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Objective Fibronectin fragments are thought to play a critical role in the initiation and progression of cartilage degradation in arthritis. In a recent study, fibronectin neoepitopes resulting from cleavage of intact fibronectin at the Ala271/Val272 scissile bond, generating an ∼30-kd fragment with the new C-terminus VRAA271 and an ∼50–85-kd fragment with the new N-terminus 272VYQP, were identified in osteoarthritis (OA) cartilage. The present study was undertaken to isolate the enzymes responsible for this cleavage from human OA chondrocytes. Methods Fibronectin-degrading activity in human OA chondrocyte–conditioned medium (OACCM) was purified using conventional chromatography. A fluorescent peptide was developed based on the fibronectin scissile bond 269RAAVal272, and this peptide was used to track fibronectinase activity during purification. Western blotting with antibodies that detect the fibronectin neoepitopes VRAA271 and 272VYQP was used to confirm cleavage of intact fibronectin by the enzymatically active fractions. Mass spectrometry was used to identify the proteins found in the fibronectinase-enriched fractions, with further confirmation by Western blotting. In addition, a recombinant enzyme identified by mass spectrometry was tested by Western blotting and dimethylmethylene blue assay for its ability to produce fibronectin neoepitopes in OA cartilage. Results Purification of OACCM by chromatography resulted in isolation of a fibronectin-degrading enzyme, and mass spectrometry identified ADAM-8 as the fibronectinase present in these preparations. Furthermore, treatment of OA cartilage with recombinant human ADAM-8 promoted cartilage catabolism. Conclusion The results of this study identify ADAM-8 as a fibronectinase in human OA chondrocytes. Because ADAM-8 is capable of producing the fibronectin neoepitopes VRAA271 and 272VYQP in human OA cartilage, this enzyme may be an important mediator of cartilage catabolism.

Summary Objective To develop an in vivo model for rapid assessment of cartilage aggrecan degradation and its pharmacological modulation. Design Tumor necrosis factor-α (TNFα) was injected intra-articularly (IA) in rat knees and aggrecan degradation was monitored at various times following challenge. Articular cartilage was assessed for aggrecan content by Safranin O staining and by immunohistochemistry for the NITEGE epitope. Synovial fluids (SFs) were analyzed for sulfated glycosaminoglycans (GAGs) using the dimethylmethylene blue dye assay and for aggrecan fragments generated by specific cleavage at aggrecanase-sensitive sites by Western blot analysis with neoepitope antibodies. Indomethacin, dexamethasone, and an aggrecanase inhibitor were evaluated for their ability to modulate TNFα-induced proteoglycan degradation in vivo . Results (1) IA injection of TNFα in the knee joint of rats resulted in transient aggrecan degradation and release of aggrecanase-generated aggrecan fragments from the articular cartilage into the SF; (2) a correlation was observed between histologically assessed depletion of aggrecan from the articular cartilage and the appearance of specific neoepitopes in the SF; (3) aggrecan degradation was inhibited by an aggrecanase inhibitor as well as by dexamethasone, but not by the non-steroidal anti-inflammatory drug (NSAID), indomethacin. Conclusion TNFα injection in the knee joints of rats results in rapid transient cartilage proteoglycan degradation, mediated by cleavage at the aggrecanase sites. Biomarker read-out of specific neoepitopes in the SF enables the use of this mechanism-based model for rapid evaluation of aggrecanase-mediated aggrecan degradation in vivo .

ADAMTS-4 and ADAMTS-5 are aggrecanases responsible for the breakdown of cartilage aggrecan in osteoarthritis. Multiple ADAMTS-4 cleavage sites have been described in several matrix proteins including aggrecan, versican, and brevican, but no concise predictive cleavage motif has been identified for this protease. By screening a 13-mer peptide library with a diversity of 10(8), we have identified the ADAMTS-4 cleavage motif E-(AFVLMY)-X(0,1)-(RK)-X(2,3)-(ST)-(VYIFWMLA), with Glu representing P1. Several 13-mer peptides containing this motif, including DVQEFRGVTAVIR and HNEFRQRETYMVF, were shown to be substrates for ADAMTS-4. These peptides were found to be specific substrates for ADAMTS-4 as they were not cleaved by ADAMTS-5. Modification of these peptides with donor (6-FAM) and acceptor (QSY-9) molecules resulted in the development of fluorescence-based substrates with a Km of approximately 35 microM. Furthermore, the role of Glu at P1 and Phe at P1' in binding and catalysis was studied by exploring substitution of these amino acids with the D-isomeric forms. Substitution of P1 with dGlu was tolerable for binding, but not catalysis, whereas substitution of P1' with dPhe precluded both binding and catalysis. Similarly, replacement of Glu with Asp at P1 abolished recognition and cleavage of the peptide. Finally, BLAST results of the ADAMTS-4 cleavage motif identified matrilin-3 as a new substrate for ADAMTS-4. When tested, recombinant ADAMTS-4 effectively cleaved intact matrilin-3 at the predicted motif at Glu435/Ala436 generating two species of 45 and 5 kDa.

ADAMTS-4 (aggrecanase 1) is synthesized as a latent precursor protein that may require activation through removal of its prodomain before it can exert catalytic activity. We examined various proteinases as well as auto-activation under a wide range of conditions for removal of the prodomain and induction of enzymatic activity. The proprotein convertases, furin, PACE4, and PC5/6 efficiently removed the prodomain through cleavage at Arg(212)/Phe(213), generating an active enzyme. Of a broad range of proteases evaluated, only MMP-9 and trypsin were capable of removing the prodomain. In the presence of mercuric compounds, removal of the prodomain through autocatalysis was not observed, nor was it observed at temperatures from 22 to 65 degrees C, at ionic strengths from 0.1 to 1M, or at acidic/neutral pH. At basic pH 8-10, removal of the prodomain by autocatalysis occurred, generating an active enzyme. In conclusion, the pro-form of ADAMTS-4 is not catalytically active and only a limited number of mechanisms mediate its N-terminal activation.

Members of the bone morphogenetic protein (BMP) group of the TGF-beta superfamily have been shown to enhance matrix synthesis and maintain cartilage phenotype in long-term culture. These proteins have also been shown to augment cartilage repair in vivo, and may be of potential therapeutic benefit in the treatment of damaged articular cartilage. The present study was undertaken to examine the effects of BMP-9 on the metabolism of juvenile and adult bovine cartilage in vitro, and to compare the effects to those produced by two previously characterized BMPs: BMP-2 and 13 (CDMP-2). BMP-9 lead to a 7-8-fold stimulation of proteoglycan synthesis at the highest concentration tested, and a 6.4-fold stimulation of collagen synthesis at a concentration of 50 ng/mL in juvenile cartilage. BMP-2 also lead to a 7-8-fold increase in proteoglycan synthesis at the highest concentration tested, and was able to induce collagen synthesis 6.4-fold, but at a concentration of 1000 ng/mL. Proteoglycans isolated from BMP-9 treated cartilage exhibited an increased hydrodynamic size possibly due to increased glycosaminoglycan substitution or decreased C-terminal proteolysis. Consistent with the idea of limited C-terminal proteolysis, BMP-9 treatment lead to a significant reduction in the turnover rate of proteoglycans in juvenile explants. Interestingly, all three BMPs were unable to induce a measurable anabolic response in adult cartilage explants.

Osteoarthritis is characterized by the loss of aggrecan and collagen from the cartilage extracellular matrix. The proteinases responsible for the breakdown of cartilage aggrecan include ADAMTS-4 (aggrecanase 1) and ADAMTS-5 (aggrecanase 2). Post-translational inhibition of ADAMTS-4/-5 activity may be important for maintaining normal homeostasis of aggrecan metabolism, and thus, any disruption to this inhibition could lead to accelerated aggrecan breakdown. To date TIMP-3 (tissue inhibitor of matrix metalloproteinases-3) is the only endogenous inhibitor of ADAMTS-4/-5 that has been identified. In the present studies we identify alpha(2)-macroglobulin (alpha(2)M) as an additional endogenous inhibitor of ADAMTS-4 and ADAMTS-5. alpha(2)M inhibited the activity of both ADAMTS-4 and ADAMTS-5 in a concentration-dependent manner, demonstrating 1:1 stoichiometry with second-order rate constants on the order of 10(6) and 10(5) m(-1) s(-1), respectively. Inhibition of the aggrecanases was mediated by proteolysis of the bait region within alpha(2)M, resulting in physical entrapment of these proteinases. Both ADAMTS-4 and ADAMTS-5 cleaved alpha(2)M at Met(690)/Gly(691), representing a novel proteinase cleavage site within alpha(2)M and a novel site of cleavage for ADAMTS-4 and ADAMTS-5. Finally, the use of the anti-neoepitope antibodies to detect aggrecanase-generated alpha(2)M-fragments in synovial fluid was investigated and found to be uninformative.

Aggrecanase-2 (a disintegrin and metalloproteinase with thrombospondin motifs-5 (ADAMTS-5)), a member of the ADAMTS protein family, is critically involved in arthritic diseases because of its direct role in cleaving the cartilage component aggrecan. The catalytic domain of aggrecanase-2 has been refolded, purified, and crystallized, and its three-dimensional structure determined to 1.4A resolution in the presence of an inhibitor. A high resolution structure of an ADAMTS/aggrecanase protein provides an opportunity for the development of therapeutics to treat osteoarthritis.

ADAMTS-4 (aggrecanase-1) is implicated in the breakdown of articular cartilage and is an attractive target for therapeutic intervention in arthritis. Cleavage of the native substrate, aggrecan, occurs through exosite interactions and peptide sequence recognition. Although expected to be competitive with aggrecan, the hydroxamic acid, SC81956, demonstrated noncompetitive inhibition kinetics with a Ki of 23 nM. The IC50 of SC81956 did not change when aggrecan was varied from 12.8 to 200 nM (0.2-3.3 times the apparent aggrecan Km of 61 nM) but was shifted as expected for a competitive inhibitor when increasing levels of a low molecular weight peptide substrate were added to a fluorogenic peptide assay system. These observations are consistent with a model for aggrecan cleavage where substrate initially binds at an exosite, followed by binding of the appropriate peptide sequence at the active site. A peptide-competitive inhibitor could bind both free enzyme and initial substrate-enzyme exosite complex but would be excluded by the final Michaelis complex. Noncompetitive appearing kinetics for such inhibitors is predicted as long as the equilibrium between the two forms of enzyme-substrate complex significantly favors the initial exosite complex. In support, hydrolysis of a low molecular weight peptide substrate and its inhibition by SC81956 were unaffected by aggrecan concentrations substantially above the Km. These observations suggest that the apparent Km for aggrecan cleavage predominately reflects the exosite interaction. Consequently, the efficacy of active-site inhibitors of ADAMTS-4 will not be limited by competition with native substrate as predicted from the Km determined by traditional kinetic models.