Articular cartilage and changes in Arthritis: Matrix degradation
© BioMed Central Ltd 2001
Received: 16 May 2001
Accepted: 7 August 2001
Published: 6 September 2001
While many proteases in articular cartilage have been described, current studies indicate that members of two families of metalloproteases – MMPs and the ADAMTSs – are responsible for the degradation of the major components of this tissue. Collagenases (MMPs) make the first cleavage in triple-helical collagen, allowing its further degradation by other proteases. Aggrecanases (ADAMTSs), in conjunction with other MMPs, degrade aggrecan, a component of the proteoglycan aggregate. Anti-neoepitope antibodies that recognize the cleavage products of collagen and aggrecan generated by these enzymes are now available and are being used to detect the sites of action and to quantitate degradation products.
Keywordsaggrecanase collagenase neoepitope protease protease inhibitor
Destruction of articular cartilage is an irreversible consequence of arthritis. Cartilage consists of two major components, a type-II-collagen-fibril network with associated small proteoglycans, and proteoglycan aggregates composed of a noncovalent association between aggrecan, hyaluronate, and link protein. In arthritis, proteoglycan degradation is thought to be an early and reversible process, whereas the breakdown of the collagen network is believed to be irreversible, contributing to loss of joint function. While free radical attack and the action of glycosidases may play a role in cartilage deterioration, the most important degradative agents are proteolytic enzymes.
Proteolytic cleavage of the major components of the cartilage extracellular matrix is effected by a number of proteases, many of which are synthesized by chondrocytes and synovial cells in response to inflammatory stimuli. Members of each of the four classes of protease – serine/threonine proteases, cysteine proteases, aspartic proteases, and metalloproteases – have been implicated in the degradation of cartilage. However, current data indicate that the initial steps in matrix breakdown are extracellular processes involving metalloproteases. This class of enzyme is characterized by the presence, within the active site, of a metal ion (usually zinc), which is required for catalytic activity. Of the metalloproteases, the members of two families, the matrix metalloproteases (MMPs) and the ADAMTSs (a disintegrin and a metalloprotease with thrombospondin motifs) family, have been implicated in the breakdown of collagen and aggrecan, respectively. These enzymes are members of the M10 and M12 peptidase families as classified in the universal protease database, MEROPS .
Cleavage of peptide bonds is a very simple chemical reaction and many proteolytic enzymes are relatively small proteins (~30 kDa) consisting simply of a binding site to accommodate about six amino acid residues of the substrate and the catalytic machinery. Other proteases, in particular the metalloproteases involved in matrix degradation, have a more sophisticated, multidomain composition, in which additional protein elements are present, giving additional functions to the protease. These include assisting in substrate binding by attachment at sites remote from where peptide bond cleavage occurs, and binding to nonsubstrate molecules in the extracellular matrix, allowing appropriate localization of the enzyme.
Control of MMP synthesis, activation, and activity is tightly regulated under physiological conditions. Thus, all the MMPs are synthesized as inactive proenzymes. Enzyme latency is maintained by ligation of a cysteine residue in the prodomain to the active-site Zn2+ ion. Activation occurs via complex, highly regulated intermolecular proteolytic cascades leading to destabilization of the Cys–Zn interaction , followed by a second cleavage that results in the release of the prodomain from the active enzyme. While many pathways for MMP activation have been demonstrated using in vitro model systems, in most cases the actual in vivo mechanisms are still not clear. MMP activity is modulated by the naturally occurring endogenous inhibitors of the MMPs, the tissue inhibitors of MMPs (TIMPs). These molecules bind tightly to the active site of activated MMPs with a 1:1 stoichiometry and have Ki values of less than 10-9 M .
The MT-MMPs represent an important control point in MMP activation. In contrast to most of the other MMPs, the MT-MMPs are activated intracellularly, in the Golgi apparatus, by the action of the serine protease furin, which cleaves pro-MT-MMPs at a specific site. Once at the cell surface, active MT1-MMP forms a trimolecular complex with pro-MMP-2 and the inhibitor TIMP-2, resulting in the activation of MMP-2.
It is apparent, then, that the accelerated turnover of collagen associated with joint diseases may result from a number of factors, including increased synthesis and activation of MMPs and/or an imbalance in levels of MMPs and their inhibitors, the TIMPs.
The ADAMTS family
Cysteine and other proteases
Members of the cysteine and aspartic protease classes may also contribute to cartilage matrix degradation. Thus the lysosomal enzymes, including cathepsins B, D, and L, are thought to play a secondary role in cartilage degradation, involving intracellular digestion of products released by other proteases. It has also been postulated that at least some of these enzymes may function extracellularly within the cartilage, with cathepsin B, for example, potentially contributing to aggrecan breakdown . The protein is expressed in situ in synovial cells attached to cartilage and bone at sites of erosion, and an enhanced transcription of the protein was observed in these synovial cells obtained from patients with rheumatoid arthritis compared with the transcription in normal fibroblasts . The relative importance of different cysteine proteases in osteoarthritis has been investigated recently, and again cathepsin B appears to be a major factor . In addition, cathepsin K, which is the principal cysteine protease of the osteoclast, has also been found in the synovium and may contribute to cartilage degradation .
Evidence for the action of specific proteases in cartilage
An important characteristic of the metalloproteases, which is thought to mediate the initial steps in the cleavage of collagen and aggrecan, is their specificity. Collagenases make an initial cleavage at a specific weak point in the collagen fibril, and aggrecanases cleave the core protein at five well characterized sites. In addition, other MMPs, such as stromelysin (MMP-3), cleave aggrecan at a well-characterized site in the aggrecan core protein . These cleavage events generate terminal neoepitopes that are antigenically distinct from the same sequence in the intact protein . Antibodies produced against such neoepitopes have been used to demonstrate the sites of collagenase and aggrecanase action as well as those of other metalloproteases such as stromelysin in normal and pathological turnover of cartilage [27,28]. In addition, since even after further processing the neoepitopes survive on smaller peptides, they can be detected in body fluids and used to evaluate the levels of ongoing matrix degradation .
Conclusions: protease inhibition as a therapeutic option
Evidence for the action of MMPs, aggrecanases, and other proteases in the degradation of cartilage associated with arthritis immediately suggests that their inhibition would be a fruitful therapeutic approach. While to date very few bioavailable cysteine protease inhibitors have been developed, a massive effort to produce MMP inhibitors over the past decade has resulted in several potent compounds. As yet, their use in arthritis therapy has been unsuccessful, in part because of an incomplete understanding of the entire metalloprotease repertoire. One of the problems in assessing the efficacy of protease inhibitor therapy is the difficulty in measuring biological outcome, since changes in cartilage metabolism are not readily apparent, particularly to the patient. The availability of methods to measure specific cartilage matrix fragments in synovial fluid, blood, or urine that are indicative of unique protease action, using specific anti-neoepitope antibodies for aggrecan and collagen fragments, promises to allow direct monitoring of the effect of different therapeutic approaches.
While the terms 'protease' and 'proteinase' were once used interchangeably, more recently 'protease' has become predominant. Except in definitions of abbreviations, which were based on the earlier usage, the term 'protease' is used in this review.
a disintegrin and a metalloprotease
a disintegrin and a metalloprotease with thrombospondin motifs
membrane-type matrix metalloproteinase
tissue inhibitor of metalloproteinases.
We thank Guylaine Bedard for artwork. The authors' research is funded by the Shriners of North America.
- The MEROPS database. [http://www.merops.ac.uk/]
- Chung L, Shimokawa K, Dinakarpandian D, Grams F, Fields GB, Nagase H: Identification of the 183 RWTNNFREY191 region as a critical segment of matrix metalloproteinase 1 for the expression of collagenolytic activity. J Biol Chem. 2000, 275: 29610-29617. 10.1074/jbc.M004039200.PubMedView Article
- Knauper V, Lopez-Otin C, Smith B, Knight CG, Murphy G: Biochemical characterization of human collagenase-3. J Biol Chem. 1996, 271: 1544-1550. 10.1074/jbc.271.3.1544.PubMedView Article
- Steffensen B, Bigg HF, Overall CM: The involvement of the fibronectin type II-like modules of human gelatinase A in cell surface localization and activation. J Biol Chem. 1998, 273: 20622-20628. 10.1074/jbc.273.32.20622.PubMedView Article
- Nagase H, Woessner JF: Matrix metalloproteinases. J Biol Chem. 1999, 274: 21491-21494. 10.1074/jbc.274.31.21491.PubMedView Article
- Ohuchi E, Imai K, Fujii Y, Sato H, Seiki M, Okada Y: Membrane type 1 matrix metalloproteinase digests interstitial collagens and other extracellular matrix macromolecules. J Biol Chem. 1997, 272: 2446-2451. 10.1074/jbc.272.4.2446.PubMedView Article
- 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. Proc Natl Acad Sci U S A. 1990, 87: 364-368.PubMedPubMed CentralView Article
- Willenbrock F, Crabbe T, Slocombe PM, Sutton CW, Docherty WJP, Cockett MI, O'Shea M, Brocklehurst K, Phillips IR, Murphy G: The activity of the tissue inhibitor of metalloproteinases is regulated by C-terminal domain interactions. A kinetic analysis of the inhibition of gelatinase A. Biochemistry. 1993, 32: 4330-4337.PubMedView Article
- Kaushal GP, Shah SV: The new kids on the block: ADAMTSs, potentially multifunctional metalloproteinases of the ADAM family. J Clin Invest. 2000, 105: 1335-1337.PubMedPubMed CentralView Article
- Wolfsberg TG, White JM: ADAMs in fertilization and development. Dev Biol. 1996, 180: 389-401. 10.1006/dbio.1996.0313.PubMedView Article
- Loulakis P, Shrikhande A, Davis G, Maniglia CA: N-Terminal sequence of proteoglycan fragments isolated from medium of interleukin-1-treated articular-cartilage cultures. Biochem J. 1992, 284: 589-593.PubMedPubMed CentralView Article
- Tortorella MD, Burn TC, Pratta MA, Abbaszade I, Hollis JM, Liu R, Rosenfeld SA, Copeland RA, Decicco CP, Wynn R, Rockwell A, Yang F, Duke JL, Solomon K, George H, Bruckner R, Nagase H, Itoh Y, Ellis DM, Ross H, Wiswall BH, Murphy K, Hillman MCJ, Hollis GF, Newton RC, Magolda RL, Trzaskos JM, Arner EC: Purification and cloning of aggrecanase-1: A member of the ADAMTS family of proteins. Science. 1999, 284: 1664-1666. 10.1126/science.284.5420.1664.PubMedView Article
- Abbaszade I, Liu RQ, Yang F, Rosenfeld SA, Ross OH, Link JR, Ellis DM, Tortorella MD, Pratta MA, Hollis JM, Wynn R, Duke JL, George HJ, Hillman MC, Murphy K, Wiswall BH, Copeland RA, Decicco CP, Bruckner R, Nagase H, Itoh Y, Newton RC, Magolda RL, Trzaskos JM, Hollis GF, Arner EC, Burn TC: Cloning and characterization of ADAMTS11, an aggrecanase from the ADAMTS family. J Biol Chem. 1999, 274: 23443-23450. 10.1074/jbc.274.33.23443.PubMedView Article
- Kuno K, Terashima Y, Matsushima K: ADAMTS-1 is an active metalloproteinase associated with the extracellular matrix. J Biol Chem. 1999, 274: 18821-18826. 10.1074/jbc.274.26.18821.PubMedView Article
- Flannery CR, Little CB, Hughes CE, Caterson B: Expression of ADAMTS homologues in articular cartilage. Biochem Biophys Res Commun. 1999, 260: 318-322. 10.1006/bbrc.1999.0909.PubMedView Article
- Kuno K, Okada Y, Kawashima H, Nakamura H, Miyasaka M, Ohno H, Matsushima K: ADAMTS-1 cleaves a cartilage proteoglycan, aggrecan. FEBS Lett. 2000, 478: 241-245. 10.1016/S0014-5793(00)01854-8.PubMedView Article
- Tortorella MD, Pratta M, Liu RQ, Abbaszade I, Ross H, Burn T, Arner E: The thrombospondin motif of aggrecanase-1 (ADAMTS-4) is critical for aggrecan substrate recognition and cleavage. J Biol Chem. 2000, 275: 25791-25797. 10.1074/jbc.M001065200.PubMedView Article
- Billington CJ, Clark IM, Cawston TE: An aggrecan-degrading activity associated with chondrocyte membranes. Biochem J. 1998, 336: 207-212.PubMedPubMed CentralView Article
- Kashiwagi M, Tortorella M, Nagase H, Brew K: TIMP-3 is a potent inhibitor of ADAM-TS4 (aggrecanase 1) and ADAM-TS5 (aggrecanase 2). J Biol Chem. 2001, 276: 12501-12504. 10.1074/jbc.C000848200.PubMedView Article
- Hashimoto G, Aoki T, Nakamura H, Tanzawa K, Okada Y: Inhibition of ADAMTS4 (aggrecanase-1) by tissue inhibitors of metalloproteinases (TIMP-1, 2, 3 and 4). FEBS Lett. 2001, 494: 192-195. 10.1016/S0014-5793(01)02323-7.PubMedView Article
- Mort JS, Magny M-C, Lee ER: Cathepsin B: an alternative protease for the generation of an aggrecan "metalloproteinase" cleavage neoepitope. Biochem J. 1998, 335: 491-494.PubMedPubMed CentralView Article
- Trabandt A, Gay RE, Fassbender H-G, Gay S: Cathepsin B in synovial cells at the site of joint destruction in rheumatoid arthritis. Arthritis Rheum. 1991, 34: 1444-1451.PubMedView Article
- Lang A, Horler D, Baici A: The relative importance of cysteine peptidases in osteoarthritis. J Rheumatol. 2000, 27: 1970-1979.PubMed
- Dodds RA, Connor JR, Drake FH, Gowen M: Expression of cathepsin K messenger RNA in giant cells and their precursors in human osteoarthritic synovial tissues. Arthritis Rheum. 1999, 42: 1588-1593. 10.1002/1529-0131(199908)42:8<1588::AID-ANR4>3.0.CO;2-S.PubMedView Article
- Flannery CR, Lark MW, Sandy JD: Identification of a stromelysin cleavage site within the interglobular domain of human aggrecan. Evidence for proteolysis at this site in vivo in human articular cartilage. J Biol Chem. 1992, 267: 1008-1014.PubMed
- Mort JS, Buttle DJ: The use of cleavage site specific antibodies to delineate protein processing and breakdown pathways. J Clin Pathol :Mol Pathol. 1999, 52: 11-18.
- Stoop R, van der Kraan PM, Buma P, Hollander AP, Billinghurst RC, Poole AR, van den Berg WB: Type II collagen degradation in spontaneous osteoarthritis in C57Bl/6 and BALB/c mice. Arthritis Rheum. 1999, 42: 2381-2389. 10.1002/1529-0131(199911)42:11<2381::AID-ANR17>3.0.CO;2-E.PubMedView Article
- Lark MW, Bayne EK, Flanagan J, Harper CF, Hoerrner LA, Hutchinson NI, Singer II, Donatelli SA, Weidner JR, Williams HR, Mumford RA, Lohmander LS: Aggrecan degradation in human cartilage. Evidence for both matrix metalloproteinase and aggrecanase activity in normal, osteoarthritic, and rheumatoid joints. J Clin Invest. 1997, 100: 93-106.PubMedPubMed CentralView Article
- Downs JT, Lane CL, Nestor NB, McLellan TJ, Kelly MA, Karam GA, Mezes PS, Pelletier JP, Otterness IG: Analysis of collagenase-cleavage of type II collagen using a neoepitope ELISA. J Immunol Methods. 2001, 247: 25-34. 10.1016/S0022-1759(00)00302-1.PubMedView Article