Juvenile idiopathic arthritis (JIA) is the most commonly diagnosed rheumatic disease in children, with a prevalence of 1–4 per 1,000 children (1, 2). It is a chronic, heterogeneous disorder characterized by persistent synovial inflammation which, if unresolved, can lead to joint destruction and permanent disability. Prior to the advent of the new biologic therapies, the incidence of cartilage erosion, detected radiographically as joint space narrowing in JIA patients with long-term disease, was significant (3–6).
While the new biologic agents have improved the management of synovial inflammation in JIA, there has been little progress in strategies for preventing cartilage damage in JIA or other arthritides. Development of new treatments for limiting cartilage erosion in JIA is hampered by the lack of fundamental information on the mechanisms involved; to date, studies on aggrecanolysis have focused exclusively on adult cartilage, predominantly in osteoarthritis (OA). A recent study by Kim and colleagues suggests that the extent of cartilage damage in JIA might be underrecognized, since new imaging modalities have revealed microstructural changes indicative of cartilage damage in JIA patients by T2 mapping (7). These microstructural changes are thought to be markers of disease progression that develop early, despite clinical improvement and before changes in cartilage morphology can be detected with conventional magnetic resonance imaging. Thus, although there have been major improvements in JIA outcomes brought about by the introduction of biologic therapies, destructive cartilage erosion leading to irreparable joint abnormalities remains a risk for these patients.
The aggrecanases (ADAMTS) have been studied intensively since their discovery in 1999 (8, 9), and it is now clear that ADAMTS-4 and/or ADAMTS-5 are the principal aggrecan-degrading enzymes in OA and inflammatory arthritis in humans and experimental animals (10–16). Cleavage in the interglobular domain (IGD) of aggrecan is the signature activity of this enzyme family and is widely regarded as the defining activity of the aggrecanases, even though cleavage at this site is preceded by cleavage at the SELE1545 and KEEE1714 sites in the chondroitin sulfate–rich region (17–20) (Figure 1). (Numbering is from 1VETS of the mature protein [NCBI accession no. P16112].) The most abundant ADAMTS-derived aggrecan fragments found in OA synovial fluid (SF) are the 374ARGS-SELE1545 fragment (fragment b in Figure 1), its 1546GRGT-G3 counterpart (fragment f in Figure 1), 2 shorter G3-containing fragments (fragments g and h in Figure 1), and a KEEE1714 fragment with an intact N-terminal G1 domain (fragment d in Figure 1) (10, 15, 21). These fragments are readily detected in human OA SF by Western blotting with neoepitope or anti-G3 antibodies.
Figure 1. Aggrecanase cleavage sites in human aggrecan detected with neoepitope antibodies. Aggrecanase cleavage sites in the human aggrecan core protein are shown, with numbering from the first amino acid (valine) of the mature protein. Circled letters (a–k) identify individual fragments corresponding to bands detected on Western blots (see Figures 3–5). The triangle marks the matrix metalloproteinase cleavage site at IPEN341342FFGV. The dotted line indicates regions of the core protein corresponding to the interglobular domain (IGD), keratan sulfate (KS)–rich region, and chondroitin sulfate (CS)–rich region. Boxed numbers 1 and 2 indicate the preferred order of aggrecanase cleavage.
Download figure to PowerPoint
Enzymes other than aggrecanases can also degrade aggrecan in vivo. Members of the matrix metalloproteinase (MMP) family cleave aggrecan in the IGD at IPEN341342FFGV, generating G1-IPEN341 and 342FFGV fragments in vivo and in vitro (15, 21, 22). The intracellular, nonlysosomal proteinase calpain (23) and the serine proteinase HtrA1 (24) also cleave aggrecan in vivo, albeit at low levels. It is estimated that in osteoarthritic cartilage ∼75% of aggrecan degradation is due to the action of ADAMTS aggrecanases and ∼25% due to MMP activity (23), with other enzyme families making only minimal contributions to total aggrecanolysis. To date there is no published information on the identity of the proteinases that degrade aggrecan in JIA or the pattern or abundance of aggrecan fragments in JIA SF. Such fragments could potentially be used as biomarkers of early cartilage erosion in children or as markers of the efficacy of cartilage-sparing treatments.
In the present study, we analyzed SF samples from a small group of JIA patients for the presence of aggrecan fragments produced by ADAMTS enzymes. We found that proteolysis by aggrecanases in the aggrecan IGD appears negligible in JIA patients compared with OA patients, despite robust levels of aggrecanase cleavage in the chondroitin sulfate–rich region. These results suggest that ADAMTS cleavage in the aggrecan IGD might not be a pathogenic event in JIA, and that cartilage-sparing drugs designed to block aggrecanase cleavage in the IGD might not be useful for arresting cartilage erosion in this disease.
- Top of page
- PATIENTS AND METHODS
- AUTHOR CONTRIBUTIONS
This is the first study to analyze aggrecan fragments in JIA SF. The apparent absence of 374ARGS fragments in the JIA samples is striking. Possible explanations are 1) that ADAMTS-4 and -5 are less active in JIA cartilage, perhaps as a consequence of processing that removes C-terminal ancilliary domains from these enzymes, 2) that JIA aggrecan is less susceptible to aggrecanase attack, due to different amounts or types of glycosylation near the TEGE373374ARGS cleavage site (36, 37), or 3) that an unidentified aggrecanase cleaves in the chondroitin sulfate–rich region, but not in the IGD (38), in JIA cartilage.
Alternatively, it is possible that the 374ARGS epitope is created and then destroyed by either aminopeptidase activity, MMP cleavage at TSED441442LVVR in the aggrecan IGD (35), or other processing events that generate low-density fragments that are not recovered in the CsCl gradients. There is currently no evidence to indicate whether 374ARGS fragments are created and then destroyed or not created at all, although we strongly favor the latter interpretation. Another reason 374ARGS fragments might not be created in JIA cartilage relates to enzyme kinetics, since the Km of ADAMTS-4/5 for aggrecan cleavage at C-terminal sites is ∼20-fold lower than at the IGD site. The progressive, age-related shortening (proteolysis) of aggrecan from the C-terminus creates adult aggrecan with fewer preferred cleavage sites. In children, with a higher proportion of preferred (C-terminal) cleavage sites acting as competing sites, the production of 374ARGS fragments from the nonpreferred (IGD) cleavage site will proceed more slowly. One final hypothesis to explain the lack of 374ARGS fragments is that hyaluronidase activity released into the matrix (39) of juvenile but not adult cartilage liberates intact aggrecan from its cartilage anchor into SF, where it is cleaved by aggrecanases at C-terminal sites.
The slowest-migrating SELE1545 and KEEE1714 fragments in some JIA SF samples are much larger than the G1-SELE1545 or G1-KEEE1714 fragments seen in OA SF, suggesting that aggrecan fragments in JIA might interact with one or more molecules present in the cartilage matrix or the SF. The interacting molecule(s) is not an experimental or biologic contaminant because, in patient E for example, the SF samples were collected over a period of 5 years. The strength of the interaction, which is resistant to dissociation by SDS and 4M GuHCl, suggests that it might be stabilized by a molecular crosslinker such as transglutaminase 2 (40). The ubiquitously expressed transglutamase 2 catalyzes transamidation of glutamine residues to lysine residues and is thought to help stabilize tissue against injury or infection. However, in other cases, inappropriately crosslinked protein aggregates may trigger inflammation, and in this context it is interesting to note the association between transglutamase 2 and disease progression in RA (40). SF levels of transglutamase 2 also correlate with knee OA in the Hartley guinea pig model of spontaneous OA (41). Thus, the potential for transglutamase 2 activity to mediate crosslinking of aggrecan fragments in JIA cartilage is intriguing and warrants further investigation.
Given the almost complete absence of 374ARGS bands in JIA samples, it will be interesting in the future to determine whether large SELE1545 and KEEE1714 fragments have an N-terminus other than 374ARGS. The putative 374ARGS N-terminus of JIA SELE1545 fragment b might be destroyed by the activity of aminopeptidases or dipeptidylpeptidases. Alternatively, the N-terminus could be generated by MMP cleavage in the IGD since large molecular weight 342FFGV fragments were indeed present in JIA SF and 342FFGV-SELE1545 fragments have been detected in OA SF (15).
We have observed that the 342FFGV neoepitope is labile in the mouse since, although the IPEN341 and 342FFGV neoepitopes are generated in equimolar amounts, the 342FFGV epitope is extremely difficult to detect compared with IPEN341; this is despite the fact that lower molar amounts of epitope are detected with the anti-FFGV antibody than with the anti-IPEN antibody (34). We suspect that aminopeptidase or dipeptidylpeptidase activity with specificity for N-terminal phenylalanine might be responsible for this apparent loss of 342FFGV immunoreactivity. Similarly, aminopeptidases removing 1 or 2 N-terminal amino acids from an 374ARGS peptide would destroy the antigenicity of the neoepitope. Neutral aminopeptidase activity has been detected in the SF and peripheral blood of RA and JIA patients (42), and inhibitors of aminopeptidase and dipeptidylpeptidase activities have been investigated as treatments for RA (43).
From a clinical perspective, the findings of this study are interesting for two reasons. First, the results suggest that the drivers of aggrecanase activity are uniform between joints during periods of active arthritis; hence, the identical fragmentation patterns in individual patients' left and right knees aspirated at the same time. This is unexpected because systemic treatments for JIA can sometimes show better efficacy in one joint than another, suggesting that the drivers (cytokines, for example) might not be identical between two joints at the one time. The second interesting finding is that in individual patients, the aggrecan fragmentation pattern changes with time. This could be due to intrinsic disease factors or therapies. It is noteworthy that for patients A and F, patient E (at ages 11 and 12 years only), and patient K (at ages 8 and 12 years only) the therapies were the same, even though the fragmentation patterns were different. This suggests that treatment regimens alone are unlikely to be the underlying cause of the changing pattern of aggrecan fragments over time.
Several studies have shown that glycosaminoglycans, and in particular keratan sulfate (KS), may have a role in modulating aggrecanase activity in cartilage explants. For example, aggrecanase cleavage in the IGD is increased in the presence of endogenous KS and reduced when KS is removed by keratanase treatment (44–46). In human aggrecan, the KS content increases from a minimal percentage at birth to more than one-quarter of the GAG content at maturity, and the extent of modification is similarly increased. For example, maturing cartilage (9–18 years) has intermediate and increasing levels of KS sulfation, fucosylation, and sialylation (47). We have shown that in pig aggrecan, KS in the IGD is uniquely undersulfated compared with KS elsewhere on aggrecan (48), and also that KS on recombinant IGD potentiates aggrecanase cleavage in vitro (46). Given the variable patterns of aggrecan fragments in JIA, the proximity of IGD KS to the aggrecanase cleavage site, and evidence that KS potentiates aggrecanase activity in vitro, further studies to explore the role of KS in the pathogenesis of JIA are warranted.
The limitations of this study are the small number of patient SF samples examined in detail and the inability to quantitate 374ARGS bands that cannot be detected on gels. Future studies with more patients to elucidate the mechanisms involved in cartilage erosion in JIA, in conjunction with the use of newer technologies for quantitating the 374ARGS neoepitope at low concentrations (49, 50), are needed for the analysis of aggrecanase fragments in JIA SF.
In summary, this is the first detailed study of aggrecanolysis in JIA. Our results suggest that compared with aggrecanases in OA cartilage, aggrecanases in JIA cleave poorly in the aggrecan IGD. Accordingly, cleavage at the TEGE373374ARGS cleavage site in the aggrecan IGD may not be an appropriate therapeutic target for management of cartilage erosion in children with JIA.
- Top of page
- PATIENTS AND METHODS
- AUTHOR CONTRIBUTIONS
All authors were involved in drafting the article or revising it critically for important intellectual content, and all authors approved the final version to be published. Dr. Fosang had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.
Study conception and design. Struglics, Allen, Fosang.
Acquisition of data. Struglics, Last, Akikusa, Allen.
Analysis and interpretation of data. Struglics, Lohmander, Last, Akikusa, Fosang.