ADAMTS-9 in Mouse Cartilage Has Aggrecanase Activity That Is Distinct from ADAMTS-4 and ADAMTS-5.

A disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)-4 and ADAMTS-5 are the principal aggrecanases in mice and humans; however, mice lacking the catalytic domain of both enzymes (TS-4/5∆cat) have no skeletal phenotype, suggesting there is an alternative aggrecanase for modulating normal growth and development in these mice. We previously identified aggrecanase activity that (a) cleaved at E↓G rather than E↓A bonds in the aggrecan core protein, and (b) was upregulated by retinoic acid but not IL-1α. The present study aimed to identify the alternative aggrecanase. Femoral head cartilage explants from TS-4/5∆cat mice were stimulated with IL-1α or retinoic acid and total RNA was analysed by microarray. In addition to ADAMTS-5 and matrix metalloproteinase (MMP)-13, which are not candidates for the novel aggrecanase, the microarray analyses identified MMP-11, calpain-5 and ADAMTS-9 as candidate aggrecanases upregulated by retinoic acid. When calpain-5 and MMP-11 failed to meet subsequent criteria, ADAMTS-9 emerged as the most likely candidate for the novel aggrecanase. Immunohistochemistry revealed ADAMTS-9 expression throughout the mouse growth plate and strong expression, particularly in the proliferative zone of the TS-4/5-∆cat mice. In conclusion, ADAMTS-9 has a novel specificity for aggrecan, cleaving primarily at E↓G rather than E↓A bonds in mouse cartilage. ADAMTS-9 might have more important roles in normal skeletal development compared with ADAMTS-4 and ADAMTS-5, which have key roles in joint pathology.

An aggrecan fragment drives osteoarthritis pain through Toll-like receptor 2.

Pain is the predominant symptom of osteoarthritis, but the connection between joint damage and the genesis of pain is not well understood. Loss of articular cartilage is a hallmark of osteoarthritis, and it occurs through enzymatic degradation of aggrecan by cleavage mediated by a disintegrin and metalloproteinase with thrombospondin motif 4 (ADAMTS-4) or ADAMTS-5 in the interglobular domain (E373-374A). Further cleavage by MMPs (N341-342F) releases a 32-amino-acid aggrecan fragment (32-mer). We investigated the role of this 32-mer in driving joint pain. We found that the 32-mer excites dorsal root ganglion nociceptive neurons, both in culture and in intact explants. Treatment of cultured sensory neurons with the 32-mer induced expression of the proalgesic chemokine CCL2. These effects were mediated through TLR2, which we demonstrated was expressed by nociceptive neurons. In addition, intra-articular injection of the 32-mer fragment provoked knee hyperalgesia in WT but not Tlr2-null mice. Blocking the production or action of the 32-mer in transgenic mice prevented the development of knee hyperalgesia in a murine model of osteoarthritis. These findings suggest that the aggrecan 32-mer fragment directly activates TLR2 on joint nociceptors and is an important mediator of the development of osteoarthritis-associated joint pain.

A Disintegrin and Metalloproteinase with Thrombospondin Motifs-5 (ADAMTS-5) Forms Catalytically Active Oligomers.

The metalloproteinase ADAMTS-5 (A disintegrin and metalloproteinase with thrombospondin motifs) degrades aggrecan, a proteoglycan essential for cartilage structure and function. ADAMTS-5 is the major aggrecanase in mouse cartilage, and is also likely to be the major aggrecanase in humans. ADAMTS-5 is a multidomain enzyme, but the function of the C-terminal ancillary domains is poorly understood. We show that mutant ADAMTS-5 lacking the catalytic domain, but with a full suite of ancillary domains inhibits wild type ADAMTS activity, in vitro and in vivo, in a dominant-negative manner. The data suggest that mutant ADAMTS-5 binds to wild type ADAMTS-5; thus we tested the hypothesis that ADAMTS-5 associates to form oligomers. Co-elution, competition, and in situ PLA experiments using full-length and truncated recombinant ADAMTS-5 confirmed that ADAMTS-5 molecules interact, and showed that the catalytic and disintegrin-like domains support these intermolecular interactions. Cross-linking experiments revealed that recombinant ADAMTS-5 formed large, reduction-sensitive oligomers with a nominal molecular mass of ∼ 400 kDa. The oligomers were unimolecular and proteolytically active. ADAMTS-5 truncates comprising the disintegrin and/or catalytic domains were able to competitively block full-length ADAMTS-5-mediated aggrecan cleavage, measured by production of the G1-EGE(373) neoepitope. These results show that ADAMTS-5 oligomerization is required for full aggrecanase activity, and they provide evidence that blocking oligomerization inhibits ADAMTS-5 activity. The data identify the surface provided by the catalytic and disintegrin-like domains of ADAMTS-5 as a legitimate target for the design of aggrecanase inhibitors.

Bioactivity in an Aggrecan 32-mer Fragment Is Mediated via Toll-like Receptor 2.

OBJECTIVE: To determine whether an aggrecan 32-mer fragment derived from dual ADAMTS and matrix metalloproteinase (MMP) cleavage in the aggrecan interglobular domain was bioactive and, if so, to elucidate its mechanism of action. METHODS: Mouse primary chondrocytes, synovial fibroblasts, or peritoneal macrophages, human primary chondrocytes, and cells or cell lines from myeloid differentiation factor 88 (MyD88)-deficient and Toll-like receptor 2 (TLR-2)-deficient mice were stimulated with synthetic mouse 32-mer peptide, human 32-mer peptide, a 32-mer scrambled peptide, or native, glycosylated 32-mer peptide. Cells stimulated with 32-mer peptide were analyzed for changes in messenger RNA (mRNA) expression by quantitative polymerase chain reaction. Conditioned medium was analyzed for levels of interleukin-6 protein by an AlphaLISA or for levels of MMP-3 and MMP-13 protein by Western blotting. NF-κB activation was measured in a luciferase reporter assay. RESULTS: Treatment of mouse cells or cartilage explants with 32-mer peptide or scrambled peptide revealed that the 32-mer peptide, but not the scrambled peptide, had antianabolic, procatabolic, and proinflammatory bioactivity in vitro. Chondrocytes, synovial fibroblasts, and macrophages from MyD88-deficient mice failed to respond to 32-mer peptide stimulation. A macrophage cell line derived from TLR-2-deficient mice also failed to respond to 32-mer peptide stimulation. Stimulation of human chondrocytes with human 32-mer peptide increased the expression of catabolic markers at the mRNA and protein levels. Mouse and human 32-mer peptide stimulated NF-κB activation in a TLR-2-dependent reporter assay, and the response of chondrocytes from both species to native, glycosylated 32-mer peptide was similar to the response to synthetic peptides. CONCLUSION: The aggrecan 32-mer fragment is a novel endogenous ligand of TLR-2 with the potential to accelerate cartilage destruction in vivo.

Evidence for lysosomal exocytosis and release of aggrecan-degrading hydrolases from hypertrophic chondrocytes, in vitro and in vivo.

The abundant proteoglycan, aggrecan, is resorbed from growth plate cartilage during endochondral bone ossification, yet mice with genetically-ablated aggrecan-degrading activity have no defects in bone formation. To account for this apparent anomaly, we propose that lysosomal hydrolases degrade extracellular, hyaluronan-bound aggrecan aggregates in growth plate cartilage, and that lysosomal hydrolases are released from hypertrophic chondrocytes into growth plate cartilage via Ca(2+)-dependent lysosomal exocytosis. In this study we confirm that hypertrophic chondrocytes release hydrolases via lysosomal exocytosis in vitro and we show in vivo evidence for lysosomal exocytosis in hypertrophic chondrocytes during skeletal development. We show that lysosome-associated membrane protein 1 (LAMP1) is detected at the cell surface following in vitro treatment of epiphyseal chondrocytes with the calcium ionophore, ionomycin. Furthermore, we show that in addition to the lysosomal exocytosis markers, cathepsin D and ß-hexosaminidase, ionomycin induces release of aggrecan- and hyaluronan-degrading activity from cultured epiphyseal chondrocytes. We identify VAMP-8 and VAMP7 as v-SNARE proteins with potential roles in lysosomal exocytosis in hypertrophic chondrocytes, based on their colocalisation with LAMP1 at the cell surface in secondary ossification centers in mouse tibiae. We propose that resorbing growth plate cartilage involves release of destructive hydrolases from hypertrophic chondrocytes, via lysosomal exocytosis.

Aggrecanase cleavage in juvenile idiopathic arthritis patients is minimally detected in the aggrecan interglobular domain but robust at the aggrecan C-terminus.

OBJECTIVE: To investigate aggrecan degradation in juvenile idiopathic arthritis (JIA). METHODS: The pattern and abundance of aggrecan fragments in synovial fluid (SF) aspirates from JIA patients were analyzed and compared with aggrecan fragments in SF from patients with other arthritides, children with knee injury, and a knee-healthy reference group. Concentrations of sulfated glycosaminoglycan (sGAG) in SF were measured by Alcian blue precipitation assay. Aggrecan fragments were purified by dissociative CsCl density-gradient centrifugation, deglycosylated, and analyzed by Western blot using antibodies specific for either aggrecanase-derived ARGS, SELE, and KEEE neoepitopes or the aggrecan G3 domain. RESULTS: The concentration of sGAG in SF from patients with JIA was significantly lower compared with that in SF from patients with osteoarthritis (OA) (P < 0.001), patients with juvenile knee injury (P = 0.006), and knee-healthy controls (P = 0.022). Western blot analysis revealed KEEE, SELE, and G3 fragments generated by aggrecanase cleavage in the chondroitin sulfate-rich region of aggrecan in patients with JIA. The pattern of aggrecan fragments in JIA patients was not identical to that in pooled OA SF, although there were notable similarities. Surprisingly, aggrecanase-derived ARGS fragments were barely detectable in JIA SF, in marked contrast to levels in OA SF. CONCLUSION: Aggrecanases appear to cleave minimally in the interglobular domain of aggrecan in JIA patients despite robust levels of cleavage in the chondroitin sulfate-rich region. These results suggest that in JIA, unlike other arthritides, aggrecanase cleavage in the aggrecan interglobular domain might not be a major pathogenic event.

Investigating ADAMTS-mediated aggrecanolysis in mouse cartilage.

Proteolysis of the cartilage proteoglycan aggrecan is a feature of arthritis. We present a method for analyzing aggrecanolysis in in vitro cultures of 3-week-old mouse femoral head cartilage based on traditional methods developed for large animal species. Investigators can choose either a simple analysis that detects several aggrecan fragments released into culture medium only or a more comprehensive study that detects all fragments present in both the medium and the cartilage matrix. The protocol comprises (i) cartilage culture and optional cartilage extraction, (ii) a quick and simple colorimetric assay for quantitating aggrecan and (iii) neoepitope western blotting to identify specific aggrecan fragments partitioning to the medium or cartilage compartments. The crucial difference between the methods for mice and larger animals is that the proportion of aggrecan in a given sample is normalized to total aggrecan rather than to tissue wet weight. This necessary break from tradition arises because tiny volumes of liquid clinging to mouse cartilage can increase the apparent tissue wet weight, causing unacceptable errors. The protocol has broad application for the in vitro analysis of transgenic mice, particularly those with mutations that affect cartilage remodeling, arthritic disease and skeletal development. The protocol is robust, reliable and takes 7-11 d to complete.

Cytokine-induced increases in ADAMTS-4 messenger RNA expression do not lead to increased aggrecanase activity in ADAMTS-5-deficient mice.

OBJECTIVE: To compare the regulation of aggrecanase messenger RNA (mRNA) and enzyme activity by proinflammatory cytokines in primary mouse chondrocytes. METHODS: Primary chondrocytes were isolated from knee epiphyses of 6-8-day-old mice and cultured as monolayers. The cells were incubated with tumor necrosis factor α (TNFα), oncostatin M (OSM), or interleukin-6 (IL-6)/soluble IL-6 receptor, and mRNA levels were measured by quantitative polymerase chain reaction at various time points. To measure aggrecanase activity, the cells were incubated with cytokine in the presence of exogenous aggrecan, and substrate cleavage was measured using antibodies to neoepitopes. RESULTS: Expression of both ADAMTS-4 and ADAMTS-5 mRNA was up-regulated by TNFα and OSM. ADAMTS-5 mRNA expression was also up-regulated by IL-6. Treatment of wild-type mouse chondrocytes with each of the 3 cytokines increased cleavage of aggrecan at Glu(373)↓(374) Ala and Glu(1670)↓(1671) Gly; in chondrocytes lacking ADAMTS-5 activity, there was negligible cleavage at either site despite increased expression of ADAMTS-4 mRNA in the presence of TNFα or OSM. None of the cytokines substantially altered mRNA expression of ADAMTS-1 or ADAMTS-9. CONCLUSION: Despite substantial increases in the expression of ADAMTS-4 mRNA induced by TNFα and OSM, these cytokines induced little if any increase in aggrecanolysis in ADAMTS-5-deficient mouse chondrocytes. Our data show a poor correlation between the level of cytokine-induced ADAMTS-4 mRNA expression and the level of aggrecan-degrading activity in cultured chondrocytes.

Neoepitope antibodies against MMP-cleaved and aggrecanase-cleaved aggrecan.

Neoepitope antibodies recognize the newly created N or C terminus of protein degradation products but fail to recognize the same sequence of amino acids present in intact or undigested protein. Aggrecan neoepitope antibodies have been pivotal in studies determining the contribution of matrix metalloproteinases (MMPs) and aggrecanases to aggrecanolysis. In particular, an antibody to the A(374)RGSV N terminus was instrumental in the landmark discovery of the aggrecanases, ADAMTS-4 and ADAMTS-5. Antibodies to neoepitopes at the major MMP cleavage site DIPEN(341)/(342)FFGVG helped to distinguish MMP-driven aggrecan loss from aggrecanase-driven aggrecan loss and identified a role for MMPs in late-stage disease. More recently, neoepitope antibodies that recognize cleavage sites in the chondroitin sulphate-rich region of aggrecan have been used to show that aggrecanase cleavage proceeds in a defined manner, beginning at the C terminus and proceeding to the signature cleavage at NITEGE(373)/(374)ARGSV in the interglobular domain. Work with the C-terminal neoepitope antibodies has underscored the need to use a suite of neoepitope antibodies to fully describe aggrecanolysis in vitro. In this chapter, we describe the production of two aggrecan neoepitope antibodies as examples: the monoclonal anti-FFGVG antibody (AF-28) and the polyclonal anti-DIPEN antisera.

ADAMTS5 is the major aggrecanase in mouse cartilage in vivo and in vitro.

Aggrecan is the major proteoglycan in cartilage, endowing this tissue with the unique capacity to bear load and resist compression. In arthritic cartilage, aggrecan is degraded by one or more 'aggrecanases' from the ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) family of proteinases. ADAMTS1, 8 and 9 have weak aggrecan-degrading activity. However, they are not thought to be the primary aggrecanases because ADAMTS1 null mice are not protected from experimental arthritis, and cleavage by ADAMTS8 and 9 is highly inefficient. Although ADAMTS4 and 5 are expressed in joint tissues, and are known to be efficient aggrecanases in vitro, the exact contribution of these two enzymes to cartilage pathology is unknown. Here we show that ADAMTS5 is the major aggrecanase in mouse cartilage, both in vitro and in a mouse model of inflammatory arthritis. Our data suggest that ADAMTS5 may be a suitable target for the development of new drugs designed to inhibit cartilage destruction in arthritis, although further work will be required to determine whether ADAMTS5 is also the major aggrecanase in human arthritis.

N-linked keratan sulfate in the aggrecan interglobular domain potentiates aggrecanase activity.

Keratan sulfate is thought to influence the cleavage of aggrecan by metalloenzymes. We have therefore produced a recombinant substrate, substituted with keratan sulfate, suitable for the study of aggrecanolysis in vitro. Recombinant human G1-G2 was produced in primary bovine keratocytes using a vaccinia virus expression system. Following purification and digestion with specific hydrolases, fluorophore-assisted carbohydrate electrophoresis was used to confirm the presence of the monosulfated Gal-GlcNAc6S and GlcNAc6s-Gal disaccharides and the disulfated Gal6S-GlcNAc6S disaccharides of keratan sulfate. Negligible amounts of fucose or sialic acid were detected, and the level of unsulfated disaccharides was minimal. Treatment with keratanases reduced the size of the recombinant G1-G2 by approximately 5 kDa on SDS-PAGE. Treatment with N-glycosidase F also reduced the size of G1-G2 by approximately 5 kDa and substantially reduced G1-G2 immunoreactivity with monoclonal antibody 5-D-4, indicating that keratan sulfate on the recombinant protein is N-linked. Cleavage of G1-G2 by aggrecanase was markedly reduced when keratan sulfate chains were removed by treatment with keratanase, keratanase II, endo-beta-galactosidase, or N-glycosidase F. These results indicate that modification of oligosaccharides in the aggrecan interglobular domain with keratan sulfate, most likely at asparagine residue 368, potentiates aggrecanase activity in this part of the core protein.

Matrix metalloproteinases are not essential for aggrecan turnover during normal skeletal growth and development.

The growth plate is a transitional region of cartilage and highly diversified chondrocytes that controls long bone formation. The composition of growth plate cartilage changes markedly from the epiphysis to the metaphysis, notably with the loss of type II collagen, concomitant with an increase in MMP-13; type X collagen; and the C-propeptide of type II collagen. In contrast, the fate of aggrecan in the growth plate is not clear: there is biosynthesis and loss of aggrecan from hypertrophic cartilage, but the mechanism of loss is unknown. All matrix metalloproteinases (MMPs) cleave aggrecan between amino acids N341 and F342 in the proteinase-sensitive interglobular domain (IGD), and MMPs in the growth plate are thought to have a role in aggrecanolysis. We have generated mice with aggrecan resistant to proteolysis by MMPs in the IGD and found that the mice develop normally with no skeletal deformities. The mutant mice do not accumulate aggrecan, and there is no significant compensatory proteolysis occurring at alternate sites in the IGD. Our studies reveal that MMP cleavage in this key region is not a predominant mechanism for removing aggrecan from growth plate cartilage.

ADAMTS4 cleaves at the aggrecanase site (Glu373-Ala374) and secondarily at the matrix metalloproteinase site (Asn341-Phe342) in the aggrecan interglobular domain.

Two major proteolytic cleavages, one at NITEGE(373)/A(374)RGSVI and the other at VDIPEN(341)/F(342)FGVGG, have been shown to occur in vivo within the interglobular domain of aggrecan. The Glu(373)-Ala(374) site is cleaved in vitro by aggrecanase-1 (ADAMTS4) and aggrecanase-2 (ADAMTS5), whereas the other site, at Asn(341)-Phe(342), is efficiently cleaved by matrix metalloproteinases (MMPs) and by cathepsin B at low pH. Accordingly, the presence of the cleavage products globular domain 1 (G1)-NITEGE(373) and G1-VDIPEN(341) in vivo has been widely interpreted as evidence for the specific involvement of ADAMTS enzymes and MMPs/cathepsin B, respectively, in aggrecan proteolysis in situ. We show here, in digests with native human aggrecan, that purified ADAMTS4 cleaves primarily at the Glu(373)-Ala(374) site, but also, albeit slowly and secondarily, at the Asn(341)-Phe(342) site. Cleavage at the Asn(341)-Phe(342) site in these incubations was due to bona fide ADAMTS4 activity (and not a contaminating MMP) because the cleavage was inhibited by TIMP-3 (a potent inhibitor of ADAMTS4), but not by TIMP-1 and TIMP-2, at concentrations that totally blocked MMP-3-mediated cleavage at this site. Digestion of recombinant human G1-G2 (wild-type and cleavage site mutants) confirmed the dual activity of ADAMTS4 and supported the idea that the enzyme cleaves primarily at the Glu(373)-Ala(374) site and secondarily generates G1-VDIPEN(341) by removal of the Phe(342)-Glu(373) peptide from G1-NITEGE(373). These results show that G1-VDIPEN(341) is a product of both MMP and ADAMTS4 activities and challenge the widely held assumption that this product represents a specific indicator of MMP- or cathepsin B-mediated aggrecan degradation.