Understanding the role of tissue degrading enzymes and their inhibitors in development and disease

Best Practice & Research Clinical Rheumatology Vol. 20, No. 5, pp. 983e1002, 2006 doi:10.1016/j.berh.2006.06.007 available online at http://www.sciencedirect.com

11 Understanding the role of tissue degrading enzymes and their inhibitors in development and disease Tim E. Cawston* Amy J. Wilson Musculoskeletal Research Group, 4th Floor Cookson Building, The Medical School, University of Newcastle upon Tyne, NE2 4HH, UK

Cartilage and the underlying bone are destroyed in severe cases of arthritis preventing joints from functioning normally. Cartilage and bone collagen can be specifically cleaved by the collagenases, members of the matrix metalloproteinase family (MMPs), whilst cartilage aggrecan is degraded by members of the ADAMTS (A Disintegrin And Metalloproteinase with ThromboSpondin repeats) family of proteinases. Intracellular cysteine proteinases are involved in bone resorption by osteoclasts and the serine proteinases are involved in activating MMPs. Together, these enzymes act in concert during normal growth and development, especially within the growth plate; however they are also involved in tissue destruction during disease. Synthetic MMP inhibitors have been investigated as a means to block tissue destruction in arthritis but have been unsuccessful, although recent trials with doxycycline suggest this may block joint

Abbreviations: ADAM, a disintegrin and metalloproteinase; ADAMTS, A Disintegrin And Metalloproteinase with ThromboSpondin motifs; Ala, alanine; BMP, bone morphogenetic protein; ECM, extracellular matrix; FDA, Food and Drug Administration; FLRG, follistatin-related gene; Glu, glutamine; GPI, glycosylphosphatidyl inositol; HDAC, histone deacetylase; HDACi, histone deacetylase inhibitor; IGF, insulin-like growth factor; IGFBP, insulin-like growth factor binding protein; IkB, Inhibitor of kappa B; IL, interleukin; Jak-STAT, janus kinase-signal tranducer and activator of transcription; Ki, kinetic inhibition constant (reversible); MAPK, mitogen-activated protein kinase; MAPK-JNK, mitogen-activated protein kinaseec-Jun NH2-terminal kinase; MMP, matrix metalloproteinases; MRI, magnetic resonance imaging; mRNA, messenger ribonucleic acid; NF-kB, nuclear factor kappa B; OA, osteoarthritis; OSM, oncostatin M; PI3, phosphatidylinositol-3 protein; RA, rheumatoid arthritis; Runx, runt transcription factor family; Sox, SRYrelated transcription factor family; TGF, transforming growth factor; TIMP, tissue inhibitors of metalloproteinases; TNF, tumour necrosis factor; tPA, tissue-type plasminogen activator; uPA, urokinase-type plasminogen activator; US, United States of America; VEGF, vascular endothelial growth factor. * Corresponding author. Tel.: þ44 191 222 5363; Fax: þ44 191 222 5455. E-mail address: [email protected] (T. E. Cawston). 1521-6942/$ - see front matter ª 2006 Elsevier Ltd. All rights reserved.

984 T. E. Cawston and A. J. Wilson

destruction in osteoarthritis. It is likely that combinations of therapy will be required to ensure that joint destruction is prevented in arthritis patients. Key words: metalloproteinase; cartilage; collagen; extracellular matrix; growth plate; arthritis; inhibitor.

INTRODUCTION Both cartilage and the underlying bone are destroyed in severe cases of arthritis preventing the joints from functioning normally. Cartilage tissue consists of a single cell type, chondrocytes1, which are embedded within a dense extracellular matrix (ECM) of aggrecan, type II collagen and other minor components. Collagen fibres are made up of rod-shaped molecules that combine in a staggered array to form cross-linked fibres giving connective tissues strength and rigidity. Trapped within these collagen fibres are aggrecan molecules2 that, in the presence of hyaluronic acid, form highly charged aggregates that attract water into the tissue and allow cartilage to resist compression. During growth and development chondrocytes within cartilage synthesise matrix components and the level of synthesis exceeds the rate of degradation. In normal adult cartilage a steady state of matrix turnover is maintained where the rate of matrix synthesis equals the rate of degradation. Any change in this steady state affects the functional integrity of the cartilage. During matrix resorption an increase in the rate of degradation occurs and/or a reduction in the rate of matrix synthesis.3 Proteinases responsible for matrix turnover The five main classes of proteinase are classified according to the chemical group that participates in the hydrolysis of peptide bonds (Figure 1).4 Cysteine, aspartate and threonine proteinases are predominantly active at acid pH and act intracellularly; the serine and metalloproteinases are active at neutral pH and act extracellularly.

Figure 1. There are five classes of proteinase, three of which act predominantly intracellularly (aspartate, cysteine and threonine) and two of which are predominantly extracellular (metallo and serine). Examples are shown of enzymes from each class.

Tissue degrading enzymes and their inhibitors 985

Some extracellular proteinases are membrane-bound (rather than secreted from the cell) and are associated with cytokine processing, receptor shedding and the removal of proteins involved in cellecell or cellematrix interactions.5 Some proteinases are not directly involved in the cleavage of matrix proteins but activate proenzymes that, in turn, degrade matrix. All classes of proteinase play a part in the turnover of connective tissues and, often, one proteinase pathway combines with another. For example, in bone the removal of the outer osteoid layer by metalloproteinases precedes the attachment of the osteoclast and the subsequent breakdown of the ECM by cysteine proteinases.6 A close apposition of intra- and extra-cellular pathways will be found in many conditions where there is connective tissue turnover. EXTRACELLULAR PROTEOLYSIS e NEUTRAL PROTEINASES The metzincin superfamily These metalloproteinases are distinguished by a highly conserved motif containing three histidine residues that bind zinc at the catalytic site and a conserved methionine turn that lies beneath the active-site zinc.7 Metalloproteinases are divided into five multigene families: the serralysins, the astacins, ADAMs (A Disintegrin And Metalloproteinase), adamalysins, the matrix metalloproteinases (MMPs)8 and the pappalysins9, which cleave insulin-like growth factor binding protein (IGFBP)-4 and e5.10 Matrix metalloproteinases This multigene family of over 23 secreted and cell surface zinc-dependent endopeptidases process or degrade numerous substrates at neutral pH.11 All MMPs contain common domains (Figure 2), all have zinc present at the catalytic centre and all are produced in a proenzyme form. Latency of the proMMP is maintained by the interaction of a conserved cysteine residue in the prodomain with the catalytic zinc in the active site.12 The MMP family are best known for their ability to cleave components of the ECM but they also cleave other proteinases, proteinase inhibitors, latent growth factors, chemotactic molecules, growth factor binding proteins, cell surface receptors and cellecell adhesion molecules.13 Traditionally MMPs were divided into different groups, according to their substrates, called the stromelysins, collagenases and gelatinises.11 MMP-3 and MMP-10 (stromelysin-1 and e2, respectively) have similar substrate specificity but have distinct tissue expression patterns. Their natural substrates are probably proteoglycans, fibronectin and laminin and both enzymes are able to activate latent collagenases. There are three mammalian collagenases: MMP-1, MMP-8 and MMP-13 (collagenase-1, -2 and e3, respectively). These enzymes cleave fibrillar collagens at a specific site, producing characteristic three-quarter- and one-quarter-sized fragments; MMP-2 and MMP-14 can also cleave in this way. The collagenases differ in their specificity for different collagen types: MMP-13 prefers to cleave type II collagen whilst MMP-1 and MMP-8 prefer type III and I, respectively. The two gelatinases cleave denatured collagen (gelatin), type IV and V collagen and elastin. MMP-2 (gelatinase A) is the most widespread of all the MMPs and can activate proMMP-13. MMP-9 (gelatinase B) is expressed in a wide variety of transformed and tumour-derived cells.

986 T. E. Cawston and A. J. Wilson Group 1

MMP-7, -26

Minimal domain

Group 2

MMP-1, -3, -8, -10, -12, -13, -18, -19, -20, -22, -27

Simple haemopexin domain

Group 3

MMP-2, -9

Gelatin binding

Group 4

MMP-11, -28

Furin activation, secreted

Group 5

MMP-21

Vitronectin insert

Group 6

MMP-14, -15, -16, -24

Transmembrane MMPs

Group 7

MMP-17, -25

GPI anchored MMPs

Group 8

MMP-23

Cys/Pro rich with Iglike domain

Figure 2. Domain structures of matrix metalloproteinases (MMPs). MMPs were originally classified according to their substrate specificity but are now more commonly grouped according to domain structure. All metalloproteinases have a catalytic, zinc-binding domain (Zn) and pro-peptide that preserves latency. Some contain a furin recognition motif (Fu) that allows intracellular activation by furin-like proteinases. Apart from MMPs -7, -26 (Group 1) and -23 (Group 8) all contain a haemopexin domain that often determines substrate specificity (Group 2). Other domains include the fibronectin-like domains (F) in MMP-2 and -9 (Group 3) and the vitronectin-like domain (V) in MMP-21 (Group 5). Some furin-activated enzymes are secreted (MMP-11 and -28; Group 4) whilst some MMPs are anchored to the cell surface via a TM with cytoplasmic tail (Cyt) (MMP-14, -15, -16 and e24: Group 6) or via a glycosylphosphatidyl inositol (GPI) anchor (MMP-17 and e25: Group 7). MMP-23 is structurally unique and contains an N-terminal TM (actually an N-terminal signal anchor), a cysteine array (CA) and a immunoglobulin-like domain (Ig-like) (Group 8).

Increasingly, however, the number, complexity and range of MMP substrates identified makes grouping by domain structure a more straightforward approach to MMP classification (Figure 2).8,14 Neither MMP-7 (matrilysin) nor MMP-26 has a haemopexin domain (Group 1) but most MMPs resemble MMP-1 (Group 2). MMP-2 and MMP-9 have fibronectin-like inserts (Group 3) whilst MMP-21 has a vitronectin-like domain insert (Group 5). Groups 4e8 all have a furin cleavage site. Group 6, MMPs 14e17 are also known as membrane-type (MT) MMP (numbered MT1-4 MMP, respectively) due to the presence of transmembrane domains, while MMP-17 and MMP-25 both have a cytoplasmic glycosylphosphatidyl inositol (GPI) anchor (Group 7). MMP23 has a C-terminal immunoglobulin-like domain (Group 8). MMPs regulate many biological processes and consequently are precisely controlled at a number of critical steps that include: synthesis and secretion, activation of the proenzymes, inhibition of active enzymes and localisation or clearance of MMPs (Figure 3). Cytokines such as interleukin (IL)-1, tumour necrosis factor (TNF)-a and IL-17 stimulate numerous cell types to produce MMPs15,16 and combinations of cytokines can be highly effective in synergistically upregulating these enzymes.17 The control of MMP proenzyme activation is an important control point in connective tissue breakdown.18e20 MMPs that have a furin recognition sequence between the propeptide and the catalytic domain (see Figure 2) are often activated within the golgi and secreted in an active form. (Figure 3) Members of the MT-MMP family are also activated in this way and some members (MMP-14e16, as well as MMP-24 and 25) can activate proMMP-2.21e25 MMP-14 can also activate proMMP-13. Plasmin and other

Tissue degrading enzymes and their inhibitors 987

Figure 3. Control of matric metalloproteinase (MMP) activity. Cytokines and growth factors can upregulate or downregulate MMP expression and sometimes act synergistically (1). Different intracellular signalling pathways combine (2) to activate or suppress transcription (3). RNA can be unstable and rapidly processed (4). ProMMPs can be activated intracellularly by furin (5) or after they have left the cell (6). Some MMPs are stored in granules within the cell (7) prior to secretion. Secreted MMP can be expressed on the cell surface (8), bound to cell surface receptor proteins (9) or sequested by extracellular matrix proteins (10). All active MMPs can be inhibited by tissue inhibitors of metalloproteinases (TIMPs) (11). Other control mechanisms include secretion to specific regions of the plasma membrane, proteolytic processing and inactivation of MMPs and endocytosis and lysosomal breakdown.

serine proteinases can activate some secreted proMMPs19 and active MMP-3 can activate the pro-collagenases and pro-MMP-9. All active MMPs are inhibited by tissue inhibitors of metalloproteinases (TIMPs)26,27 that bind tightly to active MMPs in a 1:1 ratio (Figure 3), allowing control of connective tissue breakdown. Therefore, if TIMP levels exceed those of active enzyme, connective tissue turnover is prevented. TIMP-2 is associated with the activation of proMMP-2, TIMP-3 inhibits some members of the ADAM family and is bound by the ECM after secretion, whilst TIMP-4 is predominantly localised in the heart but can also be produced by joint tissues.28 TIMP-1 and -3 are up-regulated by growth factors such as transforming growth factor (TGF), insulin-like growth factor (IGF)-1 and oncostatin M (OSM) and these agents also induce matrix synthesis.29 All active MMPs are bound and inhibited by a2-macroglobulin and the complexes formed can be measured in the circulation.30 Proteolysis often occurs in the immediate vicinity of the cell in peri-cellular pockets close to the cell membrane where MMPs can be secreted to specific areas at the cell surface.31 These localisation mechanisms allow a high degree of control and can enhance MMP activity, prevent access of MMP inhibitors, concentrate MMPs to their precise target substrate and limit the extent of proteolysis to a discrete region. MMPs with transmembrane domains (MT-MMPs) are found at the cell surface and can cleave

988 T. E. Cawston and A. J. Wilson

cell surface receptors and activate other enzymes at the cell surface. Some MMPs bind to peri-cellular matrix proteins (Figure 3). Cell surface heparan sulphate can bind MMPs such as MMP-732 and also TIMP-3, whilst MMP-1 can bind to the cell-surface protein EMMPRIN (Figure 3).33 ADAM family of proteinases To date, over 25 ADAM genes and 19 ADAMTS (A Disintegrin And Metalloproteinase with ThromboSpondin motifs) genes have been described. ADAMs are usually membrane-anchored proteinases with diverse functions conferred by the addition of different protein domains.5,34 The disintegrin domain can bind to integrins and prevent cellecell interactions. Cysteine-rich, epidermal growth factor-like, transmembrane and cytoplasmic tail domains are also found. Members of the ADAMTS family are known to be involved in proteoglycan cleavage. Serine proteinases Many in vitro experiments with tissue or cells point to a role for the plasminogene plasmin system in the activation of proMMPs.11,35 IL-1 and TNFa-induced proteoglycan release can be blocked with an inhibitor of the urokinase-type plasminogen activator (uPA).36 The inclusion of a1 proteinase inhibitor to resorbing cartilage effectively blocks the release of collagen implicating serine proteinase(s) in the activation cascades of pro-inflammatory cytokine-induced proMMPs.19 INTRACELLULAR PATHWAYS e ACID PROTEINASES Cysteine proteinases Cysteine proteinases can degrade type I collagen at acidic pH and cysteine proteinase inhibitors prevent the resorption of bone explants, suggesting an involvement of lysosomal cysteine proteinases in matrix resorption.37 The level of cathepsin B is raised in osteoarthritis (OA) tissue and raised levels of cathepsins B, L and H are found in antigen-induced rat arthritis models and within the rheumatoid arthritis (RA) affected joint. Incubation of resorbing cartilage with specific cathepsin B inhibitors blocks the release of proteoglycan fragments38 suggesting an involvement in cartilage proteoglycan breakdown. Everts et al showed that substantial amounts of fibrillar collagen accumulate intracellularly in the presence of cysteine proteinase inhibitors.39 Cathepsin K is known to play a key role in collagen turnover and subsequent bone resorption40,41 as it cleaves the N-terminal end of the triple helix at pH values as high as pH 6.542 and is produced by synovial fibroblasts, contributing to synovium-initiated bone destruction in the rheumatoid joint.43 Both cathepsins K and S are expressed in RA and OA synovial tissue44 and there is evidence that cathepsin K is localised to sites of cartilage erosion.45 Cathepsin K has potent aggrecan-degrading activity and the resulting degradation products potentiate its collagenolytic activity towards collagen types I and II.46 Calpain (a calcium-dependent neutral cysteine proteinase) can cleave proteoglycan47 and its presence correlates with arthritis and tissue destruction48 although the precise role of calpains in arthritic disease is not known.49

Tissue degrading enzymes and their inhibitors 989

Threonine proteinases Threonine proteinases represent a relatively new class of proteinases50 found in the proteosome, which is a ubiquitously expressed intracellular protease complex that performs many intracellular roles. This includes the degradation of phosphorylated and ubiquitinated inhibitor of kappa B (IkB). ROLE OF TISSUE DEGRADING ENZYMES IN THE GROWTH PLATE Tissue degrading enzymes are vital for normal physiological processes such as embryonic development, growth and tissue remodelling, where the ECM must be degraded and tissue remodelled.51e53 For example, in long-bone growth, elongation occurs at the growth plate of long bones where chondrocyte proliferation, maturation and apoptosis result in expansion of the cartilage matrix and therefore longitudinal growth. This is followed by endochondral ossification where avascular cartilage is replaced by highly vascularised, mineralised bone matrix.51 Given the complexity of limb growth and patterning it is not surprising that disturbances to the order of events, or lack of specific genes, within the process of limb bud development or endochondral ossification can cause severe skeletal abnormalities. Therefore, the series of events required for bone elongation and patterning is highly regulated and coordinated. The regulation of longitudinal growth at the growth plate is controlled through the interaction of circulating systemic hormones and locally produced peptide growth factors, which can trigger changes in gene expression by growth plate chondrocytes. Recently several cytokine families have been identified as key players in the regulation of limb formation. These families include fibroblast growth factors, bone morphogenic proteins (BMPs), parathyroid hormone and Indian Hedgehog and involve both the SRY-related transcription factor family (Sox) and the runt transcription factor family (Runx) members (Figure 4).51,52 Chondrocytes originating at the growth plate are initially proliferative and organised in short columnar rows between which they secrete type II collagen. As the chondrocytes progress away from the growth plate they stop proliferating and become prehypertrophic and finally hypertrophic cells that express predominantly type X collagen and are capable of mineralising the cartilage matrix (Figure 4). The hypertrophic chondrocytes then undergo apoptosis and blood vessels invade the newly formed cartilaginous matrix. This vascularisation is the step required for replacement of the soft tissue with trabecular bone (primary spongiosa).53 Osteoclast precursors originating from haemopoietic stem cells then migrate together with endothelial cells into the mineralised cartilage where they fuse to form large multinucleated osteoclast cells, which are able to dissolve bone mineral and degrade the matrix.54 Osteoblasts are then recruited to the sites of resorption to lay down trabecular bone. In this aspect endochondral ossification can be said to be unique in that it involves the remodelling and replacement of a template tissue (cartilage) with a distinct permanent tissue (bone)55,56, a process that requires specific enzymes from the MMP, ADAM and ADAMTS families. ELUCIDATING THE ROLES OF MMP, ADAM AND ADAMTS FAMILY MEMBERS THROUGH KNOCKOUT ANIMAL PHENOTYPES The different roles of MMPs in skeletal development have been investigated using knockout animals. Those that give skeletal phenotypes are MMP-2, -9, -13 and -14.

990 T. E. Cawston and A. J. Wilson

Articular Chondrocyte Round Proliferative Chondrocyte Flat Proliferative Chondrocyte Pre-Hypertrophic Chondrocyte Hypertrophic Chondrocyte

Cartilage

C PTHrP Y T O K I N E S M A T R I X

Bone

Sox 5/6/9 Ihh VEGF

GDF FGFR3

Runx 2/3 CX

CII

Osteocytes: MMP 3

MMP 10 M M P

TIMP 1-4

+ T I M P

CI

MMP 1-2

Osteoblasts: MMP 1, 3, 9, 10, 11, 13

MMP 9-11

TIMP 1, 2

MMP 13 MMP 14

Osteoclasts: MMP 2, 9, 10, 14 TIMP 1

Figure 4. Gene expression in developing endochondral bone. Schematic representation of gene expression in a mouse long-bone at a late stage of foetal development. Abbreviations: C, collagen; FGFR, fibroblast growth factor receptor; GDF, growth and differentiation factor; Ihh, indian hedgehog; PTHrP, perethyroid hormone-related peptide; Runx, runt transcription factor family; Sox, SRY-related transcription factor family; MMP, matrix metalloproteinase; TIMP, tissue inhibitor of MMP; VEGF, vascular endothelial growth factor. Diagram adapted from Provot & Schipani (2005).53

MMP-9 (gelatinase B) deficient mice exhibit delayed vascularisation and ossification resulting in a lengthening of the growth plate to approximately eight times that of wild-type mice.57 This aberrant skeletal development is, however, compensated for 3 weeks postnatally so that the animals ultimately have a normal skeletal phenotype.

Tissue degrading enzymes and their inhibitors 991

This finding, along with bone resorption in vitro models, indicates that MMP-9 is specifically required for osteoclast and endothelial cell recruitment into the cartilage by specifically regulating the proteolysis of non-mineralised cartilage and the release of ECM-bound vascular endothelial growth factor (VEGF).58 Engsig et al59 have found that VEGF is necessary for invasion of the hypertrophic cartilage by osteoclasts. MMP-13 (collagenase 3) knockout mice show abnormal skeletal development with dwarfism and bowing of the legs.55 These deformities are similar to those seen in patients with spondyloepimetaphyseal dysplasia-Missouri type, which is caused by a missense mutation in the MMP-13 gene.60 Interestingly these abnormalities, in both affected patients and knockout mice, resolve themselves by adolescence or 12 weeks of age, respectively. They appear to be caused by late chondrocyte exit from the growth plate despite normal differentiation. Stickens et al55 found that the two major components of cartilage extracellular matrix (type II collagen and aggrecan) are in vivo substrates of MMP-13 and that the breakdown of cartilage collagen and aggrecan is a coordinated process in which MMP-13 works synergistically with MMP-9. Mice lacking both MMP-13 and MMP-9 show a severe endochondral bone phenotype with drastically shortened long bones. These mice have diminished ECM remodelling, prolonged chondrocyte survival, delayed vascular recruitment and defective trabecular bone formation.55 MT1-MMP (MMP-14) knockout mice have severe skeletal development defects such as craniofacial dysmorphism and dwarfism due to growth plate abnormalities with marked retardation of postnatal growth and death by 3 weeks of age.61 These dwarfism abnormalities are a consequence of decreased proliferation by proliferative chondrocytes at the growth plate. Angiogenesis and vascular invasion of the cartilage also causes enlargement of the hypertrophic chondrocyte zone and delayed ossification. This lack of proper vascularisation is not restricted to the cartilage and may explain the premature mortality seen in these mice. Explants from MMP-14 null mice also show deficient activation of proMMP-2, suggesting that MMP-14 is essential for its activation.61 MMP-2 (gelatinase A) can degrade collagenous substances and is therefore important in vascular invasion, accelerating the process of matrix mineralisation62 and MMP-2 knockout mice show delayed formation of dentin and bone that are both mineralised tissues.63 Deletion of MMP-2 results in a more severe and MMP-9 in a less severe antibody-induced arthritis in animal models, indicating a suppressive role for MMP-2 and a pivotal role for MMP-9 in the development of joint disease.64 The involvement of MMPs in skeletal growth indicates the importance of proper ECM remodelling as a major limiting factor for vital parts of the long bone developmental process, including apoptosis, angiogenesis and osteoblast recruitment. ADAM enzymes are membrane-bound proteins that have been implicated in cell adhesion (both to matrix and other cells), protein ectodomain shedding, matrix degradation, cell fusion and tissue morphogenesis. Several have been implicated in skeletal formation. The cysteine-rich disintegrin domain of ADAM-8 has been implicated in the later stages of osteoclast differentiation.65 ADAM-10 is expressed predominantly by osteoblasts at sites of active bone formation and by the superficial layer of chondrocytes in articular cartilage.66 It appears that this enzyme has catalytic activity within the Golgi and may play a role in activation of Notch receptor homologs. This implicates ADAM-10 in cell-fate determination of osteoblast progenitor cells with a potential impact on skeletal development and remodelling.67 ADAM-12 has been identified as an important regulator of osteoclast differentiation66, however, its role is mediated by interaction with the cysteine-rich domain of the follistatin-related gene (FLRG),

992 T. E. Cawston and A. J. Wilson

which causes a significant reduction in the number of osteoclasts formed and in the average number of nuclei per osteoclast.68 ADAMTS enzymes are a recently identified subgroup of the metalloproteinase superfamily. ADAMTS-1 has been shown to have potent anti-angiogenic activity and to have the ability to process aggrecan, versican and brevican (all proteoglycans that are found in cartilage). Transgenic mice with inactivated ADAMTS1 show significantly reduced growth rates although the relevance of this phenotype in the context of known activities of ADAMTS-1 is so far unclear.69 ADAMTS-2 and 3 are highly homologous and can cleave the N-terminal collagen propeptide, which is required prior to assembly of collagen type I and II fibrils in vivo.70,71 ADAMTS-5 has been found to be the major aggrecanase in mice although whether it is also the major aggrecanase in humans has yet to be determined.72e74 ARTICULAR CARTILAGE AND ITS DESTRUCTION e THE ROLE OF PROTEOLYTIC ENZYMES Collagenases MMP-1 and MMP-13 are synthesised by macrophages, fibroblasts and chondrocytes when these cells are stimulated with inflammatory mediators. MMP-8 is predominantly released from neutrophils upon stimulation of the cell but is also produced by chondrocytes. All three collagenases are present in diseased cartilage75 although their control, by external stimuli, can be different: for example, retinoic acid, which downregulates MMP-1, is known to upregulate MMP-13 in some cells.76 The stromelysins (MMP-3 and MMP-10) are present in articular cartilage and synovium from patients with either RA or OA.77e79 MMP-2 and MMP-9 protein levels are elevated in RA synovial fluids and tissues80e82 and there is clear evidence of a role for MMP-9 in angiogenesis. There is an increase in levels of different MMPs in rheumatoid synovial fluid, in conditioned culture media from rheumatoid synovial tissues and cells, synovial tissue at the cartilageepannus junction from rheumatoid joints, osteoarthritic cartilage and in animal models of arthritis.26,75,83 In RA there is a marked increase in matrix breakdown. This contrasts with the situation in OA where both the rate of matrix synthesis and breakdown are increased, leading to the formation of excess matrix in some regions (such as osteophytes) with focal loss of matrix in other areas. The major aggrecan fragments from resorbing cartilage are cleaved at a specific glutamineealanine bond (Glu(373)eAla(374)).84 ADAMTS-4 and -5 are thought to be the mediators of aggrecan catabolism as they cleave proteoglycan at this bond3,85 and ADAMTS-5 null mice show reduced cartilage destruction in antigen-induced arthritis models.86 ADAMTS-1, -8 and -15 also degrade aggrecan at this site.87e89 Purified chondrocyte membranes also cleave at this GlueAla bond but it is not known if this activity is associated with a known aggrecanase or a different enzyme.90 Recently, ADAMTS-9 was also identified as an aggrecanase, but cleaved aggrecan at the Glu(1771)eAla(1772) bond.91 Proteoglycan release from cartilage occurs following stimulation with a variety of mediators such as IL-1, TNFa, IL-17, retinoic acid and fibronectin fragments.86,92,93 Levels of ADAMTS-4 are upregulated in cartilage in response to IL-1 and TNFa and in synovial fibroblasts in response to TGFb85,94, whilst ADAMTS-5 appears to be unaffected. In an immortalised chondrocyte line, ADAMTS-1, -4, -5 and -9 were all regulated by a mixture of IL-1 and OSM, although the speed of induction differed between these enzymes.95 Aggrecanase activity can be blocked by specific synthetic inhibitors96

Tissue degrading enzymes and their inhibitors 993

and by TIMP-3.97 A role for neprilysin-induced aggrecanase activity via the generation of regulatory peptides has also been proposed.98 ADAM-10, -12 -15 and -17 have also been described in cartilage99 and ADAM-17 is known for its ability to release TNFa from the cell surface.100 Both tissue- and urokinase-type plasminogen activators (tPA and uPA) are found in cartilage and cleave plasminogen to plasmin. Other serine proteinase activities have also been implicated in arthritis, for example granzyme B can initiate proteoglycan (but not collagen) degradation and granzyme B-positive cells can be detected in synovium and at the invasive front in RA.101 Many enzymes have furin recognition motifs in their prodomains and studies have shown that blocking furin can prevent the release of collagen from resorbing cartilage.20

SYNTHETIC MMP INHIBITORS AND ARTHRITIS As MMPs have an important role in ECM turnover many groups have investigated if blocking MMPs will prevent tissue destruction. Early challenges involved the development of MMP inhibitors with high potency that would be orally available, avoiding modification or destruction within the gut. Some of these compounds were found to cause musculoskeletal pain and tendonitis was identified as a reversible side-effect in treated patients.102,103 These effects commenced in the small joints of the hand and upper limbs and the symptoms were time and dose dependent but reversible. Such symptoms were seen with a compound Ro 31-9790 (Roche) and this led to its withdrawal from development as an arthritis treatment. New compounds are now screened in rodent models and compounds that cause musculoskeletal side effects are discarded at an early stage of development. The cause of these side effects is not known and Peterson103 has emphasised the need to estimate the dose required for efficacy and that producing toxicity during the development of each new inhibitor. MMPs are involved in many physiological processes that include the activation and/or release of cytokines and growth factors from the ECM.13 Inhibition of such MMPs could block processes that in normal tissues maintain the precise balance between synthetic and degradative pathways. Synthetic MMP inhibitors were initially studied for their ability to prevent joint destruction in animal models of arthritis. Sabatini et al104 showed that S-34291, a wide-spectrum MMP inhibitor with a preferential effect on MMP-13 as compared with MMP-1, prevented the loss of cartilage ex vivo and in a guinea pig model of OA. Two papers from Ishikawa et al (Fujisawa Pharmaceuticals) established that the broad-spectrum metalloproteinase inhibitors FR217840 and FR255031 suppressed joint destruction in adjuvant and collagen-induced arthritis rat models, respectively and they have suggested that these inhibitors may be novel anti-rheumatic drugs.105,106 Trocade (Ro 32-3555), a selective collagenase inhibitor has a low nanomolar inhibition constant (Ki) against MMP-1, -8 and -13 with approximately 10- to 100-fold lower potency against MMP-2, -3 and -9. It blocked IL-1a-induced collagen release from cartilage explants and, in vivo, prevented cartilage degradation in a rat granuloma model, a P. acnes-induced rat arthritis model and an OA model using the SRT/ORT mouse.107 Novartis have described an orally active hydroxamate MMP inhibitor, CGS 27023A, with a nanomolar kinetic inhibition constant (reversible) (Ki) against MMP-1, -2, -3, -9, -12 and -13 that was chondroprotective in both rabbit and guinea pig models of OA.108,109 A synthetic inhibitor, Tanomastat (Bayer: BAY 12-9566) targets MMP-2, -3, -8, -9

994 T. E. Cawston and A. J. Wilson

and -13 with low activity against MMP-1 and is effective in guinea pig and canine models of OA.110 To date 27 compounds have been tested as anti-arthritis treatments.103 Phase I clinical trials of CGS 27023A in humans were halted due to musculoskeletal side effects.111 A large scale trial of 1000 RA patients treated with Trocade was terminated after 1 year because of a lack of efficacy, although the drug was reported to be well tolerated in patients.112 OA patients treated for 3 months with BAY-12-9566 had no musculoskeletal side effects reported and the drug could be detected in the cartilage of treated patients undergoing joint replacement.113 However, this compound was withdrawn from patient phase III OA trials following negative results in cancer patients.114 A number of companies are developing MMP-13 inhibitors such as CP-544439 (Pfizer) and WAY-170523 (Wyeth Research), which were taken to phase II clinical trials. Other compounds such as CPA-926 (developed by Kureha and Sanyo for the treatment of OA) and the broad spectrum MMP inhibitor ONO-4817 (developed by ONO) have also been used in phase II or I clinical trials, respectively.103 Pharmacia, Wyeth and Proctor & Gamble have all reported on the preclinical evaluation of MMP inhibitors for the treatment of arthritis.14,103 The Bayer compound, BAY 12-9566, was withdrawn as it was associated with increased tumour growth and poor survival times in small cell lung cancer114 but no other cases of such effects have been reported with other compounds. It is not necessarily logical to assume that an effect seen with one member of this class of compounds will automatically be seen by all and there are significant differences in chemical structure and metabolism of individual inhibitors. The antibiotic doxycycline is known to inhibit MMPs. Some recent derivatives have been shown to inhibit MMPs but have no antibiotic activity and these have been proposed as treatments to prevent cartilage damage in the arthritides.115 Periostat (CollaGenex Pharmaceuticals, Inc.), a sub-antimicrobial dose of doxycycline, is currently the only US Food and Drug Administration (FDA) approved MMP inhibitor and is licensed for the treatment of periodontal disease. These compounds are effective in animal models116 but their effectiveness in patients with RA has been less clear.117,118 However, results from a trial of 430 OA patients randomly assigned to receive either doxycycline or placebo treatment look to be promising.119 When the X-rays of the two groups were compared at 30 months the results suggested that protection of the affected joint was seen in the treated patients. There was also a suggestion from this trial that early cartilage changes could be driven by different mechanisms to late cartilage loss. A variety of explanations have been offered to explain why many of the metalloproteinase inhibitors have been unsuccessful in clinical trials in patients with joint diseases and cancer.120 There is no doubt that MMPs are present and active in joint diseases, but if compounds are unable to penetrate the cartilage/bone/synovial interface they would clearly be ineffective. Early inhibitors were originally screened against a limited set of available MMPs and so may not inhibit some that have subsequently been discovered and which play important roles. Further studies are required to demonstrate the effectiveness of MMP inhibitors in the prevention of joint destruction, although the clinical evaluation of these drugs is difficult and expensive. Radiographs are still the most reliable measure of joint damage but any change in joint damage is impossible to detect over short periods of time. Whilst some progress has been made with the use of more sensitive magnetic resonance imaging (MRI) techniques to image joints this technology has still to be proven and routine centres do not have access to validated methods for quantification.

Tissue degrading enzymes and their inhibitors 995

SIGNALLING PATHWAY INHIBITORS AND MMP EXPRESSION The efficacy of anti-cytokine biotherapies in the treatment of RA patients provides supporting evidence that the inhibition of a signal-transduction pathway, involved in proteinase regulation, could be a potential therapeutic target. Cytokine-mediated transcriptional regulation has been shown to be a key mechanism in the control of expression of many MMPs. The four main pathways involved in the inflammatory response are believed to be those acting through nuclear factor kappa B (NF-kB), mitogen-activated protein kinase (MAPK), phosphatidylinositol-3 protein (PI3) kinase and janus kinase-signal transducer and activator of transcription (Jak-STAT). Both synthetic and natural inhibitors, along with biologicals, of these pathways have been developed and tested in vitro and in vivo with varying degrees of success.121 For example, SP600125, a pharmacological inhibitor of the MAPK-JNK (c-Jun N-terminal kinase) pathway decreases joint destruction in an adjuvant arthritis model, in part by diminishing the production of MMP-1.122 Gene therapy using inhibitors of both these pathways appear efficacious in arthritis animal models123,124 and represent excellent potential methodologies to prevent the induction of degradative MMPs. Acetylation is a key post-translational protein modification that controls signal transduction and gene transcription events.125 Deacetylation is mediated by a family of 11 enzymes, the histone deacetylases (HDACs). Many structurally divergent HDAC inhibitors (HDACi) have been developed as cancer therapies as they cause cancer cells specifically to undergo growth arrest, differentiation or apoptosis in vitro and in vivo.126 HDACi potently inhibited cartilage degradation with decreased levels of collagenolytic enzymes in explant-conditioned culture and blocked the cytokine (IL-1 and OSM) induction of key MMPs (e.g. MMP-1, -3, -8 and -13) and aggrecanases (e.g. ADAMTS4, ADAMTS5 and ADAMTS9) at the messenger ribonucleic acid (mRNA) level.127 Thus, HDACi function as potent repressors of metalloproteinase expression in chondrocytes and may, therefore, be a new treatment for any of the destructive arthritides and other inflammatory diseases mediated by metalloproteinases.128 MMPeSUBSTRATE INTERACTIONS As more detailed information about the interaction of MMPs with their substrates becomes available it may be possible to design inhibitors that target areas of the enzyme other than the active site. For example, the C-terminal haemopexin-like domain of collagenases is required for collagenolysis presumably because of interactions with substrate. The activation of the proenzyme is also a valid target, again requiring a detailed knowledge of the underlying biology. MODIFICATION OF TIMP FUNCTION OR EXPRESSION One further possibility for inhibiting metalloproteinase activity is to induce the expression of their natural inhibitors, the TIMPs, or exogenously deliver modified TIMPs that are targeted to inhibit specific enzymes.129e131 Both TIMP-1 and TIMP-2 are capable of preventing cartilage destruction ex vivo, while the N-terminal domain of TIMP-3 in a similar system can prevent aggrecan release. Adenoviral delivery of TIMP-1 and -3 prevents cartilage degradation and invasion by rheumatoid synovial fibroblasts in vitro132, however, their efficacy in animal arthritis models requires further confirmation.

996 T. E. Cawston and A. J. Wilson

Finally, like many metalloproteinases, TIMP-1, -3 and -4 are regulated at the transcriptional level and can be induced by a number of growth factors and cytokines. Modulation of these cytokine pathways may re-address the local balance of metalloproteinase and TIMP activities believed to be pivotal in determining the extent of ECM turnover in disease. SUMMARY Proteinases are clearly associated with growth and development and with the homeostatic maintenance of connective tissues. The MMPs are implicated as responsible for matrix turnover in the growth plate and in tissue damage in disease. Cartilage and the underlying bone are destroyed in severe cases of arthritis preventing joints from functioning normally. Inhibition of cartilage collagen destruction still remains an important and viable target to prevent joint damage in arthritic disease. Although the trials of MMP inhibitors in patients have been disappointing, new agents are still under development and these may overcome some of the problems of both delivery and side effects. A key to future success is to identify the specific MMPs that are responsible for destruction within arthritic joints in different diseases. This will allow highly specific inhibitors to be developed that target individual enzymes, either directly or via signalling pathways or epigenetic control mechanisms, potentially reducing side effects. MMPs are not alone in being implicated in joint disease. Serine proteinases are believed to be involved in MMP activation and cysteine proteinases have been shown to degrade collagen, particularly during bone resorption. Other therapeutic targets include blocking cytokine action or the signalling pathways involved in proteinase regulation. It may also be possible to block enzyme substrate interactions or increase the effectiveness of natural inhibitors. It may be necessary to combine proteinase inhibitors, either in sequence or with other agents that hit key specific steps in the pathogenesis, before the chronic cycle of joint destruction found in these diseases can be broken.

Research agenda
Discover the precise role of individual proteinases in normal cartilage and bone development and turnover to gain a greater understanding of their role in disease processes.
Investigate the mechanisms of abnormal skeletal development to understand the process of normal skeletal development.
Develop specific MMP inhibitors that block the action of key matrix metalloproteinases (MMPs) in different diseases.
Understand the mechanism of cleavage of extracellular matrix (ECM) components to target exo-sites and prevent breakdown.
Design trials to treat patients at early stages of disease using agents that inhibit multiple proteinase pathways.
Develop biomarkers related to the cleavage of the ECM that allows disease progression and the response to treatment to be effectively monitored.

Tissue degrading enzymes and their inhibitors 997

ACKNOWLEDGEMENTS We thank the arthritis research campaign, the Wellcome Trust, FARNE, Dunhill Medical Trust, JGW Pattinson Trust and the Nuffield Foundation (Oliver Bird Fund) for financial support. REFERENCES 1. Goldring MB. The role of the chondrocyte in osteoarthritis. Arthritis and Rheumatism 2000; 43(9): 1916e1926. 2. Iozzo RV. Matrix proteoglycans: from molecular design to cellular function. Annual Review of Biochemistry 1998; 67: 609e652. 3. Mort JS & Billington CJ. Articular cartilage and changes in arthritis: matrix degradation. Arthritis Research 2001; 3(6): 337e341. 4. Barrett AJ, Rawlings ND & Woessner Jr. JF. Handbook of Proteolytic Enzymes. London: Academic Press, 1998. 5. Becherer JD & Blobel CP. Biochemical properties and functions of membrane-anchored metalloprotease-disintegrin proteins (ADAMs). Current Topics in Developmental Biology 2003; 54: 101e123. 6. Everts V, Delaisse JM, Korper W et al. Degradation of collagen in the bone-resorbing compartment underlying the osteoclast involves both cysteine-proteinases and matrix metalloproteinases. Journal of Cellular Physiology 1992; 150(2): 221e231. 7. Stocker W, Grams F, Baumann U et al. The metzincins e topological and sequential relations between the astacins, adamalysins, serralysins, and matrixins (collagenases) define a superfamily of zincpeptidases. Protein Science: a Publication of the Protein Society 1995; 4(5): 823e840. 8. Egeblad M & Werb Z. New functions for the matrix metalloproteinases in cancer progression. Nature Reviews. Cancer 2002; 2(3): 161e174. 9. Boldt HB, Overgaard MT, Laursen LS et al. Mutational analysis of the proteolytic domain of pregnancyassociated plasma protein-A (PAPP-A): classification as a metzincin. The Biochemical Journal 2001; 358(2): 359e367. 10. Overgaard MT, Boldt HB, Laursen LS et al. Pregnancy-associated plasma protein-A2 (PAPP-A2), a novel insulin-like growth factor-binding protein-5 proteinase. The Journal of Biological Chemistry 2001; 276(24): 21849e21853. 11. Nagase H & Woessner Jr. JF. Matrix metalloproteinases. The Journal of Biological Chemistry 1999; 274(31): 21491e21494. 12. Springman EB, Angleton EL, Birkedal-Hansen H & Van Wart HE. Multiple modes of activation of latent human fibroblast collagenase: evidence for the role of a Cys73 active-site zinc complex in latency and a ‘‘cysteine switch’’ mechanism for activation. Proceedings of the National Academy of Sciences of the USA 1990; 87: 364e368. 13. Sternlicht MD & Werb Z. How matrix metalloproteinases regulate cell behavior. Annual Review of Cell and Developmental Biology 2001; 17: 463e516. 14. Clark IM & Parker AE. Metalloproteinases: their role in arthritis and potential as therapeutic targets. Expert Opinion on Therapeutic Targets 2003; 7(1): 19e34. 15. van den Berg WB. The role of cytokines and growth factors in cartilage destruction in osteoarthritis and rheumatoid arthritis. The Journal of Rheumatology 1999; 58(3): 136e141. 16. Koshy PJ, Henderson N, Logan C et al. Interleukin 17 induces cartilage collagen breakdown: novel synergistic effects in combination with proinflammatory cytokines. Annals of the Rheumatic Diseases 2002; 61(8): 704e713. 17. Cawston TE, Curry VA, Summers CA et al. The role of oncostatin M in animal and human connective tissue collagen turnover and its localisation within the rheumatoid joint. Arthritis and Rheumatism 1998; 41(10): 1760e1771. 18. Kleiner Jr. DE & Stetler-Stevenson WG. Structural biochemistry and activation of matrix metalloproteases. Current Opinion in Cell Biology 1993; 5(5): 891e897. 19. Milner JM, Elliott SF & Cawston TE. Activation of procollagenases is a key control point in cartilage collagen degradation: interaction of serine and metalloproteinase pathways. Arthritis and Rheumatism 2001; 44(9): 2084e2096.

998 T. E. Cawston and A. J. Wilson 20. Milner JM, Rowan AD, Elliott SF & Cawston TE. Inhibition of furin-like enzymes blocks interleukin1alpha/oncostatin M stimulated cartilage degradation. Arthritis and Rheumatism 2003; 48(4): 1057e1066. 21. Sato H, Takino T, Okada Y et al. A matrix metalloproteinase expressed on the surface of invasive tumour cells. Nature 1994; 370: 61e65. 22. Takino T, Sato H, Shinagawa A & Seiki M. Identification of the second membrane-type matrix metalloproteinase (MT-MMP-2) gene from a human placenta cDNA library e MT-MMPs form a unique membranetype subclass in the MMP family. The Journal of Biological Chemistry 1995; 270: 23013e23020. 23. Butler GS, Will H, Atkinson SJ & Murphy G. Membrane-type-2 matrix metalloproteinase can initiate the processing of progelatinase A and is regulated by the tissue inhibitors of metalloproteinases. European Journal of Biochemistry 1997; 244(2): 653e657. 24. Pei D. Identification and characterization of the fifth membrane-type matrix metalloproteinase MT5MMP. The Journal of Biological Chemistry 1999; 274(13): 8925e8932. 25. Velasco G, Cal S, Merlos-Sua´rez A et al. Human MT6-matrix metalloproteinase: identification, progelatinase A activation and expression in brain tumors. Cancer Research 2000; 60(4): 877e882. 26. Cawston TE. Metalloproteinases inhibitors and the prevention of connective tissue breakdown. Pharmacology and Therapeutics 1996; 70(3): 163e182. 27. Brew K, Dinakarpandian D & Nagase H. Tissue inhibitors of metalloproteinases: evolution, structure and function. Biochimica et Biophysica Acta 2000; 1477(1e2): 267e283. 28. Greene J, Wang M, Liu YE et al. Molecular cloning and characterization of human tissue inhibitor of metalloproteinase 4. The Journal of Biological Chemistry 1996; 271: 30375e30380. 29. Varga J, Rosenbloom J & Jimenez A. Transforming growth factor beta (TGF beta) causes a persistent increase in steady-state amounts of type I and type III collagen and fibronectin mRNAs in normal human dermal fibroblasts. The Biochemical Journal 1987; 247(3): 597e604. 30. Tchetverikov I, Verzijl N, Huizinga TW et al. Active MMPs captured by alpha 2 macroglobulin as a marker of disease activity in rheumatoid arthritis. Clinical and Experimental Rheumatology 2003; 21(6): 711e718. 31. Zucker S, Pei D, Cao J & Lopez-Otin C. Membrane type-matrix metalloproteinases (MT-MMP). Current Topics in Developmental Biology 2003; 54: 1e74. 32. Yu WH & Woessner Jr. JF. Heparan sulfate proteoglycans as extracellular docking molecules for matrilysin (matrix metalloproteinase 7). The Journal of Biological Chemistry 2000; 275(6): 4183e4191. 33. Guo H, Li R, Zucker S & Toole BP. EMMPRIN (CD147), an inducer of matrix metalloproteinase synthesis, also binds interstitial collagenase to the tumor cell surface. Cancer Research 2000; 60(4): 888e891. 34. Primakoff P & Myles DG. The ADAM gene family: surface proteins with adhesion and protease activity. Trends in Genetics 2000; 16(2): 83e87. 35. Campbell IK, Wojta J, Novak U & Hamilton JA. Cytokine modulation of plasminogen activator inhibitor-1 (PAI-1) production by human articular cartilage and chondrocytes. Down-regulation by tumor necrosis factor a and up-regulation by transforming growth factor-b and basic fibroblast growth factor. Biochimica et Biophysica Acta 1994; 1226: 277e285. 36. Bryson H, Bunning RAD, Feltell R et al. A serine proteinase inactivator inhibits chondrocyte-mediated cartilage proteoglycan breakdown occurring in response to proinflammatory cytokines. Archives of Biochemistry and Biophysics 1998; 355(1): 15e25. 37. Turk V, Turk B & Turk D. Lysosomal cysteine proteases: facts and opportunities. The EMBO Journal 2001; 20(17): 4629e4633. 38. Buttle DJ, Bramwell H & Hollander AP. Proteolytic mechanisms of cartilage breakdown: a target for arthritis therapy? Clinical Molecular Pathology 1995; 48: M167eM177. 39. Everts V, Van der Zee E, Creemers L & Beertsen W. Phagocytosis and intracellular digestion of collagen, its role in turnover and remodelling. The Histochemical Journal 1996; 28: 229e245. 40. Inaoka T, Bilbe G, Ishibashi O et al. Molecular cloning of human cDNA for cathepsin K: novel cysteine proteinase predominantly expressed in bone. Biochemical and Biophysical Research Communications 1995; 206(1): 89e96. 41. Bossard MJ, Tomaszek TA, Thompson SK et al. Proteolytic activity of human osteoclast cathepsin K. Expression, purification, activation, and substrate identification. The Journal of Biological Chemistry 1996; 271(21): 12517e12524. 42. Kafienah W, Bromme D, Buttle DJ et al. Human cathepsin K cleaves native type I and II collagens at the N-terminal end of the triple helix. The Biochemical Journal 1998; 331(Pt 3): 727e732.

Tissue degrading enzymes and their inhibitors 999 43. Hummel KM, Petrow PK, Franz JK et al. Cysteine proteinase cathepsin K mRNA is expressed in synovium of patients with rheumatoid arthritis and is detected at sites of synovial bone destruction. The Journal of Rheumatology 1998; 25(10): 1887e1894. 44. Hou WS, Li W, Keyszer G et al. Comparison of cathepsins K and S expression within the rheumatoid and osteoarthritic synovium. Arthritis and Rheumatism 2002; 46(3): 663e674. 45. Li Z, Hou WS & Bromme D. Collagenolytic activity of cathepsin K is specifically modulated by cartilageresident chondroitin sulfates. Biochemistry 2000; 39(3): 529e536. 46. Hou WS, Li Z, Gordon RE et al. Cathepsin k is a critical protease in synovial fibroblast-mediated collagen degradation. American Journal of Pathology 2001; 159(6): 2167e2177. 47. Suzuki K, Shimizu K, Hamamoto T et al. Characterization of proteoglycan degradation by calpain. The Biochemical Journal 1992; 285(Pt 3): 857e862. 48. Szomor Z, Shimizu K, Fujimori Y et al. Appearance of calpain correlates with arthritis and cartilage destruction in collagen induced arthritic knee joints of mice. Annals of the Rheumatic Diseases 1995; 54(6): 477e483. 49. Ishikawa H, Nakagawa Y, Shimizu K et al. Inflammatory cytokines induced down-regulation of m-calpain mRNA expression in fibroblastic synoviocytes from patients with osteoarthritis and rheumatoid arthritis. Biochemical and Biophysical Research Communications 1999; 266(2): 341e346. 50. Wlodawer A. Proteasome: a complex protease with a new fold and a distinct mechanism. Structure (London, England: 1993) 1995; 3(5): 417e420. 51. Ballock RT & O’Keefe RJ. Physiology and pathophysiology of the growth plate. Birth Defects Research. Part C, Embryo Today: Reviews 2003; 69(Part C): 123e143. 52. Ornitz DM & Marie PJ. Fgf signaling pathways in endochondral and intramembranous bone development and human genetic disease. Genes and Development 2002; 16(12): 1446e1465. 53. Provot S & Schipani E. Molecular mechanisms of endochondral bone development. Biochemical and Biophysical Research Communications 2005; 328(3): 658e665. 54. Vaananen HK, Zhao H, Mulari M & Halleen JM. The cell biology of osteoclast function. Journal of Cell Science 2000; 113(Pt 3): 377e381. 55. Stickens D, Behonick DJ, Ortega N et al. Altered endochondral bone development in matrix metalloproteinase 13-deficient mice. Development 2004; 131(23): 5883e5895. 56. Olsen BR, Reginato AM & Wang W. Bone development. Annual Review of Cell and Developmental Biology 2000; 16: 191e220. 57. Vu TH, Shipley JM, Bergers G et al. MMP-9/gelatinase B is a key regulator of growth plate angiogenesis and apoptosis of hypertrophic chondrocytes. Cell 1998; 93(3): 411e422. 58. Bergers G, Brekken R, McMahon G et al. Matrix metalloproteinase-9 triggers the angiogenic switch during carcinogenesis. Nature Cell Biology 2000; 2(10): 737e744. 59. Engsig MT, Chen QJ, Vu TH et al. Matrix metalloproteinase 9 and vascular endothelial growth factor are essential for osteoclast recruitment into developing long bones. The Journal of Cell Biology 2000; 151(4): 879e889. 60. Kennedy AM, Inada M, Krane SM et al. MMP13 mutation causes spondyloepimetaphyseal dysplasia, Missouri type (Semdmo). The Journal of Clinical Investigation 2005; 115(10): 2832e2842. 61. Zhou Z, Apte SS, Soininen R et al. Impaired endochondral ossification and angiogenesis in mice deficient in membrane-type matrix metalloproteinase I. Proceedings of the National Academy of Sciences of the USA 2000; 97(8): 4052e4057. 62. Ortega N, Behonick D, Stickens D & Werb Z. How proteases regulate bone morphogenesis. Annals of the New York Academy of Sciences 2003; 995: 109e116. 63. Satoyoshi M, Kawata A, Koizumi T et al. Matrix metalloproteinase-2 in dentin matrix mineralization. Journal of Endodontics 2001; 27(7): 462e466. 64. Itoh T, Matsuda H, Tanioka M et al. The role of matrix metalloproteinase-2 and matrix metalloproteinase-9 in antibody-induced arthritis. Journal of Immunology 2002; 169(5): 2643e2647. 65. Choi SJ, Han JH & Roodman GD. ADAM8: a novel osteoclast stimulating factor. Journal of Bone and Mineral Research 2001; 16(5): 814e822. 66. Verrier S, Hogan A, McKie N & Horton M. ADAM gene expression and regulation during human osteoclast formation. Bone 2004; 35(1): 34e46. 67. Dallas DJ, Genever PG, Patton AJ et al. Localization of Adam10 and Notch receptors in bone. Bone 1999; 25(1): 9e15.

1000 T. E. Cawston and A. J. Wilson 68. Bartholin L, Destaing O, Forissier S et al. FLRG, a new ADAM12-associated protein, modulates osteoclast differentiation. Biology of the cell 2005; 97(7): 577e588. 69. Shindo T, Kurihara H, Kuno K et al. ADAMTS-1: a metalloproteinase-disintegrin essential for normal growth, fertility, and organ morphology and function. The Journal of Clinical Investigation 2000; 105(10): 1345e1352. 70. Colige A, Li SW, Sieron AL et al. cDNA cloning and expression of bovine procollagen I N-proteinase: a new member of the superfamily of zinc-metalloproteinases with binding sites for cells and other matrix components. Proceedings of the National Academy of Sciences of the USA 1997; 94(6): 2374e2379. 71. Hurskainen TL, Hirohata S, Seldin MF & Apte SS. ADAM-TS5, ADAM-TS6, and ADAM-TS7, novel members of a new family of zinc metalloproteases. General features and genomic distribution of the ADAM-TS family. The Journal of Biological Chemistry 1999; 274(36): 25555e25563. 72. Tortorella MD, Burn TC, Pratta MA & Abbaszade I. Purification and cloning of aggrecanase-1: a member of the ADAMTS family of proteins. Science 1999; 284(5420): 1664e1666. 73. Matthews RT, Gary SC, Zerillo C et al. Brain-enriched hyaluronan binding (BEHAB)/brevican cleavage in a glioma cell line is mediated by a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) family member. The Journal of Biological Chemistry 2000; 275(30): 22695e22703. 74. Stanton H, Rogerson FM, East CJ et al. ADAMTS5 is the major aggrecanase in mouse cartilage in vivo and in vitro. Nature 2005; 434(7033): 648e652. 75. Tetlow LC, Adlam DJ & Woolley DE. Matrix metalloproteinase and proinflammatory cytokine production by chondrocytes of human osteoarthritic cartilage. Arthritis and Rheumatism 2001; 44(3): 585e594. 76. Shingleton WD, Ellis AJ, Rowan AD & Cawston TE. Retinoic acid combines with interleukin-1 to promote the degradation of collagen from bovine nasal cartilage: matrix metalloproteinases-1 and -13 are involved in cartilage collagen breakdown. Journal of Cellular Biochemistry 2000; 79(4): 519e531. 77. Hembry RM, Bagga MR, Reynolds JJ & Hamblen DL. Immunolocalisation studies on six matrix metalloproteinases and their inhibitors, TIMP-1 and TIMP-2, in synovia from patients with osteoand rheumatoid arthritis. Annals of the Rheumatic Diseases 1995; 54: 25e32. 78. Okada Y, Shinmei M, Tanaka O et al. Localization of matrix metalloproteinase 3 (stromelysin) in osteoarthritic cartilage and synovium. Laboratory Investigation 1992; 66: 680e690. 79. Wolfe GC, MacNaul KL, Buechel FF et al. Differential in vivo expression of collagenase messenger RNA in synovium and cartilage: quantitative comparison with stromelysin messenger RNA levels in human rheumatoid arthritis and osteoarthritis patients and in two animal models of acute inflammatory arthritis. Arthritis and Rheumatism 1993; 36: 1540e1547. 80. Ahrens D, Koch AE, Pope RM et al. Expression of matrix metalloproteinase 9 (96-kd gelatinase B) in human rheumatoid arthritis. Arthritis and Rheumatism 1996; 39(9): 1576e1587. 81. Gruber BL, Sorbi D, French DL et al. Markedly elevated serum MMP-9 (gelatinase B) levels in rheumatoid arthritis: a potential useful laboratory marker. Clinical Immunology and Immunopathology 1996; 78(2): 161e171. 82. Yoshihara Y, Nakamura H, Obata K et al. Matrix metalloproteinases and tissue inhibitors of metalloproteinases in synovial fluids from patients with rheumatoid arthritis or osteoarthritis. Annals of the Rheumatic Diseases 2000; 59: 455e461. 83. Konttinen YT, Ainola M, Valleala H et al. Analysis of 16 different matrix metalloproteinases (MMP-1 to MMP-20) in the synovial membrane: different profiles in trauma and rheumatoid arthritis. Annals of the Rheumatic Diseases 1999; 58(11): 691e697. 84. Sandy JD, Flannery CR, Neame PJ & Lohmander LS. The structure of aggrecan fragments in human synovial fluid. Evidence for the involvement in osteoarthritis of a novel proteinase which cleaves the Glu 373-Ala 374 bond. The Journal of Clinical Investigation 1992; 89(5): 1512e1516. 85. Caterson B, Flannery CR, Hughes CE & Little CB. Mechanisms involved in cartilage proteoglycan catabolism. Matrix Biology 2000; 19: 333e344. 86. Arner EC, Hughes CE, Decicco CP et al. Cytokine-induced cartilage proteoglycan degradation is mediated by aggrecanase. Osteoarthritis and Cartilage 1998; 6(3): 214e228. 87. Porter S, Clark IM, Kevorkian L & Edwards DR. The ADAMTS metalloproteinases. The Biochemical Journal 2005; 386(Pt 1): 15e27. 88. Rodriguez-Manzaneque JC, Westling J, Thai SN et al. ADAMTS1 cleaves aggrecan at multiple sites and is differentially inhibited by metalloproteinase inhibitors. Biochemical and Biophysical Research Communications 2002; 293(1): 501e508.

Tissue degrading enzymes and their inhibitors

1001

89. Collins-Racie LA, Flannery CR, Zeng W et al. ADAMTS-8 exhibits aggrecanase activity and is expressed in human articular cartilage. Matrix Biology 2004; 23(4): 219e230. 90. Billington CJ, Clark IM & Cawston TE. An aggrecan-degrading activity associated with chondrocyte membranes. The Biochemical Journal 1998; 15(336 Pt 1): 207e212. 91. Somerville RP, Longpre JM, Jungers KA et al. Characterization of ADAMTS-9 and ADAMTS-20 as a distinct ADAMTS subfamily related to Caenorhabditis elegans GON-1. The Journal of Biological Chemistry 2003; 278(11): 9503e9513. 92. Stanton H, Ung L & Fosang AJ. The 45 kDa collagen-binding fragment of fibronectin induces matrix metalloproteinase-13 synthesis by chondrocytes and aggrecan degradation by aggrecanases. The Biochemical Journal 2002; 364(Pt 1): 181e190. 93. Dudler J, Renggli-Zulliger N, Busso N & Lotz M. Effect of interleukin 17 on proteoglycan degradation in murine knee joints. Annals of the Rheumatic Diseases 2000; 59(7): 529e532. 94. Yamanishi Y, Boyle DL, Clark M et al. Expression and regulation of aggrecanase in arthritis: the role of TGF-beta. Journal of Immunology (Baltimore, Md.: 1950) 2002; 168(3): 1405e1412. 95. Koshy PJ, Lundy CJ, Rowan AD et al. The modulation of matrix metalloproteinase and ADAM gene expression in human chondrocytes by interleukin-1 and oncostatin M: a time-course study using real-time quantitative reverse transcription-polymerase chain reaction. Arthritis and Rheumatism 2002; 46(4): 961e967. 96. Ellis AJ, Curry VA, Powell EK & Cawston TE. The prevention of collagen breakdown in bovine nasal cartilage by TIMP-1, TIMP-2 and a low molecular weight synthetic inhibitor. Biochemical and Biophysical Research Communications 1994; 201: 94e101. 97. Kashiwagi M, Tortorella M, Nagase H & Brew K. TIMP-3 is a potent inhibitor of aggrecanase 1 (ADAMTS4) and aggrecanase 2 (ADAM-TS5). The Journal of Biological Chemistry 2001; 276(16): 12501e12504. 98. Chevrier A, Mort JS, Crine P et al. Soluble recombinant neprilysin induces aggrecanase-mediated cleavage of aggrecan in cartilage explant cultures. Archives of Biochemistry and Biophysics 2001; 396(2): 178e186. 99. McKie N, Edwards T, Dallas DJ et al. Expression of members of a novel membrane linked metalloproteinase family (ADAM) in human articular chondrocytes. Biochemical and Biophysical Research Communications 1997; 230(2): 335e339. 100. Black RA, Rauch CT, Kozlosky CJ et al. A metalloproteinase disintegrin that releases tumour-necrosis factor-a from cells. Nature 1997; 385: 729e733. 101. Ronday HK, van der Laan WH, Tak PP et al. Human granzyme B mediates cartilage proteoglycan degradation and is expressed at the invasive front of the synovium in rheumatoid arthritis. Rheumatology 2001; 40(1): 55e61. 102. Drummond AH, Beckett P, Brown PD et al. Preclinical and clinical studies of MMP inhibitors in cancer. Annals of the New York Academy of Sciences 1999; 30(878): 228e235. 103. Peterson JT. The importance of estimating the therapeutic index in the development of matrix metalloproteinase inhibitors. Cardiovascular Research 2006; 69: 677e687. 104. Sabatini M, Lesur C, Thomas M et al. Effect of inhibition of matrix metalloproteinases on cartilage loss in vitro and in a guinea pig model of osteoarthritis. Arthritis and Rheumatism 2005; 52(1): 171e180. 105. Ishikawa T, Nishigaki F, Miyata S et al. Prevention of progressive joint destruction in adjuvant induced arthritis in rats by a novel matrix metalloproteinase inhibitor, FR217840. European Journal of Pharmacology 2005; 508(1e3): 239e247. 106. Ishikawa T, Nishigaki F, Miyata S et al. Prevention of progressive joint destruction in collagen-induced arthritis in rats by a novel matrix metalloproteinase inhibitor, FR255031. British Journal of Pharmacology 2005; 144(1): 133e143. 107. Lewis EJ, Bishop J, Bottomley KM et al. Ro 32-3555, an orally active collagenase inhibitor, prevents cartilage breakdown in vitro and in vivo. British Journal of Pharmacology 1997; 121(3): 540e546. 108. MacPherson LJ, Bayburt EK, Capparelli MP et al. Discovery of CGS 27023A, a non-peptidic, potent, and orally active stromelysin inhibitor that blocks cartilage degradation in rabbits. Journal of Medicinal Chemistry 1997; 40(16): 2525e2532. 109. O’Byrne EM, Blancuzzi V, Singh H et al. Chondroprotective Activity of a Matrix Metalloproteinase Inhibitor, CGS 27023A in Animal Models of Osteoarthritis. Tokyo, Japan: Springer-Verlag, 1999. 110. Chau T, Jolly G, Plym MJ et al. Inhibition of articular cartilage degradation in dog and guinea-pig models of osteoarthritis by the stromelysin inhibitor, BAY-12e9566. Arthritis and Rheumatism 1998; 41: S300.

1002 T. E. Cawston and A. J. Wilson 111. Levitt NC, Eskens FA, O’Byrne KJ et al. Phase I and pharmacological study of the oral matrix metalloproteinase inhibitor, MM1270 (CGS27023A), in patients with advanced solid cancer. Clinical Cancer Research 2001; 7(7): 1912e1922. 112. Hemmings FJ, Farhan M, Rowland J et al. Tolerability and pharmacokinetics of the collagenase-selective inhibitor Trocade in patients with rheumatoid arthritis. Rheumatology (Oxford, England) 2001; 40(5): 537e543. 113. Leff RL, Elias I, Ionescu M et al. Molecular changes in human osteoarthritic cartilage after 3 weeks of oral administration of BAY 12-9566, a matrix metalloproteinase inhibitor. The Journal of Rheumatology 2003; 30(3): 544e549. 114. Bayer drug casts shadow over MMP inhibitors in cancer. Scrip 1999; 2476: 7. 115. Ryan ME, Greenwald RA & Golub LM. Potential of tetracyclines to modify cartilage breakdown in osteoarthritis. Current Opinion in Rheumatology 1996; 8(3): 238e247. 116. de Bri E, Lei W, Svensson O et al. Effect of an inhibitor of matrix metalloproteinases on spontaneous osteoarthritis in guinea pigs. Advances in Dental Research 1998; 12(2): 82e85. 117. Stone M, Fortin PR, Pacheco-Tena C & Inman RD. Should tetracycline treatment be used more extensively for rheumatoid arthritis? Metaanalysis demonstrates clinical benefit with reduction in disease activity. The Journal of Rheumatology 2003; 30(10): 2112e2122. 118. van der Laan W, Molenaar E, Ronday K et al. Lack of effect of doxycycline on disease activity and joint damage in patients with rheumatoid arthritis. A double blind, placebo controlled trial. The Journal of Rheumatology 2001; 28(9): 1967e1974. 119. Brandt KD, Mazzuca SA, Katz BP et al. Effects of doxycycline on progression of osteoarthritis: results of a randomised, placebo-controlled, double-blind trial. Arthritis and Rheumatism 2005; 52(7): 2015e2025. 120. Pavlaki M & Zucker S. Matrix metalloproteinase inhibitors (MMPIs): the beginning of phase I or the termination of phase III clinical trials. Cancer and Metastasis Reviews 2003; 22(2e3): 177e203. 121. Sweeney SE & Firestein GS. Signal transduction in rheumatoid arthritis. Current Opinion in Rheumatology 2004; 16(3): 231e237. 122. Han Z, Boyle DL, Chang L et al. c-Jun N-terminal kinase is required for metalloproteinase expression and joint destruction in inflammatory arthritis. The Journal of Clinical Investigation 2001; 108(1): 73e81. 123. Tak PP, Gerlag DM, Aupperle KR et al. Inhibitor of nuclear factor kappa B kinase beta is a key regulator of synovial inflammation. Arthritis and Rheumatism 2001; 44(8): 1897e1907. 124. Shouda T, Yoshida T, Hanada T et al. Induction of the cytokine signal regulator SOCS3/CIS3 as a therapeutic strategy for treating inflammatory arthritis. The Journal of Clinical Investigation 2001; 108(12): 1781e1788. 125. Kouzarides T. Acetylation: a regulatory modification to rival phosphorylation? The EMBO Journal 2000; 19(6): 1176e1179. 126. Johnstone RW. Histone-deacetylase inhibitors: novel drugs for the treatment of cancer. Nature Reviews. Drug Discovery 2002; 1(4): 287e299. 127. Young DA, Lakey RL, Pennington CJ et al. Histone deacetylase inhibitors modulate metalloproteinase gene expression in chondrocytes and block cartilage resorption. Arthritis Research and Therapy 2005; 7(3): R503eR512. 128. Blanchard F & Chipoy C. Histone deacetylase inhibitors: new drugs for the treatment of inflammatory diseases? Drug Discovery Today 2005; 10(3): 197e204. 129. Nagase H & Brew K. Designing TIMP (tissue inhibitor of metalloproteinases) variants that are selective metalloproteinase inhibitors. Biochemical Society Sympopsia 2003;(70): 201e212. 130. Lee MH, Rapti M, Knauper V & Murphy G. Threonine 98, the pivotal residue of tissue inhibitor of metalloproteinases (TIMP)-1 in metalloproteinase recognition. The Journal of Biological Chemistry 2004; 279(17): 17562e17569. 131. Lee MH, Rapti M & Murphy G. Total conversion of tissue inhibitor of metalloproteinase (TIMP) for specific metalloproteinase targeting: fine-tuning TIMP-4 for optimal inhibition of TNF-a converting enzyme (TACE). The Journal of Biological Chemistry 2005; 280(16): 15967e15975. 132. van der Laan WH, Quax PH, Seemayer CA et al. Cartilage degradation and invasion by rheumatoid synovial fibroblasts is inhibited by gene transfer of TIMP-1 and TIMP-3. Gene Therapy 2003; 10(3): 234e242.