Emerging roles of serine proteinases in tissue turnover in arthritis.

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: j.m.milner@ncl.ac.uk.
Submitted for publication May 1, 2008; accepted in revised
form August 15, 2008.
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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-
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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
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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
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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
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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.
ACKNOWLEDGMENTS
We are grateful to Professors Tim Cawston (Newcastle
University, Newcastle upon Tyne, UK) and Ian Clark (University of East Anglia, Norwich, UK) for critical reading of the
manuscript.
AUTHOR CONTRIBUTIONS
Dr. Milner 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.
Manuscript preparation. Milner, Patel, Rowan.
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