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Osteoarthritis and Cartilage 18 (2010) 1109e1116

Review Identifying the human aggrecanase

A.J. Fosang*, F.M. Rogerson

University of Melbourne, Department of Paediatrics and Murdoch Childrens Research Institute, Royal Children’s Hospital, Flemington Road, Parkville 3052, Australia

article info summary

Article history: It is clear that A And with motif (ADAMTS)-5 is the Received 9 April 2010 major aggrecanase in mouse cartilage, however it is not at all clear which is the major Accepted 4 June 2010 aggrecanase in human cartilage. Identifying the human aggrecanase is difficult because multiple, inde- pendent, molecular processes determine the final level of enzyme activity. As investigators, we have Keywords: good methods for measuring changes in the expression of ADAMTS mRNA, and good methods for detecting aggrecanase activity, but no methods that distinguish the source of the activity. In between Aggrecanase gene expression and enzyme action there are many processes that can potentially enhance or inhibit the Cartilage fi ADAMTS-4 nal level of activity. In this editorial we discuss how each of these processes affects ADAMTS activity and ADAMTS-5 argue that measuring any one process in isolation has little value in predicting overall ADAMTS activity Messenger RNA in vivo. Proteinase activity Crown Copyright Ó 2010 Published by Elsevier Ltd on behalf of Osteoarthritis Research Society International. All rights reserved.

Introduction activity, epigenetic modifications, regulation by non-coding RNA (ncRNA) including microRNA (miRNA), and alternative splicing. At It is now over a decade since A Disintegrin And Metal- the level, ADAMTS activity is determined by the summed loproteinase with ThromboSpondin motif (ADAMTS)-41, and effect of prodomain processing, C-terminal processing, the avail- ADAMTS-52 were identified as aggrecanases, based on their ability ability and binding of endogenous enhancers or inhibitors, and the to cleave at the Glu373 Y 374Ala bond in the aggrecan interglobular extent of glycosylation on both the aggrecan substrate and the domain (IGD). Since then ADAMTS-1, -8, -9, -15, -16 and -18 family themselves (Fig. 1). We herein discuss how each of these members have also been identified as aggrecan-degrading enzymes processes affects ADAMTS activity and argue that measuring any in vitro, but none have seriously challenged ADAMTS-4 and one parameter in isolation is an inadequate surrogate for deter- ADAMTS-5 as the candidate human aggrecanases because these mining the identity of the human aggrecanase. have substantially greater aggrecan-degrading activity in vitro. ADAMTS-4 and -5 are expressed in human cartilage and in many cases their expression levels are increased in joint disease, and at Regulating ADAMTS-4 and -5 mRNA expression sites of localised aggrecan loss. The results of a recent genome-wide association study revealed eight polymorphisms in the ADAMTS-5 Apart from activity studies that tell us a great deal about gene, two of which caused amino acid changes. However neither aggrecanases collectively, the most abundant studies on human polymorphism was associated with difference in susceptibility to ADAMTS-4 and -5 are those that have analysed mRNA expression osteoarthritis (OA) in a large cohort of patients3. either in vitro with and without catabolic stimuli, or immediately ex e It is not clear whether ADAMTS-4 or ADAMTS-5 is the aggre- vivo, from normal or OA cartilage. Most4 8 but not all9,10 the in vitro canase in human cartilage. Indeed there is evidence suggesting that studies in human cartilage or chondrocytes found that ADAMTS-4 both aggrecanases have key roles in degrading aggrecan in vivo4. mRNA was induced by catabolic cytokines such as interleukin-1b Identifying the human aggrecanase is a major challenge because (IL-1b) or Tumor necrosis factor a (TNFa) and that ADAMTS-5 the final level of enzyme activity is determined by multiple, inde- mRNA was not regulated by cytokines but expressed constitutively. pendent and interacting molecular processes, including promoter These findings differ from most animal studies which show that ADAMTS-5 mRNA expression is upregulated by catabolic cytokines (reviewed in Ref. 11). * Address correspondence and reprint requests to: University of Melbourne, The absence of ADAMTS-4 and -5 in early OA gene profiling Department of Paediatrics and Murdoch Childrens Research Institute, Royal Chil- 12e14 dren’s Hospital, Flemington Road, Parkville 3052, Australia. studies suggests that on a global scale, these enzymes are E-mail address: [email protected] (A.J. Fosang). lowly expressed. Other expression studies focussing specifically on

1063-4584/$ e see front matter Crown Copyright Ó 2010 Published by Elsevier Ltd on behalf of Osteoarthritis Research Society International. All rights reserved. doi:10.1016/j.joca.2010.06.014 1110 A.J. Fosang, F.M. Rogerson / Osteoarthritis and Cartilage 18 (2010) 1109e1116

Activation ADAMTS-5 is not regulated by this growth factor20. Another growth (N-terminal factor, FGF-2, inhibits IL-1-induced expression of ADAMTS-4 and -5 processing) in primary human chondrocytes without inhibiting IL-1 actions on mRNA C-terminal 10 expression processing other target molecules . Conversely, the stress-induced calcium- binding protein, S100A8, enhances IL-1-induced ADAMTS-5 expression in mouse cartilage, resulting in increased NITEGE373 Enhancers Inhibitors 31 of activity of activity immunoreactivity and aggrecan loss . In other studies, high ADAMTS molecular weight HA (Mre2700 kDa) can decrease IL-1a-induced expression of ADAMTS-4 mRNA and protein, without inhibiting activity ADAMTS-4 expression directly8; the mechanism is unclear. In Enzyme miRNA glycosylation modifications summary, ADAMTS-4 and -5 mRNA can be up- or down-regulated by a variety of modulators typically found in arthritic joints. However we emphasise that to date, there are no corresponding Substrate Other RNA studies linking increased mRNA expression with increased glycosylation modifications Epigenetic ADAMTS-4 or ADAMTS-5 activity. modifications In addition to modulators that are known, or thought, to act via intracellular signalling pathways, at least two transcription factors Fig. 1. Aggrecanase activity is determined by multiple molecular processes. can potentially modify aggrecanase gene expression by direct binding to the ADAMTS-4 or -5 gene promoters. The human cartilage proteinases show that ADAMTS-5 is more highly ADAMTS-4 promoter has two putative binding sites for the tran- expressed than ADAMTS-4 in both normal and OA cartilage5,15. scription factor nuclear factor of activated T cells-p (NFATp) and Some studies report increased expression of both ADAMTS-4 and eight putative binding sites for the Runx family of transcription ADAMTS-5 in knee cartilage from patients with late, but not early factors32. Over-expression of either NFATp or Runx2 in cells stage OA5,16. Other studies report that only ADAMTS-4 mRNA is expressing 4.5 kb of the ADAMTS-4 promoter resulted in trans- increased in knee cartilage7,17 or hips18 measured at the time of activation of the ADAMTS-4 promoter32. Similarly, analysis of 2.6 kb joint replacement, while others report decreases in both ADAMTS-4 of the human ADAMTS-5 promoter revealed four putative binding and -5 mRNA in patients with late stage hip OA15. Given the sites for the family of Runx transcriptions factors and Runx2 differences between the aetiopathogenesis of hip and knee OA19,it transactivated the ADAMTS-5 promoter in vitro33. The in vivo is not surprising that ADAMTS expression differs between knee and influence of Runx2 and NFATp on aggrecanase activity via the hip joints in humans15 and presumably differs also with stage of ADAMTS-4 or ADAMTS-5 promoter is unknown. disease, although this has not been systematically examined. ADAMTS expression in cartilage represents only one part of the ADAMTS-4 and -5 regulation by alternative splicing, total joint picture, since ADAMTS enzymes are expressed in joint epigenetics and ncRNA capsule and meniscus, as well as in synovium where ADAMTS e expression is particularly well documented20 28. In distinct There are other pre-translational mechanisms for modulating contrast to cartilage, both ADAMTS-4 and -5 are expressed consti- aggrecanase activity independently of mRNA expression; these tutively in synovium and their expression by synovial fibroblasts is include alternative splicing of the ADAMTS transcripts, regulation not regulated by IL-1 in vitro20,21,29. Expression profiling studies by ncRNAs including miRNA and gene silencing (or unsilencing) by show that ADAMTS-4 and -5 are significantly decreased in syno- epigenetic modifications to DNA and histones. vium from patients with hip OA, compared with the levels in An alternatively spliced transcript of ADAMTS-4 has been synovium from patients with neck of femur fractures26. A different identified in synovium from OA patients27. The splice variation study using synovium from patients with cruciate ligament injury occurs between exons 8 and 9 to create a longer protein which as the non-arthritic comparator showed that ADAMTS-4, but not diverges from wildtype ADAMTS-4 near the start of the spacer ADAMTS-5 was significantly increased in rheumatoid arthritis (RA) region, after Arg696. There is no information on the abundance of patients; however, in this study neither ADAMTS-4 nor ADAMTS-5 this transcript in synovium, its expression in cartilage and other expression levels were changed in OA synovium20. Not surprisingly, joint tissues, or its expression in other patient groups. It will be synovial fibroblasts from mouse joints express a different reper- fascinating to determine the efficacy of the ADAMTS-4 splice toire of aggrecan-degrading activities compared with mouse variant as an aggrecan-degrading enzyme. The splice variant is chondrocytes, based on the pattern of neoepitopes detected by likely to have altered enzyme activity, given the importance of the Western blotting29. During episodes of synovial hyperplasia it is spacer region in conferring maximum aggrecanolytic activity on entirely possible that synovial fibroblasts might be the major ADAMTS-434. There is no evidence for alternatively spliced tran- source of aggrecanases within the joint, and that synovial-derived scripts of ADAMTS-5. enzymes mediate aggrecanolysis in cartilage29. There is less liter- The genomes of eukaryotes studied to date are almost entirely ature available on aggrecanase activity in the meniscus, however transcribed, leading to vast numbers of ncRNAs with unknown one study has shown that mechanical compression of meniscal functions35. These ncRNAs and miRNAs represent a new frontier in tissue in vitro up-regulates ADAMTS-5 mRNA and down-regulates the molecular biology of human disease. miRNAs primarily affect ADAMTS-4 mRNA expression23. The effect of theses changes on gene expression by inhibiting protein translation, without affecting aggrecanase activity in the meniscus is not known. transcription. Much less is known about ncRNAs in terms of actions ADAMTS mRNA expression and activity can be directly regulated in vivo but they are thought to have important roles in epigenetic, by transforming growth factor-b (TGF-b)6,20 and indirectly modu- transcriptional and post-transcriptional regulation of gene lated by fibroblast growth factor-2 (FGF-2), S100A8 and high expression (reviewed in Ref. 35). molecular weight hyaluronan (HA). TGF-b is well known for its It is clear that miRNAs provide important regulatory checks in anabolic role in matrix metabolism, but TGF-b can also promote the immune system and inflammatory reactions, and a 16-miRNA joint destruction30. TGF-b strongly up-regulates ADAMTS-4 mRNA gene signature that is differentially expressed in OA has recently and protein expression in human synovial fibroblasts, whereas been described36. Two independent studies have now reported the A.J. Fosang, F.M. Rogerson / Osteoarthritis and Cartilage 18 (2010) 1109e1116 1111 down-regulation of miR140 in OA cartilage compared with healthy and/or cell surface activation has been reported for ADAMTS-550,52 controls36,37. miR140À/À mice have significantly increased levels of and both intracellular49 and cell surface51 activation has been ADAMTS-5 mRNA and increased aggrecan loss in vitro, compared reported for ADAMTS-4. with wildtype mice38. miR140 is also predicted to target ADAMTS-4 Variables in the ADAMTS activation process (which PC, which and -13 (MMP-13)36,39. There is no data cellular site) could significantly influence the efficiency of enzyme comparing the effects of miR140 on ADAMTS-4- vs ADAMTS-5- activation and alter enzyme activity, independent of mRNA levels. mediated aggrecanase activity in any species. Mimic miRNAs and Other variables include the fact that PCs themselves are subject to anti-miRNAs are of interest for the treatment of cancers however regulated expression and selective glycosylation of PCs can affect the study of ncRNAs in cartilage, and their influence in OA and their activity. inflammatory arthritis, is very much in its infancy. fi Epigenetic modi cations include changes in the methylation Regulation of ADAMTS-4 and -5 activities by C-terminal status of CpG sites in gene promoters, and the acetylation and processing deacetylation of histone . Epigenetic changes determine whether genes are silenced or transcribed and they lead to heri- Processing to remove C-terminal ancillary domains of ADAMTS-4 table and permanent alterations in gene expression. In arthritic and -5 is likely to be a common mechanism for regulating aggreca- diseases, epigenetic changes can potentially escalate disease nase activity. Truncated forms of both enzymes are consistently progression through, for example, synovial hyperplasia in RA, or found in tissue extracts and experimental systems16,17,20,21,54,55 cell proliferation within chondrocyte clusters that are typically following local proteolysis54 or autolysis56,57. For example, the seen in human OA. Recent studies have shown that aberrant major form of ADAMTS-5 in normal human synovium is the 70 kDa expression of ADAMTS-4 is associated with loss of DNA methyla- mature form, whereas synovium from RA patients is predominantly fi 18,40 tion at speci c CpG sites in the ADAMTS-4 promoter . the 53 kDa form lacking the last thrombospondin module and the Furthermore, a related study comparing young (23 years of age) spacer domain20. The minimum domain composition for a molecule e and old (80 90 years of age) OA cartilage, with non-arthritic age- containing aggrecan-degrading activity, in both ADAMTS-4 and -5, is matched comparators, suggests that ageing alone does not account the catalytic domain, the disintegrin domain and the first throm- fi for the epigenetic unsilencing in OA. This is based on the nding bospondin module (Fig. 2); recombinant truncates lacking the first e that the association between ADAMTS-4 expression and deme- thrombospondin module are inactive against aggrecan55,58 60. thylation of the ADAMTS-4 promoter was a consistent feature of Mature ADAMTS-4 and -5 containing a full suite of C-terminal 41 both the young and old OA cartilage . There are examples of ancillary domains are the most active species in vitro and, in the 42 abnormal ADAMTS-5 promoter methylation in some cancers .In absence of heparin34, engineered deletions of C-terminal domains the future it will be important to characterise the epigenetic diminish catalytic activity against both the IGD and chondroitin changes in ADAMTS-4 and -5 and correlate them with changes in sulphate (CS)-rich regions of aggrecan59. In these studies with aggrecan-degrading activity. recombinant enzymes expressed in HEK293-EBNA cells, deletion of the spacer domain of ADAMTS-4 or the cysteine-rich domain of ADAMTS-4 and -5 activation by N-terminal processing ADAMTS-5 ablates aggrecan-degrading activity by more than 95%, while having only minor effects on cleavage of non-aggrecan Two early studies pointed to the possibility that aggrecanase substrates such as fibromodulin59. activity might be controlled at a post-translational level. Flannery It is possible that C-terminal processing of ADAMTS-4 might have and colleagues showed that retinoic acid-induced aggrecanase different effects in other cell types, since Gao et al. have used a human activity was increased in bovine chondrocytes with no concomitant chondrosarcoma cell line stably transfected with ADAMTS-4 and 43 increase in mRNA levels for any ADAMTS homologues . In a sepa- MT4-MMP, to show that the most active form of ADAMTS-4 is rate study, Pratta and colleagues showed that IL-1-induced generated by MT4-MMP-mediated removal of the spacer domain54. ADAMTS-4 activity was increased in bovine chondrocytes in the The authors propose that sites at Lys694 Y 695Phe and Thr581 Y 582Phe absence of any detectable increase in ADAMTS-4 protein produc- in the spacer domain are susceptible to MT4-MMP cleavage. 44 tion ; these investigators concluded that enzyme activation was There is also evidence that C-terminal processing influences responsible for the increased activity of constitutively expressed tissue localisation of aggrecanases. ADAMTS-5 in normal human ADAMTS-4. cartilage localises around cells in the superficial zone and around ADAMTS-4 and -5 are synthesised as latent proenzymes and clonal clusters in OA cartilage16. The pericellular localisation is retained in their inactive state by interactions between their pro thought to occur via HA binding to the disintegrin domain16 and to and catalytic domains. The mechanism by which the prodomains a heparin-binding motif in the spacer domain. Other studies in maintain ADAMTS latency is unknown but it is unlikely to be via the HTB-94 chondrosarcoma cells show that the cysteine-rich domain well known cysteine switch mechanism found in the matrix met- of ADAMTS-5 is essential for binding and localisation in the 45 alloproteinase family . Instead, the ADAMTS-4 and -5 prodomains extracellular matrix59. In ADAMTS-4, it is the spacer domain that might directly inhibit enzyme activation via a non-cysteine switch mediates matrix binding56,57,60,61. Thus, C-terminal processing can 46,47 mechanism, similar to that seen for ADAM17 . influence aggrecanase activity directly via its effects on enzyme Furin, or furin-like proprotein convertases (PC), has major roles in activating ADAMTS enzymes. Furin, PC7 and PC5A activate e recombinant ADAMTS-4 or -548 51 and Malfait et al. have used silencing RNA (siRNA) to show that PACE4 activates aggrecanases in ADAMTS-5 sig Pro Cat Dis TS Cys Spac TS OA and cytokine-stimulated human cartilage in culture52. Cleavage occurs at the multibasic furin/PC recognition sites located at ADAMTS-4 sig Pro Cat Dis TS Cys Spac RRAKR212 in human ADAMTS-41 and RRRRR261 in human ADAMTS- 52,50. Importantly, N-terminal processing and activation can be Fig. 2. The domain structure of ADAMTS-4 and ADAMTS-5. The schematic shows the sequential arrangement of the signal peptide (sig), prodomain (Pro), catalytic domain mediated by more than one PC at more than one cellular location, (Cat), disintegrin domain (Dis), first thrombospondin-like motif (TS), the cysteine-rich since PCs are located in the trans Golgi network, in endosomes, at domain (Cys) and spacer domain (Spac). ADAMTS-5 has an additional thrombospondin 53 50 the cell surface and in the extracellular matrix . Extracellular motif at its C-terminus. 1112 A.J. Fosang, F.M. Rogerson / Osteoarthritis and Cartilage 18 (2010) 1109e1116 activity, and indirectly by disrupting ADAMTS localisation in C-terminal processing, their activity can be further modified by extracellular matrices. endogenous inhibitors. The Tissue Inhibitor of Proteinases that cleave ADAMTS C-terminal domains include (TIMP) family of inhibitors are expressed in joint tissues, and are MT4-MMP54, PACE452 and ADAMTS-4 and -5 themselves56.In regulated in arthritis. Of the four family members, TIMP-3 is the addition, two putative PC cleavage sites are located at RRTR565 and most potent inhibitor of both ADAMTS-470,71 and ADAMTS-570 and RKFR696 in ADAMTS-448. The presence of two established and two there is negligible inhibition of aggrecanases by TIMPs-1, -2 or -470. putative C-terminal cleavage sites, combined with apparent In the TIMP-3 null mouse there is increased aggrecan loss and susceptibility of ADAMTS enzymes to at least three different increased NITEGE373 neoepitope, concomitant with mild cartilage families of proteinases, suggests that ADAMTS-4 and -5 C-terminal degradation72, suggesting that TIMP-3 is indeed an endogenous processing is a key mechanism for regulating aggrecanase activity. aggrecanase inhibitor. TIMP-3 binds tightly to negatively charged In addition to , alternative splicing of ADAMTS-4 glycosaminoglycans73 and TIMP-3 potency against ADAMTS-4 is provides another means of disrupting C-terminal ancillary domains enhanced by ADAMTS-4 binding to aggrecan74; it appears that to modulate enzyme activity27. TIMP-3 has a greater affinity for ADAMTS-4 complexed with aggrecan, than it does for ADAMTS-4 alone74. A recent, exciting Regulation of ADAMTS-4 and -5 activities by glycosylation: finding is that TIMP-3 potency against ADAMTS-4 and -5 is greatly enzyme and substrate enhanced in the presence of calcium pentosan polysulphate (CaPPS)75. CaPPS itself inhibits aggrecanase activity via interactions Almost all secreted proteins undergo some form of post-trans- with the spacer domain of ADAMTS-4 and the cysteine-rich domain lational modification and the most common type of modification is of ADAMTS-575. The levels of TIMP-3 in cartilage are very low due to glycosylation. In many cases glycosylation confers essential func- rapid internalisation by the cell via Lipoprotein receptor-related tional properties on secreted proteins. All ADAMTS proteinases are proteins (LRP)-mediated endocytosis75. predicted to be glycoproteins carrying at least one mannose modi- Other proteinase inhibitors in cartilage include a2-macroglob- fication on tryptophan residues within prescribed motifs in the first ulin and the membrane-anchored glycoprotein, reversion-inducing 62 thrombospondin motif . This prediction has been confirmed cysteine-rich protein with Kazal motifs (RECK). a2-macroglobulin experimentally for ADAMTS-5 in studies which identified two C- inhibits ADAMTS-4 and -5 in vitro by trapping the enzymes in a 1:1 mannose substitutions as well as a glucose-fucose disaccharide on complex76, however, there is no evidence for such a complex in 62 the thrombospondin domain of recombinant enzyme . Two sites synovial fluid, nor evidence that a2-macroglobulin is cleaved by for C-mannosylation are also predicted for the thrombospondin either enzyme76. Similarly, there is no evidence that RECK inhibits domain of ADAMTS-4. N-linked glycosylation in the ADAMTS-5 ADAMTS-4 or -5. Thus, Timp-3 appears to be the only endogenous spacer domain was predicted2 and has been confirmed in a separate inhibitor of aggrecanase activity in cartilage. Interestingly, study by a change in electrophoretic mobility of recombinant ADAMTS-4 activity is blocked when the C-terminal domain of ADAMTS-5 after digestion with PNGase F55. There is no evidence fibronectin binds the ADAMTS-4 spacer domain61. Enzyme inhibi- that ADAMTS-4 carries N-linked glycosylation63 and there is no tion following interactions between fibronectin and ADAMTS-4 evidence that underglycosylation affects ADAMTS-4 or -5 activity. could be relieved by C-terminal processing of ADAMTS-4 to Mannosylation in the thrombospondin domain regulates secretion displace fibronectin or fibronectin fragments. The concentrations of of some members of the ADAMTS family62. It is possible that fibronectin and fibronectin fragments are increased in OA and secretion of ADAMTS-4 and -5 is also influenced by mannosylation. rheumatoid synovial fluids. ADAMTS enzymes degrade aggrecan extracellularly so controlling their secretion will affect aggrecanase activity directly. Enhancers of ADAMTS-4 and -5 activity Glycosylation of the aggrecan substrate is another variable that markedly affects aggrecanase activity. The signature activity of the There is potential for increased aggrecanase activity following aggrecanases is cleavage within the IGD. The IGD contains keratan interactions between ADAMTS enzymes and other cell surface or sulphate (KS) but no CS substitutions, so it is not surprising that extracellular matrix molecules. For example, the interaction of e removal of KS alone blocks aggrecanase cleavage in the IGD64 66. ADAMTS-177 and ADAMTS-578 with the matrix molecule fibulin-l The KS content of human aggrecan increases from a minimal increases catalytic activities toward aggrecan or versican percentage at birth, to over a quarter of the glycosaminoglycan substrates, respectively. Fibulin-1 itself is not a substrate for these content at maturity, and KS microstructure also changes dramati- enzymes. The C-terminal thrombospondin domain of ADAMTS-1 cally during this time67. In addition, the microstructure of KS in the interacts with fibulin-1, however, it is not clear which domain(s) of IGD is distinctly different to the microstructure of KS elsewhere on ADAMTS-5 might interact with fibulin. The epidermal growth aggrecan68, suggesting that KS microstructure might contain factor (EGF)-like repeats in domain II of fibulin-1 interact specifi- functional information. Furthermore, whereas ADAMTS-5 cleavage cally with the G3 domain of aggrecan79, and since aggrecan is in the IGD is highly efficient in both mature and juvenile cartilage, a major ADAMTS-5 substrate, the interaction between fibulin-1 and ADAMTS-4 cleavage in the IGD is inefficient in juvenile carti- ADAMTS-5 might provide a mechanism for localising ADAMTS-5 in lage64,69. Collectively these studies suggest an intriguing link close proximity with its substrate. between increased susceptibility to aggrecanolysis with ageing, Recent in vitro studies have shown that ADAMTS-5 interacts and increased KS substitution with ageing. Further work is required directly with syndecan-4, and mice deficient in syndecan-4 are to investigate this potential link and to determine whether protected against aggrecan loss and cartilage damage in a running ADAMTS-4 and -5 have age-specific functions in cartilage and OA. wheel model of joint damage80. These results suggest that synde- can-4 might enhance ADAMTS-5 activity, however the mechanism Inhibitors and enhancers of ADAMTS-4 and -5 activity is unknown. Perhaps syndecan-4 is required to tether ADAMTS-5 to the cell surface80, reminiscent of the model in which syndecan-1 is Inhibitors of ADAMTS-4 and -5 activity thought to tether ADAMTS-4 to the cell surface54. High molecular weight HA is also thought to have a role in sequestering ADAMTS-5 Once aggrecanase enzymes have been synthesised, secreted in the pericellular matrix of OA cartilage16. No complexes between from the cell, activated by prodomain removal and modified by HA and ADAMTS-4, or HA and ADAMTS-9, have been detected in A.J. Fosang, F.M. Rogerson / Osteoarthritis and Cartilage 18 (2010) 1109e1116 1113

OA cartilage. It is worth considering the possibility that HA, systems, it is important to counter that in the same or similar syndecan-4 and syndecan-1 enhance aggrecanase activity, not via systems, ADAMTS-5 is often expressed at much higher levels than their interactions with ADAMTS-4 or -5, but by virtue of anchoring ADAMTS-4, and that ADAMTS-5 has a 30-fold greater intrinsic these enzymes at their sites of action and limiting their ability to activity than ADAMTS-434. Accordingly, the dilemma of identifying diffuse away. ADAMTS-4 and -5 are certainly diffusible enzymes; the human aggrecanase remains. ADAMTS-4 and -5 synthesised by mouse synovial fibroblasts in co- cultures can penetrate and degrade aggrecan within femoral head Author contributions cartilage explants29. Together these findings suggest firstly, that there are mechanisms for helping to colocalise aggrecanase AJF role: conception, drafting, critical revision and final approval enzymes with aggrecan substrate and secondly, that ADAMTS of the manuscript. activity detected in one joint tissue might be the product of RFM role: drafting, critical revision and final approval of the ADAMTS mRNA produced in a different joint tissue. manuscript.

Perspectives and challenges for the future Conflict of interest Aggrecanase research to date has been hampered by two, major, The authors have no conflicts of interest to declare. technical limitations. The first is the inability to measure the products of aggrecanase activity in patient serum or urine. In this Acknowledgements issue of Osteoarthritis and Cartilage, Thirunavukkarasu and colleagues describe successful reverse engineering of the original The authors thank Dr Heather Stanton for critical reading of the aggrecanase neoepitope antibody, BC381. The authors have gener- manuscript. The authors are funded by the National Health and ated antibody BC3-C2 with a 4-log improvement in affinity for the Medical Research Council (Australia). 374ARGS neoepitope82. The sensitivity and specificity of this new antibody will enable the measurement of aggrecanase fragments in References human serum and urine by ELISA assay and facilitate clinical testing of aggrecanase inhibitors. However BC3-C2 will not solve the thorny 1. Tortorella MD, Burn TC, Pratta MA, Abbaszade I, Hollis JM, issue of identifying the human aggrecanase. The second technical Liu R, et al. Purification and cloning of aggrecanase-1: limitation is that we have no methods at all for distinguishing the a member of the ADAMTS family of proteins. Science products of ADAMTS-4 activity, from the products of ADAMTS-5 1999;284:1664e6. activity; clearly, because the products are identical. At present this 2. Abbaszade I, Liu RQ, Yang F, Rosenfeld SA, Ross OH, Link JR, limitation seems insurmountable; we are stymied on this front! et al. Cloning and characterization of ADAMTS11, an aggreca- And to complicate matters further, there is a third aggrecan- nase from the ADAMTS family. J Biol Chem 1999;274: degrading enzyme that cleaves at the well-recognised aggrecanase 23443e50. cleavage sites in the CS-rich region. This enzyme cleaves at the 3. Rodriguez-Lopez J, Mustafa Z, Pombo-Suarez M, Malizos KN, Glu1279 Y 1280Gly and Glu1467 Y 1468Gly aggrecanase sites in the CS- Rego I, Blanco FJ, et al. Genetic variation including non- rich region of mouse aggrecan and its activity is detected in the synonymous polymorphisms of a major aggrecanase, ADAMTS-4/5 double-deficient mouse83. While this third aggreca- ADAMTS-5, in susceptibility to osteoarthritis. Arthritis Rheum nase is unlikely to be the aggrecanase in humans, it is nevertheless 2008;58:435e41. important that the enzyme be identified and characterised. If it has 4. Song RH, Tortorella MD, Malfait AM, Alston JT, Yang Z, a role in OA pathology, then it will be necessary to inhibit this Arner EC, et al. Aggrecan degradation in human articular enzyme along with ADAMTS-4 and/or ADAMTS-5. Conversely, if it cartilage explants is mediated by both ADAMTS-4 and has a role in normal skeletal development and endochondral ossi- ADAMTS-5. Arthritis Rheum 2007;56:575e85. fication, it will be important to ensure that aggrecanase inhibitors 5. Bau B, Gebhard PM, Haag J, Knorr T, Bartnik E, Aigner T. currently in development do not inhibit this enzyme. Relative messenger RNA expression profiling of Despite intensive research over the last decade we are still and aggrecanases in human articular chondrocytes in vivo and unable to positively identify the human aggrecanase and indeed in vitro. Arthritis Rheum 2002;46:2648e57. the possibility remains that both ADAMTS-4 and ADAMTS-5 play 6. Moulharat N, Lesur C, Thomas M, Rolland-Valognes G, equally important roles in aggrecanolysis. In this editorial we have Pastoureau P, Anract P, et al. Effects of transforming growth highlighted the difficulties in finding definitive answers to this factor-beta on aggrecanase production and quandary; the regulation of ADAMTS-4 and -5 enzymes is complex degradation by human chondrocytes in vitro. Osteoarthritis and there are significant pre- and post-transcriptional events that Cartilage 2004;12:296e305. contribute to the final level of enzyme activity. These are likely to be 7. Naito S, Shiomi T, Okada A, Kimura T, Chijiiwa M, Fujita Y, et al. dynamic events that are themselves under tight control. The Expression of ADAMTS4 (aggrecanase-1) in human osteoar- simplest and most abundant studies have examined regulation of thritic cartilage. Pathol Int 2007;57:703e11. ADAMTS-4 and -5 mRNA expression, comparing OA cartilage with 8. Yatabe T, Mochizuki S, Takizawa M, Chijiiwa M, Okada A, normal cartilage, or the regulatory effects of cytokines, growth Kimura T, et al. Hyaluronan inhibits expression of ADAMTS4 factors and other exogenous modulators, in vitro. However, we (aggrecanase-1) in human osteoarthritic chondrocytes. Ann caution that no reliable conclusions about the source of the Rheum Dis 2009. aggrecanase activity can be made from mRNA studies, either in vitro 9. Young DA, Lakey RL, Pennington CJ, Jones D, Kevorkian L, or in vivo; the number and range of processes (Fig. 1) that deter- Edwards DR, et al. Histone deacetylase inhibitors modulate mine the final level of ADAMTS-4 or ADAMTS-5 activity is signifi- metalloproteinase gene expression in chondrocytes and block cant, and likely to overwhelm the effects of increased or decreased cartilage resorption. Arthritis Res Ther 2005;7:R503e12. mRNA expression, making absolute conclusions about the 10. Sawaji Y, Hynes J, Vincent T, Saklatvala J. Fibroblast growth contributing aggrecanase doubtful. Although ADAMTS-4 mRNA is factor 2 inhibits induction of aggrecanase activity in human reproducibly upregulated by cytokines in a number of experimental articular cartilage. Arthritis Rheum 2008;58:3498e509. 1114 A.J. Fosang, F.M. Rogerson / Osteoarthritis and Cartilage 18 (2010) 1109e1116

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