138,139), and elevated expression is detected in murine CIA (140). Unlike DPP-4, FAP␣ has gelatinolytic activity and thus may contribute to ECM degradation, although the exact mechanistic role of FAP␣ in arthritis (and indeed other pathologies) is unknown. A soluble plasma form of FAP␣ termed antiplasmin cleaving enzyme (APCE) cleaves 12 amino acids from the methionine N-terminal end of ␣2-antiplasmin (Met– ␣2AP) to generate Asn–␣2AP (132), which is more rapidly incorporated into fibrin. This cleaved form also inhibits plasmin-mediated fibrin digestion more efficiently than Met–␣2AP, such that the presence of APCE activity in the joint would be detrimental due to fibrin accumulation (see above). DPP-4 and FAP␣ may function together, because they can form heteromeric complexes that are localized to invadopodia, membrane protrusions at the leading edge of migrating cells. The gelatinolytic activity of FAP␣ and the ability of DPP-4 to bind to fibronectin (141) and collagen are considered to be important in cell migration and the matrix invasion that occurs during tumor invasion, angiogenesis, and metastasis (142,143). However, the catalytic domains of both DPP-4 and FAP␣ are not always required to exert their tumor suppressor or promoter properties (for review, see ref. 130), and another function of FAP␣ and DPP-4 may be as activators of cell signaling via association with membrane-bound signaling molecules such as integrins (144). Thus, therapies directed at inhibition of the catalytic activity of these enzymes may not always be the most appropriate strategy. Pro-protein convertases (PCs) PCs are a group of calcium-dependent serine proteinases that are highly homologous to bacterial subtilisin and yeast kexin endoproteinases. Members of this group, furin/PACE, PC1/PC3, PC2, PC4, PACE4, PC5/PC6, and PC7, cleave precursor proteins at basic residues with the general motif (K/R) (Xn) (K/R) 2, where n ⫽ 0, 2, 4, or 6, and X is usually not a cysteine. Several contain a transmembrane domain and cycle between the trans-Golgi network and the cell surface via endosomes, which enables them to process pro-proteins in the secretory pathway and at the cell surface. PCs cleave numerous proteins including prohormones, serum proteins, bacterial toxins, viral glycoproteins, metalloproteinases, growth factors, growth factor receptors, neuropeptides, and adhesion molecules. Consequently, these enzymes are involved in numerous physiologic and 3649 pathologic pathways including embryonic development, Alzheimer’s disease, cancer, obesity, diabetes, cardiovascular disease, and infectious disease (145,146). Furin is present exclusively in the superficial zone of normal cartilage but is also present at high levels in the deep zone of OA cartilage (147). In a model of cartilage resorption, we have shown that the addition of a chloromethylketone PC inhibitor reduces the levels of aggrecan and collagen breakdown, thus implicating a role for PCs in the cascades leading to cartilage resorption (148). In this assay, a reduction in the levels of active collagenase and MMP-2 was observed, suggesting that PCs are involved in proMMP activation (see below). ProMMP-14 can be furin-processed (149) (Figure 1), and active MMP-14 can activate proMMP-13 (39), a major collagenase implicated in cartilage collagenolysis. Furthermore, the major aggrecanases also contain the typical PC recognition motif and can be activated by furin (150). TACE (or ADAM-17) is implicated in arthritis, and furin, PACE4, PC5/PC6, PC1, and PC2 are all predicted to process proTACE (151). B lymphocyte stimulator (BLyS; trademark of Human Genome Sciences, Rockville, MD) is a member of the TNF superfamily and is an important regulator of B cell autoimmunity. Significant levels of soluble BLyS are found in RA serum and synovial fluid. TNF␣ activates the release of membrane-bound BLyS from invading neutrophils at sites of inflammation, and this shedding is furin mediated (152). Furin can also generate active TGF␤, which in turn increases furin expression (153). In fibroblastlike synoviocytes, TGF␤ increases ADAMTS-4 expression (154), such that a positive feedback loop is created, with increased levels of active ADAMTS-4 and aggrecanolysis (145). Conversely, we and other investigators have shown TGF ␤ to block cartilage resorption (104,155,156). Other PC family members are less well characterized in terms of arthritis, and it remains to be seen whether these have important roles in arthritis tissue. Serine proteinases and cell signaling The PAR family consists of 4 transmembrane GPCRs that are activated following proteolytic cleavage of their extracellular N-terminus; this unmasks a tethered ligand, which then interacts with the receptor (for review, see ref. 17). Synovial fibroblasts, chondrocytes, macrophages, neutrophils, mast cells, T cells, and dendritic cells all express PARs, which exhibit both antiinflammatory and proinflammatory properties, although 3650 recent data point toward a detrimental role especially in the context of immune-mediated effects in arthritis (157). A study in PAR-2⫺/⫺ mice confirmed PAR-2 as a key mediator of chronic joint inflammation (158), and this is likely to be a consequence of activation by mast cell–derived tryptase (86,87). PAR-2 is associated with the perpetuation of inflammation in both RA and OA, because PAR-2 levels are elevated by proinflammatory cytokines and growth factors (159,160), while PAR-2 activation in peripheral blood monocytes and chondrocytes enhances proinflammatory cytokine production (161,162). Thus, increased expression of serine proteinases capable of activating PARs (Figure 1) has the potential to exacerbate inflammation. For example, PAR-2 can be activated by trypsin, mast cell tryptase (163), tissue factor–factor VIIa and factor Xa (15), some kallikreins (164), PR3 (165), and matriptase (166). Thrombin activates all PARs except PAR-2, and a role for thrombin in arthritis has been known for some time (167). PAR-1 is also known as the thrombin receptor and appears to have a regulatory role in immunity: PAR-1⫺/⫺ mice have less severe antigeninduced arthritis with reduced synovial IL-1, IL-6, and MMP-13 expression (168); thrombin inhibition (via hirudin) ameliorates CIA via reduced synovial hyperplasia and IL-1␤ expression (11). PAR-2 is the most consistently observed chondrocyte PAR, although differing expression profiles for the other PARs have been reported (see ref. 169 and the references therein), while synovial tissues express all except PAR-4 (86,87,170). NE and CTG inactivate (or “disarm”) PAR-1 and PAR-2, while NE also inactivates PAR-3 (see ref. 171 and the references therein) such that serine proteinases may be key proteolytic regulators of PAR-mediated signaling in the context of joint inflammation as well as neutrophil infiltration. The therapeutic potential of PARs in arthritis has recently been reviewed (159). Serine proteinases can also influence cell signaling via the proteolytic “activation” of growth factors such as IGF-1 and TGF␤. Enzymes that target IGFBPs include C1s (27), HtrA1 (172), plasmin (43,173), thrombin (173), CTG (174), and NE (174), while furin, plasmin, thrombin, NE, and tryptase can all process latent TGF␤ proteins (for review, see ref. 42). Thus, multiple serine proteinases offer a potential chondroprotective role (via release of IGF-1 and/or TGF␤) to limit cartilage damage and promote new matrix synthesis. Serine proteinases and activation of procollagenases in cartilage resorption Cartilage collagen degradation is mediated by collagenases, specifically MMP-1, MMP-8, MMP-13, MILNER ET AL Figure 2. Interaction of serine proteinase and matrix metalloproteinase (MMP) cascades leading to cartilage collagen breakdown. ProMMP-14 can be activated by furin, which in turn can release active MMP-2 from a proMMP-2:tissue inhibitor of metalloproteinases 2 (TIMP-2) complex at the cell surface. MMP-14, as well as MMP-2, can also activate proMMP-13. Trypsin-like proteinases such as urokinase plasminogen activator (uPA) bind their cell surface receptor (uPAR) and promote the generation of plasmin from plasminogen. Plasmin can directly activate proMMP-1 and proMMP-13. ProMMP-3 can also be activated by plasmin, and active MMP-3 can process other proMMPs, including the collagenases proMMP-1, proMMP-8, and proMMP-13. and MMP-14. All MMPs are synthesized in an inactive form that requires the removal of a pro domain in order to generate active enzyme (for review, see ref. 175). Activation of procollagenases in cartilage and other collagenous matrices is an important control point and a rate-limiting step in collagen resorption (176–180). We have shown that both furin- and trypsin-like serine proteinases are involved in the cascades that lead to such activation (148,176), although the precise proteinases involved remain unknown. It has long been established that plasmin can activate procollagenases (36), leading to the suggestion that the PA/plasmin cascade might function in various resorbing tissues to activate proMMPs, including procollagenases (37). A role for this cascade in the activation of endogenous cartilage metalloproteinases was subsequently reported (181,182), and there is also evidence for its presence in invasive rheumatoid synovium (183). MMP-3 is a known activator of several proMMPs including collagenases (184–186), and the combined proteolysis of a trypsin-like serine proteinase and MMP-3 is required to generate fully active MMP-1 (187). MMP-3 activation of procollagenases is important in the initiation of collagenolysis (188). Intracellular activation of several MMPs (MMP-11, MMP-14, MMP15, MMP-16, MMP-17, MMP-23, MMP-24, MMP-25, and MMP-28) can occur, because they contain a furin or SERINE PROTEINASES AND TISSUE TURNOVER IN RA AND OA PC target sequence (RXKR or RRKR) between their pro and catalytic domains; this can result in secretion of active MMPs. Thus, cartilage resorption involves both metalloproteinases and serine proteinases, which are likely to function through a series of interacting cascades (Figure 2). A large number of serine proteinases have been shown to activate proMMPs, especially proMMP-3 (trypsin, chymotrypsin, tryptase, chymase, plasmin, plasma kallikrein, NE, CTG, trypsinogen 2, thrombin, and matriptase) (38,81,84,189–192). However, the precise activation mechanisms for many of the proMMPs that occur in vivo in both cartilage and other matrices are unknown (175). MMP and serine proteinase activities are further regulated by the endogenous inhibitors tissue inhibitors of metalloproteinases and serpins, respectively. The balance between activation and inhibition is an important control mechanism in proteolysis. For example, genetic deficiency of ␣1 proteinase inhibitor results in a proteinase–antiproteinase imbalance on the lung surface and susceptibility to emphysema (193). However, little is known about the regulatory roles of serpins in the context of cartilage turnover. Conclusions A large number of novel serine proteinases have now been identified, especially transmembrane enzymes (194), and many have not been studied in arthritis. The expression of several of these serine proteinases appears to be dysregulated during tumor development and progression, and their cell surface location implicates important roles in pericellular proteolysis, an important process in cartilage degradation (195). They may also have potential roles in cellular signaling via their cytoplasmic tails or association with other cell surface proteins that are known to activate signaling (e.g., integrins). It has long been established that the traditional serine proteinase pathways such as coagulation, fibrinolysis, and complement are important in the arthritic joint, but it is now beginning to be discovered that serine proteinases have much more diverse functions than previously considered, such as activation of cell surface receptors and interactions with their noncatalytic domains. Collectively, these serine proteinase cascades interact with one another as well as with MMPs to promote the characteristic cartilage destruction of arthritis (Figures 1 and 2). The failure of MMP inhibitors in clinical trials was partly attributable to lack of knowledge of the MMP family and the use of broad-spectrum inhibitors (1). It will be important that any serine proteinase inhibitor developed for clinical use has the 3651 relevant specificity. For example, although ␣1 proteinase inhibitor blocks cartilage degradation (176), its broad-spectrum inhibition profile could be detrimental, unlike the specific DPP-4 inhibitors used in diabetes (110). This highlights the need for a greater understanding of the precise roles of proteinases in tissue turnover and awareness that proteinases can promote both catabolic and anabolic cascades. Although it is still too early to know whether distinct serine proteinase cascade(s) occur in RA and OA, it is evident that there is overlap in terms of collagenolysis (148,176,183). Further understanding of the complex roles of these enzymes, their substrate specificities, and identification of novel serine proteinases in the arthritic joint will provide new opportunities to identify tractable therapeutic targets that effectively prevent pathologic tissue turnover in RA and OA. 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